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PDF file - 174K, Expression of the GDNF receptors GFRa1 and RET in specimens excised from patients with pancreatic ductal adenocarcinoma.(a) Low magnification (X20) immunofluorescence staining with anti-RET ab (green, Alexa 485), (b) anti-GFRa1 ab (red, Alexa 565) , and (c) overlay image with DAPI staining (blue). (d) Immunohistochemical staining of the same specimen with anti-CK19 ab (brown staining). (e) High magnification (X40) fluorescent microscope imaging performed with anti-RET (green, Alexa 485), (f) anti-GFRa1 antibodies (red, Alexa 565) and (g) DAPI (blue staining). The overlay image in (h) shows co-expression of the receptors by PDA cells.

PDF file - 112K, The ability of EMPhis to invade in a matrigel matrix towards a colony of PDA cancer cells.(a) An illustration showing the calculation of the invasion distance. (b) A photograph of the assay showing that the invasion of EMPhis was more prominent towards the cancer colony than to the other direction (magnification X4). Cancer cells were differentiated from EMPhi by morphologic criteria. (c) Analysis of the results of the invasion assay showing significantly higher migration towards the tumor colony (a- blue bar) compared to the opposite directions (g and beta - red bars, P

PDF file - 178K, Scratch assays of MiaPaCa2 cells in the presence or absence of EMPhi-CM. (a) Representative pictures taken after 24 and 48 hours showing increased motility of cancer cells incubated with EMPhi-CM (lower row) relative to normal medium (DMEM- upper row). (b) Summary of the data showing statistically significant increase in cancer cell motility after incubation in EMPhi-CM compared to normal media (n=6, P

PDF file - 25K, The effect of PYP1 on the PDA cells proliferation. As revealed by XTT assay, PYP1 (2mum) had no effect on cancer cell proliferation induced by the EMPhi conditioned media.

Human colonic organoids derived from adult tissue biopsies are based on the ability of isolated somatic epithelial stem cells to reconstitute the structure and function of the colon, offering new opportunities for studying the biology of the large intestine in both health and disease. These colonoids may also function as efficient platforms for drug screening and discovery. Here, we describe the establishment of human colonic organoids derived from healthy, and adenomatous polyp tissues. We then demonstrate that organoids grown from adenomas of familial adenomatous polyposis (FAP) patients harboring nonsense mutations in the tumor suppressor gene adenomatous polyposis coli (APC), can be used to establish a personalized therapeutic strategy which relies on nonsense mutation readthrough therapy.

Carboxypeptidase E (CPE) a key factor in the biosynthesis of most peptide hormones and neuropeptides, is predominantly expressed in endocrine tissues and the nervous system. This highly conserved enzyme cleaves the C-terminal basic residues of the peptide precursors to generate their bioactive form. CPE is a secreted protein; however, the Intracellular pathways leading to its secretion are still obscure. We combined live-cell microscopy and molecular analysis to examine the intracellular distribution and secretion dynamics of fluorescently tagged CPE. CPE was found to be a soluble luminal protein as it traffics from the ER via the Golgi apparatus to lysosomes. Moreover, CPE is efficiently secreted and reinternalized to lysosomes of neighboring cells. The C-terminal amphipathic helix of CPE is essential for its efficient targeting to, and secretion from lysosomes. Fluorescence resonance energy transfer demonstrated that CPE and its substrate neuropeptide Y (NPY) interact in the Golgi apparatus and Immunoprecipitation analysis demonstrated that both CPE and NPY are co-secreted. The implications of the well-defined CPE intra and extracellular routes are discussed.

The endoplasmic reticulum (ER) is involved in biogenesis, modification and transport of secreted and membrane proteins. The ER membranes are spread throughout the cell cytoplasm as well as the export domains known as ER exit sites (ERES). A subpopulation of ERES is centrally localized proximal to the Golgi apparatus. The significance of this subpopulation on ER-to-Golgi transport remains unclear. Transport carriers (TCs) form at the ERES via a COPII-dependent mechanism and move to Golgi on microtubule (MT) tracks. It was shown previously that ERES are distributed along MTs and undergo chaotic short-range movements and sporadic rapid long-range movements. The long-range movements of ERES are impaired by either depolymerization of MTs or inhibition of dynein, suggesting that ERES central concentration is mediated by dynein activity. We demonstrate that the processive movements of ERES are frequently coupled with the TC departure. Using the Sar1a[H79G]-induced ERES clustering at the perinuclear region, we identified BicaudalD2 (BicD2) and Rab6 as components of the dynein adaptor complex which drives perinuclear ERES concentration at the cell center. BicD2 partially colocalized with ERES and with TC. Peri-Golgi ERES localization was significantly affected by inhibition of BicD2 function with its N-terminal fragment or inhibition of Rab6 function with its dominant-negative mutant. Golgi accumulation of secretory protein was delayed by inhibition of Rab6 and BicD2. Thus, we conclude that a BicD2/Rab6 dynein adaptor is required for maintenance of Golgi-associated ERES. We propose that Golgi-associated ERES may enhance the efficiency of the ER-to-Golgi transport.

