Patient-derived Cell Models Research Articles

1919P - FGF19 promotes esophageal squamous cell carcinoma progression by inhibiting autophagy

BackgroundEsophageal squamous cell carcinoma (ESCC) with high mortality is particularly prevalent in China and the prognosis is poor. Although genetic amplification and overexpression of the fibroblast growth factor 19 (FGF19) gene are found in ESCC, mechanisms that contribute to such functional alterations remain elusive. MethodsGene-Panel Sequencing was used to detect genetic variants that may be associated with a risk of ESCC. The effect of proliferation inhibition of LY2874455, which is inhibitor of fibroblast growth factor receptors (FGFR), was assessed in ESCC patient-derived cell (PDC) and xenograft (PDX) models. Autophagic activity was evaluated by immunoblotting for microtubule-associated protein 1 light chain 3 (LC3) and p62. ResultsUsing Gene-Panel Sequencing of 161 FFPE tissues, we found that FGF19 copy number amplified in almost 30% ESCC. Pharmacological of FGF19/FGFR by LY2874455 significantly induces repression of FGF19 amplificated ESCC progression in either PDC or PDX models. Mechanistic study revealed that treatment with LY2874455 induced activity of autophagy by inhibiting the ERK/MAPK pathway not by AKT pathway. ConclusionsOur findings reveal that FGF19 amplification inhibits autophagic activity by increasing the phosphorylation level of ERK, which may play an essential role in the pathogenesis of ESCC. Using FGFR inhibitor may represent an effective strategy to suppress FGF19 amplificated ESCC development and progression. Legal entity responsible for the studyThe authors. FundingThe Major Project of Health Commission of Zhejiang Province of China (No. WKJ-ZJ-1902), the Public Welfare Technology Foundation of Zhejiang Province of China (No. 2017C34001), Zhejiang high-level innovative talent program, and the 1022 program of Zhejiang Cancer Hospital. DisclosureAll authors have declared no conflicts of interest.

Integrative Tumor Profiling Following a Ketogenic Diet Offers a Window into the Interplay between Metabolic Vulnerabilities and Radiation Sensitization in Glioblastoma.

Glioblastoma (GBM) continues to be an invariably fatal malignancy with limited treatment options. Although considerable progress has been made in understanding molecular alterations in these tumors, this level of knowledge has not translated to clinical improvements. Metabolic reprogramming represents a hallmark of cancer and may serve as a therapeutic target. A ketogenic diet (KD) is a clinical strategy designed to target tumor metabolism and represents an active area of research. Although both preclinical and clinical data demonstrating efficacy has been mixed, a recent report suggests an unprecedented potential of a KD to enhance radiation response (RT) in an orthotopic model using an established mouse GBM cell line. In this study, we sought to validate these findings by determining the capacity of a KD to enhance RT in an aggressive, patient-derived GBM stem cell model and perform an integrative analysis on tissue to both define metabolic changes related to a KD and provide a mechanistic framework of activity. Immune deficient mice (nu/nu) were injected orthotopically with patient derived mesenchymal GBM tumor stem cells and randomized to four treatment arms (n=8/arm). Mice were fed ad libitum standard diet (SD) or KD (Bio-Serve; 1:4) alone or in combination with hypofractionated RT (hfRT; 6 Gy x 3). LC/GC-MS will be used to perform global metabolomic and proteomic profiling of tumor, serum, and normal brain of treated mice. Blood glucose and ketones were measured using the Abbott Precision Xtra meter kit. A KD demonstrated improved survival in GBM tumor bearing mice when compared to SD (mOS 22d vs. 17d; p=0.001) and equivalent survival when compared to SD mice treated with hfRT (mOS 24d; p= 0.57). The combination of KD and hfRT led to marked improvement in overall survival (mOS not reached; p<0.001). Although studies remain ongoing, at Day 29, 7/8 mice treated with KD+hfRT remained alive, while all 8 mice treated with SD+hfRT required euthanization due to neurologic progression. Surprisingly, alterations in blood ketone levels or glucose were not observed in mice treated with a KD. Global metabolomic and proteomic profiling of tumor, serum, and normal brain are ongoing. The anti-tumor activity and potent radiation sensitization of a KD was validated in an aggressive patient-derived GBM model. Ongoing integrative profiling of tumor and serum in mice treated with a KD will be presented to provide a previously undescribed insight into mechanisms of anti-tumor activity. As a KD is challenging to implement clinically, these mechanistic-based studies will provide a unique opportunity to learn how metabolism may be therapeutically exploited to provide a framework for the identification of novel mechanisms of radiation sensitization.

