Although initial treatment of ovarian cancer is successful, tumors typically relapse and become resistant to treatment. Because of poor infiltration of effector T cells, patients are mostly unresponsive to immunotherapy. Plasma gelsolin (pGSN) is transported by exosomes (small extracellular vesicle, sEV) and plays a key role in ovarian cancer chemoresistance, yet little is known about its role in immunosurveillance. Here, we report the immunomodulatory roles of sEV-pGSN in ovarian cancer chemoresistance. In chemosensitive conditions, secretion of sEV-pGSN was low, allowing for optimal CD8+ T-cell function. This resulted in increased T-cell secretion of IFNγ, which reduced intracellular glutathione (GSH) production and sensitized chemosensitive cells to cis-diaminedichloroplatinum (CDDP)-induced apoptosis. In chemoresistant conditions, increased secretion of sEV-pGSN by ovarian cancer cells induced apoptosis in CD8+ T cells. IFNγ secretion was therefore reduced, resulting in high GSH production and resistance to CDDP-induced death in ovarian cancer cells. These findings support our hypothesis that sEV-pGSN attenuates immunosurveillance and regulates GSH biosynthesis, a phenomenon that contributes to chemoresistance in ovarian cancer.
These findings provide new insight into pGSN-mediated immune cell dysfunction in ovarian cancer chemoresistance and demonstrate how this dysfunction can be exploited to enhance immunotherapy.
Ovarian cancer is the fifth most commonly diagnosed, but most fatal gynecologic cancer worldwide. In the United States, one of 95 women will die as a result of ovarian cancer. Ovarian cancer originating from the epithelial cells comprises of 90% of all reported cases and approximately 70% of these are high-grade serous subtype (HGS; ref. 1). The standard treatment for ovarian cancer is a combination of cytoreductive surgery and chemotherapeutic treatment with platinum and taxane derivatives (1). Although treatment is initially efficient, the tumor relapses and becomes resistant to treatment. The mechanisms involved in ovarian cancer chemoresistance are multifactorial with mutation of tumor suppressor genes, activation of oncogenes, dysregulation of apoptotic signaling, and immune suppression playing key roles (2). Ovarian cancer is considered a cold tumor, hence has poor immune cell infiltration. Thus, immunotherapy is relatively ineffective to date (3–5). There is still more to learn about the molecular contributors to chemoresistance in ovarian cancer to afford new diagnosis and treatment.
The tumor microenvironment (TME) is a strong contributing factor for chemoresistance (2). Both cytolytic (CD8+, CD4+, granzyme b, and IFNγ) and suppressive (PD-1+ tumor-infiltrating lymphocyte, CD25+ FoxP3+ regulatory T cells, PD-L1+, and CTLA-4) subsets are colocalized in ovarian cancer, resulting in an immunologic stalemate and net positive association with survival (3–5). Thus, multipronged approaches for immunotherapy are needed for effectiveness and should be tailored to the baseline features of the TME. Although patients with colon, melanoma, and lung cancer respond well to immunotherapy, patients with ovarian cancer are unresponsive due, in part, to the coldness of ovarian cancer (3, 4, 6). There is thus, an urgent need to explore and target novel pathways responsible for the coldness of ovarian cancer to make them more receptive to the immune system to enhance the efficacy of alternative immunotherapies.
Plasma gelsolin (pGSN) is the secreted isoform of the gelsolin (GSN) gene and a multifunctional actin-binding protein (7, 8). Total GSN forms a complex with Fas-associated death domain-like interleukin-1β–converting enzyme (FLICE)-like inhibitory protein (FLIP) and Itch, which regulates caspase-3 activation and chemoresistance in gynecologic cancer cells (9, 10). Small extracellular vesicles (sEV; mostly called exosomes) are vesicles of approximately 30–100 nm in size and formed within endosomes by membrane invaginations, whereas large EVs (lEV; mostly called microvesicles) range from 0.1 to 1.0 μm and are produced by membrane blebbing by cells under stress (11, 12). We have previously demonstrated that pGSN is highly expressed and secreted in chemoresistant ovarian cancer cells compared with its chemosensitive counterparts (13). pGSN is transported via sEVs and upregulates HIF1α-mediated pGSN expression in chemoresistant ovarian cancer cells in an autocrine manner, and confers cisplatin resistance in otherwise chemosensitive ovarian cancer cells (13). We have also shown that elevated circulating pGSN is associated with higher levels of residual disease after surgery and allows for detection of early-stage ovarian cancer (14). However, little is known about its interaction with immune cells in the TME. Whether pGSN contributes to immunosurveillance escape in the TME remains to be examined.
Glutathione (GSH) and the nuclear factor erythroid 2-related factor (NRF2)-dependent genes help to maintain homeostasis in normal cells, but can also provide a huge advantage to cancer cells to become cis-diaminedichloroplatinum (CDDP) resistant and escape immunosurveillance (15–17). NRF2 activation results in elevated levels of intracellular GSH that inactivates and efflux toxic therapeutic agents from cancer cells as well as increase DNA repair (15–17). However, it has not been demonstrated whether and how pGSN induces GSH production in ovarian cancer cell lines. Also, as to whether NRF2, an antioxidant transcription factor, is involved is yet to be investigated in the context of pGSN-mediated ovarian cancer chemoresistance (18). NRF2-dependent genes such as cystine/glutamate transporter (xCT) and glutamate-cysteine ligase regulatory subunit (GCLM) are associated with drug resistance in cancer (18–20). xCT is a membrane amino acid transporter that regulates the influx of cysteine and the efflux of glutamate. GCLM regulates the first step of GSH synthesis from l-cysteine and l-glutamate. Although suppressing GSH biosynthesis has a potential of enhancing tumor killing, no significant clinical advantage has been achieved (15–17). Exogenous pGSN treatment increases GSH levels in the blood, which mitigates radiation-induced injury in mice (21) although the exact mechanism has not been investigated. As to whether pGSN regulates GSH biosynthesis, is not known. The purpose of this study was to investigate the immunoinhibitory and GSH regulatory role of pGSN in ovarian cancer chemoresistance.
