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Oxaliplatin resistance in colorectal cancer enhances TRAIL sensitivity via death receptor 4 upregulation and lipid raft localization

  1. Joshua D Greenlee
  2. Maria Lopez-Cavestany
  3. Nerymar Ortiz-Otero
  4. Kevin Liu
  5. Tejas Subramanian
  6. Burt Cagir
  7. Michael R King  Is a corresponding author
  1. Vanderbilt University, Department of Biomedical Engineering PMB, United States
  2. Donald Guthrie Foundation (DGF) for Research and Education Sayre, United States
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Cite this article as: eLife 2021;10:e67750 doi: 10.7554/eLife.67750

Abstract

Colorectal cancer (CRC) remains a leading cause of cancer death, and its mortality is associated with metastasis and chemoresistance. We demonstrate that oxaliplatin-resistant CRC cells are sensitized to TRAIL-mediated apoptosis. Oxaliplatin-resistant cells exhibited transcriptional downregulation of caspase-10, but this had minimal effects on TRAIL sensitivity following CRISPR-Cas9 deletion of caspase-10 in parental cells. Sensitization effects in oxaliplatin-resistant cells were found to be a result of increased DR4, as well as significantly enhanced DR4 palmitoylation and translocation into lipid rafts. Raft perturbation via nystatin and resveratrol significantly altered DR4/raft colocalization and TRAIL sensitivity. Blood samples from metastatic CRC patients were treated with TRAIL liposomes, and a 57% reduction of viable circulating tumor cells (CTCs) was observed. Increased DR4/lipid raft colocalization in CTCs was found to correspond with increased oxaliplatin resistance and increased efficacy of TRAIL liposomes. To our knowledge, this is the first study to investigate the role of lipid rafts in primary CTCs.

Introduction

Colorectal cancer (CRC) is the second leading cause of cancer death and is responsible for over 50,000 deaths annually in the United States (Siegel et al., 2019). The probability of being diagnosed with CRC in one’s lifetime is 1 in 24, and there are over 100,000 new cases diagnosed annually in the United States alone. While the 5-year survival rate of localized and regional disease is 90 and 71%, respectively, patients with metastatic disease have just a 14% 5-year survival rate (Early detection, diagnosis, and staging, 2021). Dissemination to other organs is the cause of high mortality in most cancers as nearly 90% of all cancer deaths is attributed to metastasis (Mehlen and Puisieux, 2006). The most common sites of CRC metastases include the liver, lungs, and peritoneum (peritoneal carcinomatosis). While surgery and radiation remain curative options for patients with localized disease, the standard of care for CRC patients with advanced metastatic disease is commonly combination front-line chemotherapy treatment (Werner and Heinemann, 2016). These chemotherapy regimens typically include fluorouracil (5-FU) and leucovorin (LV) in combination, which work together to inhibit DNA and RNA synthesis and modulate tumor growth, extending median survival in patients from 9 months (with palliative care) to over 12 months (Rodriguez-Bigas et al., 2003). Oxaliplatin is a chemotherapeutic agent that upon binding to DNA forms DNA adducts to cause irreversible transcriptional errors, resulting in cellular apoptosis. When oxaliplatin is administered with 5-FU/LV (FOLFOX), the objective response rate is 50% in previously untreated patients, increasing the median overall survival to 18–24 months (Rodriguez-Bigas et al., 2003; Briffa et al., 2017).

While there have been incremental advances in extending survival using FOLFOX and other oxaliplatin-containing chemotherapeutics, patients who eventually succumb to the disease frequently develop chemoresistant subpopulations of cancer cells via intrinsic or acquired mechanisms (Briffa et al., 2017; Martinez-Balibrea et al., 2015). Mechanisms of oxaliplatin resistance in tumors include alterations in responses to DNA damage, cell death pathways (e.g., apoptosis, necrosis), NF-κB signaling, and cellular transport (Martinez-Balibrea et al., 2015). Despite the robustness of these oxaliplatin-resistant cancer cells, multiple studies suggest that chemoresistant subpopulations may be increasingly susceptible to adjuvant therapies (Martinez-Balibrea et al., 2015; Sussman et al., 2007; Jeught et al., 2018; Combès et al., 2019; Ruiz de Porras et al., 2016; Cuello et al., 2001). Tumor necrosis factor‐related apoptosis‐inducing ligand (TRAIL) is a member of the TNF family of proteins and induces apoptosis in cancer cells via binding to transmembrane death receptors (von Karstedt et al., 2017). The binding of TRAIL to trimerized death receptor 4 (DR4) and 5 (DR5) initiates an intracellular apoptotic cascade beginning with the recruitment of death domains and formation of the death‐inducing signaling complex (DISC).

Lipid rafts (LRs) are microdomains in the plasma membrane lipid bilayer that are enriched in cholesterol and sphingolipids, with a propensity to assemble specific transmembrane and GPI-anchored proteins (Simons and Toomre, 2000). Mounting evidence has demonstrated that LRs play major roles in tumor progression, metastasis, and cell death (Greenlee et al., 2021). Studies have shown that translocation into LRs can augment signaling for a variety of cancer-implicated receptors, including growth factor receptors (IGFR and EGFR) and death receptors (Fas and Death receptors 4/5) (Laurentiis et al., 2007; Marconi et al., 2013; Mollinedo and Gajate, 2020; George and Wu, 2012). Translocation of death receptors into rafts enhances apoptotic signaling through the formation of clusters of apoptotic signaling molecule-enriched rafts (CASMER), which act as scaffolds to facilitate trimerization and supramolecular clustering of receptors (Mollinedo and Gajate, 2020). It has become increasingly evident that higher-order oligomerization of death receptors is necessary for effective apoptotic signaling in cancer cells (Naval et al., 2019).

Studies have shown that combination treatment of chemotherapeutic agents with TRAIL may sensitize cancer cells to TRAIL-mediated apoptosis through a variety of mechanisms, including death receptor upregulation (Nagane et al., 2000; Gibson et al., 2000; Baritaki et al., 2007), suppression of apoptotic inhibitors within the intrinsic pathway (El Fajoui et al., 2011), and redistribution of death receptors into LRs (Xu et al., 2009). However, no study has examined whether surviving oxaliplatin-resistant subpopulations of cancer cells have an enhanced sensitivity to TRAIL. In this study, we demonstrate that oxaliplatin-resistant cells show enhanced sensitivity to TRAIL-mediated apoptosis through LR translocation of DR4. Moreover, we elucidate mechanisms that drive this sensitization using chemoresistant cell lines and blood samples collected from metastatic cancer patients. The response of oxaliplatin-resistant CRC to TRAIL-based therapeutics may prove critical to establishing promising new adjuvants for patients who have exhausted conventional treatment modalities.

Results

Oxaliplatin-resistant cell lines show enhanced TRAIL sensitivity

Cell viability of four colorectal cancer cell lines after 24 hr treatment with 0.1–1000 ng/ml of TRAIL was measured and compared to the viability of oxaliplatin-resistant (OxR) cell lines (Figure 1A). Briefly, oxaliplatin-resistant cell lines were previously derived from exposure to increasing concentrations of oxaliplatin until a 10-fold increase in IC50 was achieved (Dallas et al., 2009; Yang et al., 2006; Tanaka et al., 2015). Parental and OxR cells were treated with a range of oxaliplatin concentrations to ensure that chemoresistance was conserved after multiple passages in culture (Figure 1—figure supplement 1A). Moreover, oxaliplatin-resistant cells were found to have increased invasion and motility compared to parental cells, consistent with literature reporting their derivation (Figure 1—figure supplement 1B). Interestingly, oxaliplatin-resistant HT29, SW620, and HCT116 cell lines showed increased maximum TRAIL sensitization levels compared to their parental counterparts, while SW480 cells showed similar or decreased sensitization levels (Figure 1—figure supplement 2). IC50 values demonstrate that a chemoresistant phenotype resulted in augmented TRAIL-mediated apoptosis in two cell lines (Figure 1B). Importantly, cells were not treated with any oxaliplatin in quantifying the level of TRAIL sensitization, and oxaliplatin was not supplemented in the cell culture media to exclude any possible effects from combination treatment. In SW620 cells, large differences in apoptosis were observed when treated with the highest concentration of TRAIL (1000 ng/ml) (Figure 1C). Only 33.3% of parental cells were found to be in late-stage apoptosis after 24 hr compared to 60.6% for OxR cells.

Figure 1 with 3 supplements see all
Oxaliplatin-resistant (OxR) colorectal cancer (CRC) cell lines exhibit enhanced sensitization to TRAIL-mediated apoptosis via the intrinsic pathway and mitochondrial permeabilization.

(A) Oxaliplatin-resistant SW620, SW480, HCT116, and HT29 colon cancer cell lines demonstrate similar or enhanced sensitivity to TRAIL compared to their parental counterparts after 24 hr of treatment. N = 3 (biological replicates); n = 9 (technical replicates). (B) IC50 values were calculated using a variable slope four-parameter nonlinear regression. (C) Representative Annexin-V/PI flow plots comparing SW620 parental and OxR cell viability after 24 hr of treatment with 1000 ng/ml TRAIL. The four quadrants represent viable cells (bottom left), early apoptosis (bottom right), necrosis (top left), and late apoptosis (top right). (D) Representative flow plots of JC-1 assay after treatment with 1000 ng/ml of TRAIL. Mitochondrial depolarization is evidenced by decreased red fluorescence and increased green fluorescence. (E) Mitochondrial depolarization as a function of TRAIL concentration for SW620 parental and OxR cell lines. N = 3 (n = 9). For all graphs, data are presented as mean ± SD. **p<0.01; ****p<0.0001 (unpaired two-tailed t-test).

Figure 1—source data 1

Raw viable cell counts from Annexin-V/PI assays and percent depolarized mitochondria in SW620 cells (panels A and E).

https://cdn.elifesciences.org/articles/67750/elife-67750-fig1-data1-v1.xlsx

To determine if the observed differences in apoptosis were due to enhanced mitochondrial outer membrane permeability, a JC-1 dye was used. SW620 OxR cells exhibited over a threefold increase in the population of JC-1 red (-) cells, indicating increased mitochondrial depolarization (Figure 1D). Mitochondrial depolarization was significantly enhanced in OxR cells for TRAIL concentrations of 50 ng/ml and higher (Figure 1E). Similar TRAIL-induced mitochondrial effects were observed in HCT116 cells (Figure 1—figure supplement 3). These results demonstrate that enhanced TRAIL-mediated apoptosis is occurring, at least in part, via the intrinsic pathway and mitochondrial disruption.

Oxaliplatin-resistant derivatives have decreased CASP10 that has little consequence on TRAIL sensitization

Given that enhanced TRAIL-mediated apoptosis was found to occur via the mitochondrial pathway, gene expression of apoptotic transcripts was compared between the parental and OxR cells. RT-PCR human apoptosis profiler arrays were used to analyze transcripts within the SW620 and HCT116 cell lines since these cells showed the highest degree of OxR TRAIL sensitization and exhibit different innate sensitivities to TRAIL (HCT116 cells are TRAIL-sensitive, whereas SW620 cells are TRAIL-resistant). Interestingly, upon analyzing the RNA expression of 84 apoptotic transcripts, both cell lines shared similar profiles between parental and OxR derivatives. HCT116 OxR cells showed upregulated pro-apoptotic transcripts cytochrome-c and caspase-4 (Figure 2A). Cytochrome-c is released from the mitochondria into the cytosol after mitochondrial permeabilization, binding to adaptor molecule apoptosis-protease activating factor 1 (Apaf-1) to form the apoptosome and initiate downstream caspase signaling (Garrido et al., 2006). Caspase-4 is localized to the ER and initiates apoptosis in response to ER stress (Hitomi et al., 2004). Interestingly, SW620 OxR cells had upregulated Fas, a cell surface death receptor that acts similarly in apoptotic signaling to DR4/DR5 via binding of Fas ligand (Özören and El-Deiry, 2003), and osteoprotegerin, a soluble decoy receptor that sequesters TRAIL and inhibits apoptosis (Sandra et al., 2006; Figure 2B). Upregulated Fas expression in SW620 OxR cells was confirmed via surface staining and flow cytometry; however, receptor neutralization with the ZB4 anti-Fas antibody had no effect on TRAIL sensitization when treated in combination with TRAIL (Figure 2—figure supplement 1A–C). Notably, both HCT116 and SW620 OxR cell lines had caspase-10 as the most significantly downregulated transcript. To determine whether this was of consequence to the observed TRAIL sensitization, an SW620 caspase-10 knockout cell line was created using a multi-guide sgRNA CRISPR-Cas9 approach. Knock-out (KO) efficiency was found to be 93% (Figure 2C). The TRAIL sensitivity of this caspase-10 KO cell line was compared to a control cell line treated with Cas9 only. Caspase-10 KO cells showed only a slight decrease in cell viability after 24 hr of TRAIL treatment (Figure 2D). The number of late-stage apoptotic cells remained similar between cell lines (Figure 2E), and the maximum TRAIL sensitization observed was insignificant following caspase-10 KO (Figure 2F).

Figure 2 with 1 supplement see all
Microarray profiles show that parental and oxaliplatin-resistant (OxR) colorectal cancer (CRC) cell lines have similar expression of apoptotic transcripts while OxR derivatives have significantly downregulated CASP10.

