Hypoxia-induced exosome secretion promotes survival of African-American and Caucasian prostate cancer cells

African American men in the United States have higher mortality due to prostate cancer (PCa) compared to other races. One reason for this disparity is the lack of in-depth understanding of the PCa biology in African Americans. For example, hypoxia in prostate tumor microenvironment is associated with adverse prognosis; still, no hypoxia-related studies have been reported in African Americans. Here, we compared African-American and Caucasian PCa cells for exosome secretion under normoxic (21% O2) and hypoxic (1% O2) conditions. All cell lines showed higher exosome secretion under hypoxia but it was clearly more prominent in African-American PCa cells. Further, under hypoxia, Rab5 (a biomarker for early endosome) was clustered in perinuclear region; and CD63 (a biomarker for exosomes and multivesicular endosomes) showed greater co-localization with actin cytoskeleton especially in African American PCa cells. Importantly, exosome biogenesis inhibitors GW4869 (10–20 µM) or DMA (10–20 µg/ml) significantly decreased cell viability and clonogenicity in PCa cells. Interestingly, we also observed higher level of lactic acid loaded in exosomes secreted under hypoxia. Overall, under chronic hypoxia, PCa cells secrete more exosomes as a survival mechanism to remove metabolic waste.

Next, we characterized the exosomes isolated from PCa cells cultured under normoxic and hypoxic conditions by TEM. As shown in Fig. 2, in all the cell lines, exosome size was ≤100 nm.

Hypoxia promotes early endosome formation and fusion of multivesicular endosomes (MVE)
with plasma membrane. To understand why exosome secretion was higher in PCa cells under hypoxia, we analyzed formation of early endosomes and MVEs by staining for Rab5 and CD63 in African American PCa E006AA-hT and Caucasian PCa PC3 cells, respectively. Rab5 belongs to a family of small GTPases that regulates various steps in vesicular trafficking 26 . Rab5 regulates clathrin-coated vesicle-mediated transport from the cell membrane to early endosomes and homotypic early endosome fusion 27 . In both PCa cell lines under hypoxia, Rab5 was more clustered in the perinuclear region especially in E006AA-hT cells (Fig. 3A,B).
CD63 is a member of the tetraspanin family and mostly used as a biomarker for exosomes. CD63 is also present on MVEs; therefore, we next examined CD63 expression in E006AA-hT and PC3 cells under normoxia and hypoxia. As shown in Fig. 4A, CD63 level was higher in E006AA-hT cells under hypoxia compared to normoxia. In PC3 cells there was no obvious change in CD63 level under hypoxia (Fig. 4B).
During exosome biogenesis MVEs move along the cytoskeleton and fuse with plasma membranes. Specific actin structures such as invadopodia are important in MVEs fusion and exosome release 28,29 . To explore the potential role of actin remodeling under hypoxia with respect to exosome biogenesis, we also analyzed actin expression using confocal microscopy. E006AA-hT cells showed more organized actin, and a greater colocalization of actin with CD63 under hypoxia compared to normoxia (Fig. 4A). In PC3 cells, quite similar colocalization of CD63 and actin was observed under both normoxia and hypoxia (Fig. 4B). Greater colocalization of MVEs with the actin cytoskeleton could explain relatively higher exosome secretion in E006AA-hT cells under hypoxia compared to PC3 cells.
Inhibition of exosome biogenesis reduces growth in PCa cells. Next, to determine whether exosome secretion is helpful in cell survival, we treated all cell lines with an exosome biogenesis inhibitor, GW4869, and assessed their viability under normoxic and hypoxic conditions. Cell viability decreased significantly following GW4869 treatment in all cell lines at both time points (24 and 48 h), under both normoxia and hypoxia (Fig. 5).
Next, we performed clonogenic assays to determine the effects of GW4869 on colony-forming potential of PCa cells (E006AA-hT, 22Rv1, and PC3). GW4869 (10-20 µg/ml) treatment reduced the clonogenicity of all the cell lines ( Fig. 6A-C). To further validate these results, we treated these cell lines with another exosome biogenesis inhibitor, DMA (10-20 µg/ml). Treatment strongly inhibited clonogenicity of all three cell lines under both normoxic and hypoxic conditions ( Fig. 7A-C).
