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Supplementary Figures S1-S8. S1. Glucose concentration and pH in spheroid culture medium; S2. Dose-response and time-course experiment; S3. GFP-based and TOX8 assay comparison; S4. HT-29 spheroid-based clonogenic assay; S5. Final hit compounds' effects in spheroids formed with or without medium change S6. Nitazoxanide and tizoxanide effects comparison; S7. Nitazoxanide and irinotecan combination in clonogenic assay; S8. CD44 IHC staining.

Supplementary materials, Tables S1-S2. Supplementary Table S1. List of the 12 3D-selective screening hits, Supplementary Table S2. Structures and properties of the five final hit compounds.

Because dormant cancer cells in hypoxic and nutrient-deprived regions of solid tumors provide a major obstacle to treatment, compounds targeting those cells might have clinical benefits. Here, we describe a high-throughput drug screening approach, using glucose-deprived multicellular tumor spheroids (MCTS) with inner hypoxia, to identify compounds that specifically target this cell population. We used a concept of drug repositioning—using known molecules for new indications. This is a promising strategy to identify molecules for rapid clinical advancement. By screening 1,600 compounds with documented clinical history, we aimed to identify candidates with unforeseen potential for repositioning as anticancer drugs. Our screen identified five molecules with pronounced MCTS-selective activity: nitazoxanide, niclosamide, closantel, pyrvinium pamoate, and salinomycin. Herein, we show that all five compounds inhibit mitochondrial respiration. This suggests that cancer cells in low glucose concentrations depend on oxidative phosphorylation rather than solely glycolysis. Importantly, continuous exposure to the compounds was required to achieve effective treatment. Nitazoxanide, an FDA-approved antiprotozoal drug with excellent pharmacokinetic and safety profile, is the only molecule among the screening hits that reaches high plasma concentrations persisting for up to a few hours after single oral dose. Nitazoxanide activated the AMPK pathway and downregulated c-Myc, mTOR, and Wnt signaling at clinically achievable concentrations. Nitazoxanide combined with the cytotoxic drug irinotecan showed anticancer activity in vivo. We here report that the FDA-approved anthelmintic drug nitazoxanide could be a potential candidate for advancement into cancer clinical trials. Mol Cancer Ther; 14(6); 1504–16. ©2015 AACR.

Treatment of patients with high-grade serous ovarian carcinoma (HGSOC) and triple-negative breast cancer (TNBC) includes platinum-based drugs, gemcitabine, and PARP inhibitors. However, resistance to these therapies develops in most cases, highlighting the need for novel therapeutic approaches and biomarkers to guide the optimal treatment choice. Using a CRISPR loss-of-function screen for carboplatin sensitizers in the HGSOC cell line OVCAR8, we identifiedCSNK2A2, the gene encoding for the alpha’ (α’) catalytic subunit of casein kinase 2 (CK2). Expanding on this finding, we confirmed that the CK2 inhibitors silmitasertib and SGC-CK2-1 sensitized many, but not all, TNBC and HGSOC cell lines to the drugs that perturb DNA replication, including platinum drugs, gemcitabine, and PARP inhibitors. We identified RB1 tumor suppressor deficiency as a prerequisite context for the CK2 inhibition-mediated sensitization to these therapeutics. In RB1-deficient cells, CK2 inhibition resulted in accumulation of cells in S phase of the cell cycle, associated with micronuclei formation, and accelerated PARP inhibitor-induced aneuploidy and mitotic cell death. Patient HGSOC organoids that lacked RB1 expression displayed an enhanced long-term response to carboplatin and PARP inhibitor niraparib when combined with silmitasertib, suggesting RB1-stratified efficacy in patients. As RB1 deficiency affects up to 25% of HGSOC and 40% of TNBC cases, CK2 inhibition, proven safe from previous clinical exploration with silmitasertib, is a promising approach to overcome resistance to standard therapeutics in large strata of patients.