(A, B) Volcano plots of RT-PCR Apoptosis Profiler arrays demonstrate downregulation of CASP10 in OxR phenotypes. N = 3. (C) CRISPR/Cas9 knockout of caspase-10 in SW620 parental cells was confirmed via western blot. sgRNA/Cas9 ribonucleoprotein complexes reduced caspase-10 expression by 93% compared to cells treated with Cas9 alone. (D) CASP10 knock-out (KO) cells demonstrate slight decreases in viability when treated with TRAIL compared to Cas9 control. Data are presented as mean ± SD. N = 3 (n = 9). (E) Representative Annexin-V/PI flow plots comparing SW620 parental (Cas9 only) and CASP10 KO cell viability after 24 hr of treatment with 1000 ng/ml TRAIL. (F) Depletion of caspase-10 did not have a significant effect on TRAIL sensitization (unpaired two-tailed t-test). Data are presented as mean + SEM. N = 3 (n = 9).

Figure 2—source data 1

Apoptosis microarray data in HCT116 cells with fold regulation calculations generated using the GeneGlobe Data Analysis Center (panel A).

https://cdn.elifesciences.org/articles/67750/elife-67750-fig2-data1-v1.xlsx
Figure 2—source data 2

Apoptosis microarray data in SW620 cells with fold regulation calculations generated using the GeneGlobe Data Analysis Center (panel B).

https://cdn.elifesciences.org/articles/67750/elife-67750-fig2-data2-v1.xlsx
Figure 2—source data 3

Western blot images (raw and annotated) confirming CASP10 KO (panel C).

https://cdn.elifesciences.org/articles/67750/elife-67750-fig2-data3-v1.zip
Figure 2—source data 4

Quantification of CASP10 KO from western blots (panel C).

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Figure 2—source data 5

Cell viability and TRAIL sensitization calculations in CASP10 KO cells (panels D and F).

https://cdn.elifesciences.org/articles/67750/elife-67750-fig2-data5-v1.xlsx

TRAIL-sensitized OxR cell lines have upregulated DR4

While changes in death receptor expression were insignificant at a transcriptional level, studies have demonstrated that chemoresistance can alter receptor abundance via mechanisms of translational regulation (Si et al., 2019; Just et al., 2019). Confocal microscopy showed that oxaliplatin-resistant cells have increased DR4 in both HCT116 (Figure 3A) and SW620 (Figure 3B) cell lines. To quantify receptor expression, total DR4 area per cell was analyzed for at least 70 cells. Analysis showed OxR derivative cell lines had significantly increased DR4 area per cell (Figure 3C). There were no differences in cell size between parental and OxR derivatives for all four cell lines (Figure 3—figure supplement 1). Flow cytometry staining of non-permeabilized cells was used to determine if this death receptor increase was also observed on the cell surface. Both HCT116 OxR and SW620 OxR cells showed significant increases in DR4 surface expression (Figure 3D). Total and surface DR4 expression was similar between parental and OxR derivatives in mildly sensitized HT29 cells and unsensitized SW480 cells (Figure 3—figure supplement 2A–C). To account for possible thresholding effects in area quantification, raw integrated density counts per cell were also measured and found to be consistent with changes in receptor area (Figure 3—figure supplement 3). Increases in DR4 expression of OxR derivatives were also confirmed via western blot but were only significant in SW620 cells (Figure 3E, F). OxR HCT116, SW620, and HT29 cells all displayed increases in DR5 area per cell, while SW480 OxR cells had significant decreases in total DR5 expression (Figure 3—figure supplement 4A–D). However, total receptor area per cell was considerably lower for DR5 compared to DR4. Additionally, expression of surface DR5, analyzed via flow cytometry, was only significantly upregulated in SW620 OxR cells (Figure 3—figure supplement 4E). This is further confounded by western blot data, which show no change in DR5 expression in HCT116 OxR cells, and a significant decrease in SW620 OxR cells (Figure 3—figure supplement 5A, B). Decoy receptors are surface receptors that, like death receptors, can bind to exogenous TRAIL. However, decoy receptor 1 (DcR1) and decoy receptor 2 (DcR2) are unable to activate the apoptotic pathway, making these receptors sequestering agents that competitively bind to TRAIL. While some studies have shown that chemotherapy-induced changes in TRAIL sensitivity have been linked to modulation or augmentation of decoy receptors (Toscano et al., 2008), all cell lines exhibited no meaningful difference in surface DcR1 and DcR2 expression between parental and OxR derivatives (Figure 3—figure supplement 6). Despite statistical significance in SW480 and HT29 cells, decoy receptor expression, especially DcR2, was expressed in negligible quantities in these cell lines.

Figure 3 with 7 supplements see all
Oxaliplatin-resistant (OxR) colon cancer cell lines have upregulated DR4 expression.

(A, B) Confocal micrographs of HCT116 and SW620 cells, respectively. Red channel represents DR4, green is lipid rafts, and blue is DAPI (nuclei). Scale bar = 30 μm. (C) Quantification of DR4 area per cell in HCT116 and SW620 cells. For each cell line, N = 75 cells were analyzed. Data are presented as mean + SEM from N = 3 independent experiments. ***p<0.001; ****p<0.0001 (unpaired two-tailed t-test). (D) OxR cells had increased surface expression of DR4 in non-permeabilized cells analyzed via flow cytometry. #Significant according to a chi-squared test (see Supplementary file 1). (E) Western blots for DR4 in whole cell lysates of parental and OxR cells. (F) Quantification of western blots from three independent experiments (N = 3). Data are presented as mean + SEM. *p<0.05 (unpaired two-tailed t-test). (G) Percentage of apoptotic SW620 cells after treatment with 0.01–10 µg/ml mapatumumab (sum of early and late-stage apoptotic cells from Annexin/PI staining). Data are presented as mean ± SD. N = 3 (n = 6). ****p<0.0001 (multiple unpaired two-tailed t-tests). (H) Cell viability of SW620 cells after mapatumumab treatment, determined by Annexin-V/PI staining. Data are presented as mean ± SD. N = 3 (n = 6). (I) Maximum mapatumumab sensitization within OxR cell lines compared to their parental counterparts. Data are presented as mean + SEM.

Figure 3—source data 1

Quantification of DR4 area per cell in HCT116 and SW620 cells (panel C).

https://cdn.elifesciences.org/articles/67750/elife-67750-fig3-data1-v1.xlsx
Figure 3—source data 2

Western blot images (raw and annotated) for DR4 (panel E).

https://cdn.elifesciences.org/articles/67750/elife-67750-fig3-data2-v1.zip
Figure 3—source data 3

Quantification of DR4 from western blots (panel F).

https://cdn.elifesciences.org/articles/67750/elife-67750-fig3-data3-v1.xlsx
Figure 3—source data 4

Cell viabilty and percent apoptosis in SW620 cells after mapatumumab treatment (panels G-I).

https://cdn.elifesciences.org/articles/67750/elife-67750-fig3-data4-v1.xlsx

Given the consistency in data suggesting DR4 upregulation in TRAIL-sensitized OxR cell lines, cells were treated with the DR4-agonist monoclonal antibody mapatumumab to determine DR4 specificity. SW620 OxR cells exhibited significant increases in the number of apoptotic cells after 24 hr of treatment, including a threefold increase in apoptosis at concentrations of 10 µg/ml (Figure 3G). Cell viability closely paralleled TRAIL treatments: parental cells remained resistant at high doses while OxR cells exhibited a dose-responsive decrease in cell viability (Figure 3H). HCT116 cell lines exhibited similar results as OxR cells were significantly more apoptotic at concentrations exceeding 0.1 µg/ml (Figure 3—figure supplement 7A, B). The maximum mapatumumab sensitization was calculated to be greater than 40% for oxaliplatin-resistant HCT116 and SW620 cells (Figure 3I), providing more causal evidence for a DR4-associated mechanism.

TRAIL-sensitized OxR cell lines have enhanced colocalization of DR4 into LRs

Binary projections of colocalization events between DR4 and LRs demonstrate that OxR phenotypes had enhanced DR4 translocation into LRs in HCT116 and SW620 cells (Figure 4A). Quantification of total area of colocalization events showed that HCT116 OxR and SW620 OxR cells have significantly enhanced DR4 localized into LRs, each with an over fourfold increase (Figure 4B). The areas of DR4/LR colocalized events per cell were not significantly different in HT29 and SW480 cells (Figure 3—figure supplement 2D). Other methods of colocalization analysis, including calculation of the Manders’ Correlation Coefficient (MCC), supported these results, specifically in HCT116 and SW620 cell lines where the Manders’ overlap was significantly greater in OxR cells (Figure 4—figure supplement 1). The fold change in DR4/LR colocalization area between OxR and parental cells exhibited a strong linear correlation (0.86) with TRAIL sensitization (Figure 4C). Colocalization of LRs with DR5 was significantly enhanced only in HCT116 and HT29 cells, and analysis of the correlation between DR5 colocalization and TRAIL sensitization resulted in a weaker correlation of 0.48 (Figure 4—figure supplement 2A–C). Quantification of LR area per cell revealed insignificant changes between parental and OxR derivatives in all cell lines except for HT29 cells, where parental cells showed significantly more rafts (Figure 4—figure supplement 3).

Figure 4 with 3 supplements see all
Oxaliplatin-resistant (OxR) colon cancer cell lines have enhanced colocalization of DR4 into lipid rafts.

(A) Composite images and binary projections of DR4/LR colocalization areas in HCT116 and SW620 cell lines. Lipid raft and DR4 binary images were generated for a specified threshold, then multiplied by one another to generate images with positive pixels in double-positive areas. Red is DR4, green is lipid rafts, and blue is DAPI. Scale bar = 30 μm. (B) Quantification of DR4 and lipid raft colocalization area per cell in HCT116 and SW620 cells. For each cell line, N = 75 cells were analyzed. **p<0.01; ****p<0.0001 (unpaired two-tailed t-test). (C) Correlation between the fold change in DR4/LR colocalization (OxR phenotype/parental) and maximum TRAIL sensitization observed by the OxR phenotype for each of the four cell lines (simple linear regression analysis). (D) Lipid raft fractions were isolated and analyzed for DR4 via western blot in parental and OxR cells. (E) Quantification of lipid raft DR4 blots in (D). *p<0.05 (unpaired two-tailed t-test). For all graphs, data are presented as mean + SEM. (F) Förster resonance energy transfer (FRET) efficiencies of FITC-labeled DR4 (donor) and Alexa 555-labeled lipid rafts (acceptor) in parental and OxR cells analyzed via flow cytometry. **p<0.01; ***p<0.001 (unpaired two-tailed t-test).

Figure 4—source data 1

Quantification of LR-colocalized DR4 area per cell in HCT116 and SW620 cells (panel B).

https://cdn.elifesciences.org/articles/67750/elife-67750-fig4-data1-v1.xlsx
Figure 4—source data 2

Correlation analysis of LR-colocalized DR4 area per cell with TRAIL sensitization (panel C).

https://cdn.elifesciences.org/articles/67750/elife-67750-fig4-data2-v1.xlsx
Figure 4—source data 3

Western blot images (raw and annotated) for DR4 from LR isolates (panel D).

https://cdn.elifesciences.org/articles/67750/elife-67750-fig4-data3-v1.zip
Figure 4—source data 4

Quantification of LR DR4 from western blots (panel E).

https://cdn.elifesciences.org/articles/67750/elife-67750-fig4-data4-v1.xlsx
Figure 4—source data 5

FRET efficency calculations (panel F).

https://cdn.elifesciences.org/articles/67750/elife-67750-fig4-data5-v1.xlsx

To confirm DR4 redistribution into rafts, western blots for DR4 were run on plasma membrane-derived LR fractions, isolated using non-ionic detergent and centrifugation. Isolated LR fractions exhibited significant increases in DR4 for both HCT116 OxR and SW620 OxR cells (Figure 4D, E). β-Actin was used as a loading control to compare relative DR4 expression between parental and OxR cell lines (Suprynowicz et al., 2008). There were no detectable levels of DR5 in western blots from LR isolated fractions (Figure 4—figure supplement 2D). This is consistent with studies in hematological cancers that demonstrate raft localization of DR4 but not DR5 (Marconi et al., 2013; Xiao et al., 2011). To further examine the proximity of DR4 and LRs between phenotypes, Förster resonance energy transfer (FRET) efficiency was measured using a previously described flow cytometry protocol and calculated via a donor quenching method (Ujlaky-Nagy et al., 2018). Both HCT116 and SW620 OxR cells had significantly increased FRET efficiencies compared to their parental counterparts, with an over twofold and fivefold increase, respectively (Figure 4F).

Altering LR composition affects DR4/LR colocalization and has consequential effects on TRAIL sensitization

To probe the effects of LR modulation on DR4 clustering and TRAIL sensitization, SW620 OxR and HCT116 OxR cells were treated with 5 µM of nystatin, a cholesterol-sequestering agent that inhibits LR formation, in combination with TRAIL for 24 hr (Figure 5A, G). Nystatin inhibited TRAIL-mediated apoptosis in SW620 OxR cells, significantly decreasing the maximum TRAIL sensitization from 45% to 23% (Figure 5B). Nystatin treatment was found to decrease DR4/LR colocalization by over 20-fold (Figure 5C, M). Similar results were found in HCT116 OxR cells as nystatin treatment decreased TRAIL sensitization from 62% to 1% (Figure 5H) and decreased DR4/LR colocalization by over ninefold (Figure 5I). To demonstrate that enhancing LR formation would have pro-apoptotic effects, parental cells were treated with 70 μM of resveratrol in combination with TRAIL for 24 hr (Figure 5D, J). Resveratrol has been shown to stabilize liquid-ordered domains in the plasma membrane and promote cholesterol/sphingolipid-enriched LRs (Neves et al., 2016). Resveratrol significantly sensitized parental SW620 cells to TRAIL irrespective of TRAIL concentration with a maximum TRAIL sensitization of 68% (Figure 5E). Treatment with resveratrol coincided with significant augmentation of DR4/LR colocalization area, an increase of over sixfold (Figure 5F, N). Similarly, parental HCT116 cells treated with resveratrol were sensitized 59% (Figure 5K), corresponding with a nearly sevenfold increase in DR4/LR colocalization area per cell (Figure 5L). Resveratrol and nystatin had no significant effects on DR5 LR colocalization, except in SW620 OxR cells where nystatin treatment surprisingly resulted in a slight increase in colocalization (Figure 5—figure supplement 1A, B).

