High-throughput Combinatorial Drug Screening Research Articles

Multi-Inlet Spheroid Generator for High-Throughput Combinatorial Drug Screening Based on the Tumor Microenvironment.

Tumor spheroids are powerful tools for drug screening and understanding tumor physiology. Among spheroid formation methods, the hanging drop method is considered most suitable for high-throughput screening (HTS) of anticancer drugs because it does not require surface treatment. However, it still needs to increase the liquid-holding capacity because hanging drops often fall due to the increased pressure caused by the addition of drugs, cells, etc. Here, we report a multi-inlet spheroid generator (MSG) enabling the stable addition of liquid-containing drugs or cells into a spheroid through its side inlet. The MSG was able to load additional solutions through the side inlet without increasing the force applied to the hanging drop. The volume of the additional liquid was easily controlled by varying the diameter of the side inlet. Furthermore, the sequences of the solution injections were manipulated using multiple side inlets. The feasibility of the MSG in clinical application was demonstrated by testing the efficacy of drugs in patient-derived cancer (PDC) cells and controlling the stromal cell ratio in the tumor microenvironment (TME) containing spheroids. Our results suggest that the MSG is a versatile platform for HTS of anticancer drugs and recapitulating the TME.

A Precision Clinical-Omics Approach in Predicting Therapeutic Response to Pancreatic Cancer

Background: Pancreatic ductal adenocarcinoma is one of the most aggressive solid tumours. Despite numerous advances in surgical techniques, survival has remained largely unchanged over the last decade. Pancreatic cancer is thus considered a ‘systemic disease’ where chemotherapy plays a pivotal role in both the pre- and post-surgery setting. Tools that can predict the most effective chemotherapeutic regimens for patients are urgently needed to improve survival by optimising biological effects while minimising side effects, thereby not comprising their fitness for surgery. The aim of the study was to characterise the proteome of patient derived pancreatic cancer organoids to identify biomarkers predictive of chemotherapeutic sensitivity. Method: Using quantitative mass spectrometry we performed deep proteomic profiling of patient derived pancreatic cancer organoids with matched primary tumour tissue, and performed high-throughput combinatorial drug screening on organoid lines. Results: We demonstrated that common pancreatic cancer characteristics were conserved in organoids derived from different patients, and through comparative analysis between organoids revealed individual diversity that correlated with patient specific features. By correlating personalized organoid proteomes with their matched drug sensitivity profiles derived using high-throughput combinatorial drug screening, we identified protein biomarkers that correlated with therapeutic response to both standard of care and other novel therapeutics. Candidate biomarkers were verified by immunohistochemistry of organoids, with further validation to be performed in an interventional trial using organoids to predict second-line chemotherapy in patients with metastatic disease. Conclusions: We present the integration of deep proteomic profiling with a patient derived organoid platform for therapeutic biomarker discovery in pancreatic cancer.

Multilayer architecture microfluidic network array for combinatorial drug testing on 3D-cultured cells

In vitro testing of drug compounds on cell models during the drug development process represents an indispensable step in the initial screening process. Although drug testing on three-dimensional (3D) cultured cells may provide a more accurate prediction of drug efficacy, it is relatively costly and time-consuming to perform compared with conventional 2D cultures due to the thick z-axis of the 3D models. In this study, we have presented a microfluidic platform with integrated pneumatic valves for producing a thin-gel 3D cell culture-based combinatorial drug screening array (3D-μCDS array). The multilayer architecture and microfluidic layout has a smaller device footprint than a single-layer microfluidic channel arrangement, making it well suited to scaling up for high-throughput combinatorial drug screening on 3D cell model. We performed 8 × 8 combination drug screening experiments with the device using two anti-cancer drugs (doxorubicin and paclitaxel) on MDA-MB-231 and MCF-7 breast cancer cell lines for demonstration. Our results indicate that our 3D-μCDS array device allows the successful screening of multiple drug combinations while reducing the operation time and the number of sample/reagents required, making it an ideal tool for general combinatorial drug screening, as well as for applications using valuable tissues and clinical samples.

