Osteosarcoma Microenvironment Research Articles

Spatial profiling identifies regionally distinct microenvironments and targetable immunosuppressive mechanisms in pediatric osteosarcoma pulmonary metastases.

Osteosarcoma is the most common malignant bone tumor in young patients and remains a significant clinical challenge, particularly in the context of metastatic disease. Despite extensive documentation of genomic alterations in osteosarcoma, studies detailing the immunosuppressive mechanisms within the metastatic osteosarcoma microenvironment are lacking. Our objective was to characterize the spatial transcriptional landscape of metastatic osteosarcoma to reveal these immunosuppressive mechanisms and identify promising therapeutic targets. Here, we performed spatial transcriptional profiling on a cohort of osteosarcoma pulmonary metastases from pediatric patients. We reveal a conserved spatial gene expression pattern resembling a foreign body granuloma, characterized by peripheral inflammatory signaling, fibrocollagenous encapsulation, lymphocyte exclusion, and peritumoral macrophage accumulation. We also show that the intratumoral microenvironment of these lesions lack inflammatory signaling. Additionally, we identified CXCR4 as an actionable immunomodulatory target that bridges both the intratumoral and extratumoral microenvironments and highlights the spatial heterogeneity and complexity of this pathway. Collectively, this study reveals that metastatic osteosarcoma specimens are comprised of multiple regionally distinct immunosuppressive microenvironments.

Single-cell profiling of SLC family transporters: uncovering the role of SLC7A1 in osteosarcoma

BackgroundOsteosarcoma is the most common malignant bone tumor in children and adolescents, characterized by high disability and mortality rates. Over the past three decades, therapeutic outcomes have plateaued, underscoring the critical need for innovative therapeutic targets. Solute carrier (SLC) family transporters have been implicated in the malignant progression of a variety of tumors, however, their specific role in osteosarcoma remains poorly understood.MethodsThe single-cell sequencing data from GSE152048 and GSE162454, along with RNA-seq from the TARGET and GSE21257 cohorts, were utilized for the analysis in this study. LASSO regression analysis was conducted to identify prognostic genes and construct an SLC-related prognostic signature. Survival analysis and ROC analysis evaluated the validity of the prognostic signature. The ESTIMATE and CIBERSORT Packages were utilized to assess the immune infiltration status. Pseudotime and CellChat analyses were performed to investigate the relationship between SLC7A1, malignant phenotypes, and the immune microenvironment. CCK8 assays, EdU staining, colony formation assays, Transwell assays, and co-culture systems were used to assess the effects of SLC7A1 on cell proliferation, metastasis, and macrophage polarization. Finally, virtual docking identified potential drugs targeting SLC7A1.ResultsSLCs displayed distinct expression patterns across various cell types within the osteosarcoma microenvironment, with myeloid cells exhibiting a preference for amino acid uptake. A prognostic model comprising nine genes was constructed via LASSO regression, with SLC7A1 showing the highest hazard ratio. Multiple analytical algorithms indicated that SLCs were associated with immune cell infiltration and immune checkpoint gene expression. Single-cell analysis indicated that SLC7A1 was predominantly expressed in osteosarcoma cells and correlated with various malignant tumor characteristics. SLC7A1 also regulate interactions between tumor cells and macrophages, as well as modulate macrophage function through multiple pathways. In vitro assays and survival analysis demonstrated that inhibition of SLC7A1 suppressed the malignant phenotype of osteosarcoma cells, with SLC7A1 expression correlating with poor prognosis. Co-culture models confirmed the involvement of SLC7A1 in macrophage polarization. Finally, virtual screening and CETSA identified Cepharanthine as potential inhibitors of SLC7A1.ConclusionSLC-related prognostic signatures can be utilized for the prognostic evaluation of osteosarcoma. Pharmacological inhibition of SLC7A1 may be a feasible therapeutic approach for osteosarcoma.

Osteosarcoma-targeting PtIV prodrug amphiphile for enhanced chemo-immunotherapy via Ca2+ trapping.

