Osteosarcoma is a highly malignant tumor with poor prognosis. Current CAR-T cell therapies for osteosarcoma are predominantly designed with single targets, but their efficacy remains unsatisfactory. In this study, a novel bispecific CAR-T cell was developed to provide an experimental basis for improving the therapeutic outcome of osteosarcoma. Single-cell RNA sequencing (scRNA-seq) identified two antigens highly expressed in osteosarcoma cells, ANXA2 and CD147, whose expression was further validated at the tissue level by qRT-PCR, flow cytometry, and immunohistochemistry. Based on a second-generation CAR backbone, a bispecific ANXA2/CD147 CAR-T construct was generated using magnetic bead sorting, primary T-cell culture, and lentiviral transduction, achieving a transduction efficiency of 47.1%. LDH release assays demonstrated that bispecific CAR-T cells exhibited significantly greater cytotoxicity against tumor cells than single-target and control groups. ELISA confirmed that bispecific CAR-T cells released higher levels of effector molecules, including GZMB and TNFα. In a subcutaneous CDX model, bispecific CAR-T cells displayed superior antitumor activity and greater T-cell infiltration. In a paw pad xenograft model, mice treated with bispecific CAR-T cells exhibited the smallest tumor volumes, lowest tumor weights, and reduced rates of lymph node metastasis. Furthermore, PDX models confirmed that bispecific CAR-T cells effectively suppressed osteosarcoma growth. ScRNA-seq of tumors derived from CDX models and immunohistochemistry revealed markedly increased infiltration of M1 macrophages in the bispecific group. Collectively, this study successfully generated a bispecific ANXA2/CD147 CAR-T cell with robust antitumor activity, providing a promising new strategy for the immunotherapy of osteosarcoma.

Intervertebral disc degeneration (IVDD) is a predominant cause of low back pain, and mesenchymal stem cell (MSC) transplantation represents a promising therapeutic strategy. However, its efficacy is severely limited by the harsh oxidative microenvironment of the degenerative disc, which rapidly triggers ferroptosis, an iron-dependent form of cell death, in transplanted MSCs. This review critically appraised current ferroptosis-inhibition strategies, highlighting their transient or single-axis limitations. We then synthesized a hierarchical framework for engineering robust MSC resistance, progressing from dual-target gene circuits and genetic-pharmacological alliances to smart, protective biomaterial niches. Conventional approaches provide only partial protection. In contrast, advanced multi-layered strategies, including dual-target gene circuits (e.g., the Prominin-2/FBXO22/BACH1 axis) potentiated by genetic-pharmacological alliances (e.g., with TBE56), confer superior, cell-intrinsic resilience, increasing MSC survival by approximately 1.5-fold and significantly improving regenerative outcomes in IVDD models. Furthermore, encapsulating engineered MSCs in responsive biomaterials establishes a protective niche, ensuring sustained function. The paradigm is shifting from passive protection to active cellular empowerment. Engineering MSCs with multi-layered, comprehensive ferroptosis shielding is fundamental to unlocking their full therapeutic potential. This engineered cellular empowerment strategy represents a paradigm shift from palliative care to potentially curative, regenerative treatment for IVDD, with the potential to fundamentally change clinical management by addressing the root cause of MSC therapy failure.

Macrophages contribute to osteoarthritis (OA) pathogenesis, but how specific macrophage states influence chondrocyte senescence remains unclear. Using single-cell RNA sequencing of naturally aged mouse joints, we identified a PTGS2-high macrophage subset associated with joint senescence. In a surgically induced OA model, lineage tracing revealed a marked increase of these macrophages, and their selective depletion using a diphtheria toxin receptor-based mouse model attenuated cartilage senescence and disease progression. PTGS2+ macrophage-derived THBS1 promoted chondrocyte senescence through SDC4-associated pericellular interactions and, at least in part, TGF-β-dependent signaling. Macrophage-specific deletion of Thbs1 reduced chondrocyte senescence markers and attenuated OA-like degeneration. Mechanistically, elevated glycolytic lactate enhanced THBS1 lactylation at lysine 262 (K262), which reduces NEDD4-dependent ubiquitination and stabilizes THBS1 protein. Intra-articular targeting of PTGS2-high macrophages attenuated chondrocyte senescence and OA progression. These findings link macrophage metabolic remodeling to a pro-senescent secreted pathway and suggest a locally delivered strategy for OA.

