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N.R. Little
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P1.04 - Poster Session/ Biology, Pathology, and Molecular Testing (ID 233)
- Event: WCLC 2015
- Type: Poster
- Track: Biology, Pathology, and Molecular Testing
- Presentations: 1
- Moderators:
- Coordinates: 9/07/2015, 09:30 - 17:00, Exhibit Hall (Hall B+C)
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P1.04-056 - Use of Pooled shRNA Synthetic Lethal Screens within an In Vivo Murine Model to Identify Microenvironment-Dependent Lung Cancer Genes (ID 3140)
09:30 - 09:30 | Author(s): N.R. Little
- Abstract
Background:
Lung cancer remains the leading cause of cancer-related deaths worldwide. While significant knowledge has been gained regarding the characterization of mutational drivers in NSCLC, much less is known regarding interactions between tumor cells and the surrounding microenvironment that are critical for tumor progression. Additionally, a significant limitation in current understanding is the lack of knowledge regarding which tumor gene products are necessary for promoting cell survival in the context of the tumor microenvironment. We hypothesize that the use of pooled shRNA synthetic lethal screens within an in vivo murine model will allow for the elucidation of targetable microenvironment-dependent genes.
Methods:
We generated a custom murine shRNA lentiviral library targeting 250 genes implicated in the communication between cancer cells and the microenvironment, which was used to transduce two murine cell lines: Lewis Lung carcinoma (LLC) and CMT167 cells. Following puromycin selection of cells harboring incorporated shRNA’s of interest, populations were expanded and designated for in vitro versus in vivo replication and growth. Selected cells were allocated to either in vitro passage vs direct in vivo injection into the lungs of 18 week-old syngeneic C57BL6 mice. After 4 weeks, cells were harvested and gDNA was isolated. Sequencing and quantitation of shRNA was performed using an Illumina deep-sequencing platform. Both raw and normalized read counts were assessed and analyzed to determine the relative representation of a particular shRNA within an in vitro or in vivo sample. Following quality control assessments which demonstrated adequate read count numbers per sample, and appropriate correlation of sample similarity per groups, direct comparisons between in vitro and in vivo samples were performed.
Results:
Multiple gene candidates were identified and largely reproducible via either rank analysis, mean, or t-test analyses. Candidate genes included multiple chemokines, and their receptors, matrix proteases, complement factors, and growth factor receptors.
Conclusion:
These results suggest a list of genes that are both intriguing and diverse, pointing toward gene products that would not have been previously predicted to influence cancer cell survival and growth through a lung cancer cell-autonomous fashion. Furthermore, these genes appear to potentially interact with multiple compartments of the tumor microenvironment including the extracellular matrix, cytokine milieu, vascular structures (complement factors), and the adaptive immune system. Validation of specific gene targets are ongoing through assessment of tumor growth comparing murine cell lines transfected with individual shRNA’s of interest vs control tumor cells. Furthermore, parallel pooled shRNA synthetic lethal screens within selectively adaptive immune-deficient models are currently in progress.
