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170 Background: Transcriptome-based analysis has begun to reshape the approach to prostate cancer (PC). Two different gene expression signatures have shown that PC can be divided into 3 subclasses reflecting luminal-basal biology. These subtypes point toward biological drivers that may strongly influence how care should be personalized including optimization of androgen receptor targeted therapy. The majority of work done in this area has been based on tissue-based gene expression. With the advent of newer nanotechnology platforms for isolation of circulating tumor cells (CTCs), profiling of PC gene expression from blood is now possible. Methods: We recruited 34 patients with metastatic castration resistant PC at Cedars-Sinai Medical Center who had available blood specimens prior to initiation of androgen receptor signaling inhibitor (ARSI, e.g. abiraterone, enzalutamide and apalutamide) therapy.We utilized the NanoVelcro Assays which allow for capture and release of CTCs with intact mRNA. Gene sets from the PCS and PAM50 signatures were re-reviewed to optimize signal detection in the blood and enriched for genes upregulated in PC. The NanoString nCounter platform was used for RNA profiling. Results: The final assay was tested in banked blood samples and provided classifications of patients that associated with clinical responsiveness to therapy. Validation was conducted to examine the performance of the CTC-specific PCS/PAM50 panel in public databases (including Prostate Cancer Transcriptome Atlas and GenomeDx). Our pilot study showed that the median overall survival was significantly worse in PCS1 patients. Conclusions: This study shows initial proof of principle that genomic classification in blood is possible using contemporary tool for blood component isolation and RNA profiling. Additional technical and clinical validations are needed prior to widespread implementation, but these methods may make it possible to increase the utilization of genomic classifiers in clinical studies and in practice.

Background: Bone metastasis in human prostate cancer remains a major clinical problem since no effective therapy exists. The RANKL/RANK pathway plays a predominant role in the interaction between metastasized prostate cancer cells and osteoclasts that increases the bone turnover. The current therapies, including targeting RANKL with denosumab, address the growth of prostate tumor cells that have already colonized the bone, but are largely ineffective in prolonging the survival of human prostate cancer patients with bone metastasis. Further, a major impediment to prostate cancer bone metastasis research is the lack of an animal model that spontaneously recapitulates human prostate cancer bone metastasis in the context of an intact immune system. Results: To overcome this major limitation, we have developed a novel syngeneic mouse model to study prostate cancer bone metastasis. Both the CXCL12/CXCR4 and RANKL/RANK pathways have been reported to be overexpressed / dysregulated in human prostate cancer bone metastatic samples. Data generated utilizing our immune-intact mouse model shows that the CXCL12/CXCR4 and RANKL/RANK pathways co-operate with each other to drive prostate cancer bone metastasis. Studies have shown that targeting the CXCL12/CXCR4 and RANKL/RANK pathways individually affects the immune system, thereby making our syngeneic mouse model an indispensable tool for studying the critical co-operation between these 2 pathways in the manifestation of human prostate cancer bone metastasis. Extending our earlier findings that RANKL drives PCa metastases in immune-deficient mice (Chu et al., 2014) to immune-intact C57/Bl6 mice, we found that MPC3 mouse PCa cells with RANKL overexpression (MPC3-Luc-GFP-RANKL) develop 70-80% limb and jaw within 4 weeks of intra-cardiac injection in these syngeneic mice. Control MPC3 cells had no bone metastasis. Bone lesions visualized by luciferase imaging and X-ray were confirmed by micro CT and immunohistochemistry. RANKL signaling drove bone and visceral metastases via the downstream CXCL12/CXCR4 signaling axis. MPC3-Luc-GFP-RANKL cells showed increased CXCR4 protein levels by immunohistochemistry. Metastatic bone marrow flush showed dramatically increased levels of CXCL12 mRNA compared with control mice (MPC3-Luc-GFP-EV). Conclusion: In sum, 1) circulating PCa cells induce a marked CXCL12 elevation after colonizing bone, triggering chemotaxis and recruiting CXCR4-positive PCa cells to migrate to bone; and 2) osteomimetic PCa cells with increased RANKL expression interact with osteoclasts to enhance bone resorption and turnover, releasing additional growth factors and chemokines for PCa cell growth and survival in bone. Citation Format: Srinivas Nandana, Murali Gururajan, Manisha Tripathi, Chia-Yi Chu, Haiyen Zhau, Stephen Shiao, Leland Chung. A novel syngeneic mouse model of prostate cancer bone metastasis: Mechanisms of chemotaxis and bone colonization [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 4523.

Cancer metastasis is highly inefficient and complex. Common features of metastatic cancer cells have been observed using cancer cell lines and genetically reconstituted mouse and human tumor xenograft models. These include cancer cell interaction with the tumor microenvironment, and the ability of cancer cells to sense extracellular stimuli and adapt to adverse growth conditions. This review summarizes the coordinated response of cancer cells to soluble growth factors, such as RANKL, by a unique forward feedback mechanism employing coordinated upregulation of RANKL and c-Met with downregulation of androgen receptor. The RANK-mediated signal network was found to drive epithelial to mesenchymal transition in prostate cancer cells, promote osteomimicry and the ability of prostate cancer cells to assume stem cell and neuroendocrine phenotypes, and confer the ability of prostate cancer cells to home to bone. Prostate cancer cells with activated RANK-mediated signal network were observed to recruit and even transform the non-tumorigenic prostate cancer cells to participate in bone and soft tissue colonization. The coordinated regulation of cancer cell invasion and metastasis by the forward feedback mechanism involving RANKL, c-Met, transcription factors and VEGF-neuropilin could offer new therapeutic opportunities to target prostate cancer bone and soft tissue metastases.

