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R. Nemenoff
Moderator of
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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: 8
- Moderators:S.K. Padda, R. Nemenoff
- Coordinates: 9/09/2015, 18:30 - 20:00, Four Seasons Ballroom F1+F2
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ORAL41.01 - Tumor-Infiltrating B Lymphocytes Characterized by CD79a and MUM1 Independently Predict Outcome in Patients with Non-Small Cell Lung Cancer (ID 485)
18:30 - 18:41 | Author(s): R.N. Fischer, A.H. Scheel, S.I. Rothschild, H.A. Schlößer, J. Wolf, R. Büttner, S. Ansén, M.S. Von Bergwelt-Baildon
- Abstract
- Presentation
Background:
Tumor-infiltrating lymphocytes play an important role in cell-mediated immune-destruction of cancer cells and tumor growth control. For non-small cell lung cancer (NSCLC) a prognostic role of T cell subtypes, natural killer cells and dendritic cells within the tumor stroma has been described. Here, we studied the role of tumor-infiltrating B cells characterized by CD79a (B-cell antigen receptor complex-associated protein alpha chain) and MUM1 surface expression (Multiple myeloma oncogene 1) in patients with NSCLC. To our knowledge, this study represents the so far largest cohort analyzing the prognostic impact of tumor-infiltrating B-cells.
Methods:
B cell infiltration was quantified using immunohistochemistry and antibodies to CD79a (Dako, clone JCB117) and MUM1 (Dako, clone MUM1p) on tissue microarrays (TMA) of paraffin embedded tumor sections. Genetic driver mutations were identified by next-generation sequencing and FISH analysis. SPSS version 20 (IBM Corp.) was used for statistical analysis. Chi-square test, Fisher’s exact test, Kaplan-Meier survival analysis and Cox-regression analysis were used as appropriate.
Results:
478 tissue samples from NSCLC patients were available for immunohistochemistry. 65% of patients were male, median age was 66 years. 56% had adenocarcinoma and 39% squamous cell histology. 61% of patients had localized disease (stage I/II), 30% locally advanced disease (stage III) and 6% were diagnosed with stage IV. Frequencies of genomic aberrations are listed in Table 1. CD79a and MUM1 positive cells were detected in 40.8% (195/478) and 40.2% (192/478) of the analyzed NSCLC tissue samples, respectively. B cell infiltration was not associated with clinical or histo-pathological characteristics. MUM1 expression was associated with a significantly prolonged overall survival (median OS 54 vs. 40 months, p=0.025). The expression of CD79a showed a trend towards a better outcome (median OS 49 vs. 40 months, p=0.069). In the multivariate analysis B cell infiltration characterized by CD79a/MUM1 positivity was an independent prognostic marker for survival (p=0.045) as was MUM1 expression (p=0.031). Table 1.Genomic aberration Number of patients Frequency TP53 mutation 136 28.5% KRAS mutation 65 13.6% FGFR1 amplification 28 5.9% PIK3CA mutation 17 3.6% EGFR mutation 12 2.5% ALK fusion 4 0.8% ERBB2 mutation 4 0.8% ERBB2 amplificiation 4 0.8% ROS1 fusion 2 0.4% BRAF mutation 2 0.4% DDR2 mutation 2 0.4% FGFR2 mutation 1 0.2%
Conclusion:
B cell infiltration characterized by immunohistochemical positivity for CD79a and MUM1 represents an independent prognostic marker in NSCLC. This finding supports the hypothesis of a B cell-mediated anti-tumor immunity.
