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S2. Colon carcinomas are insensitive to TGFB inhibition relative to sarcomas

S7. Modeling UPS extravasation with zebrafish xenografts

S6. The role of RHAMM in UPS migration

S5. The role of RHAMM in UPS in proliferation and TGFB signaling

Supplementary Figure Legends 1-7

S3. YAP1 target expression in human and mouse UPS

High-grade sarcomas are metastatic and pose a serious threat to patient survival. Undifferentiated pleomorphic sarcoma (UPS) is a particularly dangerous and relatively common sarcoma subtype diagnosed in adults. UPS contains large quantities of extracellular matrix (ECM) including hyaluronic acid (HA), which is linked to metastatic potential. Consistent with these observations, expression of the HA receptor, hyaluronan-mediated motility receptor (HMMR/RHAMM), is tightly controlled in normal tissues and upregulated in UPS. Moreover, HMMR expression correlates with poor clinical outcome in these patients. Deregulation of the tumor-suppressive Hippo pathway is also linked to poor outcome in these patients. YAP1, the transcriptional regulator and central effector of Hippo pathway, is aberrantly stabilized in UPS and was recently shown to control RHAMM expression in breast cancer cells. Interestingly, both YAP1 and RHAMM are linked to TGFβ signaling. Therefore, we investigated crosstalk between YAP1 and TGFβ resulting in enhanced RHAMM-mediated cell migration and invasion. We observed that HMMR expression is under the control of both YAP1 and TGFβ and can be effectively targeted with small-molecule approaches that inhibit these pathways. Furthermore, we found that RHAMM expression promotes tumor cell proliferation and migration/invasion. To test these observations in a robust and quantifiable in vivo system, we developed a zebrafish xenograft assay of metastasis, which is complimentary to our murine studies. Importantly, pharmacologic inhibition of the TGFβ–YAP1–RHAMM axis prevents vascular migration of tumor cells to distant sites. Implications: These studies reveal key metastatic signaling mechanisms and highlight potential approaches to prevent metastatic dissemination in UPS.YAP1 and TGFβ cooperatively enhance proliferation and migration/invasion of UPS and fibrosarcomas.

CD8+ T cell dysfunction is a critical barrier to anti-tumor immunity, but molecular mechanisms underlying the regulation of T cell dysfunction in solid tumors are diverse and complex. Extracellular matrix (ECM) composition facilitates solid tumor progression in part by inhibiting T cell migration/infiltration; however, the impact of individual ECM molecules on T cell function in the tumor microenvironment (TME) is virtually unknown. Moreover, upstream regulators of aberrant ECM deposition/organization in solid tumors are poorly defined. Therefore, we investigated the regulation and effects of ECM composition on CD8+ T cell function in undifferentiated pleomorphic sarcoma (UPS). This immunologically “hot” soft-tissue sarcoma exhibits durable responses to checkpoint therapy in some human patients, suggesting it may provide insights into strategies for optimizing T cell function and improving immunotherapy efficacy. Using an autochthonous model of UPS and data from multiple human patient cohorts, we discovered a multi-pronged mechanism wherein oncogene-induced remodeling of the TME promotes CD8+ T cell dysfunction, suppresses T cell-mediated cytolysis, and enhances immune evasion. Specifically, we observed that the transcriptional co-activator Yap1, which we previously linked to UPS progression, promotes the aberrant deposition of collagen VI in the UPS TME. In turn, collagen VI induces CD8+ T cell dysfunction by inhibiting T cell autophagic flux and remodeling fibrillar collagen architecture in the TME. Furthermore, collagen type I opposed ColVI in this setting, acting as tumor suppressor. Thus, our findings reveal that CD8+ T cell-mediated anti-tumor immunity in solid cancers is dependent upon oncogene-mediated ECM composition and remodeling in the TME.

Primary malignant bone tumors are rare, occur in all age groups, and include distinct entities such as osteosarcoma, Ewing sarcoma, and chondrosarcoma. Traditional treatment with some combination of chemotherapy, surgery, and radiation has reached the limit of efficacy, with substantial room for improvement in patient outcome. Furthermore, genomic characterization of these tumors reveals a paucity of actionable molecular targets. Against this backdrop, recent advances in cancer immunotherapy represent a silver lining in the treatment of primary bone cancer. Major strategies in cancer immunotherapy include stimulating naturally occurring anti-tumor T cells and adoptive transfer of tumor-specific cytotoxic T cells. Chimeric antigen receptor T cells (CAR-T cells) belong to the latter strategy and are an impressive application of both insights into T cell biology and advances in genetic engineering. In this review, we briefly describe the CAR-T approach and discuss its applications in primary bone tumors. © 2019 American Society for Bone and Mineral Research.

