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Despite recent advances in cancer immunotherapy, there have been limitations in cancer treatment and patient survival due to a lack of antigen recognition and immunosuppressive tumor microenvironment. To overcome this issue, we have shown that miRNA modified tumor-derived Extracellular Vesicles (mt-EVs) would be an advantageous prospect since they are tumor specific and associated antigen sources which cause increase in maturation and antigen-presenting function of dendritic cells. Also, miRNAs are promising candidates for cancer therapy because of their ability to control several host immune subsets to respond against cancer cells as well as tumor microenvironment remodeling. Here, we report that mt-EVs containing tumor specific antigens loaded with miRNAs (Let-7i, miR-142 and, miR-155) could increase the survival rate of tumor-bearing mice and induce reduction in tumor growth. Importantly, the administration of mt-EVs elicited cytotoxic T cells with increasing in IFNγ and Granzyme B production ability. Notably, intramuscular (IM) injection of let7i, miR142-EVs had a significant effect on dendritic cell (DC) maturation and T cell activation along with tumor shrinkage. Collectively, our findings suggest that administration of miRNA containing EVs may be effective immunotherapy against solid tumors.

Recent advances in cancer immunotherapy had a positive impact on the life expectancy of patients for a large range of clinical indications. With new treatment strategies and druggable targets being identified at an increasing pace, the number of patients eligible for cancer immunotherapy is expected to expand steadily. However, promising therapeutic developments face hurdles in translating preclinical findings into therapy since conventional 2D cancer models hold low clinical predictive value. HUB developed an innovative alternative, build on the discovery that adult stem cells proliferate and organise into three-dimensional organotypic structures when they are embedded into extracellular matrix. Patient specific organoids are generated from healthy and malignant tissues and stored as biobanks with high quality and reproducibility. HUB Organoids recapitulate complex characteristics of the original parental tissue, including molecular heterogeneity, and morphological and functional traits. Since cancer progression and responses to immunotherapy are governed by immune cell interactions in the tumour microenvironment, we developed an assay in which various types of tumour organoids are co-cultured with T cells modified with chimeric antigen receptors (CAR T cells) to assess the cytotoxic potential. CAR-T cell therapy has been demonstrated to be efficacious against many hematological malignancies, however therapeutic application to treat solid cancers remains challenging. Our organoid-T cell co-culture assay offers a good platform to study the response of tumour organoids on the CAR T cells and will greatly contribute to our understanding of the critical factors that determine a successful CAR T cell therapy. In collaboration with GSK, HUB tested the cytotoxicity of GSK CAR T cells which target specific target antigen on human colorectal cancer (CRC) organoids. Six HUB CRC organoid models were selected based on differential expression levels of target antigen that was confirmed by FACS antibody staining. To evaluate GSK CAR T cell mediated organoid killing, caspase 3/7 expression was monitored at various timepoints and IFNγ secretion was measured by ELISA. For control, staurosporine and nonspecific-CAR T cells were included. Detected CRC-organoid killing demonstrated to be in line with antigen expression levels, indicating the specificity of the tested CAR T cells. Nevertheless, some CRC-organoid models demonstrated more resilient to CAR T cell mediated killing and further evaluation revealed upregulation of check point molecules such as PD-L1. This study reveals that tumour organoid-T cell co-culture assay hold value for preclinical development of CAR T-cell products and for evaluating tumour-evasion strategies. Moreover, this platform could potentially predict patient specific responses to CAR- T-cell therapy. Citation Format: Soura Mardpour, Mirella Georgouli, Ana Pereira Puri, Ravit Oren, Joshua Audu-Evans, Farzin Pourfarzad, Sara Brett, Pleun Hombrink, Sylvia F. Boj. Tumour organoid-t cell co-culture assay to evaluate CAR t cell cytotoxicity [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 908.

