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Background: Immunotherapy has brought great progress with durable responses for cancers that were difficult to treat. However, it remains challenging to select sensitive patients upfront. PD-L1 expression, TMB and MSI/MSS status, do not optimally differentiate between the potential sensitivity to the individual immunotherapies. Purpose: We aim to improve stratification of patients for immunotherapy through classification of patient-specific tumor sensitivity based on 3D ex vivo drug measurement. Material: Our study includes 108 Patients with predominantly ovarian carcinoma (81/108). Tumor tissue was collected in ongoing clinical trials from biopsies, ascites or pleural fluid. Five immune checkpoint inhibitors and a STING pathway activator were tested. Method: Fresh tumor clusters were seeded into 384 well plates and exposed to different immunotherapies, while preserving the TME. Tumor killing and immune cell proliferation were measured using 3D image analysis. Ex vivo tumor sensitivity was classified as no response (50%), based on percentage tumor killing and statistical significance (p-value: * < 0.05, ** < 3.33e-4). Results: Differential patient response profiles were observed (Table 1). For the current cohort, we measured a highly significant immunotherapy response for ~30% of the samples. The assay has a technical success rate of 89%. Conclusion: This study reports the development of a robust ex vivo tumor testing platform that classified patient-specific sensitivity to 6 immunotherapies for over 100 patients, demonstrating the potential of ex vivo tumor testing to optimize patient stratification for immunotherapy. Discussion: The platform enables improved efficiency in clinical development of novel treatments and supports treatment decisions in the clinic. Clinical trials are ongoing to establish the correlation of our testing with patients' response to immunotherapy in lung cancer. Overview of ex vivo immunotherapy response per category 1Therapy # Samples tested % Response No response Weak Strong Very strong SEA (control) 108 43% (30%**) 62 5 19 22 CD3/CD28 Dynabeads (control) 18 33% (17%**) 12 1 5 0 Pembrolizumab 89 16% (8%**) 75 6 5 3 Nivolumab 72 10% (4%**) 65 1 5 1 Ipilimumab 97 20% (11%**) 78 4 10 5 Atezolizumab 62 15% (11%**) 53 1 4 4 Durvalumab 52 2% (0%**) 51 1 0 0 ADU-S100 60 37% (27%**) 38 5 9 8 Citation Format: Lieke Johanna Ceton, Marta Garcia Montero, Fanny Grillet, Dieudonné J. van der Meer, Willemijn Vader, Anne van Altena, Cor D. de Kroon, Nelleke Ottevanger, Judith Kroep. Classification of patient-specific sensitivity to immunotherapies by ex vivo tumor testing. [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 4113.

Simple Summary In the present work, we describe (for the first time) the use of the transmembrane protein, CD44v6, to detect CTCs from blood samples of several patients with colorectal or breast cancer. We used CD44v6 antibodies to demonstrate that live CTCs can be specifically purified from CRC patient blood samples via magnetic bead- or FACS-based isolation techniques. Finally, we demonstrated that CD44v6-positive CTCs rarely expressed EpCam, which is currently the gold standard to enumerate CTCs, suggesting the need to use a combination of markers for a more comprehensive view of CTC heterogeneity. Abstract Circulating tumor cells (CTCs) are promising diagnostic and prognostic tools for clinical use. In several cancers, including colorectal and breast, the CTC load has been associated with a therapeutic response as well as progression-free and overall survival. However, counting and isolating CTCs remains sub-optimal because they are currently largely identified by epithelial markers such as EpCAM. New, complementary CTC surface markers are therefore urgently needed. We previously demonstrated that a splice variant of CD44, CD44 variable alternative exon 6 (CD44v6), is highly and specifically expressed by CTC cell lines derived from blood samples in colorectal cancer (CRC) patients. Two different approaches—immune detection coupled with magnetic beads and fluorescence-activated cell sorting—were optimized to purify CTCs from patient blood samples based on high expressions of CD44v6. We revealed the potential of the CD44v6 as a complementary marker to EpCAM to detect and purify CTCs in colorectal cancer blood samples. Furthermore, this marker is not restricted to colorectal cancer since CD44v6 is also expressed on CTCs from breast cancer patients. Overall, these results strongly suggest that CD44v6 could be useful to enumerate and purify CTCs from cancers of different origins, paving the way to more efficacious combined markers that encompass CTC heterogeneity.

