Tumor-bone Microenvironment Research Articles

Osteoblasts are induced into cancer-associated osteoblasts to promote tumor progression in head and neck squamous cell carcinoma

Bone invasion by head and neck squamous cell carcinoma (HNSCC) significantly impacts tumor staging, treatment choice, prognosis, and quality of life. While HNSCC is known to cause osteolytic bone invasion, we found that specific HNSCC subtypes can induce osteogenic bone destruction at the tumor-bone interface. This destruction mode significantly correlated with reduced patient survival rates and increased neck lymph node metastasis. Further in vivo and in vitro experiments indicated that HNSCC cells triggered abnormal phenotypic changes in osteoblasts to remodel the tumor-bone microenvironment, facilitating tumor lymphatic metastasis. Through transcriptome analysis, we identified three genes—osteopontin (SPP1), chemokine (C-X-C motif) ligand 1 (CXCL1), and matrix metalloprotein (MMP)9 (MMP9) linked to a poorer prognosis. We discovered osteoblasts with abnormal phenotypes at the tumor-bone interface exhibiting high SPP1, MMP9, and CXCL1 expressions. Based on these characteristics, we identified this osteoblast subpopulation as “cancer-associated osteoblasts (CAOs).” HNSCC cells activated the TNF-α/NF-κB signaling pathway in osteoblasts, transforming them into “CAOs.” These CAOs significantly contributed to the progression of tumor-induced bone invasion, facilitating cancer growth and metastasis. We first provided clinical data and in vivo and in vitro evidence that HNSCC cells can promote tumor progression by manipulating osteoblasts into “CAOs” in the bone invasion.

Amino Acid Metabolism in Bone Metastatic Disease.

Breast and prostate tumors frequently metastasize to the bone, but the underlying mechanisms for osteotropism remain elusive. An emerging feature of metastatic progression is metabolic adaptation of cancer cells to new environments. This review will summarize the recent advances on how cancer cells utilize amino acid metabolism during metastasis, from early dissemination to interactions with the bone microenvironment. Recent studies have suggested that certain metabolic preferences for amino acids may be associated with bone metastasis. Once in the bone microenvironment, cancer cells encounter a favorable microenvironment, where a changing nutrient composition of the tumor-bone microenvironment may alter metabolic interactions with bone-resident cells to further drive metastatic outgrowth. Enhanced amino acid metabolic programs are associated with bone metastatic disease and may be further augmented by the bone microenvironment. Additional studies are necessary to fully elucidate the role of amino acid metabolism on bone metastasis.

Nanoparticle-mediated synergistic drug combination for treating bone metastasis

Bone metastasis at an advanced disease stage is common in most solid tumors and is untreatable. Overexpression of receptor activator of nuclear factor κB ligand (RANKL) in tumor-bone marrow microenvironment drives a vicious cycle of tumor progression and bone resorption. Biodegradable nanoparticles (NPs), designed to localize in the tumor tissue in bone marrow, were evaluated in a prostate cancer model of bone metastasis. The combination treatment, encapsulating docetaxel, an anticancer drug (TXT-NPs), and Denosumab, a monoclonal antibody that binds to RANKL (DNmb-NPs), administered intravenously regressed the tumor completely, preventing bone resorption, without causing any mortality. With TXT-NPs alone treatment, after an initial regression, the tumor relapsed and acquired resistance, whereas DNmb-NPs alone treatment was ineffective. Only in the combination treatment, RANKL was not detected in the tumor tibia, thus negating its role in tumor progression and bone resorption. The combination treatment was determined to be safe as the vital organ tissue showed no increase in inflammatory cytokine or the liver ALT/AST levels, and animals gained weight. Overall, dual drug treatment acted synergistically to modulate the tumor-bone microenvironment with encapsulation enhancing their therapeutic potency to achieve tumor regression.

