Standardization of esophageal adenocarcinoma in vitro model and its applicability for model drug testing.

FLO-1 cell line represents an important tool in esophageal adenocarcinoma (EAC) research as a verified and authentic cell line to study the disease pathophysiology and antitumor drug screenings. Since in vitro characteristics of cells depend on the microenvironment and culturing conditions, we performed a thorough characterization of the FLO-1 cell line under different culturing conditions with the aim of (1) examining the effect of serum-free growth medium and air-liquid interface (A-L) culturing, which better reflect physiological conditions in vivo and (2) investigating the differentiation potential of FLO-1 cells to mimic the properties of the in vivo esophageal epithelium. Our study shows that the composition of the media influenced the morphological, ultrastructural and molecular characteristics of FLO-1 cells, such as the expression of junctional proteins. Importantly, FLO-1 cells formed spheres at the A-L interface, recapitulating key elements of tumors in the esophageal tube, i.e., direct contact with the gas phase and three-dimensional architecture. On the other hand, FLO-1 models exhibited high permeability to model drugs and zero permeability markers, and low transepithelial resistance, and therefore poorly mimicked normal esophageal epithelium. In conclusion, the identified effect of culture conditions on the characteristics of FLO-1 cells should be considered for standardization, data reproducibility and validity of the in vitro EAC model. Moreover, the sphere-forming ability of FLO-1 cells at the A-L interface should be considered in EAC tumor biology and anticancer drug studies as a reliable and straightforward model with the potential to increase the predictive efficiency of the current in vitro approaches.

Unbiased High-Throughput Drug Combination Pilot Screening Identifies Synergistic Drug Combinations Effective against Patient-Derived and Drug-Resistant Melanoma Cell Lines.

We describe the development, optimization, and validation of 384-well growth inhibition assays for six patient-derived melanoma cell lines (PDMCLs), three wild type (WT) for BRAF and three with V600E-BRAF mutations. We conducted a pilot drug combination (DC) high-throughput screening (HTS) of 45 pairwise 4×4 DC matrices prepared from 10 drugs in the PDMCL assays: two B-Raf inhibitors (BRAFi), a MEK inhibitor (MEKi), and a methylation agent approved for melanoma; cytotoxic topoisomerase II and DNA methyltransferase chemotherapies; and drugs targeting the base excision DNA repair enzyme APE1 (apurinic/apyrimidinic endonuclease-1/redox effector factor-1), SRC family tyrosine kinases, the heat shock protein 90 (HSP90) molecular chaperone, and histone deacetylases.Pairwise DCs between dasatinib and three drugs approved for melanoma therapy-dabrafenib, vemurafenib, or trametinib-were flagged as synergistic in PDMCLs. Exposure to fixed DC ratios of the SRC inhibitor dasatinib with the BRAFis or MEKis interacted synergistically to increase PDMCL sensitivity to growth inhibition and enhance cytotoxicity independently of PDMCL BRAF status. These DCs synergistically inhibited the growth of mouse melanoma cell lines that either were dabrafenib-sensitive or had acquired resistance to dabrafenib with cross resistance to vemurafenib, trametinib, and dasatinib. Dasatinib DCs with dabrafenib, vemurafenib, or trametinib activated apoptosis and increased cell death in melanoma cells independently of their BRAF status or their drug resistance phenotypes. These preclinical in vitro studies provide a data-driven rationale for the further investigation of DCs between dasatinib and BRAFis or MEKis as candidates for melanoma combination therapies with the potential to improve outcomes and/or prevent or delay the emergence of disease resistance.

Ex vivo cultures and drug testing of primary acute myeloid leukemia samples: Current techniques and implications for experimental design and outcome.

New treatment options of acute myeloid leukemia (AML) are rapidly emerging. Pre-clinical models such as ex vivo cultures are extensively used towards the development of novel drugs and to study synergistic drug combinations, as well as to discover biomarkers for both drug response and anti-cancer drug resistance. Although these approaches empower efficient investigation of multiple drugs in a multitude of primary AML samples, their translational value and reproducibility are hampered by the lack of standardized methodologies and by culture system-specific behavior of AML cells and chemotherapeutic drugs. Moreover, distinct research questions require specific methods which rely on specific technical knowledge and skills. To address these aspects, we herein review commonly used culture techniques in light of diverse research questions. In addition, culture-dependent effects on drug resistance towards commonly used drugs in the treatment of AML are summarized including several pitfalls that may arise because of culture technique artifacts. The primary aim of the current review is to provide practical guidelines for ex vivo primary AML culture experimental design.