High-grade serous ovarian cancer (HGSC) is the most prevalent and lethal ovarian cancer type. While HGSC usually responds well to primary treatment, most cases eventually relapse. Functional precision medicine - tailoring individualized treatments based on functional in vitro assays on patient-derived cells - has been recently employed in cancer clinical trials. Cancer organoids - three-dimensional, self-organizing, self-renewing cell cultures that recapitulate original tissue structure and function - have been applied as cellular models in these trials. However, in case of HGSC, organoid derivation has proven time consuming and inefficient, hindering their application in functional precision medicine due to a short time window, in which therapy for each patient needs to be selected. To address this problem, we aimed to establish whether drug vulnerabilities at HGSC relapse could be predicted using organoids derived from the primary disease cells. We derived sequential organoid models from material sampled during primary treatment and at relapse. Then, for organoid pairs (primary-relapse), we performed large-scale drug response profiling of a library of 370 compounds (approved drugs or drugs in clinical development), in 384-well microplate format, alone or in combination with a standard HGSC chemotherapeutic agent carboplatin. First, we found that HGSC organoid responses to standard chemotherapeutics retrospectively correlated to observed clinical treatment outcomes. But further, for each patient we identified compounds with pronounced cytotoxicity both in the primary and in the relapsed model, amounting to 66% of all hits (7% were primary-specific and 27% relapse-specific). We then focused on identifying patient-specific hits rather than compounds displaying general toxicity in all patient models. Based on a potential clinical applicability, for three patients we selected compounds for validation in organoid outgrowth assay, with prolonged (>1 month) drug-free period post-treatment. In two patients, AZD4573, a selective CDK9 inhibitor in clinical development for hematological malignancies, at nanomolar concentrations caused eradication of organoids when combined with carboplatin. Organoids from the third patient were vulnerable to nitazoxanide, an approved anti-helminthic agent and an inhibitor of mitochondrial oxidative phosphorylation. Importantly, the selected final hits were identified solely based on screening in organoid models from primary disease. In summary, we here demonstrate that HGSC organoids derived from primary disease material predict a majority of patient-specific drug vulnerabilities of organoids derived from the relapsed HGSC lesions. This indicates that patient stratification in functional precision medicine for treatment of HGSC relapse could be prospectively performed at the primary disease stage. Citation Format: Wojciech Senkowski, Laura Gall-Mas, Matias M. Falco, Yilin Li, Kari Lavikka, Mette C. Kriegbaum, Jaana Oikkonen, Daria Bulanova, Elin J. Pietras, Karolin Voßgröne, Yan-Jun Chen, Erdogan P. Erkan, Mia K. Høg, Ida M. Larsen, Tarja Lamminen, Katja Kaipio, Jutta Huvila, Anni Virtanen, Lars H. Engelholm, Pernille Christiansen, Eric Santoni Rugiu, Kaisa Huhtinen, Olli Carpén, Johanna Hynninen, Sampsa Hautaniemi, Anna Vähärautio, Krister Wennerberg. A platform utilizing high-grade serous ovarian cancer organoids for prospective patient stratification in functional precision medicine. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 5779.

Nitazoxanide is a Food and Drug Administration-approved antiprotozoal drug recently demonstrated to be selectively active against quiescent and glucose-deprived tumour cells. This drug also has several characteristics that suggest its potential as a radiosensitizer. The present study aimed to investigate the interaction between nitazoxanide and radiation on human colon cancer cells cultured as monolayers, and to mimic key features of solid tumours in patients, as spheroids, as well as in xenografts in mice. In the present study, colon cancer HCT116 green fluorescent protein (GFP) cells were exposed to nitazoxanide, radiation or their combination. Cell survival was analysed by using total cell kill and clonogenic assays. DNA double-strand breaks were evaluated in the spheroid experiments, and HCT116 GFP cell xenograft tumours in mice were used to investigate the effect of nitazoxanide and radiation

Summary Spheroid cultures of primary human hepatocytes (PHH) are used in studies of hepatic drug metabolism and toxicity. The cultures are maintained under different conditions, with possible confounding results. We performed an in-depth analysis of the influence of various culture conditions to find the optimal conditions for the maintenance of an in vivo like phenotype. The formation, protein expression, and function of PHH spheroids were followed for three weeks in a high-throughput 384-well format. Medium composition affected spheroid histology, global proteome profile, drug metabolism and drug-induced toxicity. No epithelial-mesenchymal transition was observed. Media with fasting glucose and insulin levels gave spheroids with phenotypes closest to normal PHH. The most expensive medium resulted in PHH features most divergent from that of native PHH. Our results provide a protocol for culture of healthy PHH with maintained function - a prerequisite for studies of hepatocyte homeostasis and more reproducible hepatocyte research., Graphical abstract, Highlights • 3D spheroid cultures were established in 384-well format • Eight different media variants were used to optimize the 3D cultures • Optimized William's medium was as good as expensive commercial medium • The 3D cultures were used to study drug metabolism and toxicity, Biological sciences; Proteomics; Methodology in biological sciences

