Identifying specific TLS-associated genes as potential biomarkers for predicting prognosis and evaluating the efficacy of immunotherapy in soft tissue sarcoma.

Background: The tertiary lymphatic structure (TLS) is an important component of the tumor immune microenvironment and has important significance in patient prognosis and response to immune therapy. However, the underlying mechanism of TLS in soft tissue sarcoma remains unclear. Methods: A total of 256 RNAseq and 7 single-cell sequencing samples were collected from TCGA-SARC and GSE212527 cohorts. Based on published TLS-related gene sets, four TLS scores were established by GSVA algorithm. The immune cell infiltration was calculated via TIMER2.0 and "MCPcounter" algorithms. In addition, the univariate, LASSO, and multivariate-Cox analyses were used to select TLS-related and prognosis-significant hub genes. Single-cell sequencing dataset, clinical immunohistochemical, and cell experiments were utilized to validate the hub genes. Results: In this study, four TLS-related scores were identified, and the total-gene TLS score more accurately reflected the infiltration level of TLS in STS. We further established two hub genes (DUSP9 and TNFSF14) prognosis markers and risk scores associated with soft tissue sarcoma prognosis and immune therapy response. Flow cytometry analysis showed that the amount of CD3, CD8, CD19, and CD11c positive immune cell infiltration in the tumor tissue dedifferentiated liposarcoma patients was significantly higher than that of liposarcoma patients. Cytological experiments showed that soft tissue sarcoma cell lines overexpressing TNFSF14 could inhibit the proliferation and migration of sarcoma cells. Conclusion: This study systematically explored the TLS and related genes from the perspectives of bioinformatics, clinical features and cytology experiments. The total-gene TLS score, risk score and TNFSF14 hub gene may be useful biomarkers for predicting the prognosis and immunotherapy efficacy of soft tissue sarcoma.

The bispecific B7H3xCD3 antibody CC-3 induces T cell immunity against bone and soft tissue sarcomas.

Sarcomas are rare and heterogeneous malignancies that are difficult to treat. Approximately 50% of patients diagnosed with sarcoma develop metastatic disease with so far very limited treatment options. The transmembrane protein B7-H3 reportedly is expressed in various malignancies, including different sarcoma subtypes. In several cancer entities B7-H3 expression is associated with poor prognosis. In turn, B7-H3 is considered a promising target for immunotherapeutic approaches. We here report on the preclinical characterization of a B7-H3xCD3 bispecific antibody in an IgG-based format, termed CC-3, for treatment of different sarcoma subtypes. We found B7-H3 to be expressed on all sarcoma cells tested and expression on sarcoma patients correlated with decreased progression-free and overall survival. CC-3 was found to elicit robust T cell responses against multiple sarcoma subtypes, resulting in significant activation, release of cytokines and effector molecules. In addition, CC-3 promoted T cell proliferation and differentiation, resulting in the generation of memory T cell subsets. Finally, CC-3 induced potent target cell lysis in a target cell restricted manner. Based on these results, a clinical trial evaluating CC-3 in soft tissue sarcoma is currently in preparation.

Correlations between the modification patterns mediated by pyroptosis-related genes, tumor microenvironment, and immunotherapy in soft tissue sarcoma.