Platinum (PtII)-based anticancer agents exhibit a lack of selectivity in the treatment of osteosarcoma, resulting in significant toxicity. Furthermore, immune surveillance withinthe tumor microenvironment impedes the uptake of platinum drugs by osteosarcoma cells. To overcome these challenges, an oxaliplatin-based PtIV prodrug amphiphile (Lipo-OXA-ALN) was designed and synthesized by incorporatingan osteosarcoma-targeting alendronate (ALN) alongside a lipid tail. The lipid nanoparticles (ALN-OXA), which self-assemble from Lipo-OXA-ALN, enhanced intracellular platinum uptake due to their superior Ca2+ trapping ability and significantly inhibit osteosarcoma cell activity. Moreover, ALN-OXA exhibited potent targeting capabilities, effectively suppressing osteosarcoma growth while preventing bone destruction. Importantly, ALN-OXA induces a series of immune responses characterized by the activation of immune cells, maturation of dendritic cells, and secretion of related cytokines, followed by the activation and infiltration of T lymphocytes and a significant increase in the ratio of cytotoxic T cells. Additionally, the ratio of M1/M2 macrophages increased markedly after ALN-OXA treatment, suggesting potential reprogramming of the tumor microenvironment by ALN-OXA. Overall, the improved therapeutic efficacy against osteosarcoma demonstrates that the PtIV prodrug amphiphile represents a promising strategy for combining targeted chemotherapy with strategies aimed at reversing immune suppression. STATEMENT OF SIGNIFICANCE: Platinum (PtII)-based chemotherapy for osteosarcoma faces challenges due to poor tumor selectivity, leading to suboptimal efficacy and increased toxicity. Additionally, the osteosarcoma microenvironment impedes effective drug delivery. To overcome these limitations, we developed an oxaliplatin-based PtIV prodrug nanoparticle (ALN-OXA) for targeted chemo-immunotherapy. ALN-OXA showed significant in vivo efficacy, effectively preventing bone damage and enhancing the immune microenvironment to improve treatment outcomes. This innovative approach not only targets the tumor more efficiently but also boosts immune response, offering a promising strategy for tumor blockade, tumor starvation, and other therapeutic applications in osteosarcoma treatment.

Reproducibility and repeatability of quantitative T2 and T2* mapping of osteosarcomas in a mouse model

BackgroundNew immunotherapies activate tumor-associated macrophages (TAMs) in the osteosarcoma microenvironment. Iron oxide nanoparticles (IONPs) are phagocytosed by TAMs and, therefore, enable TAM detection on T2*- and T2-weighted magnetic resonance images. We assessed the repeatability and reproducibility of T2*- and T2-mapping of osteosarcomas in a mouse model.MethodsFifteen BALB/c mice bearing-murine osteosarcomas underwent magnetic resonance imaging (MRI) on 3-T and 7-T scanners before and after intravenous IONP infusion, using T2*-weighted multi-gradient-echo, T2-weighted fast spin-echo, and T2-weighted multi-echo sequences. Each sequence was repeated twice. Tumor T2 and T2* relaxation times were measured twice by two independent investigators. Repeatability and reproducibility of measurements were assessed.ResultsWe found excellent agreement between duplicate acquisitions for both T2* and T2 measurements at either magnetic field strength, by the same individual (repeatability), and between individuals (reproducibility). The repeatability concordance correlation coefficient (CCC) for T2* values were 0.99 (coefficients of variation (CoV) 4.43%) for reader 1 and 0.98 (CoV 5.82%) for reader 2. The reproducibility of T2* values between the two readers was 0.99 (CoV 3.32%) for the first acquisitions and 0.99 (CoV 6.30%) for the second acquisitions. Regarding T2 values, the repeatability of CCC was similar for both readers, 0.98 (CoV 3.64% for reader 1 and 4.45% for reader 2). The CCC of the reproducibility of T2 was 0.99 (CoV 3.1%) for the first acquisition and 0.98 (CoV 4.38%) for the second acquisition.ConclusionsOur results demonstrated high repeatability and reproducibility of quantitative T2* and T2 mapping for monitoring the presence of TAMs in osteosarcomas.Relevance statementT2* and T2 measurements of osteosarcomas on IONP-enhanced MRI could allow identifying patients who may benefit from TAM-modulating immunotherapies and for monitoring treatment response. The technique described here could be also applied across a wide range of other solid tumors.Key points• Optimal integration of TAM-modulating immunotherapies with conventional chemotherapy remains poorly elucidated.• We found high repeatability of T2* and T2 measurements of osteosarcomas in a mouse model, both with and without IONPs contrast, at 3-T and 7-T MRI field strengths.• T2 and T2* mapping may be used to determine response to macrophage-modulating cancer immunotherapies.Graphical

Prognositic value of anoikis and tumor immune microenvironment-related gene in the treatment of osteosarcoma.

