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Indiana University-Purdue University Indianapolis (IUPUI) / Pancreatic Ductal Adenocarcinoma (PDAC) is the third leading cause of cancerrelated deaths with a five-year survival rate of 11%. PDAC tumors are characterized by a dense desmoplastic stromal microenvironment, mediated in part through local cytokine production. PDAC tumors also elicit a systemic inflammatory response in the host; this, combined with a loss of body weight due to muscle and fat wasting, is characteristic of cachexia. Understanding the molecular mechanisms that drive malignant inflammation is critical to improve PDAC therapy and increase patient survival. Oncostatin M (OSM) belongs to the IL-6/GP130 family of cytokines, members of which have been shown to promote PDAC tumor development, inflammation, and cachexia. Much less is known of OSM. My central hypothesis was that OSM promotes pancreatic cancer and cachexia by inducing local and systemic inflammation, fibrosis, and wasting via OSM signaling through the receptor, OSM receptor (OSMR). We investigated effects of exogenous OSM administration in wildtype and IL-6 null mice without cancer. OSM induced systemic fibrosis, bone loss, local muscle wasting, and cardiac dysfunction in presence and absence of IL-6. We further defined the roles of OSM/OSMR in the pancreatic cancer microenvironment and macroenvironment. OSM activated genes involved in inflammation, fibrosis, and tumor progression in both tumor cells and fibroblasts and altered the tumor microenvironment, promoting a dense compaction of tumor cells and cancer associated fibroblasts. Loss of systemic OSM signaling altered tumor metabolism and reduced the stromal compartment without affecting tumor size. Loss of OSMR signaling in tumor cells reduced tumor size and promoted survival. However, systemic loss of OSM or OSMR in host cells did not halt effects of cachexia including muscle dysfunction, atrophy, or inflammation/anemia. Overall, OSM/OSMR signaling in the microenvironment is necessary in modulating tumor phenotype and promoting survival in PDAC but may not be necessary for pancreatic cancer cachexia. / 2024-08-02

<p>NK cells play a pivotal role in tumor rejection; however, once present in the tumor microenvironment, they are characterized by decreased cytotoxicity and reduced expression of activating receptors. The mechanisms governing the inactivation of NK cells within tumors remain poorly understood. Since tumor associated macrophages (TAMs) are a highly abundant and suppressive cell type within tumors, we hypothesized that they are capable of altering the function of NK cells. Following the co-culture of alternatively activated macrophages (M2) or TAMs with NK cells we observed that the expression of the cytotoxic marker CD27 on NK cells was down-regulated as well as the ability of these cells to kill YAC-1 cells in a killing assay. We have demonstrated that the mechanism by which M2 cells inhibit NK cells is TGF-β dependent. Notably, the developmental stage of NK cells after interaction with TAMs was altered and the NK cells became phenoytpically mature and potentially exhausted (CD27^lowCD11b^high). This prompted our interest in examining the developmental stage of NK cells from polyoma MT antigen (pyMT) transgenic mouse (MMTV-pMT) breast tumors. Interestingly, in contrast to the <em>in vitro</em> results, we have shown that NK cells isolated from pyMT tumors are developmentally immature; however maintain their maturity within the spleen. Their immature phenotype correlates well with their decreased expression of perforin, granzyme, and NKp46. Both our <em>in vitro</em> studies with TAMs and our <em>in vivo</em> developmental studies using the pyMT model demonstrate that NK cells are altered by their surroundings. A better understanding of how NK cells are modified by the tumor microenvironment will help to develop strategies aimed at bolstering immune responses against tumors.</p> / Master of Science (MSc)

<p>Pre-clinical and clinical data strongly support the use of immunotherapies for cancer treatment. Cancer vaccines offer a promising approach, however, the outcomes of clinical vaccine trials have been largely disappointing, prompting a need for further investigation. Using the B16F10 murine melanoma, we have investigated the local events within growing tumors following recombinant adenovirus immunization. In chapter 2, we investigated the ability of a pre-clinical vaccine to elicit only transient tumor growth suppression. We observed that tumors were initially infiltrated by a small number of highly functional tumor-specific CD8+ T cells following vaccination that instigated a rapid adaptive response in the tumor that suppressed local immune activity. In chapter 3, we questioned whether increasing the rate and magnitude of early immune attack would result in more robust tumor attack prior to tumor adaptation. Increasing the rate of tumor-specific CD8+ T cell expansion following vaccination resulted in tumor regression and durable cures in approximately 65% of treated mice. Further analysis revealed that tumor regression correlated with an early burst in immune attack that outpaced tumor adaptation. In chapter 4, we explored whether the same vaccine could be improved when combined with immunomodulatory antibodies. Vaccination combined with anti 4-1BB and anti PD-1 resulted in complete tumor regression and durable cure of >70% of treated animals and was associated with increased local immune activity. Gene expression profiling revealed a unique gene signature associated with the curative treatment, which was also associated with positive outcome in human melanoma patients. The described research sheds new light on mechanisms that limit the efficacy of therapeutic cancer vaccines. Namely, rapid tumor adaptation, triggered by early vaccine-induced CD8+ T cells, acts to suppress the local immune response prior to maximal immune attack. Strategies to overcome these adaptive processes should therefore be considered in future vaccine design.</p> / Doctor of Philosophy (Medical Science)