Soft tissue sarcoma (STS) incidence, progression, and metastasis are tightly linked to the tumor microenvironment (TME). The modification patterns mediated by pyroptosis-related genes (PRGs) in STS are unknown regarding the immune cell infiltration landscape of TME, immunotherapy effect, and prognostic value. First, we downloaded STS samples from the Cancer Genome Atlas (TCGA) and gene-expression omnibus (GEO) databases. Based on 52 PRGs, 2 pyroptosis modification patterns were analyzed, and the associations of pyroptosis modification patterns with immune cell infiltration in the TME were elucidated systematically. To quantify PRG modification patterns in STS patients, we generated a pyroptosis scoring system using principal component analysis (PCA). We identified 2 distinct pyroptosis modification patterns in STS. Compared to PRG cluster A, the prognosis of cluster B was better. These 2 pyroptosis modification patterns corresponded to different characteristics of immune cell infiltration in the TME and biological behaviors. In the pyroptosis scoring system, a high pyroptosis score was connected to higher immune cell infiltration, stronger immune surveillance, immune-killing effects on tumor cells, and better clinical benefits. The results from 3 anti-PD1/PD-L1-treated immune cohorts demonstrated that higher pyroptosis scores are also closely connected to better immunotherapy results. We demonstrated that pyroptosis modification is essential to the STS microenvironment. Moreover, the pyroptosis score is a reliable and independent prognostic factor in STS patients, enabling a richer understanding of the STS microenvironment and the screening of immunotherapy candidates, predicting the immunotherapeutic effects for individual STS patients, and guiding the use of chemotherapy drugs.

Sarcoma microenvironment cell states and ecosystems are associated with prognosis and predict response to immunotherapy.

Characterization of the diverse malignant and stromal cell states that make up soft tissue sarcomas and their correlation with patient outcomes has proven difficult using fixed clinical specimens. Here, we employed EcoTyper, a machine-learning framework, to identify the fundamental cell states and cellular ecosystems that make up sarcomas on a large scale using bulk transcriptomes with clinical annotations. We identified and validated 23 sarcoma-specific, transcriptionally defined cell states, many of which were highly prognostic of patient outcomes across independent datasets. We discovered three conserved cellular communities or ecotypes associated with underlying genomic alterations and distinct clinical outcomes. We show that one ecotype defined by tumor-associated macrophages and epithelial-like malignant cells predicts response to immune-checkpoint inhibition but not chemotherapy and validate our findings in an independent cohort. Our results may enable identification of patients with soft tissue sarcomas who could benefit from immunotherapy and help develop new therapeutic strategies.

A randomized, non-comparative phase 2 study of neoadjuvant immune-checkpoint blockade in retroperitoneal dedifferentiated liposarcoma and extremity/truncal undifferentiated pleomorphic sarcoma.

Based on the demonstrated clinical activity of immune-checkpoint blockade (ICB) in advanced dedifferentiated liposarcoma (DDLPS) and undifferentiated pleomorphic sarcoma (UPS), we conducted a randomized, non-comparative phase 2 trial ( NCT03307616 ) of neoadjuvant nivolumab or nivolumab/ipilimumab in patients with resectable retroperitoneal DDLPS (n = 17) and extremity/truncal UPS (+ concurrent nivolumab/radiation therapy; n = 10). The primary end point of pathologic response (percent hyalinization) was a median of 8.8% in DDLPS and 89% in UPS. Secondary end points were the changes in immune infiltrate, radiographic response, 12- and 24-month relapse-free survival and overall survival. Lower densities of regulatory T cells before treatment were associated with a major pathologic response (hyalinization > 30%). Tumor infiltration by B cells was increased following neoadjuvant treatment and was associated with overall survival in DDLPS. B cell infiltration was associated with higher densities of regulatory T cells before treatment, which was lost upon ICB treatment. Our data demonstrate that neoadjuvant ICB is associated with complex immune changes within the tumor microenvironment in DDLPS and UPS and that neoadjuvant ICB with concurrent radiotherapy has significant efficacy in UPS.

Intratumoral NKp46+ natural killer cells are spatially distanced from T and MHC-I+ cells with prognostic implications in soft tissue sarcoma.

