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S100A14 increases tumorigenesis in triple-negative breast cancer
EACR24 Poster Sessions / European Journal of Cancer 61, Suppl. 1 (2016) S9–S218 on glioma stemness properties and invasion. Clinical relevance of CD109 expression was investigated using glioma tumor microarrays. Results and Discussion: Based on tumor microarray analysis, CD109 expression is grade-dependent in gliomas with a trend toward poor patient survival. 90% of GBMs expressed CD109 while 80% of grade III and only 50% of grade II were positive. Western blot analysis showed high expression of CD109 in patient-derived GSC-like cell lines. Silencing of CD109 expression induced apparent morphological changes in gliospheres, decreased glioma stem cell marker expression, and self-renewal ability of the cells indicating association between CD109 and glioma cell stemness. Moreover, CD109 expressing GSC-like cells were highly tumorigenic and invasive in vivo. Importantly, immunofluorescence staining showed CD109 expression at the invasive front of the xenograft tumors. Conclusions: Our results demonstrate that CD109 is an important mediator of the tumorigenic properties in GBM. No conflict of interest. 421 S100A14 increases tumorigenesis in triple-negative breast cancer S. Ehmsen1 , K. Nørgaard2 , H.J. Ditzel1 , R. Leth-Larsen3 . 1 Molecular medicin, Cancer and inflammation research, Odense, Denmark, 2 Biochemistry and Molecular biology, Biochemistry and Molecular biology, Odense M, Denmark, 3 Institute of Regional Health Sciences, Institute of Regional Health Sciences, Odense C, Denmark Introduction: Breast cancer is the main cause of cancer-related mortality in women worldwide, in which the triple-negative breast cancer (TNBC (ER-/PR/HER2normal)) subtype accounts for about 10% of all breast cancers, and is often aggressive and associated with a higher mortality within the first 5 years than other subtypes of breast cancer. S100A14, a member of the S100 protein family, is a protein previously demonstrated to have prognostic value of metastatic breast cancer in patients with TNBC. However little is known about the cellular functional roles played by S100A14. Material and Methods: Stable knock down of S100A14 and bioluminescence labeling of two epithelial, S100A14 high expressing TNBC cell lines, HMT3909S13-AB and MDA-MB-468 were performed using shRNAs. The influence of S100A14 in tumorigenesis and in the metastatic process was elucidated by inoculation of those cells into either the fat pad or the tail vein of immune deficient NOG mice. In vitro growth rate was investigated using crystal violet assay and apoptosis level was investigated using Cell Death Detection ELISAPLUS kit (Roche). Results and Discussion: In this study we made a panel of cell lines in with stable knockdown of S100A14, with up to 95% knockdown efficiency. As S100A14 previously has been implicated to play a role in proliferation and apoptosis, we investigated the growth rate and apoptosis level in the stable knockdown cell lines. We found that S100A14 promotes cell proliferation and decreases apoptosis of TNBC. Metastasis is the main cause of cancer-related death in patients, and we have previously shown that human TNBCs with high S100A14 protein expression have shorter overall survival compared to the S100A14 low group. It was therefore inevitably to hypothesize that S100A14 is involved in the metastatic process. We found that high S100A14 expression is vital for cells to survive, proliferate, form micrometastasis and colonize after extravasation. Further, S100A14s role in primary tumor formation in fat pad was investigated. Supporting the metastasis results, S100A14 was found to promote primary tumor formation and increase tumorigenesis. Together the in vivo results complement our previous findings in TNBC patients that high protein expression of S100A14 is correlated with poor outcome, highlighting the clinical relevance of our recent findings. Conclusions: We found that high S100A14 expression promotes primary tumor formation and increases the colonization and tumor burden in lungs. Further we found that S100A14 increases proliferation with a subsequent decrease in the level of apoptosis of TNBC cells in vitro. TNBC patients with high expression of S100A14 could potentially benefit from therapeutic targeting of S100A14 to inhibit disseminated tumor cells to grow to fulminant metastasis. No conflict of interest. 422 Cell-of-origin links histotype spectrum to immune microenvironment diversity in non-small cell lung cancer driven by mutant Kras and loss of Lkb1 A. Nagaraj1 , J. Lahtela1 , A. Hemmes1 , M. Mayr ¨ anp ¨ a¨ a¨ 2 , K. Salmenkivi2 , K. Narhi ¨ 1 , E.W. Verschuren1 . 1 Institute for Molecular Medicine Finland FIMM, University of Helsinki, Helsinki, Finland, 2 Department of Pathology, Helsinki University Central Hospital and University of Helsinki, Helsinki, Finland Introduction: Lung cancers exhibit pronounced histological and genomic heterogeneity, confounding precision medicine. Hence, we addressed the need for deeper understanding of the lung cancer-related niche and progenitor cell-specific properties that lead to molecular and microenvironmental tumour heterogeneity.
