Circulating human prostate cancer cells from an orthotopic mouse model rapidly captured by immunomagnetic beads and imaged by GFP expression

Circulating tumor cells (CTCs) are potential precursors of metastasis. They are also of use in diagnosing malignancy and for prognostic purposes. Our laboratory has previously isolated CTCs from orthotopic nude mouse models of human prostate cancer cells where the PC-3 cancer cells express green fluorescent protein (GFP). It was found that orthotopic tumors produced CTCs and not subcutaneous tumors, which may explain why orthotopic tumors metastasize and subcutaneous tumors do not. However, in this previous study, CTCs were observed only after culture. In the present study, using the GFP-expressing PC-3 orthotopic model and immunomagnetic beads coated with anti-epithelial cell adhesion molecule (EpCAM) and anti-prostate specific membrane antigen (PSMA), GFP-expressing CTC were isolated within 15 minutes and were readily visualized by GFP fluorescence. It was possible to immediately place the immunomagnetic-bead-captured GFP-expressing PC-3 CTCs in 3-dimensional sponge cell culture, where they proliferated. The combination of GFP expression and the use of immunomagnetic beads is a very powerful method to obtain CTCs for either immediate analysis or for biological characterization in vivo or in 3-dimensional culture.     

原位膀胱癌动物模型的建立及应用

背景与目的:表浅性膀胱癌术后膀胱灌注丝裂霉素等药物进行化疗,肿瘤仍有较高的复发率.有研究报道往膀胱内灌注小型干扰RNA(siRNA)可抑制裸鼠膀胱肿瘤生长.本研究目的是建立荷人膀胱癌的原位动物模型,通过磁共振成像(magnetic resonance imaging,MRI)监测肿瘤生长过程,并利用此模型评价靶向Survivin的干扰质粒对丝裂霉素的增效作用.方法:直视下经尿道机械损伤BALB/c裸鼠膀胱粘膜,将人膀胱癌细胞T24经尿道种植于25只裸鼠膀胱,建立荷人膀胱癌原位动物模型.以钆-二乙三胺五乙酸作为膀胱造影剂,用MRI监测肿瘤的生长,同时取裸鼠膀胱组织标本行HE染色进行病理学检查.同法建立膀胱癌裸鼠动物模型18只,分为对照组、丝裂霉素组和联合组3组,每周两次膀胱灌注,联合组为靶向Survivin的干扰质粒和丝裂霉素交替用药;膀胱灌注6次后荷瘤膀胱称重.结果:25只裸鼠在种植T24细胞后均形成膀胱肿瘤.种植后7天裸鼠膀胱MRI检查无明显变化,14、21、28天MRI检查均可发现膀胱不同程度的充盈缺损,MRI图像与肿瘤实际大小吻合.病理检查显示:种植后7天,肿瘤生长于裸鼠膀胱粘膜或浅层肌肉;14～28天局限于肌层;35天时侵及浆膜层.丝裂霉素组和联合组的抑瘤率分别为33.45%、56.34%,联合组优于丝裂霉素组(P＜0.05).结论:成功建立了裸鼠原位膀胱癌动物模型,肿瘤生长基本模拟了人膀胱癌的发生、发展过程;MRI检查可作为对裸鼠膀胱原位肿瘤动态观察的可靠方法.靶向Survivin的干扰质粒增加了丝裂霉素的抗肿瘤作用.     

A novel mouse model for segmental orthotopic colon cancer

Spontaneous colon tumor mouse strains offer numerous advantages in modeling disease. However, the wide temporal window in which lesions form and the stochastic nature of lesion location require larger cohorts for assessment of disease modulation. Reliable, reproducible and inexpensive mouse models of early-stage and invasive cancer would add to existing transgenic models. We show a new method for the creation of orthotopic murine tumors centered in the mucosal and submucosal layers anywhere in the colon, allowing creation of lesions of known age, location and extent. The system overcomes the disadvantages of heterotopic implantation and allows evaluation of lesions distally in the colon as well as proximally, thereby providing an additional method to study the effects of regionality. Invasion, host vascularization and application to disparate cell lines are demonstrated. Noninvasive imaging with magnetic resonance and colonoscopy, allowed in part by the tumor location, show potential applications of this approach.     

