Organoids as a new approach in advancing cancer therapies for hematologic malignancies.

Introduction: Integrin signaling plays an important role in cellular proliferation and migration via interactions with extracellular matrix proteins. Prior studies indicate that integrin signaling facilitates tumor invasion and metastasis, and there are several ongoing clinical trials using agents that modulate this pathway. We recently identified clonal enrichment in missense mutations in the integrin cell surface interactions pathways in advanced chemotherapy-resistant urothelial carcinoma. An ideal strategy for investigating integrin signaling is via 3D organoid culture, maintaining intercellular interactions that replicate the epithelial microenvironment. We hypothesize that pharmacologic integrin signaling modulation will impair organoid growth in human bladder cancer cells and demonstrate a potential therapeutic utility for this approach. Methods: RT4 human bladder cancer cell line was used as well as a second cell line established from a patient-derived bladder cancer sample (PM748). Cells were grown in 3D organoid culture as previously described. For in vitro integrin modulation, defactinib (VS-6063), an orally-bioavailable selective inhibitor of focal adhesion kinase (FAK, a convergent and conserved enzyme activated by integrin ligand binding), was used. SDS-PAGE and immunoblotting were performed to show in vitro FAK inhibition. Single-cell suspensions and formed organoids were plated in the presence of various concentrations of defactinib to determine the impact on organoid formation and regression. Results: Both RT4 and PM748 bladder cancer cell lines demonstrated consistent organoid growth in three-dimensional culturing conditions. Addition of defactinib to cultured cells showed a dose-dependent decrease in autophosphorylation of FAK for both cell lines, demonstrating effective FAK inhibition. 3D culture of single cells in the presence of defactinib produced a dose-dependent decrease in organoid size after 96 hours (mean size for DMSO only, 100nM, 1uM, and 10uM were 128um, 75um, 48um, and 26um, respectively; p<0.0001 versus DMSO for all dilutions). Established bladder cancer organoids showed a dose-dependent regression in size after 72 hours of defactinib exposure (mean size for DMSO, 100nM, 1uM, and 10uM were 225um, 96um, 70um, and 34um, respectively; p<0.0001 versus DMSO). Experiments utilizing Crispr-Cas9-mediated FAK knock-out as well as in vivo studies with FAK inhibitors in mouse xenograft models are currently underway. Conclusions: Integrin modulation via FAK inhibition with defactinib causes both inhibition of organoid formation as well as regression of formed organoids, and the effects are seen at concentrations well below the cytotoxic range for the drug. This study suggests a utility for these agents in bladder cancer treatment. Citation Format: LaMont Barlow, Rebecca Meyer, Ethan Shelkey, Bishoy Faltas, Mark Rubin. Integrin signaling modulation demonstrates potential therapeutic strategy in bladder cancer using three-dimensional organoid culture [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 5941. doi:10.1158/1538-7445.AM2017-5941

PurposeRenal cell carcinoma is a heterogeneous kidney cancer, and over 403,000 cases were reported worldwide in 2018. Current methods for studying renal cell carcinoma are limited to two-dimensional (2D) culture of primary cell lines and patient-derived xenograft models. Numerous studies have suggested that 2D culture poorly represents the diversity, heterogeneity, and drug-resistance of primary tumors. The time and cost associated with patient-derived xenograft models poses a realistic barrier to their clinical utility. As a biomimetic model, patient-derived three-dimensional (3D) organoid culture can overcome these disadvantages and bridge the gap between in vitro cell culture and in vivo patient-derived xenograft models. Here, we establish a patient-derived 3D organoid culture system for clear cell renal cell carcinoma and demonstrate the biomimetic characteristics of our model with respect to both primary kidney cancer and conventional 2D culture.Materials and MethodsNormal renal tissues and tumor tissues were collected from patients with clear cell renal cell carcinoma. The dissociated cells were cultured as conventional 2D culture and 3D organoid culture. The biomimetic characteristic of the two cultures were compared.ResultsCompared with 2D culture, the 3D organoid cultures retained the characteristic lipid-rich, clear cell morphology of clear cell renal cell carcinoma. Carbonic anhydrase 9 and vimentin were validated as biomarkers of renal cell carcinoma. Expression of the two validated biomarkers was more enhanced in 3D organoid culture.ConclusionsPatient-derived 3D organoid culture retains the characteristics of renal cell carcinoma with respect to morphology and biomarker expression.