Introduction: Soft tissue sarcomas (STS) are rare, heterogenous malignancies with an unmet need for novel immunotherapies. Tumor infiltrating lymphocytes (TILs) have been linked with favorable outcomes in STS patients, though the contribution of natural killer (NK) cells and spatial relationships of TILs with MHC-I expressing cells lacks detailed characterization. Experimental design: Using archived and prospectively collected specimens, we evaluated intratumoral NK cells by immunohistochemistry (IHC), flow cytometry, and immunofluorescence (IF). We assessed spatial localization of NK and T cells by multiplex IF, analyzing the effects of MHC-I expression status on NK and T cell clustering. Results: Both intratumoral NKp46 and CD56dim expression were associated with significantly improved overall survival (P=0.05), while higher infiltrates of CD56bright NK cells predicted a worse prognosis (P=0.05). The presence of intratumoral NK cells was inversely proportional to CD3+ T cells. Spatial analyses showed NK cells preferentially clustering close to other NK cells with sparse CD3+ T and CD8+ T cells in range (P<0.0001). Additionally, CD3+ T and CD8+ T cells showed significantly greater co-localization with MHC-I+ cells, compared to NK cells (P<0.0001). After neoadjuvant radiotherapy, there was greater CD8 clustering, while after neoadjuvant chemotherapy, there was overall lower TIL clustering. Conclusion: Intratumoral NK cells are prognostic in STS and localize closer to MHC-I- cells than T cells. Although both NK and T cells are associated with improved survival in STS, their differential distribution in the TME based on MHC-I expression status may serve as a biomarker for improved immunotherapy treatment selection.

circCsnk1g3- and circAnkib1-regulated interferon responses in sarcoma promote tumorigenesis by shaping the immune microenvironment.

Exonic circular RNAs (circRNAs) produce predominantly non-coding RNA species that have been recently profiled in many tumors. However, their functional contribution to cancer progression is still poorly understood. Here, we identify the circRNAs expressed in soft tissue sarcoma cells and explore how the circRNAs regulate sarcoma growth in vivo. We show that circCsnk1g3 and circAnkib1 promote tumor growth by shaping a pro-tumorigenic microenvironment, possibly due to their capabilities to regulate tumor-promoting elements extrinsic to the tumor cells. Accordingly, circCsnk1g3 and circAnkib1 can control the expression of interferon-related genes and pro-inflammatory factors in the sarcoma cells, thus directing immune cell recruitment into the tumor mass, and hence their activation. Mechanistically, circRNAs may repress pro-inflammatory elements by buffering activation of the pathways mediated by RIG-I, the cytosolic viral RNA sensor. The current findings suggest that the targeting of specific circRNAs could augment the efficacy of tumor and immune response to mainstay therapies.

Immune-Desert Tumor Microenvironment in Thoracic SMARCA4-Deficient Undifferentiated Tumors with Limited Efficacy of Immune Checkpoint Inhibitors.

BACKGROUND: Thoracic SMARCA4-deficient undifferentiated tumors (SMARCA4-UT) are aggressive neoplasms. Data linking BAF alterations with tumor microenvironment (TME) and efficacy of immune checkpoint inhibitors (ICI) are contradictory. The TME of SMARCA4-UT and their response to ICI are unknown. MATERIALS AND METHODS: Patients diagnosed with SMARCA4-UT in our institution were included. Immunostainings for tertiary lymphoid structures (TLS), immune cell markers, and checkpoints were assessed. Validation was performed using an independent transcriptome dataset including SMARCA4-UT, non-small cell lung cancers (NSCLC) with/without SMARCA4 mutations, and unclassified thoracic sarcomas (UTS). CXCL9 and PD-L1 expressions were assessed in NSCLC and thoracic fibroblast cell lines, with/without SMARCA4 knockdown, treated with/without interferon gamma. RESULTS: Nine patients were identified. All samples but one showed no TLS, consistent with an immune desert TME phenotype. Four patients received ICI as part of their treatment, but the only one who responded, had a tumor with a TLS and immune-rich TME. Unsupervised clustering of the validation cohort using immune cell scores identified 2 clusters associated with cell ontogeny and immunity (cluster 1 enriched for NSCLC independently of SMARCA4 status (n = 9/10; P = .001); cluster 2 enriched for SMARCA4-UT (n = 11/12; P = .005) and UTS (n = 5/5; P = .0005). SMARCA4 loss-of-function experiments revealed interferon-induced upregulation of CXCL9 and PD-L1 expression in the NSCLC cell line with no effect on the thoracic fibroblast cell line. CONCLUSION: SMARCA4-UT mainly have an immune desert TME with limited efficacy to ICI. TME of SMARCA4-driven tumors varies according to the cell of origin questioning the interplay between BAF alterations, cell ontogeny and immunity.