Discovering the hidden messages within cell trajectories using a deep learning approach for in vitro evaluation of cancer drug treatments.

We describe a novel method to achieve a universal, massive, and fully automated analysis of cell motility behaviours, starting from time-lapse microscopy images. The approach was inspired by the recent successes in application of machine learning for style recognition in paintings and artistic style transfer. The originality of the method relies i) on the generation of atlas from the collection of single-cell trajectories in order to visually encode the multiple descriptors of cell motility, and ii) on the application of pre-trained Deep Learning Convolutional Neural Network architecture in order to extract relevant features to be used for classification tasks from this visual atlas. Validation tests were conducted on two different cell motility scenarios: 1) a 3D biomimetic gels of immune cells, co-cultured with breast cancer cells in organ-on-chip devices, upon treatment with an immunotherapy drug; 2) Petri dishes of clustered prostate cancer cells, upon treatment with a chemotherapy drug. For each scenario, single-cell trajectories are very accurately classified according to the presence or not of the drugs. This original approach demonstrates the existence of universal features in cell motility (a so called "motility style") which are identified by the DL approach in the rationale of discovering the unknown message in cell trajectories.

Optimization of cell viability assays to improve replicability and reproducibility of cancer drug sensitivity screens.

Cancer drug development has been riddled with high attrition rates, in part, due to poor reproducibility of preclinical models for drug discovery. Poor experimental design and lack of scientific transparency may cause experimental biases that in turn affect data quality, robustness and reproducibility. Here, we pinpoint sources of experimental variability in conventional 2D cell-based cancer drug screens to determine the effect of confounders on cell viability for MCF7 and HCC38 breast cancer cell lines treated with platinum agents (cisplatin and carboplatin) and a proteasome inhibitor (bortezomib). Variance component analysis demonstrated that variations in cell viability were primarily associated with the choice of pharmaceutical drug and cell line, and less likely to be due to the type of growth medium or assay incubation time. Furthermore, careful consideration should be given to different methods of storing diluted pharmaceutical drugs and use of DMSO controls due to the potential risk of evaporation and the subsequent effect on dose-response curves. Optimization of experimental parameters not only improved data quality substantially but also resulted in reproducible results for bortezomib- and cisplatin-treated HCC38, MCF7, MCF-10A, and MDA-MB-436 cells. Taken together, these findings indicate that replicability (the same analyst re-performs the same experiment multiple times) and reproducibility (different analysts perform the same experiment using different experimental conditions) for cell-based drug screens can be improved by identifying potential confounders and subsequent optimization of experimental parameters for each cell line.

Summary of a workshop on preclinical and translational safety assessment of CD3 bispecifics.

Currently, there is a multitude of CD3 bispecifics with different molecular designs and binding properties in preclinical and clinical development for the treatment of liquid or solid tumors. The key safety concerns with CD3 bispecifics are excessive release of cytokines, which may translate to potentially life-threating cytokine release syndrome (CRS), target organ toxicity due to redirection of T-cells to normal tissues expressing the tumor-associated antigen (TAA) (off-tumor/on-target cytotoxicity), and, in some instances, neurotoxicity. Another key challenge is to arrive at a safe clinical starting dose and an efficient escalating strategy that allows patients in early dose cohorts the potential for clinical benefit in Phase 1 trials. To expand the therapeutic index and bring more treatment options to patients, there are intense efforts to overcome these challenges through improvements in molecular design, preclinical safety assessment strategies, and clinical management practices. A recent workshop at the U.S. Food and Drug Administration (FDA) with industry, academic, and regulatory agency representation was held to discuss the challenges and explore where such improvements to the development of CD3 bispecifics can be implemented. Here, the content of the presentations and the discussion that occurred during this workshop are summarized.

A normalized drug response metric improves accuracy and consistency of anticancer drug sensitivity quantification in cell-based screening.