There is no study on the relationship between peripheral blood different lymphocyte subtypes and the prognosis of osteosarcoma (OS). Therefore, this study aims to investigate the predictive value of T cells and natural killer (NK) cells for the prognosis of OS patients. This study retrospectively analyzed the clinical data and preliminary laboratory indicators of patients with OS admitted from dual-center between January 2014 and January 2021. The receiver operating characteristic (ROC) curve was employed to determine optimal cutoff values for different lymphocyte subtypes, with T cells, NK cells, and B lymphocytes subsequently stratified into high- and low-proportion groups based on their respective optimal cutoff values. Kaplan–Meier curve was employed to analyze the impact of different lymphocyte on survival time and status. Univariate and multivariate Cox analyses were performed on clinical and laboratory indicators to identify independent prognostic factors influencing the prognosis of OS patients. After screening 277 patients with OS, a total of 106 patients were eligible for this study. The median follow-up time was 36.00 months. At the last follow-up, patients were categorized as having a good prognosis if they survived or a poor prognosis if they died: good prognosis (n = 48) and poor prognosis (n = 58). Kaplan–Meier curve revealed that patients with a high proportion of T (Median overall survival: 41 months vs. 32 months, P = 0.007) and NK (Median overall survival: 44 months vs. 32 months, P = 0.004) cells had a better prognosis compared to those with a low proportion. Univariate analysis indicated that age, body mass index (BMI), C-reactive protein (CRP), tumor size, Enneking stage, surgical method, and the proportions of T, NK, and B cells were associated with the prognosis of OS patients (P < 0.05). Multivariate analysis indicated that Enneking stage (II vs. I, HR = 12.543, P = 0.015; III vs. I, HR = 29.078, P = 0.001), and the proportions of T and NK cells (HR = 0.466, P = 0.048; HR = 0.497, P = 0.029) were independent factors influencing the prognosis of OS patients (P < 0.05). The proportions of T and NK cells may serve as efficient and practical prognostic indicators for OS patients, with higher proportions often associated with a better prognosis.

Articular cartilage, owing to the lack of undifferentiated stem cells after injury, faces significant challenges in reconstruction and repair, making it a major clinical challenge. Therefore, there is an urgent need to design a multifunctional hydrogels capable of recruiting autologous stem cells to achieve in situ cartilage regeneration. Here, our study investigated the potential of a piezoelectric hydrogel (Hyd6) for enhancing cartilage regeneration through ultrasound (US) stimulation. Hyd6 has multiple properties including injectability, self-healing capabilities, and piezoelectric characteristics. These properties synergistically promote stem cell chondrogenesis. The fabrication and characterization of Hyd6 revealed its excellent biocompatibility, biodegradability, and electromechanical conversion capabilities. In vitro and in vivo experiments revealed that Hyd6, when combined with US stimulation, significantly promotes the recruitment of autologous stem cells and enhances chondrogenesis by generating electrical signals that promote the influx of Ca2+, activating downstream CaM/CaN signaling pathways and accelerating cartilage formation. An in vivo study in a rabbit model of chondral defects revealed that Hyd6 combined with US treatment significantly improved cartilage regeneration, as evidenced by better integration of the regenerated tissue with the surrounding cartilage, greater collagen type II expression, and improved mechanical properties. The results highlight the potential of Hyd6 as a novel therapeutic approach for treating cartilage injuries, offering a self-powered, noninvasive, and effective strategy for tissue engineering and regenerative medicine.

