Application of patient-derived tumor models in anticancer drug development and individualized medicine

Malignant tumor is the second leading cause of death due to its high incidence, lack of effective treatment and poor prognosis. The evaluation of anticancer drugs used to based on NCI-60 cell line models, but the limited heterogeneity and the divorce from clinical practice of models lead to extremely low success rate of novel anticancer drugs during clinical trials (less than 10%). In recent years, because of the high heterogeneity and human derived tumor matrix, patient-derived tumor models have been gradually applied to the preclinical evaluation of various antitumor drugs, which shows certain advantages in predicting the clinical efficacy of antitumor drugs. Optimize the drug combination through patient-derived tumor models to achieve individualized medicine has gradually become an indispensable strategy in clinical cancer therapy. The current review summarized the development of patient-derived tumor models, characterized the application, advantages and challenges of them in preclinical antitumor drug evaluation and clinical precise medicine, which will provide a scientific basis and novel insights for further basic research, drug development and clinical application.

Patient-derived xenograft model in colorectal cancer basic and translational research

Colorectal cancer(CRC)is one of the most popular malignancies globally,with 930000 deaths in 2020.The evaluation of CRC-related pathogenesis and the discovery of po-tential therapeutic targets will be meaningful and helpful for improving CRC treat-ment.With huge efforts made in past decades,the systematic treatment regimens have been applied to improve the prognosis of CRC patients.However,the sensitivity of CRC to chemotherapy and targeted therapy is different from person to person,which is an important cause of treatment failure.The emergence of patient-derived xenograft(PDX)models shows great potential to alleviate the straits.PDX models possess similar genetic and pathological characteristics as the features of primary tu-mors.Moreover,PDX has the ability to mimic the tumor microenvironment of the original tumor.Thus,the PDX model is an important tool to screen precise drugs for individualized treatment,seek predictive biomarkers for prognosis supervision,and evaluate the unknown mechanism in basic research.This paper reviews the recent advances in constructed methods and applications of the CRC PDX model,aiming to provide new knowledge for CRC basic research and therapeutics.

Patient-derived non-small cell lung cancer xenograft mirrors complex tumor heterogeneity

Objective:Patient-derived xenograft(PDX)models have shown great promise in preclinical and translational applications,but their consistency with primary tumors in phenotypic,genetic,and pharmacodynamic heterogeneity has not been well-studied.This study aimed to establish a PDX repository for non-small cell lung cancer(NSCLC)and to further elucidate whether it could preserve the heterogeneity within and between tumors in patients.Methods:A total of 75 surgically resected NSCLC specimens were implanted into immunodeficient NOD/SCID mice.Based on the successful establishment of the NSCLC PDX model,we compared the expressions of vimentin,Ki67,EGFR,and PD-L1 proteins between cancer tissues and PDX models using hematoxylin and eosin staining and immunohistochemical staining.In addition,we detected whole gene expression profiling between primary tumors and PDX generations.We also performed whole exome sequencing(WES)analysis in 17 first generation xenografts to further assess whether PDXs retained the patient heterogeneities.Finally,paclitaxel,cisplatin,doxorubicin,atezolizumab,afatininb,and AZD4547 were used to evaluate the responses of PDX models to the standard-of-care agents.Results:A large collection of serially transplantable PDX models for NSCLC were successfully developed.The histology and pathological immunohistochemistry of PDX xenografts were consistent with the patients’tumor samples.WES and RNA-seq further confirmed that PDX accurately replicated the molecular heterogeneities of primary tumors.Similar to clinical patients,PDX models responded differentially to the standard-of-care treatment,including chemo-,targeted-and immuno-therapeutics.Conclusions:Our established PDX models of NSCLC faithfully reproduced the molecular,histopathological,and therapeutic characteristics,as well as the corresponding tumor heterogeneities,which provides a clinically relevant platform for drug screening,biomarker discovery,and translational research.

