Integrative Genomic Analysis of Drug Resistance in MET Exon 14 Skipping Lung Cancer Using Patient-Derived Xenograft Models

Abstract Background Non-small cell lung cancer (NSCLC) driven by MET exon 14 skipping (METex14) occurs in 3-4% of NSCLC cases and defines a subset of patients with distinct characteristics. MET targeted therapy has led to strong clinical responses, however little is known about aquired resistance to drugs in these patients. Patient derived xenograft (PDX) models are recognized as excellent preclinical models to facilitate the understanding of the mechanisms underlying drug resistance. Methods We describe a patient case harboring METex14 who exhibited drug resistance after treatment with crizotinib. Subcutaneous xenografts were generated from pretreatment and post-resistance patient specimens. PDX mice were then treated with MET inhibitors (crizotinib and tepotinib) to evaluate their drug response. DNA and RNA sequencing analysis was performed on patient tumor specimens and matching xenografts. Results PDXs preserved most of the histological and molecular profiles of the parental tumors. Drug resistance to MET targeted therapy was confirmed in PDX models through in vivo drug analysis. Newly aquired MET D1228N mutations and EGFR amplificated were detected in patient-resistant tumor specimens. Although the mutations were not detected in the PDX, EGFR overexpression was observed in RNA sequencing analysis indicating possible off target resistance through the EGFR bypass signaling pathway. Conclusions We established and characterized a pair of METex14 NSCLC patient-derived xenografts (PDXs), including the first crizotinib resistant METex14 PDX. This model will be a powerful tool for testing hypotheses of drug resistance mechanisms and investigations into novel therapeutic strategies.

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A Simple Three-Dimensional In Vitro Culture Mimicking the In Vivo-Like Cell Behavior of Bladder Patient-Derived Xenograft Models

Cancers ◽

10.3390/cancers12051304 ◽

2020 ◽

Vol 12 (5) ◽

pp. 1304

Author(s):

Robson Amaral ◽

Maike Zimmermann ◽

Ai-Hong Ma ◽

Hongyong Zhang ◽

Kamilla Swiech ◽

...

Keyword(s):

Drug Resistance ◽

In Vitro Culture ◽

Cancer Cells ◽

Low Cost ◽

Maintenance Cost ◽

Tumor Spheroids ◽

Patient Derived Xenograft ◽

Pdx Models

Patient-derived xenograft (PDX) models allow for personalized drug selection and the identification of drug resistance mechanisms in cancer cells. However, PDX models present technical disadvantages, such as long engraftment time, low success rate, and high maintenance cost. On the other hand, tumor spheroids are emerging as an in vitro alternative model that can maintain the phenotype of cancer cells long enough to perform all assays and predict a patient’s outcome. The present work aimed to describe a simple, reproducible, and low-cost 3D in vitro culture method to generate bladder tumor spheroids using human cells from PDX mice. Cancer cells from PDX BL0293 and BL0808 models, previously established from advanced bladder cancer, were cultured in 96-well round-bottom ultra-low attachment (ULA) plates with 5% Matrigel and generated regular and round-shaped spheroids (roundness > 0.8) with a diameter larger than 400 μm and a hypoxic core (a feature related to drug resistance in solid tumors). The responses of the tumor spheroids to the antineoplastic drugs cisplatin, gemcitabine, and their combination were similar to tumor responses in in vivo studies with PDX BL0293 and BL0808 mice. Therefore, the in vitro 3D model using PDX tumor spheroids appears as a valuable tool that may predict the outcome of in vivo drug-screening assays and represents a low-cost strategy for such purpose.

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Patient-Derived Nasopharyngeal Cancer Organoids for Disease Modeling and Radiation Dose Optimization

Frontiers in Oncology ◽

10.3389/fonc.2021.622244 ◽

2021 ◽

Vol 11 ◽

Author(s):

Sasidharan Swarnalatha Lucky ◽

Martin Law ◽

Ming Hong Lui ◽

Jamie Mong ◽

Junli Shi ◽

...

