Modeling and characterization of inflammatory breast cancer emboli grown in vitro.

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 TGFß. This study provides a biologically relevant in vitro model to accurately grow and study inflammatory breast cancer biology and metastasis.

Differential expression of the HMGN family of chromatin proteins during ocular development.

The HMGN proteins are a group of non-histone nuclear proteins that associate with the core nucleosome and alter the structure of the chromatin fiber. We investigated the distribution of the three best characterized HMGN family members, HMGN1, HMGN2 and HMGN3 during mouse eye development. HMGN1 protein is evenly distributed in all ocular structures of 10.5 days post-coitum (dpc) mouse embryos however, by 13.5dpc, relatively less HMGN1 is detected in the newly formed lens fiber cells compared to other cell types. In the adult, HMGN1 is detected throughout the retina and lens, although in the cornea, HMGN1 protein is predominately located in the epithelium. HMGN2 is also abundant in all ocular structures of mouse embryos, however, unlike HMGN1, intense immunolabeling is maintained in the lens fiber cells at 13.5dpc. In the adult eye, HMGN2 protein is still found in all lens nuclei while in the cornea, HMGN2 protein is mostly restricted to the epithelium. In contrast, the first detection of HMGN3 in the eye is in the presumptive corneal epithelium and lens fiber cells at 13.5dpc. In the lens, HMGN3 remained lens fiber cell preferred into adulthood. In the cornea, HMGN3 is transiently upregulated in the stroma and endothelium at birth while its expression is restricted to the corneal epithelium in adulthood. In the retina, HMGN3 upregulates around 2 weeks of age and is found at relatively high levels in the inner nuclear and ganglion cell layers of the adult retina. RT-PCR analysis determined that the predominant HMGN3 splice form found in ocular tissues is HMGN3b which lacks the chromatin unfolding domain although HMGN3a mRNA is also detected. These results demonstrate that the HMGN class of chromatin proteins has a dynamic expression pattern in the developing eye.

RhoC GTPase activation assay.

RhoC GTPase has 91% homology to RhoA GTPase. Because of its prevalence in cells, many reagents and techniques for RhoA GTPase have been developed. However, RhoC GTPase is expressed in metastatic cancer cells at relatively low levels. Therefore, few RhoC-specific reagents have been developed. We have adapted a Rho activation assay to detect RhoC GTPase. This technique utilizes a GST-Rho binding domain fusion protein to pull out active RhoC GTPase. In addition, we can harvest total protein at the beginning of the assay to determine levels of total (GTP and GDP bound) RhoC GTPase. This allows for the determination of active versus total RhoC GTPase in the cell. Several commercial versions of this procedure have been developed however, the commercial kits are optimized for RhoA GTPase and typically do not work well for RhoC GTPase. Parts of the assay have been modified as well as development of a RhoC-specific antibody.