A 1536-well quantitative high-throughput screen to identify compounds targeting cancer stem cells.
ABSTRACT: Tumor cell subpopulations called cancer stem cells (CSCs) or tumor-initiating cells (TICs) have self-renewal potential and are thought to drive metastasis and tumor formation. Data suggest that these cells are resistant to current chemotherapy and radiation therapy treatments, leading to cancer recurrence. Therefore, finding new drugs and/or drug combinations that cause death of both the differentiated tumor cells as well as CSC populations is a critical unmet medical need. Here, we describe how cancer-derived CSCs are generated from cancer cell lines using stem cell growth media and nonadherent conditions in quantities that enable high-throughput screening (HTS). A cell growth assay in a 1536-well microplate format was developed with these CSCs and used to screen a focused collection of oncology drugs and clinical candidates to find compounds that are cytotoxic against these highly aggressive cells. A hit selection process that included potency and efficacy measurements during the primary screen allowed us to efficiently identify compounds with potent cytotoxic effects against spheroid-derived CSCs. Overall, this research demonstrates one of the first miniaturized HTS assays using CSCs. The procedures described here should enable further testing of the effect of compounds on CSCs and help determine which pathways need to be targeted to kill them.
Project description:Cancer stem cells (CSCs), a rare cell population in tumors, are resistant to conventional chemotherapy and thus responsible for tumor recurrence. To screen for active compounds targeting CSCs, a good CSC-enriched model compatible with high-throughput screening (HTS) is needed. Here, we describe a new head and neck cancer stem cell-enriched spheroid model (SCESM) suitable for HTS analyses of anti-CSC compounds. We used FaDu cells, round-bottom ultra-low adherent (ULA) microplates, and stem medium. The formed spheroids displayed increased expression of all stem markers tested (qRT-PCR and protein analysis) in comparison to the FaDu cells grown in a standard adherent culture or in a well-known HTS-compatible multi-cellular tumor spheroid model (MCTS). Consistent with increased stemness of the cells in the spheroid, confocal microscopy detected fast proliferating cells only at the outer rim of the SCESM spheroids, with poorly/non-proliferating cells deeper in. To confirm the sensitivity of our model, we used ATRA treatment, which strongly reduced the expression of selected stem markers. Altogether, we developed a CSC-enriched spheroid model with a simple protocol, a microplate format compatible with multimodal detection systems, and a high detection signal, making it suitable for anti-CSC compounds' HTS.
Project description:RNA-binding proteins (RBPs) can act as stem cell modulators and oncogenic drivers, but have been largely ignored by the pharmaceutical industry as potential therapeutic targets for cancer. The MUSASHI (MSI) family has recently been demonstrated to be an attractive clinical target in the most aggressive cancers. Therefore, the discovery and development of small molecule inhibitors could provide a novel therapeutic strategy. In order to find novel compounds with MSI RNA binding inhibitory activity, we have developed a fluorescence polarization (FP) assay and optimized it for high throughput screening (HTS) in a 1536-well microtiter plate format. Using a chemical library of 6,208 compounds, we performed pilot screens, against both MSI1 and MSI2, leading to the identification of 7 molecules for MSI1, 15 for MSI2 and 5 that inhibited both. A secondary FP dose-response screen validated 3 MSI inhibitors with IC50 below 10 ?M. Out of the 25 compounds retested in the secondary screen only 8 demonstrated optical interference due to high fluorescence. Utilizing a SYBR-based RNA electrophoresis mobility shift assay (EMSA), we further verified MSI inhibition of the top 3 compounds. Surprisingly, even though several aminoglycosides were present in the library, they failed to demonstrate MSI inhibitor activity challenging the concept that these compounds are pan-active against RBPs. In summary, we have developed an in vitro strategy to identify MSI specific inhibitors using an FP HTS platform, which will facilitate novel drug discovery for this class of RBPs.
Project description:Screens for agents that specifically kill epithelial cancer stem cells (CSCs) have not been possible due to the rarity of these cells within tumor cell populations and their relative instability in culture. We describe here an approach to screening for agents with epithelial CSC-specific toxicity. We implemented this method in a chemical screen and discovered compounds showing selective toxicity for breast CSCs. One compound, salinomycin, reduces the proportion of CSCs by >100-fold relative to paclitaxel, a commonly used breast cancer chemotherapeutic drug. Treatment of mice with salinomycin inhibits mammary tumor growth in vivo and induces increased epithelial differentiation of tumor cells. In addition, global gene expression analyses show that salinomycin treatment results in the loss of expression of breast CSC genes previously identified by analyses of breast tissues isolated directly from patients. This study demonstrates the ability to identify agents with specific toxicity for epithelial CSCs.
