Notch1 and Notch3 coordinate for pericyte-induced stabilization of vasculature.

The Notch pathway regulates complex patterning events in many species and is critical for the proper formation and function of the vasculature. Despite this importance, how the various components of the Notch pathway work in concert is still not well understood. For example, NOTCH1 stabilizes homotypic endothelial junctions, but the role of NOTCH1 in heterotypic interactions is not entirely clear. NOTCH3, on the other hand, is essential for heterotypic interactions of pericytes with the endothelium, but how NOTCH3 signaling in pericytes impacts the endothelium remains elusive. Here, we use in vitro vascular models to investigate whether pericyte-induced stabilization of the vasculature requires the cooperation of NOTCH1 and NOTCH3. We observe that both pericyte NOTCH3 and endothelial NOTCH1 are required for the stabilization of the endothelium. Loss of either NOTCH3 or NOTCH1 decreases the accumulation of VE-cadherin at endothelial adherens junctions and increases the frequency of wider, more motile junctions. We found that DLL4 was the key ligand for simulating NOTCH1 activation in endothelial cells and observed that DLL4 expression in pericytes is dependent on NOTCH3. Altogether, these data suggest that an interplay between pericyte NOTCH3 and endothelial NOTCH1 is critical for pericyte-induced vascular stabilization.

Transient Support from Fibroblasts is Sufficient to Drive Functional Vascularization in Engineered Tissues.

Formation of capillary blood vasculature is a critical requirement for native as well as engineered organs and can be induced in vitro by co-culturing endothelial cells with fibroblasts. However, whether these fibroblasts are required only in the initial morphogenesis of endothelial cells or needed throughout is unknown, and the ability to remove these stromal cells after assembly could be useful for clinical translation. In this study, we introduce a technique termed CAMEO (Controlled Apoptosis in Multicellular Tissues for Engineered Organogenesis), whereby fibroblasts are selectively ablated on demand, and utilize it to probe the dispensability of fibroblasts in vascular morphogenesis. The presence of fibroblasts is shown to be necessary only during the first few days of endothelial cell morphogenesis, after which they can be ablated without significantly affecting the structural and functional features of the developed vasculature. Furthermore, we demonstrate the use of CAMEO to vascularize a construct containing primary human hepatocytes that improved tissue function. In conclusion, this study suggests that transient, initial support from fibroblasts is sufficient to drive vascular morphogenesis in engineered tissues, and this strategy of engineering-via-elimination may provide a new general approach for achieving desired functions and cell compositions in engineered organs.

In vivo detection of drug-induced apoptosis in tumors using Raman spectroscopy.

We describe a label-free approach based on Raman spectroscopy, to study drug-induced apoptosis in vivo. Spectral-shifts at wavenumbers associated with DNA, proteins, lipids, and collagen have been identified on breast and melanoma tumor tissues. These findings may enable a new analytical method for rapid readout of drug-therapy with miniaturized probes.

Salactin, a dynamically unstable actin homolog in Haloarchaea.

IMPORTANCE: Protein filaments play important roles in many biological processes. We discovered an actin homolog in halophilic archaea, which we call Salactin. Just like the filaments that segregate DNA in eukaryotes, Salactin grows out of the cell poles towards the middle, and then quickly depolymerizes, a behavior known as dynamic instability. Furthermore, we see that Salactin affects the distribution of DNA in daughter cells when cells are grown in low-phosphate media, suggesting Salactin filaments might be involved in segregating DNA when the cell has only a few copies of the chromosome.

A Miniaturized Platform for Multiplexed Drug Response Imaging in Live Tumors.

By observing the activity of anti-cancer agents directly in tumors, there is potential to greatly expand our understanding of drug response and develop more personalized cancer treatments. Implantable microdevices (IMD) have been recently developed to deliver microdoses of chemotherapeutic agents locally into confined regions of live tumors; the tissue can be subsequently removed and analyzed to evaluate drug response. This method has the potential to rapidly screen multiple drugs, but requires surgical tissue removal and only evaluates drug response at a single timepoint when the tissue is excised. Here, we describe a "lab-in-a-tumor" implantable microdevice (LIT-IMD) platform to image cell-death drug response within a live tumor, without requiring surgical resection or tissue processing. The LIT-IMD is inserted into a live tumor and delivers multiple drug microdoses into spatially discrete locations. In parallel, it locally delivers microdose levels of a fluorescent cell-death assay, which diffuses into drug-exposed tissues and accumulates at sites of cell death. An integrated miniaturized fluorescence imaging probe images each region to evaluate drug-induced cell death. We demonstrate ability to evaluate multi-drug response over 8 h using murine tumor models and show correlation with gold-standard conventional fluorescence microscopy and histopathology. This is the first demonstration of a fully integrated platform for evaluating multiple chemotherapy responses in situ. This approach could enable a more complete understanding of drug activity in live tumors, and could expand the utility of drug-response measurements to a wide range of settings where surgery is not feasible.