BACKGROUND:Our preliminary research has revealed that Prominin-2 overexpression effectively guarded against oxidative stress (OS)-induced ferroptosis by decreasing BTB and CNC homolog 1 (BACH1) expression, thus promoting bone marrow mesenchymal stem cells (BMSCs) survival under the OS microenvironments in degenerative discs. METHODS:In this study, we probed how Prominin-2 controls the BACH1 expression in OS-induced BMSC ferroptosis. We then evaluated the efficiency of targeted Prominin-2/BACH1 pathway in BMSCs in treating degenerative nucleus pulposus cells (NPCs) and intervertebral disc degeneration (IVDD). RESULTS:Using lentivirus infection and Western Blot, we observed that F-box only protein 22 (FBXO22) levels decreased in OS-induced BMSCs while overexpressing Prominin-2 restored its expression and pharmacological inhibition of FBXO22 impaired Prominin-2-mediated BACH1 degradation. The pull-down assay further confirmed the essential role of FBXO22 in the degradation of BACH1 promoted by Prominin-2. FBXO22 overexpression suppressed BMSCs' ferroptosis, and FBXO22 activity enhancer TBE56 (biotinylated TBE31) could further improve Prominin-2-overexpressed BMSCs' viability under OS circumstances. Finally, in vitro co-culture and in vivo studies illustrated that engraftment of Prominin-2-overexpressed BMSCs pre-treated by TBE56 enhanced the treatment efficiency of BMSCs for degenerative NPCs and rats' IVDD. CONCLUSIONS:Our data proposed a novel treatment strategy targeting the ferroptosis of BMSCs for treating IVDD by regulating FBXO22 in Prominin-2-overexpressed BMSCs.

BACKGROUND:Osteosarcoma is a rare malignant tumor originating from bone tissue. Despite advancements in neoadjuvant chemotherapy, the 5-year survival rate for osteosarcoma patients has plateaued around 60 % for the past fifty years, primarily due to the development of chemo-insensitivity. Cisplatin, a cornerstone in current treatment regimens, still has a low response rate in osteosarcoma patients, highlighting the need for strategies to enhance cisplatin sensitivity. PURPOSE:The purpose of this study is to explore the effects of formononetin, a bioactive compound, in sensitizing osteosarcoma cells to cisplatin. STUDY DESIGN:We utilized PDX models of osteosarcoma to evaluate the combined therapeutic effect of formononetin and cisplatin. Single-cell RNA sequencing and single-cell ATAC sequencing were performed on tumor tissues from these models to provide a detailed molecular profile of the treatment effects. METHODS:PDX models of osteosarcoma were established, followed by treatment with formononetin and cisplatin. A total of 7216 human-derived osteosarcoma cells and 89,558 mouse-derived cells were analyzed to assess their role in cisplatin sensitivity and tumor immune microenvironment changes. RESULTS:Our findings demonstrated that cisplatin insensitivity in osteosarcoma is strongly linked to ferroptosis. Formononetin sensitized osteosarcoma cells to cisplatin by inhibiting MAZ/GPX4 axis and inducing ferroptosis. Additionally, formononetin increased NK cell infiltration and immune activity, while reducing the infiltration of exhausted Cd8+ T cells and tumor-associated neutrophils, thereby reprogramming the tumor immune microenvironment and further enhancing cisplatin sensitivity. CONCLUSION:This study is the first to demonstrate that formononetin can enhance cisplatin sensitivity in osteosarcoma. By using osteosarcoma PDX models and performing comprehensive single-cell sequencing analyses, we identified formononetin as a promising sensitizer for cisplatin treatment. Our findings offer new therapeutic insights and mechanistic understanding that could help overcome cisplatin insensitivity in osteosarcoma and potentially improve patient outcomes.

Osteoarthritis (OA) is a prevalent joint disease characterized by pain, disability, and loss of physical function, posing a challenge to public health. However, molecular mechanisms of OA pathogenesis have not been fully described. We report that tripartite motif containing 15 (TRIM15) is a regulator in chondrocyte senescence and OA. Our study revealed heightened expression of TRIM15 in chondrocytes of senescent cartilage from patients with OA and in aged wild-type mice. Using gain- and loss-of-function studies, we found that TRIM15 facilitated human chondrocyte senescence. Conditional deletion of Trim15 in mouse chondrocytes severely impaired skeletal growth, partially because of impaired embryonic chondrocyte senescence. Compared with conditionally knocked out Col2a1-CreERT2/Trim15flox/flox mice, Trim15flox/flox control mice exhibited accelerated OA phenotypes, increased senescence markers, and senescence-associated secretory phenotype during aging. Mechanistically, TRIM15 bound with yes-associated protein (YAP) and mediated K48-linked YAP ubiquitination at K254, which interrupted the interaction between YAP and angiomotin, leading to enhanced YAP nuclear translocation. Dysregulation of TRIM15-YAP and transcriptional coactivator with PDZ-binding motif (TAZ) signaling promoted OA progression in both the surgery-induced and natural aging-induced mouse OA model. Intra-articular injection of adeno-associated virus 5 (AAV5)-Trim15 shRNA decelerated OA progression in mice. In particular, YAP and TAZ protein amounts were increased in chondrocytes of patients with OA. Our preclinical results demonstrated that the AAV5-TRIM15 shRNA treatment protected human OA explants against degeneration through inhibiting chondrocyte senescence. Together, our findings underscore the potential of targeting TRIM15 in reshaping the aging cartilage microenvironment and suggest a promising therapeutic avenue for OA.

