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1.
J Vis Exp ; (191)2023 Jan 06.
Artigo em Inglês | MEDLINE | ID: mdl-36688549

RESUMO

Organoids are 3D dynamic tumor models that can be grown successfully from patient-derived ovarian tumor tissue, ascites, or pleural fluid and aid in the discovery of novel therapeutics and predictive biomarkers for ovarian cancer. These models recapitulate clonal heterogeneity, the tumor microenvironment, and cell-cell and cell-matrix interactions. Additionally, they have been shown to match the primary tumor morphologically, cytologically, immunohistochemically, and genetically. Thus, organoids facilitate research on tumor cells and the tumor microenvironment and are superior to cell lines. The present protocol describes distinct methods to generate patient-derived ovarian cancer organoids from patient tumors, ascites, and pleural fluid samples with a higher than 97% success rate. The patient samples are separated into cellular suspensions by both mechanical and enzymatic digestion. The cells are then plated utilizing a basement membrane extract (BME) and are supported with optimized growth media containing supplements specific to the culturing of high-grade serous ovarian cancer (HGSOC). After forming initial organoids, the PDOs can sustain long-term culture, including passaging for expansion for subsequent experiments.


Assuntos
Cistadenocarcinoma Seroso , Neoplasias Ovarianas , Humanos , Feminino , Ascite/patologia , Neoplasias Ovarianas/patologia , Cistadenocarcinoma Seroso/patologia , Carcinoma Epitelial do Ovário/patologia , Organoides/patologia , Microambiente Tumoral
2.
Cancer Lett ; 555: 216048, 2023 Feb 28.
Artigo em Inglês | MEDLINE | ID: mdl-36603689

RESUMO

Liver cancer and disease are among the most socially challenging global health concerns. Although organ transplantation, surgical resection and anticancer drugs are the main methods for the treatment of liver cancer, there are still no proven cures owing to the lack of donor livers and tumor heterogeneity. Recently, advances in tumor organoid technology have attracted considerable attention as they can simulate the spatial constructs and pathophysiological characteristics of tumorigenesis and metastasis in a more realistic manner. Organoids may further contribute to the development of tailored therapies. Combining organoids with other emerging techniques, such as CRISPR-HOT, organ-on-a-chip, and 3D bioprinting, may further develop organoids and address their bottlenecks to create more practical models that generalize different tissue or organ interactions in tumor progression. In this review, we summarize the various methods in which liver organoids may be generated and describe their biological and clinical applications, present challenges, and prospects for their integration with emerging technologies. These rapidly developing liver organoids may become the focus of in vitro clinical model development and therapeutic research for liver diseases in the near future.


Assuntos
Carcinoma Hepatocelular , Neoplasias Hepáticas , Humanos , Carcinoma Hepatocelular/tratamento farmacológico , Carcinoma Hepatocelular/patologia , Organoides/patologia , Neoplasias Hepáticas/tratamento farmacológico , Neoplasias Hepáticas/patologia , Carcinogênese/patologia
3.
J Transl Med ; 21(1): 9, 2023 Jan 09.
Artigo em Inglês | MEDLINE | ID: mdl-36624452

RESUMO

BACKGROUNDS: Papillary thyroid cancer (PTC), which is often driven by acquired somatic mutations in BRAF genes, is the most common pathologic type of thyroid cancer. PTC has an excellent prognosis after treatment with conventional therapies such as surgical resection, thyroid hormone therapy and adjuvant radioactive iodine therapy. Unfortunately, about 20% of patients develop regional recurrence or distant metastasis, making targeted therapeutics an important treatment option. Current in vitro PTC models are limited in representing the cellular and mutational characteristics of parental tumors. A clinically relevant tool that predicts the efficacy of therapy for individuals is urgently needed. METHODS: Surgically removed PTC tissue samples were dissociated, plated into Matrigel, and cultured to generate organoids. PTC organoids were subsequently subjected to histological analysis, DNA sequencing, and drug sensitivity assays, respectively. RESULTS: We established 9 patient-derived PTC organoid models, 5 of which harbor BRAFV600E mutation. These organoids have been cultured stably for more than 3 months and closely recapitulated the histological architectures as well as mutational landscapes of the respective primary tumors. Drug sensitivity assays of PTC organoid cultures demonstrated the intra- and inter-patient specific drug responses. BRAFV600E inhibitors, vemurafenib and dabrafenib monotherapy was mildly effective in treating BRAFV600E-mutant PTC organoids. Nevertheless, BRAF inhibitors in combination with MEK inhibitors, RTK inhibitors, or chemotherapeutic agents demonstrated improved efficacy compared to BRAF inhibition alone. CONCLUSIONS: These data indicate that patient-derived PTC organoids may be a powerful research tool to investigate tumor biology and drug responsiveness, thus being useful to validate or discover targeted drug combinations.


