Carvalho, T. G. | Search bioRxiv

bioRxiv2022

A novel and simple heat-based method eliminates the highly detrimental effect of xylene deparaffinization on acid-fast stains.

ObjectivesHistopathology is an important method for the diagnosis of extrapulmonary tuberculosis, yet tissue sections are often negative for mycobacteria after acid-fast-staining (AFS). This study investigated the mechanism of AFS and the detrimental effect of histological processing, in particular xylene deparaffinization, on AFS staining and mycobacterial detection. MethodsThe target of the fluorescent Auramine O (AuO) AFS was investigated using triple staining with DNA and RNA specific dyes. The effect of xylene deparaffinization on acid-fastness of mycobacteria in cultures or tissue sections was studied using AuO fluorescence as a quantitative marker. The xylene method was compared to a novel, solvent-free projected-hot-air-deparaffinization (PHAD). ResultsColocalization of AuO with DNA/RNA stains suggests that intracellular nucleic acids are the true target of AFS, producing highly specific patterns. Xylene reduces mycobacterial fluorescence significantly (p<0,0001, moderate effect size: r = 0.33). PHAD yielded significantly higher fluorescence than xylene deparaffinization in tissues (p<0.0001, large effect size: r = 0.85). ConclusionsAuO can be applied for nucleic-acid-staining of mycobacteria in tissues producing typical beaded patterns. AFS depends crucially on the integrity of the mycobacterial cell-wall, which seems to be damaged by xylene. A solvent-free tissue deparaffinization method has the potential to increase mycobacterial detection significantly. Key PointsAcid-fast-stains for mycobacterial detection have a notoriously poor yield, due to misconceptions of the underlying staining principle, but mainly because of the possible detrimental effect of xylene. The yield of acid-fast-stains for mycobacterial detection in histology, including fluorescent stains, can be significantly increased by eliminating solvents (xylene) in the deparaffinization step. Acid-fast-stains depend on bacterial integrity because they target nucleic acids. A simple hot air deparaffinization is significantly superior to xylene, avoiding damage to the lipid-rich cell wall.

pathology↗

bioRxiv2019

Telomere shortening produces an inflammatory environment that promotes tumor invasiveness in zebrafish

Cancer incidence increases exponentially with age, when human telomeres are shorter. Similarly, telomerase mutant zebrafish (tert) have premature short telomeres and anticipate cancer incidence to younger ages. However, because short telomeres constitute a road block to cell proliferation, telomere shortening is currently viewed as a tumor suppressor mechanism and should protect from cancer. This conundrum is not fully understood. In our current study, we report that telomere shortening promotes cancer in a non-cell autonomous manner. Using zebrafish chimeras, we show increased incidence of invasive melanoma when WT tumors are generated in tert mutant zebrafish. tert zebrafish show increased levels of senescence (cdkn2a and ink4a/b) and inflammation (TNF-). In addition, we transferred second generation tert blastula cells into WT to produce embryo chimeras. Cells with very short telomeres induced senescence and increased neutrophil numbers in surrounding larval tissues in a non-cell autonomous manner, creating an inflammatory environment. Considering that inflammation is pro-tumorigenic, we transplanted melanoma-derived cells into second generation tert zebrafish embryos and observed that tissue environment with short telomeres leads to increased micrometastasis. To test if inflammation was necessary for this effect, we treated melanoma transplants with non-steroid anti-inflammatory drugs and show that higher melanoma invasiveness can be averted. Thus, apart from the cell autonomous role of short telomeres in contributing to genome instability, we propose that telomere shortening with age causes systemic chronic inflammation leading to increased tumor incidence. Significance StatementCancer incidence increases exponentially in human midlife. Even though mutation accumulation in somatic tissues results in increased tumorigenesis, it is currently not understood how aging contributes to cancer. Telomeres, the ends of eukaryotic linear chromosomes, shorten with each cell division. Here we show that telomere shortening contributes to cancer in a non-cell autonomous manner. Using embryo chimeras of telomerase deficient zebrafish generated from melanoma-prone fish, we show that tumors arise more frequently and become more invasive in animals with shorter telomeres. Telomere shortening gives rise to increased senescence and systemic inflammation. We observed increased melanoma metastasis dissemination in zebrafish larvae with very short telomeres. Thus, telomere shortening similar to human aging, generates a chronic inflammatory environment that increases cancer incidence.

cancer biology↗