Extensive utilization of organoids from high-grade serous ovarian carcinoma (HGSOC), the most common and lethal ovarian cancer, has been hampered by low success rates of long-term culture and scarcity of fresh tumor material. Here we present the development of a novel method for efficient generation, expansion and use of HGSOC organoids from cryopreserved tumor material. First, we assessed commonly used organoid media components and found that supplements such as FGF-2, R-Spondin1, Wnt or Noggin had negative impact on the HGSOC organoid derivation. But further, we found that supplementation with FGF-4, which has not been used in cancer organoid culture before, is beneficial for HGSOC organoid growth. Through extensive testing of various supplements and their combinations, we designed two novel HGSOC organoid media formulations - Medium 1 (M1) and Medium 2 (M2). Using M1 and M2 enabled generation and long-term expansion of living HGSOC organoid biobank with markedly improved success rate than in previous reports (55% vs. 23-38%). The organoids were established from cryopreserved tumor material, demonstrating the feasibility of using frozen tissue biobanks for HGSOC organoid derivation. Overall, we generated a collection of 18 expandable HGSOC organoid lines from 11 patients, encompassing samples from different tissue sites and disease progression stages. We validated the organoids using whole-genome sequencing, immunohistochemistry and single-cell RNA sequencing and demonstrated that they are genetically and phenotypically representative of original patient samples over long-term culture. Based on available patient consents, we deposited 3 organoid lines in a publicly accessible biobank. Finally, we investigated whether organoid drug responses correlate to those observed earlier in the clinic in the corresponding patients. Organoid-based drug-response profiling of clinically used HGSOC drug collection was performed in 384-well microplate format. To explore whether growth conditions impact correlation between organoid drug responses and clinical response, we compared the organoid drug responses in the nutrient-rich M1/M2 growth media to the ones observed in human plasma-like medium (HPLM), supplemented with relevant niche factors from M1/M2. Organoid drug responses correlated with clinical treatment outcomes, but only for organoids maintained in HPLM (Spearman r = 0.987, p=0.007 in HPLM vs 0.607, p=0.167 in growth medium, n=7), highlighting the importance of culture conditions in organoid-based functional assays. Taken together, we introduce a resource for efficient development and use of HGSOC organoids from cryopreserved material in ovarian cancer research. Citation Format: Wojciech Senkowski, Laura Gall Mas, Matias M. Falco, Yilin Li, Kari Lavikka, Mette C. Kriegbaum, Jaana Oikkonen, Daria Bulanova, Elin J. Pietras, Karolin Voßgröne, Erdogan P. Erkan, Terese K. Høj, Mia K. Høg, Tarja Lamminen, Katja Kaipio, Anni Virtanen, Lars H. Engelholm, Pernille Christiansen, Eric Santoni-Rugiu, Kaisa Huhtinen, Olli Carpén, Johanna Hynninen, Sampsa Hautaniemi, Anna Vähärautio, Krister Wennerberg. Efficient establishment and utilization of a high-grade serous ovarian cancer organoid biobank [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 3069.

