Protocol for 3D drug sensitivity and resistance testing of patient-derived cancer cells in 384-well plates

Publisher Copyright: © 2022Establishment of drug testing of patient-derived cancer cells (PDCs) in physiologically relevant 3-dimensional (3D) culture is central for drug discovery and cancer research, as well as for functional precision medicine. Here, we describe the detailed protocol allowing the 3D drug testing of PDCs - or any type of cells of interest - in Matrigel in 384-well plate format using automation. We also provide an alternative protocol, which does not require supporting matrices. The cancer tissue is obtained directly from clinics (after surgery or biopsy) and processed into single cell suspension. Systematic drug sensitivity and resistance testing (DSRT) is carried out on the PDCs directly after cancer cell isolation from tissue or on cells expanded for a few passages. In the 3D-DSRT assay, the PDCs are plated in 384-well plates in Matrigel, grown as spheroids, and treated with compounds of interest for 72 h. The cell viability is directly measured using a luminescence-based assay. Alternatively, prior to the cell viability measurement, drug-treated cells can be directly subjected to automated high-content bright field imaging or stained for fluorescence (live) cell microscopy for further image analysis. This is followed by the quality control and data analysis. The 3D-DSRT can be performed within a 1-3-week timeframe of the clinical sampling of cancer tissue, depending on the amount of the obtained tissue, growth rate of cancer cells, and the number of drugs being tested. The 3D-DSRT method can be flexibly modified, e.g., to be carried out with or without supporting matrices with U-bottom 384-well plates when appropriate for the PDCs or other cell models used.Peer reviewe

Genome-wide siRNA screening reveals several host receptors for the binding of human gut commensal Bifidobacterium bifidum

Bifidobacterium spp. are abundant gut commensals, especially in breast-fed infants. Bifidobacteria are associated with many health-promoting effects including maintenance of epithelial barrier and integrity as well as immunomodulation. However, the protective mechanisms of bifidobacteria on intestinal epithelium at molecular level are poorly understood. In this study, we developed a high-throughput in vitro screening assay to explore binding receptors of intestinal epithelial cells for Bifidobacterium bifidum. Short interfering RNAs (siRNA) were used to silence expression of each gene in the Caco-2 cell line one by one. The screen yielded four cell surface proteins, SERPINB3, LGICZ1, PKD1 and PAQR6, which were identified as potential receptors as the siRNA knock-down of their expression decreased adhesion of B. bifidum to the cell line repeatedly during the three rounds of siRNA screening. Furthermore, blocking of these host cell proteins by specific antibodies decreased the binding of B. bifidum significantly to Caco-2 and HT29 cell lines. All these molecules are located on the surface of epithelial cells and three out of four, SERPINB3, PKD1 and PAQR6, are involved in the regulation of cellular processes related to proliferation, differentiation and apoptosis as well as inflammation and immunity. Our results provide leads to the first steps in the mechanistic cascade of B. bifidum-host interactions leading to regulatory effects in the epithelium and may partly explain how this commensal bacterium is able to promote intestinal homeostasis.Peer reviewe

Functional temozolomide sensitivity testing of patient-specific glioblastoma stem cell cultures is predictive of clinical outcome

Serum-free culturing of patient-derived glioblastoma biopsies enrich for glioblastoma stem cells (GSCs) and is recognized as a disease-relevant model system in glioblastoma (GBM). We hypothesized that the temozolomide (TMZ) drug sensitivity of patient-derived GSC cultures correlates to clinical sensitivity patterns and has clinical predictive value in a cohort of GBM patients. To this aim, we established 51 individual GSC cultures from surgical biopsies from both treatment-naive primary and pretreated recurrent GBM patients. The cultures were evaluated for sensitivity to TMZ over a dosing range achievable in normal clinical practice. Drug efficacy was quantified by the drug sensitivity score. MGMT-methylation status was investigated by pyrosequencing. Correlative, contin-gency, and survival analyses were performed for associations between experimental and clinical data. We found a heterogeneous response to temozolomide in the GSC culture cohort. There were significant differences in the sensitivity to TMZ between the newly diagnosed and the TMZ-treated recurrent disease (pPeer reviewe

Functional diagnostics using fresh uncultured lung tumor cells to guide personalized treatments

