High-impact practices in cancer education and research: Undergraduate students' perceptions of skills and career development.

High-impact practices (HIPs) are educational practices that foster student success. HIPs have not been widely used in cancer education and research despite the need for students to develop key transferable skills and cultivate social responsibility. Our study addresses this need by implementing four community-based learning HIPs within the context of cancer education and research. Each HIP was classified as having low, moderate, or high alignment with the traits of effective HIPs. Undergraduate science students participated in one to four HIPs as a Feedback Participant, General Volunteer, Student Leader, or Cancer Undergraduate Research and Education (CURES) Class Student. We then studied the effect of these HIPs on students' development of knowledge and skills; career interest and preparedness; and social responsibility. Results from self-reported questionnaires showed that HIPs increased students' cancer knowledge and developed their transferable and technical skills. Many students reported that these HIPs strongly impacted their career preparedness; positively influenced their interest in pursuing careers in health or biomedical sciences; and encouraged them to participate in community service activities. Thus, these findings provide new insights into the perceived benefits of HIPs in cancer education and research by undergraduate students.

Addressing the Barriers to Clinical Trials Accrual in Community Cancer Centres Using a National Clinical Trials Navigator:A Cross-Sectional Analysis.

INTRODUCTION: Clinical trials, although academically accepted as the most effective treatment available for cancer patients, poor accrual to clinical trials remains a significant problem. A clinical trials navigator (CTN) program was piloted where patients and/or their healthcare professionals could request a search and provide a list of potential cancer clinical trials in which a patient may be eligible based on their current status and disease. OBJECTIVES: This study examined the outcomes of a pilot program to try to improve clinical trials accrual with a focus on patients at medium to small sized cancer programs. Outcomes examined included patient disposition (referral to and accrual to interventional trials), patient survival, sites of referral to the CTN program. METHODS: One 0.5 FTE navigator was retained. Stakeholders referred to the CTN through the Canadian Cancer Clinical Trials Network. Demographic and outcomes data were recorded. RESULTS: Between March 2019 and February 2020, 118 patients from across Canada used the program. Seven per cent of patients referred were enrolled onto treatment clinical trials. No available trial excluded 39% patients, and 28% had a decline in their health and died before they could be referred or enrolled onto a clinical trial. The median time from referral to death was 109 days in those that passed. CONCLUSION: This novel navigator pilot has the potential to increase patient accrual to clinical trials. The CTN program services the gap in the clinical trials system, helping patients in medium and small sized cancer centres identify potential clinical trials at larger centres.

Fitted filtration efficiency and breathability of 2-ply cotton masks: Identification of cotton consumer categories acceptable for home-made cloth mask construction.

The objective of this study was to characterize commercially-available cotton fabrics to determine their suitability as materials for construction of cloth masks for personal and public use to reduce infectious disease spread. The study focused on cottons because of their widespread availability, moderate performance and they are recommended for inclusion in home-made masks by international health authorities. Fifty-two cottons were analyzed by electron microscopy to determine fabric characteristics and fabric weights. Sixteen fabrics were selected to test for breathability and to construct 2-ply cotton masks of a standard design to use in quantitative fit testing on a human participant. Cotton mask fitted filtration efficiencies (FFEs) for 0.02-1 µm ambient and aerosolized sodium chloride particles ranged from 40 to 66% compared with the mean medical mask FFE of 55±2%. Pressure differentials across 2-ply materials ranged from 0.57 to > 12 mm H2O/cm2 on samples of equal surface area with 6 of 16 materials exceeding the recommended medical mask limit. Models were calibrated to predict 2-ply cotton mask FFEs and differential pressures for each fabric based on pore characteristics and fabric weight. Models indicated cotton fabrics from 6 of 9 consumer categories can produce cloth masks with adequate breathability and FFEs equivalent to a medical mask: T-shirt, fashion fabric, mass-market quilting cotton, home décor fabric, bed sheets and high-quality quilting cotton. Masks from one cloth mask and the medical mask were re-tested with a mask fitter to distinguish filtration from leakage. The fabric and medical masks had 3.7% and 41.8% leakage, respectively. These results indicate a well fitted 2-ply cotton mask with overhead ties can perform similarly to a disposable 3-ply medical mask on ear loops due primarily to the superior fit of the cloth mask which compensates for its lower material filtration efficiency.

