Bone Metastatic Microenvironment Research Articles

A Selective FGFR inhibitor AZD4547 suppresses RANKL/M-CSF/OPG-dependent ostoclastogenesis and breast cancer growth in the metastatic bone microenvironment

Aberrant activation of fibroblast growth factor receptor (FGFR) signalling contributes to progression and metastasis of many types of cancers including breast cancer. Accordingly, FGFR targeted tyrosine kinase inhibitors (TKIs) are currently under development. However, the efficacy of FGFR TKIs in the bone microenvironment where breast cancer cells most frequently metastasize and also where FGFR is biologically active, has not been clearly investigated. We investigated the FGFR-mediated interactions among cancer and the bone microenvironment stromal cells (osteoblasts and osteoclasts), and also the effects of FGFR inhibition in bone metastasis. We showed that addition of culture supernatant from the MDA-MB-134-VI FGFR-amplified breast cancer cells-activated FGFR siganalling in osteoblasts, including increased expression of RANKL, M-CSF, and osteoprotegerin (OPG). Further in vitro analyses showed that AZD4547, an FGFR TKI currently in clinical trials for breast cancer, decreased RANKL and M-CSF, and subsequently RANKL and M-CSF-dependent osteoclastogenesis of murine bone marrow monocytes. Moreover, AZD4547 suppressed osteoclastogenesis and tumor-induced osteolysis in an orthotopic breast cancer bone metastasis mouse model using FGFR non-amplified MDA-MB-231 cells. Collectively, our results support that FGFR inhibitors inhibit the bone microenvironment stromal cells including osteoblasts and osteoclasts, and effectively suppress both tumor and stromal compartments of bone metastasis.

Engineering osteoblastic metastases to delineate the adaptive response of androgen-deprived prostate cancer in the bone metastatic microenvironment

While stromal interactions are essential in cancer adaptation to hormonal therapies, the effects of bone stroma and androgen deprivation on cancer progression in bone are poorly understood. Here, we tissue-engineered and validated an in vitro microtissue model of osteoblastic bone metastases, and used it to study the effects of androgen deprivation in this microenvironment. The model was established by culturing primary human osteoprogenitor cells on melt electrowritten polymer scaffolds, leading to a mineralized osteoblast-derived microtissue containing, in a 3D setting, viable osteoblastic cells, osteocytic cells, and appropriate expression of osteoblast/osteocyte-derived mRNA and proteins, and mineral content. Direct co-culture of androgen receptor-dependent/independent cell lines (LNCaP, C4-2B, and PC3) led cancer cells to display functional and molecular features as observed in vivo. Co-cultured cancer cells showed increased affinity to the microtissues, as a function of their bone metastatic potential. Co-cultures led to alkaline phosphatase and collagen-I upregulation and sclerostin downregulation, consistent with the clinical marker profile of osteoblastic bone metastases. LNCaP showed a significant adaptive response under androgen deprivation in the microtissues, with the notable appearance of neuroendocrine transdifferentiation features and increased expression of related markers (dopa decarboxylase, enolase 2). Androgen deprivation affected the biology of the metastatic microenvironment with stronger upregulation of androgen receptor, alkaline phosphatase, and dopa decarboxylase, as seen in the transition towards resistance. The unique microtissues engineered here represent a substantial asset to determine the involvement of the human bone microenvironment in prostate cancer progression and response to a therapeutic context in this microenvironment.

Targeting the Hepatocyte Growth Factor and c-Met Signaling Axis in Bone Metastases.

Bone metastasis is the terminal stage disease of prostate, breast, renal, and lung cancers, and currently no therapeutic approach effectively cures or prevents its progression to bone metastasis. One of the hurdles to the development of new drugs for bone metastasis is the complexity and heterogeneity of the cellular components in the metastatic bone microenvironment. For example, bone cells, including osteoblasts, osteoclasts, and osteocytes, and the bone marrow cells of diverse hematopoietic lineages interact with each other via numerous cytokines and receptors. c-Met tyrosine kinase receptor and its sole ligand hepatocyte growth factor (HGF) are enriched in the bone microenvironment, and their expression correlates with the progression of bone metastasis. However, no drugs or antibodies targeting the c-Met/HGF signaling axis are currently available in bone metastatic patients. This significant discrepancy should be overcome by further investigation of the roles and regulation of c-Met and HGF in the metastatic bone microenvironment. This review paper summarizes the key findings of c-Met and HGF in the development of novel therapeutic approaches for bone metastasis.