An interventional image-guided microdevice implantation and retrieval method for in-vivo drug response assessment.

PURPOSE: Recently developed implantable microdevices can perform multi-drug response assessment of cancer drugs in-vivo, with potential to develop highly optimized personalized cancer treatment strategies. However, minimally invasive/interventional image-guided methods of in-vivo microdevice implantation, securement, and retrieval are needed for broad clinical translation. Here we demonstrate proof-of-concept of an interventional microdevice implantation and retrieval method for personalized drug response assessment, using ex-vivo phantom, ex-vivo tissue, and in-vivo murine models. METHODS: A method for minimally-invasive microdevice implantation and retrieval was developed, by which a custom-prototyped 6 mm retrievable microdevice can be implanted into a live tumor, deliver drugs into 10 discrete regions of adjacent tissue, and retrieved along with the adjacent drug-exposed tissue with a custom-prototyped retrieval needle device to allow in-vivo multi-drug response assessment. Computed tomography (CT) and ultrasound (US)-guided minimally invasive microdevice implantation and retrieval were tested in ex-vivo phantom and tissue models. Successful retrieval was defined as retrieval of the microdevice and adjacent core phantom/tissue sample containing at least 4/10 drug delivery sites. Subsequently, 10 implantation and retrieval trials in phantom models were performed using bi-axial and tri-axial retrieval needles; success rates were calculated and compared using a two-proportion z-test and the number of successfully retrieved drug release sites per microdevice was calculated and compared using a one-tailed independent t-test. Finally, five microdevices, each containing ten reservoirs preloaded with chemotherapy agent Doxorubicin, were implanted into mouse tumors in-vivo, secured for 24-h during drug release, and microdevice/tissue retrieval was performed under ultrasound guidance. Fluorescence microscopy of the retrieved tissue was used to confirm drug delivery and apoptosis staining assessed in-vivo tissue response; correlation of drug release and apoptosis staining were used to assess in-vivo drug efficacy. RESULTS: Image-guided microdevice implantation and retrieval were successful in ex-vivo phantom and tissue models with both US and CT guidance. Bi-axial retrieval success rate was significantly higher than triaxial retrieval in ex-vivo phantom trials (90% vs 50%, z = 1.95, P = 0.026), and had nonsignificantly higher number of retrieved drug-release sites per microdevice (8.3 vs 7.0, t = 1.37, P = 0.097). Bi-axial retrieval was successful in all five in-vivo mouse tumor models, and allowed in-vivo drug response assessment at up to ten discrete drug delivery sites per microdevice. An average of 6.8/10 discrete tumor sites containing micro-doses of delivered drug were retrieved per in-vivo attempt (min 5, max 10, std 1.93). Tissue regions of drug delivery, as assessed with fluorescent Doxorubicin drug signal, correlated with regions of apoptosis staining in all in-vivo models, indicating drug efficacy. No bleeding, microdevice migration, or other complications were noted during implantation, 24-h observation, or retrieval. CONCLUSIONS: The demonstrated image-guided minimally invasive microdevice implantation and retrieval method is similar to routine outpatient biopsy procedures, obviates the need for surgery, and can be performed at varying depths under CT and/or US guidance. There is potential for this method to enable clinical translation of in-vivo personalized drug response assessment/prediction in a much larger number of patients than currently possible.

Direct evidence for cancer-cell-autonomous extracellular protein catabolism in pancreatic tumors.

Mammalian tissues rely on a variety of nutrients to support their physiological functions. It is known that altered metabolism is involved in the pathogenesis of cancer, but which nutrients support the inappropriate growth of intact malignant tumors is incompletely understood. Amino acids are essential nutrients for many cancer cells that can be obtained through the scavenging and catabolism of extracellular protein via macropinocytosis. In particular, macropinocytosis can be a nutrient source for pancreatic cancer cells, but it is not fully understood how the tumor environment influences metabolic phenotypes and whether macropinocytosis supports the maintenance of amino acid levels within pancreatic tumors. Here we utilize miniaturized plasma exchange to deliver labeled albumin to tissues in live mice, and we demonstrate that breakdown of albumin contributes to the supply of free amino acids in pancreatic tumors. We also deliver albumin directly into tumors using an implantable microdevice, which was adapted and modified from ref. 9. Following implantation, we directly observe protein catabolism and macropinocytosis in situ by pancreatic cancer cells, but not by adjacent, non-cancerous pancreatic tissue. In addition, we find that intratumoral inhibition of macropinocytosis decreases amino acid levels. Taken together, these data suggest that pancreatic cancer cells consume extracellular protein, including albumin, and that this consumption serves as an important source of amino acids for pancreatic cancer cells in vivo.