Bone pain is a presenting feature of bone cancers such as osteosarcoma (OS), relayed by skeletal-innervating peripheral afferent neurons. Potential functions of tumor-associated sensory neurons in bone cancers beyond pain sensation are unknown. To uncover neural regulatory functions, a chemical-genetic approach in mice with a knock-in allele for TrkA was used to functionally perturb sensory nerve innervation during OS growth and disease progression. TrkA inhibition in transgenic mice led to significant reductions in sarcoma-associated sensory innervation and vascularization, skewed tumor associated macrophage polarization, reduced tumor growth and metastasis, and prolonged overall survival. Single-cell transcriptomics revealed that sarcoma denervation was associated with phenotypic alterations in both OS tumor cells and cells within the tumor microenvironment, and with reduced calcitonin gene-related peptide (CGRP) and vascular endothelial growth factor (VEGF) signaling. Multimodal and multiomics analyses of human OS bone samples further implicated peripheral innervation and neurotrophin signaling in OS tumor biology. Next and in two parallel approaches to inhibit nerve ingrowth, we repurposed FDA-approved bupivacaine liposomes and separately blocked CGRP signaling using FDA-approved Rimegepant. Both strategies led to significant reductions in sarcoma growth, vascularity, and sarcoma-induced hyperalgesia. In sum, TrkA-expressing peripheral neurons positively regulate key aspects of OS progression and sensory neural inhibition disrupts CGRP signaling within the sarcoma microenvironment leading to significantly reduced tumor growth and improved survival. These data suggest that interventions to prevent pathological innervation of OS represent an adjunctive therapy to improve clinical outcomes and survival.

Bone pain is a presenting feature of bone cancers such as osteosarcoma (OS), relayed by skeletal-innervating peripheral afferent neurons. Potential functions of tumor-associated sensory neurons in bone cancers beyond pain sensation are unknown. To uncover neural regulatory functions, a chemical-genetic approach in mice with a knock-in allele for TrkA was used to functionally perturb sensory nerve innervation during OS growth and disease progression. TrkA inhibition in transgenic mice led to significant reductions in sarcoma-associated sensory innervation and vascularization, tumor growth and metastasis, and prolonged overall survival. Single-cell transcriptomics revealed that sarcoma denervation was associated with phenotypic alterations in both OS tumor cells and cells within the tumor microenvironment, and with reduced calcitonin gene-related peptide (CGRP) and vascular endothelial growth factor (VEGF) signaling. Multimodal and multi-omics analyses of human OS bone samples and human dorsal root ganglia neurons further implicated peripheral innervation and neurotrophin signaling in OS tumor biology. In order to curb tumor-associated axonal ingrowth, we next leveraged FDA-approved bupivacaine liposomes leading to significant reductions in sarcoma growth, vascularity, as well as alleviation of pain. In sum, TrkA-expressing peripheral neurons positively regulate key aspects of OS progression and sensory neural inhibition appears to disrupt calcitonin receptor signaling (CALCR) and VEGF signaling within the sarcoma microenvironment leading to significantly reduced tumor growth and improved survival. These data suggest that interventions to prevent pathological innervation of osteosarcoma represent a novel adjunctive therapy to improve clinical outcomes and survival.

Supplementary Data from NELL1 Regulates the Matrisome to Promote Osteosarcoma Progression