We hypothesized that increased pGSN will suppress CD8+ T-cell function as well as promote GSH production, an outcome that contributes to ovarian cancer chemoresistance. For the first time, we report in this study that in chemosensitive condition, ovarian cancer secretes low levels of sEV-pGSN, which is unable to suppress the functions of CD8+ T cells. Thus, optimal secretion of IFNγ inhibits the production of GSH in ovarian cancer cells. In chemoresistant conditions, increased pGSN promotes NRF2-dependent production of GSH in ovarian cancer cells as well as caspase-3–dependent apoptosis in CD8+ T cells.
Materials and Methods
Ethics statement and tissue sampling
A written informed consent was obtained from all subjects. The study was conducted in accordance with the appropriate guidelines approved by the Centre hospitalier de l′Universite de Montreal (CHUM) Ethics Committee [Montreal, Quebec, Canada Institutional Review Board (IRB) approval number, BD 04-002] and the Ottawa Health Science Network Research Ethics Board (Ottawa, Ontario, Canada, IRB approval number, OHSN-REB 1999540-01H). Normal and tumor tissues were collected from patients with ovarian cancer receiving treatment from 1992 to 2012 at the CHUM (Montreal, Quebec, Canada). A total of 208 formalin-fixed, paraffin-embedded HGS and unverified ovarian cancer tissues as well as 14 normal fallopian tube samples in duplicates were used to build a tissue microarray (TMA), as described previously (22). Details of patient population are outlined in Supplementary Table S1. Patients were diagnosed, tissues examined, and clinical data gathered as described previously (22).
Interrogation of ovarian cancer public datasets
Two ovarian serous cystadenocarcinoma datasets publicly available on cBioPortal (https://www.cbioportal.org/) were interrogated: The Cancer Genome Atlas (TCGA), nature 2011 (n = 489) and TCGA, firehouse legacy (n = 530). The mRNA expression patterns of pGSN (GSN), NRF2 (NFE2L2), xCT (SLC7A11), GCLM, IFNγ (IFNG), granzyme B (GZMB), and perforin (PRF1) were evaluated in each patient and presented as heatmaps. Pearson and Spearman correlation tests were performed to assess the association between pGSN and the other genes. Significant correlations were inferred as P ≤ 0.05.
Tissue sections (TMAs) were immunostained using the BenchMark XT Automated Stainer (Ventana Medical System Inc.), as described previously (22). After deparaffinization and antigen retrieval, the tissues were stained with their respective antibodies. Details of staining are described in the Supplementary Materials and Methods.
Ovarian cancer cell pellets (TMA) were immunostained using BenchMark XT Automated Stainer (Ventana Medical System Inc.). After deparaffinization and antigen retrieval, the tissues were stained with anti-pGSN and then incubated in a secondary antibody. Details of tissue staining and antibodies used are described in the Supplementary Materials and Methods and Supplementary Table S2, respectively.
CDDP, phenylmethylsulfonyl fluoride (PMSF), aprotinin, DMSO, sodium orthovanadate (Na3VO4), CCK-8, and Hoechst 33258 were supplied by MilliporeSigma. Two preparations of pGSN siRNA (siRNA1 and 2) and scrambled sequence siRNA (control) were purchased from Integrated DNA Technologies and Dharmacon, respectively. Human CRISPR/Cas9 knockout (KO) and CRISPR activation (OX) plasmid for FLIP (short and long), and their scrambled sequence siRNA (control) were purchased from Santa Cruz Biotechnology. Recombinant human pGSN (rhpGSN) and IFNγ (rhIFNγ) were purchased from Cytoskeleton, Inc. and Life Technologies, respectively. pGSN cDNA and 3.1A vector plasmids were generously provided by Dr. Dar-Bin Shieh (National Cheng Kung University Hospital, Tainan, Taiwan). pCT-CD63-GFP was purchased from System Biosciences, LLC. See Supplementary Table S2 for details on antibodies and other reagents.
Cell lines and primary cells
Human peripheral CD8+ and CD4+ T cells were purchased from Stemcell Technologies. T cells were expanded with ImmunoCult XF T-cell expansion media and activated using ImmunoCult Human CD3/CD28/CD2 T-cell activator. Both expansion and activating media were purchased from Stemcell Technologies. Chemosensitive and chemoresistant ovarian cancer cell lines (1.6 × 106 cells) of HGS and endometrioid histologic subtypes were used for all in vitro studies. The endometrioid cell lines were generously donated by Dr. Barbara Vanderhyden (Ottawa Hospital Research Institute, Ottawa, Ontario, Canada), whereas the HGS cell lines were kindly provided by A.-M. Mes-Masson. Cell lines used were tested for Mycoplasma contamination using PlasmoTest Mycoplasma Detection Kit (InvivoGen, catalog no.: rep-pt1), authenticated, and continuously monitored for morphologic changes as well as growth rate for any batch-to-batch change. Cell lines were maintained between passages 10 and 21 during the study after thawing. Details on the histologic subtypes and mutations of the cell lines used are described in Supplementary Table S3. The HGS cell lines were cultured and maintained in OSE Medium (Wisent Inc.), whereas the endometrioid cell lines were cultured and maintained in DMEM (Gibco DMEM/F12; Life Technologies, catalog nos.: 10565-018/10313-021) and/or Gibco RPMI1640 (Life Technologies, catalog no.: 31800-022), as reported previously (23–26). Media were supplemented with 10% FBS (MilliporeSigma), 50 U/mL penicillin, 50 U/mL streptomycin, and 2 mmol/L l-glutamine (Gibco Life Technologies). All experiments were carried out in serum-free media.
