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All PC-3 scaﬀold cultures had greater pEGFR expression compared with 2D (Fig. 6). The 4 wt% CA cultures had the greatest expression of pEGFR among the scaﬀold cultures, while 6 wt% CA cultures had lowest expression, which was similar to the 2D cultures (Fig. 6). The PC-3 scaﬀold cultures had similar KRT8 expression among the CA composi-tions, with all scaﬀold compositions showing KRT8 expression in-dicating the epithelial phenotype in Trizma maleate in scaﬀold cultures. The C4-2B
2D cultures had the greatest AR expression and AR expression was downregulated in the scaﬀold cultures, with significant diﬀerences in expression between 2D and scaﬀold cultures (Fig. 6). There was no significant diﬀerence in AR expression between the scaﬀold cultures. The scaﬀold cultures demonstrated KRT8 expression in all scaﬀold culture conditions indicating epithelial phenotype of C4-2B cells grown in CA scaﬀolds (Fig. 6). The 22Rv1 cultures had similar AR and KRT8 expression between the four groups (Fig. 6). The 4 wt% CA scaﬀold cultures had the greatest AR expression compared to the other cultures. The KRT8 expression for 22Rv1 was relatively consistent among the cultures (Fig. 6). Individual IF images for each cell line and each scaf-fold composition are presented in Fig. S7-S9. Our IF results show no significant changes in cell phenotype for PC-3 and 22Rv1, but a reduced AR expression in C4-2B cells in CA scaﬀolds.
The gene expression for all three cell lines and culture conditions was evaluated at 10 d with qRT-PCR to assess the preservation of PCa cell characteristics (Fig. 7). C4-2B and 22Rv1 cell lines were evaluated for expression of AR and PSA mRNAs, while PC-3 was tested for ex-pression of LIMK1 mRNA. One of the characteristics of C4-2B and 22Rv1 cell lines is that both cell lines express AR, which is a tran-scription factor. AR specifically stimulates transcription of PSA mRNA, hence expression of PSA represents the activation status of the AR. PSA is also the clinical gold standard biomarker for PCa diagnosis . PSA expression is a marker for PCa progression as PSA downregulates cell apoptosis and increases cell proliferation . LIMK1 is an enzyme that
Fig. 5. Actin expression for PCa cultures. Immunofluorescence images of 2D, 2, 4, and 6 wt% CA cultures at 10 d. Actin is stained red and nuclei are stained blue. Scale bars are 15 μm. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)
Fig. 6. Expression of characteristic proteins for PC-3, C4-2B, and 22Rv1 cells. a) Comparison of characteristic protein expression for all PCa cell lines at 10 d. Cytokeratin 8 (KRT8) is shown in red and nuclei are shown in blue for all cell lines. The green color is androgen receptor (AR) for C4-2B and 22Rv1, while it is phospho-epidermal growth factor receptor (pEGFR) for PC-3. b) Relative florescent intensity of pEGFR normalized by 2D intensity for PC-3, c) Relative florescent intensity of AR normalized by 2D intensity for C4-2B, d) Relative florescent intensity of pEGFR normalized by 2D intensity for 22Rv1, * denotes statistically significant diﬀerences (p < 0.05). (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)
regulates the actin cytoskeleton and is overexpressed in PCa , specifically in PC-3 cells. LIMK1 is also critical for the PCa invasive growth . Because PC-3 cells are highly aggressive androgen-in-dependent PCa cells, LIMK1 expression was used as a marker for ag-gressive phenotype of PCa cells.
The PC-3 CA scaﬀold cultures had downregulated LIMK1 expression compared to the 2D cultures, with statistically significant diﬀerences between 2D and each scaﬀold composition (Fig. 7a). Among the scaf-fold cultures, LIMK1 expression showed a significant increase in 6 wt% cultures compared to 2 wt% cultures. PSA expression demonstrated diﬀerent trends for C4-2B and 22Rv1 cultures with respect to stiﬀness (Fig. 7b). PSA expression did not change in C4-2B scaﬀold cultures at 2 wt% and 4 wt% CA compared with 2D cultures (Fig. 7b). The PSA expression for the 6 wt% CA cultures was significantly lower than the 2D and 2 wt% and 4 wt% CA scaﬀold cultures. All 22Rv1 CA scaﬀold cultures had upregulated PSA expression compared with 2D cultures (Fig. 7b) with a maximum increase at 6 wt% CA compared to 2D