in Vitro and in Vivo Models to Decipher Intra-tumor Heterogeneity

Recent advances in next-generation sequencing and other omics technologies capable to map cell fate provide increasing evidence on the crucial role of intra-tumor heterogeneity (ITH) for cancer progression. The different facets of ITH, from genomic to microenvironmental heterogeneity and the hierarchical cellular architecture originating from the cancer stem cell compartment, contribute to the range of tumor phenotypes. Decoding these complex data resulting from the analysis of tumor tissue complexity poses a challenge for developing novel therapeutic strategies that can counteract tumor evolution and cellular plasticity. To achieve this aim, the development of in vitro and in vivo cancer models that resemble the complexity of ITH is crucial in understanding the interplay of cells and their (micro)environment and, consequently, in testing the efficacy of new targeted treatments and novel strategies of tailoring combinations of treatments to the individual composition of the tumor. This challenging approach may be an important cornerstone in overcoming the development of pharmaco-resistances during multiple lines of treatment. In this paper, we report the latest advances in patient-derived 3D (PD3D) cell cultures and patient-derived tumor xenografts (PDX) as in vitro and in vivo models that can retain the genetic and phenotypic heterogeneity of the tumor tissue

Glioblastoma cells express functional cell membrane receptors activated by daily used medical drugs

PURPOSE: Calcium ions are highly versatile spacial and temporal intracellular signals of non-excitable cells and have an important impact on nearly every aspect of cellular life controlling cell growth, metabolism, fluid secretion, information processing, transcription, apoptosis, and motility. Neurons and glia respond to stimuli, including neurotransmitters, neuromodulators, and hormones, which increase the intracellular calcium concentration. The function of intracellular calcium in gliomas is unknown. Lots of daily used drugs may act via receptors that can be linked to the intracellular calcium system and therefore could influence glioma biology. METHODS: Glioma cells were loaded with the calcium ion sensitive dye Fura 2-AM. Subsequently, cells were stimulated with 25 different medical drugs for 30 s. The increase of free intracellular calcium ions was measured and calculated by a microscope–camera–computer-unit. RESULTS: Except for the buffer solution HEPES that served as negative control and for the cortisol derivative dexamethasone, all other 24 tested drugs induced a rise of intracellular calcium ions. The cellular calcium responses were classified into seven functional groups. The tested substances activated several types of calcium channels and receptors. CONCLUSIONS: Our study impressively demonstrates that medical drugs are potent inducers of intracellular calcium signals. Totally unexpected, the results show a high amount of functional cellular receptors and channels on glioma cells, which could be responsible for certain biological effects like migration and cell growth. This calcium imaging study proves the usability of the calcium imaging as a screening system for functional receptors on human glioma cells

Context Matters—Why We Need to Change From a One Size Fits all Approach to Made-to-Measure Therapies for Individual Patients With Pancreatic Cancer

Pancreatic cancer is one of the deadliest cancers and remains a major unsolved health problem. While pancreatic ductal adenocarcinoma (PDAC) is associated with driver mutations in only four major genes (KRAS, TP53, SMAD4, and CDKN2A), every tumor differs in its molecular landscape, histology, and prognosis. It is crucial to understand and consider these differences to be able to tailor treatment regimens specific to the vulnerabilities of the individual tumor to enhance patient outcome. This review focuses on the heterogeneity of pancreatic tumor cells and how in addition to genetic alterations, the subsequent dysregulation of multiple signaling cascades at various levels, epigenetic and metabolic factors contribute to the oncogenesis of PDAC and compensate for each other in driving cancer progression if one is tackled by a therapeutic approach. This implicates that besides the need for new combinatorial therapies for PDAC, a personalized approach for treating this highly complex cancer is required. A strategy that combines both a target-based and phenotypic approach to identify an effective treatment, like Reverse Clinical Engineering® using patient-derived organoids, is discussed as a promising way forward in the field of personalized medicine to tackle this deadly disease

A RAS-Independent Biomarker Panel to Reliably Predict Response to MEK Inhibition in Colorectal Cancer

Background: In colorectal cancer (CRC), mutations of genes associated with the TGF-β/BMP signaling pathway, particularly affecting SMAD4, are known to correlate with decreased overall survival and it is assumed that this signaling axis plays a key role in chemoresistance. Methods: Using CRISPR technology on syngeneic patient-derived organoids (PDOs), we investigated the role of a loss-of-function of SMAD4 in sensitivity to MEK-inhibitors. CRISPR-engineered SMAD4R361H PDOs were subjected to drug screening, RNA-Sequencing, and multiplex protein profiling (DigiWest®). Initial observations were validated on an additional set of 62 PDOs with known mutational status. Results: We show that loss-of-function of SMAD4 renders PDOs sensitive to MEK-inhibitors. Multiomics analyses indicate that disruption of the BMP branch within the TGF-β/BMP pathway is the pivotal mechanism of increased drug sensitivity. Further investigation led to the identification of the SFAB-signature (SMAD4, FBXW7, ARID1A, or BMPR2), coherently predicting sensitivity towards MEK-inhibitors, independent of both RAS and BRAF status. Conclusion: We identified a novel mutational signature that reliably predicts sensitivity towards MEK-inhibitors, regardless of the RAS and BRAF status. This finding poses a significant step towards better-tailored cancer therapies guided by the use of molecular biomarkers

Age-related transcriptional changes in gene expression in different organs of mice support the metabolic stability theory of aging

Individual differences in the rate of aging are determined by the efficiency with which an organism transforms resources into metabolic energy thus maintaining the homeostatic condition of its cells and tissues. This observation has been integrated with analytical studies of the metabolic process to derive the following principle: The metabolic stability of regulatory networks, that is the ability of cells to maintain stable concentrations of reactive oxygen species (ROS) and other critical metabolites is the prime determinant of life span. The metabolic stability of a regulatory network is determined by the diversity of the metabolic pathways or the degree of connectivity of genes in the network. These properties can be empirically evaluated in terms of transcriptional changes in gene expression. We use microarrays to investigate the age-dependence of transcriptional changes of genes in the insulin signaling, oxidative phosphorylation and glutathione metabolism pathways in mice. Our studies delineate age and tissue specific patterns of transcriptional changes which are consistent with the metabolic stability–longevity principle. This study, in addition, rejects the free radical hypothesis which postulates that the production rate of ROS, and not its stability, determines life span

Cell Culture Models for Translational Research on Thymomas and Thymic Carcinomas: Current Status and Future Perspectives

Cell culture model systems are fundamental tools for studying cancer biology and identifying therapeutic vulnerabilities in a controlled environment. TET cells are notoriously difficult to culture, with only a few permanent cell lines available. The optimal conditions and requirements for the ex vivo establishment and permanent expansion of TET cells have not been systematically studied, and it is currently unknown whether different TET subtypes require different culture conditions or specific supplements. The few permanent cell lines available represent only type AB thymomas and thymic carcinomas, while attempts to propagate tumor cells derived from type B thymomas so far have been frustrated. It is conceivable that epithelial cells in type B thymomas are critically dependent on their interaction with immature T cells or their three-dimensional scaffold. Extensive studies leading to validated cell culture protocols would be highly desirable and a major advance in the field. Alternative methods such as tumor cell organoid models, patient-derived xenografts, or tissue slices have been sporadically used in TETs, but their specific contributions and advantages remain to be shown