1. Cancer Biology
Download icon

Acid-base transporters and pH dynamics in human breast carcinomas predict proliferative activity, metastasis, and survival

  1. Nicolai J Toft
  2. Trine V Axelsen
  3. Helene L Pedersen
  4. Marco Mele
  5. Mark Burton
  6. Eva Balling
  7. Tonje Johansen
  8. Mads Thomassen
  9. Peer M Christiansen
  10. Ebbe Boedtkjer  Is a corresponding author
  1. Department of Biomedicine, Aarhus University, Denmark
  2. Department of Pathology, Regionshospitalet Randers, Denmark
  3. Department of Surgery, Regionshospitalet Randers, Denmark
  4. Department of Clinical Genetics, University of Southern Denmark, Denmark
  5. Clinical Genome Center, University and Region of Southern Denmark, Denmark
  6. Department of Plastic and Breast Surgery, Department of Clinical Medicine, Aarhus University Hospital, Denmark
Research Article
  • Cited 0
  • Views 453
  • Annotations
Cite this article as: eLife 2021;10:e68447 doi: 10.7554/eLife.68447

Abstract

Breast cancer heterogeneity in histology and molecular subtype influences metabolic and proliferative activity and hence the acid load on cancer cells. We hypothesized that acid-base transporters and intracellular pH (pHi) dynamics contribute inter-individual variability in breast cancer aggressiveness and prognosis. We show that Na+,HCO3 cotransport and Na+/H+ exchange dominate cellular net acid extrusion in human breast carcinomas. Na+/H+ exchange elevates pHi preferentially in estrogen receptor-negative breast carcinomas, whereas Na+,HCO3 cotransport raises pHi more in invasive lobular than ductal breast carcinomas and in higher malignancy grade breast cancer. HER2-positive breast carcinomas have elevated protein expression of Na+/H+ exchanger NHE1/SLC9A1 and Na+,HCO3 cotransporter NBCn1/SLC4A7. Increased dependency on Na+,HCO3 cotransport associates with severe breast cancer: enlarged CO2/HCO3-dependent rises in pHi predict accelerated cell proliferation, whereas enhanced CO2/HCO3-dependent net acid extrusion, elevated NBCn1 protein expression, and reduced NHE1 protein expression predict lymph node metastasis. Accordingly, we observe reduced survival for patients suffering from luminal A or basal-like/triple-negative breast cancer with high SLC4A7 and/or low SLC9A1 mRNA expression. We conclude that the molecular mechanisms of acid-base regulation depend on clinicopathological characteristics of breast cancer patients. NBCn1 expression and dependency on Na+,HCO3 cotransport for pHi regulation, measured in biopsies of human primary breast carcinomas, independently predict proliferative activity, lymph node metastasis, and patient survival.

Introduction

Breast cancer heterogeneity is a challenge in clinical practice and calls for extensive patient stratification. Similarly, the underlying tumor biology needs evaluation in stratified patient populations. Breast cancers classify into five molecular subtypes (normal-like, luminal A and B, HER2-enriched, basal-like) that differ in metabolic and proliferative activity, metastatic potential, therapeutic responsiveness, and prognosis (Dai et al., 2015). Whereas radical surgery can cure most patients with localized breast cancer, disseminated disease requires additional systemic therapy. Available successful therapies target the receptor tyrosine-protein kinase HER2 (ErbB2/neu) and estrogen receptors. However, patients with basal-like/triple-negative breast cancer lack targeted treatment options and currently receive classical chemotherapy (e.g., anthracycline and taxane in combination) with considerable adverse effects (Ávalos-Moreno et al., 2020).

Accelerated intermediary metabolism in breast cancer tissue (Voss et al., 2020) burdens the molecular pathways for acidic waste product elimination. In solid cancer tissue, extracellular pH (pHo) can reach as low as 6.5 (Voss et al., 2020Vaupel et al., 1989), which is distinct from corresponding normal tissue with pHo around 7.3–7.4. The acidity of the extracellular tumor microenvironment challenges intracellular pH (pHi) homeostasis as it inhibits cellular net acid extrusion (Bonde and Boedtkjer, 2017). The changes in metabolic profile and proliferative rate of cancer cells contribute to the acidity of the tumor microenvironment, are important determinants of the malignant phenotype, and shape breast cancer progression (Parks et al., 2017; Persi et al., 2021; Boedtkjer and Pedersen, 2020).

Cells generally eliminate their metabolic acid load via membrane proteins that mediate H+ extrusion (e.g., Na+/H+ exchange, H+-ATPase activity) or HCO3 uptake (e.g., Na+,HCO3 cotransport) (Aalkjaer et al., 2014; Xu et al., 2018; Stransky et al., 2016; see Figure 1B). Tissue relying partly on fermentative glycolysis can also eliminate acidic waste products from metabolism through coupled transport of H+ and lactate via monocarboxylate transporters (Pérez-Escuredo et al., 2016). In human and murine breast cancer tissue analyzed without stratification by molecular subtype, Na+,HCO3 cotransport activity is elevated and protein expression of the Na+,HCO3– cotransporter NBCn1 (SLC4A7) and monocarboxylate transporters MCT1 (SLC16A1) and MCT4 (SLC16A3) are upregulated compared to normal breast tissue (Boedtkjer, 2019; Boedtkjer et al., 2013; Lee et al., 2016; Lee et al., 2018; Lee et al., 2015). Protein expression of the Na+/H+ exchanger NHE1 (SLC9A1) is more variable in primary breast cancer tissue showing unchanged or only moderately elevated levels (Lee et al., 2016; Lee et al., 2015) when compared to normal breast tissue as one unstratified group.

Figure 1 with 1 supplement see all
Cellular net acid extrusion in human breast cancer tissue and normal breast tissue relies on extracellular Na+ and is partially CO2/HCO3-dependent consistent with the expression of Na+,HCO3 cotransporter NBCn1 and Na+/H+ exchanger NHE1.

Moreover, steady-state intracellular pH (pHi) and the capacity for net acid extrusion are elevated in human breast cancer tissue compared to normal breast tissue. (A) Exemplar fluorescence images of 2',7'-bis-(2-carboxyethyl)-5-(and-6)-carboxyfluorescein (BCECF)-loaded organoids freshly isolated from human breast cancer tissue (lower panel) and normal breast tissue (upper panel). The scale bars represent 100 µm. (B) Illustration of the experimental procedure for acute enzymatic isolation of organoids from human breast biopsies and a schematic showing the acid-base transporters involved in pHi control in breast cancer cells. The image was generated with Biorender.com. CA, carbonic anhydrase. (C,D) Traces of NH4+-prepulse-induced pHi dynamics in human breast cancer tissue (C, n=75–76) and normal breast tissue (D, n=48–49). The time scale within the dotted rectangles is expanded in order to improve resolution during the pHi recovery phase. (E) Cellular net acid extrusion activities in presence and nominal absence of CO2/HCO3 were calculated in the pHi range 6.5–6.6 for human breast cancer tissue and normal breast tissue (n=48–76). Figure 1—figure supplement 1 provides a detailed analysis of the net acid extrusion capacity as function of pHi. (F) Initial steady-state pHi in human breast cancer tissue (n=79–80) and normal breast tissue (n=49–50) in presence and nominal absence of CO2/HCO3. (G–I) Representative immunohistochemical images (G) and summarized pathologist-scored protein expression data for NHE1 (H, n=79) and NBCn1 (I, n=79) in human breast carcinomas. The size bars represent 100 µm. Data in panels E and F were compared by mixed-effects analyses followed by Sidak’s multiple comparisons test. ‘Interaction’ reports whether the effect of CO2/HCO3 varies between breast cancer and normal breast tissue. *p<0.05, ***p<0.001 vs. normal breast tissue under similar conditions. Figure 1—source data 1 contains the data pertaining to this figure and the de-identified clinicopathological information used to stratify data in Figure 2, 3, 4, 5, 7 and 8 and Figure 2—figure supplement 1; this information is combined in the multiple linear and logistic regression analyses illustrated in Figure 6.

Figure 1—source data 1

Data file containing NBCn1 and NHE1 protein expression levels, steady-state intracellular pH (pHi) values, and net acid extrusion capacities linked to de-identified clinical and pathological patient characteristics.

Note that this source data file pertains to Figure 1 but it also contains the clinicopathological information used for stratification in Figures 25, 7, and 8, and Figure 2—figure supplement 1; for the multiple linear and logistic regression analyses in Figure 6; and for the plots in Figure 6—figure supplement 1.

https://cdn.elifesciences.org/articles/68447/elife-68447-fig1-data1-v2.xlsx

Net acid extrusion from cancer cells elevates the cytosolic pH and acidifies the outer cell surface and interstitial space. In various model systems, acid-base transporters can modify carcinogenesis and the behavior of cancer cells including cancer cell proliferation, migration, and invasion (Boedtkjer and Pedersen, 2020; Amith and Fliegel, 2017; Stock and Pedersen, 2017). Although detailed molecular mechanisms are not yet established, elevated pHi maintains metabolic activity (Parks et al., 2013), increases DNA and protein synthesis (Pedersen, 2006), and accelerates cell cycle progression (Flinck et al., 2018) in cultured cell lines. In accordance, mice with disrupted expression of NBCn1 show delayed tumor development and decelerated tumor growth when tested using models of carcinogen- and ErbB2-induced breast cancer (Lee et al., 2016; Lee et al., 2018).

Acidification at the outer cell surface depends on the rate of net acid transfer across the cell membrane and on diffusion hindrances that limit exchange with the bulk interstitial solution and the blood stream. Cell surface pH can modify cell-cell and cell-matrix interactions (Stock et al., 2005; Riemann et al., 2019), pH gradients from the leading to the rear end of cells can promote directional migration (Stock et al., 2007; Boedtkjer et al., 2016), and interstitial acidification of the tumor microenvironment has potential for modifying anti-cancer immune responses (Cassim and Pouyssegur, 2019). Indeed, cancer progression through metastasis and development of treatment resistance have been reported sensitive to inhibition of Na+/H+ exchangers (Amith and Fliegel, 2017; Stock and Pedersen, 2017); however, several anti-cancer effects of pharmacologically inhibiting Na+/H+ exchange appear only partly pH-dependent (Boedtkjer et al., 2012; Loo et al., 2012; Schwab et al., 2012) and can be caused by NHE1-independent toxicity due to intracellular drug accumulation (Rolver et al., 2020).

Despite the molecular and mechanistic insights from human cultured cell lines and inbred mouse models, the consequences of pH deregulation in human cancer tissue remain unclear. In particular, mechanisms of pH control in breast cancer tissue and their consequences for disease progression were previously explored in models that do not reflect the heterogeneity of human breast cancer. In the current study, we investigated an extensive cohort of human breast cancer patients, sufficiently large to reflect the heterogeneity of acid-base conditions and the variation in cellular handling of metabolic waste products. We tested the hypotheses that (a) specific clinical and pathological characteristics accelerate cellular net acid extrusion and determine the underlying molecular mechanisms of pH regulation in human breast cancer tissue; and (b) the capacity for cellular net acid extrusion, the steady-state pHi level, and the expression of acid-base transporters in human breast cancer tissue predict the severity of disease.

Results

We sampled human tissue biopsies from an extensive cohort of 110 women with breast cancer (Table 1) and evaluated pHi dynamics based on organoids freshly isolated from the breast cancer tissue and corresponding normal breast tissue (Figure 1). Within this patient population, we stratified the pHi dynamics and the NHE1 and NBCn1 expression levels by histopathology (Figure 2 and Figure 2—figure supplement 1), malignancy grade (Figure 3), estrogen receptor status (Figure 4), and HER2 status (Figure 5); and adjusted for variation in other clinical and pathological characteristics (Figure 6). We then explored how the pHi dynamics and the NHE1 and NBCn1 protein expression levels relate to cancer cell proliferation (Figure 7) and lymph node metastasis (Figure 8). Finally, we evaluated how variation in expression levels for acid-base transporters influence patient survival within individual breast cancer molecular subtypes (Figures 9 and 10, and Figure 10—figure supplements 1 and 2).

Figure 2 with 2 supplements see all
Na+,HCO3 cotransport is critical for cellular net acid extrusion and steady-state intracellular pH (pHi) in human invasive ductal and, particularly, lobular breast carcinomas.

(A,B) Traces of NH4+-prepulse-induced pHi dynamics in human invasive lobular (A, n=8–9) and ductal (B, n=60–62) breast carcinomas. The time scale within the dotted rectangles is expanded in order to improve resolution during the pHi recovery phase. (C) Cellular net acid extrusion activities in presence and nominal absence of CO2/HCO3 were calculated in the pHi range 6.5–6.6 for human invasive lobular and ductal breast carcinomas (n=8–62). Figure 2—figure supplement 2 provides a detailed analysis of the net acid extrusion capacity as function of pHi. (D) Initial steady-state pHi in human invasive lobular (n=9) and ductal (n=64–65) breast carcinomas in presence and nominal absence of CO2/HCO3. Data in panels C and D were compared by mixed-effects analyses. ‘Interaction’ reports whether the effect of CO2/HCO3 varies between human invasive lobular and ductal breast carcinomas. (E,F) Summarized pathologist-scored, immunohistochemistry-based protein expression data for NHE1 (E) and NBCn1 (F) in human invasive lobular (n=8) and ductal (n=65) breast carcinomas. Protein expression in human invasive lobular and ductal breast carcinomas was compared by χ2 tests for trend. Figure 2—figure supplement 1 provides data from mucinous adenocarcinomas.

Figure 3 with 1 supplement see all
Steady-state intracellular pH (pHi) is elevated in human breast carcinomas of high malignancy grade due to cellular CO2/HCO3-dependent net acid extrusion.

(A–C) Traces of NH4+-prepulse-induced pHi dynamics in human invasive ductal breast carcinomas of malignancy grades I (A, n=12–14), II (B, n=34), and III (C, n=14). The time scale within the dotted rectangles is expanded in order to improve resolution during the pHi recovery phase. (D) Cellular net acid extrusion activities in presence and nominal absence of CO2/HCO3 were calculated in the pHi range 6.5–6.6 for human invasive ductal breast carcinomas of malignancy grades I, II, and III (n=12–34). Figure 3—figure supplement 1 provides a detailed analysis of the net acid extrusion capacity as function of pHi. (E) Initial steady-state pHi in human invasive ductal breast carcinomas of malignancy grades I (n=14–15), II (n=35), and III (n=15) in presence and nominal absence of CO2/HCO3. Data in panels D and E were compared by mixed-effects analyses and repeated-measures one-way ANOVA with post-test for linear trend. ‘Interaction’ reports whether the effect of CO2/HCO3 varies between human breast carcinomas of malignancy grades I, II, and III. (F, G) Summarized pathologist-scored, immunohistochemistry-based protein expression data for NHE1 (F, n=65) and NBCn1 (G, n=65) in human invasive ductal carcinomas stratified by malignancy grade. Protein expression in human breast carcinomas of malignancy grades I, II, and III was compared by χ2 tests.

