20 results on '"Stephanie E. Busch"'
Search Results
2. CTCF Haploinsufficiency Destabilizes DNA Methylation and Predisposes to Cancer
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Christopher J. Kemp, James M. Moore, Russell Moser, Brady Bernard, Matt Teater, Leslie E. Smith, Natalia A. Rabaia, Kay E. Gurley, Justin Guinney, Stephanie E. Busch, Rita Shaknovich, Victor V. Lobanenkov, Denny Liggitt, Ilya Shmulevich, Ari Melnick, and Galina N. Filippova
- Subjects
Biology (General) ,QH301-705.5 - Abstract
Epigenetic alterations, particularly in DNA methylation, are ubiquitous in cancer, yet the molecular origins and the consequences of these alterations are poorly understood. CTCF, a DNA-binding protein that regulates higher-order chromatin organization, is frequently altered by hemizygous deletion or mutation in human cancer. To date, a causal role for CTCF in cancer has not been established. Here, we show that Ctcf hemizygous knockout mice are markedly susceptible to spontaneous, radiation-, and chemically induced cancer in a broad range of tissues. Ctcf+/− tumors are characterized by increased aggressiveness, including invasion, metastatic dissemination, and mixed epithelial/mesenchymal differentiation. Molecular analysis of Ctcf+/− tumors indicates that Ctcf is haploinsufficient for tumor suppression. Tissues with hemizygous loss of CTCF exhibit increased variability in CpG methylation genome wide. These findings establish CTCF as a prominent tumor-suppressor gene and point to CTCF-mediated epigenetic stability as a major barrier to neoplastic progression.
- Published
- 2014
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3. Neutrophils dominate the immune cell composition in non-small cell lung cancer
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Jesse J. Hubbard, Martin W. McIntosh, Heather E. Metz, Grace H. Y. Yang, A. McGarry Houghton, Mark L. Hanke, Kyoung-Hee Kim, Sylvia Lee, David K. Madtes, Julia Kargl, and Stephanie E. Busch
- Subjects
0301 basic medicine ,Cell type ,Science ,Cell ,General Physics and Astronomy ,chemical and pharmacologic phenomena ,Biology ,General Biochemistry, Genetics and Molecular Biology ,Flow cytometry ,03 medical and health sciences ,0302 clinical medicine ,Immune system ,medicine ,Receptor ,Lung cancer ,Multidisciplinary ,medicine.diagnostic_test ,General Chemistry ,biochemical phenomena, metabolism, and nutrition ,medicine.disease ,Immune checkpoint ,respiratory tract diseases ,3. Good health ,030104 developmental biology ,medicine.anatomical_structure ,030220 oncology & carcinogenesis ,Immunology ,bacteria ,Adenocarcinoma - Abstract
The response rate to immune checkpoint inhibitor therapy for non-small-cell lung cancer (NSCLC) is just 20%. To improve this figure, several early phase clinical trials combining novel immunotherapeutics with immune checkpoint blockade have been initiated. Unfortunately, these trials have been designed without a strong foundational knowledge of the immune landscape present in NSCLC. Here, we use a flow cytometry panel capable of measuring 51 immune cell populations to comprehensively identify the immune cell composition and function in NSCLC. The results show that the immune cell composition is fundamentally different in lung adenocarcinoma as compared with lung squamous cell carcinoma, and that neutrophils are the most prevalent immune cell type. Using T-cell receptor-β sequencing and tumour reactivity assays, we predict that tumour reactive T cells are frequently present in NSCLC. These results should help to guide the design of clinical trials and the direction of future research in this area.
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- 2017
4. Lung Cancer Subtypes Generate Unique Immune Responses
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Mark L. Hanke, Heather E. Metz, Julia Kargl, David MacPherson, A. McGarry Houghton, and Stephanie E. Busch
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0301 basic medicine ,Lung Neoplasms ,Myeloid ,Immunology ,Mice, Transgenic ,Treatment of lung cancer ,Adenocarcinoma ,CD8-Positive T-Lymphocytes ,Biology ,medicine.disease_cause ,T-Lymphocytes, Regulatory ,Article ,Mice ,03 medical and health sciences ,0302 clinical medicine ,Immune system ,medicine ,Animals ,Humans ,Immunology and Allergy ,Lung cancer ,Tumor microenvironment ,Cancer ,medicine.disease ,Small Cell Lung Carcinoma ,Neoplasm Proteins ,respiratory tract diseases ,030104 developmental biology ,medicine.anatomical_structure ,030220 oncology & carcinogenesis ,Mutation ,KRAS - Abstract
Lung cancer, the leading cause of cancer-related deaths worldwide, is a heterogeneous disease comprising multiple histologic subtypes that harbor disparate mutational profiles. Immune-based therapies have shown initial promise in the treatment of lung cancer patients but are limited by low overall response rates. We sought to determine whether the host immune response to lung cancer is dictated, at least in part, by histologic and genetic differences, because such correlations would have important clinical ramifications. Using mouse models of lung cancer, we show that small cell lung cancer (SCLC) and lung adenocarcinoma (ADCA) exhibit unique immune cell composition of the tumor microenvironment. The total leukocyte content was markedly reduced in SCLC compared with lung ADCA, which was validated in human lung cancer specimens. We further identified key differences in immune cell content using three models of lung ADCA driven by mutations in Kras, p53, and Egfr. Although Egfr-mutant cancers displayed robust myeloid cell recruitment, they failed to mount a CD8+ immune response. In contrast, Kras-mutant tumors displayed significant expansion of multiple immune cell types, including CD8+ cells, regulatory T cells, IL-17A–producing lymphocytes, and myeloid cells. A human tissue microarray annotated for KRAS and EGFR mutations validated the finding of reduced CD8+ content in human lung ADCA. Taken together, these findings establish a strong foundational knowledge of the immune cell contexture of lung ADCA and SCLC and suggest that molecular and histological traits shape the host immune response to cancer.