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Material and Methods: We studied how the cell-of-origin contributes to phenotypic and immune microenvironment heterogeneity following conditional expression of KrasG12D and loss of Lkb1 (Kras;Lkb1) in murine lungs. Cohorts of nine mice were infected with progenitor cell type-restricted adenoviral-Cre viruses targeting cells expressing Clara cell antigen 10 (CC10) or Surfactant Protein C (SPC). Cohorts were followed for signs of laboured breathing, and survival curve analyses were done. Complete lung tissues were harvested, followed by a detailed histopathological and biomarker tissue-level analysis to investigate differential transformation propensities of cells in the airways or distal alveolar space. A transcriptome analysis of the predominant tumour histopathology types arising from the airways and distal alveolar space was performed. Flow cytometry and immunohistochemistry-based analyses of tumour immune cell infiltrates were done to assess histopathology-specific immune microenvironment phenotypes. Results and Discussion: Our results show that Ad5-CC10-Cre infected mice exhibit a shorter latency compared with Ad5-SPC-Cre cohorts. We further demonstrate that CC10+ cells are the predominant progenitors of adenosquamous carcinoma (ASC) tumors, and give rise to a wider spectrum of histotypes which uniquely include mucinous and acinar invasive adenocarcinomas. ASCs show histotype-specific downregulation of Class I MHC genes, and upregulation of pro-inflammatory and immunomodulatory genes. ASC-specific immunosuppression is evidenced by recruitment of Gr-1+CD11b+ myeloid derived suppressor cells and decreased CD3+ T cell infiltration. Conclusion: We conclude that progenitor cell-specific etiology influences the Kras;Lkb1-driven tumor histopathology spectrum and histotype-specific immune microenvironment. Given the increasing appreciation of the role of an immunosuppressive microenvironment during human tumour progression and metastasis, the Kras;Lkb1-driven ASC model may represent a valuable preclinical model for the study of anti-MDSC immune therapy as a treatment for ASCs, which in humans represents a rare but aggressive type of lung cancer. No conflict of interest. 423 Visualizing ZO-1-mediated coupled migration between cancer and stromal cells in lung cancer metastasis via a 5-dimensional cell imaging system H.H. Lu1 , B.C. Chen2 , C.C. Ho1 , H.W. Chen3 , C.J. Yu1 . 1 National Taiwan University Hospital, Department of Internal Medicine, Taipei, Taiwan, 2 Academia Sinica, Research Center for Applied Science, Taipei, Taiwan, 3 National Taiwan University- College of Medicine, Institute of Toxicology, Taipei, Taiwan Background: A long-standing question concerns how cancer cells efficiently travel from primary tumor to distant metastases. The unique roles of microenvironment are also been studied, including cancer associated fibroblasts (CAFs), tumor associated macrophages, and endothelial cells. However, the initiation stage of metastasis is still unknown. In this study, we present the crucial role of ZO-1 in coupled migration regulated by CAFs in lung cancer metastasis. Methods: CD90+ CAFs were isolated from tumor tissue of lung cancer patients. A matrigel-based 3-dimentional (3-D) co-culture system was established to study the interaction between cancer cells and CAFs in vitro. To further investigate the coupled migration between cancer cells and CAFs, we used lattice light-sheet microscopy system to perform a rapid time-lapse imaging system, including three spatial-temporal model, and the excitation and imaging of several spectrally separated fluorophore labels i.e. 5D to observe the real-time motion and cell-cell coordinated migration. Results: Lung cancer cell lines and CAFs were co-cultured in the 3-D system. Cancer cells and CAFs could form a systemic structure in a very short time point (~4 h) through the direct contact interaction. Via confocal microscopy and lattice light-sheet microscopy, we found there were bridge-like structures formed by CAFs, and CAFs would carry cancer cells to move together as the coupled migration. Furthermore, CAFs not only significantly promoted invasion ability of cancer cells in in vitro assay (>4 folds), but also enhanced cancer metastasis in orthotopic injection model (>3 folds). ZO-1 in CAFs seems play a pivotal role in the coupled migration of metastasis processes. Knockdown of ZO-1 in CAFs resulted in dramatically cutback the interaction between cancer cells and CAFs. Conclusions: These results propose that CAFs play imperative roles in the initiation stage of cancer metastasis via coupled migration and the direction of movement. It also implies that the interaction between cancer cells and CAFs might be a novel treatment strategy in cancer metastasis by targeting the direct contact components between cancer cells and CAFs. No conflict of interest.
