No abstract

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.

Soft-tissue sarcomas (STS) are mesenchymal tumors having remarkably diverse histological features. Genomic studies reported that STS have low frequencies of genetic mutation, but often harboring copy number variations (CNVs). For STS, CNVs frequently happen in essential tumor suppressor genes and oncogenic transcriptional genes, neither of which are easily “druggable” targets. Moreover, in concordance with low tumor mutation burden rate, STS show low immunogenicity with less cytotoxic TIL in the TME and is unfortunately linked to a lower responsive rate to PD1/PD-L1 ICB. Using a syngeneic mouse UPS model, we previously found that tumor-associated macrophages (TAM) in the TME are promoting the growth of sarcoma. We further identified specific tumor microenvironmental elements contributing to the pro-tumorigenicity of TAMs, which represent a promising novel clinical target for myeloid-related intervention. Therefore, we hypothesize that exploration of TME is beneficial to identify clinically promising targets for STS. To study the TME in STS, we performed bulk RNA-seq and scRNA-seq on human sarcoma samples which covered different histological subtypes of STS and compare them to normal samples. Our results show that, unlike carcinomas originating from epithelial cells, extracellular matrix (ECM)-related genes are highly upregulated in both cancer-associated fibroblast (CAF) and sarcoma tumor cells. Further analysis show that upregulation of these ECM genes is associated with a worse survival outcome in sarcoma patients. In order to further explore the STS and TME orchestra, we established several syngeneic mouse models according to the genetic aberrations reported in TCGA data. We are currently characterizing these syngeneic mouse models to find out whether they recapitulate human STS samples. Our goal is to utilize these mouse models to study the relationship of ECM-related genes in both CAF and tumor cells, find out the effect of upregulated ECM-related genes on the immune compartment in TME and search for potential therapeutic interventions. Citation Format: Jin-Fen Xiao, Marina Broz, Roberta Piras, Kristin Ishaya, Emily Ko, Jlenia Guarnerio. Profiling tumor microenvironment for therapeutic intervention to soft-tissue sarcomas [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 1364.

Trafficking of T lymphocytes from the lymph nodes to the tumor microenvironment is a critical process of the tumor immunity cycle to elicit cytotoxic anti-tumor responses driven by CD8+ T cells. However, some tumors termed “immune excluded” recruit lymphocytes to the tumor site, but the lymphocytes are unable to penetrate the tumor parenchyma and localize primarily in the peritumoral region. In soft tissue sarcoma patients, most tumors are poorly infiltrated by T cells, which is associated with a poor response to immunotherapies. It has been described that cancer associated fibroblasts (CAFs) are enriched in immune excluded tumors and may directly block the migration of T cells via the production of dense extracellular matrix or by forging an immunosuppressive niche. We generated two models of undifferentiated pleomorphic sarcoma (UPS) that recapitulate the “immune excluded” and “inflamed” microenvironments observed in sarcoma patients. These syngeneic models rely on p53KO mesenchymal stem cells overexpressing either Ccne1 or Vgll3, which are frequently amplified in UPS patients. These models differ in their overall proportion of infiltrating TILs, and specifically T cells, making them ideal for comparative studies to investigate the mechanisms driving T cell exclusion in the TME. Using single-cell RNA-sequencing, we identified a population of CAFs expressing Nt5e, encoding CD73, which are spatially enriched in the peritumoral region of immune excluded Ccne1 tumors and closely associate with CD8+ T cells located at the tumor margin. Using transwell invasion assays, we show that CD73+ CAFs but not CD73- CAFs are able to block the migration of activated T cells towards tumor cells, even in the presence of CXCL10. Further, we show that Nt5e CAFs are enriched for signatures of glucose metabolism, and hypoxia, thus we hypothesized that CD73+ CAFs may block the migration of T cells into tumors by forging a nutrient poor metabolic barrier around the tumor. To test this, we treated Ccne1 tumors with BAY-876, a GLUT1 inhibitor and observed a significant accumulation of infiltrating CD8+ T cells compared to controls. GLUT1 treated CAFs expressed significantly less Nt5e, indicating that CD73 may play a role in the maintenance of glucose metabolism in CAFs. Furthermore, blockade of CD73 in CD73+ CAFs decreases the expression of the glucose transporter, Glut1. All together, these data suggest that CD73 may serve as a marker of glucose dependent CAFs that alter the metabolic niche to block T cell infiltration into tumors. Citation Format: Marina Broz, Emily Ko, Jinfen Xiao, Marco DeSimone, Roberta Piras, Kristin Ishaya, Xen Ping Hoi, Jlenia Guarnerio. Glucose dependent CD73+ CAFs enforce a tumor metabolic barrier that promotes T cell exclusion [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 1255.

Soft tissue sarcomas (STS) are tumors of mesenchymal origin and include multiple sub-types such as undifferentiated pleomorphic sarcoma (UPS) which is the most frequent classification of STS. UPS is one of the most aggressive and recurrent sarcomas. The current standard of care for UPS patients remains surgical resection, radiotherapy, and chemotherapy, but many patients still develop recurrent tumors after these interventions. In addition, anti-PD-1 therapy has only been successful in a small fraction of patients with high levels of tumor infiltrating B and T cells, while immune excluded tumors showed poor responses. To understand why these tumors are frequently recurrent, it is essential to understand the characteristics of resistant tumor cells following standard treatment regimens in both immune excluded and immune infiltrated tumor microenvironments. We generated multiple mouse models that reflect the genetic alterations frequently found in patients via overexpression of oncogenes Ccne1 or Vgll3 in p53KO mesenchymal stem cells. Classification of the immune microenvironment of these models revealed that these driver genetics promote an immune excluded or immune infiltrated tumor microenvironment, respectively. We leveraged these two tumor models to understand the mechanisms of therapeutic resistance to common agents doxorubicin or anti-PD-1. We found that despite higher levels of baseline tumor infiltrating lymphocytes in Vgll3 tumors, anti-PD-1 failed to reduce tumor growth. Doxorubicin treatment resulted in a modest reduction of tumor volume in Ccne1 tumors, and more significantly in immune infiltrated Vgll3 tumors. To understand the transcriptional profiles of resistant tumor cells we harnessed single-cell RNA-sequencing (scRNA-seq) to characterize the least responsive model to determine if treatments had an impact on tumor cell expression profiles and which tumor clusters persist after treatment. scRNA-seq analysis revealed that different clusters of tumor cells were differently affected by anti-PD-1 and doxorubicin. While doxorubicin mainly affected tumor cells expressing collagens and matrix associated adhesion proteins, treatment with anti-PD-1 selectively reduced tumor cells enriched in interferon signaling pathways. Interestingly, tumor cells expressing high levels of extracellular matrix (ECM) remodeling genes remained unaffected and, in some cases, were enriched under both treatments. These results may suggest that tumor cells capable of ECM remodeling may shield tumor cells from chemotherapy agents and immune cells from immune checkpoint inhibitors and promote tumor recurrence. Therefore, these results highlight the need for investigation of combination therapies targeting extracellular matrix proteins in addition to immune checkpoint blockade. Citation Format: Marina T Broz, Marco DeSimone, Emily Ko, Roberta Piras, Jlenia Guarnerio. Uncovering transcriptional signatures of drug resistant tumor cells: Mechanisms of therapeutic resistance and opportunities for combination therapies [abstract]. In: Proceedings of the AACR Special Conference: Tumor Immunology and Immunotherapy; 2022 Oct 21-24; Boston, MA. Philadelphia (PA): AACR; Cancer Immunol Res 2022;10(12 Suppl):Abstract nr A29.