Despite advances in the treatment of inflammatory bowel disease (IBD), current therapies have limited efficacy and can cause serious adverse reactions, which not only reduces the quality of life but also results in shorter life expectancy for patients. Therefore, it is necessary to develop new methods of diagnosis and find more effective ways of treatment. A significant breakthrough in the study of intestinal epithelial cells in various pathologies, including IBS, has been achieved by the establishment of three-dimensional (3D) organoid cultures that in vitro recreate intestinal epithelial tissue. Organoid cultures are derived from either somatic stem cells or pluripotent stem cells. These cells, grown under appropriate conditions, self-organize into specific spherical tissue structures with the properties and functions of gastrointestinal tissues. It has been shown that intestinal organoids may be a promising tool for assessing the molecular and functional impact on the intestinal epithelium of genetic and epigenetic changes associated with the gastrointestinal tract. To objectively evaluate the functional status of the intestinal epithelium in IBD, one needs to present organoid models together with the intestinal microbiota in order to determine the specific effect of bacteria on the epithelial barrier. Intestinal organoids are also being used to explore novel strategies for both direct drug exposure to the intestinal epithelium and targeted drug delivery, including nanoparticle and microparticle-based approaches, thereby reducing the limitations of systemic treatment. Finally, organoids serve as the basis of regenerative medicine for the treatment of IBD. New treatment techniques can be applied in patients with IBD in combination with immunoregulatory therapy and contribute to the achievement of prolonged clinical remission. Key words: inflammatory bowel disease, Crohn’s disease, ulcerative colitis, organoid cultures, diagnosis, stem cells, regenerative medicine

The keratinocytes are predominant cells in the epidermis of the human skin. To assess the cellular response of the keratinocytes to the genotoxic stress, we derived the skin keratinocytes from human induced pluripotent stem cells (iPSCs). Furthermore, three-dimensional (3D) organoid culture method is powerful tool to analyze the organ and tissue response against the genotoxic stress. Here we describe the method of 3D organoid culture using skin keratinocytes derived from human iPSCs.

BACKGROUND Glioblastoma (GBM) is the most common primary brain tumor with a dismal prognosis. The inherent cellular diversity and interactions within tumor microenvironments represent a significant challenge to effective treatment. Traditional culture methods may mask the complexity of such interactions while three-dimensional (3D) organoid culture systems derived from patient cancer stem cells (CSCs) can preserve cellular complexity and microenvironments. Our objective was to determine whether organoid cultures show increased patterns of resistance to potential clinical therapies compared to traditional sphere cultures. METHODS Adult and pediatric surgical specimens were collected and established as 3D organoids. We created organoid microarrays and visualized bulk and spatially defined differences in cell proliferation using immunohistochemistry (IHC) staining, as well as cell cycle analysis by flow cytometry with 3D regional labeling. We tested the response of CSCs grown in each culture method to temozolomide, ibrutinib, lomustine, ruxolitinib, and radiotherapy using proliferative and viability assays. RESULTS Compared to sphere cultures from the same patient, organoids showed diverse proliferative cell populations and broad resistance to all therapies tested, albeit with both intraspecimen and interspecimen variability in the extent of resistance. Organoid specimens demonstrated a blunt response to current GBM standard of care therapy (combination temozolomide and radiotherapy) and maintained both cellular proliferation in their outer rim and overall structure and viability compared to the matched sphere specimens. CONCLUSIONS Our results suggest that growth of tumor specimens as organoid cultures may better reflect the cellular diversity and clinical reality of GBM therapeutic response. Patient-derived GBM organoids offer a valuable complement to traditional culture methods and may have powerful predictive capability of personalized drug sensitivities and therapeutic resistance.