CD8+ T cell dysfunction, characterized by reduced effector function, impaired proliferation, and inhibitory receptor upregulation, is a fundamental barrier to anti-tumor immunity. However, molecular mechanisms underlying the regulation of CD8+ T cell dysfunction in the tumor microenvironment (TME) are incompletely understood. In solid cancers, the extracellular matrix (ECM) facilitates tumor progression in part by inhibiting T cell migration/infiltration, but the impact of individual tumor-associated ECM molecules on T cell function remains unclear. Therefore, we investigated the regulation and impact of ECM composition on CD8+ T cell function in muscle-derived undifferentiated pleomorphic sarcoma (UPS). UPS exhibits durable responses to checkpoint therapy in a subset of human patients, potentially offering valuable insights into strategies for ameliorating T cell function and improving patient responses to immunotherapy. Using the autochthonous Kras G12D/+; Trp53 fl/fl (KP) murine model of UPS, we previously showed that deletion of the central Hippo pathway effector Yap1 (Kras G12D/+; Trp53 fl/fl; Yap1 fl/fl; KPY) suppressed UPS cell proliferation and tumor progression. Given the well-established role of Yap1 in mechanotransduction, we leveraged this system to investigate the effects of Yap1 on the ECM and CD8+ T cell function in UPS. We discovered that loss of UPS-cell intrinsic Yap1 reduced the proportion of dysfunctional CD8+ T cells in the TME and enhanced T cell cytolytic capacity. Yap1 loss also downregulated expression of multiple collagen genes in UPS cells and bulk tumors, including those that encode collagen type VI (ColVI). ColVI is a beaded microfilament collagen that binds to fibril-forming collagens in the ECM, such as collagen type I (ColI), and has been implicated in the pathogenesis of skeletal muscle myopathies. These data suggest that proper ColVI structure and function are critical for normal skeletal muscle physiology, with important implications for muscle-derived tumors such as UPS. Accordingly, COL6A1 was upregulated in human UPS relative to normal skeletal muscle, and inversely associated with UPS patient survival. Moreover, loss of UPS cell-intrinsic Col6a1 suppressed tumor progression, enhanced T cell cytolytic function, and attenuated CD8+ T cell exhaustion, phenocopying the effects of Yap1 deletion. Mechanistically, Yap1-mediated ColVI deposition promoted CD8+ T cell dysfunction by remodeling ColI networks in the UPS TME, and inhibiting T cell autophagic flux. Furthermore, ColI depletion dramatically increased tumor growth in an immunocompetent setting. Our findings reveal a novel role for UPS cell-intrinsic Yap1 in immune activation, and demonstrate that ColVI and ColI have opposing functions downstream of Yap in this context. These results underscore the need to systematically evaluate the roles of individual ECM components in the regulation of immune cell function, and implicate YAP1 and/or COLVI targeting as potential strategies for improving the efficacy of immunotherapy in human patients. Citation Format: Ashley M. Fuller, Hawley C. Pruitt, Hoogeun Song, Ying Liu, Ann Devine, Rohan S. Katti, Samir Devalaraja, Gabrielle E. Ciotti, Michael Gonzalez, Erik F. Williams, Ileana Murazzi, Nicolas Skuli, Hakon Hakonarson, Kristy Weber, Malay Haldar, Joseph A. Fraietta, Sharon Gerecht, T. S. Karin Eisinger-Mathason. Oncogene-induced matrix reorganization controls CD8+ T cell immunity in the UPS microenvironment [abstract]. In: Proceedings of the AACR Special Conference: Sarcomas; 2022 May 9-12; Montreal, QC, Canada. Philadelphia (PA): AACR; Clin Cancer Res 2022;28(18_Suppl):Abstract nr PR001.

