The prevalence of post-prostatectomy incontinence is high due to the increased number of radical prostatectomies, however, the improvement of surgical techniques helped to decrease the incidence. Transurethral injection of stem cells or their secretome have been reported as an effective treatment modality for incontinence in animal models and human clinical trials. Safety with recurrent/residual tumor remains unclear. We aimed to study the role of human mesenchymal stem cells (hMSCs) and their secretome in prostate cancer functionality and survival in-vitro.

We gathered the basic information to understand the cytokines profiling of hMSCs, prostate cancer cell lines, LNCaP (androgen responsive) and PC-3 (androgen resistant). We cultured hMSCs ,LNCaP and PC-3 cell lines individually and were maintained in Dulbecco’s modified Eagle’s medium (DMEM)-low glucose media, supplemented with %10 FBS, Penicillin(100U/ml) and Streptomycin (100U/ml).The cultures were incubated at 37⁰C in a humidified atmosphere containing 5% CO2. Cells were passaged and trypsinized at 75-85% confluency. The medium was replenished every three days. After third passage, we collected the conditioned medium (containing cytokines/secretome) of various cell lines after 72 hours. We evaluated 120 Human Cytokine profiling using membrane microarray (RayBio C1000, C-series Human Cytokine Antibody Array.AAH-CYT-1000-8). We used ImageJ to compare the densitometry of each cytokine.  
We used 6-well transwell plates to co-culture 1X106 of androgen-responsive prostate cancer cells (LNCaP)/well with hMSCs in different concentrations (0.5:1, 1:1 and 1.5:1) relative to the amount of LNCaP cells. One well was left as a negative control and one well was treated with hMSCs cytokines without cells (72 hours conditioned media).  After 48 hours we checked the cell count and morphology of LNCaP cells using Countess II automated cell counter.
Prostate cancer cell lines were cultured in 96-well plates. Cells were treated with hMSCs cytokines (72 hours conditioned media) in dose-variable fashion (0%, 50%, 75% and 100%) for 24 hours. At the end of cell treatments, cells viability were evaluated using MTT proliferation assay. MTT solvent (20microliter) was added to each well and then incubated 3.5 hours at 37⁰C in a humidified atmosphere containing 5% CO2. The MTT assay is based on the cleavage of the yellow 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide for to purple formazan crystals by metabolically active cells. Optical densities were measured by using spectrophotometer at wavelengths 590nm and 630nm. The level of significance between control and dose-dependent treatments was determined using analysis of variance (ANOVA) with post hoc Fisher’s LSD test. P-value< 0.05 was considered to be statistically significant. We used IBM SPSS v.22 software for statistical analyses.

The differential cytokine profiling is shown in figure 1. Twenty-one cytokines were highly expressed in hMSCs more than 10 times their expression in LNCaP including; IL1 alpha, IL-2, IL-4, IL-6, IL-7, IL-13, IL-16, LIGHT, SDF-1, BDNF, TNF-B, HGF, bFGF, FGF-7, IGFBP-4, GDNF, TGF-B1, MCP-1, MIG, Flt-3 lig. Twenty cytokines were highly expressed in hMSCs about 5-10 times their expression in LNCaP including; IL-1ra, IL-1B, IL15, MCP-2, MCP-3, MCP-4, TGF-B3, MIP-1-delta, Leptin, axl, RANTES, Eotaxin, Eotaxin-3, TIMP-1, PARC, SCF, TARC, GCP-2, Fractalkine, MDC.
Co-culturing of hMSCs and LNCaP cells revealed regression of cancer cell count when co-cultured with equivalent (1:1) or more amount of hMSCs (2XhMSCs: 1XLNCaP). Co-culturing less cell count of hMSCs (0.5 to 1) with LNCaP cells resulted in a higher proliferation of the cancer cells than control. Treating the cancer cells with the secretome revealed marked regression (figure 2a). 
After 24 hours of treatment of prostate cancer cells with hMSCs secretome without cells, LNCaP (androgen sensitive) and PC-3 (androgen resistant) cells were evaluated by performing MTT viability assay (Figure 2b). Androgen-sensitive (LNCaP) cells were significantly declined. However, PC-3 cells increased after treatment without reaching statistical significance.

Radical prostatectomy is usually indicated in early stages of pathological progression, i.e. androgen sensitive tumor. Treatment of post-prostatectomy incontinence using hMSCs cells might be tricky if there is microscopic or macroscopic residual tumor. Our in-vitro results showed that cell therapy resulted in variable responses when co-cultured with androgen-sensitive cancer cells (LNCaP). Interestingly, secretome alone without cells might control the environment around the cancer cells leading to stabilization of tumor progression. 
We are the first to explore the differential expression in the cytokines between hMSCs and prostate cancer cells. Studying of the differential expression in the cytokines may lead to understanding the suppressor signaling pathway. Regenerative cytokines as TGF-B and SDF-1 were highly expressed in hMSCs secretome while they are known to help in regeneration of injured sphincter complex. Anti-tumor cytokines as IL2, IL6 and IL15 were highly expressed in the secretome than LNCaP cells. hMSCs may have a potential role in the immunomodulatory pathway through enhancing M1 over M2 activity to limit cancer progression. Our future research will focus on translating the experiment to in vivo model and to understand the complex healthy microenvironment and checkpoints that can regenerate the tissue without tumor progression.

Our in-vitro cell culture results revealed that hMSCs secretome (not the cells) resulted in significant suppression of the growth of androgen-sensitive prostate cancer cell line (LNCaP cells). This might support its safety and provide an additional advantage for hMSCs secretome over the hMSCs cells in future regenerative applications (e.g. post-prostatectomy incontinence). Understanding the involved signaling cascade between hMSCs and cancer cells may help patients with risk of cancer progression.