Abstract 3647: Anti-tumor actions of neutrophils are mediated via STAT5 inhibition

Abstract The overall patient survival rate drops dramatically when localized prostate cancer (PCa) progresses to bone metastatic prostate cancer (BM-PCa). The treatment for BM-PCa is limited to palliative therapy, and it is currently incurable. Neutrophils are the most prevalent immune cell in the bone, and we have found that PCa enhances their recruitment and infiltration into the prostate tumor-bone microenvironment of patients with BM-PCa. Additionally, we observed that bone marrow neutrophils directly induce apoptosis of PCa cells both in vitro and in vivo and that this is mediated by the inhibition of STAT5, a transcription factor that promotes PCa progression. Moreover, STAT5 has also been found to contribute to the development of resistance to standard care androgen deprivation therapy. Neutrophils specifically target and kill only cells that express STAT5, as stable knockdown of STAT5 in BM-PCa cells makes them resistant to neutrophil-mediated cytotoxicity, while the overexpression of STAT5 in STAT5-negative PCa cells, which are generally resistant to killing by neutrophils, sensitizes them to neutrophil-mediated killing. Furthermore, transcriptomic analyses derived from the STAT5 knockdown and STAT5 overexpressing cells suggest that STAT5 expression in PCa induces pro-inflammatory signaling and that neutrophil-mediated cytotoxicity may be exerted via STAT5-induced IL-1 and/or IL-6. Preliminary data in our laboratory show that STAT5-induced production of IL-1 by PCa cells leads to their death by neutrophils, as knocking out the IL-1 receptor on neutrophils reduces their cytotoxic potential against STAT5-positive PCa cells. Interestingly, in addition to killing BM-PCa cells, we have demonstrated that bone marrow-derived neutrophils are also capable of killing pancreatic cancer cells. Further delineation of the role of neutrophils in STAT5 signaling in BM-PCa growth holds promise for enhancing neutrophil cytotoxicity in the bone which could lead to novel therapeutic options for bone metastatic prostate cancer. Citation Format: Sanjana Rajgopal, Massar Alsamraae, Diane Costanzo-Garvey, Leah Cook. Anti-tumor actions of neutrophils are mediated via STAT5 inhibition. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 3647.

Osteoid cell-derived chemokines drive bone-metastatic prostate cancer.

One of the greatest challenges in improving prostate cancer (PCa) survival is in designing new therapies to effectively target bone metastases. PCa regulation of the bone environment has been well characterized; however, bone-targeted therapies have little impact on patient survival, demonstrating a need for understanding the complexities of the tumor-bone environment. Many factors contribute to creating a favorable microenvironment for prostate tumors in bone, including cell signaling proteins produced by osteoid cells. Specifically, there has been extensive evidence from both past and recent studies that emphasize the importance of chemokine signaling in promoting PCa progression in the bone environment. Chemokine-focused strategies present promising therapeutic options for treating bone metastasis. These signaling pathways are complex, with many being produced by (and exerting effects on) a plethora of different cell types, including stromal and tumor cells of the prostate tumor-bone microenvironment. ​This review highlights an underappreciated molecular family that should be interrogated for treatment of bone metastatic prostate cancer (BM-PCa).

The role played by ailanthone in inhibiting bone metastasis of breast cancer by regulating tumor-bone microenvironment through the RANKL-dependent pathway.

Introduction: Bone metastasis of breast cancer (BC) is a process in which the disruption of the bone homeostatic microenvironment leads to an increase in osteoclast differentiation. Ailanthus altissima shows an inhibitory effect on osteoclast differentiation. Ailanthone (AIL) refers to a natural compound isolated from Ailanthus altissima, a Chinese herbal medicine, and has effective anti-tumor activity in numerous cell lines. Its impact on bone metastases for BC is yet unclear. Methods: We measured the effect of AIL on MDA-MB-231 cells by wound healing experiments, Transwell and colony formation experiment. Using the Tartrate-resistant Acid Phosphatase (TRAP) staining tests, filamentous (F-actin) staining and bone resorption test to detect the effect of AIL on the osteoclast cell differentiation of the Bone Marrow-derived Macrophages (BMMs), activated by the MDA-MB-231 cell Conditioned Medium (MDA-MB-231 CM) and the Receptor Activator of Nuclear factor-κB Ligand (RANKL),and to explore its possibility Mechanisms. In vivo experiments verified the effect of AIL on bone destruction in breast cancer bone metastasis model mice. Results: In vitro, AIL significantly decrease the proliferation, migration and infiltration abilities of MDA-MB-231 cells at a safe concentration, and also reduced the expression of genes and proteins involved in osteoclast formation in MDA-MB-231 cells. Osteoclast cell differentiation of the BMMs, activated by MDA-MB-231 CM and RANKL, were suppressed by AIL in the concentration-dependent manner. Additionally, it inhibits osteoclast-specific gene and protein expression. It was noted that AIL inhibited the expression of the osteoclast differentiation-related cytokines RANKL and interleukin-1β (IL-1β) that were secreted by the MDA-MB-231 cells after upregulating the Forkhead box protein 3 (FOXP3) expression. Furthermore, AIL also inhibits the expression of the Mitogen-Activated Protein Kinase (MAPK), Phosphoinositide 3-kinase (PI3K)/protein kinase B (AKT), and Nuclear factor-κB Ligand (NF-κB) signaling pathways, which then suppresses the MDA-MB-231CM-induced development of Osteoclasts. Conclusion: Our study shows that AIL blocks osteoclast differentiation in the bone metastasis microenvironment by inhibiting cytokines secreted by BC cells, which may be a potential agent for the treatment of BC and its secondary bone metastasis.

