br cultures Additionally Rv cultures
cultures. Additionally, 22Rv1 cultures had statistically significant in-crease in PSA expression in 6 wt% CA compared to 2 wt% and 4 wt% CA scaﬀold cultures. There were no significant diﬀerences in AR expression observed for C4-2B and 22Rv1 scaﬀold cultures. The C4-2B AR ex-pression was downregulated in the scaﬀold cultures compared to 2D cultures, whereas no significant change in AR expression in 22Rv1 cells was observed between 2D and 3D cultures. The C4-2B and 22Rv1 AR expression PCR results support the results of IF expression analysis. The marker expression analysis with IF and qRT-PCR demonstrated dif-ferent responses to scaﬀold stiﬀness for each cell line. PC-3 cells de-monstrated upregulation of pEGFR expression and downregulation of LIMK1 expression in the CA scaﬀolds. C4-2B cultures had down-regulation of PSA and AR expression in 3D cultures, while 22Rv1 cul-tures had similar expression of AR between 2D and CA scaﬀold cultures and PSA expression was upregulated in the 6 wt% CA scaﬀolds. Col-lectively, the protein and gene expression results demonstrated that the CA scaﬀold cultures supported expression of phenotypic markers
diﬀerent expression profiles for each cell line.
Characterization of phenotype demonstrated a shift in marker ex-pression for PC-3 cells grown in CA scaﬀolds. There was a loss of ex-pression of LIMK1 but a gain in expression of pEGFR in PC-3 cells in 2 wt% and 4 wt% CA. Collectively, these observations suggest that PC-3 cells maintain their aggressive phenotype in 3D cultures. Interestingly, the PC-3 E-cadherin and FF-MAS expression was upregulated in CA scaﬀold cultures, which may be related the downregulation of LIMK1 expression. Further experiments are needed to explore this po-tential connection for CA scaﬀold cultured PC-3 cells. Similarly, there is no significant change in AR expression and activity in C4-2B and 22Rv1 cells grown in 2 wt% and 4 wt% CA scaﬀolds except PSA expression in 22Rv1 cells grown in 6 wt% CA scaﬀold culture. Similar PSA expression results for C4-2B and 22Rv1 cultures have been reported in the litera-ture. Downregulation of PSA expression like in the C4-2B cultures was observed where LNCaP cells were cultured in rotary wall vessels as 3D organoids, but the PSA expression was restored when the 3D organoids were co-cultured with human prostate fibroblast cells . This de-monstrates the importance of the stromal cell contribution to PCa malignancy that cannot be recreated with cultures only comprised of cancer cells. Upregulation of PSA expression like in the 22Rv1 cultures was observed where LNCaP cells cultured on collagen-based scaﬀolds had higher PSA expression compared with 2D cultures .
3.5. CA scaﬀolds promote mineralization in osteoblastic PCa cell lines
Osteoblastic and osteolytic responses of PCa cells have been de-monstrated both in vitro  and in vivo [64–66]. These responses arise due to factors produced by PCa cells that can act directly or in-directly to induce an osteogenic response [64,67,68]. PCa cells grown in 2D culture in normal growth media do not mineralize (Fig. S10). However, C4-2B cells were shown to mineralize in 2D cultures with osteogenic media (containing 10 mM β-glycerophosphate and 50 mg/ ml L-ascorbic acid) . To evaluate whether CA scaﬀold culture may promote mineralization of C4-2B cells in normal growth media and to determine if the rough appearance of the C4-2B spheroids was related to mineralization, Alizarin Red staining and osteocalcin IF were per-formed for all groups to assess the presence of calcium phosphates and bone matrix proteins. Alizarin Red staining indicates the presence of calcium deposits and is commonly used to assess the osteogenic dif-ferentiation of mesenchymal stem cells.
Positive Alizarin Red staining in C4-2B and 22Rv1 samples was observed (Fig. 8), denoted by the bright red deposits and arrows, while no positive staining was observed for PC-3. The faint red color observed in 2, 4 and 6 wt% scaﬀolds for PC-3 cultures and in regions of the other