Tumor-bone Interface Research Articles

MMP13 Expression and Activity Suggest Its Role in Bone Resorption in Ameloblastomas.

Ameloblastoma is a locally destructive benign odontogenic tumor. While the neoplastic cells of conventional ameloblastoma can infiltrate the connective tissue and bone, in unicystic ameloblastoma the epithelium is encapsulated. The mechanisms driving ameloblastoma's bone resorption remains unclear. RNA sequencing (RNA-seq) was performed in a discovery cohort of conventional ameloblastoma, and pathway enrichment analysis was carried out. mRNA levels of MMP13, a gene associated with bone resorption, were assessed using RT-qPCR in a larger cohort of conventional ameloblastoma and in unicystic ameloblastoma. Zymogram gels and the immunoexpression profile of collagenase 3 (encoded by MMP13 gene) were evaluated as well. Enriched pathways related to bone mineralization and upregulation of MMP13 were observed in ameloblastomas. Collagenolytic activity of collagenase 3 was detected in the tumor lysates. Collagenase 3 immunopositivity was observed in ameloblastomatous epithelium infiltrating the fibrous capsule of unicystic ameloblastoma. At the tumor-bone interface, collagenase 3 expression was detected in stromal cells, osteoblasts, and osteocytes. The results indicate a potential involvement of MMP13 in ameloblastoma-related bone resorption and progression.

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

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

Abstract 3339: TTFields for the management of spinal metastases in in vitro and in vivo models

Abstract Spine metastases often affect cancer patients, leading to significant morbidity and mortality. Their prevalence has increased over time due to the progressive improvements in cancer management which led to a longer survival of these individuals. Radiation therapy is the mainstay treatment for local control of these lesions, with surgery reserved for selected cases (e.g., spinal instability, neurological deficits secondary to neural compression requiring urgent decompression, separation surgery). Chemotherapy addresses the diffusely metastatic spread. However, when spinal metastases progress despite using all the appropriate available treatment, there follows a progressive decline in function and further treatment options are limited, highlighting the need for development of novel therapies. Tumor treating fields (TTFields) has demonstrated to impair tumor cell replication and are influenced by surrounding tissue, such as bone (presents non-conductive properties). Our goal was to investigate the effects of TTFields in in vitro and in vivo spinal metastasis models. We hypothesize that the deposition of TTFields is increased in bone, and this would ultimately result in vertebral metastasis growth suppression. A computational model using a simulated spine resection cavity with metallic hardware was used to investigate the TTFields intensity in various tissues. For in vitro studies, luciferase tagged KRIB osteosarcoma and A549 lung adenocarcinoma cell lines were cultured in demineralized human bone graft. These were treated with TTFields and cell viability was quantified. In vivo assays were performed using a previous validated murine orthotopic tumor model. The effects of TTFields in these animals were monitored with imaging (bioluminescence and MRI) and clinical findings (progressive neurological deficits milestones that corresponds to the degree of spinal cord compression). At the end of the study, tissue was collected for histologic analysis. Sham stimulation was used for control groups. Computational simulation demonstrated an elevated TTFields intensity in the tumor-bone interface of the resected vertebral body. In all experimental models, TTFields significantly suppressed tumor cell growth. In vivo models, this effect on cell proliferation prevented tumor growth into the spinal canal, delaying, or even preventing, neurological deficits. TTFields inhibited tumor growth in three-dimensional cultures and orthotopic murine models of spinal metastases, preventing functional loss. The bony environment surrounding the tumor demonstrated to enhance the intensity of electric fields on a computational simulation, which stands as an advantage with the use of TTFields for vertebral tumors. These results provide solid evidence supporting future clinical trials investigating the effects of this novel and promising technology for the treatment of spine metastases. Citation Format: Romulo Augusto Andrade de Almeida, Daniel Ledbetter, Xizi Wu, Ariel Naveh, Chirag Patel, Queena Gonzalez, Thomas H. Beckham, Robert Y. North, Laurence Rhines, Christopher A. Alvarez-Breckenridge, Claudio E. Tatsui. TTFields for the management of spinal metastases in in vitro and in vivo models [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 3339.