Yes / With an escalating incidence of breast cancer cases all over the world and the deleterious psychological impact that mastectomy has on patients along with several limitations of the currently applied modalities, it's plausible to seek unconventional approaches to encounter such a burgeoning issue. Breast tissue engineering may allow that chance via providing more personalized solutions which are able to regenerate, mimicking natural tissues also facing the witnessed limitations. This review is dedicated to explore the utilization of adipose tissue-derived mesenchymal stem cells for breast tissue regeneration among postmastectomy cases focusing on biomaterials and cellular aspects in terms of harvesting, isolation, differentiation and new tissue formation as well as scaffolds types, properties, material–host interaction and an in vitro breast tissue modeling.

β-catenin is a multifunctional protein that plays crucial roles in embryonic development, physiological homeostasis, and a wide variety of human cancers. Previously, we showed that in vivo targeted ablation of β-catenin in melanoma-associated fibroblasts after melanoma formation significantly suppressed tumor growth. However, when the expression of β-catenin was ablated in melanoma-associated fibroblasts before tumor initiation, melanoma development was surprisingly accelerated. How stromal β-catenin deficiency leads to opposite biological effects in melanoma progression is not completely understood. Here, we report that β-catenin is indispensable for the activation of primary human stromal fibroblasts and the mediation of fibroblast-melanoma cell interactions. Using coimmunoprecipitation and proximity ligation assays, we identified Yes-associated protein (YAP) as an important β-catenin-interacting partner in stromal fibroblasts. YAP is highly expressed in the nuclei of cancer-associated fibroblasts (CAFs) in both human and murine melanomas. Mechanistic investigation revealed that YAP nuclear translocation is significantly modulated by Wnt/β-catenin activity in fibroblasts. Blocking Wnt/β-catenin signaling in stromal fibroblasts inhibited YAP nuclear translocation. In the absence of YAP, the ability of stromal fibroblasts to remodel the extracellular matrix (ECM) was inhibited, which is consistent with the phenotype observed in cells with β-catenin deficiency. Further studies showed that the expression of ECM proteins and enzymes required for remodeling the ECM was suppressed in stromal fibroblasts after YAP ablation. Collectively, our data provide a new paradigm in which the β-catenin-YAP signaling axis regulates the activation and tumor-promoting function of stromal fibroblasts.