Systematic Evaluation of Tumor Cell Index and Two-Drug Combination Therapy via 3-Dimensional High-Throughput Drug Screening

Background: Tumour heterogeneity greatly limits personalised treatment of cancer. Patient-derived tumour cell (PDC) models precisely recapitulate the molecular properties and biology of the disease, making them effective preclinical tools for assessing anti-cancer drug activities. Accurately estimating tumor purity is essential for performing high-throughput drug screening (HTS). In the current study, we measured and predicted the tumour population index in PDC models for two-drug combinational strategies using an HTS system. Methods: Gastric cancer cell-lines and PDCs were subjected to multi-colour immunofluorescence analysis against EpCAM and vimentin to evaluate the tumour cell index based on EpCAM expression levels. We generated a tumour purity prediction model using five different gastric cancer cell-lines (AGS, KATO-III, MKN-45, NCI-N87, SNU-216) with fluorescence intensity-based techniques. These stage IV gastric cancer PDC models were evaluated using a micropillar/microwell chip-based HTS system. Findings: HER2/CCNE1-amplified PDCs were considerably resistant to an HER2 inhibitor, while combinational treatment consisting of an HER2 inhibitor with anti-WEE1 compound substantially suppressed tumor cell growth. Moreover, PDCs with BRCA1/2 mutations were synergistically sensitive to HER2 and PARP inhibition therapy. Finally, somatic mutations in TP53 and CDKN2A with MYC amplification rendered PDCs susceptible to the drug combination of WEE1 and HER2. Interpretation: Collectively, our systematic method of high-throughput drug sensitivity screening is an integral pre-clinical platform for evaluating potential two-drug combinational approaches for personalised treatment of cancer. Funding: This work was supported by the AZ-KHIDI (AstraZeneca-Korea Health Industry Development Institute) R & D program. Declaration of Interest: The authors declare no competing interests. Ethical Approval: This investigation was conducted in accordance with the ethical standards of the Declaration of Helsinki and national and international guidelines and was approved by the Institutional Review Board at Samsung Medical Center in Seoul, Korea.

Circular RNAs add diversity to androgen receptor isoform repertoire in castration-resistant prostate cancer.

Deregulated expression of circular RNAs (circRNAs) is associated with various human diseases, including many types of cancer. Despite their growing links to cancer, there has been limited characterization of circRNAs in metastatic castration-resistant prostate cancer, the major cause of prostate cancer mortality. Here, through the analysis of an exome-capture RNA-seq dataset from 47 metastatic castration-resistant prostate cancer samples and ribodepletion and RNase R RNA-sequencing of patient-derived xenografts (PDXs) and cell models, we identified 13 circRNAs generated from the key prostate cancer driver gene-androgen receptor (AR). We validated and characterized the top four most abundant, clinically relevant AR circRNAs. Expression of these AR circRNAs was upregulated during castration-resistant progression of PDXs. The upregulation was not due to global increase of circRNA formation in these tumors. Instead, the levels of AR circRNAs correlated strongly with that of the linear AR transcripts (both AR and AR-variants) in clinical samples and PDXs, indicating a transcriptional mechanism of regulation. In cultured cells, androgen suppressed the expression of these AR circRNAs and the linear AR transcripts, and the suppression was attenuated by an antiandrogen. Using nuclear/cytoplasmic fractionation and RNA in-situ hybridization assays, we demonstrated predominant cytoplasmic localization of these AR circRNAs, indicating likely cytoplasmic functions. Overall, this is the first comprehensive characterization of circRNAs arising from the AR gene. With greater resistance to exoribonuclease compared to the linear AR transcripts and detectability of AR circRNAs in patient plasma, these AR circRNAs may serve as surrogate circulating markers for AR/AR-variant expression and castration-resistant prostate cancer progression.

Mutation-specific Fabry disease patient-derived cell model to evaluate the amenability to chaperone therapy

BackgroundPatients with Fabry disease (FD) and amenable mutations can be treated with the chaperone migalastat to restore endogenous α-galactosidase A (AGAL) activity. However, certain amenable mutations do not respond biochemically...

Abstract 1917: High-throughput evaluation of treatment response in patient-derived glioma stem cell models

Abstract Although significant progresses in molecular oncology are being made using conventional cell lines, most therapies still fail in phase III clinical trials. Patient-derived models are being used more frequently as they are more faithfully representing the genomic features of primary tumors. However, one by one test of each model from large biobanks is extremely economy and time consuming. In this study, each of a panel of patient-derived glioblastoma stem cell (GSC) models was uniquely tagged by a lentiviral Cas9D10A and paired-gRNA targetable unique reporter (CAPTURE) barcoding system. Barcoded GSCs were then pooled evenly and following by radiation treatment (RT) in vitro. Amplicon sequencing was employed to count the barcodes distribution, which represent the relative cell number. The results showed that this approach faithfully identified the RT resistant GSCs from a mixing pool when comparing to the results from canonical clonogenic assay. In addition, a fluorescence marker will be switched by delivery of corresponding barcodes targeting CRISPR so that we can re-isolate interested cell models from the treated pool for investigating the treatment sensitivity and resistance mechanism. This study will provide a robust approach for therapeutic discovery take advantage of patient-derived models from large biobanks. Citation Format: Ze-yan Zhang, Yingwen Ding, Ravesanker Ezhilarasan, Jie Yang, Lihong Long, Lawrence Bronk, Qianghu Wang, Erik P. Sulman. High-throughput evaluation of treatment response in patient-derived glioma stem cell models [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 1917.