BackgroundHuman pluripotent stem cells (hPSCs), including human embryonic stem cells (hESCs) and induced pluripotent stem cells (hiPSCs), can undergo erythroid differentiation, offering a potentially invaluable resource for generating large quantities of erythroid cells. However, the majority of erythrocytes derived from hPSCs fail to enucleate compared with those derived from cord blood progenitors, with an unknown molecular basis for this difference. The expression of vimentin (VIM) is retained in erythroid cells differentiated from hPSCs but is absent in mature erythrocytes. Further exploration is required to ascertain whether VIM plays a critical role in enucleation and to elucidate the underlying mechanisms.MethodsIn this study, we established a hESC line with reversible vimentin degradation (dTAG-VIM-H9) using the proteolysis-targeting chimera (PROTAC) platform. Various time-course studies, including erythropoiesis from CD34+ human umbilical cord blood and three-dimensional (3D) organoid culture from hESCs, morphological analysis, quantitative real-time PCR (qRT-PCR), western blotting, flow cytometry, karyotyping, cytospin, Benzidine-Giemsa staining, immunofluorescence assay, and high-speed cell imaging analysis, were conducted to examine and compare the characteristics of hESCs and those with vimentin degradation, as well as their differentiated erythroid cells.ResultsVimentin expression diminished during normal erythropoiesis in CD34+ cord blood cells, whereas it persisted in erythroid cells differentiated from hESC. Depletion of vimentin using the degradation tag (dTAG) system promotes erythroid enucleation in dTAG-VIM-H9 cells. Nuclear polarization of erythroblasts is elevated by elimination of vimentin.ConclusionsVIM disappear during the normal maturation of erythroid cells, whereas they are retained in erythroid cells differentiated from hPSCs. We found that retention of vimentin during erythropoiesis impairs erythroid enucleation from hPSCs. Using the PROTAC platform, we validated that vimentin degradation by dTAG accelerates the enucleation rate in dTAG-VIM-H9 cells by enhancing nuclear polarization.Graphical

Background/Aims: Blocking estrogen signaling with endocrine therapies (Tamoxifen or Fulverstrant) is an effective treatment for Estrogen Receptor-α positive (ER⁺) breast cancer tumours. Unfortunately, development of endocrine therapy resistance (ETR) is a frequent event resulting in disease relapse and decreased overall patient survival. The long noncoding RNA, H19, was previously shown to play a significant role in estrogen-induced proliferation of both normal and malignant ER⁺ breast epithelial cells. We hypothesized that H19 expression is also important for the proliferation and survival of ETR cells. Methods: Here we utilized established ETR cell models; the Tamoxifen (Tam)-resistant LCC2 and the Fulvestrant and Tam cross-resistant LCC9 cells. Gain and loss of H19 function were achieved through lentiviral transduction as well as pharmacological inhibitors of the Notch and c-Met receptor signaling pathways. The effects of altered H19 expression on cell viability and ETR were assessed using three-dimensional (3D) organoid cultures and 2D co-cultures with low passage tumour-associated fbroblasts (TAFs). Results: Here we report that treating ETR cells with Tam or Fulvestrant increases H19 expression and that it’s decreased expression overcomes resistance to Tam and Fulvestrant in these cells. Interestingly, H19 expression is regulated by Notch and HGF signaling in the ETR cells and pharmacological inhibitors of Notch and c-MET signaling together significantly reverse resistance to Tam and Fulvestrant in an H19-dependent manner in these cells. Lastly, we demonstrate that H19 regulates ERα expression at the transcript and protein levels in the ETR cells and that H19 protects ERα against Fulvestrant-mediated downregulation of ERα protein. We also observed that blocking Notch and the c-MET receptor signaling also overcomes Fulvestrant and Tam resistance in 3D organoid cultures by decreasing ERα and H19 expression in the ETR cells. Conclusion: In endocrine therapy resistant breast cancer cells Fulvestrant is ineffective in decreasing ERα levels. Our data suggest that in the ETR cells, H19 expression acts as an ER modulator and that its levels and subsequently ERα levels can be substantially decreased by blocking Notch and c-MET receptor signaling. Consequently, treating ETR cells with these pharmacological inhibitors helps overcome resistance to Fulvestrant and Tamoxifen.

Three-dimensional (3D) organoid culture holds great promises in cancer precision medicine. However, Matrigel and stem cell-stimulating supplements are necessary for culturing 3D organoid cells. It costs a lot of money and consumes more time and effort compared with 2D cultured cells. Therefore, the establishment of cheaper and Matrigel-free organoid culture that can maintain the characteristics of a part of 3D organoids is demanded. In the previous study, we established a dog bladder cancer (BC) 3D organoid culture system by using their urine samples. Here, we successfully isolated cells named “2.5D organoid” from multiple strains of dog BC 3D organoids using 2.5 organoid media. The cell proliferation speed of 2.5D organoids was faster than parental 3D organoid cells. The expression pattern of stem cell markers was close to 3D organoids. Injection of 2.5D organoid cells into immunodeficient mice formed tumors and showed the histopathological characteristics of urothelial carcinoma similar to the injection of dog BC 3D organoids. The 2.5D organoids had a similar sensitivity profile for anti-cancer drug treatment to their parental 3D organoids. These data suggest that our established 2.5D organoid culture method might become a reasonable and useful tool instead of 3D organoids in dog BC research and therapy.