Assuntos
Carcinoma Papilar , Neoplasias da Glândula Tireoide , Humanos , Câncer Papilífero da Tireoide/tratamento farmacológico , Câncer Papilífero da Tireoide/genética , Neoplasias da Glândula Tireoide/tratamento farmacológico , Neoplasias da Glândula Tireoide/genética , Neoplasias da Glândula Tireoide/patologia , Proteínas Proto-Oncogênicas B-raf/genética , Radioisótopos do Iodo/uso terapêutico , Carcinoma Papilar/tratamento farmacológico , Carcinoma Papilar/genética , Mutação/genética , Inibidores de Proteínas Quinases/farmacologia , Inibidores de Proteínas Quinases/uso terapêutico , Organoides/patologia
4.
Methods Mol Biol ; 2561: 105-133, 2023.
Artigo em Inglês | MEDLINE | ID: mdl-36399267

RESUMO

Alzheimer's disease (AD) is a common neurodegenerative disorder and a mechanistically complex disease. For the last decade, human models of AD using induced pluripotent stem cells (iPSCs) have emerged as a powerful way to understand disease pathogenesis in relevant human cell types. In this review, we summarize the state of the field and how this technology can apply to studies of both familial and sporadic studies of AD. We discuss patient-derived iPSCs, genome editing, differentiation of neural cell types, and three-dimensional organoids, and speculate on the future of this type of work for increasing our understanding of, and improving therapeutic development for, this devastating disease.


Assuntos
Doença de Alzheimer , Células-Tronco Pluripotentes Induzidas , Humanos , Doença de Alzheimer/genética , Doença de Alzheimer/patologia , Neurônios/patologia , Neuroglia/patologia , Organoides/patologia
5.
Methods Mol Biol ; 2610: 167-178, 2023.
Artigo em Inglês | MEDLINE | ID: mdl-36534290

RESUMO

Studying neurological diseases have long been hampered by the lack of physiologically relevant models to resemble the complex human brain and the associated pathologies. Three-dimensional brain organoids have emerged as cutting-edge technology providing an alternative in vitro model to study healthy neural development and function as well as pathogenesis of neurological disorders and neuropathologies induced by pathogens. Nonetheless,  the absence of immune cells in current models poses a barrier to fully recapitulate brain microenvironment during the onset of HIV-1-associated neuropathogenesis. To address this and to further the brain organoid technology, we have incorporated HIV-target microglia into brain organoids, generating a complex multicellular interaction, which mimics the HIV-1-infected brain environment. Here we describe the method to generate a brain organoid consisting on neurons, astrocytes, and microglia (with and without HIV infection) that recapitulate the HIV-associated neuropathology. This model has tremendous potential to expand our knowledge on neuronal dysfunction associated with HIV-1 infection of glia.


Assuntos
Infecções por HIV , HIV-1 , Doenças do Sistema Nervoso , Humanos , Infecções por HIV/patologia , Encéfalo/patologia , Doenças do Sistema Nervoso/patologia , Organoides/patologia
6.
Oxid Med Cell Longev ; 2022: 7264649, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-36518994