Taken together, the MTT and clonogenic assay results confirmed that exosome secretion by PCa cells is a potential survival mechanism, and that inhibition of exosome biogenesis reduces cancer cell growth.
Hypoxic PCa cells secrete exosomes loaded with higher amounts of lactate. Since exosome secretion offers a survival advantage to cells, we surmised that exosomes might remove metabolic waste. Lactic acid is a major metabolic end-product in cells under hypoxia. We measured lactate levels in exosomes secreted by PCa cells (E006AA-hT, MDA PCa 2b, 22Rv1, LNCaP, and PC3). Significantly higher amount of lactate was loaded in exosomes secreted by all cell lines under hypoxia compared to normoxia (Fig. 8A). Similar to exosomes, we observed higher lactate levels in all cell lines under hypoxia compared to normoxia (Fig. 8B).
Hypothesizing that lactate secretion might improve survival of PCa cells; we treated PCa cells (E006AA-hT and PC3) with the exosome biogenesis inhibitor DMA under hypoxia, and measured intracellular levels of lactate. However, these did not change significantly (Fig. 8C,D). hypoxia, which is a key determinant of cancer aggressiveness 15,30 . We found that: (a) under hypoxia, both African American and Caucasian PCa cells secrete higher numbers of exosomes (more so in African American PCa cells), which offers a survival advantage to cells; (b) exosome biogenesis inhibitors could reduce the growth of PCa cells; and (c) hypoxic PCa exosomes have higher amount of lactic acid.
Exosome secretion increased under hypoxia in all PCa cell lines. King et al. earlier reported that under hypoxia, breast cancer cells produce higher amount of exosomes, dependent upon HIF-1α expression 25 . In another study, hypoxia-resistant multiple myeloma cells generated higher amount of exosomes than parental cells under acute hypoxic or normoxic conditions 31 . Parolini et al. suggested that an acidic microenvironment under hypoxia could cause higher exosome release and uptake by cancer cells 32 .
Several studies have shown that exosomes secreted under hypoxia offer growth advantages and promote cancer progression. For example, exosomes derived from hypoxic glioblastoma cells strongly induced angiogenesis 33 . Exosomes secreted by leukemia cells under hypoxia promoted the tube formation in human umbilical vein endothelial cells via miR-210 34 . Recently, we described that under hypoxia, PCa cells secrete exosomes that induce EMT, invasiveness, and stemness in naïve PCa cells, and promote a CAF-type phenotype in prostate fibroblasts 23 . Exosomes secreted by oral squamous cell carcinoma cells under hypoxia elicited a pro-metastatic phenotype through delivering miR-21 to normoxic cells 35 . Similarly, hypoxic exosomes promoted the bladder tumor growth via transferring long non-coding RNA-UCA1 36 . Recently, Hsu et al. reported that exosomal micro RNA from hypoxic cancer cells increases permeability and angiogenesis 37 . Together, these studies suggest that exosomes secreted under hypoxia promote tumor growth and progression. Since we observed that almost all African American PCa cells secrete more exosomes under hypoxia compared to Caucasian PCa cells, this could be the one reason for PCa aggressiveness in African Americans.
The role of lactate in tumor metabolism, tumor immunology, and remodeling of tumor microenvironment has been studied by several groups 38 . Lactic acid released by cancer cells increases acidification of the tumor microenvironment and promotes cancer progression. Further, lactic acid is considered a critical immunosuppressive metabolite 39 . Lactate inhibited the monocytes differentiation to dendritic cells and blocked cytokine release from dendritic cells 40 as well as cytotoxic T cells 41 . Similarly, lactate enhanced the survival of regulatory T lymphocytes (Treg) cells and diminished the production of cytotoxic cytokines produced by CD8+T-cells 39 . Lactate is also emerging as a possible promotor of M2 polarization of macrophages, which generates an immune-tolerant environment and promote tumor growth and metastasis 42 .