Introduction Broad application of immunotherapy to treat cancer has been hampered by low response rates in the clinics (~20%) and high failure rates of clinical trials. The lack of translational preclinical models that accurately replicate human immunological complexity is one of the reasons leading to ineffective candidates in clinical trials. There is an urgent need of advanced preclinical models that can enhance clinical trial success rate and improve clinical impact of immunotherapies. We present a novel 3D Ex vivo Patient Tissue platform combining short-term 3D ex vivo tumor culture system with high content image (HCI)-based analysis. The platform preserves tumor heterogeneity and tumor immune context, including (exhausted) tumor infiltrating lymphocytes. We report a quantification of ex vivo tumor responses to a panel of immunotherapies, including checkpoint inhibitors, tested on patient tissues from various cancers. Methods Patient tumor tissues were obtained from hospitals and tissue providers and processed within 24 hours to preserve the tumor microenvironment (TME). Tumor clusters from ovarian, cervical, breast, and non-small cell lung cancer patients were embedded in a protein-rich hydrogel and exposed to panels of immunomodulatory drugs in a 384-well format for 5-7 days. Phenotypic effects of the drugs on physiologically relevant morphological changes, such as tumor cell killing and immune cell proliferation, were measured using our proprietary automated HCI analysis platform. In addition, IHC and FACS analysis of primary samples as well as cytokine measurements were performed. Results Responses of ex vivo tissues to immunotherapies targeting various pathways (e.g., ipilimumab, pembrolizumab and STING agonists), combination treatments and selected controls were evaluated by automated phenotypic analysis platform. Based on their sensitivity, patient tissues were classified as immunotherapy responders or non-responders. A deeper investigation of the responder tissues was performed with IHC and FACS to pinpoint the critical TME components necessary for immunotherapy sensitivity. In addition, cytokine profiling was done in supernatants from treated ex vivo cultures to confirm the functional HCI readouts. Accurate and reproducible response evaluation demonstrated the feasibility of preclinical immunotherapy drug testing on primary patient material using this platform. Conclusion The 3D ex vivo patient tissue platform successfully combined drug testing protocols using fresh patient tumor tissue with preserved TME and advanced 3D HCI analysis. The platform offers a rapid, reliable and patient-relevant approach to test (clinical) immunotherapies for different solid tumours. It has the potential to significantly improve the preclinical evaluation of immunotherapies and support the decision-making process during progression of drug candidates to the clinic. Citation Format: Nataliia Beztsinna, Niels Meesters, Lidia Daszkiewicz, Fanny Grillet, Donny van der Meer, Kuan Yan, Emma Spanjaard, Willemijn Vader, Leo Price. Immunotherapy testing in 3D Ex vivo Patient Tissue Platform with preserved tumor microenvironment [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 2059.

Introduction The staggeringly high failure rate of clinical trials for oncology drugs can be attributed to many factors, including suboptimal in vitro and in vivo models that fail to recapitulate the complexity of the human tumor microenvironment (TME) or predict patient response. Translational human 3D cell culture models, such as patient-derived tumor organoids, have begun to bridge the gap between tissue culture systems and patients in the clinic. However, even in these advanced models, the endogenous cells of the TME, such as tumor infiltrating lymphocytes (TILs), fibroblasts, macrophages and other immune cells, are absent. These TME components have been shown to express important drug targets and play a critical role in both tumor progression and modulation of the response to drugs. Here we present a novel patient avatar platform that combines a short-term 3D ex vivo tumor culture system with high content image (HCI)-based analysis. Patient tumor tissues from pleural fluid, ascites, surgical resections or biopsy were tested ex vivo to preserve tumor heterogeneity and resident immune cells, removing the need for artificial co-culture systems. This study entailed a detailed quantification of tumor sensitivity to targeted therapies, standard of care, and novel (immune) drugs and drug combinations, tested on different cancer types. Methods Patient tumor tissues were obtained from ongoing clinical trials in the Netherlands as well as from commercial tissue providers, and processed within 24 hours to preserve the native tumor heterogeneity and TME. Freshly isolated tumor cells from ovarian, breast cancer and non small cell lung cancer (NSCLC) patients were embedded in a protein-rich hydrogel and exposed to panels of single and combination drug treatments at different concentrations in a 384-well format for 5-7 days. Effects of drugs and combination therapies on physiologically relevant morphological features, such as tumor cell killing, growth arrest, invasion and immune cell proliferation, were measured using our proprietary automated HCI analysis platform. Results Patient-specific drug sensitivity profiles were generated based on the response to a broad range of drugs including standard of care (e.g., platinum, paclitaxel, gemcitabine), targeted therapies (e.g., PARP and EGFR inhibitors), and activity of immunomodulatory drugs (e.g., ipilimumab, pembrolizumab and STING agonists). Accurate and reproducible response evaluation demonstrates the feasibility of preclinical drug testing on patient primary material within the platform. Conclusion Our platform successfully combined proven ex vivo drug testing protocols using fresh patient tumor tissue with preserved TME components and advanced 3D HCI analysis. Our approach offers a rapid, reliable and patient-relevant approach to test various candidate compounds (e.g., antibodies, antibody-drug conjugates and small molecules) for various cancer types. It has the potential to significantly improve the preclinical evaluation of drugs, and also to improve the success rate of clinical trials. Citation Format: Nataliia Beztsinna, Fanny Grillet, Niels Meesters, Donny van der Meer, Lidia Daszkiewicz, Kuan Yan, Emma Spanjaard, Willemijn Vader, Leo Price. Novel patient avatar platform for oncology drug testing using 3D ex vivo models derived from fresh patient tumor tissues [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2021 Oct 7-10. Philadelphia (PA): AACR; Mol Cancer Ther 2021;20(12 Suppl):Abstract nr P115.