One-Step Generation of a Drug-Releasing Hydrogel Microarray-On-A-Chip for Large-Scale Sequential Drug Combination Screening.

Large‐scale screening of sequential drug combinations, wherein the dynamic rewiring of intracellular pathways leads to promising therapeutic effects and improvements in quality of life, is essential for personalized medicine to ensure realistic cost and time requirements and less sample consumption. However, the large‐scale screening requires expensive and complicated liquid handling systems for automation and therefore lowers the accessibility to clinicians or biologists, limiting the full potential of sequential drug combinations in clinical applications and academic investigations. Here, a miniaturized platform for high‐throughput combinatorial drug screening that is “pipetting‐free” and scalable for the screening of sequential drug combinations is presented. The platform uses parallel and bottom‐up formation of a heterogeneous drug‐releasing hydrogel microarray by self‐assembly of drug‐laden hydrogel microparticles. This approach eliminates the need for liquid handling systems and time‐consuming operation in high‐throughput large‐scale screening. In addition, the serial replacement of the drug‐releasing microarray‐on‐a‐chip facilitates different drug exchange in each and every microwell in a simple and highly parallel manner, supporting scalable implementation of multistep combinatorial screening. The proposed strategy can be applied to various forms of combinatorial drug screening with limited amounts of samples and resources, which will broaden the use of the large‐scale screening for precision medicine.

BET Inhibition-Induced GSK3β Feedback Enhances Lymphoma Vulnerability to PI3K Inhibitors

The phosphatidylinositol 3 kinase (PI3K)-glycogen synthase kinase β (GSK3β) axis plays a central role in MYC-driven lymphomagenesis, and MYC targeting with bromodomain and extraterminal protein family inhibitors (BETi) is a promising treatment strategy in lymphoma. In a high-throughput combinatorial drug screening experiment, BETi enhance the antiproliferative effects of PI3K inhibitors in a panel of diffuse large B cell lymphoma (DLBCL) and Burkitt lymphoma cell lines. BETi or MYC silencing upregulates several PI3K pathway genes and induces GSK3β S9 inhibitory phosphorylation, resulting in increased β-catenin protein abundance. Furthermore, BETi or MYC silencing increases GSK3β S9 phosphorylation levels and β-catenin protein abundance through downregulating the E2 ubiquitin conjugating enzymes UBE2C and UBE2T. In a mouse xenograft DLBCL model, BETi decrease MYC, UBE2C, and UBE2T and increase phospho-GSK3β S9 levels, enhancing the anti-proliferative effect of PI3K inhibitors. Our study reveals prosurvival feedbacks induced by BETi involving GSK3β regulation, providing a mechanistic rationale for combination strategies.

MYC-Dependent PI3K and MCL-1 Feedbacks Attenuate BET Inhibitors Activity in Diffuse Large B-Cell Lymphoma