Goetze et al. showed that lactate treatment increases the random migration of head and neck carcinoma cell lines 43 . Lactate exposure in cultured fibroblasts increased the hyaluronan production, and led to a higher expression of CD44, a predominant hyaluronan receptor on cell surfaces 44 . This is interesting observation as tumor stroma generally has higher hyaluronan levels produced by CAFs, which promotes growth and motility of cancer cells 45,46 . Lactate accumulation in primary tumors has clinical relevance as high lactate accumulation in primary cervical cancer predicted metastatic spread, tumor recurrence, and limited patient survival 47 . Here, we report the presence of high lactate in exosomes secreted by PCa cells under hypoxia, which could potentially cause both local and distant remodeling of the tumor microenvironment.
By secreting exosomes, cancer cells support tumor progression and metastasis by modifying the local and systemic micro-environment; therefore, targeting exosome biogenesis in cancer cells constitutes a novel cancer therapy 48 . Several investigators have reported that exosome biogenesis inhibitors such as GW4869 and DMA inhibit cancer growth. Kosaka et al. reported cell growth inhibition following inhibition of neutral sphingomyelinase 2 by GW4869 49 . Importantly, sphingomyelinase 2 controls the ceramide biosynthesis, which triggers the inward budding of exosomes from the endosomal limiting membrane 50 . GW4869 treatment in lung cancer-bearing mice significantly reduced the lung metastases 51 . Treatment of gemcitabine-exposed CAFs with GW4869 significantly reduced the survival in co-cultured epithelial cells 52 . Similarly, in colorectal cancer cells, inhibiting the secretion of exosomes in CAFs by GW4869 decreased the clonogenicity and tumor growth 53 . Pancreatic cancer cell-derived exosomes promoted chemoresistance and migration of surrounding cancer cells, which was mitigated by GW4869 treatment 54 . Recently, Matsumoto et al. reported that GW4869-induced inhibition of exosome secretion decreased the proliferation of B16BL6 cells. In the same study, intra-tumoral injection of GW4869 suppressed the tumor growth in mice 55 .
Similar to GW4869, several studies have shown that treatment with DMA inhibits growth of cancer cells. DMA targets the H + /Na + and Na + /Ca 2+ channels, which control intracellular Ca 2+ concentrations (a regulator of exosome secretion) 56 . In mice bearing CT26 tumors, blocking these ion channels with DMA reduced the exosomes secretion 57 . Further, DMA treatment inhibited the tumor growth and decreased the proliferation of tumor cells in H6 hepatomas 58 . Another report suggested that DMA has a potent effect against peritoneal metastasis in gastric cancer 59   In accordance with the above studies, here we observed a significant decrease in colony-forming ability of both African American and Caucasian PCa cells following treatment with GW4869 and DMA. These compounds offer a novel therapeutic strategy against PCa and suggest the need for the development of new exosome biogenesis inhibitors. In this regard, recently, Datta et al. screened a drug library and identified Manumycin-A as a candidate that could inhibit exosome biogenesis and secretion in CRPC cells 63 . Cancer-specific pathways regulating exosome biogenesis need to be identified and targeted to control cancer growth and progression.
Overall, the present study found that African American and Caucasian PCa cells release higher amount of exosomes under hypoxia. Further, exosome secretion offers a survival advantage to PCa cells probably through removal of certain metabolites such as lactic acid. In addition, the present study highlights the potential value of targeting the exosome biogenesis pathway as a novel anti-cancer therapy.  Cell culture and hypoxia exposure. E006AA-hT, LNCaP, and 22Rv1 cells were grown in RPMI1640 medium supplemented with 10% FBS, 100 U/ml penicillin G and 100 µg/ml streptomycin sulfate. PC3 cells were grown in similar media as above but with 10% heat-inactivated FBS instead of FBS. RC77T/E, RC77N/E, and PWR1E cells were cultured in Keratinocyte SFM media supplemented with EGF (5 ng/ml) and bovine pituitary extract (0.05 mg/ml). MDA PCa 2b cells were cultured in BRFF-HPC1 ™ media on Poly-L-Lysine (50 µg/ml DPBS) coated flasks/plates. For normoxic (21% O 2 ) condition, cells were cultured at 37 °C in a 5% CO 2 humidified environment as an adherent monolayer. Hypoxia experiments were performed in a hypoxia chamber (Baker Ruskinn INVIVO 2 400 or BioSpherix X3 Xvivo system) at 1% O 2 at 37 °C in a 5% CO 2 humidified environment.

