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BackgroundTherapeutic management of stage II colon cancer remains difficult regarding the decision whether adjuvant chemotherapy should be administered or not. Low rates of recurrence are opposed to chemotherapy induced toxicity and current clinical features are limited in predicting disease relapse. Predictive biomarkers are urgently needed and we hypothesise that the spatial tissue composition of relapsed and non-relapsed colon cancer stage II patients reveals relevant biomarkers.MethodsThe spatial tissue composition of stage II colon cancer patients was examined by in situ sequencing technology with sub-cellular resolution. A panel of 175 genes was designed investigating specific cancer-associated processes and components of the tumour microenvironment. We identified a tumour gene signature to subclassify tissue into neoplastic and non-neoplastic tissue compartments based on spatial expression patterns generated by in situ sequencing (GTC-tool – Genes-To-Count).ResultsThe GTC-tool automatically identified tissue compartments that were used to quantify gene expression of biological processes upregulated within the neoplastic tissue in comparison to non-neoplastic tissue and within relapsed versus non-relapsed stage II colon patients. Three differentially expressed genes (FGFR2, MMP11 and OTOP2) in the neoplastic tissue compartments of relapsed patients in comparison to non-relapsed patients were identified predicting recurrence in stage II colon cancer.ConclusionsIn depth spatial in situ sequencing revealed novel potential predictive biomarkers for disease relapse in colon cancer stage II patients. Our developed open-access GTC-tool allows to accurately capture the tumour compartment and quantify spatial gene expression in colon cancer tissue.

Background Assessment of liver quality during machine perfusion (MP) is under intensive investigation. Gold standard methods either are of limited validity or evaluation is time-consuming. Hence, herein we investigate a novel real-time method to estimate liver function during sub-normothermic MP. Methods Three healthy porcine livers and one non-allocable human liver were subjected to 24h of SNMP with either Custodiol® or Custodiol MP as perfusate. Additional to liver enzymes, Flavin mononucleotide (FMN) and High-mobility group box 1 (HMGB-1) as well-established markers of liver quality, a novel 13C-Methacetin based method was evaluated. Readouts of ϭ13CO2 plots generated by the 13CORLab Prototype were calculated and compared to established parameters. Results The newly established method mirrored the pathological findings as well as the increasing changes in liver enzymes, HMGB-1 and FMN levels bearing the advantage of being a real-time, non-invasive test which is applicable on site. Conclusion This novel method is an easy to implement and reliable tool for quick on-site estimation of liver function during liver SNMP.

Cholangiocarcinoma (CCA) are characterized by their desmoplastic and hypervascularized tumor microenvironment (TME), which is mainly composed of tumor cells and cancer-associated fibroblasts (CAFs). CAFs play a pivotal role in general and CCA tumor progression, angiogenesis, metastasis, and the development of treatment resistance. To our knowledge, no continuous human in vivo-like co-culture model is available for research. Therefore, we aimed to establish a new model system (called MUG CCArly) that mimics the desmoplastic microenvironment typically seen in CCA. Proteomic data comparing the new CCA tumor cell line with our co-culture tumor model (CCTM) indicated a higher gene expression correlation of the CCTM with physiological CCA characteristics. A pro-angiogenic TME that is typically observed in CCA could also be better simulated in the CCTM group. Further analysis of secreted proteins revealed CAFs to be the main source of these angiogenic factors. Our CCTM MUG CCArly represents a new, reproducible, and easy-to-handle 3D CCA model for preclinical studies focusing on CCA-stromal crosstalk, tumor angiogenesis, and invasion, as well as the immunosuppressive microenvironment and the involvement of CAFs in the way that drug resistance develops.