Nonviral cytokine gene therapy on an orthotopic bladder cancer model.

PURPOSE: The purpose is to assess cytokine gene transfection in tumor cells and its therapeutic efficacy in an orthotopic mouse bladder cancer model after liposome-mediated gene transfer. EXPERIMENTAL DESIGN: A total of 1 x 10(5) MB49 cells was instilled into the bladder of C57BL/6 mice after electrocautery to establish the tumor model. The plasmids were constructed by inserting the coding sequences for murine IFN-alpha1 and granulocyte macrophage colony-stimulating factor into a plasmid vector pBudCE4.1. Transient transfection was performed using a cationic lipid N-[1-(2,3-dioleoyloxyl)propyl]-N,N,N-trimethylammoniummethyl sulfate and methyl-beta-cyclodextrin-solubilized cholesterol. The in vitro expression of cytokines was checked by ELISA. The expression of the transgene in situ was confirmed by immunohistochemistry and 5-bromo-4-chloro-3-indolyl-beta-D-galactopyranoside staining. Mice bearing orthotopic tumors were treated with plasmid DNA/liposome complex by intravesical instillation twice a week for 3 weeks. RESULTS: Superficial bladder tumors were established by intravesical instillation of MB49 into cauterized bladders. The expression level of cytokines in transfected cell lines was increased significantly. In situ gene transfer to bladder tumors was accomplished via intravesical instillation of plasmid DNA/N-[1-(2,3-dioleoyloxyl)propyl]-N,N,N-trimethylammoniummethyl sulfate/methyl-beta-cyclodextrin-solubilized cholesterol after a single 2 h in situ transfection. The tumor incidence in the treatment groups was dramatically decreased from 76.9% in the control group to 15.4-30.8% in the treatment groups. CONCLUSIONS: We demonstrated in the orthotopic mouse bladder cancer model that successful inhibition of tumor cell growth could be obtained with cytokine gene therapy. The results suggest that our liposome transfection system appears to be a promising method for gene therapy of bladder cancer in vivo.     

浅表性膀胱癌模型的建立及其活体荧光成像

目的:建立浅表性膀胱癌模型并以活体荧光成像系统观察肿瘤生长情况.方法:利用脂质体将增强型绿色荧光蛋白(EGFP)表达质粒转染人膀胱癌细胞株KU-7,G418筛选稳定表达EGFP的克隆.采用经尿道膀胱灌注法使肿瘤细胞种植于膀胱黏膜.活体荧光荧光成像系统直接观察肿瘤细胞生长及形成瘤体的过程.结果:建立了转染率接近100%的人膀胱癌KU-7/EGFP细胞系,在体外及裸小鼠体内均能够长期稳定表达绿色荧光蛋白.活体荧光成像观察发现,1～4周随着肿瘤体积逐渐增大,肿瘤的发光随着观察时间的延长而增加.结论:利用绿色荧光蛋白标记膀胱肿瘤细胞,建立一种浅表性膀胱癌模型,为连续动态实时观察和准确评价自然状态下肿瘤细胞生长过程提供了新的手段.     