Identification of Prognostic lncRNA Related to the Immune Microenvironment of Soft Tissue Sarcoma.

BACKGROUND: Soft tissue sarcoma is a malignant tumor with high degree of malignancy and poor prognosis, originating from mesenchymal tissue. Long noncoding RNAs (lncRNAs) are involved in various biological and pathological processes in the body. They perform preprocessing, splicing, transport, degradation, and translation of mRNA to achieve posttranscriptional level regulation, resulting in the occurrence, invasion, and metastasis of tumors. Therefore, they are highly relevant with regard to early diagnoses and as prognostic indicators. OBJECTIVE: The objective of the present study was to identify immune microenvironment-related lncRNAs that can be used to predict soft tissue sarcomas. METHODS: Clinical data and follow-up data were obtained from the cBioPortal database, and RNA sequencing data used for the model structure can be accessed from The Cancer Genome Atlas (TCGA) database. LncRNAs were screened by differential expression analysis and coexpression analysis. The Cox regression model and Kaplan-Meier analysis were used to study the association between lncRNAs and soft tissue sarcoma prognosis in the immune microenvironment. Unsupervised cluster analysis was then completed to discover the impact of screening lncRNAs on disease. We constructed an mRNA-lncRNA network by Cytoscape software. Finally, qRT-PCR was used to verify the difference in the expression of the lncRNAs in normal cells and sarcoma cells. RESULTS: Unsupervised cluster analysis revealed that the 210 lncRNAs screened showed strong correlation with the tumor immune microenvironment. Two signatures containing seven and five lncRNAs related to the tumor microenvironment were constructed and used to predict overall survival (OS) and disease-free survival (DFS). The Kaplan-Meier (K-M) survival curve showed that the prognoses of patients in the high-risk and low-risk groups differed significantly, and the prognosis associated with the low-risk group was better than that associated with the high-risk group. Two nomograms with predictive capabilities were established. qRT-PCR results showed that the expression of AC108134.3 and AL031717.1 was significantly different in normal and sarcoma cells. CONCLUSION: In summary, the experimental results showed that lncrnA associated with immune microenvironment was related to tumor, which may provide a new idea for immunotherapy of STS.

Evaluation of PD-L1 Expression in Undifferentiated Pleomorphic Sarcomas, Liposarcomas and Chondrosarcomas.

Immune checkpoint inhibitors (ICIs) such as PD1/PD-L1 blockers are an established treatment for many solid cancers. There are currently no approved ICIs for sarcomas, but satisfactory results have been seen in some patients with disseminated disease in certain histological types. Most studies on PD-L1 in sarcoma have used small specimens and there are no clear cutoff values for scoring. We investigated PD-L1 immunoreactivity in high-grade chondrosarcomas (CS), abdominal liposarcoma (LS) and undifferentiated pleomorphic sarcomas (UPS). In total, 230 tumors were stained with SP142 and SP263 assays and evaluated by two clinical pathologists. Immunoreactivity in tumor and immune cells was correlated with clinical outcome. Overall, ≥1% PD-L1 immunoreactivity in tumor cells was found in 11 CS, 26 LS and 59 UPS (SP142 assay) and in 10 CS, 26 LS and 77 UPS (SP263 assay). Most tumors exhibited ≤10% PD-L1 immunoreactivity, but a subset across all three subtypes had >50%. Kaplan-Meier survival analysis showed no significant difference in metastasis-free or overall survival in relation to PD-L1 immunoreactivity in tumor or immune cells for any subtype. As there is a lack of clinical data regarding PD-L1/PD-1 status and therapy response, it is not currently possible to establish clear cutoff values. Patients with high (>50%) PD-L1 immunoreactivity in tumor cells (TC) with the SP263 assay would be a logical group to investigate for potentially beneficial PD1/PD-L1-targeted treatment.