Abstract 3108: Inhibition of the mTOR and MAPK pathways suppresses growth, induces apoptosis, and decreases vascularity in pediatric low grade glioma

Abstract Pediatric low-grade glioma (pLGG) is the most common brain tumor in children. We and others have recently identified constitutive activation of the mTOR and MEK-pathway in pLGG. This led us to investigate the antitumor activity of TAK228, a second generation mTORC1/2-inhibitor, and the FDA approved MEK-inhibitor, trametinib, in mono- and combination therapy in pLGG. We examined antitumor activity in five different patient-derived pLGG cell models treated with TAK228, trametinib, and combination: JHH_NF1_PA1NF1mut, BT66_SV40BRAF:KIAA1549, BT40BRAFV600E, Res186PTEN-/- and Res259CDKN2A-/-. In all cell lines, as measured by western blot (WB), trametinib treatment led to suppression of phospho-ERK at low levels (1-5nM). Similarly, TAK228 treatment led to inhibition of mTORC1 and mTORC2 in the low nanomolar range. Treatment with TAK228 or trametinib reduced cell proliferation in a dose and time dependent manner (MTS-assay). The combination treatment exerted a synergistic effect at 5-20nM in JHH_NF1_PA1, Res186, and Res259 cells (by Chou-Talay method). The BT66 cell line had a 70% reduction in cell growth with 10nM TAK228 (p&amp;lt;0.001 by 1-way ANOVA), but not in combination, or mono-treatment with trametinib in clinically relevant dose range. The combination of TAK228 and trametinib increased apoptosis by up to 127% (p&amp;lt;0.001) in Res186, Res259, and JHH_NF1_PA1 cells as measured by cleaved caspase 3 immunocytochemistry. We tested trametinib and TAK228 against the mutant BT40BRAFV600E patient-derived xenograft cell line in immunodeficient mice. The combination of TAK228 with trametinib showed greater antitumor activity than that of either mono-treatment in vivo. BT40 tumor growth was significantly decreased in combination compared to vehicle or either agent alone, (p&amp;lt;0.01). Immunohistochemistry (IHC) imaging showed a significant decrease in proliferation marker Ki67 with combination treatment (p&amp;lt;0.0001), and trametinib mono-treatment (p=0.0466), compared to TAK228 and vehicle. TAK228 and combination treatment decreased the mTORC1 phosphorylation of S6 ribosomal protein (WB &amp; IHC). Combination treatment strikingly reduced the vascularization of the tumor after combination treatment analyzed by VEGF protein expression (WB, p=0.0076), compared to the vehicle control, and mono-treatment. IHC confirmed a reduction in vascularization by CD31 staining. Our study provides evidence that pLGG-derived cell lines in vitro and in vivo are sensitive to mTORC1/2 kinase inhibition and MEK inhibition. Combination treatment with TAK228 and trametinib had a significant anti-tumor activity in vivo shown in survival rate, decreased tumor size, and reduced vascularity. These results provide the first strong rationale for combination therapy with TAK228 and trametinib for clinical consideration in pLGG in the near future. Citation Format: Antje Arnold, Lauren Harris, Ming Yuan, Fausto Rodriguez, Charles Eberhart, Eric Raabe. Inhibition of the mTOR and MAPK pathways suppresses growth, induces apoptosis, and decreases vascularity in pediatric low grade glioma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 3108.

