RESUMO
BACKGROUND: Ovarian cancer is the most lethal gynecological cancer. As the primary treatment, chemotherapy has a response rate of only 60-70% in advanced stages, and even lower as a second-line treatment. Despite guideline recommendations, which drugs will be most effective remains unclear. Thus, a strategy to prioritize chemotherapy options is urgently needed. Cancer organoids have recently emerged as a method for in vitro drug testing. However, limited clinical correlations have been assessed with test results from cancer organoids, particularly in gynecological cancers. We therefore aimed to generate patient-derived organoids (PDOs) of ovarian cancer, to assess their drug sensitivities and correlations with patient clinical outcomes. METHODS: PDOs were generated from fresh tumors obtained during surgical resection, which was then cultured under matrix gel and appropriate growth factors. Morphological and molecular characterization of PDOs were assessed by phase contrast microscopy and paraffin-embedded histopathology. Expressions of PAX8, TP53, WT1, CK7, and CK20 were tested by immunohistochemical staining and compared with parental tumor tissues and the human protein atlas database. PDOs were subjected to in vitro drug testing to determine drug sensitivity using Titer-Glo® 3D Cell Viability Assay. PDO viability was measured, and area under the curve calculated, to compare responses to various compounds. Correlations were calculated between selected patients' clinical outcomes and in vitro drug testing results. RESULTS: We established 31 PDOs. Among them, 28 PDOs can be expanded, including 15, 11, and 2 from ovarian, endometrial, and cervical cancers, respectively. The PDOs preserved the histopathological profiles of their originating tumors. In vitro drug testing of 10 ovarian cancer PDOs revealed individual differential responses to recommended drugs, and interpersonal heterogeneity in drug sensitivity, even with the same histology type. Among four patients who were platinum sensitive, resistant, or refractory, PDO drug responses correlated well with their clinical courses. CONCLUSION: In vitro drug testing using ovarian cancer organoids is feasible and correlates well with patient clinical responses. These results may facilitate development of precision chemotherapy and personalized screening for repurposed or new drugs.
Assuntos
Ensaios de Seleção de Medicamentos Antitumorais , Organoides , Neoplasias Ovarianas , Humanos , Feminino , Organoides/efeitos dos fármacos , Organoides/patologia , Neoplasias Ovarianas/tratamento farmacológico , Neoplasias Ovarianas/patologia , Neoplasias Ovarianas/metabolismo , Ensaios de Seleção de Medicamentos Antitumorais/métodos , Antineoplásicos/farmacologia , Antineoplásicos/uso terapêutico , Pessoa de Meia-Idade , IdosoRESUMO
In recognition of the lethal nature of cancer, extensive efforts have been made to understand the mechanistic causation while identifying the effective therapy modality in hope to eradicate cancerous cells with minimal damage to healthy cells. In search of such effective therapeutics, establishing pathophysiologically relevant in vitro models would be of importance in empowering our capabilities of truly identifying those potent ones with significantly reduction of the preclinical periods for rapid translation. In this regard, wealthy progresses have been achieved over past decades in establishing various in vitro and in vivo tumor models. Ideally, the tumor models should maximally recapture the key pathophysiological attributes of their native counterparts. Many of the current models have demonstrated their utilities but also showed some noticeable limitations. This book chapter will briefly review some of the mainstream platforms for in vitro tumor models followed by detailed elaboration on the modular strategies to form in vitro tumor models with complex structures and spatial organization of cellular components. Clearly, with the ability to modulate the building modules it becomes a new trend to form in vitro tumor models following a bottom-up approach, which offers a high flexibility to satisfy the needs for pathophysiological study, anticancer drug screening or design of personalized treatment.