Advanced stage tumors can acquire the ability to divide uncontrollably, invade the surrounding matrix, and circulate through the bloodstream or lymphatic system to distant organs in a process known as metastasis. The brain, which is shielded from the environment by the blood brain barrier, offers an immunocompetent lodging spot for the circulating cancer cells. Therefore, it is a "popular" destination for metastasized cancers which even surpass the incidents of primary brain tumors. It is hypothesized that the disseminated cancer cells engage with the host cells of the perivascular neural niche in a poorly understood crosstalk of molecular factors, that in turn augment the metastatic colonization of cancer cells. A better understanding of this crosstalk is indispensable to apprehending the complexity of the metastasis process, and to facilitating the discovery of biomarkers that predict metastatic potential and improve patient prognosis. The larger goal of this study was to adopt a mass spectrometry-based systems biology approach to investigate the molecular mechanisms and regulatory networks that underlie the complex phenomenon of breast cancer propagation at the brain metastatic site. To achieve this, the study was divided in three sub-projects designed around the following objectives, i.e., (a) to comprehensively characterize the protein landscape of the neural niche or the brain microenvironment comprised of astrocytes, microglia and endothelial cells, (b) to explore the immunological protein networks activated in microglia cells upon stimulation with anti-inflammatory cytokines released by tumor cells in the brain, and (c) to investigate the protein-level changes elicited in HER2+ breast cancer cells when grown under conditions that simulate the brain microenvironment in-vitro. Detailed characterization of the neural niche enabled us to propose molecular mechanisms that allow for the seeding and outgrowth of metastasized cancer cells in the brain. The study further provided novel insights into the signaling networks that regulate the immune functions of the microglia and their role during cancer development. Lastly, an in-depth investigation of breast cancer cells cultured in the presence of neural niche factors revealed potential novel mechanisms of cancer cell dormancy during metastasis. Altogether, large-scale proteomics data generated in this work will help clarify the mechanisms of metastatic cancer development, and will lay the groundwork for future studies that aim at the discovery of novel biomarkers and druggable targets for the treatment of brain metastatic cancers. / Doctor of Philosophy / In the US, the incidence of breast cancer ranks only second to lung cancer, and the primary cause for almost all cancer related deaths is the development of cancer metastasis in patients. The process of metastasis involves cancer cells leaving the primary tumor, entering the bloodstream or the lymphatic system, and spreading to other parts of the body during advanced stages of the disease. The brain is a common metastatic site for cancer cells to form a secondary tumor. Previous research has found that it is the brain cells which support the progression of secondary tumors in the central nervous system by releasing protein molecules that favor the survival and growth of disseminated cancer cells. Brain cells, including endothelial cells (that form the blood vessels), astrocytes (that regulate brain development), and microglia (that are the immune cells of the brain), are the first to respond to metastasized cancer cells. A better understanding of the behavior of these cells and of their signaling molecules which support the process of cancer metastasis can help us discover new drug targets to treat cancer patients. This study had three main objectives, i.e., (a) to profile the protein make-up of brain cells and identify specific proteins that support cancer development in the brain, (b) to investigate the changes in microglial proteins when these cells are exposed to conditions that simulate cancerous growth in the brain, and (c) to explore the proteins that become active in breast cancer cells when they are subjected to conditions that simulate the brain microenvironment. To accomplish this, we used a high-throughput technology called mass spectrometry, that allows for the identification of thousands of proteins in a sample at any given time. Overall, the study provided novel insights into the biological mechanisms of secondary tumor formation, and into the early response of breast cancer cells when they encounter unfamiliar conditions in the brain. The work further supports the discovery of specific proteins that can be targeted in anti-cancer therapies.

The tissue microenvironment is a complex living system containing heterogeneous mechanical and biophysical cues. Cellular components are surrounded by extracellular matrix and interstitial fluid, while transport of nutrients and biochemical factors is achieved via the vasculature. Each constituent of the tissue microenvironment can play a significant role in its ability to function normally. Many diseases including cancer have been linked with dysfunction in the tissue microenvironment; therefore an improved understanding of interaction between components of this complex system is needed. In vitro platforms mimicking the tissue microenvironment appear to provide the most promising avenue for studies of cell-cell and cell-matrix interactions as well as elucidation of the mechanisms leading to disease phenomena such as tumor metastasis. However, successful recapitulation of all three primary components of the tissue microenvironment in three dimensions has remained challenging. In particular, matching mechanical cues and biochemical transport to in vivo conditions is difficult because of lack of quantitative characterization of the physical properties and parameters of such platforms. In this work, extensive characterization of collagen I hydrogels, popular for use as extracellular matrix mimics, was performed in order to enable tuning to specific in vivo conditions. Additionally, perfusion of blood in a 3D tissue microenvironment platform fabricated using collagen hydrogels was characterized to enable future advances in in vitro modeling of the in vivo microenvironment. Finally, the tissue microenvironment platform is modified to enable biochemical gradients within the hydrogel and used to examine directed migration (chemotaxis) of human breast cancer cells in response to gradients in growth factor combined with varied stiffness and pore diameter of the extracellular matrix. / Ph. D.

This work is an experimental study on heat transfer from a human arm and a model cylinder. Heat transfer from a human forearm to a large jet, representative of a building HVAC vent/outlet was studied using both an IR camera and a heat flux sensor. The isothermal jet was discharged horizontally from a wind tunnel, at the same temperature as the ambient air. The model cylinder was used to validate the heat transfer results with results from previous studies, using both the IR camera and heat flux sensors. Further, a study on heat transfer to impingement jets from a human forearm at various Reynolds numbers (Re = 9500-41000) and impinging distances of four and eight jet diameters was done. Heat transfer from a human arm to such impingement jets were then compared with heat transfer due to natural convection under both open and controlled environments. A significant increase in convection heat transfer with Reynolds number and distance from the jet outlet was observed. A nearly four-fold increase in convection heat transfer coefficient was obtained when a jet with Reynolds number of 9500 was impinged on a human arm when compared to that obtained under natural convection in an open environment. Empirical correlations for predicting the stagnation and average Nusselt number from a human arm were also developed with high values of correlation coefficients for future studies. Impingement jets were found to be an effective means to transfer heat from human bodies and could potentially be used for creating thermally conditioned microenvironments. / M. S.