Accurate quantification of drug effects is crucial for identifying pharmaceutically actionable cancer vulnerabilities. Current cell viability-based measurements often lead to biased response estimates due to varying growth rates and experimental artifacts that explain part of the inconsistency in high-throughput screening results. We developed an improved drug scoring model, normalized drug response (NDR), which makes use of both positive and negative control conditions to account for differences in cell growth rates, and experimental noise to better characterize drug-induced effects. We demonstrate an improved consistency and accuracy of NDR compared to existing metrics in assessing drug responses of cancer cells in various culture models and experimental setups. Notably, NDR reliably captures both toxicity and viability responses, and differentiates a wider spectrum of drug behavior, including lethal, growth-inhibitory and growth-stimulatory modes, based on a single viability readout. The method will therefore substantially reduce the time and resources required in cell-based drug sensitivity screening.

The culture conditions and outputs from breast cancer cell line in vitro experiments.

One of the major cancer types that have gained significant importance globally is the breast cancer due to its socio-economic impact. Breast cancer research is an area of considerable importance and several types of material are available for research applications. These include cancer cell lines which can be utilized in several ways. Cell lines are convenient to use and recently about 84 human breast cancer cell lines were classified by molecular sub-typing. These cells lines come under five major molecular subtypes namely the luminal A and B, HER-2+, triple- A and B subtypes. These cell lines have been well characterized and were utilized for understanding various aspects of breast cancers. Also, apart from providing an understanding of the molecular mechanisms associated with breast cancers, these cell lines have contributed significantly to areas such as drug testing. We present in this review the features of these cell lines, the studies conducted using them and the outcome of such studies. Also, the details about the culture conditions and study outcomes of the cell lines grown in 3-dimensional (3D) systems are presented.

Biological Characterization of SB3, a Trastuzumab Biosimilar, and the Influence of Changes in Reference Product Characteristics on the Similarity Assessment.

BACKGROUND: SB3 has been developed as a trastuzumab biosimilar, a therapeutic monoclonal antibody targeted to human epidermal growth factor receptor 2 (HER2), and approved by the European Commission and United States (US) Food and Drug Administration (FDA). During the developmental period of a biosimilar, setting an appropriate quality target is critical for assessing the similarity of the biosimilar product to the reference product. A stepwise approach should be taken to assessing similarity, beginning with extensive characterization of the reference product to establish the quality target. OBJECTIVE: In this study, we evaluated the similarity of SB3 to the reference product and the impact of changes in the biological profile of the reference product on similarity assessment. METHODS: Analytical similarity was assessed with defined test procedures in terms of critical quality attributes (CQAs) that could affect efficacy, potency, and safety, as well as for the non-CQAs that are related to process consistency. The quality target was established using up to 154 lots of European Union (EU)- and US-sourced Herceptin® (reference product), analyzed during the developmental period of SB3. RESULTS: Trends of the EU- and US-sourced reference product showed that the biological profile exhibited two marked changes for Fc-related attributes, and then recovered to pre-change quality level. Since the similarity range set by pre-change lots was considered most relevant, the changed lots were excluded from establishing the similarity range, which resulted in tightened acceptance criteria. As shown in the results of similarity assessment using the stringent quality target ranges, SB3 exhibits highly similar functional activities compared to the reference product in terms of both CQAs and non-CQAs. CONCLUSION: SB3 has been developed as a trastuzumab biosimilar approved in the EU and USA, and its manufacturing process is deemed to be robust and well-controlled within stringent quality target ranges.

New weapons to penetrate the armor: Novel reagents and assays developed at the NCI RAS Initiative to enable discovery of RAS therapeutics.

Development of therapeutic strategies against RAS-driven cancers has been challenging due in part to a lack of understanding of the biology of the system and the ability to design appropriate assays and reagents for targeted drug discovery efforts. Recent developments in the field have opened up new avenues for exploration both through advances in the number and quality of reagents as well as the introduction of novel biochemical and cell-based assay technologies which can be used for high-throughput screening of compound libraries. The reagents and assays developed at the NCI RAS Initiative offer a suite of new weapons that could potentially be used to enable the next generation of RAS drug discovery efforts with the hope of finding novel therapeutics for a target once deemed undruggable.

Screening drug effects in patient-derived cancer cells links organoid responses to genome alterations.

