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Epithelial-to-mesenchymal transition (EMT) is discussed to be centrally involved in invasion, stemness, and drug resistance. Experimental models to evaluate this process in its biological complexity are limited. To shed light on EMT impact and test drug response more reliably, we use a lung tumor test system based on a decellularized intestinal matrix showing more in vivo-like proliferation levels and enhanced expression of clinical markers and carcinogenesis-related genes. In our models, we found evidence for a correlation of EMT with drug resistance in primary and secondary resistant cells harboring KRAS\(^{G12C}\) or EGFR mutations, which was simulated in silico based on an optimized signaling network topology. Notably, drug resistance did not correlate with EMT status in KRAS-mutated patient-derived xenograft (PDX) cell lines, and drug efficacy was not affected by EMT induction via TGF-β. To investigate further determinants of drug response, we tested several drugs in combination with a KRAS\(^{G12C}\) inhibitor in KRAS\(^{G12C}\) mutant HCC44 models, which, besides EMT, display mutations in P53, LKB1, KEAP1, and high c-MYC expression. We identified an aurora-kinase A (AURKA) inhibitor as the most promising candidate. In our network, AURKA is a centrally linked hub to EMT, proliferation, apoptosis, LKB1, and c-MYC. This exemplifies our systemic analysis approach for clinical translation of biomarker signatures.

High attrition rates associated with drug testing in 2D cell culture and animal models stress the need for improved modeling of human tumor tissues. In previous studies, our 3D models on a decellularized tissue matrix have shown better predictivity and higher chemoresistance. A single porcine intestine yields material for 150 3D models of breast, lung, colorectal cancer (CRC) or leukemia. The uniquely preserved structure of the basement membrane enables physiological anchorage of endothelial cells and epithelial-derived carcinoma cells. The matrix provides different niches for cell growth: on top as monolayer, in crypts as aggregates, and within deeper layers. Dynamic culture in bioreactors enhances cell growth. Comparing gene expression between 2D and 3D cultures, we observed changes related to proliferation, apoptosis and stemness. For drug target predictions, we utilize tumor-specific sequencing data in our in silico model, finding an additive effect of metformin and gefitinib treatment for lung cancer in silico, validated in vitro. To analyze mode-of-action, immune therapies such as trispecific T-cell engagers in leukemia or toxicity on non-cancer cells, the model can be modularly enriched with human endothelial cells (hECs), immune cells and fibroblasts. Upon addition of hECs, transmigration of immune cells through the endothelial barrier can be investigated. In an allogenic CRC model, we observe a lower basic apoptosis rate after applying PBMCs in 3D compared to 2D, which offers new options to mirror antigen-specific immunotherapies in vitro. In conclusion, we present modular human 3D tumor models with tissue-like features for preclinical testing to reduce animal experiments.

Chimeric antigen receptor (CAR) engineering of T cells allows one to specifically target tumor cells via cell surface antigens. A candidate target in Ewing sarcoma is the ganglioside G(D2), but heterogeneic expression limits its value. Here we report that pharmacological inhibition of Enhancer of Zeste Homolog 2 (EZH2) at doses reducing H3K27 trimethylation, but not cell viability, selectively and reversibly induces G(D2) surface expression in Ewing sarcoma cells. EZH2 in Ewing sarcoma cells directly binds to the promoter regions of genes encoding for two key enzymes of G(D2) biosynthesis, and EZH2 inhibition enhances expression of these genes. G(D2) surface expression in Ewing sarcoma cells is not associated with distinct in vitro proliferation, colony formation, chemosensitivity, or in vivo tumorigenicity. Moreover, disruption of G(D2) synthesis by gene editing does not affect its in vitro behavior. EZH2 inhibitor treatment sensitizes Ewing sarcoma cells to effective cytolysis by G(D2)-specific CAR gene-modified T cells. In conclusion, we report a clinically applicable pharmacological approach for enhancing efficacy of adoptively transferred G(D2)-redirected T cells against Ewing sarcoma, by enabling recognition of tumor cells with low or negative target expression.

