Transcriptional Diversity of Regulatory T Cells in Colorectal Cancer

Colorectal cancer (CRC) is a highly heterogeneous disease with substantial global incidence and mortality. A major determinant of CRC progression and treatment response is the tumor microenvironment (TME), particularly the immune landscape. Tumor-infiltrating lymphocytes (TILs) include anti-tumor effector CD4+ and CD8+ T cells as well as immunosuppressive regulatory CD4+ T (Treg) cells. While tumor-specific T cell responses can develop, effective tumor clearance frequently fails due to the accumulation and activity of Treg cells, which suppress immune responses and promote tumor immune evasion. Increased Treg cell presence in CRC is associated with poor prognosis and resistance to immunotherapy, highlighting the need to understand their origin, maintenance, and function within tumors.
This dissertation focuses on defining the identity, phenotype, and heterogeneity of Treg cells in CRC. Chapter 1 introduces the importance of the TME in CRC, while Chapter 2 reviews the current understanding of Treg cell biology in CRC, including mechanisms driving their differentiation, recruitment, and adaptation to the TME, as well as emerging therapeutic strategies aimed at modulating Treg cell function.
Using preclinical in vitro and in vivo models, the thesis investigates how CRC tumors shape Treg cell differentiation and suppressive activity. Chapter 3 evaluates hydrogel-based three-dimensional (3D) culture systems commonly used for preclinical immunotherapy testing, including chimeric antigen receptor T (CAR-T) cell screening. The study compares chemically undefined matrices (Matrigel and basement membrane extract, BME) with a synthetic, chemically defined nanofibrillar cellulose (NFC) hydrogel. Matrigel and BME promoted increased Treg cell frequencies among murine CD4+ T cells and impaired CAR-T cell activation and proliferation. In contrast, NFC preserved human T cell proliferation, activation, and CAR-T cell function. These findings demonstrate that hydrogel composition can significantly influence T cell behavior and emphasize the importance of selecting appropriate 3D culture systems for immunotherapy research.
Further in vitro studies showed that CRC tumor organoids directly induce Treg cell differentiation. Both murine and human CRC-derived organoids generated Treg cells with enhanced suppressive capacity and transcriptional profiles associated with CRC-specific Treg cells and poor clinical outcomes. This organoid-based model provides a valuable platform to study tumor-driven immune modulation and to identify strategies aimed at disrupting Treg-mediated immunosuppression.
In Chapter 5, an in vivo CRC metastasis model was used, involving implantation of CRC organoids into the livers of Foxp3eGFP mice. Tumors displayed increased Treg cell infiltration alongside reduced effector CD4+ and CD8+ T cell populations. RNA sequencing revealed transcriptional signatures consistent with CRC-associated Treg cells observed in patients, with site-specific differences between liver and peritoneal tumors, including altered WNT signaling. Additionally, splenic Treg cells from tumor-bearing mice exhibited distinct transcriptional profiles, indicating systemic immune modulation.
Overall, this thesis advances understanding of Treg cell phenotype, heterogeneity, and tumor-driven adaptation in CRC. By integrating advanced 3D culture systems, organoid models, and in vivo studies, it provides insights into how Treg cells contribute to immune suppression, disease progression, and treatment resistance. These findings aim to inform future therapeutic strategies targeting tumor-infiltrating Treg cells to enhance anti-tumor immunity and improve CRC outcomes.