Figure 5 with 1 supplement see all
Pharmacological perturbation of DR4 localization in lipid rafts significantly alters cellular apoptosis in response to TRAIL.

(A, G) SW620 oxaliplatin-resistant (OxR) and HCT116 OxR cells, respectively, treated for 24 hr with a combination of TRAIL and 5 µM nystatin. (B, H) SW620 OxR and HCT116 OxR cells, respectively, showed a significant decrease in TRAIL sensitization when treated in combination with nystatin. N = 3 (n = 9). (C, I) Treatment with 5 µM nystatin significantly decreased DR4/LR colocalization area in SW620 OxR and HCT116 OxR cells, respectively. For each cell line, N = 40 cells were analyzed. (D, J) SW620 Par and HCT116 Par cells, respectively, treated for 24 hr with a combination of TRAIL and 70 µM resveratrol. N = 3 (n = 9). (E, K) SW620 Par and HCT116 Par cells, respectively, showed a significant increase in TRAIL sensitization when treated in combination with resveratrol. N = 3 (n = 9). (F, L) Treatment with 70 µM nystatin significantly increased DR4/LR colocalization area in SW620 Par and HCT116 Par cells, respectively. For each cell line, N = 40 cells were analyzed. (M) Representative composite images and binary projections of DR4/LR colocalization in SW620 OxR cells before and after nystatin treatment. (N) Representative composite images and binary projections of DR4/LR colocalization in parental SW620 cells before and after resveratrol treatment. Red represents DR4, green is lipid rafts, and blue is DAPI. Scale bar = 30 μm. **p<0.01; ****p<0.0001 (unpaired two-tailed t-test for all graphs). (A, D, G, J) Data are presented as mean ± SD. (B, C, E, F, H, I, K, L) Data are presented as mean + SEM.

Figure 5—source data 1

Cell viability after TRAIL combination treatments with resveratrol and nystatin (panels A, D, G, J).

https://cdn.elifesciences.org/articles/67750/elife-67750-fig5-data1-v1.xlsx
Figure 5—source data 2

TRAIL sensitization calculations after resveratrol and nystatin (panels B, E, H, K).

https://cdn.elifesciences.org/articles/67750/elife-67750-fig5-data2-v1.xlsx
Figure 5—source data 3

Quantification of LR-colocalized DR4 after resveratrol and nystatin treatment (panels C, F, I, L).

https://cdn.elifesciences.org/articles/67750/elife-67750-fig5-data3-v1.xlsx

S-Palmitoylation of DR4 is enhanced in OxR cells

Palmitoylation is the reversible, post-translational addition of the saturated fatty acid palmitate to the cystine residue of proteins. Palmitoylation of DR4 has proven to be critical for receptor oligomerization and LR translocation, both obligatory for effective TRAIL-mediated apoptotic signaling (Rossin et al., 2009). S-Palmitoylation of DR4 in SW620 parental and OxR cells was analyzed via protein precipitation, free thiol blocking, thioester cleavage of palmitate linkages, and exchange with a mass tag label to quantify the degree of palmitoylated protein. We discovered that DR4 has four distinct palmitoylated sites, the degree of which was enhanced in the oxaliplatin-resistant phenotype (Figure 6A). Quantifying the percentage of palmitoylated protein in relation to input fraction (IFC) and non-mass tag preserved controls (APC-) validated that OxR cells had a significantly higher percentage of DR4 that was palmitoylated (55% compared to 43%) (Figure 6B). To determine whether enhanced palmitoylation was specific to DR4 and not a ubiquitous characteristic of the OxR phenotype, total cellular protein palmitoylation was measured and analyzed via flow cytometry (Figure 6—figure supplement 1A). Fluorescent azide labeling of palmitic acid confirmed that total cellular palmitoylation was unchanged between parental and OxR cells (Figure 6—figure supplement 1B).

Figure 6 with 1 supplement see all
Oxaliplatin resistance enhances palmitoylation of DR4, selectively.

(A) Death receptor palmitoylation was determined by protein precipitation, thioester cleavage, and conjugation of a mass tag to enumerate and quantify the degree of S-palmitoylation between cellular phenotypes. Samples with a mass tag ‘B’ have distinct bands of equivalent increasing mass, with each mass shift indicating a palmitoylated site. Input fraction control (IFC) samples ‘A’ were collected before thioester cleavage, while the acyl preservation negative control (APC) samples were incubated with an acyl-preservation reagent to block free thiols in place of the mass tag reagent. Arrows show palmitoylation bands. (B) Quantification of the percentage of palmitoylated DR4, calculated by dividing the total palmitoylated mass shift intensity by the average intensity of IFC and APC for each sample. Data are presented as mean ± SD (N = 3). *p<0.05 (unpaired two-tailed t-test). (C) Treatment with the irreversible palmitoylation inhibitor 2BP in combination with TRAIL significantly reduced TRAIL sensitization in SW620 OxR cells. Data are presented as mean + SEM. N = 3 (n = 9). *p<0.0001 (unpaired two-tailed t-test). (D) Percentage of apoptotic SW620 parental and OxR cells after treating with 1000 ng/ml TRAIL and 3.5 μM 2BP in combination (sum of early and late-stage apoptotic cells from Annexin/PI staining). Data are presented as mean + SD. N = 3 (n = 9). *p<0.05; ****p<0.0001 (ordinary one-way ANOVA–Tukey’s multiple comparison test). (E) Cell viability determined by Annexin-V/PI staining for cells treated with 0.1–1000 ng/ml TRAIL and 3.5 μM 2BP. IC50 values were calculated using a variable slope four-parameter nonlinear regression. Data are presented as mean ± SD. N = 3 (n = 9). (F) Proposed mechanism of enhanced TRAIL-mediated apoptosis in oxaliplatin-resistant cells.

Figure 6—source data 1

Quantification of palmitoylated DR4 from western blots (panel B).

https://cdn.elifesciences.org/articles/67750/elife-67750-fig6-data1-v1.xlsx
Figure 6—source data 2

Cell viability and percent apoptosis in SW620 cells after 2BP and TRAIL combination treatment (panels C-E).

https://cdn.elifesciences.org/articles/67750/elife-67750-fig6-data2-v1.xlsx
Figure 6—source data 3

Western blot images (raw and annotated) for palmitoylated DR4.

https://cdn.elifesciences.org/articles/67750/elife-67750-fig6-data3-v1.zip

To further examine the relationship between DR4 palmitoylation and TRAIL sensitization in OxR cells, the irreversible palmitoylation inhibitor 2-bromopalmitate (2BP) was used. 2BP is a commonly used palmitate analog that is thought to bind to palmitoyl acyl transferase, forming an inhibitory enzyme complex (Draper and Smith, 2009). Treating SW620 OxR cells with 3.5 μM 2BP in combination with TRAIL significantly reduced TRAIL sensitization and increased the IC50 to over 1000 ng/ml (Figure 6C, E). 2BP significantly reduced the number of apoptotic cells in both parental and OxR cells, demonstrating the importance of DR4 palmitoylation in TRAIL signaling, particularly in chemoresistant cells (Figure 6D). These data suggest a novel mechanism for enhanced DR4/LR colocalization in OxR cells via enhanced DR4 palmitoylation (Figure 6F).

Metastatic CRC patients show sensitivity to TRAIL liposomes despite chemoresistance

Despite promising specificity for cancer cells and low off-target toxicity, TRAIL’s translational relevance has been confounded by a short half-life and ineffective delivery modalities (Stuckey and Shah, 2013). In recent studies, our lab has demonstrated that TRAIL-coated leukocytes via the administration of liposomal TRAIL can be effective in eradicating circulating tumor cells (CTCs) in the blood of metastatic cancer patients (Ortiz-Otero et al., 2020). Briefly, liposomes were synthesized as previously described using a thin-film hydration method, stepwise extrusion to 100 nm in diameter, and decoration with E-selectin and TRAIL via his-tag conjugation (Mitchell et al., 2014; Figure 7A). Undecorated ‘control’ liposomes, soluble TRAIL (290 ng/ml; at equivalent concentrations as TRAIL liposomes), and oxaliplatin (at peak plasma concentrations of 5 μM) were used as controls. Blood was collected from 13 metastatic CRC patients who had previously undergone or were currently undergoing an oxaliplatin chemotherapy regimen (Table 1). Of these, five patients were analyzed at 2–3 time points over their respective treatment regimens, representing 21 total samples. Blood samples were treated with TRAIL liposomes or control treatments under hematogenous circulatory shear conditions in a cone-and-plate viscometer. TRAIL liposomes significantly decreased the average percentage of viable CTCs in patient blood to 43% compared to just 86% when treated with oxaliplatin (Figure 7B). Interpatient variation was dominant in response to TRAIL liposome treatment as the between-patient coefficient of variation was twice as high (CoV = 0.55) as the average within-patient variation (CoV = 0.28). Viable CTCs were categorized as cells that were cytokeratin(+), DAPI(+), CD45(-), and propidium iodide(-) (Figure 7C). TRAIL liposomal therapy reduced total viable CTC counts by 58% compared to control liposomes, and over 32% compared to oxaliplatin after just 4 hr in circulation (Figure 7—figure supplement 1). Notably, in two patients (P10 and P11), there were no detectable viable CTCs in blood samples treated with TRAIL liposomes. When categorizing patients by location of metastasis, patients that presented with metastases in the liver or bone showed a greater reduction in viable CTCs (69 and 71%, respectively) than patients with both lung and liver metastases (32%) (Figure 7D). Patients had similar CTC reductions regardless of their treatment at the time of blood draw, while those undergoing FOLFOX or capecitabine + oxaliplatin had the highest reduction in CTCs (65 and 60%, respectively) (Figure 7E). When categorizing patients as either oxaliplatin-sensitive or -resistant, based on their response to 5 μM oxaliplatin under hematogenous circulatory-shear conditions (threshold 80% CTC viability), there was no significant difference in CTC response to TRAIL liposomes (Figure 7—figure supplement 2A). Likewise, grouping patients by those undergoing oxaliplatin chemotherapy and those who had failed oxaliplatin previously, there was no significant difference in reduction of viable CTCs from the administration of liposomal TRAIL (Figure 7—figure supplement 2B). This demonstrates the utility of TRAIL liposomes to eradicate CTCs in both oxaliplatin-sensitive and OxR patients.

Figure 7 with 2 supplements see all
TRAIL-conjugated liposomes neutralize circulating tumor cells (CTCs) from the blood of patients with metastatic, oxaliplatin-resistant colorectal cancer.

(A) Liposomes were synthesized using a thin-film hydration method, followed by extrusion and his-tag conjugation of TRAIL and E-selectin protein. Patient blood samples were treated in a cone-and-plate viscometer under circulatory shear conditions with either control liposomes, TRAIL liposomes, soluble TRAIL, or oxaliplatin. (B) Effects of TRAIL liposomes and control treatments on the number of viable CTCs, normalized to control liposome treatment. Bars represent the average of all patients and time points (N = 21). **p<0.001; ****p<0.0001 (ordinary one-way ANOVA–Tukey’s multiple comparison test). (C) Representative micrographs of two patients showing neutralization of CTCs in TRAIL liposomes compared to control liposomes, stained for cytokeratin (green), DAPI (blue), CD45 (red), and propidium iodide (yellow). Scale bar = 100 µm. (D, E) Reduction in viable CTCs categorized by location of metastasis and treatment administered at the time of blood draw, respectively. (F) DR4/LR colocalization area of patient CTCs plotted against the percentage of viable CTCs following TRAIL liposome treatments. Each point corresponds with one patient draw. ####p<0.0001 (simple linear regression to confirm significant deviation from zero). (G) DR4/LR colocalization area of patient CTCs plotted against the normalized percentage of viable CTCs following oxaliplatin treatment. Each point corresponds with one patient draw. ####p<0.0001 (simple linear regression to confirm significant deviation from zero). (H) CTCs of patient 7, stained for DR4 (red) and lipid rafts (green), demonstrating increased DR4/LR colocalization over the course of 10 months of FOLFOX treatment (with progressive disease despite treatment). Scale bar = 30 µm. For all graphs, data are presented as mean ± SEM.

Figure 7—source data 1

Viability of patient CTCs following treatment.

https://cdn.elifesciences.org/articles/67750/elife-67750-fig7-data1-v1.xlsx
Figure 7—source data 2

Correlation analysis of LR-colocalized DR4 area with percent viable CTCs after treatment.

https://cdn.elifesciences.org/articles/67750/elife-67750-fig7-data2-v1.xlsx
Table 1
Demographic and clinical information of metastatic colorectal cancer (CRC) patients enrolled in this study.

*Denotes missing treatment analysis for this sample. †Denotes missing DR4/LR analysis for this sample.

