Sarcoma Microenvironment Research Articles

The Impact of Extracellular Vesicles on Inflammation in the Tumor Microenvironment of Sarcomas: A Literature Review

The Impact of Extracellular Vesicles on Inflammation in the Tumor Microenvironment of Sarcomas: A Literature Review

Predictive and Dynamic Signature for Antiangiogenics in Combination with a PD1 Inhibitor in Soft-Tissue Sarcoma: Correlative Studies Linked to the IMMUNOSARC Trial.

The IMMUNOSARC trial combined an antiangiogenic agent (sunitinib) with a PD1 inhibitor (nivolumab) in advanced sarcomas. Here, we present the first correlative studies of the soft-tissue sarcoma cohort enrolled in this trial. Formalin-fixed paraffin-embedded and peripheral blood samples were collected at baseline and week 13. Formalin-fixed paraffin-embedded samples were used for transcriptomics and multiplex immunofluorescence, whereas peripheral blood samples were used for multiplexed immunoassays. Flow cytometry and Luminex assays were performed to validate translational findings in tumor-isolated cells and peripheral blood mononuclear cells derived from patients. The density of intratumoral CD8+ T cells, measured by multiplexed immunophenotyping, was significantly increased after treatment. This augment was accompanied by the dynamic significant increase in the gene expressions of CD86, CHI3L1, CXCL10, CXCL9, LAG3, and VCAM1 and the decrease in the expression levels of NR4A1. In peripheral blood, 12 proteins were significantly modulated by treatment at week 13. A score integrating the dynamic expression of the 7 genes and the 12 soluble factors separated 2 groups with distinct progression-free survival (PFS): 4.1 months [95% confidence interval, 3.5-not reached (NR)] versus 17 months (95% confidence interval, 12.0-NR), P = 0.014. This molecular score was predictive of PFS when applied to the normalized data determined in the baseline samples. Treatment with sunitinib and nivolumab inflamed the sarcoma microenvironment, increasing CD8+ T-cell density and the expression of several genes/proteins with relevance in the response to PD1 inhibitors. A molecular signature identified two groups of patients with distinct PFS for the combination of antiangiogenics plus PD1 inhibitor therapy.

Circumventing resistance within the Ewing sarcoma microenvironment by combinatorial innate immunotherapy

BackgroundPediatric patients with recurrent/metastatic Ewing sarcoma (ES) have a dismal 5-year survival. Novel therapeutic approaches are desperately needed. Natural killer (NK) cell number and function are low in ES patient...

Prognostic Value of Cancer-Associated Fibroblast Marker Expression in the Intratumoral and Marginal Areas of Soft Tissue Sarcoma.

The tumor microenvironment of sarcomas has not been studied in detail; in particular, little is known about cancer-associated fibroblasts (CAFs). Sarcoma cells are difficult to distinguish from CAFs, either histomorphologically or immunohistochemically. We scored the expression of individual CAF markers (fibroblast-activating protein [FAP], CD10, and podoplanin) in the intratumoral and marginal areas of 133 sarcomas. We also examined the association between these markers, as well as the number of CD163-positive macrophages (i.e., tumor-associated macrophages), and clinical outcome. In all cases, the log-rank test revealed that those with high marker scores and macrophage counts (except for marginal CD10+ CAFs) showed significantly worse disease-free survival (DFS). Grade 2/3 cases with high CAF scores (excluding the marginal FAP and CD10 scores) showed significantly worse DFS, whereas those with high intratumoral FAP/CD10 and marginal podoplanin scores showed significantly worse metastasis-free survival (MFS), and those with high intratumoral CD10 score showed significantly worse local recurrence-free survival (LFS). Multivariate analysis identified intratumoral CD10/podoplanin scores and marginal FAP/podoplanin scores as independent prognostic factors for DFS, intratumoral FAP/CD10 and marginal FAP/podoplanin/CD163-positive macrophage scores as independent prognostic factors for MFS, and the intratumoral podoplanin score as an independent prognostic factor for LFS. There was a weak-to-moderate correlation between each score and CD163-positive macrophage counts. Patients with high CAF marker expression in the intratumoral and marginal areas have a poorer outcome.

Clinical and translational implications of immunotherapy in sarcomas.