Figure 4 with 1 supplement see all
Intracellular pH (pHi) is elevated in estrogen receptor-negative breast cancer.

(A,B) Traces of NH4+-prepulse-induced pHi dynamics in human breast carcinomas stratified by estrogen receptor status (A: negative, n=6; B: positive, n=63–64). The time scale within the dotted rectangles is expanded in order to improve resolution during the pHi recovery phase. (C) Cellular net acid extrusion activities in presence and nominal absence of CO2/HCO3 were calculated in the pHi range 6.5–6.6 for human breast carcinomas stratified by estrogen receptor status (n=6–64). Figure 4—figure supplement 1 provides a detailed analysis of the net acid extrusion capacity as function of pHi. (D) Initial steady-state pHi in human estrogen receptor-negative (0–15% ER+, n=7–8) and -positive (90–100% ER+, n=66–67) breast carcinomas. Data in panels C and D were compared by mixed-effects analyses. ‘Interaction’ reports whether the effect of CO2/HCO3 varies between estrogen receptor-negative and -positive breast carcinomas. (E,F) Pathologist-scored, immunohistochemistry-based protein expression levels for NHE1 (E) and NBCn1 (F) in human breast carcinomas (n=73) stratified by estrogen receptor status. Protein expression in human estrogen receptor-negative and -positive breast carcinomas was compared by χ2 tests for trend.

Figure 5 with 1 supplement see all
Protein expression of NHE1 and NBCn1 is elevated in HER2-positive human breast cancer.

(A,B) Traces of NH4+-prepulse-induced intracellular pH (pHi) dynamics in human HER2-positive (A, n=9–11) and HER2-normal (B, n=58–61) breast carcinomas. The time scale within the dotted rectangles is expanded in order to improve resolution during the pHi recovery phase. (C) Cellular net acid extrusion activities in the presence and nominal absence of CO2/HCO3 were calculated in the pHi range 6.5–6.6 for human breast carcinomas stratified by HER2 status (n=9–61). Figure 5—figure supplement 1 provides a detailed analysis of the net acid extrusion capacity as function of pHi. (D) Initial steady-state pHi in HER2-positive (n=10–11) and -normal (n=63) human breast carcinomas. Data in panels C and D were compared by mixed-effects analyses. ‘Interaction’ reports whether the effect of CO2/HCO3 varies between HER2-positive and -normal breast carcinomas. (E,F) Summarized pathologist-scored, immunohistochemistry-based protein expression data for NHE1 (E) and NBCn1 (F) in human breast carcinomas (n=73) stratified by HER2 status. Protein expression in human HER2-positive and -normal breast carcinomas was compared by χ2 tests for trend.

Figure 6 with 1 supplement see all
Histology, malignancy grade, and receptor expression profiles are independent predictors of intracellular pH (pHi) dynamics and acid-base transporter expression in human invasive lobular and ductal breast carcinomas.

(A–D) Multiple linear regression analyses show the independent influences of patient age, tumor size, histology, malignancy grade, and expression of estrogen and HER2 receptors on steady-state pHi and net acid extrusion capacity in human breast cancer tissue (n=78). Data were adjusted for inter-investigator variation. Figure 6—figure supplement 1 provides plots of steady-state pHi and net acid extrusion as functions of tumor size and patient age. (E,F) Ordinal logistic regression analyses show the independent influences of patient age, tumor size, histology, malignancy grade, and expression of estrogen and HER2 receptors on protein expression levels for NHE1 (E) and NBCn1 (F) in human breast cancer tissue (n=73).

Figure 7 with 1 supplement see all
Steady-state intracellular pH (pHi) is elevated in human breast carcinomas with high proliferative activity (elevated Ki67 index).

(A,B) Traces of NH4+-prepulse-induced pHi dynamics in breast carcinomas with high (A, 35–90% Ki67+, n=19–20) and low (B, 0–30% Ki67+, n=49–51) Ki67 index. The time scale within the dotted rectangles is expanded in order to improve resolution during the pHi recovery phase. (C) Cellular net acid extrusion activities in presence and nominal absence of CO2/HCO3 were calculated in the pHi range 6.5–6.6 for human breast carcinomas stratified by Ki67 index (n=19–51). Figure 7—figure supplement 1 provides a detailed analysis of the net acid extrusion capacity as function of pHi. (D) Initial steady-state pHi in breast carcinomas with high (n=20) and low (n=53–54) Ki67 index. Data in panels C and D were compared by mixed-effects analyses. ‘Interaction’ reports whether the effect of CO2/HCO3 varies between breast carcinomas with high and low Ki67 index. (E,F) Summarized pathologist-scored, immunohistochemistry-based protein expression levels for NHE1 (E) and NBCn1 (F) in human breast carcinomas (n=73) stratified by Ki67 index. Protein expression in human breast carcinomas of low and high Ki67 index was compared by χ2 tests for trend. (G) Results of binominal logistic regression analyses where the influence of the acid-base parameters and transporter expression levels on cellular proliferation was adjusted for patient age, tumor size, histology, malignancy grade, estrogen receptor status, HER2 status, and inter-investigator variation (n=73–78). Odds ratios (OR) are given for variables showing statistically significant association.

Figure 8 with 1 supplement see all
Primary breast cancer tissue from patients with axillary lymph node metastases shows higher Na+,HCO3 cotransport activity during intracellular acidification, higher NBCn1 expression, and lower NHE1 expression than breast cancer tissue from patients without metastases.

(A,B) Traces of NH4+-prepulse-induced intracellular pH (pHi) dynamics in primary breast carcinomas from patients with (A, n=25–28) and without (B, n=42–44) axillary lymph node metastases. Time scales within the dotted rectangles are expanded to improve resolution during the pHi recovery. (C) Cellular net acid extrusion activities in presence and absence of CO2/HCO3 calculated in the pHi range 6.5–6.6 for primary breast carcinomas stratified by axillary lymph node status (n=25–44). Figure 8—figure supplement 1 provides detailed analysis of net acid extrusion capacities as function of pHi. (D) Initial steady-state pHi in primary breast carcinomas from patients with (n=28) and without (n=45–46) lymph node metastases. Data in panels C and D were compared by mixed-effects analyses. ‘Interaction’ reports whether the effect of CO2/HCO3 varies between breast carcinomas from patients with and without lymph node metastases. (E,F) Summarized pathologist-scored, immunohistochemistry-based protein expression levels for NHE1 (E) and NBCn1 (F) in primary breast carcinomas from patients with (n=24) and without (n=49) lymph node metastases. Protein expression data were compared by χ2 tests for trend. (G) Results of binominal logistic regression analyses where influences of acid-base parameters and transporter expression levels on lymph node metastasis were adjusted for patient age, tumor size, histology, malignancy grade, estrogen receptor status, HER2 status, and inter-investigator variation (n=73–78). Odds ratios (OR) are given for variables showing statistically significant association.

Figure 9 with 1 supplement see all
The levels of SLC9A1 mRNA, encoding NHE1, vary among breast cancer subtypes, and high SLC9A1 expression is associated with improved survival in patients with luminal A breast cancer.

(A) Variation in SLC9A1 mRNA levels among patients with different breast cancer subtypes (n=135–344). Expression data were compared by one-way ANOVA followed by Tukey’s post-test. Figure 9—figure supplement 1 provides data on mRNA expression of ESR1, PGR, ERBB2, MKI67, LDHA, PECAM1, and CD34 in breast cancer tissue of the different molecular subtypes. Figure 9—source data 1 provides results from correlation analyses between SLC9A1 mRNA expression and the mRNA levels for ESR1, PGR, ERBB2, SLC4A7, SLC16A1, and SLC16A3. *p<0.05, ***p<0.001. (B–G) Survival curves stratified by SLC9A1 mRNA levels in patients with different breast cancer subtypes. The ticks on the curves represent censored subjects. Survival data were compared by Mantel-Cox and Gehan-Breslow-Wilcoxon tests. HR, hazard ratio.

Figure 9—source data 1

The mRNA expression for SLC9A1 correlates with that of ESR1 and ERBB2.

When adjusted for expression of ESR1, PGR, and ERBB2, the mRNA expression of SLC9A1 does not correlate with that of SLC4A7, SLC16A1, and SLC16A3 (n=409–1162).

https://cdn.elifesciences.org/articles/68447/elife-68447-fig9-data1-v2.docx
Figure 10 with 2 supplements see all
The levels of SLC4A7 mRNA, encoding NBCn1, vary among breast cancer subtypes, and high SLC4A7 expression is associated with poor survival in patients with luminal A or basal-like breast cancer.

(A) Variation in SLC4A7 mRNA levels among patients with different breast cancer subtypes (n=135–344). Expression data were compared by one-way ANOVA followed by Tukey’s post-test. *p<0.05, ***p<0.001. Figure 10—source data 1 provides results from correlation analyses between SLC4A7 mRNA expression and the mRNA levels for ESR1, PGR, ERBB2, SLC9A1, SLC16A1, and SLC16A3. (B–G) Survival curves stratified by SLC4A7 mRNA levels in patients with different breast cancer molecular subtypes. The ticks on the curves represent censored subjects. Survival data were compared by Mantel-Cox and Gehan-Breslow-Wilcoxon tests. HR, hazard ratio. Figure 10—figure supplement 1 provides results on the expression and survival consequences of SLC16A1 mRNA. Figure 10—figure supplement 2 provides results on the expression and survival consequences of SLC16A3 mRNA.

Figure 10—source data 1

The mRNA expression for SLC4A7 correlates with that of ESR1, PGR, and ERBB2.

When adjusted for expression of ESR1, PGR, and ERBB2, the mRNA expression of SLC4A7 does not correlate with that of SLC9A1, SLC16A1, and SLC16A3 (n=409–1162).

https://cdn.elifesciences.org/articles/68447/elife-68447-fig10-data1-v2.docx
Table 1
Clinical and pathological characteristics of the patient cohort investigated for intracellular pH (pHi) dynamics and protein expression.
Number of patients110
Patient age (years; median, interquartile range)64.5 (56–74)
Tumor size (mm; median, interquartile range)18 (14–26)
Histological type
Invasive ductal carcinomas92 (84%)
Invasive lobular carcinomas92 (84%)
Mucinous adenocarcinomas5 (5%)
Tubular carcinoma2 (2%)
Pleomorphic lobular carcinoma1 (1%)
HER2 receptor status
Normal95 (86%)
Overexpression or gene amplification15 (14%)
Estrogen receptor status
90–100% ER+ cells99 (90%)
0–15% ER+ cells11 (10%)
Malignancy grade
I31 (28%)
II52 (47%)
III22 (20%)
Not graded5 (5%)
Axillary lymph node status
Negative69 (63%)
Isolated tumor cells15 (14%)
Micro-metastases5 (5%)
Macro-metastases21 (19%)
Ki67 index
0–30% Ki67+ cells82 (75%)
35–90% Ki67+ cells28 (25%)

The elevated net acid extrusion capacity is Na+- and partly CO2/HCO3-dependent in human breast cancer tissue

We freshly isolated organoids from human breast biopsies by partial collagenase digestion (Figure 1A,B). We prepared the organoids in immediate continuation of the breast-conserving surgery and investigated them directly after isolation without culture in order to avoid phenotypic changes. We previously confirmed that organoids freshly isolated from breast tissue biopsies consist predominantly of cytokeratin-19-positive epithelial cells with few smooth muscle α-actin-positive myofibroblasts (Lee et al., 2016; Lee et al., 2015).

We experimentally induced intracellular acidification by NH4+-prepulse technique (Boron and De Weer, 1976; Boedtkjer and Aalkjaer, 2012) as illustrated in Figure 1C for breast cancer tissue and Figure 1D for normal breast tissue. Addition of NH4Cl to the experimental bath solution acutely elevates pHi as NH3 traverses plasma membranes and binds H+ from the cytosol. The subsequent gradual decline of pHi occurs when NH4+ enters cells—predominantly through plasma membrane K+ conductances (e.g., K+ channels and Na+/K+-ATPases)—and base equivalents are extruded, for instance, through Cl/HCO3 exchange. Washout of NH4Cl then causes NH3 to rapidly leave the cells; and intracellular acidification ensues when H+, as a consequence, is liberated from intracellular NH4+ and accumulates in the cytosol.

Assessing the patient population as a whole, we observed that cellular net acid extrusion during intracellular acidification was almost fully dependent on extracellular Na+ and relied on both CO2/HCO3-dependent and -independent transport mechanisms (Figure 1C,D and Figure 1—figure supplement 1). As schematically illustrated in Figure 1B, these observations support previous reports (Boedtkjer et al., 2013; Lee et al., 2015) that Na+,HCO3 cotransporters and Na+/H+ exchangers are mainly responsible for cellular net acid extrusion in human breast cancer tissue. We observed upregulated capacity for Na+/H+ exchange activity during carcinogenesis based on the faster Na+-dependent pHi recovery in organoids freshly isolated from breast cancer tissue compared to normal breast tissue when evaluated in the nominal absence of CO2/HCO3 (Figure 1C,D). Likewise, we detected contribution from Na+,HCO3 cotransport as the ability to recover pHi faster and at more alkaline pHi when CO2/HCO3 was present (Figure 1C,D).

The pHi traces (Figure 1C,D) recorded from freshly processed breast tissue biopsies allowed us to evaluate (a) the initial steady-state pHi level with extracted values summarized in Figure 1F and (b) the capacity for cellular net acid extrusion during pHi recovery from NH4+-prepulse-induced acidification (dotted rectangles in Figure 1C,D) with calculated values summarized in Figure 1E. As the acid extrusion mechanisms activate at low pHi (Figure 1—figure supplement 1), their activities must be compared at equivalent pHi values: at pH6.5–6.6, we demonstrated a greater capacity for net acid extrusion in human breast cancer tissue compared to normal breast tissue, whether examined in the presence or nominal absence of CO2/HCO3 (Figure 1E). The net acid extrusion capacity in breast cancer tissue was upregulated predominantly in the near-neutral pHi range (compare Figure 1—figure supplement 1A and B), which was also reflected in an elevated steady-state pHi when human breast cancer tissue was investigated under similar experimental conditions as normal breast tissue either with or without CO2/HCO3 present (Figure 1F). The drop in steady-state pHi in response to nominal omission of CO2/HCO3 (Figure 1F) supports a greater contribution from Na+,HCO3 cotransport compared to anion exchange in the near-physiological pHi range (Figure 1B).

