Bone Colonization Research Articles

Breast cancer cells promote osteoclast differentiation in an MRTF-dependent paracrine manner.

Bone is a frequent site for breast cancer metastasis. The vast majority of breast cancer-associated metastasis is osteolytic in nature, and RANKL (receptor activator for nuclear factor κB)-induced differentiation of bone marrow-derived macrophages (BMDMs) to osteoclasts (OCLs) is a key requirement for osteolytic metastatic growth of cancer cells. In this study, we demonstrate that Myocardin-related transcription factor (MRTF) in breast cancer cells plays an important role in paracrine modulation of RANKL-induced osteoclast differentiation. This is partly attributed to MRTF's critical role in maintaining the basal cellular expression of connective tissue growth factor (CTGF), findings that align with a strong positive correlation between CTGF expression and MRTF-A gene signature in the human disease context. Luminex analyses reveal that MRTF depletion in breast cancer cells has a broad impact on OCL-regulatory cell-secreted factors that extend beyond CTGF. Experimental metastasis studies demonstrate that MRTF depletion diminishes OCL abundance and bone colonization breast cancer cells in vivo, suggesting that MRTF inhibition could be an effective strategy to diminish OCL formation and skeletal involvement in breast cancer. In summary, this study highlights a novel tumor-extrinsic function of MRTF relevant to breast cancer metastasis.

Breast cancer cells promote osteoclast differentiation in an MRTF-dependent paracrine manner.

Bone is a frequent site for breast cancer metastasis. Conditioning of the local tumor microenvironment (TME) through crosstalk between tumor cells and bone resident cells in the metastatic niche is a major driving force for bone colonization of breast cancer cells. The vast majority of breast cancer-associated metastasis is osteolytic in nature, and RANKL-induced differentiation of bone marrow-derived macrophages to osteoclasts (OCLs) is a key requirement for osteolytic metastatic growth of cancer cells. In this study, we demonstrate that breast cancer cell-secreted factors stimulate RANKL-induced OCL differentiation of BMDMs requiring the function of Myocardin-related transcription factor (MRTF) in tumor cells. This is partly attributed to the critical role of MRTF in maintaining the basal cellular expression of connective tissue growth factor (CTGF), a pro-osteoclastogenic matricellular factor known to promote bone metastasis in human breast cancer. Supporting these in vitro findings, bioinformatics analyses of multiple human breast cancer transcriptome datasets reveal a strong positive correlation between CTGF expression and MRTF gene signature further establishing the relevance of our findings in a human disease context. By Luminex analyses, we show that MRTF depletion in breast cancer cells has a broad impact on OCL-regulatory cell-secreted factors that extends beyond CTGF. These findings, taken together with demonstration of MRTF-dependence for bone colonization breast cancer cells in vivo, suggest that MRTF inhibition could be an effective strategy to diminish OCL formation and skeletal involvement in breast cancer. In summary, this study highlights a novel tumor-extrinsic function of MRTF relevant to breast cancer metastasis.

Abstract PO2-27-02: Re-sensitizing the Refractory Breast Cancer Bone Metastasis to Endocrine Therapies

