Bone Metastatic Microenvironment Research Articles

Abstract 5285: DKK1 blockade inhibits progression of bone metastases via multiple mechanisms and synergize with zoledronate treatment

Abstract Background: Despite the important breakthroughs of immune checkpoint blockade (ICB) therapy in multiple primary cancers, responses of ICB therapy in bone metastases remain extremely poor, largely due to the highly immunosuppressive bone metastasis microenvironment. However, the current understanding of bone metastasis microenvironment is still incomplete, and specific treatment targets for patients with bone metastasis are still lacking. Methods: Transcriptome sequencing data of gastric and breast cancer bone metastasis samples from The TCGA and GEO databases were obtained. The correlation between DKK1 expression and prognosis, intertumoral immune infiltration of bone-metastatic patients was analyzed. Mouse bone metastasis models from gastric, breast and lung cancer were established, and in vivo treatment efficacy of DKK1 and/or zoledronic acid was evaluated by micro-computed tomography (CT), micro-magnetic resonance imaging (MRI) and immunohistochemistry (IHC) staining. The changes of immune components wthin bone-tumor microenvironment were explored via flow cytometry, CyTOF and scRNA-seq analysis. The impact of DKK1 on different immune cell types was analyzed via in vitro co-culture models. Results: Serum detection of gastric cancer patients from our center showed that compared with non-metastatic patients, DKK1 concentration in patients with bone metastasis was the significantly increased (p<0.0001); and compared with other metastatic sites of breast cancer patients, bone metastases had the highest level of DKK1 mRNA expression (p<0.05). Using multiple bone metastasis mouse models, we found that DKK1 blockade significantly controlled the tumor burden of bone metastases. Results of micro-CT and TRAP staining showed that the bone destruction and osteoclast activity within bone lesions were significantly inhibited after DKK1 blockade. In addition, results of flow cytometry and immunofluorescence staining on bone metastasis samples showed that both innate and adaptive anti-immune responses were obviously improved after DKK1 blockade, including increased infiltration of CD8+ T cells, M1 macrophages and decreased infiltration of M2 macrophages. Mechanistically, DKK1 directly induced inhibition of CD8+ T cell activation, M2 polarization of macrophages and maturation of osteoclasts, which all contributes to the immune suppression in the bone metastasis microenvironment. Finally, combined therapy of DKK1 blockade and zoledronic acid had synergistic treatment effects in bone metastases. Conclusion: Our study provides novel insights into the multiple role of DKK1 in the bone metastasis immune microenvironment. DKK1 is a potential immunotherapeutic target for bone metastases, and dual blockade of DKK1 and zoledronic acid is a promising combination immunotherapy strategy for patients with bone metastasis. Citation Format: Tao Shi, Kaijie Liang, Yipeng Zhang, Xiaoyu Zhou, Baorui Liu, Jia Wei. DKK1 blockade inhibits progression of bone metastases via multiple mechanisms and synergize with zoledronate treatment [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 5285.

Abstract 2815: Immunotherapy development landscape for bone metastasis - need of predictive preclinical efficacy evaluation for de-risking clinical development

Abstract Most cancer deaths are due to metastases, and bone metastases are challenge especially in breast and prostate cancer, being developed in 70-90% of advanced-stage patients. Bone metastases remain incurable causing high mortality, skeletal-related effects and decreased quality of life. Currently used immunotherapies are ineffective in bone metastases. This creates a need to improve understanding immune microenvironment in bone metastases, namely interactions between tumor, immune and bone cells according to the osteoimmuno-oncology (OIO) concept, and ultimately to develop immunotherapies that primarily target bone metastases. We used 1stOncology database, a cancer drug development resource to identify novel immunotherapies in development for breast and prostate cancer bone metastasis. Twenty-four immunotherapies that included evaluation of effects on bone metastasis were identified. Efficacy of only three of the identified therapies had been evaluated in preclinical bone metastasis models, one reason being that such models have not been commonly available. The use of clinically relevant and predictive preclinical bone metastasis models would significantly de-risk decision making when moving to clinical development in bone metastasizing cancers. To support predictive preclinical evaluation of immunotherapies for bone metastatic cancers, we describe a preclinical bone metastasis technology platform for evaluating efficacy of immunotherapies. The platform utilizes tumors growing in bone microenvironment, mimicking growth of bone metastases in patients, and generates tumor-induced bone effects in a cancer-type specific manner. Syngeneic and/or humanized mouse models with tumor and immune cells of the same species are needed for development of immunotherapies, allowing interactions of tumor and immune cells in the bone metastatic microenvironment to support mode-of-action studies. The platform is clinically predictive, as demonstrated with observed effects that align with clinical findings. As an example, anti-PD-1 antibody had no effects on breast and prostate cancer bone metastases, predicting recent clinical findings that demonstrate lack of efficacy in clinical prostate cancer trials. We conclude that tumor microenvironment in bone metastases has unique characteristics and is understudied as a potential primary cause of lack of efficacy of many immunotherapies, especially in breast and prostate cancer. It is prime time to focus on bone metastases by increasing understanding of the immune landscape in the bone tumor microenvironment according to the OIO concept. The biologically relevant and clinically predictive preclinical bone metastasis technology platform should be routinely used to study the mechanism-of-action and efficacy of therapies before entering clinical development in bone metastasizing cancers. Citation Format: Tiina E. Kähkönen, Jussi M. Halleen, Gary MacRitchie, Ronnie M. Andersson, Jenni Bernoulli. Immunotherapy development landscape for bone metastasis - need of predictive preclinical efficacy evaluation for de-risking clinical development [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 2815.

