It has been demonstrated that divalent zinc ions packaged with insulin in β-cell granules can be detected by MRI during glucose-stimulated insulin secretion using a gadolinium-based Zn 2+sensitive agent. This study was designed to evaluate whether a simpler agent design having single Zn 2+-sensing moieties but with variable Zn 2+ binding affinities might also detect insulin secretion from the pancreas. Using an implanted MR-compatible window designed to hold the pancreas in a fixed position for imaging, we now demonstrate that focally intense "hot spots" can be detected in the tail of the pancreas using these agents after administration of glucose to stimulate insulin secretion. Histological staining of the same tissue verified that the hot spots identified by imaging correspond to clusters of islets, perhaps reflecting first-responder islets that are most responsive to a sudden increase in glucose. A comparison of images obtained when using a high-affinity Zn 2+ sensor versus a lower-affinity sensor showed that the lower-affinity sensors produced the best image contrast. An equilibrium model that considers all possible complexes formed between Zn 2+ , the GdL sensor, and HSA predicts that a GdL sensor with lower affinity for Zn 2+ generates a lower background signal from endogenous Zn 2+ prior to glucose-stimulated insulin secretion (GSIS) and that the weaker binding affinity agent is more responsive to a further increase in Zn 2+ concentration near β-cells after GSIS. These model predictions are consistent with the in vivo imaging observations.

Cancer cells rely on extensive support from the stroma in order to survive, proliferate and invade. The tumor stroma is thus an important potential target for anti-cancer therapy. Typical changes in the stroma include a shift from the quiescence promoting- antiangiogenic extracellular matrix to a provisional matrix that promotes invasion and angiogenesis. These changes in the extracellular matrix are induced by changes in the secretion of extracellular matrix proteins and glucose amino glycans, extravasation of plasma proteins from hyperpermeable vessels and release of matrix modifying enzymes resulting in cleavage and crosslinking of matrix macromolecules. These in turn alter the rigidity of the matrix and the exposure and release of cytokines. Changes in matrix rigidity and vessel permeability affect drug delivery and mediate resistance to cytotoxic therapy. These stroma changes are brought about not only by the cancer cells, but also through the action of many cell types that are recruited by tumors including immune cells, fibroblasts and endothelial cells. Within the tumor, these normal host cells are activated resulting in loss of inhibitory and induction of cancer promoting activities. Key to the development of stroma targeted therapies, selective biomarkers were developed for specific imaging of key aspects of the tumor stroma.

The ovary is a dynamic organ that undergoes dramatic remodeling throughout the ovulatory cycle. Maturation of the ovarian follicle, release of the oocyte in the course of ovulation as well as formation and degradation of corpus luteum involve tightly controlled remodeling of the extracellular matrix and vasculature. Ovarian tumors, regardless of their tissue of origin, dynamically interact with the ovarian microenvironment. Their activity in the tissue encompasses recruitment of host stroma and immune cells, attachment of tumor cells to mesothelial layer, degradation of the extracellular matrix and tumor cell migration. High-resolution dynamic imaging of such processes is particularly challenging for internal organs. The implementation of a novel imaging window as reported here enabled longitudinal microscopy of ovarian physiology and orthotopic tumor invasion.

Thin section histology is limited in providing 3D structural information, particularly of the intricate morphology of the vasculature. Availability of high spatial resolution imaging for thick samples, would overcome the restriction dictated by low light penetration. Our study aimed at optimizing the procedure for efficient and affordable tissue clearing, along with an appropriate immunofluorescence labeling that will be applicable for high resolution imaging of blood and lymphatic vessels. The new procedure, termed whole organ blood and lymphatic vessels imaging (WOBLI), is based on two previously reported methods, CLARITY and ScaleA2. We used this procedure for the analysis of isolated whole ovary, uterus, lung and liver. These organs were subjected to passive clearing, following fixation, immunolabeling and embedding in hydrogel. Cleared specimens were immersed in ScaleA2 solution until transparency was achieved and imaged using light sheet microscopy. We demonstrate that WOBLI allows detailed analysis and generation of structural information of the lymphatic and blood vasculature from thick slices and more importantly, from whole organs. We conclude that WOBLI offers the advantages of morphology and fluorescence preservation with efficient clearing. Furthermore, WOBLI provides a robust, cost-effective method for generation of transparent specimens, allowing high resolution, 3D-imaging of blood and lymphatic vessels networks.

Angiogenesis and lymphangiogenesis are key processes during embryogenesis as well as under physiological and pathological conditions. Vascular endothelial growth factor C (VEGFC), the ligand for both VEGFR2 and VEGFR3, is a central lymphangiogenic regulator that also drives angiogenesis. Here, we report that members of the highly conserved BACH (BTB and CNC homology) family of transcription factors regulate VEGFC expression, through direct binding to its promoter. Accordingly, down-regulation of bach2a hinders blood vessel formation and impairs lymphatic sprouting in a Vegfc-dependent manner during zebrafish embryonic development. In contrast, BACH1 overexpression enhances intratumoral blood vessel density and peritumoral lymphatic vessel diameter in ovarian and lung mouse tumor models. The effects on the vascular compartment correlate spatially and temporally with BACH1 transcriptional regulation of VEGFC expression. Altogether, our results uncover a novel role for the BACH/VEGFC signaling axis in lymphatic formation during embryogenesis and cancer, providing a novel potential target for therapeutic interventions.