Highlights • Secretion of progastrin is a signature event of early malignant transformation in the colon. • In the healthy epithelium, progastrin is produced by a subset of enteroendocrine cells expressing both Bmi1 and Prox1. • LGR5-high intestinal stem cells are a primary source of progastrin production in early mouse and human intestinal adenomas., Progastrin is an unprocessed soluble peptide precursor with a well-described tumor-promoting role in colorectal cancer. It is expressed at small levels in the healthy intestinal mucosa, and its expression is enhanced at early stages of intestinal tumor development, with high levels of this peptide in hyperplastic intestinal polyps being associated with poor neoplasm-free survival in patients. Yet, the precise type of progastrin-producing cells in the healthy intestinal mucosa and in early adenomas remains unclear. Here, we used a combination of immunostaining, RNAscope labelling and retrospective analysis of single cell RNAseq results to demonstrate that progastrin is produced within intestinal crypts by a subset of Bmi1+/Prox1+/LGR5low endocrine cells, previously shown to act as replacement stem cells in case of mucosal injury. In contrast, our findings indicate that intestinal stem cells, specified by expression of the Wnt signaling target LGR5, become the main source of progastrin production in early mouse and human intestinal adenomas. Collectively our results suggest that the previously identified feed-forward mechanisms between progastrin and Wnt signaling is a hallmark of early neoplastic transformation in mouse and human colonic adenomas.

International audience; Colorectal cancer lethality usually results from post-treatment relapse in the majority of stage II-IV patients, due to the enhanced resistance of Cancer Stem Cells (CSCs). Here, we show that the nuclear receptor Pregnane X Receptor (PXR, NR1I2), behaves as a key driver of CSC-mediated tumor recurrence. First, PXR is specifically expressed in CSCs, where it drives the expression of genes involved in self-renewal and chemoresistance. Clinically, high levels of PXR correlate with poor recurrence-free survival in a cohort of >200 stage II/III colorectal cancer patients treated with chemotherapy, for whom finding biomarkers of treatment outcome is an urgent clinical need. shRNA silencing of PXR increased the chemo-sensitivity of human colon CSCs, reduced their self-renewal and tumor-initiating potential, and drastically delayed tumor recurrence in mice following chemotherapy. This study uncovers PXR as a key factor for CSC self-renewal and chemoresistance and targeting PXR thus represents a promising strategy to minimize colorectal cancer relapse by selectively sensitizing CSCs to chemotherapy.

Resume Le processus metastatique est defini comme « une dissemination de cellules neoplasiques dans un site secondaire non contigu et distant, au sein duquel ces cellules proliferent pour former une masse extravasculaire de cellules incompletement differenciees ». La majorite des deces imputables aux cancers solides est la consequence de l’apparition et du developpement de metastases disseminees qui sont responsables a terme de la dysfonction d’organes vitaux. Dans cette revue un certains de nombre de points, soit recemment decouverts soit sujets a controverse, sont abordes. En effet il sera discute de la transition epithelio-mesenchymateuse (EMT), des cellules tumorales circulantes, de la dormance tumorale, de la colonisation dans un organe distant et enfin des cellules souches cancereuses.

