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...

With a 42% and 18% 5-and 10-year respective disease-free survival rate, inflammatory breast cancer (IBC) is arguably the deadliest form of breast cancer. IBC invades the dermal lymphatic vessels of the skin overlying the breast and as a consequence nearly all women have lymph node involvement and $1/3 have gross distant metastases at the time of diagnosis. One year after diagnosis $90% of patients have detectable metastases, making IBC a paradigm for lymphovascular invasion. Understanding the underlying mechanisms of the IBC metastatic phenotype is essential for new therapies. Work from our laboratory and others show distinct molecular differences between IBC and non-IBCs (nIBCs). Previously we showed that RhoC GTPase is a metastatic switch responsible for the invasive phenotype of IBC. In this study we integrate observations made in IBC patients with in vitro analysis. We show that the PI3K/Akt signaling pathway is crucial in IBC invasion. Key molecules involved in cytoskeletal control and cell motility are specifically upregulated in IBC patients compared with stage and cell-typeof-origin matched nIBCs patients. Distinctively, RhoC GTPase is a substrate for Akt1 and its phosphorylation is absolutely essential for IBC cell invasion. Further our data show that Akt3, not Akt1 has a role in IBC cell survival. Together our data show a unique and targetable pathway for IBC invasion and survival.

Esophageal squamous cell carcinoma (ESCC) is an aggressive malignancy with a poor prognosis due to its highly invasive and metastatic potential. The molecular pathogenesis underlying the invasive mechanism of ESCC is not well known because of the lack of existing models to study this disease. p120-Catenin (p120ctn) and the epidermal growth factor receptor (EGFR) have each been implicated in several cancers, including ESCC. p120ctn is down-regulated in 60% of ESCC tumors, whereas EGFR is the most commonly overexpressed oncogene in ESCC. For these reasons, we investigated the cooperation between p120ctn and EGFR and its effect on ESCC invasion. We show that p120ctn down-regulation is commonly associated with EGFR overexpression. By using a three-dimensional culture system, we demonstrate that the inverse relationship between p120ctn and EGFR has biological implications. Specifically, p120ctn down-regulation coupled with EGFR overexpression in human esophageal keratinocytes (EPC1-PE) was required to promote invasion. Morphological comparison of EPC1-PE cells grown in three-dimensional culture and human ESCC revealed identical features, including significantly increased cellularity, nuclear grade, and proliferation. Molecular characteristics were measured by keratin expression patterns, which were nearly identical between EPC1-PE cells in three-dimensional culture and ESCC samples. Altogether, our analyses have demonstrated that p120ctn down-regulation and EGFR overexpression are able to mimic human ESCC in a relevant three-dimensional culture model.