Assuntos
Ensaios de Seleção de Medicamentos Antitumorais , Neoplasias , Humanos , Neoplasias/tratamento farmacológico , Neoplasias/patologia , Animais , Ensaios de Seleção de Medicamentos Antitumorais/métodos , Antineoplásicos/farmacologia , Antineoplásicos/uso terapêutico , Modelos BiológicosRESUMO
Breast cancer is the most common malignancy in women worldwide, and major challenges in its treatment include drug resistance and metastasis. Three-dimensional cell culture systems have received widespread attention in drug discovery studies but existing models have limitations, that warrant the development of a simple and repeatable three-dimensional culture model for high-throughput screening. In this study, we designed a simple, reproducible, and highly efficient microencapsulated device to co-culture MCF-7 cells and HUVECs in microcapsules to establish an in vitro vascularized micro-tumor model for chemotherapeutic drug screening. First, to construct a three-dimensional micro-tumor model, cell encapsulation devices were created using three different sizes of flat-mouthed needles. Immunohistochemistry and immunofluorescence assays were conducted to determine vascular lumen formation. Cell proliferation was detected using the Cell Counting Kit-8 assay. Finally, to observe the drug reactions between the models, anticancer drugs (doxorubicin or paclitaxel) were added 12â h after the two-dimensional cultured cells were plated or 7 days after cell growth in the core-shell microcapsules. Vascularized micro-tumors were obtained after 14 days of three-dimensional culture. The proliferation rate in the three-dimensional cultured cells was slower than that of two-dimensional cultured cells. Three-dimensional cultured cells were more resistant to anticancer drugs than two-dimensional cultured cells. This novel sample encapsulation device formed core-shell microcapsules and can be used to successfully construct 3D vascularized micro-tumors in vitro. The three-dimensional culture model may provide a platform for drug screening and is valuable for studying tumor development and angiogenesis.
Assuntos
Antineoplásicos , Proliferação de Células , Técnicas de Cocultura , Ensaios de Seleção de Medicamentos Antitumorais , Células Endoteliais da Veia Umbilical Humana , Humanos , Proliferação de Células/efeitos dos fármacos , Antineoplásicos/farmacologia , Ensaios de Seleção de Medicamentos Antitumorais/métodos , Neovascularização Patológica/tratamento farmacológico , Neovascularização Patológica/patologia , Feminino , Células MCF-7 , Neoplasias da Mama/patologia , Neoplasias da Mama/tratamento farmacológico , Paclitaxel/farmacologia , Doxorrubicina/farmacologia , Técnicas de Cultura de Células em Três Dimensões/métodosRESUMO
Metastasis contributes to the increased mortality rate of cancer, but the intricate mechanisms remain unclear. Cancer cells from a primary tumor invade nearby tissues and access the lymphatic or circulatory system. If these cells manage to survive and extravasate from the vasculature into distant tissues and ultimately adapt to survive, they will proliferate and facilitate malignant tumor formation. Traditional two-dimensional (2D) cell cultures offer a rapid and convenient method for validating the efficacy of anticancer drugs within a reasonable cost range, but their utility is limited because of tumors' high heterogeneity in vivo and spatial complexities. Three-dimensional (3D) cell cultures that mimic the physiological conditions of cancer cells in vivo have gained considerable interest. In these cultures, cells assemble into spheroids through gravity, magnetic forces, or their low-adhesion to the plates. Although these approaches address some of the limitations of 2D cultures, they often require a considerable amount of time and cost. Therefore, this study aims to enhance the effectiveness of 3D culture techniques by using microfluidic systems to provide a high-throughput and sensitive pipeline for drug screening. Using these systems, we studied the effects of lanthanide elements, which have garnered interest in cancer treatment, on spheroid formation and cell spreading. Our findings suggest that these elements alter the compactness of cell spheroids and decrease cell mobility.
Assuntos
Elementos da Série dos Lantanídeos , Esferoides Celulares , Esferoides Celulares/efeitos dos fármacos , Humanos , Elementos da Série dos Lantanídeos/toxicidade , Elementos da Série dos Lantanídeos/farmacologia , Técnicas de Cultura de Células/métodos , Linhagem Celular Tumoral , Antineoplásicos/farmacologia , Antineoplásicos/toxicidade , Técnicas Analíticas Microfluídicas/instrumentação , Técnicas Analíticas Microfluídicas/métodos , Sobrevivência Celular/efeitos dos fármacos , Técnicas de Cultura de Células em Três Dimensões/métodos , Ensaios de Seleção de Medicamentos Antitumorais/métodosRESUMO
Cancer is a leading cause of death worldwide. Around one-third of the total global cancer incidence and mortality are related to gastrointestinal (GI) cancers. Over the past few years, rapid developments have been made in patient-derived organoid (PDO) models for gastrointestinal cancers. By closely mimicking the molecular properties of their parent tumors in vitro, PDOs have emerged as powerful tools in personalized medicine and drug discovery. Here, we review the current literature on the application of PDOs of common gastrointestinal cancers in the optimization of drug treatment strategies in the clinic and their rising importance in pre-clinical drug development. We discuss the advantages and limitations of gastrointestinal cancer PDOs and outline the microfluidics-based strategies that improve the throughput of PDO models in order to extract the maximal benefits in the personalized medicine and drug discovery process.