Netrin-1 Role in Nociceptive Neuron Sprouting through Activation of DCC Signaling in a Rat Model of Bone Cancer Pain.

Bone cancer pain (BCP) is a common primary or metastatic bone cancer complication. Netrin-1 plays an essential role in neurite elongation and pain sensitization. This study aimed to determine the role of netrin-1 from the metastatic bone microenvironment in BCP development and identify the associated signaling pathway for the strategy of BCP management. The rat BCP model was established by intratibial implantation of Walker 256 cells. Von Frey filaments measured the mechanical pain threshold. Movement-induced pain was assessed using limb use scores. Expressions of associated molecules in the affected tibias or dorsal root ganglia (DRG) were measured by immunofluorescence, immunohistochemistry, real-time quantitative polymerase chain reaction, or western blotting. Transduction of deleted in colorectal cancer (DCC) signaling was inhibited by intrathecal injection of DCC-siRNA. In BCP rats, the presence of calcitonin gene-related peptide (CGRP)-positive nerve fibers increased in the metastatic bone lesions. The metastatic site showed enrichment of well-differentiated osteoclasts and expressions of netrin-1 and its attractive receptor DCC. Upregulation of DCC and increased phosphorylation levels of focal adhesion kinase (FAK) and Rac family small GTPase 1/Cell division cycle 42 (Rac1/Cdc42) were found in the DRG. Intrathecal administration of DCC-siRNA led to a significant reduction in FAK and Rac1/Cdc42 phosphorylation levels in the DRG, decreased nociceptive nerve innervation, and improved pain behaviors. Netrin-1 may contribute to the activation of the BCP by inducing nociceptive nerve innervation and improving pain behaviors.

Multiple influence of immune cells in the bone metastatic cancer microenvironment on tumors.

Bone is a common organ for solid tumor metastasis. Malignant bone tumor becomes insensitive to systemic therapy after colonization, followed by poor prognosis and high relapse rate. Immune and bone cells in situ constitute a unique immune microenvironment, which plays a crucial role in the context of bone metastasis. This review firstly focuses on lymphatic cells in bone metastatic cancer, including their function in tumor dissemination, invasion, growth and possible cytotoxicity-induced eradication. Subsequently, we examine myeloid cells, namely macrophages, myeloid-derived suppressor cells, dendritic cells, and megakaryocytes, evaluating their interaction with cytotoxic T lymphocytes and contribution to bone metastasis. As important components of skeletal tissue, osteoclasts and osteoblasts derived from bone marrow stromal cells, engaging in 'vicious cycle' accelerate osteolytic bone metastasis. We also explain the concept tumor dormancy and investigate underlying role of immune microenvironment on it. Additionally, a thorough review of emerging treatments for bone metastatic malignancy in clinical research, especially immunotherapy, is presented, indicating current challenges and opportunities in research and development of bone metastasis therapies.

Abstract A007: Preclinical bone metastasis technology platform – Predictive evaluation of experimental therapies on bone metastasis