In congruence with the functional observations, we identified prominent protein expression of NHE1 and NBCn1 in the human breast cancer tissue and considerable inter-individual variation within the evaluated patient population (Figure 1G–I).

Na+,HCO3 cotransport is more pronounced in invasive lobular than ductal breast carcinomas

Breast cancer is histopathologically diverse, but invasive ductal and lobular breast carcinomas are most frequent (Table 1). We evaluated whether the mechanisms of net acid extrusion differ between tumors of separate histopathologies (Figure 2 and Figure 2—figure supplement 1). Invasive lobular (Figure 2A and Figure 2—figure supplement 2A) and ductal (Figure 2B and Figure 2—figure supplement 2B) breast carcinomas both showed dual dependency on Na+/H+ exchange and Na+,HCO3 cotransport for pHi regulation. We observed a tendency toward greater relative capacity for net acid extrusion via Na+,HCO3 cotransport during intracellular acidification in invasive lobular than ductal breast carcinomas (Figure 2C). This increased influence of Na+,HCO3 cotransport in invasive lobular carcinomas was more pronounced with respect to steady-state pHi control where it reached statistical significance both before (Figure 2D) and after (Figure 6B) we adjusted for variation in other clinicopathological characteristics (i.e., patient age, tumor size, malignancy grade, estrogen receptor expression, and HER2 status).

Expression of NHE1 and NBCn1 protein was generally moderate in invasive lobular carcinomas and showed less inter-individual variation than observed for invasive ductal carcinomas (Figures 2E,F and 6E,F).

Although our functional data cover too few patients with mucinous adenocarcinomas (n=5) to perform a formal comparison with the other histopathologies, the pattern of pHi regulation (Figure 2—figure supplement 1A) was similar to that observed in invasive lobular and ductal breast carcinomas (Figure 2A,B). Notably, the net acid extrusion capacity (Figure 2—figure supplement 1B,C) and steady-state pHi (Figure 2—figure supplement 1D) in mucinous adenocarcinomas confirmed the dual dependency on Na+/H+ exchange and Na+,HCO3 cotransport.

Na+,HCO3 cotransport elevates pHi more in breast carcinomas of high malignancy grade

Malignancy grading of breast cancer tissue provides valuable prognostic information (Rakha et al., 2010), and we show here that the malignancy grade is also reflected in the pHi dynamics of the breast cancer tissue (Figure 3A–C). We observed a tendency toward a greater net acid extrusion capacity in higher malignancy grade breast carcinomas (Figure 3D, Figure 3—figure supplement 1, and Figure 6D). More prominently, we found that the Na+,HCO3 cotransport activity established an increasingly elevated steady-state pHi in breast carcinomas with higher malignancy grade, and this effect reached statistical significance both before (Figure 3E) and after (Figure 6B) adjustment for other clinicopathological characteristics.

Protein expression levels for NHE1 and NBCn1 did not significantly differ between breast cancer tissue of different malignancy grades (Figures 3F,G and 6E,F).

Na+/H+ exchange elevates pHi more in breast carcinomas with low estrogen receptor expression

Estrogen receptors play important roles in mammary gland development (Shyamala, 1997). The expression of estrogen receptors in breast cancer tissue varied considerably between patients: as shown in Table 1, there was a clear distinction between a large group of patients with tumors showing widespread estrogen receptor expression (≥90% positive cells) and a smaller subset of patients with tumors showing very limited estrogen receptor expression (≤15% positive cells). Previous studies have found that estrogen receptors are expressed in around 10% of the cells in normal breast epithelium (Oh et al., 2017).

We detected no obvious effect of estrogen receptor expression on the net acid extrusion capacity of human breast cancer tissue during intracellular acidification (Figure 4A–C, Figure 4—figure supplement 1, and Figure 6C,D). However, as illustrated in Figure 4D, we observed elevated steady-state pHi in breast cancer tissue with no or very low expression (0–15% ER+) compared to breast cancer tissue with high expression (90–100% ER+) of estrogen receptors. This effect was explained by a greater contribution of Na+/H+ exchange activity—as it was evident both in the presence and absence of CO2/HCO3—and became statistically significant after adjustment for other clinicopathological characteristics (Figure 6A).

The protein expression levels for NHE1 and NBCn1 were not significantly influenced by estrogen receptor status (Figures 4E,F and 6E,F).

HER2 receptors increase expression of NBCn1 and NHE1 protein

Growth factor input facilitates cancer cell proliferation and the malignant phenotype of cancer cells; and the functional implications of HER2 receptors are amplified by overexpression or gene amplification in 10–20% of breast cancer patients (Table 1) and less commonly by activating somatic mutations (Connell and Doherty, 2017).

HER2 receptor status did not significantly influence the net acid extrusion capacity of breast cancer tissue during intracellular acidification (Figure 5A–C and Figure 5—figure supplement 1). Whereas we observed a strong tendency toward a higher CO2/HCO3-dependent rise in steady-state pHi in HER2-positive tumors (Figure 5D), this effect was substantially attenuated when adjusted for other clinicopathological characteristics (Figure 6B).

The protein expression levels of NHE1 as well as NBCn1 were elevated in breast carcinomas with HER2 overexpression or gene amplification both before (Figure 5E,F) and after (Figure 6E,F) adjustment for other clinicopathological characteristics.

Patient age and tumor size

We next plotted steady-state pHi levels (Figure 6—figure supplement 1A,B) and capacities for net acid extrusion during intracellular acidification (Figure 6—figure supplement 1C,D) as functions of tumor size (Figure 6—figure supplement 1A,C) and patient age (Figure 6—figure supplement 1B,D). The CO2/HCO3-independent net acid extrusion capacity decreased as function of patient age before (Figure 6—figure supplement 1D) but not after (Figure 6C) adjustment for other clinicopathological characteristics, whereas none of the other plots revealed significant correlations (Figure 6A–D and Figure 6—figure supplement 1A–D).

Clinicopathological characteristics independently predict acid-base dynamics and expression of acid-base transporters

The patient groups illustrated in Figures 25 are stratified by individual clinicopathological parameters. We next performed multiple linear and logistic regression analyses to control for multiple comparisons, take into account unbalanced distributions within individual groups, and identify clinical and pathological characteristics that independently predict steady-state pHi (Figure 6A,B), net acid extrusion capacity (Figure 6C,D), or acid-base transporter expression (Figure 6E,F).

Estrogen receptor status was the predominant influence on steady-state pHi in absence of CO2/HCO3 (Figure 6A), whereas histology and malignancy grade were the predominant modifiers of the CO2/HCO3-dependent increase in steady-state pHi (Figure 6B).

None of the tested clinical or pathological parameters (i.e., patient age, tumor size, histology, malignancy grade, estrogen receptor status, HER2 status) showed independent value to predict the capacity for CO2/HCO3-independent net acid extrusion mediated by Na+/H+ exchange (Figure 6C) or the capacity for CO2/HCO3-dependent net acid extrusion mediated by Na+,HCO3 cotransport (Figure 6D) during intracellular acidification.

The NHE1 and NBCn1 protein expression levels in the human breast cancer tissue were independently elevated by HER2 overexpression or gene amplification (Figure 6E,F).

Elevated pHi predicts high proliferative activity

The accentuated metabolism of cancer cells supplies chemical intermediates and energy for cell proliferation. However, the accelerated metabolism also leads to a higher cellular acid load and risk of intracellular acidification, which can limit further cell proliferation (Boedtkjer and Pedersen, 2020).

When we stratified the patient cohort by expression of proliferative markers (Figure 7A–D and Figure 7—figure supplement 1), tumors with high Ki67 index had more elevated steady-state pHi than tumors with low Ki67 index under experimental conditions where Na+,HCO3 cotransport was active (Figure 7D). In contrast, we saw no difference in steady-state pHi between the groups in nominal absence of CO2/HCO3 (Figure 7D). The measured CO2/HCO3-dependent rise in pHi carried independent predictive value (odds ratio of 3.12 for a 0.1 increase in pH) to the identification of patients with elevated Ki67 index (Figure 7G). The rate of pHi recovery and capacity for net acid extrusion during intracellular acidification did not differ between tumors with high and low Ki67 index (Figure 7C and Figure 7—figure supplement 1).

We demonstrated elevated NBCn1 protein expression in the patient group with high compared to low Ki67 index (Figure 7F) although this effect was not quite significant after we adjusted for other clinicopathological characteristics (Figure 7G). We found no difference in NHE1 protein expression between tumors with high and low Ki67 index (Figure 7E).

Increased Na+,HCO3 cotransport capacity and NBCn1 expression predict lymph node metastasis

Breast cancer prognosis critically depends on the invasive potential of the cancer cells, and acid-base transporters are implicated in key metastatic steps including cell migration and extracellular matrix degradation (Boedtkjer and Pedersen, 2020). Thus, we evaluated whether expression and function of acid-base transporters varied between primary breast cancer tissue from patients with and without lymph node metastases (Figure 8 and Figure 8—figure supplement 1). We compared primary breast cancer tissue from patients without detectable tumor cells in the axillary lymph nodes to primary breast cancer tissue from patients with axillary lymph nodes containing macro-metastases, micro-metastases, or isolated tumor cells.

The initial CO2/HCO3-dependent pHi recovery from NH4+-prepulse-induced intracellular acidification (Figure 8A,B) and the corresponding Na+,HCO3 cotransport activity (Figure 8C and Figure 8—figure supplement 1) were accelerated in primary breast cancer tissue from patients with axillary lymph node metastases. The Na+,HCO3– cotransport activity quantified at pH6.5–6.6 carried independent predictive value to identify patients with axillary lymph node metastases (odds ratio of 1.54 for each 10 mM/min increase; Figure 8G). The capacity of the human primary breast carcinomas for Na+/H+ exchange activity was not significantly different between patients with and without detected lymph node metastases (Figure 8C,G and Figure 8—figure supplement 1). We also did not observe any differences in steady-state pHi—in the presence or absence of CO2/HCO3—between primary breast carcinomas from patients with or without identified axillary lymph node metastases (Figure 8D,G).

In congruence with the enhanced Na+,HCO3 cotransport activity, we identified increased NBCn1 protein expression in primary breast cancer tissue from patients with lymph node metastases (Figure 8F). Thus, elevated NBCn1 protein expression was an independent predictor of lymph node metastasis with an odds ratio of 1.72 for a single-unit step increase on the applied 6-point expression scale (Figure 8G). The selective importance of Na+,HCO3 cotransport and NBCn1 expression was supported by our finding that NHE1 protein expression was reduced in primary breast cancer tissue from patients with lymph node metastases (Figure 8E) and qualified as an independent predictor negatively related to metastatic risk with an odds ratio of 0.60 (Figure 8G).

mRNA expression levels for acid-base transporters predict patient survival

We next evaluated whether the biological implications of acid-base transporters in breast cancer tissue have consequences for patient prognosis. We used transcriptomics data to stratify a patient cohort of nearly 1500 breast cancer patients by their mRNA expression levels for acid-base transporters. We then studied how the survival proportions developed over time in the entire patient population and in subpopulations with well-defined molecular subtypes (i.e., normal-like, luminal A and B, HER2-enriched, and basal-like breast cancer). We expect no straightforward proportionality between mRNA, protein, and function when comparing across molecular subtypes or clinicopathological characteristics driven by different carcinogenic mechanisms. This should be kept in mind particularly when interpreting survival analyses performed across the whole unstratified patient population (Figures 9B and 10B, and Figure 10—figure supplements 1B and 2B). Focusing on individual molecular subtypes, the relationship from mRNA to protein and function is likely much simpler, and our emphasis is therefore on the survival analyses performed after molecular subtype stratification (Figure 9C–G, Figure 10C–G, and Figure 10—figure supplements 1C–G and 2C–G).

The breast cancer molecular subtypes reflect differences in sex hormone (estrogen and progesterone; Figure 9—figure supplement 1A,B) and growth factor (HER2; Figure 9—figure supplement 1C) receptor expression. The individual breast cancer molecular subtypes also showed systematic differences in expression of proliferative markers (MKI67 mRNA; Figure 9—figure supplement 1D) and lactate dehydrogenase involved in fermentative glycolysis (LDHA mRNA; Figure 9—figure supplement 1E), consistent with the notion that malignancy gradually increases from normal-like and luminal A across luminal B and HER2-enriched to basal-like breast cancer (Dai et al., 2015). In contrast, the angiogenic markers PECAM1 and CD34 showed the highest mRNA expression in normal-like breast cancer (Figure 9—figure supplement 1F,G).

The level of SLC9A1 mRNA, encoding NHE1, was most abundant in normal-like, luminal A, and HER2-enriched breast cancer, lower in luminal B breast cancer, and lowest in basal-like breast cancer (Figure 9A). The SLC9A1 mRNA expression level was not associated with altered survival for the whole population of breast cancer patients (Figure 9B) or for breast cancer patients suffering from normal-like (Figure 9C), luminal B (Figure 9E), HER2-enriched (Figure 9F), or basal-like (Figure 9G) breast cancer. However, patients with high SLC9A1 mRNA expression suffering from luminal A breast cancer showed significantly improved survival (hazard ratio 0.552) compared to patients with low SLC9A1 mRNA expression (Figure 9D), which is consistent with our observation of reduced lymph node metastasis among patients with high NHE1 protein expression (Figure 8G).