Summary The regulation of monocyte differentiation in the tumor microenvironment is of significant interest to tumor immunologists. Monocytes injected into the circulation may not track into tumors in sufficient numbers, making intratumoral injections a preferred experimental approach. Monocyte enrichment with antibody-based positive selection may activate downstream signaling, while cell sorters expose monocytes to mechanical stress. Here, we describe an approach of intratumoral monocyte transfer that circumvents these limitations by using negative selection and fluorescent reporter mice. For complete details on the use and execution of this protocol, please refer to Devalaraja et al. (2020)., Graphical Abstract, Highlights • Isolation of bone marrow monocytes from fluorescent reporter mice • Transfer of monocytes directly into tumors generated in syngeneic hosts • Analysis of intratumorally transferred monocytes using flow cytometry, The regulation of monocyte differentiation in the tumor microenvironment is of significant interest to tumor immunologists. Monocytes injected into the circulation may not track into tumors in sufficient numbers, making intratumoral injections a preferred experimental approach. Monocyte enrichment with antibody-based positive selection may activate downstream signaling, while cell sorters expose monocytes to mechanical stress. Here, we describe an approach of intratumoral monocyte transfer that circumvents these limitations by using negative selection and fluorescent reporter mice.

Summary The immunosuppressive tumor microenvironment (TME) is a major barrier to immunotherapy. Within solid tumors, why monocytes preferentially differentiate into immunosuppressive tumor-associated macrophages (TAMs) rather than immunostimulatory dendritic cells (DCs) remains unclear. Using multiple murine sarcoma models, we find that the TME induces tumor cells to produce retinoic acid (RA), which polarizes intratumoral monocyte differentiation toward TAMs and away from DCs via suppression of DC-promoting transcription factor Irf4. Genetic inhibition of RA production in tumor cells or pharmacologic inhibition of RA signaling within TME increases stimulatory monocyte-derived cells, enhances T cell-dependent anti-tumor immunity, and synergizes with immune checkpoint blockade. Furthermore, an RA-responsive gene signature in human monocytes correlates with an immunosuppressive TME in multiple human tumors. RA has been considered as an anti-cancer agent, whereas our work demonstrates its tumorigenic capability via myeloid-mediated immune suppression and provides proof of concept for targeting this pathway for tumor immunotherapy.

Soft tissue sarcomas (STS) comprise a heterogeneous group of solid tumors arising from mesodermal tissues. With few effective systemic therapies, advanced-stage STS is frequently fatal. Immune checkpoint blockade (ICB) is currently under investigation for the treatment of STS. However, STS harbor a highly immunosuppressive tumor microenvironment (TME) that inhibits effective antitumor T cell responses, with only a subset of patients responding to ICB. While the major focus of immunotherapy to date has been on the role of T cells, targeting innate immune cells such as tumor associated macrophages (TAMs) within the TME holds great promise. TAMs are the most abundant leukocytes in STS, and contribute to the establishment of an immunosuppressive TME through multiple mechanisms. We identified the endothelin B receptor (EDNRB) as a potential novel therapeutic target on TAMs based on an analysis of TCGA data. EDNRB is highly expressed in “immunologically quiet” tumors with high macrophage to T cell ratios and M2-like gene expression. EDNRB encodes a G-protein-coupled receptor that binds the vasoactive peptides endothelin (EDN) 1, 2, or 3. EDNRB is upregulated during macrophage differentiation, and is highly expressed by certain macrophages subsets, including TAMs. However, the role of the EDN-EDNRB axis in regulating macrophage function and antitumor immunity is unknown. Using a murine syngeneic transplant model with cell lines derived from methylcholanthrene-induced fibrosarcomas, we first genetically deleted or overexpressed the ligand Edn1 in sarcoma cells prior to transplant. Genetic deletion of Edn1 using CRISPR-Cas9 resulted in significantly increased major histocompatibility complex class II (MHCII) expression in TAMs, indicative of a more immunostimulatory phenotype. RNA-seq of sorted TAMs from Edn1 CRISPR tumors revealed enrichment for gene sets involved in antitumor immunity, including interferon gamma and NF-kB. Conversely, overexpression of Edn1 dramatically reduced TAM MHCII expression. Furthermore, knockout of Edn1 or pharmacologic blockade of EDNRB significantly enhanced responses to anti-PD1. To confirm these effects were mediated by macrophage EDNRB, we proceeded to generate a conditional deletion of Ednrb in TAMs by crossing Ednrbflox/flox and LysM-Cre mice. Consistent with our hypothesis, TAMs from sarcomas in LysM-Cre:Ednrbflox/flox mice also took on an immunostimulatory phenotype, with increased MHCII expression and reduced M2 markers. Taken together, these findings support the existence of a novel regulatory axis, whereby tumor-derived endothelins promote a suppressive TAM phenotype through macrophage EDNRB. Combining endothelin receptor antagonists with immune checkpoint blockade has the potential to enhance antitumor immune responses in STS and other solid tumors by targeting both innate and adaptive immune cells within the TME. Citation Format: Ian Wesley Folkert, Tsun Ki Jerrick To, Samir Devalaraja, Robert J. Norgard, Malay Haldar. Tumor-derived endothelins regulate antitumor immune responses through macrophage endothelin B receptor [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1776.