Rotator cuff tear (RCT) is a common musculoskeletal disease that poses challenges for functional regeneration of the tendon-bone interface (TBI). The transition of TBI between soft and hard tissues determines its structural and physiological environment complexity. Here, we present an injectable biopiezoelectric material PVA/CNF/BTO@PDA (Piezoelectric) hydrogel based on three-dimensional (3D) printing inspired by the "muscle-electrical coupling". This Piezoelectric hydrogel indicated desirable piezoelectric and mechanical properties, excellent biodegradability, and biosafety. In vitro, electrical stimulation from Piezoelectric hydrogel by the Flexcell Tissue Train system promoted the polarization of macrophages to the M2 phenotype, directing the targeted aggregation and zonal-specific differentiation of bone mesenchymal stem cells (BMSCs) for TBI formation. Also, optimal piezoelectric stimulation of the Piezoelectric hydrogel could alleviate inflammatory factor expression and regulate the osteotendinogenic differentiation of BMSCs under an H2O2/IL-1β inflammation environment. Furthermore, in vivo application of injectable Piezoelectric hydrogel demonstrates its regenerative potential, indicating that physiological repair with Piezoelectric hydrogel significantly accelerates and promotes TBI healing in a chronic RCT model. Therefore, our findings propose a new therapeutic strategy for functional TBI regeneration and enhance the treatment outcomes for RCT.

Resistance to and associated toxic side effects of neoadjuvant chemotherapy remain major obstacles to improving the prognosis of osteosarcoma patients. Consequently, there is an urgent need to discover effective therapeutic agents with lower toxicity. In this study, the patient-derived xenograft (PDX) model was established and single-cell multi-omics sequencing was performed to comprehensively analyze changes in cellular heterogeneity and gene expression patterns of under formononetin treatment. We found that formononetin can significantly inhibit tumor growth in the osteosarcoma PDX model, on which the single-cell sequencing identified MYO1B as a key target mediating the anti-osteosarcoma effects of formononetin. In vitro experiments demonstrated that MYO1B overexpression enhanced the proliferation, invasion, and migration of osteosarcoma cells, while MYO1B silencing exhibited the opposite effects. Further investigation revealed that formononetin treatment markedly downregulated MYO1B expression, effectively suppressing the proliferative, invasive, and migratory phenotypes of osteosarcoma cells. Moreover, single-cell transcriptomic analysis of murine-derived cells showed that formononetin enhanced the cytotoxic activity of NK cells, promoted M1 macrophage polarization and inhibited M2 polarization, and reduced the proportion of senescent neutrophils, thereby alleviating the immunosuppressive state of the tumor microenvironment. Overall, our findings provide a comprehensive single-cell-level elucidation of the molecular mechanisms underlying the anti-osteosarcoma effects of formononetin, primarily involving downregulating the expression of MYO1B and remodeling the tumor immune microenvironment.

