Hypoxia-induced exosomes contribute to a more aggressive and chemoresistant ovarian cancer phenotype: a novel mechanism linking STAT3/Rab proteins

Abstract

Hypoxia-mediated tumor progression, metastasis, and drug resistance are major clinical challenges in ovarian cancer. Exosomes released in the hypoxic tumor microenvironment may contribute to these challenges by transferring signaling proteins between cancer cells and normal cells. We observed that ovarian cancer cells exposed to hypoxia significantly increased their exosome release by upregulating Rab27a, downregulating Rab7, LAMP1/2, NEU-1, and also by promoting a more secretory lysosomal phenotype. STAT3 knockdown in ovarian cancer cells reduced exosome release by altering the Rab family proteins Rab7 and Rab27a under hypoxic conditions. We also found that exosomes from patient-derived ascites ovarian cancer cell lines cultured under hypoxic conditions carried more potent oncogenic proteins-STAT3 and FAS that are capable of significantly increasing cell migration/invasion and chemo-resistance in vitro and tumor progression/metastasis in vivo. Hypoxic ovarian cancer cells derived exosomes (HEx) are proficient in re-programming the immortalized fallopian tube secretory epithelial cells (FT) to become pro-tumorigenic in mouse fallopian tubes. In addition, cisplatin efflux via exosomes was significantly increased in ovarian cancer cells under hypoxic conditions. Co-culture of HEx with tumor cells led to significantly decreased dsDNA damage and increased cell survival in response to cisplatin treatment. Blocking exosome release by known inhibitor Amiloride or STAT3 inhibitor and treating with cisplatin resulted in a significant increase in apoptosis, decreased colony formation, and proliferation. Our results demonstrate that HEx are more potent in augmenting metastasis/chemotherapy resistance in ovarian cancer and may serve as a novel mechanism for tumor metastasis, chemo-resistance, and a point of intervention for improving clinical outcomes.

Fig 1. Isolation and characterization of exosomes

A,B) The concentration of exosomes released is higher in immortalized and primary ovarian cancer cells than normal epithelial and FT cells normalized to the respective cell counts (n=3±SD) as measured by NanoSight analysis. The Size mode: 105 nm averaged from three technical replicates for each sample(n=3±SD). C) Cryo-TEM images of the exosomes isolated from OVCAR8 and TR127 cells (Scale bar-100nm) D) Confirmation of exosome specific markers such as EPCAM, CD-63, TSG-101 and GM130 and a cis-Golgi apparatus protein for validation of vesicle purity in the exosome preparation, confirmed by Western Blot. E) Fold change in the exosome concentration isolated from different ovarian cancer cell lines and primary ovarian cancer cells (POCC) cultured in normoxia (20%O₂) and hypoxia (1%O₂) (n=3±SD; *p-value,0.05, ** 0.005, *** 0.0001). F) Gene Ontology (GO) Term analysis of the exosome protein data set shows that most of the exosomal proteins are related to cellular processes, metabolic processes and biological regulation in the hypoxia exosomes when compared with the normoxia exosomes from OVCAR8 cells(p<0.05). G) Ingenuity pathway analysis of the exosome proteome data set shows the list of biomolecules identified in the dataset to be associated in top six different diseases and functions. Fisher’s exact test was applied to calculate significance (p<0.05).

Fig.2. Hypoxia favors increased lysosomal docking to the plasma membrane

A&B) The lysosomes labelled using the lyso-tracker Red dye visualized by confocal microscopy in ovarian cancer cell lines (A2780 and OVCAR-8) show a perinuclear localization in normoxia and a peripheral orientation favoring the docking to the plasma membrane in hypoxia, confirming a more secretory phenotype (60×) with higher fluorescence intensity in hypoxic cells than the normoxic cells as quantified by Image J (n=3±SD; p<0.01). C). B-Hexosaminidase A concentration as a measure of lysosomal exocytosis by Competitve ELISA method shows increased concentration in Hypoxia and was further confirmed by the treatment of OVCAR8 with Bafilomycin –A1(autophagy inhibitor) (1μM for 24 hr) Vs Normoxia (n=3±SD; p<0.05 using student t-test). D) The ovarian surface epithelial cells (OSE) show a perinuclear localization of the lysosomes in normoxia. E) The peripheral lysosome co-localization (red) with LAMP-1(green) was further confirmed in cells isolated from patient ascites, which is a hypoxic environment. F) The real time gene expressions of LAMP1&2 in 48 hr period in hypoxia (HC-48) were increased when compared to normoxia (NC-48). Further the downregulated mRNA expression of Neuraminidase 1 (a negative regulator of lysosomal exocytosis) as observed in hypoxia causes the accumulation of oversialylated LAMP’s favoring the movement of lysosomes to the periphery of the cell (n=3±SD; p<0.001) using student t-test).