Abstract Most cancer deaths are due to metastases, and bone metastases are a considerable problem especially in breast and prostate cancer, being developed in 70-90% of advanced-stage patients. Despite major investments in oncology drug development, bone metastases are currently incurable and a high unmet medical need with only 5% of patients being alive 5 years after the diagnosis. Development of therapies for bone metastasis has been challenging due to lack of appropriate preclinical models available to support decision making in next phases of drug development. In the absence of clinically relevant preclinical bone metastasis models, the current strategy is to rely on preclinical efficacy data obtained with subcutaneous models that lack the clinically relevant local tissue microenvironment, which has a major impact on tumor growth. The use of clinically non-relevant models in preclinical-stage development may be one important reason for the current >95% failure rate of oncology drugs in clinical trials. To support predictive evaluation of therapies for bone metastatic cancers, we describe a preclinical bone metastasis technology platform for evaluating efficacy of novel therapies on bone metastases. The platform utilizes tumors growing in bone microenvironment, mimicking growth of bone metastases in patients. Syngeneic or humanized mouse models with tumor and immune cells of same species are needed for supporting development of immunotherapies, allowing interactions of tumor and immune cells in the bone metastatic microenvironment, according to the novel osteoimmuno-oncology concept. The platform provides a predictive tool for studying unique biological features associated with different types of bone metastases in cancer-type specific manner. Here we summarize case examples where results from preclinical bone metastasis studies align with clinical findings of different therapies approved or evaluated for bone metastasis. Zoledronic acid, an anti-resorptive bisphosphonate that is currently used in breast cancer patients with bone metastases to prevent cancer-induced bone loss, showed improved bone health but no effects on tumor growth. Radium-223 dichloride, an approved treatment for bone metastatic castration-resistant prostate cancer in patients, showed reduced prostate cancer growth and decreased tumor-induced bone changes. As for immunotherapies, an IDO1 inhibitor had no effects on breast cancer bone metastases and the anti-PD-1 antibody pembrolizumab had no effects on breast and prostate cancer bone metastases, predicting recent clinical findings that demonstrate lack of efficacy of anti-PD-1 in clinical prostate cancer trials. We conclude that the bone metastasis technology platform is a biologically relevant tool for preclinical evaluation of the efficacy of experimental therapies on bone metastasis, and it has been validated with positive and negative case examples, demonstrating its clinically predictive power. Citation Format: Tiina E Kähkönen, Jussi M Halleen, Jenni Bernoulli. Preclinical bone metastasis technology platform – Predictive evaluation of experimental therapies on bone metastasis [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2023 Oct 11-15; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2023;22(12 Suppl):Abstract nr A007.

Abstract A069: Microscale modeling of the human prostate bone metastatic niche

Abstract Difficulties in modeling the human bone metastasis microenvironment (BMME) niche contribute to limitations in our understanding of the cellular interactions that promote treatment resistance in prostate cancer. To address this, we have developed a multicellular, vascularized, and fully humanized 3D tissue chip model of bone metastases that will allow us to identify drivers of treatment resistance and test emerging cancer therapies in the context of multicellular networks. We utilized LumeNEXT, a closed microfluidics platform with a removable center rod allowing vessel formation, to create this bone microenvironment. To obtain major stromal and bone cell types found in the BMME, we differentiated primary bone marrow-derived mesenchymal stem cells (MSCs) into osteoblasts, fibroblasts, and adipocytes and differentiated patient-derived monocytes from peripheral blood into M2-polarized macrophages and osteoclasts. The primary stromal and bone cells were seeded into LumeNEXT devices and combined in co-cultured with primary prostate cancer tumor organoids in a collagen-based matrix. and deposited into LumeNEXT devices. After polymerization of the hydrogel, the center rod in each device was removed and the tunnels were seeded with endothelial cells to create functional microvasculature. We successfully differentiated the patient-derived cell types, optimized a novel multi-phenotype media (MPM) for co-culture, and maintained greater than 80% viability of all seven cell types in co-culture for at least 14 days in the LumeNEXT platform. qPCR analysis of the model confirmed that expression of differentiation marker genes was maintained after 14 days of co-culture for all cell types. Viability assays and time-lapse microscopy of cell tracker staining also showed viability and growth of prostate cancer organoids in co-culture. Integrated multi-analyte assay platforms were developed for cellular and molecular analysis of patient-derived primary tumor organoids including assessment of cellular phenotype, transcriptomic and epigenetic patterns in the context of multicellular networks. Additionally, we have aimed to develop assays to test emerging cancer-targeting therapies, such as antibody-drug conjugate (ADC). Functional testing of this model using ADC has demonstrated drug perfusion into the stroma through the microvessels, and ADC-specific tumor killing. In summary, we created a multicellular microfluidics model of human bone metastases. High viability of all seven cell types included in our model was maintained for 14 days and early ADC testing demonstrates functional microvasculature and tumor cell killing. The duration of viable co-culture multicellular cultures and promising functional testing demonstrates the suitability of this model for rapid drug testing. Future studies will focus on testing novel chemo-, hormone, and radiation therapies in this multicellular BMME model to determine mechanisms of microenvironment-mediated treatment resistance in prostate cancer. Citation Format: Adeline Ding, Cristina Sanchez de Diego, Sheena C. Kerr, Ravi Chandra Yada, Erika Heninger, Nan Sethakorn, Shannon Reese, Maria Virumbrales-Muñoz, Pete Geiger, Xavier Hazelberg, Joshua M. Lang, Dave Beebe. Microscale modeling of the human prostate bone metastatic niche [abstract]. In: Proceedings of the AACR Special Conference: Advances in Prostate Cancer Research; 2023 Mar 15-18; Denver, Colorado. Philadelphia (PA): AACR; Cancer Res 2023;83(11 Suppl):Abstract nr A069.

Heterogeneity in the immune microenvironment of bone metastasis in driver-positive non-small cell lung cancer.