Assuntos
Neoplasias Gastrointestinais , Organoides , Medicina de Precisão , Humanos , Medicina de Precisão/métodos , Organoides/efeitos dos fármacos , Organoides/patologia , Neoplasias Gastrointestinais/tratamento farmacológico , Neoplasias Gastrointestinais/patologia , Avaliação Pré-Clínica de Medicamentos/métodos , Descoberta de Drogas/métodos , Ensaios de Seleção de Medicamentos Antitumorais/métodosRESUMO
Three-dimensional (3D) ex vivo cultures allow the study of cancer progression and drug resistance mechanisms. Here, we present a protocol for measuring on-target drug sensitivity in a scaffold-free 3D culture system through quantification of apoptotic tumor cells. We provide detailed steps for sample processing, immunofluorescence staining, semi-high-throughput confocal imaging, and imaged-based quantification of 3D cultures. This protocol is versatile and can be applied in principle to any patient-derived material.
Assuntos
Neoplasias Ovarianas , Humanos , Neoplasias Ovarianas/patologia , Neoplasias Ovarianas/tratamento farmacológico , Feminino , Antineoplásicos/farmacologia , Técnicas de Cultura de Células em Três Dimensões/métodos , Resistencia a Medicamentos Antineoplásicos , Apoptose/efeitos dos fármacos , Ensaios de Seleção de Medicamentos Antitumorais/métodos , Células Tumorais Cultivadas , Linhagem Celular TumoralRESUMO
Combining drugs can enhance their clinical efficacy, but the number of possible combinations and inter-tumor heterogeneity make identifying effective combinations challenging, while existing approaches often overlook clinically relevant activity. We screen one of the largest cell line panels (N = 757) with 51 clinically relevant combinations and identify responses at the level of individual cell lines and tissue populations. We establish three response classes to model cellular effects beyond monotherapy: synergy, Bliss additivity, and independent drug action (IDA). Synergy is rare (11% of responses) and frequently efficacious (>50% viability reduction), whereas Bliss and IDA are more frequent but less frequently efficacious. We introduce "efficacious combination benefit" (ECB) to describe high-efficacy responses classified as either synergy, Bliss, or IDA. We identify ECB biomarkers in vitro and show that ECB predicts response in patient-derived xenografts better than synergy alone. Our work here provides a valuable resource and framework for preclinical evaluation and the development of combination treatments.
Assuntos
Protocolos de Quimioterapia Combinada Antineoplásica , Sinergismo Farmacológico , Neoplasias , Humanos , Linhagem Celular Tumoral , Animais , Neoplasias/tratamento farmacológico , Neoplasias/patologia , Protocolos de Quimioterapia Combinada Antineoplásica/farmacologia , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapêutico , Ensaios de Seleção de Medicamentos Antitumorais/métodos , Ensaios Antitumorais Modelo de Xenoenxerto , Camundongos , Antineoplásicos/farmacologia , Antineoplásicos/uso terapêutico , Biomarcadores Tumorais/metabolismoRESUMO
Over the past three decades, high-throughput phenotypic cancer cell line screens have revealed unanticipated small-molecule activities and illuminated connections between tumor genotypes and anticancer efficacy. Founded in 1984, the National Cancer Institute's "NCI60" screen laid the conceptual groundwork for the contemporary landscape of phenotypic drug discovery. NCI60 first operated as a primary bioactivity screen, but molecular characterization of the NCI60 cell line panel and development of a small-molecule sensitivity pattern recognition algorithm (called "COMPARE") have enabled subsequent studies into drug mechanisms of action and biomarker identification. In this issue of Cancer Research, Kunkel and colleagues report an updated version of the NCI60 screen, dubbed "HTS384" NCI60, that better aligns with current cell proliferation assay standards and has higher throughput. Changes include the use of a 384-well plate format, automated laboratory equipment, 3 days of compound exposure, and a CellTiter-Glo luminescent endpoint. To confirm that data from the HTS384 and classic NCI60 screen are comparable, the authors tested a library of 1,003 anticancer agents using both protocols and applied COMPARE to analyze patterns of cell line sensitivities. More than three dozen groups of targeted therapies showed high comparability between screens. Modernization of NCI60, and closer integration with other large-scale pharmacogenomic screens and molecular feature sets, will help this public screening service remain pertinent for cancer drug discovery efforts for years to come. See related article by Kunkel et al., p. 2403.