Recent advances in cancer immunotherapy had a positive impact on the life expectancy of patients for a large range of clinical indications. With new treatment strategies and druggable targets being identified at an increasing pace, the number of patients eligible for cancer immunotherapy is expected to expand steadily. However, promising therapeutic developments face hurdles in translating preclinical findings into therapy since conventional 2D cancer models hold low clinical predictive value. We developed an innovative alternative, built on the discovery that adult stem cells proliferate and organise into three-dimensional organotypic structures when they are embedded into extracellular matrix. Patient specific organoids are generated from healthy and malignant tissues and stored as biobanks with high quality and reproducibility. HUB Organoids recapitulate complex characteristics of the original parental tissue, including molecular heterogeneity, and morphological and functional traits. Since cancer progression and responses to immunotherapy are governed by immune cell interactions in the tumor microenvironment, we developed an assay in which colorectal (CRC) and non-small cell lung cancer (NSCLC) tumor organoids are co-cultured with simultaneously expanded paired tumor infiltrating lymphocytes (TIL) as a source of tumor reactive cells. Our system allows robust expansion of TIL with preserved T-cell receptor repertoires and has an establishment rate of 75%. Paired resources allow for screening of immune-modulatory therapies under physiologic conditions not depending on peptide-pulsing and allo-reactivity. For this purpose, we developed a organoid-TIL co-culture system which quantifies and characterizes tumor organoid apoptosis by combining image-based analysis with flow cytometry and cytokine release assays as measurements of T-cell cytotoxicity against tumor (and normal-matched) organoids. Our system offers a powerful tool for the development and validation of cancer immunotherapy and may help to stratify cancer patients for susceptibility of various types of immunotherapies. Citation Format: Soura Mardpour, Claudia Beaurivage, Jane Sun, Lorenz Jahn, Rene Overmeer, Lars-Eric Fielmich, Pleun Hombrink, Farzin Pourfarzad, Sylvia F. Boj. Autologous organoids and T cell co-cultures as a powerful personalized platform for immunotherapy development [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 188.

Various somatic tissue-derived mesenchymal stromal cells (MSCs) have been considered as an attractive therapeutic tool for treatment of liver diseases in which the secretion of soluble factors or extracellular vesicles (EVs) is the most probable mechanism. The experimental application of human embryonic stem cell-derived MSC (ES-MSC) increased rapidly and showed promising results, in vitro and in vivo. However, possible therapeutic effects of human ES-MSC and their EVs on Thioacetamide (TAA)-induced chronic liver injury have not been evaluated yet. Our data indicated that human ES-MSC can significantly suppress the proliferation of peripheral blood mononuclear cells compared to bone marrow (BM)-MSC and adipose (AD)-MSC. Moreover, ES-MSC increased the secretion of anti-inflammatory cytokines (i.e., TGF-β and IL-10) and decreased IFN-γ, compared to other MSCs. ES-MSC EVs demonstrated immunomodulatory activities comparable to parental cells and ameliorated cirrhosis in TAA-induced chronic rat liver injury, that is, reduction in fibrosis and collagen density, necrosis, caspase density, portal vein diameter, and transaminitis. The gene expression analyses also showed upregulation in collagenases (MMP9 and MMP13), anti-apoptotic gene (BCL-2) and anti-inflammatory cytokines (TGF-β1 and IL-10) and down-regulation of major contributors to fibrosis (Col1α, αSMA, and TIMP1), pro-apoptotic gene (BAX) and pro-inflammatory cytokines (TNFα and IL-2) following treatment with ES-MSC and ES-MSC-EV. These results demonstrated that human ES-MSC and ES-MSC EV as an off-the-shelf product, that needs further assessment to be suggested as an allogeneic product for therapeutic applications for liver fibrosis.

Natural killer (NK) cell therapy is one of the most promising treatments for Glioblastoma Multiforme (GBM). However, this emerging technology is limited by the availability of sufficient numbers of fully functional cells. Here, we investigated the efficacy of NK cells that were expanded and treated by interleukin-2 (IL-2) and heat shock protein 70 (HSP70), both in vitro and in vivo. Proliferation and cytotoxicity assays were used to assess the functionality of NK cells in vitro, after which treated and na&iuml, ve NK cells were administrated intracranially and systemically to compare the potential antitumor activities in our in vivo rat GBM models. In vitro assays provided strong evidence of NK cell efficacy against C6 tumor cells. In vivo tracking of NK cells showed efficient homing around and within the tumor site. Furthermore, significant amelioration of the tumor in rats treated with HSP70/Il-2-treated NK cells as compared to those subjected to nontreated NK cells, as confirmed by MRI, proved the efficacy of adoptive NK cell therapy. Moreover, results obtained with systemic injection confirmed migration of activated NK cells over the blood brain barrier and subsequent targeting of GBM tumor cells. Our data suggest that administration of HSP70/Il-2-treated NK cells may be a promising therapeutic approach to be considered in the treatment of GBM.