Immunotherapy has emerged as promising treatment in sarcomas, but the high variability in terms of histology, clinical behavior and response to treatments determines a particular challenge for its role in these neoplasms. Tumor immune microenvironment (TiME) of sarcomas reflects the heterogeneity of these tumors originating from mesenchymal cells and encompassing more than 100 histologies. Advances in the understanding of the complexity of TiME have led to an improvement of the immunotherapeutic responsiveness in sarcomas, that at first showed disappointing results. The proposed immune-classification of sarcomas based on the interaction between immune cell populations and tumor cells showed to have a prognostic and potential predictive role for immunotherapies. Several studies have explored the clinical impact of immune therapies in the management of these histotypes leading to controversial results. The presence of Tumor Infiltrating Lymphocytes (TIL) seems to correlate with an improvement in the survival of patients and with a higher responsiveness to immunotherapy. In this context, it is important to consider that also immune-related genes (IRGs) have been demonstrated to have a key role in tumorigenesis and in the building of tumor immune microenvironment. The IRGs landscape in soft tissue and bone sarcomas is characterized by the connection between several tumor-related genes that can assume a potential prognostic and predictive therapeutic role. In this paper, we reviewed the state of art of the principal immune strategies in the management of sarcomas including their clinical and translational relevance.

TrkA + sensory neurons regulate osteosarcoma proliferation and vascularization to promote disease progression.

Bone pain is a presenting feature of bone cancers such as osteosarcoma (OS), relayed by skeletal-innervating peripheral afferent neurons. Potential functions of tumor-associated sensory neurons in bone cancers beyond pain sensation are unknown. To uncover neural regulatory functions, a chemical-genetic approach in mice with a knock-in allele for TrkA was used to functionally perturb sensory nerve innervation during OS growth and disease progression. TrkA inhibition in transgenic mice led to significant reductions in sarcoma-associated sensory innervation and vascularization, tumor growth and metastasis, and prolonged overall survival. Single-cell transcriptomics revealed that sarcoma denervation was associated with phenotypic alterations in both OS tumor cells and cells within the tumor microenvironment, and with reduced calcitonin gene-related peptide (CGRP) and vascular endothelial growth factor (VEGF) signaling. Multimodal and multi-omics analyses of human OS bone samples and human dorsal root ganglia neurons further implicated peripheral innervation and neurotrophin signaling in OS tumor biology. In order to curb tumor-associated axonal ingrowth, we next leveraged FDA-approved bupivacaine liposomes leading to significant reductions in sarcoma growth, vascularity, as well as alleviation of pain. In sum, TrkA-expressing peripheral neurons positively regulate key aspects of OS progression and sensory neural inhibition appears to disrupt calcitonin receptor signaling (CALCR) and VEGF signaling within the sarcoma microenvironment leading to significantly reduced tumor growth and improved survival. These data suggest that interventions to prevent pathological innervation of osteosarcoma represent a novel adjunctive therapy to improve clinical outcomes and survival.

Oncogene-induced matrix reorganization controls CD8+ T cell function in the soft-tissue sarcoma microenvironment.

CD8+ T cell dysfunction impedes antitumor immunity in solid cancers, but the underlying mechanisms are diverse and poorly understood. Extracellular matrix (ECM) composition has been linked to impaired T cell migration and enhanced tumor progression; however, impacts of individual ECM molecules on T cell function in the tumor microenvironment (TME) are only beginning to be elucidated. Upstream regulators of aberrant ECM deposition and organization in solid tumors are equally ill-defined. Therefore, we investigated how ECM composition modulates CD8+ T cell function in undifferentiated pleomorphic sarcoma (UPS), an immunologically active desmoplastic tumor. Using an autochthonous murine model of UPS and data from multiple human patient cohorts, we discovered a multifaceted mechanism wherein the transcriptional coactivator YAP1 promotes collagen VI (COLVI) deposition in the UPS TME. In turn, COLVI induces CD8+ T cell dysfunction and immune evasion by remodeling fibrillar collagen and inhibiting T cell autophagic flux. Unexpectedly, collagen I (COLI) opposed COLVI in this setting, promoting CD8+ T cell function and acting as a tumor suppressor. Thus, CD8+ T cell responses in sarcoma depend on oncogene-mediated ECM composition and remodeling.

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.

Impact of metronomic trabectedin combined with low-dose cyclophosphamide on sarcoma microenvironment and correlation with clinical outcome: results from the TARMIC study.

