br Fig While the C B cells formed spheroids in
(Fig. 4). While the C4-2B cells formed spheroids in CA scaﬀolds, the lower E-cadherin expression compared to 2D cultures indicates that another mechanism may be promoting spheroid formation. PC-3 cells vary their E-cadherin expression in suspension cultures compared with adherent cultures, with cell attachment and cell spreading needed for dynamic regulation of E-cadherin and N-cadherin expression . The involvement of β1 integrin mediated cell adhesions to the matrix is a key component in promoting N-cadherin expression . The β1 in-tegrin is the most commonly occurring integrin heterodimer that pro-motes interaction with collagen and fibronectin . It has been shown to support anchorage-independent growth of PCa cells and may influ-ence the size of PCa colonies grown in Matrigel . These results demonstrate that integrin binding and focal adhesion activation may be needed to promote N-cadherin expression and downregulate E-cadherin expression in the CA scaﬀolds.
To further evaluate the PCa interaction with the CA scaﬀolds, the N,N-Dimethylsphingosine cytoskeleton was evaluated with IF. All 3D cultures had greater actin expression than 2D cultures (Fig. 5). Actin was localized in the cytoplasm surrounding the nucleus in all 3D cultures, not in stress fi-bers. This indicates that the PCa cells are not engaging integrins or forming focal adhesions to bind to the CA scaﬀold matrix. The focal adhesion development is a multistep process that begins upon cell contact with the matrix, followed by cellular contraction that organizes the actin fibers and promotes focal adhesion reinforcements, finally resulting in a mature focal adhesion with pronounced actin stress fibers . The focal adhesions enable force transmission from the matrix to the cell and the mechanotransduction cellular responses . These results suggest that another molecule is promoting adhesion to the CA scaﬀold, which is not surprising as the CA scaﬀolds do not contain proteins, only polysaccharides, so no ligands for integrins are present. Further experiments are needed to address the mechanism for cell ad-hesion to the CA scaﬀolds. Such studies may examine the influence of CD44 or other hyaluronic acid (HA) binding proteins, since the CA scaﬀolds are comprised of molecules that resemble the HA structure. Elucidating this cell adhesion mechanism to the CA scaﬀolds will pro-vide insights into the PCa responses observed with increasing CA scaﬀold stiﬀness. Additionally, experiments incorporating proteins or peptides on to the CA scaﬀolds could promote integrin binding and focal adhesion formation.
3.4. Evaluation of PCa phenotype in scaﬀold cultures
IF staining and qRT-PCR were used to evaluate the expression of biomarkers to quantify the phenotype of the CA scaﬀold cultures. IF assessed the expression of pEGFR, AR, and cytokeratin 8 (KRT8) in the
Fig. 4. E-cadherin expression for PCa cultures. Immunofluorescence images of 2D, 2, 4, and 6 wt% CA cultures at 10 d. E-cadherin (E-cad) is stained green 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.)
samples. KRT8 was examined in all three cell lines, while AR was only examined in C4-2B and 22Rv1 as PC-3 has an inactive AR. As a result, PC-3 was examined with pEGFR in place of AR. EGFR is regulated in normal cells but constantly stimulated in tumor cells, where it can fa-cilitate cancer cell growth, metastasis, and invasion . EGFR over-expression was shown to be correlated with the hormone refractory PCa phenotype  and increased EGFR expression was observed in the majority of androgen independent PCa patients . Androgen is the primary molecule that stimulates PCa progression by promoting pro-liferation and preventing apoptosis via binding to and activating AR . AR is present in the majority of primary and metastatic PCa cases, spanning many stages, grades, and even hormone refractory cancers . The AR provides a primary target for PCa treatment until the PCa becomes androgen independent, often due mutation into a con-stitutively active AR mutant . KRT8 is an intermediate filament protein found in normal prostate epithelial cells, but it is also found on the cell membrane of some carcinoma cells . KRT8 expression was used as an epithelial cell marker.