Spontaneous xenogeneic GvHD in Wilms'tumor Patient-Derived xenograft models and potential solutions

Severely immunocompromised NOD.Cg-PrkdcIl2rg(NOG)mice are among the ideal animal recipients for generation of human cancer models.Transplantation of human solid tumors having abundant tumor-i nfiltrating lymphocytes(TILs)can induce xenogeneic graft-versus-host disease(xGvHD)following engraftment and expansion of the TILs inside the animal body.Wilms’tumor(WT)has not been recognized as a lymphocyte-predominant tumor.However,3 consecutive generations of NOG mice bearing WT patient-derived xenografts(PDX)xenotransplanted from a single donor showed different degrees of inflammatory symptoms after transplantation before any therapeutic intervention.In the initial generation,dermatitis,auto-amputation of digits,weight loss,lymphadenopathy,hepatitis,and interstitial pneumonitis were observed.Despite antibiotic treatment,no response was noticed,and thus the animals were prematurely euthanized(day 47 posttransplantation).Laboratory and histopathologic evaluations revealed lymphoid infiltrates positively immunostained with anti-human CD3 and CD8 antibodies in the xenografts and primary tumor,whereas no microbial infection or lymphoproliferative disorder was found.Mice of the next generation that lived longer(91 days)developed sclerotic skin changes and more severe pneumonitis.Cutaneous symptoms were milder in the last generation.The xenografts of the last 2 generations also contained TILs,and lacked lymphoproliferative transformation.The systemic immunoinflammatory syndrome in the absence of microbial infection and posttransplant lymphoproliferative disorder was suggestive of xGvHD.While there are few reports of xGvHD in severely immunodeficient mice xenotransplanted from lymphodominant tumor xenografts,this report for the first time documented serial xGvHD in consecutive passages of WT PDX-bearing models and discussed potential solutions to prevent such an undesired complication.

Advantages and limitations in the establishment and utilization of patient-derived xenografts in gastric cancer

Owing to the high genetic heterogeneity of tumors, small number of therapeutic strategies available, and frequent presentation of drug resistance, the prognosis for patients with advanced gastric cancer(AGC) are unsatisfactory. The utility of traditional cancer cell lines in translational research is limited by their poor correspondence to the genomic alterations and expression profiles that occur in actual patient tumors. In the last decade, increasing attention has been given to patient-derived tumor xenografts(PDTXs), which can faithfully recapitulate the histopathology, molecular characteristics, and therapeutic responses of the patient's tumor. However, the widespread development and utilization of PDTXs is restricted by factors such as the timeframe of establishment, lymphoma transformation during passaging, the immunodeficient microenvironment, and pharmacokinetic differences between mice and humans. In this review, we summarize the establishment and characterization of PDTX models for gastric cancer(GC). We then weigh the advantages and limitations of PDTXs when used to evaluate novel compounds, identify effective biomarkers, demonstrate resistance mechanisms, and predict clinical outcomes.

Molecular phenotypes reveal heterogeneous engraftments of patient-derived hepatocellular carcinoma xenografts

Objective:Patient-derived xenograft(PDX)models provide a promising preclinical platform for hepatocellular carcinoma(HCC).However,the molecular features associated with successful engraftment of PDX models have not been revealed.Methods:HCC tumor samples from 76 patients were implanted in immunodeficient mice.The molecular expression was evaluated by immunohistochemistry.Patient and tumor characteristics as well as tumor molecular expressions were compared for PDX engraftment using the Chi-square test.The independent prediction parameters were identified by logistic regression analyses.Results:The engraftment rate for PDX models from patients with HCC was 39.47%(30/76).Tumors from younger patients and patients with elevated preoperative alpha-fetoprotein level had higher engraftment rates.Tumors with poor differentiation and vascular invasion were related to engraftment success.The positive expression of CK19,CD133,glypican-3(GPC3),and Ki67 in tumor samples was associated with engraftment success.Logistic regression analyses indicated that GPC3 and Ki67 were two of the strongest predictors of PDX engraftment.Tumors with GPC3/Ki67 phenotypes showed heterogeneous engraftment rates,with 71.9%in GPC3^(+)/Ki67^(+)tumors,30.8%in GPC3^(-)/Ki67^(+)tumors,15.0%in GPC3^(+)/Ki67^(-)tumors,and 0 in GPC3^(-)/Ki67^(-)tumors.Conclusions:Successful engraftment of HCC PDXs was significantly related to molecular features.Tumors with the GPC3+/Ki67+phenotype were the most likely to successfully establish HCC PDXs.

