Bone Colonization Research Articles

Massive hyper-progression during anti-PD1 immunotherapy in a young patient with metastatic mucinous adenocarcinoma of the right colon: a case report and literature review

: Immune checkpoint inhibitors (ICIs) have dramatically changed the treatment landscape of both solid and hematological malignancies, including tumors historically considered “non-immunogenic”, such as colorectal cancer (CRC). The increasing use of immunotherapy brought to light novel patterns of response due to its intrinsic mechanism of action. Besides the “pseudo-progression”, another peculiar phenomenon linked to ICIs activity is the “hyper-progression” (HP), namely a paradoxical disease acceleration during immunotherapy. This event, which suggests potentially deleterious effects of immunotherapy, has not been yet completely understood and lacks strict definition criteria, pathogenetic characterization as well as predictive factors. In this report, we present a case of an atypical massive progression in a 40-years old man with metastatic mucinous right colon cancer harboring high microsatellites instability (MSI-H), occurring after 2 cycles of pembrolizumab as first line therapy. Unfortunately, he experienced a widespread cancer dissemination for massive bone colonization and both numerical and volumetric increase of pre-existing node metastasis associated with rapid clinical worsening, which were suggestive for HP. To our knowledge, this is one of the few cases of HP in metastatic CRC that has been reported, particularly with a so rapid clinical deterioration and massive skeletal involvement. Other experiences and further studies are warranted to better understand this phenomenon and anticipate its recognition.

Neuroendocrine neoplasia and bone (Review).

This is a narrative review focusing on neuroendocrine neoplasia (NEN) and bone status, in terms of metastases and osteoporosis/fractures. One fifth of NEN have skeletal dissemination, this affinity being regulated by intrinsic tumor factors such as the C-X-C chemokine receptor 4 (CXCR4). Bone colonization impairs the patient quality of life, representing a surrogate of reduced survival. Patients with NEN without bone metastases may exhibit low bone mineral density, perhaps carcinoid-related osteoporosis, yet not a standardized cause of osteoporosis. Case-finding strategies to address bone health in NEN with a good prognosis are lacking. Contributors to fractures in NEN subjects may include: menopausal status and advanced age, different drugs, induced hypogonadism, malnutrition, malabsorption (due to intestinal resection, carcinoid syndrome), hypovitaminosis D, impaired glucose profile (due to excessive hormones such as glucagon, somatostatinoma or use of somatostatin analogues), various corticoid regimes, and high risk of fall due to sarcopenia. Pheocromocytoma/paraganglioma involve bone through malignant forms (bone is an elective site) and potential secondary osteoporosis due to excessive hormonal content and increased sympathetic activity which is a key player of bone microarchitecture/quality as reflected by low Trabecular Bone Score. Glucocorticoid osteoporosis is related to NEN-associated ectopic Cushing syndrome. Currently, there are a lack of studies to emphasis that excessive gut-derivate serotonin in NENs with carcinoid syndrome is a specific activator of bone loss thus a contributor to carcinoid-related osteoporosis.

Abstract 2878: HIF signaling in breast tumors controls spontaneous tumor dissemination in a site-specific manner