Assuntos
Antineoplásicos , Descoberta de Drogas , Ensaios de Seleção de Medicamentos Antitumorais , Ensaios de Triagem em Larga Escala , Humanos , Descoberta de Drogas/métodos , Ensaios de Seleção de Medicamentos Antitumorais/métodos , Ensaios de Triagem em Larga Escala/métodos , Linhagem Celular Tumoral , Antineoplásicos/farmacologia , National Cancer Institute (U.S.) , Fenótipo , Neoplasias/tratamento farmacológico , Neoplasias/patologia , Neoplasias/genética , Estados Unidos , Proliferação de Células/efeitos dos fármacosRESUMO
Gemcitabine is a nucleoside analog widely used as an anticancer agent against several types of cancer. Although gemcitabine sometimes shows excellent effectiveness, cancer cells are often poorly responsive to or resistant to the drug. Recently, specific strains or dysbiosis of the human microbiome were correlated with drug reactivity and resistance acquisition. Therefore, we aimed to identify antibiotic compounds that can modulate the microbiome to enhance the responsiveness to gemcitabine. To achieve this, we confirmed the gemcitabine responsiveness based on public data and conducted drug screening on a set of 250 antibiotics compounds. Subsequently, we performed experiments to investigate whether the selected compounds could enhance the responsiveness to gemcitabine. First, we grouped a total of seven tumor cell lines into resistant and sensitive group based on the IC50 value (1 µM) of gemcitabine obtained from the public data. Second, we performed high-throughput screening with compound treatments, identifying seven compounds from the resistant group and five from the sensitive group based on dose dependency. Finally, the combination of the selected compound, puromycin dihydrochloride, with gemcitabine in gemcitabine-resistant cell lines resulted in extensive cell death and a significant increase in cytotoxic efficacy. Additionally, mRNA levels associated with cell viability and stemness were reduced. Through this study, we screened antibiotics to further improve the efficacy of existing anticancer drugs and overcome resistance. By combining existing anticancer agents and antibiotic substances, we hope to establish various drug combination therapies and ultimately improve cancer treatment efficacy.
Assuntos
Antibacterianos , Desoxicitidina , Resistencia a Medicamentos Antineoplásicos , Ensaios de Seleção de Medicamentos Antitumorais , Gencitabina , Ensaios de Triagem em Larga Escala , Desoxicitidina/análogos & derivados , Desoxicitidina/farmacologia , Humanos , Ensaios de Triagem em Larga Escala/métodos , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Linhagem Celular Tumoral , Antibacterianos/farmacologia , Ensaios de Seleção de Medicamentos Antitumorais/métodos , Puromicina/farmacologia , Antimetabólitos Antineoplásicos/farmacologia , Sinergismo Farmacológico , Antineoplásicos/farmacologia , Bibliotecas de Moléculas Pequenas/farmacologiaRESUMO
Tumor organoids have emerged as a crucial preclinical model for multiple cancer research. Their high establishment rates, stability, and ability to replicate key biological features of original tumor cells in vivo render them invaluable for exploring tumor molecular mechanisms, discovering potential anti-tumor drugs, and predicting clinical drug efficacy. Here, we review the establishment of tumor organoid models and provide an extensive overview of organoid culturing strategies. We also emphasize the significance of integrating cellular components of the tumor microenvironment and physicochemical factors in the organoid culturing system, highlighting the importance of artificial intelligence technology in advancing organoid construction. Moreover, we summarize recent advancements in utilizing organoid systems for novel anti-cancer drug screening and discuss promising trends for enhancing advanced organoids in next-generation disease modeling.