Biological Effects of Cyclin-Dependent Kinase Inhibitors Ribociclib, Palbociclib and Abemaciclib on Breast Cancer Bone Microenvironment.

The CDK4/6 inhibitors (CDKi) palbociclib, ribociclib, and abemaciclib are currently approved in combination with anti-estrogen therapy for the treatment of advanced and/or metastatic hormone receptor-positive/HER2-neu-negative breast cancer patients. Given the high incidence of bone metastases in this population, we investigated and compared the potential effects of palbociclib, ribociclib, and abemaciclib on the breast cancer bone microenvironment. Primary osteoclasts (OCs) and osteoblasts (OBs) were obtained from human monocyte and mesenchymal stem cells, respectively. OC function was evaluated by tartrate-resistant acid phosphatase assay and real-time PCR; OB activity was assessed by an alizarin red assay. OB/breast cancer co-culture models were generated via the seeding of MCF-7 cells on a layer of OBs, and tumor cell proliferation was analyzed using flow cytometry. Here, we showed that ribociclib, palbociclib, and abemaciclib exerted similar inhibitory effects on the OC differentiation and expression of bone resorption markers without affecting OC viability. On the other hand, the three CDKi did not affect the ability of OB to produce bone matrix, even if the higher doses of palbociclib and abemaciclib reduced the OB viability. In OB/MCF-7 co-culture models, palbociclib demonstrated a lower anti-tumor effect than ribociclib and abemaciclib. Overall, our results revealed the direct effects of CDKi on the tumor bone microenvironment, highlighting differences potentially relevant for clinical practice.

Construction and evaluation of detachable bone-targeting MOF carriers for the delivery of proteasome inhibitors.

Tumor bone metastasis is an important cause of tumor recurrence and death. Although bone-targeting nanoparticles decorated with targeting ligands have shown good affinity for bone tissues with the properties of adhesion to the bone matrix, it is not easy to detach from the surface of the bone matrix in the tumor-bone microenvironment, attributed to the robust coordination force between the targeting ligands, such as bisphosphates with bone-deposited calcium. This may hinder the transport of nanoparticles from bone tissue to bone metastatic tumors. In this research, we designed a bone-targeting nanocarrier with detachable bone-targeting character for the therapy of bone metastases. The nanoparticles were constructed by using ZIF-8 and bone-targeting and MMP enzyme sensitive polypeptide-modified hyaluronic acid as a carrier and proteasome inhibitor Bortezomib (BTZ) as cargo. The results show that the constructed D8-M3-HA-ZIF8@BTZ nanoparticles possessed several favorable properties such as good colloidal stability, acid-sensitive drug release, D8 peptide mediated bone targeting and MMP enzyme-responsive desorption. Besides, nanoparticle endocytosis and cytotoxicity were enhanced through HA-mediated targeting to CD44 over-expressing tumor cells. Altogether, this study provides a potential cascade targeting strategy for improving the delivery effects of bone targeted nanoparticles for the delivery of proteasome inhibitors.

Emerging roles for myeloid immune cells in bone metastasis.