Cancer drug screening in patient-derived cells holds great promise for personalized oncology and drug discovery but lacks standardization. Whether cells are cultured as conventional monolayer or advanced, matrix-dependent organoid cultures influences drug effects and thereby drug selection and clinical success. To precisely compare drug profiles in differently cultured primary cells, we developed DeathPro, an automated microscopy-based assay to resolve drug-induced cell death and proliferation inhibition. Using DeathPro, we screened cells from ovarian cancer patients in monolayer or organoid culture with clinically relevant drugs. Drug-induced growth arrest and efficacy of cytostatic drugs differed between the two culture systems. Interestingly, drug effects in organoids were more diverse and had lower therapeutic potential. Genomic analysis revealed novel links between drug sensitivity and DNA repair deficiency in organoids that were undetectable in monolayers. Thus, our results highlight the dependency of cytostatic drugs and pharmacogenomic associations on culture systems, and guide culture selection for drug tests.

Culture methods of diffuse intrinsic pontine glioma cells determine response to targeted therapies.

Diffuse intrinsic pontine glioma (DIPG) is an aggressive type of brainstem cancer occurring mainly in children, for which there currently is no effective therapy. Current efforts to develop novel therapeutics for this tumor make use of primary cultures of DIPG cells, maintained either as adherent monolayer in serum containing medium, or as neurospheres in serum-free medium. In this manuscript, we demonstrate that the response of DIPG cells to targeted therapies in vitro is mainly determined by the culture conditions. We show that particular culture conditions induce the activation of different receptor tyrosine kinases and signal transduction pathways, as well as major changes in gene expression profiles of DIPG cells in culture. These differences correlate strongly with the observed discrepancies in response to targeted therapies of DIPG cells cultured as either adherent monolayers or neurospheres. With this research, we provide an argument for the concurrent use of both culture conditions to avoid false positive and false negative results due to the chosen method.

Reproducible pharmacogenomic profiling of cancer cell line panels.

The use of large-scale genomic and drug response screening of cancer cell lines depends crucially on the reproducibility of results. Here we consider two previously published screens, plus a later critique of these studies. Using independent data, we show that consistency is achievable, and provide a systematic description of the best laboratory and analysis practices for future studies.

Recommendations for Benchmarking Preclinical Studies of Nanomedicines.

Nanoparticle-based delivery systems provide new opportunities to overcome the limitations associated with traditional small-molecule drug therapy for cancer and to achieve both therapeutic and diagnostic functions in the same platform. Preclinical trials are generally designed to assess therapeutic potential and not to optimize the design of the delivery platform. Consequently, progress in developing design rules for cancer nanomedicines has been slow, hindering progress in the field. Despite the large number of preclinical trials, several factors restrict comparison and benchmarking of different platforms, including variability in experimental design, reporting of results, and the lack of quantitative data. To solve this problem, we review the variables involved in the design of preclinical trials and propose a protocol for benchmarking that we recommend be included in in vivo preclinical studies of drug-delivery platforms for cancer therapy. This strategy will contribute to building the scientific knowledge base that enables development of design rules and accelerates the translation of new technologies.

Benchmarking of gastric cancer sensitivity to anti-cancer drugs ex vivo as a basis for drug selection in systemic and intraperitoneal therapy.

BACKGROUND: The choice of drugs for treatment of advanced gastric cancer (GC) is empirical. The purpose of the current study was to benchmark ex vivo the sensitivity of GC tumor cells from patients to standard cytotoxic and some newly introduced targeted drugs (TDs), as a basis for drug selection in the treatment of GC. METHODS: Tumor cell samples from patients with GC were analyzed for sensitivity to 5-fluorouracil, cisplatin, oxaliplatin, irinotecan, mitomycin C, doxorubicin and docetaxel as well as for the targeted drugs bortezomib, sorafenib, sunitinib and rapamycin using a short-term in vitro assay based on retention of viable tumor cells of fluorescent fluorescein. Samples of normal mononuclear cells, chronic lymphocytic leukemia, ovarian cancer and colorectal cancer were included for comparison. RESULTS: The GC samples were essentially as sensitive to the standard drugs and the TDs as those from colorectal cancer whereas the ovarian cancer samples were more sensitive. The individual GC samples varied considerably in sensitivity to increasing concentrations of the clinically used standard drugs. In GC, cisplatin was cross-resistant to oxaliplatin and 5-fluorouracil which, on the other hand, was not cross-resistant to the other cytotoxic drugs. The activity of sunitinib did not obviously correlate to that of the standard drugs. CONCLUSION: Ex vivo assessment of drug sensitivity of tumor cells from patients with GC is feasible and may provide information that could be useful for selection of drugs for treatment. Drug sensitivity varies considerably between and within individual samples arguing for individualized selection of drugs for chemotherapy.