Calvarial Bone Implantation and in vivo Imaging of Tumor Cells in Mice

Bone is one of common metastasis sites for many types of cancer. In bone metastatic microenvironment, tumor-bone interactions play a significant role in the regulation of osteolytic or osteoblastic bone metastasis. In order to investigate the direct interaction between tumor cells and bone tissue, it is essential to generate appropriate animal models that mimic the behavior of tumor cells in bone metastatic lesions. Calvarial implantation model (bone invasion model) is a newly-established animal model that accurately recapitulates the behavior of tumor cells in the tumor-bone microenvironment. The surgical technique for tumor cell implantation is simpler than intracardiac, intra-arterial, or intraosseous injection techniques. This model can be useful for the identification of key factors driving tumor-induced osteolytic or osteoblastic changes.

Bioengineered Microtissue Models of the Human Bone Metastatic Microenvironment: A Novel In Vitro Theranostics Platform for Cancer Research.

One of the major limitations of studying cancer in distant sites is the lack of representative laboratory models that mimic the biological processes occurring in vivo. In this protocol, we demonstrate the application of melt electrowriting technology (MEW) to provide 3D microfiber scaffolds suitable for this purpose. Using primary human cells, MEW scaffolds support the reproducible formation of human bone-like 3D microenvironments. Co-culture with human cancer cells provides an in vitro bioengineered model of metastases in bone, suitable for investigating cell-cell and cell-matrix interactions between bone and cancer cells. By proposing variations to standard tissue histology, immunohistochemistry, immunofluorescence, and 3D imaging techniques, we show how to characterize cell morphology and protein expression in a reproducibly engineered bone metastatic microtissue.

Abstract 111: The CX3CR1-Fractalkine axis drives both circulating prostate cancer cells and macrophages to the bone metastatic microenvironment

Abstract Prostate cancer (PCa) is the second most frequently diagnosed cancer and its progression into metastatic disease reduces the 5-year survival rate to 29% in diagnosed men. A better understanding of the mechanisms underpinning metastatic dissemination and the role played by the tumor microenvironment at distant sites is essential. Our lab has shown that PCa cells express the chemokine receptor CX3CR1 and seed the skeleton when attracted by bone-derived Fractalkine (FKN) (a.k.a. CX3CL1). A crucial component of the bone microenvironment consists of heterogeneous populations of tumor-associated macrophages (TAMs), which can polarize into M1 or M2 phenotypes and are likely implicated in metastatic progression. TAMs migrate in close proximity of cancer cells and release growth factors, chemokines, and other inflammatory mediators that influence tumor growth. Using cell biology tools, we show that both PCa cells and macrophages express the CX3CR1 receptor, which signal to downstream targets when engaged by FKN and is inhibited by a novel small-molecule inhibitor. We also employed a mouse model of cancer cell dissemination and metastasis to show that our novel CX3CR1 antagonist impairs tumor seeding to the skeleton. Furthermore, as macrophages also express CX3CR1, targeting this receptor may potentially hinder TAMs' accumulation into the metastatic microenvironment, depriving it of crucial pro-metastatic conditioning. In conclusion, we propose that the CX3CR1-FKN axis drives both cancer cells and TAMs to metastatic sites and that the mitigation of the functional interactions between this receptor and its unique chemokine ligand by antagonists of CX3CR1 will impair the clinical progression of PCa metastasis. Citation Format: Ramanpreet Kaur, Chen Qian, Joseph Salvino, Olimpia Meucci, Alessandro Fatatis. The CX3CR1-Fractalkine axis drives both circulating prostate cancer cells and macrophages to the bone metastatic microenvironment [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 111.