Osteosarcoma is a highly aggressive primary malignant bone tumor commonly seen in children and adolescents, with a poor prognosis. Anchorage-dependent cell death (anoikis) has been proven to be indispensable in tumor metastasis, regulating the migration and adhesion of tumor cells at the primary site. However, as a type of programmed cell death, anoikis is rarely studied in osteosarcoma, especially in the tumor immune microenvironment. This study aims to clarify prognostic value of anoikis and tumor immune microenvironment-related gene in the treatment of osteosarcoma. Anoikis-related genes (ANRGs) were obtained from GeneCards. Clinical information and ANRGs expression profiles of osteosarcoma patients were sourced from the therapeutically applicable research to generate effective therapies and Gene Expression Omnibus (GEO) databases. ANRGs highly associated with tumor immune microenvironment were identified by the estimate package and the weighted gene coexpression network analysis (WGCNA) algorithm. Machine learning algorithms were performed to construct long-term survival predictive strategy, each sample was divided into high-risk and low-risk subgroups, which was further verified in the GEO cohort. Finally, based on single-cell RNA-seq from the GEO database, analysis was done on the function of signature genes in the osteosarcoma tumor microenvironment. A total of 51 hub ANRGs closely associated with the tumor microenvironment were identified, from which 3 genes (MERTK, BNIP3, S100A8) were selected to construct the prognostic model. Significant differences in immune cell activation and immune-related signaling pathways were observed between the high-risk and low-risk groups based on tumor microenvironment analysis (all P<0.05). Additionally, characteristic genes within the osteosarcoma microenvironment were identified in regulation of intercellular crosstalk through the GAS6-MERTK signaling pathway. The prognostic model based on ANRGs and tumor microenvironment demonstrate good predictive power and provide more personalized treatment options for patients with osteosarcoma.

Enhanced pyroptosis induction with pore-forming gene delivery for osteosarcoma microenvironment reshaping

Osteosarcoma is the most common malignant bone tumor without efficient management for improving 5-year event-free survival. Immunotherapy is also limited due to its highly immunosuppressive tumor microenvironment (TME). Pore-forming gasdermins (GSDMs)-mediated pyroptosis has gained increasing concern in reshaping TME, however, the expressions and relationships of GSDMs with osteosarcoma remain unclear. Herein, gasdermin E (GSDME) expression is found to be positively correlated with the prognosis and immune infiltration of osteosarcoma patients, and low GSDME expression was observed. A vector termed as LPAD contains abundant hydroxyl groups for hydrating layer formation was then prepared to deliver the GSDME gene to upregulate protein expression in osteosarcoma for efficient TME reshaping via enhanced pyroptosis induction. Atomistic molecular dynamics simulations analysis proved that the hydroxyl groups increased LPAD hydration abilities by enhancing coulombic interaction. The upregulated GSDME expression together with cleaved caspase-3 provided impressive pyroptosis induction. The pyroptosis further initiated proinflammatory cytokines release, increased immune cell infiltration, activated adaptive immune responses and create a favorable immunogenic hot TME. The study not only confirms the role of GSDME in the immune infiltration and prognosis of osteosarcoma, but also provides a promising strategy for the inhibition of osteosarcoma by pore-forming GSDME gene delivery induced enhanced pyroptosis to reshape the TME of osteosarcoma.

Transcriptome and single-cell analysis reveal disulfidptosis-related modification patterns of tumor microenvironment and prognosis in osteosarcoma

Osteosarcoma (OS) is the most common malignant bone tumor with high pathological heterogeneity. Our study aimed to investigate disulfidptosis-related modification patterns in OS and their relationship with survival outcomes in patients with OS. We analyzed the single-cell-level expression profiles of disulfidptosis-related genes (DSRGs) in both OS microenvironment and OS subclusters, and HMGB1 was found to be crucial for intercellular regulation of OS disulfidptosis. Next, we explored the molecular clusters of OS based on DSRGs and related immune cell infiltration using transcriptome data. Subsequently, the hub genes of disulfidptosis in OS were screened by applying multiple machine models. In vitro and patient experiments validated our results. Three main disulfidptosis-related molecular clusters were defined in OS, and immune infiltration analysis suggested high immune heterogeneity between distinct clusters. The in vitro experiment confirmed decreased cell viability of OS after ACTB silencing and higher expression of ACTB in patients with lower immune scores. Our study systematically revealed the underlying relationship between disulfidptosis and OS at the single-cell level, identified disulfidptosis-related subtypes, and revealed the potential role of ACTB expression in OS disulfidptosis.

