Strong Correlation of Gene Counts and Differentially Expressed Genes Between a 3′ RNA-Seq and RNA Hybridisation Platform in Transcriptome Analyses from Canine Archival Tissue [nCounter]

Canine cutaneous histiocytoma (CCH) is a tumor originating from dermal Langerhans cells, especially affecting young dogs. The common spontaneous regression of CCH makes it an interesting model in comparative oncology research. Previous studies have indicated that anti-tumor immune responses may be involved but details remain speculative to date. Here, we asked which specific immuno-oncological dynamics underlie spontaneous regression of CCH on mRNA and protein levels. QuantSeq 3' mRNA sequencing with functional overrepresentation analysis and an nCounter® RNA hybridization assay were employed on 21 formalin-fixed, paraffin-embedded CCH samples representing three different tumor stages. Nine additional samples were subjected to matrix-assisted laser desorption ionization mass spectrometry imaging (MALDI-MSI). Surprisingly, only minor stage-specific differences were found. When we investigated expression of B7 family ligands and CD28 family receptors holding co-stimulatory and -inhibitory functions respectively, we found higher abundance of CD80, CD86 and CD28, which trigger activation of lymphocyte-mediated immune responses. CD80 and CD86 expressing cells were further quantified by in situ hybridization and compared with data from three cases of canine histiocytic sarcoma (HS), a malignant tumor variant originating from antigen presenting cells. A stage-specific increase of CD80 expression was recorded in CCH from the tumor bottom to the top, while CD86 was continuously and homogenously expressed at high levels. Expression of CD80 in CCH was similar to that in HS (73.3 ± 37.4% versus 62.1 ± 46.4% positive cells), while CD86 was significantly higher expressed in CCH (94.7 ± 10.3%) when compared to HS (57.6 ± 11.0%). Our data suggest that major immuno-oncological pathways are seemingly not regulated during regression of CCH on the mRNA or protein levels as detectable by the methods used. We speculate that key signals leading to tumor regression may occur at an interactome level or at an earlier time point than examined here. Our data further supports a role of immune stimulatory B7 family ligands and CD28 family receptors in CCH regression. To determine the correlation of gene expression data generated by the nCounter® Canine IO Panel and QuantSeq 3' from three types of canine tumours. FFPE tissue samples were obtained from the archive (spanning the years 2013 to 2021) and total RNA was extracted. After QuantSeq 3' and hybridisation to the nCounter® Canine IO Panel with bioinformatic processing, counts and differential gene expression data were compared to calculate correlations between the two technologies employed. Attention: This GEO submission includes the nCounter® data from the stage-contrasting comparison (n = 10). The data for this comparison from 3' RNA-Seq (QuantSeq 3') was submitted separately to GEO (262022). The other entity-contrasting tumour samples (n = 25) used in the publication were also submitted separately to GEO (nCounter®: 261791; 3' RNA-Seq: 261790).