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ORAL41.02 - Novel Mechanism of Immune-Tolerance and Cancer Metastasis Due to Aberrant Expression of Natural Killer Immunoglobulin-Like Receptors (KIRs) (ID 2199)
18:41 - 18:52 | Author(s): D.C. Chan, Z. Zhang, D. Zheng, T. Chan, J. Dipaola, B. Warren, M. Berg, K. Horwitz, N. Ahn, L. Lanier, P.A. Bunn, Jr
- Abstract
- Presentation
Background:
Natural Killer (NK) cells are a major defense to eliminate cancer cells. Cancer cells and metastases may have aberrantly expressed KIRs to prevent killing by NK cells. In addition, platelets may inhibit NK killing of cancer cells. Metastatic cancer cells spread through blood vessels where they constantly interact with platelets by forming tumor microemboli and thereby protected from otherwise rapid elimination from host immune defense cells such as NK cells. Here, using an in vivo model of cancer metastasis in athymic nude mice by directly injecting cancer cells into the blood stream, we study the ability of platelets and KIRs in helping cancer cells to escape from immune surveillance and promote metastasis.
Methods:
GFP-luciferase tagged human lung adenocarcinoma cell line, H2122-GL, was further transfected with KIR2DL1 (LL454) plasmids. Stable transformants were enriched by cell sorting. In vivo experimental metastasis were performed in both athymic nude mice and in Nbeal2 knockout and wild type C57 black mice, by tail vein injections of H2122 parental and KIR expressing cells, with and without pre-infusion of human platelets. Levels of tumor cells detected in the lung and other sites were closely monitored by bioluminescence imaging at various time intervals, using an IVIS200 imager.
Results:
24 hours after tail vein injection of a million parental H2122-GL, as low as 0.4 million photons were detectable in the lungs of nude mice (n=5), while those mice injected with a same number of H2122-GL-KIR2DL1 cells, they produced 1.85 million photons in the lungs, showing a 4.6 fold increase in accumulation of KIR-expressed cancer cells than those parental cells in the lung. When the nude mice were pre-infused with iv injection of human platelets followed by tail vein injection of parental or KIR-expressed H2122 cells, enhancement up to 7 fold of lung metastases of KIR expressed H2122 were detected relative to the parental cells as early as 24 hours. 5 weeks post injection, an enhancement up to 190 fold in bioluminescence intensity was found with KIR expressed cells relative to the parental cells. Interestingly, the enhancement of lung metastases was abrogated when similar experiments were repeated in the NBeal2 knockout mice, whose platelets were nonfunctional due to defective alpha-granules and deficiency in their cargo, including von Willebrand factor, thrombospondin-1, and platelet factor 4. One hour after tail vein injection, both parental and KIR expressed H2122 cells produced same but low number of lung metastases, indicating that the defective platelets in the ko mice had failed to promote lung metastases. In the wild type mice, significantly more KIR expressed H2122 cells were detected in the lung relative to parental cells. However, as expected, these early lung metastases were rejected later by the host intact immune cells.
Conclusion:
Our studies demonstrated that metastatic cancer cells acquire immune-resistance by aberrantly express Natural Killer-Cell Immunoglobulin-like Receptors (KIRs) on their surface and that KIR-expressing cancer cells interact more strongly with platelets leading to significantly increase in NK tolerance and enhancing cancer metastases in pre-clinical models.
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ORAL41.03 - Myeloid Derived Supressor Cells and Their Clinical Relevance in Non-Small Cell Lung Cancer (ID 2946)
18:52 - 19:03 | Author(s): O. Arrieta Rodriguez, R. Morales-Flores, A. Garcia-Vicente, E. Montes-Servín, F. Salinas-Parra, L. Barrera
- Abstract
- Presentation
Background:
Lung cancer is the leading cause of cancer death worldwide and most of the patients are diagnosed with advanced disease. Myeloid-derived suppressor cells (MDSCs) are major contributors to tumor immune tolerance and targeting them can improve antitumor activity.
Methods:
We investigated the CD33[+]CD11b[+]CD66b[+]CD15[+]VEGFR-1[hi] MDSCs frequency in 120 non-small cell lung cancer (NSCLC) treatment-naive patients, with stage IIIB and IV of disease. We analyzed 1-year survival and its prognostic significance in relation to outcome analysis as well as its potential immunosuppression over cytotoxic CD8[+] T lymphocytes. The immunophenotyping of cell population was performed with multiparametric technique by flow cytometry.
