Research
Investigating Cancer Associated Fibroblasts' (CAFs) role in promoting tumor growth and immune exclusion
Tumors have heterogeneous environments with some areas exhibiting high infiltration of cytotoxic T cells, while others remain T cell excluded. Tumors with an overall high T cell infiltration have improved survival rates and responsiveness to immunotherapies, including immune checkpoint inhibitors and CAR-T cell therapies. However, a significant challenge arises as most tumors display extensive areas with poor T cells infiltration; consequently, the applicability of immune cell-based therapies remains limited for most patients. The question of how to enhance T cell trafficking into immune-excluded areas is of utmost clinical relevance.
By using innovative tumor mouse models, we recently revealed that glycolytic cancer-associated fibroblasts (glyCAFs) are enriched at the margins of immune-excluded tumors to a greater extent than the immune-infiltrated ones. Through functional studies, we demonstrated that glyCAFs play a direct role in impeding the trafficking of T cells into the tumor parenchyma through the Cxcr6/Cxcl16 axis. We found that reprogramming CAFs and blocking the interactions between CAFs and T cells had the potential to enhance T cell infiltration into the tumor mass, resulting in improved tumor control when combined with chemotherapy.
Moving forward, our research will focus on the following objectives:
a. Elucidating the intrinsic mechanisms operating within tumor cells that contribute to the spatial accumulation of CAFs with distinct transcriptional programs, while exploring their functional impact on tumor growth.
b. Evaluating the therapeutic potential of CAF reprogramming as a strategy to enhance tumor immune infiltration.
Figure 1. Schematic representation of the role of glyCAFs in blocking T cells tumor infiltration through Cxcl16/Cxcr6.
Circular RNAs as novel molecular mechanisms of tumor immune exclusion
Most transcripts in mammalian cells are non-coding RNAs that lack protein-coding capacity. Circular RNAs (circRNAs) have emerged as a distinct class of non-coding RNAs that are generated through alternative splicing events, where the 3'-end of an exon forms a circular structure by back-splicing to the 5'-end of an upstream exon. Repetitive and highly complementary sequences (e.g., ALU, SINE, LINE) located in the introns between the circularizing exons favor the back-splicing events. CirRNAs are extremely stable in the cells and, despite being single-stranded, form double-stranded RNA (dsRNA) secondary structures that make them alike viral dsRNAs. Accordingly, like viral dsRNAs, the circRNAs can interact with dsRNA binding proteins, including those involved in viral recognition and immune activation (e.g., RIG-I, PKR, and MDA5). Through functional investigations conducted in mouse and human tumor cells, we have discovered that specific circRNAs, (e.g., circCsnk1g3 and circAnkib1), promote tumor growth by suppressing interferons and pro-inflammatory factors within tumor cells. This suppression ultimately hinders the recruitment and activation of cytotoxic T cells into the tumor mass, shaping the immune landscape of the TME. Targeting circRNAs represents a promising and novel approach to enhance the presence of cytotoxic T cells within tumors, holding strong clinical potential.
Moving forward, our investigation will pursue the following objectives:
a. Elucidating the underlying mechanisms by which circRNAs regulate interferon and inflammatory responses in tumor and immune cells.
b. Expanding the functional exploration of highly immune-suppressive circRNAs in leukemia, sarcoma, and other immune-excluded tumors.
c. Conducting preclinical evaluations of circRNA-targeting strategies (e.g., antisense oligonucleotides (ASOs) and CRISPR-Cas13-based methodologies) in combination with standard cancer treatments and immunotherapy.
d. Exploit circRNA-engineering to generate anti-tumor RNA-based cancer vaccines.
Figure 2. (A) biogenesis of circular RNAs. (B) Schematic representation of the circRNAs role to regulate inflammatory responses and recruit immune cells to the tumor mass.
Tumor-associated macrophage (TAM) reprogramming and engineering for novel anti-tumor therapies
Monocytes are highly recruited to the tumor parenchyma, where they become tumor-associated macrophages (TAMs). TAMs could, potentially, eliminate tumor cells through tumor cells engulfment and by facilitating the recruitment and activation of cytotoxic T cells. However, prolonged exposure to inhibitory signals in the TME leads to dysfunctional behavior in TAMs. This dysfunction is characterized by a loss of phagocytic properties and the acquisition of tumor-promoting characteristics, such as angiogenic signaling and enhanced fatty acid oxidation, which create an anti-inflammatory environment. Through single-cell analyses, we have identified four distinct clusters of TAMs in sarcoma, covering a functional spectrum from tumor-promoting to tumor-restraining, based on their activation states. By targeting specific elements of interaction between tumor cells and TAMs (e.g. MIF/CD74), we demonstrated that TAMs-reprogramming can restrains tumor growth.
Moving forward, our investigation will pursue the following objectives:
a. Use functional genomics approaches to elucidate novel mechanisms underpinning interactions between tumor cells and TAMs, which drive TAMs’ dysfunction.
b. Reprogramming and engineering of TAMs to unleash their anti-tumor properties.
Figure 3. Clusters of myeloid cells found in mouse sarcoma models. Schematic representation of MIF role in directing myeloid cells pro-tumorigenic functions by changing the myeloid cells expression profiles.
Defining druggable sarcoma vulnerabilities
Figure 4. Generation of murine sarcoma cells from normal mesenchymal cells upon genetic manipulation of oncogenes and tumor suppressor genes.
Soft-tissue sarcomas comprise a heterogeneous group of aggressive tumors that originate from cells belonging to the mesenchymal lineages. Compared to epithelial carcinomas, sarcomas have been less studied and lack effective therapeutic alternatives to radiation and chemotherapy. With the aim of identifying new and more effective therapies for these tumors, the Guarnerio Laboratory has developed a genetic platform to generate immune-competent mouse models of sarcoma, which differ from previous models based on the use of human sarcoma cell lines xenografted into immunocompromised mice. Our models closely mimic various aspects of human sarcoma, including the relevant genetic alterations found in patients, the specific cell of origin for different sarcoma subtypes, and the heterogeneous composition of the TME, ranging from high to poor T cell infiltration.
Moving forward, our investigation will pursue the following objectives:
a. Generate models of distinct sarcoma sub-types (e.g. liposarcoma and leiomyosarcoma)
b. Study the molecular mechanisms of sarcomagenesis, including mechanisms leading to generation of tumor stem cells.
c. Test in pre-clinical trials new therapeutic avenues to cure these tumors.