Abstract As tumors outgrow their blood supply, they frequently become hypoxic, activating hypoxia-inducible factor (HIF) signaling. Previous studies suggest that HIF signaling in breast cancer cells promotes lung dissemination in genetic models and bone colonization following intracardiac inoculation, but the impact of HIF signaling in the primary tumor on spontaneous dissemination to bone has never been evaluated. Thus, we hypothesized that Hif1α or Hif2α deletion in the primary tumor would reduce spontaneous dissemination to lung and bone, while deletion of Vhl (resulting in constitutive HIF signaling) would increase dissemination to distant metastatic sites. To test this, we generated MMTV-Cre.Hif1αf/f.PyMT, MMTV-Cre.Hif2αf/f.PyMT, and MMTV-Cre.Vhlf/f.PyMT mice, which spontaneously develop mammary carcinomas with conditional deletion of Hif1a, Hif2a, or Vhl, respectively. Littermate and cousin controls were used for all experiments. All mice were euthanized when the largest tumor reached 1cm in any dimension. Tumor growth was significantly delayed in Hif1α-/- (n=21) and Vhl-/- mice (n=19), while Hif2α-/- mice (n=18) had similar tumor progression compared to wildtype controls (n=19-20). Despite the delay in tumor growth, Hif1α-/- mice had greater total tumor weight (71%, p<0.01) and Vhl-/- mice had reduced total tumor weight (31%, p<0.05) on average compared to wildtype mice, with no difference in Hif2α-/- mice. Surprisingly, histological inspection of lung sections revealed that Hif1α-/- mice had greater incidence of macroscopic tumor nodules (n=15/21 Hif1α-/-, n=6/19 Hif1αf/f, p<0.05), and greater average lesion number and area (1.9- and 2.2-fold, respectively, p<0.05), but there was no difference in Ki67 staining in the lung nodules. In contrast, bone dissemination was significantly reduced in Hif1α-/- (56%, p<0.05) and Hif2α-/- mice (43%, p<0.01) and significantly increased in Vhl-/- mice (238%, p<0.01) as assessed by flow cytometry. However, there was no change in the expression of an 89-gene panel of metastasis associated genes or markers of EMT (Cdh1/2, Egfr, Notch1, Snai1-3, Twist1, Zeb1/2) or autophagy (Atg7, Becn1, Map1lc3a/b, Sqstm1, Ulk1) in Hif1α-/- primary tumors. None of the three transgenic strains showed a difference in dissemination to liver or brain. These data indicate that while both HIF1α and HIF2α drive tumor dissemination to bone, HIF1α is also essential for preventing metastatic outgrowth in the lung. It is therefore likely that the increase in bone dissemination when mammary fat pad VHL is deleted is due to elevated HIF1α and HIF2α signaling. Taken together, these data indicate a critical, yet divergent, role of HIF signaling in dissemination to bone and lung. Thus, targeted inhibition of HIF1α or HIF2α could be beneficial in preventing bone metastasis but may promote the outgrowth of lung-disseminated breast cancer cells. Citation Format: Vera M. Todd, Lawrence A. Vecchi, Katherine P. Snow, Lauren Himmel, Marjan Rafat, Rachelle W. Johnson. HIF signaling in breast tumors controls spontaneous tumor dissemination in a site-specific manner [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2878.

Abstract 2904: Mechanical loading induced osteogenic response does not interfere with the colonization of breast cancer in bone

