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Background: The need to better understand the molecular underpinnings of the heterogeneous outcomes of patients with prostate cancer is a pressing global problem and a key research priority for Movember. To address this, the Movember Global Action Plan 1 Unique tissue microarray (GAP1-UTMA) project constructed a set of unique and richly annotated tissue microarrays (TMA) from prostate cancer samples obtained from multiple institutions across several global locations. Methods: Three separate TMA sets were built that differ by purpose and disease state. Results: The intended use of TMA1 (Primary Matched LN) is to validate biomarkers that help determine which clinically localized prostate cancers with associated lymph node metastasis have a high risk of progression to lethal castration-resistant metastatic disease, and to compare molecular properties of high-risk index lesions within the prostate to regional lymph node metastases resected at the time of prostatectomy. TMA2 (Pre vs. Post ADT) was designed to address questions regarding risk of castration-resistant prostate cancer (CRPC) and response to suppression of the androgen receptor/androgen axis, and characterization of the castration-resistant phenotype. TMA3 (CRPC Met Heterogeneity)'s intended use is to assess the heterogeneity of molecular markers across different anatomic sites in lethal prostate cancer metastases. Conclusions: The GAP1-UTMA project has succeeded in combining a large set of tissue specimens from 501 patients with prostate cancer with rich clinical annotation. Impact: This resource is now available to the prostate cancer community as a tool for biomarker validation to address important unanswered clinical questions around disease progression and response to treatment.

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.

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.

Extracellular matrix (ECM) interactions contribute to cancer metastasis and chemotherapy resistance by regulating invasive cancer growth and apoptosis evasion. However, the key ECM pathways activated during cancer evolution, including tumor metastasis and tissue responses to chemotherapy, remain to be systematically identified. Using a unique longitudinal high-grade serous ovarian carcinoma (HGSOC) cohort to understand the ECM deposition and remodeling in response to disease development and chemotherapeutics, we conducted ECM proteome, i.e., matrisome, analysis on HGSOC tissues of distinct anatomic sites from treatment-naïve and post-chemotherapy primary samples. Together with an unbiased functional ECM screen for platinum-treatment responses, we report how the solid tumor ECM evolves with disease progression and how specific stromal ECM proteins, including fibronectin, promote ovarian cancer cell motility, invasion, and resistance to platinum treatment. Importantly, we observed these specific stromal ECM proteins to be expressed already at HGSOC omental microlesions with remarkable desmoplastic reaction, as shown by immunohistochemistry of longitudinally collected patient cohort samples. Further, by using clinical The Cancer Genome Atlas (TCGA) microarray data we found that at transcriptional level the overexpression of a specific ECM protein, with platinum-induced cancer cell spreading and migratory response, also associated with poor survival in the treatment-naïve TCGA patient data, and was specifically upregulated after chemotherapy in our longitudinal cohort. In ovarian cancer cell cultures and in patient-derived 3D HGSOC organoid cultures, this specific ECM-rich microenvironment associated with an epithelial-to-mesenchymal transition transcriptional markers coupled to a stem-like morphology and properties with increased resistance. These results underline the importance of cell-matrix communication in invasion and chemotherapy response, revealing certain ECM proteins as important promoters of aggressiveness and chemotherapy resistance in the metastatic HGSOC lesions. Citation Format: Elina A. Pietilä, Jordi Gonzales-Molina, Lidia Moyano-Galceran, Laura Lehtinen, Pauliina Turunen, Tomas Santos Martins, Ville Rantanen, Antti Häkkinen, Sanaz Jamalzadeh, Kaiyang Zhang, Tarja Lamminen, Katja Kaipio, Johanna Hynninen, Sakari Hietanen, Seija Grénman, Rainer Lehtonen, Sampsa Hautaniemi, Olli Carpén, Kaisa Lehti. Extracellular matrix proteins increase invasive growth and chemotherapy resistance of ovarian cancer cells [abstract]. In: Proceedings of the AACR Special Conference on Advances in Ovarian Cancer Research; 2019 Sep 13-16, 2019; Atlanta, GA. Philadelphia (PA): AACR; Clin Cancer Res 2020;26(13_Suppl):Abstract nr A64.

