类器官在肿瘤转化医学中的应用和进展

随着精准医学概念的提出，肿瘤作为一类高度异质性的疾病，其个体化治疗已成为精准医学的一个关键领域也受到更多关注。近期提出的类器官模型为肿瘤的基础研究和个体化治疗带来了新突破。类器官指从干细胞或器官祖细胞来源，以类似体内细胞分化的方式组织成的器官特异性的细胞集合。肿瘤类器官指利用原代恶性细胞经过体外3D培养构建的恶性细胞团，在体外培养条件下可持续增殖，一定程度上保留了原代肿瘤的病理学形态特征、基因组与转录组特征、药物敏感性及恶性细胞间异质性，为体外肿瘤研究提供了新方法，尤其在预测患者药物敏感性、药物高通量筛选等方面有巨大潜力，为肿瘤个体化治疗作出了贡献。但该模型仍存在不足，如无法重现体内肿瘤微环境等，使其在临床等方面的应用受限。目前将肿瘤类器官与其他肿瘤成分共培养、与微流控设备和生物打印技术等联合应用将有望弥补其缺陷、突破肿瘤治疗领域的瓶颈。本文将常用肿瘤研究模型的特点进行对比，总结部分肿瘤类器官的培养方法，并描述了肿瘤类器官的临床相关应用，最后对其与其他技术联合应用进行了叙述，并对未来肿瘤类器官的发展方向予以展望。     

胃肠胰神经内分泌肿瘤临床前模型的研究进展

胃肠胰神经内分泌肿瘤(gastroenteropancreatic neuroendocrine neoplasm,GEP-NEN)是一种近年来发病率逐年升高的罕见异质性肿瘤,根据其分化程度,可分为神经内分泌瘤(neuroendocrine tumor,NET)和神经内分泌癌(neuroendocrine carcinoma,NEC)。随着对其发病机制和治疗手段的探索逐渐加深,临床前模型的重要性日渐凸显。本文就目前已发表的细胞系、类器官模型、人源肿瘤异种移植物模型及基因工程小鼠模型等临床前模型进行综述,并探讨这些模型目前的实际应用情况及其优缺点,旨在为未来研究提供新的思路和方向。     

食管癌小鼠模型研究进展

食管癌小鼠模型有三大类：化学致癌模型、异种移植瘤模型和遗传工程模型。化学致癌模型很好地模拟了人食管癌发生发展的病理过程,制备方法简单;异种移植瘤模型是目前最常用的食管癌动物模型,具有成瘤快、成瘤率高、成本低的优点;遗传工程模型涉及的基因主要有p53、Cyclin D1、Brcal和p120ctn,有助于了解肿瘤的生物学特征,但其价格昂贵且制备时间长。     

肿瘤类器官技术及其在耐药研究中的应用

类器官，作为自问世以来便飞速发展并在各大实验平台广泛应用的三维结构微器官，其相比于传统的实验模型如2D细胞、人源性组织异种移植（patient-derived xenografts,PDX）等，更具有与真实器官较为相似的复杂结构以及多种细胞形态，并能较大程度地契合来源组织或器官的生理机能。此外，随着类器官培养技术及分析手段在研究中的创新改进，日益丰富的培养介质方案及高通量筛选等方法的建立使得类器官的应用更趋完备化和多样化。同时，新兴的类器官技术在肿瘤研究中扮演重要角色，尤其在药物筛选、疾病建模等领域可作为可靠度极高的实验模型。本篇综述概括了近年来肿瘤类器官在培养、分析方法上的部分改进和细化，以及肿瘤类器官在发展应用中相比于传统实验模型的优势，最后总结了肿瘤类器官在耐药性应用中的研究进展。本文对肿瘤类器官的耐药性概述将有利于为肿瘤耐药研究提供较为可靠的理论依据和方法参考。     