Translational pancreatic cancer research:a comparative study on patient-derived xenograft models

AIM To assess the viability of orthotopic and heterotopic patient-derived pancreatic cancer xenografts implanted into nude mice.METHODS This study presents a prospective experimental analytical follow-up of the development of tumours in mice upon implantation of human pancreatic adenocarcinoma samples. Specimens were obtained surgically from patients with a pathological diagnosis of pancreatic adenocarcinoma. Tumour samples from pancreatic cancer patients were transplanted into nude mice in three different locations(intraperitoneal, subcutaneous and pancreatic). Histological analysis(haematoxylin-eosin and Masson's trichrome staining) and immunohistochemical assessment of apoptosis(TUNEL), proliferation(Ki-67), angiogenesis(CD31) and fibrogenesis(α-SMA) were performed. When a tumour xenograft reached the target size, it was reimplanted in a new nude mouse. Three sequential tumour xenograft generations were generated(F1, F2 and F3).RESULTS The overall tumour engraftment rate was 61.1%. The subcutaneous model was most effective in terms of tissue growth(69.9%), followed by intraperitoneal(57.6%) and pancreatic(55%) models. Tumour development was faster in the subcutaneous model(17.7 ± 2.6 wk) compared with the pancreatic(23.1 ± 2.3 wk) and intraperitoneal(25.0 ± 2.7 wk) models(P = 0.064). There was a progressive increase in the tumour engraftment rate over successive generations for all three models(F1 28.1% vs F2 71.4% vs F3 80.9%, P < 0.001). There were no significant differences in tumour xenograft differentiation and cell proliferation between human samples and the three experimental models among the sequential generations of tumour xenografts. However, a progressive decrease in fibrosis, fibrogenesis, tumour vascularisation and apoptosis was observed in the three experimental models compared with the human samples. All three pancreatic patient-derived xenograft models presented similar histological and immunohistochemical characteristics.CONCLUSION In our experience, the faster development andgreatest number of viable xenografts could make the subcutaneous model the best option for experimentation in pancreatic cancer.

Specific bone region localization of osteolytic versus osteoblastic lesions in a patient-derived xenograft model of bone metastatic prostate cancer

Objective:Bone metastasis occurs in up to 90%of men with advanced prostate cancer and leads to fractures,severe pain and therapy-resistance.Bone metastases induce a spectrum of types of bone lesions which can respond differently to therapy even within individual prostate cancer patients.Thus,the special environment of the bone makes the disease more complicated and incurable.A model in which bone lesions are reproducibly induced that mirrors the complexity seen in patients would be invaluable for pre-clinical testing of novel treatments.The microstructural changes in the femurs of mice implanted with PCSD1,a new patient-derived xenograft from a surgical prostate cancer bone metastasis specimen,were determined.Methods:Quantitative micro-computed tomography(micro-CT)and histological analyses were performed to evaluate the effects of direct injection of PCSD1 cells or media alone(Control)into the right femurs of Rag2
/
gc
/
male mice.Results:Bone lesions formed only in femurs of mice injected with PCSD1 cells.Bone volume(BV)was significantly decreased at the proximal and distal ends of the femurs(p<0.01)whereas BV(p<0.05)and bone shaft diameter(p<0.01)were significantly increased along the femur shaft.Conclusion:PCSD1 cells reproducibly induced bone loss leading to osteolytic lesions at the ends of the femur,and,in contrast,induced aberrant bone formation leading to osteoblastic lesions along the femur shaft.Therefore,the interaction of PCSD1 cells with different bone region-specific microenvironments specified the type of bone lesion.Our approach can be used to determine if different bone regions support more therapy resistant tumor growth,thus,requiring novel treatments.