Mutations in epidermal growth factor receptor and anaplastic lymphoma kinase are common driver events in non-small cell lung cancer (NSCLC), which are associated with a high frequency of bone metastases (BMs). While the bone marrow represents a specialized immune microenvironment, the immune repertoire of BMs remains unknown. Considering the higher incidence of BMs in driver gene-positive NSCLCs, and the unique biology of the bone, herein, we assessed the infiltrating immune cells and T cell receptor (TCR) profile of BMs in driver-positive NSCLCs. Immune profile of BMs in driver gene-positive NSCLC were assessed in 10 patients, where 6 had driver gene-positive mutation. TCR and bulk RNA sequencing were performed on malignant bone samples. The diversity and clonality of the TCR repertoire were analyzed. The cellular components were inferred from bulk gene expression profiles computationally by CIBERSORT. Although BMs were generally regarded as immune-cold tumors, immune cell composition analyses showed co-existence of cytotoxic and suppressor immune cells in driver-positive BM samples, as compared to primary lung. Analysis of the TCR repertoire indicated a trend of higher diversity and similar clonality in the driver-positive compared with the driver-negative subsets. In addition, we identified two cases that showed the opposite response to immune checkpoint blockade. A comparison of these two patients' BM samples showed more highly amplified clones, fewer M2 macrophages and more activated natural killer cells in the responder. In summary, BMs in NSCLC are heterogeneous in their immune microenvironment, which might be related to differential clinical outcomes to immune checkpoint blockade.

Abstract 1189: MERTK is a target in the microenviroenment of osteolytic bone metastasis

Abstract Metastatic bone disease often induces osteolytic lesions caused by an imbalance of osteoblastic bone formation and osteoclastic bone resorption. We could previously show that TAM family receptor tyrosine kinase MERTK exerts an oncogenic function in multiple myeloma, NSCLC and breast cancer bone metastasis, providing a rationale for using MERTK inhibitors in bone metastasis therapy. Furthermore, we reported a thus far unrecognized role of MERTK as a novel negative regulator of osteoblast function and showed that MERTK represents an osteoanabolic target in the tumor microenvironment of bone metastasis by targeting osteoblasts. It is described that MERTK exerts a prominent role in phagocytosis of macrophages. Nevertheless, the role of MERTK in osteoclasts is unknown. To assess a potential role of MERTK in physiological bone remodeling by osteoclasts in vivo, we performed microcomputed tomography (μCT) of tibia of LysM-cre+;Mertkflox/flox mice and observed increased bone volume. Histomorphometry revealed reduced osteoclast number in LysM-cre+;Mertkflox/flox mice, indicating that increased bone volume induced by loss of MERTK in the myeloid lineage is mediated by decreased osteoclast formation in vivo. Interestingly, analysis of TRAP+ mononuclear cells in the metaphyseal bone marrow showed decreased numbers and increased distance to closest bone surface, suggesting dysfunctional osteoclast precursor cell differentiation and migration in LysM-cre+;Mertkflox/flox mice. In vitro assays demonstrated that loss of Mertk results in decreased osteoclast precursor cell migration towards its ligand PROS1 and decreased osteoclast differentiation through a RHOA dependent mechanism. To elucidate if MERTK represents a target in the tumor microenvironment of osteolytic bone metastasis we utilized syngeneic EO771 breast cancer bone metastasis model and injected luciferase transduced EO771 cells into Mertkflox/flox and LysM-cre+;Mertkflox/flox C57BL/6J mice. Metastatic spread was monitored by bioluminescence imaging. Analysis of bone metastasis by μCT showed higher bone volume in EO771 tumor bearing LysM-cre+;Mertkflox/flox mice. Furthermore, osteolysis zones in the cortical bone were markedly decreased. Histomorphometry of TRAP/Hematoxylin staining indicated decreased osteoclast numbers in EO771 tumor bearing LysM-cre+;Mertkflox/flox mice. Furthermore, intratumoral TRAP+ mononuclear cells in breast cancer bone metastases were decreased, indicating that osteolytic bone metastasis recruit osteoclast precursors from its microenvironment in the bone marrow via MERTK to induce osteolysis. In summary, tumor-induced activation of MERTK suppresses and transforms osteoblasts, which counteracts bone formation and recruits osteoclast precursors to form osteoclasts and mediate osteolysis. Hence, MERTK is a novel target in the tumor microenvironment to inhibit osteolytic bone metastasis. Citation Format: Janik Engelmann, Jennifer Zarrer, Kristoffer Riecken, Jonas Waizenegger, Maria Elena Vargas-Delgado, Lara Meier, Carsten Bokemeyer, Klaus Pantel, Emily Alberto, Sourav Ghosh, Carla Rothlin, Eric Hesse, Hanna Taipaleenmäki, Isabel Ben-Batalla, Sonja Loges. MERTK is a target in the microenviroenment of osteolytic bone metastasis [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 1189.

Macrophages and bone metastasis.

