Modeling Tumor: Lymphatic Interactions in Lymphatic Metastasis of Triple Negative Breast Cancer.

Breast cancer frequently metastasizes to lymphatics and the presence of breast cancer cells in regional lymph nodes is an important prognostic factor. Delineating the mechanisms by which breast cancer cells disseminate and spatiotemporal aspects of interactions between breast cancer cells and lymphatics is needed to design new therapies to prevent lymphatic metastases. As triple-negative breast cancer (TNBC) has a high incidence of lymphatic metastasis, we used a three-dimensional (3D) coculture model of human TNBC cells and human microvascular lymphatic endothelial cells (LECs) to analyze TNBC:LEC interactions. Non-invasive analyses such as live-cell imaging in real-time and collection of conditioned media for secretomic analysis were facilitated by our novel microfluidic chambers. The volumes of 3D structures formed in TNBC:LEC cocultures are greater than that of 3D structures formed by either LEC or TNBC monocultures. Over 4 days of culture there is an increase in multicellular invasive outgrowths from TNBC spheroids and an association of TNBC spheroids with LEC networks. The increase in invasive phenotype also occurred when TNBC spheroids were cultured in LEC-conditioned media and in wells linked to ones containing LEC networks. Our results suggest that modeling spatiotemporal interactions between TNBC and LECs may reveal paracrine signaling that could be targeted to reduce lymphatic metastasis.

Spatio-temporal modeling and live-cell imaging of proteolysis in the 4D microenvironment of breast cancer.

Cells grown in three dimensions (3D) within natural extracellular matrices or synthetic scaffolds more closely recapitulate the phenotype of those cells within tissues in regard to normal developmental and pathobiological processes. This includes degradation of the surrounding stroma as the cells migrate and invade through the matrices. As 3D cultures of tumor cells predict efficacy of, and resistance to, a wide variety of cancer therapies, we employed tissue-engineering approaches to establish 3D pathomimetic avatars of human breast cancer cells alone and in the context of both their cellular and pathochemical microenvironments. We have shown that we can localize and quantify key parameters of malignant progression by live-cell imaging of the 3D avatars over time (4D). One surrogate for changes in malignant progression is matrix degradation, which can be localized and quantified by our live-cell proteolysis assay. This assay is predictive of changes in spatio-temporal and dynamic interactions among the co-cultured cells and changes in viability, proliferation, and malignant phenotype. Furthermore, our live-cell proteolysis assay measures the effect of small-molecule inhibitors of proteases and kinases, neutralizing or blocking antibodies to cytokines and photodynamic therapy on malignant progression. We suggest that 3D/4D pathomimetic avatars in combination with our live-cell proteolysis assays will be a useful preclinical screening platform for cancer therapies. Our ultimate goal is to develop 3D/4D avatars from an individual patient's cancer in which we can screen "personalized medicine" therapies using changes in proteolytic activity to quantify therapeutic efficacy.

Pathomimetic avatars reveal divergent roles of microenvironment in invasive transition of ductal carcinoma in situ.

BACKGROUND: The breast tumor microenvironment regulates progression of ductal carcinoma in situ (DCIS) to invasive ductal carcinoma (IDC). However, it is unclear how interactions between breast epithelial and stromal cells can drive this progression and whether there are reliable microenvironmental biomarkers to predict transition of DCIS to IDC. METHODS: We used xenograft mouse models and a 3D pathomimetic model termed mammary architecture and microenvironment engineering (MAME) to study the interplay between human breast myoepithelial cells (MEPs) and cancer-associated fibroblasts (CAFs) on DCIS progression. RESULTS: Our results show that MEPs suppress tumor formation by DCIS cells in vivo even in the presence of CAFs. In the in vitro MAME model, MEPs reduce the size of 3D DCIS structures and their degradation of extracellular matrix. We further show that the tumor-suppressive effects of MEPs on DCIS are linked to inhibition of urokinase plasminogen activator (uPA)/urokinase plasminogen activator receptor (uPAR)-mediated proteolysis by plasminogen activator inhibitor 1 (PAI-1) and that they can lessen the tumor-promoting effects of CAFs by attenuating interleukin 6 (IL-6) signaling pathways. CONCLUSIONS: Our studies using MAME are, to our knowledge, the first to demonstrate a divergent interplay between MEPs and CAFs within the DCIS tumor microenvironment. We show that the tumor-suppressive actions of MEPs are mediated by PAI-1, uPA and its receptor, uPAR, and are sustained even in the presence of the CAFs, which themselves enhance DCIS tumorigenesis via IL-6 signaling. Identifying tumor microenvironmental regulators of DCIS progression will be critical for defining a robust and predictive molecular signature for clinical use.