肺癌防治动物模型研究进展

精准模拟肺癌发病病理过程的动物模型是促进肺癌基础和转化研究的关键,而目前常用的皮下移植瘤模型难以体现肺脏的基质环境特征。小动物活体成像技术可客观、动态地评价肺癌的肿瘤负荷变化及转移发生情况,在此基础上建立的肺癌原位移植瘤动物模型可解决以上难题,充分体现肺癌的发病学特征。而人源性肿瘤组织异种移植(patient derived tumor xenograft,PDTX)原位模型和类器官模型也可部分体现肿瘤微环境和肿瘤细胞的异质性,均已成为该领域的重要发展方向。     

类器官在肿瘤中的研究现状及应用前景

类器官是由正常组织或肿瘤的特异性干细胞,在体外三维培养条件下,形成的三维体外细胞结构,具有自我组织形成类似于原始组织的"微型器官"的能力,能够较好地概括来源组织的特征和细胞异质性.类器官作为肿瘤研究的临床前模型,可用于药物筛选,预测患者对治疗的反应,为患者选择最合适的治疗方案.此外,还可对其进行基因修饰,用于肿瘤的发生...     

类器官培养系统在卵巢癌药物筛选与治疗靶点研究中的应用前景

卵巢癌是一种致命的妇科肿瘤,目前卵巢癌的研究平台主要使用2D细胞系培养和异种移植,两者均有其相应的局限性。类器官是一种体外3D培养模型,能够高度还原患者体内病变部位的组织学和基因组特征,维持肿瘤细胞的高度异质性。除此之外,类器官在基因分析、药物筛选和药物敏感性测定等方面具有强大优势,可以用作卵巢癌研究的临床前模型。     

肿瘤免疫研究新平台——肿瘤组织微环境类器官研究进展

肿瘤免疫研究新平台——肿瘤组织微环境类器官,因其保留了体内肿瘤微环境（TME）的特征,在肿瘤免疫治疗领域的应用有明显的优势。根据是否需要添加外源免疫细胞以及基质细胞可以将模拟TME的类器官模型分为初始TME类器官培养模型（native TME model）和重建TME类器官培养模型（reconstituted TME model）两种。介绍两种模型的体外培养方法和其在肿瘤免疫治疗临床前研究中的应用,如鉴定生物标志物、评估免疫联合治疗策略的疗效、制备肿瘤特异性T细胞和筛选免疫治疗新方法等,并对该平台目前存在的短板和局限性以及未来的发展前景等进行初步分析和展望,以期为该领域研究提供参考和帮助。     

患者源性膀胱癌类器官模型的应用及研究进展

目的 总结类器官在膀胱癌领域的研究现状及应用进展。方法 在PubMed、Web of Science、CNKI等数据库中,分别以“膀胱癌、类器官、癌症研究、三维培养”为中文关键词,“organoids, bladder cancer, precision treatment, drug screening”为英文关键词检索2000年至2022年肿瘤类器官和患者来源类器官相关文献。纳入标准：(1)类器官培养的进展;(2)类器官在膀胱癌中的研究;(3)类器官研究面临的问题。排除标准：重复性、数据陈旧的相关文献。最终纳入48篇文献进行分析。结果 患者源性的肿瘤类器官在药物研发、药物毒性和药效实验、精准医疗等方面取得有价值的进展,为临床研究提供参考。结论 肿瘤类器官培养作为一种新型的肿瘤体外模型,既能够很好的反应肿瘤异质性、模拟肿瘤微环境,又需要较少时间成本和经济成本,在肿瘤生物学研究、药物研发、精准医疗等方面具有显著优势。     

小鼠Lewis肺癌模型制备方法的比较研究

目的 探讨不同方法制备小鼠Lewis肺癌模型的可行性及相关技术的改良.方法 采用体外培养细胞法、胰酶消化法、胶原酶法和匀浆法制备的不同浓度的细胞悬液皮下注射C57BL/6小鼠,观察不同方法获得的细胞数、成瘤率、成瘤时间、肿瘤体积、离体肿瘤重量、荷瘤鼠的生活习性等,判定改良技术的可行性.结果 体外培养细胞法可以获得所需细胞,成瘤率100%.而胰酶消化法、匀浆法获得的细胞较少,而且成瘤率较低,约80%～90%和60%～75%.胶原酶法则是几种方法中获得细胞数最多的,成瘤率100%.结论 改良后的胶原酶法制备小鼠Lewis肺癌模型具有方法简便、成瘤率高、经济实惠的特点,为肿瘤的实验研究奠定了基础.     