- Published
- 2016
5. Chronic obstructive pulmonary disease alters immune cell composition and immune checkpoint inhibitor efficacy in non-small cell lung cancer
- Author
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Sudhakar Pipavath, A. McGarry Houghton, Stephanie E. Busch, Jesse J. Hubbard, Huajia Zhang, Heather E. Metz, Grace H. Y. Yang, Nicholas M. Mark, Julia Kargl, and David K. Madtes
- Subjects
Oncology ,Male ,Molecular composition ,Lung Neoplasms ,Immune checkpoint inhibitors ,medicine.medical_treatment ,CD8-Positive T-Lymphocytes ,Critical Care and Intensive Care Medicine ,Pathogenesis ,Cohort Studies ,Pulmonary Disease, Chronic Obstructive ,0302 clinical medicine ,Carcinoma, Non-Small-Cell Lung ,Tumor Microenvironment ,030212 general & internal medicine ,Prospective Studies ,Prospective cohort study ,Pneumonectomy ,education.field_of_study ,COPD ,General Medicine ,T-Lymphocytes, Helper-Inducer ,Middle Aged ,Flow Cytometry ,Editorial Commentary ,medicine.anatomical_structure ,030220 oncology & carcinogenesis ,Female ,Non small cell ,Immunosuppressive Agents ,Signal Transduction ,Pulmonary and Respiratory Medicine ,medicine.medical_specialty ,Population ,Pulmonary disease ,Sensitivity and Specificity ,03 medical and health sciences ,Immune system ,Internal medicine ,medicine ,Humans ,education ,Lung cancer ,Aged ,Tumor microenvironment ,Lung ,business.industry ,Original Articles ,medicine.disease ,respiratory tract diseases ,030228 respiratory system ,Immunology ,business - Abstract
Chronic obstructive pulmonary disease (COPD) and non-small cell lung cancer (NSCLC) are interrelated diseases with substantial mortality, and the pathogenesis of both involves aberrant immune functioning.To profile immune cell composition and function in patients with NSCLC and describe the effects of COPD on lung and tumor microenvironments.We profiled resected lung and tumor tissue using flow cytometry and T-cell receptor sequencing in patients with and without COPD from a prospective cohort of patients undergoing resection of NSCLC. A murine cigarette smoke exposure model was used to evaluate the effect on pulmonary immune populations. A separate retrospective cohort of patients who received immune checkpoint inhibitors (ICIs) was analyzed, and their survival was quantified.We observed an increased number of IFN-γ-producing CD8In patients with COPD, Th1 cell populations were expanded in both lung and tumor microenvironments, and the presence of COPD was associated with longer progression-free intervals in patients treated with ICIs. This has implications for understanding the immune mediators of COPD and developing novel therapies for NSCLC.
- Published
- 2019
6. CTCF Haploinsufficiency Destabilizes DNA Methylation and Predisposes to Cancer
- Author
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Denny Liggitt, Kay E. Gurley, James M. Moore, Russell Moser, Galina N. Filippova, Victor V. Lobanenkov, Leslie E. Smith, Stephanie E. Busch, Brady Bernard, Natalia A. Rabaia, Ari Melnick, Justin Guinney, Ilya Shmulevich, Christopher J. Kemp, Rita Shaknovich, and Matt Teater
- Subjects
CCCTC-Binding Factor ,Mice, Transgenic ,Haploinsufficiency ,Biology ,medicine.disease_cause ,General Biochemistry, Genetics and Molecular Biology ,Article ,Epigenesis, Genetic ,03 medical and health sciences ,Mice ,0302 clinical medicine ,Cell Line, Tumor ,Neoplasms ,medicine ,Animals ,Humans ,Genes, Tumor Suppressor ,Genetic Predisposition to Disease ,Epigenetics ,lcsh:QH301-705.5 ,030304 developmental biology ,Regulation of gene expression ,0303 health sciences ,Mutation ,Cancer ,DNA Methylation ,medicine.disease ,Survival Analysis ,3. Good health ,Chromatin ,Gene Expression Regulation, Neoplastic ,Mice, Inbred C57BL ,Repressor Proteins ,lcsh:Biology (General) ,CTCF ,030220 oncology & carcinogenesis ,DNA methylation ,Cancer research ,Protein Binding - Abstract
SummaryEpigenetic alterations, particularly in DNA methylation, are ubiquitous in cancer, yet the molecular origins and the consequences of these alterations are poorly understood. CTCF, a DNA-binding protein that regulates higher-order chromatin organization, is frequently altered by hemizygous deletion or mutation in human cancer. To date, a causal role for CTCF in cancer has not been established. Here, we show that Ctcf hemizygous knockout mice are markedly susceptible to spontaneous, radiation-, and chemically induced cancer in a broad range of tissues. Ctcf+/− tumors are characterized by increased aggressiveness, including invasion, metastatic dissemination, and mixed epithelial/mesenchymal differentiation. Molecular analysis of Ctcf+/− tumors indicates that Ctcf is haploinsufficient for tumor suppression. Tissues with hemizygous loss of CTCF exhibit increased variability in CpG methylation genome wide. These findings establish CTCF as a prominent tumor-suppressor gene and point to CTCF-mediated epigenetic stability as a major barrier to neoplastic progression.