Spatiotemporal evolutionary process of osteosarcoma immune microenvironment remodeling and C1QBP-driven drug resistance deciphered through single-cell multi-dimensional analysis.

The tumor immune microenvironment has manifested a crucial correlation with tumor occurrence, development, recurrence, and metastasis. To explore the mechanisms intrinsic to osteosarcoma (OS) initiation and progression, this study synthesizes multiple single-cell RNA sequencing data sets, constructing a comprehensive landscape of the OS microenvironment. Integrating single-cell RNA sequencing with bulk RNA sequencing data has enabled the identification of a significant correlation between heightened expression of the fatty acid metabolism-associated gene (C1QBP) and patient survival in OS. C1QBP not only amplifies the proliferation, migration, invasion, and anti-apoptotic properties of OS but also instigates cisplatin resistance. Subsequent investigations suggest that C1QBP potentially promotes macrophage polarization from monocytes/macrophages toward M2 and M3 phenotypes. Consequently, C1QBP may emerge as a novel target for modulating OS progression and resistance therapy.

Single-cell RNA-seq reveals T cell exhaustion and immune response landscape in osteosarcoma.

T cell exhaustion in the tumor microenvironment has been demonstrated as a substantial contributor to tumor immunosuppression and progression. However, the correlation between T cell exhaustion and osteosarcoma (OS) remains unclear. In our present study, single-cell RNA-seq data for OS from the GEO database was analysed to identify CD8+ T cells and discern CD8+ T cell subsets objectively. Subgroup differentiation trajectory was then used to pinpoint genes altered in response to T cell exhaustion. Subsequently, six machine learning algorithms were applied to develop a prognostic model linked with T cell exhaustion. This model was subsequently validated in the TARGETs and Meta cohorts. Finally, we examined disparities in immune cell infiltration, immune checkpoints, immune-related pathways, and the efficacy of immunotherapy between high and low TEX score groups. The findings unveiled differential exhaustion in CD8+ T cells within the OS microenvironment. Three genes related to T cell exhaustion (RAD23A, SAC3D1, PSIP1) were identified and employed to formulate a T cell exhaustion model. This model exhibited robust predictive capabilities for OS prognosis, with patients in the low TEX score group demonstrating a more favorable prognosis, increased immune cell infiltration, and heightened responsiveness to treatment compared to those in the high TEX score group. In summary, our research elucidates the role of T cell exhaustion in the immunotherapy and progression of OS, the prognostic model constructed based on T cell exhaustion-related genes holds promise as a potential method for prognostication in the management and treatment of OS patients.

Osteosarcoma through the Lens of Bone Development, Signaling, and Microenvironment.

In this work, we review the multifaceted connections between osteosarcoma (OS) biology and normal bone development. We summarize and critically analyze existing research, highlighting key areas that merit further exploration. The review addresses several topics in OS biology and their interplay with normal bone development processes, including OS cell of origin, genomics, tumor microenvironment, and metastasis. We examine the potential cellular origins of OS and how their roles in normal bone growth may contribute to OS pathogenesis. We survey the genomic landscape of OS, highlighting the developmental roles of genes frequently altered in OS. We then discuss the OS microenvironment, emphasizing the transformation of the bone niche in OS to facilitate tumor growth and metastasis. The role of stromal and immune cells is examined, including their impact on tumor progression and therapeutic response. We further provide insights into potential development-informed opportunities for novel therapeutic strategies.

RARRES2 is involved in the “lock-and-key” interactions between osteosarcoma stem cells and tumor-associated macrophages