Prostate cancer (PCa) metastasis to bone is lethal and there is no adequate animal model for studying the mechanisms underlying the metastatic process. Here, we report that receptor activator of NF-κB ligand (RANKL) expressed by PCa cells consistently induced colonization or metastasis to bone in animal models. RANK-mediated signaling established a premetastatic niche through a feed-forward loop, involving the induction of RANKL and c-Met, but repression of androgen receptor (AR) expression and AR signaling pathways. Site-directed mutagenesis and transcription factor (TF) deletion/interference assays identified common TF complexes, c-Myc/Max, and AP4 as critical regulatory nodes. RANKL–RANK signaling activated a number of master regulator TFs that control the epithelial-to-mesenchymal transition (Twist1, Slug, Zeb1, and Zeb2), stem cell properties (Sox2, Myc, Oct3/4, and Nanog), neuroendocrine differentiation (Sox9, HIF1α, and FoxA2), and osteomimicry (c-Myc/Max, Sox2, Sox9, HIF1α, and Runx2). Abrogating RANK or its downstream c-Myc/Max or c-Met signaling network minimized or abolished skeletal metastasis in mice. RANKL-expressing LNCaP cells recruited and induced neighboring non metastatic LNCaP cells to express RANKL, c-Met/activated c-Met, while downregulating AR expression. These initially non-metastatic cells, once retrieved from the tumors, acquired the potential to colonize and grow in bone. These findings identify a novel mechanism of tumor growth in bone that involves tumor cell reprogramming via RANK–RANKL signaling, as well as a form of signal amplification that mediates recruitment and stable transformation of non-metastatic bystander dormant cells.

Identification of factors that mediate visceral and bone metastatic spread and subsequent bone remodeling events is highly relevant to successful therapeutic intervention in advanced human prostate cancer. TBX2, a T-box family transcription factor that negatively regulates cell-cycle inhibitor p21, plays critical roles during embryonic development, and recent studies have highlighted its role in cancer. Here, we report that TBX2 is overexpressed in human prostate cancer specimens and bone metastases from xenograft mouse models of human prostate cancer. Blocking endogenous TBX2 expression in PC3 and ARCaPM prostate cancer cell models using a dominant-negative construct resulted in decreased tumor cell proliferation, colony formation, and invasion in vitro. Blocking endogenous TBX2 in human prostate cancer mouse xenografts decreased invasion and abrogation of bone and soft tissue metastasis. Furthermore, blocking endogenous TBX2 in prostate cancer cells dramatically reduced bone-colonizing capability through reduced tumor cell growth and bone remodeling in an intratibial mouse model. TBX2 acted in trans by promoting transcription of the canonical WNT (WNT3A) promoter. Genetically rescuing WNT3A levels in prostate cancer cells with endogenously blocked TBX2 partially restored the TBX2-induced prostate cancer metastatic capability in mice. Conversely, WNT3A-neutralizing antibodies or WNT antagonist SFRP-2 blocked TBX2-induced invasion. Our findings highlight TBX2 as a novel therapeutic target upstream of WNT3A, where WNT3A antagonists could be novel agents for the treatment of metastasis and for skeletal complications in prostate cancer patients. Cancer Res; 77(6); 1331–44. ©2017 AACR.

Aim Dentin hypersensitivity, caused by the exposure and patency of dentinal tubules, can affect patients' quality of life. The aim of this study was to undertake a systematic review and a network meta-analysis, comparing the effectiveness in resolving dentin hypersensitivity among different in-office desensitizing treatments. Materials and Methods A literature search was performed with electronic databases and by hand until December 2011. The included trials were divided into six treatment groups as placebo, physical occlusion, chemical occlusion, nerve desensitization, laser therapy and combined treatments. The treatment effects between groups were estimated with standardized mean differences by using a Bayesian network meta-analysis. Results Forty studies were included. The standardized mean difference between placebo and physical occlusion was −2.57 [95% credible interval (CI): −4.24 to −0.94]; placebo versus chemical occlusion was −2.33 (95% CI: −3.65 to −1.04); placebo versus nerve desensitization was −1.72 (95% CI: −4.00 to 0.52); placebo versus laser therapy was −2.81 (95% CI: −4.41 to −1.24); placebo versus combined treatment was −3.47 (95% CI: −5.99 to −0.96). The comparisons of the five active treatments showed no significant differences. Conclusions The results from network meta-analysis showed that most active treatment options had significantly better treatment outcome than placebo.

Overexpression of Fas ligand (FasL) in cancer cells elicits potential antitumor effects via recruitment of neutrophils. Conversely, FasL-expressing tumors may counterattack tumor-infiltrating lymphocytes by delivering apoptotic death signals via Fas/FasL interactions, which may lead to tumor escape. In order to distinguish the role of FasL in antitumor activity and tumor progression, Lewis lung carcinoma cells (LLC-1) were used to establish the cell line LLC-FasL, in which FasL expression was repressed by doxycycline (Dox) treatment and induced in the absence of Dox. LLC-FasL cells promote tumor regression when expressing FasL, whereas tumor outgrowth is observed by depletion of FasL expression. To investigate whether initial expression of FasL during tumor formation is critical for FasL-mediated tumor regression, Dox-treated LLC-FasL cells were inoculated into Dox-treated mice, but Dox treatment was stopped 5 days after inoculation. When low cell numbers were inoculated, we observed 80% survival and no tumor formation, whereas no mice survived inoculation with high cell numbers, despite the delayed induction of FasL by Dox withdrawal. The inoculation of a high density of cells may establish a favorable tumor microenvironment before the expression of FasL. Our findings demonstrate that FasL may elicit antitumor activity when it is initially present on injected cancer cells and thus can act prior to tumor microenvironment formation. Furthermore, a well-established tumor microenvironment abrogates FasL-mediated antitumor activity. © 2012 Wiley Periodicals, Inc.