Functional profiling of a cancer patient's tumor cells holds potential to tailor personalized cancer treatment. Here, we report the utility of fresh uncultured tumor-derived EpCAM(+) epithelial cells (FUTCs) for ex vivo drug response interrogation. Analysis of murine Kras mutant FUTCs demonstrates pharmacological and adaptive signaling profiles comparable to subtype-matched cultured cells. By applying FUTC profiling on non-small cell lung cancer patient samples, we report robust drug-response data in 19 of 20 cases, with cells exhibiting targeted drug sensitivities corresponding to their oncogenic drivers. In one of these cases, an EGFR mutant lung adenocarcinoma patient refractory to osimertinib, FUTC profiling is used to guide compassionate treatment. FUTC profiling identifies selective sensitivity to disulfiram and the combination of carboplatin plus etoposide, and the patient receives substantial clinical benefit from treatment with these agents. We conclude that FUTC profiling provides a robust, rapid, and actionable assessment of personalized cancer treatment options.Peer reviewe

Ex Vivo Drug Testing in Patient-derived Papillary Renal Cancer Cells Reveals EGFR and the BCL2 Family as Therapeutic Targets

BACKGROUND Immune checkpoint inhibitors and antiangiogenic agents are used for first-line treatment of advanced papillary renal cell carcinoma (pRCC) but pRCC response rates to these therapies are low. OBJECTIVE To generate and characterise a functional ex vivo model to identify novel treatment options in advanced pRCC. DESIGN, SETTING, AND PARTICIPANTS We established patient-derived cell cultures (PDCs) from seven pRCC samples from patients and characterised them via genomic analysis and drug profiling. OUTCOME MEASUREMENTS AND STATISTICAL ANALYSIS Comprehensive molecular characterisation in terms of copy number analysis and whole-exome sequencing confirmed the concordance of pRCC PDCs with the original tumours. We evaluated their sensitivity to novel drugs by generating drug scores for each PDC. RESULTS AND LIMITATIONS PDCs confirmed pRCC-specific copy number variations such as gains in chromosomes 7, 16, and 17. Whole-exome sequencing revealed that PDCs retained mutations in pRCC-specific driver genes. We performed drug screening with 526 novel and oncological compounds. Whereas exposure to conventional drugs showed low efficacy, the results highlighted EGFR and BCL2 family inhibition as the most effective targets in our pRCC PDCs. CONCLUSIONS High-throughput drug testing on newly established pRCC PDCs revealed that inhibition of EGFR and BCL2 family members could be a therapeutic strategy in pRCC. PATIENT SUMMARY We used a new approach to generate patient-derived cells from a specific type of kidney cancer. We showed that these cells have the same genetic background as the original tumour and can be used as models to study novel treatment options for this type of kidney cancer

High-throughput compound screening identifies navitoclax combined with irradiation as a candidate therapy for HPV-negative head and neck squamous cell carcinoma

Conventional chemotherapeutic agents are nonselective, often resulting in severe side effects and the development of resistance. Therefore, new molecular-targeted therapies are urgently needed to be integrated into existing treatment regimens. Here, we performed a high-throughput compound screen to identify a synergistic interaction between ionizing radiation and 396 anticancer compounds. The assay was run using five human papillomavirus (HPV)-negative head and neck squamous cell carcinoma (HNSCC) cell lines cultured on the human tumor-derived matrix Myogel. Our screen identified several compounds with strong synergistic and antagonistic effects, which we further investigated using multiple irradiation doses. Navitoclax, which emerged as the most promising radiosensitizer, exhibited synergy with irradiation regardless of the p53 mutation status in all 13 HNSCC cell lines. We performed a live cell apoptosis assay for two representative HNSCC cell lines to examine the effects of navitoclax and irradiation. As a single agent, navitoclax reduced proliferation and induced apoptosis in a dose-dependent manner, whereas the navitoclax-irradiation combination arrested cell cycle progression and resulted in substantially elevated apoptosis. Overall, we demonstrated that combining navitoclax with irradiation resulted in synergistic in vitro antitumor effects in HNSCC cell lines, possibly indicating the therapeutic potential for HNSCC patients.Peer reviewe

Effects of Wee1 inhibitor adavosertib on patient-derived high-grade serous ovarian cancer cells are multiple and independent of homologous recombination status