Metastasis, especially bone metastasis, is a major cause of cancer-related deaths, which is associated with long-term pain due to skeletal-related events and poor quality of life. Tumor cells alter the bone microenvironment through aberrant activation of osteoclasts and osteoblasts which induces bone osteolysis and release of growth factors leading to cancer growth. Though this phenomenon has been well characterized, bone-targeted therapies have shown little improvement in patient survival. Recent evidence indicates a growing appreciation for the complex bone environment, in addition to bone-remodeling stromal cells, which includes an abundance of myeloid immune cells that can either protect against or contribute to the progression of the disease within the bone cavity. Additionally, myeloid cells are recruited into primary tumor sites, where they promote development of the pre-metastatic niche and also can regulate tumor progression within the tumor-bone microenvironment through a milieu of complex mechanisms and involving heterogeneous myeloid populations. In this review, we have highlighted the complex roles of myeloid immunity in bone metastasis and hope to bring attention to the potential of novel immunotherapeutic interventions for the elimination of bone metastasis.

Modeling the Human Bone-Tumor Niche: Reducing and Replacing the Need for Animal Data.

ABSTRACTBone is the most common site for cancer metastasis. Understanding the interactions within the complex, heterogeneous bone–tumor microenvironment is essential for the development of new therapeutics. Various animal models of tumor‐induced bone disease are routinely used to provide valuable information on the relationship between cancer cells and the skeleton. However, new model systems exist that offer an alternative approach to the use of animals and might more accurately reveal the cellular interactions occurring within the human bone–tumor niche. This review highlights replacement models that mimic the bone microenvironment and where cancer metastases and tumor growth might be assessed alongside bone turnover. Such culture models include the use of calcified regions of animal tissue and scaffolds made from bone mineral hydroxyapatite, synthetic polymers that can be manipulated during manufacture to create structures resembling trabecular bone surfaces, gel composites that can be modified for stiffness and porosity to resemble conditions in the tumor–bone microenvironment. Possibly the most accurate model system involves the use of fresh human bone samples, which can be cultured ex vivo in the presence of human tumor cells and demonstrate similar cancer cell–bone cell interactions as described in vivo. In addition, the use of mathematical modeling and computational biology approaches provide an alternative to preliminary animal testing. The use of such models offers the capacity to mimic significant elements of the human bone–tumor environment, and complement, refine, or replace the use of preclinical models. © 2020 The Authors. JBMR Plus published by Wiley Periodicals, Inc. on behalf of American Society for Bone and Mineral Research.

Measuring Bone Remodeling and Recreating the Tumor-Bone Microenvironment Using Calvaria Co-culture and Histomorphometry.

Bone is a connective tissue constituted of osteoblasts, osteocytes, and osteoclasts and a mineralized extracellular matrix, which gives it its strength and flexibility and allows it to fulfill its functions. Bone is continuously exposed to a variety of stimuli, which in pathological conditions can deregulate bone remodeling. To study bone biology and diseases and evaluate potential therapeutic agents, it has been necessary to develop in vitro and in vivo models. This manuscript describes the dissection process and culture conditions of calvarias isolated from neonatal mice to study bone formation and the bone tumor microenvironment. In contrast to in vitro and in vivo models, this ex vivo model allows preservation of the three-dimensional environment of the tissue as well as the cellular diversity of the bone while culturing under defined conditions to simulate the desired microenvironment. Therefore, it is possible to investigate bone remodeling and its mechanisms, as well as the interactions with other cell types, such as the interactions between cancer cells and bone. The assays reported here use calvarias from 5-7 day old BALB/C mice. The hemi-calvarias obtained are cultured in the presence of insulin, breast cancer cells (MDA-MB-231), or conditioned medium from breast cancer cell cultures. After analysis, it was established that insulin induced new bone formation, while cancer cells and their conditioned medium induced bone resorption. The calvarial model has been successfully used in basic and applied research to study bone development and cancer-induced bone diseases. Overall, it is an excellent option for an easy, informative, and low-cost assay.

