Diagnosis of ductal carcinoma in situ (DCIS) presents a challenge as we cannot yet distinguish between those lesions that remain dormant from cases that may progress to invasive ductal breast cancer (IDC) and require therapeutic intervention. Our overall interest is to develop biomimetic three-dimensional (3D) models that more accurately recapitulate the structure and characteristics of pre-invasive breast cancer in order to study the underlying mechanisms driving malignant progression. These models allow us to mimic the microenvironment to investigate many aspects of mammary cell biology, including the role of the extracellular matrix (ECM), the interaction between carcinoma-associated fibroblasts (CAFs) and epithelial cells, and the dynamics of cytoskeletal reorganization. In this review article, we outline the significance of 3D culture models as reliable pre-clinical tools that mimic the in vivo tumor microenvironment and facilitate the study of DCIS lesions as they progress to invasive breast cancer. We also discuss the role of CAFs and other stromal cells in DCIS transition as well as the clinical significance of emerging technologies like tumor-on-chip and co-culture models.

Supplementary Figures 1-5, Tables 1-3 from Bicarbonate Increases Tumor pH and Inhibits Spontaneous Metastases

AVI file - 4.8MB, Degradation of collagen I by 4T1.2 CTSBkd cells

PDF file - 241K, Histological analysis of LC3 spatial expression patterns in relation to pimonidazole hydrochloride (Hypoxia) and Glut1 expression. MDA-MB-231 MFP tumor was histologically stained for LC3, Glut1, and pimonidazole hydrochloride. Pixel analysis was performed to highlight the areas of highest expression. High spatial concordance was observed for all three histological markers. Pixel analysis was performed using AperioTM Positive Pixel Count v9 (Red - strong positive)

Supplementary Table S2 from Hu/Mu ProtIn Oligonucleotide Microarray: Dual-Species Array for Profiling Protease and Protease Inhibitor Gene Expression in Tumors and Their Microenvironment

Supplementary Legends for Figure 1, Movies 1-3 from Fibroblast Hepatocyte Growth Factor Promotes Invasion of Human Mammary Ductal Carcinoma In situ

Abstract Metastasis to bone is a major cause of morbidity in breast cancer patients, emphasizing the importance of identifying molecular drivers of bone metastasis for new therapeutic targets. The endogenous cysteine cathepsin inhibitor stefin A is a suppressor of breast cancer metastasis to bone that is coexpressed with cathepsin B in bone metastases. In this study, we used the immunocompetent 4T1.2 model of breast cancer which exhibits spontaneous bone metastasis to evaluate the function and therapeutic targeting potential of cathepsin B in this setting of advanced disease. Cathepsin B abundancy in the model mimicked human disease, both at the level of primary tumors and matched spinal metastases. RNA interference–mediated knockdown of cathepsin B in tumor cells reduced collagen I degradation in vitro and bone metastasis in vivo. Similarly, intraperitoneal administration of the highly selective cathepsin B inhibitor CA-074 reduced metastasis in tumor-bearing animals, a reduction that was not reproduced by the broad spectrum cysteine cathepsin inhibitor JPM-OEt. Notably, metastasis suppression by CA-074 was maintained in a late treatment setting, pointing to a role in metastatic outgrowth. Together, our findings established a prometastatic role for cathepsin B in distant metastasis and illustrated the therapeutic benefits of its selective inhibition in vivo. Cancer Res; 72(5); 1199–209. ©2012 AACR.