Project description:Glioblastoma (GBM) is the most aggressive primary brain cancer with an average survival time after diagnosis of only 12-14 months, with few (<5%) long-term survivors. A growing body of work suggests that GBMs contain a small population of glioma stem cells (GSCs) that are thought to be major contributors to treatment resistance and disease relapse. Identifying compounds that modulate GSC proliferation would provide highly valuable molecular probes of GSC-directed signaling. However, targeting GSCs pharmacologically has been challenging. Patient-derived GSCs can be cultured as neurospheres, and in vivo these cells functionally recapitulate the heterogeneity of the original tumor. Using patient-derived GSC-enriched cultures, we have developed a 1536-well spheroid-based proliferation assay and completed a pilot screen, testing ~3300 compounds comprising approved drugs. This cytotoxic and automation-friendly assay yielded a signal-to-background (S/B) ratio of 161.3 ± 7.5 and Z' of 0.77 ± 0.02, demonstrating its robustness. Importantly, compounds were identified with anti-GSC activity, demonstrating the applicability of this assay for large-scale high-throughput screening (HTS).
Project description:Glioblastoma multiforme (GBM) is the most aggressive and lethal type of brain tumor. Both therapeutic resistance and restricted permeation of drugs across the blood-brain barrier (BBB) play a major role in the poor prognosis of GBM patients. Accumulated evidence suggests that in many human cancers, including GBM, therapeutic resistance can be attributed to a small fraction of cancer cells known as cancer stem cells (CSCs). CSCs have been shown to have stem cell-like properties that enable them to evade traditional cytotoxic therapies, and so new CSC-directed anti-cancer therapies are needed. Nanoparticles have been designed to selectively deliver payloads to relevant target cells in the body, and there is considerable interest in the use of nanoparticles for CSC-directed anti-cancer therapies. Recent advances in the field of nanomedicine offer new possibilities for overcoming CSC-mediated therapeutic resistance and thus significantly improving management of GBM. In this review, we will examine the current nanomedicine approaches for targeting CSCs and their therapeutic implications. The inhibitory effect of various nanoparticle-based drug delivery system towards CSCs in GBM tumors is the primary focus of this review.
Project description:Combinations of antiangiogenic agents (AAs) with cytotoxic agents have shown significant promise in cancer treatment, and several such clinical trials are currently underway. We have designed, synthesized, and evaluated two compounds that each inhibit vascular endothelial growth factor receptor-2 (VEGFR-2) and platelet-derived growth factor receptor-beta (PDGFR-beta) for antiangiogenic effects and also inhibit human thymidylate synthase (hTS) for cytotoxic effects in single agents. The synthesis of these compounds involved the nucleophilic displacement of the common intermediate 5-chloro-9H-pyrimido[4,5-b]indole-2,4-diamine with appropriate benzenethiols. The inhibitory potency of both these single agents against VEGFR-2, PDGFR-beta, and hTS is better than or close to standards. In a COLO-205 xenograft mouse model, one of the analogs significantly decreased tumor growth (tumor growth inhibition (TGI) = 76% at 35 mg/kg), liver metastases, and tumor blood vessels compared with a standard drug and with control and thus demonstrated potent tumor growth inhibition, inhibition of metastasis, and antiangiogenic effects in vivo. These compounds afford combination chemotherapeutic potential in single agents.
Project description:Therapeutic resistance is a major clinical challenge in oncology. Evidence identifies cancer stem cells (CSCs) as a driver of tumor evolution. Accordingly, the key stemness property unique to CSCs, may represent a reservoir of therapeutic target to improve cancer treatment. Here, we carried out a genome-wide RNA interference screen to identify genes that regulates breast CSCs-fate (bCSC). Using an interactome/regulome analysis, we integrated screen results in a functional mapping of the CSC-related processes. This network analysis uncovered potential therapeutic targets controlling bCSC-fate. We tested a panel of 15 compounds targeting these regulators. We showed that mifepristone, salinomycin, and JQ1 represent the best anti-bCSC activity. A combination assay revealed a synergistic interaction of salinomycin/JQ1 association to deplete the bCSC population. Treatment of primary breast cancer xenografts with this combination reduced the tumor-initiating cell population and limited metastatic development. The clinical relevance of our findings was reinforced by an association between the expression of the bCSC-related networks and patients prognosis. Targeting bCSCs with salinomycin/JQ1 combination provides the basis for a new therapeutic approach for breast cancer care.
