The aim of this thesis is to develop a better understanding of the mechanisms and modalities exploited by breast cancer cells during invasion, paying particular attention to those mechanisms employed during cell migration in 3D spheroid culture, a context that more accurately recapitulates the complexities seen in vivo. Using a hydrogel-based model system, we investigated the roles of cellular blebs in cancer migration, developed a novel protocol that enhances optical microscopy images and facilitates high content imaging techniques including mass spectrometry imaging, and then used this new technology to investigate gain of function activity of mutant p53 in breast cancer cell migration in terms of both actomyosin contractility and in lipid abundances and distributions. Chapter 1 begins with an overview of breast cancer, an introduction to cancerous behavior and migration, a discussion of the importance of 3D cell culture and interactions of breast cancer cell and the extracellular matrix, and an overview of the various imaging techniques used in this work. In Chapter 2 we describe a novel mode of breast cancer migration that we term bleb – driven migration. This migratory mode is characterized by invasive cells that are both round and bleb bearing. This migratory mode is highly dependent on actomyosin contractility, a mechanism that is imperative to bleb formation. We show that blebs can actively attach to and rearrange the extracellular matrix via accumulations of β1 integrins at the bleb necks. We show both polarization of blebs as well as collagen alignment in regions of the cell enriched in blebs, both of which are dependent on the expression of β1 integrins by the cell. The discovery of this new migratory mode is important, as many cancers are able to overcome cancer therapies by using escape mechanisms, such as alternate migratory mechanisms. As such, developing a fuller understanding of the migratory modes used by cancer cells is vital in our fight to prevent cancer deaths. Chapter 3 tackles a significant problem associated with working in 3D spheroid culture: obtaining high quality, high resolution images. 3D samples tend to be highly refractive and poorly diffusive, and image acquisition can be severely hindered due to these factors. Further, the depth at which 3D samples can be imaged is limited by the low working distances of high numerical aperture objectives. As such, we developed a protocol that enables the sectioning of invasive spheroids, which we term DISC-3D (dual hydrogel invasive cryosectioning of spheroids in 3D). This protocol enables us to capture images that are higher in resolution and signal to background noise, removes imaging depth related constraints, and enables images to be acquired using dyes and techniques that have not previously been demonstrated in invasive 3D in vitro samples. Using this technology, Chapter 4 then examines the gain of function activity mutant p53 imparts on invasive breast cancer cells. We show that mutant p53 plays a role in increasing actomyosin contractility by promoting targeting of RhoA to the cell membrane. We also show an alteration in lipid expression in mutant p53 bearing cells, a feat that is made possible through use of the DISC-3D technology. Collectively, this work provides insights in to the invasive mechanisms exploited by breast cancer cells. We repeatedly demonstrate the importance of in vitro, 3D cell culture in the study of breast cancer migration. Using such cell culture techniques, we outline previously unknown aspects of breast cancer cellular migration both in regards to the importance of blebs and to the gain of function activity of mutant p53, the latter of which was made possible through use of the DISC-3D protocol. We argue that continued study in this area will provide insights into how breast cancer cells migrate, providing paths and new treatment strategies for preventing such migration.

The purpose of this work was to develop novel optical imaging technology and algorithms as a nondestructive method for detection and diagnosis of cancer in breast specimens. There are many ways in which the diagnosis of disease can benefit from fast and intelligent optical imaging technology. Our existing ability to provide this diagnosis depends on time-consuming pathology analysis. Optical coherence tomography (OCT) is a non-invasive optical imaging modality that provides depth-resolved, high-resolution images of tissue microstructure in real-time. OCT could provide a rapid evaluation of specimens while patients are still in the office, and has strong potential to improve the efficiency in evaluation of breast pathology specimens (biopsy or surgical). In this work, we demonstrate an imaging system to address this unmet clinical need, artificial intelligence algorithms to interpret the images, and early work towards miniaturizing the technology. We present an OCT system that achieves a line scan rate of 250kHz, meaning we can image a pathology cassette in 41 seconds, which is more than double the fastest scan rate in the field. By utilizing a multiplexed superluminescent diode (SLD) light source, which has strong noise performance over imaging speed, we achieve high resolution imaging under 5 um in tissue (axially and laterally). The system features a 1.1 mm 6-dB sensitivity fall-off range when imaging at 250 kHz. The scanner features large-area scanning with the implementation of a 2-axis motorized stage, enabling visualization of areas up to 10 cm x 10 cm (prior work visualizes 3 mm x 3mm). We showcase the results of demonstrating the performance of this system on a 100-patient clinical imaging study of breast biopsies, as well as imaging of clinical pathology specimens from the breast, prostate, lung, and pancreas in an IRB-approved study. Further, we show our work towards developing artificial intelligence (AI) for cancer detection within OCT images. Using retrospective data, we developed a type of AI algorithm known as a convolutional neural network (CNN) to classify OCT images of breast tissue from 49 patients. The binary cancer classification achieved 94% accuracy, 96% sensitivity, and 92% specificity. This framework had higher accuracy than the 88% accuracy of 7 clinician readers combined in our lab’s earlier multi-reader study. Lastly, we demonstrate a supercontinuum light source based on a 1 mm2 Si3N4 photonic chip for OCT imaging that has better performance than the state-of-the-art laser. Existing broadband laser sources for OCT are large, bulky, and have high excess noise. Our Si3N4 chip fundamentally eliminates the excess noise common to lasers and achieves 105 dB sensitivity and 1.81 mm 6-dB sensitivity roll-off with only 300 µW power on the sample.