Abstract 38: NSCLC patient-derived organoids to guide personalized therapy

Abstract Purpose: Translational research in non-small cell lung cancer (NSCLC) has been largely hampered by relatively low success rates for establishing patient-derived cell model or patient-derived xenograft model. Recently, NSCLC patient-derived organoid model (PDO) has been introduced, showing a high take rate and stable growth in long-term in vitro cultures. Translational relevance of NSCLC PDOs remains unclear. In this study, we evaluated whether NSCLC PDOs can predict anti-cancer treatment responses and provide therapeutic strategies to overcome drug resistance. Experimental design: Nineteen malignant effusions and 13 biopsied/surgical specimens were obtained from 31 patients with lung adenocarcinoma. Eleven patients (35%) had no prior therapy, 7 (23%) chemotherapy or radiotherapy, 6 (19%) EGFR-tyrosine kinase inhibitors (TKI), 4 (13%) ALK/ROS1-TKIs, and 3 (10%) immunotherapy. Samples were processed and cultured as reported previously. In brief, specimens were reviewed by pathologists to confirm malignancy. Then, samples were cultured in Advanced DMEM/F12 containing 10% R-spondin 1 conditioned medium, 25 ng/mL FGF7, 100 ng/mL FGF10, 100 ng/mL noggin, 500 nM A83-01, 500 nM SB202190, 1X B27, 1.25 mM N-acetylcysteine, 5 mM nicotinamide, 1X glutamax, 10 mM HEPES, and 1X primocin. For efficient establishment, we stained organoids with H&amp;E at early passages to confirm histological features of NSCLC. Cell viability assay was performed using CellTiter-Glo 3D. PDOs (&amp;lt;P10) were analyzed by whole-exome sequencing and RNA-seq and cryopreserved to establish a biobank. Results: A total of 22 PDOs derived from NSCLC patients was established. Take rates from malignant effusions and biopsied/surgical specimens were 89% and 38%. Of these established PDOs, 9 were wild-type, 9 EGFR-mutant, 3 ROS1-rearranged, and 1 ALK-rearranged. Genomic and transcriptomic profile of established PDOs were concordant to those of matching parental tumors. Of note, we established clinically important models progressing to targeted therapy or immunotherapy as follows: osimertinib-resistant EGFR mutant PDOs (n=4), repotrectinib-resistant ROS1-rearranged PDO (n=1), and pembrolizumab-resistant PDO (n=1). For example, YUO-2 was established from progressing pleural effusion after 5 months of osimertinib and had lost EGFR T790M but maintained EGFR exon 19 deletion. YUO-2 was resistant to gefitinib, afatinib, and osimertinib with IC50 values of 27300 nM, 9296 nM, and 5691 nM, respectively, implicating clinical relevance of NSCLC PDO models. TKI-resistant PDOs were screened with combinations of TKI targeting the driver mutation of the established models and each drug from the FDA-approved drug library. Conclusions: NSCLC PDO models could recapitulate the characteristics of corresponding NSCLC tumors and clinical response. PDO-based co-clinical trial will accelerate translational research in NSCLC. Further results will be presented at 2019 AACR meeting. Citation Format: Seok-Young Kim, Dong Hwi Kim, Hyeong-Seok Joo, Mi Ran Yun, Ji Yeon Lee, Sang Min Kim, Hyunki Kim, Min Hee Hong, Hye Ryun Kim, Byoung Chul Cho. NSCLC patient-derived organoids to guide personalized therapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 38.

Tracing Clonal Dynamics Reveals that Two- and Three-dimensional Patient-derived Cell Models Capture Tumor Heterogeneity of Clear Cell Renal Cell Carcinoma

BackgroundExtensive DNA sequencing has led to an unprecedented view of the diversity of individual genomes and their evolution among patients with clear cell renal cell carcinoma (ccRCC). ObjectiveTo understand subclonal architecture and dynamics of patient-derived two-dimensional (2D) and three-dimensional (3D) ccRCC models in vitro, in order to determine whether they mirror ccRCC inter- and intratumor heterogeneity. Design, setting, and participantsWe have established a comprehensive platform of living renal cancer cell models from ccRCC surgical specimens. Outcome measurements and statistical analysisWe confirmed the concordance of 2D and 3D patient-derived cell (PDC) models with the original tumor tissue in terms of histology, biomarker expression, cancer driver mutations, and copy number alterations. We addressed inter- and intrapatient heterogeneity by analyzing clonal dynamics during serial passaging. Results and limitationsIn-depth genetic characterization verified the presence of heterogeneous cell populations, and revealed a high degree of similarity between subclonal compositions of monolayer and organoid cell cultures and the corresponding parental ccRCCs. Clonal dynamics were evident during serial passaging of cells in vitro, suggesting that PDC cultures can offer insights into evolutionary potential and treatment susceptibility of ccRCC subclones in vivo. Proof-of-concept drug profiling using selected ccRCC-targeted therapy agents highlighted patient-specific vulnerabilities in PDC models that could not be anticipated by interrogating commercially available cell lines. ConclusionsWe demonstrate that PDC models mirror inter- and intratumor heterogeneity of ccRCC in vitro. Based on our findings, we envision that the use of these models will advance our understanding of the trajectories that cause genetic diversity and their consequences for treatment on an individual level. Patient summaryIn this study, we developed two- and three-dimensional patient-derived models from clear cell renal cell carcinoma (ccRCC) as “mini-tumors in a dish.” We show that these cell models retain important features of the human ccRCCs such as the profound tumor heterogeneity, thus highlighting their importance for cancer research and precision medicine.

