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Metastasis of primary mammary tumors to vital secondary organs is the primary cause of breast cancer-associated death, with no effective treatment. Metastasis is a highly selective process that requires cancer cells to overcome multiple barriers to escape the primary tumor, survive in circulation, and eventually colonize distant secondary organs. One of the important aspects of metastatic cancers is the ability to undergo epithelial-mesenchymal transition (EMT) and the reverse process mesenchymal-epithelial transition (MET) process. Constant interconversion of tumor cells between these phenotypes creates epithelial-mesenchymal heterogeneity (EMH) and interaction between these tumor cell types and the stromal cell compartment is clearly important to metastasis. In healthy tissues, stromal cells maintain the composition and structure of the tissue through the production of extracellular matrix (ECM) proteins and paracrine signaling with epithelial cells. However, little is known about how EMH promotes changes in the ECM to promote breast cancer progression and metastasis. Cancer cells also secret exosomes, nano-size extracellular vesicles, to establish intercellular communication with distant organs in order to induce metastasis. These exosomes contain a plethora of different proteins including extracellular matrix proteins and matrix crosslinking enzymes. Fibronectin, an important ECM protein, plays an active role in tumor progression and is often crosslinked by tissue transglutaminase 2 (TGM2) to promote fibrosis in cancer. Both FN and TGM2 exist in exosomes and are expressed by heterogenous breast tumors. Although FN and TGM2 have been reported to play essential roles in cancer, their involvement in metastasis remains unclear. This work utilizes a variety of approaches to investigate the role of tumor heterogeneity and ECM proteins in promoting breast cancer metastasis. In this dissertation, we establish that mesenchymal cells expressing intracellular FN are held in a stable non-metastatic mesenchymal phenotype and produce cellular fibrils containing functionalized FN capable of supporting the growth of metastatic competent epithelial cells. We introduce a novel 3D culture system consisting of a tessellated scaffold which is capable of recapitulating cellular and matrix phenotypes in vivo. Further, we also demonstrate breast tumor cells secrete exosomes containing TGM2 crosslinked FN fibrils to promote premetastatic niche formation and induction of metastasis. Using genetic approaches, we establish TGM2 is essential and sufficient to drive metastasis. Finally, we demonstrate pharmacological inhibition of TGM2 offers a potential therapeutic strategy to treat metastatic breast cancer. Altogether, our research provides insights into the mechanism through which TGM2 promotes metastatic breast cancer. This work will help in developing new drugs to target TGM2 aimed at reducing breast cancer metastasis.

Figure S1. PC expression is not correlated with primary tumor size. Patient samples within the METABRIC dataset were analyzed for PC expression in relation to primary tumor size. Data are analyzed by a nonparametric Spearman correlation resulting in the indicated r and P values. The linear regression for these data is also shown. (PDF 235 kb)

Figure S4. PC is not required for cell migration. (A) Control (shScram) and PC depleted (shPC28) cells were plated into 8 μm transwell inserts in serum-free media containing 1 or 4 mM pyruvate. Cell migration was quantified 12 h after plating using calcein AM. SFM represents wells where the bottom chamber was filled with serum-free media as negative control. Values are presented as mean relative fluorescence units, ±SEM. (B) Control (shScram) and PC-depleted (shPC28) 4 T1 monolayers were wounded and closure was measured 15 and 24 h later. Values are presented as mean percent would closure, ±SEM. (PDF 372 kb)

Acid peptic disease is the most common condition affecting the gastrointestinal tract. H. pylori, which colonizes the gastric submucosa, being the most prolific etiological agent and responsible for various gastrointestinal lesions like gastritis to gastric ulcer and sometime malignant lesion. The present study deals with the spectrum of lesions caused by H. pylori and their diagnosis by rapid urease test & histopathological examination of gastroduodenal biopsies. Total 100 patients with symptoms of acid peptic disease were evaluated and endoscopic biopsies from gastroduodenal site were obtained. The biopsy specimens were processed for rapid urease test & histopathological examination using H&E and special staining (Warthin-starry and Giemsa stain) techniques. Among the 100 cases, common age group of presentation was 31-40 years & male to female ratio was 1.78:1. On histopathological examination the most common lesion was chronic gastritis (33 cases) followed by duodenal ulcer (21 cases), gastric ulcer (12 cases) & gastric neoplastic lesion (14 cases). Among neoplastic lesion, adenocarcinoma was most common finding. Rapid urease test showed positive results for H. pylori in 61% cases and on histopathology in 63% of cases. H. pylori infection has significant role in the pathogenesis of acid peptic disease, of which gastritis was most common condition. Rapid urease test is simple and cost-effective. Histopathological examination using H&E and special stains are the gold standard tools for the diagnosis of H. pylori infection.