The therapeutic effect of miR-125b is enhanced by the prostaglandin endoperoxide synthase 2/cyclooxygenase 2 blockade and hampers ETS1 in the context of the microenvironment of bone metastasis

Bone is the most common site for breast cancer spread. In the pro-metastatic cell line 1833, derived from MDA-MB-231 breast adenocarcinoma cells, both hypoxia and hepatocyte growth factor (HGF) influence the effect of miR-125b on ETS proto-oncogene 1 transcription factor (ETS1). The effect of hypoxia inducible factor 1 alpha subunit (HIF1A), known to promote metastatic spread by upregulating prostaglandin endoperoxide synthase 2 (PTGS2), may be dampened by miR-125b targeting PTGS2. Here, we investigated whether miR-125b plays a role in breast cancer metastasis by measuring its activity in response to the chemotherapeutic agent NS-398 in a xenograft model. NS-398 is typically used in the clinic to target PTGS2. We also aimed to describe the molecular mechanisms in vitro, since the enhancement of epithelial properties may favor the efficacy of therapies. We report that in the xenograft model, miR-125b reduced metastasis to the bone. We also report suppression of PTGS2 enhanced survival by decreasing HIF1A in cells within the bone marrow. In 1833 cells transfected with a miR-125b mimic we observed several phenotypic changes including enhancement of the epithelial marker E-cadherin, a reduction of mesenchymal-associated genes and a reduction of WNT-associated stem cell signaling. Our findings suggest that in vivo, key players of the bone microenvironment promoting breast cancer spread are regulated by miR-125b. In future, biological molecules imitating miR-125b may enhance the sensitivity of chemotherapeutic agents used to counteract bone metastases.

Targeting the Metastatic Bone Microenvironment by MicroRNAs

Bone metastases are a common and devastating feature of late-stage breast cancer. Metastatic bone disease is a consequence of disturbed bone remodeling due to pathological interactions between cancer cells and the bone microenvironment (BME). In the BME, breast cancer cells severely alter the balanced bone formation and bone resorption driven by osteoblasts and osteoclasts. The complex cellular cross talk in the BME is governed by secreted molecules, signaling pathways and epigenetic cues including non-coding RNAs. MicroRNAs (miRNAs) are small non-coding RNAs that reduce protein abundance and regulate several biological processes, including bone remodeling. Under pathological conditions, abnormal miRNA signaling contributes to the progression of diseases, such as bone metastasis. Recently miRNAs have been demonstrated to regulate several key drivers of bone metastasis. Furthermore, miRNAs are implicated as important regulators of cellular interactions within the metastatic BME. As a consequence, targeting the BME by miRNA delivery or antagonism has been reported to limit disease progression in experimental and preclinical conditions positioning miRNAs as emerging novel therapeutic tools in metastatic bone disease. This review will summarize our current understanding on the composition and function of the metastatic BME and discuss the recent advances how miRNAs can modulate pathological interactions in the bone environment.

Abstract P3-06-02: Tackling bone metastatic breast cancer growth with novel bone-seeking matrix metalloproteinase-2 inhibitors

