Cancer-Associated Fibroblasts Modify the Response of Prostate Cancer Cells to Androgen and Anti-Androgens in Three-Dimensional Spheroid Culture

Abstract

Androgen receptor (AR) targeting remains the gold standard treatment for advanced prostate cancer (PCa); however, treatment resistance remains a major clinical problem. To study the therapeutic effects of clinically used anti-androgens we characterized herein a tissue-mimetic three-dimensional (3D) in vitro model whereby PCa cells were cultured alone or with PCa-associated fibroblasts (CAFs). Notably, the ratio of PCa cells to CAFs significantly increased in time in favor of the tumor cells within the spheroids strongly mimicking PCa in vivo. Despite this loss of CAFs, the stromal cells, which were not sensitive to androgen and even stimulated by the anti-androgens, significantly influenced the sensitivity of PCa cells to androgen and to the anti-androgens bicalutamide and enzalutamide. In particular, DuCaP cells lost sensitivity to enzalutamide when co-cultured with CAFs. In LAPC4/CAF and LNCaP/CAF co-culture spheroids the impact of the CAFs was less pronounced. In addition, 3D spheroids exhibited a significant increase in E-cadherin and substantial expression of vimentin in co-culture spheroids, whereas AR levels remained unchanged or even decreased. In LNCaP/CAF spheroids we further found increased Akt signaling that could be inhibited by the phosphatidyl-inositol 3 kinase (PI3K) inhibitor LY294002, thereby overcoming the anti-androgen resistance of the spheroids. Our data show that CAFs influence drug response of PCa cells with varying impact and further suggest this spheroid model is a valuable in vitro drug testing tool.

Cancer-associated fibroblasts promote prostate tumor growth and progression through upregulation of cholesterol and steroid biosynthesis

“…In a previous study we showed that PCa cells become less responsive to enzalutamide when they are cocultured as tumor spheroids in a 3-dimensional environment together with cancer-associated fibroblasts (CAFs) [11]. In this study, we performed gene expression profiling of these co-culture spheroids and revealed that CAFs induce a significant upregulation of cholesterol metabolism and steroid biosynthesis in PCa cells.…”

“…The androgenablated subline LNCaPabl was previously established by long-term culture in androgen-ablated medium [12] and maintained in RPMI 1640 (Lonza) with 10% charcoalstripped (CS) FCS and 1% penicillin and streptomycin. Immortalized CAFs [13] stably expressing green fluorescent protein (GFP) have been previously established [11] and were grown in DMEM with 10% FCS and 1% penicillin and streptomycin and 1x GlutaMAX™ (Gibco). The enzalutamide resistant (EnzaR) cell lines DuCaP EnzaR and LNCaPabl EnzaR have been previously established by long-term treatment with 8 μM enzalutamide [14].…”

3D tumor spheroids as in vitro models to mimic in vivo human solid tumors resistance to therapeutic drugs

“…fibroblasts (CCD-18Co;Jeong, Lee, Shin, Chung, & Kuh, 2016); prostate cancer cells (LNCaP and DuCaP) and CAF (Eder et al, 2016) and SU6668 [Joyce, 2005]) have also been studied.…”

“…fibroblasts (CCD-18Co;Jeong, Lee, Shin, Chung, & Kuh, 2016); prostate cancer cells (LNCaP and DuCaP) and CAF (Eder et al, 2016); breast cancer cells (MDA-MB-231) and microvascular endothelial cells (HMEC-1) (Kassim, Al Tawil, Buquet, Le Cerf, & PierreVannier, 2017); pancreatic cancer cells (PANC-1) and pancreatic stellate cells (myofibroblast-like cells; J.-H. .…”

Inhibition of Nox4‐dependent ROS signaling attenuates prostate fibroblast activation and abrogates stromal‐mediated protumorigenic interactions

“…1 While epithelial in origin, the tumor microenvironment plays a critical role in prostate adenocarcinoma pathogenesis, for example, stromal signaling is required for tumor initiation, tumor cell proliferation, angiogenesis, metastasis and diminishes therapy response. [2][3][4] These protumorigenic actions of the tumor microenvironment are largely mediated via the secretion of…”