Exploring the impact of PDGFD in osteosarcoma metastasis through single-cell sequencing analysis.

PURPOSE: The overall survival rate for metastatic osteosarcoma hovers around 20%. Responses to second-line chemotherapy, targeted therapies, and immunotherapies have demonstrated limited efficacy in metastatic osteosarcoma. Our objective is to validate differentially expressed genes and signaling pathways between non-metastatic and metastatic osteosarcoma, employing single-cell RNA sequencing (scRNA-seq) and additional functional investigations. We aim to enhance comprehension of metastatic mechanisms and potentially unveil a therapeutic target. METHODS: scRNA-seq was performed on two primary osteosarcoma lesions (1 non-metastatic and 1 metastatic). Seurat package facilitated dimensionality reduction and cluster identification. Copy number variation (CNV) was predicted using InferCNV. CellChat characterized ligand-receptor-based intercellular communication networks. Differentially expressed genes underwent GO function enrichment analysis and GSEA. Validation was achieved through the GSE152048 dataset, which identified PDGFD-PDGFRB as a common ligand-receptor pair with significant contribution. Immunohistochemistry assessed PDGFD and PDGFRB expression, while multicolor immunofluorescence and flow cytometry provided insight into spatial relationships and the tumor immune microenvironment. Kaplan-Meier survival analysis compared metastasis-free survival and overall survival between high and low levels of PDGFD and PDGFRB. Manipulation of PDGFD expression in primary osteosarcoma cells examined invasion abilities and related markers. RESULTS: Ten clusters encompassing osteoblasts, osteoclasts, osteocytes, fibroblasts, pericytes, endothelial cells, myeloid cells, T cells, B cells, and proliferating cells were identified. Osteoblasts, osteoclasts, and osteocytes exhibited heightened CNV levels. Ligand-receptor-based communication networks exposed significant fibroblast crosstalk with other cell types, and the PDGF signaling pathway was activated in non-metastatic osteosarcoma primary lesion. These results were corroborated by the GSE152048 dataset, confirming the prominence of PDGFD-PDGFRB as a common ligand-receptor pair. Immunohistochemistry demonstrated considerably greater PDGFD expression in non-metastatic osteosarcoma tissues and organoids, correlating with extended metastasis-free and overall survival. PDGFRB expression showed no significant variation between non-metastatic and metastatic osteosarcoma, nor strong correlations with survival times. Multicolor immunofluorescence suggested co-localization of PDGFD with PDGFRB. Flow cytometry unveiled a highly immunosuppressive microenvironment in metastatic osteosarcoma. Manipulating PDGFD expression demonstrated altered invasive abilities and marker expressions in primary osteosarcoma cells from both non-metastatic and metastatic lesions. CONCLUSIONS: scRNA-seq illuminated the activation of the PDGF signaling pathway in primary lesion of non-metastatic osteosarcoma. PDGFD displayed an inhibitory effect on osteosarcoma metastasis, likely through the suppression of the EMT signaling pathway.

Diagnostic value of H3F3A mutation and clinicopathological features of giant cell tumours in non-long bones.

Aims: A histone H3F3A (H3.3) mutation involving a substitution in H3.3 G34 recently has been reported in GCTB within the frequency range (from 69 % to 96 %) and is a helpful diagnostic indicator of GCTB. However, the relationship between H3F3A mutations and the clinicopathological feature of GCTB involving non-long bones (irregular bones and small bones) is unclear. Methods and results: H3F3A mutations were observed in a cohort of specimens (230 samples of GCTB) using immunohistochemistry and Sanger sequencing. The relationship between H3F3A mutations and the clinicopathological characteristics of patients with GCTB occurring in the non-long bones of the appendicular skeleton was investigated. No significant difference between H3F3A mutations in GCTB arising in non-long bones and the classic sites was found (P = 0.483). GCTB in non-long bones occurred more common in female (31/49, 63.3 %) than in male patients (P = 0.016). GCTB with H3.3 G34L/V/R mutation occurred more often in younger patients compared with those with H3.3 G34W mutation (P = 0.009). The majority of GCTB with soft tissue extension developed in irregular bones but not in small bones (P = 0.061). The H3.3 G34L/V/R mutations rate (7/45) in the non-long bones was significantly higher than that in long bones. The recurrence rate of the GCTB in long bones and non-long bones was 23.3 % (45/193) including 43 cases with local recurrene and 2 cases with lung metastasis. No recurrence occurred in cases with G34V/L/R mutations. Conclusions: H3F3A was an effective diagnostic marker for GCTB of the non-long bones. The younger patients with GCTB of the non-long bones harboured H3.3 G34L/V/R mutations and may had a female preference and rarely recurrent.