MYC overexpression is a poor prognostic predictor in Diffuse Large B-Cell Lymphoma (DLBCL). MYC-targeting with bromodomain and extraterminal protein family (BET) inhibitors is a promising strategy for the treatment of MYC-driven cancers, including lymphomas. However, preclinical and emerging data from early clinical trials demonstrated a modest antiproliferative activity in vitro and in vivo. We hypothesized that BET inhibition may induce feedback survival mechanisms preventing or attenuating cell death that could be exploited for designing future, more effective, combination strategies. In a high-throughput combinatorial drug screening experiment, we found that phosphatidylinositol 3-kinase (PI3K) pathway inhibitors enhanced the antiproliferative effects of BET inhibitors (JQ1, I-BET 151, CPI-203) with a strong class effect. JQ1 upregulated the mRNA expression of several upstream components of the PI3K pathway, including PIK3CA, PIK3R1, PDK1 in a large panel of DLBCL and Burkitt lymphoma cell lines. These effects translated in increased pathway activation as demonstrated by increased levels of the phosphorylated forms of downstream targets GSK3α/β, TSC2, P70S6K, and by increased concentrations of chemokines known to be regulated by PI3K in cell culture supernatants (CCL3 and CCL4). This effect was reversed by submicromolar doses of the PI3K inhibitor BKM-120. MYC silencing recapitulated the effects of BET inhibitors on PI3K pathway gene expression, activation and chemokine secretion. These data indicate that BET inhibition induces PI3K activation by a MYC-dependent feedback. We also observed transcriptional upregulation of the antiapoptotic gene Myeloid Leukemia 1 (MCL-1) following BET inhibition or MYC depletion, suggesting a second MYC-dependent mechanism. RNAi-mediated MCL-1 silencing or co-treatment with a small molecule MCL-1 inhibitor (UMI-77) enhanced the effects of BET inhibitors in DLBCL cell lines by inducing apoptosis. Using SILAC-based quantitative mass spectrometry, we found that BET inhibitors at submicromolar doses downregulated several E2 ubiquitin conjugating enzymes including UBE2C. RNAi mediated UBE2C knockdown induced MCL-1 upregulation in DLBCL cells. The enhanced in vitro effect of combining BETi and PI3Ki was reproduced in TMD8 mouse xenografts. To our knowledge, this is the first study demonstrating MYC-dependent regulation of the PI3K pathway, MCL-1 and the ubiquitin system upon BET inhibition. Our study revealed previously unknown mechanisms of action of BET inhibitors uncovering novel MYC-dependent survival feedback loops, and providing a framework for future combination strategies. DisclosuresZelenetz:Gilead Sciences: Research Funding.

A novel hanging spherical drop system for the generation of cellular spheroids and high throughput combinatorial drug screening.

We propose a novel hanging spherical drop system for anchoring arrays of droplets of cell suspension based on the use of biomimetic superhydrophobic flat substrates, with controlled positional adhesion and minimum contact with a solid substrate. By facing down the platform, it was possible to generate independent spheroid bodies in a high throughput manner, in order to mimic in vivo tumour models on the lab-on-chip scale. To validate this system for drug screening purposes, the toxicity of the anti-cancer drug doxorubicin in cell spheroids was tested and compared to cells in 2D culture. The advantages presented by this platform, such as feasibility of the system and the ability to control the size uniformity of the spheroid, emphasize its potential to be used as a new low cost toolbox for high-throughput drug screening and in cell or tissue engineering.

Abstract 695: p70S6 kinase is a critical node that integrates HER-family and PI3 kinase signaling

Abstract Targeted cancer therapies often induce compensatory adaptive responses that blunt drug effectiveness, resulting in therapeutic resistance - intrinsic or acquired. Adaptive responses are characteristic of the complex nature of the cancer cell signaling network. Within this network, nodes of convergence between signaling modules are critical mediators of adaptive responses to targeted drugs. The critical nodes in an oncogenically-activated signaling network represent potential therapeutic vulnerabilities because their inhibition could result in collapse of the network and hence enhanced cytotoxicity. We have previously used high-throughput combinatorial drug screening to empirically identify adaptive survival responses to targeted therapies. We found that HER-family and PI3K represented compensatory signaling pathways, and combination therapy caused synergistic cytotoxicity in cases where inhibition of neither target was effective as a monotherapy. RPPA analysis identified ribosomal protein S6 as being synergistically down-regulated upon HER-family and PI3K co-inhibition. Expression of a constitutively active construct of the upstream activator of S6, p70S6K, was protective against apoptosis induced by combined HER-family and PI3K inhibition. Direct inhibition of p70S6K using small molecule inhibitors phenocopied the growth inhibition and apoptosis caused by HER-family and PI3K inhibition. These data implicate p70S6K as a critical node and druggable target in the HER-family / PI3K signaling network. The ability of direct inhibitors of p70S6K to phenocopy the co-inhibition of upstream signaling indicates that identification and targeting of critical nodes may be a way to overcome adaptive resistance to targeted therapies. Citation Format: Mark J. Axelrod, Rolando E. Mendez, Daniel G. Gioeli, Mark J. Jameson, Michael J. Weber. p70S6 kinase is a critical node that integrates HER-family and PI3 kinase signaling. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 695. doi:10.1158/1538-7445.AM2014-695