Human pluripotent stem cell (hPSC)-derived microglia for the study of brain disorders. A comprehensive review of existing protocols

3D-organoid culture supports differentiation of human CAR+ iPSCs into highly functional CAR Tcells.

Proper patient-tailoring strategy and the validation of novel therapeutic targets remain enormous challenges during drug discovery processes. Patient-derived three-dimensional organoid cell culture models possess great potential to associate compound sensitivity and disease complexity in order to provide a key missing link between compound screening and clinical trials. Abemaciclib is a reversible, ATP competitive, selective inhibitor of the kinase activity of both CDK4 and CDK6 and is currently undergoing advanced clinical testing. In this study, we established and characterized three-dimensional organoid cultures from primary colorectal cancer patients and validated their use as drug sensitivity models. We aimed to explore the antitumor activity of abemaciclib in colon cancer organoid cultures by assessing markers for cell viability, proliferation, cell cycle, senescence and apoptosis. Single cell suspension of patient-derived samples were precultured for four days to allow for complete morphogenesis of three-dimensional organoid structures. Subsequently, the cultures were treated for at least two population doubling times and analyzed by luminescent cell viability, immunohistochemistry and flow cytometry assays. Our data suggest that abemaciclib treatment decreased the cell viability of patient-derived colorectal cancer organoid cultures characterized by G1 cell cycle arrest and reduced Ki-67-positive cells. Furthermore, treated cultures showed elevated levels of reactive oxygen species and increased markers for early and late apoptosis. In summary, complex organoid models have the potential to further evaluate the antitumor activity of abemaciclib in various tumor types by enabling mechanistic studies in a patient-specific preclinical setting. Citation Format: Karsten Boehnke, Bruna Calsina, Joaquín Amat, Ana Hermoso, Raquel Torres, Christoph Reinhard, Juan A. Velasco, Philip W. Iversen, Alfonso De Dios, Sean Buchanan, Richard P. Beckmann, Dirk Schumacher, Christian RA Regenbrecht, Marie-Laure Yaspo, Hans Lehrach, María José Lallena. Preclinical analysis and characterization of abemaciclib using three-dimensional patient-derived colorectal cancer organoid cultures. [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 2829.

Three-dimensional organoid culture unveils resistance to clinical therapies in adult and pediatric glioblastoma

Invariant NKT (iNKT) cells have a T-cell receptor that is common to all individuals and are activated by recognizing glycolipids on MHC class I-like CD1d molecules. Activated iNKT cells are known to exert anti-tumor effects through the activation of other immune cells and have attracted attention as promising T cells for eliciting anti-tumor immunity. However, securing a sufficient number of iNKT cells is an obstacle to treatment because iNKT cells are a very small cell population, less than 0.1% of the peripheral blood lymphocytes. Although previous studies have demonstrated redifferentiation of a large number of CD4-CD8- double-negative iNKT cells from induced pluripotent stem (iPS) cells in two-dimensional monolayer cultures, CD4+ single-positive (CD4SP) iNKT cells could not be induced. Here we show CD4SP iNKT cells can be obtained by three-dimensional (3D) organoid culture (3D-CD4+ iNKT cell). We additionally describe 3D-CD4+ iNKT cells show antigen-specific helper functions, as they proliferate, produce interferon-γ/interleukin-4 (IFN-γ/IL-4), and induce dendritic cell maturation in response to α-galactosylceramide. Furthermore, they reverse the inhibition of T cell proliferation induced by immunosuppressive macrophages in an antigen-specific manner. Collectively, 3D-CD4+ iNKT cells may become an adjuvant T-cell source to enhance current T-cell immunotherapy against solid tumors.

A Simple Bioreactor-Based Method to Generate Kidney Organoids fromPluripotent Stem Cells.

Lumen formation of salivary glands has been investigated using in vivo or ex vivo rudiment culture models. In this study, we used a three-dimensional (3D) salivary gland organoid culture system and demonstrated that lumen formation could be recapitulated in mouse SMG organoids. In our organoid culture system, lumen formation was induced by vasoactive intestinal peptide and accelerated by treatment with RA. Furthermore, lumen formation was observed in branching duct-like structure when cultured in combination of fibroblast growth factors (FGF) in the presence of retinoic acid (RA). We suggest RA signaling-mediated regulation of VIPR1 and KRT7 as the underlying mechanism for lumen formation, rather than apoptosis in the organoid culture system. Collectively, our results support a fundamental role for RA in lumen formation and demonstrate the feasibility of 3D organoid culture as a tool for studying salivary gland morphogenesis.