Immunologic constant of rejection signature is prognostic in soft-tissue sarcoma and refines the CINSARC signature.

BACKGROUND: Soft-tissue sarcomas (STSs) are heterogeneous and aggressive tumors, with high metastatic risk. The immunologic constant of rejection (ICR) 20-gene signature is a signature of cytotoxic immune response. We hypothesized that ICR might improve the prognostic assessment of early-stage STS. METHODS: We retrospectively applied ICR to 1455 non-metastatic STS and searched for correlations between ICR classes and clinicopathological and biological variables, including metastasis-free survival (MFS). RESULTS: Thirty-four per cent of tumors were classified as ICR1, 27% ICR2, 24% ICR3, and 15% ICR4. These classes were associated with patients' age, pathological type, and tumor depth, and an enrichment from ICR1 to ICR4 of quantitative/qualitative scores of immune response. ICR1 class was associated with a 59% increased risk of metastatic relapse when compared with ICR2-4 class. In multivariate analysis, ICR classification remained associated with MFS, as well as pathological type and Complexity Index in Sarcomas (CINSARC) classification, suggesting independent prognostic value. A prognostic clinicogenomic model, including the three variables, was built in a learning set (n=339) and validated in an independent set (n=339), showing greater prognostic precision than each variable alone or in doublet. Finally, connectivity mapping analysis identified drug classes potentially able to reverse the expression profile of poor-prognosis tumors, such as chemotherapy and targeted therapies. CONCLUSION: ICR signature is independently associated with postoperative MFS in early-stage STS, independently from other prognostic features, including CINSARC. We built a robust prognostic clinicogenomic model integrating ICR, CINSARC, and pathological type, and suggested differential vulnerability of each prognostic group to different systemic therapies.

Immunological and prognostic significance of novel ferroptosis-related genes in soft tissue sarcoma.

BACKGROUND: Ferroptosis has exhibited great potential in the treatment of cancer and has gained widespread attention in soft tissue sarcoma (STS). The aim was to explore the immunological and prognostic significance of novel ferroptosis-related genes in STS. METHODS: We identified ferroptosis-related differentially expressed genes (DEGs) in STS to construct the networks of enrichment analysis and protein-protein interaction. Subsequently, hub genes with prognostic significance were localized and a series of prognostic and immune analyses were performed. RESULTS: 40 ferroptosis-related DEGs were identified, of which HELLS, STMN1 EPAS1, CXCL2, NQO1, and IL6 were classified as hub genes and were associated with the prognosis in STS patients. In the results of the immune analysis, PDCD1, CTLA4, TIGIT, IDO1 and CD27 exhibited consistent intense correlations as immune checkpoint genes, as well as macrophage, neutrophil, cytotoxic cell, dendritic cell, interdigitating dendritic cell and plasmacytoid dendritic cell as immune cells. EPAS1 and HELLS might be independent prognostic factors for STS patients, and separate prognostic models were constructed by using them. CONCLUSIONS: We recognized novel ferroptosis-related genes with prognostic value in STS. Furthermore, we searched out potential immune checkpoints and critical immune cells.

A seven-gene signature and the C-C motif chemokine receptor family genes are the sarcoma-related immune genes.