RESUMO

The basic technology of stem cells has been developed and created organoids, which have established a strong interest in regenerative medicine. Different cell types have been used to generate cerebral organoids, which include interneurons and oligodendrocytes (OLs). OLs are fundamental for brain development. Abundant studies have displayed that brain organoids can recapitulate fundamental and vital features of the human brain, such as cellular regulation and distribution, neuronal networks, electrical activities, and physiological structure. The organoids contain essential ventral brain domains and functional cortical interneurons, which are similar to the developing cortex and medial ganglionic eminence (MGE). So, brain organoids have provided a singular model to study and investigate neurological disorder mechanisms and therapeutics. Furthermore, the blood brain barrier (BBB) organoids modeling contributes to accelerate therapeutic discovery for the treatment of several neuropathologies. In this review, we summarized the advances of the brain organoids applications to investigate neurological disorder mechanisms such as neurodevelopmental and neurodegenerative disorders, mental disorders, brain cancer, and cerebral viral infections. We discussed brain organoids' therapeutic application as a potential therapeutic unique method and highlighted in detail the challenges and hurdles of organoid models.


Assuntos
Doenças Neurodegenerativas , Organoides , Humanos , Organoides/metabolismo , Organoides/patologia , Barreira Hematoencefálica , Encéfalo/patologia , Interneurônios , Doenças Neurodegenerativas/patologia
7.
Expert Rev Mol Med ; 25: e3, 2022 Dec 15.
Artigo em Inglês | MEDLINE | ID: mdl-36517884

RESUMO

Alzheimer's disease (AD) is a progressive neurodegenerative disorder characterised by gradual memory loss and declining cognitive and executive functions. AD is the most common cause of dementia, affecting more than 50 million people worldwide, and is a major health concern in society. Despite decades of research, the cause of AD is not well understood and there is no effective curative treatment so far. Therefore, there is an urgent need to increase understanding of AD pathophysiology in the hope of developing a much-needed cure. Dissecting the cellular and molecular mechanisms of AD pathogenesis has been challenging as the most commonly used model systems such as transgenic animals and two-dimensional neuronal culture do not fully recapitulate the pathological hallmarks of AD. The recent advent of three-dimensional human brain organoids confers unique opportunities to study AD in a humanised model system by encapsulating many aspects of AD pathology. In the present review, we summarise the studies of AD using human brain organoids that recapitulate the major pathological components of AD including amyloid-ß and tau aggregation, neuroinflammation, mitochondrial dysfunction, oxidative stress and synaptic and circuitry dysregulation. Additionally, the current challenges and future directions of the brain organoids modelling system are discussed.


Assuntos
Doença de Alzheimer , Animais , Humanos , Doença de Alzheimer/etiologia , Doença de Alzheimer/patologia , Peptídeos beta-Amiloides/metabolismo , Encéfalo/metabolismo , Encéfalo/patologia , Neurônios/metabolismo , Neurônios/patologia , Organoides/metabolismo , Organoides/patologia
8.
Klin Onkol ; 35(6): 473-481, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-36513514

RESUMO

BACKGROUND: Current in vitro model systems do not fully reflect the bio-logical and clinical diversity of prostate cancer (PCa). Organoids are 3D in vitro cell cultures that may better recapitulate disease heterogeneity and retain parental tumor characteristics. Short-term ex vivo culture of PCa tissues may also facilitate drug testing in personalized medicine. MATERIALS AND METHODS: For organoid culture, we have processed both cancer and normal tissues from 50 patients who underwent radical prostatectomy or transurethral resection of the prostate. In addition, we exploited the ex vivo tissue culture technique and performed short-term chemotherapy assay using gemcitabine and Chk1 inhibitor MU380 in 10 patient samples. RESULTS: In total, we were able to cultivate organoids from 58% of tumors (29/50) and 69% of normal tissue (20/29). Immunohistochemical staining of two representative cases revealed cell positivity for pan-cytokeratin confirming the presence of epithelial cells. However, the overexpression of AMACR and ERG proteins in tumors was not recapitulated in organoids. Another limitation was the propagation of organoids only up to 3 weeks till the first passage. Next, a short-term drug test was performed for ten patients using ex vivo tissue culture. Samples from prostatectomies mostly presented a low proliferation rate as assessed by Ki-67 staining. Another drawback of this ap-proach was inconsistent tissue morphology among particular tissue fragments. Only one case showed a high proliferation rate for drug testing and tumor tissue was present in all tested samples. In our work, we also provide an overview of recent studies and a detailed comparison of culture conditions. CONCLUSION: We have established cultures of both organoids and tissue fragments from PCa patient samples. However, the expression of tumor markers was not recapitulated in organoids. Inconsistent morphology among tissue fragments and low proliferation hampered the interpretation of the drug testing in most cases. Still, these approaches may be promising using tissues from metastatic castration-resistant prostate cancer.


