Renal cancer secretome induces migration of mesenchymal stromal cells

Background: Advanced Renal cell carcinoma (RCC) is therapeutically challenging. RCC progression is facilitated by mesenchymal stem/stromal cells (MSCs) that exert remarkable tumor tropism. The specific mechanisms mediating MSCs’ migration to RCC remain unknown. Here, we comprehensively analyzed RCC secretome to identify MSCs attractants. Methods: Conditioned media (CM) were collected from five RCC-derived cell lines (Caki-1, 786-O, A498, KIJ265T, KIJ308T) and non-tumorous control cell line (RPTEC/TERT1) and analyzed using cytokine arrays targeting 274 cytokines in addition to global CM proteomics. MSCs were isolated from bone marrow of patients undergoing standard orthopedic surgeries. RCC CM and the selected recombinant cytokines were used to analyze their influence on MSCs migration and microarray-targeted gene expression. The expression of genes encoding cytokines was evaluated in 100 matched-paired control-RCC tumor samples. Results: When compared with normal cells, CM from advanced RCC cell lines (Caki-1, KIJ265T) were the strongest stimulators of MSCs migration. Targeted analysis of 274 cytokines and global proteomics of RCC CM revealed decreased DPP4 and EGF, as well as increased AREG, FN1, and MMP1, with consistently altered gene expression in RCC cell lines and tumors. AREG and FN1 stimulated, while DPP4 attenuated MSCs migration. RCC CM induced MSCs’ transcriptional reprogramming, stimulating the expression of CD44, PTX3, and RAB27B. RCC cells secreted hyaluronic acid (HA), a CD44 ligand mediating MSCs’ homing to the kidney. AREG emerged as an upregulator of MSCs’ transcription. Conclusions: advanced RCC cells secrete AREG, FN1 and HA to induce MSCs migration, while DPP4 loss prevents its inhibitory effect on MSCs homing. RCC secretome induces MSCs’ transcriptional reprograming to facilitate their migration. The identified components of RCC secretome represent potential therapeutic targets. We used microarrays to determine the effect of the conditioned media (CM) collected from two RCC-derived cell lines (Caki-1, KIJ265T) and non-tumorous control cell line (RPTEC/TERT1) on the transcriptome change in mesenchymal stem/stromal cells (MSCs). Conditioned media (CM) were collected from renal cancer-derived cell lines (Caki-1, KIJ265T) and non-tumorous control cell line (RPTEC/TERT1). Per each cell line, CM was collected from cells independently cultured in three separate cell culture flasks. Mesenchymal stromal cells were isolated from bone-marrow collected during standard orthopedic surgeries with the agreement of the Local Bioethics Committee (approval no. KB/115/2016) and written informed consent of patients and used for treatment of mesenchymal stromal cells (MSCs). For the CM treaments, 50,000 MSC cells were seeded on a well of 12-well plate, cultured for 24h, rinsed twice with DMEM (low glucose, no glutamine, no phenol red) (Gibco/Thermo Fisher Scientific, Paisley, UK) supplemented with GLUTAMAX (Gibco/Thermo Fisher Scientific, Paisley, UK), and cultured for 24h in medium without FBS. Next, the cells were rinsed once with PBS and four times with DMEM supplemented with GLUTAMAX and cultured for 24h in CM from RPTEC/TERT1, Caki-1 or KIJ265T cells. MSCs were treated with CM collected from Caki-1 (n=3), KIJ265T (n=3), and RPTEC/TERT1 (n=3). RNA was isolated from the treated MSCs and subjected to microarray analysis using Microarray analysis was performed using Affymetrix Gene Atlas System.