Live-Cell Imaging of Protease Activity: Assays to Screen Therapeutic Approaches.

Methodologies to image and quantify the activity of proteolytic enzymes have been developed in an effort to identify protease-related druggable pathways that are involved in malignant progression of cancer. Our laboratory has pioneered techniques for functional live-cell imaging of protease activity in pathomimetic avatars for breast cancer. We analyze proteolysis in the context of proliferation and formation of structures by tumor cells in 3-D cultures over time (4D). In order to recapitulate the cellular composition and architecture of tumors in the pathomimetic avatars, we include other tumor-associated cells (e.g., fibroblasts, myoepithelial cells, microvascular endothelial cells). We also model noncellular aspects of the tumor microenvironment such as acidic pericellular pH. Use of pathomimetic avatars in concert with various types of imaging probes has allowed us to image, quantify, and follow the dynamics of proteolysis in the tumor microenvironment and to test interventions that impact directly or indirectly on proteolytic pathways. To facilitate use of the pathomimetic avatars for screening of therapeutic modalities, we have designed and fabricated custom 3D culture chambers with multiple wells that are either individual or connected by a channel to allow cells to migrate between wells. Optical glass microscope slides underneath an acrylic plate allow the cultures to be imaged with an inverted microscope. Fluid ports in the acrylic plate are at a level above the 3D cultures to allow introduction of culture media and test agents such as drugs into the wells and the harvesting of media conditioned by the cultures for immunochemical and biochemical analyses. We are using the pathomimetic avatars to identify druggable pathways, screen drug and natural product libraries and accelerate entry of validated drugs or natural products into clinical trials.

Cabozantinib (XL184) Inhibits Growth and Invasion of Preclinical TNBC Models.

PURPOSE: Triple-negative breast cancer (TNBC) is an aggressive breast cancer subtype that is associated with poor clinical outcome. There is a vital need for effective targeted therapeutics for TNBC patients, yet treatment strategies are challenged by the significant intertumoral heterogeneity within the TNBC subtype and its surrounding microenvironment. Receptor tyrosine kinases (RTK) are highly expressed in several TNBC subtypes and are promising therapeutic targets. In this study, we targeted the MET receptor, which is highly expressed across several TNBC subtypes. EXPERIMENTAL DESIGN: Using the small-molecule inhibitor cabozantinib (XL184), we examined the efficacy of MET inhibition in preclinical models that recapitulate human TNBC and its microenvironment. To analyze the dynamic interactions between TNBC cells and fibroblasts over time, we utilized a 3D model referred to as MAME (Mammary Architecture and Microenvironment Engineering) with quantitative image analysis. To investigate cabozantinib inhibition in vivo, we used a novel xenograft model that expresses human HGF and supports paracrine MET signaling. RESULTS: XL184 treatment of MAME cultures of MDA-MB-231 and HCC70 cells (± HGF-expressing fibroblasts) was cytotoxic and significantly reduced multicellular invasive outgrowths, even in cultures with HGF-expressing fibroblasts. Treatment with XL184 had no significant effects on MET(neg) breast cancer cell growth. In vivo assays demonstrated that cabozantinib treatment significantly inhibited TNBC growth and metastasis. CONCLUSIONS: Using preclinical TNBC models that recapitulate the breast tumor microenvironment, we demonstrate that cabozantinib inhibition is an effective therapeutic strategy in several TNBC subtypes.

Imaging Sites of Inhibition of Proteolysis in Pathomimetic Human Breast Cancer Cultures by Light-Activated Ruthenium Compound.

The cysteine protease cathepsin B has been causally linked to progression and metastasis of breast cancers. We demonstrate inhibition by a dipeptidyl nitrile inhibitor (compound 1) of cathepsin B activity and also of pericellular degradation of dye-quenched collagen IV by living breast cancer cells. To image, localize and quantify collagen IV degradation in real-time we used 3D pathomimetic breast cancer models designed to mimic the in vivo microenvironment of breast cancers. We further report the synthesis and characterization of a caged version of compound 1, [Ru(bpy)2(1)2](BF4)2 (compound 2), which can be photoactivated with visible light. Upon light activation, compound 2, like compound 1, inhibited cathepsin B activity and pericellular collagen IV degradation by the 3D pathomimetic models of living breast cancer cells, without causing toxicity. We suggest that caged inhibitor 2 is a prototype for cathepsin B inhibitors that can control both the site and timing of inhibition in cancer.