Advances in prostate cancer research models:From transgenic mice to tumor xenografting models

The identification of the origin and molecular characteristics of prostate cancer(PCa)has crucial implications for personalized treatment.The development of effective treatments for PCa has been limited;however,the recent establishment of several transgenicmouse lines and/or xenografting models is better reflecting the disease in vivo.With appropriate models,valuable tools for elucidating the functions of specific genes have gone deep into prostate development and carcinogenesis.In the present review,we summarize a number of important PCa research models established in our laboratories(PSA-Cre-ERT2/PTEN transgenic mouse models,AP-OX model,tissue recombination-xenografting models and PDX models),which represent advances of translational models from transgenic mouse lines to human tumor xenografting.Better understanding of the developments of these models will offer new insights into tumor progression and may help explain the functional significance of genetic variations in PCa.Additionally,this understanding could lead to new modes for curing PCa based on their particular biological phenotypes.

Circulating tumor DNA dynamics analysis in a xenograft mouse model with esophageal squamous cell carcinoma

BACKGROUND It remains unclear which factors,such as tumor volume and tumor invasion,influence circulating tumor DNA(ctDNA),and the origin of ctDNA in liquid biopsy is always problematic.To use liquid biopsies clinically,it will be very important to address these questions.AIM To assess the origin of ctDNA,clarify the dynamics of ctDNA levels,assess ctDNA levels by using a xenograft mouse after treatment,and to determine whether tumor volume and invasion are related to ctDNA levels.METHODS Tumor xenotransplants were established by inoculating BALB/c-nu/nu mice with the TE11 cell line.Groups of mice were injected with xenografts at two or four sites and sacrificed at the appropriate time point after xenotransplantation for ctDNA analysis.Analysis of ctDNA was performed by droplet digital PCR,using the human telomerase reverse transcriptase(hTERT)gene.RESULTS Mice given two-site xenografts were sacrificed for ctDNA at week 4 and week 8.No hTERT was detected at week 4,but it was detected at week 8.However,in four-site xenograft mice,hTERT was detected both at week 4 and week 6.These experiments revealed that both tumor invasion and tumor volume were asso ciated with the detection of ctDNA.In resection experiments,hTERT was detected at resection,but had decreased by 6 h,and was no longer detected 1 and 3 d after resection.CONCLUSION We clarified the origin and dynamics of ctDNA,showing that tumor volume is an important factor.We also found that when the tumor was completely resected,ctDNA was absent after one or more days.

Establishment,functional and genetic characterization of three novel patient-derived rectal cancer cell lines

AIM To establish patient-individual tumor models of rectal cancer for analyses of novel biomarkers, individual response prediction and individual therapy regimens.METHODS Establishment of cell lines was conducted by direct in vitro culturing and in vivo xenografting with subsequent in vitro culturing. Cell lines were in-depth characterized concerning morphological features, invasive and migratory behavior, phenotype, molecular profile including mutational analysis, protein expression, and confirmation of origin by DNA fingerprint. Assessment of chemosensitivity towards an extensive range of current chemotherapeutic drugs and of radiosensitivity was performed including analysis of a combined radioand chemotherapeutic treatment. In addition, glucose metabolism was assessed with 18 F-fluorodeoxyglucose(FDG) and proliferation with 18 F-fluorothymidine.RESULTS We describe the establishment of ultra-low passage rectal cancer cell lines of three patients suffering from rectal cancer. Two cell lines(HROC126, HROC284 Met) were established directly from tumor specimens while HROC239 T0 M1 was established subsequent to xenografting of the tumor. Molecular analysis classified all three cell lines as CIMP-0/non-MSI-H(sporadic standard) type. Mutational analysis revealed following mutational profiles: HROC126: APC^(wt), TP53^(wt), KRAS^(wt), BRAF^(wt), PTEN^(wt); HROC239 T0 M1: APC^(mut), P53^(wt), KRAS^(mut), BRAF^(wt), PTEN^(mut) and HROC284 Met: APC^(wt), P53^(mut), KRAS^(mut), BRAF^(wt), PTEN^(mut). All cell lines could be characterized as epithelial(EpCAM+) tumor cells with equivalent morphologic features and comparable growth kinetics. The cell lines displayed a heterogeneous response toward chemotherapy, radiotherapy and their combined application. HROC126 showed a highly radio-resistant phenotype and HROC284 Met was more susceptible to a combined radiochemotherapy than HROC126 and HROC239 T0 M1. Analysis of 18 F-FDG uptake displayed a markedly reduced FDG uptake of all three cell lines after combined radiochemotherapy. CONCLUSION These newly established and in-depth characterized ultra-low passage rectal cancer cell lines provide a useful instrument for analysis of biological characteristics of rectal cancer.