P70S6 kinase is a critical node that integrates HER-family and PI3 kinase signaling networks

Therapies targeting oncogenic drivers rapidly induce compensatory adaptive responses that blunt drug effectiveness, contributing to therapeutic resistance. Adaptive responses are characteristic of robust cell signaling networks, and thus there is increasing interest in drug combinations that co-target the driver and the adaptive response. An alternative approach to co-inhibiting oncogenic and adaptive targets is to identify a critical node where the activities of these targets converge. Nodes of convergence between signaling modules represent potential therapeutic vulnerabilities because their inhibition could result in the collapse of the network, leading to enhanced cytotoxicity. In this report we demonstrate that p70S6 kinase (p70S6K) can function as a critical node linking HER-family and phosphoinositide-3-kinase (PI3K) pathway signaling. We used high-throughput combinatorial drug screening to identify adaptive survival responses to targeted therapies, and found that HER-family and PI3K represented compensatory signaling pathways. Co-targeting these pathways with drug combinations caused synergistic cytotoxicity in cases where inhibition of neither target was effective as a monotherapy. We utilized Reverse Phase Protein Arrays and determined that phosphorylation of ribosomal protein S6 was synergistically down-regulated upon HER-family and PI3K/mammalian target of rapamycin (mTOR) co-inhibition. Expression of constitutively active p70S6K protected against apoptosis induced by combined HER-family and PI3K/mTOR inhibition. Direct inhibition of p70S6K with small molecule inhibitors phenocopied HER-family and PI3K/mTOR co-inhibition. These data implicate p70S6K as a critical node in the HER-family/PI3K signaling network. The ability of direct inhibitors of p70S6K to phenocopy co-inhibition of two upstream signaling targets indicates that identification and targeting of critical nodes can overcome adaptive resistance to targeted therapies.

Abstract B29: Combinatorial screens with targeted inhibitors reveal diverse compensatory responses and mechanisms of adaptive resistance to therapy

Abstract The effectiveness of targeted inhibition of cell signaling can be blunted by compensatory signaling, which generates adaptive resistance mechanisms and reduces therapeutic responses. We have performed high-throughput combinatorial drug screening as a discovery tool to identify compensatory pathways that confer resistance to the cytotoxic effects of targeted therapy. We screened subsets of over 1,000 drug combinations in 14 different epithelial cell lines representing three distinct cancer lineages, and 19 melanoma cell lines, and assessed the ability of each combination to cause synergistic cytotoxicity. We focused on synergistic combinations because they point to mechanistic linkages between the signaling pathways, and also because of the possibility of improved therapeutic index in vivo. Drug substitution studies were used to validate the functionally important drug targets. Of the 84 combinations that caused robust synergy in multiple epithelial cell lines, none were synergistic in more than half of the lines tested, and we observed no pattern with respect to lineage specificity or mutational status of commonly altered oncogenes in the observed synergies. Within the melanoma panel, BRAF mutational status predicted response to single-agent BRAF inhibition, but did not predict synergistic drug combinations, which were different for each cell line. These results reflect the heterogeneity of genetic alterations and the plasticity of cell signaling networks even among cell lines of the same tissue of origin that contain the same predominant driver mutations. We suggest that there is not a sharp dichotomy between “driver” and “passenger” mutations, and that the biological responses to combination therapies are determined by functionally important modifier mutations that we term “back-seat drivers.” This hypothesis is supported by analysis of the transcriptomes and phosphoproteomes of cells treated with drugs singly and in combination, and by exome sequencing. Such analyses also can reveal critical nodes with the potential to function as effective single targets. We found that co-inhibition of EGFR and PI3 Kinase causes synergistic cytotoxicity in some epithelial cancer cell lines, and that phosphoproteomic analysis of signaling pathway responses revealed concordant synergistic inhibition of p70S6 Kinase in KU-7 bladder cancer cells. Using an epistasis paradigm, restoration of p70S6 Kinase signaling by expression of mutationally activated p70S6 Kinase resulted in protection from cytotoxicity, indicating that p70S6 Kinase is a critical node for enhanced cytotoxicity due to combination treatment. AT7867, a potent inhibitor of p70S6 Kinase, was able to inhibit phosphorylation of ribosomal protein S6 and induce cytotoxicity as effectively as the combination drug treatment. We suggest that p70S6 Kinase acts as a functionally important node within the EGFR/PI3 Kinase signaling network and is an attractive target for therapeutic intervention. Citation Format: Mark J. Axelrod, Devin Roller, Brian Capaldo, Aaron J. Mackey, Mark Conaway, Daniel Gioeli, Michael J. Weber. Combinatorial screens with targeted inhibitors reveal diverse compensatory responses and mechanisms of adaptive resistance to therapy. [abstract]. In: Proceedings of the AACR Precision Medicine Series: Synthetic Lethal Approaches to Cancer Vulnerabilities; May 17-20, 2013; Bellevue, WA. Philadelphia (PA): AACR; Mol Cancer Ther 2013;12(5 Suppl):Abstract nr B29.