Improved treatment strategies for sarcoma rely on clarification of the molecular mediators of disease progression. Recently, we reported that the secreted glycoprotein NELL-1 modulates osteosarcoma (OS) disease progression in part via altering the sarcomatous extracellular matrix (ECM) and cell-ECM interactions. Of known NELL-1 interactor proteins, Contactin-associated protein-like 4 (Cntnap4) encodes a member of the neurexin superfamily of transmembrane molecules best known for its presynaptic functions in the central nervous system. Here, CRISPR/Cas9 gene deletion of CNTNAP4 reduced OS tumor growth, sarcoma-associated angiogenesis, and pulmonary metastases. CNTNAP4 knockout (KO) in OS tumor cells largely phenocopied the effects of NELL-1 KO, including reductions in sarcoma cell attachment, migration, and invasion. Further, CNTNAP4 KO cells were found to be unresponsive to the effects of NELL-1 treatment. Transcriptomic analysis combined with protein phospho-array demonstrated notable reductions in the MAPK/ERK signaling cascade with CNTNAP4 deletion, and the ERK1/2 agonist isoproterenol restored cell functions among CNTNAP4 KO tumor cells. Finally, human primary cells and tissues in combination with sequencing datasets confirmed the significance of CNTNAP4 signaling in human sarcomas. In summary, our findings demonstrate the biological importance of NELL-1/CNTNAP4 signaling axis in disease progression of human sarcomas and suggest that targeting the NELL-1/CNTNAP4 signaling pathway represents a strategy with potential therapeutic benefit in sarcoma patients.

The purpose of our retrospective article is to review the CT imaging features of chondrosarcomas of the chest wall with pathologic correlation. For 26 subjects with biopsy-proven chondrosarcomas of the chest wall, two musculoskeletal radiologists retrospectively reviewed 26 CT scans in consensus. Descriptive statistics were performed. The mean tumor size was 57 mm. Twenty (20/26, 77%) chondrosarcomas were located in the ribs and six (6/26, 23%) in the sternum. The majority were lytic (19/26, 73%) with <25% calcification (15/26, 58%), and with a soft tissue mass (22/27, 85%). In this study CT features of grade 1 chondrosarcoma overlapped with those of grade 2 tumors. In conclusion, chondrosarcomas of the chest wall are generally lytic with an associated soft tissue mass, showing little calcified matrix and low-to-intermediate grade.

Bone regeneration following injury is initiated by inflammatory signals and occurs in association with infiltration by sensory nerve fibers. Together, these events are believed to coordinate angiogenesis and tissue reprogramming, but the mechanism of coupling immune signals to reinnervation and osteogenesis is unknown. Here, we found that nerve growth factor (NGF) is expressed following cranial bone injury and signals via p75 in resident mesenchymal osteogenic precursors to affect their migration into the damaged tissue. Mice lacking Ngf in myeloid cells demonstrated reduced migration of osteogenic precursors to the injury site with consequently delayed bone healing. These features were phenocopied by mice lacking p75 in Pdgfra+ osteoblast precursors. Single-cell transcriptomics identified mesenchymal subpopulations with potential roles in cell migration and immune response, altered in the context of p75 deletion. Together, these results identify the role of p75 signaling pathway in coordinating skeletal cell migration during early bone repair.

A subset of clear cell chondrosarcomas may contain focal areas of low-grade conventional chondrosarcoma; however, it is rare to find foci resembling clear cell chondrosarcoma admixed with areas otherwise typical conventional chondrosarcoma. We report two patients with conventional chondrosarcoma with clear cell features occurring in the rib, one in the setting of multiple hereditary exostoses (MHE) and the other without MHE. Both patients were found to have a destructive rib mass with a soft tissue component and underwent en bloc resection. Histologic examination revealed predominantly grade 2 conventional chondrosarcomas; however, multiple foci containing large cells with pale eosinophilic to clear cytoplasm, distinct cell borders, centrally located nuclei, and conspicuous nucleoli, resembling clear cell chondrosarcoma were identified throughout the specimen. The significance of clear cell features in an otherwise typical conventional chondrosarcoma, to our knowledge, is unknown and deserves recognition. Finally, these tumors highlight the need for careful histologic examination and proper classification as unexpected findings may impact management.