Abstract Breast cancer is the most commonly diagnosed cancer and frequently metastasizes to bone. Previous evidence has suggested that cancer cells target the osteoblastic lineage cells to establish colonization of bone. It is well established that mechanical loading (e.g. load bearing exercise) elevates bone remodeling via stimulating osteoblastic activities. Whether mechanical loading facilitates the colonization of breast cancer cells in bone is not fully understood. To test this, we have firstly applied a murine tibial mechanical loading model and determined the effects of mechanical loading on bone remodeling in vivo. Six 10-week-old BALB/c nude female mice were subjected to a right tibial dynamic load (9N) onto a 2N pre-load using the ElectroForce 5500 Test Instrument, 40 load cycles with 10 sec intervals, 3 times a week for 1 week. Mice were then euthanized with additional 4 mice euthanized before mechanical loading as baseline controls. Quantitative micro-CT analysis suggested that bone mass related parameters significantly increased in the loaded tibias compared to contra-lateral non-loaded controls, while data in the baseline group showed no differences between left and right tibias. As previously published 2-week loading regimens, one week of loading was enough to induce a 24.3% increase in trabecular bone volume (P=0.001, paired t-test), a 9.0% increase in trabecular thickness (P= 0.01), and a 17.9% increase in cortical bone volume (P<0.0001). Using this model, we tested whether mechanical loading affects the arrival of breast cancer cells in bone. Human and mouse breast cancer cells (MDA-MB-231 and 4T1) were firstly labelled with vital fluorescent lipophilic dyes (Vybrant DiD) to allow tracking of individual tumor cells arriving in bone ex vivo. Cells were injected into the circulation (intracardiac) after mice (BALB/c nude and BALB/c) subjected to 1-week of mechanical loading. Mice were then loaded 3 times more to maintain bone mass before euthanized 7 days post-injection. DiD labelled cancer cells were examined and quantified in tibial bone marrow using multiphoton microscopy ex vivo. In both xenograft and syngeneic models, there were no significant differences found in the number of breast cancer cells arriving in bone marrow (MDA: P=0.32; 4T1: P=0.67) and their relative location to bone surfaces (MDA: P= 0.45; 4T1: P= 0.91) in loaded vs non-loaded bones. There was no significant difference in size of objectives (indicating size of micro tumors) detected in the MDA model (P= 0.21), comparing loaded to non-loaded control tibias but a significant 17.7% reduction in the 4T1 model in the loaded tibias (P=0.02). These pre-clinical data demonstrate that mechanical loading induced osteoblastic bone formation does not affect the initial colonization of breast cancer cells in bone, and suggest that load bearing exercise will not increase the risk of breast cancer bone metastasis in patients. Citation Format: Alexandria R. Sprules, Norain B. Latif, Janet E. Brown, Alison Gartland, Ning Wang. Mechanical loading induced osteogenic response does not interfere with the colonization of breast cancer in bone [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2904.

CD8+ T cells from experimental in situ breast carcinoma interfere with bone homeostasis.

Before bone colonization, immune cells primed by breast primary tumor cells actively modify the bone microenvironment, disturbing the complex and tightly homeostatic signaling network regulated by osteoblasts and osteoclasts. Indeed, we have shown that RANKL+ CD4+ T cells specific for the 4T1 mammary carcinoma cell line, arrive at the bone marrow (BM) before metastatic cells and set the pre-metastatic niche. In the absence of RANKL expressed by T cells, there is no pre-metastatic osteolytic disease and bone metastases are blocked. Adding to the role of T cells, we have recently demonstrated that dendritic cells (DCs) provide a positive feedback loop to the osteolytic profile induced by the metastatic tumor. In this setting, DCs are able to differentiate into potent bone resorbing osteoclast-like cells keeping their antigen-presenting cell (APC) properties to maintain RANKL+ CD4+ Th17 T cells activities, via IL-23 expression. Here we show that 67NR non-metastatic tumor cells, a sibling of 4T1 tumor cell line, induce an increase in trabecular bone mass on day 11 post-tumor implant. This observation was associated with an expansion of the osteoblastic lineage cells accompanied by a reduction of osteoclasts numbers. Moreover, BM derived CD8+ T cells from 67NR tumor-bearing mice, express an anti-osteoclastogenic cytokine milieu enriched by IFN-γ, IL-10 and producing low levels of RANKL. The frequency of BM derived CD8+ FoxP3+ regulatory T cells, known as potent suppressors of osteoclastogenesis both in vitro and in vivo, was also increased in such animals. This milieu was capable to suppress 4T1 tumor-specific CD4+ T cells phenotype in vivo and in vitro and strongly inhibited bone metastases establishment, restoring trabecular bone mass volume. We concluded that the 67NR+ tumor derived CD8+ T cells phenotypes, either contributing to bone homeostasis and/or control of 4T1 breast tumor pre-metastatic disease, interfere with osteoclasts and osteoblasts activities inside BM. Our study highlights the opposing roles of subverted tumor CD4+ and CD8+ T cell subtypes in directing breast cancer progression and bone metastases establishment. For non-metastatic tumors, the role of T cells regarding bone remodeling has never been addressed before. As far as we know, this is the first description that an in situ carcinoma can modify distant sites. In the case showed here, modification of the distant bone site disfavors pre-metastatic bone niche formation.