Abstract Background. Despite medical advances, currently there is no treatment for breast to bone metastasis. The progression of bone metastatic breast cancer is critically dependent on interactions with the surrounding microenvironment. Therefore, identifying the underpinning molecular mechanisms is vital for the development of new therapies. Rationale. Gene expression analysis and validation in human and murine specimens of bone metastases revealed matrix metalloproteinases, such as MMP-2, are highly expressed in the bone metastatic microenvironment. Genetic ablation of MMP-2 demonstrated the importance of this MMP in driving the growth of the osteolytic bone metastatic breast cancer by regulating the bioavailability of transforming growth factor β (TGFβ). These data support the rationale for the development of a highly specific MMP-2 inhibitor for the eradication of active bone metastatic breast cancer. Methods. We utilized a novel chemical approach to synthesize bone seeking MMP inhibitors (BMMPIs) on a bisphosphonic backbone, with specificity for MMP-2 in the nanomolar range (IC50=140 nM). In vitro, we tested the effect of BMMPIs at varying doses (1nM-100μM) on the viability of the major cellular components of the cancer-bone microenvironment, namely breast cancer cells (PyMT, 4T1), osteoblasts (MC3T3) and osteoclasts (primary monocytes and RAW 264.7). In vivo, mice were intratibially inoculated with either luciferase expressing 4T1 or PyMT (1x105) cells. Mice (n=10/group) then received vehicle, zoledronate (1 mg/kg) or BMMPIs (1 mg/kg). Tumor growth was determined via luminescence quantitation. Cancer induced bone disease was measured ex vivo by μCT, Xray and histomorphometry. MMP activity in vivo and ex vivo was determined via specific activatable MMP probes. Pharmacokinetic and pharmacodynamic studies were performed. Plasma and bone marrow supernatants were collected from PyMT-R221A tumor bearing mice treated with ML115 (5mg/Kg) at 0.25, 0.5, 1, 2, 4, 8, 24 hours and three weeks (n=3 mice/time point). Currently, we are investigating the BMMPIs ability to impact the metastatic process through an in vivo model of intracardiac inoculation. Results. BMMPIs significantly impacted the viability of breast cancer cells and osteoclasts in vitro (p<0.05) compared to control. In vivo, BMMPIs significantly reduced the growth of bone metastatic breast cancer compared to control and the standard of care bisphosphonate, zoledronate. MMP activity was also lower in the BMMPI treated groups (using tumor burden to normalize values). μCT/Xray/Histomorphometry analysis also illustrated the significant beneficial effects of the BMMPIs in reducing the size of osteolytic lesions (up to 80% by μCT; p<0.05). ML115 is rapidly cleared from the plasma and accumulates selectively in the bone marrow microenvironment over time. Conclusions. MMP-2 specific BMMPIs prevent bone metastatic breast cancer growth by impacting cancer cell viability and cancer induced osteolysis. Given that bisphosphonates are well tolerated in the clinical setting, we predict that BMMPIs could be translated to the clinical setting for the treatment and eradication of bone metastatic breast cancer. Citation Format: Tauro M, Laghezza A, Tortorella P, Soliman HH, Lynch CC. Tackling bone metastatic breast cancer growth with novel bone-seeking matrix metalloproteinase-2 inhibitors [abstract]. In: Proceedings of the 2017 San Antonio Breast Cancer Symposium; 2017 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2018;78(4 Suppl):Abstract nr P3-06-02.

C-C motif ligand 5 promotes migration of prostate cancer cells in the prostate cancer bone metastasis microenvironment.

Chemokines and their receptors have key roles in cancer progression. The present study investigated chemokine activity in the prostate cancer bone metastasis microenvironment. Growth and migration of human prostate cancer cells were assayed in cocultures with bone stromal cells. The migration of LNCaP cells significantly increased when co‐cultured with bone stromal cells isolated from prostate cancer bone metastases. Cytokine array analysis of conditioned medium from bone stromal cell cultures identified CCL5 as a concentration‐dependent promoter of LNCaP cell migration. The migration of LNCaP cells was suppressed when C‐C motif ligand 5 (CCL5) neutralizing antibody was added to cocultures with bone stromal cells. Knockdown of androgen receptor with small interfering RNA increased the migration of LNCaP cells compared with control cells, and CCL5 did not promote the migration of androgen receptor knockdown LNCaP. Elevated CCL5 secretion in bone stromal cells from metastatic lesions induced prostate cancer cell migration by a mechanism consistent with CCL5 activity upstream of androgen receptor signaling.

Cancer-secreted hsa-miR-940 induces an osteoblastic phenotype in the bone metastatic microenvironment via targeting ARHGAP1 and FAM134A

Bone metastatic lesions are classified as osteoblastic or osteolytic lesions. Prostate and breast cancer patients frequently exhibit osteoblastic-type and osteolytic-type bone metastasis, respectively. In metastatic lesions, tumor cells interact with many different cell types, including osteoblasts, osteoclasts, and mesenchymal stem cells, resulting in an osteoblastic or osteolytic phenotype. However, the mechanisms responsible for the modification of bone remodeling have not been fully elucidated. MicroRNAs (miRNAs) are transferred between cells via exosomes and serve as intercellular communication tools, and numerous studies have demonstrated that cancer-secreted miRNAs are capable of modifying the tumor microenvironment. Thus, cancer-secreted miRNAs can induce an osteoblastic or osteolytic phenotype in the bone metastatic microenvironment. In this study, we performed a comprehensive expression analysis of exosomal miRNAs secreted by several human cancer cell lines and identified eight types of human miRNAs that were highly expressed in exosomes from osteoblastic phenotype-inducing prostate cancer cell lines. One of these miRNAs, hsa-miR-940, significantly promoted the osteogenic differentiation of human mesenchymal stem cells in vitro by targeting ARHGAP1 and FAM134A Interestingly, although MDA-MB-231 breast cancer cells are commonly known as an osteolytic phenotype-inducing cancer cell line, the implantation of miR-940-overexpressing MDA-MB-231 cells induced extensive osteoblastic lesions in the resulting tumors by facilitating the osteogenic differentiation of host mesenchymal cells. Our results suggest that the phenotypes of bone metastases can be induced by miRNAs secreted by cancer cells in the bone microenvironment.