Abstract Approximately 90% of Breast Cancer (BrCa) related deaths are caused by metastasis and bone is the first and most frequent site of breast cancer metastatic expansion, which can lead to severe pain, immobility, fractures, and nerve damage, as well as the development of metastases in other organs, hence; a poor quality of life in patients. Currently, treatment options for these bone metastases (BoM) are limited because most patients with BoM shows reduced ER dependency which leads to resistance to standard endocrine or hormonal therapies for estrogen receptor-positive (ER+) BrCa. The long-term goal of the proposed research is to develop a strategy where metastatic cancer cells in bone can be dependent on ER signaling again thereby restoring their sensitivity to endocrine therapies. We observed a transient loss of ER expression in concert with enhanced activity of the transcriptional coactivators YAP/TAZ - the downstream effectors of the Hippo pathway (responsible for regulating cell proliferation and death) during bone colonization by BrCa cells. We hypothesize that the interaction between cancer cells and osteoblasts in the bone microenvironment leads to the activation of calcium signaling in cancer cells, resulting in the de-phosphorylation and nuclear localization of YAP/TAZ which subsequently suppresses the transcription of the estrogen receptor gene (ESR). Our research strategy is as presented below. First, we will employ a combination of 2D/3D co-culture models of BoM along with a mechanical loading system and determine the molecular mechanism driving the dynamical activation of YAP/TAZ in BrCa BoM. Using a unique ex vivo BM model called Bone In Culture Array (BICA) which replicates the in-vivo conditions, we will determine if verteporfin, a YAP/TAZ antagonist, can synergize and possibly enhance the effects of Fulvestrant, an ER antagonist in current use for certain types of BrCa in preventing BoM outgrowths and treating established BoM in mice. This research could ultimately lead to a favorable combination strategy to treat endocrine-resistant BoM in BrCa patients and prolong their survival. Citation Format: Anindita Das, Cheyenne Ernst. Re-sensitizing the Refractory Breast Cancer Bone Metastasis to Endocrine Therapies [abstract]. In: Proceedings of the 2023 San Antonio Breast Cancer Symposium; 2023 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2024;84(9 Suppl):Abstract nr PO2-27-02.

Abstract PO5-05-05: Clinical outcomes of CDK4/6 inhibitors in patients with bone only metastatic breast cancer

Abstract Background: CDK4/6 inhibitors (CDK 4/6i) have significantly advanced the treatment of hormone receptor-positive (HR+) metastatic breast cancer in the last decade. Several clinical trials including PALOMA, MONALEESA, and MONARCH have shown improved progression-free survival (PFS) rates when CDK4/6i were combined with endocrine therapy (ET). These results led to recommending their use in combination with ET as the first line of therapy in the metastatic settings. Bone metastasis is the most common site of disease in patients with HR+ metastatic breast cancer, about 40% of whom have bone as the only site of metastasis (bone-only disease [BoD]). While previous studies have shown that BoD can have a unique clinical course and a more favorable prognosis compared to non-BoD metastasis, most of the aforementioned CDK 4/6i clinical trials have not shown significant improvements in PFS in BoD subset of patients. Here we aimed to investigate the survival outcomes of BoD patients at a single institution center and evaluate CDK 4/6i activity in preclinical models of bone metastasis in breast cancer. Patients and methods: Patients with HR+, HER2-negative metastatic breast cancer from a retrospective CDK4/6i study at Washington University School of Medicine were included in this analysis. We selected all the patients who received CDK4/6i in the first- or second-line settings from June 2014 to March 2020. Outcomes were collected through June 10, 2023. The Kaplan-Meier method with log rank test and multivariable cox model were used for survival analysis. Results: 114 female patients (n=75 first line, n=39 second line, n=20 African American, n=94 Caucasian) were included in this study. All patients received Palbociclib as the first exposure to CDK4/6 inhibitor in combination with ET for at least two months. Median age was 60 years (range, 28-87) and forty-seven patients (41%) were in the BoD group. Survival outcomes for BoD group were compared to the rest of patients who had either bone and visceral or visceral only metastatic lesions (BV group). Our results demonstrated a significantly reduced median PFS in BoD group compared to BV group (13.1 months [95%CI:8-18.2] vs 19.6 months [95%CI:6.8-32.4], p=0.01) respectively. There was no significant difference in overall survival between BoD and BV groups (36.6 months [95%CI:32.2-41] vs 41.7 months [95%CI 36.1-47.2] p=0.74) respectively. In multivariate analysis regarding the associated risk factors with PFS, after adjusting for age and race, BoD had a HR of 1.62 (95%CI:1.04-2.54) for PFS. Among the 75 patients who received CDK4/6i as their first line of therapy, BoD (n=32 patients [42.7%]) also showed an inferior (p=0.05) median PFS (20 months [95%CI 12.9-27]) compared to BV group (n=43 patients [57.3%], median PFS=28.4 months [95%CI: 16.3-40.4]). To investigate potential treatment resistance mechanisms in bone metastases, we used the triple negative 4T1 and the ER+ luminal B PyMT Bo1 breast tumor cell line derived mammary gland models in immunocompetent mice. Single agent CDK4/6i, ribociclib, decreased primary mammary gland tumor burden. This effect was abrogated when CD8+ T-cells were depleted. In the bone colonization model using these cell lines, ribociclib had little effect on bone tumor burden. Arg1+ immune suppressive macrophages were significantly decreased in the primary mammary tumor but not in the bone tumor after ribociclib administration, suggesting a relative treatment resistance and immune suppression in the bone tumor microenvironment compared to the primary mammary gland. Conclusion: CDK4/6i play a significant role in the treatment of patients with HR+ metastatic breast cancer. Our single institution, retrospective study and preclinical data suggests that CDK4/6i may not be as effective for the treatment of tumors residing in the bone microenvironment, warranting future studies to understand the mechanisms of CDK4/6 inhibitors action in bone versus visceral metastases. Citation Format: Mahta Mardani, Suleyman Noordeen, Katherine Clifton, Cynthia Ma, Jingqin Luo, Jing Xi, Nusayba Bagegni, Foluso Ademuyiwa, Rama Suresh, Ashley Frith, Andrew Davis, Ron Bose, Lindsay Peterson, Shana Thomas, Yu Tao, Takayuki Kobayashi, Jingyu Xiang, Yalin Xu, Xinming Su, Katherine Weilbaecher. Clinical outcomes of CDK4/6 inhibitors in patients with bone only metastatic breast cancer [abstract]. In: Proceedings of the 2023 San Antonio Breast Cancer Symposium; 2023 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2024;84(9 Suppl):Abstract nr PO5-05-05.