Biomechanical regulation of breast cancer metastasis and progression

Physical activity has been consistently linked to decreased incidence of breast cancer and a substantial increase in the length of survival of patients with breast cancer. However, the understanding of how applied physical forces directly regulate breast cancer remains limited. We investigated the role of mechanical forces in altering the chemoresistance, proliferation and metastasis of breast cancer cells. We found that applied mechanical tension can dramatically alter gene expression in breast cancer cells, leading to decreased proliferation, increased resistance to chemotherapeutic treatment and enhanced adhesion to inflamed endothelial cells and collagen I under fluidic shear stress. A mechanistic analysis of the pathways involved in these effects supported a complex signaling network that included Abl1, Lck, Jak2 and PI3K to regulate pro-survival signaling and enhancement of adhesion under flow. Studies using mouse xenograft models demonstrated reduced proliferation of breast cancer cells with orthotopic implantation and increased metastasis to the skull when the cancer cells were treated with mechanical load. Using high throughput mechanobiological screens we identified pathways that could be targeted to reduce the effects of load on metastasis and found that the effects of mechanical load on bone colonization could be reduced through treatment with a PI3Kγ inhibitor.

Targeted Therapy to β3 Integrin Reduces Chemoresistance in Breast Cancer Bone Metastases.

Breast cancer bone metastases are common and incurable. Tumoral integrin β3 (β3) expression is induced through interaction with the bone microenvironment. Although β3 is known to promote bone colonization, its functional role during therapy of established bone metastases is not known. We found increased numbers of β3+ tumor cells in murine bone metastases after docetaxel chemotherapy. β3+ tumor cells were present in 97% of post-neoadjuvant chemotherapy triple-negative breast cancer patient samples (n = 38). High tumoral β3 expression was associated with worse outcomes in both pre- and postchemotherapy triple-negative breast cancer groups. Genetic deletion of tumoral β3 had minimal effect in vitro, but significantly enhanced in vivo docetaxel activity, particularly in the bone. Rescue experiments confirmed that this effect required intact β3 signaling. Ultrastructural, transcriptomic, and functional analyses revealed an alternative metabolic response to chemotherapy in β3-expressing cells characterized by enhanced oxygen consumption, reactive oxygen species generation, and protein production. We identified mTORC1 as a candidate for therapeutic targeting of this β3-mediated, chemotherapy-induced metabolic response. mTORC1 inhibition in combination with docetaxel synergistically attenuated murine bone metastases. Furthermore, micelle nanoparticle delivery of mTORC1 inhibitor to cells expressing activated αvβ3 integrins enhanced docetaxel efficacy in bone metastases. Taken together, we show that β3 integrin induction by the bone microenvironment promotes resistance to chemotherapy through an altered metabolic response that can be defused by combination with αvβ3-targeted mTORC1 inhibitor nanotherapy. Our work demonstrates the importance of the metastatic microenvironment when designing treatments and presents new, bone-specific strategies for enhancing chemotherapeutic efficacy.

Abstract IA009: Dact1 biomolecular condensates mediate TGFβ-Wnt crosstalk in bone metastasis