Introduction While systemic treatment has been quite static in the past decade in ovarian cancer, the challenge for the coming years is to stratify patients for optimal and new treatment strategies, such as targeting DNA damage response pathways, or immune modulation. Patient primary tumor from resections, biopsy or ascites can be cultured such that tumor heterogeneity and resident immune cells are preserved. These can be used to predict treatment response in patients or to test new cancer therapies. Here we present a high throughput short-term 3D ex-vivo culture platform combined with high content image-based analysis. This platform allows visualization and quantification of standard of care therapy and new drugs on fresh and cryopreserved patient derived ovarian cancer tumoroids. Methods Patients with advanced primary or relapsing ovarian carcinoma are included in ongoing multi-center clinical trials in the Netherlands. Ovarian cancer derived tumoroids, freshly isolated or cryopreserved, are embedded in a protein-rich hydrogel and exposed to up to 20 drugs during a short term 3D culture. The automated high content imaging analysis platform measures a large panel of tumoroid morphological features, and responses such as tumor cell killing, growth arrest and local invasion are measured to define the response for each drug. In addition, clinical response data from the ongoing trials are collected to be correlated to clinical outcome of patients treated with systemic therapy. Results We present results of drug sensitivity testing in patient-derived primary tumor cells of fresh and cryopreserved ascites and solid tumor. Patient-specific drug sensitivity is identified for up to 20 drugs including standard of care (e.g. carboplatin, paclitaxel) and novel treatment strategies (e.g. PARP inhibitors). Activity of immunomodulatory drugs were also monitored successfully in vitro. Accurate response evaluation demonstrates the feasibility of high-throughput drug screening on patient primary material within OcellO's platform. Our results show a high reproducibility in testing freshly isolated or cryopreserved material, highlighting the potential of a cryopreserved collection of patient derived primary material to test new drugs. Conclusion OcellO's advanced 3D image analysis of patient primary material represents a rapid and biologically relevant approach to test various candidate compounds (e.g. antibodies, antibody-drug conjugates and small molecules) for ovarian cancer. The availability of drug response data combined with patient clinical data, including BRCA mutation and cytology/histology pathologist evaluation is expected to support drug discovery, improve the efficiency of clinical trials and establish predictive testing in the clinical setting. Citation Format: Fanny Grillet, Abbas Jariani, Juul Overkamp, Lidia Daszkiewicz, Kuan Yan, Leo S. Price, Willemijn Vader. Ovarian cancer ex vivo 3D tumor cultures predict patient specific drug sensitivity [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 4159.

Posttreatment recurrence of colorectal cancer, the third most lethal cancer worldwide, is often driven by a subpopulation of cancer stem cells (CSC). The tight junction (TJ) protein claudin-2 is overexpressed in human colorectal cancer, where it enhances cell proliferation, colony formation, and chemoresistance in vitro. While several of these biological processes are features of the CSC phenotype, a role for claudin-2 in the regulation of these has not been identified. Here, we report that elevated claudin-2 expression in stage II/III colorectal tumors is associated with poor recurrence-free survival following 5-fluorouracil–based chemotherapy, an outcome in which CSCs play an instrumental role. In patient-derived organoids, primary cells, and cell lines, claudin-2 promoted colorectal cancer self-renewal in vitro and in multiple mouse xenograft models. Claudin-2 enhanced self-renewal of ALDHHigh CSCs and increased their proportion in colorectal cancer cell populations, limiting their differentiation and promoting the phenotypic transition of non-CSCs toward the ALDHHigh phenotype. Next-generation sequencing in ALDHHigh cells revealed that claudin-2 regulated expression of nine miRNAs known to control stem cell signaling. Among these, miR-222-3p was instrumental for the regulation of self-renewal by claudin-2, and enhancement of this self-renewal required activation of YAP, most likely upstream from miR-222-3p. Taken together, our results indicate that overexpression of claudin-2 promotes self-renewal within colorectal cancer stem-like cells, suggesting a potential role for this protein as a therapeutic target in colorectal cancer. Significance: Claudin-2-mediated regulation of YAP activity and miR-222-3p expression drives CSC renewal in colorectal cancer, making it a potential target for therapy. Cancer Res; 78(11); 2925–38. ©2018 AACR.