A Hierarchical Machine Learning Model to Discover Gleason Grade-Specific Biomarkers in Prostate Cancer.

(1) Background:One of the most common cancers that affect North American men and men worldwide is prostate cancer. The Gleason score is a pathological grading system to examine the potential aggressiveness of the disease in the prostate tissue. Advancements in computing and next-generation sequencing technology now allow us to study the genomic profiles of patients in association with their different Gleason scores more accurately and effectively. (2) Methods: In this study, we used a novel machine learning method to analyse gene expression of prostate tumours with different Gleason scores, and identify potential genetic biomarkers for each Gleason group. We obtained a publicly-available RNA-Seq dataset of a cohort of 104 prostate cancer patients from the National Center for Biotechnology Information's (NCBI) Gene Expression Omnibus (GEO) repository, and categorised patients based on their Gleason scores to create a hierarchy of disease progression. A hierarchical model with standard classifiers in different Gleason groups, also known as nodes, was developed to identify and predict nodes based on their mRNA or gene expression. In each node, patient samples were analysed via class imbalance and hybrid feature selection techniques to build the prediction model. The outcome from analysis of each node was a set of genes that could differentiate each Gleason group from the remaining groups. To validate the proposed method, the set of identified genes were used to classify a second dataset of 499 prostate cancer patients collected from cBioportal. (3) Results: The overall accuracy of applying this novel method to the first dataset was 93.3%; the method was further validated to have 87% accuracy using the second dataset. This method also identified genes that were not previously reported as potential biomarkers for specific Gleason groups. In particular, PIAS3 was identified as a potential biomarker for Gleason score 4 + 3 = 7, and UBE2V2 for Gleason score 6. (4) Insight: Previous reports show that the genes predicted by this newly proposed method strongly correlate with prostate cancer development and progression. Furthermore, pathway analysis shows that both PIAS3 and UBE2V2 share similar protein interaction pathways, the JAK/STAT signaling process.

Transcriptomics Signature from Next-Generation Sequencing Data Reveals New Transcriptomic Biomarkers Related to Prostate Cancer.

Prostate cancer is one of the most common types of cancer among Canadian men. Next-generation sequencing using RNA-Seq provides large amounts of data that may reveal novel and informative biomarkers. We introduce a method that uses machine learning techniques to identify transcripts that correlate with prostate cancer development and progression. We have isolated transcripts that have the potential to serve as prognostic indicators and may have tremendous value in guiding treatment decisions. Analysis of normal versus malignant prostate cancer data sets indicates differential expression of the genes HEATR5B, DDC, and GABPB1-AS1 as potential prostate cancer biomarkers. Our study also supports PTGFR, NREP, SCARNA22, DOCK9, FLVCR2, IK2F3, USP13, and CLASP1 as potential biomarkers to predict prostate cancer progression, especially between stage II and subsequent stages of the disease.

IL-10 correlates with the expression of carboxypeptidase B2 and lymphovascular invasion in inflammatory breast cancer: The potential role of tumor infiltrated macrophages.