(CTG)n repeat-mediated dysregulation of MBNL1 and MBNL2 expression during myogenesis in DM1 occurs already at the myoblast stage.

Myotonic dystrophy type 1 (DM1) is a severe neuromuscular disorder caused by the expression of trinucleotide repeat-containing DMPK transcripts. Abnormally expanded (CUG)n repeats in these transcripts form hairpin-like structures that cause the RNA to accumulate in the cell nucleus by sequestering isoforms of the Muscleblind (MBNL) family, tissue-specific regulators of developmentally programmed, post-transcriptional processes in RNA metabolism. Through this mechanism, the function of MBNL in RNA processing becomes dominantly perturbed, which eventually leads to aberrant alternative splicing and the expression of foetal splice variants of a wide variety of proteins, including the MBNL isoforms themselves. Here, we employ a patient-derived muscle cell model for DM1 to examine in detail the expression of MBNL RNA and protein variants during myogenic differentiation. This DM1 model consists of a panel of isogenic myoblast cell lines that either contain a pathogenic DMPK allele with a congenital mutation of 2600 triplets, or lack this expanded repeat through CRISPR/Cas9-mediated gene editing. We found that the temporal expression levels of MBNL1, MBNL2 and MBNL3 RNAs are not influenced by presence of the (CTG)2600 repeat during myogenesis in vitro. However, throughout myoblast proliferation and differentiation to myotubes a disproportionate inclusion of MBNL1 exon 5 and MBNL2 exons 5 and 8 occurs in cells with the (CTG)2600 repeat. As a consequence, a reduced quantity and imbalanced collection of splice variants of MBNL1 and MBNL2 accumulates in both the cytoplasm and the nucleus of DM1 myoblasts and myotubes. We thus propose that both the quantitative and qualitative changes in the intracellular partitioning of MBNL proteins are a pivotal cause of skeletal muscle problems in DM1, starting already in muscle progenitor cells.

Targeted degradation of aberrant tau in frontotemporal dementia patient-derived neuronal cell models.

Tauopathies are neurodegenerative diseases characterized by aberrant forms of tau protein accumulation leading to neuronal death in focal brain areas. Positron emission tomography (PET) tracers that bind to pathological tau are used in diagnosis, but there are no current therapies to eliminate these tau species. We employed targeted protein degradation technology to convert a tau PET-probe into a functional degrader of pathogenic tau. The hetero-bifunctional molecule QC-01-175 was designed to engage both tau and Cereblon (CRBN), a substrate-receptor for the E3-ubiquitin ligase CRL4CRBN, to trigger tau ubiquitination and proteasomal degradation. QC-01-175 effected clearance of tau in frontotemporal dementia (FTD) patient-derived neuronal cell models, with minimal effect on tau from neurons of healthy controls, indicating specificity for disease-relevant forms. QC-01-175 also rescued stress vulnerability in FTD neurons, phenocopying CRISPR-mediated MAPT-knockout. This work demonstrates that aberrant tau in FTD patient-derived neurons is amenable to targeted degradation, representing an important advance for therapeutics.

Pluripotent Stem Cells to Model Degenerative Retinal Diseases: The RPE Perspective.

Pluripotent stem cell technology, including human-induced pluripotent stem cells (hiPSCs) and human embryonic stem cells (hESCs), has provided a suitable platform to investigate molecular and pathological alterations in an individual cell type using patient's own cells. Importantly, hiPSCs/hESCs are amenable to genome editing providing unique access to isogenic controls. Specifically, the ability to introduce disease-causing mutations in control (unaffected) and conversely correct disease-causing mutations in patient-derived hiPSCs has provided a powerful approach to clearly link the disease phenotype with a specific gene mutation. In fact, utilizing hiPSC/hESC and CRISPR technology has provided significant insight into the pathomechanism of several diseases. With regard to the eye, the use of hiPSCs/hESCs to study human retinal diseases is especially relevant to retinal pigment epithelium (RPE)-based disorders. This is because several studies have now consistently shown that hiPSC-RPE in culture displays key physical, gene expression and functional attributes of human RPE in vivo. In this book chapter, we will discuss the current utility, limitations, and plausible future approaches of pluripotent stem cell technology for the study of retinal degenerative diseases. Of note, although we will broadly summarize the significant advances made in modeling and studying several retinal diseases utilizing hiPSCs/hESCs, our specific focus will be on the utility of patient-derived hiPSCs for (1) establishment of human cell models and (2) molecular and pharmacological studies on patient-derived cell models of retinal degenerative diseases where RPE cellular defects play a major pathogenic role in disease development and progression.