Background: Treatment of prostate cancer by hormone suppression leads to the appearance of aggressive variants of metastatic castration-resistant prostate cancer (mCRPC) with variable or no dependence on the androgen receptor (AR). Here we identify the neural transcription factor ONECUT2 as a negative regulator of the AR axis, that emerges in aggressive PC variants to control transcriptional networks linked to CRPC and neuroendocrine (NE) differentiation. We further demonstrate that ONECUT2 can be targeted with a small molecule that inhibits mCRPC metastasis in mice. Methods: ONECUT2 was confirmed as a mCRPC-relevant protein and to be targetable by computational modeling and bioinformatics, enforced expression, silencing, microarray, ChIP-Seq, immunohistochemistry, immunofluorescence, quantitative imaging, functional assays, in vivo experiments, and surface plasmon resonance. Results: We have performed a master regulator analysis using 260 mCRPC transcriptome profiles and developed a model transcription factor network for mCRPC that associates ONECUT2 for the first time with metastatic progression. Gene expression profiling of ONECUT2-engineered PC cell lines has allowed us to generate a ONECUT2 activity signature that reveals high positive correlation with pro-neural and aggressive PC signatures, and a negative correlation with AR activation pathways. We find that ONECUT2 is a negative regulator of AR expression and a repressor of its transcriptional program through direct binding to AR target genes. We also find that ONECUT2 is significantly increased in human NEPC and confers NE properties to CRPC through direct downregulation of the NEPC inhibitor FOXA1 and direct upregulation of the NEPC driver PEG10. Finally, we show that ONECUT2 is required for cell growth and survival and that it can be targeted with a small molecule that, by binding to the ONECUT2 C-terminal DNA binding domain, inhibits mCRPC metastasis in mice. Conclusions: OC2 is a master regulator of aggressive mCRPC variants that drives AR-dependent adenocarcinoma toward NEPC differentiation by blocking AR/FOXA1-activity and inducing PEG10. OC2 can be targeted with a drug-like small molecule that inhibits CRPC metastasis in mice. Patients with OC2 active tumors may benefit from OC2 inhibitor therapy. Citation Format: Mirja Rotinen, Sungyong You, Julie Yang, Simon Coetzee, Wen-Chin Huang, Fangjin Huang, Xinlei Pan, Alberto Yáñez, Dennis Hazelett, Chia-Yi Chu, Leland Chung, Stephen Freedland, Dolores Di Vizio, Isla Garraway, Ramachandran Murali, Beatrice Knudsen, Michael Freeman. ONECUT2 is a targetable master regulator of aggressive variants of castration-resistant prostate cancer [abstract]. In: Proceedings of the AACR Special Conference: Prostate Cancer: Advances in Basic, Translational, and Clinical Research; 2017 Dec 2-5; Orlando, Florida. Philadelphia (PA): AACR; Cancer Res 2018;78(16 Suppl):Abstract nr A047.

Introduction and Objective: Keratins (KRTs) were reported to interact with multiple cellular proteins and initiate signaling cascades that promote cell proliferation, migration and metastasis in cancers. Keratin13 (KRT13), a member of the intermediate filament proteins, plays important role in breast cancer progression and metastasis. The objectives of this study are to determine the role and molecular mechanism of KRT13 in promoting breast cancer growth and metastatic progression. Methods and Results: We evaluated KRT13 protein expression in 58 primary breast cancer tissues by immunohistochesmistry and found that KRT13 is overexpressed in metastatic breast cancer specimens (12/18) (p Conclusion: KRT13 exerts a novel function in the regulation of breast cancer cell growth, progression and metastasis via alterations of nuclear translocation of plakoglobin that modulates downstream c-Myc-dependent signaling, inducing stem cell-like phenotype and metastasis of breast cancer. Our findings could reveal potential novel targeting of breast cancer progression and metastasis. Funded by an NCI P01 CA098912 and a Board of Governors Chair of Cancer Research fund from Cedars-Sinai Medical Center to LWK Chung. Citation Format: Lijuan Yin, Gina Chia-Yi Chu, Qinlong Li, Liyuan Yin, Michael Lewis, Mourad Tighiouart, Leland W. k. Chung, Haiyen E. Zhau. KRT13 promotes stemness and drives metastasis in breast cancer through direct interaction with plakoglobin-desmoplakin complexes regulating c-Myc signaling pathway [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 5002.

BACKGROUND: Cisplatin and tyrosine kinase inhibitors (TKIs) are recommended to treat non-small-cell lung cancer (NSCLC). However, ubiquitous acquired drug resistance in NSCLC patients diminishes their therapeutic efficacy. Overcome cisplatin and TKIs resistance is an unmet medical need. DZ-191, a novel synthetic statin derivative, is capable of targeting specifically tumor cells, bypassing the liver. We observed this agent inhibited tumor cell growth in vitro, tumor growth in vivo and accelerated tumor regression in mice. METHODS: Cell viability was examined by crystal violet assay in human NSCLC A549, A549DDP, H1650, and H1975 cells. DZ-191-induced apoptosis in NSCLC cells was detected by Annexin V-FITC/PI staining followed by FACS and confirmed with western blot of apoptosis-associated proteins. Mitochondrial membrane potential was determined by rhodamine-123 followed by flow cytometry. H1650 tumor growth in mice was assessed and autophagy markers were analyzed by western blotting. RESULTS: Treatment with DZ-191 alone inhibited the growth of A549, H1650 and H1975 NSCLC cells, with IC50 values in the range of 8±1μM. In the cisplatin-resistant A549DDP cells, DZ-191 exposure at the IC10, markedly sensitized the cisplatin-resistant A549DDP cells by lowering its IC50 value from 87.6±1.0 to 13.6±1.2 μM. We observed DZ-191, but not statins, significantly increased the apoptosis of A549DDP and H1650 cells. These results are consistent with the observation that DZ191, but not statins, accumulates in mitochondria and lysosomes, resulting in depolarized mitochondrial membrane potential, decreased autophagy and lysosomal protein degradation. We observed decreased autophagy markers, assessed by decreased LC3 conversion and increased p62 accumulation. Combining with our RNA-seq data, DZ191 could induce NSCLC cell death by reducing the removal of damaged mitochondria through mitophagy. Furthermore, we observed DZ-191, but not statins, could resensitize anti-tumor responses of geftinib in a TKI-resistant NSCLC tumor xenografts in nude mice. CONCLUSIONS: These results demonstrated that DZ-191 is superior to statins in inhibiting and resensitizing the anti-tumor responses of cisplatin- and geftinib-resistant NSCLC cells by targeting mitochondria-mediated autophagy and downstream lysosome-related protein degradation. DZ-191 can be employed as a promising sensitizer to overcome cisplatin- and geftinib-resistance in NSCLC patients. Key words: DZ-191, statins, cisplatin- geftinib-resistance, autophagy, NSCLC, mitochondria, lysosomes This work is support in part by US NCI P01 CA098912 and a Board of Governors Chair of Cancer Research fund from Cedars-Sinai Medical Center to LWK Chung and China National 863 Lung Cancer Research Grant (2012AA02A502) and International Technology Targeted Therapeutics Grant (2016YEE0103400) to Q. Zhou Citation Format: Liyuan Yin, Gina Chia-Yi Chu, Ruoxiang Wang, Anisha Madhav, Lijuan Yin, Neil Bhowmick, Haiyen E. Zhau, Qinghua Zhou, Leland W.K. Chung. A novel mitochondria-based targeting to restore therapeutic responsiveness in cisplatin- and geftinib-resistant human lung cancer cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 1267.