Heterotopic ossification (HO) is a pathologic process characterized by the formation of bone tissue in extraskeletal locations. The hip is a common location of HO, especially as a complication of arthroplasty. Here, we devise a first-of-its-kind mouse model of post-surgical hip HO and validate expected cell sources of HO using several HO progenitor cell reporter lines. To induce HO, an anterolateral surgical approach to the hip was used, followed by disclocation and acetabular reaming. Animals were analyzed with high-resolution roentgenograms and micro-computed tomography, conventional histology, immunohistochemistry, and assessments of fluorescent reporter activity. All the treated animals' developed periarticular HO with an anatomical distribution similar to human patients after arthroplasty. Heterotopic bone was found in periosteal, inter/intramuscular, and intracapsular locations. Further, the use of either PDGFRα or scleraxis (Scx) reporter mice demonstrated that both cell types gave rise to periarticular HO in this model. In summary, acetabular reaming reproducibly induces periarticular HO in the mouse reproducing human disease, and with defined mesenchymal cellular contributors similar to other experimental HO models. This protocol may be used in the future for further detailing of the cellular and molecular mediators of post-surgical HO, as well as the screening of new therapies.

Sarcomas produce abnormal extracellular matrix (ECM) which in turn provides instructive cues for cell growth and invasion. Neural EGF Like-Like molecule 1 (NELL1) is a secreted glycoprotein characterized by its non-neoplastic osteoinductive effects, yet highly expressed in skeletal sarcomas. Here, NELL1 gene deletion markedly reduced invasive behavior across human osteosarcoma (OS) cell lines. This resulted in reduced OS disease progression, inhibited metastatic potential and improved survival in a xenograft model. These observations were recapitulated with Nell1 conditional knockout in mouse models of p53/Rb driven sarcomagenesis, including reduced tumor frequency, and extended tumor free survival. Transcriptomic and phospho-proteomic analysis demonstrated that NELL1 loss skews the expression of matricellular proteins associated with reduced FAK signaling. Culture on OS enriched matricellular proteins reversed phenotypic and signaling changes among NELL1 knockout sarcoma cells. These findings in mouse and human models suggest that NELL1 expression alters the sarcoma matrix, thereby modulating cellular invasive potential and prognosis. Disruption of NELL1 signaling may represent a novel therapeutic approach to short circuit sarcoma disease progression.

Abstract Sarcomas produce an abnormal extracellular matrix (ECM), which in turn provides instructive cues for cell growth and invasion. Neural EGF like-like molecule 1 (NELL1) is a secreted glycoprotein characterized by its nonneoplastic osteoinductive effects, yet it is highly expressed in skeletal sarcomas. Here, we show that genetic deletion of NELL1 markedly reduces invasive behavior across human osteosarcoma (OS) cell lines. NELL1 deletion resulted in reduced OS disease progression, inhibiting metastasis and improving survival in a xenograft mouse model. These observations were recapitulated with Nell1 conditional knockout in mouse models of p53/Rb-driven sarcomagenesis, which reduced tumor frequency and extended tumor-free survival. Transcriptomic and phosphoproteomic analyses demonstrated that NELL1 loss skews the expression of matricellular proteins associated with reduced FAK signaling. Culturing NELL1 knockout sarcoma cells on wild-type OS-enriched matricellular proteins reversed the phenotypic and signaling changes induced by NELL1 deficiency. In sarcoma patients, high expression of NELL1 correlated with decreased overall survival. These findings in mouse and human models suggest that NELL1 expression alters the sarcoma ECM, thereby modulating cellular invasive potential and prognosis. Disruption of NELL1 signaling may represent a novel therapeutic approach to short-circuit sarcoma disease progression. Significance: NELL1 modulates the sarcoma matrisome to promote tumor growth, invasion, and metastasis, identifying the matrix-associated protein as an orchestrator of cell–ECM interactions in sarcomagenesis and disease progression.