Pro-carboxypeptidase B2 (pro-CPB2) or thrombin-activatable fibrinolysis inhibitor (TAFI) is a glycoprotein encoded by the CPB2 gene and deregulated in several cancer types, including breast cancer. Thrombin binding to thrombomodulin (TM), encoded by THBD, is important for TAFI activation. CPB2 gene expression is influenced by genetic polymorphism and cytokines such as interleukin 10 (IL-10). Our previous results showed that tumor infiltrating monocytes/macrophages (CD14+/CD16+) isolated from inflammatory breast cancer (IBC) patients' secrete high levels of IL-10. The aim of the present study is to test genetic polymorphism and expression of CPB2 in healthy breast tissues and carcinoma tissues of non-IBC and IBC patients. Furthermore, to investigate whether IL-10 modulates the expression of CPB2 and THBD in vivo and in-vitro. We tested CPB2 Thr325Ile polymorphism using restriction fragment length polymorphism, (RFLP) technique in healthy and carcinoma breast tissues. The mRNA expression of CPB2, THBD and IL10 were assessed by RT-qPCR. Infiltration of CD14+ cells was assessed by immunohistochemistry. In addition, we investigated the correlation between infiltration of CD14+ cells and expression of IL10 and CPB2. Furthermore, we correlated IL10 expression with the expression of both CPB2 and THBD in breast carcinoma tissues. Finally, we validated the role of recombinant IL-10 in regulating the expression of CPB2 and THBD using different breast cancer cell lines. Our results showed that CPB2 genotypes carrying the high-risk allele [Thr/Ile (CT) and Ile/Ile (TT)] were more frequent in both IBC and non-IBC patients compared to control group. CPB2 genotypes did not show any statistical correlation with CPB2 mRNA expression levels or patients' clinical pathological properties. Interestingly, CPB2 and IL10 expression were significantly higher and positively correlated with the incidence of CD14+ cells in carcinoma tissues of IBC as compared to non-IBC. On the other hand, THBD expression was significantly lower in IBC carcinoma versus non-IBC tissues. Based on molecular subtypes, CPB2 and IL10 expression were significantly higher in triple negative (TN) as compared to hormonal positive (HP) carcinoma tissues of IBC. Moreover, CPB2 expression was positively correlated with presence of lymphovascular invasion and the expression of IL10 in carcinoma tissues of IBC patients. Furthermore, recombinant human IL-10 stimulated CPB2 expression in SUM-149 (IBC cell line) but not in MDA-MB-231 (non-IBC cell line), while there was no significant effect THBD expression. In conclusion, carcinoma tissues of IBC patients are characterized by higher expression of CPB2 and lower expression of THBD. Moreover, CPB2 positively correlates with IL10 mRNA expression, incidence of CD14+ cells and lymphovascular invasion in IBC patients. IL-10 stimulated CPB2 expression in TN-IBC cell line suggests a relevant role of CPB2 in the aggressive phenotype of IBC.

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.

Activated thrombin-activatable fibrinolysis inhibitor attenuates the angiogenic potential of endothelial cells: potential relevance to the breast tumour microenvironment.

Thrombin-activatable fibrinolysis inhibitor (TAFI) is a basic carboxypeptidase zymogen present in blood plasma. Proteolytic activation of TAFI by thrombin, thrombin in complex with the endothelial cell cofactor thrombomodulin, or plasmin results in an enzyme (TAFIa) that removes carboxyl-terminal lysine residues from protein and peptide substrates, including cell-surface plasminogen receptors. TAFIa is therefore capable of inhibiting plasminogen activation in the pericellular milieu. Since plasminogen activation has been linked to angiogenesis, TAFIa could therefore have anti-angiogenic properties, and indeed TAFIa has been shown to inhibit endothelial tube formation in a fibrin matrix. In this study, the TAFI pathway was manipulated by providing exogenous TAFI or TAFIa or by adding a potent and specific inhibitor of TAFIa. We found that TAFIa elicited a series of anti-angiogenic responses by endothelial cells, including decreased endothelial cell proliferation, cell invasion, cell migration, tube formation, and collagen degradation. Moreover, TAFIa decreased tube formation and proteolysis in endothelial cell culture grown alone and in co-culture with breast cancer cell lines. In accordance with these findings, inhibition of TAFIa increased secretion of matrix metalloprotease proenzymes by endothelial and breast cancer cells. Finally, treatment of endothelial cells with TAFIa significantly inhibited plasminogen activation. Taken together our results suggest a novel role for TAFI in inhibiting tumour angiogenic behaviors in breast cancer.

Activated thrombin-activatable fibrinolysis inhibitor (TAFIa) attenuates breast cancer cell metastatic behaviors through inhibition of plasminogen activation and extracellular proteolysis.