EGCG regulated osteolytic microenvironment to enhance the antitumor effect of DOX on orthotopic osteosarcoma

Here, we demonstrated that EGCG could enhance the anti-osteosarcoma effect of DOX in vivo and in vitro. The combination of DOX and EGCG could inhibit the activity of osteoclasts at the tumor-bone interface, thereby preventing tumor-induced osteolysis and blocking the vicious circle. Taken together, EGCG may be used as an adjuvant chemotherapy drug to treat osteosarcoma. • EGCG enhanced DOX-induced proliferation inhibition of 143B Cells. • EGCG in synergy with DOX inhibited osteoclast differentiation and bone resorption in vitro. • EGCG in synergy with DOX inhibited AKT phosphorylation in 143B cells and BMMs. • EGCG could enhance the anti-osteosarcoma and anti- osteolysis effect of DOX in vivo. Osteosarcoma is the most common malignant bone tumor with the pathological essence of osteolysis. Doxorubicin (DOX) is common in adjuvant chemotherapy of osteosarcoma. However, it is required to address the conflict between enhancing its dose to improve efficacy and reducing its complications and chemotherapy resistance. Previous studies have demonstrated that EGCG has an antitumor and osteoclastogenesis inhibition effect. However, the combined effect of EGCG and DOX on osteosarcoma is still unclear. Here, EGCG was demonstrated to enhance the 143B cell proliferation inhibition and anti-osteoclastogenesis effect of DOX in vitro. The results suggest that EGCG in synergy with DOX inhibited AKT phosphorylation, which was involved in 143B cells proliferation and osteoclastogenesis. In vivo, the combination of DOX and EGCG could reduce tumor volume and osteolysis destruction compared with the DOX treatment group. This present study raised the possibility that EGCG may be used as an adjuvant chemotherapy drug to treat osteosarcoma.

Epithelial-mesenchymal transition related to bone invasion in oral squamous cell carcinoma

IntroductionBone invasion is an important prognostic factor in oral squamous cell carcinoma, leading to a lower survival rate and the use of aggressive treatment approaches. Epithelial-mesenchymal transition (EMT) is possibly involved in this process, because it is often related to mechanisms of cell motility and invasiveness. This study examined whether a panel of epithelial-mesenchymal markers are present in cases of oral squamous cell carcinoma with bone invasion and whether these proteins have any relationship with patients’ clinical-pathological parameters and prognostic factors. MethodsImmunohistochemical analysis of E-cadherin, twist, vimentin, TGFβ1, and periostin was performed in paraffin-embedded samples of 62 oral squamous cell carcinoma cases. ResultsThe analysis revealed that most cases (66%) presented with a dominant tumor infiltrative pattern in bone tissue, associated with lower survival rates, when compared with cases with a dominant erosive invasion pattern (P = 0.048). Twenty-seven cases (43%) expressed markers that were compatible with total or partial EMT at the tumor-bone interface. There was no association between evidence of total or partial EMT and other demographic or prognostic features. E-cadherin-positive cases were associated with tobacco smoking (P = 0.022); vimentin-positive cases correlated with tumors under 4 cm (P = 0.043). Twistexpression was observed in tumors with a dominant infiltrative pattern (P = 0.041) and was associated with the absence of periostin (P = 0.031). ConclusionWe observed evidence of total or partial EMT in oral squamous cell carcinoma bone invasion. The transcription factor twist appears to be involved in bone invasion and disease progression.

Abstract P033: Bone microenvironment-suppressed T cells increase osteoclast formation and the development of osteolytic bone metastases in mice