OBJECTIVES:Epithelioid sarcoma (ES) rarely arises in the nerve. To increase our understanding of this unusual tumor originating in the nerve, we describe the features of three cases and review the literature.METHODS:Clinical data, imaging, pathology, treatment, and follow-up are detailed. A systematic literature review was conducted.RESULTS:Two patients were male and one female; the median age was 24 years. The patients had neurologic symptoms, and the tumors arose in large nerves and ranged from 2.4 to 5.8 cm. The tumors were avid on positron emission tomography-computed tomography and showed increased signal intensity on T2-weighted magnetic resonance imaging. Centered in the nerve, the tumors grew with an infiltrative pattern and encased the nerve fascicles. All were treated with wide resection, and adjuvant treatment included combinations of chemotherapy and radiation. One recurred, and the limb was amputated. Metastases were documented to lymph nodes, lung, pleura, and skin. One patient died of disease after 54 months. Literature review including our cases showed that tumors stained with pancytokeratin (9/9), EMA (4/4), and CD34 (7/7); there was loss of INI1 in all six cases tested.CONCLUSIONS:ES rarely arises in the peripheral nerve, and its infiltrative nature often requires morbid surgery. The differential includes a variety of benign and malignant epithelioid neoplasms.

Abstract Diffuse-type tenosynovial giant cell tumor (TGCT) is a neoplasm that exhibits infiltrative growth, tends to recur locally, and is often located within a joint. We describe a challenging diagnosis and clinical course of a 14-year-old girl with malignant diffuse TGCT, highlighting the difficulty of acquiring a representative biopsy. The patient presented with a painful foot mass, which was diagnosed initially through open biopsy as chronic osteomyelitis. Because her pain persisted, additional open biopsies were performed 1 and 2.5 years after the initial biopsy, which also showed features of benign TGCT. At age 16 years, she underwent marginal resection but developed local recurrence with metastatic disease 1 year later. Core needle biopsy of the foot mass again confirmed diffuse TGCT and showed no evidence of atypical or malignant features. Malignant features were confirmed only through excisional biopsy of chest wall metastasis. The patient started chemotherapy and underwent below-the-knee amputation at age 17 years. Amputation specimen showed malignant diffuse TGCT. She died of disease progression approximately 1 year later. Metastasis of diffuse TGCT with benign histology is challenging to diagnose. Previous studies have also found that benign diffuse TGCT can transform into an aggressive, malignant tumor. This case highlights that biopsy sampling can be challenging in histologically heterogeneous tumors. Initial evaluation by a multidisciplinary team, as well as image-guided biopsy techniques, may increase diagnostic accuracy of the biopsy.

Case: A 30-year-old man presented with progressive lower right extremity pain and swelling, initially diagnosed as a deep venous thrombosis. He returned 18 months later after 2 episodes of gross hemoptysis, with chest computed tomography angiography findings concerning for tumor thrombus in the left pulmonary artery. Subsequent advanced imaging showed a lesion arising from his right femoral vein, which open biopsy revealed to be a primary intravascular mesenchymal chondrosarcoma. He underwent medical therapy, with improvement of pain and swelling and successful return to work. Conclusion: Mesenchymal chondrosarcoma is a rare pathology, and its intravascular origin makes this case extraordinarily uncommon.

Chondroblastoma is a rare benign tumor of immature cartilage cells that generally occurs in an epiphyseal location of skeletally immature individuals. However, a few studies have reported cases in older patients. The purpose of this study was to evaluate the clinical, radiographic, and pathologic features of chondroblastoma in an adult population. The pathology archives of our institution were searched for cases of chondroblastoma diagnosed in patients ≥25 years of age. Of 14 patients identified, 8 were male and 6 were female with a median age of 34 years (range = 29-54 years). Most lesions occurred in short bones of hands and feet (N = 7, 50%), followed by the long tubular bones (N = 4, 28%). All demonstrated typical histologic features of chondroblastoma, but more extensive calcification, necrosis, and degenerative changes were also seen. At follow-up (median = 73.5 months), 2 patients (17%) had local recurrence. None had metastasis. In summary, chondroblastoma in adults tends to involve the short bones of the hands and feet and demonstrate histologic changes associated with long-standing growth of a benign tumor.