- Published
- 2014
7. Insulin receptor substrate-1 deficiency drives a proinflammatory phenotype in KRAS mutant lung adenocarcinoma
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Jay K. Kolls, Stephanie E. Busch, Heather E. Metz, Morris F. White, Shira Abberbock, Brenda F. Kurland, Julia Kargl, Kyoung-Hee Kim, A. McGarry Houghton, Julie Randolph-Habecker, and Sue E. Knoblaugh
- Subjects
0301 basic medicine ,Adult ,Male ,Lung Neoplasms ,Insulin Receptor Substrate Proteins ,Kaplan-Meier Estimate ,Adenocarcinoma ,medicine.disease_cause ,Proinflammatory cytokine ,Proto-Oncogene Proteins p21(ras) ,03 medical and health sciences ,Growth factor receptor ,Cell Line, Tumor ,medicine ,Animals ,Humans ,Aged ,Aged, 80 and over ,Mice, Knockout ,Multidisciplinary ,biology ,Receptors, Interleukin ,Middle Aged ,Biological Sciences ,IRS1 ,Insulin receptor ,030104 developmental biology ,Phenotype ,A549 Cells ,Mutation ,biology.protein ,Cancer research ,Female ,KRAS ,Signal transduction ,Janus kinase ,Signal Transduction - Abstract
Insulin receptor substrate-1 (IRS-1) is a signaling adaptor protein that interfaces with many pathways activated in lung cancer. It has been assumed that IRS-1 promotes tumor growth through its ability to activate PI3K signaling downstream of the insulin-like growth factor receptor. Surprisingly, tumors with reduced IRS-1 staining in a human lung adenocarcinoma tissue microarray displayed a significant survival disadvantage, especially within the Kirsten rat sarcoma viral oncogene homolog (KRAS) mutant subgroup. Accordingly, adenoviral Cre recombinase (AdCre)-treated LSL-Kras/Irs-1(fl/fl) (Kras/Irs-1(-/-)) mice displayed increased tumor burden and mortality compared with controls. Mechanistically, IRS-1 deficiency promotes Janus kinase/signal transducers and activators of transcription (JAK/STAT) signaling via the IL-22 receptor, resulting in enhanced tumor-promoting inflammation. Treatment of Kras/Irs-1(+/+) and Kras/Irs-1(-/-) mice with JAK inhibitors significantly reduced tumor burden, most notably in the IRS-1-deficient group.
- Published
- 2016
8. ARF suppresses hepatic vascular neoplasia in a carcinogen-exposed murine model
- Author
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Kay E. Gurley, Christopher J. Kemp, Russell Moser, and Stephanie E. Busch
- Subjects
Pathology ,medicine.medical_specialty ,Endothelium ,Genetic strain ,Biology ,medicine.disease ,Malignancy ,Pathology and Forensic Medicine ,medicine.anatomical_structure ,Hemangiosarcoma ,p14arf ,CDKN2A ,medicine ,Signal transduction ,Carcinogen - Abstract
Hepatic haemangiosarcoma is a deadly malignancy whose aetiology remains poorly understood. Inactivation of the CDKN2A locus, which houses the ARF and p16INK4a tumour suppressor genes, is a common event in haemangiosarcoma patients, but the precise role of ARF in vascular tumourigenesis is unknown. To determine the extent to which ARF suppresses vascular neoplasia, we examined the incidence of hepatic vascular lesions in Arf-deficient mice exposed to the carcinogen urethane [intraperitoneal (i.p.), 1 mg/g]. Loss of Arf resulted in elevated morbidity and increased the incidence of both haemangiomas and incipient haemangiosarcomas. Suppression of vascular lesion development by ARF was heavily dependent on both Arf gene-dosage and the genetic strain of the mouse. Trp53-deficient mice also developed hepatic vascular lesions after exposure to urethane, suggesting that ARF signals through a p53-dependent pathway to inhibit the development of hepatic haemangiosarcoma. Our findings provide strong evidence that inactivation of Arf is a causative event in vascular neoplasia and suggest that the ARF pathway may be a novel molecular target for therapeutic intervention in haemangiosarcoma patients. Copyright © 2012 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.
- Published
- 2012
9. ARF Inhibits the Growth and Malignant Progression of Non-Small Cell Lung Carcinoma
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H D Liggitt, Christopher J. Kemp, Stephanie E. Busch, Kay E. Gurley, Russell Moser, and Karen S. Kelly-Spratt
- Subjects
Cancer Research ,Lung Neoplasms ,Biology ,medicine.disease_cause ,Article ,Mice ,p14arf ,CDKN2A ,Carcinoma, Non-Small-Cell Lung ,Genetics ,medicine ,Carcinoma ,Neoplasm ,Animals ,Neoplasm Invasiveness ,Neoplasm Metastasis ,Sarcomatoid carcinoma ,Molecular Biology ,Cyclin-Dependent Kinase Inhibitor p16 ,Cell Proliferation ,Mice, Knockout ,Cell cycle ,medicine.disease ,respiratory tract diseases ,Genes, ras ,Mutation ,Cancer research ,Disease Progression ,KRAS ,Tumor Suppressor Protein p53 ,Carcinogenesis ,DNA Damage - Abstract
Non-small-cell lung carcinoma (NSCLC) is among the deadliest of human cancers. The CDKN2A locus, which houses the INK4a and ARF tumor suppressor genes, is frequently altered in NSCLC. However, the specific role of ARF in pulmonary tumorigenesis remains unclear. KRAS and other oncogenes induce the expression of ARF, thus stabilizing p53 activity and arresting cell proliferation. To address the role of ARF in Kras-driven NSCLC, we compared the susceptibility of NIH/Ola strain wild-type and Arf-knockout mice to urethane-induced lung carcinogenesis. Lung tumor size, malignancy and associated morbidity were significantly increased in Arf(-/-) compared with Arf(+/+) animals at 25 weeks after induction. Pulmonary tumors from Arf-knockout mice exhibited increased cell proliferation and DNA damage compared with wild-type mice. A subgroup of tumors in Arf(-/-) animals presented as dedifferentiated and metastatic, with many characteristics of pulmonary sarcomatoid carcinoma, a neoplasm previously undocumented in mouse models. Our finding of a role for ARF in NSCLC is consistent with the observation that benign adenomas from Arf(+/+) mice robustly expressed ARF, while ARF expression was markedly reduced in malignant adenocarcinomas. ARF expression also frequently colocalized with the expression of p21(CIP1), a transcriptional target of p53, arguing that ARF induces the p53 checkpoint to arrest cell proliferation in vivo. Taken together, these findings demonstrate that induction of ARF is an early response in lung tumorigenesis that mounts a strong barrier against tumor growth and malignant progression.