Osteosarcoma (OS) is a type of tumor. Osteosarcoma stem cells (OSCs) are responsible for drug resistance, recurrence, and immunosuppression in OS. We aimed to determine the heterogeneity of OSCs and the immunosuppression mechanisms underlying the interactions between OSCs and tumor-associated macrophages (TAMs). The cell components, trajectory changes, and cell communication profiles of OS cells were analyzed by transcriptomics at the single-cell level. The intercellular communication patterns of OSCs were verified, and the role of the cell hub genes was revealed. Hub geneS are genes that play important roles in regulating certain biological processes; they are often defined as the genes with the strongest regulatory effect on differentially expressed gene sets. Moreover, various cellular components of the OS microenvironment were identified. Malignant cells were grouped, and OSCs were identified. Further regrouping and communication analysis revealed that the genes in the stemness maintenance and differentiation subgroups were involved in communication with macrophages. Key receptor–ligand pairs and target gene sets for cell communication were obtained. Transcriptome data analysis revealed the key gene RARRES2, which is involved in intercellular communication between OSCs and TAMs. In vitro studies confirmed that macrophages promote RARRES2-mediated stemness maintenance in OSCs via the TAM-secreted cytokine insulin-like growth factor 1. Patient studies confirmed that RARRES2 could be a biomarker of OS. OSCs are highly heterogeneous, and different subgroups are responsible for proliferation and communication with other cells. The IGF-RARRES2 axis plays a key role in maintaining OSC stemness through communication with TAMs.

Nerve growth factor promote VCAM-1-dependent monocyte adhesion and M2 polarization in osteosarcoma microenvironment: Implications for larotrectinib therapy.

Osteosarcoma is the most prevalent form of primary malignant bone tumor, primarily affecting children and adolescents. The nerve growth factors (NGF) referred to as neurotrophins have been associated with cancer-induced bone pain; however, the role of NGF in osteosarcoma has yet to be elucidated. In osteosarcoma samples from the Genomic Data Commons data portal, we detected higher levels of NGF and M2 macrophage markers, but not M1 macrophage markers. In cellular experiments, NGF-stimulated osteosarcoma conditional medium was shown to facilitate macrophage polarization from the M0 to the M2 phenotype. NGF also enhanced VCAM-1-dependent monocyte adhesion within the osteosarcoma microenvironment by down-regulating miR-513c-5p levels through the FAK and c-Src cascades. In in vivo xenograft models, the overexpression of NGF was shown to enhance tumor growth, while the oral administration of the TrK inhibitor larotrectinib markedly antagonized NGF-promoted M2 macrophage expression and tumor progression. These results suggest that larotrectinib could potentially be used as a therapeutic agent aimed at mitigating NGF-mediated osteosarcoma progression.

Abstract A150: Effects of radioimmunotherapy on human and canine osteosarcoma microenvironment

Abstract Introduction: Osteosarcoma (OS) is a type of primary bone cancer that predominantly affects children and adolescents with an unacceptably high mortality rate of 30% within 5 years of diagnosis. Though several treatment options including radiotherapy, chemotherapy, and amputation surgery exist, these are either ineffective or very invasive. Also, no significant advancements in treatment options have occurred in the last 30 years. The incidence of OS among companion dogs is much higher than in humans and phenotypically OS is very similar between humans and canines. We are using a comparative oncology approach to OS treatment by developing radioimmunotherapy (RIT) which targets the insulin-like growth factor receptor type 2 (IGF2R) on OS cells. IGF2R is abundant in human and canine OS and has shown promise a novel target for RIT of OS using a comparative oncology approach. It is therefore important to investigate the mechanistic underpinnings of RIT efficacy by investigating its interaction with OS microenvironment to develop improved RIT regimens for OS. Materials and Methods: Severe combined immunodeficiency (SCID) female mice bearing human and canine patient-derived OS xenografts were treated with RIT utilizing IF3, a novel full-length human mAb targeting IGF2R obtained by phage-display technology. The IF3 antibody was radiolabeled with an alpha emitter 225Actinium (225Ac) or a beta emitter 177Lutetim (177Lu) radionuclides. The tumors were removed at 1, 3 and 7 days post-RIT and the effects of RIT on IGF2R-positive tumor cells, OS stem cells, and immune components of the tumor microenvironment such as natural killer (NK) cells and tumor-associated macrophages (TAMs) were assessed using quantitative immunohistochemistry (IHC). Results: In the untreated tumors IHC detected the presence of IGF2R-positive tumor cells, OS stem cells, and immune components of the tumor microenvironment such as NK cells and TAMs, in both human and canine OS tumors obtained from mouse models. Quantitative analysis of IHC results in the tumors post-RIT demonstrated a time-dependent decrease in IGF2R-positive OS tumor cells and OS stem cells and an increase in TAMs and NK cells. Conclusions: Deciphering the mechanisms of RIT interaction with tumor microenvironment can assist in the design of the clinical trials in companion dogs with OS, which will be followed by the trials in children with this cancer. Also, it supports combining RIT with immunotherapy targeting OS stem cells and other immune components of tumor microenvironments such as NK cells and TAMS, which can result in improved patient outcomes. Citation Format: Sabeena Giri, Kevin J.H. Allen, Maruti Uppalapati, Ekaterina Dadachova. Effects of radioimmunotherapy on human and canine osteosarcoma microenvironment [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2023 Oct 11-15; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2023;22(12 Suppl):Abstract nr A150.