Soft tissue sarcomas (STS) are diverse mesenchymal tumors with few therapeutic options in advanced stages. Trabectedin has global approval for treating STS patients resistant to anthracycline-based regimens. Recent pre-clinical data suggest that trabectedin's antitumor activity extends beyond tumor cells to influencing the tumor microenvironment (TME), especially affecting tumor-associated macrophages and their pro-tumoral functions. We present the phase I/II results evaluating a combination of metronomic trabectedin and low-dose cyclophosphamide on the TME in patients with advanced sarcomas. 50 patients participated: 20 in phase I and 30 in phase II. Changes in the TME were assessed in 28 patients using sequential tumor samples at baseline and day two of the cycle. Treatment notably decreased CD68 + CD163 + macrophages in biopsies from tumor lesions compared to pre-treatment samples in 9 of the 28 patients after 4 weeks. Baseline CD8 + T cell presence increased in 11 of these patients. In summary, up to 57% of patients exhibited a positive immunological response marked by reduced M2 macrophages or increased CD8 + T cells post-treatment. This positive shift in the TME correlated with improved clinical benefit and progression-free survival. This study offers the first prospective evidence of trabectedin's immunological effect in advanced STS patients, highlighting a relationship between TME modulation and patient outcomes.This study was registered with ClinicalTrial.gov, number NCT02406781.

The Role of Macrophages in Sarcoma Tumor Microenvironment and Treatment.

Sarcomas are a heterogeneous group of malignant mesenchymal tumors, including soft tissue and bone sarcomas. Macrophages in the tumor microenvironment, involved in immunosuppression and leading to tumor development, are called tumor-associated macrophages (TAMs). TAMs are very important in modulating the microenvironment of sarcomas by expressing specific markers and secreting factors that influence immune and tumor cells. They are involved in many signaling pathways, such as p-STAT3/p-Erk1/2, PI3K/Akt, JAK/MAPK, and JAK/STAT3. TAMs also significantly impact the clinical outcomes of patients suffering from sarcomas and are mainly related to poor overall survival rates among bone and soft tissue sarcomas, for example, chondrosarcoma, osteosarcoma, liposarcoma, synovial sarcoma, and undifferentiated pleomorphic sarcoma. This review summarizes the current knowledge on TAMs in sarcomas, focusing on specific markers on sarcoma cells, cell-cell interactions, and the possibly involved molecular pathways. Furthermore, we discuss the clinical significance of macrophages in sarcomas as a potential target for new therapies, presenting clinical relevance, possible new treatment options, and ongoing clinical trials using TAMs in sarcoma treatment.

Immune microenvironment of intimal sarcomas: Adaptive immune resistance with potential therapeutic implications.

Intimal sarcomas are rare, aggressive neoplasms that arise from large blood vessels. Characterization of the tumor immune microenvironment may suggest new treatment strategies. Seventeen specimens from 7 patients were labeled by immunohistochemistry for programmed cell death 1 ligand 1 (PD-L1), CD45, CD8, CD4, FOXP3, CD20, CD68, and LAG3. The immune cell density was scored as a percentage of the tumor area (1+ [<5%], 2+ [5%-50%], 3+ [>50%]); PD-L1 expression was scored on tumor cells and on intratumoral immune cells. Immune marker density was quantified using image analysis software. All intimal sarcomas showed immune cell infiltration (41% were 1+, 53% were 2+, 6% were 3+). Tumor and immune cell PD-L1 labeling was seen in 35% and 76% of cases, respectively; PD-L1+ intimal sarcomas had higher CD45+, CD8+, FOXP3+, CD68+, and leukocyte activation gene 3 (LAG3)+ cell densities (P ≤ .01). Similarly, PD-L1 expression on immune cells correlated with higher densities of CD8+ and FOXP3+ cells (P < .04). Higher LAG3+ cell density correlated with higher CD68+ cell density and necrosis (P < .05). One patient with prolonged survival had the highest immune cell density and PD-L1 expression. These data show that intimal sarcomas have an active tumor microenvironment with an adaptive pattern of PD-L1 expression. Our results suggest that immunotherapy may be an effective treatment option.

Breaking down the tumor immune infiltration within pediatric sarcomas.

Immunotherapies are a promising therapeutic option, yet for a variety of reasons, these treatments have achieved limited success against sarcomas. The immunosuppressive tumor microenvironment (TME) of sarcomas as well as lack of predictive biomarkers, decreased T-cell clonal frequency, and high expression of immunosuppressive infiltrating cells has thus far prevented major success using immunotherapies. By breaking down the TME into its individual components and understanding how the various cell types interact with each other as well as in the context of the complex immune microenvironment, can lead to effective therapeutic immunotherapy treatments, potentially improving outcomes for those with metastatic disease.

