Abstract 1517: Tc1 cytotoxic T-cells and Th1 helper T-cell rich tumor microenvironment is a hallmark of MSI colorectal cancers
Authors: Mandelkow Tim , Huang Zhihao , Bady Elena , Müller Jan H. , Sauter Guido , Ebner Julia , Lennartz Maximilian , Gorbokon Natalia , Simon Ronald , Kluth Martina , Hube-Magg Claudia , Minner Sarah , Blessin Niclas C. 
Published: March 2024

Background: Microsatellite instability (MSI-h) is a strong biomarker to predict response to immune checkpoint therapy and patient’s outcome in colorectal cancer. Although enrichment of distinct T-cell subpopulations, such as type 1 cytotoxic T-cells (Tc1) or type 1 helper T-cells (Th1), have been identified to impact patient’s outcome and response to immune checkpoint therapy, only little is known about the underlying changes in the composition of the immune tumor microenvironment.

Material and Methods: To assess the density, composition, degree of functional marker expression, and spatial interplay of T-cell subpopulations in 93 MSI and 1153 microsatellite stable (MSS) colorectal cancers, a tissue microarray as well as large sections were stained with antibodies directed against CD3, CD4, CD8, FOXP3, T-bet, GATA3, RORyT, BCL6, CD27, CD56, TIM3, PD-1, CTLA-4 Granzym B, and Ki67 using our BLEACH&STAIN multiplex fluorescence immunohistochemistry approach. A deep learning-based framework comprising two different convolutional neuronal networks (U-Net and DeepLabv3+) was used for image analysis.

Results: The composition of Type 1 (T-bet+), Type 2 (GATA3+), Type 17 (RORyT+), NKT-like (CD56+), regulatory (FOXP3+), and follicular (BCL6+) cytotoxic (CD3+CD8+) or helper (CD3+CD4+) T-cells showed marked differences between MSI and MSS patients. For instance, the density and proportion of Tc1 as well as Th1 was significantly higher (p&lt;0.001 each) in MSI compared to MSS patients. In contrast, the density and composition of Tregs, Th2, Th17 T-cells was significantly lower in MSI compared to MSS patients in the intraepithelial tumor compartment. In addition, among the MSI colorectal cancers, NKT-cells showed the highest level of Granzyme B that was significantly higher than in MSS patients (p&lt;0.05). The degree of immune checkpoint expression (i.e., TIM3, PD-1, CTLA-4) was significantly higher in MSI compared to MSS patients for most T-cell subpopulations (p&lt;0.05 each). The additional analysis of 10 large sections confirmed the observations from the TMA analysis and revealed that the alterations in the T-cell composition seen in the center of the tumor largely reflect the T-cell composition at the invasive margin in both MSI and MSS patients.

Conclusion: This study identified a higher proportion of Tc1 cytotoxic T-cells and Th1 helper T-cells accompanied by a paucity of regulatory T-cell subpopulations, Th17 and Th2 T- helper cells in MSI colorectal cancers compared to MSS patients.

Citation Format: Tim Mandelkow, Zhihao Huang, Elena Bady, Jan H. Müller, Guido Sauter, Julia Ebner, Maximilian Lennartz, Natalia Gorbokon, Ronald Simon, Martina Kluth, Claudia Hube-Magg, Sarah Minner, Niclas C. Blessin. Tc1 cytotoxic T-cells and Th1 helper T-cell rich tumor microenvironment is a hallmark of MSI colorectal cancers [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 1517.