SLC4A7 mRNA, encoding NBCn1, was expressed at the highest level in luminal A breast cancer, at intermediate level in normal-like and luminal B breast cancer, and at lowest level in HER2-enriched and basal-like breast cancer (Figure 10A). The high overall SLC4A7 expression level in luminal A breast cancer argues for a prominent role of NBCn1 in this breast cancer molecular subtype and suggests that the upregulation of NBCn1 in this molecular subtype largely occurs due to transcriptional regulation. Indeed, we observed significantly shortened survival times (hazard ratio 2.18) for the luminal A breast cancer patients with the highest compared to the lowest SLC4A7 mRNA levels (Figure 10D). Even though expression of SLC4A7 mRNA was relatively low in basal-like breast cancer, we also observed a significantly worse prognosis (hazard ratio 2.14) for patients with the highest SLC4A7 mRNA levels within this breast cancer subtype (Figure 10G). The importance of NBCn1 in basal-like breast cancer is likely explained by the high proliferative and glycolytic activity (Figure 9—figure supplement 1D,E), which will expectedly elevate the cellular acid load and thus the requirement for net acid extrusion. Shorter survival of breast cancer patients with high expression of SLC4A7 mRNA (Figure 10D,G) is consistent with the greater tendency for lymph node metastasis among patients with high NBCn1 protein expression and accelerated Na+,HCO3 cotransport (Figure 8G) as well as with the enhanced proliferative activity in patients with elevated steady-state pHi due to Na+,HCO3 cotransport (Figure 7G). For patients with normal-like (Figure 10C), luminal B (Figure 10E), and HER2-enriched (Figure 10F) breast cancer, we saw no association of SLC4A7 mRNA expression with survival. We also observed no overall survival differences according to the SLC4A7 mRNA expression level across the whole unstratified population of breast cancer patients (Figure 10B) where the survival effects of differences in SLC4A7 transcript levels within specific molecular subgroups are diluted in the larger patient population.

Monocarboxylate transporters play roles in eliminating lactate and associated H+ from cells relying on fermentative glycolysis and allow neighboring cells to take up and metabolize the lactate through oxidation (Pérez-Escuredo et al., 2016). Thus, the monocarboxylate transporters could influence intra- and extracellular acid-base conditions as well as cellular energy levels in solid cancer tissue (Boedtkjer and Pedersen, 2020). SLC16A1 mRNA (Figure 10—figure supplement 1A), encoding MCT1, and SLC16A3 mRNA (Figure 10—figure supplement 2A), encoding MCT4, both showed high expression levels in basal-like breast cancer characterized by high proliferative (Figure 9—figure supplement 1D) and glycolytic (Figure 9—figure supplement 1E) activity. In contrast, SLC16A3 mRNA expression was high (Figure 10—figure supplement 2A) and SLC16A1 mRNA expression very low (Figure 10—figure supplement 1A) in luminal B breast cancer tissue. Notably, although SLC16A1 mRNA levels showed no association with breast cancer survival (Figure 10—figure supplement 1B–G), high SLC16A3 mRNA levels were significantly associated with—or showed tendency toward—poor survival in all breast cancer molecular subtypes (Figure 10—figure supplement 2B,C and E–G) except for luminal A (Figure 10—figure supplement 2D).

Even though the expression of the investigated acid-base transporters vary systematically across the molecular subtypes (Figures 9A and 10A, Figure 10—figure supplements 1A and 2A), the transcript levels for SLC4A7, SLC9A1, SLC16A1, and SLC16A3 showed no significant pairwise correlation when controlled for the patterns of ERBB2, ESR1, and PGR mRNA expression (Figure 9—source data 1, Figure 10—source data 1, Figure 10—figure supplement 1—source data 1 and Figure 10—figure supplement 2—source data 1). This observation supports that the acid-base transporters independently modify survival when analyzed for each of the breast cancer molecular subtypes separately.

Discussion

We report here on the first large-scale study of pHi dynamics in human cancer samples. We demonstrate that acid-base transporters play key pathophysiological roles by counteracting intracellular acidification and setting the steady-state pHi in human breast cancer tissue (Figure 1). Furthermore, the level of cellular acidity and the capacity for net acid extrusion in human primary breast carcinomas can account for variation in proliferative activity (Figure 7) and are predictive for the occurrence of lymph node metastasis (Figure 8). The functional recordings of pHi dynamics carry predictive value that is complementary to and independent from information based on protein expression analyses (Figures 7G and 8G).

We detect substantial inter-patient heterogeneity in acid-base conditions of breast carcinomas and explore the underlying modifiers and biological consequences. As illustrated in Figure 11, we show that (a) Na+,HCO3 cotransport raises pHi more in invasive lobular than ductal breast carcinomas and particularly in breast cancer tissue of high malignancy grade (Figures 2, 3 and 6); (b) Na+/H+ exchange raises pHi more in estrogen receptor-negative breast carcinomas (Figures 4 and 6); (c) protein expression of NBCn1 and NHE1 is elevated by HER2 overexpression or gene amplification (Figures 5 and 6); (d) elevated steady-state pHi particularly due to Na+,HCO3 cotransport predicts high proliferative activity in primary breast carcinomas (Figure 7); (e) elevated capacity for Na+,HCO3 cotransport, high NBCn1 protein expression, and low NHE1 protein expression predict lymph node metastasis (Figure 8); and (f) high SLC4A7 and/or low SLC9A1 mRNA expression are associated with shorter survival in patients with luminal A and basal-like/triple-negative breast cancer (Figures 9 and 10).

Schematics summarizing identified interactions and associated functional implications of NHE1 (upper panel) and NBCn1 (lower panel) in human breast cancer.

The image was generated with BioRender.com.

Supporting the validity of our study, the findings are based on two distinct patient populations evaluated by separate experimental approaches: (a) the links between pHi dynamics, protein expression of acid-base transporters, and clinical and pathological patient characteristics (Figures 18) come from a cohort of 110 breast cancer patients with information derived from medical records, pHi recordings, and immunohistochemical staining; and (b) the prognostic evidence linking transcriptomics data to patient survival (Figures 9 and 10) comes from a separate cohort of 1457 breast cancer patients.

As depicted schematically in Figure 1B, HCO3 uptake via NBCn1 is functionally equivalent to H+ extrusion via NHE1 when the CO2/HCO3 buffer is in equilibrium (Boedtkjer et al., 2012). However, as summarized in Figure 11, it is clear from our analyses that NBCn1 and NHE1 have very different consequences in human breast cancer tissue: Na+,HCO3 cotransport sets the steady-state pHi associated with proliferative activity (Figure 7G); and the capacity for Na+,HCO3 cotransport and protein expression of NBCn1 predict lymph node metastasis (Figure 8G). In contrast, steady-state pHi in absence of CO2/HCO3 and the capacity for Na+/H+ exchange show no relation to proliferation (Figure 7G) or metastasis (Figure 8G); and the protein expression of NHE1 is even negatively related to axillary lymph node metastasis (Figure 8G). Accordingly, high SLC4A7 but low SLC9A1 mRNA expression is associated with poor survival in select breast cancer molecular subtypes (Figures 9 and 10). Although the reason for these marked differences between NBCn1 and NHE1 is not yet entirely clear, distinct cellular and subcellular expression patterns (Boedtkjer et al., 2013; Lauritzen et al., 2012), allosteric regulation by pHi and pHo (Bonde and Boedtkjer, 2017; Boedtkjer et al., 2013; Hulikova et al., 2011), molecular interacting partners, and responses to auto-, para-, and endocrine signals (Boedtkjer et al., 2012; Boedtkjer and Aalkjaer, 2009; Danielsen et al., 2013) likely play important roles.

The impact of global steady-state pHi on cell proliferation (Figure 7G) is consistent with earlier observations from cultured cell lines that cell cycle progression requires a permissive pHi in the slightly alkaline range (Flinck et al., 2018). Low pHi inhibits the enhanced DNA and protein synthesis preceding cell division although the exact molecular mechanisms have not yet been conclusively identified (Boedtkjer, 2019; Boedtkjer and Aalkjaer, 2013; Pouysségur et al., 1985; Ober and Pardee, 1987). We previously found that knockout of NBCn1 inhibits cell proliferation in murine breast cancer tissue, particularly in deep tumor regions and larger-sized tumors where fermentative glycolysis dominates and the cellular metabolic acid load is elevated (Lee et al., 2016; Lee et al., 2018).

Variation in the rate of acidic metabolic waste production and in the capacity for net acid extrusion shapes the local chemical environment and thereby influences the cancer cell phenotype important for disease progression. There is evidence that local pHi induces cytoskeletal rearrangements (Bernstein et al., 2000; Pope et al., 2004) and that cell surface pH modifies cell-cell and cell-matrix interactions (Riemann et al., 2019; Stock et al., 2007), which in combination with acid-induced degradation of the extracellular matrix (Rofstad et al., 2006) can promote directional migration and metastasis. Differences in cell surface pH along the axis of migration have been reported for various migrating cells in culture based on differences in expression and activity of acid-base transporters between leading and rear ends (Schwab et al., 2012). We have previously found that migrating vascular smooth muscle cells generate NBCn1-dependent spatial pH gradients that are critical for directional migration (Boedtkjer et al., 2016), and the role of NBCn1 was subsequently supported by a study on a lung adenocarcinoma cell line (Hwang et al., 2020). Our current findings (Figure 8G) suggest that NBCn1-mediated Na+,HCO3 cotransport plays a similar role for the invasiveness of human breast cancer cells. Interestingly, the cellular capacity for net acid extrusion was not associated with other of the tested clinicopathological characteristics (Figure 6C,D), which supports that NBCn1 independently impact the metastatic potential of breast cancer cells (Figure 8G).

Based on the negative relationship between NHE1 expression and lymph node metastasis (Figure 8G) and considering the improved survival of luminal A breast cancer patients with high SLC9A1 expression (Figure 9D), we propose that NHE1 is a metastasis suppressor in human breast cancer. Metastasis suppressor proteins are frequently upregulated in early cancer disease, as their expression is lost during cancer progression rather than malignant transformation (Smith and Theodorescu, 2009; Guo et al., 1996). The spatiotemporal regulation of NHE1 expression during breast cancer development has not been investigated in detail. In studies of murine carcinogen-induced breast cancer, NHE1 protein levels were unchanged in the primary cancer tissue compared to normal breast tissue and showed a tendency to decline with increasing tumor size (Lee et al., 2016). In previous studies on human breast cancer tissue, NHE1 protein expression was very high in ductal carcinoma in situ lesions and elevated in early primary breast carcinomas, yet showed a tendency to decrease during metastatic progression (Boedtkjer et al., 2013; Lee et al., 2015). In the same human biopsy material, NBCn1 showed a continuous trend for progressively increasing protein levels from normal breast tissue, over primary breast carcinomas, to metastatic lesions (Boedtkjer et al., 2013). Previous investigations in MCF7 human breast cancer cells support the functional consequences of NHE1 observed in our human breast cancer cohort: NHE1 protein expression in the MCF7 cells markedly increased during heterologous overexpression of an amino-truncated ErbB2 receptor (Lauritzen et al., 2010), yet pharmacological inhibition of NHE1 under these conditions stimulated cell migration (Lauritzen et al., 2012).

Acid-base transporters play a key role for eliminating metabolic acidic waste, and hence their pathophysiological impact depends on the metabolic activity in the specific cancer tissue. This is particularly evident in triple-negative breast cancer that shows an overall low expression of SLC4A7 mRNA (Figure 10A). Nonetheless, the survival of triple-negative breast cancer patients is sensitive to variation in SLC4A7 transcript levels (Figure 10G) most likely due to high proliferative and metabolic activities (Figure 9—figure supplement 1) that challenge pHi homeostasis. This observation supports that selectivity of anti-cancer therapies targeting acid-base transporters can be conferred not only by dramatic overexpression in cancer tissue relative to other tissues in the body but also by a greater functional dependency on net acid extrusion capacity in the cancer tissue. Pharmacological inhibitors of acid-base transporters have not yet reached clinical use. Previously described small molecule inhibitors of NBCn1 (e.g., S0859) do not provide the selectivity or pharmacokinetic properties necessary for systemic therapy (Boedtkjer et al., 2012; Larsen et al., 2012; Steinkamp et al., 2015); and whereas NHE1 inhibitors (e.g., cariporide, eniporide) reached phase 3 clinical studies for ischemic heart disease (Mentzer et al., 2008; Zeymer et al., 2001), they have not been sufficiently explored for cancer therapy. Recently developed inhibitors of monocarboxylate transporters show initial experimental promise as anti-cancer drugs (Beloueche-Babari et al., 2017).

Most current diagnostic procedures and selection of patients for targeted therapy (e.g., based on HER2 or estrogen receptors) rely on analysis of protein expression in fixed tissue. The current study validates that quantitative protein activity measurements and evaluation of functional contribution from specific molecular targets in viable tissue preparations provide additional clinically and prognostically important information. The independent information carried by RNA expression, protein expression, and acid-base transport activity suggests that considerable regulation occurs at translational and post-translational levels. The pHi dynamics in the tumor tissue can therefore not be directly deduced from data on protein or gene expression. It is especially noticeable that the protein expression levels of NBCn1 and NHE1 (Figures 5E,F and 6E,F) are elevated in breast cancer tissue with HER2 overexpression or gene amplification despite low SLC4A7 and intermediate SLC9A1 mRNA levels (Figures 9A and 10A). For NBCn1, this inverse relationship between mRNA and protein expression is in congruence with previous reports from murine breast cancer tissue where ErbB2 overexpression is associated with significantly elevated NBCn1 protein levels despite a drastically decreased Slc4a7 mRNA level compared to normal breast tissue (Lee et al., 2018). Thus, increased translational activity or protein stability must be responsible for the raised NBCn1 protein levels in HER2-enriched breast cancer tissue. Because the SLC4A7 mRNA level—based on the abovementioned considerations—does not reflect the NBCn1 protein level or functional capacity in HER2-enriched breast cancer, the survival analysis based on SLC4A7 mRNA expression (Figure 10F) should be interpreted with caution for this molecular subtype. Even without a link between SLC4A7 mRNA expression and survival, the NBCn1 protein expression level and functional capacity could influence patient prognosis.

In light of the widespread use of HER2-targeted breast cancer therapy, it is intriguing that HER2 signaling shows apparently opposing effects on breast cancer progression by concurrently upregulating the protein expression of NBCn1 and NHE1 (Figures 5E,F and 8G). Our findings indicate that (a) additional detailed analysis of downstream signaling effects (e.g., relative upregulation of NBCn1 vs. NHE1) may provide more accurate predictive value than simple evaluation of HER2 overexpression and gene amplification and (b) selective targeting of individual HER2-activated effectors—including acid-base transporters—could optimize the current therapeutic approach based on direct HER2 inhibition.