BACKGROUND: Thrombin activatable fibrinolysis inhibitor (TAFI) is a plasma zymogen, which can be converted to activated TAFI (TAFIa) through proteolytic cleavage by thrombin, plasmin, and most effectively thrombin in complex with the endothelial cofactor thrombomodulin (TM). TAFIa is a carboxypeptidase that cleaves carboxyl terminal lysine and arginine residues from protein and peptide substrates, including plasminogen-binding sites on cell surface receptors. Carboxyl terminal lysine residues play a pivotal role in enhancing cell surface plasminogen activation to plasmin. Plasmin has many critical functions including cleaving components of the extracellular matrix (ECM), which enhances invasion and migration of cancer cells. We therefore hypothesized that TAFIa could act to attenuate metastasis. METHODS: To assess the role of TAFIa in breast cancer metastasis, in vitro migration and invasion assays, live cell proteolysis and cell proliferation using MDA-MB-231 and SUM149 cells were carried out in the presence of a TAFIa inhibitor, recombinant TAFI variants, or soluble TM. RESULTS: Inhibition of TAFIa with potato tuber carboxypeptidase inhibitor increased cell invasion, migration and proteolysis of both cell lines, whereas addition of TM resulted in a decrease in all these parameters. A stable variant of TAFIa, TAFIa-CIIYQ, showed enhanced inhibitory effects on cell invasion, migration and proteolysis. Furthermore, pericellular plasminogen activation was significantly decreased on the surface of MDA-MB-231 and SUM149 cells following treatment with various concentrations of TAFIa. CONCLUSIONS: Taken together, these results indicate a vital role for TAFIa in regulating pericellular plasminogen activation and ultimately ECM proteolysis in the breast cancer microenvironment. Enhancement of TAFI activation in this microenvironment may be a therapeutic strategy to inhibit invasion and prevent metastasis of breast cancer cells.

A novel model used to detect differential splice junctions as biomarkers in prostate cancer from RNA-Seq data.

BACKGROUND: In cancer alternative RNA splicing represents one mechanism for flexible gene regulation, whereby protein isoforms can be created to promote cell growth, division and survival. Detecting novel splice junctions in the cancer transcriptome may reveal pathways driving tumorigenic events. In this regard, RNA-Seq, a high-throughput sequencing technology, has expanded the study of cancer transcriptomics in the areas of gene expression, chimeric events and alternative splicing in search of novel biomarkers for the disease. RESULTS: In this study, we propose a new two-dimensional peak finding method for detecting differential splice junctions in prostate cancer using RNA-Seq data. We have designed an integrative process that involves a new two-dimensional peak finding algorithm to combine junctions and then remove irrelevant introns across different samples within a population. We have also designed a scoring mechanism to select the most common junctions. CONCLUSIONS: Our computational analysis on three independent datasets collected from patients diagnosed with prostate cancer reveals a small subset of junctions that may potentially serve as biomarkers for prostate cancer. AVAILABILITY: The pipeline, along with their corresponding algorithms, are available upon request.

Pathomimetic cancer avatars for live-cell imaging of protease activity.

Proteases are essential for normal physiology as well as multiple diseases, e.g., playing a causative role in cancer progression, including in tumor angiogenesis, invasion, and metastasis. Identification of dynamic alterations in protease activity may allow us to detect early stage cancers and to assess the efficacy of anti-cancer therapies. Despite the clinical importance of proteases in cancer progression, their functional roles individually and within the context of complex protease networks have not yet been well defined. These gaps in our understanding might be addressed with: 1) accurate and sensitive tools and methods to directly identify changes in protease activities in live cells, and 2) pathomimetic avatars for cancer that recapitulate in vitro the tumor in the context of its cellular and non-cellular microenvironment. Such avatars should be designed to facilitate mechanistic studies that can be translated to animal models and ultimately the clinic. Here, we will describe basic principles and recent applications of live-cell imaging for identification of active proteases. The avatars optimized by our laboratory are three-dimensional (3D) human breast cancer models in a matrix of reconstituted basement membrane (rBM). They are designated mammary architecture and microenvironment engineering (MAME) models as they have been designed to mimic the structural and functional interactions among cell types in the normal and cancerous human breast. We have demonstrated the usefulness of these pathomimetic avatars for following dynamic and temporal changes in cell:cell interactions and quantifying changes in protease activity associated with these interactions in real-time (4D). We also briefly describe adaptation of the avatars to custom-designed and fabricated tissue architecture and microenvironment engineering (TAME) chambers that enhance our ability to analyze concomitant changes in the malignant phenotype and the associated tumor microenvironment.