Purpose: Ovarian cancer is the most lethal gynecological cancer. Histologically, about 90% of the ovarian cancers have epithelial origin and more than 75% are characterized as high-grade serous ovarian cancer (HGSOC). Despite having a good primary response to the standard platinum-taxane based chemotherapy, almost all HGSOC patients experience a relapsed disease. The 5-year survival rate of ovarian cancer is only 38% globally, indicating need for the development of novel efficient treatments to fight the resistance. Here we have established a personalized drug screening and evaluation platform using HGSOC patient samples and medicines approved for treatment of different types of cancers. Methods: Tissues collected from ovarian cancer patients were grown as 3-dimensional (3D) ex vivo long-term tumor organoid cultures. Seven HGSOC cell lines were additionally grown as spheroids or as xenograft tumors to establish them as 3D tumor organoid cultures for drug efficiency evaluation. Cancer stemness and further classification of samples were determined via immunofluorescent staining with relevant antibodies (pax8, Ki67, acetylated tubulin, p53, cytokeratin-8) and consequent high-throughput imaging using the ImageXpress Micro Confocal microscope (Molecular Devices). The images were analyzed and quantified using MetaXpress software. Organoid growth and response to tested treatments was validated via invasive growth and survival assays. Both assays were used to monitor resistance of organoids towards cisplatin and carboplatin treatment and towards suggested potential new treatments. Cancer drugs were selected according to suggestions based on RNA seq, DNA and/or drug screenings done in cancer cell lines. A preclinical mouse model (in immunodeficient NOD/Shi-scid/IL-2Rγnull (NOG) mice) was set up to be used for in vivo validation by monitoring the tumor growth in response to the treatment. Results: Currently, 20 patient tumor samples received from Turku University Hospital, Finland (KH, JH) have been cultured in 4 different culture media to obtain highest possible survival rates for each sample. Organoids from ovarian cancer cell lines are characterized for their resistance to increasing concentrations of platinum treatment. Notably, organoid analysis protocols and screening conditions have been setup for the high-throughput microscopy. These include organoid growth, survival, and invasiveness/aggressiveness. We are currently set up for screening organoids with the first drugs suggested by bioinformatics-based genetic pathway analysis and drug screening studies in cancer cell lines. The results of the screen will be presented. This study is a part of a large EU-funded ovarian cancer project DECIDER (https://www.deciderproject.eu/). Citation Format: Aikaterini Skorda, Kaisa Huhtinen, Wojciech Senkowski, Krister Wennerberg, Sampsa Hautaniemi, Johanna Hynninen, Tuula Kallunki. Ex vivo screening and analysis of novel effective treatments for ovarian cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 192.

We and others have previously reported a correlation between high phosphodiesterase 3A (PDE3A) expression and selective sensitivity to phosphodiesterase (PDE) inhibitors. This indicates that PDE3A could serve both as a drug target and a biomarker of sensitivity to PDE3 inhibition. In this report, we explored publicly available mRNA gene expression data to identify cell lines with different PDE3A expression. Cell lines with high PDE3A expression showed marked in vitro sensitivity to PDE inhibitors zardaverine and quazinone, when compared with those having low PDE3A expression. Immunofluorescence and immunohistochemical stainings were in agreement with PDE3A mRNA expression, providing suitable alternatives for biomarker analysis of clinical tissue specimens. Moreover, we here demonstrate that tumor cells from patients with ovarian carcinoma show great variability in PDE3A protein expression and that level of PDE3A expression is correlated with sensitivity to PDE inhibition. Finally, we demonstrate that PDE3A is highly expressed in subsets of patient tumor cell samples from different solid cancer diagnoses and expressed at exceptional levels in gastrointestinal stromal tumor (GIST) specimens. Importantly, vulnerability to PDE3 inhibitors has recently been associated with co-expression of PDE3A and Schlafen family member 12 (SLFN12). We here demonstrate that high expression of PDE3A in clinical specimens, at least on the mRNA level, seems to be frequently associated with high SLFN12 expression. In conclusion, PDE3A seems to be both a promising biomarker and drug target for individualized drug treatment of various cancers.

There is a need for preclinical models that can enable identification of novel radiosensitizing drugs in clinically relevant high-throughput experiments. We used a new high-throughput compatible total cell kill spheroid assay to study the interaction between drugs and radiation in order to identify compounds with radiosensitizing activity. Experimental drugs were compared to known radiosensitizers and cytotoxic drugs clinically used in combination with radiotherapy. VLX600, a novel iron-chelating inhibitor of oxidative phosphorylation, potentiated the effect of radiation in tumor spheroids in a synergistic manner. This effect was specific to spheroids and not observed in monolayer cell cultures. In conclusion, the total cell kill spheroid assay is a feasible high-throughput method in the search for novel radiosensitizers. VLX600 shows encouraging characteristics for development as a novel radiosensitizer.