Measuring Bone Remodeling and Recreating the Tumor-Bone Microenvironment Using Calvaria Co-culture and Histomorphometry

Measuring Bone Remodeling and Recreating the Tumor-Bone Microenvironment Using Calvaria Co-culture and Histomorphometry

GP130 Cytokines in Breast Cancer and Bone

Breast cancer cells have a high predilection for skeletal homing, where they may either induce osteolytic bone destruction or enter a latency period in which they remain quiescent. Breast cancer cells produce and encounter autocrine and paracrine cytokine signals in the bone microenvironment, which can influence their behavior in multiple ways. For example, these signals can promote the survival and dormancy of bone-disseminated cancer cells or stimulate proliferation. The interleukin-6 (IL-6) cytokine family, defined by its use of the glycoprotein 130 (gp130) co-receptor, includes interleukin-11 (IL-11), leukemia inhibitory factor (LIF), oncostatin M (OSM), ciliary neurotrophic factor (CNTF), and cardiotrophin-1 (CT-1), among others. These cytokines are known to have overlapping pleiotropic functions in different cell types and are important for cross-talk between bone-resident cells. IL-6 cytokines have also been implicated in the progression and metastasis of breast, prostate, lung, and cervical cancer, highlighting the importance of these cytokines in the tumor–bone microenvironment. This review will describe the role of these cytokines in skeletal remodeling and cancer progression both within and outside of the bone microenvironment.

Bone metastases in the era of targeted treatments: insights from molecular biology.

Bone metastases remain a common feature of advanced cancers and are associated with significant morbidity and mortality. Recent research has identified promising novel treatment targets to improve current treatment strategies for bone metastatic disease. This review summarizes the well-known and recently discovered molecular biology pathways in bone that govern normal physiological remodeling or drive the pathophysiological changes observed when bone metastases are present. In the rapidly changing world of targeted cancer treatments, it is important to recognize the specific treatment effects induced in bone by these agents and the potential impact on common imaging strategies. The osteoclastic targets (bisphosphonates, LGR4, RANKL, mTOR, MET-VEGFR, cathepsin K, Src, Dock 5) and the osteoblastic targets (Wnt and endothelin) are discussed, and the emerging field of osteo-immunity is introduced as potential future therapeutic target. Finally, a summary is provided of available trial data for agents that target these pathways and that have been assessed in patients. The ultimate goal of research into novel pathways and targets involved in the tumor-bone microenvironment is to tackle one of the great remaining unmet needs in oncology, that is finding a cure for bone metastatic disease.

Abstract LB-099: Prostate cancer-secreted CCN3/NOV promotes osteoblast differentiation via Wnt signal pathway and miR-608 suppression

Abstract Objective: NOV/CCN3 is a secreted protein that interacts with the extracellular matrix and thereby regulates many cellular functions, including cell division, chemotaxis, apoptosis, adhesion, motility, and ion transport. Recently, CCN3 expression have been implicated in progression of various cancer, and our previous study indicates that CCN3 increases the migration and invasion of prostate cancer (PCa) cells by regulation of ICAM-1 expression. Our research group also review the pivotal role CCN3 in bone metastasis of PCa by modulating the tumor-bone microenvironment. Moreover, evidence has highlighted the pivotal role of osteoblasts in osteoblastic bone metastasis in PCa. Material and Methods: MC3T3-E1 osteoblast cells were incubated with conditioned media collected from PCa cells. The cell proliferation and osteogenic differentiation were evaluated by MTT assay and Alizarin red S staining. The osteogenic markers were also examined by Western blot and QPCR. Furthermore, the Wnt activation was tested by Western blot and luciferase reporter assay. Pretreatment with chemical inhibitor of Wnt was used to determine the regulatory role of Wnt in osteogenic differentiation in response to PCa CM. Finally, expression of miR-608 was investigated by QPCR and mimic miR-608 was used to certify the role of miR-608 in osteogenic differentiation in response to PCa CM. Results: Here, we show that PCa-secreted CCN3 increases expression of osteoblastic differentiation factors including BMPs, Runx2 and Osterix. Furthermore, this effect is regulated by Wnt signal pathway or miR-608 repression. The present study supposes that PCa-secreted CCN3 can promote osteoblastic bone metastasis in PCa by activating Wnt signal and inhibition of miR-608 expression. Our present study provides an opportunity to develop CCN3 as a therapeutic target that prevents bone metastasis in PCa. Citation Format: Po-Chun Chen, Chia-Chia Chao, An-Chen Chang, Chih-Hsin Tang. Prostate cancer-secreted CCN3/NOV promotes osteoblast differentiation via Wnt signal pathway and miR-608 suppression [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 LB-099.