T24-多柔比星原位膀胱癌多药耐药模型的建立及检测

 目的　建立T24-多柔比星（ADM）模拟人类原位膀胱癌多药耐药模型并行耐药性检测。方法　采用细胞移植法将T24-ADM耐药株及T24细胞敏感株移植到两组裸鼠膀胱后定期观察其一般情况。28 d后处死裸鼠,称成瘤膀胱质量、测体积,行组织病理学和细胞学检测,反转录聚合酶链反应（RT-PCR）检测多药耐药基因-1（MDR-1）的表达,MTT法测瘤组织制成的细胞悬液耐药性。结果　T24-ADM组及T24组裸鼠膀胱的成瘤率分别为80 %（8／10）和90 %（9／10）,成瘤膀胱平均体质量和体积分别为 （0.8±0.3）g、（1.0±0.5）g、（875±158）mm3、（903±192）mm3,组间比较差异均无统计学意义（t＝1.332和t＝1.215,P＞0.05）。组织病理学：T24-ADM组4只出现右肾增大,T24组2只出现右肾增大,1.0 cm×1.8 cm,色暗灰到暗红。膀胱肿瘤组织切片细胞结构排列较乱,形态不规则,向肌层不同深度浸润,未突破浆膜层,两组间差异不明显。RT-PCR测MDR-1表达两组间差异有统计学意义（t＝3.612,P＜0.01）。MTT检测T24-ADM组裸鼠肿瘤细胞较T24组对ADM有明显的耐药性,差异有统计学意义（F＝412.107,P＜0.01）,且对其他几种药物也有耐药性,差异有统计学意义（P＜0.05）。结论　细胞移植法成功建立了T24-ADM原位膀胱癌耐药模型,并具有多药耐药特性。     

Establishment of orthotopic transplantation model of human bladder cancer and detection by MRI

Objective： To establish an orthotopic bladder cancer model bearing human bladder cancer for experimental research, and monitor tumor progression by magnetic resonance imaging （MRI）. Methods： The mucosa was mechanically damaged transurethrally under direct vision, and then human bladder cancer cell line T24 was inoculated into the bladders of BALB/c nude mice to establish orthotopic bladder cancer model. To find a suitable concentration of Gd-DTPA for this research. MRI was performed weekly to assess tumor growth, using Gd-DTPA as contrast agent. The pathologic morphology of the bladders and other specimens were observed with HE stain. Results： All the 25 mice developed bladder cancer after inoculation. The best concentration of Gd-DTPA was 1.408 mg/mL. On MRI, no change in the bladders was observed on day 7 after inoculation, filling defect in the bladders, accordant to actual tumor size, was detected on days 14, 21 and 28. Pathologic examination showed that tumor grew in the mucosa or superficial muscle of bladder on day 7, confined in muscle layer on days 14-28, and invaded serosa on day 35. Conclusion： Transurethrally damaged bladder mucosa under direct vision and instilled bladder cancer cell T24, we successfully established an orthotopic bladder cancer model. Tumor growth simulated the progression of human bladder cancer approximately. MRI was a reliable way for dynamic detection of murine orthotopic bladder tumor.     

人膀胱癌裸鼠原位移植瘤动物模型的建立和MRI检测

Objective:To establish an orthotopic bladder cancer model bearing human bladder cancer for experimental research, and monitor tumor progression by magnetic resonance imaging (MRI). Methods: The mucosa was mechanically damaged transurethrally under direct vision, and then human bladder cancer cell line T24 was inoculated into the bladders of BALB/c nude mice to establish orthotopic bladder cancer model. To find a suitable concentration of Gd-DTPA for this research. MRI was performed weekly to assess tumor growth, using Gd-DTPA as contrast agent. The pathologic morphology of the bladders and other specimens were observed with HE stain. Results: All the 25 mice developed bladder cancer after inoculation. The best concentration of Gd-DTPAwas 1.408 mg/mL. On MRI, no change in the bladders was observed on day 7 after inoculation, filling defect in the bladders, accordant to actual tumor size, was detected on days 14, 21 and 28. Pathologic examination showed that tumor grew in the mucosa or superficial muscle of bladder on day 7, confined in muscle layer on days 14-28, and invaded serosa on day 35. Conclusion: Transurethrally damaged bladder mucosa under direct vision and instilled bladder cancer cell T24, we successfully established an orthotopic bladder cancer model. Tumor growth simulated the progression of human bladder cancer approximately. MRI was a reliable way for dynamic detection of murine orthotopic bladder tumor.     

Complementarity of ultrasound and fluorescence imaging in an orthotopic mouse model of pancreatic cancer

Background  

Pancreatic cancer is a devastating disease characterized by dismal 5-year survival rates and limited treatment options. In an effort to provide useful models for preclinical evaluation of new experimental therapeutics, we and others have developed orthotopic mouse models of pancreatic cancer. The utility of these models for pre-clinical testing is dependent upon quantitative, noninvasive methods for monitoring in vivo tumor progression in real time. Toward this goal, we performed whole-body fluorescence imaging and ultrasound imaging to evaluate and to compare these noninvasive imaging modalities for assessing tumor burden and tumor progression in an orthotopic mouse model of pancreatic cancer.     