Luminal A breast cancer differs from other breast cancer molecular subtypes in the profile whereby expression of acid-base transporters influences mortality rates. Elevated mRNA expression of SLC4A7 impedes (Figure 10D), SLC9A1 improves (Figure 9D), whereas SLC16A1 and SLC16A3 show no influence on (Figure 10—figure supplements 1D and 2D) patient survival in luminal A breast cancer. The observation regarding SLC16A3 is notable because high expression worsens (or tends to worsen) patient prognosis in the other breast cancer molecular subtypes (Figure 10—figure supplement 2C and E–G). Luminal A breast cancer comprises 50–70% of breast cancer cases in the United States and Europe (Kulkarni et al., 2019; Acheampong et al., 2020; Valla et al., 2016) and is clinically interesting because distant metastasis occurs throughout follow-up for as long as 25 years after initial diagnosis (Yu et al., 2019). This is different from, for instance, luminal B breast cancer where the risk of metastasis is high the first 5 years after diagnosis but then markedly declines (Yu et al., 2019). Given the identified link between acid-base conditions and metastasis (Figure 8), the distinct prognostic dependency of luminal A breast cancer patients on expression of acid-base transporters could hold a key to the underlying pathophysiology resulting in protracted metastatic risk.

Invasive lobular carcinomas show diffusely infiltrative growth patterns that differ from the dominant masses typical for invasive ductal carcinomas (Thomas et al., 2019). Lost expression of the cell-cell adhesion molecule E-cadherin can explain the tendency for cancer cells from lobular carcinomas to invade in single file (Gamallo et al., 1993; Pai et al., 2013), which is more difficult to delineate by mammography, and thus account for the higher positive surgical margin rates following lumpectomy procedures (van Deurzen, 2016). More efficient net acid extrusion in lobular compared to ductal breast carcinomas is evident from the greater elevation of pHi by Na+,HCO3 cotransport (Figures 2 and 6) and likely translates to enhanced extracellular acidification. Thus, based on the downregulation of E-cadherin expression in response to extracellular acidosis—previously identified in cultured cell lines (Riemann et al., 2019; Suzuki et al., 2014)—it is an intriguing possibility that cellular acid-base handling in human breast carcinomas shapes the histology characteristic growth patterns.

To our knowledge, the current study reports from the largest existing human cancer cohort (Table 1) with detailed cellular acid-base information. Still, the size of the cohort comes with some limitations. Mortality rates for breast cancer patients undergoing breast-conserving surgery are generally low (Onitilo et al., 2015). Therefore, at currently 2–5 years of follow-up, we are unable to perform meaningful survival analyses linking directly to measurements of pH or protein expression. However, our meta-analysis based on transcriptomics data from larger cohorts with longer follow-up partly compensates for this limitation (Figures 9 and 10, and Figure 10—figure supplements 1 and 2). In addition, despite the large overall cohort size, we are for the individual subgroup analyses limited by the natural incidence of specific clinicopathological characteristics. For some of the less frequent characteristics—e.g., rarer histological types (Table 1)—the subgroups are too small for statistical comparison.

Whereas the short delay from tissue isolation to functional evaluation is a unique strength of the current study—because it minimizes the risk of phenotypical changes and thereby strengthens the connection to the clinical condition—it limits the experimental possibilities for detailed mechanistic and molecular studies. The experimental setup allows for manipulation and precise control of the buffer compositions; but with half-lives of protein degradation of 76 and 48 hr for NBCn1 and NHE1, respectively (Olesen et al., 2018), we cannot realistically reduce overall cellular protein levels by interfering with their expression—e.g., by RNAi knockdown technologies (Boedtkjer et al., 2006)—in the human biopsy material. Also, the available pharmacological options are too unspecific and without selectivity for individual Na+,HCO3 cotransporters (Boedtkjer et al., 2016; Boedtkjer et al., 2012; Larsen et al., 2012). Still, our previous studies confirm that the Na+,HCO3 cotransport in human breast cancer tissue is of low 4,4′-diisothiocyano-2,2′-stilbenedisulfonic acid sensitivity (Boedtkjer et al., 2013), which is a pharmacological characteristic of NBCn1 relative to other Na+,HCO3 cotransporters (Boedtkjer et al., 2012; Choi et al., 2000; Romero et al., 2004). This pharmacological profile thus corroborates strong recent molecular evidence—based on gene knockout technology—that the upregulated Na+,HCO3 cotransport in two different murine breast cancer models completely depends on NBCn1 (Lee et al., 2016; Lee et al., 2018).

In conclusion, we identify distinct patterns of pHi dynamics as well as mRNA and protein expression of acid-base transporters among breast cancer patients based on clinical and pathological characteristics and molecular subtypes. The mechanisms of acidic waste product elimination reflect the heterogeneity in human breast cancer tissue. Dependency on Na+,HCO3 cotransport for steady-state pHi regulation independently predicts proliferative activity, whereas the capacity for Na+,HCO3 cotransport activity and the expression of NBCn1 predict lymph node metastasis and patient survival. In contrast, NHE1 expression negatively predicts lymph node metastasis and patient survival. Together, these findings underscore the important pathophysiological role of acid-base homeostasis in human breast cancer tissue and emphasize the potential of acid-base transporters as anti-cancer targets.

Materials and methods

Key resources table
Reagent type
(species) or resource
DesignationSource or referenceIdentifiersAdditional information
Gene (Homo sapiens)SLC4A7GenBankGene ID: 9497Encodes NBCn1
Gene (Homo sapiens)SLC9A1GenBankGene ID: 6548Encodes NHE1
Gene (Homo sapiens)SLC16A1GenBankGene ID: 6566Encodes MCT1
Gene (Homo sapiens)SLC16A3GenBankGene ID: 9123Encodes MCT4
Biological sample (Homo sapiens)Surgical breast biopsiesRegionshospitalet Randers, DenmarkThis study cohortCancer and matched normal tissue
Chemical compound, drugCollagenase type 3Worthington Biochemical CorporationCat. #: LS004182450 IU/mL
Chemical compound, drugBCECF-AMThermo Fisher ScientificCat. #: B11703 µM
AntibodyAnti-NBCn1 (Rabbit polyclonal)Jeppe Praetorius, Aarhus University, Denmark Damkier et al., 2006ReferenceIHC (1:100)
AntibodyAnti-NHE1 (Mouse monoclonal)Santa Cruz BiotechnologyCat. #: sc-136239; RRID:AB_2191254IHC (1:100)
Commercial assay, kitOptiView DAB IHC detection kitRoche DiagnosticsRRID:AB_2833075Goat anti-rabbit and anti-mouse
Software, algorithmSPSSIBMRRID:SCR_002865
Software, algorithmPrismGraphPadRRID:SCR_002798Version 9.1.1

Human breast biopsies

Request a detailed protocol

Viable tissue biopsies of human breast cancer tissue and matched normal breast tissue were obtained from breast-conserving lumpectomies at the Department of Surgery, Regionshospitalet Randers, Denmark, essentially as previously described (Boedtkjer et al., 2013; Lee et al., 2015). We collected samples only from primary breast cancer resections, that is, no samples were acquired from recurrent tumors or metastatic sites. None of the included patients had received pre-operative radiation or chemotherapy. The studies included women, who were at least 18 years of age and presented with operable primary breast cancer (>10 mm) diagnosed by triple test including clinical examination, mammography combined with ultrasonography, and fine-needle aspiration cytology and/or core-needle biopsy. Table 1 summarizes the clinical and pathological characteristics of the 110 included patients. We obtained information regarding patient age, tumor size, histology, malignancy grade, expression of estrogen receptors, HER2 overexpression or gene amplification, Ki67 index, and lymph node metastasis from the medical records of the standard diagnostic care.

Preparation of freshly isolated organoids

Request a detailed protocol

We freshly isolated epithelial organoids—multicellular conglomerates of approximately 150 µm diameter dominated by cytokeratin-19-positive epithelial cells (Lee et al., 2016; Lee et al., 2018; Lee et al., 2015)—from breast cancer tissue and normal breast tissue through partial collagenase digestion of the collected breast biopsies (Figure 1A,B). The tissue samples were first cut into 1 mm pieces in phosphate-buffered saline and then transferred to Tissue Culture Flat Tubes (Techno Plastic Products AG, Switzerland) containing advanced DMEM/F12 culture medium (Life Technologies, Denmark) added 10% fetal bovine serum (Biochrom AG, Germany), 1% GlutaMAX (Thermo Fisher Scientific, Denmark) and 450 IU/mL collagenase type 3 (Worthington Biochemical Corporation, Lakewood, NJ). After continuous overnight shaking at 60 rpm in an incubator with 5% CO2 at 37°C, the isolated organoids sedimented for 20 min by gravitational forces and were then used directly for experiments without culture in order to best retain the functional characteristics of the sampled breast tissue.

pHi measurements

Request a detailed protocol

We studied pHi dynamics in epithelial organoids—freshly isolated from human breast cancer tissue and normal breast tissue—loaded with the pH-sensitive fluorophore 2',7'-bis-(2-carboxyethyl)-5-(and-6)-carboxyfluorescein (BCECF). We added 3 µM acetoxymethyl ester form of BCECF to a physiological saline solution containing 0.1% dimethyl sulfoxide, and loaded the organoids at 37°C for approximately 20 min before they were investigated on the stage of a Nikon Diaphot 200 microscope (Nikon, Japan) equipped with an SRV CCD Retiga camera (QImaging, Canada) and VisiView software (Visitron Systems, Germany). Emission light was collected at 530 nm during alternating excitation at 440 and 495 nm. After background subtraction, the F495/F440 BCECF fluorescence ratio was calibrated to pHi using the high-[K+] nigericin technique (Thomas et al., 1979).

Intracellular acidification was induced with the NH4+-prepulse technique (Boron and De Weer, 1976). Using the equation: β=Δ[NH4+]/ΔpHi, we calculated the intracellular intrinsic buffering capacity from the change in pHi upon addition or washout of NH4Cl under CO2/HCO3-free conditions. The contribution of CO2/HCO3 to the intracellular buffering power was calculated based on the formula βCO2/HCO3=2.3·[HCO3]i (Roos and Boron, 1981). Concentrations of NH4+ and HCO3 were computed from the Henderson-Hasselbalch equation. We calculated net acid extrusion activity as the product of the total intracellular buffering capacity and the rate of pHi recovery from NH4+-prepulse-induced intracellular acidification. Net acid extrusion activities were generally calculated for 30 s intervals covering the full pHi recovery phase; but in cases with extreme intracellular acidification (reaching below pHi 6.4), we omitted the most acidic phase while maintaining a pHi span of no less than 0.1. We plotted net acid extrusion as function of the average pHi within the individually analyzed intervals and calculated for each experiment the transport rate at pH6.5–6.6. A few organoids were excluded from the analyses because of insufficient NH4+-prepulse-induced acidification.

The CO2/HCO3-containing physiological saline solution contained (in mM): 119 NaCl, 22 NaHCO3, 10 HEPES, 1.2 MgSO4, 2.82 KCl, 5.5 glucose, 1.18 KH2PO4, 0.03 EDTA, 1.6 CaCl2. CO2/HCO3-free solutions were produced by substitution of HCO3 with Cl, whereas Na+-free solutions were produced by substitution of Na+ with N-methyl-D-glucammonium (NMDG+), except for the NaHCO3 that was substituted with choline-HCO3. All solutions contained 5 mM probenecid to inhibit the organic anion transporter and avoid extrusion of BCECF from the cancer cells (Lee et al., 2015). The buffer solutions were aerated with 5% CO2/balance air (for CO2/HCO3-containing solutions) or nominally CO2-free air (for CO2/HCO3-free solutions) and pH adjusted to 7.40 at 37°C.

Immunohistochemistry

Request a detailed protocol

Histological sections were prepared from paraffin-embedded tissue blocks and immunohistochemically stained on a BenchMark ULTRA automated staining system (Roche Diagnostics, Indianapolis, IN). After deparaffinization, slides were heated to 100°C and pretreated using BenchMark ULTRA CC1 conditioning solution (Roche Diagnostics). The slides were then incubated with primary antibody diluted 1:100 in Dako REAL Antibody Diluent (S2022; Agilent Technologies, Inc, Santa Clara, CA) for 32 min. The rabbit anti-NH2-terminal NBCn1 antibody was generously provided by Dr Jeppe Praetorius (Damkier et al., 2006). The mouse anti-NHE1 antibody (#sc-136239, RRID:AB_2191254) was from Santa Cruz Biotechnology (Dallas, TX). Finally, endogenous peroxidase activity was inhibited, and bound antibody was detected with the OptiView DAB IHC detection kit (Roche Diagnostics, RRID:AB_2833075). Stained slides were imaged with a Hamamatsu NanoZoomer S60 digital slide scanner (Japan). Staining intensity was scored by an experienced breast pathologist.

Information on estrogen receptor, HER2, and Ki67 expression was obtained from the standard diagnostic procedures. We observed a clear distinction between one group with very high estrogen receptor expression (≥90% positive cells) and another group with low estrogen receptor expression (≤15% positive cells) similar to values (median of 10%) previously reported for normal breast tissue (Oh et al., 2017). The absence of intermediate expression levels provided a clear and obvious separation between the groups. As previously noted by others (Coates et al., 2015), Ki67 expression displays a continuous distribution with no clear separation between groups of high and low expression. The median Ki67 index was 20% in our study cohort as a whole and also in the patient group with estrogen receptor-positive disease (see Figure 1—source data 1). Therefore, we followed the guidelines of the 2015 St Gallen International Expert Consensus recommending a cut-off setting of 30% for identification of patients with a clearly high Ki67 index (Coates et al., 2015).

Transcript levels and survival data in human breast cancer

Request a detailed protocol

We retrieved seven normalized microarray datasets from studies by van de Vijver and co-workers (van de Vijver et al., 2002), Guo and co-workers (Guo et al., 2005), Calza and co-workers (Calza et al., 2006), and from the Gene Expression Omnibus series: GSE1992 (Hu et al., 2006), GSE2034 (Wang et al., 2005), GSE11121 (Schmidt et al., 2008), and GSE3143 (Bild et al., 2006). These studies collectively cover 1457 breast cancer patients with information on our genes of interest. The majority of the studies measure gene expression with multiple probes per gene; and therefore, we collapsed the multiple expression values by gene symbol using the maximum mean probe intensity. Next, we assigned each sample to one of the five well-defined breast cancer molecular subtypes (normal-like, luminal A, luminal B, HER2-enriched, and basal-like) identified in previous studies (Sørlie et al., 2001; Perou et al., 2000) using the PAM50 Breast Cancer Intrinsic Classifier (Parker et al., 2009).

