Integrated transcriptomic analysis systematically reveals the heterogeneity and molecular characterization of cancer-associated fibroblasts in osteosarcoma.

BACKGROUND: Osteosarcoma (OS), with a peak incidence during the adolescent growth spurt, is correlated with poor prognosis for its high malignancy. The tumor microenvironment (TME) is highly complicated, with frequent interactions between tumor and stromal cells. The cancer-associated fibroblasts (CAFs) in the TME have been considered to actively involve in the progression, metastasis, and drug resistance of OS. This study aimed to characterize cellular heterogeneity and molecular characterization in CAFs subtypes and explore the potential targeting therapeutic strategies to improve the prognosis of OS patients. METHODS: The single-cell atlas of human OS tumor lesions were constructed from the GEO database. Then significant marker genes and potential biological functions for each CAFs subtype were identified and explored using the Seurat R package. Next, by performing the survival analyses and constructing the risk scores for CAFs subtypes, we aimed to identify and characterize the prognostic values of specific marker genes and different CAFs subtypes. Furthermore, we explored the therapeutic targets and innovative drugs targeting different CAFs subtypes based on the GDSC database. Finally, prognoses related CAFs subtypes were further validated through immunohistochemistry (IHC) on clinical OS specimens. RESULTS: Overall, nine main cell clusters and five subtypes of CAFs were identified. The differentially expressed marker genes for each CAFs clusters were then identified. Moreover, through Gene Ontology (GO) enrichment analysis, we defined the CAFs_2 (upregulated CXCL14 and C3), which was closely related to leukocyte migration and chemotaxis, as inflammatory CAFs (iCAFs). Likewise, we defined the CAFs_4 (upregulated CD74, HLA-DRA and HLA-DRB1), which was closely related to antigen process and presentation, as antigen-presenting CAFs (apCAFs). Furthermore, Kaplan-Meier analyses showed that CAFs_2 and CAFs_4 were correlated with poor clinical prognosis of OS patients. Meanwhile, therapeutic drugs targeting CAFs_2 and CAFs_4, such as 17-AAG/Docetaxel/Bleomycin and PHA-793887/NG-25/KIN001-102, were also explored, respectively. Finally, IHC assay confirmed the abundant CAFs_2 and CAFs_4 subtypes infiltration in the OS microenvironment compared with adjacent tissues. CONCLUSION: Our study revealed the diversity, complexity, and heterogeneity of CAFs in OS, and complemented the single-cell atlas in OS TME.

Sulfatinib, a novel multi-targeted tyrosine kinase inhibitor of FGFR1, CSF1R, and VEGFR1-3, suppresses osteosarcoma proliferation and invasion via dual role in tumor cells and tumor microenvironment.

Introduction: Tumor progression is driven by intrinsic malignant behaviors caused by gene mutation or epigenetic modulation, as well as crosstalk with the components in the tumor microenvironment (TME). Considering the current understanding of the tumor microenvironment, targeting the immunomodulatory stromal cells such as cancer-associated fibroblasts (CAFs) and tumor-associated macrophages (TAMs) could provide a potential therapeutic strategy. Here, we investigated the effect of sulfatinib, a multi-targeted tyrosine kinase inhibitor (TKI) of FGFR1, CSF1R, and VEGFR1-3, on the treatment of osteosarcoma (OS). Methods: In vitro, the antitumor effect was tested by clony formation assay and apoptosis assay.The inhibition of tumor migration and invasion was detected by Transwell assay, and the de-polarization of macrophage was detected by flow cytometry.In vivo, subcutaneous and orthotopic tumor models were established to verify antitumor effect, and the underlying mechanism was verified by immunohistochemistry(IHC), immunofluorescence(IF) and flow cytometry. Results: Sulfatinib suppressed OS cell migration and invasion by inhibiting epithelial-mesenchymal transition (EMT) by blocking the secretion of basic fibroblast growth factor (bFGF) in an autocrine manner. In addition, it regulated immune TME via inhibition of the migration of skeletal stem cells (SSCs) to the TME and the differentiation from SSCs to CAFs. Moreover, sulfatinib can suppress OS by modulation of the TME by inhibiting M2 polarization of macrophages. Systemic treatment of sulfatinib can reduce immunosuppression cells M2-TAMs, Tregs, and myeloid-derived suppressor cells (MDSCs) and increase cytotoxic T-cell infiltration in tumors, the lungs, and the spleens. Discussion: Our preclinical experiments have shown that sulfatinib can inhibit the proliferation, migration, and invasion of OS by playing a dual role on tumor cells and the tumor microenvironment simultaneously and systematically reverse immunosuppression to immune activation status, which could be translated into clinical trials.