Keyword(s):

Rna Sequencing ◽

Survival Data ◽

Disease Modeling ◽

Oxygen Deficiency ◽

Nasopharyngeal Cancer ◽

Sequencing Analysis ◽

Boost Dose ◽

Pdx Models

Effective radiation treatment (RT) for recurrent nasopharyngeal cancers (NPC), featuring an intrinsic hypoxic sub-volume, remains a clinical challenge. Lack of disease‐specific in-vitro models of NPC, together with difficulties in establishing patient derived xenograft (PDX) models, have further hindered development of personalized therapeutic options. Herein, we established two NPC organoid lines from recurrent NPC PDX models and further characterized and compared these models with original patient tumors using RNA sequencing analysis. Organoids were cultured in hypoxic conditions to examine the effects of hypoxia and radioresistance. These models were then utilized to determine the radiobiological parameters, such as α/β ratio and oxygen enhancement ratio (OER), characteristic to radiosensitive normoxic and radioresistant hypoxic NPC, using simple dose-survival data analytic tools. The results were further validated in-vitro and in-vivo, to determine the optimal boost dose and fractionation regimen required to achieve effective NPC tumor regression. Despite the differences in tumor microenvironment due to the lack of human stroma, RNA sequencing analysis revealed good correlation of NPC PDX and organoid models with patient tumors. Additionally, the established models also mimicked inter-tumoral heterogeneity. Hypoxic NPC organoids were highly radioresistant and had high α/β ratio compared to its normoxic counterparts. In-vitro and in-vivo fractionation studies showed that hypoxic NPC was less sensitive to RT fractionation scheme and required a large bolus dose or 1.4 times of the fractionated dose that was effective against normoxic cells in order to compensate for oxygen deficiency. This study is the first direct experimental evidence to predict optimal RT boost dose required to cause sufficient damage to recurrent hypoxic NPC tumor cells, which can be further used to develop dose-painting algorithms in clinical practice.

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Cisplatin loaded multiwalled carbon nanotubes reverse drug resistance in NSCLC by inhibiting EMT

Cancer Cell International ◽

10.1186/s12935-021-01771-9 ◽

2021 ◽

Vol 21 (1) ◽

Author(s):

Yuxin Qi ◽

Wenping Yang ◽

Shuang Liu ◽

Fanjie Han ◽

Haibin Wang ◽

...

Keyword(s):

Lung Cancer ◽

Carbon Nanotubes ◽

Drug Resistance ◽

Western Blot ◽

Multiwalled Carbon Nanotubes ◽

Cell Lines ◽

Nude Mice ◽

Expression Level ◽

Multiwalled Carbon

Abstract Background Lung cancer is one of the important health threats worldwide, of which 5-year survival rate is less than 15%. Non-small-cell lung cancer (NSCLC) accounts for about 80% of all lung cancer with high metastasis and mortality. Methods Cisplatin loaded multiwalled carbon nanotubes (Pt-MWNTS) were synthesized and used to evaluate the anticancer effect in our study. The NSCLC cell lines A549 (cisplatin sensitive) and A549/DDP (cisplatin resistant) were used in our in vitro assays. MTT was used to determine Cancer cells viability and invasion were measured by MTT assay and Transwell assay, respectively. Apoptosis and epithelial-mesenchymal transition related marker proteins were measured by western blot. The in vivo anti-cancer effect of Pt-MWNTs were performed in male BALB/c nude mice (4-week old). Results Pt-MWNTS were synthesized and characterized by X-ray diffraction, Raman, FT-IR spectroscopy and scan electron microscopy. No significant cytotoxicity of MWNTS was detected in both A549/DDP and A549 cell lines. However, Pt-MWNTS showed a stronger inhibition effect on cell growth than free cisplatin, especially on A549/DDP. We found Pt-MWNTS showed higher intracellular accumulation of cisplatin in A549/DDP cells than free cisplatin and resulted in enhanced the percent of apoptotic cells. Western blot showed that application of Pt-MWNTS can significantly upregulate the expression level of Bax, Bim, Bid, Caspase-3 and Caspase-9 while downregulate the expression level of Bcl-2, compared with free cisplatin. Moreover, the expression level of mesenchymal markers like Vimentin and N-cadherin was more efficiently reduced by Pt-MWNTS treatment in A549/DDP cells than free cisplatin. In vivo study in nude mice proved that Pt-MWNTS more effectively inhibited tumorigenesis compared with cisplatin, although both of them had no significant effect on body weight. Conclusion Pt-MWNT reverses the drug resistance in the A549/DDP cell line, underlying its possibility of treating NSCLC with cisplatin resistance.

