Abstract 4305: Analysis of tumor heterogeneity in syngeneic models; CT26.WT colon carcinoma and 4T1-Luc2-1A4 breast carcinoma in female BALB/cAnNHsd mice
Authors: Snider Justin , Germain Derrik , Kucharczyk Lauren , Zaitouna Anita , Barnes Sheri , Draper David , Wise Scott 
Published: March 2024

Abstract
Cancer is a complex disease that involves both malignant cells and the surrounding microenvironment. The tumor microenvironment (TME) is comprised of various components, including cancer-associated fibroblasts, immune cells, blood vessels, and extracellular matrix elements, all of which interact with cancer cells. It is increasingly recognized that the components of the TME are a critical determinant of therapeutic response and resistance, and as such targeting the TME has emerged as a promising strategy in cancer therapy.
Syngeneic models are necessary to study the TME, and more specifically the tumor infiltrating immune cell populations, as these models allow us to monitor various aspects of the immune response. Another important aspect to consider is tumor heterogeneity, which can cause issues during analysis due to immune cell populations not being evenly distributed throughout the tumor. The decision of whether to analyze whole tumors or halved tumors when monitoring changes in immune cell populations is an important consideration. When considering multiple endpoints from a single tumor sample, halved tumors are oftentimes considered as we remain mindful of the 3Rs (replacement, reduction and refinement) in animal research. As such, it is important to understand the effects of splitting samples and how it can affect the data that is generated.
To determine the effects of analyzing whole and halved tumor samples on immune cell subsets, studies were designed to subcutaneously implant either CT26.WT or 4T1-Luc2-1A4 cells into female BALB/cAnNHsd mice and harvest tumor samples after the completion of treatment. Mice were treated with isotype control (Clone MPC-11) or anti-mCTLA-4 (Clone 9D9). At the time of sample collection, tumors were kept whole intact or halved and analyzed via flow cytometry to quantify lymphoid and myeloid immune cell subsets within tumor samples.
The data revealed differences in immune cell subset populations for both isotype vs. anti-CTLA-4 treated tumors and whole vs. halved tumors. Changes in the lymphoid cell compartment were observed for whole vs. halved tumors of ± ~25-100% and ± ~0-40% for isotype treated and of ± ~0-30% and ± ~25-2600% for anti-CTLA-4 treated CT26.WT or 4T1-Luc2-1A4 tumors, respectively. Changes in the myeloid cell compartment of ± ~5-45% and ± ~10-65% for isotype treated and of ± ~15-100% and ± ~0-70% for anti-CTLA-4 treated CT26.WT or 4T1-Luc2-1A4 tumors, respectively, were also observed. Additionally, differences were observed between the two tumor halves, not just between whole and halved tumors.
Taken together, the data support that splitting tumor samples can negatively impact the analysis of the immune cell populations. Tumors can be split if required to perform multiple analyses, however, this should be done with caution, as it can result in trend differences and increases variability within the dataset.
Citation Format: Justin Snider, Derrik Germain, Lauren Kucharczyk, Anita Zaitouna, Sheri Barnes, David Draper, Scott Wise. Analysis of tumor heterogeneity in syngeneic models; CT26.WT colon carcinoma and 4T1-Luc2-1A4 breast carcinoma in female BALB/cAnNHsd mice [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 4305.