Targeting epigenetic aberrations of sarcoma in CRISPR era.

Sarcomas are rare malignancies that exhibit diverse biological, genetic, morphological, and clinical characteristics. Genetic alterations, such as gene fusions, mutations in transcriptional machinery components, histones, and DNA methylation regulatory molecules, play an essential role in sarcomagenesis. These mutations induce and/or cooperate with specific epigenetic aberrations required for the growth and maintenance of sarcomas. Appropriate mouse models have been developed to clarify the significance of genetic and epigenetic interactions in sarcomas. Studies using the mouse models for human sarcomas have demonstrated major advances in our understanding the developmental processes as well as tumor microenvironment of sarcomas. Recent technological progresses in epigenome editing will not only improve the studies using animal models but also provide a direct clue for epigenetic therapies. In this manuscript, we review important epigenetic aberrations in sarcomas and their representative mouse models, current methods of epigenetic editing using CRISPR/dCas9 systems, and potential applications in sarcoma studies and therapeutics.

Combined analysis of expression, prognosis and immune infiltration of GINS family genes in human sarcoma.

Objective: This study was undertaken to explore the expression and prognostic value of GINS family in human sarcoma, as well as the association between the expression levels of the GINS family and sarcoma immune infiltration.Results: We discovered that the mRNA expression levels of GINS1, GINS2, GINS3, and GINS4 were all higher in the majority of tumor tissues than in normal samples, of course, including sarcoma. Through the CCLE, all the four members expression were observed in high levels in sarcoma cell lines. In Gene Expression Profiling Analysis (GEPIA) and Kaplan-Meier Plotter, our results indicated that the poor overall survival (OS), disease-free survival (DFS) and relapse free survival (RFS) were tightly associated with the increased expression of GINS genes. In TIMER database, we found that highly expressed GINS was significantly correlated with the low infiltration level of CD4+ T cell and macrophage.Conclusions: The four GINS family members were all the prognostic biomarkers for the prognosis of human sarcoma and can reduce the level of immune cell infiltration in the sarcoma microenvironment.Methods: In terms of the expression levels of mRNA for GINS family members, a particular contrast in various cancers, especially human sarcoma, was conducted through ONCOMINE and GEPIA and CCLE databases. Kaplan-Meier Plotter was used to identify the prognostic value of GINS family in sarcoma. The relationship between the expression level of GINS and the infiltration of immune cells was analyzed in TIMER database.

Abstract 5609: Investigating the undifferentiated pleomorphic sarcoma microenvironment through cytokine profiles of primary tumor and immune infiltrates

Abstract Sarcoma is a heterogeneous group of connective tissue cancers with over 60 subtypes. Curative treatment relies on surgical resection combined with radiation. In the event of metastasis, chemotherapy is used but generally only with palliative intent. Given the heterogeneity of sarcomas, targeted therapies are limited, which has contributed to the relatively stagnant survival rates seen in the past decades. Objective: Personalized treatments, such as adoptive cell therapy (ACT), may be promising for select sarcoma patients, specifically those with Undifferentiated Pleomorphic Sarcoma (UPS). To better understand ACT’s feasibility for UPS, we aim to investigate the intersection of tumor-infiltrating lymphocytes (TILs) and tumor cells by examining their cytokine output, whose role remains largely unstudied. Methods: Tissue samples from untreated sarcoma patients have been collected and viably preserved by our group. The samples were processed for in vitro expansion of tumor or TIL cell lines. Tumor cultures were validated with Whole Exome Sequencing (WES) to ensure tumor-specific variations were retained. TILs were isolated with tumor fragment (TF) processing and magnetic bead coated anti-CD3/CD28 Rapid Expansion Protocol (REP). After validating tumor cultures, both tumor and TIL-secreted cytokines were evaluated using Luminex assays. Results: Of the 11 primary UPS samples cultured, 10 generated viable cell cultures as defined by the presentation of stable growth beyond passage 5. A total of 3 cell lines were selected for further validation, and through WES, retained tumor specific variations beyond passage 5. On the other hand, the sarcoma-optimized TF and REP protocols expanded TILs from 7 of 8 UPS cases and yielded up to 6 x 10^7 cells. Although IL-2 is commonly used to facilitate TIL growth, it can also cause activation-induced cell death which is problematic for therapy. We demonstrated that an alternative γ-chain interleukin, IL-15, can support UPS TIL growth comparable to IL-2. Unbiased cytokine screening detected anti-tumoral CXCL9/IFN-γ and pro-tumoral IL-10/TGF-Beta as highly secreted by TIL cultures while some tumor cultures secreted relatively high concentrations of IL-6 and CCL2. Conclusion: We developed a robust in vitro pipeline to expand sarcoma tumor cultures and TILs for downstream characterization. Functional assays aimed at dissecting the consequences of tumor-mediated IL-6 secretion are underway. These include siRNA-induced knock downs of genes hypothesized to augment the cytokine profile of tumor cells. Future evaluation of TILs will identify their clinical efficacy via flow cytometry and intracellular cytokine staining. Understanding cytokine secretion from both the tumor and immune components of sarcoma will aid in elucidating interactions within the microenvironment that may be therapeutically relevant. Citation Format: Victoria Coward, Jacky Chen, Nalan Gokgoz, Kim Tsoi, Jay S. Wunder, Irene L. Andrulis. Investigating the undifferentiated pleomorphic sarcoma microenvironment through cytokine profiles of primary tumor and immune infiltrates [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 5609.

