Search

Abstract Background FFPE tissue samples are commonly used in biomedical research and are a valuable source for next-generation sequencing in oncology, however, extracting RNA from these samples can be difficult the quantity and quality achieved can impact the downstream analysis. This study compared the effectiveness of seven different commercially available RNA extraction kits specifically designed for use with FFPE samples in terms of the quantity and quality of RNA recovered. Methods This study used 9 samples of FFPE tissue from three different types of tissue (Tonsil, Appendix and lymph node of B-cell lymphoma) to evaluate RNA extraction methods. Three sections of 20 µm of each sample were combined per sample. The slices were distributed in a systematic manner to prevent any biases. Each of the 7 commercially available RNA extraction kits were used according to manufacturer's instructions, with each sample being tested in triplicate resulting in a total of 189 extractions. The concentration, RNA quality score (RQS) and DV200 of each extraction was analysed using a nucleic acid analyser to determine the quantity and quality of the recovered RNA. Results This study found that despite processing the FFPE samples in the same standardized way, there were disparities in the quantity and quality of RNA recovered across the different tissue types. Additionally, the study found notable differences in the quantity of RNA recovered when using different extraction kits. In terms of quality, three of the kits performed better than the other four in terms of RQS and DV200 values. Conclusion Though many laboratories have developed their own protocols for specific tissue types, using commercially available kits is still a popular option. Although these kits use similar processes and extraction procedures, the amount and quality of RNA obtained can vary greatly between kits. In this study, among the kits tested, while the Roche kit, provided a nearly systematic better-quality recovery than other kits, the ReliaPrep FFPE Total RNA miniprep from Promega yielded the best ratio of both quantity and quality on the tested tissue samples.

IntroductionColorectal cancer (CRC) is a prevalent malignancy with significant morbidity and mortality worldwide. A deeper understanding of the interaction of cancer cells with other cells in the tumor microenvironment is crucial to devise effective therapeutic strategies. MUC2, a major component of the protective mucus layer in the gastrointestinal tract, has been implicated in CRC progression and immune response regulation.MethodIn this study, we sought to elucidate the relationship between MUC2 expression and immune infiltration within CRC using in vitro models involving two well-established cell lines, HT-29 and LS-174T. By employing CRISPR-mediated MUC2 knockout, we investigated the influence of MUC2 on tumor immune infiltration and its interplay with T cells and NK cells enriched peripheral blood mononuclear cells (PBMCs) in 3D spheroid cultures.ResultsWhile MUC2 was more abundant in LS-174T cell line compared to HT-29, its knockout resulted in increased immune infiltration solely in the HT-29 cell line, but not in the LS-174T cell line. We revealed that the removal of MUC2 protein was compensated in LS-174T by the expression of other gel-forming mucin proteins (MUC6, MUC5B) commonly expressed in the gastrointestinal epithelium, while this was not observed in HT-29 cell line.ConclusionOur study is the first to demonstrate that MUC2 functions as a physical barrier to immune infiltration in colorectal cancer (CRC) in vitro. In HT-29 cells, MUC2 knockout increased immune infiltration, while in LS-174T cells, compensatory expression of other mucins (MUC6, MUC5B) maintained the barrier. These findings reveal the complexity of mucin biology in CRC and suggest that targeting mucin pathways could be a novel therapeutic approach.

Abstract Background Lack of Schlafen family member 11 (SLFN11) expression has been recently identified as a dominant genomic determinant of response to DNA damaging agents in numerous cancer types. Thus, several strategies aimed at increasing SLFN11 are explored to restore chemosensitivity of refractory cancers. In this study, we examined various approaches to elevate SLFN11 expression in breast cancer cellular models and confirmed a corresponding increase in chemosensitivity with using the most successful efficient one. As oncogenic transcriptomic downregulation is often driven by methylation of the promotor region, we explore the demethylation effect of 5-aza-2′-deoxycytidine (decitabine), on the SLFN11 gene. Since SLFN11 has been reported as an interferon inducible gene, and interferon is secreted during an active anti-tumor immune response, we investigated the in vitro effect of IFN-γ on SLFN11 expression in breast cancer cell lines. As a secondary approach to pick up cross talk between immune cells and SLFN11 expression we used indirect co-culture of breast cancer cells with activated PBMCs and evaluated if this can drive SLFN11 upregulation. Finally, as a definitive and specific way to modulate SLFN11 expression we implemented SLFN11 dCas9 (dead CRISPR associated protein 9) systems to specifically increase or decrease SLFN11 expression. Results After confirming the previously reported correlation between methylation of SLFN11 promoter and its expression across multiple cell lines, we showed in-vitro that decitabine and IFN-γ could increase moderately the expression of SLFN11 in both BT-549 and T47D cell lines. The use of a CRISPR-dCas9 UNISAM and KRAB system could increase or decrease SLFN11 expression significantly (up to fivefold), stably and specifically in BT-549 and T47D cancer cell lines. We then used the modified cell lines to quantify the alteration in chemo sensitivity of those cells to treatment with DNA Damaging Agents (DDAs) such as Cisplatin and Epirubicin or DNA Damage Response (DDRs) drugs like Olaparib. RNAseq was used to elucidate the mechanisms of action affected by the alteration in SLFN11 expression. In cell lines with robust SLFN11 promoter methylation such as MDA-MB-231, no SLFN11 expression could be induced by any approach. Conclusion To our knowledge this is the first report of the stable non-lethal increase of SLFN11 expression in a cancer cell line. Our results show that induction of SLFN11 expression can enhance DDA and DDR sensitivity in breast cancer cells and dCas9 systems may represent a novel approach to increase SLFN11 and achieve higher sensitivity to chemotherapeutic agents, improving outcome or decreasing required drug concentrations. SLFN11-targeting therapies might be explored pre-clinically to develop personalized approaches.