Abstract Bone metastases are a highly debilitating complication in more than 70% of patients with advanced breast and prostate cancer, causing fractures, nerve compression, hypercalcemia. Currently approved treatments fail to cure bone metastases or increase patient survival. Immunotherapies activating T cells to fight cancer cells are changing cancer treatment, causing a durable response in some patients. However, it remains unclear whether immunotherapy could benefit patients with bone metastases. The bone microenvironment combines various immunosuppressive factors that could limit its efficacy. Also, T cells could increase bone resorption releases pro-metastatic growth factors from the bone matrix that can increase cancer cell growth. Using syngeneic mouse models, our study revealed that bone metastases from 4T1 breast cancer contain tumor-infiltrating lymphocyte (TILs) and that their development is increased in normal mice compared to immunodeficient and T-cell depleted mice (x3.6 and x1.6, respectively, p<0.05). This effect seemed caused by the TILs in bone, as T-cell depletion did not affect bone metastases from RM-1 prostate cancer cells that lack TILs and increased the volume of 4T1 orthotopic tumors. T cells from bone metastases expressed the pro-osteoclastic genes Rankl and Tnfa, and increased osteoclast formation ex vivo and in vivo, at the tumor-bone interface, contributing to bone metastasis development. This pro-osteoclastic effect is specific to inactivated T cells, since activated T cells, secreting IFNγ and IL-4, actually suppressed osteoclastogenesis, which could benefit patients. We confirmed that T cells in bone metastases were not activated, as >85% of T cells lacked the activation marker CD69. In addition, T cells from 4T1 bone metastases could not be activated in ex vivo cultures confirming the presence of immunosuppressive factors in this microenvironment. 4T1 bone metastases were associated with an increase of polymorphonuclear- and monocytic-MDSCs, metabolically active as they produced elevated levels of ROS and NO, respectively. While effective in other models, the PDE-5 inhibitor sildenafil and the bisphosphonate zoledronic acid did not affect the levels of MDSCs in bone metastases or their production of ROS and NO. Seeking other therapeutic targets, we found that 80% of monocytic-MDSCs are PD-L1+ in bone, which could trigger T-cell suppression since 70% express its receptor, PD-1. Collectively, our findings identified a new mechanism by which suppressed T cells increase osteoclastogenesis and bone metastases, and also provide a rationale for using immune checkpoint inhibitors since T-cell activation would increase their anti-cancer and their anti-osteoclastic properties. Citation Format: Danna L. Arellano, Patricia Juárez, Paloma S. Almeida-Luna, Felipe Olvera, Samanta Jiménez, Pierrick G. J. Fournier. Bone microenvironment-suppressed T cells increase osteoclast formation and the development of osteolytic bone metastases in mice [abstract]. In: Abstracts: AACR Virtual Special Conference: Tumor Immunology and Immunotherapy; 2021 Oct 5-6. Philadelphia (PA): AACR; Cancer Immunol Res 2022;10(1 Suppl):Abstract nr P033.

Percutaneous management of bone metastases: State of the art

Interventional radiology is playing an increasingly important role in the local treatment of bone metastases; this treatment is usually done with palliative intent, although in selected patients it can be done with curative intent. Two main groups of techniques are available.The first group, centered on bone consolidation, includes osteoplasty/vertebroplasty, in which polymethyl methacrylate (PMMA) is injected to reinforce the bone and relieve pain, and percutaneous osteosynthesis, in which fractures with nondisplaced or minimally bone fragments are fixed in place with screws. The second group centers on tumor ablation. tumor ablation refers to the destruction of tumor tissue by the instillation of alcohol or by other means. Thermoablation is the preferred technique in musculoskeletal tumors because it allows for greater control of ablation. Thermoablation can be done with radiofrequency, in which the application of a high frequency (450 Hz–600 Hz) alternating wave to the tumor-bone interface achieves high temperatures, resulting in coagulative necrosis. Another thermoablation technique uses microwaves, applying electromagnetic waves in an approximate range of 900 MHz–2450 MHz through an antenna that is placed directly in the core of the tumor, stimulating the movement of molecules to generate heat and thus resulting in coagulative necrosis. Cryoablation destroys tumor tissue by applying extreme cold. A more recent, noninvasive technique, magnetic resonance-guided focused ultrasound surgery (MRgFUS), focuses an ultrasound beam from a transducer placed on the patient's skin on the target lesion, where the waves’ mechanical energy is converted into thermal energy (65 °C–85 °C). Treatment should be planned by a multidisciplinary team. Treatment can be done with curative or palliative intent. Once the patient is selected, a preprocedural workup should be done to determine the most appropriate technique based on a series of factors. During the procedure, protective measures must be taken and the patient must be closely monitored. After the procedure, patients must be followed up.

The Combination of Radium-223 Dichloride and Bortezomib Effectively Reduces Tumor Burden and Lesion Area in the Syngeneic, Systemic 5TGM1 Multiple Myeloma Mouse Model