Organoid culture system for patient-derived lung metastatic osteosarcoma.

Osteosarcoma (OS) is the most common primary bone malignancy with high rates of recurrence and metastasis. OS often spreads to lungs, an optimized model for studying lung metastatic OS cells may help develop potential therapies for patients with lung metastasis. Here we firstly report an organoid culture system for lung metastatic OS tissues. We provided a fully described formula that was required for establishing lung metastatic OS organoids (OSOs). Using this protocol, the lung OSOs were able to be maintained and serially propagated for at least six months; the OSOs can also be generated from cryopreserved patient samples without damaging the morphology. The patient-derived lung OSOs retained the cellular morphology and expression of OS markers (Vimentin and Sox9) that recapitulate the histological features of the human OS. The microenvironment of primary lung metastatic OSOs preserved a similar T cell distribution with the human lung OS lesions; this provided a possible condition to explore how OS cells may react to immunotherapy. OSOs established from this protocol can be further utilized for studying various aspects of OS biology (e.g., tumorigenesis and drug screen/discovery) for precision medicine.

Clonality analysis and IDH1 and IDH2 mutation detection in both components of dedifferentiated chondrosarcoma, implicated its monoclonal origin.

Dedifferentiated chondrosarcoma (DDCS) is a highly malignant tumor that belongs to an uncommon subtype of chondrosarcoma with a poor prognosis. Microscopically, it is composed of highly differentiated chondrosarcoma and highly malignant noncartilaginous sarcomas with an abrupt interface. The question of whether the two components originated from the same archaeocyte has not yet been clarified. To further investigate this issue, DNA was separately extracted from the two components of the same patient. In total, 18 DDCS patients were analyzed. A portion of DNA samples from 9 female patients was used for clonality analysis. Another portion of DNA from 9 female and DNA from 9 male patients was used for isocitrate dehydrogenase 1(IDH1) and IDH2 gene mutation detection. The results of clonality analysis showed that the same X chromosome inactivation and consistent mutation states of the IDH1 and IDH2 genes in the two DDCS components. We conclude that the two DDCS components originate from the same primitive cell and that DDCS is monoclonal in origin.

CDKL3 promotes osteosarcoma progression by activating Akt/PKB.

Osteosarcoma (OS) is a primary malignant bone neoplasm with high frequencies of tumor metastasis and recurrence. Although the Akt/PKB signaling pathway is known to play key roles in tumorigenesis, the roles of cyclin-dependent kinase-like 3 (CDKL3) in OS progression remain largely elusive. We have demonstrated the high expression levels of CDKL3 in OS human specimens and comprehensively investigated the role of CDKL3 in promoting OS progression both in vitro and in vivo. We found that CDKL3 regulates Akt activation and its downstream effects, including cell growth and autophagy. The up-regulation of CDKL3 in OS specimens appeared to be associated with Akt activation and shorter overall patient survival (P = 0.003). Our findings identify CDKL3 as a critical regulator that stimulates OS progression by enhancing Akt activation. CDKL3 represents both a biomarker for OS prognosis, and a potential therapeutic target in precision medicine by targeting CDKL3 to treat Akt hyper-activated OS.

Hepatoprotective effect of exosomes from human-induced pluripotent stem cell-derived mesenchymal stromal cells against hepatic ischemia-reperfusion injury in rats.