Gene interference and transient transfection
Cells were transfected (50 nmol/L, 24 hours) with CRISPR/Cas9-KO plasmids (2 μg, 24 hours) and siRNAs (empty vector as controls) using Lipofectamine 2000. Cells were transfected (2 μg, 24 hours) with pGSN cDNA and FLIP activation plasmid (empty vector as controls). Cells were then treated with CDDP (10 μmol/L, 24 hours) or sEV-pGSN (40 μg/4 × 105 cells, 24 hours) and harvested for analysis, as described previously (23, 24, 27). Two different siRNAs were used for each target to exclude off-target effects. Successful knockdown/KO and overexpression were confirmed by Western blotting (9). See Supplementary Table S2 for details on antibodies.
EVs isolation, characterization, and nanoparticle tracking analysis
Serum-free conditioned media (CM) from cultured cells were used for EV isolation and characterization, as described previously (28). sEVs were isolated by differential ultracentrifugation: 300 × g for 10 minutes at room temperature to remove cells; 2 × 104 × g for 20 minutes, room temperature to remove lEVs (microparticles); and then 10 × 104 × g for 90 minutes at 4°C for sEVs (exosomes). Details on EV characterization and nanoparticle tracking analysis are provided in the Supplementary Materials and Methods.
sEV-GFP tagging and uptake
Chemosensitive and chemoresistant ovarian cancer cell lines (1.6 × 106 cells) were transfected with exosome cytotracer, pCT-CD63-GFP (SBI System Biosciences; CYTO120-PA-1; 1 μg) for 24 hours in serum-free RPM11640. CM were collected and sEVs isolated. Activated human peripheral CD8+ T cells were treated with sEVs (40 μg/4 × 105 cells, 24 hours). Cells were collected and Western blot analysis was used to assess pGSN and GFP contents. Details of antibodies are described in Supplementary Table S2.
Protein extraction and Western blot analysis
Western blotting procedure for proteins was carried out as described previously (9, 23, 24). After protein transfer, membranes were incubated with primary antibodies (1:1,000) in 5% (w/v) blotto and subsequently with the appropriate horseradish peroxidase–conjugated secondary antibody (1:2,000) in 5% (w/v) blotto. See Supplementary Table S2 for details of antibodies used. Chemiluminescent Kit (Amersham Biosciences) was used to visualize the peroxidase activity. Signal intensities generated on the film were measured densitometrically using Image J.
Concentrations of IL2, TGFβ, TNFα, IL12, IL4, and IFNγ in cell-free CM from human peripheral CD8+ and CD4+ T cells treated with sEVs and rhpGSN were measured by Multi-Analyte ELISArray Kit (Qiagen) and pGSN (patient plasma) by sandwich ELISA (Aviscera Bioscience, Inc.), as determined previously (14). Details of the assay are provided in the Supplementary Materials and Methods.
Intracellular GSH detection
Intracellular GSH in cell lysates (100 μL) were colorimetrically determined at 450 nm, using a GSH detection assay kit (ab239727) and a microtiter plate reader, as per the manufacturer's instructions. A total of 1.6 × 106 cells (cultured in DMEM) were used in each experiment in triplicates. Concentrations were reported as μmol/L/mg protein.
Assessment of cell proliferation and apoptosis
Apoptosis and cell proliferation were assessed morphologically with Hoechst 33258 nuclear stain and colorimetrically with the CCK-8 assay, respectively. “Blinded” counting approach was used to prevent experimental bias with the Hoechst 33258 nuclear staining.
Statistical analyses were performed using the SPSS Software version 25 (SPSS Inc.), PRISM Software version 8.3 (GraphPad), Student t test, one- or two-way ANOVA, and Bonferroni post hoc tests (to determine the differences between multiple experimental groups). Two-sided P ≤ 0.05 was inferred as statistically significant. The relationship of variables to other clinicopathologic correlates was examined using Fisher exact test, t test, and Kruskal–Wallis test, as appropriate. Survival curves [progression-free survival (PFS) and overall survival (OS)] were plotted with Kaplan–Meier and P values calculated using the log-rank test. Univariate and multivariate Cox proportional hazard models were used to assess the HR for age, residual disease, stage [Federation Internationale des Gynaecologistes et Obstetristes (FIGO)], pGSN, and CD8+ T cells as well as corresponding 95% confidence intervals (CI).
Tumor staging and pathology was performed by a certified gynecologic oncology team. The characteristics of patients with ovarian cancer (N = 208) are described in Supplementary Table S1. The median age of patients was 62 years (range, 36–89 years) and classified as FIGO stages I (N = 13), II (N = 20), III (N = 153), and IV (N = 22). Patients in this study received no neoadjuvant chemotherapy or radiotherapy prior to sample collection at surgery. Eighty-six patients received complete/optimal cytoreduction. The median PFS and OS were 18 and 49 months, respectively.
pGSN expression and CD8+ T-cell infiltration in normal fallopian tube and HGS ovarian cancer tissues
Although increased pGSN expression renders HGS cancer cell lines resistant to CDDP treatment (13), the role of pGSN in the TME is not known. To investigate this, we first examined pGSN expression and CD8+ T-cell infiltration using immunofluorescence (IF) on a TMA constructed with 208 ovarian cancer specimens (174 HGS, 33 unverified, and one endometrioid) and 14 normal fallopian tube tissues (Fig. 1A; Supplementary Table S1). Prior to the study, staining of pGSN expression was optimized in nine HGS ovarian cancer cell pellets (Western blotting and IHC, TMA) as well as in a test TMA comprising of 15 HGS ovarian cancer tissues and seven normal fallopian tube tissues (IF; Supplementary Fig. S1A and S1B). We then determined by digital image analysis and measure of mean fluorescence intensity (MFI), the relative levels of pGSN expression in the epithelial and stromal areas of the tissues as well as their prognostic impact on the survival of the patients. pGSN was rarely expressed in epithelial and stromal areas of the fallopian tube tissue although CD8+ T-cell infiltration was detectable (Fig. 1A; Supplementary Table S4). In contrast, pGSN expression was clearly detectable in HGS ovarian cancer tissues, with the stromal levels [mean ± SD (717.4 ± 303.4)] being higher (P = 0.0001) than the epithelial levels [mean ± SD (586.9 ± 323.2)]. HGS ovarian cancer tissues were positive for CD8+ T cells with stromal localization [mean ± SD (39.3 ± 35.9 vs. 20.3 ± 23.2)] being higher (P = 0.0001; Fig. 1B). Increased expression of pGSN was significantly associated with PFS in the stroma region (P = 0.029), but not in the epithelial region (P = 0.232; Fig. 1C). Patients with increased epithelial pGSN expression had significantly poorer OS compared with patients with lower pGSN expression (P = 0.001; Fig. 1D). However, no significant difference was observed in OS between patients with lower and higher stromal pGSN expression (P = 0.131; Fig. 1D).