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A Novel Multidrug-Resistant Cell Line from an Italian Intrahepatic Cholangiocarcinoma Patient

Cancers ◽

10.3390/cancers13092051 ◽

2021 ◽

Vol 13 (9) ◽

pp. 2051

Author(s):

Caterina Peraldo-Neia ◽

Annamaria Massa ◽

Francesca Vita ◽

Marco Basiricò ◽

Chiara Raggi ◽

...

Keyword(s):

Drug Resistance ◽

Cell Line ◽

Intrahepatic Cholangiocarcinoma ◽

Biological Behavior ◽

Population Doubling ◽

Sequencing Analysis ◽

Tumor Type ◽

Clinical Issue ◽

Mesenchymal Transition

Chemotherapy resistance is a relevant clinical issue in tumor treatment, in particular in biliary tract carcinoma (BTC), for which there are no effective therapies, neither in the first nor in the second line. The development of chemoresistant cell lines as experimental models to investigate the mechanisms of resistance and identify alternative druggable pathways is mandatory. In BTC, in which genetics and biological behavior depend on the etiology, ethnicity, and anatomical site of origin, the creation of models that better recapitulate these characteristics is even more crucial. Here we have established and characterized an intrahepatic cholangiocarcinoma (iCCA) cell line derived from an Italian patient, called 82.3. Cells were isolated from a patient-derived xenograft (PDX) and, after establishment, immunophenotypic, biological, genetic, molecular characteristics, and tumorigenicity in vivo in NOD/SCID mice were investigated. 82.3 cells exhibited epithelial morphology and cell markers (EPCAM, CK7, and CK19); they also expressed different cancer stem markers (CD44, CD133, CD49b, CD24, Stro1, PAX6, FOXA2, OCT3/4), α–fetoprotein and under anchorage-independent and serum-free conditions were capable of originating cholangiospheres. The population doubling time was approximately 53 h. In vitro, they demonstrated a poor ability to migrate; in vivo, 82.3 cells retained their tumorigenicity, with a long latency period (16 weeks). Genetic identity using DNA fingerprinting analysis revealed 16 different loci, and the cell line was characterized by a complex hyperdiploid karyotype. Furthermore, 82.3 cells showed cross-resistance to gemcitabine, 5-fluorouracil, carboplatin, and oxaliplatin; in fact, their genetic profile showed that 60% of genes (n = 168), specific for drug resistance and related to the epithelial-mesenchymal transition, were deregulated in 82.3 cells compared to a control iCCA cell line sensitive to chemotherapeutics. RNA sequencing analysis revealed the enrichment for genes associated with epithelial to mesenchymal transition (EMT), vasculature development, and extracellular matrix (ECM) remodeling, underlining an aggressive phenotype. In conclusion, we have created a new iCCA cell line of Caucasian origin: this could be exploited as a preclinical model to study drug resistance mechanisms and to identify alternative therapies to improve the prognosis of this tumor type.

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Cell membrane based biomimetic nanocomposites for targeted therapy of drug resistant EGFR-mutated lung cancer

Nanoscale ◽

10.1039/c9nr05791a ◽

2019 ◽

Vol 11 (41) ◽

pp. 19520-19528 ◽

Cited By ~ 9

Author(s):

Pengying Wu ◽

Dongtao Yin ◽

Jiaming Liu ◽

Huige Zhou ◽

Mengyu Guo ◽

...

Keyword(s):

Lung Cancer ◽

Drug Resistance ◽

Targeted Therapy ◽

Cell Membrane ◽

Cancer Cell ◽

Egfr Mutation ◽

Drug Resistant ◽

Egfr Mutated

A cancer cell membrane-based biomimetic strategy was developed by loading doxorubicin and icotinib to overcome drug-resistance of EGFR-mutation lung cancer.

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Network Pharmacology and Experiment Verification to Explore the Potential Mechanism of Yin-Huo-Tang for Lung Adenocarcinoma Recurrence

10.21203/rs.3.rs-956330/v1 ◽

2021 ◽

Author(s):

Dianna Liu ◽

Shicheng Lin ◽

Yuan Li ◽

Tian Zhou ◽

Kaiwen Hu ◽

...