Establishment of a Novel PDX Mouse Model and Evaluation of the Tumor Suppression Efficacy of Bortezomib Against Liposarcoma

The patient-derived xenograft (PDX) model has been adopted as a major tool for studying tumorigenesis and differentiation in various carcinomas. In addition, it has been used in the development of anticancer agents. PDX models have been among the most meaningful tools used to understand the role of stromal cells and vascular cells in the body, which are major factors in cancer development and the application of therapeutic agents. Also, the establishment of PDX models from liposarcoma patients is considered to be important for understanding lipomagenesis and following drugs development. For these reasons, we developed patient-derived cell (PDC) and PDX models derived from 20 liposarcoma patients. The tissues of these patients were obtained in accordance with the principles of the Samsung Medical Center's ethics policy, and cell culture and xenografting onto the mice were performed under these principles. High-throughput drug screening (HTS) was carried out using established PDCs to select candidate drugs. Among the different candidate anticancer drugs, we tested the effect of bortezomib, which was expected to inhibit MDM2 amplification. First, we confirmed that the PDCs maintained the characteristics of liposarcoma cells by assessing MDM2 amplification and CDK4 overexpression using fluorescence in situ hybridization. Analysis of short tandem repeats and an array using comparative genomic hybridization confirmed that the PDX model exhibited the same genomic profile as that of the patient. Immunohistochemistry for MDM2 and CDK4 showed that the overexpression patterns of both proteins were similar in the PDX models and the PDCs. Specifically, MDM2 amplification was observed to be significantly correlated with the successful establishment of PDX mouse models. However, CDK4 expression did not show such a correlation. Of the anticancer drugs selected through HTS, bortezomib showed a strong anticancer effect against PDC. In addition, we observed that bortezomib suppressed MDM2 expression in a dose-dependent manner. In contrast, p21 tended to elicit an increase in PDC expression. Treatment of the PDX model with bortezomib resulted in an anticancer effect similar to that seen in the PDCs. These results support that PDCs and PDX models are among the most powerful tools for the development and clinical application of anticancer drugs for the treatment of liposarcoma patients.

Exosomal microRNAs as potential biomarkers for cancer cell migration and prognosis in hepatocellular carcinoma patient-derived cell models.

Hepatocellular carcinoma (HCC) is one of the most common malignant tumors worldwide, and current treatments exhibit limited efficacy against advanced HCC. The majority of cancer-related deaths are caused by metastasis from the primary tumor, which indicates the importance of identifying clinical biomarkers for predicting metastasis and indicating prognosis. Patient-derived cells (PDCs) may be effective models for biomarker identification. In the present study, a wound healing assay was used to obtain 10 fast-migrated and 10 slow-migrated PDC cultures from 36 HCC samples. MicroRNA (miRNA) signatures in PDCs and PDC-derived exosomes were profiled by microRNA-sequencing. Differentially expressed miRNAs between the low- and fast-migrated groups were identified and further validated in 372 HCC profiles from The Cancer Genome Atlas (TCGA). Six exosomal miRNAs were identified to be differentially expressed between the two groups. In the fast-migrated group, five miRNAs (miR-140-3p, miR-30d-5p, miR-29b-3p, miR-130b-3p and miR-330-5p) were downregulated, and one miRNA (miR-296-3p) was upregulated compared with the slow-migrated group. Pathway analysis demonstrated that the target genes of the differentially expressed miRNAs were significantly enriched in the ‘focal adhesion’ pathway, which is consistent with the roles of these miRNAs in tumor metastasis. Three miRNAs, miR-30d, miR-140 and miR-29b, were significantly associated with patient survival. These findings indicated that these exosomal miRNAs may be candidate biomarkers for predicting HCC cell migration and prognosis and may guide the treatment of advanced HCC.

Modelling inherited cardiac disease using human induced pluripotent stem cell-derived cardiomyocytes: progress, pitfalls, and potential.

In the past few years, the use of specific cell types derived from induced pluripotent stem cells (iPSCs) has developed into a powerful approach to investigate the cellular pathophysiology of numerous diseases. Despite advances in therapy, heart disease continues to be one of the leading causes of death in the developed world. A major difficulty in unravelling the underlying cellular processes of heart disease is the extremely limited availability of viable human cardiac cells reflecting the pathological phenotype of the disease at various stages. Thus, the development of methods for directed differentiation of iPSCs to cardiomyocytes (iPSC-CMs) has provided an intriguing option for the generation of patient-specific cardiac cells. In this review, a comprehensive overview of the currently published iPSC-CM models for hereditary heart disease is compiled and analysed. Besides the major findings of individual studies, detailed methodological information on iPSC generation, iPSC-CM differentiation, characterization, and maturation is included. Both, current advances in the field and challenges yet to overcome emphasize the potential of using patient-derived cell models to mimic genetic cardiac diseases.