PatientAgeSexCancerMetastatic locationTreatment history at
draw 1
Draw 2Draw 3
P0159FColonParaaortic lymph nodesFOLFOX (2016), FOLFIRI, 5-FU + Avastin+2 months
5-FU + Avastin
* +7 months
5-FU + Avastin
P0283FColonLiverFOLFOX, FOLFIRI, FOLFOX + Avastin
P0453FRectalPelvis, mesenteric lymph nodesFOLFOX + Avastin, capecitabine +radiation,
regorafenib + nivolumab
P0568MRectalPulmonaryCapecitabine + oxaliplatin
P0668FRectalLung, boneFOLFOX, FOLFIRI+6 months
FOLFIRI
+7 months
FOLFIRI
P0764MCecumPeritoneal carcinomatosisFOLFOX (1st cycle)+7 months
FOLFOX (progression)
+10 months
FOLFOX (progression)
Started FOLFIRI
P0869MColonLung, abdomenFOLFOX + Avastin (progression) capecitabine + Avastin,
5-FU + cetuximab +
panitumumab
P0973MSigmoidLiver, mesenteryFOLFOX, capecitabine, FOLFIRI, Lonsurf+7 months
FOLFIRI
N/A
(patient deceased)
P1052MRectalLungRadiation + capecitabine
capecitabine + oxaliplatin
P1170MColonLiverFOLFOX
P1259MColonLiver, lungs, R adrenalFOLFOX + Avastin, FOLFIRI + AvastinCetuximab + encorafenib
(progression)
+3 months cetuximab + encorafenib
(progression)
P1363FColonAdnexa pelvisFOLFOX, irinotecan + panitumumab, capecitabine + oxaliplatin
P1579MColonLiverFOLFOX (1st cycle)

CTC DR4-LR colocalization corresponds with TRAIL liposome treatment efficacy and oxaliplatin resistance

Patient CTCs were also stained for DR4 and LRs to examine the relationships between raft colocalization, treatment efficacy, and oxaliplatin resistance. Decreasing LR colocalization with DR4 coincided with reduced efficacy of TRAIL liposomes (higher percentage of viable CTCs after treatment), with a negative slope that significantly deviated from zero (Figure 7F). Additionally, increasing LR DR4 corresponded with increasing resistance to oxaliplatin (higher percentage of viable CTCs after oxaliplatin treatment), with a positive slope that significantly deviated from zero (Figure 7G). These same trends were observed for total DR4 area (Figure 7—figure supplement 2C, D). Despite the small size of the patient cohort, these results are encouraging and support our in vitro data in OxR cell lines. Five patients provided multiple blood samples over the course of their treatment, as shown in Table 1. Of these, P07 was the only patient being treated with oxaliplatin (FOLFOX) over the course of all three blood draws. Patient 7 was undergoing the first cycle of FOLFOX at the time of draw 1 and progressed while on FOLFOX for draws 2 and 3. However, DR4 and LR staining of CTCs revealed increased DR4/LR colocalization despite progression (Figure 7H). This same trend of enhanced CTC DR4/LR colocalization with treatment was observed in patients undergoing 5FU + Avastin (P01) and FOLFIRI (P09), while P06 (FOLFIRI) exhibited a bimodal response (Figure 7—figure supplement 2E). Interestingly, P12 exhibited decreased colocalization in CTCs over the course of treatment. This is hypothesized to be a result of a switch in treatment (FOLFIRI + Avastin to cetuximab + encorafenib) due to progression after the first draw.

Discussion

Our lab has demonstrated the utility of TRAIL nanoparticles to treat a variety of cancer types in vitro (Mitchell et al., 2014), in vivo (Jyotsana et al., 2019), and in clinical samples (Ortiz-Otero et al., 2020). While front-line chemotherapy remains a viable option for patients with metastatic CRC, long-term treatment frequently leads to chemoresistance, consequently yielding a more aggressive, robust phenotype that is unresponsive to many systemic treatments (Martinez-Balibrea et al., 2015). Our results demonstrate that OxR CRC cells are particularly susceptible to TRAIL-mediated apoptosis. Additionally, the ability to eradicate over 57% of OxR CTCs in patient blood demonstrates the utility of TRAIL liposomes clinically. Moreover, two patient samples exhibited 100% neutralization of all viable CTCs following ex vivo TRAIL liposomal treatment. This demonstrates the natural cancer cell targeting ability and low toxicity of TRAIL-based therapeutics, presenting a promising cancer management strategy for patients who have exhausted traditional treatment modalities. TRAIL’s apoptotic affect has been shown to be sensitized by circulatory shear stress, further supporting its use as an antimetastatic therapy in the blood of patients (Mitchell and King, 2013). Multiple other studies have demonstrated that platin-based chemotherapeutics, including oxaliplatin, are able to sensitize cancer cells to TRAIL-mediated apoptosis when treated in combination (Cuello et al., 2001; Nagane et al., 2000; Gibson et al., 2000; Toscano et al., 2008). However, no study has investigated the effects of oxaliplatin resistance on TRAIL-mediated apoptosis, and importantly, no study has demonstrated that OxR cancers can be exploited with TRAIL therapies.

Elucidating the mechanisms that drive OxR TRAIL sensitization will be key in establishing personalized treatment strategies in patients. Interestingly, genetic analysis of TRAIL-sensitized OXR cells demonstrated that OxR cells consistently exhibited downregulated caspase-10. This may seem counterintuitive generally since caspase-10 is a caspase-8 analog that initiates the apoptotic pathway after binding to FADD. However, studies have demonstrated the potential antiapoptotic effects of high caspase-10 expression (Mühlethaler-Mottet et al., 2011; Sprick et al., 2002). One recent study in particular demonstrated that upon activation with Fas ligand caspase-10 reduced DISC association and activation of caspase-8, rewiring DISC signaling toward the NF-κB pathway and cell survival (Horn et al., 2017). However, this noncanonical caspase-10 signaling was found to have an insignificant effect on TRAIL sensitization as evidenced in experiments where caspase-10 was depleted in parental cells. This establishes that the observed augmentation of TRAIL sensitivity is likely a result of a translational or post-translational effect induced by oxaliplatin resistance, rather than a transcriptional change within the apoptotic pathway.

We have demonstrated that augmentation of death receptors, particularly DR4, in OxR cells is one of the drivers of enhanced sensitization. One study found that cisplatin and 5-FU-resistant side populations of colon cancer cells had upregulated DR4, consistent with our results (Sussman et al., 2007). While microscopy data suggest that DR5 is upregulated in TRAIL-sensitized OxR cell lines, DR5 area per cell was considerably lower than DR4. Additionally, conflicting western blot and flow cytometry data make the case for DR5 augmentation in OxR cells less convincing. Treatment with the DR4 agonist antibody mapatumumab validated the role of DR4 in the TRAIL sensitization of OxR cells as the differential treatment responses were analogous to that observed from TRAIL treatment. Interestingly, DR4 augmentation appears to be independent from transcriptional upregulation as there was no significant change in mRNA expression between OxR and parental cell lines. Increasing evidence demonstrates that chemoresistance affects small non-coding microRNA (miRNA) expression, which modulates transcriptional and translational processes (Si et al., 2019; Just et al., 2019). More specifically, studies have shown that oxaliplatin treatment and subsequent resistance in CRC cells alter miRNA expression, affecting signaling pathways within p53, epithelial-to-mesenchymal transition, and cell migration (Tanaka et al., 2015; Gasiulė et al., 2019; Evert et al., 2018). Moving forward, future studies should examine the role of miRNA attenuation post-oxaliplatin resistance on the expression of death receptors, particularly DR4.

While sufficient DR4 expression is important for sustained apoptotic signaling, DR4 localization and compartmentalization within LRs is unequivocally vital. LRs enhance the signaling capacity of surface receptors through a multitude of mechanisms (Greenlee et al., 2021). For example, LRs promote death receptor trimerization, which is needed for signal transduction, act as concentrating platforms for DISC assembly and the recruitment of death domains, and protect DRs from internalization or enzymatic degradation (Simons and Toomre, 2000). Additionally, juxtaposition of multiple DR trimers forms supramolecular entities, recently termed ‘CASMER’ (Mollinedo and Gajate, 2020), capable of multivalent TRAIL signaling via extracellular pre-ligand assembly domains (PLADs) (Naval et al., 2019). Altering raft integrity via cholesterol sequestration using nystatin had profound impacts on reducing TRAIL sensitization within the OxR phenotype. Moreover, raft stabilization with resveratrol was able to enhance TRAIL sensitization within the parental phenotype, mirroring that observed in OxR cells. These changes in sensitivity were confirmed to coincide specifically with enhanced clustering of DR4 within rafts. These results are consistent with other studies, which have shown that pharmacological alterations of LRs have profound impacts on Fas and TRAIL toxicity (George and Wu, 2012; Delmas et al., 2004). Other studies have demonstrated that DR4 localization into LRs is obligatory for TRAIL-induced apoptosis in hematological malignancies and non-small cell lung cancer, whereas DR5 has no dependence on raft translocation (Marconi et al., 2013; Naval et al., 2019; Ouyang et al., 2013; Song et al., 2007), consistent with our correlative data and receptor contents from LR-isolated membrane fractions. Additionally, one study found that oxaliplatin combination treatment with TRAIL in gastric cancer cells enhances apoptotic signaling through casitas B-lineage lymphoma (CBL) regulation and death receptor redistribution into LRs (Xu et al., 2009). While it is evident that rafts promote CASMER formation, death receptor oligomerization, and TRAIL-mediated apoptosis, the mechanism linking the OxR phenotype and enhanced DR4 localization within rafts has yet to be studied.

We have demonstrated that a mechanism for this phenomenon is via enhanced DR4 palmitoylation. Palmitoylation is the post-translational covalent attachment of a fatty acid tail to cysteine residues in the protein transmembrane domain, influencing protein trafficking and signaling. There is evidence that both Fas receptor and DR4 are palmitoylated, while DR5 is not (Rossin et al., 2009; Chakrabandhu et al., 2007). Furthermore, this post-translational modification has proven to be mandatory for DR4 oligomerization, LR localization, and TRAIL-mediated apoptotic signaling (Rossin et al., 2009). Interestingly, in a sensory neuron study in rats, palmitoylation of δ-catenin in dorsal root ganglion was significantly increased after chronic oxaliplatin treatment (Zhang et al., 2018). This is analogous to our results as OxR CRC cells that have undergone chronic oxaliplatin treatment exhibited a higher percentage of palmitoylated DR4. Inhibiting palmitoylation with 2BP abrogated the TRAIL-sensitizing effects within OxR cells, demonstrating the mandatory role palmitoylation has on DR4-mediated TRAIL signaling. Additionally, the fact that palmitoylation is inherent to DR4 and not DR5 explains why TRAIL sensitization of OxR cells strongly correlated with LR translocation of DR4, but not DR5. Further studies probing the differences in palmitoylation between parental and OxR phenotypes are warranted to provide a more detailed understanding of oxaliplatin-induced palmitoylation of specific membrane proteins.

We have also shown that these results translate clinically as DR4 expression and LR colocalization of patient CTCs coincided with increased oxaliplatin resistance and increased neutralization of CTCs from TRAIL liposome treatment. Additionally, some metastatic CRC patients exhibited increased DR4/LR colocalization with ongoing chemotherapy cycles despite metastatic progression and worsening prognosis. To our knowledge, this is the first study investigating LR/protein interactions in primary CTCs (Greenlee et al., 2021). Overall, our results demonstrate a novel mechanism for TRAIL sensitization in chemoresistant CRC cells via death receptor upregulation and localization within LRs. However, since this sensitization was only observed in two of the four CRC cell lines tested, future studies should investigate genetic and phenotypic differences between these cell lines that may make some more susceptible than others to DR4 palmitoylation, augmentation, and localization. For the scope of this study, we chose to focus on the use of TRAIL treatment alone given its low toxicity and given our previous work in engineering TRAIL-conjugated delivery vehicles. However, since patients are treated with combination therapies, it would be valuable to investigate other therapeutics, such as curcumin or oxaliplatin, that synergize with TRAIL to treat OxR cancer cells (Ruiz de Porras et al., 2016; El Fajoui et al., 2011). Additionally, future studies should examine the TRAIL sensitization of OxR cells in vivo in orthotopic models of CRC metastasis (Tseng et al., 2007). Examining the efficacy of TRAIL and TRAIL-conjugated nanoparticles to curb metastasis of OxR cells in humanized mouse models will provide translational evidence to support the mechanisms elucidated in this study. Moving forward, leveraging the enhanced signaling of death receptors in LRs through mechanisms of drug delivery and LR antagonization will be instrumental in therapeutic development for chemoresistant cancers.