Increased pGSN expression is associated with reduced survival impact of tumor-infiltrated CD8+ T cells on patient survival
After demonstrating in HGS ovarian cancer tissues that patients with increased levels of pGSN are highly associated with poor chemoresponsiveness and survival, we hypothesized that pGSN expression may influence the prognostic impact of infiltrated CD8+ T cells. Thus, we analyzed the MFI of pGSN expression and CD8+ T-cell density in the epithelial and stromal regions of the HGS ovarian cancer TMAs and determined the prognostic impact of pGSN and CD8+ T-cell colocalization in patients with HGS ovarian cancer (Fig. 2; Supplementary Table S4). pGSN expression and CD8+ T-cell infiltration were clearly detectable in the epithelial and stromal regions of 95% of the HGS ovarian cancer TMAs investigated (Fig. 2A). Chemosensitive patients [progression-free interval (PFI) > 12 months; N = 135] had increased CD8+ T-cell infiltration regardless of the tissue location [mean ± SD; epithelium: 21.8 ± 25.6 (chemosensitive) vs. 18.0 ± 17.1 (chemoresistance) and stroma: 43.5 ± 40.0 (chemosensitive) vs. 30.9 ± 23.1 (chemoresistance)] compared with chemoresistant patients (PFI ≤ 12 months; N = 58), although the difference was only significant in the stromal compartment (epithelium: P = 0.23; stroma: P = 0.03; Fig. 2B). Patients with increased epithelial CD8+ T-cell infiltration had improved PFS (P = 0.005) and OS (P = 0.042) compared with patients with lower CD8+ T-cell infiltration (Fig. 2C). Patients with lower pGSN expression but higher CD8+ T-cell infiltration had improved PFS and OS compared with higher pGSN but lower CD8+ T-cell infiltration. Interestingly, increased pGSN expression appeared to be associated with lower protective effect of elevated levels of CD8+ T-cell infiltration as observed in the OS (P = 0.05) and PFS (P = 0.019) of patients with high pGSN and high CD8+ T cells (Fig. 2C). To determine why most infiltrated CD8+ T cells stained positive for pGSN and their survival impact on patients was hindered by increased pGSN expression, we immunostained the HGS ovarian cancer tissues for activated caspase-3, a marker for apoptotic cell death (Supplementary Fig. S1C). Caspase-3 activation was observed in both CD8 T cells and epithelial cells and was difficult attributing the caspase activation to only CD8+ T cells (Supplementary Fig. S1C). T cells barely express pGSN at both mRNA and protein levels (Supplementary Fig. S2A and S2B), hence it was surprising that CD8+ T cells stained positive for pGSN in both the epithelial [N = 195; mean ± SD (739.4 ± 352.7)] and stromal regions [N = 196; mean ± SD (798.9 ± 336.4)], although there was no significant difference between the locations (P = 0.09; Fig. 2D). We further analyzed the correlation between circulatory pGSN and infiltrated CD8+ T cells in the epithelium and stroma (Fig. 2E). Circulatory pGSN of patients were analyzed and dichotomized into low and high groups. The epithelial and stromal CD8+ T-cell infiltration were quantitated and compared. Interestingly, patients with low levels of circulatory pGSN had increased infiltration of CD8+ T cells compared with patients with higher circulatory pGSN levels in both the epithelial [P = 0.026; N = 92; (mean ± SD, 25.7 ± 28.0 vs. 15.1 ± 15.6)] and stroma [P = 0.02; N = 92; (mean ± SD, 47.0 ± 42.7 vs. 28.0 ± 29.7)] compartments (Fig. 2E). This supports the hypothesis that secreted pGSN regulates immune cell infiltration and function.
Prognostic impact of epithelial pGSN and relationship with other clinicopathologic parameters
The prognostic impact of epithelial pGSN and CD8+ T-cell density together with other clinicopathologic parameters (age, residual disease, and stage) were evaluated using uni- and multivariate Cox regression analyses, as demonstrated in Supplementary Tables S5 and S6, respectively. The optimal (using Fisher exact test) cutoffs for the markers were used to predict PFS and OS. From the univariate Cox regression analysis, only stage (FIGO), residual disease, and CD8+ T-cell density demonstrated a significant association with PFS (Supplementary Table S5). Unlike PFS, all parameters analyzed showed a significant association with OS. In the multivariate Cox regression analysis (Supplementary Table S6), only residual disease (HR, 0.552; 95% CI, 0.359–0.850; P = 0.007) significantly predicted PFS. In predicting increased risk of death, only pGSN (HR, 0.185; 95% CI, 0.089–0.373; P < 0.001) was significantly associated with OS (Supplementary Table S6).
sEV-pGSN induces CD8+ T-cell death via FLIP downregulation and caspase-3 activation
We had earlier detected that elevated pGSN expression in HGS ovarian cancer tissues was associated with shortened survival of patients with tumors highly infiltrated by CD8+ T cells (Fig. 2). Although activated caspase-3 was detected in the tissues, it was difficult attributing it to infiltrated CD8+ T cells or the epithelial cells (Supplementary Fig. S1C). We, therefore, hypothesized that sEV-pGSN induced apoptosis in CD8+ T cells via caspase-3 activation. Using standard in vitro techniques, we validated the proapoptotic role of pGSN in CD8+ T cells as well as the mechanism involved. CM from chemoresistant and chemosensitive ovarian cancer cells were collected and sEVs isolated. sEVs were characterized as determined previously (13); pGSN knockdown and overexpression in sEVs was confirmed in chemoresistant and chemosensitive ovarian cancer cells, respectively (Supplementary Fig. S3A and S3B).
