One-fits-all pretreatment protocol facilitating Fluorescence in Situ Hybridization on formalin-fixed paraffin-embedded, fresh frozen and cytological slides

Background: The Fluorescence In Situ Hybridization (FISH) technique is a very useful tool for diagnostic and prognostic purposes in molecular pathology. However, clinical testing on patient tissue is challenging due to variables of tissue processing that can influence the quality of the results. This emphasizes the necessity of a standardized FISH protocol with a high hybridization efficiency. We present a pretreatment protocol that is easy, reproducible, cost-effective, and facilitates FISH on all types of patient material simultaneously with good quality results. During validation, FISH analysis was performed simultaneously on formalin-fixed paraffin-embedded, fresh frozen and cytological patient material in combination with commercial probes using our optimized one-fits-all pretreatment protocol. An optimally processed sample is characterized by strong specific signals, intact nuclear membranes, non-disturbing autofluorescence and a homogeneous DAPI staining. Results: In our retrospective cohort of 3881 patient samples, overall 93% of the FISH samples displayed good quality results leading to a patient diagnosis. All FISH were assessed on quality aspects such as adequacy and consistency of signal strength (brightness), lack of background and / or cross-hybridization signals, and additionally the presence of appropriate control signals were evaluated to assure probe accuracy. In our analysis 38 different FISH probes from 3 commercial manufacturers were used (Cytocell, Vysis and ZytoLight). The majority of the patients in this cohort displayed good signal quality and barely non-specific background fluorescence on all tissue types independent of which commercial probe was used. Conclusion: The optimized one-fits-all FISH method is robust, reliable and reproducible to deliver an accurate result for patient diagnostics in a lean workflow and cost-effective manner. This protocol can be used for widespread application in cancer and non-cancer diagnostics and research

Cardiomyocytes stimulate angiogenesis after ischemic injury in a ZEB2-dependent manner

The disruption in blood supply due to myocardial infarction is a critical determinant for infarct size and subsequent deterioration in function. The identification of factors that enhance cardiac repair by the restoration of the vascular network is, therefore, of great significance. Here, we show that the transcription factor Zinc finger E-box-binding homeobox 2 (ZEB2) is increased in stressed cardiomyocytes and induces a cardioprotective cross-talk between cardiomyocytes and endothelial cells to enhance angiogenesis after ischemia. Single-cell sequencing indicates ZEB2 to be enriched in injured cardiomyocytes. Cardiomyocyte-specific deletion of ZEB2 results in impaired cardiac contractility and infarct healing post-myocardial infarction (post-MI), while cardiomyocyte-specific ZEB2 overexpression improves cardiomyocyte survival and cardiac function. We identified Thymosin β4 (TMSB4) and Prothymosin α (PTMA) as main paracrine factors released from cardiomyocytes to stimulate angiogenesis by enhancing endothelial cell migration, and whose regulation is validated in our in vivo models. Therapeutic delivery of ZEB2 to cardiomyocytes in the infarcted heart induces the expression of TMSB4 and PTMA, which enhances angiogenesis and prevents cardiac dysfunction. These findings reveal ZEB2 as a beneficial factor during ischemic injury, which may hold promise for the identification of new therapies