Patient-Derived Stem Cell Models in SPAST HSP: Disease Modelling and Drug Discovery.

Hereditary spastic paraplegia is an inherited, progressive paralysis of the lower limbs first described by Adolph Strümpell in 1883 with a further detailed description of the disease by Maurice Lorrain in 1888. Today, more than 100 years after the first case of HSP was described, we still do not know how mutations in HSP genes lead to degeneration of the corticospinal motor neurons. This review describes how patient-derived stem cells contribute to understanding the disease mechanism at the cellular level and use this for discovery of potential new therapeutics, focusing on SPAST mutations, the most common cause of HSP.

Loss of tuberous sclerosis complex 2 sensitizes tumors to nelfinavir\u2212bortezomib therapy to intensify endoplasmic reticulum stress-induced cell death

Cancer cells lose homeostatic flexibility because of mutations and dysregulated signaling pathways involved in maintaining homeostasis. Tuberous Sclerosis Complex 1 (TSC1) and TSC2 play a fundamental role in cell homeostasis, where signal transduction through TSC1/TSC2 is often compromised in cancer, leading to aberrant activation of mechanistic target of rapamycin complex 1 (mTORC1). mTORC1 hyperactivation increases the basal level of endoplasmic reticulum (ER) stress via an accumulation of unfolded protein, due to heightened de novo protein translation and repression of autophagy. We exploit this intrinsic vulnerability of tumor cells lacking TSC2, by treating with nelvinavir to further enhance ER stress while inhibiting the proteasome with bortezomib to prevent effective protein removal. We show that TSC2-deficient cells are highly dependent on the proteosomal degradation pathway for survival. Combined treatment with nelfinavir and bortezomib at clinically relevant drug concentrations show synergy in selectively killing TSC2-deficient cells with limited toxicity in control cells. This drug combination inhibited tumor formation in xenograft mouse models and patient-derived cell models of TSC and caused tumor spheroid death in 3D culture. Importantly, 3D culture assays differentiated between the cytostatic effects of the mTORC1 inhibitor, rapamycin, and the cytotoxic effects of the nelfinavir/bortezomib combination. Through RNA sequencing, we determined that nelfinavir and bortezomib tip the balance of ER protein homeostasis of the already ER-stressed TSC2-deficient cells in favor of cell death. These findings have clinical relevance in stratified medicine to treat tumors that have compromised signaling through TSC and are inflexible in their capacity to restore ER homeostasis.

Abstract 2848: Radiographic and genomic evolution of individual metastases during HER2 blockade in colorectal cancer

Abstract Targeting HER2 with trastuzumab and lapatinib is effective in ERBB2 amplified metastatic colorectal cancer (mCRC). Although at least 30% of the patients initially respond, secondary resistance occurs in most of the cases. Since the drivers of secondary resistance to trastuzumab and lapatinib in ERBB2 amplified mCRC are unknown, we exploited longitudinal plasma collections and patient-derived cell models to define the molecular bases of resistance to HER2 blockade. Levels of ERBB2 amplification in plasma circulating tumor DNA (ctDNA) paralleled response and relapse. The emergence of EGFR, ERBB2, RAS, BRAF and PIK3CA variants in ctDNA was associated with resistance. Radiographic measurements of individual metastases coupled with longitudinal liquid biopsies unveiled lesion-specific patterns of heterogeneous response in several patients. Phylogenetic tracking and functional analyses on tissue samples and patient-derived cell models established from eight metastases of a single case revealed new druggable oncogenic dependencies and genomic evolution associated with resistance. These data highlight the relevance of coupling imaging and liquid biopsies analyses in precision oncology and provide the rationale for additional lines of therapies in HER2 positive mCRC relapsing upon HER2 blockade. Citation Format: Giulia Siravegna, Luca Lazzari, Andrea Sartore-Bianchi, Giovanni Crisafulli, Benedetta Mussolin, Andrea Cassingena, Cosimo Martino, Richard Lanman, Rebecca Nagy, Giorgio Corti, Alice Bartolini, Pamela Arcella, Monica Montone, Francesca Lodi, Alice Vanzati, Emanuele Valtorta, Giovanni Cappello, Andrea Bertotti, Sara Lonardi, Vittorina Zagonel, Francesco Leone, Mariangela Russo, Antonella Balsamo, Mauro Truini, Federica Di Nicolantonio, Alessio Amatu, Erica Bonazzina, Silvia Ghezzi, Daniele Regge, Angelo Vanzulli, Livio Trusolino, Salvatore Siena, Silvia Marsoni, Alberto Bardelli. Radiographic and genomic evolution of individual metastases during HER2 blockade in colorectal cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 2848.