Construction of in vitro patient-derived tumor models to evaluate anticancer agents and cancer immunotherapy

An in vitro assay system using patient-derived tumor models represents a promising preclinical cancer model that replicates the disease better than traditional cell culture models. Patient-derived tumor organoid (PDO) and patient-derived tumor xenograft (PDX) models have been previously established from different types of human tumors to recapitulate accurately and efficiently their tissue architecture and function. However, these models have low throughput and are challenging to construct. Thus, the present study aimed to establish a simple in vitro high-throughput assay system using PDO and PDX models. Furthermore, the current study aimed to evaluate different classes of anticancer drugs, including chemotherapeutic, molecular targeted and antibody drugs, using PDO and PDX models. First, an in vitro high-throughput assay system was constructed using PDO and PDX established from solid and hematopoietic tumors cultured in 384-well plates to evaluate anticancer agents. In addition, an in vitro evaluation system of the immune response was developed using PDO and PDX. Novel cancer immunotherapeutic agents with marked efficacy have been used against various types of tumor. Thus, there is an urgent need for in vitro functional potency assays that can simulate the complex interaction of immune cells with tumor cells and can rapidly test the efficacy of different immunotherapies or antibody drugs. An evaluation system for the antibody-dependent cellular cytotoxic activity of anti-epidermal growth factor receptor antibody and the cytotoxic activity of activated lymphocytes, such as cytotoxic T lymphocytes and natural killer cells, was constructed. Moreover, immune response assay systems with bispecific T-cell engagers were developed using effector cells. The present results demonstrated that in vitro assay systems using PDO and PDX may be suitable for evaluating anticancer agents and immunotherapy potency with high reproducibility and simplicity.     

人源免疫重建PDX模型构建及其在肿瘤免疫治疗中的应用进展

免疫治疗已成为肿瘤治疗领域最具潜力的手段之一。由于肿瘤存在高度异质性等特点,导致免疫治疗响应率偏低,疗效有限。因此,迫切需要具有预测性的临床前模型,用于推动合理、安全的免疫治疗策略开发。传统体内、外肿瘤研究模型受限于人源免疫系统的缺失。因此,构建同时具有人源免疫系统和肿瘤的研究模型成为热点。将患者来源肿瘤移植入人源免疫系统重建小鼠体内肿瘤、免疫双人源化模型,具有可重现人体内免疫细胞与肿瘤间相互作用的优势,成为生理和病理条件下模拟人体肿瘤免疫治疗疗效的新模型。本文主要对多种类型人源化免疫重建PDX模型构建、优缺点及其在肿瘤免疫治疗研究中的最新应用进展进行综述。      

卵巢癌临床前模型:从细胞系到类器官体

卵巢癌作为妇科三大恶性肿瘤之一,严重威胁着女性的生命健康。因其早期临床症状隐匿,往往在晚期甚至已经转移才被诊断出来。所以卵巢癌的总生存率一直不是很理想,寻找和开发新的治疗方案一直是卵巢癌的研究重点。在此过程中,准确模拟体内肿瘤生物学特征的临床前模型是必不可少的。细胞系因其培养简单,可大规模生产等优点,曾经是卵巢癌临床前研究的金标准。之后由于精准医学强调患者衍生材料的重要性,患者来源的异体移植模型(PDX)逐渐受到大家的重视。随后又出现了三维结构的球状体和更加能体现肿瘤异质性的类器官体。类器官体是利用干细胞直接诱导生成的三维组织模型,为人类生物学研究提供了新的方法,此项研究正在不断发展进步中。类器官体可以被应用到卵巢肿瘤的基因分析和药物筛选等各方面研究,为卵巢癌临床前研究提供了一个新的平台。     

Using heterogeneity of the patient-derived xenograft model to identify the chemoresistant population in ovarian cancer