Inhibition of cathepsin B activity attenuates extracellular matrix degradation and inflammatory breast cancer invasion.

INTRODUCTION: Inflammatory breast cancer (IBC) is an aggressive, metastatic and highly angiogenic form of locally advanced breast cancer with a relatively poor three-year survival rate. Breast cancer invasion has been linked to proteolytic activity at the tumor cell surface. Here we explored a role for active cathepsin B on the cell surface in the invasiveness of IBC. METHODS: We examined expression of the cysteine protease cathepsin B and the serine protease urokinase plasminogen activator (uPA), its receptor uPAR and caveolin-1 in two IBC cell lines: SUM149 and SUM190. We utilized a live cell proteolysis assay to localize in real time the degradation of type IV collagen by IBC cells. IBC patient biopsies were examined for expression of cathepsin B and caveolin-1. RESULTS: Both cell lines expressed comparable levels of cathepsin B and uPA. In contrast, levels of caveolin-1 and uPAR were greater in SUM149 cells. We observed that uPA, uPAR and enzymatically active cathepsin B were colocalized in caveolae fractions isolated from SUM149 cells. Using a live-cell proteolysis assay, we demonstrated that both IBC cell lines degrade type IV collagen. The SUM149 cells exhibit predominantly pericellular proteolysis, consistent with localization of proteolytic pathway constitutents to caveolar membrane microdomains. A functional role for cathepsin B was confirmed by the ability of CA074, a cell impermeable and highly selective cathepsin B inhibitor, to significantly reduce pericellular proteolysis and invasion by SUM149 cells. A statistically significant co-expression of cathepsin B and caveolin-1 was found in IBC patient biopsies, thus validating our in vitro data. CONCLUSION: Our study is the first to show that the proteolytic activity of cathepsin B and its co-expression with caveolin-1 contributes to the aggressiveness of IBC.

Cathepsin B: a potential prognostic marker for inflammatory breast cancer.

BACKGROUND: Inflammatory breast cancer (IBC) is the most aggressive form of breast cancer. In non-IBC, the cysteine protease cathepsin B (CTSB) is known to be involved in cancer progression and invasion; however, very little is known about its role in IBC. METHODS: In this study, we enrolled 23 IBC and 27 non-IBC patients. All patient tissues used for analysis were from untreated patients. Using immunohistochemistry and immunoblotting, we assessed the levels of expression of CTSB in IBC versus non-IBC patient tissues. Previously, we found that CTSB is localized to caveolar membrane microdomains in cancer cell lines including IBC, and therefore, we also examined the expression of caveolin-1 (cav-1), a structural protein of caveolae in IBC versus non-IBC tissues. In addition, we tested the correlation between the expression of CTSB and cav-1 and the number of positive metastatic lymph nodes in both patient groups. RESULTS: Our results revealed that CTSB and cav-1 were overexpressed in IBC as compared to non-IBC tissues. Moreover, there was a significant positive correlation between the expression of CTSB and the number of positive metastatic lymph nodes in IBC. CONCLUSIONS: CTSB may initiate proteolytic pathways crucial for IBC invasion. Thus, our data demonstrate that CTSB may be a potential prognostic marker for lymph node metastasis in IBC.

Interleukin-6 increases expression and secretion of cathepsin B by breast tumor-associated monocytes.