Reduction-responsive nucleic acid nanocarrier-mediated miR-22 inhibition of PI3K/AKT pathway for the treatment of patient-derived tumor xenograft osteosarcoma

miRNAs are important regulators of gene expression and play key roles in the development of cancer, including osteosarcoma. During the development of osteosarcoma, the expression of miR-22 is significantly downregulated, making miR-22 as a promising therapeutic target against osteosarcoma. To design and fabricate efficient delivery carriers of miR-22 into osteosarcoma cells, a hydroxyl-rich reduction-responsive cationic polymeric nanoparticle, TGIC-CA (TC), was developed in this work, which also enhanced the therapeutic effects of Volasertib on osteosarcoma. TC was prepared by the ring-opening reaction between amino and epoxy groups by one-pot method, which had the good complexing ability with nucleic acids, reduction-responsive degradability and gene transfection performance. TC/miR-22 combined with volasertib could inhibit proliferation, migration and promote apoptosis of osteosarcoma cells in vitro. The anti-tumor mechanisms were revealed as TC/ miR-22 and volasertib could inhibit the PI3K/Akt signaling pathway synergistically. Furthermore, this strategy showed outstanding tumor suppression performance in animal models of orthotopic osteosarcoma, especially in patient-derived chemo-resistant and chemo-intolerant patient-derived xenograft (PDX) models, which reduced the risk of tumor lung metastasis and overcame drug resistance. Therefore, it has great potential for efficient treatment of metastasis and drug resistance of osteosarcoma by the strategy of localized, sustained delivery of miR-22 using the cationic nanocarriers combined with non-traditional chemotherapy drugs.

靶向FOLR1的脱氧核酶提高鼻咽癌移植瘤对紫杉醇的敏感性

目的:探讨裸鼠鼻咽癌移植瘤能否通过靶向FOLR1的脱氧核酶(DrzE)提高其对紫杉醇的敏感性。方法:取CNE-1鼻咽癌亲本细胞及CNE-1/taxol紫杉醇耐药细胞进行裸鼠成瘤实验,分别予靶向FOLR1的“10-23”型DrzE单用和紫杉醇单用及紫杉醇-DrzE联用后比较各组鼻咽癌移植瘤体的抑制情况。RT-qPCR及Western blot检测用药前后各组瘤体FOLR1 mRNA及蛋白的表达。结果:CNE-1瘤体较CNE-1/taxol瘤体生长速度快,瘤体体积更大(P<0.05);CNE-1瘤体生长能被紫杉醇单药抑制,但紫杉醇对CNE-1/taxol瘤体无效;两种瘤体生长均能被DrzE单药抑制,其中耐药瘤体更显著(P<0.05);紫杉醇与DrzE联用较分别单用两药对移植瘤的抑制作用更显著(P<0.05);给药前CNE-1/taxol移植瘤中FOLR1的表达量显著高于CNE-1(P<0.01),用药后DrzE单药组及紫杉醇与DrzE联用组中两种移植瘤体FOLR1表达量均显著低于对照组(P<0.05)。结论:靶向FOLR1的脱氧核酶DrzE转染裸鼠移植瘤体后可减慢鼻咽癌瘤体的生长,提高鼻咽癌耐药移植瘤体对紫杉醇的敏感性。