Il-6 signaling between ductal carcinoma in situ cells and carcinoma-associated fibroblasts mediates tumor cell growth and migration.

BACKGROUND: Ductal carcinoma in situ (DCIS) is a non-obligate precursor lesion of invasive breast cancer in which approximately half the patients will progress to invasive cancer. Gaining a better understanding of DCIS progression may reduce overtreatment of patients. Expression of the pro-inflammatory cytokine interleukin-6 increases with pathological stage and grade, and is associated with poorer prognosis in breast cancer patients. Carcinoma associated fibroblasts (CAFs), which are present in the stroma of DCIS patients are known to secrete pro-inflammatory cytokines and promote tumor progression. METHODS: We hypothesized that IL-6 paracrine signaling between DCIS cells and CAFs mediates DCIS proliferation and migration. To test this hypothesis, we utilized the mammary architecture and microenvironment engineering or MAME model to study the interactions between human breast CAFs and human DCIS cells in 3D over time. We specifically inhibited autocrine and paracrine IL-6 signaling to determine its contribution to early stage tumor progression. RESULTS: Here, DCIS cells formed multicellular structures that exhibited increased proliferation and migration when cultured with CAFs. Treatment with an IL-6 neutralizing antibody inhibited growth and migration of the multicellular structures. Moreover, selective knockdown of IL-6 in CAFs, but not in DCIS cells, abrogated the migratory phenotype. CONCLUSION: Our results suggest that paracrine IL-6 signaling between preinvasive DCIS cells and stromal CAFs represent an important factor in the initiation of DCIS progression to invasive breast carcinoma.

Cathepsin B-deficient mice as source of monoclonal anti-cathepsin B antibodies.

Cathepsin B has been demonstrated to be involved in several proteolytic processes that support tumor progression and metastasis and neurodegeneration. To further clarify its role, defined monoclonal antibodies are needed. As the primary structure of human cathepsin B is almost identical to that of the mouse, cathepsin B-deficient mice were used in a novel approach for generating such antibodies, providing the chance of an increased immune response to the antigen, human cathepsin B. Thirty clones were found to produce cathepsin B-specific antibodies. Seven of these antibodies were used to detect cathepsin B in MCF10-DCIS human breast cancer cells by immunocytochemistry and immunoblotting. Five different binding sites were identified by epitope mapping giving the opportunity to combine these antibodies in oligoclonal antibody mixtures for an improved detection of cathepsin B.

Cathepsin B contributes to Na+ hyperabsorption in cystic fibrosis airway epithelial cultures.

In cystic fibrosis (CF) lung disease, the absence of functional CF transmembrane conductance regulator results in Cl(-)/HCO3 (-) hyposecretion and triggers Na(+) hyperabsorption through the epithelial Na(+) channel (ENaC), which contribute to reduced airway surface liquid (ASL) pH and volume. Prostasin, a membrane-anchored serine protease with trypsin-like substrate specificity has previously been shown to activate ENaC in CF airways. However, prostasin is typically inactive below pH 7.0, suggesting that it may be less relevant in acidic CF airways. Cathepsin B (CTSB) is present in both normal and CF epithelia and is secreted into ASL, but little is known about its function in the airways. We hypothesized that the acidic ASL seen in CF airways may stimulate CTSB to activate ENaC, contributing to Na(+) hyperabsorption and depletion of CF ASL volume. In Xenopus laevis oocytes, CTSB triggered α- and γENaC cleavage and induced an increase in ENaC activity. In bronchial epithelia from both normal and CF donor lungs, CTSB localized to the apical membrane. In normal and CF human bronchial epithelial cultures, CTSB was detected at the apical plasma membrane and in the ASL. CTSB activity was significantly elevated in acidic ASL, which correlated with increased abundance of ENaC in the plasma membrane and a reduction in ASL volume. This acid/CTSB-dependent activation of ENaC was ameliorated with the cell impermeable, CTSB-selective inhibitor CA074, suggesting that CTSB inhibition may have therapeutic relevance. Taken together, our data suggest that CTSB is a pathophysiologically relevant protease that activates ENaC in CF airways.

Deficiency in metabolic regulators PPARγ and PTEN cooperates to drive keratinizing squamous metaplasia in novel models of human tissue regeneration.