Abstract 1159: Development of uterine leiomyoma 3D in vitro models for high-throughput drug and chemical compound screenings: Towards personalized medicine

Abstract Uterine leiomyomas (ULs) are extremely common smooth muscle tumors, occurring in an estimated 77% of women of reproductive age. Although benign, ULs form a major burden to women's health and are the leading cause of hysterectomy worldwide. Recent findings show that ULs can be classified into at least three distinct molecular subclasses, each with a characteristic genetic driver aberration and global gene expression profile: MED12 (mediator complex subunit 12) mutation-positive, HMGA2 (high mobility group AT-hook 2)-overexpressing, FH (fumarate hydratase)-deficient. However, ULs are currently treated without taking into account the possible different subclasses. The aim of this project is to develop and characterize 3D in vitro models of ULs. The models will be used to examine responses to existing treatments in different subclasses of UL and to identify subclass-specific novel lead compounds for drugs. The work is based on the hypothesis that different molecular UL subclasses have distinct sensitivity and resistance patterns to existing treatment options. Therefore, ULs should not be treated as a single entity. The study material consists of UL tissue belonging to different subclasses and myometrium, collected from hysterectomy patients during surgery in Helsinki University Hospital, Finland. Freshly operated samples are processed to initiate primary cell cultures. The primary cells are seeded on Ultra-Low Attachment (ULA) multiwell plates and grown to form 3D spheroids. In case of MED12-mutated tumors, the spheroids are screened for MED12 mutations by Sanger sequencing. MED12-wild type samples and spheroids are analyzed for overexpression of HMGA2 and FH-deficiency. Immunofluorescence staining is performed to characterize different cell types in the spheroid structure. The presence of live/dead cells in spheroids is monitored during the drug treatments in 384-well plates with high-content confocal microscopy. The approach will first focus on the most common subclasses (MED12-mutated and HMGA2-overexpressing tumors) and most common treatment options. Here, we have set up novel patient-derived cell models for ULs. We have characterized myometrium and UL-derived spheroids and show that they retain the characteristics and genetic driver aberration of the original samples. Furthermore, we show they are composed mostly of live cells. Subsequent in vitro high-throughput drug screening will lead to characterization of drug sensitivities and identification of novel lead compounds for drugs. This information is important for rational drug design, where the effects of candidate drugs can be evaluated in light of the molecular class of the respective tumors. This work will lead to new knowledge towards precision medicine, which has potential for scientific breakthrough, since it would avoid exposing the patients to ineffective treatment options. Citation Format: Simona Bramante, Vilja Pietiäinen, Lassi Paavolainen, Annukka Pasanen, Netta Mäkinen, Hanna-Riikka Heinonen, Pirjo Ikonen, Oskari Heikinheimo, Jari Sjöberg, Ralf Bützow, Lauri Aaltonen. Development of uterine leiomyoma 3D in vitro models for high-throughput drug and chemical compound screenings: Towards personalized medicine [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 1159.

Nitration of microtubules blocks axonal mitochondrial transport in a human pluripotent stem cell model of Parkinson's disease.

Neuronal loss in Parkinson's disease (PD) is associated with aberrant mitochondrial function in dopaminergic (DA) neurons of the substantia nigra pars compacta. An association has been reported between PD onset and exposure to mitochondrial toxins, including the agrochemicals paraquat (PQ), maneb (MB), and rotenone (Rot). Here, with the use of a patient-derived stem cell model of PD, allowing comparison of DA neurons harboring a mutation in the α-synuclein (α-syn) gene ( SNCA-A53T) against isogenic, mutation-corrected controls, we describe a novel mechanism whereby NO, generated from SNCA-A53T mutant neurons exposed to Rot or PQ/MB, inhibits anterograde mitochondrial transport through nitration of α-tubulin (α-Tub). Nitration of α-Tub inhibited the association of both α-syn and the mitochondrial motor protein kinesin 5B with the microtubules, arresting anterograde transport. This was, in part, a result of nitration of α-Tub in the C-terminal domain. These effects were rescued by inhibiting NO synthesis with the NOS inhibitor Nω-nitro-L-arginine methyl ester. Collectively, our results are the first to demonstrate a gene by environment interaction in PD, whereby agrochemical exposure selectively triggers a deficit in mitochondrial transport by nitrating the microtubules in neurons harboring the SNCA-A53T mutation.-Stykel, M. G., Humphries, K., Kirby, M. P., Czaniecki, C., Wang, T., Ryan, T., Bamm, V., Ryan, S. D. Nitration of microtubules blocks axonal mitochondrial transport in a human pluripotent stem cell model of Parkinson's disease.