To conduct a meta-analysis, we cross-sample standardized each dataset separately, and then combined all seven datasets into one expression matrix that was subjected to a second round of cross-sample standardization. We used this standardized expression matrix to compare and test for differential gene expression levels of CD34, ESR1, HER2, LDHA, MKI67, PECAM1, PGR, SLC4A7, SLC9A1, SLC16A1, and SLC16A3 between the five molecular subtypes and for survival analyses. Kaplan-Meier survival curves were constructed for groups with high and low mRNA expression defined by z-scores above 0.5 and below –0.5, respectively. In cases where either the group of high or low mRNA expression included less than 10 deaths, we used z-score cut-off thresholds of 0.3 and –0.3. For z-score calculation, the difference between the raw score and the population mean was divided by the population standard deviation.

Statistics

Data are given as mean ± SEM unless otherwise specified; n equals number of patients (i.e., biological replicates) and experiments were performed one time for each experimental condition. No explicit power calculations were performed. Within the study periods, tissue was sampled from all patients, who fulfilled the inclusion criteria, and allocated to the appropriate groups based on information from the standard diagnostic procedures. The investigators were blinded during the experiments and analyses, as the clinicopathological information was not collected until later. We considered p-values smaller than 0.05 as statistically significant. Acid-base transport activities and steady-state pHi values were compared between groups by mixed-effects analyses followed by Sidak’s multiple comparisons test. We compared categorical protein expression levels by χ2-tests for trend. mRNA expression data were compared between multiple groups by one-way ANOVA followed by Tukey’s post-test. Relationships between patient age, tumor size, acid-base transport activity, and steady-state pHi were tested by Spearman’s correlation analyses. We identified associations between multiple clinical and pathological independent variables and continuous dependent variables based on multiple linear regression analyses, whereas we identified associations with dichotomous and ordered categorical dependent variables using binomial and ordinal logistic regression analyses, respectively. Kaplan-Meier survival curves were compared by Mantel-Cox and Gehan-Breslow-Wilcoxon tests. We tested pairwise correlations between mRNA expression of acid-base transporters, angiogenic markers, and sex hormone and growth factor receptors based on Pearson and partial correlation analyses. Statistical analyses were performed with GraphPad Prism 9.0.0 (RRID:SCR_002798) and IBM SPSS Statistics (RRID:SCR_002865) software.

Study approval

Request a detailed protocol

All participants gave written informed consent. The Mid-Jutland regional division of the Danish Committee on Health Research Ethics (M-20100288) and the Danish Data Protection Agency (1-16-02-191-16) approved the procedures for tissue sampling and data handling, respectively.

Data availability

In order to comply with the ethical approval, we share the human data presented in Figure 1–8 and corresponding figure supplements (data on acid-base transport activity, intracellular pH, and protein expression of transporters linked to clinicopathological information) in de-identified form. Following consultation with the legal team at the Regional Committee on Health Research Ethics, we have generated dataset files where restricted information is grouped in intervals each consisting of no less than five individuals. To provide the reader with the best possible data insight, we also show figure supplements with more detailed and advanced plots of the data and include the corresponding de-identified dataset. The meta analyses presented in Figure 9, 10, and corresponding figure supplements (data on RNA expression linked to patient survival) are based on data that have previously been published by other investigators, as detailed in the manuscript and the dataset list.

The following previously published data sets were used
    1. Calza S
    2. Hall P
    3. Auer G
    4. Bjöhle J
    5. Klaar S
    6. Kronenwett U
    7. Liu ET
    8. Miller L
    9. Ploner A
    10. Smeds J
    11. Bergh J
    12. Pawitan Y
    (2006) Karolinska Institutet
    ID Intrinsic_Signature.zip. Intrinsic molecular signature of breast cancer in a population-based cohort of 412 patients.
    1. Schmidt M
    2. Böhm D
    3. von Törne C
    4. Steiner E
    5. Puhl A
    6. Pilch H
    7. Lehr H-A
    8. Hengstler JG
    9. Kölbl H
    10. Gehrmann M
    (2008) NCBI Gene Expression Omnibus
    ID GSE11121. The humoral immune system has a key prognostic impact in node-negative breast cancer.

References

    1. Dai X
    2. Li T
    3. Bai Z
    4. Yang Y
    5. Liu X
    6. Zhan J
    7. Shi B
    (2015)
    Breast cancer intrinsic subtype classification, clinical use and future trends
    American Journal of Cancer Research 5:2929–2943.
    1. Gamallo C
    2. Palacios J
    3. Suarez A
    4. Pizarro A
    5. Navarro P
    6. Quintanilla M
    7. Cano A
    (1993)
    Correlation of E-cadherin expression with differentiation grade and histological type in breast carcinoma
    The American Journal of Pathology 142:987–993.
    1. Guo X
    2. Friess H
    3. Graber HU
    4. Kashiwagi M
    5. Zimmermann A
    6. Korc M
    7. Büchler MW
    (1996)
    KAI1 expression is up-regulated in early pancreatic cancer and decreased in the presence of metastases
    Cancer Research 56:4876–4880.
    1. Vaupel P
    2. Kallinowski F
    3. Okunieff P
    (1989)
    Blood flow, oxygen and nutrient supply, and metabolic microenvironment of human tumors: a review
    Cancer Research 49:6449–6465.

Decision letter

  1. Caigang Liu
    Reviewing Editor; Shengjing Hospital of China Medical University, China
  2. Maureen E Murphy
    Senior Editor; The Wistar Institute, United States
  3. Caigang Liu
    Reviewer; Shengjing Hospital of China Medical University, China

In the interests of transparency, eLife publishes the most substantive revision requests and the accompanying author responses.

Acceptance summary:

Dysregulated acid-base microenvironment has been revealed to be associated with several adverse outcomes, such as drug resistance and enhanced aggression. In this research a large-scale cohort comprised of patient-derived organoids were employed mimicking exactly the condition under clinical setting to observe effect of dysregulated acid-base transporter on clinical parameters and survival. This work provides potential novel therapeutic avenue through targeting acid-base transporter regulating pH.

Decision letter after peer review:

Thank you for submitting your article "Acid-base transporters and pH dynamics in human breast carcinomas predict proliferative activity, metastasis, and survival" for consideration by eLife. Your article has been reviewed by 3 peer reviewers, including Caigang Liu as the Reviewing Editor and Reviewer #1, and the evaluation has been overseen by Maureen Murphy as the Senior Editor.

The reviewers have discussed their reviews with one another, and the Reviewing Editor has drafted this to help you prepare a revised submission.

Essential revisions:

1) Most of the conclusions could be well supported by clinical data statistics. However, there still exist some concerns expected to be explained, especially, detailed mechanism or therapeutic strategies description regarding pH dysregulation are deficient.

2) There are several contradictory and confusing conclusions in the manuscript remaining to be discussed. It would be better to reorganize the manuscript in a clearer and more convincing manner to present the data in the study.

Reviewer #1:

The authors launched an observational study on 110 breast cancer organoids, measured the cancer and normal cell net acid extrusion capacity to correlate different clinicopathological parameters with intracellular pH regulator (NHE1 and NBCn1) level, and unravel the relationship between them and tumor proliferation, lymph node metastasis together with the impact of cotransporter mRNA on patient's survival, thus highlighting the role of pH regulator in malignancy. This study was performed on a large-scale cohort based on tissue samples to comprehensively reflect the heterogeneity setting. However, there lacks detailed mechanism or therapeutic strategies description regarding pH dysregulation. The data has been sufficient to hold back the views proposed and the results will provide better understanding for the role of dysregulated pH in cancer progression and help future studies regarding targeting pH regulator in managing cancer.

1. In Figure 9 and 10, SLC9A1 and SLC4A7 mRNA level was analyzed for their correlation with survival outcome, it would be better to supplement survival analysis regarding the expression of SLC9A1 and SLC4A7 protein based on your study cohort.

2. Please specify the reasons for ER and Ki-67 cut-off value setting and include them in Method or insert supporting reference literature.

3. It is described in lines 320-321 that the protein expression level of NHE1 and NBCn1 was not significantly influenced by estrogen receptor status. And it is described in lines 333-335 that the protein expression levels of NHE1 as well as NBCn1 were elevated in breast carcinomas with HER2 overexpression or gene amplification both before and after adjustment for other clinicopathological characteristics. But the survival figures of Figure 9, 10 indicated that 'For patients with HER2-enriched breast cancer, we saw no association of SLC4A7 or SLC9A1 mRNA expression with survival; The high overall SLC4A7 expression level in Luminal A breast cancer suggests a prominent role of NBCn1 in this breast cancer molecular subtype. And patients with high SLC9A1 mRNA expression suffering from Luminal A breast cancer showed significantly improved survival (hazard ratio 0.552) compared to patients with low SLC9A1 mRNA expression'. The conclusions seem to be contradictory, please explain this.

4. In lines 75-81: 'In human and murine breast cancer tissue analyzed without stratification by molecular subtype, Na+, HCO3- cotransport activity is elevated and protein expression of the NBCn1 (SLC4A7) and MCT1 (SLC16A1) and MCT4 (SLC16A3) are upregulated compared to normal breast tissue. Protein expression of the NHE1 (SLC9A1) is either unchanged or moderately upregulated when breast cancer tissue as one unstratified group is compared to normal breast tissue.' However, worse prognosis was observed in patients suffering from Luminal A or Basal-like/triple-negative breast cancer with high SLC4A7 and/or low SLC9A1 mRNA expression. The expression level of NHE1 (SLC9A1) and the prognostic data are conflicting, please discuss it in detail.

5. The authors found that the expressions of NBCn1 and NHE1 were related to lymph node metastasis and cancer cell proliferation. However, lymph node metastasis and cancer cell proliferation are both key factors predicting worse prognosis. The existing data cannot indicate whether intratumor PH directly affects the patient's prognosis, or indirectly affects the prognosis through lymph nodes metastasis or cancer cell proliferation. Please add survival charts of patients in different subgroups to illustrate it.

6. The authors separately verified the effects of NBCn1 (SLC4A7), MCT1 (SLC16A1), MCT4 (SLC16A3) and NHE1 (SLC9A1) on the prognosis of patients, but whether there exist synergistic or antagonistic effect among the four genes, or whether there is mutual regulation relationship should be addressed.

7. This manuscript requires a more comprehensive summary regarding previous reported role of abnormal pH in promoting cancer in Introduction, and the same about mechanism description and future interfering strategies in Discussion to help readers comprehend this field more conveniently.

8. Breast cancer organoids are mainly composed of cytokeratin 19-positive epithelial cells while the authors only provide the microscopic pictures. It would be better to provide immunofluorescence or IHC staining pictures of cytokeratin 19-positive epithelial cells.

9. Several references date 20 years ago, new references supporting the authors' findings would strengthen the hypothesis. Meanwhile, please arrange it in accordance with the reference format required by eLife.

Reviewer #2:

In this extensive study, authors have shown that Na+,HCO3- cotransport and Na+/H+-exchange transporters predominantly mediate overall cellular acid extrusion in breast cancer microenvironment. In addition, authors demonstrate that higher activity of Na+,HCO3--cotransport correlates with increased growth and lymph node metastasis of breast cancer. The mechanistic insights gained from the study will help in the development of therapeutic strategies for breast cancers. The strengths of this study include the use of organoid culture system, and detailed cellular acid-base homeostasis analyses in an extensive cohort of breast cancer patients. Overall, this study is of interest to the peers working in the breast cancer field.

1. It would be interesting if the authors have used genetic knockdown or specific inhibitors against NBCn1 and NHE1 in breast cancer organoids to avoid the redundant functions of other ion exchangers.

2. Did the authors analyze the expression of angiogenic markers such as CD31 or CD34 in the patient samples?

3. For the ease of readers, in Figure 1B, authors should also highlight the observations on expression and correlation of NBCn1 and NHE1 with overall survival in different breast cancer subtypes.

4. Authors should discuss the possible limitations of their study in the Discussion part of the manuscript.

5. Please correct the spelling of Maier to Kaplan-Meier in methods.

6. In Figure 1 E, authors should use only one asterisk to show the statistical significance between the groups.

Reviewer #3:

Nicolai J. Toft et al. investigated the feature of acid-base metabolism in breast cancer tissue from several aspects, revealing the underlying mechanism of pH regulation according to the heterogeneity. Moreover, the acid-base transport and intracellular pH state were proved capable of predicting the proliferation ability and lymph node metastasis risk of breast cancer, and were associated with the survival in specific breast cancer subtypes. The conclusions of this paper are mostly well supported by data. However, it would have been clearer and more convincing to present the clinical significance combining all data in this study, especially in a more direct way (using tables or graphs).

1. An exposition for combined analysis of the results in figure 1-6 and figure 7-8 is expected to let the readers better understand the clinical significance (like prediction for proliferation and lymph node metastasis) of the various situations in breast cancer with different acid-base metabolism features.

2. There are some questions of how to select different indicators to evaluate the prognosis tendency. For example, data in figure 5E-F and 6E-F showed that Her2 receptor increased the expression of both NBCn1 and NHE1, which exert opposite predictive function in lymph node metastasis according to the data in figure 8G. In this condition, how to evaluate the risk of metastasis? What is the clinical significance of the data on Her2-enriched subtype?

3. How to confirm the statistical significance of the difference in the figures showing "traces of NH+4-prepulse-induced pHi dynamics" (like figure 2A-B)? Meanwhile, the p value is better to be marked in the figures.

4. The standard to define estrogen receptor-negative breast cancer and estrogen receptor-positive breast cancer should be pointed out. The results in figure 4 is confusing.

5. The information in figure 10A and 10F showed that the mRNA of SLC4A7, which encodes protein NBCn1, expressed in a very low level and could not predict the survival prognosis in HER2-riched breast cancer subtype. However, in figure 5F and figure 6F, the data indicated that HER2 expression level was positively associated with higher NBCn1 frequency. So, a further explanation is needed for the contradiction of these data and their clinical significance.

6. The authors should add more biological and molecular experiments to better support the conclusions.

https://doi.org/10.7554/eLife.68447.sa1

Author response

Essential revisions:

1) Most of the conclusions could be well supported by clinical data statistics. However, there still exist some concerns expected to be explained, especially, detailed mechanism or therapeutic strategies description regarding pH dysregulation are deficient.

We have now very substantially expanded the introduction (l. 82-99), results (l. 380-387), and discussion (l. 452-469 and 486-565) sections in order to better explain how our findings relate to molecular mechanisms of breast carcinogenesis and further discuss the prognostic and possible therapeutic impact of pH dysregulation. Please also see the responses to the individual reviewer comments below.

2) There are several contradictory and confusing conclusions in the manuscript remaining to be discussed. It would be better to reorganize the manuscript in a clearer and more convincing manner to present the data in the study.