Tumor heterogeneity in high-grade serous ovarian cancer (HGSOC) stems from highly variable copy number changes that lack shared actionable drug targets and contribute to inevitable recurrence and death. To unveil the vast heterogeneity in HGSOC, and understand the molecular changes that occur during chemotherapy, we analyzed single-cell transcriptomes of 49 dissociated HGSOC tumor or ascites specimens, along with six normal postmenopausal fimbria/tuba specimens as controls. This set contains 13 pairs of samples from patients who underwent neoadjuvant chemotherapy, taken before treatment (primary), and after three cycles of standard carboplatin-paclitaxel chemotherapy (interval). Clustering single-cell transcriptomes based on acknowledged cell type markers revealed epithelial cancer cells, as well as diverse stromal and immune cell types. The cell type proportions displayed high variability across HGSOC tumors, intriguingly with lowest tumor purity in patients with short platinum-free interval. To identify the impact of chemotherapy on transcriptional programs, we extracted shared transcriptomic differences in the cancer cells of seven primary-interval pairs. Regardless of the clinical response, three cycles of chemotherapy induced an oxidative stress response program that inhibits apoptosis, while inducing genes involved in MHC II antigen presentation to T helper cells. Next, to study how cell states change during chemotherapy, we ordered the cells in pseudotime based on their transcriptomic similarity, revealing a mixture of primary and interval cells in all but two primary-interval pairs. Furthermore, based on inferred copy number analysis, we estimate that genomic differences contribute variably, between 20%, to the overall transcriptomic differences between the primary and interval pairs. These results suggest that pre- and post-chemotherapy states are not completely separate and that genomic evolution has a highly variable effect on the state differences, providing support for an intrinsic chemoresistance model with genomic and nongenomic components. To further the understanding of intrinsic resistance, we next focused on patients’ clinical response mechanisms in HGSOC cancer cells. Genes encoding well-known serum markers of HGSOC, WFDC2 and MUC16, were expressed at higher levels in primary specimens of patients with incomplete response, whereas patients with complete response displayed higher expression of genes related to chromatin repression/heterochromatin. Finally, we focused on tumor resectability to understand the transcriptomic patterns underlying HGSOC metastasis. Already prior to chemotherapy, patients with residual disease showed induced FOXO3-driven regulon linked to quiescence, stress resistance, and stemness, whereas patients with no residual disease express increased level of E2F1/DP1-driven regulon linked to BRCAness, high proliferation, and high metabolic rate. Citation Format: Erdogan Pekcan Erkan, Jun Dai, Kaiyang Zhang, Katja Kaipio, Tarja Lamminen, Naziha Mansuri, Lasse Suominen, Amjad Alkodsi, Kaisa Huhtinen, Sakari Hietanen, Johanna Hynninen, Seija Grénman, Olli Carpén, Sampsa Hautaniemi, Anna Vähärautio. Singling out tumor heterogeneity and chemoresistance in high-grade serous ovarian cancer [abstract]. In: Proceedings of the AACR Special Conference on Advances in Ovarian Cancer Research; 2019 Sep 13-16, 2019; Atlanta, GA. Philadelphia (PA): AACR; Clin Cancer Res 2020;26(13_Suppl):Abstract nr A40.

High-grade serous ovarian cancer (HGSOC) is the most common cause of gynecologic-cancer-associated death. Although HGSOC is initially extremely sensitive to platinum-based chemotherapy, relapse and progression to chemotherapy resistance are frequently seen, resulting in the low 5-year survival rate of this disease. Therefore, we aim to establish a feasible methodology that combines genome editing and single-cell RNA-seq to high-throughput screen new potential targets that could be utilized alone or combined with platinum for HGSOC treatment. Firstly, we constructed several inducible Cas9-expressed HGSOC cell lines, including both platinum-sensitive and -resistant cells. Secondly, we transduced these cell lines with a pooled sgRNA lentivirus consisting of 105 non-target scrambled sgRNAs and sgRNAs against 188 genes (10 sgRNAs per gene) that are currently targets of FDA/EMA approved drugs or drugs undergoing clinical trials in ovarian cancer treatment. Our analysis of multiple HGSOC dissociated tumors by single-cell RNA-seq has revealed intertumoral heterogeneity in expression of these drug targets in vivo. The majority of these 188 genes, approximately two thirds, are expressed in low level in most epithelial ovarian cancer (EOC) cells. Only 4 genes (AKT1, IL6, C1QA, INHBA) show relatively high expression, and only in a small subset of EOC cells. Also, the remaining drug target genes that are expressed show intertumoral heterogeneity, e.g., high expression of PARP-1 in tumors that are potentially deficient in homologous recombination repair. To elucidate the functionality of these drug targets, we will next analyze the transcriptomic effects of specific gene CRISPR/Cas9 knockouts in these HGSOC cell lines by droplet-based single-cell RNA-seq. From the data, we will compare the differences between platinum-sensitive and -resistant cell lines after the gene knockouts to identify the correlations between specific genetic perturbations and platinum resistance. As a future perspective, we will continue optimizing our platform for screening other potential targets. Overall, this study provides a comprehensive and unbiased approach for potential HGSOC target screening. Note: This abstract was not presented at the conference. Citation Format: Jun Dai, Erdogan Pekcan Erkan, Kaiyang Zhang, Katja Kaipio, Tarja Lamminen, Kaisa Huhtinen, Johanna Hynninen, Seija Grenman, Olli Mikael Carpen, Sampsa Hautaniemi, Anna Vähärautio. High-throughput screening of new potential targets for high-grade serous ovarian cancer treatment. [abstract]. In: Proceedings of the AACR Conference: Addressing Critical Questions in Ovarian Cancer Research and Treatment; Oct 1-4, 2017; Pittsburgh, PA. Philadelphia (PA): AACR; Clin Cancer Res 2018;24(15_Suppl):Abstract nr B40.