Osteoarthritis (OA) is a whole-joint disorder that interferes with the quality of life in older individuals. Here we report that ZDHHC11 is highly expressed in articular chondrocytes but is downregulated in the degenerated cartilage of aged mice and patients with OA. ZDHHC11 prevents chondrocyte senescence and promotes cartilage anabolism, culminating in an improved OA phenotype. The deletion of Zdhhc11 in mice (Zdhhc11fl/fl) exacerbates OA progression in a destabilized medial meniscus model. Specifically, we identify ZDHHC11 as a key palmitoyltransferase whose depletion leads to a GNB2-dependent E3 ubiquitin ligase-mediated proteasomal degradation of APOD. Mechanistically, ZDHHC11-mediated palmitoylation alleviates OA progression by deactivating the GATA4–P65 signaling pathway. We also propose an original lipid nanoparticle-based platform for Zdhhc11 mRNA delivery to rejuvenate impaired cartilage by specifically targeting chondrocytes in vivo. Collectively, ZDHHC11-dependent palmitoylation is essential for ameliorating OA, and the targeted delivery of ZDHHC11 may serve as a promising strategy for future OA treatment. Wang, He, Gong and colleagues identify an age-related decline in the palmitoyltransferase ZDHHC11 in chondrocytes that leads to senescence and the pathogenesis of osteoarthritis, highlighting the potential of targeted ZDHHC11 delivery as a therapeutic strategy for osteoarthritis.

OBJECTIVES:To investigate the inhibitory effect of polyphyllin VII (PP7) on osteosarcoma xenograft growth in mice and explore the underlying molecular mechanism. METHODS:Ultra‑performance liquid chromatography‑tandem mass spectrometry was used to analyze the main active components of Paris polyphylla. Six nude mice bearing patient‑derived xenograft (PDX) were randomized into two groups for treatment with 2 mg/kg PP7 gavage or saline every other day for 28 days, and the changes in tumor volume and mass were measured. In cultured 143B and HOS cells, the effect of PP7 treatment (0, 1.25, 2.5, 5, and 10 μmol/L) on cell proliferation was assessed with CCK‑8 assay, and Transwell assays were employed to examine the changes in cell migration and invasion. The target of PP7 was predicted by integrated analyses with single‑cell RNA sequencing (scRNA‑seq), bulk RNA sequencing (bulk RNA‑seq) and molecular docking and verified using Western blotting. In osteosarcoma cells transfected with SOHLH1 siRNAs or a negative control sequence, the effects of PP7 treatment (5 μmol/L) on cell migration, invasion, ferroptosis, reactive oxygen species (ROS) production and lipid peroxidation (LPO) were analyzed. RESULTS:PP7 was identified as one of the major active constituents of Paris polyphylla. In the tumor-bearing mice, PP7 treatment significantly lower the tumor volume and mass. In 143B and HOS cells, PP7 concentration‑dependently inhibited cell proliferation, and at 5 μmol/L, PP7 significantly inhibited cell proliferation, migration and invasion. Multi‑omics analysis identified SOHLH1 as a potential target of PP7, and Western blotting confirmed that PP7 upregulated SOHLH1 expressions at both the mRNA and protein levels. SOHLH1 silencing obviously attenuated the inhibitory effects of PP7 on cell migration and invasion and reduced PP7‑induced ferroptosis. CONCLUSIONS:PP7 suppresses osteosarcoma xenograft growth in mice by inducing ferroptosis via upregulating SOHLH1 expression.

As the most common primary malignant bone tumor, osteosarcoma (OS) is characterized by drug resistance and poor prognosis, highlighting the urgent need for promising therapeutic agents. Formononetin (FMN), a natural product derived from Spatholobi Caulis, has been reported to possess anti-tumor properties. However, its role in OS has not yet been elucidated. In the present study, we established an OS patient-derived xenograft model to investigate the effects of FMN and the underlying mechanisms of its effects on OS. When FMN treatment was completed, bulk transcriptome sequencing was conducted, and the analyses were combined with OS single-cell RNA sequencing (scRNA-seq) data. Results indicated that GTSF1 was up-regulated in OS but down-regulated after FMN intervention, which may regulate the apoptosis of OS cells. Furthermore, the qRT-PCR and IHC results demonstrated that GTSF1 expression was significantly up-regulated in OS cells, whereas FMN expression was down-regulated both in vitro and in vivo. Moreover, in vitro experiments revealed that FMN effectively promoted apoptosis and suppressed the proliferation, migration, and invasion of OS cells. Therefore, this study demonstrated that FMN exerts anti-OS effects by down-regulating GTSF1 expression, thus effectively promoting the apoptosis and inhibiting the proliferation of OS cells, making FMN a promising anti-OS drug.