Assuntos
Neoplasias da Próstata , Ressecção Transuretral da Próstata , Masculino , Humanos , Medicina de Precisão/métodos , Organoides/metabolismo , Organoides/patologia , Neoplasias da Próstata/patologia
9.
Biomaterials ; 291: 121883, 2022 12.
Artigo em Inglês | MEDLINE | ID: mdl-36343611

RESUMO

Complex 3D bioengineered tumour models provide the opportunity to better capture the heterogeneity of patient tumours. Patient-derived organoids are emerging as a useful tool to study tumour heterogeneity and variation in patient responses. Organoid cultures typically require a 3D microenvironment that can be manufactured easily to facilitate screening. Here we set out to create a high-throughput, "off-the-shelf" platform which permits the generation of organoid-containing engineered microtissues for standard phenotypic bioassays and image-based readings. To achieve this, we developed the Scaffold-supported Platform for Organoid-based Tissues (SPOT) platform. SPOT is a 3D gel-embedded in vitro platform that can be produced in a 96- or 384-well plate format and enables the generation of flat, thin, and dimensionally-defined microgels. SPOT has high potential for adoption due to its reproducible manufacturing methodology, compatibility with existing instrumentation, and reduced within-sample and between-sample variation, which can pose challenges to both data analysis and interpretation. Using SPOT, we generate cultures from patient derived pancreatic ductal adenocarcinoma organoids and assess the cellular response to standard-of-care chemotherapeutic compounds, demonstrating our platform's usability for drug screening. We envision 96/384-SPOT will provide a useful tool to assess drug sensitivity of patient-derived organoids and easily integrate into the drug discovery pipeline.


Assuntos
Carcinoma Ductal Pancreático , Neoplasias Pancreáticas , Humanos , Organoides/patologia , Carcinoma Ductal Pancreático/patologia , Neoplasias Pancreáticas/patologia , Avaliação Pré-Clínica de Medicamentos/métodos , Descoberta de Drogas , Microambiente Tumoral
10.
Zhonghua Kou Qiang Yi Xue Za Zhi ; 57(11): 1141-1146, 2022 Nov 09.
Artigo em Chinês | MEDLINE | ID: mdl-36379893

RESUMO

Objective: To establish an in vitro organoid model of human salivary gland basal cell adenoma (BCA). Methods: Fresh tumor sample from a 66-year-old female patient diagnosed with salivary gland BCA was collected from the Dpartment of Oral pathology, Ninth People's Hospital Affiliated to Shanghai Jiao Tong University School of Medicine in October 2021. And the organoid culture was performed in vitro in a culture medium based on solid droplets of matrix gel, and the growth of the organoid was observed by inverted microscopy. After 14 days, the organoid was fixed in 10% neutral formalin and made into paraffin blocks by agar pre-embedding paraffin embedding method, sectioned. HE staining, morphological observation and immunohistochemical staining of p63, Ki-67, cytokeratin14 (CK14), ß-catenin, S-100 and calponin were used for organoids identification. Results: The established BCA organoids were lobulated nodular locally under light microscopy, with deposition of eosinophilic glass-like material around the nests of organoid cells, similar to the morphological architectures of the parental BCA. Immunohistochemistry showed that organoids expressed CK14, p63, and ß-catenin in various degree, which was consistent with the immunophenotypic characteristics of the parental BCA tumor cells. Conclusions: An in vitro culture system of BCA organoids was preliminarily established which provides a new model for the study of the pathogenesis of salivary gland tumors.