头颈部恶性肿瘤研究模型的演化

恶性肿瘤发生发展的机制探索以及抗癌药物治疗疗效的评估均有赖于各种体内与体外研究模型的建立。近几十年间,随着生物医学技术的快速发展,恶性肿瘤的体内外研究模型也发生了巨大的变化。基因检测技术从单基因到多基因的进展促进了生物信息学飞速发展和恶性肿瘤概念的转变;体外细胞研究模型从单层的二维培养、原代培养向立体的三维构型发展,从而更好地重现肿瘤组织的细胞间交互作用与功能;体内动物研究模型由传统的致癌物诱导、细胞或组织形成移植瘤逐渐演变为基因编辑的动物模型或人源性肿瘤异种移植模型,从而可以针对性地研究相关基因在肿瘤发生发展中的作用;传统的临床研究也从简单的临床回顾性研究更多地向前瞻性研究转变,Ⅰ期/Ⅱ期/Ⅲ期临床研究,研究者发起的临床研究以及真实世界临床研究,这些研究为临床研究增添了活力。目前恶性肿瘤研究模型存在的主要不足包括模型的单一性、对肿瘤微环境的模拟不足、动物肿瘤模型与人类肿瘤差异性,以及缺乏对个性化医疗的考量。未来仍需要进一步研发和优化研究模型,并更有效地将不同模型整合起来,形成一个优化的整体实验模型系统。本文将系统回顾恶性肿瘤研究模型的演化并对相关模型进行阐述,为科研工作者进行恶性肿瘤的研究提供合理的研究模型。

The evolution of cancer genomic medicine in Japan and the role of the National Cancer Center Japan

The journey to implement cancer genomic medicine(CGM)in oncology practice began in the 1980s,which is considered the dawn of genetic and genomic cancer research.At the time,a variety of activating oncogenic alterations and their functional significance were unveiled in cancer cells,which led to the development of molecular targeted therapies in the 2000s and beyond.Although CGM is still a relatively new discipline and it is difficult to predict to what extent CGM will benefit the diverse pool of cancer patients,the National Cancer Center(NCC)of Japan has already contributed considerably to CGM advancement for the conquest of cancer.Looking back at these past achievements of the NCC,we predict that the future of CGM will involve the following:1)A biobank of paired cancerous and non-cancerous tissues and cells from various cancer types and stages will be developed.The quantity and quality of these samples will be compatible with omics analyses.All biobank samples will be linked to longitudinal clinical information.2)New technologies,such as whole-genome sequencing and artificial intelligence,will be introduced and new bioresources for functional and pharmacologic analyses(e.g.,a patient-derived xenograft library)will be systematically deployed.3)Fast and bidirectional translational research(bench-to-bedside and bedside-to-bench)performed by basic researchers and clinical investigators,preferably working alongside each other at the same institution,will be implemented;4)Close collaborations between academia,industry,regulatory bodies,and funding agencies will be established.5)There will be an investment in the other branch of CGM,personalized preventive medicine,based on the individual's genetic predisposition to cancer.