Maintenance of glucose homeostasis is essential for normal physiology. Deviation from normal glucose levels, in either direction, increases susceptibility to serious medical complications such as hypoglycemia and diabetes. Maintenance of glucose homeostasis is achieved via functional interactions among various organs: liver, skeletal muscle, adipose tissue, brain, and the endocrine pancreas. The liver is the primary site of endogenous glucose production, especially during states of prolonged fasting. However, enhanced gluconeogenesis is also a signature feature of type 2 diabetes (T2D). Thus, elucidating the signaling pathways that regulate hepatic gluconeogenesis would allow better insight into the process of normal endogenous glucose production as well as how this process is impaired in T2D. Here we demonstrate that the TGF-β1/Smad3 signaling pathway promotes hepatic gluconeogenesis, both upon prolonged fasting and during T2D. In contrast, genetic and pharmacological inhibition of TGF-β1/Smad3 signals suppressed endogenous glucose production. TGF-β1 and Smad3 signals achieved this effect via the targeting of key regulators of hepatic gluconeogenesis, protein phosphatase 2A (PP2A), AMP-activated protein kinase (AMPK), and FoxO1 proteins. Specifically, TGF-β1 signaling suppressed the LKB1-AMPK axis, thereby facilitating the nuclear translocation of FoxO1 and activation of key gluconeogenic genes, glucose-6-phosphatase and phosphoenolpyruvate carboxykinase. These findings underscore an important role of TGF-β1/Smad3 signaling in hepatic gluconeogenesis, both in normal physiology and in the pathophysiology of metabolic diseases such as diabetes, and are thus of significant medical relevance.

Sarcoma is a rare but heterogeneous collection of fatal malignancies that arise from mesenchymal tissue such as fat, muscle, cartilage, etc. Recent efforts to utilize immunotherapies such as immune checkpoint blockade in sarcoma have demonstrated efficacy only in a small percentage of patients, underscoring the importance of elucidating additional immune evasion mechanisms. Immunosuppressive tumor associated macrophages (TAMs) are abundant in the solid tumor microenvironment (TME) and pose a major barrier to effective anti-tumor immunity. Though circulating monocytes are a major reservoir for TAMs, how the TME governs intratumoral monocyte differentiation is poorly understood. In multiple types of mouse and human sarcoma, we found that the TME induced tumor cells to produce high levels of the tissue metabolite retinoic acid (RA). RA promoted intratumoral monocytes to differentiate into immunosuppressive TAMs and inhibited monocyte differentiation into dendritic cells. Sarcomas genetically modified to produce limited RA harbored a more stimulatory myeloid compartment and enhanced T cell dependent anti-tumor immunity. Notably, RA inhibition demonstrated robust synergy with immune checkpoint blockade therapy. Together, our findings suggest that RA is a local tissue metabolite that promotes myeloid-mediated immune suppression in sarcoma.

Erythroid function and development is intimately linked to macrophages. The primary function of erythrocytes is oxygen delivery, which is mediated by iron-containing hemoglobin. The major source of this iron is a recycling pathway where macrophages scavenge old and damaged erythrocytes to release iron contained within the heme moiety. Macrophages also promote erythropoiesis by providing a supportive niche in the bone marrow as an integral component of “erythorblastic islands.” Importantly, inflammation leads to alterations in iron handling by macrophages with significant impact on iron homeostasis and erythropoiesis. The importance of macrophages in erythropoiesis and iron homeostasis is well established and has been extensively reviewed. However, this developmental relationship is not one way, and erythrocytes can also regulate macrophage development and function. Erythrocyte-derived heme can induce the development of iron-recycling macrophages from monocytes, engage pattern recognition receptors to activate macrophages, and act as ligand for specific nuclear receptors to modulate macrophage function. Here, we discuss the role of heme as a signaling molecule impacting macrophage homeostasis. We will review these actions of heme within the framework of our current understanding of the role of micro-environmental factors in macrophage development and function.

