Hypoxia alters the release and size distribution of extracellular vesicles in pancreatic cancer cells to support their adaptive survival.

Pancreatic tumors are highly desmoplastic and poorly-vascularized, and therefore must develop adaptive mechanisms to sustain their survival under hypoxic condition. Extracellular vesicles (EV) play vital roles in pancreatic tumor pathobiology by facilitating intercellular communication. Here we studied the effect of hypoxia on the release of EVs and examined their role in adaptive survival of pancreatic cancer (PC) cells. Hypoxia promoted the release of EV in PC cell lines, MiaPaCa and AsPC1, wherein former exhibited a far greater induction. Moreover, a time-dependent, measurable and significant increase was recorded for small EV (SEV) in both the cell lines with only minimal induction observed for medium (MEV) and large EVs (LEV). Similarly, noticeable changes in size distribution of SEV were also recorded with a shift toward smaller average size under extreme hypoxia. Thrombospondin (apoptotic bodies marker) was exclusively detected on LEVs, while Arf6 (microvesicles marker) was mostly present on MEV with some expression in LEV as well. However, CD9 and CD63 (exosome markers) were expressed in both SEV and MEVs with a decreased expression recorded under hypoxia. Among all subfractions, SEV was the most bioactive in promoting the survival of hypoxic PC cells and hypoxia-inducible factor-1α stabilization was involved in heightened EV release under hypoxia and for their potency to promote hypoxic cell survival. Altogether, our findings provide a novel mechanism for the adaptive hypoxic survival of PC cells and should serve as the basis for future investigations on broader functional implications of EV.

Exosomes confer chemoresistance to pancreatic cancer cells by promoting ROS detoxification and miR-155-mediated suppression of key gemcitabine-metabolising enzyme, DCK.

BACKGROUND: Chemoresistance is a significant clinical problem in pancreatic cancer (PC) and underlying molecular mechanisms still remain to be completely understood. Here we report a novel exosome-mediated mechanism of drug-induced acquired chemoresistance in PC cells. METHODS: Differential ultracentrifugation was performed to isolate extracellular vesicles (EVs) based on their size from vehicle- or gemcitabine-treated PC cells. Extracellular vesicles size and subtypes were determined by dynamic light scattering and marker profiling, respectively. Gene expression was examined by qRT-PCR and/or immunoblot analyses, and direct targeting of DCK by miR-155 was confirmed by dual-luciferase 3'-UTR reporter assay. Flow cytometry was performed to examine the apoptosis indices and reactive oxygen species (ROS) levels in PC cells using specific dyes. Cell viability was determined using the WST-1 assay. RESULTS: Conditioned media (CM) from gemcitabine-treated PC cells (Gem-CM) provided significant chemoprotection to subsequent gemcitabine toxicity and most of the chemoresistance conferred by Gem-CM resulted from its EVs fraction. Sub-fractionation grouped EVs into distinct subtypes based on size distribution and marker profiles, and exosome (Gem-Exo) was the only sub-fraction that imparted chemoresistance. Gene expression analyses demonstrated upregulation of SOD2 and CAT (ROS-detoxifying genes), and downregulation of DCK (gemcitabine-metabolising gene) in Gem-Exo-treated cells. SOD/CAT upregulation resulted, at least in part, from exosome-mediated transfer of their transcripts and they suppressed basal and gemcitabine-induced ROS production, and partly promoted chemoresistance. DCK downregulation occurred through exosome-delivered miR-155 and either the functional suppression of miR-155 or restoration of DCK led to marked abrogation of Gem-Exo-mediated chemoresistance. CONCLUSIONS: Together, these findings establish a novel role of exosomes in mediating the acquired chemoresistance of PC.

DNA microarray analysis of the heat shock transcriptome of the obligate intracytoplasmic pathogen Rickettsia prowazekii.

Here we present the first oligonucleotide DNA microarray analysis of global gene expression changes in the obligate intracytoplasmic pathogen Rickettsia prowazekii using temperature upshift as a model stress condition, and we describe a methodology for isolating highly purified rickettsial RNA. In toto, 23 transcripts were significantly increased by temperature upshift (> or = 2.0-fold; P < 0.05), and no transcripts demonstrated reproducible decreases. Array results for three heat shock-inducible mRNAs were confirmed using quantitative reverse transcription-PCR.

Exosomal Markers (CD63 and CD9) Expression Pattern Using Immunohistochemistry in Resected Malignant and Nonmalignant Pancreatic Specimens.

OBJECTIVES: Exosomes are important mediators in intercellular communications and play a role in cancer progression and metastasis. Exosomal membranes are enriched in endosome-specific tetraspanins (CD9 and CD63). Here, we explored the expression of CD63 and CD9 utilizing immunohistochemistry in malignant and nonmalignant cells in 29 resected pancreatic specimens (RPSs) of mixed racial background. METHODS: The pathologic tissues (PTs) and adjacent normal tissues (ANTs) in each RPS were stained for CD63 and CD9. Two pathologists independently scored the expression of CD63 and CD9. Staining intensity was graded from 1 to 3. Staining percentage was estimated in 10% increments. An average Quick score (Q score) (intensity × percentage of staining) was calculated. Unpaired t test was used for statistical analysis. RESULTS: The mean multiplicative Q score for CD63 and CD9 expression is higher in PTs (209 and 72) compared with ANTs (154 and 24) (P = 0.0041, P = 0.0018), respectively. The Mean Q score for CD63 and CD9 expression is higher in the malignant PTs (231 and 85) compared with ANTs (129 and 25) (P < 0.0001 and P < 0.0124). CONCLUSIONS: Exosomal markers (CD63 and CD9) expression assessment using immunohistochemistry is feasible in RPS. The expression of CD63 and CD9 is higher in PTs and malignant PTs compared with their ANTs.