SummaryCOPII and COPI are considered to be analogous sets of vesicle coat protein heterocomplexes. Coupled to cargo selection, they mediate the formation of membrane vesicles translocating in opposite directions to differ rent destinations within the secretory pathway. Here, live cell and electron microscopy provided evidence for a different localization and mode of function of the COPII coat during protein export from the endoplasmic reticulum (ER). Pharmaceutical and genetic perturbations of ER-Golgi transport were used to demonstrate that COPII is recruited to membranes defining the boundary of ER-ER Exit Sites (ERES) where it facilitates selective cargo concentration. Uncoating of COPII membranes precedes cargo accumulation and fission of Golgi-bound carriers. Moreover, we report what may be direct transfer of cargo to the Golgi apparatus from Golgi-associated BFA sensitive ERESs. Finally, in ldlF cells the stably expressed functional ε-COPI-EYFP labeled both ERESs and anterograde carriers. These findings change our understanding of the role of coat proteins in ER to Golgi transport.

Live-cell microscopy reveals a novel mode of function for COPII: COPII binds the membrane at the boundary between the ER and ER exit sites, where it executes cargo sorting and concentration into ERES and does not coat cargo exporting containers., COPII and COPI mediate the formation of membrane vesicles translocating in opposite directions within the secretory pathway. Live-cell and electron microscopy revealed a novel mode of function for COPII during cargo export from the ER. COPII is recruited to membranes defining the boundary between the ER and ER exit sites, facilitating selective cargo concentration. Using direct observation of living cells, we monitored cargo selection processes, accumulation, and fission of COPII-free ERES membranes. CRISPR/Cas12a tagging, the RUSH system, and pharmaceutical and genetic perturbations of ER-Golgi transport demonstrated that the COPII coat remains bound to the ER–ERES boundary during protein export. Manipulation of the cargo-binding domain in COPII Sec24B prohibits cargo accumulation in ERES. These findings suggest a role for COPII in selecting and concentrating exported cargo rather than coating Golgi-bound carriers. These findings transform our understanding of coat proteins’ role in ER-to-Golgi transport.

Export out of the endoplasmic reticulum (ER) involves the Sar1 and COPII machinery acting at ER exit sites (ERES). Whether and how cargo proteins are recruited upstream of Sar1 and COPII is unclear. Two models are conceivable, a recruitment model where cargo is actively transported through a transport factor and handed over to the Sar1 and COPII machinery in ERES, and a capture model, where cargo freely diffuses into ERES where it is captured by the Sar1 and COPII machinery. Using the novel secretion inhibitor FLI-06, we show that recruitment of the cargo VSVG to ERES is an active process upstream of Sar1 and COPII. Applying FLI-06 before concentration of VSVG in ERES completely abolishes its recruitment. In contrast, applying FLI-06 after VSVG concentration in ERES does not lead to dispersal of the concentrated VSVG, arguing that it inhibits recruitment to ERES as opposed to capture in ERES. FLI-06 also inhibits export out of the trans-Golgi network (TGN), suggesting that similar mechanisms might orchestrate cargo selection and concentration at the ER and TGN. FLI-06 does not inhibit autophagosome biogenesis and the ER-peroxisomal transport route, suggesting that these rely on different mechanisms.

Perineural invasion of cancer cells (CPNI) is found in most patients with pancreatic adenocarcinomas (PDAs), prostate or head and neck cancers. These patients undergo palliative rather than curative treatment due to dissemination of cancer along nerves, well beyond the extent of any local invasion. Although CPNI is a common source of distant tumor spread and a cause of significant morbidity, its exact mechanism is undefined. Immunohistochemical analysis of specimens excised from patients with PDAs showed a significant increase in the number of endoneurial macrophages (EMΦs) which lie around nerves invaded by cancer compared to normal nerves. Video microscopy and time-lapse analysis revealed that EMΦs are recruited by the tumor cells in response to colony stimulated factor-1 secreted by invading cancer cells. Conditioned medium (CM) of tumor-activated EMΦs (tEMΦs) induced a 5-fold increase in migration of PDA cells compared to controls. Compared to resting EMΦs, tEMΦs secreted higher levels of glial-derived neurotrophic factor (GDNF), inducing phosphorylation of RET and downstream activation of extracellular signal-regulated kinases (ERK) in PDA cells. Genetic and pharmacologic inhibition of the GDNF receptors GFRA1 and RET abrogated the migratory effect of EMΦ-CM and reduced ERK phosphorylation. In an in-vivo CPNI model, CCR2-deficient mice which have reduced macrophages recruitment and activation showed minimal nerve invasion, while wild-type mice developed complete sciatic nerve paralysis due to massive CPNI. Taken together, our results identify a paracrine response between EMΦs and PDA cells which orchestrates the formation of cancer nerve invasion. Citation Format: Oren Cavel, Richard J. Wong, Olga Shomron, Moran Amit, Ziv Gil. Endoneurial macrophages induce perineural invasion of pancreatic cancer cells by secretion of GDNF and activation of RET tyrosine kinase receptor. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 1529. doi:10.1158/1538-7445.AM2013-1529 Note: This abstract was not presented at the AACR Annual Meeting 2013 because the presenter was unable to attend.