Radium-223 dichloride (radium-223, Xofigo®), a targeted alpha therapy, is currently used for the treatment of patients with castration-resistant prostate cancer with bone metastases. It is a calcium-mimetic that binds to hydroxyapatite in the bone and targets areas of increased bone turnover surrounding cancer lesions. It has been shown that osteoblasts incorporate Radium-223 into the bone matrix in vitro . Radium-223 inhibits disease progression by reducing tumor growth and tumor-induced pathological bone reaction in breast and prostate cancer mouse models. Multiple myeloma (MM) is a plasma-cell malignancy characterized by increased osteoclast and reduced or no osteoblast activity. Bortezomib (Velcade®), an approved treatment for MM, restores the impaired osteoblast activity in MM. Lenalidomide (Revlimid®) induces apoptosis in MM cells and disrupts the interaction between the myeloma cell and its microenvironment. Here, we report results including the effects of radium-223, bortezomib and their combination on mouse MM 5TGM1 cell proliferation in vitro . Furthermore, the anti-tumor effect of the combination of radium-223 with either bortezomib or lenalidomide on the bone lesions and tumor burden in the syngeneic 5TGM1 mouse MM model has been evaluated.Proliferation assays were performed with the mouse MM 5TGM1 cells using CellTiter-Glo® Luminescent Cell Viability Assay. In vivo experiments were performed in the syngeneic 5TGM1 mouse MM model. This model recapitulates many features of human MM including monoclonal paraprotein as well as pronounced osteolytic bone lesions. In the combination study radium-223 and bortezomib, female 7-week-old C57BL/KaLwRij mice (n=15/group) were inoculated with 5TGM1 cells via tail vein. The administration of radium-223 (300 kBq/kg, single i.v. injection) and/or bortezomib (1 mg/kg i.p., twice a week) or vehicle control was initiated 26 days later. In addition, the effect of the combination of radium-223 and lenalidomide was tested in a separate experiment using an early therapeutic setting in the syngeneic 5TGM1 MM model. The development of osteolytic lesions was monitored by radiography. Tumor cell apoptosis and necrosis were analyzed in histological samples of the hind limbs. TRACP-stained osteoclasts were counted at the tumor-bone interface. Hind limbs were also used for total activity measurement performed by a gamma-counter. The whole-body anti-tumor effect was determined measuring paraprotein (IgG2b) concentration in serum samples in the in vivo studies.Radium-223 inhibited proliferation of 5TGM1 cancer cells at 800 Bq/ml and bortezomib at 25 nM concentrations. Combination of radium-223 with bortezomib showed increased inhibition of cell proliferation in vitro . The 5TGM1 in vivo model demonstrated that both bortezomib and radium-223 decreased the tumor-induced osteolytic bone lesion area as monotherapy (p<0.05 and p<0.01, respectively), with the combination being more effective than either monotherapy alone (p<0.001). Bortezomib decreased the number of osteoclasts at the tumor-bone interface (p<0.05) and an additive decrease was observed with combination therapy with radium-223 (p<0.01) resulting in almost complete eradication of osteoclasts. The number of apoptotic cells was increased in the radium-223 group, and an increase of necrotic area was observed in the combination group of radium-223 and bortezomib. Tumor burden was lower in groups receiving either bortezomib or bortezomib and radium-223 combination as determined by serum paraprotein (IgG2b). Incorporation of radium-223 to bone was higher with combination therapy based on total activity measurements. All treatments were well tolerated in terms of fatal toxicities, cachexia and body weight loss.In conclusion, radium-223 therapy in combination with bortezomib decreased the osteolytic lesion area, tumor burden and almost completely eradicated tumor-associated osteoclasts in the 5TGM1 mouse model of myeloma bone disease. The incorporation of radium-223 to bone matrix around tumor lesions was improved, possibly via induction of osteoblast activity by bortezomib. These data provide a strong rationale for combination of radium-223 and bortezomib as possible effective therapy in MM. This is currently investigated in a Phase Ib/II trial in patients with early relapsed MM (NCT02928029). DisclosuresSuominen:Pharmatest Services Ltd.: Employment, Equity Ownership. Kähkönen:Pharmatest Services Ltd.: Employment. Mäki-Jouppila:Pharmatest Services Ltd.: Employment. Rissanen:Pharmatest Services Ltd.: Equity Ownership. Luostarinen:Pharmatest Services Ltd.: Employment. Fagerlund:Pharmatest Services Ltd.: Employment, Equity Ownership. Sjöholm:Aurexel Life Sciences Ltd.: Employment. Alhoniemi:Avoltus Oy: Employment. Kakonen:Aurexel Life Sciences Ltd.: Employment, Equity Ownership. Siemeister:Bayer AG: Employment, Equity Ownership, Patents & Royalties. Mumberg:Bayer AG: Employment, Equity Ownership, Patents & Royalties. Halleen:Pharmatest Services Ltd.: Employment, Equity Ownership. Ziegelbauer:Bayer AG: Employment, Equity Ownership, Patents & Royalties. Scholz:Bayer AG: Employment, Equity Ownership, Patents & Royalties.