Combinatorial drug screening identifies compensatory pathway interactions and adaptive resistance mechanisms

Constitutively activated signaling molecules are often the primary drivers of malignancy, and are favored targets for therapeutic intervention. However, the effectiveness of targeted inhibition of cell signaling can be blunted by compensatory signaling which generates adaptive resistance mechanisms and reduces therapeutic responses. Therefore, it is important to identify and target these compensatory pathways with combinations of targeted agents to achieve durable clinical benefit. In this report, we demonstrate the use of high-throughput combinatorial drug screening as a discovery tool to identify compensatory pathways that generate resistance to the cytotoxic effects of targeted therapy. We screened 420 drug combinations in 14 different cell lines representing three cancer lineages, and assessed the ability of each combination to cause synergistic cytotoxicity. Drug substitution studies were used to validate the functionally important drug targets. Of the 84 combinations that caused robust synergy in multiple cell lines, none were synergistic in more than half of the lines tested, and we observed no pattern of lineage specificity in the observed synergies. This reflects the plasticity of cell signaling networks, even among cell lines of the same tissue of origin. Mechanistic analysis of one novel synergistic combination identified in the screen, the multi-kinase inhibitor Ro31-8220 and lapatinib, demonstrated compensatory crosstalk between the p70S6 kinase and EGF receptor pathways. In addition, we identified BAD as a node of convergence between these two pathways that may be playing a role in the enhanced apoptosis observed upon combination treatment.

Microfluidic Devices for Bioapplications

Harnessing the ability to precisely and reproducibly actuate fluids and manipulate bioparticles such as DNA, cells, and molecules at the microscale, microfluidics is a powerful tool that is currently revolutionizing chemical and biological analysis by replicating laboratory bench-top technology on a miniature chip-scale device, thus allowing assays to be carried out at a fraction of the time and cost while affording portability and field-use capability. Emerging from a decade of research and development in microfluidic technology are a wide range of promising laboratory and consumer biotechnological applications from microscale genetic and proteomic analysis kits, cell culture and manipulation platforms, biosensors, and pathogen detection systems to point-of-care diagnostic devices, high-throughput combinatorial drug screening platforms, schemes for targeted drug delivery and advanced therapeutics, and novel biomaterials synthesis for tissue engineering. The developments associated with these technological advances along with their respective applications to date are reviewed from a broad perspective and possible future directions that could arise from the current state of the art are discussed.