癌胚性TUFT1过表达及基因转录干预对肝细胞癌的抑制作用

目的分析肝细胞癌(hepatocellular carcinoma,HCC)釉丛蛋白(tuftelin,TUFT1)表达及基因转录干预的抑制作用。方法收集术后HCC组织和癌旁组织,分析TUFT1表达;从不同肝癌细胞和对照肝LO2细胞株中提取TUFT1,以免疫印迹法筛选TUFT1低或过表达的癌细胞株;将已构建含TUFT1基因或干扰TUFT1-shRNA3的慢病毒质粒,分别转染低或过表达TUFT1的癌细胞株,以CCK-8法、划痕、Transwell法、流式细胞术等分析细胞增殖、侵袭、迁移等生物学行为;将稳定表达TUFT1的两种细胞株皮下接种裸鼠,分析对移植瘤生长的影响。结果肝癌组织TUFT1过表达,主要定位于细胞浆和细胞膜。肝癌组TUFT1阳性率为87.1%,均显著高于癌旁组(χ^(2)=18.563,P<0.001);肝癌细胞株中TUFT1表达明显高于LO2细胞(P<0.001)。体外研究,以有效的TUFT1-shRNA3质粒,转染高TUFT1表达的MHCC-97H细胞,在TUFT1功能丧失后癌细胞的增殖、侵袭和迁移能力显著抑制,凋亡率升高;以插入TUFT1基因的质粒,转染低TUFT1表达的Hep3B细胞,在TUFT1功能获得后癌细胞增殖、侵袭和迁移能力均显著增强。体内研究,将功能丧失的MHCC-97H细胞于裸鼠皮下接种,移植瘤中TUFT1下调,并显著抑制瘤体生长(Z=12.000,P<0.01)。结论TUFT1为癌胚性蛋白,对其干预有望成为潜在的HCC治疗的分子靶目标。

肺癌恶性胸腔积液来源肿瘤细胞的小鼠PDX模型构建及实验验证

目的·构建肺癌患者恶性胸腔积液(malignant pleural effusion,MPE)肿瘤细胞来源的肿瘤异种移植(patientderived tumor xenograft,PDX)模型,并进行实验验证。方法·从基因表达综合数据集(Gene Expression Omnibus,GEO)下载人肺癌伴MPE单细胞转录组测序公共数据GSE131907和人肺癌实体瘤单细胞转录组测序公共数据GSE203360,对数据进行聚类、差异基因本体功能富集分析,明确应用MPE建模的可行性。同时收集肺癌患者的MPE样本,经离心、裂解红细胞等富集细胞操作后,将其植入非肥胖型糖尿病重症联合免疫缺陷(non-obese diabetic/severe combined immunodeficient,NOD/SCID)小鼠皮下,待移植瘤生长至1000 mm³时进行瘤体传代及保存。对稳定传代移植瘤进行组织病理学检测,通过苏木精-伊红染色(hematoxylin-eosin staining,H-E染色)观察细胞组织形态,免疫组织化学法(immunohistochemistry,IHC)检测肺癌标志物表达情况。结果·经单细胞数据分析发现MPE中肿瘤细胞的增殖功能更强,提示MPE中肿瘤细胞PDX建模或具备更佳成瘤效果;共收集35例肺癌MPE样本,成功构建13例PDX模型,成功率达37.14%;在组织病理学检测中,H-E染色可见移植瘤组织细胞异型性明显,IHC检测显示细胞角蛋白7(cytokeratin 7,CK7)、甲状腺转录因子1(thyroid transcription factor-1,TTF1)和天冬氨酸蛋白酶A(Napsin A)等肺癌标志物均呈阳性表达。结论·通过富集肺癌患者MPE中的肿瘤细胞,成功构建了更为简便高效、可实时动态建模的PDX模型。该模型保留了肺癌患者肿瘤细胞的恶性特征及蛋白表达特性,为肺癌伴MPE患者的基础研究和临床用药指导提供了重要的实验模型工具。