Oral Squamous Cell Carcinoma Bone Invasion: Possible Roles of E-Cadherin in Osteoclastogenesis and Bone Infiltration

Introduction: Squamous cell carcinoma is the most common cancer of the oral cavity. When the tumor invades the bone tissue, the prognostic and survival rates decrease a lot, and the treatment becomes more aggressive, with several damages to the patient and health system. Many of the molecular mechanisms of bone invasion process are not understood yet, but it is already known that one of central processes of tumor evolution – adjacent tissues invasion and metastasis – is a large spectrum of phenotypic changes in epithelial cells to mesenchymal, in a process named as epithelial-mesenchymal transition (EMT). Loss of E-cadherin, an important epithelial cell adhesion protein, is a hallmark of this phenomenon. The objective of this retrospective study is to evaluate the expression of E-cadherin protein, comparing its distribution with clinical characteristics of the patients and possibly relation to EMT. Methods: Sixty-two cases with respective clinical data were analyzed by comparing immunohistochemical, H and E staining, and clinical data, observing the tumor-bone interface (TBI) and the surrounding tumor that had no direct contact with the bone surface (ST). Results: Forty cases were positive for E-cadherin (64%) with a heterogeneous pattern. Statistical analysis showed a significant difference between the presence of E-cadherin expression and tobacco smokers. Also, the equal or weaker protein expression in the ST than TBI is related to a worse overall survival. No statistically significant difference in other prognostic factors was observed. Conclusion: Our results suggest that the tumor cells that interact with the bone tissue could gain molecular changes, like partial EMT and osteoclastogenesis induction, which facilitate their migration and increase the bone resorption, resulting in a worse patient’s prognosis.

Additive Benefits of Radium-223 Dichloride and Bortezomib Combination in a Systemic Multiple Myeloma Mouse Model.

Osteolytic bone disease is a hallmark of multiple myeloma (MM) mediated by MM cell proliferation, increased osteoclast activity, and suppressed osteoblast function. The proteasome inhibitor bortezomib targets MM cells and improves bone health in MM patients. Radium-223 dichloride (radium-223), the first targeted alpha therapy approved, specifically targets bone metastases, where it disrupts the activity of both tumor cells and tumor-supporting bone cells in mouse models of breast and prostate cancer bone metastasis. We hypothesized that radium-223 and bortezomib combination treatment would have additive effects on MM. In vitro experiments revealed that the combination treatment inhibited MM cell proliferation and demonstrated additive efficacy. In the systemic, syngeneic 5TGM1 mouse MM model, both bortezomib and radium-223 decreased the osteolytic lesion area, and their combination was more effective than either monotherapy alone. Bortezomib decreased the number of osteoclasts at the tumor–bone interface, and the combination therapy resulted in almost complete eradication of osteoclasts. Furthermore, the combination therapy improved the incorporation of radium-223 into MM-bearing bone. Importantly, the combination therapy decreased tumor burden and restored body weights in MM mice. These results suggest that the combination of radium-223 with bortezomib could constitute a novel, effective therapy for MM and, in particular, myeloma bone disease.

A Role for TGFβ Signaling in Preclinical Osteolytic Estrogen Receptor-Positive Breast Cancer Bone Metastases Progression.

While tumoral Smad-mediated transforming growth factor β (TGFβ) signaling drives osteolytic estrogen receptor α-negative (ER-) breast cancer bone metastases (BMETs) in preclinical models, its role in ER+ BMETs, representing the majority of clinical BMETs, has not been documented. Experiments were undertaken to examine Smad-mediated TGFβ signaling in human ER+ cells and bone-tropic behavior following intracardiac inoculation of estrogen (E2)-supplemented female nude mice. While all ER+ tumor cells tested (ZR-75-1, T47D, and MCF-7-derived) expressed TGFβ receptors II and I, only cells with TGFβ-inducible Smad signaling (MCF-7) formed osteolytic BMETs in vivo. Regulated secretion of PTHrP, an osteolytic factor expressed in >90% of clinical BMETs, also tracked with osteolytic potential; TGFβ and E2 each induced PTHrP in bone-tropic or BMET-derived MCF-7 cells, with the combination yielding additive effects, while in cells not forming BMETs, PTHrP was not induced. In vivo treatment with 1D11, a pan-TGFβ neutralizing antibody, significantly decreased osteolytic ER+ BMETs in association with a decrease in bone-resorbing osteoclasts at the tumor-bone interface. Thus, TGFβ may also be a driver of ER+ BMET osteolysis. Moreover, additive pro-osteolytic effects of tumoral E2 and TGFβ signaling could at least partially explain the greater propensity for ER+ tumors to form BMETs, which are primarily osteolytic.