Purpose: β2-Microglobulin (β2M) has been shown to promote osteomimicry and the proliferation of human prostate cancer cells. The objective of this study is to determine the mechanism by which targeting β2M using anti-β2M antibody inhibited growth and induced apoptosis in prostate cancer cells.Experimental Design: Polyclonal and monoclonal β2M antibodies were used to interrupt β2M signaling in human prostate cancer cell lines and the growth of prostate tumors in mice. The effects of the β2M antibody on a survival factor, androgen receptor (AR), and its target gene, prostate-specific antigen (PSA) expression, were investigated in cultured cells and in tumor xenografts.Results: The β2M antibody inhibited growth and promoted apoptosis in both AR-positive and PSA-positive, and AR-negative and PSA-negative, prostate cancer cells via the down-regulation of the AR in AR-positive prostate cancer cells and directly caused apoptosis in AR-negative prostate cancer cells in vitro and in tumor xenografts. The β2M antibody had no effect on AR expression or the growth of normal prostate cells.Conclusions: β2M downstream signaling regulates AR and PSA expression directly in AR-positive prostate cancer cells. In both AR-positive and AR-negative prostate cancer cells, interrupting β2M signaling with the β2M antibody inhibited cancer cell growth and induced its apoptosis. The β2M antibody is a novel and promising therapeutic agent for the treatment of human prostate cancers.

Epithelial-mesenchymal transition (EMT) in cancer describes the phenotypic and behavioral changes of cancer cells from indolent to virulent forms with increased migratory, invasive and metastatic potential. EMT can be induced by soluble proteins like transforming growth factor beta1 (TGFbeta1) and transcription factors including Snail and Slug. We utilized the ARCaP(E)/ARCaP(M) prostate cancer progression model and LNCaP clones stably overexpressing Snail to identify novel markers associated with EMT. Compared to ARCaP(E) cells, the highly tumorigenic mesenchymal ARCaP(M) and ARCaP(M1) variant cells displayed a higher incidence of bone metastasis after intracardiac administration in SCID mice. ARCaP(M) and ARCaP(M1) expressed mesenchymal stromal markers of vimentin and N-cadherin in addition to elevated levels of Receptor Activator of NF-kappaB Ligand (RANKL). We observed that both epidermal growth factor (EGF) plus TGFbeta1 treatment and Snail overexpression induced EMT in ARCaP(E) and LNCaP cells, and EMT was associated with increased expression of RANKL protein. Finally, we determined that the RANKL protein was functionally active, promoting osteoclastogenesis in vitro. Our results indicate that RANKL is a novel marker for EMT during prostate cancer progression. RANKL may function as a link between EMT, bone turnover, and prostate cancer skeletal metastasis.

Prostate cancer remains the most frequently diagnosed cancer in men in North America, and despite recent advances in treatment patients with metastatic disease continue to have poor five-year survival rates. Recent studies in prostate cancer have revealed the critical role of the tumor microenvironment in the initiation and progression to advanced disease. Experimental data have uncovered a reciprocal relationship between the cells in the microenvironment and malignant tumor cells in which early changes in normal tissue microenvironment can promote tumorigenesis and in turn tumor cells can promote further pro-tumor changes in the microenvironment. In the tumor microenvironment, the presence of persistent immune infiltrates contributes to the recruitment and reprogramming of other non-immune stromal cells including cancer-associated fibroblasts and a unique recently identified population of metastasis-initiating cells (MICs). These MICs, which can also be found as part of the circulating tumor cell (CTC) population in PC patients, promote cancer cell transformation, enhance metastatic potential and confer therapeutic resistance. MICs act can on other cells within the tumor microenvironment in part by secreting exosomes that reprogram adjacent stromal cells to create a more favorable tumor microenvironment to support continued cancer growth and progression. We review here the current data on the intricate relationship between inflammation, reactive stroma, tumor cells and disease progression in prostate cancer.

The effects of microgravity and spaceflight on cancer development and progression have not been defined. We provide an introductory review of our studies of microgravity and spaceflight on human prostate cancer cell growth, with emphasis on the investigation of cancer–stromal interaction under 3-dimensional (3-D) rotary wall vessel (RWV) bioreactor and space flight conditions.

