Necroptosis activation is associated with greater methylene blue-photodynamic therapy-induced cytotoxicity in human pancreatic ductal adenocarcinoma cells.

Pancreatic ductal adenocarcinomas (PDAC) are the fourth leading cause of death due to neoplasms. In view of the urgent need of effective treatments for PDAC, photodynamic therapy (PDT) appears as a promising alternative. However, its efficacy against PDAC and the mechanisms involved in cell death induction remain unclear. In this study, we set out to evaluate PDT's cytotoxicity using methylene blue (MB) as a photosensitizer (PS) (MB-PDT) and to evaluate the contribution of necroptosis in its effect in human PDAC cells. Our results demonstrated that MB-PDT induced significant death of different human PDAC models presenting two different susceptibility profiles. This effect was independent of MB uptake or its subcellular localization. We found that the ability of triggering necroptosis was determinant to increase the treatment efficiency. Analysis of single cell RNA-seq data from normal and neoplastic human pancreatic tissues showed that specific necroptosis proteins RIPK1, RIPK3 and MLKL presented significant higher expression levels in cells displaying a transformed phenotype providing further support to the use of approaches that activate necroptosis, like MB-PDT, as useful adjunct to surgery of PDAC to tackle the problem of microscopic residual disease as well as to minimize the chance of local and metastatic recurrence.

Reverse transcription lesion-induced DNA amplification: An instrument-free isothermal method to detect RNA.

One challenge in point-of-care (POC) diagnostics is the lack of room-temperature methods for RNA detection based on enzymatic amplification and visualization steps. Here we perform reverse transcription lesion-induced DNA amplification (RT-LIDA), an isothermal amplification method that only requires T4 DNA ligase. RT-LIDA involves the RNA-templated ligation of DNA primers to form complementary DNA (cDNA) followed by toehold-mediated strand displacement of the cDNA and its exponential amplification via our isothermal ligase chain reaction LIDA. Each step is tuned to proceed at 28 °C, which falls within the range of global room temperatures. Using RT-LIDA, we can detect as little as ∼100 amol target RNA and can distinguish RNA target from total cellular RNA. Finally, we demonstrate that the resulting DNA amplicons can be detected colorimetrically, also at room temperature, by rapid, target-triggered disassembly of DNA-modified gold nanoparticles. This integrated amplification/detection platform requires no heating or visualization instrumentation, which is an important step towards realizing instrument-free POC testing.