Manejo percutáneo de las metástasis óseas

Interventional radiology is playing an increasingly important role in the local treatment of bone metastases; this treatment is usually done with palliative intent, although in selected patients it can be done with curative intent. Two main groups of techniques are available.The first group, centered on bone consolidation, includes osteoplasty / vertebroplasty, in which polymethyl methacrylate (PMMA) is injected to reinforce the bone and relieve pain, and percutaneous osteosynthesis, in which fractures with nondisplaced or minimally bone fragments are fixed in place with screws. The second group centers on tumor ablation. Tumor ablation refers to the destruction of tumor tissue by the instillation of alcohol or by other means. Thermoablation is the preferred technique in musculoskeletal tumors because it allows for greater control of ablation. Thermoablation can be done with radiofrequency, in which the application of a high frequency (450 Hz–600Hz) alternating wave to the tumor-bone interface achieves high temperatures, resulting in coagulative necrosis. Another thermoablation technique uses microwaves, applying electromagnetic waves in an approximate range of 900MHz to 2450MHz through an antenna that is placed directly in the core of the tumor, stimulating the movement of molecules to generate heat and thus resulting in coagulative necrosis. Cryoablation destroys tumor tissue by applying extreme cold. A more recent, noninvasive technique, magnetic resonance-guided focused ultrasound surgery (MRgFUS), focuses an ultrasound beam from a transducer placed on the patient's skin on the target lesion, where the waves’ mechanical energy is converted into thermal energy (65°C–85°C). Treatment should be planned by a multidisciplinary team. Treatment can be done with curative or palliative intent. Once the patient is selected, a preprocedural workup should be done to determine the most appropriate technique based on a series of factors. During the procedure, protective measures must be taken and the patient must be closely monitored. After the procedure, patients must be followed up.

EphA2 Is a Clinically Relevant Target for Breast Cancer Bone Metastatic Disease.

ABSTRACTEphA2 receptor tyrosine kinase (RTK) is highly expressed in breast tumor cells across multiple molecular subtypes and correlates with poor patient prognosis. In this study, the potential role of EphA2 in this clinically relevant phenomenon is investigated as metastasis of breast cancer to bone is a major cause of morbidity and mortality in patients. It was found that the EphA2 function in breast cancer cells promotes osteoclast activation and the development of osteolytic bone disease. Blocking EphA2 function molecularly and pharmacologically in breast tumors reduced the number and size of bone lesions and the degree of osteolytic disease in intratibial and intracardiac mouse models, which correlated with a significant decrease in the number of osteoclasts at the tumor–bone interface. EphA2 loss of function in tumor cells impaired osteoclast progenitor differentiation in coculture, which is mediated, at least in part, by reduced expression of IL‐6. EPHA2 transcript levels are enriched in human breast cancer bone metastatic lesions relative to visceral metastatic sites; EphA2 protein expression was detected in breast tumor cells in bone metastases in patient samples, supporting the clinical relevance of the study's findings. These data provide a strong rationale for the development and application of molecularly targeted therapies against EphA2 for the treatment of breast cancer bone metastatic disease. © 2021 The Authors. JBMR Plus published by Wiley Periodicals LLC. on behalf of American Society for Bone and Mineral Research.

Transforming growth factor-β-regulated fractalkine as a marker of erosive bone invasion in oral squamous cell carcinoma.

Patients with oral squamous cell carcinoma (OSCC) bone invasion are surgically treated with bone resection, which results in severe physical and psychological damage. Here, we investigated the potential of fractalkine (CX3CL1), which is regulated by transforming growth factor (TGF-β), as a novel biomarker for correct prediction and early detection of OSCC-associated bone invasion. TGF-β knockdown and treatment with a TGF-β-neutralizing antibody decreased the level of fractalkine in the culture media of HSC-2 and YD10B OSCC cells. Treatment with a fractalkine-neutralizing antibody reduced TGF-β-stimulated invasion by HSC-2 and YD10B cells. Fractalkine treatment increased the viability, invasion, and uPA secretion of both OSCC cell lines. Furthermore, OSCC cell bone invasion was assessed following subcutaneous inoculation of wild-type or TGF-β knockdown OSCC cells in mouse calvaria. TGF-β knockdown prevented erosive bone invasion, reduced the number of osteoclasts at the tumor-bone interface, and downregulated fractalkine expression in mouse tumor tissues. Our results indicate that the production of fractalkine is stimulated by TGF-β and mediates TGF-β-induced cell invasion in several OSCC cell lines showing an erosive pattern of bone invasion. Fractalkine may be a useful predictive marker and therapeutic target for OSCC-induced bone destruction.