Mebendazole (MBZ), a drug commonly used for helminitic infections, has recently gained substantial attention as a repositioning candidate for cancer treatment. However, the mechanism of action behind its anticancer activity remains unclear. To address this problem, we took advantage of the curated MBZ-induced gene expression signatures in the LINCS Connectivity Map (CMap) database. The analysis revealed strong negative correlation with MEK/ERK1/2 inhibitors. Moreover, several of the most upregulated genes in response to MBZ exposure were related to monocyte/macrophage activation. The MBZ-induced gene expression signature in the promyeloblastic HL-60 cell line was strongly enriched in genes involved in monocyte/macrophage pro-inflammatory (M1) activation. This was subsequently validated using MBZ-treated THP-1 monocytoid cells that demonstrated gene expression, surface markers and cytokine release characteristic of the M1 phenotype. At high concentrations MBZ substantially induced the release of IL-1β and this was further potentiated by lipopolysaccharide (LPS). At low MBZ concentrations, cotreatment with LPS was required for MBZ-stimulated IL-1β secretion to occur. Furthermore, we show that the activation of protein kinase C, ERK1/2 and NF-kappaB were required for MBZ-induced IL-1β release. MBZ-induced IL-1β release was found to be dependent on NLRP3 inflammasome activation and to involve TLR8 stimulation. Finally, MBZ induced tumor-suppressive effects in a coculture model with differentiated THP-1 macrophages and HT29 colon cancer cells. In summary, we report that MBZ induced a pro-inflammatory (M1) phenotype of monocytoid cells, which may, at least partly, explain MBZ's anticancer activity observed in animal tumor models and in the clinic.

Cancer cell lines grown as two-dimensional (2D) cultures have been an essential model for studying cancer biology and anticancer drug discovery. However, 2D cancer cell cultures have major limitations, as they do not closely mimic the heterogeneity and tissue context of in vivo tumors. Developing three-dimensional (3D) cell cultures, such as multicellular tumor spheroids, has the potential to address some of these limitations. Here, we combined a high-throughput gene expression profiling method with a tumor spheroid-based drug-screening assay to identify context-dependent treatment responses. As a proof of concept, we examined drug responses of quiescent cancer cells to oxidative phosphorylation (OXPHOS) inhibitors. Use of multicellular tumor spheroids led to discovery that the mevalonate pathway is upregulated in quiescent cells during OXPHOS inhibition, and that OXPHOS inhibitors and mevalonate pathway inhibitors were synergistically toxic to quiescent spheroids. This work illustrates how 3D cellular models yield functional and mechanistic insights not accessible via 2D cultures.

Poorly vascularized areas of solid tumors contain quiescent cell populations that are resistant to cell cycle-active cancer drugs. The compound VLX600 was recently identified to target quiescent tumor cells and to inhibit mitochondrial respiration. We here performed gene expression analysis in order to characterize the cellular response to VLX600. The compound-specific signature of VLX600 revealed a striking similarity to signatures generated by compounds known to chelate iron. Validation experiments including addition of ferrous and ferric iron in excess, EXAFS measurements, and structure activity relationship analyses showed that VLX600 chelates iron and supported the hypothesis that the biological effects of this compound is due to iron chelation. Compounds that chelate iron possess anti-cancer activity, an effect largely attributed to inhibition of ribonucleotide reductase in proliferating cells. Here we show that iron chelators decrease mitochondrial energy production, an effect poorly tolerated by metabolically stressed tumor cells. These pleiotropic features make iron chelators an attractive option for the treatment of solid tumors containing heterogeneous populations of proliferating and quiescent cells. Funding Agencies|Swedish Research Council; Knut och Alice Wallenbergs Stiftelse; Cancerforeningen in Stockholm; Cancerfonden; Vetenskapsradet; Barncancerfonden

In vitro-based search for promising anti-cancer drug combinations may provide important leads to improved cancer therapies. Currently there are no integrated computational-experimental methods specifically designed to search for combinations, maximizing a predefined therapeutic index (TI) defined in terms of appropriate model systems. Here, such a pipeline is presented allowing the search for optimal combinations among an arbitrary number of drugs while also taking experimental variability into account. The TI optimized is the cytotoxicity difference (in vitro) between a target model and an adverse side effect model. Focusing on colorectal carcinoma (CRC), the pipeline provided several combinations that are effective in six different CRC models with limited cytotoxicity in normal cell models. Herein we describe the identification of the combination (Trichostatin A, Afungin, 17-AAG) and present results from subsequent characterisations, including efficacy in primary cultures of tumour cells from CRC patients. We hypothesize that its effect derives from potentiation of the proteotoxic action of 17-AAG by Trichostatin A and Afungin. The discovered drug combinations against CRC are significant findings themselves and also indicate that the proposed strategy has great potential for suggesting drug combination treatments suitable for other cancer types as well as for other complex diseases.