Adipose tissue:Critical contributor to the development of prostate cancer

The prostate is surrounded by periprostatic adipose tissue. Although adipose tissue was thought to play limited physiological roles, it has recently been recognized as an active endocrine organ, secreting growth factors and adipokines. Epidemiologically, obesity is associated with prostate cancer progression. A major mechanism to explain the link between obesity and cancer includes the insulin and insulin-like growth factor (IGF)-1 axis, sex steroids, and adipokines. When prostate cancer cells invade periprostatic adipose tissue, adipose tissue contributes to create the tumor microenvironment, mainly via adipokine secretion. Furthermore, direct crosstalk between adipocytes and cancer cells can exist. We showed that fatty acid-binding protein 4 (FABP4) released from adipocytes was taken up into prostate cancer cells and may act as a carrier of an energy source for the invasion. Bone is an adipocyte-rich organ and is the common metastatic site of prostate cancer. In the microenvironment of bone metastases, tumor cells, osteoblasts, osteoclasts, adipocytes, and other stromal cells are interacting with one another and organizing a complex system. Thus, growing evidence implicates adipose tissue as a critical contributor to the development of prostate cancer. A deeper understanding of the mechanisms leads to more effective therapeutic strategies for prostate cancer. J. Med. Invest. 65:9-17, February, 2018.

Development of a Human Preclinical Model of Osteoclastogenesis from Peripheral Blood Monocytes Co-cultured with Breast Cancer Cell Lines.

The crosstalk between tumor cells and bone cells in the bone microenvironment is crucial to understanding the mechanism of bone metastasis formation. We developed an in vitro fully human preclinical model of a co-culture of breast cancer cells and monocytes undergoing differentiation towards osteoclasts. We optimized a model of osteoclastogenesis starting from a sample of peripheral blood collected from healthy donors. Peripheral blood mononuclear cells (PBMCs) were first separated by density gradient centrifugation, seeded at a high density and induced to differentiate by adding two growth factors (GFs): receptor activator of nuclear factor-κB ligand (RANKL) and macrophage colony-stimulating factor (MCSF). The cells were left in culture for 14 days and then fixed and analyzed by downstream analysis. In osteolytic bone metastases, one of the effects of cancer cell arrival in bone is the induction of osteoclastogenesis. We thus challenged our model with co-cultures of breast cancer cells to study the differentiation power of cancer cells with respect to GFs. A straightforward way of studying cancer cell-osteoclast interaction is to perform indirect co-cultures based on the use of conditioned medium collected from breast cancer cell cultures and mixed with fresh medium. This mixture is then used to induce osteoclast differentiation. We also optimized a method of direct co-culture in which cancer cells and monocytes undergoing differentiation share the medium and exchange secreted factors. This is a significant improvement over the original indirect co-culture method as researchers can observe the reciprocal interactions of the two cell types and perform downstream analyses for both cancer cells and osteoclasts. This method enables us to study the effect of drugs on the metastatic bone microenvironment and to seed cell lines other than those derived from breast cancer. The model can also be used to study other diseases such as osteoporosis or other bone conditions.