The Role of Cathepsins in the Growth of Primary and Secondary Neoplasia in the Bone

The upregulation of proteolytic enzymes has been demonstrated to promote primary tumor development and metastatic bone cancer. The secreted proteases increase tumor growth and angiogenesis, and potentiate neoplastic cell dissemination. This article reviews the role and mechanisms of cathepsins in normal physiology, cancer, bone remodeling, and the tumor–bone interface, with a specific focus on cathepsins B, D, H, G, L, and K. In this review, we highlight the role of cathepsins in primary bone cancer (i.e., osteosarcoma (OS)), as well as metastatic breast (BCa) and prostate (PCa) cancer. In addition, we discuss the clinical utility and therapeutic potential of cathepsin-targeted treatments in primary and secondary bone cancers.

Bone microenvironment-suppressed T cells increase osteoclast formation and breast cancer bone metastases

Abstract Bone metastases is a frequent (&amp;gt;75%) complication of advanced breast cancer (BCa). BCa cells in bone are supported by cytokines released during the osteoclastic resorption, and remain untreatable. T cells during immunotherapy could turn against BCa cells in bone but could also increase osteoclasts and bone metastases. Comparing the development of bone metastases from 4T1 BCa cells between Balb/C and SCID or T cell-depleted mice, the absence of T cells decreased bone metastases (−72% and −38%, respectively), while orthotopic tumors were increased. Histology confirmed that the osteoclast number was decreased at the tumor-bone interface of mice lacking T cells. Ex vivo addition of non-activated T cells increased osteoclast formation, and flow cytometry confirmed that &amp;gt;85% of T cells in bone metastases were not expressing activation markers. In sharp contrast, activated T cells were potent inhibitors of osteoclast formation, which could be beneficial for the treatment of patients. Although activated T cells expressed the anti-osteoclastic Ifng and Il4, their effect was not reversed by neutralizing antibodies, or due to a bystander effect by cytotoxic T cells. Thus, we sought to activate T cells of bone metastases. However, it was not possible to activate T cells in bone marrow cultures ex vivo. This could be due to an increase of MDSCs in 4T1 bone metastases, including M-MDSCs that were PD-L1+ (&amp;gt;85%) and could suppress PD-1+ T cells (&amp;gt;70%) in bone. Our results suggest that while T cells under the influence of the bone microenvironment promote BCa bone metastases, immunotherapy-activated T cells could suppress bone resorption and eliminate BCa cells in patients with bone metastases.

Myeloma Cells Down-Regulate Adiponectin in Bone Marrow Adipocytes Via TNF-Alpha.

ABSTRACTMultiple myeloma is caused by abnormal plasma cells that accumulate in the bone marrow and interact with resident cells of the bone microenvironment to drive disease progression and development of an osteolytic bone disease. Bone marrow adipocytes (BMAds) are emerging as having important endocrine functions that can support myeloma cell growth and survival. However, how BMAds respond to infiltrating tumor cells remains poorly understood. Using the C57BL/KaLwRij murine model of myeloma, bone marrow adiposity was found to be increased in early stage myeloma with BMAds localizing along the tumor‐bone interface at later stages of disease. Myeloma cells were found to uptake BMAd‐derived lipids in vitro and in vivo, although lipid uptake was not associated with the ability of BMAds to promote myeloma cell growth and survival. However, BMAd‐derived factors were found to increase myeloma cell migration, viability, and the evasion of apoptosis. BMAds are a major source of adiponectin, which is known to be myeloma‐suppressive. Myeloma cells were found to downregulate adiponectin specifically in a model of BMAds but not in white adipocytes. The ability of myeloma cells to downregulate adiponectin was dependent at least in part on TNF‐α. Collectively our data support the link between increased bone marrow adiposity and myeloma progression. By demonstrating how TNF‐α downregulates BMAd‐derived adiponectin, we reveal a new mechanism by which myeloma cells alter the bone microenvironment to support disease progression. © 2019 The Authors. Journal of Bone and Mineral Research published by American Society for Bone and Mineral Research.

Notch3 promotes prostate cancer-induced bone lesion development via MMP-3.