FYN is a SRC family kinase (SFK) that has been shown to be up-regulated in human prostate cancer (PCa) tissues and cell lines. In this study, we observed that FYN is strongly up-regulated in human neuroendocrine PCa (NEPC) tissues and xenografts, as well as cells derived from a NEPC transgenic mouse model. In silico analysis of FYN expression in prostate cancer cell line databases revealed an association with the expression of neuroendocrine (NE) markers such as CHGA, CD44, CD56, and SYP. The loss of FYN abrogated the invasion of PC3 and ARCaPM cells in response to MET receptor ligand HGF. FYN also contributed to the metastatic potential of NEPC cells in two mouse models of visceral metastasis with two different cell lines (PC3 and TRAMPC2-RANKL). The activation of MET appeared to regulate neuroendocrine (NE) features as evidenced by increased expression of NE markers in PC3 cells with HGF. Importantly, the overexpression of FYN protein in DU145 cells was directly correlated with the increase of CHGA. Thus, our data demonstrated that the neuroendocrine differentiation that occurs in PCa cells is, at least in part, regulated by FYN kinase. Understanding the role of FYN in the regulation of NE markers will provide further support for ongoing clinical trials of SFK and MET inhibitors in castration-resistant PCa patients.

Brain tumors and brain metastases are among the deadliest malignancies of all human cancers, largely due to the cellular blood–brain and blood–tumor barriers that limit the delivery of imaging and therapeutic agents from the systemic circulation to tumors. Thus, improved strategies for brain tumor visualization and targeted treatment are critically needed. Here we identified and synthesized a group of near-infrared fluorescence (NIRF) heptamethine carbocyanine dyes and derivative NIRF dye-drug conjugates for effective imaging and therapeutic targeting of brain tumors of either primary or metastatic origin in mice, which is mechanistically mediated by tumor hypoxia and organic anion-transporting polypeptide genes. We also demonstrate that these dyes, when conjugated to chemotherapeutic agents such as gemcitabine, significantly restricted the growth of both intracranial glioma xenografts and prostate tumor brain metastases and prolonged survival in mice. These results show promise in the application of NIRF dyes as novel theranostic agents for the detection and treatment of brain tumors.

Purpose: MicroRNAs in the delta-like 1 homolog–deiodinase, iodothyronine 3 (DLK1-DIO3) cluster have been shown to be critical for embryonic development and epithelial to mesenchymal transition (EMT). DLK1-DIO3 cluster miRNAs are elevated in the serum of patients with metastatic cancer. However, the biologic functions of these miRNAs in the EMT and metastasis of cancer cells are poorly understood. We previously demonstrated the oncogenic and metastatic role of miR-409-3p/5p, a member of this cluster, in prostate cancer. In this study, we defined the role of miR-154* and miR-379, two key members of this cluster, in prostate cancer progression and bone metastasis in both cell line models and clinical specimens. Experimental Design: Genetic manipulation of miR-154* and miR-379 was performed to determine their role in tumor growth, EMT, and bone metastasis in mouse models. We determined the expression of miR-154* in prostate cancer clinical samples and bone metastasis samples using in situ hybridization and quantum dot labeling. Results: Elevated expression of miR-154* and miR-379 was observed in bone metastatic prostate cancer cell lines and tissues, and miR-379 expression correlated with progression-free survival of patients with prostate cancer. Intracardiac inoculation (to mimic systemic dissemination) of miR-154* inhibitor-treated bone metastatic ARCaPM prostate cancer cells in mice led to decreased bone metastasis and increased survival. Conclusion: miR-154* and miR-379 play important roles in prostate cancer biology by facilitating tumor growth, EMT, and bone metastasis. This finding has particular translational importance because miRNAs in the DLK1-DIO3 cluster can be attractive biomarkers and possible therapeutic targets to treat bone metastatic prostate cancer. Clin Cancer Res; 20(24); 6559–69. ©2014 AACR.

Purpose: miR-409-3p/-5p is a miRNA expressed by embryonic stem cells, and its role in cancer biology and metastasis is unknown. Our pilot studies demonstrated elevated miR-409-3p/-5p expression in human prostate cancer bone metastatic cell lines; therefore, we defined the biologic impact of manipulation of miR-409-3p/-5p on prostate cancer progression and correlated the levels of its expression with clinical human prostate cancer bone metastatic specimens. Experimental Design: miRNA profiling of a prostate cancer bone metastatic epithelial-to-mesenchymal transition (EMT) cell line model was performed. A Gleason score human tissue array was probed for validation of specific miRNAs. In addition, genetic manipulation of miR-409-3p/-5p was performed to determine its role in tumor growth, EMT, and bone metastasis in mouse models. Results: Elevated expression of miR-409-3p/-5p was observed in bone metastatic prostate cancer cell lines and human prostate cancer tissues with higher Gleason scores. Elevated miR-409-3p expression levels correlated with progression-free survival of patients with prostate cancer. Orthotopic delivery of miR-409-3p/-5p in the murine prostate gland induced tumors where the tumors expressed EMT and stemness markers. Intracardiac inoculation (to mimic systemic dissemination) of miR-409-5p inhibitor–treated bone metastatic ARCaPM prostate cancer cells in mice led to decreased bone metastasis and increased survival compared with control vehicle–treated cells. Conclusion: miR-409-3p/-5p plays an important role in prostate cancer biology by facilitating tumor growth, EMT, and bone metastasis. This finding bears particular translational importance as miR-409-3p/-5p appears to be an attractive biomarker and/or possibly a therapeutic target to treat bone metastatic prostate cancer. Clin Cancer Res; 20(17); 4636–46. ©2014 AACR.