Osteoclasts in Osteosarcoma: Mechanisms, Interactions, and Therapeutic Prospects.

Osteosarcoma is an extremely malignant tumor, and its pathogenesis is complex and remains incompletely understood. Most cases of osteosarcoma are accompanied by symptoms of bone loss or result in pathological fractures due to weakened bones. Enhancing the survival rate of osteosarcoma patients has proven to be a long-standing challenge. Numerous studies mentioned in this paper, including in-vitro, in-vivo, and in-situ studies have consistently indicated a close association between the symptoms of bone loss associated with osteosarcoma and the presence of osteoclasts. As the sole cells capable of bone resorption, osteoclasts participate in a malignant cycle within the osteosarcoma microenvironment. These cells interact with osteoblasts and osteosarcoma cells, secreting various factors that further influence these cells, disrupting bone homeostasis, and shifting the balance toward bone resorption, thereby promoting the onset and progression of osteosarcoma. Moreover, the interaction between osteoclasts and various other cells types, such as tumor-associated macrophages, myeloid-derived suppressor cells, DCs cells, T cells, and tumor-associated fibroblasts in the osteosarcoma microenvironment plays a crucial role in disease progression. Consequently, understanding the role of osteoclasts in osteosarcoma has sparked significant interest. This review primarily examines the physiological characteristics and functional mechanisms of osteoclasts in osteosarcoma, and briefly discusses potential therapies targeting osteoclasts for osteosarcoma treatment. These studies provide fresh ideas and directions for future research on the treatment of osteosarcoma.

Mesenchymal Stem/Stromal Cells: Immunomodulatory and Bone Regeneration Potential after Tumor Excision in Osteosarcoma Patients.

Osteosarcoma (OS) is a type of bone cancer that is derived from primitive mesenchymal cells typically affecting children and young adults. The current standard of treatment is a combination of neoadjuvant chemotherapy and surgical resection of the cancerous bone. Post-resection challenges in bone regeneration arise. To determine the appropriate amount of bone to be removed, preoperative imaging techniques such as bone and CT scans are employed. To prevent local recurrence, the current standard of care suggests maintaining bony and soft tissue margins from 3 to 7 cm beyond the tumor. The amount of bone removed in an OS patient leaves too large of a deficit for bone to form on its own and requires reconstruction with metal implants or allografts. Both methods require the bone to heal, either to the implant or across the allograft junction, often in the setting of marrow-killing chemotherapy. Therefore, the issue of bone regeneration within the surgically resected margins remains an important challenge for the patient, family, and treating providers. Mesenchymal stem/stromal cells (MSCs) are potential agents for enhancing bone regeneration post tumor resection. MSCs, used with scaffolds and growth factors, show promise in fostering bone regeneration in OS cases. We spotlight two MSC types-bone marrow-derived (BM-MSCs) and adipose tissue-derived (ASCs)-highlighting their bone regrowth facilitation and immunomodulatory effects on immune cells like macrophages and T cells, enhancing therapeutic outcomes. The objective of this review is two-fold: review work demonstrating any ability of MSCs to target the deranged immune system in the OS microenvironment, and synthesize the available literature on the use of MSCs as a therapeutic option for stimulating bone regrowth in OS patients post bone resection. When it comes to repairing bone defects, both MB-MSCs and ASCs hold great potential for stimulating bone regeneration. Research has showcased their effectiveness in reconstructing bone defects while maintaining a non-tumorigenic role following wide resection of bone tumors, underscoring their capability to enhance bone healing and regeneration following tumor excisions.