In the prostate bone metastasis microenvironment, macrophages activate a cascade that involves Activin A, the extracellular matrix, and SRC kinase and drives resistance to anti-androgen therapy. These findings (Li et al., 2023. J. Exp. Med.https://doi.org/10.1084/jem.20221007) have broad implications, including metastasis diversity in different tissue milieus and the interplay between hormones and immunity.

Detection of Immune Microenvironment Changes and Immune-Related Regulators in Langerhans Cell Histiocytosis Bone Metastasis.

The inflammation/immune response pathway is considered a key contributor to the development of Langerhans cell histiocytosis (LCH) bone metastasis. However, the dynamic changes in the immune microenvironment of LCH bone metastasis and critical regulators are still unclear. Expression profiling by arrays of GSE16395, GSE35340, and GSE122476 was applied to detect the immune microenvironment changes in the development of LCH bone metastasis. The single-cell high-throughput sequencing of GSE133704, involved in LCH bone lesions, was analyzed. The online database Metascape and gene set variation analysis (GSVA) algorithms were used to detect the gene function of Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG). The protein-protein interaction (PPI) network of hub regulators was constructed by the STRING database. In these results, key immune cells, such as Tem cells, NK T cells, CD8(+) T cells, and Th1 cells, were identified in LCH bone metastasis. These genes, which include LAG3, TSPAN5, LPAR5, VEGFA, CXCL16, CD74, and MARCKS, may significantly correlate with the cellular infiltration of B cells, aDCs, pDCs, cytotoxic cells, T cells, CD8+ T cells, T helper cells, and Tcm cells. In conclusion, our study constructed an atlas of the immune microenvironment of LCH bone metastasis. Genes including LAG3, TSPAN5, LPAR5, VEGFA, CXCL16, CD74, and MARCKS may be involved in the development of LCH bone metastasis. The hub gene-immune cell interactive map may be a potential prognostic biomarker for the progression of LCH bone metastasis and synergetic targets for immunotherapy in LCH patients.

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.

The impacts of exosomes on bone metastatic progression and their potential clinical utility

Bone is one of the most common sites of cancer metastasis. Once cancer metastasizes to the bone, the mortality rate of cancer patients dramatically increases. Although the exact mechanisms for this observation remain elusive, recent studies have revealed that the complex crosstalk between bone marrow microenvironment and bone metastatic cancer cells is responsible for the induction of treatment resistance. Consequently, bone metastasis is currently considered incurable. Bone metastasis not only impairs the patients' survival, but also negatively affects their quality of life by causing painful complications. It has recently been implicated the regulatory role of exosomes in cancer development and/or progression as a delivery biomaterial between cancer cells and tumor microenvironment. However, little is known as to how exosomes contribute to the progression of bone metastasis by impaction on the crosstalk between bone metastatic cancer cells and bone marrow microenvironment. Here, we highlighted the emerging roles of cancer-derived exosomes in (i) the process of dissemination and bone colonization of bone metastatic cancer cells, (ii) the enhancement of crosstalk between bone marrow microenvironment and bone metastatic cancer cells, (iii) the development of its resultant painful complications, and (iv) the clinical applications of exosomes in the bone metastatic setting.

Abstract 2191: Alteration of anti-tumoral immunity in the pre-metastatic bone microenvironment via autonomic nerve system dysfunction

Abstract Bone metastasis is a major cause of morbidity and mortality for breast cancer patients. Bone is a unique metastatic microenvironment because of complex interactions among numerous distinct cell types such as osteo-blasts, -clasts, -cytes and marrow immune cells in physiological and pathological conditions. Imbalanced bone homeostasis by diverse factors such as the sympathetic nerve system (SNS) activation, potentially contribute to the progression of bone metastasis, yet precise mechanisms remain unclear. We investigated the effects of beta 2 adrenergic receptor (β2AR) activation in osteoblasts induced by prolonged SNS stimulation on anti-tumoral immunity in bone metastasis. We treated female Balb/C with chronic immobilization stress (CIS; 2 h. daily) and found that CIS increased syngeneic intra-tibial 4T1 bone metastasis growth as well as the number of CD11b+Gr1+ myeloid-derived suppressor cells (MDSC) counterbalancing anti-tumoral cytotoxic T cells in bone microenvironment. A β2AR-specific inhibitor (ICI-118551) or MDSC depletion using anti-Gr1 antibodies reversed CIS-induced bone metastatic tumor growth. More importantly, 2-week CIS pre-treatment increased EDU+ MDSC proliferation in the bone marrow, contributing to subsequent bone metastatic tumor growth and micro-metastatic seeding in intra-tibial and intra-cardiac tumor injection models, respectively. When CIS was administered after the establishment of bone metastatic tumors, the pro-tumorigenic effects were marginal. In osteoblast-specific β2AR knockout mice (Adrb2flox/flox; murine 2.3kb Col1-CreC57BL6 mice), CIS did not increase MDSC proliferation compared with littermate floxed controls, suggesting that β2AR in osteoblasts upregulates MDSC in bone microenvironment. RNA-sequencing transcriptome analysis of saline- or clenbuterol (a selective β2AR agonist)-treated murine osteoblasts showed that interleukin-6 (IL6) was the most significantly increased cytokine. Indeed, CIS or clenbuterol treatment increased IL-6 expression in vivo (immunohistochemistry) and in vitro (cultured osteoblasts), respectively. MDSC isolated from the bone marrow of CIS-treated mice had significantly increased STAT3 phosphorylation by flow cytometry and the expression of immuno-suppressive functional molecules such as arginase 1 (Arg1) and indolamine 2,3-dioxygenase 1 (Ido1) compared to those from control mice. Our data collectively demonstrated that SNS-dependent β2AR in osteoblasts expands and activates MDSC in the pre-metastatic bone microenvironment via the IL6-STAT3-Arg1/IDO1 pathway, creating a fertile soil for bone metastasis progression. Our data provided the first direct evidence for anti-tumoral immunity and pro-tumorigenic effects of autonomous nerve system dysfunction specific to the metastatic bone microenvironment. Citation Format: Eun Jung Lee, Da Hyeon Yun, Seungpil Jung, Serk In Park. Alteration of anti-tumoral immunity in the pre-metastatic bone microenvironment via autonomic nerve system dysfunction [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2191.

