<p>Breast cancer remains the leading cause of death among females worldwide. While systemic therapy for breast cancer may work effectively in the early phases, for more than 10% of primary and 50% of metastatic cases, the disease eventually progresses, resisting treatments. To overcome this issue, recognizing markers of resistance as early as possible is critical. However, the underlying mechanisms of resistance remains elusive. The influence of microenvironmental factors of the extracellular matrix (ECM) on tumor behavior has been revealed relatively recently and increased stiffness of ECM is associated with cancer progression. Additionally, impacts of other matrix components such as non-neoplastic epithelial cells (that may constitute an important portion of the tumor microenvironment -TME) are suspected to influence tumors but they have not been investigated in detail. Besides, it is not known whether the response to increasing stiffness depends on the subtypes of breast cancer. Here, using breast models in 3D cell culture we have shown that the non-neoplastic epithelial compartment can influence the effect of matrix stiffness even for tumors recognized as highly aggressive. The degree of tumor aggressiveness recognizable via tumor architecture is associated with a differential behavior when ECM stiffness changes. In a 3D microenvironmental context, which provides an optimal level of constraints for tumors to display their phenotype, we report stiffness and paracrine influence impact on cisplatin-mediated cytotoxicity, which correlates with distinct nuclear morphometry and distribution pattern associated with population heterogeneity. The response pattern varies across cell lines representing higher and lower levels of aggressiveness in the basal-like subtypes of breast cancer. Our results also highlight the need for integrating biochemical and physical components of the TME in future designs of <i>in vitro</i> drug screening platforms.</p>

<p>  </p> <p>Metastases are responsible for approximately 90% of all cancer-related deaths, with metastatic breast cancer (BC) holding a 5-year survival rate of only 27%. Recent research has highlighted a complex dynamic between cancer cells and the tumor microenvironment as essential for the formation of macrometastases. Within this field, tissue stiffening through matrix accumulation and altered matrix organization at the primary tumor site were recently linked with sustained proliferation and increased migration of tumor cells. Separately, elevated levels of the glycoprotein, fibronectin, were correlated to poor patient survival in BC and were linked to enhanced seeding of disseminated tumor cells at metastatic sites. Through my doctoral work, we have identified several mechanisms through which accumulated fibronectin impacts the metastatic potential of BC cells. First, we identified a transient increase in extracellular fibronectin in the lungs, which peaked before overt metastasis, coupled with a non-transient increase in total lung volume. To better recapitulate physiological conditions, we then developed a novel magnetically-actuated platform with the ability to apply tensile strain on cells at various amplitudes and frequencies in a high-throughput multi-well culture plate using suspended fibrillar fibronectin for 3D cell culture that is not reliant on a synthetic substrate. Using this as a biomimetic lung model, we found that cyclic mechanical force acted as a suppressor of cancer cell growth in a biomimetic lung model, implicating the accumulation and reorganization of extracellular matrix as an attempt by the cancer cells to alter the mechanical properties of the lung tissue and resist entering dormancy. However, our results showed that BC cells could not organize extracellular fibronectin independently. Instead, BC cells altered the accumulation and architecture of fibronectin by conditioning fibroblasts through soluble factors and extracellular vesicles. We observed that the fibronectin produced by conditioned fibroblasts varied as an effect of both the method of conditioning and the phenotype of the BC cell as the conditioning source. Taken together, these results have increased our knowledge of the relationship between disseminated breast cancer cells, fibroblasts, and fibronectin architecture in the early metastatic lung niche that paves the way for further investigation on targeting disseminated BC cells during early disease intervention in order to inhibit later overt metastatic outgrowth.</p>