Materials and Methods
Exosome isolation. Cells were cultured in exosome-depleted media under either normoxia (21% O 2 ) or hypoxia (1% O 2 ). After 48 hrs, conditioned media was collected and exosomes isolated by ultracentrifugation method, as reported by us previously 23,24 . In brief, the collected cell culture media was centrifuged at 300 g at 4 °C for 10 minutes; supernatant filtered through 0.22 µm filters (Merck Millipore); and filtrate concentrated using concentrators (150 K MWCO/20 ml, Thermo Scientific). The supernatants were then subjected to ultracentrifugation at 100,000 g for 90 min (L-80 Ultracentrifuge, 70.1 Ti fixed angel rotor, Beckman Coulter). Finally, the pelleted exosomes were suspended in DPBS. As indicated, exosomes were also isolated using a commercially available ExoQuick ™ reagent (System Biosciences) according to the vendor's protocol. Nanoparticle tracking analyses (NTA). Exosome size distribution and concentrations were analyzed using a Nano sight LM10 system (Nano sight Ltd, Navato, CA) as reported by us earlier 23,24 . In each case, exosome concentration was normalized with corresponding cell number.

Transmission electron microscopy (TEM).
A 400 mesh Formvar ® /carbon-coated copper grid was soaked in 100% ethanol for 1 min. A 20 µl droplet of exosome samples (in PBS) was applied on a sheet of Parafilm. Then the grid was floated with the dark-coated side of the grid facing the sample and incubated for 90 minutes. Residual sample was washed from the grid by dipping it into 20 µl drops of molecular biology-grade water three times. Then grids were stained for 1 minute with 2% uranyl acetate. After allowing 60 minutes for At the end, cells were processed and colony number was counted as detailed in methods. Representative images are presented and data is presented as mean ± SE (n = 3). *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. drying, the grids were imaged with a TecnaiTM G2 Spirit BIOTWIN TEM equipped with an AMT Image capture 2Vu camera system. MTT assays. ~2,000 cells were plated in a 96-well plate, followed by exposure to GW4869 under normoxia or hypoxia. At the end, MTT (5 mg/mL in PBS) was added for 2 hrs and then formazone formed in each well was dissolved in DMSO. Finally, absorbance was measured at 550 and 660 nm. Clonogenic assays. PCa cells (1,000 cells per well) were plated in 6-well plates, treated with exosome biogenesis inhibitors (DMA or GW 4869) and cultured either under normoxia or hypoxia for 7-8 days. Cells were then fixed with 10% formalin, stained with crystal violet solution, and then washed with deionized water. Plates were dried, scanned to obtain images and colonies (≥50 cells) were scored.
Lactic acid measurements. Lysates (cells or exosomes) were prepared in RIPA buffer supplemented with Halt Protease and Phosphatase Inhibitor. Lactate levels were determined using a lactate assay kit II (Sigma-Aldrich, MAK065) following vendor's protocol.
Immunofluorescence. PCa cells were cultured in chamber slides (Nunc ™ Lab-Tek ™ II Chamber Slide ™ System) under normoxia or hypoxia for 48 hrs. Thereafter, cells were fixed, permeabilized, and incubated overnight with primary antibodies (1:500; CD63, actin, and Rab5). Cells were then incubated with secondary anti-rabbit and/or anti-mouse antibodies conjugated with Alexa Fluor, followed by staining with Prolong ® Gold Antifade Reagent with DAPI. Fluorescent images were captured using an Olympus FV1200 SPECTRAL Laser scanning Confocal Microscope at 60× objective lens (Olympus IX83 inverted platform). Statistical analyses. Statistical analyses were performed using Graphpad Prism 7.0 software (La Jolla, CA) by parametric unpaired t-tests. Significance was considered at p < 0.05. Data availability. The datasets generated during and/or analyzed during the current study are available from the corresponding author on reasonable request.