ObjectiveA major challenge in the treatment of platinum-resistant high-grade serous ovarian cancer (HGSOC) is lack of effective therapies. Much of ongoing research on drug candidates relies on HGSOC cell lines that are poorly documented. The goal of this study was to screen for effective, state-of-the-art drug candidates using primary HGSOC cells. In addition, our aim was to dissect the inhibitory activities of Wee1 inhibitor adavosertib on primary and conventional HGSOC cell lines. MethodsA comprehensive drug sensitivity and resistance testing (DSRT) on 306 drug compounds was performed on three patient-derived genetically unique HGSOC cell lines and two commonly used ovarian cancer cell lines. The effect of adavosertib on the cell lines was tested in several assays, including cell-cycle analysis, apoptosis induction, proliferation, wound healing, DNA damage, and effect on nuclear integrity. ResultsSeveral compounds exerted cytotoxic activity toward all cell lines, when tested in both adherent and spheroid conditions. In further cytotoxicity tests, adavosertib exerted the most consistent cytotoxic activity. Adavosertib affected cell-cycle control in patient-derived and conventional HGSOC cells, inducing G2/M accumulation and reducing cyclin B1 levels. It induced apoptosis and inhibited proliferation and migration in all cell lines. Furthermore, the DNA damage marker gamma H2AX and the number of abnormal cell nuclei were clearly increased following adavosertib treatment. Based on the homologous recombination (HR) signature and functional HR assays of the cell lines, the effects of adavosertib were independent of the cells' HR status. ConclusionOur study indicates that Wee1 inhibitor adavosertib affects several critical functions related to proliferation, cell cycle and division, apoptosis, and invasion. Importantly, the effects are consistent in all tested cell lines, including primary HGSOC cells, and independent of the HR status of the cells. Wee1 inhibition may thus provide treatment opportunities especially for patients, whose cancer has acquired resistance to platinum-based chemotherapy or PARP inhibitors.Peer reviewe

KIT pathway upregulation predicts dasatinib efficacy in acute myeloid leukemia

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Human Tumor–Derived Matrix Improves the Predictability of Head and Neck Cancer Drug Testing

In vitro cancer drug testing carries a low predictive value. We developed the human leiomyoma–derived matrix “Myogel” to better mimic the human tumor microenvironment (TME). We hypothesized that Myogel could provide an appropriate microenvironment for cancer cells, thereby allowing more in vivo–relevant drug testing. We screened 19 anticancer compounds, targeting the epidermal growth factor receptor (EGFR), MEK, and PI3K/mTOR on 12 head and neck squamous cell carcinoma (HNSCC) cell lines cultured on plastic, mouse sarcoma–derived Matrigel (MSDM), and Myogel. We applied a high-throughput drug screening assay under five different culturing conditions: cells in two-dimensional (2D) plastic wells and on top or embedded in Matrigel or Myogel. We then compared the efficacy of the anticancer compounds to the response rates of 19 HNSCC monotherapy clinical trials. Cancer cells on top of Myogel responded less to EGFR and MEK inhibitors compared to cells cultured on plastic or Matrigel. However, we found a similar response to the PI3K/mTOR inhibitors under all culturing conditions. Cells grown on Myogel more closely resembled the response rates reported in EGFR-inhibitor monotherapy clinical trials. Our findings suggest that a human tumor matrix improves the predictability of in vitro anticancer drug testing compared to current 2D and MSDM methods

Human Tumor–Derived Matrix Improves the Predictability of Head and Neck Cancer Drug Testing

In vitro cancer drug testing carries a low predictive value. We developed the human leiomyoma–derived matrix “Myogel” to better mimic the human tumor microenvironment (TME). We hypothesized that Myogel could provide an appropriate microenvironment for cancer cells, thereby allowing more in vivo–relevant drug testing. We screened 19 anticancer compounds, targeting the epidermal growth factor receptor (EGFR), MEK, and PI3K/mTOR on 12 head and neck squamous cell carcinoma (HNSCC) cell lines cultured on plastic, mouse sarcoma–derived Matrigel (MSDM), and Myogel. We applied a high-throughput drug screening assay under five different culturing conditions: cells in two-dimensional (2D) plastic wells and on top or embedded in Matrigel or Myogel. We then compared the efficacy of the anticancer compounds to the response rates of 19 HNSCC monotherapy clinical trials. Cancer cells on top of Myogel responded less to EGFR and MEK inhibitors compared to cells cultured on plastic or Matrigel. However, we found a similar response to the PI3K/mTOR inhibitors under all culturing conditions. Cells grown on Myogel more closely resembled the response rates reported in EGFR-inhibitor monotherapy clinical trials. Our findings suggest that a human tumor matrix improves the predictability of in vitro anticancer drug testing compared to current 2D and MSDM methods