Assuntos
Neoplasias , Organoides , Microambiente Tumoral , Organoides/efeitos dos fármacos , Organoides/patologia , Humanos , Neoplasias/patologia , Neoplasias/tratamento farmacológico , Animais , Inteligência Artificial , Ensaios de Seleção de Medicamentos Antitumorais/métodos , Antineoplásicos/farmacologiaRESUMO
PURPOSE: Brain metastases represent the most common intracranial tumors in adults and are associated with a poor prognosis. We used a personalized in vitro drug screening approach to characterize individual therapeutic vulnerabilities in brain metastases. METHODS: Short-term cultures of cancer cells isolated from brain metastasis patients were molecularly characterized using next-generation sequencing and functionally evaluated using high-throughput in vitro drug screening to characterize pharmacological treatment sensitivities. RESULTS: Next-generation sequencing identified matched genetic alterations in brain metastasis tissue samples and corresponding short-term cultures, suggesting that short-term cultures of brain metastases are suitable models for recapitulating the genetic profile of brain metastases that may determine their sensitivity to anti-cancer drugs. Employing a high-throughput in vitro drug screening platform, we successfully screened the cultures of five brain metastases for response to 267 anticancer compounds and related drug response to genetic data. Among others, we found that targeted treatment with JAK3, HER2, or FGFR3 inhibitors showed anti-cancer effects in individual brain metastasis cultures. CONCLUSION: Our preclinical study provides a proof-of-concept for combining molecular profiling with in vitro drug screening for predictive evaluation of therapeutic vulnerabilities in brain metastasis patients. This approach could advance the use of patient-derived cancer cells in clinical practice and might eventually facilitate decision-making for personalized drug treatment.
Assuntos
Antineoplásicos , Neoplasias Encefálicas , Humanos , Neoplasias Encefálicas/tratamento farmacológico , Neoplasias Encefálicas/secundário , Neoplasias Encefálicas/patologia , Neoplasias Encefálicas/genética , Antineoplásicos/farmacologia , Antineoplásicos/uso terapêutico , Células Tumorais Cultivadas , Ensaios de Seleção de Medicamentos Antitumorais/métodos , Sequenciamento de Nucleotídeos em Larga Escala , Feminino , Masculino , Pessoa de Meia-Idade , Ensaios de Triagem em Larga Escala/métodosRESUMO
The understanding of anti-tumor drug effects requires specific experimental settings which model clinical scenarios. We describe a protocol for 10-day treatment of lowly aggressive tumor cell lines with antineoplastic agents at concentrations which do not affect cell growth. We describe steps for seeding cells and treating cells with anti-tumor drugs. We then detail steps for cell sensitivity, cell proliferation, and mRNA and protein expression assays. We also detail assays to determine modifications in compound efflux. For complete details on the use and execution of this protocol, please refer to Rios Medrano et al.1.
Assuntos
Antineoplásicos , Proliferação de Células , Humanos , Linhagem Celular Tumoral , Antineoplásicos/farmacologia , Proliferação de Células/efeitos dos fármacos , Neoplasias/tratamento farmacológico , Neoplasias/patologia , Neoplasias/metabolismo , Ensaios de Seleção de Medicamentos Antitumorais/métodosRESUMO
Before molecular pathways in cancer were known to a depth that could predict targets, drug development relied on phenotypic screening, where the effectiveness of candidate chemicals is judged from functional readouts without considering the mechanisms of action. The unraveling of tumor-specific pathways has brought targets for molecularly driven drug discovery, but precedents in the field have shown that awareness of pathways does not necessarily predict therapeutic efficacy, and many cancers still lack druggable targets. Phenotypic screening therefore retains a niche in drug development where a targeted approach is not informative. We analyze the unique advantages of phenotypic screens, and how technological advances have improved their discovery power. Notable advances include the use of larger biological panels and refined protocols that address the disease-relevance and increase data content with imaging and omic approaches.
