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T.T. Nguyen
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ORAL 41 - Immune Biology, Microenvironment and Novel Targets (ID 159)
- Event: WCLC 2015
- Type: Oral Session
- Track: Biology, Pathology, and Molecular Testing
- Presentations: 1
- Moderators:S.K. Padda, R. Nemenoff
- Coordinates: 9/09/2015, 18:30 - 20:00, Four Seasons Ballroom F1+F2
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ORAL41.06 - Transcriptional Profiling of Distinct Macrophage Subsets in Lung Tumor Microenvironment Reveals Their Functional Heterogeneity (ID 3181)
18:30 - 20:00 | Author(s): T.T. Nguyen
- Abstract
Background:
Lung cancer is the leading cause of cancer-related deaths in both men and women. While extensive research has focused on genetic mutations in neoplastic epithelial cells, it has now become apparent that cancer progression and metastasis involve complex interactions between cancer cells and the cells of the tumor microenvironment. Myeloid cells of mononuclear phagocyte lineage are a significant component of the tumor microenvironment in lung cancer. Depending on the activation state, myeloid cells have been implicated in tumor – promoting processes such angiogenesis, tissue remodeling and immunosuppression, but also in anti-tumor immunity such as supporting immune surveillance and direct cytotoxicity. The goal of this study was to identify distinct populations of monocyte/macrophage cells and to gain insight into their functions through transcriptional profiling.
Methods:
We used an orthotopic immunocompetent mouse model, in which Lewis Lung carcinoma cells, a cell line derived from mouse adenocarcinoma, were injected directly into the left lung lobe of syngeneic C57BL/6 mice. Whole left lung lobes bearing primary tumors were harvested at 2 and at 3 weeks after cancer cell injection, together with lungs from uninjected mice. Tissues were processed into single-cell suspensions and analyzed by multi-color flow cytometry. The flow cytometry strategy employed a combination of myeloid specific surface markers such as CD11b, CD11c, CD64, and SiglecF to identify distinct monocyte/macrophage subpopulations. We recovered these cell populations by flow cytometry-based cell sorting, isolated RNA, and performed transcriptional profiling by RNA-seq. Sequencing data were analyzed by TopHat/Cufflinks/CuffDiff software package and EdgeR. To define the lineage of the isolated cells we correlated their transcriptional profiles to published profiles of immune cells from blood and lung of naïve mice. Further, we used hierarchical clustering and web-based bioinformatic pathway analysis tool to discover functions and pathways enriched in specific myeloid populations.
Results:
Based on the combination of myeloid markers and transcriptional profiling, we identified 4 distinct populations of monocyte/macrophage cells: MacA, which represent alveolar macrophages, MacB1, which represent a mixture of dendritic cells and Ly6C- monocytes, MacB2, which represent Ly6C+ monocytes, and MacB3, which represent interstitial/infiltrating macrophages. While the numbers of MacA and MacB1 remain unchanged with cancer progression, MacB2 and MacB3 expand rapidly. Pathway analysis indicated that each population of cells regulates distinct functions in the tumor microenvironment, such as lipid metabolism, cytokine or chemokine secretion, production and remodeling of extracellular matrix, antigen presentation.
Conclusion:
These data provide critical insights into the heterogeneous nature and diverse functions of myeloid cells in tumor microenvironment of lung cancer. This study has the potential for development of therapeutics that target specific subsets of myeloid cells that could complement conventional cancer-cell-targeted therapies.
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P2.04 - Poster Session/ Biology, Pathology, and Molecular Testing (ID 234)
- Event: WCLC 2015
- Type: Poster
- Track: Biology, Pathology, and Molecular Testing
- Presentations: 1
- Moderators:
- Coordinates: 9/08/2015, 09:30 - 17:00, Exhibit Hall (Hall B+C)
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P2.04-007 - In Vitro and in Vivo Efficacy of AZD9291 Is Enhanced by Combination with AZD4547 in EGFR Mutant Lung Cancer Cells (ID 2456)
09:30 - 17:00 | Author(s): T.T. Nguyen
- Abstract
Background:
EGFR-specific tyrosine kinase inhibitors (TKIs) provide marked clinical responses in patients bearing EGFR mutated lung tumors, although acquired resistance limits the durability of the response. In light of the frequent emergence of erlotinib and gefitinib-resistant EGFR T790M mutations upon tumor progression, 3[rd] generation EGFR-specific TKIs have been developed that specifically inhibit gain-of-function EGFR mutants irrespective of T790M status. Recently, we reported a distinct mechanism of acquired resistance whereby specific EGFR mutant lung cancer cell lines including H1650 and HCC4006 cells, but not PC9 cells, undergo an epithelial-mesenchymal transition (EMT) upon chronic in vitro treatment with gefitinib. As a result, the adapted cells acquire vulnerability to FGFR inhibitors by virtue of EMT-mediated FGF2 and FGFR1 induction. Herein, we have tested the hypothesis that combination of the FGFR inhibitor, AZD4547, with the 3[rd] generation EGFR TKI, AZD9291 will yield superior anti-tumor activity relative to AZD9291 alone.
Methods:
Lung cancer cell lines bearing gain-of-function EGFR mutations (HCC4006, H1650 and PC9) were submitted to in vitro clonogenic growth assays in the presence of AZD9291 and/or AZD4547 over concentration ranges for 1 to 300 nM for each drug. For in vivo measurement of the activity of these drugs, flank xenografts were established in Nu/Nu mice with the 3 lung cancer cell lines and treated by daily oral gavage (5 days on, 2 days off) with diluent, AZD9291 (5 mg/kg), AZD4547 (12.5 mg/kg) and the combination of the two drugs at these doses. Tumor size was measured with calipers and volume was calculated using the formula, Volume=3.14(short diameter)[2](long diameter)/6.
Results:
HCC4006, H1650 and PC9 cells were highly sensitive to ZD9291 in vitro with IC~50~ values of 1.6, 7.4 and 3.3 nM, respectively. In a 2 week clonogenic growth assay, AZD9291 reduced growth of all cell lines by >95%, although viable drug resistant persisters clearly remained. While none of these cell lines exhibited significant growth inhibition in response to AZD4547 alone, combination of AZD9291 and AZD4547 further reduced clonogenic growth of HCC4006 and H1650 cells, but not PC9 cells. In flank xenograft studies, AZD9291 monotherapy induced marked tumor shrinkage (H1650, ~80% at day 10; HCC4006, ~90% at day 30; PC9, 89% at day 25), although regrowth of the tumors occurred with all three xenografts. AZD4547 yielded little or no growth inhibition as a monotherapy, but significantly enhanced the degree of tumor shrinkage and delayed the time to tumor progression in H1650 and HCC4006 tumors, but not PC9 tumors.
Conclusion:
Combination of the FGFR inhibitor AZD4547 with AZD9291 affords greater growth suppression relative to AZD9291 alone in HCC4006 and H1650 cells that undergo EMT and induction of an FGF2-FGFR1 pathway. Predictably, this combination was not more effective compared to AZD9291 alone in PC9 cells that fail to undergo EMT in response to EGFR TKI treatment. The studies support the efficacy of combined AZD9291 and AZD4547 treatment of a subset of lung tumors driven by mutated EGFR, although the features of these particular lung tumors that predict this response is unknown at this time.