AVI file - 4.7MB, Degradation of collagen I by 4T1.2 cells + CA-074 inhibitor

PDF file - 344K, (A) Reference guide for the phospho-protein array. (B) Representative fluorescent arrays for whole cell lysates from MDA-MB-231 cells cultured at pH 7.4 or pH 6.7 for 72 hours. (C-D) Quantitative analysis of fluorescence intensity for all phosphorylated proteins. Data represent the mean S.D. of two replicate arrays for each sample

Supplementary Figure 1 from Lysosomal Cathepsin B Participates in the Podosome-Mediated Extracellular Matrix Degradation and Invasion via Secreted Lysosomes in v-Src Fibroblasts

PDF file - 287K, Figure S1: Photomicrographs of cells in this study. HMEC, HCT116/GFP and MDAmb231 cells were grown to 70-80% confluency. Samples were then viewed with an automated Zeiss Observer Z.1 inverted microscope through a 10x /0.3NA or 20x/0.4NA objective and a phase contrast ring. Images were produced using the AxioCam MRm CCD camera and Axiovision version 4.6 software suite. (Carl Zeiss Inc., Germany).

Supplementary Movie 3 from Lysosomal Cathepsin B Participates in the Podosome-Mediated Extracellular Matrix Degradation and Invasion via Secreted Lysosomes in v-Src Fibroblasts

Supplementary Figure S1 from Hu/Mu ProtIn Oligonucleotide Microarray: Dual-Species Array for Profiling Protease and Protease Inhibitor Gene Expression in Tumors and Their Microenvironment

Supplementary Figure and Movie Legends 1-3 from Lysosomal Cathepsin B Participates in the Podosome-Mediated Extracellular Matrix Degradation and Invasion via Secreted Lysosomes in v-Src Fibroblasts

PDF file - 431K, Histological analysis of in-vivo LC3 expression in HS766T subcutaneous tumors buffered with sodium bicarbonate. (A) Whole cell lysates from HS766T cells cultured at neutral pH 7.4 or pH 6.7 for 48 hours were analyzed for LC3-II expression. (B) Positive pixel analysis was completed for LC3 staining on whole tissue cross sections from HS766T tumors treated with tap or NaHCO3. An overall significant decrease in total positive and strong positive LC3 pixels was observed in NaHCO3 treated samples. The data are plotted as the mean standard deviation of three whole tumor cross sections from each treatment group. (C) A 5x magnification of representative tissue regions from HS766T tumors stained for LC3

Supplementary Movie 2 from Lysosomal Cathepsin B Participates in the Podosome-Mediated Extracellular Matrix Degradation and Invasion via Secreted Lysosomes in v-Src Fibroblasts

Supplementary Methods; Figure S1: Immunoblot and immunofluorescence analysis of MET and HGF in TNBC and fibroblast cell lines. Figure S2: Met inhibition has minimal effect on ERK5 signaling in human TNBC cells. Figure S3: XL184 and U0126 significantly reduce volume and outgrowths of MDA-MB-231 structures. Figure S4: Minimal growth recovery is observed after XL184 removal Figure S5: MET inhibition in C3H-SCID mice.

Abstract The external pH of solid tumors is acidic as a consequence of increased metabolism of glucose and poor perfusion. Acid pH has been shown to stimulate tumor cell invasion and metastasis in vitro and in cells before tail vein injection in vivo. The present study investigates whether inhibition of this tumor acidity will reduce the incidence of in vivo metastases. Here, we show that oral NaHCO3 selectively increased the pH of tumors and reduced the formation of spontaneous metastases in mouse models of metastatic breast cancer. This treatment regimen was shown to significantly increase the extracellular pH, but not the intracellular pH, of tumors by 31P magnetic resonance spectroscopy and the export of acid from growing tumors by fluorescence microscopy of tumors grown in window chambers. NaHCO3 therapy also reduced the rate of lymph node involvement, yet did not affect the levels of circulating tumor cells, suggesting that reduced organ metastases were not due to increased intravasation. In contrast, NaHCO3 therapy significantly reduced the formation of hepatic metastases following intrasplenic injection, suggesting that it did inhibit extravasation and colonization. In tail vein injections of alternative cancer models, bicarbonate had mixed results, inhibiting the formation of metastases from PC3M prostate cancer cells, but not those of B16 melanoma. Although the mechanism of this therapy is not known with certainty, low pH was shown to increase the release of active cathepsin B, an important matrix remodeling protease. [Cancer Res 2009;69(6):2260–8]