Fig.3. Alteration of Rabs and LAMPs expression under hypoxia: STAT3 regulated Rab7& 27a in vesicular trafficking

A, B) The protein and mRNA expression of Rab GTPase 7 and 27a were found to be inversely proportional in both Normoxia and hypoxia observed after 48 hr. The reduced Rab7 might reduce the endo-lysosomal fusion augmenting the lysosomal exocytosis which is further supported by increased Rab27a to release the exosomes in hypoxia (n=3±SD; p<0.05 using student t-test). C) The role of STAT3 in the exosome pathway was confirmed by measuring the exosome concentration isolated from STAT3 knockdown OVCAR-8 cells cultured in hypoxia for 48 hour and compared with the wild type OVCAR8 in Hypoxia (n=3±SD; p<0.005 using student t-test). D) The knockdown of STAT3 significantly increased the expression of RAb7 and decreased Rab27a when compared to OVCAR8 WT (hypoxia) demonstrating its role regulating the Rab GTPases involved in vesicle trafficking. E) Exosome concentration measured in STAT3-OE and KD OVCAR8 ells show the significant increase and decrease in the exosome concentration (n=3±SD). F) Also STAT3 knockdown cells showed a significant decrease of lysosomal numbers indicating its putative role in lysosome biogenesis under hypoxia. G. Exosomes concentrations were measured in HIF1α KD and STAT3 KD, no significance changes observed in HIF1 KD cells (n=3±SD).

Fig. 4. Exosome internalization and its influence on the tumor migration and metastasis in the recipient cells

A) Exosomes isolated from hypoxic ovarian cancer cells were labelled with exo-glow- green, which labels the proteins inside the exosomes, and co-cultured with the OVCAR-8 ovarian cancer cells in conditioned medium. The exosome internalization after 24 hrs was confirmed by confocal microscopy. The differential contrast image (DIC) shows that exosomes are internalized. B) The percentage of migration was quantified using Image J software in normal (OSE and FT-33 cells) and ovarian cancer cell lines (OVCAR-8) with and without HEx co-culture (n=3±SD; p<0.05 using student t-test). C &D) Invasive potential of the OVCAR-8 cells was analyzed using cell invasion transwell assay kit. The number of cells invading the membrane was significantly increased after 48 and 72hr when co-cultured with HEx and quantified as percentage of invasion as shown (n=3±SD; p<0.005 using student t-test. E) NEx and Hex(20μg on alternate days) was co-cultured with A2780 and OVCAR-8 cells in conditioned medium under normoxia for a week and observed an increase in the protein expression of activated STAT3 and proteins involved in tumor progression and metastasis-MMP2. F) To investigate the transport of STAT3 via exosomes the expression of STAT3 in the IOSE and cancer cell exosomes showed the presence of pSTAT3 in the exosomes derived from cancer cell line(OVCAR8) but absent in normal cell (IOSE) exosomes, though the total STAT3 was seen in both normal and cancer cell exosomes. G), The primary cells isolated from different patient ascites (n=6± SD, Stage IV) were cultured in conditioned medium for 48hrs and the exosome concentration measured by NTA showed higher magnitude (10¹¹) of exosome release when compared to the magnitude released from immortalize cancer cells(10⁹) in Normoxia, normalized to their respective cell counts(10⁷). H) pSTAT3 expression in primary ovarian tumor tissues, ascites and metastatic tissues (n=3±SD, *p<0.05, ** p<0.005) from the same patient show increased STAT3 activation in the ascites and metastatic sites than the primary tumor site implying the role of STAT3 in tumor progression and metastasis. I) The expression of oncoproteins such as STAT3, pSTAT3, and FAS were confirmed in the exosomes isolated from patient ascites normalized to the EpCam expression. J) Migration assay proved the role of exosomal STAT3 using exosomes isolated from both STAT3 WT and STAT3 knock down (KD) cells. The OVCAR8 cells co-cultured with exosomes from STAT3 KD cells for 48 hrs significantly decreased the migration potential of OVCAR- 8 cells.

