Abstract
Genetically engineered mouse models (GEMMs), in which autochthonous tumors develop into advanced-stage disease in the presence of a functional immune system, have contributed significantly to the understanding of most types of cancer. Using a GEMM of lung adenocarcinoma, we have found that immune cells are present in complex, highly organized, lymph node (LN)-like structures known as the tumor-associated tertiary lymphoid structures (TA-TLS). TA-TLS have been characterized in human lung cancer patients, but not in animal tumor models, and hence remain untapped targets for therapeutic interventions. We have shown that TA-TLS emerge as a result of tumor growth and that therapeutically depleting regulatory T cells (Tregs) from TA-TLS results in tumor elimination. Hence, a strong antitumor immune response exists but is suppressed in TA-TLS. Here, we describe a high-throughput immunofluorescence (IF) analysis pipeline for visualization and quantification of TA-TLS. Imaging the relatively small size of TA-TLS within tumor-bearing lung lobes using confocal microscopy is a labor-intensive process that can take up to 1 month. We have optimized this process and reduced the time required per lung lobe to 1–2 weeks using automated microscopy methods. Combining IF with multicolor fluorescence-activated cell sorting (FACS), we are able to interrogate not only the size and location of TA-TLS but also the activation status of immune cells within these structures. Using these techniques, investigation of TLS in lung adenocarcinoma combines cutting-edge technological tools in cancer biology and immunology to interrogate a fundamental, but poorly understood, tumor-associated immune structure.
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Acknowledgements
We thank Professor Tyler Jacks (Massachusetts Institute of Technology) and Jacks lab members for their generous gift of reagents, advice, and time.
Funding: This work was supported by the National Cancer Institute K22 Transition Career Award.
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Connolly, K.A., Nader, M., Joshi, N. (2018). Investigating Tumor-Associated Tertiary Lymphoid Structures in Murine Lung Adenocarcinoma. In: Dieu-Nosjean, MC. (eds) Tertiary Lymphoid Structures. Methods in Molecular Biology, vol 1845. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-8709-2_15
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DOI: https://doi.org/10.1007/978-1-4939-8709-2_15
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