Abstract 934: γδ CAR T-cell therapy significantly mitigates bone metastatic castrate-resistant prostate cancer

Abstract Bone metastasis is a frequent complication in advanced prostate cancer, with the resultant lesions significantly contributing to patient morbidity and mortality. While next generation hormone ablation therapies and bone protecting bisphosphonates alleviate these symptoms, the disease remains incurable, and new therapeutic approaches are of urgent need. In this regard, we have focused on tapping the tumor-seeking potential of chimeric antigen receptor (CAR) T cells. CAR T cells have shown remarkable anti-tumor responses in hematologic malignancies, but unique physical and biological challenges have hindered their activity in solid tumors. Interestingly, patients treated with bisphosphonates such as zoledronate exhibit enhanced recruitment and activation of the γδ subset of T cells in bone due intracellular accumulation of isopentenyl pyrophosphate (IPP) phosphoantigen. Whereas conventional CAR T cell therapies utilize αβ T cells, we posit that designing γδ CAR T cells could improve homing to bone metastases when administered with bisphosphonates. First, to test the impact of prostate specific stem cell antigen (PSCA)-specific γδ CAR T cells against bone metastatic prostate cancer in vivo, NSG mice (n=10) were intratibially injected with PSCA/luciferase-expressing C4-2B (2x105) castrate resistant prostate cancer cells. Tumors were allowed to establish for 10 days and then randomized into control or γδ CAR T (1.5x107 via tail vein) groups. Subsequent bioluminescent imaging indicated a rapid and significant (p=0.0006) regression of tumors in the γδ CAR T cell group, leading to increased overall survival (5/5 γδ CAR T vs. 0/5 control after 68 days, p=0.0002). Ex vivo bone morphometry analysis also demonstrated the significant protective effect of γδ CAR T associated bone disease. To determine whether bisphosphonates could further enhance the homing of γδ CAR T to bone, NSG mice (n=30) were intratibially injected with C4-2B (2x105), and randomized into control and zoledronate (30µg/kg) groups. After 10 days, mice received γδ T cells (3x106). Subsets were sacrificed at 1, 3, and 5 days post-T cell administration, and peripheral blood, tibia bone marrow, and spleens isolated. CD3-Vδ2 flow cytometry indicated increased γδ T cells in the tibia bone marrow from zoledronate groups (Day 1=61%, Day 3=32%, and Day 5=57%). Furthermore, decreased tumor growth rates were observed in the zoledronate group, suggesting that increased homing of γδ T cells induced anti-tumor effects. Our data to date demonstrate that γδ CAR T cells significantly mitigate bone metastatic prostate cancer and associated bone disease. Further, bisphosphonates (already used in the clinical setting) enhance the homing of γδ CAR T cells to the tumor-bone microenvironment. We posit that γδ CAR T will be an effective immunotherapy approach for the treatment of men with incurable bone metastatic prostate cancer. Citation Format: Jeremy Steven Frieling, Maria Cecilia Ramello, Ismahene Benzaid, Emiliano Roselli, Chen Hao Lo, Conor C. Lynch, Daniel Abate-Daga. γδ CAR T-cell therapy significantly mitigates bone metastatic castrate-resistant prostate cancer [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 934.

Abstract 1087: Bone-resident neutrophils are mediators of prostate cancer growth in bone

