Abstract 4997: Using three-dimensional (3D) culture systems to delineate the role of RANKL in metastatic prostate cancer.
Authors: Ziaee Shabnam , Sieh Shirly , Chu Chia-Yi , Wang Ruoxiang , Hutmacher Dietmar W. , Nelson Colleen , Guo Chin-Lin , Chung Leland W.K. 
Published: August 2014

Introduction and Objective: While prostate cancer (PCa) is curable at early diagnosis, metastatic PCa is terminal. Recent studies have shown that Receptor Activator of NF kappa-B Ligand, RANKL, markedly enhances bone metastasis (70-100%) by the LNCaP human PCa cell line in mice compared to neo controls (0%). Multivariable tumor and microenvironmental factors in vivo make it difficult to identify the specific cellular response induced by RANKL causing PCa cell homing and growth in bone. Current in vitro 2D cultures lack mechanical and biochemical properties mimicking the bone microenvironment. Our preliminary data showed integrin alpha2 is elevated in RANKL-over-expressing LNCaP cells. PCa interaction with the major bone matrix protein Col1 may contribute to the metastatic potential of LNCAP-RANKL cells. This study used 3D systems to explore the role of RANKL in PCa-bone matrix interactions.

Methods: 3D cultures of RANKL-expressing (highly metastatic) or non-metastatic LNCaP cells were evaluated in 2D, suspension cultures, on type 1 collagen (Col1) coated polymeric meshes, embedded in Col1 gel or hydrogel mixed with Col1 for growth, morphology, motility, and gene expression. Time-dependent cell migration was assessed by a time-lapse imaging system.

Results: LNCAP-RANKL cells and Neo-expressing controls grew as aggregates in all 3D cultures but not 2D cultures. Col1 promoted less compact and more widespread aggregation of LNCAP-RANKL cells in the 3D cultures, exhibiting more motile and directional migratory behavior. A molecular signature of 3D growth was generated and compared among different conditions. Consistent with 2D studies, 3D qRT-PCR data showed RANKL drove epithelial-to-mesenchymal transition (EMT), shown by increased expression of vimentin and N-cadherin but decreased E-cadherin. Decreased expression of androgen receptor and PSA was observed in RANKL-expressing PCa cells. Supporting the migration data, the presence of Col1 drove RANKL-expressing PCa cells to express higher levels of c-Met protooncogene, sensitizing PCa cell responses to the c-Met ligand, hepatocyte growth factor (HGF).

Conclusions: 3D culture systems were established to study interactions between Col1 and metastatic or non-metastatic PCa cells. 3D culture systems supported PCa cell growth, while maintaining their classical markers, validating the reliability of our 3D systems. Bone metastatic PCa cells preferentially exhibited EMT, increased expression of α2 integrin, and c-Met protooncogene. These 3D systems will be used to assess liquid biopsy specimens harvested from human PCa patients to improve prognosis and therapeutic follow-up.

Citation Format: Shabnam Ziaee, Shirly Sieh, Chia-Yi Chu, Ruoxiang Wang, Dietmar W. Hutmacher, Colleen Nelson, Chin-Lin Guo, Leland W.K. Chung. Using three-dimensional (3D) culture systems to delineate the role of RANKL in metastatic prostate cancer. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 4997. doi:10.1158/1538-7445.AM2013-4997