SOST knockdown promotes differentiation of osteoblasts MG63 and mesenchymal stem cells C3H10 in an in vitro model of bone metastasis of breast cancer

To investigate whether SOST is involved in breast cancer MDA-MB-231 cells-induced suppression of differentiation of osteoblast MG63 cells and mesenchymal stem C3H10 cells. SOST-specific small interfering RNA (siRNA) was transfected into breast cancer MDA-MB-231 cells, and the interfering efficiency was verified by RT-PCR. The supernatants were collected from MDA-MB-231 cells in routine culture, cells transfected with SOST siRNA via adenovirus, and cells transfected with empty adenoviral vectors and added in MG63 or C3H10 cell cultures. The changes in the expressions of OPG, OCN, OPN and IBSP in MG63 and C3H10 cells were detected using quantitative real-time PCR, and ALP activity was detected with ALP reading and ALP staining with the cells cultured in routine culture medium and cells in osteogenic induction medium as the negative and positive controls. The adenovirus Ad-siSOST effectively knocked down the expression of SOST in MDA-MB-231 cells. MG63 cells and C3H10 cells cultured in osteogenic medium showed significantly upregulated expressions of the osteoblast markers OPG, OPN, OCN and IBSP (P<0.01), while co-culture with the supernatant of MDA-MB-231 cells obviously reduced the expressions of the osteoblast markers (P<0.01); the expression of the markers increased again in MG63 and C3H10 cells after treatment with the supernatant of MDA-MB-231 cells transfected with ad-siSOST (P<0.01). ALP activity in MG63 and C3H10 cells exhibited a similar pattern of variations in response to the treatments (P<0.01). In the in vitro model of bone metastasis of breast cancer, the differentiation of MG63 or C3H10 cells is suppressed, which can be partly reversed by knocking down the expression of SOST in the bone metastasis microenvironment.

Bone morphogenetic proteins, breast cancer, and bone metastases: striking the right balance.

Bone morphogenetic proteins (BMPs) belong to the TGF-β super family, and are essential for the regulation of foetal development, tissue differentiation and homeostasis and a multitude of cellular functions. Naturally, this has led to the exploration of aberrance in this highly regulated system as a key factor in tumourigenesis. Originally identified for their role in osteogenesis and bone turnover, attention has been turned to the potential role of BMPs in tumour metastases to, and progression within, the bone niche. This is particularly pertinent to breast cancer, which commonly metastasises to bone, and in which studies have revealed aberrations of both BMP expression and signalling, which correlate clinically with breast cancer progression. Ultimately a BMP profile could provide new prognostic disease markers. As the evidence suggests a role for BMPs in regulating breast tumour cellular function, in particular interactions with tumour stroma and the bone metastatic microenvironment, there may be novel therapeutic potential in targeting BMP signalling in breast cancer. This review provides an update on the current knowledge of BMP abnormalities and their implication in the development and progression of breast cancer, particularly in the disease-specific bone metastasis.

Abstract LB-145: Study of prostate cancer &amp; endosteal niche adhesive interactions can aid understanding of metastatic behavior