Cells of the tumor microenvironment exert a vital influence on sarcoma prognosis. This study aimed to analyze and identify differentially expressed genes (DEGs) related to immunity and their significance as immune biomarkers for the accurate prediction of overall survival of patients with sarcoma. The Cancer Genome Atlas was adopted for obtaining sarcoma gene microarray and corresponding clinical information. ESTIMATE algorithm was used to calculate tumor immune microenvironment indices. Immune-associated DEGs were identified using the limma packages and were further analyzed using the ClusterProfiler package and STRING website. Based on the results of these analyses, we constructed a prognostic model. Furthermore, we assessed the prognosis prediction model through functional evaluation and analysis of GSE17674. The functional analysis revealed that the upregulated immune DEGs were related to immune-related aspects. Chemokine ligands/receptors and immune-related genes were found to be vital for sarcoma formation and progression. We established a prognostic signature of seven genes, which indicated that high-risk cases exhibit poor prognostic outcome. The prognostic signature constructed in this study can accurately predict the overall prognosis in patients with sarcoma. Moreover, the novel immune gene expression analysis may provide clinical guidance for predicting prognosis in patients with sarcoma.

Tumor-propagating side population cells are a dynamic subpopulation in undifferentiated pleomorphic sarcoma.

Sarcomas contain a subpopulation of tumor-propagating cells (TPCs) with enhanced tumor-initiating and self-renewal properties. However, it is unclear whether the TPC phenotype in sarcomas is stable or a dynamic cell state that can derive from non-TPCs. In this study, we utilized a mouse model of undifferentiated pleomorphic sarcoma (UPS) to trace the lineage relationship between sarcoma side population (SP) cells that are enriched for TPCs and non-SP cells. By cotransplanting SP and non-SP cells expressing different endogenous fluorescent reporters, we show that non-SP cells can give rise to SP cells with enhanced tumor-propagating potential in vivo. Lineage trajectory analysis using single-cell RNA sequencing from SP and non-SP cells supports the notion that non-SP cells can assume the SP cell phenotype de novo. To test the effect of eradicating SP cells on tumor growth and self-renewal, we generated mouse sarcomas in which the diphtheria toxin receptor is expressed in the SP cells and their progeny. Ablation of the SP population using diphtheria toxin did not impede tumor growth or self-renewal. Altogether, we show that the sarcoma SP represent a dynamic cell state and targeting TPCs alone is insufficient to eliminate tumor progression.

High Levels of LINC01140 Expression Predict a Good Prognosis and Improve Radiotherapy in Sarcoma Patients.

Long intergenic non-protein coding RNA has an important biological role in tumors. But, LINC01140 in sarcoma has not been studied yet. This study investigates the expression and prognosis role of LINC01140 in sarcoma. LINC01140 was lower in metastatic sarcoma, and low LINC01140 expression predicted poor overall survival, disease-free survival, and disease-specific survival in sarcoma. High LINC01140 expression and radiotherapy could promote survival of sarcoma. Gene set enrichment analysis showed LINC01140 was involved in interferon-gamma response, epithelial-mesenchymal transition, the interaction between cytokine receptors, and cholesterol homeostasis. Gene ontology enrichment analysis showed LINC01140 was involved in immunity, fatty acid metabolism, amino acid metabolism, cell division, serine/threonine-protein kinase. LINC01140 expression was negatively correlated with various epithelial-mesenchymal transition factors and positively correlated with the expression of anti-cancer factor hypermethylated-in-cancer 1. These results confirmed that LINC01140 may be a potential novel prognostic molecule in sarcoma.

Associations of PD-L1, PD-L2, and HLA class I expression with responses to immunotherapy in patients with advanced sarcoma: post hoc analysis of a phase 1/2 trial