Transfer of microRNA-22-3p by M2 macrophage-derived extracellular vesicles facilitates the development of ankylosing spondylitis through the PER2-mediated Wnt/β-catenin axis

Pathological osteogenesis and inflammation possess critical significance in ankylosing spondylitis (AS). The current study aimed to elucidate the mechanisms regarding extracellular vesicle (EV)-packaged microRNA-22-3p (miR-22-3p) from M2 macrophages in the osteogenic differentiation of mesenchymal stem cells (MSCs) in AS. EVs were initially isolated from M2 macrophages, which had been treated with either restored or depleted miR-22-3p. AS-BMSCs were subsequently treated with M2 macrophage-derived EVs to detect osteogenic differentiation in BMSCs using gain- or loss-of-function experiments. The binding affinity among miR-22-3p, period circadian protein 2 (PER2), and Wnt7b was identified. Finally, AS mouse models were established for testing the effects of M2-EV-miR-22-3p on the bone metastatic microenvironment in vivo. miR-22-3p from M2 macrophages could be transferred into BMSCs via EVs, which promoted the osteogenic differentiation of AS-BMSCs. miR-22-3p inhibited PER2, while PER2 blocked the Wnt/β-catenin signaling pathway via Wnt7b inhibition. M2-EV-shuttled miR-22-3p facilitated alkaline phosphatase activity and extracellular matrix mineralization via PER2-regulated Wnt/β-catenin axis, stimulating the BMSC osteogenic differentiation. Taken together, these findings demonstrate that miR-22-3p in M2 macrophage-released EVs downregulates PER2 to facilitate the osteogenesis of MSCs via Wnt/β-catenin axis.

GDF15 promotes prostate cancer bone metastasis and colonization through osteoblastic CCL2 and RANKL activation

Bone metastases occur in patients with advanced-stage prostate cancer (PCa). The cell-cell interaction between PCa and the bone microenvironment forms a vicious cycle that modulates the bone microenvironment, increases bone deformities, and drives tumor growth in the bone. However, the molecular mechanisms of PCa-mediated modulation of the bone microenvironment are complex and remain poorly defined. Here, we evaluated growth differentiation factor-15 (GDF15) function using in vivo preclinical PCa-bone metastasis mouse models and an in vitro bone cell coculture system. Our results suggest that PCa-secreted GDF15 promotes bone metastases and induces bone microarchitectural alterations in a preclinical xenograft model. Mechanistic studies revealed that GDF15 increases osteoblast function and facilitates the growth of PCa in bone by activating osteoclastogenesis through osteoblastic production of CCL2 and RANKL and recruitment of osteomacs. Altogether, our findings demonstrate the critical role of GDF15 in the modulation of the bone microenvironment and subsequent development of PCa bone metastasis.

LncRNA SNHG3 regulates the BMSC osteogenic differentiation in bone metastasis of breast cancer by modulating the miR-1273g-3p/BMP3 axis

BackgroundResearch on the role of lncRNAs in the process of bone metastasis in breast cancer (BM-BCa) has just begun at an early stage, and an increasing number of lncRNAs have been proved to play a regulatory role in the process of BM-BCa. Our study focused on the balance of osteogenic-osteoclast regulated by lncRNA-SNHG3 in bone metastasis microenvironment. MethodsSNHG3 level of clinical tissues and breast cancer cell lines was determined by RT-qPCR. ALP staining, ALP activity identification and western blotting of OPG, OSX, RUNX2, BMP2 together with BMP3 was performed to verify the osteogenesis of bone marrow mesenchymal stem cells (BMSCs) both in vitro and in vivo. Exosomes derived from MDA-MB-231 were characterized and sequenced, followed by RT-qPCR. Dual luciferase reporter gene assay was utilized to analyze the binding sites of miR-1273g-3p on SNHG3 and BMP3. ResultsExpression of BMP3 was positively regulated by SNHG3 via exosomal miR-1273g-3p. ConclusionThe overexpression of SNHG3 in breast cancer cells may be responsible for osteolytic metastasis Thus, knockdown of SNHG3 might be a potential target for improvement of BM-BCa Treatment.