Single-cell RNA-seq of a soft-tissue sarcoma model reveals the critical role of tumor-expressed MIF in shaping macrophage heterogeneity.

SUMMARYThe standard of care is unsuccessful to treat recurrent and aggressive soft-tissue sarcomas. Interventions aimed at targeting components of the tumor microenvironment have shown promise for many solid tumors yet have been only marginally tested for sarcoma, partly because knowledge of the sarcoma microenvironment composition is limited. We employ single-cell RNA sequencing to characterize the immune composition of an undifferentiated pleiomorphic sarcoma mouse model, showing that macrophages in the sarcoma mass exhibit distinct activation states. Sarcoma cells use the pleiotropic cytokine macrophage migration inhibitory factor (MIF) to interact with macrophages expressing the CD74 receptor to switch macrophages’ activation state and pro-tumorigenic potential. Blocking the expression of MIF in sarcoma cells favors the accumulation of macrophages with inflammatory and antigen-presenting profiles, hence reducing tumor growth. These data may pave the way for testing new therapies aimed at re-shaping the sarcoma microenvironment, in combination with the standard of care.

Neoadjuvant Therapy Induces a Potent Immune Response to Sarcoma, Dominated by Myeloid and B Cells.

To characterize changes in the soft-tissue sarcoma (STS) tumor immune microenvironment induced by standard neoadjuvant therapy with the goal of informing neoadjuvant immunotherapy trial design. Paired pre- and postneoadjuvant therapy specimens were retrospectively identified for 32 patients with STSs and analyzed by three modalities: multiplexed IHC, NanoString, and RNA sequencing with ImmunoPrism analysis. All 32 patients, representing a variety of STS histologic subtypes, received neoadjuvant radiotherapy and 21 (66%) received chemotherapy prior to radiotherapy. The most prevalent immune cells in the tumor before neoadjuvant therapy were myeloid cells (45% of all immune cells) and B cells (37%), with T (13%) and natural killer (NK) cells (5%) also present. Neoadjuvant therapy significantly increased the total immune cells infiltrating the tumors across all histologic subtypes for patients receiving neoadjuvant radiotherapy with or without chemotherapy. An increase in the percentage of monocytes and macrophages, particularly M2 macrophages, B cells, and CD4+ T cells was observed postneoadjuvant therapy. Upregulation of genes and cytokines associated with antigen presentation was also observed, and a favorable pathologic response (≥90% necrosis postneoadjuvant therapy) was associated with an increase in monocytic infiltrate. Upregulation of the T-cell checkpoint TIM3 and downregulation of OX40 were observed posttreatment. Standard neoadjuvant therapy induces both immunostimulatory and immunosuppressive effects within a complex sarcoma microenvironment dominated by myeloid and B cells. This work informs ongoing efforts to incorporate immune checkpoint inhibitors and novel immunotherapies into the neoadjuvant setting for STSs.

Immunotherapy in soft tissue and bone sarcoma: unraveling the barriers to effectiveness.

Sarcomas are uncommon malignancies of mesenchymal origin that can arise throughout the human lifespan, at any part of the body. Surgery remains the optimal treatment modality whilst response to conventional treatments, such as chemotherapy and radiation, is minimal. Immunotherapy has emerged as a novel approach to treat different cancer types but efficacy in soft tissue sarcoma and bone sarcoma is limited to distinct subtypes. Growing evidence shows that cancer-stroma cell interactions and their microenvironment play a key role in the effectiveness of immunotherapy. However, the pathophysiological and immunological properties of the sarcoma tumor microenvironment in relation to immunotherapy advances, has not been broadly reviewed. Here, we provide an up-to-date overview of the different immunotherapy modalities as potential treatments for sarcoma, identify barriers posed by the sarcoma microenvironment to immunotherapy, highlight their relevance for impeding effectiveness, and suggest mechanisms to overcome these barriers.

