Abstract A008: Relationship between CD4+ T cells and cancer stem cells in squamous cancers of the oro-esophageal tract

Abstract Esophageal and oral squamous cell carcinomas (ESCC and OSCC, respectively) are the most common cancers of the oro-esophageal tract. They share common risk factors, including alcohol and tobacco use, a low mutational burden, and frequent mutations in key genes such as TP53 in the vast majority of cases, followed by CDKN2A and NOTCH1. While surgery and chemotherapy are the main treatment options, prognosis worsens when surgery is not feasible. These cancers often display strong resistance to therapy, partly due to cancer stem cells (CSCs), which promote tumor recurrence and treatment failure. While the interactions between CSCs and the tumor microenvironment have attracted growing interest, the role of CD4+ T cells in this context remains largely understudied, despite their well-established immunomodulatory functions. This study aims to analyze the crosstalk between CSCs and CD4+ T cells in a mouse model of ESCC and OSCC. Tumors are induced in C57BL/6J mice using 4-nitroquinoline-1-oxide (4NQO), a chemical carcinogen that promotes squamous tumorigenesis in the esophagus and oral cavity. The tumors are then isolated, dissociated, and cultured as organoids. At the same time, naïve CD4+ T cells are isolated and then co-cultured with tumor cells at a ratio of 1:5. Naïve CD4+ T cells can also be polarized into Th0, Th1, Th2, Th9, Th17 or Treg subtypes before co-culture. The impact of CD4+ T cells on CSCs is assessed by measuring the expression of stemness-associated markers such as CD44+CD133+ by flow cytometry and immunofluorescence, as well as through organoid formation assays evaluating self-renewal capacity. Preliminary results suggest that the impact of CD4+ T cells on the frequency of CD44+CD133+ cancer stem cells and their stemness properties varies according to the type of cancer cell line and the specific CD4+ T cell subset involved. In ESCC, co-culture with naïve CD4+ T cells appears to reduce the frequency of CD44+CD133+ CSCs, along with a significant decrease in stemness-associated properties. Conversely, in OSCC, co-culture with naïve CD4+ T cells appears to increase the frequency of CD44+CD133+ CSCs and significantly enhance stemness potential. We hypothesized that these results could be explained by a polarization process occurring during co-culture. To explore this, we began testing the effects of polarized CD4+ T cells on the stemness capacity of CSCs. Preliminary data from ESCC organoid lines suggest that the impact of CD4+ T cells on stemness varies according to their polarization status. Specifically, Th0 cells appear to enhance CSC stemness, whereas Th1, Th2, and Th9 cells tend to reduce it. In contrast, Th17 and Treg cells show no significant effect on stemness properties. These results suggest that each CD4+ T cell subtype exerts a distinct effect on CSCs. This study therefore provides new insights into their immunomodulatory role in ESCC and OSCC. Further investigations are needed to elucidate the underlying mechanisms and assess their clinical relevance. Citation Format: Maxence Plateau, Véronique Giroux. Relationship between CD4+ T cells and cancer stem cells in squamous cancers of the oro-esophageal tract [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Mechanisms of Cancer Immunity and Cancer-related Autoimmunity; 2025 Sep 24-27; Montreal, QC, Canada. Philadelphia (PA): AACR; Cancer Immunol Res 2025;13(9 Suppl):Abstract nr A008.