Roughly 80% of advanced prostate cancer (PCa) patients develop bone metastases. Once bone metastasis forms, the patient’s 5-year survival rate drops from 99%, in organ-confined disease, to 30%. Prostate-specific membrane antigen (PSMA) is found on the surface of prostate epithelial cells. It is a well-known prognostic biomarker that indicates PCa progression within the primary tumor and metastasis to bone. However, PSMA’s biological and physiological impact within the metastatic bone microenvironment has yet to be elucidated. It is of interest to understand the role PSMA plays in sustaining “the vicious cycle” which advances metastatic aggression. Our current study attempts to address this issue by first establishing the metastatic and osteogenic potential of the metastatic PCa epithelial cell line C42B, followed by observing the interactions of C42B cells with human osteoblastic (hFOB1.19) cells in conditioned media co-culture experiments. The opposite effect was also tested to verify if PSMA+ PCa cells impacted the osteogenic abilities of osteoblasts.
Experimental preparation involved knocking out PSMA in C42B cells using CRISPR techniques. Characterization of hFOB1.19 cells was done to confirm maturation in culture over 8 days. We also confirmed that the C42B cell line could form bone-like minerals if treated in growth media supplemented with 10 mM β-glycerophosphate and 50 μg/mL L-ascorbic acid. Co-culture experiments used immature hFOB1.19 conditioned media (72hr) to investigate whether PSMA+ C42B cells elevated calcium deposition. RNA extraction, RT-qPCR, and Alizarin Red staining (ARS) were performed for all experimental procedures. Mature hFOB1.19 cells had high expression of different bone markers, DKK1 and ALPL, on different days, day 2 and day 4, respectively. Calcium deposition also gradually increased in these cells. Osteogenic treatment of the C42B cells showed higher calcium deposition for PSMA+ C42Bs and significant changes in the bone markers tested (DKK1 and RUNX2). RT-qPCR results of co-culture experiments with immature hFOB 1.19 conditioned media on PCa cells showed increased calcium in PSMA+ C42B cells compared to control (PSMA+ C42B cells without treatment), as well as a decrease in metastatic markers, VIM and TWIST1. hFOB1.19 showed an increase in osteogenic factors when cultured in C42B-SCR conditioned media treatment compared to the C42B-KO. Overall, PSMAs presence in PCa epithelial cells appears more impactful than when knocked out when looking at calcium formation and changes in bone marker expressions aligning with “the vicious cycle” phenomenon.