Assuntos
Antineoplásicos , Descoberta de Drogas , Neoplasias , Fenótipo , Humanos , Descoberta de Drogas/métodos , Neoplasias/tratamento farmacológico , Antineoplásicos/farmacologia , Animais , Ensaios de Seleção de Medicamentos Antitumorais/métodosRESUMO
BACKGROUND/AIM: Precision medicine aims to revolutionize healthcare by tailoring treatment regimens. This study aimed to integrate comprehensive tumor genomic profiling (CTGP) by targeted-gene panel sequencing and drug screening by circulating tumor cell-derived organoids (CTOs) into clinical practice for the treatment of gastrointestinal (GI) cancers. PATIENTS AND METHODS: Nine patients with various GI cancers underwent CTGP and CTO drug sensitivity testing. CTGP results guided targeted therapy and immunotherapy, while CTO drug sensitivity predicted response to chemotherapy and targeted agents. The drug recommendations from two platforms were correlated with the treatment response to the suggested medications retrospectively. RESULTS: Five patients received therapies aligned with CTGP, including HER2-targeted treatment, immunotherapy, and BRAF/MEK dual inhibition, showing positive responses. CTO drug sensitivity predicted progression under regorafenib (low potential benefit) and good response to chemotherapy with high potential benefit. The combination of CTGP and CTO drug sensitivity may exhibit significant correlation with clinical outcomes during treatment with candidate drugs, demonstrating a sensitivity of 79% and an accuracy of 75%. This encompasses various treatment modalities, such as chemotherapy, targeted therapy, and immunotherapy. CONCLUSION: The present investigation elucidated the integration of CTGP and CTO drug sensitivity screening into clinical practice in a complementary manner, showcasing a significant correlation between treatment response and testing outcomes. Additional prospective evaluation of these two testing modalities in a large cohort is warranted to confirm whether the inclusion of CTO drug sensitivity screening confers enhanced survival benefits compared to utilizing CTGP alone.
Assuntos
Neoplasias Gastrointestinais , Células Neoplásicas Circulantes , Organoides , Humanos , Neoplasias Gastrointestinais/genética , Neoplasias Gastrointestinais/tratamento farmacológico , Neoplasias Gastrointestinais/patologia , Neoplasias Gastrointestinais/sangue , Feminino , Masculino , Organoides/patologia , Organoides/efeitos dos fármacos , Pessoa de Meia-Idade , Idoso , Células Neoplásicas Circulantes/patologia , Células Neoplásicas Circulantes/metabolismo , Células Neoplásicas Circulantes/efeitos dos fármacos , Medicina de Precisão/métodos , Genômica/métodos , Estudos Retrospectivos , Perfilação da Expressão Gênica/métodos , Biomarcadores Tumorais/genética , Terapia de Alvo Molecular/métodos , Adulto , Ensaios de Seleção de Medicamentos Antitumorais/métodosRESUMO
The efficacy of chemotherapy varies significantly among patients with gastric cancer (GC), and there is currently no effective strategy to predict chemotherapeutic outcomes. In this study, we successfully establish 57 GC patient-derived organoids (PDOs) from 73 patients with GC (78%). These organoids retain histological characteristics of their corresponding primary GC tissues. GC PDOs show varied responses to different chemotherapeutics. Through RNA sequencing, the upregulation of tumor suppression genes/pathways is identified in 5-fluorouracil (FU)- or oxaliplatin-sensitive organoids, whereas genes/pathways associated with proliferation and invasion are enriched in chemotherapy-resistant organoids. Gene expression biomarker panels, which could distinguish sensitive and resistant patients to 5-FU and oxaliplatin (area under the dose-response curve [AUC] >0.8), are identified. Moreover, the drug-response results in PDOs are validated in patient-derived organoids-based xenograft (PDOX) mice and are consistent with the actual clinical response in 91.7% (11/12) of patients with GC. Assessing chemosensitivity in PDOs can be utilized as a valuable tool for screening chemotherapeutic drugs in patients with GC.
