IntroductionWe describe a novel 3D co-culture model using non-small cell lung cancer (NSCLC) cell lines in combination with lung fibroblasts. This model allows the investigation of tumour-stroma interactions and addresses the importance of having a more in vivo like cell culture model.MethodsAutomation-compatible multi-well hanging drop microtiter plates were used for the production of 3D mono- and co-cultures. In these hanging drops the two NSCLC cell lines A549 and Colo699 were cultivated either alone or co-cultured with lung fibroblasts. The viability of tumour spheroids was confirmed after five and ten days by using Annexin V/Propidium Iodide staining for flow-cytometry. Tumour fibroblast spheroid formation was characterized by scanning electron microscope (SEM), semi-thin sections, fluorescence microscope and immunohistochemistry (IHC). In addition to conventional histology, protein expression of E-Cadherin, vimentin, Ki67, fibronectin, cytokeratin 7 and α-smooth muscle actin (α-SMA) was investigated by IHC.ResultsLower viability was observed in A549 monocultures compared to co-cultures, whereas Colo699 monocultures showed better viability compared to co-cultures. Ki67 expression varied significantly between mono- and co-cultures in both tumour cell lines. An increase of vimentin and decreased E-Cadherin expression could be detected during the course of the cultivation suggesting a transition to a more mesenchymal phenotype. Furthermore, the fibroblast cell line showed an expression of α-SMA only in co-culture with the cancer cell line A549, thereby indicating a mesenchymal to mesenchymal shift to an even more myofibroblast phenotype.ConclusionWe demonstrate that our method is a promising tool for the generation of tumour spheroid co-cultures. Furthermore, these spheroids allow the investigation of tumour-stroma interactions and a better reflection of in vivo conditions of cancer cells in their microenvironment. Our method holds potential to contribute to the development of anti-cancer agents and support the search for biomarkers.

IntroductionPrognosis of biliary tract cancers (BTC) remains dismal and novel treatment strategies are needed to improve survival. BRCA mutations are known to occur in BTC but their frequency and the molecular landscape in which they are observed in distinct sites of BTC remain unknown.Material and methodsTumour samples from 1292 patients with BTC, comprising intrahepatic cholangiocarcinoma (IHC, n=746), extrahepatic cholangiocarcinoma (EHC, n=189) and gallbladder cancer (GBC, n=353), were analysed using next-generation sequencing (NGS). Tumour mutational burden (TMB) was calculated based on somatic non-synonymous missense mutations. Determination of tumour mismatch repair (MMR) or microsatellite instability (MSI) status was done by fragment analysis, immunohistochemistry and the evaluation of known microsatellite loci by NGS. Programmed death ligand 1 expression was analysed using immunohistochemistry.ResultsOverall, BRCA mutations were detected in 3.6% (n=46) of samples (BRCA1: 0.6%, BRCA2: 3%) with no significant difference in frequency observed based on tumour site. In GBC and IHC, BRCA2 mutations (4.0% and 2.7%) were more frequent than BRCA1 (0.3% and 0.4%, p<0.05) while in EHC, similar frequency was observed (2.6% for BRCA2 vs 2.1% for BRCA1). BRCA mutations were associated with a higher rate in subjects with MSI-H/deficient mismatch repair (19.5% vs 1.7%, p<0.0001) and tumours with higher TMB, regardless of the MMR or MSI status (p<0.05).ConclusionsBRCA mutations are found in a subgroup of patients with BTC and are characterised by a distinct molecular profile. These data provide a rationale testing poly(ADP-ribose)polymeraseinhibitors and other targeted therapies in patients with BRCA-mutant BTC.

During the last decade, novel immunotherapeutic strategies, in particular antibodies directed against immune checkpoint inhibitors, have revolutionized the treatment of different malignancies leading to an improved survival of patients. Identification of immune-related biomarkers for diagnosis, prognosis, monitoring of immune responses and selection of patients for specific cancer immunotherapies is urgently required and therefore areas of intensive research. Easily accessible samples in particular liquid biopsies (body fluids), such as blood, saliva or urine, are preferred for serial tumor biopsies.Although monitoring of immune and tumor responses prior, during and post immunotherapy has led to significant advances of patients’ outcome, valid and stable prognostic biomarkers are still missing. This might be due to the limited capacity of the technologies employed, reproducibility of results as well as assay stability and validation of results. Therefore solid approaches to assess immune regulation and modulation as well as to follow up the nature of the tumor in liquid biopsies are urgently required to discover valuable and relevant biomarkers including sample preparation, timing of the collection and the type of liquid samples. This article summarizes our knowledge of the well-known liquid material in a new context as liquid biopsy and focuses on collection and assay requirements for the analysis and the technical developments that allow the implementation of different high-throughput assays to detect alterations at the genetic and immunologic level, which could be used for monitoring treatment efficiency, acquired therapy resistance mechanisms and the prognostic value of the liquid biopsies.

The tumour microenvironment and tumour angiogenesis play a critical role in the development and therapy of many cancers, but in vitro models reflecting these circumstances are rare. In this study, we describe the development of a novel tri-culture model, using non-small cell lung cancer (NSCLC) cell lines (A549 and Colo699) in combination with a fibroblast cell line (SV 80) and two different endothelial cell lines in a hanging drop technology. Endothelial cells aggregated either in small colonies in Colo699 containing microtissues or in tube like structures mainly in the stromal compartment of microtissues containing A549. An up-regulation of hypoxia and vimentin, ASMA and a downregulation of E-cadherin were observed in co- and tri-cultures compared to monocultures. Furthermore, a morphological alteration of A549 tumour cells resembling “signet ring cells” was observed in tri-cultures. The secretion of proangiogenic growth factors like vascular endothelial growth factor (VEGF) was measured in supernatants. Inhibition of these proangiogenic factors by using antiangiogenic drugs (bevacizumab and nindetanib) led to a significant decrease in migration of endothelial cells into microtissues. We demonstrate that our method is a promising tool for the generation of multicellular tumour microtissues and reflects in vivo conditions closer than 2D cell culture.