Human peripheral CD4+ and CD8+ T cells were activated (using ImmunoCult Human CD3/CD28/CD2 T-cell activator) and expanded (using ImmunoCult XF T-cell expansion media purchased from Stemcell Technologies). Activated CD8+ T cells were treated with exogenous pGSN and sEV-pGSN derived from chemosensitive (OV2295), chemoresistant (OV90), and pGSN-knockdown (siRNA) chemoresistant (OV90-pGSN-KD) ovarian cancer cell lines. The influence of pGSN on apoptotic cell death and cell proliferation of T cells was assessed morphologically (Hoechst nuclear staining) and colorimetrically (CCK-8), respectively. Only exogenous pGSN and chemoresistant cell–derived sEV-pGSN induced apoptosis and decreased proliferation in CD8+ T cells (Fig. 3A), responses that were concentration dependent (Supplementary Fig. S4A). In contrast, CD4+ T-cell survival was not affected by sEV-pGSN treatment irrespective of the resistance status of donor cells (Supplementary Fig. S4B); however, were polarized into type 2 helper CD4+ T cells, as evident by increased IL4/IFNγ ratio (Supplementary Fig. S4C and S4D).
To further investigate the mechanism involved, activated CD8+ T cells were treated with exogenous pGSN, and CD8+ T-cell death and caspase-3–dependent signaling proteins were assessed upon sEVs uptake (Supplementary Fig. S2A). The apoptotic response of CD8+ T cell to treatment with chemoresistant cell–derived sEVs was associated with decreased FLIP content and activation of caspase-8 and caspase-3 (Fig. 3B). To demonstrate the role of FLIP in the action of pGSN, this cell survival protein was force expressed or knocked out in activated CD8+ T cells and then treated with chemoresistant cell–derived sEVs (Fig. 3C and D). Forced expression of FLIP attenuated pGSN-induced apoptosis, whereas FLIP KO further sensitized CD8+ T cells to pGSN-induced apoptosis (Fig. 3C and D). Coculturing activated CD8+ T cells with pGSN-KD–chemoresistant cells (OV90 and A2780cp) failed to induce caspase-3 activation and apoptosis (Fig. 3E). pGSN-cDNA–chemosensitive cells (OV2295 and A2780s) induced caspase-3 activation and apoptosis in activated CD8+ T cells after coculturing for 24 hours (Fig. 3F), suggesting the proapoptotic role of pGSN in activated CD8+ T cells.
Increased pGSN expression promotes NRF2-dependent GSH production
NRF2 is a transcription factor that controls the body's homeostatic balance by regulating the production of antioxidant proteins such as GSH and xCT (18). Elevated GSH production is associated with CDDP resistance in ovarian and other cancer types (15–17); however, the role of pGSN in regulating NRF2-dependent GSH biosynthesis is yet to be demonstrated. To investigate the expression and possible role of GSH in the regulation of chemoresponsiveness in ovarian cancer, we assessed the intracellular GSH production of chemoresistant and -sensitive ovarian cancer cells after treatment with CDDP (10 μmol/L, 24 hours). Chemoresistant ovarian cancer cell lines (OV90 and A2780cp) produced higher levels of intracellular GSH compared with their sensitive counterparts (OV2295 and A2780s; Fig. 4A and B). This phenomenon was associated with increased pGSN, total NRF2, and phospho-NRF2 (Fig. 4A and B). Increased γH2AX (an indicator of DNA damage as a result of CDDP accumulation) and CDDP-induced apoptosis were detected in the chemosensitive cells, but not in their resistant counterparts (Fig. 4A and B). To investigate the role of pGSN in GSH production, pGSN was knocked down in chemoresistant cell (OV90) using siRNA (50 nmol/L, 24 hours) and then treated with CDDP (10 μmol/L, 24 hours). pGSN downregulation resulted in a decrease in total NRF2, phospho-NRF2, and intracellular GSH, as well as sensitized the resistant cells to CDDP-induced γH2AX content and apoptosis (Fig. 4C). Forced expression of pGSN (2 μg cDNA, 24 hours) in chemosensitive ovarian cancer cells (OV2295) increased total NRF2, phospho-NRF2, and intracellular GSH; responses that were not affected by CDDP treatment. pGSN forced expression also attenuated CDDP-induced γH2AX and apoptosis in the sensitive ovarian cancer cells (Fig. 4D). Our interrogation of cBioPortal public datasets (TCGA firehouse legacy) revealed a positive and significant correlation between pGSN and NFE2L2 (NRF2) and SLC7A11 (xCT) involved in GSH production (Fig. 4E and F). Our interrogation of TCGA nature 2011 datasets also revealed a weak, but significant association between pGSN mRNA expression and NFE2L2(NRF2) and SLC7A11 (xCT; Supplementary Fig. S5A–S5C), providing supporting evidence that pGSN mediates GSH production via activation of the NRF2 pathway.