Background: Many new drugs and treatment strategies are in development for ovarian cancer. While systemic treatment has been quite static in the past decade, the challenge for the coming years is to stratify ovarian cancer patients for optimal treatment strategies, like targeting DNA damage response pathways or immune modulatory approaches. Patient primary tumor material collection offers cryopreserved viable primary tumor cell clusters, containing the whole heterogeneity found in the patient ascites (including potential immune cells) to predict treatment response in patients or to test new cancer therapies. Here we present a high throughput short-term 3D ex-vivo culture platform combined with high content image-based analysis. This platform allows visualization and quantification of standard of care therapy and new drugs on fresh and cryopreserved patient derived ovarian cancer tumoroids. Methods: Patients with ovarian carcinoma are included in ongoing multi-center clinical trials in the Netherlands. Ovarian cancer derived primary tumoroids, freshly isolated or cryopreserved, are embedded in a protein-rich hydrogel and exposed to up to 20 drugs during a short term 3D culture. The automated high content imaging analysis platform measures a large panel of tumoroid morphological features, and responses such as tumor cell killing, growth arrest and local invasion are measured to define the response for each drug. In addition, clinical response data from the ongoing trials will be correlated to clinical outcome of patients treated with systemic therapy. Results:We present first results of drug sensitivity in patient-derived primary tumor cells of fresh and cryopreserved ascites. Patient-specific drug sensitivity is identified for up to 20 drugs including standard of care (e.g. carboplatin, paclitaxel) and novel treatment strategies (e.g. PARP inhibitors). Accurate response evaluation demonstrates the feasibility of high-throughput drug screening on patient primary material within OcellO’s platform. Our results show a high reproducibility in testing freshly isolated or cryopreserved material, highlighting the potential of a cryopreserved collection of patient derived primary material to test new drugs. Conclusion: OcellO’s advanced 3D image analysis of patient primary material represents a rapid and biologically relevant approach to test various candidate compounds (e.g. antibodies, antibody-drug conjugates and small molecules) for ovarian cancer. The availability of drug response data combined with patient clinical data, including BRCA mutation and cytology/histology pathologist evaluation is expected to support drug discovery, improve the efficiency of clinical trials and establish predictive testing in the clinical setting. Citation Format: Fanny Grillet, Abbas Jariani, Juul Overkamp, Lidia Daszkiewicz, Kuan Yan, Leo Price, Willemijn Vader. Patient specific drug sensitivity to novel treatment approaches in ovarian cancer ex vivo 3D tumor cultures [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference on Molecular Targets and Cancer Therapeutics; 2019 Oct 26-30; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2019;18(12 Suppl):Abstract nr B139. doi:10.1158/1535-7163.TARG-19-B139

Background Malignant pleural mesothelioma (MPM) is almost exclusively linked to asbestos exposure with a latency period that is usually more than 30 years. Once diagnosis is made, the prognosis is poor. Systemic treatment combines an antifolate combined with platinum, resulting in modest responses for 40% of the patients. Recently, the addition of bevacizumab to this combination has shown to be of benefit. To date it is impossible to identify the patients who will respond before start of treatment. Second line treatments (either gemcitabine or vinorelbine) have shown to be of benefit in only a small percentage of patients. Tools to improve stratification for systemic treatment in the clinic would be of great value. Our technology based on image analysis of 3D tumor cultures accommodates accurate evaluation of drug sensitivity with small amounts of heterogeneous tumor material (tumor, pleural effusion). We have initiated clinical trials to establish the value of the platform for treatment response prediction for cancer patients, including mesothelioma. Based on the results from these trials we plan to develop diagnostics to predict drug responses for cancer patients. Methods 3D mesothelioma cultures embedded in a protein-rich hydrogel are generated from pleural effusion of chemo naïve patients, and exposed to standard-of-care therapies, targeted therapies and drug combinations. An automated high content screening platform measures cell and tissue morphology, and reports responses such as tumor cell killing, growth arrest, apoptosis, and local invasion. Morphological features are per drug selected as standard read-outs for the response. We correlate clinical response on standard of care drugs with drug response of patients’ tumor cultures. Results We present first results of drug sensitivity in patient derived ex vivo 3D pleural effusion cultures. Results are generated within three weeks after the pleural fluid is obtained from the patients. Take rates are >80%. Standard-of-care therapies were tested and results are compared with clinical response. Patient specific drug responses are identified. Conclusion Ex vivo 3D mesothelioma cultures enable drug sensitivity testing in weeks. Measuring patient-specific treatment responses to standard-of-care drugs, and potential novel therapeutic approaches offer opportunities to personalize treatment for mesothelioma patients. Ongoing trials will reveal the correlation of our in vitro test with treatment responses and relevant diagnostics parameters in the clinic. Keywords: predictive test, mesothelioma, ex vivo 3D tumor cultures Citation Format: Robin Cornelissen, Daphne Dumouline, Jan vd Thusen, Sander Basten, Fanny Grillet, Kuan Yan, Leo Price, Willemijn Vader. Predicting drug sensitivity in patient derived ex vivo 3D mesothelioma cultures [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 55.