Browsing by Subject "Tumor Microenvironment"
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Item Cooperative Invasion Between Tumor Cell Subpopulations(2013-01-17) Prechtl, Amanda Miya; Pearson, Gray W.Breast cancer is responsible for over 40,000 deaths each year in the United States. The majority of these deaths are not attributable to the primary breast tumor, but to metastases in vital organs. Tumor cell invasion is an early step in the metastatic cascade which can occur collectively by multiple cells cooperatively invading into the surrounding stroma. Primary patient breast tumors and patient-derived breast cancer cells can collectively invade yet how cells collectively invade is still largely unknown. It is well known that tumors contain heterogenous populations of cells yet traditional metastasis models focus on the ability of a rare population of neoplastic cells to autonomously invade past the basement membrane surrounding the tumor, intravasate into blood vessels and disseminate throughout the body to colonize foreign tissues. We hypothesized that there is a stable subpopulation of tumor cells that is capable of initiating the invasion of another population. Using organotypic culture models, which provide a three dimensional environment that models stromal conditions, and real-time imaging, a technique in which cell behavior can be imaged in real time at a single cell resolution, we determined that breast cancer cell lines can contain populations of cells with differential invasive potential. Furthermore, we concluded that one population of invasvie cells is sufficient to induce the invasion of other noninvasvie cells. This suggests a new mechanism for breast cancer metastasis, in which subpopulations of cells can cooperate with each other as opposed to competing against each other, to invade and potentially metastasize. Future studies will focus on determining the requirements for the leader cells to induce invasion and the follower cells to migrate behind the leader cells, with the eventual goal of targeting specific tumor populations for diagnostic and therapeutic treatment.Item Dual Mechanisms Regulating Alpha Subunit-Specific Activity in Hypoxia-Inducible Factor Signaling(2015-12-02) Nagati, Jason Sharif; Terada, Lance; Liu, Zhi-Ping; Yanagisawa, Hiromi; Munshi, Nikhil; Garcia, Joseph A.The ability to adapt to and protect from environmental stresses is essential to survival and has played a major role in fitness selection during evolution. As oxygen is essential to most life, many organisms have developed a response to conditions of low oxygen availability. Throughout the animal kingdom, hypoxia-inducible factor has emerged as a master regulator of this response. These bHLH transcription factors enhance transcription of a variety of genes that work to maintain oxygen homeostasis and allow adaptation to decreased oxygen availability. Two homologues, HIF-1α and HIF-2α, have been extensively studied in this field. Though they have similar domain structures and amino acid sequences, display overlap in some gene targets, and share regulatory mechanisms, they also perform distinct roles. They differ in tissue expression patterns, both temporally during development and spatially, hypoxia-driven expression kinetics, target genes, and fold induction. To elucidate mechanisms of this differential behavior, I investigated two aspects of HIF-2α-specific regulation. Firstly, I explored the contribution of early growth response transcription factors, EGRs, to HIF-2α-directed erythropoietin expression. Through reporter assays and chromatin immunoprecipitation, these factors were determined to occupy the erythropoietin enhancer adjacent to the HIF-2α binding site. Overexpression analysis showed they could amplify HIF-2α transactivation of erythropoietin, while knockdown experiments showed they were necessary for full, endogenous expression. And co-immunoprecipitation studies revealed a physical interaction between EGRs and HIF-2α that was necessary for cooperative activity. Secondly, I investigated the mechanism by which modulation of HIF-2α activity by CBP/SIRT1-dependent acetylation was signaled. Our studies revealed ACSS2, an acetyl CoA synthetase, as the source of acetyl CoA required for HIF-2α complex formation with the acetyltransferase CBP, subsequent HIF-2α acetylation, and target gene activation. The ACSS2 substrate acetate is produced during hypoxia, and exogenous acetate