Materials and methods

Key resources table
Reagent type
(species) or
resource
DesignationSource or
reference
IdentifiersAdditional
information
Cell line (Homo sapiens)SW620 adenocarcinoma,colorectal, Dukes' type CATCC#CCL-227RRID:CVCL_0547
L15 Media
Cell line (Homo sapiens)SW480 adenocarcinoma,colorectal, Dukes' type BATCC#CCL-228RRID:CVCL_0546
L15 Media
Cell line (Homo sapiens)HT29
adenocarcinoma, colorectal
ATCC#HTB-38RRID:CVCL_0320
McCoy’s 5A Media
Cell line (Homo sapiens)HCT116
carcinoma, colorectal
ATCC#CCL-247RRID:CVCL_0291
McCoy’s 5A Media
Cell line (Homo sapiens)SW620 OxRadenocarcinoma,colorectal, Dukes' type CKobe Pharmaceutical University#CCL-227RRID:CVCL_4V77
L15 Media
Cell line (Homo sapiens)SW480 OxRadenocarcinoma, colorectal, Dukes' type BMD Anderson Cancer Center Characterized Cell Line Core#CCL-228RRID:CVCL_AU18
L15 Media
Cell line (Homo sapiens)HT29 OxR
adenocarcinoma, colorectal
MD Anderson Cancer Center Characterized Cell Line Core#HTB-38RRID:CVCL_ 5949
McCoy’s 5A Media
Cell line (Homo sapiens)HCT116 OxR
carcinoma, colorectal
MD Anderson Cancer Center Characterized Cell Line Core#CCL-247RRID:CVCL_4V73
McCoy’s 5A Media
Chemical compound, drugOxaliplatinMedChemExpressCat#
HY-17371
CAS No: 61825-94-3
Commercial assay or kitMTT Assay KitAbcamCat# ab211091
Peptide, recombinant proteinRecombinant Human sTRAIL/Apo2LPeproTechCat# 310-04
AntibodyMouse Anti-TNFRSF10A Recombinant Antibody (clone mAY4)Creative BiolabsCat#
HPAB-1616-FY
Mapatumumab (0.01–10 μg/ml)
Commercial assay or kitFITC Annexin-V Apoptosis Detection Kit IBD PharmingenCat#
556547
Includes propidium iodide
Software, algorithmFlowJo v10.7.1FlowJoRRID:SCR_008520
Commercial assay or kitJC1 – Mitochondrial Membrane Potential Assay KitAbcamCat# ab113850
Commercial assay or kitRNeasy Plus Mini KitQiagenCat#
74134
Commercial assay or kitRT2 First Strand KitQiagenCat#
330404
Commercial assay or kitRT2 Profiler PCR Human Apoptosis ArrayQiagenCat#
PAHS-012Z
Software, algorithmCFX Maestro SoftwareBio-RadRRID:SCR_018064
Software, algorithmGeneGlobe Data Analysis CenterQiagenRRID:SCR_021211
Commercial assay or kitGene Knockout Kit v2 – Human CASP10 with Cas9 2NLS NucleaseSynthegosgRNA:Cas9 (90 pmol:10 pmol)
Commercial assay or kitVybrant Alexa Fluor 488 Lipid Raft Labeling KitInvitrogenCat# V34403
Commercial assay or kitVybrant Alexa Fluor 555 Lipid Raft Labeling KitInvitrogenCat# V34404
AntibodyMouse anti-human CD261 (DR4) Monoclonal Antibody (clone DJR1)InvitrogenCat#
14-6644-82
RRID:AB_468188
(1:50 IF)
AntibodyMouse anti-human CD262 (DR5) Monoclonal Antibody (clone DJR2-4)InvitrogenCat#
14-9908-82
RRID:AB_468592
(1:50 IF)
AntibodyAlexa Fluor 555 goat anti-mouse IgG (H+L)InvitrogenCat# A28180RRID:AB_2536164
(1:1000 IF)
OtherDAPIInvitrogenCat# D1306RRID:AB_2629482
(1 µg/ml)
Software, algorithmFiji – ImageJFIJIRRID:SCR_002285 JaCOP plugin
AntibodyHuman TruStain FcXBioLegendCat# 422301RRID:AB_2818986
AntibodyPE mouse anti-human CD261 (DR4) (clone DJR1)BioLegendCat#
307206
RRID:AB_2287472
(5 µl per sample, FC)
AntibodyPE mouse anti-human CD262 (DR5) (clone DJR2-4)BioLegendCat#
307406
RRID:AB_2204926
(5 µl per sample, FC)
AntibodyPE mouse anti-human TRAILR3 (DcR1) (clone DJR3)BioLegendCat#
307006
RRID:AB_2205089
(5 µl per sample, FC)
AntibodyPE mouse anti-human TRAILR4 (DcR2) (clone 104918)BioLegendCat#
FAB633P
RRID:AB_2205217
(5 µl per sample, FC)
AntibodyPE Mouse IgG1 κ Isotype Control (clone MOPC-21)BioLegendCat#
400114
RRID:AB_326435
(5 µl per sample, FC)
AntibodyFITC mouse anti-human DR4 (clone DR-4-02)Thermo Fisher ScientificCat# MA1-19757RRID:AB_1955203
(5 µl per sample, FC)
Chemical compound, drugResveratrolSigma-AldrichCat# R5010-100MGRRID:AB_309682
CAS: 501-36-0
Chemical compound, drugNystatinThermo Fisher ScientificCat# BP29495CAS: 1400-61-9
Chemical compound, drug2-BromopalmitateSigma-AldrichCat# 21604-1GCAS: 18263-25-7
AntibodyMouse Anti-Fas Antibody (human, neutralizing) (clone ZB4)Sigma-AldrichCat# 05-338RRID:AB_309682
500 ng/ml (neutralization)
Commercial assay or kitMinute Plasma Membrane-Derived Lipid Raft Isolation KitInvent BiotechnologiesCat#
LR-042
AntibodyDR4 Rabbit monoclonal antibody (clone D9S1R)Cell Signalling TechnologiesCat# 42533RRID:AB_2799223
(1:500 WB)
AntibodyDR5 Rabbit polyclonal antibodyThermo Fisher ScientificCat#
PA1-957
RRID:AB_2303474
(1:500 WB)
AntibodyCaspase-10 Rabbit polyclonal antibodyThermo Fisher ScientificCat#
PA5-29649
RRID:AB_2547124
(1:1000 WB)
AntibodyMouse anti-human GAPDH (clone 6C5)MilliporeSigmaCat# MAB374RRID:AB_2107445
(1:2000 WB)
AntibodyMouse Anti-β-Actin monoclonal antibody (clone C4)Santa CruzCat# sc-47778RRID:AB_2714189 (1:1000 WB)
AntibodyIRDye 800CW goat anti-rabbit secondary antibodyLICORCat# 926-32211RRID:AB_621843
(2:15,000 WB)
AntibodyIRDye 800CW goat anti-mouse secondary antibodyLICORCat#
926-32210
RRID:AB_621842
(2:15,000 WB)
Software, algorithmLICOR housekeeping protein normalization protocolLICOR
Odyssey Fc
RRID:SCR_013715
Commercial assay or kitSiteCounter S-Palmitoylated Protein KitBadrillaCat# K010312
Commercial assay or kitEZClick Palmitoylated Protein Assay KitBioVisionCat# K416-100
Commercial assay or kitCD45 magnetic beads (human)Mylteni BiotechCat#
130-045-801
AntibodyBiotin mouse anti-human CD45 Antibody (clone HI30)BioLegendCat# 304004RRID:AB_314392
(1:50 IF)
AntibodyStreptavidin-conjugated Alexa Fluor 647Thermo Fisher ScientificCat# S21374RRID:AB_2336066 (1:200 IF)
AntibodyFITC Mouse Anti-Human Cytokeratin (clone CAM5.2)BD PharmingenCat# 347653(20 µl per sample, IF)
AntibodyGoat anti-mouse Alexa Fluor 647Thermo Fisher ScientificCat# A21235RRID:AB_2535804 (1:200 IF)

Cell culture

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CRC cell lines SW620 (ATCC, #CCL-227), SW480 (ATCC, #CCL-228), HCT116 (ATCC, #CCL-247), and HT29 (ATCC, #HTB-38) were purchased from American Type Culture Collection. SW620 and SW480 cells were cultured in Leibovitz's L-15 cell culture medium (Gibco). HCT116 and HT29 cells were cultured in McCoy’s 5A cell culture medium (Gibco). Media was supplemented with 10% (v/v) fetal bovine serum and 1% (v/v) PenStrep, all purchased from Invitrogen. SW480 OxR, HCT116 OxR, and HT29 OxR cells were obtained from MD Anderson Cancer Center Characterized Cell Line Core, supplied and generated by the Dr. Lee Ellis laboratory. SW620 OxR cells were obtained from Dr. Mika Hosokawa at Kobe Pharmaceutical University in Japan. OxR derivative cell lines were cultured in the same medium as their parental counterparts. To prevent phenotypic drift of OxR lines, cells were used within six passages from the time they were received. To prevent chemotherapy-induced cytotoxicity in downstream experiments, oxaliplatin was not supplemented in OxR cell culture media. All cell lines were maintained in a humidified incubation chamber at 37°C and 5% CO2. All cell lines were screened for mycoplasma contamination and tested negative.

MTT assay

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SW620, SW620 OxR, HCT116, HCT116 OxR, HT29, HT29 OxR, SW480, and SW480 OxR cell lines were plated into tissue culture 96-well black-walled plates at a concentration of 3000 cells/well and incubated overnight at 37°C. A 10 mM stock oxaliplatin suspension was created by dissolving oxaliplatin (MedChemExpress) in molecular grade water via sonication and heating. Cell culture media was replaced with oxaliplatin treatments ranging from 0 to 1000 µM and incubated for 72 hr. Following treatments, an MTT assay (Abcam) was carried out according to the manufacturer’s protocol. The plates were then read using a plate reader (BioTek µQuant) at 590 nm absorbance using gen5 software. Control wells containing the MTT solution without cells were used for background subtraction.

Transwell assay

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Transwell inserts (6-well with 8 µm pores) (Greiner Bio-one) were evenly coated with 75 µl of a 1 mg/ml collagen solution composed of 3 mg/ml rat tail collagen (Gibco), serum-free media, and 0.2% 1 N NaOH for crosslinking. Inserts were incubated for 20 min at 37°C. After crosslinking, 2.5 ml of 10% FBS media was added to the bottom of the well plate while the top was filled with 1 ml of serum-free media until cells were ready for seeding. Parental and OxR SW480, SW620, HT29, and HCT116 cells were seeded in the collagen-coated inserts at a concentration of 200,000 cells/ml in serum-free media. The transwell inserts were replaced with new serum-free media after 2 days. On day 4, the number of cells that had migrated into the bottom plate was counted using a Thermo Fisher Countess II Automated Cell Counter.

Annexin-V/PI apoptosis assay

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Parental and OxR cell lines were plated at 100,000 cells/well onto 24-well plates and incubated overnight at 37°C. Wells were treated in triplicate with soluble human TRAIL (PeproTech) or treated with the anti-DR4 agonist antibody mapatumumab (Creative Biolabs, clone mAY4) and incubated for 24 hr. All cells were collected by recovering the supernatant and lifting the remaining adhered cells using 0.25% Trypsin-EDTA (Gibco). Cells were washed thoroughly with HBSS with calcium and magnesium (Gibco). Cells were incubated for 15 min with FITC-conjugated Annexin-V and propidium iodide (PI) (BD Pharmingen) at room temperature (RT) in the absence of light and immediately analyzed using a Guava easyCyte 12HT benchtop flow cytometer (MilliporeSigma). Viable cells were identified as being negative for both Annexin-V and PI, early apoptotic cells as positive for Annexin-V only, late apoptotic cells were positive for both Annexin-V and PI, and necrotic cells were positive for PI only. Flow cytometry plots were analyzed using FlowJo v10.7.1 software. Control samples included unstained negative control with no Annexin-V/PI to adjust for background and autofluorescence, and Annexin-V-only and PI-only samples for gating apoptotic and necrotic populations.

The change in cell viability in response to TRAIL treatments between parental and OxR cells for each of the four CRC cell lines was calculated using the following TRAIL Sensitization equation:

TRAILSensitization=(%ViableParentalCells)(%ViableOxRCells)(%ViableParentalCells)100%

where the percentage of viable cells was normalized to untreated controls for each trial. TRAIL sensitization was calculated for each concentration of TRAIL, where the ‘Maximum TRAIL Sensitization’ was the highest sensitization observed among all concentrations. Since this sensitization equation is based on a percent reduction formula, small changes in viability can yield large TRAIL sensitizations when cell viability is low. To account for this, both cell viability and TRAIL sensitization are reported to provide a complete perspective on treatment responses between cell lines.

JC-1 (mitochondrial membrane potential) assay

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SW620 and HCT116 cells (parental and OxR) were plated at 100,000 cells/well onto 24-well plates and incubated overnight. Cells were treated in triplicate with TRAIL for 24 hr. Following treatment, cells were collected, washed thoroughly with HBSS without calcium and magnesium, and incubated for 15 min with JC-1 dye (Abcam) in accordance with the manufacturer’s protocol. JC-1 fluorescence was assessed via flow cytometry. Cells with healthy mitochondria were identified as having higher red fluorescence while those with depolarized mitochondria had lower red JC-1 fluorescence.

RT-PCR profiler array

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2 × 106 SW620 and HCT116 (parental and OxR) cells were seeded into a 100-mm-diameter cell culture dish for 24 hr. Cells were lifted using a cell scraper and washed with HBSS with calcium and magnesium. RNA was isolated using the RNeasy Plus Mini Kit (Qiagen) according to the manufacturer’s protocol. RNA yield following isolation was determined using a UV5Nano spectrophotometer (Mettler Toledo). cDNA synthesis was completed using the RT2 First Strand Kit (Qiagen, 330404) using 0.5 μg RNA per sample. RNA expression of 84 apoptotic genes was analyzed using the RT2 Profiler PCR Human Apoptosis Array (Qiagen, PAHS-012Z). Arrays were prepared according to the manufacturer’s protocols applied to the prepared cDNA samples. Profiler array plates were run on a CFX96 Touch Real Time PCR (Bio-Rad) using the following protocol: 1 cycle for 10 min at 95°C, 40 cycles of 95°C for 15 s followed by 60°C for 60 s at a rate of 1°C/s. Melt curves were generated immediately following the PCR protocol. Cycle threshold (Ct) values were calculated using CFX Maestro Software (Bio-Rad). Data analysis was completed using the GeneGlobe Data Analysis Center (Qiagen). Volcano plots were generated in GraphPad Prism using calculated fold changes in gene expression between OxR and parental cells and their corresponding p-values.

