Immune exhaustion panel v1

Representative tumor material of the primary tumor was retrieved by a 1 mm core. The RNA was isolated using the truXTRAC FFPE total NA Kit (Covaris, Woburn, MA, USA) based on focused ultrasonification and column purification according to the manufacturer’s instructions. Nanostring nCounter^® Platform and Immune Exhaustion Panel v1 were used to enrich a commercially available function-specific panel of 798 genes by hybrid capture technique (Nanostring, Seattle, WA, USA). Nanostring nSolver™ software v4 and implemented nCounter^® Advanced Analysis module v2.0.115 were used for subsequent raw data processing and normalization by internal controls following differential supervised analysis between MUC1-positive (n = 7) and -negative CCA patients indicated by previous immunohistochemistry. ClueGO v.2.5.6 and CluePedia v.1.5.6 functional classification and network annotation was applied in order to identify enriched genes that were associated to overrepresented gene ontologies based on REACTOME_pathways (updated 17.08.2022) [21 (link),22 (link)]. The Ingenuity Pathway Analysis (IPA v.76765844; QIAGEN, Hilden, Germany) tool was additionally used to predict network-associated biomarkers as well as functional terms (Fx) and therapeutical agents (Rx).

RNA isolation was conducted as described recently [27 (link)]. Immune-exhaustion expression analysis was performed using the Nanostring nCounter^® Platform, the Immune Exhaustion Panel v1, Nanostring nSolver™ software v4 as well as the nCounter^® Advanced Analysis module v2.0.115 (Nanostring, Seattle, WA, USA), as indicated previously [27 (link)]. iCCA patients were classified into two groups based on the presence or absence of active autophagy, as determined by prior IHC analysis. To identify enriched genes associated with overrepresented gene ontologies based on REACTOME_pathways (updated 15 November 2022), functional classification and network annotation were performed using ClueGO v.2.5.6 and CluePedia v.1.5.6. [30 (link),31 (link)].