Tumor cells are inherently heterogeneous and often exhibit diminished adhesion, resulting in the shedding of tumor cells into the circulation to form circulating tumor cells (CTCs). A fraction of these are live CTCs with potential of metastatic colonization whereas others are at various stages of apoptosis making them likely to be less relevant to understanding the disease. Isolation and characterization of live CTCs may augment information yielded by standard enumeration to help physicians to more accurately establish diagnosis, choose therapy, monitor response, and provide prognosis. We previously reported on a group of near-infrared (NIR) heptamethine carbocyanine dyes that are specifically and actively transported into live cancer cells. In this study, this viable tumor cell-specific behavior was utilized to detect live CTCs in prostate cancer patients. Peripheral blood mononuclear cells (PBMCs) from 40 patients with localized prostate cancer together with 5 patients with metastatic disease were stained with IR-783, the prototype heptamethine cyanine dye. Stained cells were subjected to flow cytometric analysis to identify live (NIR(+)) CTCs from the pool of total CTCs, which were identified by EpCAM staining. In patients with localized tumor, live CTC counts corresponded with total CTC numbers. Higher live CTC counts were seen in patients with larger tumors and those with more aggressive pathologic features including positive margins and/or lymph node invasion. Even higher CTC numbers (live and total) were detected in patients with metastatic disease. Live CTC counts declined when patients were receiving effective treatments, and conversely the counts tended to rise at the time of disease progression. Our study demonstrates the feasibility of applying of this staining technique to identify live CTCs, creating an opportunity for further molecular interrogation of a more biologically relevant CTC population.

Introduction: Burkitt's lymphoma (BL) is is composed of highly proliferating mature B cells expressing a germinal center phenotype (Cai, Oncotarget 2015). It is one of the fastest growing human tumors and presents in extranodal as well as nodal sites. The genetic hallmark of BL is the MYC translocation with MYC and TP53 mutations in ∼60% and 40% of the cases, respectively (Ott, Blood 2014). While prompt administration of multiagent immunochemotherapy regimens is associated with favorable outcomes for the majority of patients, prognosis of refractory or relapsed disease is poor (Jacobson, Blood 2014). We previously identified a novel near-infrared heptamethine carbocyanine fluorescent dye (NIR-dye) that has tumor homing property through cell surface transporters, organic anion transporting peptides (OATPs), and shows preferential uptake in tumor but not normal cells as demonstrated in cell culture, mouse and dog tumor models, patient-derived xenografts (PDXs), and perfused kidney cancers in patients (Wu, Biomaterials 2014). Here, we describe the effects of a newly synthesized NIR-dye-cisplatin conjugate (NIR-dye-Cis) on MYC-driven TP53 mutated aggressive B-cell BL cell lines in culture and tumors in mice. Methods:The growth and viability of immortalized B-cell lymphoma cell lines, Namalwa, Raji, CA46, Ramos, Daudi, were evaluated by MTT upon 72 h exposure to conventional cisplatin (Cis) and NIR-dye-Cis. Induction of apoptosis was assesed using a caspase 3/7 luminescent assay. Cleavage of apoptosis substrates and inhibition of c-myc was determined by western blotting, with images quantified by ImageJ after normalization with GAPDH. NIR-dye-Cis uptake in cells and tumors were imaged by NIRF microscopy and IVIS Lumina XR Imaging System, respectively. Male NCr nude and NOD/SCID mice bearing s.c. Namalwa tumors were injected i.p. with equimolar doses of NIR-dye-Cis and conventional Cis 3 times a week for 4 weeks. Formalin-fixed tumor and kidney sections were stained with hematoxylin and esosin and kidney sections scored using a semiquantitative scale designed to assess AKI-associated tubular injury (tubular epithelial cell loss, necrosis, tubular epithelial simplification, intratubular debris, and casts). Results: 1) All BL cell lines tested are sensitive to growth inhibition by NIR-dye-Cis but not Cis with IC50 ranging between 720 nM - 2.53 µM and >64 µM (p Conclusion: Aggressive B-cell BL can be treated safely by NIR-dye-Cis. This agent was found to accumulate in BL cells and tumor xenografts and in contrast to Cis, NIR-dye-Cis induced apoptosis and shrinked BL tumor xenografts in mice. NIR-dye-Cis induced apoptosis, downregulated c-myc and overcame Cis-resistance in MYC-driven TP53 mutated aggressive B-cell BL. This agent could have potential clinical benefit in relapsed/refractory heavily pretreated patients for improved efficacy and reduced kidney toxicity. We proposed NIR-dye-Cis could be employed as the secondary agent for the treatment of relapsed/refractory agressive B-cell non Hodgkin lymphomas. Disclosures No relevant conflicts of interest to declare.

FYN is a SRC family kinase (SFK) that has been shown to be up-regulated in human prostate cancer (PCa) tissues and cell lines. In this study, we observed that FYN is strongly up-regulated in human neuroendocrine PCa (NEPC) tissues and xenografts, as well as cells derived from a NEPC transgenic mouse model. In silico analysis of FYN expression in prostate cancer cell line databases revealed an association with the expression of neuroendocrine (NE) markers such as CHGA, CD44, CD56, and SYP. The loss of FYN abrogated the invasion of PC3 and ARCaPM cells in response to MET receptor ligand HGF. FYN also contributed to the metastatic potential of NEPC cells in two mouse models of visceral metastasis with two different cell lines (PC3 and TRAMPC2-RANKL). The activation of MET appeared to regulate neuroendocrine (NE) features as evidenced by increased expression of NE markers in PCa cells lines treated with HGF. Importantly, the overexpression of FYN protein in DU145 cells was directly correlated with the increase of CHGA. Thus, our data demonstrated that the neuroendocrine differentiation that occurs in PCa cells is, at least in part, regulated by FYN kinase. Understanding the role of FYN in the regulation of NE markers will provide further support for ongoing clinical trials of SFK and MET inhibitors in castration-resistant PCa patients. Citation Format: Karen Cavassani, Murali Gururajan, Margarit Sievert, Peng Duan, Michael Freeman, Gina Chia-Yi Chu, Neil Bhowmick, Leland Chung, Edward Posadas. Src family kinase Fyn promotes the neuroendocrine phenotype and visceral metastasis in advanced prostate cancer. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 177.

