Project description:Variable responses to platinum chemotherapy and the development of resistant disease drive high mortality in high-grade serous ovarian cancer (HGSOC), the deadliest gynecological malignancy. Here, we developed the organoid drug resistance assay (ODR-test) to quantify the patient-specific efficacy of platinum treatment and generated post-platinum lines (ptGPC) as an in vitro model of platinum-resistant ovarian cancer (PROC). We observed a gradual increase in resistance of ptGPC lines, mirroring the clinical outcomes of patients. We have performed RNA-seq analysis from two different donor organoid lines, before carboplatin exposure and paired ptGPC lines of long-term expandable organoids that successfully overcame in vitro drug treatment. By differential expression analysis, we have identified target genes that mediate the cellular response to platinum and play a role in the development of resistant phenotype in ovarian cancer. We have found that carboplatin treatment triggers sustained reprogramming and ptGPC organoids from different donors show common changes in cell adhesion and cytoskeletal organization, indicating the existence of conserved mechanisms that lead to an increase in resistance.
Project description:A BRAF V600E colorectal organoid which is sensitive to MAP kinase inhibition was mutagenised with the chemical mutagen ENU and then drug selected using a combination of Trametinib, Dabrafenib and Cetuximab. Single cell derived organoids were then manually picked and expanded in drug. Resistance was confirmed in a 14 day assay and DNA was collected. These then underwent targeted amplicon-based sequencing to confirm candidate resistance effectors from a screen in 2 2D BRAF V600E colorectal cell lines. Pools of resistant clones were also sequenced.
Project description:We present TORNADO-seq —a high-throughput, a high-content drug discovery platform that for the first time uses next-generation sequencing (NGS) based, targeted RNA-seq in organoids monitoring the expression of 206 genes for the evaluation of complex mixtures of cellular phenotypes. TORNADO-seq is a fast, highly-reproducible, time- and cost-effective (5$ per sample including sequencing cost) method that we used to find drugs that enrich for differentiated cell phenotypes in intestinal organoids. These drugs are highly efficacious against cancer compared to wild type organoids and may therefore become promising candidates in CRC treatment. Further, TORNADO-seq facilitated in-depth insight on the mode of action of these drugs.
Project description:We treated APOB-mutant organoids with different anti-NAFLD drug candidates that were effective at resolving steatosis. To understand the mechanisms of drug action and adverse drug effects we performed bulk RNA-sequencing on drug-treated and vehicle-treated APOB mutant organoids.
Project description:This is a single-centre study based on the Simon 2-stage optimax design: 12 patients will be enrolled initially (Stage I), which will then be expanded to a further 13 patients (Stage II) if 3 or more patients enrolled in stage I of the study achieve an objective response with the chemotherapeutic agent selected by the drug screen assay. A total of 25 patients will be included in both stages of study.
Patients enrolled on study will undergo a fresh biopsy of tumour lesion to obtain cells that will be used to generate patient-derived tumour organoids based on the Invitrocue technology. Organoids will then be subjected to a 10-drug panel screening including: 5-fluorouracil, carboplatin, cyclophosphamide, docetaxel, doxorubicin, gemcitabine, irinotecan, oxaliplatin, paclitaxel and vinorelbine. A further 5 drugs (etoposide, ifosfamide, methotrexate, pemetrexed and topotecan) will be screened if sufficient organoids are grown from the biopsy samples within the screening period.
Physicians will be informed of the results, and choice of chemotherapy will be based on an IRS score of 70% or above. If more than 1 candidate drug with IRS of 70% or above is identified, the physician will exercise his/her discretion to select the most suitable drug based on patient’s comorbidities and organ function.
Project description:Variable responses to platinum chemotherapy and the development of resistant disease drive high mortality in high-grade serous ovarian cancer (HGSOC). Here, we developed the organoid drug resistance assay (ODR-test) to quantify the patient-specific efficacy of platinum treatment and generated post-platinum lines as an in vitro model of platinum-resistant ovarian cancer (PROC). We observed a gradual increase in resistance, mirroring the clinical outcomes of patients. Transcriptional and proteomic analyses of post-platinum phenotypes revealed changes in cell adhesion and differentiation as adaptive mechanisms irrespective of the basal resistance level. We found that Keratin 17 (KRT17) is a mediator of developing resistance and validated its function by CRISPR/Cas9 and overexpression. Additionally, we determined KRT17 (K-score) as a significant negative prognostic factor for survival based on analysis of a tumor microarray (N=377) of patients with advanced HGSOC. In organoids, increased KRT17 levels enhanced sensitivity to PI3K/Akt inhibitors Alpelisib and Afuresertib, highlighting the potential of KRT17 as a stratification biomarker for targeted therapies. These findings quantify patient-specific platinum response, identify PROC via KRT17 as a biomarker, and propose alternative targeted therapy for this challenging group of patients.
Project description:aCGH of human melanoma cell lines comparing parental (drug sensitve) vs isogenic drug resistant-derived subline Two condition experiment: two BRAF-V600E mutant cell lines (drug sensitive - parental baseline) vs two derived sublines after chronic exposure to the MEK inhibitor trametinib (drug resistant) are compared