Results:
We found a significant increase compared with controls in: Percentage of CD33[+]CD14[-]CD11b[+]CD66b[+]CD15[+ ](10.4 ± 5.01% vs. 3.1 ± 1.7% P<0.0001); Mean Fluorescence intensity (MFI) of VEGFR on MDSCs (P<0.001); plasma levels of arginase-1 (P<0.01); arginase-1 enzymatic activity (P<0.05); plasma levels of TGF-β (P<0.0001), IL-10 (P=0.0027) and IL-6 (P<0.0001). On the other hand, we found a significant decrease compared with controls in: Plasma levels of IFN-γ (P<0.0001); CD8[+] T cells (P<0.001); CD8[+]T cells IFN-γ production co-cultured with MDSCs (N=10; P<0.001) and MFI of CD3ζ chain (N=10; P<0.05). The percentage of MDSCs was negatively related to the percentage of CD8[+] T cells in the peripheral blood (N=155, R=-0.3045, P=0.0167). Finally, we found an inverse correlation between circulating MDSCs percentages and overall survival (P=0.09).
Conclusion:
Our study provides evidence of an increased pool of CD33+CD11b+CD66b+CD15+VEGFR-1hi MDSCs in the peripheral blood of NSCLC patients. The suppressive effect, of MDSCs on CD8+ T lymphocytes, suggests an important role in mediating immunosuppression in NSCLC that should enable the development of a novel biomarker and thus might represent a potential target for therapeutic intervention.
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ORAL41.04 - Discussant for ORAL41.01, ORAL41.02, ORAL41.03 (ID 3439)
19:03 - 19:13 | Author(s): N.A. Rizvi
- Abstract
- Presentation
Abstract not provided
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ORAL41.05 - Targeting Phosphatidylserine-Mediated Immune Suppression Enhances the Efficacy of Immune Checkpoint Blockade in Pre-Clinical Tumor Models (ID 1011)
19:13 - 19:24 | Author(s): R.A. Brekken, B. Freimark, J. Gong, C. Baldwin, V. Nguyen, M. Gray, S. Yin, J. Hutchins, A. Schroit, X. Huang
- Abstract
- Presentation
Background:
Despite substantial progress, only a subset of cancer patients benefit from blockade of the PD-1 immune checkpoint. Multifocal immune suppression in the tumor microenvironment is the underlying cause for the limited efficacy of immune checkpoint blockade. Persistent immune suppression prevents the development of a robust T cell response to tumor specific antigens that is required for effective downstream immune checkpoint blockade. An underappreciated but significant contributor to immune suppression in tumors is the expression of the membrane phospholipid phosphatidylserine (PS) on the surface of tumor cells and tumor-derived microvesicles. PS is recognized by receptors on immune cells where it triggers the secretion of immune suppressive cytokines, prevents the differentiation of myeloid-derived suppressor cells (MDSCs) and inhibits dendritic cell (DC) maturation; events that prevent a productive anti-tumor T cell response. Bavituximab, a chimeric monoclonal antibody that targets PS and inhibits PS-mediated immunosuppressive signaling, drives immune activation by reducing the levels of MDSCs, by polarizing tumor-associated macrophages towards an M1 phenotype and by promoting the maturation of dendritic cells (DCs).
Methods:
The efficacy of bavituximab, anti-PD-1 and combination therapy was evaluated in multiple syngeneic, pre-clinical tumor models. Treatment efficacy was determined by inhibition of tumor growth and by immunophenotyping of spleen and tumor infiltrating leukocytes.
Results:
The combination of antibody-mediated PS and PD-1 blockade was significantly more effective in reducing tumor burden and promoting immune activation than single agent therapy. Combination therapy increased tumor infiltration of effector T-cells (Teff), increased the Teff:T regulatory cell ratio in the tumor and enhanced Teff function as determined by IFN-γ, TNFα and granzyme B levels associated with Teff cells in the spleen and tumor. Furthermore combined blockade of PS and PD-1 signaling reduced the level of immune suppressive cells (e.g., MDSCs, M2 macrophages, and Treg) in the tumor microenvironment.