Analysis of intercellular communication in the osteosarcoma microenvironment based on single cell sequencing data

Osteosarcoma (OS) is the most common primary bone cancer in children and young adults, patient survival rates have not improved in recent decades. To further understand the interrelationship between different cell types in the tumor microenvironment of osteosarcoma, we comprehensively analyzed single-cell sequencing data from six patients with untreated osteosarcoma. Nine major cell types were identified from a total of 46,046 cells based on unbiased clustering of gene expression profiles and canonical markers. Osteosarcoma from different patients display heterogeneity in cellular composition. Myeloid cells were the most commonly represented cell type, followed by osteoblastic and TILs. Copy number variation (CNV) results identified amplifications and deletions in malignant osteoblastic cells and fibroblasts. Trajectory analysis based on RNA velocity showed that osteoclasts in the OS microenvironment could be differentiated from myeloid cells. Furthermore, we explored the intercellular communications in OS microenvironment and identified multiple ligand-receptor pairs between myeloid cells, osteoblastic cells and their cells, including 21 ligand-receptor pair genes that significantly associated with survival outcomes. Importantly, we found chemotherapy may have an effect on cellular communication in the OS microenvironment by analyzing single-cell sequencing data from seven primary osteosarcoma patients who received chemotherapy. We believe these observations will improve our understanding of potential mechanisms of microenvironment contributions to OS progression and help identify potential targets for new treatment development in the future.

Integrative analyses of ferroptosis and immune related biomarkers and the osteosarcoma associated mechanisms

Osteosarcoma (OS) is the most common primary malignant bone tumor with high metastatic potential and relapse risk. To study the regulatory mechanism of the OS microenvironment, a complex regulatory network involving the ferroptosis- and immune response-related genes remains to be established. In the present study, we determined the effect of a comprehensive evaluation system established on the basis of ferroptosis- and immune-related genes on the immune status, related biomarkers, prognosis, and the potential regulatory networks underlying OS based on the TARGET and Gene Expression Omnibus databases that contain information on OS patients by bioinformatics analyses. We first characterized individual ferroptosis scores and immune scores through gene set variation analysis (GSVA) against TARGET-OS datasets. We then identified differentially expressed genes by score groups. Weighted gene co-expression network analysis was performed to identify the most relevant ferroptosis-related and immune-related gene modules, which facilitated the identification of 327 ferroptosis gene and 306 immune gene candidates. A 4-gene (WAS, CORT, WNT16, and GLB1L2) signature was constructed and valuation using the least absolute shrinkage and selection operator-Cox regression models to effectively predict OS prognosis. The prediction efficiency was further validated by GSE39055. We stratified patients based on the prognostic scoring systems. Eight hub genes (namely CD3D, CD8A, CD3E, IL2, CD2, MYH6, MYH7, and MYL2) were identified, and TF–miRNA target regulatory networks were constructed. Furthermore, Gene Ontology, Kyoto Encyclopedia of Genes and Genomes, gene set enrichment analysis, and GSVA were used to determine the signature’s potential pathways and biological functions, which showed that the hub genes were enriched in ferroptosis-associated biological functions and immune-associated molecular mechanisms. Thereafter, we investigated the proportion and infiltration extent of 22 infiltrating immune cells by using CIBERSORT, which revealed significant subgroup differences in CD8 + T cells, M0 macrophages, and M2 macrophages. In conclusion, we determined a new ferroptosis-related and immune-related gene signature for predicting OS patients’ prognosis and further explored the ferroptosis and immunity interactions during OS development, which provides insights into the exploration of molecular mechanisms and targeted therapies in patients with OS.

Abstract 2331: Loss of LRRC15 in osteosarcoma cells disrupts extracellular matrix integration, intercellular communication, and differentiation