BACKGROUND: This study aimed to evaluate the effect of exosomes produced by human-induced pluripotent stem cell-derived mesenchymal stromal cells (hiPSC-MSCs-Exo) on hepatic ischemia-reperfusion (I/R) injury. METHODS: Exosomes were isolated and concentrated from conditioned medium using ultracentrifugation and ultrafiltration. hiPSC-MSCs-Exo were injected systemically via the inferior vena cava in a rat model of 70% warm hepatic I/R injury, and the therapeutic effect was evaluated. The serum levels of transaminases (aspartate aminotransferase [AST] and alanine aminotransferase [ALT]) were measured using an automatic analyzer. The expression of inflammatory factors was measured using enzyme-linked immunosorbent assay (ELISA). Histological changes indicated changes in pathology and inflammatory infiltration in liver tissue. Apoptosis of hepatic cells in liver tissue was measured using terminal-deoxynucleoitidyl transferase mediated nick end labeling (TUNEL) staining along with apoptotic markers. RESULTS: hiPSCs were efficiently induced into hiPSC-MSCs with typical MSC characteristics. hiPSC-MSCs-Exo had diameters ranging from 50 to 60 nm and expressed exosomal markers (CD9, CD63 and CD81). Hepatocyte necrosis and sinusoidal congestion were markedly suppressed with a lower Suzuki score after hiPSC-MSCs-Exo administration. The levels of the hepatocyte injury markers AST and ALT were significantly lower in the treated group than in the control group. Inflammatory markers, such as tumor necrosis factor (TNF)-α, interleukin (IL)-6 and high mobility group box 1 (HMGB1), were significantly reduced after administration of hiPSC-MSCs-Exo, which suggests that the exosomes have a role in suppressing the inflammatory response. Additionally, in liver tissues from the experimental group, the levels of apoptotic markers, such as caspase-3 and bax, were significantly lower and the levels of oxidative markers, such as glutathione (GSH), glutathione peroxidase (GSH-Px) and superoxide dismutase (SOD), were significantly higher than in the control group. These data point to an anti-apoptotic, anti-oxidative stress response role for hiPSC-MSCs-Exo. CONCLUSIONS: Our results demonstrated that hiPSC-MSCs-Exo alleviate hepatic I/R injury, possibly via suppression of inflammatory responses, attenuation of the oxidative stress response and inhibition of apoptosis.

SUMO-specific protease 1 regulates pancreatic cancer cell proliferation and invasion by targeting MMP-9.

SUMOylation is a dynamic process which can be reversed by a family of sentrin/SUMO-specific protease (SENPs). Recently, SENP1, a member of SENPs family was shown to have a pro-oncogenic role in many types of cancer. Here, we showed that SENP1 was upregulated in pancreatic ductal adenocarcinoma (PDAC) tissues compared with adjacent normal tissues. Moreover, clinical data showed that SENP1 was positively associated with lymph node metastasis and TNM stage. Furthermore, knockdown of SENP1 by SENP1-siRNA inhibited pancreatic cancer cell proliferation, migration, and invasion, suggesting that SENP1 played an important role in PDAC progression and metastasis. Mechanistically, silencing of SENP1 results in downregulation of MMP-9, which is pivotal for PDAC cell growth and migration. Taken together, these results suggest that SENP1 may serve as a potential novel diagnostic and therapeutic target of PDAC.

miR-212 promotes pancreatic cancer cell growth and invasion by targeting the hedgehog signaling pathway receptor patched-1.

BACKGROUND: microRNAs (miRNAs) are a class of small non-coding RNAs that play important roles in carcinogenesis. In the present study, we investigated the effect of miR-212 on pancreatic ductal adenocarcinoma (PDAC) and its target protein. METHODS: Quantitative real-time PCR(qRT-PCR) was performed to detect the expression of miR-212 in PDAC tissues and pancreatic cancer cell lines. miR-212 mimic, miR-212 inhibitor and negative control were transfected into pancreatic cancer cells and the effect of miR-212 up-regulation and down-regulation on the proliferation, migration and invasion of cells were investigated. Furthermore, the mRNA and protein levels of Patched-1(PTCH1) were measured. Meanwhile, luciferase assays were performed to validate PTCH1 as miR-212 target in PDAC. RESULTS: miR-212 was up-regulated in PDAC tissues and cells.Using both gain-of function and loss-of function experiments, a pro-oncogenic function of miR-212 was demonstrated in PDAC. Moreover, up-regulated of PTCH1 could attenuate the effect induced by miR-212. CONCLUSION: These data suggest that miR-212 could facilitate PDAC progression and metastasis through targeting PTCH1, implicating a novel mechanism for the progression of PDAC.