Conclusion:
These results raise the possibility that PS blockade with bavituximab can enhance the efficacy of anti-PD-1 therapy even in patients with tumors that are unresponsive to single agent immune checkpoint therapy.
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ORAL41.06 - Transcriptional Profiling of Distinct Macrophage Subsets in Lung Tumor Microenvironment Reveals Their Functional Heterogeneity (ID 3181)
19:24 - 19:35 | Author(s): J.M. Poczobutt, S. De, V. Yadav, H. Li, J. Kwak, T. Sippel, D. Hanson, T.T. Nguyen, M.C. Weiser-Evans, R. Nemenoff
- 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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ORAL41.07 - The Identification of Therapeutic Targets in Lung Cancer Based on Transcriptomic and Proteomic Characterization of Cancer-Testis Antigens (ID 1555)
19:35 - 19:46 | Author(s): D. Djureinovic, B. Hallström, J.S.M. Mattsson, L. La Fleur, J. Botling, L. Fagerberg, H. Brunnström, S. Ekman, E. Ståhle, H. Koyi, M. Lambe, E. Branden, C. Lindskog, F. Pontén, M. Uhlén, P. Micke
- Abstract
- Presentation
Background:
Most immunotherapeutic modalities are based on the concept that the immune system can attack targets that are specifically expressed in cancer cells. Cancer testis antigens (CTAs) are a group of genes with a broad expression in cancers including non-small cell lung cancer (NSCLC). In normal tissues the expression of CTAs is restricted to immune privileged organs such as testis and placenta. This limited expression in somatic tissues renders CTAs as a valuable group of genes for the exploration of potential immunotherapeutic targets. The aim of this study was to comprehensively explore the CTA repertoire in NSCLC and to try identifying new CTAs.
Methods:
RNA sequencing (RNAseq) was performed on 202 NSCLC samples from a consecutive clinical cohort of surgically resected patients. For the analysis of the comprehensive CTA expression profile in NSCLC we used Cancer Testis (CT) Database containing all genes reported as CTAs in the literature. The NSCLC transcriptome was compared to the normal transcriptome comprising of 22 paired normal lung tissues as well as to 122 samples from 32 different normal human tissues. Corresponding protein expression was evaluated by using immunohistochemistry (IHC) on tissue microarrays (TMAs) containing tumor tissue from the same patients as used in the RNA sequencing.
Results:
Of the 276 established CTAs, 155 genes (56%) were restricted to testis and placenta among normal tissues and were identified as CTAs. One third (35%) was expressed in at least one of the 202 individual NSCLC cases and 28 of these genes were previously not reported to be expressed as CTAs in NSCLC. Applying stringent analysis criteria on our RNA sequencing data set we identified 61 genes that were expressed in NSCLC and testis or placenta, but not in other normal tissues. Thus, these genes present potential new CTAs. The specific cancer/testis expression of selected genes (ZNF560, TGIF2LX, TFPI2, HMGB3, TKTL1 and STK31) from this group was confirmed on protein level using IHC. Additional analysis revealed that most CTAs were concurrently expressed in adenocarcinoma and squamous cell carcinoma. The expression of a subset of genes was histology dependent, with predominant expression in adenocarcinoma (e.g. XAGE family members) and in squamous cell carcinoma (e.g. MAGE family members).
Conclusion:
Our study provides deep sequencing mRNA expression profiles of the whole CTA repertoire in NSCLC. Several CTAs previously identified in other cancers but not analyzed in NSCLC have been identified on both mRNA and protein level. Additionally, we have identified 61 novel genes as CTAs in NSCLC that previously have not been reported as CTAs and several of these were also confirmed on protein level. This data offers the opportunity to design individual therapy options to target single CTAs or CTA clusters.