Assuntos
Fluoruracila , Organoides , Medicina de Precisão , Neoplasias Gástricas , Neoplasias Gástricas/tratamento farmacológico , Neoplasias Gástricas/patologia , Neoplasias Gástricas/genética , Humanos , Organoides/efeitos dos fármacos , Organoides/patologia , Organoides/metabolismo , Animais , Medicina de Precisão/métodos , Fluoruracila/farmacologia , Fluoruracila/uso terapêutico , Camundongos , Antineoplásicos/farmacologia , Antineoplásicos/uso terapêutico , Masculino , Feminino , Ensaios Antitumorais Modelo de Xenoenxerto , Ensaios de Seleção de Medicamentos Antitumorais/métodos , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Resistencia a Medicamentos Antineoplásicos/genética , Oxaliplatina/farmacologia , Oxaliplatina/uso terapêutico , Pessoa de Meia-Idade , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Biomarcadores Tumorais/metabolismo , Biomarcadores Tumorais/genética , Idoso , Relevância ClínicaRESUMO
Three-dimensional (3D) cell culture creates a more physiologically relevant environment for enhanced drug screening capabilities using microcarriers. An automated 3D system that integrates robotic manipulators, liquid handling systems, sensors, and environment control systems has the capacity to handle multiple samples in parallel, perform repetitive tasks, and provide real-time monitoring and analysis. This chapter describes a potential 3D cell culture drug screening model by combining renal proximal tubule cells as a representative normal cell line with cancer cell lines. This combination is subjected to drug screening to evaluate the drug's efficacy in suppressing cancer cells while minimizing impact on normal cells with the added benefit of having the ability to separate the two cell types by magnetic isolation for high content screens including mass spectrometry-based proteomics. This study presents advancements in 3D cell culture techniques, emphasizing the importance of automation and the potential of microcarriers in drug screening and disease modeling.
Assuntos
Técnicas de Cultura de Células em Três Dimensões , Humanos , Técnicas de Cultura de Células em Três Dimensões/métodos , Linhagem Celular Tumoral , Avaliação Pré-Clínica de Medicamentos/métodos , Ensaios de Seleção de Medicamentos Antitumorais/métodos , Túbulos Renais Proximais/citologia , Túbulos Renais Proximais/efeitos dos fármacos , Túbulos Renais Proximais/metabolismo , Técnicas de Cultura de Células/métodos , Antineoplásicos/farmacologia , Automação , Automação Laboratorial/métodos , Neoplasias/patologia , Neoplasias/tratamento farmacológicoRESUMO
Drug resistance is currently the biggest challenge in cancer chemotherapy. Here, we present a protocol to develop a chemotherapy drug screening process by constructing a cancer prognostic model (PM) using public databases. We describe steps for downloading code and data, preparing the expression matrix and metadata for analysis, screening modeling genes, and constructing a PM. We then detail procedures for constructing predictive websites for cancer patients' survival based on their age, tumor stage, gene expression levels, and risk scores. For complete details on the use and execution of this protocol, please refer to Bai et al.1.
Assuntos
Neoplasias , Humanos , Neoplasias/tratamento farmacológico , Neoplasias/genética , Prognóstico , Antineoplásicos/uso terapêutico , Antineoplásicos/farmacologia , Bases de Dados Factuais , Ensaios de Seleção de Medicamentos Antitumorais/métodosRESUMO
The NCI60 human tumor cell line screen has been in operation as a service to the cancer research community for more than 30 years. The screen operated with 96-well plates, a 2-day exposure period to test agents, and following cell fixation, a visible absorbance endpoint by the protein-staining dye sulforhodamine B. In this study, we describe the next phase of this important cancer research tool, the HTS384 NCI60 screen. Although the cell lines remain the same, the updated screen is performed with 384-well plates, a 3-day exposure period to test agents, and a luminescent endpoint to measure cell viability based upon cellular ATP content. In this study, a library of 1,003 FDA-approved and investigational small-molecule anticancer agents was screened by the two NCI60 assays. The datasets were compared with a focus on targeted agents with at least six representatives in the library. For many agents, including inhibitors of EGFR, BRAF, MEK, ERK, and PI3K, the patterns of GI50 values were very similar between the screens with strong correlations between those patterns within the dataset from each screen. However, for some groups of targeted agents, including mTOR, BET bromodomain, and NAMPRTase inhibitors, there were limited or no correlations between the two datasets, although the patterns of GI50 values and correlations between those patterns within each dataset were apparent. Beginning in January 2024, the HTS384 NCI60 screen became the free screening service of the NCI to facilitate drug discovery by the cancer research community. Significance: The new NCI60 cell line screen HTS384 shows robust patterns of response to oncology agents and substantial overlap with the classic screen, providing an updated tool for studying therapeutic agents. See related commentary by Colombo and Corsello, p. 2397.