Hindgut-derived endoderm can differentiate into rectal, prostatic, and bladder phenotypes. Stromal-epithelial interactions are crucial for this development; however, the precise mechanisms by which epithelium responds to stromal cues remain unknown. We have previously reported ectopic expression of peroxisome proliferator-activated receptor-γ2 (PPARγ2) increased androgen receptor expression and promoted differentiation of mouse prostate epithelium. PPARγ is also implicated in urothelial differentiation. Herein we demonstrate that knockdown of PPARγ2 in benign human prostate epithelial cells (BHPrEs) promotes urothelial transdifferentiation. Furthermore, in vitro and in vivo heterotypic tissue regeneration models with embryonic bladder mesenchyme promoted urothelial differentiation of PPARγ2-deficient BHPrE cells, and deficiency of both PPARγ isoforms 1 and 2 arrested differentiation. Because PTEN deficiency is cooperative in urothelial pathogenesis, we engineered BHPrE cells with combined knockdown of PPARγ and PTEN and performed heterotypic recombination experiments using embryonic bladder mesenchyme. Whereas PTEN deficiency alone induced latent squamous differentiation in BHPrE cells, combined PPARγ and PTEN deficiency accelerated the development of keratinizing squamous metaplasia (KSM). We further confirmed via immunohistochemistry that gene expression changes in metaplastic recombinants reflected human urothelium undergoing KSM. In summary, these data suggest that PPARγ isoform expression provides a molecular basis for observations that adult human epithelium can be transdifferentiated on the basis of heterotypic mesenchymal induction. These data also implicate PPARγ and PTEN inactivation in the development of KSM.

RNA-Seq of human breast ductal carcinoma in situ models reveals aldehyde dehydrogenase isoform 5A1 as a novel potential target.

Breast ductal carcinoma in situ (DCIS) is being found in great numbers of women due to the widespread use of mammography. To increase knowledge of DCIS, we determined the expression changes that are common among three DCIS models (MCF10.DCIS, SUM102 and SUM225) compared to the MCF10A model of non-tumorigenic mammary epithelial cells in three dimensional (3D) overlay culture with reconstituted basement membrane (rBM). Extracted mRNA was subjected to 76 cycles of deep sequencing (RNA-Seq) using Illumina Genome Analyzer GAIIx. Analysis of RNA-Seq results showed 295 consistently differentially expressed transcripts in the DCIS models. These differentially expressed genes encode proteins that are associated with a number of signaling pathways such as integrin, fibroblast growth factor and TGFß signaling, show association with cell-cell signaling, cell-cell adhesion and cell proliferation, and have a notable bias toward localization in the extracellular and plasma membrane compartments. RNA-Seq data was validated by quantitative real-time PCR of selected differentially expressed genes. Aldehyde dehydrogenase 5A1 (ALDH5A1) which is an enzyme that is involved in mitochondrial glutamate metabolism, was over-expressed in all three DCIS models at both the mRNA and protein levels. Disulfiram and valproic acid are known to inhibit ALDH5A1 and are safe for chronic use in humans for other disorders. Both of these drugs significantly inhibited net proliferation of the DCIS 3D rBM overlay models, but had minimal effect on MCF10A 3D rBM overlay models. These results suggest that ALDH5A1 may play an important role in DCIS and potentially serve as a novel molecular therapeutic target.

MAME models for 4D live-cell imaging of tumor: microenvironment interactions that impact malignant progression.

