Pancreatic stellate cells (PSCs) are the key precursor cells for cancer-associated fibroblasts (CAFs) in pancreatic tumor stroma. In this study, we explored miRNA as therapeutic targets in tumor ...stroma and found miR-199a-3p and miR-214-3p induced in patient-derived pancreatic CAFs and TGF-β-activated human PSCs (hPSCs). Inhibition of miR-199a/-214 using hairpin inhibitors significantly inhibited TGFβ-induced differentiation markers (e.g. α-SMA, collagen, PDGFβR), migration and proliferation. Furthermore, heterospheroids of Panc-1 and hPSCs attained smaller size with hPSCs transfected with anti-miR-199a/-214 compared to control anti-miR. The conditioned medium obtained from TGFβ-activated hPSCs induced tumor cell growth and endothelial cell tube formation. Interestingly, these inductions were abrogated in hPSCs transfected with anti-miR-199a or miR-214. Moreover, IPA analyses revealed signaling pathways related to miR-199a (TP53, mTOR, Smad1) and miR-214 (PTEN, Bax, ING4). Taken together, this study reveals miR-199a-3p and miR-214-3p as major regulators of PSC activation and PSC-induced pro-tumoral effects, representing them as key therapeutic targets in pancreatic cancer.
Tumors initiate by mutations in cancer cells, and progress through interactions of the cancer cells with non-malignant cells of the tumor microenvironment. Major players in the tumor microenvironment ...are cancer-associated fibroblasts (CAFs), which support tumor malignancy, and comprise up to 90% of the tumor mass in pancreatic cancer. CAFs are transcriptionally rewired by cancer cells. Whether this rewiring is differentially affected by different mutations in cancer cells is largely unknown. Here we address this question by dissecting the stromal landscape of BRCA-mutated and BRCA Wild-type pancreatic ductal adenocarcinoma. We comprehensively analyze pancreatic cancer samples from 42 patients, revealing different CAF subtype compositions in germline BRCA-mutated vs. BRCA Wild-type tumors. In particular, we detect an increase in a subset of immune-regulatory clusterin-positive CAFs in BRCA-mutated tumors. Using cancer organoids and mouse models we show that this process is mediated through activation of heat-shock factor 1, the transcriptional regulator of clusterin. Our findings unravel a dimension of stromal heterogeneity influenced by germline mutations in cancer cells, with direct implications for clinical research.
Cancer-related mortality is primarily a consequence of metastatic dissemination and associated complications. Pancreatic ductal adenocarcinoma (PDAC) is one of the most lethal malignancies and tends ...to metastasize early, especially in the liver. Emerging evidence suggests that organs that develop metastases exhibit microscopic changes that favor metastatic growth, collectively known as “pre-metastatic niches”. By definition, a pre-metastatic niche is chronologically established before overt metastatic outgrowth, and its generation involves the release of tumor-derived secreted factors that modulate cells intrinsic to the recipient organ, as well as recruitment of additional cells from tertiary sites, such as bone marrow—all orchestrated by the primary tumor. The pre-metastatic niche is characterized by tumor-promoting inflammation with tumor-supportive and immune-suppressive features, remodeling of the extracellular matrix, angiogenic modulation and metabolic alterations that support growth of disseminated tumor cells. In this paper, we review the current state of knowledge of the hepatic pre-metastatic niche in PDAC and attempt to create a framework to guide future diagnostic and therapeutic studies.
The role of the epithelial-mesenchymal transition (EMT) in cancer has been studied extensively in vitro, but involvement of the EMT in tumorigenesis in vivo is largely unknown. We investigated the ...potential of microRNAs as clinical markers and analyzed participation of the EMT-associated microRNA-200-ZEB-E-cadherin pathway in cancer progression. Expression of the microRNA-200 family was quantified by real-time RT-PCR analysis of fresh-frozen and microdissected formalin-fixed paraffin-embedded primary colorectal tumors, normal colon mucosa, and matched liver metastases. MicroRNA expression was validated by in situ hybridization and after in vitro culture of the malignant cells. To assess EMT as a predictive marker, factors considered relevant in colorectal cancer were investigated in 98 primary breast tumors from a treatment-randomized study. Associations between the studied EMT-markers were found in primary breast tumors and in colorectal liver metastases. MicroRNA-200 expression in epithelial cells was lower in malignant mucosa than in normal mucosa, and was also decreased in metastatic compared to non-metastatic colorectal cancer. Low microRNA-200 expression in colorectal liver metastases was associated with bad prognosis. In breast cancer, low levels of microRNA-200 were related to reduced survival and high expression of microRNA-200 was predictive of benefit from radiotheraphy. MicroRNA-200 was associated with ER positive status, and inversely correlated to HER2 and overactivation of the PI3K/AKT pathway, that was associated with high ZEB1 mRNA expression. Our findings suggest that the stability of microRNAs makes them suitable as clinical markers and that the EMT-related microRNA-200-ZEB-E-cadherin signaling pathway is connected to established clinical characteristics and can give useful prognostic and treatment-predictive information in progressive breast and colorectal cancers.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