Keyword(s):

Lung Cancer ◽

Molecular Docking ◽

Lung Adenocarcinoma ◽

Signaling Pathway ◽

Rna Sequencing ◽

Network Pharmacology ◽

Phytochemical Analysis ◽

Sequencing Analysis ◽

Lewis Lung ◽

Hub Genes

Abstract BackgroundLung adenocarcinoma (LUAD) is one of the most common malignancies with a rise in new cases worldwide each year. Recurrence significantly influences the survival in patients with LUAD. Yin-Huo-Tang (YHT) is a classic traditional Chinese prescription, used to prevent lung cancer relapse by “nourishing yin and clearing heat”. MethodsIn this study, the mechanism of YHT in LUAD recurrence was investigated. Firstly, the bioactive compounds-targets network and the protein–protein interaction network were constructed, and functional annotation and pathway enrichment analyses were performed. Pivotal compounds and hub genes were selected from the networks. Subsequently, the effectiveness of YHT was confirmed in lewis lung carcinoma mice. RNA sequencing was used to explore the mRNA expression differences between tumor tissues in the model mouses and YHT-treated mouses. The pathways screened by network pharmacology and RNA sequencing analysis at the same time were considered the most important pathways. At last, qualitative phytochemical analysis, molecular docking technology, PCR and WB analysis were used to validate the pivotal active ingredients, hub genes and main pathways.ResultsThere were 128 active compounds, 419 targets interacting with LUAD recurrence. Network analysis identified 4 pivotal compounds, 28 hub genes and 30 main pathways. Target genes mainly focused on inflammation, metabolism, immune responses and apoptosis. We confirmed that YHT could inhibit the recurrence of lung adenocarcinoma through animal experimental study. Sphingolipid signaling pathway was the common main pathway in network pharmacology and RNA sequencing results. The hub genes related with the sphingolipid signaling pathway was S1PR5. Qualitative phytochemical analysis of the water extract of YHT confirmed the presence of 3 pivotal compounds, namely stigmasterol, nootkatone and ergotamine. The results of molecular docking verified the pivotal compounds of YHT could good affinity with the S1PR5. The PCR and WB analysis verified YHT suppressed lewis lung cancer cells proliferation by inhibiting S1P/S1PR5/Gi/Ras/Raf/MEK/ERK pathway, and inhibited migration through S1P/S1PR5/Gi/PI3K/RAC pathway.ConclusionThe results confirmed the therapeutic effect of YHT on the recurrence of LUAD by multi-component-multi-target mode, the sphingolipid signaling pathway was one of the most relevant potential signaling pathways.

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EXTH-67. THERAPEUTIC EFFICACY OF GC1118, A NOVEL ANTI-EGFR ANTIBODY, AGAINST GLIOBLASTOMA WITH HIGH EGFR AMPLIFICATION IN PATIENT-DERIVED XENOGRAFTS

Neuro-Oncology ◽

10.1093/neuonc/noab196.706 ◽

2021 ◽

Vol 23 (Supplement_6) ◽

pp. vi178-vi178

Author(s):

Seung Won Choi

Keyword(s):

Therapeutic Efficacy ◽

Growth Factor Receptor ◽

Genomic Analysis ◽

Screening Assay ◽

Egfr Amplification ◽

High Throughput Drug Screening ◽

Number Variation ◽

Drug Screening Assay ◽

Pdx Models

Abstract We aimed to evaluate the preclinical efficacy of GC1118, a novel anti-epidermal growth factor receptor (EGFR) monoclonal antibody (mAb), against glioblastoma (GBM) tumors using patient-derived xenograft (PDX) models. A total of 15 distinct GBM PDX models were used to evaluate the therapeutic efficacy of GC1118. Genomic data derived from PDX models were analyzed to identify potential biomarkers associated with the anti-tumor efficacy of GC1118. A patient-derived cell-based high-throughput drug screening assay was performed to further validate the efficacy of GC1118. Compared to cetuximab, GC1118 exerted comparable growth inhibitory effects on the GBM tumors in the PDX models. We confirmed that GC1118 accumulated within the tumor by crossing the blood–brain barrier in in vivo specimens and observed the survival benefit in GC1118-treated intracranial models. Genomic analysis revealed high EGFR amplification as a potent biomarker for predicting the therapeutic efficacy of GC1118 in GBM tumors. In summary, GC1118 exerted a potent anti-tumor effect on GBM tumors in PDX models, and its therapeutic efficacy was especially pronounced in the tumors with high EGFR amplification. Our study supports the importance of patient stratification based on EGFR copy number variation in clinical trials for GBM. The superiority of GC1118 over other EGFR mAbs in GBM tumors should be assessed in future studies.