Assuntos
Adenoma , Neoplasias das Glândulas Salivares , beta Catenina , Idoso , Feminino , Humanos , Adenoma/diagnóstico , Adenoma/patologia , China , Organoides/patologia , Glândulas Salivares
11.
Cells ; 11(22)2022 Nov 15.
Artigo em Inglês | MEDLINE | ID: mdl-36429040

RESUMO

Monitoring tumor growth dynamics is crucial for understanding cancer. To establish an in vitro method for the continuous assessment of patient-specific tumor growth, tumor organoids were generated from patients with intrahepatic CCA (iCCA). Organoid growth was monitored for 48 h by label-free live brightfield imaging. Growth kinetics were calculated and validated by MTS assay as well as immunohistochemistry of Ki67 to determine proliferation rates. We exposed iCCA organoids (iCCAOs) and non-tumor intrahepatic cholangiocyte organoids (ICOs) to sub-therapeutic concentrations of sorafenib. Monitoring the expansion rate of iCCAOs and ICOs revealed that iCCAO growth was inhibited by sorafenib in a time- and dose-dependent fashion, while ICOs were unaffected. Quantification of the proliferation marker Ki67 confirmed inhibition of iCCAO growth by roughly 50% after 48 h of treatment with 4 µM sorafenib. We established a robust analysis pipeline combining brightfield microscopy and a straightforward image processing approach for the label-free growth monitoring of patient-derived iCCAOs. Combined with bioanalytical validation, this approach is suitable for a fast and efficient high-throughput drug screening in tumor organoids to develop patient-specific systemic treatment options.


Assuntos
Neoplasias dos Ductos Biliares , Colangiocarcinoma , Humanos , Organoides/patologia , Sorafenibe/farmacologia , Sorafenibe/uso terapêutico , Antígeno Ki-67 , Colangiocarcinoma/diagnóstico por imagem , Colangiocarcinoma/tratamento farmacológico , Colangiocarcinoma/patologia , Neoplasias dos Ductos Biliares/diagnóstico por imagem , Neoplasias dos Ductos Biliares/tratamento farmacológico , Neoplasias dos Ductos Biliares/patologia , Ductos Biliares Intra-Hepáticos/patologia
12.
PLoS One ; 17(10): e0276195, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-36301950

RESUMO

Ulcerative colitis and Crohn's disease are chronic inflammatory bowel diseases (IBD) of unknown cause characterized by a relapsing-remitting behavior. Growing evidence supports the idea that the epithelial barrier plays a central role in the pathogenesis of IBD as well as in its evolution over time, thus representing a potential target for novel therapeutic options. In the last decade, the introduction of 3D epithelial cultures from ex vivo-expanded intestinal adult stem cells (ASCs) has impacted our ability to study the function of the epithelium in several gastrointestinal disorders, including IBD. Here, we describe in detail a reproducible protocol to generate Matrigel-embedded epithelial organoids from ASCs of non-IBD and IBD donors using small colonic biopsies, including steps for its optimization. A slightly modified version of this protocol is also provided in case surgical samples are used. With this method, epithelial organoids can be expanded over several passages, thereby generating a large quantity of viable cells that can be used in multiple downstream analyses including genetic, transcriptional, proteomic and/or functional studies. In addition, 3D cultures generated using our protocol are suitable for the establishment of 2D cultures, which can model relevant cell-to-cell interactions that occur in IBD mucosa.


Assuntos
Colite Ulcerativa , Doenças Inflamatórias Intestinais , Adulto , Humanos , Organoides/patologia , Mucosa Intestinal/patologia , Proteômica , Doenças Inflamatórias Intestinais/patologia , Colo/patologia , Colite Ulcerativa/patologia
13.
Int J Mol Sci ; 23(19)2022 Sep 24.
Artigo em Inglês | MEDLINE | ID: mdl-36232574

RESUMO

Sarcomas are malignant tumors of mesenchymal origin that can occur at any age. The rarity of these tumors in combination with the vast number of histological subtypes render the study of sarcomas challenging. Organoids represent complex three-dimensional cell culture systems, deriving from stem cells and preserving the capacity to differentiate into the cell types of their tissue of origin. The aim of the present review is to study the current status of patient-derived organoids, as well as their potential to model tumorigenesis and perform drug screenings for sarcomas. In order to identify relevant studies, a literature review was conducted and we were able to identify 16 studies published between 2019 and 2022. The current manuscript represents the first comprehensive review of the literature focusing on the use of organoids for disease modelling and drug sensitivity testing in diverse sarcoma subtypes.