Influence of Titanium Surface Residual Stresses on Osteoblastic Response and Bacteria Colonization.

Grit basting is the most common process applied to titanium dental implants to give them a roughness that favors bone colonization. There are numerous studies on the influence of roughness on osseointegration, but the influence of the compressive residual stress associated with this treatment on biological behavior has not been determined. For this purpose, four types of surfaces have been studied using 60 titanium discs: smooth, smooth with residual stress, rough without stress, and rough with residual stress. Roughness was studied by optic interferometry; wettability and surface energy (polar and dispersive components) by contact angle equipment using three solvents; and residual stresses by Bragg-Bentano X-ray diffraction. The adhesion and alkaline phosphatase (ALP) levels on the different surfaces were studied using Saos-2 osteoblastic cultures. The bacterial strains Streptococcus sanguinis and Lactobacillus salivarius were cultured on different surfaces, determining the adhesion. The results showed that residual stresses lead to increased hydrophilicity on the surfaces, as well as an increase in surface energy, especially on the polar component. From the culture results, higher adhesion and higher ALP levels were observed in the discs with residual stresses when compared between smooth and roughened discs. It was also found that roughness was the property that mostly influenced osteoblasts' response. Bacteria colonize rough surfaces better than smooth surfaces, but no changes are observed due to residual surface tension.

Development of a precision tumor bone metastasis model by a magnetic micro-living-motor system

An ideal bone metastasis animal model is critical and fundamental for mechanistic research and following development of new drug and treatment. Caudal artery (CA) injection allows bone metastasis in the hindlimb, while in-depth targeted and quantitative studies of bone metastasis require a new model to overcome its limitations. Here, we developed a targeted, quantitative, and highly consistent method for the modeling of bone metastasis with cell-based magnetic micro-living-motor (MLM) system created by effectively combining Fe3O4-PDA-Au with biosafety. The MLM system can achieve efficient migration, target site colonization and control tumorigenesis in bone precisely with the application of a magnetic field. In vivo, day 3 post cell injection, tumor bone metastasis signals were observed locally in the injected femur among 82.76% mice of the MLM group as compared to the 56.82% in the CA group, and the signal intensity was 45.1 and 95.9 times stronger than that in the left and right lower limbs of the CA group, respectively. Post-injection day 28, metastasis in vital organs was reduced by approximately 90% in the MLM group compared to the CA group. Our innovative use of the MLM system in the field of tumor modeling opens a new avenue for exploring the mechanisms of tumor bone metastasis, recurrence and drug resistance.