Abstract TGF-β signaling is a well established pathway driving different steps of bone metastasis progression. Wnt signaling plays a dichotomous role in bone metastasis, with recent studies showing that inhibition of Wnt signaling by DKK1 is critical for avoidance of immune clearance of dormant bone micrometastasis and activation of osteoclast activity during osteolytic outgrowth. On the other hand, engagement of bone vascular E-selectin by disseminated tumor cells (DTCs) promotes metastatic colonization in bone by activating Wnt signaling to enhance tumor initiating activity of DTCs. These findings imply a complex interplay between TGF-b and Wnt signaling in different stages of bone metastasis development that allows efficient colonization, dormancy and outgrowth of secondary tumors in bone. What mediates the dynamic transition of TGF-β and Wnt signaling states during metastasis is currently unknown. Here we present data describing a novel phase-separated organelle organized by bone metastasis promoting protein Dact1 which mediates the crosstalk of TGF-β and Wnt signaling. We found that Dact1 is induced by TGF-β signaling and forms dynamically regulated biomolecular condensates that suppress Wnt signaling. A high level of Dact1 expression is associated with an elevated risk of bone metastasis in breast cancer patients and Dact1 is necessary for bone metastasis in mouse models of breast and prostate cancers. Citation Format: Yibin Kang. Dact1 biomolecular condensates mediate TGFβ-Wnt crosstalk in bone metastasis [abstract]. In: Proceedings of the AACR Virtual Special Conference on the Evolving Tumor Microenvironment in Cancer Progression: Mechanisms and Emerging Therapeutic Opportunities; in association with the Tumor Microenvironment (TME) Working Group; 2021 Jan 11-12. Philadelphia (PA): AACR; Cancer Res 2021;81(5 Suppl):Abstract nr IA009.

Hematopoietic Stem Cell Colonization of Fetal Bones is Controlled by the Integrity and Oxidative Status of the Developing Stromal-Vascular Niches

Hematopoietic stem cell (HSC) maintenance throughout life and engraftment after transplantation during anticancer treatment, depend on support from various bone marrow (BM) niche compartments. Here, by studying physiological homing of fetal HSCs, we show the critical requirement of balanced crosstalk within the multi-component BM niche for enabling HSC-lodgment. Transgene-induced over-production of VEGF by skeletal progenitor cells elicited stromal and endothelial hyper-activation, profoundly impacting the stromal-vessel interface and vascular architecture. Concomitantly, HSC seeding, settlement and survival were drastically impaired. Transcriptome-profiling, flow-cytometry and high-resolution imaging indicated alterations in peri-/endothelial characteristics, vascular permeability, and cellular metabolism, associated with a higher oxidative status and increased oxidative stress within the VEGF-enriched BM-environment. Thus, developmental HSC-homing to bone is controlled by local inter-niche communication regulating stromal-vascular integrity and theoxidative and metabolic milieu.Since irradiation of adult mice also induced stromal VEGF-expression and similar osteo-angiogenic niche changes, our findings can contribute to improving stem cell therapy.

Systemic PPARγ Antagonism Reduces Metastatic Tumor Progression in Adipocyte-Rich Bone in Excess Weight Male Rodents.

Primary tumors are widely associated with an excess in body fat. The role of adipose tissue on tumor cell homing to bone is yet poorly defined. In this study, we aimed to assess whether bone colonization by tumor cells is favored by an adipocyte-rich bone marrow. We delineated the accompanying alterations of the bone microenvironment and established a treatment approach that interferes with high fat diet (HFD)-induced bone metastasis formation. We were able to show that adipocytes affect skeletal tumor growth in a metastatic model of breast cancer in male rats and melanoma in male mice as well as in human breast cancer bone biopsies. Indeed, HFD-induced bone marrow adiposity was accompanied by accelerated tumor progression and increased osteolytic lesions. In human bone metastases, bone marrow adiposity correlated with tumor cell proliferation. By antagonization of the adipocyte differentiation and storage pathway linked to the peroxisome proliferator-activated receptor gamma (PPARγ) with bisphenol-A-diglycidylether (BADGE), we were able to decelerate tumor progression and subsequent osteolytic damage in the bones of two distinct metastatic animal models exposed to HFD. Overall these data show that adipose tissue is a critical factor in bone metastases and cancer-induced bone loss. © 2021 The Authors. Journal of Bone and Mineral Research published by Wiley Periodicals LLC on behalf of American Society for Bone and Mineral Research (ASBMR).

