Polony gels enable amplifiable DNA stamping and spatial transcriptomics of chronic pain.

Methods for acquiring spatially resolved omics data from complex tissues use barcoded DNA arrays of low- to sub-micrometer features to achieve single-cell resolution. However, fabricating such arrays (randomly assembled beads, DNA nanoballs, or clusters) requires sequencing barcodes in each array, limiting cost-effectiveness and throughput. Here, we describe a vastly scalable stamping method to fabricate polony gels, arrays of ∼1-micrometer clonal DNA clusters bearing unique barcodes. By enabling repeatable enzymatic replication of barcode-patterned gels, this method, compared with the sequencing-dependent array fabrication, reduced cost by at least 35-fold and time to approximately 7 h. The gel stamping was implemented with a simple robotic arm and off-the-shelf reagents. We leveraged the resolution and RNA capture efficiency of polony gels to develop Pixel-seq, a single-cell spatial transcriptomic assay, and applied it to map the mouse parabrachial nucleus and analyze changes in neuropathic pain-regulated transcriptomes and cell-cell communication after nerve ligation.

Fortilin interacts with TGF-ß1 and prevents TGF-ß receptor activation.

Fortilin is a 172-amino acid multifunctional protein present in both intra- and extracellular spaces. Although fortilin binds and regulates various cellular proteins, the biological role of extracellular fortilin remains unknown. Here we report that fortilin specifically interacts with TGF-ß1 and prevents it from activating the TGF-ß1 signaling pathway. In a standard immunoprecipitation-western blot assay, fortilin co-immunoprecipitates TGF-ß1 and its isoforms. The modified ELISA assay shows that TGF-ß1 remains complexed with fortilin in human serum. Both bio-layer interferometry and surface plasmon resonance (SPR) reveal that fortilin directly bind TGF-ß1. The SPR analysis also reveals that fortilin and the TGF-ß receptor II (TGFßRII) compete for TGF-ß1. Both luciferase and secreted alkaline phosphatase reporter assays show that fortilin prevents TGF-ß1 from activating Smad3 binding to Smad-binding element. Fortilin inhibits the phosphorylation of Smad3 in both quantitative western blot assays and ELISA. Finally, fortilin inhibits TGFß-1-induced differentiation of C3H10T1/2 mesenchymal progenitor cells to smooth muscle cells. A computer-assisted virtual docking reveals that fortilin occupies the pocket of TGF-ß1 that is normally occupied by TGFßRII and that TGF-ß1 can bind either fortilin or TGFßRII at any given time. These data support the role of extracellular fortilin as a negative regulator of the TGF-ß1 signaling pathway.

Potential role of HIF-1-responsive microRNA210/HIF3 axis on gemcitabine resistance in cholangiocarcinoma cells.

MicroRNA-210 (miR-210) is a robust target for hypoxia-inducible factor, and its overexpression has been detected in a variety of solid tumors. However, the role of miR-210 in the development, progression and response to therapy in cholangiocarcinoma (CCA) remains undefined. We report here that high miR-210 expression was significantly correlated with the shorter survival of CCA patients. Overexpression of miR-210 inhibited CCA cell proliferation at the G2/M phase and reduced the gemcitabine sensitivity in CCA cells under CoCl2-induced pseudohypoxia. Concomitantly, inhibition of endogenous miR-210 activity using miRNA sponges increased cell proliferation under CoCl2-induced pseudohypoxia, resulting in an increase in gemcitabine sensitivity in CCA cells. We showed that HIF-3α, a negative controller of HIF-1α, was a target of miR-210 constituting a feed-forward hypoxic regulatory loop. Our data suggest an important role of miR-210 in sustaining HIF-1α activity via the suppression of HIF-3α, regulating cell growth and chemotherapeutic drug resistance in CCA.

Antifibrotic effect of xanthohumol in combination with praziquantel is associated with altered redox status and reduced iron accumulation during liver fluke-associated cholangiocarcinogenesis.