Because dormant cancer cells in hypoxic and nutrient-deprived regions of solid tumors provide a major obstacle to treatment, compounds targeting those cells might have clinical benefits. Here, we describe a high-throughput drug screening approach, using glucose-deprived multicellular tumor spheroids (MCTS) with inner hypoxia, to identify compounds that specifically target this cell population. We used a concept of drug repositioning-using known molecules for new indications. This is a promising strategy to identify molecules for rapid clinical advancement. By screening 1,600 compounds with documented clinical history, we aimed to identify candidates with unforeseen potential for repositioning as anticancer drugs. Our screen identified five molecules with pronounced MCTS-selective activity: nitazoxanide, niclosamide, closantel, pyrvinium pamoate, and salinomycin. Herein, we show that all five compounds inhibit mitochondrial respiration. This suggests that cancer cells in low glucose concentrations depend on oxidative phosphorylation rather than solely glycolysis. Importantly, continuous exposure to the compounds was required to achieve effective treatment. Nitazoxanide, an FDA-approved antiprotozoal drug with excellent pharmacokinetic and safety profile, is the only molecule among the screening hits that reaches high plasma concentrations persisting for up to a few hours after single oral dose. Nitazoxanide activated the AMPK pathway and downregulated c-Myc, mTOR, and Wnt signaling at clinically achievable concentrations. Nitazoxanide combined with the cytotoxic drug irinotecan showed anticancer activity in vivo. We here report that the FDA-approved anthelmintic drug nitazoxanide could be a potential candidate for advancement into cancer clinical trials. (C) 2015 AACR. Funding Agencies|Swedish Cancer Society; Swedish Foundation for Strategic Research; Swedish Research Council; Lions Cancer Research Fund; Strategiska Forskningsstiftelsen (SSF); Lions Cancerforskningsfond; Cancerfonden; Barncancerfonden; Radiumhemmets Forskningsfonder; Vetenskapsradet; SSF

Monolayer, two-dimensional (2D) cell cultures have been a predominant in vitro model in anticancer drug discovery and high-throughput screening (HTS). However, 2D cultures of cancer cells lack numerous properties of in vivo tumors, such as tissue-like structure, cell-cell interactions and nutrient/oxygen gradients. Thus, in recent years there has been an increased interest in 3D cell cultures, such as multicellular tumors spheroids (MCTS), to address some of these limitations. Recently, we and others have applied MCTS for HTS and identified oxidative phosphorylation (OXPHOS) as a selective vulnerability of quiescent cancer cells persisting in hypoxic and nutrient-deprived milieu. However, prolonged continuous exposure to OXPHOS inhibitors is necessary for the cytotoxic effect. Thus, there is a need to identify processes that could be co-targeted for enhanced anticancer activity. Here, we present two distinct HTS approaches to identify combination partner molecules for OXPHOS inhibitors. Since we were interested in targeting non-dividing nutrient-deprived cancer cells, we used quiescent MCTS (Q-MCTS), as an in vitro model. Cells in Q-MCTS experience glucose concentrations and pH similar to those observed in deep tumor parenchyma in vivo. In our first screening approach, we have applied high-throughput gene-expression profiling to study drug effects in MCTS at a large scale. Using L1000 Gene Expression Profiling method, we generated a dataset of over 1000 drug-induced gene-expression profiles and found that co-targeting of OXPHOS and the mevalonate pathway results in selective synergistic toxicity in quiescent cancer cells. In the other approach, we screened a library of 1650 biologically active compounds, with or without addition of the FDA-approved anthelmintic agent nitazoxanide (an OXPHOS inhibitor with high drug repurposing potential). After the screen, we selected molecules that demonstrated pronounced synergy when combined with nitazoxanide, but not when used alone. Then, we validated the hits in an extensive dose-response combination experiments in Q-MCTS and chose 14 compounds that demonstrated strong synergistic interaction with nitazoxanide at broad range of concentrations. These included antifungal agents, kinase inhibitors and others. In summary, we here report on novel approaches, utilizing 3D cell cultures, to identify drug combinations targeting quiescent cancer cells. By high-throughput gene-expression profiling and large-scale combinatorial drug screens, we were able to identify drug combinations preferentially toxic to quiescent cells. This work also demonstrates how 3D cell cultures yield functional insights that are not accessible through standard 2D cultures. Citation Format: Wojciech Senkowski, Madiha Nazir, Malin Jarvius, Jenny Rubin, Johan Lengqvist, Mats G. Gustafsson, Peter Nygren, Kim Kultima, Rolf Larsson, Mårten Fryknäs. High-throughput drug combination screening in tumor spheroids identifies context-dependent synthetic lethalities [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 4990. doi:10.1158/1538-7445.AM2017-4990