To better convey the methodological approach and conclusions of the study and avoid confusion, we have now improved the illustration in Figure 1B and included additional schematic illustrations as a new Figure 11.

Additionally, we have reorganized the supplementary material as figure supplements linked up to the regular figures in order to create a clearer structure.

We have also expanded the discussion very substantially (l. 452-469 and 500-565). Particularly, we have added more background and explanations in the areas highlighted as confusing or contradictory by the reviewers. With the included additional information, we hope the editors and reviewers agree that the findings and conclusions of the manuscript are now more convincingly presented.

For more details, see the responses to the individual reviewer comments below.

Reviewer #1:The authors launched an observational study on 110 breast cancer organoids, measured the cancer and normal cell net acid extrusion capacity to correlate different clinicopathological parameters with intracellular pH regulator (NHE1 and NBCn1) level, and unravel the relationship between them and tumor proliferation, lymph node metastasis together with the impact of cotransporter mRNA on patient's survival, thus highlighting the role of pH regulator in malignancy. This study was performed on a large-scale cohort based on tissue samples to comprehensively reflect the heterogeneity setting. However, there lacks detailed mechanism or therapeutic strategies description regarding pH dysregulation. The data has been sufficient to hold back the views proposed and the results will provide better understanding for the role of dysregulated pH in cancer progression and help future studies regarding targeting pH regulator in managing cancer.

1. In Figure 9 and 10, SLC9A1 and SLC4A7 mRNA level was analyzed for their correlation with survival outcome, it would be better to supplement survival analysis regarding the expression of SLC9A1 and SLC4A7 protein based on your study cohort.

We appreciate this suggestion. However, even though our cohort used for protein expression analyses and pH measurements – to our knowledge – is the largest existing with functional data on acid-base regulation for any type of human cancer, at this duration (2-5 years) of follow-up, it is not large enough for survival outcome analyses. The number of fatalities is too low to provide the statistical strength needed, and we have now included a comment regarding this limitation in the discussion (l. 540-546).

2. Please specify the reasons for ER and Ki-67 cut-off value setting and include them in Method or insert supporting reference literature.

This is an important point, which we now address on l. 651-661 and 206-212 of the revised manuscript:

Regarding ER (l. 652-656 and 206-212): “We observed a clear distinction between one group of patients with very high estrogen receptor expression (≥90% positive cells) and another group of patients with low estrogen receptor expression (≤15% positive cells) in the breast tumors. […] The absence of intermediate expression levels provided a clear and obvious cut-off value for separation between the two groups.”

Regarding Ki67 (l. 656-661): “As previously noted by others (reference 74), Ki67 expression displays a continuous distribution with no clear separation between groups of high and low expression. The median Ki67 index in our study cohort was 20%; and we therefore followed the guidelines of the 2015 St Gallen International Expert Consensus (reference 74) that in a similar population recommend a cut-off setting of 30% for identification of patients with a clearly high Ki67 index.”

3. It is described in lines 320-321 that the protein expression level of NHE1 and NBCn1 was not significantly influenced by estrogen receptor status. And it is described in lines 333-335 that the protein expression levels of NHE1 as well as NBCn1 were elevated in breast carcinomas with HER2 overexpression or gene amplification both before and after adjustment for other clinicopathological characteristics. But the survival figures of Figure 9, 10 indicated that 'For patients with HER2-enriched breast cancer, we saw no association of SLC4A7 or SLC9A1 mRNA expression with survival; The high overall SLC4A7 expression level in Luminal A breast cancer suggests a prominent role of NBCn1 in this breast cancer molecular subtype. And patients with high SLC9A1 mRNA expression suffering from Luminal A breast cancer showed significantly improved survival (hazard ratio 0.552) compared to patients with low SLC9A1 mRNA expression'. The conclusions seem to be contradictory, please explain this.

The point raised by the reviewer is important and underscores that there is not always straightforward proportionality between mRNA, protein, and function. This is particularly important, when making comparisons across molecular subtypes or clinicopathological characteristics driven by distinct carcinogenic mechanisms. The relationship between mRNA, protein, and function is likely much simpler when compared between individuals within the same molecular subtype; and therefore, we focus primarily on survival analyses performed following molecular subtype stratification (Figure 9, 10 and Figure 10—figure supplement 1 and 2). We now discuss this point on l. 317-324 of the revised manuscript.

The impact of estrogen and HER2 receptors on protein expression is described in Figure 4-6, and we have now included additional analyses to evaluate how ESR1, PGR, and ERBB2 expression affects SLC4A7 and SLC9A1 mRNA levels (Figure 9-Source Data 1 and Figure 10-Source Data 1). The lower SLC4A7 mRNA but higher NBCn1 protein level in HER2-enriched breast cancer imply that the HER2-dependent regulation occurs at post-transcriptional level (i.e., by an increased translational activity or higher protein stability). This relationship between mRNA and protein expression for NBCn1 is in line with previous findings in murine breast cancer tissue induced by HER2 overexpression (reference 16), where the SLC4A7 mRNA level was found low despite an elevated NBCn1 protein level. The predominant post-transcriptional regulation of NBCn1 expression by HER2 complicates the interpretation of the corresponding survival analysis (Figure 10F), and we have now included a comment regarding this point in the manuscript (l. 496-506).

The high SLC4A7 mRNA level in Luminal A breast cancer is consistent with a prominent regulation of NBCn1 expression at transcriptional level in this molecular subtype; and in congruence, the SLC4A7 mRNA level carries predictive prognostic value for patients with Luminal A breast cancer (Figure 10D). We have now clarified and expanded this point on l. 346-351 of the revised manuscript.

4. In lines 75-81:'In human and murine breast cancer tissue analyzed without stratification by molecular subtype, Na+, HCO3- cotransport activity is elevated and protein expression of the NBCn1 (SLC4A7) and MCT1 (SLC16A1) and MCT4 (SLC16A3) are upregulated compared to normal breast tissue. Protein expression of the NHE1 (SLC9A1) is either unchanged or moderately upregulated when breast cancer tissue as one unstratified group is compared to normal breast tissue.' However, worse prognosis was observed in patients suffering from Luminal A or Basal-like/triple-negative breast cancer with high SLC4A7 and/or low SLC9A1 mRNA expression. The expression level of NHE1 (SLC9A1) and the prognostic data are conflicting, please discuss it in detail.

We have now included a much more detailed discussion of NHE1 expression in breast cancer tissue on l. 452-469 of the revised manuscript. Our data establish NHE1 as a metastasis suppressor gene in human breast cancer. Although the spatio-temporal relationships for NHE1 expression need further examination in future studies, it is not unusual that metastasis-suppressor genes are upregulated during early cancer development but downregulated during cancer progression thereby promoting cancer cell metastasis to distant regions (references 49 and 50). We have now expanded the discussion of how NHE1 expression is influenced during breast carcinogenesis and included the existing knowledge regarding NHE1 expression changes during breast cancer progression (l. 452-465). With this additional information, we hope the reviewer agrees that the conclusions based on expression, function, and survival are clearer and more consistent.

We also now emphasize (l. 465-469) that our findings are in accordance with previous studies based on the human MCF7 cancer cell line where NHE1 is upregulated in response to HER2 overexpression (reference 51) but inhibits cell migration (reference 38).

5. The authors found that the expressions of NBCn1 and NHE1 were related to lymph node metastasis and cancer cell proliferation. However, lymph node metastasis and cancer cell proliferation are both key factors predicting worse prognosis. The existing data cannot indicate whether intratumor pH directly affects the patient's prognosis, or indirectly affects the prognosis through lymph nodes metastasis or cancer cell proliferation. Please add survival charts of patients in different subgroups to illustrate it.

We have now included a much more detailed discussion of NHE1 expression in breast cancer tissue on l. 452-469 of the revised manuscript. Our data establish NHE1 as a metastasis suppressor gene in human breast cancer. Although the spatio-temporal relationships for NHE1 expression need further examination in future studies, it is not unusual that metastasis-suppressor genes are upregulated during early cancer development but downregulated during cancer progression thereby promoting cancer cell metastasis to distant regions (references 49 and 50). We have now expanded the discussion of how NHE1 expression is influenced during breast carcinogenesis and included the existing knowledge regarding NHE1 expression changes during breast cancer progression (l. 452-465). With this additional information, we hope the reviewer agrees that the conclusions based on expression, function, and survival are clearer and more consistent.

We also now emphasize (l. 465-469) that our findings are in accordance with previous studies based on the human MCF7 cancer cell line where NHE1 is upregulated in response to HER2 overexpression (reference 51) but inhibits cell migration (reference 38).

6. The authors separately verified the effects of NBCn1 (SLC4A7), MCT1 (SLC16A1), MCT4 (SLC16A3) and NHE1 (SLC9A1) on the prognosis of patients, but whether there exist synergistic or antagonistic effect among the four genes, or whether there is mutual regulation relationship should be addressed.

Our meta-analysis covers data from seven different original studies. Because of variation in the genes covered by the different studies, the n-value drops drastically if only studies covering all four genes are included in a combined analysis of synergism/antagonism. Instead, as suggested by the reviewer, we now examine whether there is mutual regulation relationships between the four genes. This can be done in pair-wise correlation analyses that maintain the original high n-values. As shown in Figure 9-Source Data 1, Figure 10-Source Data 1, Figure 10—figure supplement 1-Source Data 1, and Figure 10—figure supplement 2-Source Data 1, the gene expression levels of the four acid-base transporters vary in response to differences in ERBB2, ESR1, and PGR expression – but when corrected for these effects, we find no independent correlation between the expression levels for any pair of transporters. These findings – described and discussed in the revised manuscript (l. 380-387) – support that the survival effects of the individual transporters are independent.

7. This manuscript requires a more comprehensive summary regarding previous reported role of abnormal pH in promoting cancer in Introduction, and the same about mechanism description and future interfering strategies in Discussion to help readers comprehend this field more conveniently.

Our meta-analysis covers data from seven different original studies. Because of variation in the genes covered by the different studies, the n-value drops drastically if only studies covering all four genes are included in a combined analysis of synergism/antagonism. Instead, as suggested by the reviewer, we now examine whether there is mutual regulation relationships between the four genes. This can be done in pair-wise correlation analyses that maintain the original high n-values. As shown in Figure 9-Source Data 1, Figure 10-Source Data 1, Figure 10—figure supplement 1-Source Data 1, and Figure 10—figure supplement 2-Source Data 1, the gene expression levels of the four acid-base transporters vary in response to differences in ERBB2, ESR1, and PGR expression – but when corrected for these effects, we find no independent correlation between the expression levels for any pair of transporters. These findings – described and discussed in the revised manuscript (l. 380-387) – support that the survival effects of the individual transporters are independent.

8. Breast cancer organoids are mainly composed of cytokeratin 19-positive epithelial cells while the authors only provide the microscopic pictures. It would be better to provide immunofluorescence or IHC staining pictures of cytokeratin 19-positive epithelial cells.

Our meta-analysis covers data from seven different original studies. Because of variation in the genes covered by the different studies, the n-value drops drastically if only studies covering all four genes are included in a combined analysis of synergism/antagonism. Instead, as suggested by the reviewer, we now examine whether there is mutual regulation relationships between the four genes. This can be done in pair-wise correlation analyses that maintain the original high n-values. As shown in Figure 9-Source Data 1, Figure 10-Source Data 1, Figure 10—figure supplement 1-Source Data 1, and Figure 10—figure supplement 2-Source Data 1, the gene expression levels of the four acid-base transporters vary in response to differences in ERBB2, ESR1, and PGR expression – but when corrected for these effects, we find no independent correlation between the expression levels for any pair of transporters. These findings – described and discussed in the revised manuscript (l. 380-387) – support that the survival effects of the individual transporters are independent.

9. Several references date 20 years ago, new references supporting the authors' findings would strengthen the hypothesis. Meanwhile, please arrange it in accordance with the reference format required by eLife.

We have now included additional, newer references. The references dating back more than 20 years now comprise less than 12% of the total number of references and are restricted to seminal papers that lay the theoretical or technical groundwork for the research field.

Our impression – based on the instructions to authors – was that no special reference format is required by eLife. We apologize if we have misunderstood this point and now use a reference format that specifies all authors on each paper. We would appreciate further instructions if a different reference format is more convenient – in which case, we will happily modify.

Reviewer #2:

In this extensive study, authors have shown that Na+,HCO3- cotransport and Na+/H+-exchange transporters predominantly mediate overall cellular acid extrusion in breast cancer microenvironment. In addition, authors demonstrate that higher activity of Na+,HCO3--cotransport correlates with increased growth and lymph node metastasis of breast cancer. The mechanistic insights gained from the study will help in the development of therapeutic strategies for breast cancers. The strengths of this study include the use of organoid culture system, and detailed cellular acid-base homeostasis analyses in an extensive cohort of breast cancer patients. Overall, this study is of interest to the peers working in the breast cancer field.

1. It would be interesting if the authors have used genetic knockdown or specific inhibitors against NBCn1 and NHE1 in breast cancer organoids to avoid the redundant functions of other ion exchangers.

It is important to note that the current study is based on freshly isolated organoids that are studied after only the delay (hours) needed for enzymatic digestion of the human tissue biopsies. Since the half-lives of protein degradation for NBCn1 and NHE1 in MCF7 human breast cancer cells are around 76 and 48 hours (reference 67), respectively, genetic knockdown techniques would not be possible within the time available. This is now described on l. 551-558 of the revised manuscript. We apologize if the freshly isolated organoids used in this manuscript and their difference from organoid cultures used in other studies were inadequately described. The organoids in our studies were investigated directly after enzymatic isolation without tissue culture because it is well known that culture processes dramatically influence acid-base transporter expression and function. Had the tissue been cultured, it is very likely that it would no longer resemble the human clinical condition. To further emphasize this important point, we have now expanded Figure 1B to include an ultra-brief illustration of the methodological approach and expanded the description on l. 602-603, 125-127, and 551-554 of the revised manuscript.

Regarding specific inhibitors, no such compounds currently exist for NBCn1, which we now mention on l. 558-559 of the revised manuscript. We have previously shown that the Na+,HCO3-cotransport in human breast cancer tissue has low sensitivity to 4,4′-diisothiocyano-2,2′-stilbenedisulfonic acid (DIDS), which is a pharmacological characteristic of NBCn1 compared to other Na+,HCO3-cotransporters. A comment and reference to this information has been included on l. 559-565 of the revised manuscript.