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MicroRNA in Lung Cancer Metastasis

Cancers ◽

10.3390/cancers11020265 ◽

2019 ◽

Vol 11 (2) ◽

pp. 265 ◽

Cited By ~ 15

Author(s):

Shang-Gin Wu ◽

Tzu-Hua Chang ◽

Yi-Nan Liu ◽

Jin-Yuan Shih

Keyword(s):

Lung Cancer ◽

Targeted Therapy ◽

Cancer Metastasis ◽

Epithelial To Mesenchymal Transition ◽

Cancer Treatments ◽

Mesenchymal Transition ◽

Lung Cancer Metastasis ◽

Targeted Treatments

Tumor metastasis is a hallmark of cancer, with distant metastasis frequently developing in lung cancer, even at initial diagnosis, resulting in poor prognosis and high mortality. However, available biomarkers cannot reliably predict cancer spreading sites. The metastatic cascade involves highly complicated processes including invasion, migration, angiogenesis, and epithelial-to-mesenchymal transition that are tightly controlled by various genetic expression modalities along with interaction between cancer cells and the extracellular matrix. In particular, microRNAs (miRNAs), a group of small non-coding RNAs, can influence the transcriptional and post-transcriptional processes, with dysregulation of miRNA expression contributing to the regulation of cancer metastasis. Nevertheless, although miRNA-targeted therapy is widely studied in vitro and in vivo, this strategy currently affords limited feasibility and a few miRNA-targeted therapies for lung cancer have entered into clinical trials to date. Advances in understanding the molecular mechanism of metastasis will thus provide additional potential targets for lung cancer treatment. This review discusses the current research related to the role of miRNAs in lung cancer invasion and metastasis, with a particular focus on the different metastatic lesions and potential miRNA-targeted treatments for lung cancer with the expectation that further exploration of miRNA-targeted therapy may establish a new spectrum of lung cancer treatments.

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Induction of Acquired Resistance towards EGFR Inhibitor Gefitinib in a Patient-Derived Xenograft Model of Non-Small Cell Lung Cancer and Subsequent Molecular Characterization

Cells ◽

10.3390/cells8070740 ◽

2019 ◽

Vol 8 (7) ◽

pp. 740 ◽

Cited By ~ 5

Author(s):

Julia Schueler ◽

Cordula Tschuch ◽

Kerstin Klingner ◽

Daniel Bug ◽

Anne-Lise Peille ◽

...

Keyword(s):

Lung Cancer ◽

Small Cell Lung Cancer ◽

Cell Lung Cancer ◽

Xenograft Model ◽

Small Cell ◽

Small Cell Lung ◽

Protein Ratio ◽

Patient Derived Xenograft ◽

Resistant Lines

In up to 30% of non-small cell lung cancer (NSCLC) patients, the oncogenic driver of tumor growth is a constitutively activated epidermal growth factor receptor (EGFR). Although these patients gain great benefit from treatment with EGFR tyrosine kinase inhibitors, the development of resistance is inevitable. To model the emergence of drug resistance, an EGFR-driven, patient-derived xenograft (PDX) NSCLC model was treated continuously with Gefitinib in vivo. Over a period of more than three months, three separate clones developed and were subsequently analyzed: Whole exome sequencing and reverse phase protein arrays (RPPAs) were performed to identify the mechanism of resistance. In total, 13 genes were identified, which were mutated in all three resistant lines. Amongst them the mutations in NOMO2, ARHGEF5 and SMTNL2 were predicted as deleterious. The 53 mutated genes specific for at least two of the resistant lines were mainly involved in cell cycle activities or the Fanconi anemia pathway. On a protein level, total EGFR, total Axl, phospho-NFκB, and phospho-Stat1 were upregulated. Stat1, Stat3, MEK1/2, and NFκB displayed enhanced activation in the resistant clones determined by the phosphorylated vs. total protein ratio. In summary, we developed an NSCLC PDX line modelling possible escape mechanism under EGFR treatment. We identified three genes that have not been described before to be involved in an acquired EGFR resistance. Further functional studies are needed to decipher the underlying pathway regulation.

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