Fig. 5. HEx enhance tumor metastasis and is capable of reprogramming normal cells

A, B) The ovarian bursa of mice were injected with POCC exosome treated OVCAR-8 cells after three weeks of co-culturing with NEx and Hex (20 μg on alternate days). The tumor weights were significantly increased after four weeks in OV8HEx injected mice than NEx or control OVCAR-8 injected mice as represented by dot plots (n=4±SD). C, D) The number of metastatic nodules were significantly higher in mice injected with OVCAR-8 cells, which were co-cultured HEx than in NEx and the control in the dot plot (n=3±SD, * p<0.05, **p<0.005). E) A similar experiment was done in mice injected with fallopian tube secretory cells that were co-cultured with NEx and Hex (20 μg on alternate days) for three weeks to see the capability of the exosomes to reprogram FT cells. No tumors were observed after four weeks but a significant change in the morphology of the fallopian tube was observed in all the mice injected with FT cells previously co-cultured with HEx for four weeks. F) The weight of the fallopian tube with the ovary was significantly higher in the HEx when compared to the NEx or control as represented by dot plots (n=3±SD). G) Hematoxylin and eosin (H&E) stained sections of (i) control mouse fallopian tube (FT) injected with immortalized FT cells; surface epithelium and glands spaced by stroma are seen. There is no cellular atypia; (ii) H&E stained sections of FT after injection of HEx treated FT cells. There is confluent epithelial proliferation with absence of intervening stroma. Atypical pleomorphic nuclei are identified. Ki67 staining of (iii) control mouse FT injected with immortalized FT cells shows scant nuclear labeling when compared to iv) FT section after injection of HEx treated FT cells mice. Each image shown at 40× magnification. H) Immunofluorescent double-labeling was performed in frozen tissue sections from normal and HEx treated fallopian tube using monoclonal antibodies specific for Ki67 (green)/CK8 (red) and ki67(green)/CD31(red). Hoechst 3342 staining was performed to display all cell nuclei. I) Merged channels showing Immuno-fluorescent staining of the mouse FT- tissue sections shows an increase in the IL-6 (DAPI-blue/IL-6-red) and pSTAT3 (DAPI-blue/pSTAT3-red) expressions in mice injected with FT- cells co-cultured with HEx when compared to the control tissue.

Fig.6. HEx involved in chemo-resistance

A) Quantification of serum exosome concentration in ovarian cancer (before cisplatin treatment), cisplatin sensitive and resistant patient (after Cisplatin treatment) samples (n=5±SD, **p<0.005) show increased exosomal concentration/ml of serum. B) The measurement of cisplatin concentration in exosomes after treatment with cisplatin for 24 hr using ICP-MS in different ovarian cancer cell lines cultured in normoxia and hypoxia shows a significant efflux of the platinum via exosomes in hypoxia than normoxia (n=8±SD) (**p<0.005, ***p<0.0001using student t-test). C) The percentage of cell proliferation determined by sulphorhodamine assay was found to be significantly increased by co-culturing the OVCAR-8 cells with Hex (20μg) when compared to the control group (n=8±SD) (p<0.005 using student t-test). D, E) The OVCAR8 cells without any exosome treatment show an increase in ɣH2Ax positive cells on treatment with cisplatin at 10 μM for 24 hr when compared to the HEx pre-treated OVCAR-8 cells and was quantified based on the number of ɣH2ax positive cells (n=3±SD; **p<0.005, ***p<0.0001). F) A reduction in cisplatin sensitivity, despite STAT3 inhibition by HO-3867 treatment for 3hrs prior to cisplatin treatment, was observed by via reduced apoptotic protein expression of cleaved PARP and caspases 3&7 in HEx treated OVCAR-8 cells, when compared the OVCAR-8 cells without any prior exosome treatment, Lane: 1 (Control), Lane:2 (DMSO), Lane:3 (HO3867-10μM, STAT3 inhibitor), Lane: 4 (Cisplatin-10μM), Lane: 5 (HO3867+CP). G) The exosome concentration under hypoxia in OVCAR-8 cells quantified by NTA showed a significant decrease upon treatment with 100 μM amiloride (n=3±SD; **p<0.005). H) The treatment of OVCAR-8 cells in hypoxia with a known exosome inhibitor, amiloride, reduced the peripheral localization of the lysosomes labelled with lyso-tracker red dye when compared to cisplatin treatment where more peripherally oriented lyosomes were observed as captured by Confocal microscopy. I, J) The cell survival and colony formation assay using OVCAR-8 cells shows that amiloride (100 μM), when treated alone, is not cytotoxic when compared to control, but when co-treated with cisplatin (10 μM) did increase in the cytotoxic effects in 24 hr as compared with cisplatin treatment alone (n=3±SD; **p<0.005).

Fig. 7

Schematic overview in hypoxic ovarian cancer cells representing STAT3 mediated signaling cascade of exosome release, contributing metastasis and chemo resistance and as well showing promising options for therapeutic interventions (Green arrow) with standard chemotherapy.