Investigating Tumor-Associated Tertiary Lymphoid Structures in Murine Lung Adenocarcinoma.

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 (T _regs ) 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.