Breast cancer remodels the bone marrow immune microenvironment to favor bone metastasis

Abstract Bone is the most common site of breast cancer (BC) metastasis, occurring in 70% of patients, and an indication of short-term prognosis. Understanding the environmental and host factors involved in the induction of metastasis is essential to develop therapies that extend patient’s lifespan. Bone colonization by cancer cells is a stepwise process that relies on interactions between cancer cells and the cells that comprise the bone microenvironment, such as osteoclasts, osteoblasts, hematopoietic cells, immune cells, and adipocytes. Calcium, growth factors, and chemokines facilitate bone metastasis. However, the role of immune cells in bone metastasis remains poorly understood. Further, how physiological stressors such as nutrition contribute to pro- or anti-metastatic bone immunity remains unclear. Using mouse models of BC, we found that BC profoundly transforms the bone marrow (BM) immune microenvironment to support metastasis. BC induces bone lymphoid cell loss, with significant reduction in B cells, T cells and NK cells. On the other hand, mammary tumors are associated with enhanced monocytes and eosinophils within the BM. These results support the idea that BC cells crosstalk with the bone and remodel the immune microenvironment to favor tumor cell colonization. We have previously showed that caloric restriction (CR) induces T cell migration to the BM and remodels the bone microenvironment, favoring T cell function and a protective environment. Of note, CR-mediated targeting of the bone microenvironment restores the immune homeostatic pool and contributes to an anti-tumor bone immunity. Thus, nutritional interventions my represent potential therapeutic strategies to target bone metastasis. This work was supported in part by intramural funds of NIAID, NIH. A.T. is supported by the EMBO Postdoctoral Fellowship Programme (ALTF 1014-2021).

Osteosarcoma tumors maintain intra-tumoral transcriptional heterogeneity during bone and lung colonization

BackgroundTumors are complex tissues containing collections of phenotypically diverse malignant and nonmalignant cells. We know little of the mechanisms that govern heterogeneity of tumor cells nor of the role heterogeneity plays in overcoming stresses, such as adaptation to different microenvironments. Osteosarcoma is an ideal model for studying these mechanisms—it exhibits widespread inter- and intra-tumoral heterogeneity, predictable patterns of metastasis, and a lack of clear targetable driver mutations. Understanding the processes that facilitate adaptation to primary and metastatic microenvironments could inform the development of therapeutic targeting strategies.ResultsWe investigated single-cell RNA-sequencing profiles of 47,977 cells obtained from cell line and patient-derived xenograft models as cells adapted to growth within primary bone and metastatic lung environments. Tumor cells maintained phenotypic heterogeneity as they responded to the selective pressures imposed during bone and lung colonization. Heterogenous subsets of cells defined by distinct transcriptional profiles were maintained within bone- and lung-colonizing tumors, despite high-level selection. One prominent heterogenous feature involving glucose metabolism was clearly validated using immunofluorescence staining. Finally, using concurrent lineage tracing and single-cell transcriptomics, we found that lung colonization enriches for multiple clones with distinct transcriptional profiles that are preserved across cellular generations.ConclusionsResponse to environmental stressors occurs through complex and dynamic phenotypic adaptations. Heterogeneity is maintained, even in conditions that enforce clonal selection. These findings likely reflect the influences of developmental processes promoting diversification of tumor cell subpopulations, which are retained, even in the face of selective pressures.

Abstract P6-14-03: Oestrogen represses Noggin expression by interfering BMP/Smad signalling in ER positive breast cancer cells