Background Although immunotherapy is thought to be a promising cancer treatment, most patients do not respond to immunotherapy. In this post hoc analysis of a phase 1/2 study, associations of programmed death ligand 1 (PD-L1), PD-L2, and HLA class I expressions with responses to dendritic cells (DCs)-based immunotherapy were investigated in patients with advanced sarcoma. Methods This study enrolled 35 patients with metastatic and/or recurrent sarcomas who underwent DC-based immunotherapy. The associations of PD-L1, PD-L2, and HLA class I expressions in tumor specimens, which were resected before immunotherapy, with immune responses (increases of IFN-γ and IL-12) and oncological outcomes were evaluated. Results Patients who were PD-L2 (+) showed lower increases of IFN-γ and IL-12 after DC-based immunotherapy than patients who were PD-L2 (−). The disease control (partial response or stable disease) rates of patients who were PD-L1 (+) and PD-L1 (−) were 0% and 22%, respectively. Disease control rates of patients who were PD-L2 (+) and PD-L2 (−) were 13% and 22%, respectively. Patients who were PD-L1 (+) tumors had significantly poorer overall survival compared with patients who were PD-L1 (−). No associations of HLA class I expression with the immune response or oncological outcomes were observed. Conclusions This study suggests that PD-L1 and PD-L2 are promising biomarkers of DC-based immunotherapy, and that addition of immune checkpoint inhibitors to DC-based immunotherapy may improve the outcomes of DC-based immunotherapy (AU)

Molecular Determinants of Soft Tissue Sarcoma Immunity: Targets for Immune Intervention.

Soft tissue sarcomas (STSs) are a family of rare malignant tumors encompassing more than 80 histologies. Current therapies for metastatic STS, a condition that affects roughly half of patients, have limited efficacy, making innovative therapeutic strategies urgently needed. From a molecular point of view, STSs can be classified as translocation-related and those with a heavily rearranged genotype. Although only the latter display an increased mutational burden, molecular profiles suggestive of an "immune hot" tumor microenvironment are observed across STS histologies, and response to immunotherapy has been reported in both translocation-related and genetic complex STSs. These data reinforce the notion that immunity in STSs is multifaceted and influenced by both genetic and epigenetic determinants. Cumulative evidence indicates that a fine characterization of STSs at different levels is required to identify biomarkers predictive of immunotherapy response and to discover targetable pathways to switch on the immune sensitivity of "immune cold" tumors. In this review, we will summarize recent findings on the interplay between genetic landscape, molecular profiling and immunity in STSs. Immunological and molecular features will be discussed for their prognostic value in selected STS histologies. Finally, the local and systemic immunomodulatory effects of the targeted drugs imatinib and sunitinib will be discussed.

The KrasG12D;Trp53fl/fl murine model of undifferentiated pleomorphic sarcoma is macrophage dense, lymphocyte poor, and resistant to immune checkpoint blockade.

Sarcomas are rare, difficult to treat, mesenchymal lineage tumours that affect children and adults. Immunologically-based therapies have improved outcomes for numerous adult cancers, however, these therapeutic strategies have been minimally effective in sarcoma so far. Clinically relevant, immunologically-competent, and transplantable pre-clinical sarcoma models are essential to advance sarcoma immunology research. Herein we show that Cre-mediated activation of KrasG12D, and deletion of Trp53, in the hindlimb muscles of C57Bl/6 mice results in the highly penetrant, rapid onset undifferentiated pleomorphic sarcomas (UPS), one of the most common human sarcoma subtypes. Cell lines derived from spontaneous UPS tumours can be reproducibly transplanted into the hindlimbs or lungs of naïve, immune competent syngeneic mice. Immunological characterization of both spontaneous and transplanted UPS tumours demonstrates an immunologically-'quiescent' microenvironment, characterized by a paucity of lymphocytes, limited spontaneous adaptive immune pathways, and dense macrophage infiltrates. Macrophages are the dominant immune population in both spontaneous and transplanted UPS tumours, although compared to spontaneous tumours, transplanted tumours demonstrate increased spontaneous lymphocytic infiltrates. The growth of transplanted UPS tumours is unaffected by host lymphocyte deficiency, and despite strong expression of PD-1 on tumour infiltrating lymphocytes, tumours are resistant to immunological checkpoint blockade. This spontaneous and transplantable immune competent UPS model will be an important experimental tool in the pre-clinical development and evaluation of novel immunotherapeutic approaches for immunologically cold soft tissue sarcomas.