Abstract P6-02-05: A novel culturing 3-D model to evaluate the role of tumor microenvironment in IBC.

Abstract

Abstract Introduction: Inflammatory breast cancer (IBC) is a highly aggressive form of breast cancer associated with extremely poor outcomes. The clinical and pathological characteristics of the disease are the peculiar invasion of the dermal lymphatics as tumor emboli and the development of early recurrences. We aimed to establish a 3D model to evaluate the role of tumor microenvironment. Methods: We used human tumor-associated fibroblasts (or fibroblasts derived from metastatic skin) from IBC patients to build a multilayer extracellular matrix structure which effectively mimics aspects of the mesenchymal microenvironment of IBCs. Using this in vivo-like microenvironment we proceeded to test both matrix effects upon IBC's phenotypes and IBC modifications upon the cell-derived 3D matrices. We seeded the IBC cells into the matrix and cultured for 3 days, then tested the characterization markers cancer cells and ECM e.g. Phalloidin, E-cadherin, Ki67, α5β1 integrin and fibronectin by immunofluorescence and the expression of E-cadherin and vimentin as marker of epithelial-mesenchymal transition (EMT) by western blot. Results: We divided six IBC cell lines into 2 groups depending on the phenotypes acquired when cultured in the IBC fibroblast-derived ECM. SUM149 (EGF receptor positive and aggressive phenotype), BR016 and LG018 (harvested from patient's pleural effusions) presented a single cell organization with a spindle-like or mesenchymal type (as opposed to cluster) morphology. In comparison, SUM190 (HER2 positive and non aggressive tumorigenesis), MDA-IBC-3 and FC-IBC-02 (abstracted from patient's pleural effusion) presented a phenotype resembling mammospheres or in vivo emboli. Moreover, this last group of cells showed a peculiar capability for ECM modifications which greatly differed from the ECM modifications that were apparent following 3 day culturing of the above mentioned group represented by SUM149. In addition, proliferation measurements by Ki67 expression demonstrated a significant increased in 3D culture for SUM149, BR016 and LG018 compared with that in 2D culture, while no differences in proliferation were observed in the other three cell lines. Moreover, the expression of E-cadherin known to be upregulated in IBC tumors was increased in all cancer cells when seeded into the human fibroblast-derived 3D matrix indicating a potential role of the microenvironment in promoting proliferation, growth and invasion. Conclusion: The present study demonstrated the establishment of a novel IBC stromal 3D model using extracellular matrix produced from human fibroblasts of patients with advanced IBC. We showed a dynamic interaction between cancer cells and the microenvironment and potential sorting of IBC cells into two discrete groups which also correlate with their aggressive behaviors in vivo. We believe that these system may serve to predict levels of IBC tumorigenesis. We will proceed to further study the two identified responsive phenotypes with the goal of uncovering mechanisms of IBC tumor-stromal interactions and better understand ECM influences upon IBC development and progression. The ultimate goal will be to use the system to study IBC biology and better design drugs that will specifically affect the newly identified phenotypes. Citation Information: Cancer Res 2012;72(24 Suppl):Abstract nr P6-02-05.