Deep Tissue Imaging

Real-Time, High-Resolution Microscopy of Invasive Cancer Cells in the Tumor Microenvironment Context – Bojana Gligorijevic:

Tumor cell structures that have long been hypothesized as necessary for metastasis are invadopodia, invasive protrusions rich in structural and adhesion proteins, as well as metalloproteases. Using our unique intravital imaging approaches (Perrin et al, 2019 Canc Rep; Bayarmagnai et al, 2018 Meth Mol Biol.), we previously demonstrated that invadopodia in vivo are necessary for intravasation and consequent lung metastasis (Gligorijevic et al, Plos Bio 2014). In primary breast carcinoma, we found that cells which assemble invadopodia migrate at slow speeds, in perivascular niches where the ECM is cross-linked. Outside of these niches, no invadopodia were observed and cells migrated at high speeds, via contact guidance along collagen fibers. The invadopodia-driven motility can be switched to contact guidance by reducing the ECM cross-linking or by knocking down Tks5, which in turn reduces intravasation and metastasis. We next deduced that invadopodia-driven motility consists of two oscillating states: i. Invadopodia state, in which a cell is relatively sessile while it assembles invadopodia and degrades ECM; ii. Locomotion state. State balance is regulated by integrin β1 activation levels (Esmaeili et al 2018 Biophys J). Importantly, the Invadopodia state only occurs in early G1, whereas the Locomotion state can be seen throughout the entire cell cycle, suggesting that the cell cycle controls invadopodia assembly. Using FUCCI markers (Esmaeili et al 2018 APL BioE), we next show that Invadopodia state occurs during the G1 phase of the cell cycle (Bayarmagnai et al, 2019 JCS). A close look at the regulators of G1 revealed that the cell cycle regulator p27kip1 localizes to the sites of invadopodia assembly and overexpression of p27kip1 causes faster turnover of invadopodia and increased ECM degradation. Taken together, these findings suggest that invadopodia assembly in the perivascular niche, is necessary for lung metastasis and function is controlled by the cell cycle.

Control of Brain Capillary Flow Revealed by Optical Imaging and Manipulation of Pericytes – Andy Shih: 

The vast majority of the brain’s vasculature is comprised of intricate capillary networks adorned by capillary pericytes. It is unclear whether capillary pericytes regulate blood flow through capillaries, where blood cells flow in a single-file fashion. Using two-photon microscopy to observe and manipulate the microvasculature in vivo, we find that optogenetic stimulation of single capillary pericytes decreased lumen diameter and blood flow, but with slower kinetics than stimulation of mural cells lining upstream pre-capillary arterioles. This optogenetically-induced vasoconstriction could be inhibited by the clinically-used vasodilator fasudil, a Rho kinase inhibitor that blocks contractile machinery. In a complementary approach, optical ablation of single capillary pericytes led to sustained local dilation and augmented blood cell flux. This dilation was most prominent when pericytes were ablated from the network’s smallest diameter vessels. Thus, capillary pericytes exhibit slow contractility that supports their role in maintaining basal flow resistance through brain capillary networks.