Breast cancer (BC) is one of the deadliest forms of cancers with high incidence and mortality rates, especially in women. Encouragingly, targeted therapies have improved the overall<br>survival and quality of life in patients with various subtypes of BC. Unfortunately, these first-line therapies often fail due to inherent as well as acquired resistance of cancer cells. Treatment evading cancer cells can exhibit systemic dormancy in patients over a long period of time without manifesting any symptoms. In a suitable environment, these undetected disseminated tumor cells can relapse in the form of metastasis. Therefore, it is essential to understand the mechanisms of<br><div>BC recurrence and to develop durable therapeutic interventions to improve patient’s survival. In this dissertation work, we studied fibroblast growth factor receptors (FGFR), as therapeutic targets to treat the recurrence of drug-resistant and immune-dormant BC metastasis. <br></div><div><br></div><div>The HER2 subtype of BC is characterized by the overexpression of human epidermal growth factor receptor 2 (HER2), which drives elevated downstream signaling promoting tumorigenesis. Trastuzumab emtansine (T-DM1) is an antibody-drug conjugate in which an anti-HER2 antibody targets HER2 overexpressing tumor cells and delivers a highly potent microtubule inhibitor. Using novel models of minimal residual disease (MRD) following T-DM1 treatments, we found that epithelial to mesenchymal transition is a critical process for cells to persist the TDM1 treatments. The upregulation of FGFR1 may facilitate insensitivity to T-DM1. Our data also showed that FGFR1 overexpression in HER2+ tumors leads to a higher incidence of recurrence, and these recurrent tumors show sensitivity towards covalent inhibition of FGFR. <br></div><div><br></div><div>In addition to drug-induced MRD in the primary tumor sites, disseminated tumor cells (DTCs) can demonstrate dormant phenotype via maintaining an equilibrium with immunemediated tumor clearance. Factors affecting such equilibrium may contribute to the recurrence of breast cancers metastasis. We show that such immune-mediated dormancy can be modeled with the 4T07 tumors. These tumors display immune-exclusion phenotypes in metastatic pulmonary organs. The inhibition of FGFR modulates the immune cell compositions of pulmonary organs favoring anti-tumor immunity. However, inhibition of FGFR may also affect T cell receptor downstream signaling, resulting in the inhibition of cytolytic T cell’s function. Finally, we report that combination therapy using the FGFR kinase inhibitor and an immune checkpoint blockade showed effective targeting of metastatic 4T07 tumors. <br></div><div><br></div><div>FGFR signaling as a therapeutic target in various tumors has been an active focus of cancer research. In this dissertation work, we have expanded our understanding of the role of FGFR in the recurrence of drug-resistant breast cancers as well as in the maintenance of an immune evasive microenvironment promoting pulmonary growth of tumors. Moreover, we presented evidence that it is possible to repurpose FGFR targeted therapy alone or in combination with checkpoint blockades to target recurrent metastatic BCs. In the future, our novel models of minimal residual diseases and systemic immune dormancy may act as valuable biological tools to expand our understanding of the minimal residual disease and dormant tumor cells.</div>

Models using 3D cell culture techniques are increasingly accepted as the most biofidelic in vitro representations of tissues for research. These models are generated using biomatrices and bulk populations of cells derived from tissues or cell lines. This thesis study focuses on an alternate method to culture individually selected cells in relative isolation from the rest of the population under physiologically relevant matrix conditions. Matrix gel islands are spotted on a cell culture dish to act as support for receiving and culturing individual single cells; a glass capillary-based microfluidic setup is used to extract each desired single cell from a population and seed it on top of an island. Using examples of breast and colorectal cancers, we show that individual cells evolve into tumors or aspects of tumors displaying different characteristics of the initial cancer type and aggressiveness. By implementing a morphometry assay with luminal A breast cancer, we demonstrate the potential of the proposed approach to studying phenotypic heterogeneity. Results reveal that intertumor heterogeneity increases with time in culture and that varying degrees of intratumor heterogeneity may originate from individually seeded cells. Moreover, we observe a positive correlation between fast-growing tumors and the size and heterogeneity of their nuclei.