Metronomic oral paclitaxel shows anti-tumor effects in an orthotopic mouse model of ovarian cancer

Objective

The purpose of this study was to compare the in vivo anti-tumor efficacy of a mucoadhesive, lipid-based, oral paclitaxel formulation (DHP107) with traditional, intraperitoneal (IP) paclitaxel using an orthotopic mouse model of chemotherapy-sensitive SKOV3ip1 ovarian cancer.

Methods

To determine the optimal therapeutic dose of oral paclitaxel, DHP107 was administered per os to female athymic nude mice at 0, 25, or 50 mg/kg twice per week. Control mice received 100 µL saline once per week. IP injections of paclitaxel at 5 mg/kg once per week were used for comparison. To evaluate the potential therapeutic effect of metronomic DHP107 chemotherapy, mice received DHP107 50 mg/kg once per week per os, which was compared with 25 mg/kg twice per week and with vehicle-treated controls.

Results

Low-dose DHP107 (25 mg/kg) twice per week was as effective as IP paclitaxel (5 mg/kg once a week) but high-dose DHP107 (50 mg/kg once per week) was less effective at inhibiting tumor growth in an orthotopic mouse model (88%, 82%, and 36% decrease in tumor weight, respectively). Mice that received 25 mg/kg DHP107 twice per week or 50 mg/kg DHP107 once per week per os had a significant decrease in tumor weight compared with vehicle-treated controls (p<0.01, both doses).

Conclusion

Metronomic oral chemotherapy with DHP107 showed anti-tumor efficacy in vivo similar to IP paclitaxel in an orthotopic mouse model.     

Real-time imaging of tumor progression in a fluorescent orthotopic mouse model of thyroid cancer

There is a need for a clinically relevant mouse model of thyroid cancer that enables real-time, non-invasive monitoring of tumor growth, progression, and drug response over time. Human thyroid cancer cell lines NPA (papillary) and KAK-1 (anaplastic) were stably transfected to express either red or green fluorescent protein. Cancer cells were injected into the thyroid glands of 8-week-old athymic mice. The animals were imaged with whole-body fluorescence imaging weekly and sacrificed when premorbid. At necropsy, the primary tumor was resected en bloc with the respiratory system for processing and analysis. Histology was performed on fixed tissue specimens for review of morphologic findings. Both anaplastic and papillary thyroid cancer cell lines led to robust development of orthotopic fluorescent tumors in nude mice. Injection of 5×10(5) cancer cells was sufficient for tumor development. Tumors were visualized for both cell lines via non-invasive imaging as early as 3 weeks post-implantation and were monitored over time. Time to premorbid condition varied between mice and was associated with a primary tumor growth pattern (early local compression of the esophagus vs. late metastatic disease) rather than tumor size. At necropsy, tumor fluorescence demonstrated metastases in the lungs, lymph nodes and vessels that were not visible under white light. Thus an orthotopic mouse model of thyroid cancer has been developed that replicates the major clinical features of thyroid cancer and enables real-time, non-invasive monitoring of tumor progression. This model should permit preclinical evaluation of novel thyroid cancer therapeutics.     

Bioluminescence imaging correlates with tumor progression in an orthotopic mouse model of lung cancer

Background and ObjectivesTo determine whether bioluminescence imaging of human lung cancer cells growing in an orthotopic murine model provides a sensitive tool for monitoring tumor progression in athymic nude mice.MethodsHuman lung cancer (A549) cells were stably transfected with the firefly luciferase gene and inoculated into the right lung of athymic nude mice. Seven days after inoculation tumor growth was evaluated using the Kodak in-vivo Imaging System FX and continued to be monitored on a weekly basis.ResultsIn duplicate experiments, human lung cancer tumors formed in 90% of animal’s injected orthotopically. The mean intensity of the bioluminescence signal emitted from the lung cancer cells increased logarithmically during the course of study. Mice with positive bioluminescence signaling had confirmed tumors by microscopic histological analysis. Bioluminescence activity had a strong correlation with the tumor volume as determined histologically.ConclusionsBioluminescence intensity directly correlates with tumor volume and therefore offers a reliable approach for detecting and monitoring the growth of human lung cancer cells in orthotopic murine models.     