The prevalence and severity of obesity, type 2-diabetes, and the resultant metabolic syndrome are rapidly increasing. As successful preventive and therapeutic strategies for these life-threatening health ailments often come with adverse side effects, nutritional elements are widely used in many countries as preventive therapies to prevent or manage metabolic syndrome. Fruits are important dietary components, and contain various bioactive constituents. Many of these constituents have been proven to be useful to manage and treat various chronic diseases such as diabetes, obesity, cancer and cardiovascular diseases. Although exotic fruits are understudied throughout the world due to their limited regional presence, many studies reveal their potent ability to ameliorate metabolic derangements and the resultant conditions i.e. diabetes and obesity. The aim of this article is to review the role of exotic fruits and their constituents in the regulation of metabolic functions, which can beneficially alter diabetes and obesity pathophysiology.

Sarcomas are rare and heterogeneous cancer of mesenchymal origin. Treatment options have largely remained unchanged over decades and metastatic sarcoma is essentially incurable. There are more than 60 distinct diagnostic subtypes of sarcoma, many of which do not harbor consistent mutations. These factors make the development of targeted therapy particularly challenging in sarcomas. Therefore, a new therapeutic approach broadly targeting sarcomas of various subtypes is highly desirable. Immunotherapy offers a viable approach in cancer treatment, which has not been adequately explored in sarcomas. The immune system can distinguish malignant from normal cells and mount an antitumor T-cell response. However, tumors create a highly immunosuppressive microenvironment, which counteracts both endogenous as well as therapy-induced antitumor immune responses. Therefore, understanding the cellular and molecular basis of immunosuppression in sarcoma microenvironment is important for the development and success of any immunotherapeutic approach. Macrophages (MACs) and dendritic cells (DC) are antigen-presenting cells (APC) of the innate immune system that play key role in shaping immune responses. Studies in carcinomas have shown that immunosuppressive MACs greatly outnumber immunostimulatory DCs in the tumor microenvironment. Currently, it is unclear whether sarcomas also harbor such polarized distribution of APC in their microenvironment. Tumor-associated MACs can develop from circulating monocytes or from preexisting tissue macrophages. Likewise, DCs can develop from circulating monocytes or bone marrow-derived precursors. However, the origin of sarcoma-infiltrating MACs and DCs is unclear. Insights into the distribution and origin of tumor-infiltrating APCs is critical for devising therapeutic approaches targeting these cells to counteract immunosuppression in tumor microenvironment. We have addressed this issue by using several distinct murine models of sarcomas. Based on genetic background sarcomas can be divided into (1) translocation-associated sarcomas, (2) sarcomas harboring mutations in oncogenes and tumor suppressors, and (3) sarcomas with complex karyotypes displaying significant genomic instability. We selected one murine model to represent each of the three subtypes. An SYT-SSX fusion oncogene-driven mouse model of synovial sarcoma represented group 1, loss of P53 coupled with activation of oncogenic KRAS-driven mouse model of undifferentiated pleomorphic sarcoma represented group 2, and syngeneic transplant of sarcoma cell lines derived from methylcholanthrene-induced fibrosarcomas represented group 3. In all three murine models, APCs were found to comprise the majority of leukocyte infiltrates in tumors. Using multiparametric flow cytometry, gene-expression profiling, and lineage labeling we found that the vast majority of APCs in sarcoma were MACs with DCs comprising a very small subset. Next, using a combination of in vivo lineage tracing and lineage ablation we uncovered that the majority of MACs and DCs were derived from circulating monocytes. These results suggest that monocytes preferentially differentiate into macrophages but not DCs in sarcoma microenvironment. These findings lay the groundwork for future efforts in our laboratory aimed at understanding the molecular basis for such polarized macrophage differentiation in the tumor microenvironment. The overarching goal is to translate our findings into therapeutic approaches aimed at increasing the frequency of immunostimulatory DCs while reducing immunosuppressive MACs in sarcoma microenvironment. The clinical benefits of such an approach will be two-fold: (1) reduced immunosuppression in the tumor microenvironment increasing the efficacy of therapeutically generated T cells, and (2) increased probability of generating endogenous antitumor immune responses via the activity of DCs in the tumor microenvironment. Citation Format: Samir Devalaraja, Ian Folkert, Minghong Li, Yuma Tada, Kanika Jain, Malay Haldar. Sarcoma microenvironment blocks DC but promotes macrophage differentiation from tumor-infiltrating monocytes [abstract]. In: Proceedings of the AACR Conference on Advances in Sarcomas: From Basic Science to Clinical Translation; May 16-19, 2017; Philadelphia, PA. Philadelphia (PA): AACR; Clin Cancer Res 2018;24(2_Suppl):Abstract nr B26.