IFNγ secreted by activated CD8+ T cells reduces GSH production and sensitizes chemoresistant ovarian cancer to CDDP-induced death
The functionality of intratumoral CD8+ T cells is central to the efficiency of cancer treatment (3, 4, 29). Although increased pGSN induces apoptosis in CD8+ T cells as well as increases GSH production in ovarian cancer cells (Fig. 4), whether and how decreased viability of CD8+ T cells affects intracellular GSH production and ovarian cancer chemoresistance is not known. To examine whether intracellular GSH production in ovarian cancer cells could be altered by CD8+ T cells, chemoresistant (OV90) and -sensitive (OV2295) ovarian cancer cells were cultured with CM from activated CD8+ T cells (24 hours) and then treated with or without CDDP (10 μmol/L, 24 hours; Fig. 4G). Although treating sensitive ovarian cancer cells with CM from activated CD8+ T cells decreased intracellular GSH production, cell death was not observed (Fig. 4G). However, a significantly greater decrease in intracellular GSH production and a further increase in apoptosis were noted in the chemosensitive ovarian cancer cells treated with CDDP following pretreatment with CD8+ T-cell CM (Fig. 4G). Intracellular GSH remained elevated in the resistant cells regardless of treatment with CD8+ T-cell CM, a response associated with CDDP resistance (Fig. 4G). CDDP treatment of resistant cells following pretreatment with CD8+ T-cell CM was associated with a significant decrease in intracellular GSH and increased CDDP-induced apoptosis (Fig. 4G).
To further investigate the factor involved in reducing intracellular GSH and sensitizing resistant cells to CDDP-induced apoptosis, the cytokine profile in the CM from activated CD8+ T cells was assessed. IFNγ levels were elevated to the greatest extent compared with IL2, TGFβ, TNFα, and IL12 (Supplementary Fig. S6A). The functionality of activated CD8+ T cells by way of IFNγ secretion was tested by coculturing with chemosensitive (OV2295 and A2780s) and -resistant (OV90 and A2780cp) ovarian cancer cells (Fig. 4H). The presence of chemoresistant ovarian cancer cells markedly reduced IFNγ production compared with chemosensitive cells (Fig. 4H), suggesting a potential immunosuppressive function of pGSN. This phenomenon was validated in ovarian cancer public datasets (TCGA firehouse legacy and 2011) where negative significant, although weak, correlation was observed between pGSN and IFNγ mRNA expressions (Supplementary Fig. S6B–S6D). Using heatmap presentations, patients with low pGSN mRNA were observed to also express low levels of key antitumor soluble factors (granzyme b and perforin) derived from activated CD8+ T cells (Supplementary Fig. S6B–S6D).
IFNγ-mediated suppression of GSH production is via STAT1 phosphorylation
JAK/STAT signaling pathway mediates cellular responses upon IFNγ stimulation (30). Thus, we investigated whether JAK/STAT1 pathway is involved in IFNγ-mediated suppression of GSH production, using rhIFNγ instead of CD8+ T-cell CM (Fig. 5A). Chemoresistant (OV90) and -sensitive (OV2295) ovarian cancer cells were stimulated with IFNγ (10 pg/mL, 24 hours) and then treated with or without CDDP (10 μmol/L, 24 hours). In the chemosensitive condition, CDDP and IFNγ alone reduced intracellular GSH; however, apoptosis was only observed with treatment with CDDP, but not with IFNγ (Fig. 5A). In the chemoresistant condition, IFNγ treatment reduced intracellular GSH production, but not apoptosis (Fig. 5A). CDDP and IFNγ together significantly decreased intracellular GSH content and increased apoptosis (Fig. 5A). In both chemosensitive and -resistant conditions, STAT1 phosphorylation was only associated with either rhIFNγ stimulation alone or in combination with CDDP treatment.
We were also interested to know whether pGSN-induced chemoresistance could be mediated through suppression of IFNγ secretion in CD8+ T cells. To investigate this possibility, we primed activated CD8+ T cells with sEVs (40 μg/4 × 105 cells, 24 hours) derived from either chemosensitive (OV2295) or chemoresistant ovarian cancer cells (OV90; Fig. 5B). CM from the primed CD8+ T cells were collected and added to OV90 ovarian cancer cultures with/without IFNγ (10 pg/mL, 24 hours) and/or CDDP (10 μmol/L, 24 hours). CM from CD8+ T cells primed with sEVs from OV2295 cells failed to decrease intracellular GSH and attenuated apoptosis (Fig. 5B). CDDP treatment alone decreased intracellular GSH, which was associated with increased apoptosis (Fig. 5B). Combined IFNγ and CDDP treatment drastically reduced intracellular GSH and significantly increased apoptosis, responses that were associated with increased STAT1 phosphorylation (Fig. 5B). Following pretreatment with sEVs derived from chemoresistant cells, IFNγ and CDDP alone failed to decrease intracellular GSH; CDDP-induced apoptosis was also attenuated (Fig. 5B). However, combined treatment with CDDP and IFNγ resulted in marked decrease in intracellular GSH, increased STAT1 phosphorylation, and increased apoptosis (Fig. 5B).
To further investigate the role of IFNGR/JAK/STAT1 pathway in IFNγ-induced GSH production, chemoresistant ovarian cancer cells lines (OV90 and A2780cp) pretreated with anti-IFNGR1 blocking antibody (2 μg/mL, 24 hours) and/or JAK inhibitor 1 (5 μmol/L, 24 hours) were treated with either IFNγ (10 pg/mL, 24 hours) or/and CDDP (10 μmol/L, 24 hours; Fig. 5C). In both OV90 and A2780cp cell lines, only combined treatment with IFNγ and CDDP increased the phosphorylation of STAT1, reduced intracellular GSH, and increased apoptosis (Fig. 5C). These effects were attenuated in the presence of IFNγ receptor–blocking antibody as well as JAK1 inhibitor (Fig. 5C), suggesting the possible role of IFNGR1 and JAK1 in IFNγ-induced GSH production.