Real-time bioluminescence and tomographic imaging of gastric cancer in a novel orthotopic mouse model

Gastric cancer is the second leading cause of cancer mortality worldwide. Understanding the multistep process of carcinogenesis of gastric cancer is pivotal to develop novel therapeutic strategies. Molecular imaging in preclinical cancer models bridges the gap of laboratory-based experiment and clinical translation. To this end, the human gastric cancer cell line SGC-7901 was established to stably express luciferase and GFP by lentiviral transduction (SGC7901-Luc-GFP). Preclinical models were developed by orthotopic transplantation of SGC-7901-Luc-GFP into the sub-serosal layer of the stomach of immunocompromised mice. Tumor progression and therapeutic responses were dynamically tracked by bioluminescence imaging (BLI). Bioluminescence tomography (BLT) was used to monitor stereoscopic morphological and signal changes during tumor progression. Good correlation between cell number and bio-luminescence/fluorescence intensity was observed (R(2)=0.9983/r(2)=0.9974) in vitro. Tumor progression and therapeutic response could be successfully followed directly by BLI. Importantly, BLT provided a more accurate spatial location and tomographic quantification of the internal lesion. In conclusion, our novel bioluminescence-based preclinical gastric cancer models enable superior, noninvasive monitoring gastric cancer progression and their drug responses. The BLT technique in particular, may have great potential for future oncological studies.     

A novel mouse model of rectal cancer established by orthotopic implantation of colon cancer cells

A novel intraluminal colon tumor model was established in mice by intrarectal instillation of colon cancer cells followed by short-term induction of colitis by an irritant agent. Male BALB/c mice were fed a diet containing 3% (w/w) dextran sulfate sodium (DSS) for 7 days to induce colitis, and colon 26 cells (1-2 x 10(6) cells/mouse) were infused intrarectally after the mice had been deprived of food for the last 18 h of DSS treatment. The tumor incidence (%) and size (mean volume +/- SD, mm(3)) at the rectal mucosa were 35% (2 +/- 3), 95% (96 +/- 79), 95% (141 +/- 137) and 94% (325 +/- 270) at 1, 2, 3 and 4 weeks after instillation of tumor cells, respectively. Histopathological analyses revealed that a solid tumor was formed initially at the rectal mucosa at 1 week after instillation, then became invasive into the submucosal and muscular tissues at 3 weeks after implantation. Intrarectal instillation of human colon cancer cells, LS174T (1 x 10(7) cells/mouse), mixed with "Matrigel" (0.5 mg/mouse), an extracellular matrix solution, in SCID mice led to formation of rectal tumors at 4 weeks after instillation, and immunohistochemical analysis revealed that the tumor cells expressed human carcinoembryonic antigen, suggesting that the tumor nodule was derived from the instilled LS174T cells. Oral or intravenous administration of a camptothecin (CPT) derivative, CPT-11, resulted in a significant reduction in tumor incidence and tumor volume in the colon 26-intraluminal implantation system. In conclusion, it was suggested that the present intraluminal colon tumor model is useful for examination of chemotherapeutic agents and also intraluminal factors (dietary compounds, intestinal microflora, etc.) that might function to suppress or enhance the growth of colorectal cancer in situ.     

A novel orthotopic and metastatic mouse model of breast cancer in human mammary microenvironment

The studies of breast cancer heavily rely on the availability of experimental animal models. An ideal model of breast cancer is not only required to mimic the whole processes of tumor progress and metastasis, but also required to provide a normal human mammary microenvironment for the breast cancer cells to proliferate and metastasize. Numerous mouse models have been introduced in the literature but failed to achieve the two requirements simultaneously. In this study, we developed a novel human breast tissue-derived orthotopic and metastatic (BOM) mouse model of breast cancer, in which the normal human breast tissues were implanted subcutaneously to create a normal human mammary microenvironment, after which the human breast cancer cells were inoculated into the implants. The BOM model not only mimicked the whole processes of tumor progress and metastasis, but also allowed the orthotopic human breast cancer cells to proliferate in the normal human mammary microenvironment, and finally metastasize preferentially to the distant human tissues. Consequently, the BOM model contributed to the orthotopic tumor formation of 100% (11/11) and the metastatic tumor formation of 72.7% (8/11).     