Cancer-associated fibroblasts support bone tropic metastasis by acting as coordinators between the tumor microenvironment and bone matrix in breast cancer.

Bone is a common site of metastasis for various types of cancer cells, including breast cancer, and the consequent skeleton-related events observed in patients are severe and often fatal. Currently, it is widely accepted that cancer-associated fibroblasts (CAFs) confer a metastasis-promoting property to breast cancer cells. Furthermore, clinical observations suggest that CAFs mediate the bone tropism of metastatic breast cancer cells. Therefore, a deeper understanding of the mechanism by which CAFs are involved in the bone-tropic metastasis of breast cancer can facilitate the study of the novel and effective therapeutic drugs for the corresponding targets. In this review, we focused on the coordinator role of CAFs in remolding breast cancer cells and remodeling the bone marrow during metastasis. We discussed the potential roles of the CXCL12/CXCR4 axis, the CAFs-CSCs reinforcing loop, and exosomes in this malignant process. In summary, in agreement with Paget's theory, CAFs play a pivotal role in bone colonization by breast cancer cells by providing a "fertile soil" for the "selected seeds" by influencing tumor-intrinsic characteristics and microenvironment (ME).

Retrospective analysis of bacterial colonization of necrotic bone and antibiotic resistance in 98 patients with medication-related osteonecrosis of the jaw (MRONJ)

ObjectivesThe aim of our study was to describe microbial flora associated with MRONJ and characterize the susceptibility of pathogens to help guide an effective empiric antibiotic treatment in these patients.Materials and methodsA retrospective, single-center analysis was performed, using 116 bone samples from 98 patients. The bone samples were homogenized and subjected to routine culture methods. Growing bacteria were differentiated to the species level using whole-cell mass spectrometry and subjected to susceptibility testing.ResultsA highly diverse microbial flora was detected in necrotic bone, with a simultaneous presence of two or more bacterial species in 79% of all patients. In at least 65% of samples, gram-negative isolates were detected. Therefore, bacterial species resistant against β-lactamase inhibitors were present in at least 70% of all patients.ConclusionsThe empiric choice of antibiotics in MRONJ patients should consider the high rate of gram-negative bacteria and resistance against β-lactam antibiotics.Clinical relevanceAccording to recent guidelines and recommendations, systemic antibiotic treatment is a key component in the treatment of all stage 2 and 3 MRONJ patients. We recommend using fluoroquinolones for empiric treatment and emphasize the use of bacterial cultivation and susceptibility testing to enable an effective antibiotic treatment.

Unique cellular protrusions mediate breast cancer cell migration by tethering to osteogenic cells

Migration and invasion are key properties of metastatic cancer cells. These properties can be acquired through intrinsic reprogramming processes such as epithelial-mesenchymal transition. In this study, we discovered an alternative “migration-by-tethering” mechanism through which cancer cells gain the momentum to migrate by adhering to mesenchymal stem cells or osteoblasts. This tethering is mediated by both heterotypic adherens junctions and gap junctions, and leads to a unique cellular protrusion supported by cofilin-coated actin filaments. Inhibition of gap junctions or depletion of cofilin reduces migration-by-tethering. We observed evidence of these protrusions in bone segments harboring experimental and spontaneous bone metastasis in animal models. These data exemplify how cancer cells may acquire migratory ability without intrinsic reprogramming. Furthermore, given the important roles of osteogenic cells in early-stage bone colonization, our observations raise the possibility that migration-by-tethering may drive the relocation of disseminated tumor cells between different niches in the bone microenvironment.