- Published
- 2013
10. Abstract C15: Neutrophil elastase (NE) induces epithelial-mesenchymal transition (EMT) via upregulation of inhibitor of DNA binding 1 (ID1)
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A. McGarry Houghton, Heather E. Metz, Julia Kargl, Stephanie E. Busch, Hye Youn Yang, and Alyssa D. Gregory
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Cancer Research ,chemistry.chemical_compound ,Oncology ,Biochemistry ,chemistry ,biology ,Downregulation and upregulation ,Neutrophil elastase ,biology.protein ,Epithelial–mesenchymal transition ,DNA ,Cell biology - Abstract
Neutrophil elastase is a neutrophil-derived serine protease with broad substrate specificity. We have previously shown that NE promotes lung tumor growth in vitro and in vivo and that NE is capable of entering tumor cell endosomes in a clathrin-dependent fashion. Epithelial-mesenchymal transition (EMT) is a fundamental event for primary tumors to metastasize to distant sites. Altered gene and protein expression causes changes in cell morphology and behavior resulting in the loss of attachment to neighboring cells, intravasation, and migration into distant tissue. However, the entities residing within the tumor microenvironment that drive EMT are poorly understood. We identified NE, expressed and released by tumor-associated neutrophils, as such an entity. We found that NE induces EMT-like morphology and behavior in several cancer cell lines as well as upregulates genes (Snail and Twist) and proteins (N-cadherin, α-SMA, Fibronectin and Vimentin) associated with EMT. Furthermore, NE provokes tumor cell migration and invasion in vitro and is required for metastasis formation in vivo. Besides known EMT markers we identified significant upregulation of inhibitor of DNA binding 1 (ID1) in tumor cells in the presence of NE. Increased ID1 expression was previously shown to correlate with tumor progression and metastasis. Clathrin-dependent NE internalization is required for both ID1 expression and the EMT-like phenotype. We localized NE in different compartments of tumor cells and found it predominantly in the nuclear fraction; more specifically, NE is located in the chromatin-bound fraction, indicating a potential role in chromatin remodeling and transcriptional regulation. Additionally, the proteolytic activity of NE was measured in the nuclear and chromatin-bound fractions and is required for induction of ID1 expression and EMT-like behavior. Taken together, these data suggest the importance of NE-induced ID1 expression in metastasis formation. Citation Format: Julia Kargl, Alyssa Gregory, Hye Youn Yang, Stephanie Busch, Heather Metz, A. McGarry Houghton. Neutrophil elastase (NE) induces epithelial-mesenchymal transition (EMT) via upregulation of inhibitor of DNA binding 1 (ID1). [abstract]. In: Proceedings of the AACR Special Conference: Function of Tumor Microenvironment in Cancer Progression; 2016 Jan 7–10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2016;76(15 Suppl):Abstract nr C15.
- Published
- 2016
11. Abstract B26: Novel pro-host role for insulin receptor substrate-1 in lung adenocarcinoma
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Stephanie E. Busch, McGarry Houghton, Heather E. Metz, Kyoung-Hee Kim, and Julia Kargl
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Cancer Research ,biology ,business.industry ,Cancer ,Context (language use) ,medicine.disease ,medicine.disease_cause ,IRS1 ,Insulin receptor ,Oncology ,Immunology ,medicine ,biology.protein ,Cancer research ,Adenocarcinoma ,KRAS ,Lung cancer ,business ,Survival rate - Abstract
Lung cancer is the leading cause of cancer deaths worldwide. In the United States alone, lung cancer accounts for ~160,000 deaths per year while the five-year survival rate remains stagnant at ~15%. Lung adenocarcinoma (ADCA) accounts for over 50% of non-small lung cancer cases. Within this subset, K-ras is the most common activating mutation accounting for ~35% of cases. Kras mutant tumors have remained a largely un-targetable subtype and a better understanding of the signaling pathway involved in the different ADCA subtypes will be necessary for the development of therapeutics. Insulin Receptor Substrate-1 (IRS-1) is a signaling adaptor protein that interfaces with many of the pathways that are activated in lung cancer. We have undertaken a controlled study of this protein within the context of lung ADCA. Analysis of a well-annotated human lung ADCA TMA (n=136) revealed that positive IRS-1 staining provided a 75-month median survival advantage among all ADCA cases (p=0.01) and an 81-month median advantage within the K-ras subtype (p=0.006). EGFR and non-K-ras/non-EGFR cases did not display differences. To focus in on the K-ras subtype, we generated LSL-K-ras/IRS-1fl/fl mice and studied them over a time course. LSL-K-ras/IRS-1-/- mice displayed highly significant early mortality (p Citation Format: Heather E. Metz, Julia Kargl, Stephanie Busch, Kyoung-Hee Kim, McGarry Houghton. Novel pro-host role for insulin receptor substrate-1 in lung adenocarcinoma. [abstract]. In: Proceedings of the AACR Special Conference: Function of Tumor Microenvironment in Cancer Progression; 2016 Jan 7–10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2016;76(15 Suppl):Abstract nr B26.