We have developed 3D coculture models, which we term MAME (mammary architecture and microenvironment engineering), and used them for live-cell imaging in real-time of cell:cell interactions. Our overall goal was to develop models that recapitulate the architecture of preinvasive breast lesions to study their progression to an invasive phenotype. Specifically, we developed models to analyze interactions among pre-malignant breast epithelial cell variants and other cell types of the tumor microenvironment that have been implicated in enhancing or reducing the progression of preinvasive breast epithelial cells to invasive ductal carcinomas. Other cell types studied to date are myoepithelial cells, fibroblasts, macrophages and blood and lymphatic microvascular endothelial cells. In addition to the MAME models, which are designed to recapitulate the cellular interactions within the breast during cancer progression, we have developed comparable models for the progression of prostate cancers. Here we illustrate the procedures for establishing the 3D cocultures along with the use of live-cell imaging and a functional proteolysis assay to follow the transition of cocultures of breast ductal carcinoma in situ (DCIS) cells and fibroblasts to an invasive phenotype over time, in this case over twenty-three days in culture. The MAME cocultures consist of multiple layers. Fibroblasts are embedded in the bottom layer of type I collagen. On that is placed a layer of reconstituted basement membrane (rBM) on which DCIS cells are seeded. A final top layer of 2% rBM is included and replenished with every change of media. To image proteolysis associated with the progression to an invasive phenotype, we use dye-quenched (DQ) fluorescent matrix proteins (DQ-collagen I mixed with the layer of collagen I and DQ-collagen IV mixed with the middle layer of rBM) and observe live cultures using confocal microscopy. Optical sections are captured, processed and reconstructed in 3D with Volocity visualization software. Over the course of 23 days in MAME cocultures, the DCIS cells proliferate and coalesce into large invasive structures. Fibroblasts migrate and become incorporated into these invasive structures. Fluorescent proteolytic fragments of the collagens are found in association with the surface of DCIS structures, intracellularly, and also dispersed throughout the surrounding matrix. Drugs that target proteolytic, chemokine/cytokine and kinase pathways or modifications in the cellular composition of the cocultures can reduce the invasiveness, suggesting that MAME models can be used as preclinical screens for novel therapeutic approaches.

3D/4D functional imaging of tumor-associated proteolysis: impact of microenvironment.

Proteases play causal roles in many aspects of the aggressive phenotype of tumors, yet many of the implicated proteases originate from tumor-associated cells or from responses of tumor cells to interactions with other cells. Therefore, to obtain a comprehensive view of tumor proteases, we need to be able to assess proteolysis in tumors that are interacting with their microenvironment. As this is difficult to do in vivo, we have developed functional live-cell optical imaging assays and 3D and 4D (i.e., 3D over time) coculture models. We present here a description of the probes used to measure proteolysis and protease activities, the methods used for imaging and analysis of proteolysis and the 3D and 4D models used in our laboratory. Of course, all assays have limitations; however, we suggest that the techniques discussed here will, with attention to their limitations, be useful as a screen for drugs to target the invasive phenotype of tumors.

Cathepsin B inhibition limits bone metastasis in breast cancer.

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.

Live-cell imaging of tumor proteolysis: impact of cellular and non-cellular microenvironment.

Our laboratory has had a longstanding interest in how the interactions between tumors and their microenvironment affect malignant progression. Recently, we have focused on defining the proteolytic pathways that function in the transition of breast cancer from the pre-invasive lesions of ductal carcinoma in situ (DCIS) to invasive ductal carcinomas (IDCs). We use live-cell imaging to visualize, localize and quantify proteolysis as it occurs in real-time and thereby have established roles for lysosomal cysteine proteases both pericellularly and intracellularly in tumor proteolysis. To facilitate these studies, we have developed and optimized 3D organotypic co-culture models that recapitulate the in vivo interactions of mammary epithelial cells or tumor cells with stromal and inflammatory cells. Here we will discuss the background that led to our present studies as well as the techniques and models that we employ. This article is part of a Special Issue entitled: Proteolysis 50 years after the discovery of lysosome.

Imaging and quantifying the dynamics of tumor-associated proteolysis.

The roles of proteases in cancer are dynamic. Furthermore, the roles or functions of any one protease may differ from one stage of cancer to another. Proteases from tumor-associated cells (e.g., fibroblasts, inflammatory cells, endothelial cells) as well as from tumor cells make important contributions to 'tumor proteolysis'. Many tumors exhibit increases in expression of proteases at the level of transcripts and protein; however, whether those proteases play causal roles in malignant progression is known for only a handful of proteases. What the critical substrate or substrates that are cleaved in vivo by any given protease is also known for only a few proteases. Therefore, the recent development of techniques and reagents for live cell imaging of protease activity, in conjunction with informed knowledge of critical natural substrates, should help to define protease functions. Here we describe live cell assays for imaging proteolysis, protocols for quantifying proteolysis and the use of such assays to follow the dynamics of proteolysis by tumor cells alone and tumor cells interacting with other cells found in the tumor microenvironment. In addition, we describe an in vitro model that recapitulates the architecture of the mammary gland, a model designed to determine the effects of dynamic interactions with the surrounding microenvironment on 'tumor proteolysis' and the respective contributions of various cell types to 'tumor proteolysis'. The assays and models described here could serve as screening platforms for the identification of proteolytic pathways that are potential therapeutic targets and for further development of technologies and imaging probes for in vivo use.