<p dir="ltr">Metastatic breast cancer (BC) remains a dauting therapeutic challenge due to the heterogeneity and cellular plasticity that exists. Because of these, BC resistance to targeted therapies and immune checkpoint blockade (ICB) present major challenges in the clinical setting. As a result, incomplete clearance of BC during a therapeutic regimen can lead to the persistence of minimal residual disease (MRD) which greatly contributes to tumor relapse. Here we develop a powerful in vivo model of lung metastasis in which we can achieve robust pulmonary tumor regression in response to the fibroblast growth factor receptor (FGFR) inhibitor, pemigatinib.</p><p dir="ltr">To enhance the efficacy of ICB, tumors must first be converted from an immune “cold” environment to an immune “hot” environment. Using our in vivo model of lung metastasis, we demonstrated that pemigatinib can significantly increase the presence of infiltrating T-cells into the lungs while suppressing the presence of MDSCs both locally in the lungs and systemically. Taken together, pemigatinib is an ideal candidate to prime these immune “cold” tumors for combination with ICB.</p><p dir="ltr">Upon establishment of MRD by pemigatinib in our in vivo model we observe upregulation of an alternate growth factor receptor, platelet-derived growth factor receptor (PDGFR). Functionally, upon FGFR inhibition, there is increased response to pulmonary fibroblast derived PDGF ligand, fueling survival of MRD. We demonstrated that knockdown of PDGFR significantly delayed tumor growth reinitiation in an in vitro 3D culture following pemigatinib as well as delayed tumor relapse in our pulmonary metastasis model.</p><p dir="ltr">To limit cellular plasticity and reduce survival of MRD, we propose a novel dual-targeted approach utilizing pemigatinib, in conjunction with inhibition of DNMT1 using the reversible inhibitor GSK3484862. We used our in vivo model of lung metastasis after treatment with pemigatinib as a model of cellular plasticity to targeted therapy. This combination therapy prevented growth factor plasticity and delayed tumor recurrence. Through prevention of PDGFR upregulation induced by pemigatinib.</p><p dir="ltr">In the present dissertation works, our study demonstrates pemigatinib’s robust ability to increase infiltrating T-cells in addition to its strong antitumor effects on pulmonary tumors. Despite the robust effects of pemigatinib, acquired mechanism of resistance through upregulation of PDGFR allows survival of MRD and are supported by PDGF secreting fibroblasts. Using an approach of limiting cellular plasticity through DNA methylation inhibition combined with pemigatinib, we achieved a more durable therapeutic response. Our findings underscore the significance of understanding adaptive responses to targeted therapies and provide a tangible therapeutic strategy to prolong treatment response in metastatic breast cancer.</p>

<p>With 2.1 million new cases, breast cancer is the most common cancer in women. Triple negative breast cancer (TNBC), which is 15-20% of these breast cancer cases is clinically negative for expression of estrogen and progesterone receptors (ER/PR) and human epidermal growth factor receptor 2 (HER2) receptors<a>.</a> It is characterized by its unique molecular profile, aggressive behavior, distinct patterns of metastasis, and lack of targeted therapies. TNBCs utilize glycolysis for growth, proliferation, invasiveness, chemotherapeutic resistance and hence has poor therapeutic response. There is an urgent need for novel/alternate therapeutic strategies beyond current standard of treatment for this subset of high-risk patients. Electrical pulse-based chemotherapy, known as electrochemotherapy (ECT) could be a viable option for TNBC therapy. ECT involves the local application of precisely controlled electrical pulses to reversibly permeabilize the cell membrane for enhanced uptake. ECT can increase the cytotoxicity of the chemotherapeutics up-to 1000 times, facilitating a potent local cytotoxic effect. </p> <p>The high cost and severe side-effects of conventional chemotherapeutics motivate the application of effective natural compounds. Combining electrical pulses with natural compounds will enhance the treatment efficacy. This dissertation focuses on curcumin, the yellow pigment of natural herb turmeric, that has been used for over 5000 years for its excellent anticancer properties. Previous studies have demonstrated the effectiveness of curcumin for treating multiple cancers, including TNBC, with limited side effects. The potency of curcumin can be enhanced further by combining it with ECT to provide an attractive and cost-effective alternative for TNBC treatment. </p> <p>Towards this we studied the effect of ECT with curcumin on MDA-MB-231 cell line, a human adenocarcinoma epithelial TNBC cell line. We performed various assays, including cell viability, colony forming, cell cycle, apoptosis, H₂O₂ reactive oxygen species (ROS), immunoblotting, real time quantitative PCR (qPCR), and cellular metabolites detection to study the impact of ECT with curcumin on MDA-MB-231 cells. In addition, to better understand the underlying mechanisms, we used high throughput, label-free quantitative proteomics. While several studies have attempted to define the mechanism of action of curcumin on cancer cells, little is known on the action mechanism of the curcumin delivered with electrical pulses. This work unravels the molecular mechanism behind the enhanced effects observed under the ECT-based curcumin therapy in TNBC cells, employing a high-throughput, quantitative, label-free mass spectroscopy-based proteomics approach. The proteomics approach provides information on the thousands of cellular proteins involved in the cellular process, allowing a comprehensive understanding of the electro-curcumin-therapy mechanism. Similar studies were also performed for ECT with cisplatin to compare the efficacy of the electro-curcumin-therapy to the standard stand-alone cisplatin-based therapy.</p> <p>Our results revealed a switch in the metabolism from glycolysis to mitochondrial metabolic pathways. This metabolic switch caused an excessive production of H₂O₂ ROS to inflict apoptotic cell death in MDA-MB-231 cells, demonstrating the potency of this ECT based curcumin therapy. These results encourage further studies to extend the application of ECT for clinical practice.</p>