灵菌红素对耐阿霉素人乳腺癌细胞MCF-7/ADR的作用研究

目的探讨灵菌红素对耐阿霉素人乳腺癌细胞MCF-7/ADR的作用。方法采用平板发酵粘质沙雷氏菌WA12-1-18生产灵菌红素,CCK-8测定灵菌红素的体外活性及MCF-7/ADR的耐药指数。构建裸鼠皮下移植瘤模型,灵菌红素高、低剂量组分别腹腔注射5.0、2.5 mg/kg灵菌红素,生理盐水对照位腹腔注射等体积生理盐水,每4天1次,连续用药24 d,观察移植瘤的体积、质量及裸鼠体质量,HE染色观察移植瘤组织及裸鼠主要脏器的病理情况,免疫组织化学检测移植瘤Ki-67的表达。结果获得的灵菌红素质量分数为95.18%,对MCF-7和MCF-7/ADR的IC50分别为0.484μg/mL和0.264μg/mL。MCF-7/ADR耐药指数13,符合细胞耐药株的要求。灵菌红素处理组裸鼠移植瘤增长速度较生理盐水组慢,肿瘤细胞排列稀疏,核小且着色较浅,Ki-67染色后棕色明显减少。灵菌红素2.5 mg/kg组和灵菌红素5 mg/kg组,在荷MCF-7/ADR裸鼠中抑瘤率分别为37.23%和53.72%。另外,各组裸鼠体质量差异无统计学意义,主要脏器无明显病理变化。结论灵菌红素可抑制裸鼠体内耐阿霉素人乳腺癌细胞MCF-7/ADR的生长,对心、肝、脾、肺和肾等主要脏器无明显毒副作用,为灵菌红素在耐阿霉素乳腺癌的临床应用提供了实验依据。

转导蛋白β样1X连接受体1表达对卵巢癌A2780细胞增殖和迁移的影响

目的:研究转导蛋白β样1X连接受体1(transducin beta-like 1X-linked receptor,TBL1XR1)在卵巢癌患者组织中表达,及其对卵巢癌A2780细胞增殖和迁移的影响。方法:采用实时荧光定量PCR(qRT-PCR)检测10对卵巢癌组织、癌旁组织中及人卵巢癌IOSE80、A2780、CP70、SKOV-3中TBL1XR1 mRNA表达,筛选TBL1XR1 mRNA高表达细胞株。选择4~6周龄雌性BALB/C裸鼠,建立卵巢癌人源肿瘤异种移植(patient-derived tumor xenografts,PDTX)模型;将10只模型鼠均分为siR-NC组和si-TBL1XR1组,每组5只,分别给予siR-NC、si-TBL1XR1局部注射,10 mg/kg,每3 d注射1次,18 d后取各组瘤组织,计算其体积与重量。取卵巢癌A2780细胞,将其分为siR-NC组、si-TBL1XR1组、pcDNA3.1组和pcDNA3.1-TBL1XR1组,分别予以siR-NC、si-TBL1XR1、pcDNA3.1空载质粒和pcDNA3.1-TBL1XR1质粒处理;采用蛋白免疫印迹法检测各组卵巢癌细胞周期蛋白表达,MTT比色法检测细胞活力,流式细胞术检测细胞周期和凋亡细胞比例,以及Transwell细胞迁移实验检测细胞迁移能力。结果:卵巢癌组织中TBL1XR1 mRNA表达明显高于癌旁组织(P<0.05);人卵巢癌A2780细胞系TBL1XR1 mRNA表达明显高于卵巢癌IOSE80、CP70、SKOV-3细胞系(P<0.05)。与siR-NC组相比,第18天si-TBL1XR1组瘤体积明显减小(P<0.05),重量明显降低(P<0.05)。与siR-NC组相比,si-TBL1XR1组促癌细胞周期蛋白表达明显降低(P<0.05),与pcDNA3.1组相比,pcDNA3.1-TBL1XR1组表达则明显升高(P<0.05);与siR-NC组相比,si-TBL1XR1组卵巢癌细胞迁移数明显降低(P<0.05),早期凋亡和晚期凋亡细胞比例明显升高(P<0.05);与pcDNA3.1组相比,pcDNA3.1-TBL1XR1组卵巢癌细胞迁移数明显增多(P<0.05),早期凋亡和晚期凋亡细胞比例明显降低(P<0.05)。结论:TBL1XR1在卵巢癌组织中呈高表达,降低TBL1XR1 mRNA表达可抑制卵巢癌A2780细胞增殖和迁移。