Selected Articles from This Issue

Radium-223 dichloride is a radiopharmaceutical used to treat cancer that has metastasized to the bones, and whose effects are derived from both direct action of radiation on tumor cells as well as bystander effects caused by irradiation of both tumor and non-tumor cells in the bone microenvironment. Here, Canter and colleagues quantify the biological effects derived from both of these mechanisms of action. Direct effects from alpha particle irradiation (yellow arrows in the image at left) contribute to biological changes detected histologically in disseminated tumor cells and osteocytes within the range of the alpha particles. Bystander effects also contribute to biological changes in tumor cells beyond the range of the alpha particles emitted from the endosteal surface of the bone. These radiation-induced bystander effects can arise from tumor cells (white outlined arrow) and marrow cells (pink outlined arrow) within the range of alpha particles, and from bone cells (brown outlined arrow). The data suggest that bystander effects in the bone environment likely contribute to growth delay of disseminated tumor cells in bone marrow.Regulation of cancer stemness has emerged as a key tumor-intrinsic property underlying disease progression and therap. resistance. Recently, gain-of-function (GOF) mutations in p53 have been shown to promote increased stemness characteristics, but the mechanism for this is not well understood. Here, Ghatak and colleagues identify the microRNA miR-324-5p as the key effector of GOF mutant p53 that controls this phenomenon in non–small cell lung cancer (NSCLC) cells. GOF p53 isoforms expressed in NSCLC cells caused upregulation of c-Myc, which bound to and activated transcription of miR-324. The authors demonstrate that expression of CUEDC2—the direct target of miR-324-5p in this context—was ablated in cells expressing GOF p53 isoforms, and the loss of CUEDC2 expression was associated with activation of NF-κB and increased expression of stemness markers. CUEDC2 downregulation was further shown to be characteristic of human tumors bearing GOF p53 mutations. The authors conclude that TP53 mutations coupled with high miR-324-5p expression is associated with worse prognosis in lung cancer patients. Taken together, the data identify miR-324-5p as a novel epigenetic regulator of stemness properties in NSCLC.Balanced MDM2:p53 expression maintains proper cell cycle progression, whereas aberrant p53 activity is associated with developmental defects and pathologic states. Here, Mohibi and colleagues demonstrate that the RNA binding protein Zfp871 is a direct transcriptional target of p53 in vivo. Zfp871 functions to provoke a negative feedback loop regulating p53 activation by binding to the 3′UTR of MDM2 mRNA and stabilizing it. This interaction increases MDM2 expression and, by consequence, promotes degradation of p53. Mice deficient in Zfp871 were prone to early death, and heterozygous mice experienced a shortened lifespan and prominent steatohepatitis. The early death seen in Zfp871-KO mice, however, was ameliorated by ablation of Trp53. Gene expression analyses unveiled that p53 hyperactivation caused by Zfp871 loss was associated with alterations in lipid metabolism and inflammatory signaling as well as key developmental pathways. While the precise mechanism underlying the control of these pathways is yet to be determined, the data clearly demonstrate that precise control of p53 expression and activity—in part through the novel Zfp871-MDM2-p53 axis—is required to prevent the onset of pathologic conditions.Micro-metastatic lesions in the bone are often latent and can persist undetected for years in a quiescent state. However, the mechanisms by which rapidly proliferating tumor cells suddenly become quiescent upon metastasis are poorly defined. Here, Shupp and colleagues demonstrate that soluble signals from the bone metastatic microenvironment may play a key role in regulating this process. In particular, small extracellular vesicles (sEV) released by osteoblasts in proximity to breast cancer cells were shown to harbor factors such as miR-148a-3p, which had implications for breast cancer cell biology. Among other effects, the uptake of these sEV by metastatic breast cancer cells resulted in disruptions of both ERK1/2 signaling and cell cycle progression, thereby slowing cancer cell proliferation and contributing toward their latency. Taken together, the data elucidate a novel mechanism by which stromal cells influence cancer cell progression in the metastatic niche and underscore the importance of extracellular vesicles in mediating tumor–stromal cell interactions.