Abstract Background Bone morphogenetic proteins (BMPs) are members of Transforming Growth Factor β (TGF-β) superfamily. BMPs are actively involved in the disease progression and bone metastasis of breast cancer. As a natural antagonist of BMP, Noggin plays important roles in the regulation of BMP signalling. It can facilitate spread of breast cancer cells to bone and subsequent colonisation and formation of osteolytic bone lesions. The present study aimed to investigate the regulatory mechanism for Noggin expression in oestrogen receptor (ER) positive breast cancer cells. Method Noggin expression was analysed in The Cancer Genome Atlas (TCGA ER (+) n=763, ER (-) n=216) and E-MTAB-6703 (ER (+) n=733, ER (-) n=421) cohorts. Correlation between Noggin and ER was evaluated using Spearman test. The expression of Noggin in an ER positive breast cancer cell line MCF-7 was determined by depriving the cells from oestrogen using phenol red-free DMEM supplemented with 10% charcoal stripped foetal calf serum or adding 10-10M 17-β-oestradiol. Activation of Smad-1/5/8 and involvement of BMP receptors in the oestrogen repressed Noggin expression were further examined using recombinant human BMP7 and a BMP receptor inhibitor (LDN-193189). Influence of Noggin on cellular functions was evaluated in MCF-7 cells with Noggin overexpression using a lentiviral Noggin expression vector. Results Noggin expression was negatively correlated with ERα in both TCGA BRCA (r=-0.162, p<0.01, n=1093) and E-MTAB-6703 (r=-0.078, p<0.01, n=2302) cohorts. The expression of Noggin was increased in the MCF-7 cells upon a deprivation from oestrogen which was further validated by adding 17-β-oestradiol. This is in line with the increased expression of Noggin observed in the MCF-7 cells with ER silencing (GSE27473). Furthermore, an increased level of phosphorylated Smad1/5/8 was seen in the MCF-7 being hungered from oestrogen which was prevented by adding 17-β-oestradiol and LDN-193189, respectively. As a result, the oestrogen hunger induced Noggin expression was also decreased by adding with 17-β-oestradiol and LDN-193189. To further investigate the influence of oestrogen on BMP-Smad signalling regulated Noggin expression, Noggin expression and phosphorylation of Smad1/5/8 and Smad3 were determined in MCF-7 cells which were treated with rhBMP7 and in combination with 17-β-oestradiol and LDN-193189, respectively. BMP7 induced Noggin expression and activation of Smad1/5/8 can be diminished by 17-β-oestradiol and LDN-193189. Noggin overexpression in MCF-7 cells resulted in an increase of proliferation. Conclusion Noggin expression can be repressed by oestrogen through an inference of the BMP- Smad signalling. Overexpression of Noggin promoted proliferation of MCF-7 cells. Further investigation is required to clarify the exact role of Noggin in ER positive breast cancer and its implication in the disease progression and current therapies. Citation Format: Ming Liu, Lin Ye, Wen G. Jiang. Oestrogen represses Noggin expression by interfering BMP/Smad signalling in ER positive breast cancer cells [abstract]. In: Proceedings of the 2022 San Antonio Breast Cancer Symposium; 2022 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2023;83(5 Suppl):Abstract nr P6-14-03.

Marine sponge bioerosion in the forensic taphonomy of terrestrial bone

Bone and teeth, specialised bio-mineralized connective tissues, are left after the typical decomposition process of any vertebrate organism. Their analysis can reveal insights into an organism's life and retrace the history of the remains after death (also known as taphonomy), which ultimately evolves to destruction or fossilization. Studies on the taphonomy of terrestrial mammalian bio-mineralized tissues have mostly focussed on terrestrial depositional environments. Here, samples submerged in the marine environment are investigated.Five archaeological bones of terrestrial mammalian species (pig and oxen) with historically known post-mortem submersion interval (PMSI) (69–316 years) and recovery sites, were analysed macroscopically, microscopically and by microCT. The aim was to characterize for the first time the alterations produced by marine bioeroding sponges, and to discuss their potential interdisciplinary application, with special focus on forensic investigations.The pig samples showed microanatomical preservation (Oxford Histological Index = 3–5), increased total porosity, the presence of old tissue flakes with sponge spicules and traces of bioerosion, such as papillary holes, canals and chambers with microsculptured walls. The presence of such tissue flakes suggested that, at the time of recovery, they may have been free of sediment and inhabited by live sponges. The shape of one internal chamber was identified as the ichnospecies Entobia convoluta as typically produced by shallow, warm-water Cliothosa spp. Surface analyses for further biological evidence remained inconclusive.The taphonomy of skeletal remains has always been relevant in anthropological, natural and forensic studies. In forensics, the role of taphonomy is to contribute to personal identification, cause of death and post-mortem interval (PMI). This study detected the past colonization of terrestrial mammalian bone by marine bioeroding sponges, and aimed to link the taphonomic findings to natural processes and environments. Bioeroding sponges are for the first time confirmed to colonize terrestrial mammalian bone submerged in marine environments, and to promote diagenesis through bioerosion.