Assuntos
Sarcoma , Neoplasias de Tecidos Moles , Transformação Celular Neoplásica , Humanos , Organoides/patologia , Sarcoma/diagnóstico , Sarcoma/patologia , Sarcoma/terapia , Células-Tronco/patologia
14.
Pancreas ; 51(6): 608-616, 2022 07 01.
Artigo em Inglês | MEDLINE | ID: mdl-36206467

RESUMO

ABSTRACT: Pancreatic cancer is one of the most lethal cancer types, estimated to become the second leading cause of cancer-related deaths in the United States in 2030. The use of 3-dimensional culture systems has greatly expanded over the past few years, providing a valuable tool for the study of pancreatic cancer. In this review, we highlight some of the preclinical in vitro and in vivo models used in pancreatic cancer research, each with its own advantages and disadvantages, and focus on one of the recently used 3-dimensional culture models: organoids. Organoids are multicellular units derived from tissue samples and embedded within extracellular matrix gels after mechanical and enzymatic digestion. We define organoids, differentiate them from other 3-dimensional culture systems such as spheroids, and describe some applications of this model that have recently advanced our understanding of pancreatic cancer and its tumor microenvironment. Organoids have provided valuable insights into pancreatic cancer progression, heterogeneity, and invasion, and they have enabled the creation of biobanks, providing a platform for drug screening. In addition, we discuss some of the future directions and challenges in this model when addressing research questions.


Assuntos
Organoides , Neoplasias Pancreáticas , Humanos , Organoides/patologia , Pâncreas/patologia , Neoplasias Pancreáticas/patologia , Microambiente Tumoral
15.
Clin Transl Med ; 12(11): e1095, 2022 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-36314067

RESUMO

BACKGROUND: Transketolase (TKT), a key rate-limiting enzyme in the non-oxidative branch of the pentose phosphate pathway (PPP), provides more than 85% of the ribose required for de novo nucleotide biosynthesis and promotes the development of hepatocellular carcinoma (HCC). Pharmacologic inhibition of TKT could impede HCC development and enhance treatment efficacy. However, no safe and effective TKT inhibitor has been approved. METHODS: An online two-dimensional TKT protein immobilised biochromatographic system was established for high-throughput screening of TKT ligands. Oroxylin A was found to specifically bind TKT. Drug affinity responsive target stability, cellular thermal shift assay, surface plasmon resonance, molecular docking, competitive displacement assay, and site mutation were performed to identify the binding of oroxylin A with TKT. Antitumour effects of oroxylin A were evaluated in vitro, in human xenograft mice, diethylnitrosamine (DEN)-induced HCC mice, and patient-derived organoids (PDOs). Metabolomic analysis was applied to detect the enzyme activity. Transcriptome profiling was conducted to illustrate the anti-HCC mechanism of oroxylin A. TKT knocking-down HCC cell lines and PDOs were established to evaluate the role of TKT in oroxylin A-induced HCC suppression. RESULTS: By targeting TKT, oroxylin A stabilised the protein to proteases and temperature extremes, decreased its activity and expression, resulted in accumulation of non-oxidative PPP substrates, and activated p53 signalling. In addition, oroxylin A suppressed cell proliferation, induced apoptosis and cell-cycle arrest, and inhibited the growth of human xenograft tumours and DEN-induced HCC in mice. Crucially, TKT depletion exerted identical effects to oroxylin A, and the promising inhibitor also exhibited excellent therapeutic efficacy against clinically relevant HCC PDOs. CONCLUSIONS: These results uncover a unique role for oroxylin A in TKT inhibition, which directly targets TKT and suppresses the non-oxidative PPP. Our findings will facilitate the development of small-molecule inhibitors of TKT and novel therapeutics for HCC.


