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style="background-image:url(https://www.thermofisher.com/blog/behindthebench/wp-content/uploads/sites/9/2024/10/COL024234-Cancer-Imagery-Global-F-scaled.jpg)"> </section> <div class="site-inner"><div class="content-sidebar-wrap"><main class="content"><article class="post-18570 post type-post status-publish format-standard has-post-thumbnail category-cancer-research entry" aria-label="Cancer organoids in CAR T cell therapy research"><header class="entry-header"><h1 class="entry-title">Cancer organoids in CAR T cell therapy research</h1> <span class="post-info-author"> By <a href="https://www.thermofisher.com/blog/author/behind-the-bench-staff/" title="Posts by Behind The Bench Staff" rel="author">Behind The Bench Staff</a> </span> <span class="post-info-date"> 10.28.2024 </span> </header><div class="entry-content"><p>Chimeric Antigen Receptor T (CAR T) cell therapy has been a revolutionary advancement in oncology, particularly for the treatment of hematological malignancies. CAR T cell therapy involves the genetic modification of a patient’s T cells to express a synthetic receptor that specifically targets antigens on tumor cells and becomes activated upon recognizing those antigens to kill cancer cells. Clinically, CAR T cell therapy has demonstrated remarkable success in treating relapsed or refractory B cell malignancies and multiple myeloma, with CD19 and BCMA being the most commonly targeted antigens. The promising outcomes in hematological cancers have spurred extensive research into extending the application of CAR T cell therapy to solid tumors, despite the inherent challenges posed by the complex tumor microenvironment and antigen heterogeneity in these cancers.</p> <h2>Potential of cancer organoids in CAR T cell therapy</h2> <p>The use of patient-derived cancer organoid (or tumoroid) models during the development of CAR T cell therapies may spur the effectiveness of this class of therapies for solid cancers. Cancer organoids provide a more accurate representation of the tumor microenvironment compared to traditional two-dimensional (2D) cell cultures, better reflecting mutational patterns, gene expression levels, and the three-dimensional (3D) architecture of tumors. Additionally, the heterogeneity of cancer cells inherent in these patient-derived models may enable researchers to gain important insights into mixed cancer cell response and resistance to cell therapy in solid tumors.</p> <h2>Methods for evaluating CAR T cytotoxicity with cancer organoids</h2> <p>R&D scientists at Thermo Fisher have leveraged our well-characterized tumoroid biobank established in <a href="https://www.thermofisher.com/us/en/home/life-science/cell-culture/mammalian-cell-culture/cancer-cell-culture/tumoroid-culture/tumoroid-medium.html?icid=bid_clb_gme_r01_co_cp1610_pjt12011_bidcrtblg_0so_blg_op_awa_kt_siz_cncrorgndblg1">OncoPro™ Tumoroid Culture Medium</a> to demonstrate how these models can be used for the assessment of allogeneic CAR T cell cytotoxicity. Mesothelin, a GPI-anchored glycoprotein that is overexpressed in many cancers and is expressed on the cell surface, was chosen as the target antigen. The expression level of mesothelin was profiled using RNA sequencing, and tumoroid lines with high (endometrial tumoroids HuEn070722) and low (lung tumoroids HuLu051921) <em>MSLN3</em> expression were selected (<strong>Figure 1A</strong>). CAR T cells targeting mesothelin were engineered by knocking out endogenous T cell receptors via electroporation of Cas9 protein-gRNA ribonucleoprotein (RNP) complexes, followed by insertion of a Meso3-TCR CAR cassette using adenoviral transduction. As a control, CAR T cells targeting CD19 were also generated; neither of the tumoroid lines tested expresses <em>CD19</em>.</p> <p>To test the specificity of CAR T killing, CAR T cells were co-cultured with the selected tumoroid models at various effector:target (E:T) ratios in the presence of CellEvent™ Caspase-3/7 Red apoptosis indicator and monitored over time. In this assay, dissociated tumoroids cells were seeded and incubated in OncoPro Tumoroid Culture Medium for four days to form tumoroids. CAR T cells were then added, and co-cultures were imaged over the following 27 hours. Tumoroid killing was indicated by an increase in the fluorescence intensity of the Caspase-3/7 Red signal over time.