We extended our study to examine how intracellular GSH regulates the response of ovarian cancer cells to CDDP and IFNγ treatments and how pGSN could play a role in the mechanism. The GSH precursor N-acetyl cysteine was used to promote GSH production in chemosensitive cells, whereas buthionine sulfoximine, an inhibitor of the first rate-limiting enzyme (glutamate-cysteine ligase) of GSH synthesis, was used to deplete GSH production in chemoresistance ovarian cancer cells. Chemosensitive cells (A2780s and OV2295) were pretreated with N-acetyl cysteine (200 μmol/L, 12 hours) and then treated with or without IFNγ (10 pg/mL, 24 hours), CDDP (10 μmol/L, 24 hours), or CM from activated CD8+ T cells (3 mL, 24 hours; Fig. 5D). N-acetyl cysteine alone significantly induced intracellular GSH production without increasing apoptosis. CDDP failed to decrease N-acetyl cysteine–induced GSH production, a response associated with reduced apoptosis (Fig. 5D). Combining IFNγ or CM with CDDP and N-acetyl cysteine significantly reduced GSH production and increased apoptosis (Fig. 5D). GSH production in chemoresistant ovarian cancer cells (A2780cp and OV90) was decreased with buthionine sulfoximine (6 μmol/L, 12 hours) and were then treated with or without IFNγ (10 pg/mL, 24 hours), CDDP (10 μmol/L, 24 hours), or CM from activated CD8+ T cells (3 mL, 24 hours; Fig. 5E). Although GSH production was significantly reduced, apoptosis was unaffected by buthionine sulfoximine alone (Fig. 5E). CDDP, however, increased apoptosis in cells pretreated with buthionine sulfoximine (Fig. 5E). The addition of IFNγ or CM with buthionine sulfoximine and CDDP significantly and further reduced GSH production in the chemoresistant cells, a response associated with marked apoptosis (Fig. 5E).
To investigate the role of pGSN in the above phenomena, pGSN was knocked down using two siRNAs (50 nmol/L, 24 hours) in OV90 cells and treated with or without buthionine sulfoximine (6 μmol/L, 12 hours), IFNγ (10 pg/mL, 24 hours), CDDP (10 μmol/L, 24 hours), or CM from activated CD8+ T cells (3 mL, 24 hours; Fig. 5F). Silencing pGSN expression decreased GSH level with no effect on apoptosis (Fig. 5F). Buthionine sulfoximine treatment further reduced GSH levels in pGSN-KD OV90 cells without affecting apoptosis. CDDP treatment following pGSN KD and buthionine sulfoximine treatment further reduced GSH production and increased apoptosis, phenomena markedly enhanced by the addition of IFNγ or CM from activated CD8+ T cells (Fig. 5F).
In this study, we have demonstrated that the functionality, but not the mere presence, of infiltrated CD8+ T cells plays a significant role in prolonging the survival of patients with ovarian cancer. pGSN expression was significantly associated with poor chemoresponsiveness and shortened OS in patients with HGS ovarian cancer, making pGSN a poor prognostic marker. This is consistent with our previous study (13) as well as supports other studies where total GSN was shown as a poor prognostic marker in gynecologic, colon, pancreatic, breast, and head and neck cancers (9, 10, 31, 32). Reliable prognostic factors are crucial to achieving therapeutic success and improving patient survival. In our Cox regression analysis, Residual disease (RD) was observed to be an independent predictor of ovarian cancer recurrence, whereas epithelial pGSN presented as independent predictor for ovarian cancer death. This is consistent with our earlier findings where circulatory pGSN and RD significantly predicted patient survival (14). Combining RD and pGSN in both blood and tissues could provide a reliable prognostic index to enhance ovarian cancer patient survival and optimal management.
Tumor infiltration by CD8+ T cells provide survival benefits to patients with cancer (3, 4) and our findings that increased infiltrated CD8+ T cells are associated with reduced tumor recurrence are consistent with this notion. Interestingly, the prognostic benefits of infiltrated CD8+ T cells on patients' survival were inversely associated with increased pGSN levels. pGSN is highly secreted in patients with ovarian cancer compared with normal subjects (14) and transported via sEVs (13). Patients with higher levels of circulatory pGSN had decreased infiltration of CD8+ T cells compared with the cohorts with lower levels. This suggests that circulatory pGSN levels could predict the immunologic status of patients with ovarian cancer that could inform physicians on the possible therapeutic options.
We have previously shown that caspase-3, a mediator of apoptotic cell death, could be regulated by GSN (9). We thus examined caspase-3 activation in the same clinical samples and observed immunoreactivity. As to whether the caspase-3 activation was CD8+ T-cell specific or not was difficult to interpret because IF does not provide a time lapse to examine such mechanism. sEVs derived from chemoresistant, but not chemosensitive, ovarian cancer cells induced apoptosis in CD8+ T cells via FLIP downregulation and caspase-8/3 activations, responses that were aborted when pGSN was silenced in the sEVs. This is consistent with our previous study where the interaction of GSN with FLIP and Itch had a direct effect on caspase-3 activation (9). To date, this is the first study to provide mechanistic detail on how sEV-pGSN induces apoptosis in CD8+ T cells via activation of the FLIP/caspase-8 axis. This is consistent with the findings of Wieckowski and colleagues who have shown that tumor-derived vesicles promote apoptosis of activated CD8+ T cells (33) as well as other studies where GSN induced death in natural killer/T cells (34). Interestingly, sEV-pGSN had no apoptotic effect on CD4+ T cells, which were, however, polarized toward type 2 helper cells, as evident by increased secretion of IL4. Our findings are consistent with the findings by Chen and colleagues showing secreted gelsolin in prostate cancer desensitizes and induces apoptosis in infiltrated effector T cells, but not CD4 T cells (31) and provide new insights into how pGSN transported by sEVs induces apoptosis of CD8+ T cells in ovarian cancer, a phenomenon with significant impact on chemoresponsiveness. The decrease in IFNγ secretion as observed in the CD8+ T cells cocultured with resistant cells and granzyme b and perforin in public datasets might be as a result of the decreased number of T cells left after pGSN-induced death. Detailed investigations are, however, needed to demonstrate whether pGSN directly affects their gene expression independent of pGSN-induced CD8+ T-cell death. This could explain, in part, why most immunotherapy trials in patients with ovarian cancer have not achieved significant success (35–37). Silencing pGSN expression and secretion could hold the key to maximizing immunotherapy efficiency in patients with ovarian cancer and other solid tumors. Whether this indeed is true requires further investigation.
