Introduction: Prostate cancer (PCa) ranks among the most prevalent cancers in developed nations, yet treatments for advanced stages offer palliative relief. The existing research models for PCa lack fidelity in replicating crucial aspects of its biology, impeding translational potential to clinical applications. Our project's primary objective was to create a human-derived 3D PCa model utilizing tissue engineering techniques.

Methods: Our methodology involves the cultivation of human prostate fibroblasts using the self-assembly method, creating stroma, and subsequent seeding of prostate epithelial cells on this stroma to mature into an epithelium. Specific cell culture conditions, including testosterone incorporation, replicate the hormone-responsive nature of the prostate. We integrated invasive PCa cell lines and spheroids to analyze invasion dynamics, offering a comprehensive platform for studying the tumor microenvironment.

In our process, fibroblasts are initially seeded at confluence in six well plates with a circular paper anchor, held down with a round stainless-steel ingot, and cultured in DMEM medium supplemented with ascorbate to promote collagen deposition over 14 days. Subsequently, these sheets are reseeded with fibroblasts for another 14 days and stacked, aided by rectangular stainless-steel ingots to facilitate stromal layers’ fusion. Half of these stromas are cultured using a mixed media, incorporating conditioned media from the DU145 cell line to promote the induction of the fibroblasts into cancer-associated fibroblasts (CAF). Spheroids produced from the LNCAP and DU145 PCa cell lines were deposited onto the stromas. The DU145 line represents invasive PCa cells while LNCAP denotes a non-invasive cell line.

Results: We successfully created manipulatable stromas formed from prostate fibroblasts and from CAFs. Atomic force microscopy (AFM) was performed on the stromas to measure tissue rigidity, and thus ascertain the presence of CAF in the CAF induced stromas. Spheroids from the DU145 and LNCAP PCa cell lines were deposited on these stromas and their invasion of the tissues was analyzed by AFM and immunofluorescence. These techniques were done to allow better visualization of the spheroid invasion into the stroma and its impact on the neighboring cell’s rigidity, mimicking the cancer microenvironment, and the CAFs’ induction, leading to a higher rigidity.

Conclusion: This groundbreaking model shows great promise in discovering new treatment targets, which could improve patient outcomes and reduce the economic strain of PCa on healthcare systems. Our work in creating a 3D model of prostate cancer using human cells marks a significant advancement in PCa research.