Tumor‐infiltrating immune cells play a crucial role in tumor progression and response to treatment. However, the limited studies on infiltrating immune cells have shown inconsistent and even controversial results for osteosarcoma (OS). In addition, the dynamic changes of infiltrating immune cells after neoadjuvant chemotherapy are largely unknown. We downloaded the RNA expression matrix and clinical information of 80 OS patients from the TARGET database. CIBERSORT was used to evaluate the proportion of 22 immune cell types in patients based on gene expression data. M2 macrophages were found to be the most abundant immune cell type and were associated with improved survival in OS. Another cohort of pretreated OS samples was evaluated by immunohistochemistry to validate the results from CIBERSORT analysis. Matched biopsy and surgical samples from 27 patients were collected to investigate the dynamic change of immune cells and factors before and after neoadjuvant chemotherapy. Neoadjuvant chemotherapy was associated with increased densities of CD3+ T cells, CD8+ T cells, Ki67 + CD8+ T cells and PD‐L1+ immune cells. Moreover, HLA‐DR‐CD33+ myeloid‐derived suppressive cells (MDSC) were decreased after treatment. We determined that the application of chemotherapy may activate the local immune status and convert OS into an immune “hot” tumor. These findings provide rationale for investigating the schedule of immunotherapy treatment in OS patients in future clinical trials.

Cooperation between primary malignant cells and stromal cells can mediate the establishment of lung metastatic niches. Here, we characterized the landscape of cell populations in the tumor microenvironment in treatment-naïve osteosarcoma using single-cell RNA sequencing and identified a stem cell–like cluster with tumor cell–initiating properties and prometastatic traits. CXCL14 was specifically enriched in the stem cell–like cluster and was also significantly upregulated in lung metastases compared with primary tumors. CXCL14 induced stromal reprogramming and evoked a malignant phenotype in fibroblasts to form a supportive lung metastatic niche. Binding of CXCL14 to heterodimeric integrin α11β1 on fibroblasts activated actomyosin contractility and matrix remodeling properties. CXCL14-stimulated fibroblasts produced TGFβ and increased osteosarcoma invasion and migration. mAbs targeting the CXCL14-integrin α11β1 axis inhibited fibroblast TGFβ production, enhanced CD8+ T cell–mediated antitumor immunity, and suppressed osteosarcoma lung metastasis. Taken together, these findings identify cross-talk between osteosarcoma cells and fibroblasts that promotes metastasis and demonstrate that targeting the CXCL14-integrin α11β1 axis is a potential strategy to inhibit osteosarcoma lung metastasis. Significance: Cooperation between stem-like osteosarcoma cells and fibroblasts mediated by a CXCL14-integrin α11β1 axis creates a tumor-supportive lung metastatic niche and represents a therapeutic target to suppress osteosarcoma metastasis.

The premetastatic niche (PMN) contributes to lung-specific metastatic tropism in osteosarcoma. However, the crosstalk between primary tumor cells and lung stromal cells is not clearly defined. Here, we dissect the composition of immune cells in the lung PMN and identify granulocytic myeloid-derived suppressor cell (gMDSC) infiltration as positively associated with immunosuppressive PMN formation and tumor cell colonization. Osteosarcoma-cell-derived extracellular vesicles (EVs) activate lung interstitial macrophages to initiate the influx of gMDSCs via secretion of the chemokine CXCL2. Proteomic profiling of EVs reveals that EV-packaged S100A11 stimulates the Janus kinase 2/signal transducer and activator of transcription 3 signaling pathway in macrophages by interacting with USP9X. High level of S100A11 expression or circulating gMDSCs correlates with the presentation of lung metastasis and poor prognosis in osteosarcoma patients. In summary, we identify a key role of tumor-derived EVs in lung PMN formation, providing potential strategies for monitoring or preventing lung metastasis in osteosarcoma.

BACKGROUND: Osteosarcoma (OSA), the most common primary bone malignancy in children and adolescents, is prone to metastases and unfavourable prognosis. Owing to its strong genomic heterogeneity, traditional chemotherapy or targeted immunotherapy has not effectively improved the related overall survival for decades. Since the landscape of the OSA tumour immune microenvironment is scarcely known, despite it playing a crucial role in predicting clinical outcomes and therapeutic efficacies, we aimed to elucidate its molecular characteristics. METHODS: The immune signature of 101 OSA samples was explored using transcriptome profiling and clinical characteristics retrieved from the Therapeutically Applicable Research to Generate Effective Treatments (TARGET) program. Correlations between the prognostic immune markers and their clinical chemotherapy responses were assessed and verified based on 45 OSA primary tumours. FINDINGS: We identified the heterogeneity underlying tumour immune signature in OSA, and found CD4+ T cells and macrophage markers CD4/IFNGR2/CD68 to be feasible prognostic factors, exerting significantly positive correlation with each other. Specifically, CSF1R, which plays an essential role in the regulation of proliferation and differentiation of macrophages, was found to be a specific signature associated with CD4/CD68, with improved OSA clinical outcomes. INTERPRETATION: The immune landscape based on CD4/CD68/CSF1R gene signatures showed considerable promise for prognostic and therapeutic stratification in OSA patients. A specific immune signature for OSA, abundantly consisting of Th1-polarised CD4+ T cells and CSF1R-related CD68+ macrophages, may improve the predictive efficacy of chemotherapy and improve prognosis in patients with OSA.