Three-dimensional (3D) cell culture platforms are increasingly being used in cancer research and drug development. Using patient derived xenograft (PDX) as tumor cell source the clinical relevance of those platforms increases significantly. In combination with live cell imaging capabilities a powerful tool for dynamic studies of drug-cell interactions arises. In the current study we determined the penetration of anti-EGFR antibodies cetuximab and panitumumab in NSCLC spheroids in different culture conditions (w/wo human dermal fibroblasts, HDF) and in combination with the permeabilization-enhancing peptide iRGD. Six different NSCLC PDX models were selected based on their EGFR protein expression level (ranging from high to low determined by immunohistochemistry on PDX tissue). Cetuximab and panitumumab, were labelled with an Alexa-Fluor488 dye and their real-time uptake kinetics were quantified in an IncuCyte S3 device (1 image/well/h, Sartorius). Apart from the absolute fluorescence within the spheroid, we developed a mathematical description, fitting intensity levels in separate regions of the spheroids to estimate spatial dependent penetration of the compound in a time-resolved manner. The accumulation of EGFR antibodies over time was positively correlated with the target expression on the corresponding PDX. In addition, the invasion of the antibodies was significantly influenced by the compactness of the spheroids. The compactness of the tumor spheroid was model immanent and dependent on the molecular make-up of the respective tumor line. Enhancing the compactness by adding HDF to the tumor spheroid reduced the infiltration of both tested antibodies markedly. The simultaneous treatment of anti-EGFR antibodies with iRGD enhanced the penetration capacity in a dose dependent manner. The data of the current study highlight the importance of the tumor microenvironment (TME) for the sensitivity of tumor cells towards anticancer treatment. The modulation of the TME will be an important approach to increase antitumoral activity by enhancing the compound concentration in the tumor tissue. In general, this screening approach can be applied to fluorescence-labelled large molecules as well as compounds with intrinsic fluorescence characteristics. The combination of life cell imaging and image analysis is a promising tool in preclinical drug development to optimize compounds with regard to tissue penetration and binding efficiency. Citation Format: Kanstantsin Lashuk, Hagen Klett, Agon Kolica, Claudia Göttlich, Julia Schüler. Co-administration of iRGD and therapeutic EGFR-antibodies Cetuximab and Panitumumab enhances drug penetration in NSCLC PDX based 3D multicellular spheroids [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 3041.

In the present study, we combined an in vitro 3D lung tumor model with an in silico model to optimize predictions of drug response based on a specific mutational background. The model is generated on a decellularized porcine scaffold that reproduces tissue-specific characteristics regarding extracellular matrix composition and architecture including the basement membrane. We standardized a protocol that allows artificial tumor tissue generation within 14 days including three days of drug treatment. Our article provides several detailed descriptions of 3D read-out screening techniques like the determination of the proliferation index Ki67 staining's, apoptosis from supernatants by M30-ELISA and assessment of epithelial to mesenchymal transition (EMT), which are helpful tools for evaluating the effectiveness of therapeutic compounds. We could show compared to 2D culture a reduction of proliferation in our 3D tumor model that is related to the clinical situation. Despite of this lower proliferation, the model predicted EGFR-targeted drug responses correctly according to the biomarker status as shown by comparison of the lung carcinoma cell lines HCC827 (EGFR -mutated, KRAS wild-type) and A549 (EGFR wild-type, KRAS-mutated) treated with the tyrosine-kinase inhibitor (TKI) gefitinib. To investigate drug responses of more advanced tumor cells, we induced EMT by long-term treatment with TGF-beta-1 as assessed by vimentin/pan-cytokeratin immunofluorescence staining. A flow-bioreactor was employed to adjust culture to physiological conditions, which improved tissue generation. Furthermore, we show the integration of drug responses upon gefitinib treatment or TGF-beta-1 stimulation - apoptosis, proliferation index and EMT - into a Boolean in silico model. Additionally, we explain how drug responses of tumor cells with a specific mutational background and counterstrategies against resistance can be predicted. We are confident that our 3D in vitro approach especially with its in silico expansion provides an additional value for preclinical drug testing in more realistic conditions than in 2D cell culture.