- Published
- 2016
12. Abstract PR04: Inflammatory responses within the lung tumor microenvironment correlate with oncogenic mutation and histologic subtype
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Heather E. Metz, Kyoung-Hee Kim, Mark L. Hanke, Julia Kargl, Stephanie E. Busch, and A. McGarry Houghton
- Subjects
Cancer Research ,Tumor microenvironment ,Myeloid ,business.industry ,Cancer ,medicine.disease ,medicine.disease_cause ,respiratory tract diseases ,medicine.anatomical_structure ,Immune system ,Oncology ,Tumor progression ,Immunology ,medicine ,Cancer research ,Small Cell Lung Carcinoma ,KRAS ,business ,Lung cancer - Abstract
Lung cancer is a heterogeneous disease comprised of multiple histologic subtypes that harbor disparate mutational landscapes. Small cell lung carcinoma (SCLC), in which RB1 and TP53 mutations are common, accounts for 10-20% of lung cancer diagnoses. Approximately half of non-small cell lung carcinoma cases are classified as lung adenocarcinomas (ADCA), in which KRAS, EGFR, and TP53 mutations are the predominant genetic drivers. Novel immunotherapeutic strategies have offered new hope for the management of ADCA and SCLC, but the clinical success of immunomodulatory agents will depend on a strong foundational knowledge of the cells that comprise the lung tumor microenvironment (TME) in these molecularly and histologically distinct diseases. We therefore sought to determine whether the host immune response to lung cancer is predicated, at least in part, by histologic and genetic differences, as such correlations would have important clinical ramifications. Using three mouse models of lung ADCA KrasLSL-G12D, KrasLSL-G12D;Trp53Fl/Fl, and EgfrL858R as well as the Rb1Fl/Fl;Trp53Fl/Fl model of SCLC, we show that SCLC and ADCA tumors exhibit unique immune cell composition. Lung specimens from tumor-bearing and control animals (n ≥ 5 per cohort) were harvested at multiple time points and subjected to histological assessment and comprehensive flow cytometric immunophenotyping. Tumor-associated inflammation was discernibly lower in SCLC compared to all ADCA models. Moreover, the leukocyte composition of SCLC was dominated by cells of the adaptive rather than innate arm of the host immune system, a finding subsequently validated in 2 human SCLC surgical specimens. We further identified key differences in leukocyte content of mouse ADCA that correlated with oncogenic mutation. Although Egfr-mutant cancers displayed robust myeloid cell recruitment, they failed to mount a CD8+ cellular immune response. In contrast, Kras-mutant tumors displayed significant expansion of multiple immune cell types, including CD8+ cells, regulatory T cells, IL17-producing lymphocytes, and myeloid cells. Although loss of Trp53 promoted malignancy, it had minimal effect on immune cell composition within the Kras TME. Pre-clinical in vivo investigations with immune checkpoint inhibitor compounds are currently being conducted to assess the importance of these distinct immune responses to tumor progression and survival. To determine whether the differential inflammatory responses observed in mice are clinically relevant, we conducted an immune profiling study of a large lung cancer patient cohort that included 55 ADCA specimens. Notably, patients with EGFR mutations (n = 5) exhibited fewer total CD8+ T cells, as well as decreased exhausted CD8+PD1+ and CD8+TIM3+ content, compared to all other ADCA cases, thereby substantiating the murine phenotype. Taken together, these findings establish a strong foundational knowledge of the immune cell contexture of lung ADCA and SCLC and suggest that molecular and histological traits shape the host immune response to cancer. This abstract is also presented as Poster A01. Citation Format: Stephanie E. Busch, Julia Kargl, Mark L. Hanke, Heather E. Metz, Kyoung-Hee Kim, A McGarry Houghton. Inflammatory responses within the lung tumor microenvironment correlate with oncogenic mutation and histologic subtype. [abstract]. In: Proceedings of the AACR Special Conference: Function of Tumor Microenvironment in Cancer Progression; 2016 Jan 7–10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2016;76(15 Suppl):Abstract nr PR04.
- Published
- 2016
13. ARF suppresses hepatic vascular neoplasia in a carcinogen-exposed murine model
- Author
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Stephanie E, Busch, Kay E, Gurley, Russell D, Moser, and Christopher J, Kemp
- Subjects
Mice, Knockout ,Time Factors ,Hemangiosarcoma ,Liver Neoplasms ,Gene Dosage ,Urethane ,Article ,Mice, Inbred C57BL ,Mice ,Cell Transformation, Neoplastic ,Phenotype ,Carcinogens ,Animals ,Genetic Predisposition to Disease ,Tumor Suppressor Protein p53 ,Cyclin-Dependent Kinase Inhibitor p16 ,Signal Transduction - Abstract
Hepatic haemangiosarcoma is a deadly malignancy whose aetiology remains poorly understood. Inactivation of the CDKN2A locus, which houses the ARF and p16(INK4a) tumour suppressor genes, is a common event in haemangiosarcoma patients, but the precise role of ARF in vascular tumourigenesis is unknown. To determine the extent to which ARF suppresses vascular neoplasia, we examined the incidence of hepatic vascular lesions in Arf-deficient mice exposed to the carcinogen urethane [intraperitoneal (i.p.), 1 mg/g]. Loss of Arf resulted in elevated morbidity and increased the incidence of both haemangiomas and incipient haemangiosarcomas. Suppression of vascular lesion development by ARF was heavily dependent on both Arf gene-dosage and the genetic strain of the mouse. Trp53-deficient mice also developed hepatic vascular lesions after exposure to urethane, suggesting that ARF signals through a p53-dependent pathway to inhibit the development of hepatic haemangiosarcoma. Our findings provide strong evidence that inactivation of Arf is a causative event in vascular neoplasia and suggest that the ARF pathway may be a novel molecular target for therapeutic intervention in haemangiosarcoma patients.