Project description:Therapeutic resistance is a major clinical challenge in oncology. Evidence identifies cancer stem cells (CSCs) as a driver of tumor evolution. Accordingly, the key stemness property unique to CSCs may represent a reservoir of therapeutic target to improve cancer treatment. Here, we carried out a genome-wide RNA interference screen to identify genes that regulate breast CSCs-fate (bCSC). Using an interactome/regulome analysis, we integrated screen results in a functional mapping of the CSC-related processes. This network analysis uncovered potential therapeutic targets controlling bCSC-fate. We tested a panel of 15 compounds targeting these regulators. We showed that mifepristone, salinomycin, and JQ1 represent the best anti-bCSC activity. A combination assay revealed a synergistic interaction of salinomycin/JQ1 association to deplete the bCSC population. Treatment of primary breast cancer xenografts with this combination reduced the tumor-initiating cell population and limited metastatic development. The clinical relevance of our findings was reinforced by an association between the expression of the bCSC-related networks and patient prognosis. Targeting bCSCs with salinomycin/JQ1 combination provides the basis for a new therapeutic approach in the treatment of breast cancer.
Project description:Shifting the balance away from tumor-mediated immune suppression toward tumor immune rejection is the conceptual foundation for a variety of immunotherapy efforts currently being tested. These efforts largely focus on activating antitumor immune responses but are confounded by multiple immune cell populations, including myeloid-derived suppressor cells (MDSCs), which serve to suppress immune system function. We have identified immune-suppressive MDSCs in the brains of GBM patients and found that they were in close proximity to self-renewing cancer stem cells (CSCs). MDSCs were selectively depleted using 5-flurouracil (5-FU) in a low-dose administration paradigm, which resulted in prolonged survival in a syngeneic mouse model of glioma. In coculture studies, patient-derived CSCs but not nonstem tumor cells selectively drove MDSC-mediated immune suppression. A cytokine screen revealed that CSCs secreted multiple factors that promoted this activity, including macrophage migration inhibitory factor (MIF), which was produced at high levels by CSCs. Addition of MIF increased production of the immune-suppressive enzyme arginase-1 in MDSCs in a CXCR2-dependent manner, whereas blocking MIF reduced arginase-1 production. Similarly to 5-FU, targeting tumor-derived MIF conferred a survival advantage to tumor-bearing animals and increased the cytotoxic T cell response within the tumor. Importantly, tumor cell proliferation, survival, and self-renewal were not impacted by MIF reduction, demonstrating that MIF is primarily an indirect promoter of GBM progression, working to suppress immune rejection by activating and protecting immune suppressive MDSCs within the GBM tumor microenvironment. Stem Cells 2016;34:2026-2039.
Project description:Glutathione S-transferases (GSTs) are a superfamily of enzymes that conjugate glutathione to a wide variety of both exogenous and endogenous compounds for biotransformation and/or removal. Glutathione S-tranferase omega 1 (GSTO1) is highly expressed in human cancer cells, where it has been suggested to play a role in detoxification of chemotherapeutic agents. Selective inhibitors of GSTO1 are, however, required to test the role that this enzyme plays in cancer and other (patho)physiological processes. With this goal in mind, we performed a fluorescence polarization activity-based protein profiling (fluopol-ABPP) high-throughput screen (HTS) with GSTO1 and the Molecular Libraries Small Molecule Repository (MLSMR) 300K+ compound library. This screen identified a class of selective and irreversible ?-chloroacetamide inhibitors of GSTO1, which were optimized to generate an agent KT53 that inactivates GSTO1 with excellent in vitro (IC(50) = 21 nM) and in situ (IC(50) = 35 nM) potency. Cancer cells treated with KT53 show heightened sensitivity to the cytotoxic effects of cisplatin, supporting a role for GSTO1 in chemotherapy resistance.