A cornerstone of preclinical cancer research has been the use of clonal cell lines. However, this resource has underperformed in its ability to effectively identify novel therapeutics and evaluate the heterogeneity in a patient''s tumor. The patient-derived xenograft (PDX) model retains the heterogeneity of patient tumors, allowing a means to not only examine efficacy of a therapy, but also basic tenets of cancer biology in response to treatment. Herein we describe the development and characterization of an ovarian-PDX model in order to study the development of chemoresistance. We demonstrate that PDX tumors are not simply composed of tumor-initiating cells, but recapitulate the original tumor''s heterogeneity, oncogene expression profiles, and clinical response to chemotherapy. Combined carboplatin/paclitaxel treatment of PDX tumors enriches the cancer stem cell populations, but persistent tumors are not entirely composed of these populations. RNA-Seq analysis of six pair of treated PDX tumors compared to untreated tumors demonstrates a consistently contrasting genetic profile after therapy, suggesting similar, but few, pathways are mediating chemoresistance. Pathways and genes identified by this methodology represent novel approaches to targeting the chemoresistant population in ovarian cancer     

Modeling rectal cancer to advance neoadjuvant precision therapy

Progress in rectal cancer therapy has been hindered by the lack of effective disease-specific preclinical models that account for the unique molecular profile and biology of rectal cancer. Thus, we developed complementary patient-derived xenograft (PDX) and subsequent in vitro tumor organoid (PDTO) platforms established from preneoadjuvant therapy rectal cancer specimens to advance personalized care for rectal cancer patients. Multiple endoscopic samples were obtained from 26 Stages 2 and 3 rectal cancer patients prior to receiving 5FU/RT and implanted subcutaneously into NSG mice to generate 15 subcutaneous PDXs. Second passaged xenografts demonstrated 100% correlation with the corresponding human cancer histology with maintained mutational profiles. Individual rectal cancer PDXs reproduced the 5FU/RT response observed in the corresponding human cancers. Similarly, rectal cancer PDTOs reproduced significant heterogeneity in cellular morphology and architecture. PDTO in vitro 5FU/RT treatment response replicated the clinical 5FU/RT neoadjuvant therapy pathologic response observed in the corresponding patient tumors (p < 0.05). The addition of cetuximab to the 5FU/RT regiment was significantly more sensitive in the rectal cancer PDX and PDTOs with wild-type KRAS compared to mutated KRAS (p < 0.05). Considering the close relationship between the patient's cancer and the corresponding PDX/PDTO, rectal cancer patient-derived research platforms represent powerful translational research resources as population-based tools for biomarker discovery and experimental therapy testing. In addition, our findings suggest that cetuximab may enhance RT effectiveness by improved patient selection based on mutational profile in addition to KRAS or by developing a protocol using PDTOs to identify sensitive patients.     

Steroid Hormone Receptor Positive Breast Cancer Patient-Derived Xenografts

The vast majority of breast cancers are positive for estrogen receptor (ER) and depend on estrogens for growth. These tumors are treated with a variety of ER-targeted endocrine therapies, although eventual resistance remains a major clinical problem. Other steroid hormone receptors such as progesterone receptor (PR) and androgen receptor (AR) are emerging as additional prospective targets in breast cancer. The fundamental mechanism of action of these steroid receptors in gene regulation has been defined mainly by several breast cancer cell lines that were established in the late 1970s. More recently, breast cancer patient-derived xenografts (PDX) have been developed by multiple groups at institutions in several countries. These new models capture the large degree of heterogeneity between patients and within tumors and promise to advance our understanding of steroid hormone receptor positive breast cancer and endocrine resistance. Unfortunately, steroid hormone receptor positive breast cancers are much more difficult than their receptor negative counterparts to establish into sustainable PDX. Herein we discuss the derivation of steroid hormone receptor positive breast cancer PDX, several pitfalls in their genesis, and their utility in preclinical and translational steroid hormone receptor research.     