Abstract Introduction: Prostate cancer (PCa) is the second leading cause of cancer related death in men in the US and remains incurable in the metastatic stage. Recent reports have shown that the adhesion molecule N-cadherin, a mesenchymal cadherin associated with epithelial-to-mesenchymal transition (EMT), is expressed in a large percentage of castration resistant prostate cancer tumors, allows the interaction between N-cadherin expressing osteoblasts (OSB) at the endosteal niche and prostate cancer cells via homophilic binding, and can promote osteoclastogenesis and osteolytic disease state. There is also evidence that direct adhesive interactions between tumor cells and the bone marrow stroma are responsible for the development of drug resistance and relapse. This phenomenon termed "cell adhesion-mediated drug resistance" (CAM-DR) is thought to be one of the major mechanisms by which PCa, as well as other tumors that reside at the endosteal niche, escape the cytotoxic effects of therapeutic agents. In this study we evaluated the adhesive interaction of patient-derived primary PCa cells (i.e., conditionally reprogrammed PCa cells (Liu, X et al., Am J Pathol, 2012)) with the bone metastatic microenvironment as a first step to: 1) identify potential differences in patient response to therapy using physical cues (i.e., adhesion to the tumor microenvironment) as “physical biomarkers” of putative response to treatment, and 2) potentially target these interactions therapeutically. Materials and Methods: The human OSB cell line hFOB 1.19 was used to model the endosteal niche. Conditionally reprogrammed patient-derived PCa cells: 1) PP8T; A Gleason Score 8 CRC line from a primary site tumor and, 2) PCA3-CRC cells, which we derived from a metastatic lymph node organoid (a kind gift from Drs Charles Sawyers and Yo Chen (Gao, D, et al., Cell, 2014)), were co-cultured in a microfluidic device, layered with OSB to monitor PCa-OSB interactions using time lapse fluorescence microscopy. In separate experiments, PCa cells were labeled with the cell proliferation dye eFluor® 670 to quantify and compare proliferation of PCa as a function of their adhesive interaction with OSB. Finally, N-cadherin (CDH2) and cadherin-11 (CDH11) protein expressions were also quantified for each of the PCa lines studied as well as the OSB line utilized. Results and Conclusions: Real-time imaging showed that PCA3 cells (i.e., metastatic line) preferentially adhered to OSB whereas PP8T cells (from primary site) presented with a more invasive, yet not adherent, behavior. In addition, PP8T cells proliferated at a significantly faster rate than PCA3 cells when co-cultured with OSB. Concomitantly with these results we also found that PCA3 cells expressed significantly higher levels of N-cadherin and cadherin-11 than PP8T cells, corroborating the presence of EMT in the metastatic line. These data suggest that examination of the PCa-OSB physical interface maybe useful in determining the mechanisms by which PCa cells interact with the bone niche. Our studies represent a potential first step to predicting and targeting these interactions, prior to the development of CAM-DR. Citation Format: Jenny Zilberberg, Leah Dziopa, Eugenia Dziopa, Maia Iyer, Qiaoling Sun, Saba Choudhary, Woo Y. Lee, Lucas Tricoli, Shain Assefnia, Christopher Albanese. Study of prostate cancer &amp; endosteal niche adhesive interactions can aid understanding of metastatic behavior [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr LB-145. doi:10.1158/1538-7445.AM2017-LB-145

Abstract 5779: Building a novel 3D hyaluronan-based tri-culture model of the bone metastatic microenvironment for cancer studies

Abstract The high attrition rate of potential anti-cancer drugs entering clinical trials predicates a need for more predictive pre-clinical model systems. Three-dimensional (3D) culture systems provide cells a more physiological microenvironment than traditional cell culture methods—including relevant extracellular matrix (ECM) cues and cell-cell interactions—while allowing greater experimental control and higher-throughput studies than animal models. Prevalent cancers, including breast and prostate cancer, metastasize preferentially to bone, where they become incurable. The bone marrow microenvironment is a complex network of interacting bone homeostatic and hematopoietic niches that contribute to osteotropic cancer homing, proliferation, and treatment resistance. We created a 3D in vitro tri-culture model of the bone metastatic microenvironment to serve as a new platform to study the “trialogue” between bone metastatic cancer, bone marrow stroma, and bone marrow endothelial cells. This tool can be used to inform drug discovery and to combat treatment resistance. Within the present study, the utility of peptide-functionalized hyaluronan hydrogels for supporting the self-organization of 3D bone marrow microvascular networks was explored. Gel crosslinking density was varied to examine the effect of matrix porosity on cell migration and organization over time. Tubule length and diameter were quantified and the endothelial structures were assessed for indicators of mature vasculature, including lumen formation, basement membrane production, and the recruitment of supporting stromal cells. Breast/prostate cancer cell growth, survival, and physical association with bone marrow stromal and bone marrow microvascular endothelial cell networks was examined in tri-culture models. Ongoing studies seek to identify and disrupt key cell signaling pathways that mediate cell-cell interaction and promote cancer cell proliferation and survival, providing new tools to combat bone metastatic disease. Citation Format: Lindsey K. Sablatura, Daniel A. Harrington, Mary C. Farach-Carson. Building a novel 3D hyaluronan-based tri-culture model of the bone metastatic microenvironment for cancer studies [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 5779. doi:10.1158/1538-7445.AM2017-5779

Decreased sensory nerve excitation and bone pain associated with mouse Lewis lung cancer in TRPV1-deficient mice.

