br factors including the presence of necrotic
factors, including the presence of necrotic cells in spheroids, metabolic re-programming of the cells aﬀecting the Alamar blue assay readings, or other factors. Decreased Ki-67 expression was observed in all cell lines between 10 and 15 d (Fig. S4), indicating decreased cell proliferation and suggesting the formation of necrotic regions or Bradykinin (acetate) arrest within the tumor spheroids. The cell number was proportional with CA scaﬀold stiﬀness only at 15 d for all three cell lines, where 6 wt% CA had greatest cell number, followed by 4 wt% and 2 wt% CA. This result of cell number increasing proportionally with scaﬀold stiﬀness needs further investigation to examine the cell number over longer culture times. Other than at 15 d, there were no clear trends in the PCa cell growth with scaﬀold stiﬀness. Stiﬀness independent growth has been observed for PC-3 and 22Rv1 cells in 2D cultures . The PC-3 and 22Rv1 cells showed diﬀerences in the growth with respect to scaﬀold stiﬀness during the 15 d culture, demonstrating diﬀerent responses of the cells in 3D porous CA scaﬀolds compared to 2D cultures. All of the CA scaﬀold compositions supported PCa growth, but there was not a strong diﬀerence in cell number with respect to scaﬀold stiﬀness.
The cell morphology of the three cell lines on the CA scaﬀolds at 5, 10, and 15 d time points is presented in Fig. 3. All three cell lines had a rounded cell morphology on the 2, 4, and 6 wt% CA scaﬀolds. Despite the common rounded cell morphology, there were diﬀerences in the cell appearance in the CA scaﬀold cultures. The PC-3 cells formed grape-like cell clusters, while C4-2B and 22Rv1 cells formed multi-cellular spheroids (Fig. 3). The PC-3 cell morphology was not influ-enced by the CA scaﬀold stiﬀness, as the cells formed clusters on all scaﬀold compositions and maintained a consistent appearance throughout the cultures (Fig. 3a). The PC-3 cell morphology may have been influenced by the CA scaﬀold composition, as others have de-monstrated the influence of β1 integrin in promoting a stellate mor-phology and its inhibition resulting in a grape-like cell morphology . The CA scaﬀolds are comprised of polysaccharides and do not inherently provide binding sites for β1 integrin, potentially con-tributing to the observed grape-like cell clusters. C4-2B cells formed spheroids by 5 d, with the greatest number of spheroids observed on 2 wt% CA, and fewer spheroids on the 4 wt% and 6 wt% CA scaﬀolds (Fig. 3b). The C4-2B spheroid size decreased with increasing scaﬀold concentration from 2 to 6 wt% CA, particularly at 5 d. The C4-2B spheroids developed an increasingly rough appearance with increasing culture time from 5 to 15 d. The 6 wt% CA C4-2B cultures had a much rougher spheroid surface than the 2 and 4 wt% CA cultures at 10 d 22Rv1 cells formed spheroids with a smooth surface on all CA scaﬀold compositions (Fig. 3c). 22Rv1 cultures on 6 wt% CA at 10 and 15 d had much smoother spheroid surfaces than 2 and 4 wt% CA. The 22Rv1 spheroids had hole-like features present on the spheroids in the 4 wt% and 6 wt% CA scaﬀold cultures, with the most prominent features ob-served in the 6 wt% CA cultures at 15 d, indicated with white arrows in Fig. 3. It is unclear what these hole-like features are, but they may be the formation of duct-like structures within the spheroids.
3.3. Evaluation of PCa cell interaction with CA scaﬀolds
The PCa cells cultured on the CA scaﬀolds were characterized with immunofluorescence (IF) staining to assess the cell-cell and the cell-material interactions. The PCa cells were analyzed for E-cadherin ex-pression to characterize the cell-cell interaction in the 3D cultures. The CA scaﬀold cultured cells for all three cell lines displayed altered cell morphologies compared with the 2D cultures: the cells had rounded morphologies in the CA scaﬀolds and spread morphologies in 2D cul-tures. The rounded cell morphologies observed in the CA scaﬀold cul-tures (Fig. 3) suggested the upregulation of E-cadherin compared to 2D cultures. The E-cadherin expression increased for PC-3 and 22Rv1 cells in the CA scaﬀolds, while it decreased for C4-2B cells in the CA scaﬀolds