CSF1/CSF1R Signaling Inhibitor Pexidartinib (PLX3397) Reprograms Tumor-Associated Macrophages and Stimulates T-cell Infiltration in the Sarcoma Microenvironment.

Colony-stimulating factor 1 (CSF1) is a primary regulator of the survival, proliferation, and differentiation of monocyte/macrophage that sustains the protumorigenic functions of tumor-associated macrophages (TAMs). Considering current advances in understanding the role of the inflammatory tumor microenvironment, targeting the components of the sarcoma microenvironment, such as TAMs, is a viable strategy. Here, we investigated the effect of PLX3397 (pexidartinib) as a potent inhibitor of the CSF1 receptor (CSF1R). PLX3397 was recently approved by the Food and Drug Administration (FDA) to treat tenosynovial giant cell tumor and reprogram TAMs whose infiltration correlates with unfavorable prognosis of sarcomas. First, we confirmed by cytokine arrays of tumor-conditioned media (TCM) that cytokines including CSF1 are secreted from LM8 osteosarcoma cells and NFSa fibrosarcoma cells. The TCM, like CSF1, stimulated ERK1/2 phosphorylation in bone marrow-derived macrophages (BMDMs), polarized BMDMs toward an M2 (TAM-like) phenotype, and strikingly promoted BMDM chemotaxis. In vitro administration of PLX3397 suppressed pERK1/2 stimulation by CSF1 or TCM, and reduced M2 polarization, survival, and chemotaxis in BMDMs. Systemic administration of PLX3397 to the osteosarcoma orthotopic xenograft model significantly suppressed the primary tumor growth and lung metastasis, and thus improved metastasis-free survival. PLX3397 treatment concurrently depleted TAMs and FOXP3+ regulatory T cells and, surprisingly, enhanced infiltration of CD8+ T cells into the microenvironments of both primary and metastatic osteosarcoma sites. Our preclinical results show that PLX3397 has strong macrophage- and T-cell-modulating effects that may translate into cancer immunotherapy for bone and soft-tissue sarcomas.

Effects of moxibustion on Treg cells in sarcoma microenvironment

ObjectiveTo investigate the therapeutic effect of moxibustion on sarcomas from mesenchymal tissues, which have a low response rate to chemotherapy and radiotherapy. MethodsS180 sarcoma cell line was inoculated in C57BL/6 mice to form transplanted tumor. Moxibustion therapy was directly applied at the transplanted tumor sites, at a distance of 3.0 cm, 10 min per session, till skin temperature reached 45 °C, once a day, for 14 consecutive days of intervention. After the mice were killed, serum was collected and used to detect concentrations of interleukin-10 (IL-10), transforming growth factor-β1 (TGF-β1), IL-4 and interferon-γ (IFN-γ) by Luminex liquid suspension chip. The numbers of Treg+ T cells and CD4+CD25+Forkhead Box P3 (Foxp3)+ T cells were detected by flow cytometry. Fluorescence in situ hybridization was used to analyze the changes of CD4, CD8, Foxp3 and TGF-β1 in the tumor microenvironment (TME). ResultsWeight of S180 transplanted tumor in the control group was (2.03 ± 0.54) g, and that in the moxibustion group was (1.27 ± 0.29) g, which was statistically different (P = 0.023). The mean value of Foxp3+ T cells in the normal group was 2.01%, which increased to 3.63% after the formation of transplanted tumor, and decreased to 1.48% after moxibustion treatment. The moxibustion group also had reduced numbers of CD4+CD25+Foxp3+ T cells in the spleen of mice with transplanted tumor. The concentrations of IL-10, TGF-β1 and IL-4 decreased in the serum of mice with transplanted tumor, while the concentration of IFN-γ increased. Moxibustion was associated with downregulation in expression of Foxp3, IL-10 and TGF-β1 genes in the transplanted tumor, and increases in the gene expression of CD4+ T cells and CD8+ T cells in the TME. ConclusionMoxibustion may have therapeutic effects on sarcomas by reducing the number of Treg cells in the blood and controlling the infiltration of Treg cells in the TME.