ObjectiveThe best method for femoral fixation in anterior cruciate ligament reconstruction (ACLR) remains controversial. The study assesses the bone tunnel enlargement and clinical outcome in hamstring ACLR using cortical suspension or hybrid (cortical suspension and compression) femoral fixation.MethodsFrom January 2010 to December 2021, 102 patients who underwent quadruple hamstring ACLR using cortical suspension (39 patients) or hybrid (63 patients) fixation on the femoral side were retrospectively analyzed. Clinical evaluation was conducted using the international knee documentation committee score, the Lysholm score, the Tegner activity level scale, the knee injury and osteoarthritis outcome score (quality of life score), the Lachman test, and the side‐to‐side difference by the KT‐1000 arthrometer. The complications after the surgery were also evaluated. These data were compared at baseline and last follow‐up. The diameters of the femoral tunnel were calculated at three sites: the width of the entrance of the femoral tunnel, 1 cm proximal to the entrance of the femoral tunnel and the largest diameter of the femoral tunnel on magnetic resonance imaging (MRI) coronal images. Bone tunnel widening data were contrasted between MRI images conducted at least 2 years and within 2 weeks after surgery. The morphology of bone tunnel enlargement was also observed and recorded. The categorical parameters were analyzed using the χ2‐test and Fisher's exact test. The continuous variables conforming to a normal distribution were analyzed using Student's t‐test, and the Mann–Whitney U‐test was undertaken between the two groups without normal distribution.ResultsBoth cortical suspension and hybrid femoral fixation in quadruple hamstring ACLR achieved significantly improved patient‐reported outcome scores and knee stability compared to preoperative data. However, no significant differences were found between these two methods in clinical evaluations, postoperative complications, and patient‐reported outcome scores. Although the mean diameter of the enlarged bone tunnel was lowered by an additional bioabsorbable interference screw fixation near the joint line, a statistically insignificant difference was found between the hybrid and cortical suspension fixation on the femoral side. There was no statistical difference in the distribution of enlarged bone tunnel morphology between groups.ConclusionsNo significant difference was found in the bone tunnel enlargement and clinical outcome between cortical suspension and hybrid femoral fixation in ACLR using hamstring autograft.

Background Due to the unclear pathogenesis of osteoarthritis (OA), effective treatment for this ailment is presently unavailable. Accumulating evidence points to chondrocyte senescence as a key driver in OA development. This study aims to identify OA-specific microRNAs (miRNAs) targeting chondrocyte senescence to alleviate OA progression.Methods We screened and identified miRNAs differentially expressed in OA and normal cartilage, then confirmed the impact of miR-653-5p on chondrocyte functions and senescence phenotypes through in vitro experiments with overexpression/silencing. We identified interleukin 6 (IL-6) as the target gene of miR-653-5p and confirmed the regulatory influence of miR-653-5p on the IL-6/JAK/STAT3 signaling pathway through gain/loss-of-function studies. Finally, we assessed the therapeutic efficacy of miR-653-5p on OA using a mouse model with destabilization of the medial meniscus.Results MiR-653-5p was significantly downregulated in cartilage tissues and chondrocytes from OA patients. Overexpression of miR-653-5p promoted chondrocyte matrix synthesis and proliferation while inhibiting chondrocyte senescence. Furthermore, bioinformatics target prediction and the luciferase reporter assays identified IL-6 as a target of miR-653-5p. Western blot assays demonstrated that miR-653-5p overexpression inhibited the protein expression of IL-6, the phosphorylation of JAK1 and STAT3, and the expression of chondrocyte senescence phenotypes by regulating the IL-6/JAK/STAT3 signaling pathway. More importantly, the cartilage destruction was significantly alleviated and chondrocyte senescence phenotypes were remarkably decreased in the OA mouse model treated by agomiR-653-5p compared to the control mice.Conclusions MiR-653-5p showed a significant decrease in cartilage tissues of individuals with OA, leading to an upregulation of chondrocyte senescence phenotypes in the articular cartilage. AgomiR-653-5p emerges as a potential treatment approach for OA. These findings provide further insight into the role of miR-653-5p in chondrocyte senescence and the pathogenesis of OA.