- Published
- 2011
14. Abstract 2354: Insulin receptor substrate-1 regulates immune cell content in lung adenocarcinoma
- Author
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Stephanie E. Busch, Julia Kargl, Mark L. Hanke, Kyoung-Hee Kim, A. McGarry Houghton, and Heather E. Metz
- Subjects
Cancer Research ,Lung ,biology ,business.industry ,Cancer ,medicine.disease ,IRS1 ,Insulin receptor ,medicine.anatomical_structure ,Immune system ,Oncology ,medicine ,biology.protein ,Cancer research ,Adenocarcinoma ,Lung cancer ,business ,Survival rate - Abstract
Lung cancer is the leading cause of cancer deaths worldwide. In the United States, lung cancer accounts for ∼160,000 deaths per year while the five-year survival rate remains stagnant at ∼15%. A better understanding of the pathobiology for this disease is imperative for development of novel therapeutics to improve mortality rates. Lung adenocarcinoma (ADCA) accounts for over 50% of non-small lung cancer cases. Within this subset, K-ras is the most common activating mutation accounting for ∼35% of cases. Our group previously showed that K-ras activation generates an inflammatory response in lung tumors, mediated largely through the increased production of CC/CXC chemokines by tumor cells. The corresponding influx of neutrophils increased tumor growth, which was associated with degradation of the intracellular protein insulin receptor substrate-1 (IRS-1). Since IRS-1 is generally thought of as a pro-tumor entity, we decided to undertake a controlled study to further elucidate its pro-host role. We performed a study using a well-annotated human lung ADCA TMA (n = 136) and found that positive IRS-1 staining provided a 75-month median survival advantage among all cases (p = 0.01) and an 81-month median advantage within the K-ras subtype (p = 0.006). EGFR and non-K-ras/non-EGFR cases did not display differences. To further dissect the role of IRS-1 in tumor growth we generated LSL-K-ras/IRS-1fl/fl mice and studied them over a time course. LSL-K-ras/IRS-1-/- mice displayed highly significant early mortality (p Citation Format: Heather Metz, Stephanie E. Busch, Julia Kargl, Mark L. Hanke, Kyoung-Hee Kim, A. McGarry Houghton. Insulin receptor substrate-1 regulates immune cell content in lung adenocarcinoma. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 2354. doi:10.1158/1538-7445.AM2015-2354
- Published
- 2015
15. Abstract 3181: EGFR and KRAS activation generate discrete inflammatory responses within the lung tumor microenvironment
- Author
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Julia Kargl, Kyoung Hee Kim, Stephanie E. Busch, Mark L. Hanke, and A. McGarry Houghton
- Subjects
Oncology ,Cancer Research ,medicine.medical_specialty ,business.industry ,Internal medicine ,medicine ,Cancer research ,Lung tumor ,KRAS ,business ,medicine.disease_cause - Abstract
Despite decades of research, non-small cell lung carcinoma (NSCLC) remains one of the world's deadliest cancers. Approximately half of NSCLC cases are classified as lung adenocarcinomas (ADCA), in which KRAS, EGFR, and TP53 mutations are the predominant genetic drivers. Although patients with EGFR mutations are initially responsive to targeted therapies, drug resistance uniformly occurs within one year. KRAS mutant ADCA have proven even more intractable, as scant progress has been made in the development of therapeutics for this patient population. Recent breakthroughs in the field of immunotherapy have offered new hope for the management of NSCLC, but the clinical success of immunomodulatory agents will depend on a strong foundational knowledge of the immune cells that comprise the ADCA microenvironment. Activating mutations in KRAS and EGFR induce the aberrant function of numerous signaling pathways that may influence the host immune response in different ways. However, the discrete impact of KRAS and EGFR mutations on the immune composition of ADCA remains largely undefined. To address this question, we profiled the tumor microenvironments (TME) of four mouse models of ADCA: adenoviral Cre-infected KrasLSL-G12D and KrasLSL-G12D;Trp53Fl/Fl mice, as well as TetO-EgfrL858R;Ccsp-rtTA and TetO-EgfrL858R;T790M;Ccsp-rtTA mice. Lung specimens from tumor-bearing and control animals (n ≥ 5 per cohort) were harvested at multiple time points and subjected to histological assessment, gene expression analysis by qRT-PCR, and flow cytometric immunophenotyping, in which 23 markers were used to identify 12 unique cell types. The Kras subtype exhibited robust inflammation and expansion of both the lymphoid and myeloid cell compartments. In particular, significant increases were observed in the number of CD8+ T cells (3-fold), B cells (2.5-fold), and macrophages (16-fold). Although loss of Trp53 promoted tumor growth and malignancy, it had little effect on cytokine expression or immune cell recruitment into the Kras TME. In contrast, while EgfrL858R mutations produced similar total levels of inflammation, leukocyte recruitment into the Egfr TME was largely restricted to macrophage expansion (increased 8-fold). Introduction of the T790M resistance mutation to Egfr-mutant animals further amplified the macrophage dominant signal (14-fold). Expression of the cytokine IL-6 was correspondingly elevated in the Egfr but not Kras subtypes, a finding replicated in EGFR-transfected primary human endobronchial cells. Most notably, CD8+ cytotoxic T cell numbers were unaltered at all time points examined in the Egfr animals, a finding which raises concerns about the efficacy of most currently developed immune checkpoint blockers in EGFR patients. In summary, KRAS and EGFR mutations give rise to distinct immune responses that may require oncogene-specific immunotherapy regimens. Citation Format: Stephanie E. Busch, Mark L. Hanke, Kyoung Hee Kim, Julia Kargl, A. McGarry Houghton. EGFR and KRAS activation generate discrete inflammatory responses within the lung tumor microenvironment. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 3181. doi:10.1158/1538-7445.AM2015-3181
- Published
- 2015
16. Abstract 1290: Distinct immune cell composition for lung squamous cell carcinoma (SCCA) and adenocarcinoma (ADCA)
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Heather E. Metz, Stephanie E. Busch, Julia Kargl, A. McGarry Houghton, Martin W. McIntosh, Kyoung-Hee Kim, and Mark L. Hanke