Objective: High-grade serous ovarian cancer (HGSOC) remains the most lethal gynecologic malignancy. More than 50% of high-grade serous ovarian cancer (HGSOC) patients die due to recurrent disease within 5 years. Recurrent tumor clones are selected by chemotherapy treatment from heterogeneous subpopulations of cancer cells, and differ phenotypically. The objective of current study is to develop a high-throughput chemical biology approach to analyze phenotypic heterogeneity in HGSOC following progression of the disease. Methods: We utilize the Drug Sensitivity and Resistance Testing (DSRT) platform to assess the effects of 528 approved and investigational anti-cancer agents on primary HGSOC isolates. Samples are collected from ascites and/or solid high-grade ovarian tumors from the patients at different disease stages. DSRT profiling of the samples integrates viability and toxicity readouts with high-content imaging in order to assess the drug responses of phenotypically different cell subpopulations of epithelial cancer cells and stromal component. Results: Drug sensitivity profiling of ten primary and six relapse patient samples revealed heterogeneous drug sensitivity profiles characterized by common responsiveness to HDAC inhibitors, CDK inhibitors, and PI3K/mTOR inhibitors (mean drug sensitivity score (DSS) > 10). Relapse HGSOC samples demonstrated general trend towards resistance to a broad spectrum of chemotherapeutics and targeted drugs. Imaging-based DSRT profiling revealed that in both primary and relapse ascites-derived cancer cells the response to platinum drugs and DNA damaging agents associated with upregulation of transcriptional regulators of stemness, including BMI1, SOX2 and NANOG. The ascites-derived CK18-expressing epithelial cells collected from the same patient a) before chemotherapy, b) after 6 cycles of Carboplatin treatment and c) at the relapse demonstrated an increasing sensitivity to aldehyde dehydrogenase inhibitor disulfiram. These preliminary findings suggest that increase in a subpopulation of cancer cells with stem-like characteristics may accompany recurrence in HGSOC. Conclusions: Compound sensitivity testing combined with high-content imaging appears an informative tool to explore therapeutic vulnerabilities in chemoresistant subpopulations of ovarian cancer cells, and improves the accuracy of drug sensitivity profiling. Chemical biology screening of prospectively collected primary HGSOC samples can also help identifying phenotypic changes associated with chemoresistance development. Citation Format: Daria R. Bulanova, Katja Kaipio, Tarja Lamminen, Aleksandr Ianevski, Tero Aittokallio, Olli Carpen, Krister Wennerberg. Chemical biology approach to phenotypic intra-tumor heterogeneity in high-grade serous ovarian cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 875.