Abstract Background Osteosarcoma (OS) is the most common primary malignant bone tumor and is highly prone to metastasis. OS can metastasize to the lymph node (LN) through the lymphatics, and the metastasis of tumor cells reestablishes the immune landscape of the LN, which is conducive to the growth of tumor cells. However, the mechanism of LN metastasis of osteosarcoma and remodeling of the metastatic lymph node (MLN) microenvironment is not clear. Methods Single-cell RNA sequencing of 18 samples from paracancerous, primary tumor, and lymph nodes was performed. Then, new signaling axes closely related to metastasis were identified using bioinformatics, in vitro experiments, and immunohistochemistry. The mechanism of remodeling of the LN microenvironment in tumor cells was investigated by integrating single-cell and spatial transcriptomics. Results From 18 single-cell sequencing samples, we obtained 117,964 cells. The pseudotime analysis revealed that osteoblast(OB) cells may follow a differentiation path from paracancerous tissue (PC) → primary tumor (PT) → MLN or from PC → PT, during the process of LN metastasis. Next, in combination of bioinformatics, in vitro and in vivo experiments, and immunohistochemistry, we determined that ETS2/IBSP, a new signal axis, might promote LN metastasis. Finally, single-cell and spatial dissection uncovered that OS cells could reshape the microenvironment of LN by interacting with various cell components, such as myeloid, cancer-associated fibroblasts (CAFs), and NK/T cells. Conclusions Collectively, our research revealed a new molecular mechanism of LN metastasis and clarified how OS cells influenced the LN microenvironment, which might provide new insight for blocking LN metastasis.

Osteoclasts (OCs) and regulatory CD4(+) T cells (CD4(+)Tregs) are important components in the tumor microenvironment (TME) of osteosarcoma. In this study, we collected six osteosarcoma samples from our previous study (GSE162454). We also integrated a public database (GSE152048), which included single cell sequencing data of 11 osteosarcoma patients. We obtained 138,192 cells and then successfully identified OCs and CD4(+)Tregs. Based on the interaction gene set between OCs and CD4(+)Tregs, patients from GSE21257 were distinguished into two clusters by consensus clustering analysis. Both the tumor immune microenvironment and survival prognosis between the two clusters were significantly different. Subsequently, five model genes were identified by protein-protein interaction network based on differentially upregulated genes of cluster 2. Quantitative RT-PCR was used to detect their expression in human osteoblast and osteosarcoma cells. A prognostic model was successfully established using these five genes. Kaplan-Meier survival analysis found that patients in the high-risk group had worse survival (p = 0.029). Therefore, our study first found that cell-cell communication between OCs and CD4(+)Tregs significantly alters TME and is connected to poor prognosis of OS. The model we constructed can accurately predict prognosis for osteosarcoma patients.

Accumulating evidence suggests that microRNAs (miRNAs) play an important role in intervertebral disc degeneration (IDD), but the precise role of specific miRNAs involved in this disease remains elusive. The purpose of this study was to identify IDD-specific miRNAs, followed by functional validation of results. MiRNA expression profile was determined in nucleus pulposus (NP) tissues from patients with IDD and controls, employing Solexa sequencing and quantitative real-time PCR (qRT-PCR). Biological functions of differential expression miRNAs were further investigated in vitro and in vivo. Luciferase reporter assays and Western blotting were performed to determine miRNA targets. We identified 28 miRNAs that were differentially expressed in patients compared with controls. Following qRT-PCR confirmation, miR-193a-3p was significantly down-regulated in degenerative NP tissues. Moreover, its level was correlated with grade of disc degeneration. Through gain- and loss-of-function studies, miR-193a-3p was demonstrated to significantly promote type II collagen expression in NP cells. Knockdown of MMP14 induced effects on NP cells similar to those induced by miR-193a-3p. Bioinformatics target prediction identified MMP14 as a putative target of miR-193a-3p. Furthermore, luciferase reporter assays and Western blotting demonstrated that miR-193a-3p directly targets MMP14. MiR-193a-3p inhibited IDD in vitro and in vivo. The downregulation of miR-193a-3p induces the expression of MMP14, which promotes loss of type II collagen and thereby contributes to the development of human IDD. Our findings extend the role of miR-193a-3p in the pathogenesis of IDD and provide a potential novel therapeutic target for degenerative disc disease.