Recent advances in cancer immunotherapy had a positive impact on the life expectancy of patients with a large range of clinical indications. With new treatment strategies and druggable targets being identified at an increasing pace, the number of patients eligible for cancer immunotherapy is expected to expand steadily. However, promising therapeutic developments face hurdles in translating preclinical findings into therapy. Challenges range from lack of confidence in animal cancer models, inter-tumor heterogeneity and the absence of cellular microenvironment. Hubrecht Organoid Technology (HUB) offers an innovative alternative, build on the discovery that adult stem cells proliferate and organize into three-dimensional organotypic structures when they are embedded into Matrigel. HUBOrganoids™ are generated from healthy and disease tissues and stored as “living” biobanks with high quality and reproducibility. HUB Organoids recapitulate complex characteristics of the original parental tissue, including molecular heterogeneity, and morphological and functional traits. HUB has developed organoid-T-cell co-culture systems that allow a physiologic representation of the complexity of interactions in the tumor microenvironment, which is important for evaluating the effects of novel therapies. HUB offers an innovative platform combining image-based analysis with flow cytometry to visualize and quantify T-cell-tumor interactions as well as measurements of T-cell cytotoxicity against tumor (and normal-matched) organoids for colorectal and several other cancers. Our platform offers a powerful strategy for the development and validation of cancer immunotherapy and may help to screen the efficacy of immune checkpoint blockade or bispecific antibodies. In addition, cancer organoid-T-cell co-culture may also predict the functionality of tumor-infiltrating lymphocytes (TIL) and chimeric antigen receptor (CAR)-engineered T cells. As such, our platform offers opportunities in preclinical development and could become a diagnostic tool to predict individual patient response to immunotherapy. Citation Format: Pleun Hombrink, Soura Mardpour, Ewout Spaan, Jarmil Hanrath, Farzin Pourfarzad, Jasper Mullenders, Rene Overmeer, G.J Robert, Vries, F Sylvia, Boj. Organoid-T-cell co-cultures for preclinical testing of adoptive cell and cancer immunotherapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2977.

Pre-clinical disease models are essential for the development of novel therapies and help better understand the molecular processes. In recent years, a novel groundbreaking technology to generate patient specific in vitro cultures based on adult stem-cell organoids, has gained widespread interest for the development of new therapies and as a predictive diagnostic tool. ASC-organoids can be generated at high efficiency from both healthy and diseased tissue (resections as well as biopsies) including most carcinomas. These patient derived cultures can be expanded for prolonged periods of time while maintaining many properties of the tissue they were derived from. At HUB, we have created comprehensive ‘living biobanks' from tumors of different origin like colon, lung, breast, pancreas and ovary. These biobanks have been characterized at the genetic and transcriptional level. These analyses have shown that our ‘living biobanks' capture disease heterogeneity at the genetic and molecular level. Because organoids can be expanded long term, drug screening is feasible in these models. HUB has developed procedures for (high-throughput) compound screening in organoids including small molecules and biologics. In this setting, organoids have been used to identify novel targets, identify lead compounds and stratify potential responders based on their genetic or molecular profile. Recent advances in the field of immuno-oncology have revolutionized the treatment for cancer patients. Despite of the improvements and due to the complex and highly regulated nature of the immune system, treatment does not have a similar effect on all tumors. We have created a co-culture model of tumor derived organoids and immune cells. In these co-cultures, the cytotoxic effect of the immune cell on the tumor can be measured in a reproducible and quantitative manner. Currently we are expanding existing co-culture models to incorporate different immune cells and organoid tumor types. In addition, organoids can serve as a patient avatar in clinical development or diagnostics. In this setting, organoids could add a functional test for therapy response in a patient centered setting providing a unique tool for personalized medicine. Citation Format: Jasper Mullenders, Annemarie Buijs, Inge Van Maanen, Carol Piani, Soura Mardpour, Hans Clevers, Rob Vries, Sylvia Boj. Patient derived organoids: Bringing the "patient in the lab" for pre-clinical and clinical development [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 LB-008.