supplementation to cell culture media induced this pathway independent of hypoxia. Acetate administration in mice also augmented the HIF-2α-influenced pathways of red blood cell production and tumor growth in an ACSS2-dependent manner. Thus, EGRs represent novel HIF-2α cofactors in erythropoietin induction, while acetate, through ACSS2, regulates HIF-2α acetylation-dependent activity.Item Exploiting Multi-Cell Type Cultures to Elucidate Tumor Cell Features That Impact Macrophage Phenotype(December 2021) Voth Park, Josiah Malachi; Kim, James; Minna, John D.; Brekken, Rolf A.; Akbay, Esra A.; Malter, James; Huang, LilyLung cancer is expected to kill ~150,000 people this year, encompassing 25% of all cancer related deaths making lung cancer the leading cause of cancer-related mortality in men and women. Lung cancer is divided into non-small cell lung cancer (NSCLC) and small cell lung cancer (SCLC) which represent 80-85% and 15-20% of cases, respectively. My dissertation project focused on understanding how to model the interactions between lung cancer cells, fibroblasts and immune cells. Immune cells are critical components of the tumor microenvironment (TME) that contribute to tumorigenesis, angiogenesis and metastasis. Macrophages are key regulators of the immune landscape within the TME. The plasticity of macrophage phenotypes in the TME correlates with prognosis of NSCLC. Depending on their phenotype, macrophages in the TME can secrete pro-tumor cytokines and chemokines, ultimately suppressing the function of anti-tumor immune cells in the TME. The purpose of my project was to investigate if and how NSCLC cells alter macrophage phenotype in multi-cellular co-cultures and to relate effects on macrophages to the molecular characteristics of different NSCLCs. The central hypothesis of the project is, tumor cell characteristics drive macrophage polarization in the TME, and this can be captured using a multicellular co-culture model. Given the central importance of macrophages to the TME and the immune landscape of NSCLC, an understanding of the tumor cell characteristics associated with immune suppressive or immune stimulatory macrophage phenotype could be exploited from a therapy perspective in the future. To address this hypothesis, an in vitro co-culture system (NSCLC tumor cells, human cancer associated fibroblasts (CAFs), and mouse macrophages) was developed to interrogate cancer cell features driving heterogeneity of macrophage phenotypes across a panel of NSCLCs. We measured: mRNA expression in mouse macrophages with a panel of qPCR probes for genes associated with distinct macrophage phenotypes (Arg1, iNOS, Il-1β, Il-6, Ym-1, Socs3). This system was validated by comparison of macrophage phenotypes represented in the TME of lung cancer xenografts grown in athymic nude mice. Using our platform, we evaluated ~80 NSCLC patient derived lines for their effect on mouse macrophage phenotype. We identified three main macrophage phenotypes across this panel of NSCLCs. To identify cancer cell biomarkers for macrophage polarization, we interrogated molecular characteristics of the cancer lines. Additionally, we expanded the functionality of the platform to assess the effects of pharmacologic agents on macrophage phenotype. As a proof of principle, a small panel of known immune stimulating compounds was tested in the in vitro co-culture platform and validated in human tumor xenografts. Finally, we identified a few novel compounds that show selective cancer cell toxicity and reprogram macrophage phenotype. In conclusion, we built a reproducible in vitro platform to interrogate macrophage polarization in the TME. We leveraged this platform to identify three dominant macrophage phenotypes induced by NSCLC cells and CAFs. We found that no cancer cell molecular characteristic alone drives macrophage polarization. Finally, we illustrate the significance of this platform for immune stimulating drug identification; we identified two novel chemicals that repolarize macrophages and kill cancer cells simultaneously.Item FishATLAS: Assessment of Organotropism Determination Through Imaging Informatics of Xenografted Zebrafish(December 2021) Saucier, David Hamilton; Fiolka, Reto; Danuser, Gaudenz; Amatruda, James F.; Whitehurst, Angelique Wright; Brekken, Rolf A.; Mason, Ralph P.Ewing sarcoma patients with metastatic disease have a 5-year survival rate of approximately 28%. The hallmark of this disease