Assuntos
Antineoplásicos , Ensaios de Seleção de Medicamentos Antitumorais , Humanos , Linhagem Celular Tumoral , Antineoplásicos/farmacologia , Antineoplásicos/uso terapêutico , Ensaios de Seleção de Medicamentos Antitumorais/métodos , Ensaios de Triagem em Larga Escala/métodos , Bibliotecas de Moléculas Pequenas/farmacologia , Neoplasias/tratamento farmacológico , Neoplasias/patologia , Neoplasias/metabolismo , Sobrevivência Celular/efeitos dos fármacosRESUMO
Patient-derived organoids (PDOs) have emerged as a promising platform for clinical and translational studies. A strong correlation exists between clinical outcomes and the use of PDOs to predict the efficacy of chemotherapy and/or radiotherapy. To standardize interpretation and enhance scientific communication in the field of cancer precision medicine, we revisit the concept of PDO-based drug sensitivity testing (DST). We present an expert consensus-driven approach for medication selection aimed at predicting patient responses. To further standardize PDO-based DST, we propose guidelines for clarification and characterization. Additionally, we identify several major challenges in clinical prediction when utilizing PDOs.
Assuntos
Antineoplásicos , Consenso , Desenvolvimento de Medicamentos , Neoplasias , Organoides , Medicina de Precisão , Organoides/efeitos dos fármacos , Humanos , Medicina de Precisão/métodos , Neoplasias/tratamento farmacológico , Desenvolvimento de Medicamentos/métodos , Antineoplásicos/farmacologia , Antineoplásicos/uso terapêutico , Ensaios de Seleção de Medicamentos Antitumorais/métodosRESUMO
BACKGROUND: Functional drug testing (FDT) with patient-derived tumor cells in microfluidic devices is gaining popularity. However, the majority of previously reported microfluidic devices for FDT were limited by at least one of these factors: lengthy fabrication procedures, absence of tumor progenitor cells, lack of clinical correlation, and mono-drug therapy testing. Furthermore, personalized microfluidic models based on spheroids derived from oral cancer patients remain to be thoroughly validated. Overcoming the limitations, we develop 3D printed mold-based, dynamic, and personalized oral stem-like spheroids-on-a-chip, featuring unique serpentine loops and flat-bottom microwells arrangement. RESULTS: This unique arrangement enables the screening of seven combinations of three drugs on chemoresistive cancer stem-like cells. Oral cancer patients-derived stem-like spheroids (CD 44+) remains highly viable (> 90%) for 5 days. Treatment with a well-known oral cancer chemotherapy regimen (paclitaxel, 5 fluorouracil, and cisplatin) at clinically relevant dosages results in heterogeneous drug responses in spheroids. These spheroids are derived from three oral cancer patients, each diagnosed with either well-differentiated or moderately-differentiated squamous cell carcinoma. Oral spheroids exhibit dissimilar morphology, size, and oral tumor-relevant oxygen levels (< 5% O2). These features correlate with the drug responses and clinical diagnosis from each patient's histopathological report. CONCLUSIONS: Overall, we demonstrate the influence of tumor differentiation status on treatment responses, which has been rarely carried out in the previous reports. To the best of our knowledge, this is the first report demonstrating extensive work on development of microfluidic based oral cancer spheroid model for personalized combinatorial drug screening. Furthermore, the obtained clinical correlation of drug screening data represents a significant advancement over previously reported personalized spheroid-based microfluidic devices. Finally, the maintenance of patient-derived spheroids with high viability under oral cancer relevant oxygen levels of less than 5% O2 is a more realistic representation of solid tumor microenvironment in our developed device.