CRISPR-Cas9 KO

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KO of the CASP10 gene in SW620 cells was completed using the Gene Knockout Kit v2–Human CASP10 kit with Cas9 2NLS Nuclease (Synthego). Ribonucleoprotein (RNP) complexes were made at a 9:1 ratio of sgRNA:Cas9 (90 pmol:10 pmol) in Gene Pulser Electroporation Buffer (Bio-Rad, 1652677) and incubated for 10 min at RT. Cas9 control samples consisted of 10 pmol Cas9 with no sgRNA. RNP complexes were added to 200,000 cells in 200 µl electroporation buffer (0.2 cm cuvette) and electroporated via the Gene Pulser Xcell Electroporation System (Bio-Rad) using exponential decay pulses (145 V, 500 µF, 1000 ohm). Cells were immediately cultured in 12-well plates and allowed to recover for 7 days before measuring KO efficiency.

Confocal microscopy and image analysis

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Parental and OxR cells were seeded onto polystyrene cell culture slides (Thermo Fisher Scientific). Cells were allowed to grow for 48 hr at 37°C. In samples treated with nystatin or resveratrol, cells were plated for 24 hr then treated for 24 hr before staining. Cells were washed and LRs were stained using the Vybrant Alexa Fluor 488 Lipid Raft Labeling Kit (Invitrogen, V34403) according to the manufacturer’s protocol. Briefly, cells were incubated with Alexa488-conjugated cholera toxin subunit B (CT-B) followed by an anti-CT-B antibody to crosslink CT-B labeled rafts. Slides were fixed for 15 min with 4% paraformaldehyde (PFA) (Electron Microscopy Sciences) in PBS (Gibco) and then permeabilized using 1% Triton X-100 (MilliporeSigma) in PBS at RT. Slides were blocked for 2 hr with 5% goat serum (Thermo Fisher Scientific) and 5% bovine serum albumin (BSA, Sigma) in HBSS. Primary staining was done overnight at 4°C with either DR4 monoclonal antibody (Invitrogen, clone DJR1) or DR5 monoclonal antibody (Invitrogen, clone DJR2-4) in the blocking serum at a ratio of 1:50. Secondary staining was carried out with Alexa Fluor 555 goat anti-mouse IgG (H+L) (Invitrogen, A28180) for 30 min at RT (1:1000). Slides were stained with DAPI (Invitrogen, D1306) for 30 min at RT in the blocking solution at 1:1000. Washes were done twice between each step for 5 min each using 0.02% Tween20 in PBS. Slides were assembled using 10 μl of Vectrashield antifade mounting media (Vector Laboratories). Confocal imaging was performed using an LSM 880 (Carl Zeiss) with a 63×/1.40 Plan-Apochromat Oil, WD = 0.19 mm objective. At least five images were taken per sample.

Image analysis was performed in FIJI using a macro to automate quantification of raft and DR contents per cell. Briefly, all images were adjusted for background using the same thresholding specifications. The ‘analyze particles’ feature was used to quantify the total area of LRs and DR per outlined cell. Colocalization events were quantified by creating binary masks of DR and LR events. For each gated cell, the LR and DR binary masks were multiplied to create a binary projection of colocalized events. Raw integrated density and cell area (ROI area) were also measured. Cells with areas outside of three times the standard deviation from the mean were considered outliers and not included in the analysis. Colocalization analysis was also performed using the JACoP plugin in FIJI (Bolte and Cordelières, 2006). The MCC was calculated as the fraction of LR colocalized DR4.

Flow cytometry

Surface DR expression

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Parental and OxR cell lines were cultured to 70% confluency upon collection and split into 250,000 cells per sample. Cells were fixed in 4% PFA in HBSS for 15 min at RT, then blocked in a 100 μl 1% BSA solution for 30 min at 4°C, with 2× HBSS washes between each step. Cells suspensions of 100 μl were incubated for 15 min at RT with 2 μl Human TruStain FcX (BioLegend, 422301) to prevent nonspecific Fc receptor binding. Samples were immediately stained with 5 μl of either PE anti-human CD261 (DR4) (BioLegend, clone DJR1), PE anti-human CD262 (DR5) (BioLegend, clone DJR2-4), PE anti-human TRAILR3 (DcR1) (BioLegend, clone DJR3), PE anti-human TRAILR4 (DcR2) (R&D Systems, clone 104918), or PE Mouse IgG1 κ Isotype Control (BioLegend, clone MOPC-21) for 30 min at 4°C. Samples were washed twice with HBSS and analyzed using a Guava easyCyte flow cytometer. A chi-squared test was performed using FlowJo v10.7.1, where significance in histogram distribution was confirmed if T(x) between parental and OxR stained samples was greater than T(x) between background (unstained) parental and OxR samples (see Supplementary file 1).

FRET

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Cells were prepared as described above, but without fixation or permeabilization. Samples were stained for LRs using the Vybrant Alexa Fluor 555 Lipid Raft Labeling Kit (Invitrogen, V34404). Samples were then stained with 5 µl FITC anti-human DR4 (Thermo Fisher Scientific, clone DR-4-02) for 30 min at 4°C. Samples were washed twice with HBSS and analyzed using a Guava easyCyte flow cytometer. Donor quenching FRET efficiency was calculated using the following formula:

E=1-FILR+DR-FIBFIDR-FIB

where E is the FRET efficiency, FILR+DR is the mean fluorescence intensity of the double-stained LR/DR4 sample (acceptor + donor), FIDR is the mean fluorescence intensity of the DR4-only stain (donor only), and FIB is the fluorescence intensity of an unstained sample (background). Fluorescence intensity was recorded in the donor (FITC) channel.

TRAIL combination treatments

Resveratrol

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Parental SW620 and HCT116 cells were plated at 100,000 cells/well onto 24-well plates and incubated overnight at 37°C. Cells were treated with 70 µM resveratrol (Sigma) in combination with 0.1–1000 ng/ml of TRAIL for 24 hr. Following treatment, cells were collected for Annexin-V/PI apoptosis assay. TRAIL sensitization was calculated using the following equation:

TRAILSensitizationresveratrol=(%ViableParentalCells)(%ViableParentalCells+resv)(%ViableParentalCells)100%

where TRAIL + resv treatments were normalized to resveratrol treatment in the absence of TRAIL to account for any resveratrol-associated cytotoxicity.

Nystatin

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SW620 OxR and HCT116 OxR cells were plated at 100,000 cells/well onto 24-well plates and incubated overnight at 37°C. Cells were treated with 5 µM nystatin (Thermo Fisher Scientific) in combination with 0.1–1000 ng/ml of TRAIL. Following treatment, cells were collected for Annexin-V/PI apoptosis assay. TRAIL sensitization was calculated using the following equation:

TRAILSensitizationnystatin=(%ViableOxRCells+nys)(%ViableOxRCells)(%ViableOxRCells+nys)100%

where TRAIL + nys treatments were normalized to nystatin treatment in the absence of TRAIL to account for any nystatin-associated cytotoxicity.

2-Bromopalmitate

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SW620 parental and OxR cells were plated at 100,000 cells/well onto 24-well plates and incubated overnight at 37°C. Cells were treated with 3.5 µM 2BP (MilliporeSigma) in combination with 0.1–1000 ng/ml of TRAIL. Following treatment, cells were collected for Annexin-V/PI apoptosis assay. TRAIL sensitization was calculated as described above.

Anti-Fas (ZB4)

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SW620 OxR cells were plated at 100,000 cells/well onto 24-well plates and incubated overnight at 37°C. Cells were treated with 500 ng/ml human anti-Fas (MilliporeSigma, Clone ZB4) with and without 1000 ng/ml of TRAIL. Following treatment, cells were collected for Annexin-V/PI apoptosis assay. TRAIL sensitization was calculated as described above.

Western blot

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LRs were isolated according to the manufacturer’s protocol using the Minute Plasma Membrane-Derived Lipid Raft Isolation Kit (Invent Biotech, LR-042). Cell lysates and LR protein isolates were prepared by sonication in 4× Laemmli sample buffer (Bio-Rad, 1610747) and then loaded into 10% SDS-polyacrylamide gels for electrophoresis. Protein transfer onto a PVDF membrane was carried out overnight, and then blocked with Intercept (TBS) Blocking Buffer (LICOR, 927-60001) at RT for an hour. Primary antibody incubation occurred overnight at 4°C for DR4 (Cell Signaling Technology, 42533) and DR5 (Thermo Fisher Scientific, PA1-957) at 1:500 dilution and caspase-10 (Thermo Fisher Scientific, PA5-29649) at a 1:1000 dilution in LICOR buffer. Cell lysate protein bands were normalized to GAPDH (EMD Millipore, MAB347) at 1:2000 dilution, while LR isolates were normalized to β-actin (Santa Cruz, 47778) at 1:1000 dilution in LICOR blocking buffer. Western blots were quantified using the Licor Odyssey Fc with IRDye 800CW goat anti-rabbit secondary antibody (LICOR, 926-32211) and IRDye 800CW goat anti-mouse secondary antibody (LICOR, 926-32210) at a dilution of 2:15,000. Quantification was done following the LICOR housekeeping protein normalization protocol.

Palmitoylation assay

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Cells were grown to 70% confluency in a 100 mm tissue culture dish. Palmitoylation of DR4 was measured using the SiteCounter S-Palmitoylated Protein Kit (Badrilla, K010312) according to the manufacturer’s protocol. Input fraction controls (IFC) were obtained prior to thioester cleavage. Acyl preservation negative controls (APC-) were obtained by using an acyl preserving reagent instead of mass-tag conjugation. Western blots were run for DR4 following the ‘western blot’ protocol described above. The percentage of DR4 palmitoylation was calculated by dividing the total intensity of all palmitoylated bands (mass tag) divided by the average intensity of the IFC and APC(-) bands for that sample.

To measure the amount of total palmitoylated protein, cells were cultured in 96-well plates at a concentration of 20,000 cells/well. The EZClick Palmitoylated Protein Assay Kit (BioVision, K416-100) was used in accordance with the manufacturer’s protocol. Cells were incubated overnight with either 1× EZClick Palmitic Acid label in media or culture media with no label (background control). Cells were recovered and stained using EZClick Fluorescent Azide, then analyzed via flow cytometry for shifts in FL2-H intensity. Median fluorescence intensity (MFI) was calculated by subtracting the background intensity from each sample (Palmitic Acid label [-]/ Fluorescent Azide [+]).

Patient blood samples

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Peripheral whole blood samples of 10 ml were collected from 13 metastatic CRC patients after informed consent. Patient criteria for this study included the following: presenting with metastatic CRC at the time of blood draw and undergone (or undergoing) oxaliplatin-containing chemotherapy (i.e., FOLFOX). Additionally, five patients had samples collected through their respective chemotherapy regimens. De-identified blood samples were transported from the Guthrie Clinic to Vanderbilt University and processed within 24 hr. Blood samples were split for treatment (8 ml) and death receptor/LR staining (1–2 ml).

Ex vivo treatment of CRC patient blood samples

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For the treated samples, 2 ml of blood were treated with either 40 µl of control liposomes, 40 µl TRAIL/E-selectin conjugated liposomes (290 ng/ml of TRAIL), 6 µl (290 ng/ml) of soluble TRAIL, or 2 µl (5 µM) of oxaliplatin. Liposomes were synthesized using a thin-film hydration method followed by extrusion and his-tag conjugation as described previously (Mitchell et al., 2014). The aliquots were sheared for 4 hr in a cone-and-plate viscometer (Brookfield LVDVII) at a shear rate of 120 s−1. Prior to incubation, the cone-and-plate viscometers were blocked using 5% BSA for 30 min. After 4 hr, the blood aliquots were washed from the viscometer’s spindle and cup by using twice the volume of HBSS without calcium and magnesium. Blood aliquots were placed over twice the volume of Ficoll (GE Healthcare) to separate out mononuclear cells within the buffy coat. CTCs were enriched using a negative selection kit with CD45 magnetic beads (Mylteni Biotech, 130-045-801) following the manufacturer’s protocol (Ortiz-Otero et al., 2020).

The resulting isolated CTCs were placed in cell culture overnight using RPMI media supplemented with 10% FBS. After 1 day in culture, the cells were recovered from the tissue culture plate and stained with 100 µl of propidium iodide for 15 min. Cells were washed, fixed with 4% PFA, and cytospun onto microscope slides using a Cytospin 3 (Shandon). Samples were then permeabilized and blocked with 100 µl of 0.25% Triton-X (Sigma) for 15 min and 100 µl of blocking solution (5% BSA and 5% goat serum) for 1 hr, respectively. Cells were stained with anti-CD45 conjugated with biotin (BioLegend, clone HI30) for 45 min at 1:50 dilution. Finally, cells were stained with 100 µl of streptavidin-conjugated Alexa Fluor 647 (Thermo Fisher Scientific, S21374) at 1:200 dilution and 20 µl per sample of anti-cytokeratin conjugated with FITC (BD Pharmingen, clone CAM5.2) for 45 min. Cells were washed 3× after each staining incubation using 200 µl of 0.02% Tween20 in PBS. Cells were stained with 10 µl of DAPI mounting media (Vector Laboratories), covered with a coverslip (no. 1.5, VWR), and sealed with nail polish.