</p> <h2>Results of CAR T cytotoxicity assessment</h2> <p>Tumoroid cytotoxicity assays revealed specificity of CAR T cell killing that correlated with target expression. Minimal cell death was observed when CD19-CAR T cells were used, or when Meso3-CAR T cells were introduced to MSLN-low HuLu051921 tumoroids (<strong>Figure 1B</strong>). In contrast, Meso3-CAR T cells efficiently targeted HuEn070722 tumoroids, and dose-dependent apoptosis of the tumoroid cells was observed (<strong>Figure 1B</strong>). While this article aims to give a brief overview of this study, more details, including cytokine release profiling and controls using non-transduced T cells, can be found <a href="https://assets.thermofisher.com/TFS-Assets/BID/posters/evaluation-car-t-cytotoxicity-solid-tumors-poster.pdf?icid=bid_clb_gme_r01_co_cp1610_pjt12011_bidcrtblg_0so_blg_op_awa_kt_siz_cncrorgndblg2">in this poster</a>.</p> <h2>Conclusions on cancer organoids in cell therapy</h2> <p>This initial study demonstrates how 3D patient-derived cancer organoid models can be used to test the specificity and functional cytotoxicity of CAR T cells using physiologically-relevant cancer cell targets. Learnings from these assays could be employed not only to optimize CAR designs but also to test the impact of process development changes on immune cell functionality. Additionally, tumoroid models can be used to test the cytotoxicity of other emerging cell therapies, such as <a href="https://assets.thermofisher.com/TFS-Assets/BID/Application-Notes/functional-phenotypic-characterization-human-nk-cells-app-note.pdf?icid=bid_clb_gme_r01_co_cp1610_pjt12011_bidcrtblg_0so_blg_op_awa_kt_siz_cncrorgndblg3">expanded natural killer (NK) cells</a>. In the future, sophisticated assays to model immune cell invasion, exhaustion, and rechallenge could facilitate CAR T design and manufacturing to make these therapies more effective against solid tumors.</p> <p><img decoding="async" class="alignnone wp-image-18571 size-large" src="https://www.thermofisher.com/blog/behindthebench/wp-content/uploads/sites/9/2024/10/CAR-T-blog-figure_final-1024x677.png" alt="Cancer organoid models: 3D structures of lung and endometrial cancer cells co-cultured with CAR T cells, showing caspase activity at different cell ratios" width="760" height="502" srcset="https://www.thermofisher.com/blog/behindthebench/wp-content/uploads/sites/9/2024/10/CAR-T-blog-figure_final-1024x677.png 1024w, https://www.thermofisher.com/blog/behindthebench/wp-content/uploads/sites/9/2024/10/CAR-T-blog-figure_final-300x198.png 300w, https://www.thermofisher.com/blog/behindthebench/wp-content/uploads/sites/9/2024/10/CAR-T-blog-figure_final-768x508.png 768w, https://www.thermofisher.com/blog/behindthebench/wp-content/uploads/sites/9/2024/10/CAR-T-blog-figure_final-1536x1016.png 1536w, https://www.thermofisher.com/blog/behindthebench/wp-content/uploads/sites/9/2024/10/CAR-T-blog-figure_final-2048x1355.png 2048w" sizes="(max-width: 760px) 100vw, 760px" /></p> <p><strong>Figure 1. Patient-derived cancer organoid models for functional CAR T cell cytotoxicity assays. </strong>(A) RNA sequencing was used to quantify gene expression levels in reads per million (RPM) of patient-derived cancer organoid lines HuLu051921 (lung cancer model) and HuEn070722 (endometrial cancer model). These tumoroid lines grow as 3D cell structures with similar morphologies. (B) Representative micrographs and quantification of caspase indicator intensity over time for co-culture of tumoroid models and CAR T cells targeting mesothelin or CD19 at various effector to target (E:T) ratios. Scale bar = 800 µm.</p> <h2>Related Content</h2> <ol> <li><a href="https://www.thermofisher.com/blog/behindthebench/how-3d-cell-culture-models-work/">How 3D Cell Culture Models Work</a></li> <li><a href="https://www.thermofisher.com/blog/behindthebench/cancer-organoids-vs-cancer-spheroids/">Cancer organoids vs cancer spheroids</a></li> </ol> <p>For Research Use Only. 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