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Material and Methods: We studied how the cell-of-origin contributes to phenotypic and immune microenvironment heterogeneity following conditional expression of KrasG12D and loss of Lkb1 (Kras;Lkb1) in murine lungs. Cohorts of nine mice were infected with progenitor cell type-restricted adenoviral-Cre viruses targeting cells expressing Clara cell antigen 10 (CC10) or Surfactant Protein C (SPC). Cohorts were followed for signs of laboured breathing, and survival curve analyses were done. Complete lung tissues were harvested, followed by a detailed histopathological and biomarker tissue-level analysis to investigate differential transformation propensities of cells in the airways or distal alveolar space. A transcriptome analysis of the predominant tumour histopathology types arising from the airways and distal alveolar space was performed. Flow cytometry and immunohistochemistry-based analyses of tumour immune cell infiltrates were done to assess histopathology-specific immune microenvironment phenotypes. Results and Discussion: Our results show that Ad5-CC10-Cre infected mice exhibit a shorter latency compared with Ad5-SPC-Cre cohorts. We further demonstrate that CC10+ cells are the predominant progenitors of adenosquamous carcinoma (ASC) tumors, and give rise to a wider spectrum of histotypes which uniquely include mucinous and acinar invasive adenocarcinomas. ASCs show histotype-specific downregulation of Class I MHC genes, and upregulation of pro-inflammatory and immunomodulatory genes. ASC-specific immunosuppression is evidenced by recruitment of Gr-1+CD11b+ myeloid derived suppressor cells and decreased CD3+ T cell infiltration. Conclusion: We conclude that progenitor cell-specific etiology influences the Kras;Lkb1-driven tumor histopathology spectrum and histotype-specific immune microenvironment. Given the increasing appreciation of the role of an immunosuppressive microenvironment during human tumour progression and metastasis, the Kras;Lkb1-driven ASC model may represent a valuable preclinical model for the study of anti-MDSC immune therapy as a treatment for ASCs, which in humans represents a rare but aggressive type of lung cancer. No conflict of interest. 423 Visualizing ZO-1-mediated coupled migration between cancer and stromal cells in lung cancer metastasis via a 5-dimensional cell imaging system H.H. Lu1 , B.C. Chen2 , C.C. Ho1 , H.W. Chen3 , C.J. Yu1 . 1 National Taiwan University Hospital, Department of Internal Medicine, Taipei, Taiwan, 2 Academia Sinica, Research Center for Applied Science, Taipei, Taiwan, 3 National Taiwan University- College of Medicine, Institute of Toxicology, Taipei, Taiwan Background: A long-standing question concerns how cancer cells efficiently travel from primary tumor to distant metastases. The unique roles of microenvironment are also been studied, including cancer associated fibroblasts (CAFs), tumor associated macrophages, and endothelial cells. However, the initiation stage of metastasis is still unknown. In this study, we present the crucial role of ZO-1 in coupled migration regulated by CAFs in lung cancer metastasis. Methods: CD90+ CAFs were isolated from tumor tissue of lung cancer patients. A matrigel-based 3-dimentional (3-D) co-culture system was established to study the interaction between cancer cells and CAFs in vitro. To further investigate the coupled migration between cancer cells and CAFs, we used lattice light-sheet microscopy system to perform a rapid time-lapse imaging system, including three spatial-temporal model, and the excitation and imaging of several spectrally separated fluorophore labels i.e. 5D to observe the real-time motion and cell-cell coordinated migration. Results: Lung cancer cell lines and CAFs were co-cultured in the 3-D system. Cancer cells and CAFs could form a systemic structure in a very short time point (~4 h) through the direct contact interaction. Via confocal microscopy and lattice light-sheet microscopy, we found there were bridge-like structures formed by CAFs, and CAFs would carry cancer cells to move together as the coupled migration. Furthermore, CAFs not only significantly promoted invasion ability of cancer cells in in vitro assay (>4 folds), but also enhanced cancer metastasis in orthotopic injection model (>3 folds). ZO-1 in CAFs seems play a pivotal role in the coupled migration of metastasis processes. Knockdown of ZO-1 in CAFs resulted in dramatically cutback the interaction between cancer cells and CAFs. Conclusions: These results propose that CAFs play imperative roles in the initiation stage of cancer metastasis via coupled migration and the direction of movement. It also implies that the interaction between cancer cells and CAFs might be a novel treatment strategy in cancer metastasis by targeting the direct contact components between cancer cells and CAFs. No conflict of interest.