Abstract Bone metastatic prostate cancer (BM-PCa) significantly reduces overall patient survival and is currently incurable. Current standard immune therapies have shown promising results for patients with less advanced disease (fewer than 20 bone lesions) suggesting that PCa growth in bone contributes to immunotherapeutic response. Gaining a better understanding of the immunogenicity of BM-PCa may present a new therapeutic approach for treating mCRPC. In bone, prostate cancer cells hijack the behavior of bone resident cells, inducing excessive bone degradation and release of bone-sequestered growth factors, such as transforming growth factor beta (TGFβ), that promote a “vicious cycle” of tumor progression. Findings from my lab revealed that: 1) BM-PCa stimulates bone stromal expression of Interleukin 8 (IL-8), a potent chemoattractant for polymorphonuclear neutrophils (PMNs), innate immune cells generated in bone marrow, and 2) PMNs heavily infiltrate the prostate tumor-bone microenvironment. Recent studies demonstrated the existence of two distinct PMN populations: anti-tumoral (N1) and immunosuppressive pro-tumoral (N2) PMNs, with emergence of the latter regulated by TGFβ. RealTime qPCR revealed that bone metastatic C42B prostate cancer significantly increases neutrophil expression of TGFβ receptor and MMP9, a trend previously identified as a marker of pro-tumoral neutrophils. Based upon these findings, we hypothesized that bone metastatic prostate cancer skews neutrophils to a pro-tumoral phenotype in a TGFβ-dependent manner. To test this, bone marrow-derived primary neutrophils were treated with conditioned media from metastatic metastatic prostate cancer cells (C42B) and poorly metastatic LNCaP and gene expression and oxidative burst were measured. Similar to previous findings, C42B stimulated reactive oxygen species (ROS) production by PMNs, a mechanism of immunosuppression utilized by N2 PMNs, and induced anti-inflammatory gene expression. In contrast, LNCaP cells failed to stimulate this response. C42B media promoted PMN expansion, a phenomenon that is reversed by TGFβ-blockade. Surprisingly, direct contact with neutrophils significantly increased luminescence of luciferase-expressing C42B cells independently of cell growth, when compared to LNCaP, demonstrating that neutrophils activate alternate protein signaling in metastatic cells compared to non-metastatic. In vivo intratibial models utilizing antibody-mediated neutrophil depletion validated our hypothesis that neutrophils are critical mediators of prostate cancer growth in bone. These results demonstrate the significant role of neutrophils in the progression of bone metastatic prostate cancer. Citation Format: Leah M. Cook, Diane Costanzo-Garvey, Tyler Keeley. Bone-resident neutrophils are mediators of prostate cancer growth in bone [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 1087.

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.

Multifaceted Roles for Macrophages in Prostate Cancer Skeletal Metastasis.

Bone-metastatic prostate cancer is common in men with recurrent castrate-resistant disease. To date, therapeutic focus has largely revolved around androgen deprivation therapy (ADT) and chemotherapy. While second-generation ADTs and combination ADT/chemotherapy approaches have been successful in extending overall survival, the disease remains incurable. It is clear that molecular and cellular components of the cancer-bone microenvironment contribute to the disease progression and potentially to the emergence of therapy resistance. In bone, metastatic prostate cancer cells manipulate bone-forming osteoblasts and bone-resorbing osteoclasts to produce growth and survival factors. While osteoclast-targeted therapies such as bisphosphonates have improved quality of life, emerging data have defined important roles for additional cells of the bone microenvironment, including macrophages and T cells. Disappointingly, early clinical trials with checkpoint blockade inhibitors geared at promoting cytotoxic T cell response have not proved as promising for prostate cancer compared to other solid malignancies. Macrophages, including bone-resident osteomacs, are a major component of the bone marrow and play key roles in coordinating normal bone remodeling and injury repair. The role for anti-inflammatory macrophages in the progression of primary prostate cancer is well established yet relatively little is known about macrophages in the context of bone-metastatic prostate cancer. The focus of the current review is to summarize our knowledge of macrophage contribution to normal bone remodeling and prostate-to-bone metastasis, while also considering the impact of standard of care and targeted therapies on macrophage behavior in the tumor-bone microenvironment.

Myeloma and Bone Disease.

Bone disease is a defining characteristic of multiple myeloma (MM) and the major cause of morbidity. It manifests as lytic lesions or osteopenia and is often associated with severe pain, pathological fracture, spinal cord compression, vertebral collapse, and hypercalcemia. Here, we have reviewed recent data on understanding its biology and treatment. The imbalance between bone regeneration and bone resorption underlies the pathogenesis of osteolytic bone disease. Increased osteoclast proliferation and activity accompanied by inhibition of bone-forming osteoblasts leads to progressive bone loss and lytic lesions. Although tremendous progress has been made, MM remains an incurable disease. Novel agents targeting bone disease are under investigation with the goal of not only preventing bone loss and improving bone quality but also harnessing MM tumor growth. Current data illustrate that the interactions between MM cells and the tumor-bone microenvironment contribute to the bone disease and continued MM progression. A better understanding of this microenvironment is critical for novel therapeutic treatments of both MM and associated bone disease.