Intravesical administration of exogenous microRNA-145 as a therapy for mouse orthotopic human bladder cancer xenograft

We previously reported that the level of microRNA (miR)-145 is attenuated in human bladder cancer cells. In this current study, we investigated whether intravesical administration of miR-145 could be a potential therapeutic strategy for controlling bladder cancer by using an orthotopic human bladder cancer xenograft model. Following transfection of 253J B-V cells with miR-145, the effects of the ectopic expression of miR-145 were examined by performing MTT, Western blotting analysis, Hoechst33342 staining, and wound healing assay in vitro. Also, a mouse orthotopic human bladder cancer model was established by inoculating 253J B-V cells into the bladder wall of mice. The anti-cancer effects of intravesical injections of miR-145 into these mice were then assessed. Transfection of 253J B-V cells with miR-145 induced apoptosis and suppression of cell migration in vitro. Western blotting showed that the levels of c-Myc, socs7, FSCN1, E-cadherin, β-catenin, and catenin δ-1 were decreased and that the PI3K/Akt and Erk1/2 signaling pathways were increased in compensatory fashion. In vivo, mice treated with miR-145 showed 76% inhibition of tumor growth, with a significant prolongation of animal survival (p = 0.0183 vs. control). Western blotting showed that both apoptosis and cell motility-related genes were significantly decreased as seen in vitro. Furthermore, PI3k/Akt and Erk1/2 signaling pathways, which were activated in a compensatory manner in vitro, were decreased in vivo. Intravesical administration of exogenous miR-145 was thus concluded to be a valid therapy for bladder cancer in this human bladder cancer xenograft model.     

An orthotopic mouse model of remetastasis of human colon cancer liver metastasis.

Whether liver metastases from colon cancer are capable of metastasizing to other sites is an important question in surgical oncology. To answer this question, we have developed a highly metastatic orthotopic transplant model of a liver metastasis from a human colon cancer patient in nude mice that targets the liver and lymph nodes. The metastatic human tumor was transplanted in athymic nude mice by surgical orthotopic implantation (SOI) of a liver metastasis from a colon cancer patient. The human colon tumor was then subsequently implanted in the colon by SOI or, in an additional series of nude mice, in the liver by surgical hepatic implantation (SHI). The mice were then explored over time for lymph node involvement beginning 10 days after implantation. After SOI, 100% of the animals had liver metastasis within 10 days, and subsequently, 19 days after SOI, all lymph nodes draining the liver were involved with metastasis without any retroperitoneal or lung tissue involvement. After SHI, all sites of lymphatic drainage of the liver, including portal, celiac, and mediastinal lymph nodes, were massively involved by metastasis in 100% of the animals as early as 10 days after tumor implantation on the liver. The results of this study demonstrate that liver metastases from colon cancer are capable of remetastasizing to other sites. This study thus suggests that in colon cancer patients with liver metastasis, mediastinal, celiac, and portal lymph node metastases originate from the liver metastasis and not, as previously thought, from primary colon cancer.     

The establishment of a new mouse model with orthotopic esophageal cancer showing the esophageal stricture

We established a promising new experimental animal model with an orthotopic xenograft of esophageal cancer that successfully represents poor oral intake, a major clinical feature of esophageal cancer. The advantage of this model is that no surgical technique is required, only the injection of a cell suspension by a needle and syringe via the esophageal lumen from the mouth, which provides a high reproducibility of tumor implantation and a rapid progress of outcome. We propose that this model is useful to study cancer-related outcomes and for developing new therapies for esophageal cancer, and we expect it to make a contribution to clinical practice.