br Corresponding author Department of Materials Science
* Corresponding author. Department of Materials Science and Engineering, University of Central Florida, Orlando, FL, 32816-2455, USA. E-mail address: [email protected] (S.J. Florczyk).
perform poorly. Consequently, researchers have developed three di-mensional (3D) cultures of cancer ABT-888 (Veliparib) in several formats that de-monstrate responses that better match those observed in vivo. The use of 3D cultures and particularly 3D biomaterial scaﬀolds for culture of cancer cells results in cultures that are more drug resistant and release more growth factors and extracellular matrix proteins than 2D cultures [10–12]. Therefore, further development and characterization of 3D biomaterial scaﬀold cultured cancer cells under multiple conditions that recapitulate the in vivo 3D tumor microenvironment may lead to tumor models with predictive response of cancer therapies in patients.
The stiﬀness and composition of the microenvironment influences cell morphology, cytoskeletal structure, signaling, and function for many normal and cancer cell types [13–15]. The stiﬀness of the ex-tracellular matrix (ECM) increases during the malignant progression of cancer, promoting more aggressive and metastatic phenotypes in sev-eral cancers [16–18]. Prostate tissue stiﬀness increases as PCa develops and progresses, with stiﬀness of 42 kPa for the normal prostate tissue and 88 kPa for the PCa tumor , while the stiﬀness of mineralized bone in the metastatic niche is ~106 kPa . The diﬀerent tissue stiﬀness during the stages of PCa malignant progression provide an opportunity to model the stages with diﬀerent stiﬀness biomaterial scaﬀolds.
Chitosan is natural polymer with a similar chemical structure to glycosaminoglycans and is commonly derived from crustacean shells . Chitosan is widely used due to its beneficial properties including biocompatibility, biodegradability, and hydrophilicity [22,23]. Ad-ditionally, chitosan is cationic, allowing it to form a polyelectrolyte complex (PEC) with anionic polymers through electrostatic interactions . PECs provide the benefits of each polymer in the complex, while limiting their respective drawbacks. Alginate is a natural polymer ob-tained from brown seaweed with beneficial biomedical properties like biocompatibility and ionotropic gelation with divalent cations . Alginate is anionic when dissolved in water and forms PECs with chitosan when solutions are mixed in the appropriate pH range. Chit-osan-alginate (CA) 3D porous scaﬀolds were developed for bone tissue engineering . The CA scaﬀolds supported culture of several cancers [27,28] and enriched the cancer stem cell population in several cancers, including PCa [29,30]. The interaction of cancer cells with the CA scaﬀolds promoted cancer stem cell enrichment for several cancers while poly ε-caprolactone coated CA scaﬀolds did not, indicating that the CA material chemistry influenced the cell response and promoted more malignant cultures than 2D cultures or 3D cultures with synthetic polymer scaﬀolds [29,30]. Despite these promising CA scaﬀold results, only one composition (4 wt%) was evaluated, leaving the influence of CA scaﬀold stiﬀness unexplored. The CA scaﬀold processing was opti-mized , providing guidelines to produce diﬀerent CA scaﬀold compositions.
Herein, we present the development of two new CA scaﬀold com-positions (2 wt% and 6 wt%) and the use of those scaﬀolds, along with 4 wt% CA scaﬀolds, to assess the influence of CA scaﬀold stiﬀness on PCa response. The three CA scaﬀold compositions approximate the stiﬀness of the stages of PCa malignant progression, including normal prostate tissue, primary PCa tumor, and bone metastatic niche. Our hypothesis was that CA scaﬀold cultures will recapitulate the PCa cell phenotype indicated by expression of biomarkers, including phospho-epidermal growth factor receptor (pEGFR), LIM domain kinase 1 (LIMK1), prostate specific antigen (PSA) and androgen receptor (AR), as well as promote mineralization for the osteoblastic cell lines in a stiﬀness dependent manner. These biomarkers were selected as they are upregulated in malignant prostate tissues and correlated with bone metastases [32–35]. Confirming this hypothesis would demonstrate the link between increased CA scaﬀold stiﬀness and PCa characteristics, which is anticipated based on the stiﬀening of the tumor micro-environment during progression of PCa. The physical properties of the CA scaﬀolds were characterized with mechanical testing, scanning electron microscopy (SEM) imaging, Fourier Transform Infrared (FTIR)