Enhancing reproducibility in cancer drug screening: how do we move forward?

Large-scale pharmacogenomic high-throughput screening (HTS) studies hold great potential for generating robust genomic predictors of drug response. Two recent large-scale HTS studies have reported results of such screens, revealing several known and novel drug sensitivities and biomarkers. Subsequent evaluation, however, found only moderate interlaboratory concordance in the drug response phenotypes, possibly due to differences in the experimental protocols used in the two studies. This highlights the need for community-wide implementation of standardized assays for measuring drug response phenotypes so that the full potential of HTS is realized. We suggest that the path forward is to establish best practices and standardization of the critical steps in these assays through a collective effort to ensure that the data produced from large-scale screens would not only be of high intrastudy consistency, so that they could be replicated and compared successfully across multiple laboratories.

Better therapeutic trials in ovarian cancer.

The Ovarian Task Force of the Gynecologic Cancer Steering Committee convened a clinical trials planning meeting on October 28-29, 2011, with the goals to identify key tumor types, associated molecular pathways, and biomarkers for targeted drug intervention; review strategies to improve early-phase screening, therapeutic evaluation, and comparison of new agents; and optimize design of randomized trials in response to an evolving landscape of scientific, regulatory, and funding priorities. The meeting was attended by international clinical and translational investigators, pharmaceutical industry representatives, government regulators, and patient advocates. Panel discussions focused on disease types, early-phase trials, and randomized trials. A manuscript team summarized the discussions and assisted with formulating key recommendations. A more integrated and efficient approach for screening new agents using smaller selective randomized trials in specific disease-type settings was endorsed, together with collaborative funding models between industry and the evolving national clinical trials network, as well as efforts to enhance public awareness and study enrollment through advocacy.

Unbiased compound screening identifies unexpected drug sensitivities and novel treatment options for gastrointestinal stromal tumors.

Most gastrointestinal stromal tumors (GIST) are caused by oncogenic KIT or platelet-derived growth factor receptor activation, and the small molecule kinase inhibitor imatinib mesylate is an effective first-line therapy for metastatic or unresectable GIST. However, complete remissions are rare and most patients ultimately develop resistance, mostly because of secondary mutations in the driver oncogenic kinase. Hence, there is a need for novel treatment options to delay failure of primary treatment and restore tumor control in patients who progress under therapy with targeted agents. Historic data suggest that GISTs do not respond to classical chemotherapy, but systematic unbiased screening has not been performed. In screening a compound library enriched for U.S. Food and Drug Administration (FDA)-approved chemotherapeutic agents (NCI Approved Oncology Drugs Set II), we discovered that GIST cells display high sensitivity to transcriptional inhibitors and topoisomerase II inhibitors. Mechanistically, these compounds exploited the cells' dependency on continuous KIT expression and/or intrinsic DNA damage response defects, explaining their activity in GIST. Mithramycin A, an indirect inhibitor of the SP1 transcription factor, and mitoxantrone, a topoisomerase II inhibitor, exerted significant antitumor effects in mouse xenograft models of human GIST. Moreover, these compounds were active in patient-derived imatinib-resistant primary GIST cells, achieving efficacy at clinically relevant concentrations. Taken together, our findings reveal that GIST cells have an unexpectedly high and specific sensitivity to certain types of FDA-approved chemotherapeutic agents, with immediate implications for encouraging their clinical exploration.

An unappreciated challenge to oncology drug discovery: pitfalls in preclinical research.

Unfortunately, preclinical research studies frequently suffer from a lack of rigor and robustness that precludes their use as a foundation for a drug-development program. Too often they lack the characteristics that typically are expected in high-quality clinical studies, yet despite that, they are published in top-tier scientific journals. The key attributes that are missing include lack of blinding of investigators, failure to repeat experiments, lack of positive and negative controls, use of nonvalidated reagents, inappropriate use of statistical tests, and data selection (ignoring results that do not fit the hypothesis). Physicians and scientists should view preclinical findings that lack these characteristics with skepticism and should proceed very cautiously in applying such findings to the clinic.