Bone Metastasis Initiation Is Coupled with Bone Remodeling through Osteogenic Differentiation of NG2+ Cells.

The bone colonization of cancer cells occurs in an environment that undergoes constant remodeling. Our study provides mechanistic insights into how bone homeostasis and pathologic repair lead to the outgrowth of disseminated cancer cells, thereby opening new directions for further etiologic and epidemiologic studies of tumor recurrences. This article is highlighted in the In This Issue feature, p. 247.

A microfluidic model of human vascularized breast cancer metastasis to bone for the study of neutrophil-cancer cell interactions.

The organ-specific metastatization of breast cancer to bone is driven by specific interactions between the host microenvironment and cancer cells (CCs). However, it is still unclear the role that circulating immune cells, including neutrophils, play during bone colonization (i.e. pro-tumoral vs. anti-tumoral). Here, we aimed at analyzing the migratory behavior of neutrophils when exposed to breast CCs colonizing the bone and their contribution to the growth of breast cancer micrometastases. Based on our previous bone metastasis models, we designed a microfluidic system that allows to independently introduce human vascularized breast cancer metastatic seeds within a bone-mimicking microenvironment containing osteo-differentiated mesenchymal stromal cells and endothelial cells (ECs). ECs self-assembled into microvascular networks and connected the bone-mimicking microenvironment with the metastatic seed. Compared to controls without CCs, metastatic seeds compromised the architecture of microvascular networks resulting in a lower number of junctions (5.7 ​± ​1.2 vs. 18.8 ​± ​4.5, p ​= ​0.025) and shorter network length (10.5 ​± ​1.0 vs. 13.4 ​± ​0.8 [mm], p ​= ​0.042). Further, vascular permeability was significantly higher with CCs (2.60 ​× ​10−8 ​± ​3.59 ​× ​10−8 ​vs. 0.53 ​× ​10−8 ​± ​0.44 ​× ​10−8 [cm/s], p ​= ​0.05). Following metastatic seed maturation, neutrophils were injected into microvascular networks resulting in a higher extravasation rate when CCs were present (27.9 ​± ​13.7 vs. 14.7 ​± ​12.4 [%], p ​= ​0.01). Strikingly, the percentage of dying CCs increased in presence of neutrophils, as confirmed by confocal imaging and flow cytometry on isolated cells from the metastatic seeds. The biofabricated metastatic niche represents a powerful tool to analyze the mechanisms of interaction between circulating immune cells and organ-specific micrometastases and to test novel drug combinations targeting the metastatic microenvironment.

Identification of Staphylococcus aureus Penicillin Binding Protein 4 (PBP4) Inhibitors.

Methicillin-resistant Staphylococcus aureus (MRSA) is a global healthcare concern. Such resistance has historically been attributed to the acquisition of mecA (or mecC), which encodes an alternative penicillin binding protein, PBP2a, with low β-lactam affinity. However, recent studies have indicated that penicillin binding protein 4 (PBP4) is also a critical determinant of S. aureus methicillin resistance, particularly among community-acquired MRSA strains. Thus, PBP4 has been considered an intriguing therapeutic target as corresponding inhibitors may restore MRSA β-lactam susceptibility. In addition to its role in antibiotic resistance, PBP4 has also recently been shown to be required for S. aureus cortical bone osteocyte lacuno-canalicular network (OLCN) invasion and colonization, providing the organism with a niche for re-occurring bone infection. From these perspectives, the development of PBP4 inhibitors may have tremendous impact as agents that both reverse methicillin resistance and inhibit the organism’s ability to cause chronic osteomyelitis. Accordingly, using a whole-cell high-throughput screen of a 30,000-member small molecule chemical library and secondary assays we identified putative S. aureus PBP4 inhibitors. Quantitative reverse transcriptase mediated PCR and PBP4 binding assays revealed that hits could be further distinguished as compounds that reduce PBP4 expression versus compounds that are likely to affect the protein’s function. We also showed that 6.25 µM (2.5 µg/mL) of the lead candidate, 9314848, reverses the organism’s PBP4-dependent MRSA phenotype and inhibits its ability to traverse Microfluidic-Silicon Membrane-Canalicular Arrays (µSiM-CA) that model the OLCN orifice. Collectively, these molecules may represent promising potential as PBP4-inhibitors that can be further developed as adjuvants for the treatment of MRSA infections and/or osteomyelitis prophylactics.

