Modeling and characterization of inflammatory breast cancer emboli grown in vitro

Abstract

Inflammatory breast cancer (IBC) is the deadliest form of breast cancer, presenting as intralymphatic emboli. Emboli within the dermal lymphatic vessels are thought to contribute to rapid metastasis. The lack of appropriate in vitro models has made it difficult to accurately study how IBC emboli metastasize. To date, attempts at creating IBC tumor emboli in vitro have used 3D culture on a solid layer of Matrigel TM , which does not resemble the physical properties of the lymphatic system. Dermal lymphatic fluid produces oscillatory fluid shear forces and is 1.5-1.7-fold more viscous than water with a pH range of 7.5-7.7. We have established a method for forming tumor emboli by culturing the IBC cell lines in suspension with either polyethylene glycol-or hyaluronic acid-containing medium and oscillatory fluid shear forces. Non-IBC cells do not form emboli under identical conditions. In vitro IBC emboli were analyzed for expression of markers associated with patient emboli and their ability to undergo invasion. In a direct comparison, the in vitro IBC emboli closely resemble IBC patient emboli with respect to size, composition and E-cadherin expression. Further, cells from the emboli are able to invade in clusters via RhoC GTPase-dependent amoeboid movement. Invasion by clusters of IBC cells is disrupted by exposure to TGFb. This study provides a biologically relevant in vitro model to accurately grow and study inflammatory breast cancer biology and metastasis.Inflammatory breast cancer (IBC) is a phenotypically distinct form of locally advanced breast cancer.1,2 IBC carries a guarded prognosis with 5-and 10-year disease-free survival rates approaching 45 and 20%, respectively. The morbidity of this disease is presumably due to the presence of tumor emboli in the dermal lymphatic vessels where they rapidly disseminate throughout the body. 1 It is suggested that emboli disseminate by a process of ''passive metastasis.'' The idea that IBC disseminates passively by free-floating in the lymphatic vessels comes from studies involving palpation of IBC tumors in a mouse model, then measuring circulating tumor cells in the blood and measuring resultant pulmonary metastasis. 3 However, this model does not explain the rapid spread of IBC, nor does it explain the patterns of IBC dissemination counter-current to lymphatic flow. To date, no model of IBC exists to accurately test the properties of emboli in the laboratory. Previously, we demonstrated that RhoC GTPase is overexpressed in IBC cells and is responsible for the invasive phenotype of individual IBC cells by controlling the actin cytoskeleton. [4][5][6][7][8][9][10] Current data suggest that RhoC plays a role in amoeboid movement of tumors cells.11 Whether RhoC plays a role in IBC metastasis by amoeboid movement of the embolus is presently unknown. Furthermore, low TGFb levels are associated with metastasis of groups of cells as opposed to individual cells. 12 Recent evidence suggests that IBC patients have low TGFb levels. 13To date, the majority of in vitro IBC stud...

Inflammatory breast cancer tumor emboli express high levels of anti-apoptotic proteins: use of a quantitative high content and high-throughput 3D IBC spheroid assay to identify targeting strategies

“…These cells have the ability to spontaneously form 3D tumor spheroids when seeded in ultra-low attachment plates similar to previous studies [19, 27, 30]. Representative images of SUM149 tumor spheroids are shown in in Figure 2A.…”

“…To study this, we used a recently reported method [27] wherein IBC cells are cultured in media supplemented with PEG-8000 and grown on an orbital shaker, simulating the viscosity and shear force of the lymphatic system. These conditions allow IBC cells to form tight tumor cell clusters with similar phenotype and histological aspects observed in tumor emboli found in patient samples.…”

“…Herein, we also observed efficacy of DSF-Cu in disrupting emboli formation in a biologically relevant culture model reported to mimic the physical properties and environment of the dermal lymphatic system such that only IBC cells like SUM149 and SUM190 compared to other molecular-subtype matched non-IBC cells lines form the 3D structures when grown under these culture conditions [27]. Use of DSF-Cu as a therapeutic strategy is gaining momentum based on recent studies in various cancer types revealing the ability to target tumor cells while having minimal effect on normal cells [34].…”

Three-dimensional culture systems in cancer research: Focus on tumor spheroid model

“…IBC is marked by the presence of dermal and stromal tumor emboli in breast tissue, a hallmark of the disease. Upon obstructing lymphatic vessels, tumor emboli prevent proper drainage of the lymph fluid, causing skin reddening and painful swelling of breast tissue (Vermeulen et al , 2010; Lehman et al , 2013). IBC cells display high ALDH positivity, express high levels of E-cadherin, and interestingly, continue to express elevated levels of epithelial cell markers like E-cadherin, while gaining mesenchymal and stem-like characteristics (Nguyen et al , 2006; Charafe-jauffret et al , 2010; Cohen et al , 2015).…”

“…Serum free media is added to the bottom chamber and media supplemented with growth factors that act as a chemoattractant is added to the top chamber. Within few hours cells begin to disseminate from the spheroid, proteolytically cleave through the matrix and migrate towards the upper chamber (Lehman et al , 2013). The invasive potential of cells through cellular barrier can be evaluated by further modifying the assay where endothelial cells are plated atop the porous membrane.…”