Cholangiocarcinoma (CCA) caused by infection of the liver fluke Opisthorchis viverrini, (Ov) is the major public health problem in northeast Thailand. Following Ov infection the subsequent molecular changes can be associated by reactive oxygen species (ROS) induced chronic inflammation, advanced periductal fibrosis, and cholangiocarcinogenesis. Notably, resistance to an activation of cell death in prolonged oxidative stress conditions can occur but some damaged/mutated cells could survive and enable clonal expansion. Our study used a natural product, xanthohumol (XN), which is an anti-oxidant and anti-inflammatory compound, to examine whether it could prevent Ov-associated CCA carcinogenesis. We measured the effect of XN with or without praziquantel (PZ), an anti-helminthic treatment, on DNA damage, redox status change including iron accumulation and periductal fibrosis during CCA genesis induced by administration of Ov and N-dinitrosomethylamine (NDMA) in hamsters. Animals were randomly divided into four groups: group I, Ov infection and NDMA administration (ON); group II, Ov infection and NDMA administration and PZ treatment (ONP); the latter 2 groups were similar to group I and II, but group III received additional XN (XON) and group IV received XN plus PZ (XONP). The results showed that high 8-oxodG (a marker of DNA damage) was observed throughout cholangiocarcinogenesis. Moreover, increased expression of CD44v8-10 (a cell surface in regulation of the ROS defense system), whereas decreased expression of phospho-p38MAPK (a major ROS target), was found during the progression of the bile duct cell transformation. In addition, high accumulation of iron and expression of transferrin receptor-1 (TfR-1) in both malignant bile ducts and inflammatory cells were detected. Furthermore, fibrosis also increased with the highest level being on day 180. On the other hand, the groups of XN with or without PZ supplementations showed an effective reduction in all the markers examined, including fibrosis when compared with the ON group. In particular, the XONP group, in which a significant reduction DNA damage occurred, was also found to have iron accumulation and fibrosis compared to the other groups. Our results show that XN administered in combination with PZ could efficiently prevent CCA development and hence provide potential chemopreventive benefits in Ov-induced cholangiocarcinogenesis.

Urinary microRNA-192 and microRNA-21 as potential indicators for liver fluke-associated cholangiocarcinoma risk group.

Opisthorchis viverrini infection induces chronic inflammation in the bile ducts, leading to periductal fibrosis (PDF), which possibly associates to cholangiocarcinoma (CCA). Patients with CCA have a poor prognosis, which is linked to asymptomatic disease and late diagnosis. Hence, detecting early stage CCA is essential. Secretory miRNAs have been promoted as biomarkers for pathological changes associated with parasitic infections, fibrosis and/or cancer. We aimed to determine levels of miR-192 and miR-21 in the urine of O. viverrini infected, periductal fibrosis (PDF) and CCA groups using qRT-PCR. We found that miR-192 was significantly higher in O. viverrini infected, PDF and also CCA groups (p<0.05) than in healthy controls. By utilizing the Receiver Operation Characteristics (ROC) analysis, miR-192 differentiated patients with opisthorchiasis (the area under the curve; AUC=0.766), PDF subjects (AUC=0.781) and CCA patients (AUC=0.682) from healthy controls. MiR-21 was significantly higher in PDF and CCA groups (p<0.05) than in healthy controls. MiR-21 discriminated PDF subjects (AUC=0.735) and CCA patients (AUC=0.682) from healthy controls. Combined levels of these two miRNAs revealed an increased AUC of 0.812 for separating opisthorchiasis, AUC of 0.815 in discriminating PDF subjects, and AUC of 0.849 in differentiating CCA from healthy controls. Odds ratios (OR) indicated high levels of miR-192/miR-21 as risk predictors for opisthorchiasis, PDF and CCA. Levels of these miRNAs declined significantly for patients following praziquantel treatment. In conclusion, urinary miR-192/miR-21 have potential as risk indicators for opisthorchiasis and PDF-associated CCA in the endemic region.

Circulating miR-192 in liver fluke-associated cholangiocarcinoma patients: a prospective prognostic indicator.

BACKGROUND: This study aimed to investigate the miR-192 levels in patients' sera of liver fluke-associated cholangiocarcinoma (CCA) for a prospective prognostic indicator. METHODS: MicroRNA polymerase chain reaction (PCR) array was performed using pooled serum samples from 11 CCA patients and nine healthy subjects. Selected miRNAs were verified for the differential levels in both sera and tumor tissues (of patients and Opisthorchis viverrini (Ov)-induced CCA model) using TaqMan miRNA expression assay. RESULTS: Our results demonstrated that miR-192 was significantly higher in the serum of CCA patients than that in healthy subjects giving a sensitivity of 74% and specificity of 72% (area under the curve [AUC] = 0.803; 95% confidence interval [CI], 0.708-0.897, P < 0.0001). Serum miR-192 examined in Ov infected subjects and subjects with periductal fibrosis were increased but not statistically significantly when compared with healthy subjects. High levels of serum miR-192 were significantly correlated with lymph node metastasis (P = 0.047) and shorter survival compared with individuals with low levels of serum miR-192 (hazard ratio [HR] 2.076, 95% CI 1.004-4.291, P = 0.049). We also found that the expression levels of miR-192 appeared to be elevated in both CCA tissues of patients and in Ov-induced CCA tissues of a hamster model. CONCLUSIONS: This finding indicates that elevated levels of miR-192 may be involved in CCA genesis and have a potential utility as a noninvasive prognostic indicator for CCA patients.