Role of miRNAs in Breast Cancer-induced Bone Disease

AbstractBone is the most common site of breast cancer recurrence. Despite the increasing knowledge about the metastatic process and treatment advances, the disease still remains incurable once the cancer cells actively proliferate in bone. Complex interactions between cancer cells and cells of the bone microenvironment (BME) regulate the initiation and progression of metastatic tumor growth in bone. In particular, breast cancer cells shift the otherwise tightly balanced bone remodeling towards increased bone resorption by osteoclasts. Cellular interactions in the metastatic BME are to a large extent regulated by secreted molecules. These include various cytokines as well as microRNAs (miRNAs), small non-coding RNAs that post transcriptionally regulate protein abundance in several cell types. Through this mechanism, miRNAs modulate physiological and pathological processes including bone remodeling, tumorigenesis and metastasis. Consequently, miRNAs have been identified as important regulators of cellular communication in the metastatic BME. Disruption of the crosstalk between cancer cells and the BME has emerged as a promising therapeutic target to prevent the establishment and progression of breast cancer bone metastasis. In this context, miRNA mimics or antagonists present innovative therapeutic approaches of high potential for interfering with pathological bone – cancer cell interactions. This review will discuss the role of miRNAs in the tumor-BME crosstalk in vivo and will emphasize how this could be targeted by miRNAs to improve therapeutic outcome for patients with breast cancer bone metastases.

The nonreceptor protein tyrosine kinase Src participates in every step of cancer-induced bone pain.

Cancer-induced bone pain (CIBP) is a refractory form of pain that has a high incidence in advanced tumors. Src protein tyrosine kinase is mainly composed of six domains, with two states of automatic inhibition and activation. The modular domain allows Src to conveniently regulate by and communicate with a variety of proteins, directly or indirectly participate in each step of the CIBP process. Src is beneficial to the growth and proliferation of tumor cells, and it can promote the metastases of primary tumors to bone. In the microenvironment of bone metastasis, it mainly mediates bone resorption, activates related peripheral receptors to participate in the formation of pain signals, and may promote the generation of pathological sensory nerve fibers. In the process of pain signal transmission, it mainly mediates NMDAR and central glial cells to regulate pain signal intensity and central sensitization, but it is not limited to these two aspects. Both basic experimentation and clinical research have shown encouraging potential, providing new ideas and inspiration for the prevention and treatment of CIBP.

LncRNA nuclear-enriched abundant transcript 1 shuttled by prostate cancer cells-secreted exosomes initiates osteoblastic phenotypes in the bone metastatic microenvironment via miR-205-5p/runt-related transcription factor 2/splicing factor proline- and glutamine-rich/polypyrimidine tract-binding protein 2 axis.

Prostate cancer (PCa) patients commonly present with osteoblastic‐type bone metastasis. Exosomes derived from tumor cells possess biological significance and can mediate intercellular communication in the tumor microenvironment. Long noncoding RNA (lncRNA) nuclear‐enriched abundant transcript 1 (NEAT1) is also implicated in the stability in tumorigenesis and the development of PCa, but the underlying mechanism remains elusive. Hence, the current study set out to investigate the physiological mechanisms by which exosomes‐encapsulated NEAT1 affects the progression of PCa. First, after isolation, we found PCa cell‐derived exosomes induced the osteogenic differentiation of human bone marrow‐derived mesenchymal stem cells (hBMSCs). Besides, NEAT1 in PCa cells could be transferred into hBMSCs via exosomes. Further gain‐ and loss‐of‐function experimentation revealed that NEAT1 acted as a competing endogenous RNA (ceRNA) of microRNA (miR)‐205‐5p to upregulate the runt‐related transcription factor 2 (RUNX2) levels. Moreover, NEAT1 could promote the RUNX2 expression via the splicing factor proline‐ and glutamine‐rich (SFPQ)/polypyrimidine tract‐binding protein 2 (PTBP2) axis. Functional assays uncovered that NEAT1 shuttled by PCa‐exosomes facilitated the activity of alkaline phosphatase (ALP) and mineralization of extracellular matrix, and continuously upregulated the levels of RUNX2, ALP, alpha‐1 type 1 collagen, and osteocalcin by regulating RUNX2, to induce the osteogenic differentiation of hBMSCs. Furthermore, in vivo experimentation confirmed that upregulated NEAT1 induced osteogenesis. Collectively, our findings indicated that PCa‐derived exosomes‐loaded NEAT1 upregulated RUNX2 to facilitate the osteogenesis of hBMSCs by competitively binding to miR‐205‐5p via the SFPQ/PTBP2 axis, therefore providing a potential therapeutic target to treat osteogenesis of hBMSCs in PCa. PCa cells secrete exosomes containing NEAT1, and NEAT1 exerts effects on osteogenic differentiation of hBMSCs in PCa. NEAT1 shuttled by PCa‐derived exosomes could be transferred into hBMSCs, where NEAT1 exerted inductive properties in osteogenic differentiation of hBMSCs through the upregulation of RUNX2 by competitively binding to miR‐205‐5p and regulating SFPQ/PTBP2 in vitro and in vivo.