Inhibition of mitochondrial oxidative phosphorylation (OXPHOS) has recently emerged as a promising strategy for treatment of therapy-resistant cancer cells. These cells often reside within hypoxic tumor regions, where nutrient concentrations are low. Recently, OXPHOS inhibitors have been demonstrated to be toxic to quiescent, nutrient-deprived cells in multicellular tumor spheroids. Such spheroids, formed without medium exchange over the culture period, can serve as an appropriate model to mimic quiescent in vivo tumor regions. These spheroids exhibit low cell proliferation and comprise necrotic cores, contrary to commonly used spheroids cultured with frequent medium change. We here aimed to characterize how quiescent cells respond to OXPHOS inhibition and thereby identify processes that could be co-targeted for enhanced toxicity. We treated HCT116 colon cancer cell line, grown as monolayer cultures and spheroids, with a range of OXPHOS inhibitors (n = 10, including FDA-approved drugs, e.g. nitazoxanide) and other compounds (n = 14) at escalating doses and in 4 biological replicates. Then, we obtained global gene expression profiles (n = 1149, including 144 vehicle controls) of all treatment conditions using L1000 Gene Expression Profiling method. We found that upon exposure to OXPHOS inhibitors cells grown as nutrient-deprived spheroids significantly and in dose-dependent manner upregulate expression of genes involved in biosynthesis of cholesterol. This response was not observed for spheroids cultured with medium change or monolayer cell cultures. Thus, we were interested if simultaneous exposure to OXPHOS inhibitors and statins, inhibitors of mevalonate (cholesterol precursor) synthesis, would result in enhanced cytotoxic effects in quiescent, metabolically stressed cells. We here demonstrate that combination of OXPHOS inhibitors and statins results in pronounced synergistic cytotoxicity in metabolically stressed spheroids. This effect was observed for various classes of OXPHOS inhibitors and different types of statins, indicating that the observed synergy was not a result of off-target effects. This notion was further strengthened by the finding that mevalonate largely abrogated the synergistic effects. In conclusion, we here report that statins enhance the toxic effects of OXPHOS inhibitors in quiescent, metabolically stressed cells. Our results can serve as a foundation for further studies on targeting therapy-resistant and nutrient-deprived cancer cells by inhibition of OXPHOS. We also demonstrate, for the first time, that the L1000 Gene Expression Profiling can be used to study 3D cell cultures. Importantly, our findings underscore the importance of using a relevant cellular model for target discovery endeavors. Citation Format: Wojciech Senkowski, Malin Jarvius, Kim Kultima, Jenny Rubin, Mats Gustafsson, Peter Nygren, Rolf Larsson, Mårten Fryknäs. Mitochondrial inhibitors and statins: a lethal combination for metabolically stressed cancer cells. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 213.

In recent years, cell-based evaluation of drug-induced phenotypic changes has become an important strategy in high throughput drug screening (HTS). However, due to the lack of clinically relevant in vitro models, phenotype-based screening has not resulted in the identification of as many successful compounds as initially assumed. Thus, an application of the long-known multicellular tumor spheroid (MCTS) model for HTS has recently gained attention, as MCTS mimic the complex in vivo situation more closely than standard monolayer cell cultures. However, due to major difficulties with formation of uniform spheroids in a large scale, application of MCTS for HTS has been limited. We focused our efforts on the development of spheroid-based HTS systems in 384-well format for the identification of molecules targeting different tumor microenvironment characteristics. For spheroid formation we used two colorectal cancer cell lines, HCT116 and HT-29, both constitutively expressing green fluorescent protein (GFP). During the spheroid formation process, culture medium was either changed every 4 days or left unchanged. This approach resulted in the generation of two different spheroid models from the each cell line - one representing tumor regions with the sufficient access to nutrients; the other simulating parts with more harsh conditions. Spheroids formed with medium change were more proliferative and less dependent on oxygen. By measuring spheroid GFP fluorescence intensity we could non-invasively monitor drug action over time. First screens revealed a group of drugs particularly effective in quiescent spheroids compared with proliferating spheroids and monolayer cell cultures. All of the compounds inhibited mitochondrial oxidative phosphorylation. The screen confirmed mitochondrial respiration as a promising target for treatment of dormant cells in nutrient-deprived tumor regions and identified molecules with previously unrecognized potential for further clinical advancement. Another screening system was developed to identify drugs that directly influence the immune components of tumor microenvironment. Spheroids are formed by co-culturing cancer cells with human primary monocytes. Over the culture period, monocytes differentiate into tumor-associated macrophages. First pilot experiments showed promising results and more extensive screens are planned. For characterization of drug effects at the transcriptome level, a spheroid-based gene expression profiling method is under development. The method will enable high throughput analysis of transcripts from spheroids treated with chemical libraries. In conclusion, we here present a novel approach to HTS, which by utilizing diverse MCTS models has a potential to identify molecules directly targeting tumor-specific microenvironment components. Citation Format: Wojciech Senkowski, Xiaonan Zhang, Peter Nygren, Maria Hägg Olofsson, Mats Gustafsson, Stig Linder, Rolf Larsson, Mårten Fryknäs. Spheroid-based high throughput screening for identification of molecules targeting different tumor microenvironment characteristics. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 323. doi:10.1158/1538-7445.AM2015-323