Glioblastoma multiforme (GBM) is among the most incurable cancers. GBMs survival rate has not markedly improved, despite new radical surgery protocols, the introduction of new anticancer drugs, new treatment protocols, and advances in radiation techniques. The low efficacy of therapy, and short interval between remission and recurrence, could be attributed to the resistance of a small fraction of tumorigenic cells to treatment. The existence and importance of cancer stem cells (CSCs) is perceived by some as controversial. Experimental evidences suggest that the presence of therapy-resistant glioblastoma stem cells (GSCs) could explain tumor recurrence and metastasis. Some scientists, including most of the authors of this review, believe that GSCs are the driving force behind GBM relapses, whereas others however, question the existence of GSCs. Evidence has accumulated indicating that non-tumorigenic cancer cells with high heterogeneity, could undergo reprogramming and become GSCs. Hence, targeting GSCs as the "root cells" initiating malignancy has been proposed to eradicate this devastating disease. Most standard treatments fail to completely eradicate GSCs, which can then cause the recurrence of the disease. To effectively target GSCs, a comprehensive understanding of the biology of GSCs as well as the mechanisms by which these cells survive during treatment and develop into new tumor, is urgently needed. Herein, we provide an overview of the molecular features of GSCs, and elaborate how to facilitate their detection and efficient targeting for therapeutic interventions. We also discuss GBM classifications based on the molecular stem cell subtypes with a focus on potential therapeutic approaches.

Background In this study, we intend to assess the safety and tolerability of intra-articular knee implantation of autologous bone marrow–derived mesenchymal stromal cells (MSCs) in patients with rheumatoid arthritis (RA) and to determine the preliminary clinical efficacy data in this population. The trial registration numbers are as follows: Royan Institute Ethics Committee: AC/91/1133; NCT01873625. Methods This single-center, randomized, triple-blind, placebo-controlled phase 1/2 clinical trial randomized RA patients with knee involvement to receive either an intra-articular knee implantation of 40 million autologous bone marrow–derived MSCs per joint or normal saline (placebo). Patients were followed up for 12 months to assess therapy outcomes. Results A total of 30 patients, 15 in the MSC group and 15 in the placebo group, enrolled in this study. There were no adverse effects reported after MSC administration or during follow-up. Patients who received MSCs had superior findings according to the Western Ontario and McMaster Universities Arthritis Index (WOMAC), visual analogue scale (VAS), time to jelling and pain-free walking distance. However, this improvement could not be significantly sustained beyond 12 months. The MSC group exhibited improved standing time (P = 0.01). In addition, the MSCs appeared to contribute to reductions in methotrexate and prednisolone use. Conclusion Intra-articular knee implantation of MSCs appeared to be safe and well tolerated. In addition, we observed a trend toward clinical efficacy. These results, in our opinion, have justified the need for further investigations over an extended assessment period with larger numbers of RA patients who have knee involvement.

Background aims Recent studies have proposed that cellular transplantation may have some regenerative and functional efficacy in the treatment of cerebral palsy (CP); however, much remains to be understood regarding its safety, feasibility and efficacy. This study was initiated to evaluate the safety of autologous bone marrow–derived CD133+ cell intrathecal injection. Methods Children (n = 12), aged 4 to 12 years, who were diagnosed with different types of CP underwent BM aspiration. CD133+ cells were enriched from the BM samples and intrathecally injected. The Gross Motor Function Measure (GMFM-66), Gross Motor Function Classification System (GMFCS), UK FIM+FAM, Functional Independence Measure (FIM) and Functional Assessment Measure (FAM) were assessed at baseline and 6 months after the procedure. Patients' ability to balance was measured by the Berg Balance Scale (BBS), and severity of spasticity was evaluated by the Modified Ashworth Scale. Magnetic resonance imaging was done at baseline and 6 months after therapy. This study was registered in ClinicalTrials.gov ( NCT01404663 ). Results There were no adverse events detected by clinical and laboratory tests or imaging studies, with the exception of a seizure in 1 patient. A significant improvement was observed 6 months after cell transplantation versus baseline according to GMFM, GMFCS, FIM+FAM, Ashworth Scale, and BBS outcomes. Conclusions Subarachnoid injection of CD133-positive enriched bone marrow progenitor cells in children with CP is a safe approach. The results suggest a possible short-term improvement in neurological function.