is an aberrant transcription factor made by a fusion of Chromosomes 11 and 22 called EWSFLI1. EWSFLI1 expression levels have been correlated with differing responses in cell metastatic propensity, but much remains to be elucidated. Indeed, many current models fail to meet the statistical rigor that is needed for exceedingly spontaneous, rare events like metastasis. To address this need, FishATLAS utilizes zebrafish human cancer cell xenograft images after high fidelity registration using a novel diffeomorphic transformation to display metastatic hot spots of different cancer cell conditions to begin to grasp the deeper underpinnings of organotropism in vivo with individual cancers. Utilizing a suite of statistical tests, FishATLAS determines at a global whole-fish scale and the local microenvironment, if there are statistically different cell hotspots when comparing two or more different conditions. As it stands, data for EWSFLI1, its target SOX6, a non-transformed cell line NIH3T3, TC32 subclones, and melanoma cell lines have all shown unique distributions of metastatic hot spots. These findings serve as a tool for drug discovery and later environmental re-mapping in FishATLAS by allowing transgenic fish images (such as vasculature and lymphatics) to be overlayed onto any historical data set. These can then be used to determine a given microenvironment's contributions to secondary sites of metastasis. In the case of EWSFLI1 and its target SOX6, there was a marked difference upon shRNA-mediated knockdown that removed a population of hotspots in the upper somitic veins while some were persistent post genetic perturbation. SOX6 shRNA KD data indicates that the somite and inter-somitic arteries are more sensitive for metastatic colonization. Previous studies and our current accumulator data suggest these to be regions with higher oxidative stress, guiding insights for oxidative-mechanistic therapy. These and other conditional accumulations of sparse metastatic hotspots demonstrate the power of FishATLAS as a longitudinal assay of cellular and genetic conditions across all cancers.Item Heterogeneity in Human NSCLC Tumor Glucose Metabolism: On the Origins and Role of Pyruvate Metabolism(2015-07-22) Hensley, Christopher Thomas; Zhu, Hao; DeBerardinis, Ralph J.; Tu, Benjamin; Shay, Jerry W.In 1956 Otto Warburg, the most prominent scientist in cancer metabolism, stated that "the problem of cancer is...to discover the differences between cancer cells and normal growing cells" (Warburg, 1956a). Over fifty years later, the field still lacks a valid experimental framework to discover such metabolic differences in human tumors. A major limitation is the inability to faithfully recapitulate the microenvironment of primary human tumors in model systems. As a result, fundamental questions about tumor metabolism, including the suppression of pyruvate oxidation upon transformation proposed by Warburg more than 50 years ago, have only rarely been subjected to direct experimental assessment. To provide a direct readout of primary human tumor metabolism in vivo, we have used intra-operative 13C-glucose infusions in non-small cell lung cancer (NSCLC) patients to compare metabolism between tumors and non-cancerous lung. Pre-surgical imaging, including non-invasive assessment of tissue perfusion using dynamic contrast enhanced magnetic resonance imaging (DCE-MRI), allowed us to select areas of microenvironment-based heterogeneity, to guide sample acquisition. Specifically, this microenvironment-based heterogeneity was assessed relative to the oncogenotype, histological parameters, and metabolism of glucose through glycolysis and the TCA cycle. Diverse tumors displayed enhanced glycolysis and glucose oxidation. Furthermore, we discovered that due to the low enrichment in acetyl-CoA and other TCA cycle intermediates, all tumors had evidence for oxidation of multiple nutrients. We identified lactate as a carbon source for tumor oxidative metabolism. Additionally, metabolically heterogeneous regions were identified within and between tumors using DCE-MRI. Regions of lesser contrast enhancement demonstrated higher 13C enrichment, likely reflecting contributions of non-glucose nutrients to central carbon metabolism in well-perfused areas, or the cause or consequence of aberrant proliferation of aggressive clones resulting in inadequate perfusion. The data indicate that the heterogeneous metabolism of these tumors