Real-time evaluation of glioblastoma growth in patient-specific zebrafish xenografts

BackgroundPatient-derived xenograft (PDX) models of glioblastoma (GBM) are a central tool for neuro-oncology research and drug development, enabling the detection of patient-specific differences in growth, and in vivo drug response. However, existing PDX models are not well suited for large-scale or automated studies. Thus, here, we investigate if a fast zebrafish-based PDX model, supported by longitudinal, AI-driven image analysis, can recapitulate key aspects of glioblastoma growth and enable case-comparative drug testing.MethodsWe engrafted 11 GFP-tagged patient-derived GBM IDH wild-type cell cultures (PDCs) into 1-day-old zebrafish embryos, and monitored fish with 96-well live microscopy and convolutional neural network analysis. Using light-sheet imaging of whole embryos, we analyzed further the invasive growth of tumor cells.ResultsOur pipeline enables automatic and robust longitudinal observation of tumor growth and survival of individual fish. The 11 PDCs expressed growth, invasion and survival heterogeneity, and tumor initiation correlated strongly with matched mouse PDX counterparts (Spearman R = 0.89, p < 0.001). Three PDCs showed a high degree of association between grafted tumor cells and host blood vessels, suggesting a perivascular invasion phenotype. In vivo evaluation of the drug marizomib, currently in clinical trials for GBM, showed an effect on fish survival corresponding to PDC in vitro and in vivo marizomib sensitivity.ConclusionsZebrafish xenografts of GBM, monitored by AI methods in an automated process, present a scalable alternative to mouse xenograft models for the study of glioblastoma tumor initiation, growth, and invasion, applicable to patient-specific drug evaluation.     

Application of plasma circulating KRAS mutations as a predictive biomarker for targeted treatment of pancreatic cancer

Kirsten rat sarcoma viral oncogene homolog (KRAS) mutations in circulating tumor deoxyribonucleic acid (ctDNA) have been reported as representative noninvasive prognostic markers for pancreatic ductal adenocarcinoma (PDAC). Here, we aimed to evaluate single KRAS mutations as prognostic and predictive biomarkers, with an emphasis on potential therapeutic approaches to PDAC. A total of 128 patients were analyzed for multiple or single KRAS mutations (G12A, G12C, G12D, G12R, G12S, G12V, and G13D) in their tumors and plasma using droplet digital polymerase chain reaction (ddPCR). Overall, KRAS mutations were detected by multiplex ddPCR in 119 (93%) of tumor DNA and 68 (53.1%) of ctDNA, with a concordance rate of 80% between plasma ctDNA and tumor DNA in the metastatic stage, which was higher than the 44% in the resectable stage. Moreover, the prognostic prediction of both overall survival (OS) and progression-free survival (PFS) was more relevant using plasma ctDNA than tumor DNA. Further, we evaluated the selective tumor-suppressive efficacy of the KRAS G12C inhibitor sotorasib in a patient-derived organoid (PDO) from a KRAS G12C-mutated patient using a patient-derived xenograft (PDX) model. Sotorasib showed selective inhibition in vitro and in vivo with altered tumor microenvironment, including fibroblasts and macrophages. Collectively, screening for KRAS single mutations in plasma ctDNA and the use of preclinical models of PDO and PDX with genetic mutations would impact precision medicine in the context of PDAC.     

HER2 as a novel therapeutic target for cervical cancer

Surgery and radiation are the current standard treatments for cervical cancer. However, there is no effective therapy for metastatic or recurrent cases, necessitating the identification of therapeutic targets. In order to create preclinical models for screening potential therapeutic targets, we established 14 patient-derived xenograft (PDX) models of cervical cancers using subrenal implantation methods. Serially passaged PDX tumors retained the histopathologic and genomic features of the original tumors. Among the 9 molecularly profiled cervical cancer patient samples, a HER2-amplified tumor was detected by array comparative genomic hybridization and targeted next-generation sequencing. We confirmed HER2 overexpression in the tumor and serially passaged PDX. Co-administration of trastuzumab and lapatinib in the HER2-overexpressed PDX significantly inhibited tumor growth compared to the control. Thus, we established histopathologically and genomically homologous PDX models of cervical cancer using subrenal implantation. Furthermore, we propose HER2 inhibitor-based therapy for HER2-amplified cervical cancer refractory to conventional therapy.