- Subjects
Cancer Research ,Cell type ,Tumor microenvironment ,biology ,CD3 ,Cell ,Cancer ,medicine.disease ,Immune system ,medicine.anatomical_structure ,Oncology ,Immunology ,medicine ,biology.protein ,Adenocarcinoma ,Lung cancer - Abstract
Lung cancer is the leading cause of cancer deaths worldwide, with ∼15% of lung cancer patients surviving for five years. Recent approaches in cancer therapy target immunosuppressive factors in the tumor microenvironment (TME) capable of derailing an effective immune response, using checkpoint inhibitors (e.g. anti-PD-1/PDL-1). Unfortunately, only ∼20% of patients benefit from these treatments and appropriate studies are of importance to determine the underlying molecular mechanisms. Lung cancer is a heterogeneous disease classified by multiple histologic subtypes, with ADCA and SCCA representing the majority of non-small cell lung cancer (NSCLC). To evaluate the complexity of the immune cell composition of the TME of human NSCLC we examined tumor and non-adjacent lung tissue from the same patients. Tissue specimen (n = 43) with corresponding clinicopathologic information were analyzed by flow cytometry and immunohistochemistry. Next generation sequencing for TCRs was performed to assess the TCR repertoire. Surprisingly we found that immune cells comprise ∼80% of the tumor mass in NSCLC. Many immune cell types are significantly increased in tumor tissue, when compared to normal lung, such as CD3+ and CD4+ T-cells, as well as B-cells. Notably, CD4+ cell subtypes including Th17 and regulatory T cells (Treg), and IL-17-expressing γδ T cells are increased. In contrast, decreased presence of Th1 cells was observed in tumor tissue. Remarkably, immune cell compositions are unique for lung ADCA and SCCA. In SCCA we observed a Treg (p In contrast, ADCA represents a Th17 signature, revealing elevated levels of IL17-expressing CD4+ T-cells and γδ T cells. While SCCA immune cell content clusters well, immune cell composition in ADCA is very heterogeneous. Preliminary data suggest that driver-mutations in ADCA such as EGFR predict distinct immune cell composition. These data provide evidence that the immune cell composition present within different NSCLC subtypes displays unique phenotypes and identifying the immunosuppressive factors in different subsets will be important for successful immune-based therapy. Citation Format: Julia Kargl, Kyoung-Hee Kim, Stephanie E. Busch, Mark L. Hanke, Heather E. Metz, Martin W. McIntosh, A McGarry Houghton. Distinct immune cell composition for lung squamous cell carcinoma (SCCA) and adenocarcinoma (ADCA). [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 1290. doi:10.1158/1538-7445.AM2015-1290
- Published
- 2015
17. Abstract 3182: Reduced tumor burden and mortality in IL-17RA-deficient EGFR mutant mice
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Julia Kargl, Kyoung Hee Kim, Stephanie E. Busch, Mark L. Hanke, and A. McGarry Houghton
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Cancer Research ,Oncology ,business.industry ,Mutant ,Cancer research ,Tumor burden ,Medicine ,business - Abstract
Lung cancer is the leading cause of cancer death in the United States, with the five-year survival rates currently around 15%. Resistance to cancer therapies, like those that target epidermal growth factor receptor (EGFR), is an important clinical problem. The most common mechanism of resistance to targeted therapies in EGFR-mutant lung cancer is the T790M mutation. Given the lack of efficacy of the targeted therapy combinations tested so far, the ideal partner to an EGFR targeted therapy may be a drug that acts by an entirely different mechanism, possibly one that targets the tumor microenvironment. To dissect the role of the tumor microenvironment in lung cancer pathology we utilized mice engineered to express the L858R activating mutation within EGFR (TetO-EgfrL858R;Ccsp-rtTA) or the T790M resistance mutation (TetO-EgfrL858R/T790M;Ccsp-rtTA). Previous studies from our group indicate that KRAS mutant NSCLC is associated with a robust inflammatory response characterized by infiltrating immune cells, particularly macrophages and neutrophils. Neutrophils, which require IL-17 for recruitment and T cell suppression, promote tumor growth; and the infiltration of Th17 cells in human cancers is associated with a poor prognosis, making IL-17 an attractive therapeutic target for NSCLC. Activation of the L858R or the T790M Egfr mutation within the lung was characterized by an abundant infiltration of alveolar macrophages, 8- and 14-fold increases respectively. In contrast, the number of neutrophils and CD8 T cells remained relatively unchanged after the expression of either Egfr mutation. In addition, the few CD8 T cells that were present within the tumor microenvironment appeared to exhibit a more exhausted phenotype within mice expressing the T790M mutation. The lack of expanded CD8 T cell number was also observed in a limited number of human EGFR lung specimens (n = 3). EGFR activation did however result in amplified numbers of both T regulatory cells and IL-17 producing T cells. Increased expression of the IL-17 differentiating cytokines TGF-β and IL-6, as well as the Th17 proliferative cytokine IL-1β was also observed by qRT-PCR gene expression analysis within L858R EGFR mutant mice. To further dissect the role of IL-17 in tumor growth TetO-EgfrL858R;Ccsp-rtTA mice were crossed to IL-17RA deficient mice. IL-17RA deficient EGFR mutant mice displayed enhanced survival and decreased tumor burden when compared to IL-17RA sufficient controls. IL-17RA loss also resulted in a less robust immune response within EGFR mutant mice as the bronchial alveolar lavage fluid showed decreased infiltration of macrophages, neutrophils and lymphocytes. Further examination of EGFR mutant lung tissue displayed alterations in immune cell-recruiting chemokines and cytokines after the loss of IL-17RA. Taken together, our data demonstrate a role for IL-17 in EGFR mediated lung tumorigenesis and suggest the use of IL-17 antagonism as a possible therapy for EGFR patients. Citation Format: Mark L. Hanke, Stephanie E. Busch, Julia Kargl, Kyoung Hee Kim, A McGarry Houghton. Reduced tumor burden and mortality in IL-17RA-deficient EGFR mutant mice. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 3182. doi:10.1158/1538-7445.AM2015-3182
