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

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 (pH_o) can reach as low as 6.5 (Voss et al., 2020; Vaupel et al., 1989), which is distinct from corresponding normal tissue with pH_o around 7.3–7.4. The acidity of the extracellular tumor microenvironment challenges intracellular pH (pH_i) 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.

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Figure 1—source data 1

Data file containing NBCn1 and NHE1 protein expression levels, steady-state intracellular pH (pH_i) 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 2–5, 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.

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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 pH_i 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 pH_i level, and the expression of acid-base transporters in human breast cancer tissue predict the severity of disease.

We sampled human tissue biopsies from an extensive cohort of 110 women with breast cancer (Table 1) and evaluated pH_i 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 pH_i 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 pH_i 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).

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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).

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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).

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We report here on the first large-scale study of pH_i dynamics in human cancer samples. We demonstrate that acid-base transporters play key pathophysiological roles by counteracting intracellular acidification and setting the steady-state pH_i 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 pH_i 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 pH_i 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 pH_i 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 pH_i 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).

Figure 11

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Supporting the validity of our study, the findings are based on two distinct patient populations evaluated by separate experimental approaches: (a) the links between pH_i dynamics, protein expression of acid-base transporters, and clinical and pathological patient characteristics (Figures 1–8) come from a cohort of 110 breast cancer patients with information derived from medical records, pH_i 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 pH_i 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 pH_i 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 pH_i and pH_o (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 pH_i on cell proliferation (Figure 7G) is consistent with earlier observations from cultured cell lines that cell cycle progression requires a permissive pH_i in the slightly alkaline range (Flinck et al., 2018). Low pH_i 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 pH_i 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 pH_i 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 pH_i 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 pH_i 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 pH_i 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 pH_i 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.

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.

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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

   "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)

    "This ORCID iD identifies the author of this article:" 0000-0002-5078-9279

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