In the search for novel anticancer agents, the established principle is to harness differences between normal and tumor tissue. Thus, most of current chemotherapeutic agents target fast-proliferating cancerous cells. However, over the years such treatment strategies have proven less effective than initially expected. One of the main reasons for this is that conditions present in solid tumors, such as hypoxia, low glucose availability and high concentrations of metabolites, promote quiescent, highly resistant cell phenotypes. In our research, we aimed to target and exploit these tumor-specific conditions. To mimic the harsh conditions in a tumor, we used multicellular tumor spheroids (MCTS), which are known to simulate tumor microenvironment in vitro. We developed a novel method to easily form MCTS in 384-well format. There is only one spheroid per well formed and all are comparable in terms of size and shape. For the MCTS formation, we used colon carcinoma cell line, HCT116, with constitutive expression of green fluorescent protein (GFP). Then, we performed spheroid-based high-throughput drug screening using 1600 clinically active compounds. As a surrogate marker for cell viability, we measured mean spheroid GFP fluorescence intensity complemented with a standard resazurin-based assay. Active hits were tested in dose-response experiments in both spheroid and monolayer setup. We identified 12 compounds, which showed preferential activity against the MCTS model. We tested them in spheroid-based clonogenic assay and identified five compounds, which after 72 hrs treatment resulted in no clonogenicity at concentrations equal to monolayer-based IC50-values. Interestingly, all of these compounds have been previously reported to impair mitochondrial function. Three of them have been also reported as uncouplers of oxidative phosphorylation. We tested the influence of all five compounds on oxygen consumption rate. All, except one compound, caused irreversible shutdown of mitochondrial function. For further experiments, we decided to choose nitazoxanide - a clinically used anti-parasitic agent with excellent pharmacokinetics, bioavailability and safety profile. We show that treatment with nitazoxanide, at concentrations well below what is reached clinically, results in down regulation of c-myc, mTOR and Wnt signaling. Preliminary in vivo experiments show encouraging results. We conclude that MCTS-based screening identifies mitochondria as a potential target for cancer treatment and the anthelmintic drug nitazoxanide as a promising candidate. Citation Format: Wojciech Senkowski, Xiaonan Zhang, Maria Hägg Olofsson, Stig Linder, Rolf Larsson, Mårten Fryknäs. A spheroid-based screen identifies mitochondrial targeting as a promising strategy for cancer treatment and drug repositioning. [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 3781. doi:10.1158/1538-7445.AM2014-3781

The broad research use of organoids from high-grade serous ovarian carcinoma (HGSC) has been hampered by low culture success rates and limited availability of fresh tumor material. Here we describe a method for generation and long-term expansion of HGSC organoids with efficacy markedly improved over previous reports (55% vs. 23-38%). We established organoids from cryopreserved material, demonstrating the feasibility of using viably biobanked tissue for HGSC organoid derivation. Genomic, histologic and single-cell transcriptomic analyses revealed that organoids recapitulated genetic and phenotypic features of original tumors. Organoid drug responses correlated with clinical treatment outcomes, although in culture conditions-dependent manner and only in organoids maintained in human plasma-like medium (HPLM). Organoids from consenting patients are available to the research community through a public biobank and organoid genomic data explorable through an interactive online tool. Taken together, this resource facilitates the application of HGSC organoids in basic and translational ovarian cancer research.