Cathepsin B protein levels in endometrial cancer: Potential value as a tumour biomarker.

OBJECTIVE: Lysosomal cysteine protease Cathepsin-B has been implicated in the progression of various human tumours. We examined Cathepsin-B protein levels in endometrial carcinoma patients-mainly post-menopausal-and investigated their possible association with clinical and pathological parameters in order to assess Cathepsin-B's significance as a potential tumour biomarker. METHODS: The indirect immunoperoxidase method was used for Cathepsin-B immunohistochemical staining of 64 paraffin-embedded endometrial tumour tissues, having follow-up period of 18-240 months. Steroid hormone receptors were measured as well. Tissue samples were staged following the FIGO criteria. RESULTS: Positive Cathepsin-B immunostaining was observed in 27 patients (42.2%) and was significantly associated with the FIGO stage of the disease (p=0.006), as well as cervical and stromal invasion (p=0.001 and p=0.037, respectively) and progesterone receptor status (p=0.027). Positive Cathepsin-B expression was also inversely related to Disease-free Survival (p=0.034) and Overall Survival (p=0.035) in univariate analysis, as well as in multivariate analysis (p=0.022 and p=0.035, respectively). CONCLUSION: Increased Cathepsin-B expression was found to be predictive of more aggressive tumour behaviour over time and can be regarded as an unfavourable and independent tumour marker for endometrial cancer patients with a long follow-up.

Visualizing protease activity in living cells: from two dimensions to four dimensions.

Proteolytic degradation of extracellular matrix (ECM) components by cells is an important metabolic activity as cells grow, remodel, and migrate through the ECM. The ability to analyze ECM degradation can be valuable in the study of developmental processes as well as pathologies, such as cancer. In this unit we describe an in vitro live cell-based method to image and quantitatively measure the degradation of ECM components by live cells. Cells are grown in the presence of fluorescent dye-quenched protein substrates (DQ-gelatin, DQ-collagen I, and DQ-collagen IV) that are mixed with protein matrices. Upon proteolytic cleavage, fluorescence is released that directly reflects the level of proteolysis by the cells. Using confocal microscopy and advanced imaging software, the fluorescence is detected and accurate measurements of proteolytic degradation in three and four dimensions can be assessed.

Functional live-cell imaging demonstrates that beta1-integrin promotes type IV collagen degradation by breast and prostate cancer cells.

The ability of tumor cells to adhere to, migrate on, and remodel extracellular matrices is mediated by cell surface receptors such as beta1-integrins. Here we conducted functional live-cell imaging in real time to investigate the effects of modulating beta1-integrin expression and function on proteolytic remodeling of the extracellular matrix. Human breast and prostate cancer cells were grown on reconstituted basement membrane containing a quenched fluorescent form of collagen IV. Generation of cleavage products and the resulting increases in fluorescence were imaged and quantified. Decreases in the expression and activity of beta1-integrin reduced digestion of quenched fluorescent-collagen IV by the breast and prostate cancer cells and correspondingly their invasion through and migration on reconstituted basement membrane. Decreased extracellular matrix degradation also was associated with changes in the constituents of proteolytic pathways: decreases in secretion of the cysteine protease cathepsin B, the matrix metalloproteinase (MMP)-13, and tissue inhibitors of metalloproteinases (TIMP)-1 and 2; a decrease in expression of MMP-14 or membrane type 1 MMP; and an increase in secretion of TIMP-3. This is the first study to demonstrate through functional live-cell imaging that downregulation of beta1-integrin expression and function reduces proteolysis of collagen IV by breast and prostate cancer cells.

Functional imaging of tumor proteolysis.

The roles of proteases in cancer are now known to be much broader than simply degradation of extracellular matrix during tumor invasion and metastasis. Furthermore, proteases from tumor-associated cells (e.g., fibroblasts, inflammatory cells, endothelial cells) as well as tumor cells are recognized to contribute to pathways critical to neoplastic progression. Although elevated expression (transcripts and proteins) of proteases, and in some cases protease inhibitors, has been documented in many tumors, techniques to assess functional roles for proteases require that we measure protease activity and inhibition of that activity rather than levels of proteases, activators, and inhibitors. Novel techniques for functional imaging of protease activity, both in vitro and in vivo, are being developed as are imaging probes that will allow us to determine protease activity and in some cases to discriminate among protease activities. These should be useful clinically as surrogate endpoints for therapies that alter protease activities.