FP16.01 Development and Characterization of Two Novel Squamous Cell Lung Cancer Mouse Cell Lines

Abstract

Lung cancer is the leading cause of cancer-related death worldwide. The incidence and mortality associated with lung cancer is a major Public Health challenge in advanced societies. About 20-30% of all lung cancers are classified as lung squamous cell carcinoma (LUSC), also known as squamous cell carcinoma (SSC or SqCC) or epidermoid carcinoma. The absence of preclinical models makes LUSC disease analysis and research more challenging. The development of well-characterized cellular and molecular tools is essential for the advance in lung cancer research. In the present work, we have performed an exhaustive molecular and functional characterization of a new LUSC research model, recently developed in our laboratory. This model consists of two novel A/J mouse-derived syngeneic cell lines from NTCU-induced LUSC.In our opinion, these cell lines may become a robust tool for the study of squamous cell lung cancer in a reliable and reproducible manner. Carcinogenesis and cell line generation: LUSC tumors were induced by N-nitroso-tris-chloroethylurea (NTCU) (Toronto Research Chemicals) treatment applying 0.04 M NTCU by skin painting twice a week for 20 weeks to 8-week-old A/J mice. Immunohistochemical staining: Dissected tumors were fixed in 4% paraformaldehyde and embedded in paraffin. Three-micrometer-thick sections were used for tumors immunohistochemical phenotypic characterization. Exome sequencing: Whole exome sequencing was performed on extracted DNA from the tumor-derived cell lines. RNA sequencing analysis: Samples were prepared with the Illumina TruSeq Stranded mRNA kit and sequenced as reverse paired-end (50 bp) runs on the Nextseq sequencer. Tumor immunotherapy experiments: 2x106 cells were subcutaneously inoculated in one flank of 8-week-old female A/J mice. When the tumors reached a volume of 75 mm3, mice were randomized and treated intraperitoneally with 3 doses of 100 μg of anti-PD1 or anti-CTLA4. Tumor immune landscape analysis: 2x106 cells were subcutaneously inoculated in one flank of 6 8-week-old female A/J mice . Thirteen days after cell inoculation, tumors were collected and processed for flow cytometry analysis. Metastasis in vivo model: Cells were transduced with a triple modality construct containing a triple fusion protein GFP, luciferase and thymidine kinase. Eight-week old female mice were inoculated in the left cardiac ventricle with 1 × 105 cells. Cells were followed by bioluminescence. We have generated two transplantable LUSC cell lines (UN-SCC679 and UN-SCC680) derived from an N-nitroso-tris-chloroethylurea (NTCU) chemically-induced mouse model in A/J mice. Both cells lines expressed cytokeratins and p40, and lacked thyroid transcription factor 1 (TTF1) expression. Furthermore, we genetically characterized the LUSC cell lines by performing whole exome and RNA sequencing. In addition, we characterized the immune landscape of both tumors in vivo and assessed their response to immunotherapy studies combining the gold-standard checkpoint inhibitors in clinic. Finally, we studied the metastatic potential of each of these two models and confirmed that they reflect the human LUSC organotropism to the brain, bones, liver and adrenal glands. We have generated a very valuable tool for LUSC research that recapitulates the complexity of the human disease.