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 6010: Mouse hind limb tumor metastatic model to evaluate prophylaxis and treatment

Abstract We have established and optimized a robust tumor bone metastasis mouse model, which recapitulates the tumor metastasis progression from circulation to bone colonization, and provides windows for both prophylaxis and treatment. Specifically, we inoculated two breast cancer cell lines, JIMT-1 and MDA-MB-231, each with high metastatic potential, into mouse intra-caudal artery. By detection of steadily incremental bioluminescence signal, prophylaxis was observed to precede metastases, which were found exclusively on mouse hind limbs. These mouse models exhibited swollen hind limbs and even paralysis at late stage. Gross necropsy and pathology analysis confirmed the exclusive hind limb localization, the invasion and spreading of tumor cells to muscles and bone marrows, and the lesions in tibias and femurs. Our robust bone metastases models thus imitate the multiple stages of tumor metastasis, leading to exclusion bone metastasis and opening up windows for prophylaxis and treatment. Citation Format: Yao Shuai, Chaomeng Wang, Ling Tong, Qiao Liu, Jie Xu, Qunsheng Ji. Mouse hind limb tumor metastatic model to evaluate prophylaxis and treatment [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 6010.

Abstract 3643: The role of HBP1 in controlling breast cancer dormancy reawakening

Abstract At least 22% of breast cancer (BrCa) cases recur after 3 to 15 years, typically at metastatic sites such as the bone. Bone metastases derive from early disseminated tumor cells that become dormant because of interaction with specific cells that define the endosteal niche (EN) microenvironment. However, outside knowing that dormancy is driven by a p38MAPKhi, ERK1/2lo activation profile, our understanding of what factors regulate dormancy maintenance and reawakening is poor. We used a genomic shRNA screen to identify HBP1 as a suppressor of BrCa dormancy reawakening in a 3D-EN culture system. Our data indicate that under 3D-EN conditions, HBP1 is upregulated by p38MAPK, interacts with the metastasis suppressors, BRMS1 and BRMS1L, and with the histone modifiers, SIN3A and HDAC1-3. We hypothesize that HBP1 suppresses p38MAPK-mediated dormancy reawakening by complexing with BRMS1/BRMS1L/SIN3A to alter cell cycle gene expression through histone landscape remodeling. HBP1 suppresses the expression of cell cycle progression genes, such as MYC and CCND1, and concomitantly upregulates cyclin kinase inhibitors, p16 and p21. HBP1 RNA and protein levels are decreased in BrCa metastases relative to primary tumors, and this downregulation correlates with poorer overall survival. We are investigating whether the anti-histiminic drug, cyproheptadine, which upregulates HBP1 expression through the activation of p38MAPK, can suppress dormancy reawakening of BrCa cells in 3D-EN cultures and in in vivo models of bone dormancy using mouse mammary carcinoma D2A1 cell lines with varying abilities to form active or dormant metastatic bone colonization. Data will be shown addressing how cell-cell interactions and secretory factors between BrCa and bone EN cells facilitates dormancy signaling through a p38MAPK/HBP1/BRMS1 axis, and how drugs such as cyproheptadine agonize that pathway in order to prevent the reawakening that drives the growth of BrCa metastases in the bone, thereby increasing overall survival. Citation Format: Irwin H. Gelman, Seamus Degan, Henry G. Withers, Subrahmanya Anirudh Kaligotla Venkata. The role of HBP1 in controlling breast cancer dormancy reawakening [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 3643.

Extracellular Vesicle-Packaged CDH11 and ITGA5 Induce the Premetastatic Niche for Bone Colonization of Breast Cancer Cells.

This study provides mechanistic insights into the generation of an osteogenic premetastatic niche by breast cancer-derived EVs and identifies potential EV-derived diagnostic biomarkers and targets for breast cancer bone metastasis.

Fracture-related infection in osteoporotic bone causes more severe infection and further delays healing.