Five images per sample were taken at random locations using an LSM 710 (Carl Zeiss) with a 20×/0.8 objective. The cell number in the sample was scaled up by multiplying by the relative area (slide area/frame area). Viable tumor cells were identified using the following criteria: (1) positive for DAPI, (2) negative for CD45, (3) positive for cytokeratin, and (4) negative for propidium iodide.

Staining of LR and death receptors in primary CTCs

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CTCs from the remainder of the patient blood were isolated and cytospun onto slides as described above. Death receptors and LRs were stained and analyzed as detailed above in ‘Confocal microscopy and image analysis.’ LRs were stained using the Vybrant Alexa Fluor 555 Lipid Raft Labeling Kit (Invitrogen, V34404) after CTCs were spun onto slides. Secondary staining for DR4 and DR5 was completed using goat anti-mouse Alexa Fluor 647 (Thermo Fisher Scientific, A21235) at a 1:200 dilution. Cells were also stained with FITC-conjugated cytokeratin, as described above, to positively identify CTCs for analysis.

Statistical analysis

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Data sets were plotted and analyzed using GraphPad Prism 9. When comparing two groups, a symmetric unpaired t-test was used with p<0.05 considered significant. One-way ANOVA with multiple comparisons was used for multiple groups with p<0.05 considered significant. At least three independent biological replicates were used for each experiment unless otherwise stated.

Data availability

Raw data has been deposited to Dryad, available here: https://doi.org/10.5061/dryad.3xsj3txg3.

The following data sets were generated
    1. Greenlee JD
    2. Cavestany ML
    3. Ortiz-Otero N
    4. Liu K
    5. Subramanian T
    6. Cagir B
    7. King MR
    (2021) Dryad Digital Repository
    Oxaliplatin Resistance in Colorectal Cancer Enhances TRAIL Sensitivity Via DR4 Upregulation and Lipid Raft Localization.
    https://doi.org/10.5061/dryad.3xsj3txg3

References

    1. Nagane M
    2. Pan G
    3. Weddle JJ
    4. Dixit VM
    5. Cavenee WK
    6. Huang HJ
    (2000)
    Increased death receptor 5 expression by chemotherapeutic agents in human gliomas causes synergistic cytotoxicity with tumor necrosis factor-related apoptosis-inducing ligand in vitro and in vivo
    Cancer Research 60:847–853.
  1. Book
    1. Rodriguez-Bigas MA
    2. Lin EH
    3. Crane CH
    4. Cancer S
    (2003)
    Holl-Frei Cancer Med (Sixth Edition)
    National Library of Medicine.

Decision letter

  1. Maureen E Murphy
    Senior and Reviewing Editor; The Wistar Institute, United States

Our editorial process produces two outputs: i) public reviews designed to be posted alongside the preprint for the benefit of readers; ii) feedback on the manuscript for the authors, including requests for revisions, shown below. We also include an acceptance summary that explains what the editors found interesting or important about the work.

Acceptance summary:

This manuscript focuses on a potentially new therapeutic vulnerability of colorectal cancer (CRC) cells that are resistant to oxaliplatin treatment. Identifying new therapeutic strategies for metastatic CRC is a critical and urgent need. This study identifies a potentially new therapeutic approach, whereby Oxaliplatin resistant (OxR) cells show increased sensitivity to TRAIL-mediated apoptosis that is mediated by increased palmitoylation of DR4 and localization to lipid rafts. These findings serve as a proof of concept that patients who become resistant to Oxaliplatin therapy may benefit from TRAIL-based therapeutics.

Decision letter after peer review:

Thank you for submitting your article "Oxaliplatin Resistance in Colorectal Cancer Enhances TRAIL Sensitivity Via DR4 Upregulation and Lipid Raft Localization" for consideration by eLife. Your article has been reviewed by 3 peer reviewers, and the evaluation has been overseen by a Reviewing Editor and Maureen Murphy as the Senior Editor. The reviewers have opted to remain anonymous.

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:

While the findings of the authors are interesting, there are several major concerns with regard to data interpretation and experimental rigor.

1. The authors have not rigorously tested the role of DR4 in the sensitization to TRAIL for example through use of DR4 agonist antibodies used to treat oxaliplatin-resistant cells or through studying the effect of DR4 knockdown on the observed TRAIL sensitization. Similarly, the effect of resveratrol has not been rigorously attributed to DR4. Some data implicates DR5 in the sensitization (Supplementary Figure 6) but in the end the authors emphasize DR4. There could be other mechanisms involved, and this should be addressed.

2. The palmitoylation data in Figure 6 are equivocal. Since DR4 palmitoylation is required for its raft localization and its ability to oligomerize (Ref 35), the manuscript would be greatly enhanced if the authors could perturb palmitoylation and look at the effects on TRAIL sensitization, in order to strengthen their conclusions and render them less correlative.

3. There is very limited data on DR4 expression in CTCs from patient samples. The data included in Figure 7 with regard to DR4 is, to this reviewer, uninterpretable. Furthermore, the role of DR4 expression or its association with oxaliplatin resistance in the clinical samples is very unclear. The histograms looking at DR5 expression in Supplementary Figure 4 do not appear to support the statements about DR5 upregulation in oxaliplatin-resistant cells. A similar experiment is more convincing for DR4 in HCT116 that are oxaliplatin resistant but less convincing for oxaliplatin-resistant SW620 in Figure 3D. The quantifications in Figure 3D do not match the flow data, with oxaliplatin-resistant SW620 appearing to have more significantly increased DR5 than oxaliplatin-resistant HCT116 as comparing to their respective parental lines. These issues need to be thoroughly and rigorously corrected.

4. There is no mechanistic understanding for increased DR4 expression in oxaliplatin-resistant CRC cells. The authors suggest DR4 expression is increased in stem cells and this may be relevant given previous work that implicated a role for c-myc in DR4 induction in CSCs (ref 8). The role of Myc in the resistance to oxaliplatin or the sensitization to TRAIL is not explored here.

5. Can the authors confirm that the OxR cells have increased invasion and motility compared to parental cells? What is their morphology compared to parental cells? Before comparing area of stain positivity per cell (e.g. of DR4, LR, or DR4/LR co-localization), it should be demonstrated that there is no difference in cell size compared to parental cells.

6. To solidify the difference in DR4 expression, the authors should compare the intensity distribution per cell in the confocal microscopy images instead of the area per cell. To determine the area of DR4 per cell, the authors use a threshold for background. To demonstrate that the results are independent of the threshold and the morphological differences between OxR and parental cells, it is necessary to show the intensity distribution (Figure 3 and all following figures). The difference in intensity distribution derived from flow cytometry (Figure 3D) is small. Can the authors confirm significance in deviation using statistical tools (e.g. Kolmogorov-Smirnov test)? The western blot results are encouraging. Can the authors quantify and compare the observed differences from the western blots with statistical significance?

7. Figure 4 shows enhanced co-localization of DR4 and lipid rafts. Similar to the previous comments, the authors use binary masks (which is threshold-dependent) and compare the area per cell, which can be biased by differences in cell size between the two groups. This measure should be normalized by the total cell area. Additionally, the authors should strengthen their statements by using standard methods for co-localization analysis, such as pixel intensity spatial correlation (plugins available with Fiji). In Figure 4A, it appears that the lipid raft staining is not specific to the membrane for SW620-OxR, can the authors comment on this? There should be information on antibody optimization with control tissue in the methods section.

8. There is a lot of variability in the effect of reduced cell viability of circulating tumor cells across patients and draws. Have the authors considered inter- and intra-patient variation when comparing the cell viability with statistical tests?

9. The results in Figure 1 are not firmly supportive of the conclusions. Specifically, 2/4 (50%) of the cell lines tested show no difference in TRAIL sensitivity regardless of Oxaliplatin resistance. This needs to be addressed, and the conclusions toned down. In addition, the claims made on mitochondrial permeabilization are done on only 1 cell line. In the interest of rigor this should be reproduced in at least 2 other cell lines.

10. Volcano plots in Figure 2B show that Fas is dramatically affected in SW620 cells, yet the authors do not pursue this. What is the impact of Fas on sensitivity to TRAIL in this cell line? This is important because these cells show the greatest sensitivity.

11. The authors claim that OxR CRC cells have enhanced co-localization of DR4 into lipid rafts. While this appears to be the case in the imaging in Figure 4A-B, the Western blot data in Figure 4D does not really support this conclusion, as there appear to be extremely modest differences of Lipid Raft DR4 between parental and OxR cells. Can the authors quantify these blots using a western blot approach that uses linear range quantification, such as LiCor, and at the same time, the authors should look at a negative control, such as Lipid Raft DR5 and decoy receptors to show specificity of this result.

https://doi.org/10.7554/eLife.67750.sa1

Author response

Essential Revisions (for the authors):

While the findings of the authors are interesting, there are several major concerns with regard to data interpretation and experimental rigor.

1. The authors have not rigorously tested the role of DR4 in the sensitization to TRAIL for example through use of DR4 agonist antibodies used to treat oxaliplatin-resistant cells or through studying the effect of DR4 knockdown on the observed TRAIL sensitization. Similarly, the effect of resveratrol has not been rigorously attributed to DR4. Some data implicates DR5 in the sensitization (Supplementary Figure 6) but in the end the authors emphasize DR4. There could be other mechanisms involved, and this should be addressed.

To further demonstrate the role of DR4 in TRAIL sensitization, we have now treated parental and OxR cells with the DR4 agonist antibody Mapatumumab (Figure 3G-I, Figure 3—figure supplement 7). Mapatumumab is a fully human agonistic monoclonal antibody that has undergone Phase II clinical testing for use in non-small-cell lung cancer patients. OxR cells had a significant increase in the number of apoptotic cells and exhibited decreases in cell viability in a dose-dependent manner that was similar to the effects observed from treating cells with TRAIL. Likewise, the maximum Mapatumumab sensitizations seen in HCT116 and SW620 cells were similar to that observed from treating with TRAIL (Figure 3I).

To investigate the role of DR5 in sensitization from resveratrol/nystatin, we have measured colocalization changes of DR5 from these treatments. We found that resveratrol and nystatin had no significant effects on DR5 lipid raft colocalization, except in SW620 OxR cells where nystatin treatment surprisingly resulted in a slight increase in DR5 colocalization (Figure 5—figure supplement 1).

2. The palmitoylation data in Figure 6 are equivocal. Since DR4 palmitoylation is required for its raft localization and its ability to oligomerize (Ref 35), the manuscript would be greatly enhanced if the authors could perturb palmitoylation and look at the effects on TRAIL sensitization, in order to strengthen their conclusions and render them less correlative.

This is a great suggestion. To provide more evidence for the role of DR4 palmitoylation in sensitization, the well characterized palmitoylation inhibitor 2-bromopalmitate (2BP) was used. Parental and OxR cells were treated in combination with TRAIL and 2BP for 24 hours. 2BP increased the IC50 of SW620 OxR cells from 108 ng/ml to >1000 ng/ml, and significantly reduced the number of apoptotic cells in both parental and OxR cells (Figure 6D-E). The effects of palmitoylation inhibition were most pronounced in OxR cells, and the maximum %TRAIL sensitization was significantly decreased by 2BP treatment (Figure 6C). Treating with the palmitate analogue 2BP has been shown to inhibit DR4 palmitoylation, while DR5 is not palmitoylated (see ref 40 from Rossin et al.,).

3. There is very limited data on DR4 expression in CTCs from patient samples. The data included in Figure 7 with regard to DR4 is, to this reviewer, uninterpretable. Furthermore, the role of DR4 expression or its association with oxaliplatin resistance in the clinical samples is very unclear.

We have provided more data from patient CTCs on the relationships between DR4 expression, DR4/LR colocalization, oxaliplatin resistance, and treatment response from TRAIL liposomes (Figure 7F-G and Figure 7—figure supplement 2C-D). We demonstrate that both DR4 and DR4/LR colocalization correspond with increasing resistance to oxaliplatin (higher normalized percentage of viable CTCs after treating with oxaliplatin) with a positive slope that significantly deviates from zero. Additionally, we show that decreasing DR4 and DR4/LR colocalization coincide with reduced efficacy of TRAIL liposomes (higher percentage of viable CTCs after treatment), with a negative slope that significantly deviates from zero. The intrapatient treatment effects (between blood draws) in relation to DR4 colocalization was moved to the supplement (Figure 7—figure supplement 2E) as well as the effects of oxaliplatin resistance on TRAIL liposome efficacy (Figure 7—figure supplement 2A-B). This was done to ensure we did not overstate the significance of DR4 colocalization with treatment draw, as this relationship was heterogeneous between patients. Likewise, the data showing insignificant changes in treatment response between oxaliplatin sensitive and resistant patients was for a relatively small patient size. Therefore, this was moved to the supplement so that its insignificance is not overstated.

The histograms looking at DR5 expression in Supplementary Figure 4 do not appear to support the statements about DR5 upregulation in oxaliplatin-resistant cells. A similar experiment is more convincing for DR4 in HCT116 that are oxaliplatin resistant but less convincing for oxaliplatin-resistant SW620 in Figure 3D. The quantifications in Figure 3D do not match the flow data, with oxaliplatin-resistant SW620 appearing to have more significantly increased DR5 than oxaliplatin-resistant HCT116 as comparing to their respective parental lines. These issues need to be thoroughly and rigorously corrected.

