Salivary Organotypic Tissue Culture: An Ex-vivo 3D Model for Studying Radiation-Induced Injury of Human Salivary Glands.

An organotypic tissue culture model can maintain the cellular and molecular interactions, as well as the extracellular components of a tissue ex vivo. Thus, this 3D model biologically mimics in vivo conditions better than commonly used 2D culture in vitro models. Here, we provide a detailed workflow for generating live 3D organotypic tissue slices from patient-derived freshly resected salivary glandular tissues. We also cover the processing of these tissues for various downstream applications like live-dead viability/cytotoxicity assay, FFPE sectioning and immunostaining, and RNA and protein extraction with a focus on the salivary gland radiation injury model. These procedures can be applied extensively to various solid organs and used for disease modeling for cancer research, radiation biology, and regenerative medicine.

Isolation of extracellular vesicles from human plasma samples: The importance of controls.

BACKGROUND: Extracellular vesicles (EV) are enriched with proteins and RNA cargo, promoting cell-to-cell communication. Biofluid derived EV cargo is used for discovering disease specific markers for diagnosis and disease monitoring. RATIONAL: Blood is a complex fluid with an abundance of protiens and thus isolation of EVs is challenging. Therefore, methods for EV isolation, including commercial kits use thromboplastin D (TP-D) for pretreatment of plasma to increase EV purity and yield. This pretreatment can introduce contaminants. METHOD AND RESULTS: We performed a comparative study to evaluate the effect of EV isolation methods focusing on (a) pretreatment of plasma with additives, which include: rabbit TP (rTP) versus human recombinant thromboplastin (huTP), to increase purity and yield (b) an additional centrifugation step prior to freezing plasma and (c) comparison of frozen versus fresh plasma EV isolations. Pretreatment with rTP generated a dynamic range of proteins, however, most of these proteins were contaminants, introduced from the rTP (99.1% purity). As an alternative, huTP was used, which did not introduce any significant contaminants, however, this did not increase yield or purity. Additionally, an extra 10,000 g centrifugation did not improve either EV yield or purity. Finally, comparison of fresh or frozen plasma showed no significant difference, an important factor when sourcing plasma from biobanks. CONCLUSION: Appropriate controlsare required when adding any additives during EV isolation as even a small percentage of contaminants can have a major effect on results. Furthermore, biobanked plasma can be used with no major changes to processing.

Angiopoietin-1 Upregulates Cancer Cell Motility in Colorectal Cancer Liver Metastases through Actin-Related Protein 2/3.

Resistance to anti-angiogenic therapy is a major challenge in the treatment of colorectal cancer liver metastases (CRCLMs). Vessel co-option has been identified as a key contributor to anti-angiogenic therapy resistance in CRCLMs. Recently, we identified a positive correlation between the expression of Angiopoietin1 (Ang1) in the liver and the development of vessel co-opting CRCLM lesions in vivo. However, the mechanisms underlying its stimulation of vessel co-option are unclear. Herein, we demonstrated Ang1 as a positive regulator of actin-related protein 2/3 (ARP2/3) expression in cancer cells, in vitro and in vivo, which is known to be essential for the formation of vessel co-option in CRCLM. Significantly, Ang1-dependent ARP2/3 expression was impaired in the cancer cells upon Tie2 or PI3K/AKT inhibition in vitro. Taken together, our results suggest novel mechanisms by which Ang1 confers the development of vessel co-option in CRCLM, which, targeting this pathway, may serve as promising therapeutic targets to overcome the development of vessel co-option in CRCLM.

Cancer Cells Promote Phenotypic Alterations in Hepatocytes at the Edge of Cancer Cell Nests to Facilitate Vessel Co-Option Establishment in Colorectal Cancer Liver Metastases.

Vessel co-option is correlated with resistance against anti-angiogenic therapy in colorectal cancer liver metastases (CRCLM). Vessel co-opting lesions are characterized by highly motile cancer cells that move toward and along the pre-existing vessels in the surrounding nonmalignant tissue and co-opt them to gain access to nutrients. To access the sinusoidal vessels, the cancer cells in vessel co-opting lesions must displace the hepatocytes and occupy their space. However, the mechanisms underlying this displacement are unknown. Herein, we examined the involvement of apoptosis, autophagy, motility, and epithelial-mesenchymal transition (EMT) pathways in hepatocyte displacement by cancer cells. We demonstrate that cancer cells induce the expression of the proteins that are associated with the upregulation of apoptosis, motility, and EMT in adjacent hepatocytes in vitro and in vivo. Accordingly, we observe the upregulation of cleaved caspase-3, cleaved poly (ADP-ribose) polymerase-1 (PARP-1) and actin-related protein 2/3 (ARP2/3) in adjacent hepatocytes to cancer cell nests, while we notice a downregulation of E-cadherin. Importantly, the knockdown of runt-related transcription factor 1 (RUNX1) in cancer cells attenuates the function of cancer cells in hepatocytes alterations in vitro and in vivo. Altogether, our data suggest that cancer cells exploit various mechanisms to displace hepatocytes and access the sinusoidal vessels to establish vessel co-option.