Prostate cancer metastases primarily localize in the bone where they induce a unique osteoblastic response. Elevated Notch activity is associated with high-grade disease and metastasis. To address how Notch affects prostate cancer bone lesions, we manipulated Notch expression in mouse tibia xenografts and monitored tumor growth, lesion phenotype, and the bone microenvironment. Prostate cancer cell lines that induce mixed osteoblastic lesions in bone expressed 5–6 times more Notch3, than tumor cells that produce osteolytic lesions. Expression of active Notch3 (NICD3) in osteolytic tumors reduced osteolytic lesion area and enhanced osteoblastogenesis, while loss of Notch3 in osteoblastic tumors enhanced osteolytic lesion area and decreased osteoblastogensis. This was accompanied by a respective decrease and increase in the number of active osteoclasts and osteoblasts at the tumor-bone interface, without any effect on tumor proliferation. Conditioned medium from NICD3-expressing cells enhanced osteoblast differentiation and proliferation in vitro, while simultaneously inhibiting osteoclastogenesis. MMP-3 was specifically elevated and secreted by NICD3-expressing tumors, and inhibition of MMP-3 rescued the NICD3-induced osteoblastic phenotypes. Clinical osteoblastic bone metastasis samples had higher levels of Notch3 and MMP-3 compared to patient matched visceral metastases or osteolytic metastasis samples. We identified a Notch3-MMP-3 axis in human prostate cancer bone metastases that contributes to osteoblastic lesion formation by blocking osteoclast differentiation, while also contributing to osteoblastogenesis. These studies define a new role for Notch3 in manipulating the tumor microenvironment in bone metastases.

Rictor ablation in BMSCs inhibits bone metastasis of TM40D cells by attenuating osteolytic destruction and CAF formation

The mTOR complex 2 (mTORC2) is recognized as a promising target for breast cancer treatment. As mTORC2-specific inhibitors do not yet exist, studies into the role of mTORC2 in cancer are performed by deleting Rictor or by RNAi-mediated Rictor silencing. The purpose of this study was to explore the effects of Rictor ablation in bone mesenchymal stromal cells (BMSCs) on bone metastasis of breast cancer. First, female mice with the genotype of Prx1-Cre;Rictorf/f (hereafter RiCKO) or Rictorf/f (as control) were injected intratibially with cells of the breast cancer cell line (TM40D) at 4 months of age. Three weeks later, osteolytic bone destruction was detected in metastatic legs by X-ray and micro-CT. We found that Rictor ablation in BMSCs inhibited TM40D-induced osteolytic bone destruction and resulted in greater bone volume maintenance in vivo. Lower CTX-I serum level, a decreased number of TRAP+ osteoclasts and lower Cathepsin-K expression observed at the tumor-bone interface indicated that osteoclastogenesis was inhibited in RiCKO mice. Additionally, co-culture experiments confirmed that Rictor deletion in BMSCs diminished osteoclast differentiation partly via down regulation of RANKL expression. Furthermore, Rictor deficiency was found to reduce the transition of BMSCs to CAFs coupled with decreased secretion of cytokines (IL-6, RANKL, TGFβ), which resulted in lower chemotaxis and less proliferation in TM40D cells. These results suggest that Rictor ablation in BMSCs plays dual roles in breast cancer bone metastasis: (1) repression of osteolytic bone destruction; (2) inhibition of tumor growth.

Knockdown of Bone Morphogenetic Proteins Type 1a Receptor (BMPR1a) in Breast Cancer Cells Protects Bone from Breast Cancer-Induced Osteolysis by Suppressing RANKL Expression

Background/Aims: Bone morphogenetic proteins (BMPs) and BMP receptors widely participate in osteolytic metastasis of breast cancer, while their role in tumor-stromal interaction is largely unknown. In this study, we investigated whether BMP receptor type 1a (BMPR1a) can alter the interaction between metastatic cancer cells and osteoclast precursors. Methods: Adenovirus-mediated RNA interference was used to interrupt target genes of human breast cancer cell lines and nude mice were injected intratibially with the cancer cells. Tumor-bearing mice were examined by bioluminescence imaging and microCT. Sections of metastatic legs were measured by a series of staining methods. Murine bone marrow mononuclear cells or RAW264.7 cells were cultured with conditioned media of breast cancer cells. RT-PCR, Western blotting and ELISA were used to test mRNA and protein expressions of target molecules. Results: Expression of BMPR1a of MDA-MB-231-luc cells at tumor-bone interface was apparently stronger than that of cancer cells distant from the interface. Mice injected with BMPR1a-knockdown MDA-MB-231-luc cells showed reduced tumor growth and bone destruction compared with control groups. Knockdown (KD) of BMPR1a of MDA-MB-231-luc cells or MCF-7 cells decreased the level of receptor activator for NF-κB ligand (RANKL). Level of RANKL in MDA-MB-231-luc cells or MCF-7 cells was reduced by p38 inhibitor. Compared with control group, knockdown of p38 of breast cancer cells decreased cancer-induced osteoclastogenesis. Conclusion: Knockdown of BMPR1a of breast cancer cells suppresses their production of RANKL via p38 pathway and inhibits cancer-induced osteoclastogenesis, which indicates that BMPR1a might be a possible target in breast cancer-induced osteolytic metastasis.