Background: Current treatment in Alzheimer’s disease (AD) is initiated at a stage where the brain already has irreversible structural deteriorations. Therefore, the concept of treatment prior to obvious cognitive deficits has become widely accepted, and simple biochemical tests to discriminate normal aging from prodromal or demented stages are now common practice. Objective: The objective of the study was the differentiation of controls, mild cognitive impairment (MCI) and AD patients by novel blood-based assays in combination with neuropsychological tests. Methods: In a cross-sectional study, 143 subjects aged 18 to 85 years were recruited. All participants were classified by a comprehensive neuropsychological assessment. Blood samples were analyzed for several amyloid-β (Aβ) species, pro-inflammatory markers, anti-Aβ autoantibodies, and ApoE allele status, respectively. Results: Plasma Aβ1-42 was significantly decreased in MCI and AD compared to age-matched controls, whereas Aβ1-40 did not differ, but increases with age in healthy controls. The Aβ1-42 to Aβ1-40 ratio was stepwise decreased from age-matched controls via MCI to AD, and shows a clear correlation with memory scores. Reduced Aβ1-42 and Aβ1-42 to Aβ1-40 ratio have strongly correlated with carrying ApoE ɛ4 allele. Autoantibodies against pyroglutamate-modified Aβ, but only a certain subclass, were significantly decreased in AD compared to MCI and age-matched controls, whereas autoantibodies against the unmodified N-terminus of Aβ did not differ. Conclusion: Comprehensive sample preparation and assay standardization enable reliable usage of plasma Aβ for diagnosis of MCI and AD. Anti-pGlu-Aβ autoantibodies correlate with cognition, but not with ApoE, supporting the associated plasma Aβ analysis with additional and independent information.

For the development of new treatment strategies against cancer, understanding signaling networks and their changes upon drug response is a promising approach to identify new drug targets and biomarker profiles. Pre‐requisites are tumor models with multiple read‐out options that accurately reflect the clinical situation. Tissue engineering technologies offer the integration of components of the tumor microenvironment which are known to impair drug response of cancer cells. We established three‐dimensional (3D) lung carcinoma models on a decellularized tissue matrix, providing a complex microenvironment for cell growth. For model generation, we used two cell lines with (HCC827) or without (A549) an activating mutation of the epidermal growth factor receptor (EGFR), exhibiting different sensitivities to the EGFR inhibitor gefitinib. EGFR activation in HCC827 was inhibited by gefitinib, resulting in a significant reduction of proliferation (Ki‐67 proliferation index) and in the induction of apoptosis (TUNEL staining, M30‐ELISA). No significant effect was observed in conventional cell culture. Results from the 3D model correlated with the results of an in silico model that integrates the EGFR signaling network according to clinical data. The application of TGFβ1 induced tumor cell invasion, accompanied by epithelial–mesenchymal transition (EMT) both in vitro and in silico. This was confirmed in the 3D model by acquisition of mesenchymal cell morphology and modified expression of fibronectin, E‐cadherin, β‐catenin and mucin‐1. Quantitative read‐outs for proliferation, apoptosis and invasion were established in the complex 3D tumor model. The combined in vitro and in silico model represents a powerful tool for systems analysis., Highlights Combination of a human 3D lung tumor tissue model with a Boolean in silico Model.Establishment of in silico signaling network topology for personalized medicine.Significant decrease of tumor proliferation and induction of apoptosis upon in vitro treatment with tyrosine kinase inhibitors.Decreased proliferation of tumor cells in the 3D model compared to 2D conditions.Induction of invasion with EMT by TGFβ stimulation.