Triple-negative breast cancers (TNBCs) are highly refractive to current treatment strategies, and new multi-targeted treatments need to be elucidated. Combination therapy that includes targeting the murine double minute 2 (Mdm2) signaling axis offers a promising approach. Protein-protein interaction inhibitors such as Nutlin-3a block the binding of key signaling molecules such as p53, p73α, and E2F1 to the hydrophobic pocket of Mdm2 and can lead to activation of cell-death signaling pathways. Since clinical trials for TNBC are evaluating the DNA damaging agent carboplatin, the objective of this thesis was to evaluate the therapeutic potential and mechanism of action of combination carboplatin and Nutlin-3a to treat TNBC. In TNBC cell lines with a mutant p53 background, we determined if modulation of Mdm2 function in the context of carboplatin-mediated DNA damage resulted in a synergistic inhibition of cell growth. Several ratios of carboplatin:Nutlin-3a were strongly synergistic in increasing cell death, with combination indices of 0.5 and lower. Mechanistic studies indicated that drug sensitivity and Mdm2 expression were dependent on p73. Mdm2 localized to a larger degree in the chromatin fraction isolated from cells treated with the combination treatment consistent with observations by others that Mdm2 binds to the Mre11/Rad50/Nbs1 complex, inhibits the DNA damage response, and increases drug sensitivity. In vivo efficacy experiments were conducted in the TMD231 orthotopic mammary fat pad model in NOD.Cg-PrkdcscidIl2rgtm1Wjl/SzJ (NSG) mice. For assessment of baseline tumor burden and randomization, fluorescent imaging of E2-Crimson expressing TMD231 cells was performed. Following Nutlin-3a and carboplatin combination treatment, there was a statistically significant reduction in primary tumor volume as well as lung metastases with significantly increased probability of survival compared to Vehicle and single drug treatments (p<0.001). While there was a decrease in bone-marrow cellularity, this did not lead to bone-marrow aplasia, and body weights recovered to normal levels within 7 days post-treatment. The present studies demonstrate the promise of Mdm2 as a therapeutic target in combination with conventional therapy, increase our understanding of how to potentiate DNA damage in cancers, and may lead to new clinical therapies for triple-negative primary and metastatic breast cancer.

Indiana University-Purdue University Indianapolis (IUPUI) / Mammary ductal epithelial cell growth is controlled by microenvironmental signals in serum under both normal physiological settings and during breast cancer progression. Importantly, the effects of several of these microenvironmental signals are mediated by the activities of the tumor suppressor protein kinases of the Hippo pathway. Canonically, Hippo protein kinases inhibit cellular growth through the phosphorylation and inactivation of the oncogenic transcriptional co-activator Yes-Associated Protein (YAP). This study defines an alternative mechanism whereby Hippo protein kinases induce growth arrest via the phosphorylation of the long isoform of Angiomotin (Amot130). Specifically, serum starvation is found to activate the Hippo protein kinase, Large Tumor Suppressor (LATS), which phosphorylates the adapter protein Amot130 at serine-175. Importantly, wild-type Amot130 potently inhibits mammary epithelial cell growth, unlike the Amot130 serine-175 to alanine mutant, which cannot be phosphorylated at this residue. The growth-arrested phenotype of Amot130 is likely a result of its mechanistic response to LATS signaling. Specifically, LATS activity promotes the association of Amot130 with the ubiquitin ligase Atrophin-1 Interacting Protein 4 (AIP4). As a consequence, the Amot130-AIP4 complex amplifies LATS tumor suppressive signaling by stabilizing LATS protein steady state levels via preventing AIP4-targeted degradation of LATS. Additionally, AIP4 binding to Amot130 leads to the ubiquitination and stabilization of Amot130. In turn, the Amot130-AIP4 complex signals the ubiquitination and degradation of YAP. This inhibition of YAP activity by Amot130 requires both AIP4 and the ability of Amot130 to be phosphorylated by LATS. Together, these findings significantly modify the current view that the phosphorylation of YAP by Hippo protein kinases is sufficient for YAP inhibition and cellular growth arrest. Based upon these results, the inhibition of cellular growth in the absence of serum more accurately involves the stabilization of Amot130 and LATS, which together inhibit YAP activity and mammary epithelial cell growth.