In accordance with this comment and comment #6, new statistical tests were performed to measure differences in histograms and DR surface expression between parental and OxR cells (see response to comment #6 for more details on the statistical methods used). The flow cytometry histogram data in Supplementary Figure 4E (Now Figure 3—figure supplement 4E) display surface expression of DR5 (for these experiments, cells were not permeabilized as they were in the confocal analysis in panels A-D). That is, the quantification data measuring DR4/DR5 area is from analysis of microscopy images and distinct from the flow cytometry data. Only SW620 OxR cells were found to have significantly increased surface DR5 expression compared to their parental cell lines (See Supplementary File 1). The text within the Results section has been revised to reflect this finding, and to clarify the difference between data showing total death receptor expression and surface expression. We also provide quantifications of western blot data, showing no increase in DR5 expression in HCT116 OxR and SW620 OxR cells. We have provided a possible explanation for this discrepancy, that microscopy data shows that the total receptor area per cell was considerably lower for DR5 compared to DR4. This may result in statistical significance from relatively small changes within receptor content.

In terms of flow cytometry data for surface DR4 expression, we have added that both HCT116 OxR and SW620 OxR cells have significantly increased expression (See Supplementary File 1).

4. There is no mechanistic understanding for increased DR4 expression in oxaliplatin-resistant CRC cells. The authors suggest DR4 expression is increased in stem cells and this may be relevant given previous work that implicated a role for c-myc in DR4 induction in CSCs (ref 8). The role of Myc in the resistance to oxaliplatin or the sensitization to TRAIL is not explored here.

The reviewer raises a good point in suggesting to measure c-Myc expression between parental and OxR cell lines. We measured the changes in c-Myc expression using western blots from three independent experiments and found no increase in c-Myc expression in OxR phenotypes (see Author response image 1).

Author response image 1

In fact, SW620 OxR cells actually had a slight decrease in c-Myc expression. Within the Discussion section, we now mention that future studies should examine mechanisms behind this DR4 increase in oxaliplatin-resistant cells. Studies from the labs that derived these cell lines have previously shown that the expression of several microRNAs were significantly changed in OxR phenotypes (see ref 28 Tanaka et al.,). While it may be outside of the scope for this study to examine microRNA expression between cell lines, future studies using these cells could examine these effects in relation to DR4 upregulation. We also emphasize both within the introduction and discussion that changes in DR4 expression alone are often not sufficient to alter TRAIL sensitivity, while lipid raft localization can have much greater effects on apoptosis (see ref 17-20).

5. Can the authors confirm that the OxR cells have increased invasion and motility compared to parental cells? What is their morphology compared to parental cells? Before comparing area of stain positivity per cell (e.g. of DR4, LR, or DR4/LR co-localization), it should be demonstrated that there is no difference in cell size compared to parental cells.

To confirm OxR cells retain their phenotypes in our hands, we have performed Transwell invasion assays to demonstrate the enhanced migration within OxR cells (Figure 1—figure supplement 1B). Studies from the labs that derived these cell lines have demonstrated OxR cells have a notable spindle shape morphology compared to parental cells (see ref 26-28). Despite OxR cells having an increasingly spindle shaped morphology, we calculated that the total cell area remained unchanged between parental and OxR derivatives (Figure 3—figure supplement 1).

6. To solidify the difference in DR4 expression, the authors should compare the intensity distribution per cell in the confocal microscopy images instead of the area per cell. To determine the area of DR4 per cell, the authors use a threshold for background. To demonstrate that the results are independent of the threshold and the morphological differences between OxR and parental cells, it is necessary to show the intensity distribution (Figure 3 and all following figures).

We have demonstrated that parental and OxR cells have no significant changes in cell area (Figure 3—figure supplement 1). We also measured integrated density per cell on images with no thresholding, which yielded very similar results to our DR4 area per cell data (Figure 3—figure supplement 3). That is, HCT116 OxR and SW620 OxR cells had significantly upregulated DR4 whereas minimally sensitized HT29 and SW480 OxR cells did not.

The difference in intensity distribution derived from flow cytometry (Figure 3D) is small. Can the authors confirm significance in deviation using statistical tools (e.g. Kolmogorov-Smirnov test)?

For all death receptor stains using flow cytometry, we added statistics comparing deviations between parental and OxR cells. A chi-squared test was performed and significance in histogram distribution was confirmed if T(x) between parental and OxR stained samples was greater than T(x) between background (unstained) parental and OxR samples. Supplementary File 1 contains T(x) values calculated in FlowJo from chi-squared test analysis. Both HCT116 OxR and SW620 OxR cells were confirmed to have increased surface DR4 expression (Figure 3D). The methods (Flow cytometry: Surface DR expression) were modified to incorporate these analyses.

The western blot results are encouraging. Can the authors quantify and compare the observed differences from the western blots with statistical significance?

For all western blot analyses, we have added quantification and statistics across three independent experiments. While both HCT116 and SW620 OxR cells had increases in DR4 expression from western blots, only SW620 OxR cells were significant from their parental counterparts (Figure 3F).

7. Figure 4 shows enhanced co-localization of DR4 and lipid rafts. Similar to the previous comments, the authors use binary masks (which is threshold-dependent) and compare the area per cell, which can be biased by differences in cell size between the two groups. This measure should be normalized by the total cell area. Additionally, the authors should strengthen their statements by using standard methods for co-localization analysis, such as pixel intensity spatial correlation (plugins available with Fiji).

As we described in responses to comments #5 and #6, we have added analysis that shows parental and OxR cells have no significant changes in cell area between all four cell lines (Figure 3—figure supplement 1). Additionally, we have added supplementary colocalization analysis using the FIJI plugin JACoP (see methods section Confocal Microscopy and Image Analysis). The Manders’ Correlation Coefficient was calculated as the fraction of lipid raft colocalized DR4 (Figure 4—figure supplement 1). HCT116 OxR and SW620 OxR cells were the only cell lines with increased lipid raft colocalized DR4, consistent with our area per cell analysis using binary colocalization projects (Figure 4A-B). To further strengthen our claims of increased DR4/LR colocalization within HCT116 OxR and SW620 OxR cells, we have added new FRET data measured using flow cytometry (See methods section Flow Cytometry: FRET). FRET was calculated using a previously described flow cytometry donor quenching method (see ref 38 from Ujlaky-Nagy et al.). OxR cell lines had significantly increased DR4/LR FRET efficiency compared to their parental counterparts (Figure 4F).

In Figure 4A, it appears that the lipid raft staining is not specific to the membrane for SW620-OxR, can the authors comment on this? There should be information on antibody optimization with control tissue in the methods section.

We have changed the SW620 OxR image shown in Figure 4A to one that is more representative of the data. For lipid raft samples, best results were obtained when staining was completed in accordance with the manufacturer’s protocol, including the recommended antibody concentrations. Additionally, we have added data comparing lipid raft area per cell in parental and OxR cells (Figure 4—figure supplement 3). All cell lines, with exception to the HT29 cells, had insignificant changes in lipid raft quantities.

8. There is a lot of variability in the effect of reduced cell viability of circulating tumor cells across patients and draws. Have the authors considered inter- and intra-patient variation when comparing the cell viability with statistical tests?

We used a mixed effects repeated measures statistical model that separates between-subject variability from within-patient variability (variability between patient draws). Matching was effective, and while the effects of different treatments were significant (p<0.001), the fixed effects of draw number across treatments were insignificant (p=0.37). We found the average intrapatient coefficient of variation was low (0.279) compared to the interpatient coefficient of variation (0.555), and we have added these findings to the Results section. To further demonstrate that the lower intrapatient variation had no significant effect on statistical significance between treatments, we repeated the ANOVA with multiple comparisons analysis after averaging treatment responses for within-patient repeated draws (see Author response image 2).

Author response image 2

These analyses help to establish that the intrapatient variation from repeated draws were not artificially inflating the statistical significance between treatments.

9. The results in Figure 1 are not firmly supportive of the conclusions. Specifically, 2/4 (50%) of the cell lines tested show no difference in TRAIL sensitivity regardless of Oxaliplatin resistance. This needs to be addressed, and the conclusions toned down.

We have added a statement at the end of the discussion that reinforces that these results were only seen in two of the cell lines tested. Consistent with this, we also mention that future studies should examine genetic and phenotypic differences between these cell lines that make some susceptible and others resistant to this mechanism of TRAIL sensitization.

In addition, the claims made on mitochondrial permeabilization are done on only 1 cell line. In the interest of rigor this should be reproduced in at least 2 other cell lines.

We have repeated the JC-1 assay shown for the SW620 cell lines in the parental and OxR HCT116 cells. Similar to the SW620 OxR cells, HCT116 OxR cells had increasingly depolarized mitochondria at high TRAIL concentrations (Figure 1—figure supplement 3). We chose to perform JC-1 assays in these 2 cell lines as they were the only cells that exhibited high TRAIL sensitizations and decreases in IC50 in OxR cells.

10. Volcano plots in Figure 2B show that Fas is dramatically affected in SW620 cells, yet the authors do not pursue this. What is the impact of Fas on sensitivity to TRAIL in this cell line? This is important because these cells show the greatest sensitivity.

This is a good suggestion and an oversight on our part for not including previously. We have added analysis confirming FasR is upregulated in SW620 OxR cells using surface staining and flow cytometry (Figure 2—figure supplement 1A). We then tested the effects of treating SW620 OxR cells with the FasR neutralizing antibody ZB4 at high concentrations (500 ng/ml). FasR neutralization had no significant effects on the number of apoptotic cells or the maximum TRAIL sensitization, confirming that upregulation of Fas was inconsequential on TRAIL sensitivity in SW620 OxR cells (Figure 2—figure supplement 1B-C).

11. The authors claim that OxR CRC cells have enhanced co-localization of DR4 into lipid rafts. While this appears to be the case in the imaging in Figure 4A-B, the Western blot data in Figure 4D does not really support this conclusion, as there appear to be extremely modest differences of Lipid Raft DR4 between parental and OxR cells. Can the authors quantify these blots using a western blot approach that uses linear range quantification, such as LiCor, and at the same time, the authors should look at a negative control, such as Lipid Raft DR5 and decoy receptors to show specificity of this result.

As mentioned in the response to comment #6, we added quantifications for all western blot data. We quantified blots using LiCor software followed by normalization to a β-actin control. We found that both HCT116 OxR and SW620 OxR cells had significantly increased DR4 in lipid raft isolated fractions (Figure 4D-E). We also analyzed DR5 expression in lipid raft isolates as a negative control and found no detectable lipid raft DR5 in either cell line (Figure 4—figure supplement 2D). This is consistent with other studies which have shown that the function of DR4, but not DR5, is reliant on its translocation to lipid rafts (see ref 17, 20, 53 and 54). We have also added corroborating evidence for DR4/LR colocalization using flow cytometry FRET data (see response to comment #7).

https://doi.org/10.7554/eLife.67750.sa2

Article and author information

Author details

  1. Joshua D Greenlee

    Vanderbilt University, Department of Biomedical Engineering PMB, Nashville, United States
    Contribution
    Conceptualization, Data curation, Formal analysis, Validation, Investigation, Methodology, Writing - original draft, Writing - review and editing
    Competing interests
    No competing interests declared
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0002-1088-727X
  2. Maria Lopez-Cavestany

    Vanderbilt University, Department of Biomedical Engineering PMB, Nashville, United States
    Contribution
    Investigation, Methodology
    Competing interests
    No competing interests declared
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0001-5358-3746
  3. Nerymar Ortiz-Otero

    Vanderbilt University, Department of Biomedical Engineering PMB, Nashville, United States
    Contribution
    Investigation, Methodology
    Competing interests
    No competing interests declared
  4. Kevin Liu

    Vanderbilt University, Department of Biomedical Engineering PMB, Nashville, United States
    Contribution
    Investigation
    Competing interests
    No competing interests declared
  5. Tejas Subramanian

    Vanderbilt University, Department of Biomedical Engineering PMB, Nashville, United States
    Contribution
    Investigation
    Competing interests
    No competing interests declared
  6. Burt Cagir

    Donald Guthrie Foundation (DGF) for Research and Education Sayre, Sayre, United States
    Contribution
    Resources
    Competing interests
    No competing interests declared
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0002-9682-3807
  7. Michael R King

    Vanderbilt University, Department of Biomedical Engineering PMB, Nashville, United States
    Contribution
    Conceptualization, Supervision, Funding acquisition, Project administration, Writing - review and editing
    For correspondence
    mike.king@vanderbilt.edu
    Competing interests
    No competing interests declared
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0002-0223-7808

Funding

National Institutes of Health (R01CA203991)

  • Michael R King

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

Acknowledgements

This work was funded by the National Institutes of Health, Grant No. R01CA203991 to MRK. We thank all the cancer patients who donated blood samples for this study. We also thank the Oncology Research Coordinator at Guthrie Clinic, Michelle Hunter, for supervising blood sample collection and shipment. Finally, we thank Dr. Mika Hosokawa and Dr. Lee Ellis for providing us with the oxaliplatin-resistant cell lines, as well as Matthew R Zanotelli for assistance in writing the colocalization macro in FIJI.

Ethics

Human subjects: All of the experiments were done in accordance with the U.S Federal Policy for the Protection of Human Subjects and approved by the Institutional Review Board of the Guthrie Clinic (IRB#1909-42; Approved 10/01/2019). The participants in this study were fully informed regarding the objective of the current study and written consent was obtained.

Senior and Reviewing Editor

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

Publication history

  1. Received: February 22, 2021
  2. Preprint posted: March 5, 2021 (view preprint)
  3. Accepted: July 12, 2021
  4. Version of Record published: August 3, 2021 (version 1)

Copyright

© 2021, Greenlee 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.

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