Development of 3D culture models of plexiform neurofibroma and initial application for phenotypic characterization and drug screening.

Plexiform neurofibromas (PNs), which may be present at birth in up to half of children with type 1 neurofibromatosis (NF1), can cause serious loss of function, such as quadriparesis, and can undergo malignant transformation. Surgery is the first line treatment although the invasive nature of these tumors often prevents complete resection. Recent clinical trials have shown promising success for some drugs, notably selumetinib, an inhibitor of MAP kinase kinase (MEK). We have developed three-dimensional (3D) cell culture models of immortalized cells from NF1 PNs and of control Schwann cells (SCs) that we believe mimic more closely the in vivo condition than conventional two-dimensional (2D) cell culture. Our goal is to facilitate pre-clinical identification of potential targeted therapeutics for these tumors. Three drugs, selumetinib (a MEK inhibitor), picropodophyllin (an IGF-1R inhibitor) and LDN-193189 (a BMP2 inhibitor) were tested with dose-response design in both 2D and 3D cultures for their abilities to block net cell growth. Cell lines grown in 3D conditions showed varying degrees of resistance to the inhibitory actions of all three drugs. For example, control SCs became resistant to growth inhibition by selumetinib in 3D culture. LDN-193189 was the most effective drug in 3D cultures, with only slightly reduced potency compared to the 2D cultures. Characterization of these models also demonstrated increased proteolysis of collagen IV in the matrix by the PN driver cells as compared to wild-type SCs. The proteolytic capacity of the PN cells in the model may be a clinically significant property that can be used for testing the ability of drugs to inhibit their invasive phenotype.

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.