The interplay between the tumor cells and the surrounding stroma creates inflammation, which promotes tumor growth and spread. The inflammation is a hallmark for pancreatic adenocarcinoma (PDAC) and ...is to high extent driven by IL-1α. IL-1α is expressed and secreted by the tumor cells and exerting its effect on the stroma, i.e. cancer associated fibroblasts (CAF), which in turn produce massive amount of inflammatory and immune regulatory factors. IL-1 induces activation of transcription factors such as nuclear factor-κβ (NF-κβ), but also activator protein 1 (AP-1) via the small G-protein Ras. Dysregulation of Ras pathways are common in cancer as this oncogene is the most frequently mutated in many cancers. In contrast, the signaling events leading up to the expression of IL-1α by tumor cells are not well elucidated. Our aim was to examine the signaling cascade involved in the induction of IL-1α expression in PDAC. We found p38MAPK, activated by the K-Ras signaling pathway, to be involved in the expression of IL-1α by PDAC as blocking this pathway decreased both the gene and protein expression of IL-1α. Blockage of the P38MAPK signaling in PDAC also dampened the ability of the tumor cell to induce inflammation in CAFs. In addition, the IL-1α autocrine signaling regulated the migratory capacity of PDAC cells. Taken together, the blockage of signaling pathways leading to IL-1α expression and/or neutralization of IL-1α in the PDAC microenvironment should be taken into consideration as possible treatment or complement to existing treatment of this cancer.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
AIM:To investigate whether nitrite administered prior to ischemia/reperfusion(I/R)reduces liver injury.METHODS:Thirty-six male Sprague-Dawley rats were randomized to 3 groups,including sham ...operated(n=8),45-min segmental ischemia of the left liver lobe(IR,n=14)and ischemia/reperfusion(I/R)preceded by the administration of 480 nmol of nitrite(n=14).Serum transaminases were measured after 4 h of reperfusion.Liver microdialysate(MD)was sampled in 30-min intervals and analyzed for glucose,lactate,pyruvate and glycerol as well as the total nitrite and nitrate(NOx).The NOx was measured in serum.RESULTS:Aspartate aminotransferase(AST)at the end of reperfusion was higher in the IR group than in the nitrite group(40±6.8μkat/L vs 22±2.6μkat/L,P=0.022).Similarly,alanine aminotransferase(ALT)was also higher in the I/R group than in the nitrite group(34±6μkat vs 14±1.5μkat,P=0.0045).The NOx in MD was significantly higher in the nitrite group than in the I/R group(10.1±2.9μmol/L vs 3.2±0.9μmol/L,P=0.031)after the administration of nitrite.During ischemia,the levels decreased in both groups and then increased again during reperfusion.At the end of reperfusion,there was a tendency towards a higher NOx in the I/R group than in the nitrite group(11.6±0.7μmol/L vs 9.2±1.1μmol/L,P=0.067).Lactate in MD was significantly higher in the IR group than in the nitrite group(3.37±0.18 mmol/L vs 2.8±0.12 mmol/L,P=0.01)during ischemia and the first 30 min of reperfusion.During the same period,glycerol was also higher in the IRI group than in the nitrite group(464±38μmol/L vs 367±31μmol/L,P=0.049).With respect to histology,there were more signs of tissue damage in the I/R group than in the nitrite group,and29%of the animals in the I/R group exhibited necrosis compared with none in the nitrite group.Inducible nitric oxide synthase transcription increased between early ischemia(t=15)and the end of reperfusion in both groups.CONCLUSION:Nitrite administered before liver ischemia in the rat liver reduces anaerobic metabolism and cell necrosis,which could be important in the clinical setting.
Characterization of isolated extracellular vesicles and particles (EVPs) is crucial for determining functions and biomarker potential. Here, we present a protocol to analyze size, number, morphology, ...and EVP protein cargo and to validate EVP proteins in both humans and mice. We describe steps for nanoparticle tracking analysis, transmission electron microscopy, single-EVP immunodetection, EVP proteomic mass spectrometry and bioinformatic analysis, and EVP protein validation by ExoELISA and western blot analysis. This allows for EVP cross-validation across different platforms.
For complete details on the use and execution of this protocol, please refer to Hoshino et al.1
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•Cross-validation of isolated EVPs across different platforms•EVP evaluation using nanoparticle tracking analysis and transmission electron microscopy•LC-MS/MS and subsequent bioinformatic analysis for EVP protein cargo identification•Validation of EVP proteins by single-particle analysis, western blot, and exoELISA
Publisher’s note: Undertaking any experimental protocol requires adherence to local institutional guidelines for laboratory safety and ethics.