[Application of rotationplasty in treatment of osteosarcoma of distal femur in children].

OBJECTIVE: To investigate the effectiveness of rotationplasty in treating osteosarcoma of distal femur in children. METHODS: A clinical data of 10 children with osteosarcoma of distal femur treated with rotationplasty between March 2014 and June 2016 was retrospectively analyzed. There were 7 boys and 3 girls with an average age of 6.7 years (range, 4-10 years). There were 4 cases of osteoblastic osteosarcoma, 4 cases of mixed osteosarcoma, and 2 cases of chondroblastic osteosarcoma. All children were staged as Enneking stage ⅡB. The disease duration ranged from 3.5 to 6.0 months (mean, 4.6 months). The lower limb functional scoring system of 1993 Musculoskeletal Tumor Society (MSTS93), Toronto Extremity Salvage Score (TESS), and knee mobility were used to evaluate postoperative function. Tumor recurrence and metastases were monitored by radiograph. RESULTS: Poor superficial incision healing occurred in 1 patient, and healed after dressing change. The other incisions healed by first intention. All children were followed up 24-72 months (mean, 52.6 months). No local recurrence was observed during follow-up. Three of the ten patients suffered from metastases including 1 dying of multiple organ dysfunction syndrome, 1 alive with tumor, and 1 tumor free survival. Painful callosities and ulcers which related to prosthetic wear occurred in 2 patients and turned up after optimizing prosthetic fit and physiotherapy. The fracture healing time was 2.5-5.0 months (mean, 3.5 months). All children could walk independently at 4 months postoperatively. At last follow-up, the MSTS93 score was 19-25 (mean, 22) and the TESS score was 87-93 (mean, 90). The extension of knee joint mobility with artificial limbs was 0°-10° (mean, 5°), and the flexion of knee joint mobility with artificial limbs was 85°-95° (mean, 90.5°). CONCLUSION: Rotationplasty in treating osteosarcoma of distal femur in children with limb salvage difficulties can effectively preserve the limb function and improve the quality of life, and it can be used as an alternative to amputation.

[Evaluation of total scapular arthroplasty after total scapulectomy for scapular tumors].

OBJECTIVE: To evaluate the effectiveness of total scapular arthroplasty after total scapulectomy for scapular tumors. METHODS: A clinical data of 17 patients with scapular tumors treated with total scapulectomy and total scapular arthroplasty between January 2010 and December 2017 were retrospectively reviewed. There were 9 males and 8 females with an average age of 34.4 years (range, 13-64 years). Seven patients were diagnosed with chondrosarcoma, 3 with osteosarcoma, 2 with Ewing's sarcoma, 1 with high-grade sarcoma, 1 with polymorphic dedifferentiated sarcoma, 1 with fibrosarcoma, 1 with plasmacytoma, and 1 with bone giant cell tumor. According to the surgical staging system described by Enneking et al, 1 patient was rated as stage 3, 8 as stageⅠB, 8 as stageⅡB. According to the classifications of shoulder girdle resections of Malawer et al, 11 patients were type ⅢB, 5 were type ⅣB, 1 was type ⅥB. The disease duration ranged from 0.5 to 8.0 months (mean, 3.2 months) and tumor size ranged from 11.0 cm×7.5 cm×6.0 cm to 18.5 cm×18.0 cm×12.5 cm. The 1993 Musculoskeletal Tumor Society (MSTS) upper limb function scoring system and shoulder mobility were used to evaluate postoperative shoulder joint function. Tumor recurrence and metastases were monitored by radiograph. RESULTS: Poor superficial incision healing occurred in 1 patient, the rest incisions achieved healing by first intention. All patients were followed up 20-72 months (mean, 45.4 months). Two of the 17 patients died of multiple organ dysfunction syndrome caused by tumor metastases; 3 patients suffered from pulmonary metastases and were alive with disease. No local recurrence occurred in all patients. The overall survival rate was 88.2% (15/17) and the disease-free survival rate was 70.6% (12/17). Rib fracture after trauma, aseptic loosening, and atrophy of the deltoid muscle occurred in 1, 1, and 1 case, respectively. The other related complication was not observed. At last follow-up, the MSTS score was 26.1±1.4, and the flexion, extension, and abduction range of motion of shoulder joint were (70.0±7.5), (31.2±11.3), and (54.4 ±12.5) °, respectively. CONCLUSION: Reconstruction with total scapular arthroplasty after total scapulectomy can obtain a satisfactory shoulder contour and an acceptable functional outcomes in patients with scapular tumors.