AimsWith the ageing population, fragility fractures have become one of the most common conditions. The objective of this study was to investigate whether microbiological outcomes and fracture-healing in osteoporotic bone is worse than normal bone with fracture-related infection (FRI).MethodsA total of 120 six-month-old Sprague-Dawley (SD) rats were randomized to six groups: Sham, sham + infection (Sham-Inf), sham with infection + antibiotics (Sham-Inf-A), ovariectomized (OVX), OVX + infection (OVX-Inf), and OVX + infection + antibiotics (OVX-Inf-A). Open femoral diaphysis fractures with Kirschner wire fixation were performed. Staphylococcus aureus at 4 × 104 colony-forming units (CFU)/ml was inoculated. Rats were euthanized at four and eight weeks post-surgery. Radiography, micro-CT, haematoxylin-eosin, mechanical testing, immunohistochemistry (IHC), gram staining, agar plating, crystal violet staining, and scanning electron microscopy were performed.ResultsAgar plating analysis revealed a higher bacterial load in bone (p = 0.002), and gram staining showed higher cortical bone colonization (p = 0.039) in OVX-Inf compared to Sham-Inf. OVX-Inf showed significantly increased callus area (p = 0.013), but decreased high-density bone volume (p = 0.023) compared to Sham-Inf. IHC staining showed a significantly increased expression of TNF-α in OVX-Inf compared to OVX (p = 0.049). Significantly reduced bacterial load on bone (p = 0.001), enhanced ultimate load (p = 0.001), and energy to failure were observed in Sham-Inf-A compared to Sham-Inf (p = 0.028), but not in OVX-Inf-A compared to OVX-Inf.ConclusionIn osteoporotic bone with FRI, infection was more severe with more bone lysis and higher bacterial load, and fracture-healing was further delayed. Systemic antibiotics significantly reduced bacterial load and enhanced callus quality and strength in normal bone with FRI, but not in osteoporotic bone.Cite this article: Bone Joint Res 2022;11(2):49–60.

Osteocytes and Cancer.

While the function of osteocytes under physiologic conditions is well defined, their role and involvement in cancer disease remains relatively unexplored, especially in a context of non-bone metastatic cancer. This review will focus on describing the more advanced knowledge regarding the interactions between osteocytes and cancer. We will discuss the involvement of osteocytes in the onset and progression of osteosarcoma, with the common bone cancers, as well as the interaction that is established between osteocytes and multiple myeloma. Mechanisms responsible for cancer dissemination to bone, as frequently occur with advanced breast and prostate cancers, will be reviewed. While a role for osteocytes in the stimulation and proliferation of cancer cells has been reported, protective effects of osteocytes against bone colonization have been described as well, thus increasing ambiguity regarding the role of osteocytes in cancer progression and dissemination. Lastly, supporting the idea that skeletal defects can occur also in the absence of direct cancer dissemination or osteolytic lesions directly adjacent to the bone, our recent findings will be presented showing that in the absence of bone metastases, the bone microenvironment and, particularly, osteocytes, can manifest a clear and dramatic response to the distant, non-metastatic tumor. Our observations support new studies to clarify whether treatments designed to preserve the osteocytes can be combined with traditional anticancer therapies, even when bone is not directly affected by tumor growth.

Evolving cancer-niche interactions and therapeutic targets during bone metastasis.

Many cancer types metastasize to bone. This propensity may be a product of genetic traits of the primary tumour in some cancers. Upon arrival, cancer cells establish interactions with various bone-resident cells during the process of colonization. These interactions, to a large degree, dictate cancer cell fates at multiple steps of the metastatic cascade, from single cells to overt metastases. The bone microenvironment may even influence cancer cells to subsequently spread to multiple other organs. Therefore, it is imperative to spatiotemporally delineate the evolving cancer-bone crosstalk during bone colonization. In this Review, we provide a summary of the bone microenvironment and its impact on bone metastasis. On the basis of the microscopic anatomy, we tentatively define a roadmap of the journey of cancer cells through bone relative to various microenvironment components, including the potential of bone to function as a launch pad for secondary metastasis. Finally, we examine common and distinct features of bone metastasis from various cancer types. Our goal is to stimulate future studies leading to the development of a broader scope of potent therapies.