Cell-penetrating peptides (CPPs) are known as efficient transporters of molecular cargo across cellular membranes. Their properties make them ideal candidates for in vivo applications. However, challenges in the development of effective CPPs still exist: CPPs are often fast degraded by proteases and large concentration of CPPs required for cargo transporting can cause cytotoxicity. It was previously shown that restricting peptide flexibility can improve peptide stability against enzymatic degradation and limiting length of CPP peptide can lower cytotoxic effects. Here, we present peptides (30-mers) that efficiently penetrate cellular membranes by combining very short CPP sequences and collagen-like folding domains. The CPP domains are hexa-arginine (R6) or arginine/glycine (RRGRRG). Folding is achieved through multiple proline-hydroxyproline-glycine (POG [proline-hydroxyproline-glycine])n repeats that form a collagen-like triple helical conformation. The folded peptides with CPP domains are efficiently internalized, show stability against enzymatic degradation in human serum and have minimal toxicity. Peptides lacking correct folding (random coil) or CPP domains are unable to cross cellular membranes. These features make triple helical cell-penetrating peptides promising candidates for efficient transporters of molecular cargo across cellular membranes.

Figure S5. HIF-1α expression that characterizes primary tumors is lost in pulmonary metastases. 4 T1 cells were engrafted onto the mammary fat pad via an intraductal injection and grown as primary tumors. These tumors gave rise to spontaneous pulmonary metastases. Upon necropsy both primary and metastatic tumors were analyzed by immunohistochemistry for the expression of HIF-1α. Nuclear expression of HIF-1α is very high in viable primary tumor tissue. In contrast, nuclear HIF-1α is drastically reduced in pulmonary metastases. Data are representative of three separate mice bearing primary tumors and metastases. (PDF 1389 kb)

Figure S5. HIF-1α expression that characterizes primary tumors is lost in pulmonary metastases. 4 T1 cells were engrafted onto the mammary fat pad via an intraductal injection and grown as primary tumors. These tumors gave rise to spontaneous pulmonary metastases. Upon necropsy both primary and metastatic tumors were analyzed by immunohistochemistry for the expression of HIF-1α. Nuclear expression of HIF-1α is very high in viable primary tumor tissue. In contrast, nuclear HIF-1α is drastically reduced in pulmonary metastases. Data are representative of three separate mice bearing primary tumors and metastases. (PDF 1389 kb)

Figure S2. PC expression in primary breast tumors. PC expression within tumor cells was rated as high, medium, low, or negative (nâ =â 10). Representative sections from each group are shown. Data was obtained from the protein atlas dataset (www. proteinatlas.org ) [33]. (PDF 2805 kb)

Figure S3. Transient induction of EMT does not induce expression of PC. (A) Photomicrographs showing EMT-like changes in cellular morphology of the control (scram) and PC-depleted (shPC25 and shPC28) 4 T1 cells upon treatment with exogenous TGF-β1 (5 ng/ml) for 4 days. (B) Immunoblot analyses for PC in cells shown in Panel A. β-tubulin served as a loading control. (C) The RAS transformed MCF-10A-T1 k cells were constructed to express a doxycycline-inducible vector encoding the EMT transcription factor Snail. Expression of PC was analyzed upon addition of doxycycline (Dox) at the indicated concentrations. Expression of Snail and β-tubulin (β-Tub) served as loading controls. (PDF 1401 kb)

Figure S3. Transient induction of EMT does not induce expression of PC. (A) Photomicrographs showing EMT-like changes in cellular morphology of the control (scram) and PC-depleted (shPC25 and shPC28) 4 T1 cells upon treatment with exogenous TGF-β1 (5 ng/ml) for 4 days. (B) Immunoblot analyses for PC in cells shown in Panel A. β-tubulin served as a loading control. (C) The RAS transformed MCF-10A-T1 k cells were constructed to express a doxycycline-inducible vector encoding the EMT transcription factor Snail. Expression of PC was analyzed upon addition of doxycycline (Dox) at the indicated concentrations. Expression of Snail and β-tubulin (β-Tub) served as loading controls. (PDF 1401 kb)

Figure S2. PC expression in primary breast tumors. PC expression within tumor cells was rated as high, medium, low, or negative (nâ =â 10). Representative sections from each group are shown. Data was obtained from the protein atlas dataset (www. proteinatlas.org ) [33]. (PDF 2805 kb)