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ORAL41.08 - Discussant for ORAL41.05, ORAL41.06, ORAL41.07 (ID 3440)
19:46 - 19:56 | Author(s): R. Herbst
- Abstract
- Presentation
Abstract not provided
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Author of
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ORAL 23 - Prevention and Cancer Risk (ID 121)
- Event: WCLC 2015
- Type: Oral Session
- Track: Prevention and Tobacco Control
- Presentations: 1
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ORAL23.03 - Role of Inflammatory Infiltrates in Promoting Persistence or Regression of Bronchial Dysplasia (ID 3026)
11:07 - 11:18 | Author(s): R. Nemenoff
- Abstract
- Presentation
Background:
Inflammatory infiltrates show differing capacities to eliminate malignant cells. This capacity is related to the polarization of key inflammatory cells in tumor infiltrates. A pathway analysis of genes that are differentially expressed between persistent and regressive bronchial dysplasia (BD) identified 13 pathways associated with persistence of which 8 were related to inflammation. We have hypothesized that differences in inflammatory infiltrate polarization may contribute to lung carcinogenesis and have employed gene expression and in situ analyses to characterize differences in inflammatory infiltrates related to persistence and regression of pre-malignant BD.
Methods:
Normalized gene expression levels (Affymetrix Hu 1.0) of selected genes related to inflammatory cell polarization features were analyzed to find differences associated with follow-up histology for BD. Validational analyses of these relationships were undertaken in studies of baseline biopsies selected to represent persistent (n=43) and regressive BD (n=39). These biopsies were analyzed by quantitative immunohistochemistry and dual immunofluorescence studies to characterize the overall proportion of subsets of T-lymphocytes and macrophages in each of the groups. Image analysis tools (Aperio) were used to characterize the density of inflammatory cell subsets in the stromal and epithelial compartments of biopsy tissue within defined areas.
Results:
Analysis of expression levels for a subset of inflammatory cell related genes assessed in a global gene expression analysis indicated significantly higher levels of expression of macrophage M1 markers HLA-DRA (p=0.01) and inducible nitric oxide synthetase (iNOS; p=0.02) and T-helper lymphocyte marker CD4 (p=0.04) in regressive BD compared to persistent BD. There was also a trend toward higher expression of cytotoxic T-lymphocyte marker CD8 in regressive BD (p=0.25). Expression of B-lymphocyte and neutrophil markers were not different between regressive and persistent BD. CD68 immunohistochemical stains (IHC) demonstrated a trend toward an increase in macrophages per area of combined dysplastic epithelium and underlying stroma with a mean increase in IHC positivity of 1.75-fold in regressive versus persistent BD (p=0.08). CD4 and CD8 IHC showed 1.36- and 1.19-fold increases, respectively, in regressive BD but these changes were not statistically significant (p=0.36 and p=0.43 respectively). Dual immunofluorescence was undertaken to determine if polarization specific subsets of macrophages correlated with regression or persistence of BD. Analysis of a preliminary subset of regressive (n=3) and persistent (n=3) BD demonstrates a wide range of M1 to M2 ratios (range = 0.84 – 4.82 for ratio of HLA-DRA-CD68 dual positive M1 to CD206-CD68 dual positive M2 macrophages per high power field, 400X). Additional analyses of macrophages are ongoing to determine if the polarization status is related to regression or persistence of BD, and analysis of markers of T-helper lymphocyte subsets are planned.
Conclusion:
Gene expression analyses indicate that increased expression of markers of M1 macrophages and T-helper lymphocytes are associated with regression, and in situ analyses suggest that differences in the amount of inflammatory cell subsets may be related to outcome in BD. These studies could have implications for predicting the behavior of premalignant disease and manipulating inflammatory activity in preventing progression of BD to invasive lung cancer.
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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)
19:24 - 19:35 | Author(s): R. Nemenoff
- 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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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): R. Nemenoff
- 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.