Assuntos
Carcinoma Hepatocelular , Neoplasias Hepáticas , Humanos , Animais , Camundongos , Carcinoma Hepatocelular/tratamento farmacológico , Carcinoma Hepatocelular/genética , Carcinoma Hepatocelular/metabolismo , Transcetolase/genética , Transcetolase/metabolismo , Via de Pentose Fosfato , Neoplasias Hepáticas/tratamento farmacológico , Neoplasias Hepáticas/genética , Organoides/metabolismo , Organoides/patologia , Simulação de Acoplamento Molecular
16.
Oral Oncol ; 135: 106186, 2022 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-36265373

RESUMO

OBJECTIVE: Three-dimensional organoid cell cultures have been established for a variety of human cancers. For most rare cancers, including salivary gland cancer (SGC), these models are lacking, despite the great unmet need to study cancer biology in these diseases. Therefore, we aimed to develop patient-derived organoid (PDO) models for different subtypes of SGC. METHODS: Tumor samples of SGC patients were processed and embedded in Matrigel. Successful PDOs (expandable > 1*106 cells) were phenotypically characterized using immunohistochemistry (IHC) and genotypically by gene fusion analysis and by targeted and whole-exome sequencing. Successfully established PDOs were subjected to small-scale drug screening. RESULTS: Out of 37 attempts, 7 viable short-term PDOs were established (19 % success rate; 3 salivary duct carcinoma, 3 adenoid cystic carcinoma and 1 mucoepidermoid carcinoma). Each PDO showed close phenotypical mimicry to parental tissue. Genotypic characterization revealed that in each PDO > 97.6 % of all COSMIC annotated variants and all MYB, MYBL1 and NFIB gene rearrangements were retained. Drug screening was proven feasible in all PDOs. CONCLUSION: We present the first comprehensively characterized short-term SGC PDO models for three subtypes of SGC with close phenotypic and genotypic resemblance to parental tissue, which can be used for drug screening applications.


Assuntos
Carcinoma Adenoide Cístico , Neoplasias das Glândulas Salivares , Humanos , Organoides/patologia , Proteínas de Fusão Oncogênica/genética , Neoplasias das Glândulas Salivares/genética , Neoplasias das Glândulas Salivares/patologia , Carcinoma Adenoide Cístico/genética , Carcinoma Adenoide Cístico/patologia , Fusão Gênica
17.
Nat Commun ; 13(1): 6183, 2022 Oct 19.
Artigo em Inglês | MEDLINE | ID: mdl-36261438

RESUMO

Human organoids could facilitate research of complex and currently incurable neuropathologies, such as age-related macular degeneration (AMD) which causes blindness. Here, we establish a human retinal organoid system reproducing several parameters of the human retina, including some within the macula, to model a complex combination of photoreceptor and glial pathologies. We show that combined application of TNF and HBEGF, factors associated with neuropathologies, is sufficient to induce photoreceptor degeneration, glial pathologies, dyslamination, and scar formation: These develop simultaneously and progressively as one complex phenotype. Histologic, transcriptome, live-imaging, and mechanistic studies reveal a previously unknown pathomechanism: Photoreceptor neurodegeneration via cell extrusion. This could be relevant for aging, AMD, and some inherited diseases. Pharmacological inhibitors of the mechanosensor PIEZO1, MAPK, and actomyosin each avert pathogenesis; a PIEZO1 activator induces photoreceptor extrusion. Our model offers mechanistic insights, hypotheses for neuropathologies, and it could be used to develop therapies to prevent vision loss or to regenerate the retina in patients suffering from AMD and other diseases.


Assuntos
Degeneração Macular , Organoides , Humanos , Actomiosina , Fator de Crescimento Semelhante a EGF de Ligação à Heparina , Canais Iônicos , Degeneração Macular/patologia , Organoides/patologia , Células Fotorreceptoras , Retina/patologia , Fatores de Necrose Tumoral
18.
Ann Med ; 54(1): 2581-2597, 2022 12.
Artigo em Inglês | MEDLINE | ID: mdl-36194178

