Abstract 358: Cancer cell derived TNF mRNA as a marker of cancer cell budding in colorectal cancer

Abstract Tumor necrosis factor alpha (TNF) is a potent cytokine reported to have several functions, including stimulation of inflammation, directing cell death and cell proliferation. The cellular origin of TNF has generally believed to be the inflammatory cells especially because macrophages are prone to induction of TNF. Using in situ hybridization (ISH) based on TNF mRNA probes (RNAscope) we recently reported that TNF mRNA is also seen in cytokeratin positive cancer cells (CC) and is associated with morphologic appearance interpreted as CC branching (Møller et al, IJMS 2019, doi: 10.3390/ijms20081907). To determine the fraction of CC-derived TNF mRNA signal at different areas in colon cancers we developed a cell and color segmentation-based image analysis tool to process fluorescence signals in digital slides. The approach allowed quantification of the TNF mRNA-positive CC and TNF mRNA-positive stromal cells in the digital slides previously reported, that were also stained for cytokeratin, microRNA-21 (miR-21) and DAPI nuclear stain. In the two cases with most abundant TNF mRNA ISH signal, both showing moderate-to-high budding index, the fraction of TNF-mRNA positive CC in the outer periphery (approximately 500 µm) was 8% and 20%, respectively. The frequency of the TNF-positive CC increased towards the invasive front from 7% (33/500 CC per 0.25 mm2) to 35% (48/137 CC), and from 8% (127/1536 CC per 0.50 mm2) to 28% (108/389 CC), respectively. The upregulation is in agreement with initial qualitative evaluation. In the same areas, the fraction of TNF mRNA positive stromal cells among all TNF mRNA-positive cells was only 8% in both cases, indicating that the majority of the TNF mRNA originates from CC. The fraction of the TNF mRNA-positive CC that were also miR-21 positive was 85% and 43%, respectively, which was higher than the prevalence of the overall miR-21 positive CC, 55% and 30%, suggesting frequent co-expression with miR-21. In conclusion, we have developed a method to obtain quantitative estimates from digital multiplex-stained slides with RNAscope and LNA fluorescence ISH and immunofluorescence. More samples will need to be processed to better understand the importance of TNF expression in cancer cell invasion. Citation Format: Boye Schnack Nielsen, Kim Holmstrom. Cancer cell derived TNF mRNA as a marker of cancer cell budding in colorectal cancer [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 358.