is highly and predictably related to the microenvironment. In summary, we have made novel, significant progress in assaying and analyzing primary human tumor metabolism and its relation to the microenvironment in vivo. I close with a separate project for future directions to begin to dissect the cellular origins of the whole tumor fragment signal that is amenable to direct assays in patients.Item The Immunosuppressive Function of VEGF Signaling in the Tumor Microenvironment(December 2021) Zhang, Yuqing; Aguilera, Todd A.; Brekken, Rolf A.; Castrillon, Diego H.; Dellinger, Michael T.Angiogenesis, a hallmark of cancer, is induced by vascular endothelial growth factor-A (VEGF). As a result, anti-VEGF therapy is commonly employed for cancer treatment. However, anti-VEGF therapy generally provides modest efficacy in cancer patients and therapy-induced hypoxia results in a less differentiated mesenchymal-like tumor cell phenotype, which reinforces the need for effective companion therapies. Cyclooxygenase-2 (COX-2) inhibition has been shown to promote tumor cell differentiation and improve standard therapy response in pancreatic cancer. Here, I evaluate the efficacy of COX-2 inhibition and VEGF blockade in preclinical models of pancreatic cancer and identity it as a strategy to overcome therapy-induced resistance in pancreatic cancer. Combination therapy reverses anti-VEGF-induced epithelial-mesenchymal transition, collagen deposition and promotes an immune stimulatory microenvironment. Recent studies have also found that VEGF expression is also associated with immune suppression in cancer patients. This connection has been investigated in preclinical and clinical studies by evaluating the therapeutic effect of combining anti-angiogenic reagents with immune therapy. However, the mechanisms of how anti-VEGF strategies enhance immune therapy are not fully understood. We and others have shown selective elevation of VEGFR2 expression on tumor-associated myeloid cells in tumor-bearing animals. I further investigate the function of VEGFR2+ myeloid cells in regulating tumor immunity and find VEGF induces an immunosuppressive phenotype in VEGFR2+ myeloid cells including directly upregulating the expression of programmed cell death 1-ligand 1 (PD-L1). Moreover, I demonstrate that VEGF blockade inhibits the immunosuppressive phenotype of VEGFR2+ myeloid cells, increases T cell activation and enhances the efficacy of immune checkpoint blockade. These studies highlight the function of VEGFR2 on myeloid cells and provide mechanistic insight on how VEGF inhibition potentiates immune checkpoint blockade.Item pH Transistor Nanoprobe Advances Cancer Detection and Surgery(2015-11-24) Zhao, Tian; Li, Wen-Hong; Gao, Jinming; Sumer, Baran; Brekken, Rolf A.Cancer exhibits profound genetic and phenotypic differences, therefore broad yet cancer-specific detection of malignant tumors is challenging. Anatomy-based imaging modalities (e.g., CT, MRI) have good spatial resolution but provide little disease-specific information. 2-Deoxy-2-[18F]fluoroglucose positron emission tomography (FDG-PET) allows near universal tumor detection by leveraging altered tumor metabolism, but it is limited by low spatial resolution and high false positive rates. Here we report a near infrared fluorescent pH transistor nanoprobe targeting extracellular tumor acidosis from dysregulated pH that drives many invasive properties of cancer. The nanoprobes delineated tumors with high spatial resolution (<1 mm) for a broad range of in vivo tumor models using different clinical cameras. Our results show targeting tumor pH downstream from deregulated metabolism provides a broad strategy with improved cancer specificity which makes the nanoprobe a useful adjuvant method to reduce false rates after FDG-PET. To validate the ability of nanoprobes to provide real-time, highly sensitive and specific illumination of cancer, we performed tumor acidosis guided detection of occult disease as well as surgery and demonstrated significantly improved long-term survival benefit in head/neck and breast cancers. This binary nanotransistor design achieves digitization of an analog biologic signal (pH) for signal amplification and noise reduction that improves the accuracy of cancer detection, intraoperative tumor visualization, and imaging of therapeutic response while providing a powerful tool for understanding dysregulated pH and cellular energetics in cancer.