Protein scavenging by macropinocytosis can serve as a source of nutrients for pancreatic cancer cells. We provide direct evidence that extracellular protein is a fuel source for pancreatic cancer cells in vivo. We demonstrate that albumin-derived peptides and amino acids accumulate in tumors in a Kras-driven mouse model of pancreatic ductal adenocarcinoma. In addition, we implement a device to deliver large molecules directly into the tumor and observe protein catabolism and macropinocytosis by cancer cells within pancreatic tumors. Local release of a macropinocytosis inhibitor leads to a drastic reduction in amino acids levels in tumor tissue arguing that the direct uptake and catabolism of extracellular protein is necessary to provide amino acids to pancreatic cancer cells in tumors. These data provide evidence for albumin catabolism by tumors and also suggest a method for testing therapies that take advantage of the propensity of pancreatic cancer cells to scavenge extracellular protein.
Formation of capillary blood vasculature is a critical requirement for native as well as engineered organs and can be induced in vitro by coculturing 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 can be useful for clinical translation. In this study, a technique termed CAMEO (Controlled Apoptosis in Multicellular Tissues for Engineered Organogenesis) is introduced, whereby fibroblasts are selectively ablated on demand, and it is utilized 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, the use of CAMEO to vascularize a construct containing primary human hepatocytes that improved tissue function is demonstrated. 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.
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 cooperation of NOTCH1 and NOTCH3. We observe that both pericyte NOTCH3 and endothelial NOTCH1 are required for stabilization of the endothelium. Loss of either NOTCH3 or NOTCH1 decreases 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.
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 multidrug 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 invivo 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,...
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