Resistance to chemotherapy poses a major clinical problem in the treatment of ovarian cancer. The efficacy of platinum- based chemotherapy relies on defective DNA repair mechanisms, and increased expression of homologous recombination (HR)/DNA repair genes in primary tumor samples is associated with improved chemoresponse. However, the effects of neo-adjuvant chemotherapy (NACT) on the expression DNA repair genes in cancer cells are poorly understood. In addition, high-grade serous ovarian cancers (HGSOCs) display significant inter- and intratumoral heterogeneity, and the identification of mechanisms of drug resistance has been hampered by the lack of data at the single cell resolution. We collected 34 primary and 20 interval tumor samples from 15 patients treated for HGSOC at the Turku University Hospital, Finland. Tumor tissue total RNA was sequenced with Illumina HiSeq. For this study, we selected 363 genes selected due to their validated role in DNA repair pathways and compared their expression profiles between primary and interval samples. The treatment naïve primary tumor samples highly expressed genes related to HR, such as BRCA2, RAD54, PARPBP, FANCD2, and POLQ. Whereas after NACT the interval samples highly expressed genes regulating cell cycle, inflammatory response, and the drug efflux pump ABCB1. Moreover, higher expression of BRCA2 or POLQ in the interval samples correlated with poor primary therapy outcome (p To reveal the transcriptomics landscapes of treatment naïve and NACT treated HGSOCs at the single cell level, we performed single cell mRNA sequencing of primary and interval samples using the Fluidigm C1 Platform. Unsupervised clustering revealed eight distinct cell subpopulations, of which two clusters were annotated HGSOC cells based on enriched expression of cancer specific genes. In global gene expression analysis of 98 HGSOC cells, the most differentially expressed genes in primary HGSOC cells were heat shock proteins reflecting active protein synthesis. By contrast, the cancer cells from interval samples highly expressed genes regulating the cell cycle and immune response. Of the DNA repair pathway genes, the interval samples expressed lower levels of HES1, a transcription factor that is known to promote cancer progression and stemness. The herein elucidated DNA repair pathway transcriptomics landscape at single-cell and whole tumor level provides novel targets for overcoming chemoresistance in HGSOC. Citation Format: Anniina Farkkila, Kaiyang Zhang, Katja Kaipio, Tarja Lamminen, Rainer Lehtonen, Johanna Hynninen, Seija Grénman, Olli Carpén, Sampsa Hautaniemi, Anna Vähärautio. Single-cell transcriptomics reveals differential DNA repair signatures after chemotherapy in high-grade serous ovarian cancer [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 1428. doi:10.1158/1538-7445.AM2017-1428

Ovarian cancer is the most lethal gynecologic malignancy in developed countries. The incidence is 1 in 70 women, and 5-year survival rate below 50 %. Although major achievements have been reached in understanding the molecular alterations underlying the disease, the discoveries need yet to be integrated into clinical practice. A major obstacle in this process may be the translation of in vitro findings into diagnostic tests and novel therapeutic approaches. Well-characterized and representative preclinical mouse models that recapitulate the heterogeneity of tumors in patients are crucial in narrowing the gap between experimental and clinical studies. The aim of this work was to test and validate various mouse xenograft models for their applicability in ovarian cancer research. We tested several xenograft mouse models using (1) established commercially available ovarian cancer cell lines, (2) primary cell lines originating from high grade serous ovarian cancers or ascitic fluid, and (3) primary or metastatic tumor tissue directly from operation. Cell growth, tumor take rate and histopathological correlation between mouse and human tumors were applied for selection of commercial cell lines in xenograft experiments into athymic nude mice. Intrabursal orthotopic inoculations were used to model early, and intraperitoneal inoculations to model late stage ovarian cancer. Translational aspects were studied by comparing the histopathology between mouse tumors and human ovarian cancer specimens and by testing the utility of serum biomarkers CA-125 and HE-4 in mouse models. The mouse xenograft models faithfully represented the morphology of human serous ovarian cancer and many of the tissue markers were similarly expressed in human and mouse tumors. Importantly, the serum biomarker analysis developed in this study allowed follow-up of the tumor burden ex vivo. Such biomarker will be useful in studying drug responses and other tumor modifying therapies. The primary cell lines were inoculated subcutaneously to immunodeficient NOD-SCID and NOG mice in order to enrich and characterize ovarian cancer initiating cells (OCICs). Due to the low rate of tumor formation of in vitro cultured OCICs, we have started to use patient derived tumor xenograft (PDTX) transplantations. In this model fresh surgical tissue, sectioned in app. 3 mm3 pieces are implanted into the flanks of immunodeficient NOG mice. The preliminary results show a take rate of 75 % with PDTXs. Histopathological comparisons of original human specimen to mouse tumors reveals that this model also recapitulates the human microenvironment in PDTX modelling. Also, the PDTX model appears to increase the probability for successfully establishing novel primary cell lines. In conclusion, mouse xenograft models offer translational and important validation methods for pre-clinical cancer drug development and diagnostics. Citation Format: Tarja Lamminen, Katja Kaipio, Piia Mikkonen, Pia Roering, Olli Carpén. Xenograft mouse models for ovarian cancer. [abstract]. In: Proceedings of the AACR Special Conference on Advances in Ovarian Cancer Research: From Concept to Clinic; Sep 18-21, 2013; Miami, FL. Philadelphia (PA): AACR; Clin Cancer Res 2013;19(19 Suppl):Abstract nr B43.