In the tumor microenvironment, monocytes respond to paracrine stimuli from breast cancer cells by secreting molecules that participate in breast cancer growth, invasion, intravasation and metastasis. Here we examined the effects of media conditioned by MDA-MB-231 human breast carcinoma cells (231-CM) on expression and secretion of proteases and secretion of cytokines by U937 human monocytes. We found that 231-CM increased U937: 1) proliferation; 2) expression, activity and secretion of the cysteine protease cathepsin B (CTSB); 3) secretion of matrix metalloproteinases (MMP)-2 and -9; and 4) secretion of interleukin-6 (IL-6) and insulin-like growth factor binding protein-1 (IGFBP-1). We further demonstrated by western blotting and enzymatic activity assays that the increases in CTSB secretion and activity induced by 231-CM could be reduced by neutralizing antibodies against IL-6. Our data suggest a role for IL-6 in increased monocyte expression and secretion of CTSB in response to soluble factors secreted by breast cancer cells.

Live-cell imaging demonstrates extracellular matrix degradation in association with active cathepsin B in caveolae of endothelial cells during tube formation.

Localization of proteases to the surface of endothelial cells and remodeling of the extracellular matrix (ECM) are essential to endothelial cell tube formation and angiogenesis. Here, we partially localized active cathepsin B and its cell surface binding partners, S100A/p11 (p11) of the annexin II heterotetramer (AIIt), to caveolae of human umbilical vein endothelial cells (HUVEC). Via a live-cell proteolysis assay, we observed that degradation products of quenched-fluorescent (DQ)-proteins (i.e. gelatin and collagen IV) colocalized intracellularly with caveolin-1 (cav-1) of HUVEC grown in either monolayer cultures or in vitro tube formation assays. Activity-based probes that bind covalently to active cysteine cathepsins and degradation products of DQ-collagen IV partially localized to intracellular vesicles that contained cav-1 and active cysteine cathepsins. Biochemical analyses revealed that the distribution of active cathepsin B in caveolar fractions increased during in vitro tube formation. Pro-uPA, uPAR, MMP-2 and MMP-14, which have been linked with cathepsin B to ECM degradation pathways, were also found to increase in caveolar fractions during in vitro tube formation. Our findings are the first to demonstrate through live-cell imaging ECM degradation in association with active cathepsin B in caveolae of endothelial cells during tube formation.

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.

Lysosomal cathepsin B participates in the podosome-mediated extracellular matrix degradation and invasion via secreted lysosomes in v-Src fibroblasts.

Podosomes mediate cell migration and invasion by coordinating the reorganization of actin cytoskeleton and focal matrix degradation. MMP and serine proteases have been found to function at podosomes. The lysosomal cysteine cathepsins, a third major class of matrix-degrading enzymes involved in tumor invasion and tissue remodeling, have yet to be linked to podosomes with the exception of cathepsin K in osteoclasts. Using inhibitors and shRNA-mediated depletion, we show that cathepsin B participates in podosomes-mediated focal matrix degradation and invasion in v-Src-transformed fibroblasts. We observed that lysosomal marker LAMP-1 localized at the center of podosome rosettes protruding into extracellular matrix using confocal microscopy. Time-lapse live-cell imaging revealed that lysosomal vesicles moved to and fused with podosomes. Disruption of lysosomal pH gradient with Bafilomycin A1, chloroquine, or ammonium chloride greatly enhanced the formation of podosomes and increased the matrix degradation. Live-cell imaging showed that actin structures, induced shortly after Bafilomycin A1 treatment, were closely associated with lysosomes. Overall, our results suggest that cathepsin B, delivered by lysosomal vesicles, is involved in the matrix degradtion of podosomes.

Human monocytes augment invasiveness and proteolytic activity of inflammatory breast cancer.

Inflammatory breast cancer (IBC) is the most aggressive form of breast cancer, and here, we examined in vitro the interactions between the human IBC cell line SUM149 and U937 human naive monocytes. We found an altered morphology, enhanced invasiveness and proteolytic activity of SUM149 cells when cultured with U937 cells or in U937-conditioned media (U937-CM). Increases in expression and activity of the cysteine protease cathepsin B and expression of caveolin-1 were also detected in SUM149 cells grown in U937-CM, thus suggesting a contribution of these proteins to the augmented invasion through and proteolysis of the extracellular matrix by the IBC cells.