Abstract LB-002: ER alpha-positive human cell line xenograft models recapitulate metastatic dissemination and endocrine response of invasive lobular breast carcinoma

Abstract Invasive lobular breast carcinoma (ILC) is the most common special histological subtype of breast cancer, accounting for 10-15% of all cases, and characterized by the loss of adherens junctions through inactivating mutations in E-cadherin. Although currently existing genetically engineered mouse models of ILC faithfully capture its dyscohesive, single-file growth and expression of estrogen receptor alpha (ER), they do not fully recapitulate all aspects of human ILC such as unique sites of metastases and endocrine response. While recent work on human ILC cell lines has proven them useful for studying ILC in vitro, there is limited data on their growth as xenografts in mice and their fidelity as in vivo models. Using dual bioluminescent and fluorescent labeled ER-positive human ILC cell lines (MDA-MB-134, SUM44PE, MDA-MB-330, BCK4), herein we characterized their growth orthotopically and at secondary sites following spontaneous or experimental metastasis. Mammary fat pad xenografts gave rise to primary tumors with single-file infiltration and cytoplasmic translocation of p120-catenin, characteristic of human ILC. The tumor microenvironment exhibited deposition of collagen fibers and infiltration by fibroblasts and neutrophils. In this orthotopic model, we observed spontaneous metastases of most cell lines to bones, brain and ovaries, closely mirroring the clinical patterns of human ILC dissemination. In contrast, experimental metastases were only observed following intravenous or intracardiac xenografts of MDA-MB-134 cells. Interestingly, tail vein injections of this cell line lead to colonization of bones and lymph nodes but not of lungs, while intracardiac injections resulted in brain, bone and lymph node metastases. Importantly, we observed high ER expression in the primary tumors and the metastatic lesions, along with a significant response to the selective ER down-regulator fulvestrant in both the mammary fat pad and intracardiac models. Ongoing work focused on genomic and transcriptional analyses of primary tumors and metastases, as well as of cell lines isolated from these lesions, will reveal additional mechanistic insights into the biology of ILC dissemination. This is the first report of ER-positive and endocrine responsive human cell line xenografts faithfully representing unique ILC features such as ovarian metastases. These versatile models will serve as an invaluable pre-clinical platform for validating candidate ILC genetic drivers and testing novel therapeutics towards translation into the clinic. Citation Format: Nilgun Tasdemir, Laura Savariau, Julie Scott, Joseph D. Latoche, Weizhou Hou, Kyle Biery, Minji Chung, Emily A. Bossart, Sreeja Sreekumar, Daniel D. Brown, Azadeh Nasrazadani, Ye Qin, Jagmohan Hooda, Fangyuan Chen, Carlos A. Castro, Carolyn J. Anderson, Jennifer Atkinson, Peter C. Lucas, Nancy E. Davison, Adrian V. Lee, Steffi Oesterreich. ER alpha-positive human cell line xenograft models recapitulate metastatic dissemination and endocrine response of invasive lobular breast carcinoma [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr LB-002.

Enhanced recruitment and hematopoietic reconstitution of bone marrow-derived mesenchymal stem cells in bone marrow failure by the SDF-1/CXCR4.

Aplastic anemia (AA) is a bone marrow failure disease. It is difficult to treat AA, and in addition, relapses are common because of its complex disease pathogenesis. Allogeneic bone marrow-derived mesenchymal stem cells (BMSCs) infusion is an effective and safe treatment option for the AA patients. However, it found that BMSCs infusion in AA patients is less than 30% effective. Therefore, the key to improve the efficacy of BMSCs treatment in these patients is to enhance their homing efficiency to the target sites. Studies have shown that stromal cell-derived factor-1 (SDF-1)/CXC chemokine receptor 4 (CXCR4) axis plays an important role in promoting BMSCs homing. In this study, human BMSCs were transduced with lentivirus stably expressing CXCR4-BMSCs. Transduced BMSCs resemble normal BMSCs in many ways. Migration ability of CXCR4-BMSCs toward SDF-1 was increased because of the overexpression of CXCR4. In the mice with bone marrow failure, the migration and colonization ability of CXCR4-BMSCs to the bone marrow was significantly improved as seen by IVIS imaging and FACS. The SDF-1 level in the bone marrow failure mice was significantly higher than in the normal mice. Thus, from our study, it is clear that after CXCR4-BMSCs were infused into mice with bone marrow failure, SDF-1 interacted with CXCR4 receptor, leading cells to migrate and colonize to bone marrow. Because of the high SDF-1 expression in mouse bone marrow and CXCR4 receptor expression in cells, BMSCs homing was increased.