RANKL-elicited RANK activity plays critical roles in many biological and pathological conditions, including osteoclast differentiation/bone remodeling, lymph node/thymic development, central thermoregulation and progesterone-driven mammary gland maturation, differentiation and carcinogenesis. RANKL can be derived from osteoblasts, infiltrating inflammatory cells and stromal fibroblasts. We previously showed that malignant prostate cancer (PCa) cells expressed RANKL and that its expression was correlated with clinical PCa progression and bone metastasis. RANKL expression at the single cell level in primary PCa specimens predicts PCa patient survival. We demonstrated RANKL overexpression promoted epithelial-to-mesenchymal transition (EMT), stem cells, and neuroendocrine (NE) phenotypes of PCa cells through transactivation of c-Myc/Max and AP4 via RANK-mediated signaling and conferred PCa bone and soft tissue metastases in mice. Blockade of RANKL-RANK signaling as well as their downstream regulators abolished the metastasis of PCa induced by RANKL. Furthermore, recombinant RANKL protein alone is sufficient to induce bone colonization and growth of RANKL- and non-metastatic PCa and breast cancer cells. Even a few RANKL+ PCa cells are sufficient to initiate the in vivo metastatic cascade by recruiting non-tumorigenic RANKL- PCa cells to participate in the metastatic process via downstream signaling amplification, implicating metastasis-initiating properties of RANKL-expressing PCa cells. Recently, we further demonstrated that RANKL rendered metastatic potential to breast, lung, renal, and liver cancer cells and promote their metastases to bone and other soft tissues in mice. Collectively, these results demonstrated RANKL is a uniform factor that drives bone metastasis of many different cancers, including prostate, breast, lung, renal, and liver cancers. The autocrine/paracrine RANKL-RANK signaling in cancer cells establishes a premetastatic niche through a “vicious cycle”, inducing RANKL and c-Met expression via activation of c-Myc/Max, and this promotes PCa EMT progression, stem and NE cell properties, and bone and soft tissue metastases. RANKL-expressing cancer cells possess metastasis-initiating potential that recruits and reprograms bystander non-tumorigenic cells to participate in the metastatic process. RANKL expression status therefore offers new insights for dissecting the mechanism by which PCa cells exhibit propensity for bone colonization/metastasis. Citation Format: Gina Chia-Yi Chu, Haiyen E. Zhau, Ruoxiang Wang, Leland W.K. Chung. Autocrine/paracrine RANKL-RANK signaling promotes cancer bone metastasis and establishes premetastatic niche recruiting bystander cancer cells to participate in the metastatic process. [abstract]. In: Proceedings of the AACR Special Conference on Tumor Metastasis; 2015 Nov 30-Dec 3; Austin, TX. Philadelphia (PA): AACR; Cancer Res 2016;76(7 Suppl):Abstract nr B42.

We assessed the application of near infrared heptamethine carbocyanine dyes, including IR-783 and the synthetic analogue MHI-148, as optical imaging agents for the rapid detection of human kidney cancer.The uptake, retention and subcellular localization of these organic dyes were investigated in cultured kidney cancer cells. Tumor specificity of dye uptake and retention was evaluated by whole body imaging of mice bearing human kidney cancer xenografts or freshly harvested clinical kidney cancer specimens. In addition, dye accumulation at the tissue and cellular levels was confirmed by ex vivo studies with results confirmed by fluorescence imaging of frozen tissue sections. Peripheral blood spiked with kidney cancer cells was stained to simulate the detection of circulating tumor cells.Preferential uptake and retention of carbocyanine near infrared dyes was observed in cultured human kidney cancer cells, human kidney cancer cell spiked whole blood, human kidney cancer xenografts and freshly harvested human kidney cancer tissues compared to normal kidney epithelial cells and normal host organs.We describe a new class of near infrared heptamethine carbocyanine dyes that show potential for detecting kidney cancer cells in circulating blood and kidney cancer cells in clinical specimens. Near infrared carbocyanine dyes can be further developed as dual modality agents for deep tissue imaging of localized and disseminated kidney cancer in patients.

An injection-locked push-push oscillator is developed for millimeter-wave signal source. A V-band push-push CMOS voltage controlled oscillator (VCO) is proposed in this study. A new core complementary Colpitts structure was adopted in a 90 nm CMOS process of FET induced capacitance of LC tank. The measured phase noise at 1 MHz offset is at 59 GHz. The power consumption of the VCO core is only 28.8 mW. The total chip size is 0.719 × 0.633 mm2.

We have previously shown that human prostate cancer cells are capable of acquiring malignant attributes through interaction with stromal cells in the tumor microenvironment, while the interacting stromal cells can also become affected with both phenotypic and genotypic alterations. This study used a co-culture model to investigate the mechanism underlying the co-evolution of cancer and stromal cells. Red fluorescent androgen-dependent LNCaP prostate cancer cells were cultured with a matched pair of normal and cancer-associated prostate myofibroblast cells to simulate cancer-stromal interaction, and cellular changes in the co-culture were documented by tracking the red fluorescence. We found frequent spontaneous fusions between cancer and stromal cells throughout the co-culture. In colony formation assays assessing the fate of the hybrid cells, most of the cancer-stromal fusion hybrids remained growth-arrested and eventually perished. However, some of the hybrids survived to form colonies from the co-culture with cancer-associated stromal cells. These derivative clones showed genomic alterations together with androgen-independent phenotype. The results from this study reveal that prostate cancer cells are fusogenic, and cancer-stromal interaction can lead to spontaneous fusion between the two cell types. While a cancer-stromal fusion strategy may allow the stromal compartment to annihilate invading cancer cells, certain cancer-stromal hybrids with increased survival capability may escape annihilation to form a derivative cancer cell population with an altered genotype and increased malignancy. Cancer-stromal fusion thus lays a foundation for an incessant co-evolution between cancer and the cancer-associated stromal cells in the tumor microenvironment.