- Published
- 2015
18. Abstract 4860: Insulin receptor substrate-1 regulates immune cell content in lung adenocarcinoma
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Mark L. Hanke, A. McGarry Houghton, Heather E. Metz, Stephanie E. Busch, Julia Kargl, and Kyoung-Hee Kim
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Cancer Research ,Chemokine ,biology ,business.industry ,medicine.disease ,IRS1 ,CXCL1 ,CXCL2 ,Immune system ,Oncology ,Immunology ,medicine ,biology.protein ,STAT protein ,Signal transduction ,Lung cancer ,business - Abstract
Lung cancer is the leading cause of cancer deaths worldwide. In the United States, lung cancer accounts for ∼160,000 deaths per year while the five-year survival rate remains stagnant at ∼15%. A better understanding of the pathobiology for this disease is imperative for development of novel therapeutics to improve mortality rates. One of the most common activating mutations in lung cancer is K-ras. Our group previously showed that K-ras activation generates an inflammatory response in lung tumors, mediated largely through the increased production of neutrophil chemokines by tumor cells. The corresponding increase in neutrophil recruitment in this model increased tumor growth, which was associated with degradation of the intracellular protein insulin receptor substrate-1 (IRS-1). A study of non-small cell lung cancer demonstrated that IRS-1 was low or absent in 46% of cases. To further dissect the role of IRS-1 in tumor growth we generated LSL-K-ras/IRS-1 fl/fl mice and subjected them to adenoviral cre. LSL-K-ras/IRS-1-/- mice displayed early mortality and increased tumor burden when compared to LSL-K-ras/IRS-1+/+ controls. Surprisingly, IRS-1 loss in tumor cells generated a robust immune response. The bronchial alveolar lavage fluid of LSL-K-ras/IRS-1-/- mice showed increased infiltration of macrophages, neutrophils and lymphocytes. Further examination of the chemokine profiles of LSL-K-ras/IRS-1-/- and LSL-K-ras/IRS-1+/+ mice showed a significant increase in many immune cell-recruiting chemokines in the LSL-K-ras/IRS-1-/- mice including CCL2, CCL3, CCL4, CXCL1, CXCL2 and CXCl12. Many cancers are able to manipulate the host immune response through signaling pathways that interface with IRS-1, including: phosphoinositol 3-kinase (PI3K), extracellular signal regulated kinase (MEK/ERK) and Janus kinase/signal transducer and activator of transcription (JAK/STAT). As some cytokines can activate IRS-1 via JAK/STAT signaling, we suspected that IL-17 and IL-22 might function in this regard. Loss of IRS-1 increases pSTAT3 production, which triggers chemokine release and the generation of a pro-tumor immune response. Preliminary data to support this hypothesis include: 1) presence of IL-17 and IL-22 producing cells (e.g. Th17) at sites of tumor in LSL-K-ras mice, 2) induction of pTyr-IRS-1 upon IL-17 and IL-22 stimulation in A549 cells, and 3) increased CC and CXC chemokines in IRS-1 deficient cells upon IL-17 and IL-22 stimulation. Experimentation is ongoing to further elucidate the mechanism behind this phenotype. Citation Format: Heather E. Metz, Stephanie E. Busch, Mark L. Hanke, Julia Kargl, Kyoung-Hee Kim, A McGarry Houghton. Insulin receptor substrate-1 regulates immune cell content in lung adenocarcinoma. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 4860. doi:10.1158/1538-7445.AM2014-4860
- Published
- 2014
19. Abstract A34: Arf suppresses the growth and progression of Kras-driven lung tumors
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Christopher J. Kemp, Kay E. Gurley, Karen S. Kelly-Spratt, Russell Moser, and Stephanie E. Busch
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Cancer Research ,Pathology ,medicine.medical_specialty ,Lung ,business.industry ,Cell ,medicine.disease ,medicine.disease_cause ,respiratory tract diseases ,medicine.anatomical_structure ,Oncology ,CDKN2A ,medicine ,Carcinoma ,Cancer research ,Tumor promotion ,KRAS ,Carcinogenesis ,business ,Carcinogen - Abstract
Non-small cell lung carcinoma (NSCLC) is among the deadliest of human cancers, with an overall five-year survival rate of 10–20%. Activating mutations in the Kras proto-oncogene occur in approximately one-third of NSCLC patients, but tumor suppression in the lung remains poorly elucidated. The Cdkn2a locus, which houses both the p16lnk4a and Arf tumor suppressor genes, is frequently altered in NSCLC; however, the specific role of Arf in lung tumorigenesis has not been well characterized. Several lines of evidence indicate that Kras and other oncogenes induce the expression of Art, which in turn stabilizes p53 activity and arrests the growth of transformed cells. To determine the extent to which Arf suppresses NSCLC development in vivo, we employed the urethane chemical carcinogenesis model of Kras-driven NSCLC. We treated both wild-type and Arf-deficient mice with urethane and found that early-stage lung adenomas in wild-type mice robustly expressed Arf, In contrast to adenomas, malignant lung adenocarcinomas exhibited little induction of Art. Moreover, Arf-deficient mice exposed to urethane displayed accelerated lung tumor-associated morbidity and significantly increased lung tumor size compared to Arf wild-type and heterozygous littermates. Malignant progression of adenomas to adenocarcinomas was also markedly elevated in Arf-deficient mice. These data demonstrate that Arf functions as a barrier to tumor promotion and progression in a carcinogen model of Kras-driven NSCLC. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the Second AACR International Conference on Frontiers in Basic Cancer Research; 2011 Sep 14-18; San Francisco, CA. Philadelphia (PA): AACR; Cancer Res 2011;71(18 Suppl):Abstract nr A34.
- Published
- 2011
20. Maternal activation of gap genes in the hover fly Episyrphus
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Steffen Lemke, Stephanie E. Busch, Dionysios A. Antonopoulos, Folker Meyer, Marc H. Domanus, and Urs Schmidt-Ott
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Molecular Biology ,Developmental Biology - Published
- 2010
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