Characterizing the postmortem human bone microbiome from surface-decomposed remains.

Microbial colonization of bone is an important mechanism of postmortem skeletal degradation. However, the types and distributions of bone and tooth colonizing microbes are not well characterized. It is unknown if microbial communities vary in abundance or composition between bone element types, which could help explain differences in human DNA preservation. The goals of the present study were to (1) identify the types of microbes capable of colonizing different human bone types and (2) relate microbial abundances, diversity, and community composition to bone type and human DNA preservation. DNA extracts from 165 bone and tooth samples from three skeletonized individuals were assessed for bacterial loading and microbial community composition and structure. Random forest models were applied to predict operational taxonomic units (OTUs) associated with human DNA concentration. Dominant bacterial bone colonizers were from the phyla Proteobacteria, Actinobacteria, Firmicutes, Bacteroidetes, and Planctomycetes. Eukaryotic bone colonizers were from Ascomycota, Apicomplexa, Annelida, Basidiomycota, and Ciliophora. Bacterial loading was not a significant predictor of human DNA concentration in two out of three individuals. Random forest models were minimally successful in identifying microbes related to human DNA concentration, which were complicated by high variability in community structure between individuals and body regions. This work expands on our understanding of the types of microbes capable of colonizing the postmortem human skeleton and potentially contributing to human skeletal DNA degradation.

Bone-in-culture Array to Model Bone Metastasis in ex vivo Condition

Bone is the most frequently affected organ by metastases of breast cancer and prostate cancer. Our knowledge on bone metastasis is extremely limited due to the lack of potent and efficient experimental models. We developed the "Bone-In-Culture Array (BICA)" platform to model the bone colonization of cancer cells in ex vivo cultures. The use of the BICA platform will facilitate in-depth mechanistic studies and high-throughput screening of drugs for bone metastasis.

Bone Tropism in Cancer Metastases.

Bone is a frequent site of metastases in many cancers. Both bone properties and the tumor-intrinsic traits are associated with the metastatic propensity to bone (i.e., the bone tropism). Whereas an increasing body of mechanistic studies expanded our understanding on bone tropism, they also revealed complexity across the bone lesions originated from different cancer types. In this review, we will discuss the physical, chemical, and biological properties of bone microenvironment, identify potential players in every stage of bone metastases, and introduce some of the known mechanisms regulating the bone colonization. Our objectives are to integrate the knowledge established in different biological contexts and highlight the determinants of bone tropism.

The CXCL5/CXCR2 axis is sufficient to promote breast cancer colonization during bone metastasis

Bone is one of the most common sites for metastasis across cancers. Cancer cells that travel through the vasculature and invade new tissues can remain in a non-proliferative dormant state for years before colonizing the metastatic site. Switching from dormancy to colonization is the rate-limiting step of bone metastasis. Here we develop an ex vivo co-culture method to grow cancer cells in mouse bones to assess cancer cell proliferation using healthy or cancer-primed bones. Profiling soluble factors from conditioned media identifies the chemokine CXCL5 as a candidate to induce metastatic colonization. Additional studies using CXCL5 recombinant protein suggest that CXCL5 is sufficient to promote breast cancer cell proliferation and colonization in bone, while inhibition of its receptor CXCR2 with an antagonist blocks proliferation of metastatic cancer cells. This study suggests that CXCL5 and CXCR2 inhibitors may have efficacy in treating metastatic bone tumors dependent on the CXCL5/CXCR2 axis.

Abstract 4523: A novel syngeneic mouse model of prostate cancer bone metastasis: Mechanisms of chemotaxis and bone colonization

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