RESUMO

PROPOSE: Neoadjuvant chemotherapy has been widely used in locally advanced and inflammatory breast cancer. Generally, complete pathological response after neoadjuvant chemotherapy treatment predicts survival. Studies have shown that patient-derived organoids can be used in cancer research and drug development. Therefore, we aimed to generate a living organoid biobank from biopsy samples to predict the response of patients to neoadjuvant chemotherapy. METHOD: We generated a living organoid biobank from locally advanced breast cancer patients receiving neoadjuvant chemotherapy. When the patient received neoadjuvant chemotherapy, the organoids were treated with similar drugs, thereby simulating the situation of the patient receiving treatment. RESULT: We successfully constructed organoids from breast cancer biopsies, demonstrating that organoids can be generated from a small sample of tissue. The phenotype of breast cancer organoid often agreed with the original breast cancer according to the blinded histopathological analysis of H&E stain tissue and organoid sections. In addition, our data confirm that the patient's response to chemotherapy closely matches the organoids' response to drugs. CONCLUSION: Our data indicate that patient-derived organoids can be used to predict the clinical response of breast cancer patients to neoadjuvant chemotherapy in vitro and to screen drugs that have different effects on different patients. Key messageComplete pathological response (pCR) after adjuvant chemotherapy can predict, survival, therefore, predicting patient response to neoadjuvant chemotherapy is critical.Patient-derived organoids (PDOs) matched the original tumour in terms of histopathology, hormone receptor levels and HER2 receptor status.Patient-derived organoids can predict the responsiveness of patient to neoadjuvant chemotherapy.


Assuntos
Neoplasias da Mama , Terapia Neoadjuvante , Biópsia , Neoplasias da Mama/tratamento farmacológico , Quimioterapia Adjuvante , Feminino , Hormônios/farmacologia , Hormônios/uso terapêutico , Humanos , Organoides/patologia
19.
Stem Cell Reports ; 17(11): 2421-2437, 2022 Nov 08.
Artigo em Inglês | MEDLINE | ID: mdl-36240775

RESUMO

Usher syndrome-associated retinitis pigmentosa (RP) causes progressive retinal degeneration, which has no cure. The pathomechanism of Usher type 1B (USH1B)-RP caused by MYO7A mutation remains elusive because of the lack of faithful animal models and limited knowledge of MYO7A function. Here, we analyzed 3D retinal organoids generated from USH1B patient-derived induced pluripotent stem cells. Increased differential gene expression occurred over time without excessive photoreceptor cell death in USH1B organoids compared with controls. Dysregulated genes were enriched first for mitochondrial functions and then proteasomal ubiquitin-dependent protein catabolic processes and RNA splicing. Single-cell RNA sequencing revealed MYO7A expression in rod photoreceptor and Müller glial cells corresponding to upregulation of stress responses in NRL+ rods and apoptotic signaling pathways in VIM+ Müller cells, pointing to the defensive mechanisms that mitigate photoreceptor cell death. This first human model for USH1B-RP provides a representation of patient retina in vivo relevant for development of therapeutic strategies.


Assuntos
Organoides , Retinite Pigmentosa , Animais , Humanos , Miosina VIIa , Organoides/patologia , Patologia Molecular , Miosinas/genética , Miosinas/metabolismo , Retina/metabolismo , Retinite Pigmentosa/genética , Retinite Pigmentosa/patologia , Células Fotorreceptoras Retinianas Bastonetes/patologia
20.
Cell Rep Med ; 3(9): 100738, 2022 09 20.
Artigo em Inglês | MEDLINE | ID: mdl-36103879

RESUMO

Endometrial cancer is one of the most frequently diagnosed gynecological cancers worldwide, and its prevalence has increased by more than 50% over the last two decades. Despite the understanding of the major signaling pathways driving the growth and metastasis of endometrial cancer, clinical trials targeting these signals have reported poor outcomes. The heterogeneous nature of endometrial cancer is suspected to be one of the key reasons for the failure of targeted therapies. In this study, we perform a sequential window acquisition of all theoretical fragment ion spectra (SWATH)-based comparative proteomic analysis of 63 tumor biopsies collected from 20 patients and define differences in protein signature in multiple regions of the same tumor. We develop organoids from multiple biopsies collected from the same tumor and show that organoids capture heterogeneity in endometrial cancer growth. Overall, using quantitative proteomics and patient-derived organoids, we define the heterogeneous nature of endometrial cancer within a patient's tumor.


Assuntos
Neoplasias do Endométrio , Proteômica , Neoplasias do Endométrio/tratamento farmacológico , Feminino , Humanos , Organoides/patologia
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