Characterization of isolated extracellular vesicles and particles (EVPs) is crucial for determining functions and biomarker potential. Here, we present a protocol to analyze size, number, morphology, and EVP protein cargo and to validate EVP proteins in both humans and mice. We describe steps for nanoparticle tracking analysis, transmission electron microscopy, single-EVP immunodetection, EVP proteomic mass spectrometry and bioinformatic analysis, and EVP protein validation by exoELISA and western blot analysis. This allows for EVP cross-validation across different platforms.
AIM:To study the effects of preconditioning on inducible nitric oxide synthase(iNOS)and interleukin 1(IL-1)receptor transcription in rat liver ischemia/reperfusion injury(IRI).METHODS:Seventy-two ...male rats were randomized into 3 groups:the one-hour segmental ischemia(IRI,n=24)group,the ischemic preconditioning(IPC,n=24)group or the remote ischemic preconditioning(RIPC,n=24)group.The IPC and R-IPC were performed as 10 min of ischemia and 10 min of reperfusion.The iNOS and the IL-1 receptor mRNA in the liver tissue was analyzed with real time PCR.The total Nitrite and Nitrate(NOx)in continuously sampled microdialysate(MD)from the liver was analyzed.In addition,the NOx levels in the serum were analyzed.RESULTS:After 4 h of reperfusion,the iNOS mRNA was significantly higher in the R-IPC(ΔCt:3.44±0.57)group than in the IPC(ΔCt:5.86±0.82)group(P=0.025).The IL-1 receptor transcription activity was reduced in the IPC group(ΔCt:1.88±0.53 to 4.81±0.21),but not in the R-IPC group,during reperfusion(P=0.027).In the MD,a significant drop in the NOx levels was noted in the R-IPC group(12.3±2.2 to 4.7±1.2μmol/L)at the end of ischemia compared with the levels in early ischemia(P=0.008).A similar trend was observed in the IPC group(11.8±2.1 to 6.4±1.5μmol/L),although this difference was not statistically significant.The levels of NOx rose quickly during reperfusion in both groups.CONCLUSION:IPC,but not R-IPC,reduces iNOS and IL-1 receptor transcription during early reperfusion,indicating a lower inflammatory reaction.NOx is consumed in the ischemic liver lobe.
Circulating extracellular vesicles and particles (EVPs) are being investigated as potential biomarkers for early cancer detection, prognosis, and disease monitoring. However, the suboptimal purity of ...EVPs isolated from peripheral blood plasma has posed a challenge of in‐depth analysis of the EVP proteome. Here, we compared the effectiveness of different methods for isolating EVPs from healthy donor plasma, including ultracentrifugation (UC)‐based protocols, phosphatidylserine‐Tim4 interaction‐based affinity capture (referred to as “PS”), and several commercial kits. Modified UC methods with an additional UC washing or size exclusion chromatography step substantially improved EVP purity and enabled the detection of additional proteins via proteomic mass spectrometry, including many plasma membrane and cytoplasmic proteins involved in vesicular regulation pathways. This improved performance was reproduced in cancer patient plasma specimens, resulting in the identification of a greater number of differentially expressed EVP proteins, thus expanding the range of potential biomarker candidates. However, PS and other commercial kits did not outperform UC‐based methods in improving plasma EVP purity. PS yielded abundant contaminating proteins and a biased enrichment for specific EVP subsets, thus unsuitable for proteomic profiling of plasma EVPs. Therefore, we have optimized UC‐based protocols for circulating EVP isolation, which enable further in‐depth proteomic analysis for biomarker discovery.
We developed a modified protocol, based on differential ultracentrifugation (dUC), to isolate extracellular vesicles and particles (specifically exomeres) (EVPs) from various human and murine ...sources, including cell lines, surgically resected tumors and adjacent tissues, and bodily fluids, such as blood, lymphatic fluid, and bile. The diversity of these samples requires robust and highly reproducible protocols and refined isolation technology, such as asymmetric-flow field-flow fractionation (AF4). Our isolation protocol allows for preparation of EVPs for various downstream applications, including proteomic profiling.
For complete details on the use and execution of this protocol, please refer to Hoshino et al. (2020).
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•EVP isolation from human cell lines, tissues, and bodily fluids•First protocol describing EVP isolation from human tumor tissues and matched adjacent tissues•Improved EVP isolation and total EVP protein yield by dissecting tissues into pieces•AF4 fractionation to further analyze EVP samples
We developed a modified protocol, based on differential ultracentrifugation (dUC), to isolate extracellular vesicles and particles (specifically exomeres) (EVPs) from various human and murine sources, including cell lines, surgically resected tumors and adjacent tissues, and bodily fluids, such as blood, lymphatic fluid, and bile. The diversity of these samples necessitates robust and highly reproducible protocols and refined isolation technology, such as asymmetric-flow field-flow fractionation (AF4). Our isolation protocol allows for preparation of EVPs for various downstream applications, including proteomic profiling.
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