Overexpression of Fas ligand (FasL) in cancer cells elicits potential antitumor effects via recruitment of neutrophils. Conversely, FasL-expressing tumors may counterattack tumor-infiltrating lymphocytes by delivering apoptotic death signals via Fas/FasL interactions, which may lead to tumor escape. In order to distinguish the role of FasL in antitumor activity and tumor progression, Lewis lung carcinoma cells (LLC-1) were used to establish the cell line LLC-FasL, in which FasL expression was repressed by doxycycline (Dox) treatment and induced in the absence of Dox. LLC-FasL cells promote tumor regression when expressing FasL, whereas tumor outgrowth is observed by depletion of FasL expression. To investigate whether initial expression of FasL during tumor formation is critical for FasL-mediated tumor regression, Dox-treated LLC-FasL cells were inoculated into Dox-treated mice, but Dox treatment was stopped 5 days after inoculation. When low cell numbers were inoculated, we observed 80% survival and no tumor formation, whereas no mice survived inoculation with high cell numbers, despite the delayed induction of FasL by Dox withdrawal. The inoculation of a high density of cells may establish a favorable tumor microenvironment before the expression of FasL. Our findings demonstrate that FasL may elicit antitumor activity when it is initially present on injected cancer cells and thus can act prior to tumor microenvironment formation. Furthermore, a well-established tumor microenvironment abrogates FasL-mediated antitumor activity.

Androgen refractory cancer of the prostate (ARCaP) cells contain androgen receptor (AR) and synthesize and secrete prostate specific antigen (PSA). We isolated epithelia-like ARCaP(E) from parental ARCaP cells and induced them to undergo epithelial-mesenchymal transition (EMT) by exposing these cells to soluble factors including TGFbeta1 plus EGF, IGF-1, beta2-microglobulin (beta2-m), or a bone microenvironment. The molecular and behavioral characteristics of the resultant ARCaP(M) were characterized extensively in comparison to the parental ARCaP(E) cells. In addition to expressing mesenchymal biomarkers, ARCaP(M) gained 100% incidence of bone metastasis. ARCaP(M) cells express receptor activator of NF-kappaB ligand (RANKL), which was shown to increase tartrate-resistant acid phosphatase (TRAP)-positive osteoclasts in culture, and when metastatic to bone in vivo. We provide evidence that RANKL expression was promoted by increased cell signaling mediated by the activation of Stat3-Snail-LIV-1. RANKL expressed by ARCaP(M) cells is functional both in vitro and in vivo. The lesson we learned from the ARCaP model of EMT is that activation of a specific cell signaling pathway by soluble factors can lead to increased bone turnover, mediated by enhanced RANKL expression by tumor cells, which is implicated in the high incidence of prostate cancer bone colonization. The ARCaP EMT model is highly attractive for developing new therapeutic agents to treat prostate cancer bone metastasis.

Introduction: FYN is a Src family kinase (SFK) member that has been shown to be upregulated in human prostate cancer tissues and cell lines (PCa). The majority of the studies on SRC kinases have focused on the classical c-SRC. We demonstrated previously that knockdown of FYN in PCa cell lines leads to reduced tumor growth in vitro and in vivo. We hypothesized that MET, an oncogenic tyrosine kinase receptor implicated in metastasis and therapeutic resistance of a variety of solid tumors is regulated by FYN expression in prostate cancer. Methods: We performed in vitro growth assays, migration and invasion assays and studied PCa cell response to HGF in wildtype, FYN-depleted and FYN-reconstituted cells. Additionally, we studied the role of FYN in promoting metastasis in vivo in xenograft mouse models. Summary: In this study, we demonstrate that knockdown of FYN leads to reduced migration, invasion and metastatic dissemination of PCa cells in vitro and in vivo. Conversely, ectopic expression of FYN promotes enhanced tumor growth and invasion. Hepatocyte growth factor (HGF) induced activation of MET is perturbed in FYN knockdown cells and overexpression of FYN leads to excessive MET activation suggesting a possible feedback loop between FYN and MET in prostate cancer. Consistent these findings, PC3 prostate cancer cells overexpressing FYN are resistant to treatment with a MET inhibitor upon ligand stimulation. More importantly, we show that FYN promotes epithelial to mesenchymal transition (EMT) of PCa cells and knockdown of FYN leads to reversal of EMT and alters radiation sensitivity of PCa cells. To further identify the molecular mechanism of FYN mediated regulation of tumor growth, we performed integrated RNA sequencing analysis of FYN knockdown cells and found MET and a network of molecular targets that appear to regulate FYN mediated tumor progression and metastasis. Conclusions: We demonstrate that FYN mediated MET activation promotes PCa invasion, EMT and metastasis. None of the SFK inhibitors currently in clinical trials are specific for SFKs alone. Therefore, it is imperative to develop and test novel inhibitors that selectively target FYN and other SFKs. Our findings have therapeutic implications since clinical trials are being performed for both SFK inhibitors and MET inhibitors in castration resistant PCa patients. Our studies reveal that FYN and MET interact in PCa cells and this interaction is clinically relevant given the success of XL-184 (a MET inhibitor) in phase III clinical trials in human prostate cancer. Clinical studies with translational endpoints are the need of the hour with agents that effectively inhibit FYN and MET in a variety of settings. Citation Format: Murali Gururajan, Margarit Sievert, Sheldon Mink, Chia-Yi (Gina) Chu, Matteo Morello, Jake Lichterman, Michael R. Freeman, Edwin M. Posadas. SRC family kinase FYN promotes MET tyrosine kinase activation, epithelial to mesenchymal transition and metastasis in human prostate cancer. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 3459. doi:10.1158/1538-7445.AM2014-3459

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

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 Hutmacher, Colleen Nelson, Chin-Lin Guo, Leland W.K. Chung. Using three-dimensional (3-D) culture systems to delineate the role of RANKL in metastatic prostate cancer. [abstract]. In: Proceedings of the AACR Special Conference on Tumor Invasion and Metastasis; Jan 20-23, 2013; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2013;73(3 Suppl):Abstract nr B11.