Bone pain is one of the most common and life-limiting complications of cancer metastasis to bone. Although the mechanism of bone pain still remains poorly understood, bone pain is evoked as a consequence of sensitization and excitation of sensory nerves (SNs) innervating bone by noxious stimuli produced in the microenvironment of bone metastases. We showed that bone is innervated by calcitonin gene-related protein (CGRP)+ SNs extending from dorsal root ganglia (DRG), the cell body of SNs, in mice. Mice intratibially injected with Lewis lung cancer (LLC) cells showed progressive bone pain evaluated by mechanical allodynia and flinching with increased CGRP+ SNs in bone and augmented SN excitation in DRG as indicated by elevated numbers of pERK- and pCREB-immunoreactive neurons. Immunohistochemical examination of LLC-injected bone revealed that the tumor microenvironment is acidic. Bafilomycin A1, a selective inhibitor of H+ secretion from vacuolar proton pump, significantly alleviated bone pain, indicating that the acidic microenvironment contributes to bone pain. We then determined whether the transient receptor potential vanilloid 1 (TRPV1), a major acid-sensing nociceptor predominantly expressed on SNs, plays a role in bone pain by intratibially injecting LLC cells in TRPV1-deficient mice. Bone pain and SN excitation in the DRG and spinal dorsal horn were significantly decreased in TRPV1 -/- mice compared with wild-type mice. Our results suggest that TRPV1 activation on SNs innervating bone by the acidic cancer microenvironment in bone contributes to SN activation and bone pain. Targeting acid-activated TRPV1 is a potential therapeutic approach to cancer-induced bone pain.

Simvastatin and MBCD Inhibit Breast Cancer-Induced Osteoclast Activity by Targeting Osteoclastogenic Factors

ABSTRACTPrevious reports have documented that cholesterol-lowering simvastatin prevented osteolytic metastasis of breast cancer in animal model in which cancer cells were placed into blood circulation. Thus, simvastatin treatment might have a preventive effect in inhibiting osteoclast activity of metastatic bone microenvironment. This study documented that both simvastatin and MBCD (cholesterol depleting drug) blocked the breast cancer-induced TRAP and MMP activity, and expressions of various osteoclastogenic genes (TRAP, Cathepsin K, and NFATc1) in pre-osteoclast RAW264.7 cells, and osteoclastogenic CSF-1 and RANKL expressions in breast cancer MCF-7 cells. Thus, these findings unravel a molecular mechanism of simvastatin-/MBCD-mediated inhibition of breast cancer-driven osteoclast activity.

Bone-Seeking Matrix Metalloproteinase-2 Inhibitors Prevent Bone Metastatic Breast Cancer Growth.

Bone metastasis is common during breast cancer progression. Matrix metalloproteinase-2 (MMP-2) is significantly associated with aggressive breast cancer and poorer overall survival. In bone, tumor- or host-derived MMP-2 contributes to breast cancer growth and does so by processing substrates, including type I collagen and TGFβ latency proteins. These data provide strong rationale for the application of MMP-2 inhibitors to treat the disease. However, in vivo, MMP-2 is systemically expressed. Therefore, to overcome potential toxicities noted with previous broad-spectrum MMP inhibitors (MMPIs), we used highly selective bisphosphonic-based MMP-2 inhibitors (BMMPIs) that allowed for specific bone targeting. In vitro, BMMPIs affected the viability of breast cancer cell lines and osteoclast precursors, but not osteoblasts. In vivo, we demonstrated using two bone metastatic models (PyMT-R221A and 4T1) that BMMPI treatment significantly reduced tumor growth and tumor-associated bone destruction. In addition, BMMPIs are superior in promoting tumor apoptosis compared with the standard-of-care bisphosphonate, zoledronate. We demonstrated MMP-2-selective inhibition in the bone microenvironment using specific and broad-spectrum MMP probes. Furthermore, compared with zoledronate, BMMPI-treated mice had significantly lower levels of TGFβ signaling and MMP-generated type I collagen carboxy-terminal fragments. Taken together, our data show the feasibility of selective inhibition of MMPs in the bone metastatic breast cancer microenvironment. We posit that BMMPIs could be easily translated to the clinical setting for the treatment of bone metastases given the well-tolerated nature of bisphosphonates. Mol Cancer Ther; 16(3); 494-505. ©2017 AACR.