Abstract Background: Leucine rich repeat containing 15 (LRRC15) is a 581 amino acid type 1 transmembrane protein that contains 15 leucine rich repeats. Cysteine clusters flank the LRR domain that functions as the collagen binding domain. LRRC15 has been found to be upregulated during osteogenic differentiation of mesenchymal stem cells, induced by TGFβ, and highly expressed in multiple solid tumors, notably in osteosarcoma (1). Antibody drug conjugates targeting LRRC15 have shown promising activity in pre-clinical studies (2). Results: To investigate the function of LRRC15 in the osteosarcoma microenvironment, we generated inducible shRNA knockdown (KD) derivatives targeting 3’UTR and ORF regions of LRRC15 of four osteosarcoma cell lines. Using these models, we characterized the biological properties of LRRC15 knockdown under 2-D and 3-D growth conditions. We found that LRRC15 KD in 2-D culture resulted in reduced adhesion to extracellular matrix (ECM) components and impaired cell migration. In 3-D spheroid culture, LRRC15 KD severely impacted scaffold-free spheroid compaction and invasion of spheroids embedded in Matrigel. Dysregulation of multiple ECM, cell adhesion and communication pathways were revealed by transcriptomic analysis from samples collected at time points during the LRRC15 KD. Of interest, several key transcription factors including RUNX2, CTNNB1, and SP7 were downregulated in LRRC15 deficient cells. Conclusion: Our comprehensive results establish the importance of LRRC15 as a critical cell-adhesion molecule essential for the maintenance of the osteogenic intercellular communication, invasion and differentiation revealing novel aspects of osteosarcoma cell-biology. Reference:1.Purcell JW, Tanlimco SG, Hickson J, Fox M, Sho M, Durkin L, et al. LRRC15 Is a Novel Mesenchymal Protein and Stromal Target for Antibody-Drug Conjugates. Cancer Res 2018;78:4059-4072.2.Slemmons KK, Mukherjee S, Meltzer P, Purcell JW, Helman LJ. LRRC15 antibody-drug conjugates show promise as osteosarcoma therapeutics in preclinical studies. Pediatr Blood Cancer 2020:e28771. Citation Format: Sanjit Mukherjee, Ankit Jajoo, Jack Zhu, Marbin Pineda, Robert Walker, James Purcell, Lee J Helman, Paul S. Meltzer. Loss of LRRC15 in osteosarcoma cells disrupts extracellular matrix integration, intercellular communication, and differentiation [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 2331.

The Effects of Resveratrol Targeting MicroRNA-4325P/PDGF-B to Regulate Tumor Angiogenesis in Osteosarcoma Microenvironment

Objectives Here we sought to determine the specific mechanism by which resveratrol targets microRNA-4325p and PDGFB in an osteosarcoma (OS) cell line and investigate its effect on the OS cell line. Methods As a result of the study related to the effect of resveratrol on the conditioned media of microRNA-4325p mimic transfected MG-63 and HOS (a fibroblast and epithelial-like cell line) cells, we determined that resveratrol decreased survival, and growth, migration, and aortic ring formation of endothelial cells. A combination of miRNA mimics and HOS and MG-63 cell lines were transfected with inhibitors to explore the mechanisms behind angiogenesis formation induced by resveratrol. Results We found that resveratrol improved the activity of microRNA-4325p mimic transfection in OS, thus promoting angiogenesis, as demonstrated by angiogenic parameters and gene expression in OS. Following these results, it is evident that resveratrol can modulate angiogenesis by regulating microRNA-4325p expression by targeting PDGFB genes in OS cells. Conclusion The results demonstrate that resveratrol promotes tumor angiogenesis of OS cells through microRNA-4325p/PDGBB-mediated pathways and provides theoretical and experimental support for the discovery of new bioactive phytochemicals that are effective therapeutic agents in the prevention and treatment of OS diseases.

TREM2 as a Prognostic Biomarker for Osteosarcoma Microenvironment Remodeling.

The tumor microenvironment (TME) acts as a crucial role in the occurrence and development of osteosarcoma (OS). Despite this, the mechanism controlling the components of immunity and stroma in the tumor microenvironment remains a mystery. To conduct this study, we download and collate transcriptome data from the TARGET database, whose full name is Therapeutically Applicable Research to Generate Effective Treatments, as well as available clinical information of OS. The CIBERSORT and ESTIMATE methodology are used to acquire the proportions of components of immunity and stroma and tumor-infiltrating immune cells (TICs). Protein-protein interaction (PPI) networks and Cox regression analysis are used to select differentially expressed genes (DEGs). A prognostic biomarker is determined by intersecting univariate COX and PPI results, which lead to the finding of Triggering receptor expressed on myeloid cells-2 (TREM2). Based on the next analysis, TREM2 expression is positively correlated with OS survival time. Immune function-related genes have enrichment in the group with high expression of TREM2, according to gene set enrichment analysis (GSEA). The percentage of TICs by CIBERSORT methodology revealed that the expression of TREM2 is positively associated with follicular helper T cells, CD8-positive T cells, and M2 macrophages and negatively correlated with plasma cells, M0 macrophages, and naive CD4-positive T cells. All results suggest a possible integral role of TREM2 in the immune-related events of TME. Therefore, TREM2 may be a potential indicator of remodeling of TME in osteosarcoma, which is useful and helpful in predicting the clinical prognostic outcome of OS patients and provide a unique perspective for immunotherapy for OS.