2. Did the authors analyze the expression of angiogenic markers such as CD31 or CD34 in the patient samples?

For our cohort evaluated with pH dynamics and protein expression levels for NBCn1 and NHE1, we did not analyze the expression of angiogenic markers. In addition to the protein expression analyses for NBCn1 and NHE1, the pathological information stems from the standard diagnostic pathology evaluation, which did not include CD31/PECAM or CD34. However, to accommodate the request from the reviewer, we have now assessed how CD31 and CD34 mRNA expression relates to the different breast cancer molecular subtypes evaluated in the meta-analysis. These data are now included in Figure 9—figure supplement 1 and mentioned on l. 331-333 of the revised manuscript.

3. For the ease of readers, in Figure 1B, authors should also highlight the observations on expression and correlation of NBCn1 and NHE1 with overall survival in different breast cancer subtypes.

This is a point well taken. Instead of expanding Figure 1B – which has already been expanded with methodological details, see point 1 above – we have included new summary schematics of our conclusions regarding NHE1 and NBCn1 in a new Figure 11 in order to best maintain the natural progression of the manuscript.

4. Authors should discuss the possible limitations of their study in the Discussion part of the manuscript.

We now discuss limitations of the study on l. 540-550 of the revised manuscript. These include considerations regarding n-values and follow-up time in our cohort. We also discussion that the sizes of individual subgroups are determined by the natural incidence of the various clinicopathological characteristics, which results in a less balanced distribution for some breast cancer characteristics.

On l. 551-565, we also emphasize that the current approach based on freshly isolated organoids provides the closest possible evaluation of in vivo characteristics under conditions where experimental buffer compositions can be modified and controlled. In order to study detailed molecular mechanisms and their influence on the cellular biology, future studies in model systems will be important.

5. Please correct the spelling of Maier to Kaplan-Meier in methods.

Thank you for pointing this out – corrected twice in the methods section (l. 678 and 700).

6. In Figure 1 E, authors should use only one asterisk to show the statistical significance between the groups.

The asterisks were meant to indicate the statistical difference relating to two different comparisons between the cancer tissue and the normal breast tissue: one in the presence of CO2/HCO3, the other in the absence of CO2/HCO3. We apologize if this was not clear and have now changed the layout of the figure panel to avoid confusion.

Reviewer #3:

Nicolai J. Toft et al. investigated the feature of acid-base metabolism in breast cancer tissue from several aspects, revealing the underlying mechanism of pH regulation according to the heterogeneity. Moreover, the acid-base transport and intracellular pH state were proved capable of predicting the proliferation ability and lymph node metastasis risk of breast cancer, and were associated with the survival in specific breast cancer subtypes. The conclusions of this paper are mostly well supported by data. However, it would have been clearer and more convincing to present the clinical significance combining all data in this study, especially in a more direct way (using tables or graphs).

1. An exposition for combined analysis of the results in figure 1-6 and figure 7-8 is expected to let the readers better understand the clinical significance (like prediction for proliferation and lymph node metastasis) of the various situations in breast cancer with different acid-base metabolism features.

As suggested, we have now markedly expanded the explanation and discussion of the analyses on l. 452-469, and 507-565 of the revised manuscript. The additional discussion highlights the clinical significance and several pathological characteristics in breast cancer that based on our findings are likely to be at least partly due to the acid-base features of the individual molecular subtypes.

2. There are some questions of how to select different indicators to evaluate the prognosis tendency. For example, data in figure 5E-F and 6E-F showed that Her2 receptor increased the expression of both NBCn1 and NHE1, which exert opposite predictive function in lymph node metastasis according to the data in figure 8G. In this condition, how to evaluate the risk of metastasis? What is the clinical significance of the data on Her2-enriched subtype?

This is an excellent point, which we now discuss in greater detail on l. 507-514 of the revised manuscript. The dual effect of HER2 on expression of NBCn1 and NHE1 is intriguing as it suggests that more detailed analyses (e.g., relative regulation of NBCn1 vs NHE1) may provide more accurate predictive value than simple evaluation of HER2 overexpression/gene amplification. Our findings furthermore suggest that selective targeting of individual HER2-activated downstream signaling pathways (including individual acid-base transporters) could optimize the current therapeutic approach based on direct HER2 inhibition.

3. How to confirm the statistical significance of the difference in the figures showing "traces of NH4+-prepulse-induced pHi dynamics" (like figure 2A-B)? Meanwhile, the p value is better to be marked in the figures.

We have now included a paragraph (l. 151-155) that explains the relationship between the different panels, i.e., how data extracted from the pHi traces (like Figure 2A,B) were summarized and statistically compared in the associated panels (like Figure 2C,D and Figure 2—figure supplement 2). The statistical analyses compared the net acid extrusion capacities (Figure 2C) and steady-state pHi levels (Figure 2D) derived from the pHi traces. We include the traces in order to give the reader a better feel for the recordings underlying our analyses, which is in line with the recommendation in the eLife instructions to authors that traces should be included whenever possible.

4. The standard to define estrogen receptor-negative breast cancer and estrogen receptor-positive breast cancer should be pointed out. The results in figure 4 is confusing.

We apologize if the description of the nomenclature was inadequate and confusing. We have now described in detail how we distinguished between breast cancer samples with highly elevated estrogen receptor expression (ER-positive breast cancer: ≥90% ER+ cells) and samples where the estrogen receptor expression was in a range similar to that previously reported for normal breast tissue (ER-normal breast cancer: ≤15% ER+ cells) on l. 206-212 and 652-656 of the revised manuscript.

5. The information in figure 10A and 10F showed that the mRNA of SLC4A7, which encodes protein NBCn1, expressed in a very low level and could not predict the survival prognosis in HER2-riched breast cancer subtype. However, in figure 5F and figure 6F, the data indicated that HER2 expression level was positively associated with higher NBCn1 frequency. So, a further explanation is needed for the contradiction of these data and their clinical significance.

This is a very good point. The lower SLC4A7 mRNA but higher NBCn1 protein level in HER2-enriched breast cancer imply that the HER2-dependent regulation occurs at post-transcriptional level (i.e., by an increased translational activity or higher protein stability). This relationship between mRNA and protein expression for NBCn1 is in line with previous findings in murine breast cancer tissue induced by HER2 overexpression (reference 16), where the SLC4A7 mRNA level was found low despite an elevated NBCn1 protein level. The predominant post-transcriptional regulation of NBCn1 expression by HER2 complicates the interpretation of the corresponding survival analysis (Figure 10F), and we have now included a comment regarding this point in the manuscript (l. 496-506).

6. The authors should add more biological and molecular experiments to better support the conclusions.

The unique strength of the current study is the clinically relevant functional data on pH dynamics from a human cohort of previously unparalleled size. The data set is unique because it so clearly links up to the human clinical condition and embraces its heterogeneity and complexity, thereby allowing us to establish relationships between pH dynamics and clinical and pathological characteristics, which has not been possible before. To establish this link convincingly, we performed the functional recordings immediately after organoid isolation from the sampled tissue biopsies in order to avoid culture-induced phenotypical changes. The short time period between tissue sampling and the functional experiments, however, makes the approach unsuited for molecular interventions as the half-lives of protein degradation is ~76 hours for NBCn1 and ~48 hours for NHE1 (reference 81). We now discuss these points in the revised manuscript (l. 551-565).

Although beyond the scope of the current study, the results from the current study has already prompted new biological and molecular investigations in our group based on murine breast cancer models that are further away from the human clinical condition but much better suited for molecular intervention. Undoubtedly, the current study will also stimulate new molecular studies from colleagues in the field.

https://doi.org/10.7554/eLife.68447.sa2

Article and author information

Author details

  1. Nicolai J Toft

    Department of Biomedicine, Aarhus University, Aarhus, Denmark
    Contribution
    Data curation, Formal analysis, Investigation, Methodology, Writing - review and editing
    Competing interests
    No competing interests declared
  2. Trine V Axelsen

    Department of Biomedicine, Aarhus University, Aarhus, Denmark
    Contribution
    Data curation, Formal analysis, Investigation, Writing - review and editing
    Competing interests
    No competing interests declared
  3. Helene L Pedersen

    Department of Pathology, Regionshospitalet Randers, Randers, Denmark
    Contribution
    Data curation, Formal analysis, Writing - review and editing
    Competing interests
    No competing interests declared
  4. Marco Mele

    Department of Surgery, Regionshospitalet Randers, Randers, Denmark
    Contribution
    Data curation, Writing - review and editing
    Competing interests
    No competing interests declared
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0002-8156-7804
  5. Mark Burton

    1. Department of Clinical Genetics, University of Southern Denmark, Odense, Denmark
    2. Clinical Genome Center, University and Region of Southern Denmark, Odense, Denmark
    Contribution
    Data curation, Formal analysis, Writing - review and editing
    Competing interests
    No competing interests declared
  6. Eva Balling

    Department of Surgery, Regionshospitalet Randers, Randers, Denmark
    Contribution
    Data curation, Writing - review and editing
    Competing interests
    No competing interests declared
  7. Tonje Johansen

    Department of Pathology, Regionshospitalet Randers, Randers, Denmark
    Contribution
    Data curation
    Competing interests
    No competing interests declared
  8. Mads Thomassen

    1. Department of Clinical Genetics, University of Southern Denmark, Odense, Denmark
    2. Clinical Genome Center, University and Region of Southern Denmark, Odense, Denmark
    Contribution
    Data curation, Formal analysis, Writing - review and editing
    Competing interests
    No competing interests declared
  9. Peer M Christiansen

    1. Department of Surgery, Regionshospitalet Randers, Randers, Denmark
    2. Department of Plastic and Breast Surgery, Department of Clinical Medicine, Aarhus University Hospital, Aarhus, Denmark
    Contribution
    Data curation, Writing - review and editing
    Competing interests
    No competing interests declared
  10. Ebbe Boedtkjer

    Department of Biomedicine, Aarhus University, Aarhus, Denmark
    Contribution
    Conceptualization, Formal analysis, Supervision, Funding acquisition, Visualization, Methodology, Writing - original draft, Project administration, Writing - review and editing
    For correspondence
    eb@biomed.au.dk
    Competing interests
    is an inventor on patents covering NBCn1 as target for cancer therapy (EP 3271402).
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0002-5078-9279

Funding

Sundhed og Sygdom, Det Frie Forskningsråd (7025-00050B)

  • Ebbe Boedtkjer

Novo Nordisk Fonden (NNF18OC0053037)

  • Ebbe Boedtkjer

Danish Cancer Society (R136-A8670)

  • Nicolai J Toft

The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.

Acknowledgements

The authors would like to thank doctors and nurses, especially Dr Ida E Holm, at Regionshospitalet Randers for their contribution to tissue collection and pathology evaluation. We thank Jette K Jensen and Karen L Kristensen, Department of Pathology, Regionshospitalet Randers, for expert technical assistance. Funding: This work was financially supported by the Independent Research Fund Denmark (7025-00050B to Boedtkjer), the Novo Nordisk Foundation (NNF18OC0053037 to Boedtkjer), and the Danish Cancer Society (R136-A8670 to Toft).

Ethics

Human subjects: All participants gave written informed consent. The Mid-Jutland regional division of the Danish Committee on Health Research Ethics (M-20100288) and the Danish Data Protection Agency (1-16-02-191-16) approved the procedures for tissue sampling and data handling, respectively.

Senior Editor

  1. Maureen E Murphy, The Wistar Institute, United States

Reviewing Editor

  1. Caigang Liu, Shengjing Hospital of China Medical University, China

Reviewer

  1. Caigang Liu, Shengjing Hospital of China Medical University, China

Publication history

  1. Received: March 16, 2021
  2. Accepted: July 2, 2021
  3. Accepted Manuscript published: July 5, 2021 (version 1)
  4. Version of Record published: July 15, 2021 (version 2)

Copyright

© 2021, Toft et al.

This article is distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use and redistribution provided that the original author and source are credited.

Metrics

  • 453
    Page views
  • 62
    Downloads
  • 0
    Citations

Article citation count generated by polling the highest count across the following sources: Crossref, PubMed Central, Scopus.

Download links

A two-part list of links to download the article, or parts of the article, in various formats.

Downloads (link to download the article as PDF)

Download citations (links to download the citations from this article in formats compatible with various reference manager tools)

Open citations (links to open the citations from this article in various online reference manager services)

Further reading

    1. Cancer Biology
    2. Cell Biology
    Veethika Pandey et al.
    Research Article

    The development of pancreatic cancer requires recruitment and activation of different macrophage populations. However, little is known about how macrophages are attracted to the pancreas after injury or an oncogenic event, and how they crosstalk with lesion cells or other cells of the lesion microenvironment. Here, we delineate the importance of CXCL10/CXCR3 signaling during the early phase of murine pancreatic cancer. We show that CXCL10 is produced by pancreatic precancerous lesion cells in response to IFNγ signaling, and that inflammatory macrophages are recipients for this chemokine. CXCL10/CXCR3 signaling in macrophages mediates their chemoattraction to the pancreas, enhances their proliferation and maintains their inflammatory identity. Blocking of CXCL10/CXCR3 signaling in vivo shifts macrophage populations to a tumor promoting (Ym1+, Fizz+, Arg1+) phenotype, increases fibrosis and mediates progression of lesions, highlighting the importance of this pathway in PDA development. This is reversed when CXCL10 is overexpressed in PanIN cells.

    1. Biochemistry and Chemical Biology
    2. Cancer Biology
    Liz Hernandez Borrero et al.
    Research Article

    Mutations in TP53 occur commonly in the majority of human tumors and confer aggressive tumor phenotypes, including metastasis and therapy resistance. CB002 and structural-analogs restore p53 signaling in tumors with mutant-p53 but we find that unlike other xanthines such as caffeine, pentoxifylline, and theophylline, they do not deregulate the G2 checkpoint. Novel CB002-analogs induce pro-apoptotic Noxa protein in an ATF3/4-dependent manner, whereas caffeine, pentoxifylline, and theophylline do not. By contrast to caffeine, CB002-analogs target an S-phase checkpoint associated with increased p-RPA/RPA2, p-ATR, decreased Cyclin A, p-histone H3 expression, and downregulation of essential proteins in DNA-synthesis and DNA-repair. CB002-analog #4 enhances cell death, and decreases Ki-67 in patient-derived tumor-organoids without toxicity to normal human cells. Preliminary in vivo studies demonstrate anti-tumor efficacy in mice. Thus, a novel class of anti-cancer drugs shows the activation of p53 pathway signaling in tumors with mutated p53, and targets an S-phase checkpoint.