Intracellular GSH at a basal level provides antioxidant functions to cells by detoxifying and removing carcinogens (15–17). However, elevated levels of intracellular GSH chelate chemotherapeutic agents, which are subsequently effluxed from the cell, thus reducing their cellular accumulation needed for tumor cell death (15–17). This conference of resistance to chemotherapeutic agents provides protection to ovarian cancer cells and many cancers (15–17). It is yet to be shown whether pGSN plays a role in regulating intracellular GSH production in ovarian cancer cells. We observed that increased pGSN activates the NRF2–antioxidant pathway, leading to increased production of intracellular GSH. GSH therefore chelates, detoxifies, and effluxes CDDP, reducing γH2AX levels (an indicator of CDDP accumulation and DNA double-strand break) in tumor cells and protecting them from CDDP-induced apoptosis. It has been demonstrated that GSH secreted by fibroblasts is taken up by cancer cells, which protects them from CDDP-induced apoptosis (30) and marked increased GSH synthesis in ovarian cancer cells is a contributing factor to CDDP resistance (16, 17). Moreover, depleting GSH in human neuroblastoma MNS cells inhibits gelsolin expression although this response was not sustained (38). As to whether GSN has a positive feedback effect on GSH production is yet to be investigated. pGSN treatment provides an antioxidant advantage by way of elevating circulating GSH levels to mitigate radiation injury (21). Our current data are consistent with these findings and also advances an intrinsic regulatory mechanism by which GSH is involved in pGSN-mediated ovarian cancer chemoresistance.
Infiltration of the tumor crest by effector T cells is significantly associated with prolonged survival in patients with ovarian cancer treated with CDDP and other treatment modalities (4, 35, 36). Thus, we investigated the potential involvement of CD8+ T cells in sEV-pGSN–mediated regulation of intracellular GSH production. We observed that CD8+ T-cell–derived IFNγ activated the IFNGR1/JAK/STAT1 pathway in ovarian cancer cells, leading to reduced intracellular GSH level. As a result, ovarian cancer cells became sensitized to CDDP-induced apoptosis. This is consistent with a report that CD8+ T-cell–derived IFNγ regulates GSH and cysteine metabolism in fibroblasts via the JAK/STAT1 pathway (30). GSH antagonists, such as buthionine sulfoximine, phytochemical B-phenylethyl isothiocyanate, gossypol, 3-bromopyruvate, and acetaminophen, have shown therapeutic promises (17) although no significant clinical advantage has been achieved to date due, partly, to their adverse side effects. In a phase III clinical trial (NCT00350948), the GSH analogue Telcyta (TLK286) together with doxorubicin recorded poorer outcomes compared with the standard treatment regimen (39, 40). The role of sEV-pGSN in regulating both intracellular GSH biosynthesis and immunosurveillance could hold the key to maximizing treatment efficiency in patients with ovarian cancer.
In summary, we have demonstrated for the first time, the immunomodulatory role of sEV-pGSN in ovarian cancer chemoresistance. To facilitate future investigations, we offer the following hypothetical model for ovarian cancer cell–CD8+ T cell interaction in the TME in the regulation of chemosensitivity in human ovarian cancer (Fig. 6). In chemosensitive condition, sEV-pGSN secretion is relatively low in ovarian cancer cells, hence CD8+ T-cell function is minimally affected. Functional CD8+ T cells can, therefore, secrete higher levels of IFNγ, which reduces NRF2-dependent GSH production in ovarian cancer cells and sensitizes chemosensitive cells to CDDP-induced apoptosis. In the chemoresistant condition, marked sEV-pGSN secretion from ovarian cancer cells induce cell death in CD8+ T cells. With a small population of CD8+ T cells left, IFNγ secretion is reduced, hence NRF2-dependent GSH production in ovarian cancer cells remain high, resulting in resistance to CDDP-induced death. The above findings provide useful mechanistic and clinical information to maximize immunotherapy efficiency in chemoresistant ovarian cancer, as well as establish a reliable prognostic index for patients with ovarian cancer.
Disclosure of Potential Conflicts of Interest
No potential conflicts of interest were disclosed.
The funding agencies were not involved in the design, conduct, analysis, or interpretation of the study.
M. Asare-Werehene: Conceptualization, data curation, formal analysis, funding acquisition, validation, investigation, visualization, methodology, writing-original draft, writing-review and editing. L. Communal: Data curation, formal analysis, validation, investigation, visualization, methodology, writing-review and editing. E. Carmona: Resources, data curation, project administration, writing-review and editing. Y. Han: Writing-review and editing. Y.S. Song: Writing-review and editing. D. Burger: Conceptualization, resources, investigation, project administration, writing-review and editing. A.-M. Mes-Masson: Conceptualization, resources, data curation, supervision, funding acquisition, writing-review and editing. B.K. Tsang: Conceptualization, resources, supervision, writing-original draft, project administration, writing-review and editing.
This work was supported by the Canadian Institutes of Health Research (PJT-168949 to B.K. Tsang) and the Ovarian Cancer Canada (to M. Asare-Werehene). Tumor banking was supported by the Banque de tissus et de données of the Réseau de recherche sur le cancer du Fonds de recherche du Québec – Santé (FRQS), associated with the Canadian Tissue Repository Network. A.-M. Mes-Masson is a researcher of the CRHUM who receives support from the FRQS.
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