Efficacy and cardiotoxicity observation of different doses of capecitabine in patient-derived tumor xenograft of mice with colorectal cancer / 肿瘤研究与临床

Objective:To explore the correlation of the dose of capecitabine with the efficacy and cardiotoxicity in patient-derived tumor xenograft (PDX) model of mice with colorectal cancer.Methods:The fresh cancer tissues of 1 colorectal cancer patient were transplanted into the bilateral axillary subcutaneous of immunodeficient NOG mice to establish PDX model and passage stably. And then the morphology of tumor cells in primary generation and the second-generation tumor tissues was observed by using HE staining. The expression of tumor markers was detected by using immunohistochemistry method, and the model was evaluated. Mice were intragastrically infused with 200, 300 and 400 mg/kg capecitabine once a day, which were treated as low, middle and high dose groups respectively, 5 rats in each group; in the control group, 0.9% NaCl solution was perfused into the stomach; 14 d in total, use stop for 7 d, consecutively administered in this way. The body weight was measured every day and the tumor volume was measured every 3 days. After 100 days of observation, the mice were killed, and the tumor tissue was taken to measure the tumor weight and then the tumor volume, tumor volume inhibition rate and tumor inhibition rate were calculated. The morphology of tumor tissues was observed by using HE staining. The protein levels of anti-tumor effect indexes like rasP21, cyclooxygenase 2 (COX2), prostaglandin E2 (PGE2), cardiac troponin Ⅰ (cTn-Ⅰ) and brain natriuretic peptide (BNP) in serum of mice were detected by using enzyme linked immunosorbent assay (ELISA).Results:PDX model of mice with colorectal cancer was successfully constructed, and the histological characteristics of the primary tumor in the model were well preserved. During administration, 1 mouse died in the capecitabine high dose group; a slow down in tumor volume growth could be found with the increased dose of capecitabine. There was no statistically significant difference in body weight among 4 groups until all mice were killed ( P > 0.05). The tumor volume and tumor weight in the low, middle and high dose groups were lower than those in the control group (all P < 0.05), and the tumor volume and tumor weight showed an obvious decrease with the increase in dose. The tumor volume inhibition rates of low, middle and high dose groups were 42.61%, 67.61% and 77.27%, respectively, and the tumor inhibition rates were 35.53%, 67.77% and 75.09%, respectively. The serum anti-tumor effect indexes rasP21, COX2 and PGE2 in the middle and high dose groups were decreased compared with those in the control group (all P < 0.05), while cTn-Ⅰ and BNP levels were increased compared with those in the control group (all P < 0.05). Conclusions:The established PDX model of mice with colorectal cancer can better retain the histological characteristics of the original tumor. After treatment of middle and high dose of capecitabine, the tumor inhibition effect is obvious, but the risk of myocardial damage should be noticed.

Application and expectation of patient-derived tumor xenograft models in the treatment of pancreatic cancer / 国际外科学杂志

Pancreatic cancer has a poor prognosis, systemic comprehensive therapy including chemotherapeutic and molecular targeted therapy is the key to improve the postoperative prognosis of pancreatic cancer, as well as to prolong the survival time of advanced pancreatic cancer. However, due to the drug resistance and heterogeneity of pancreatic cancer cells, systemic comprehensive treatment still does not reach the ideal effect, and personalized therapy will be an important approach to solve this problem. The personalized therapy for pancreatic cancer will become possible with the application of patient-derived tumor xenograft (PDX) models. Here, this article will review the progress of the application of PDX models in comprehensive therapy of pancreatic cancer based on reviewing the methods of establishing pancreatic cancer PDX models, aiming to provide new ideas for personalized therapy of pancreatic cancer.

Demethylation effect of emodin combined with 5AzA-cdR on tumor suppressor gene p16, RASSF1A and ppENK in subcutaneous xenografts of pancreatic cancer nude mice model / 中华胰腺病杂志

Objective To investigate whether emodin combined with 5AzA-cdR can enhance the demethylation of tumor suppressor genes p16,RASSF1A and ppENK in nude mice with subcutaneously transplanted pancreatic cancer.Methods Pancreatic cancer cells Panc1 burdened subcutaneous xenograft nude mice model was established,which were randomly divided into control group,emodin group,5AzA-cdR group and emodin combined 5AzA-cdR group (combined group).The growth of transplanted tumors wasobserved in each group.Methylation specific PCR (MSP) was used to detect the methylation levels of p16,RASSF1A and ppENK in the xenograft tumor tissue among three groups.The mRNA and protein expression of three tumor suppressor genes were detected by FQ-PCR and Western blotting,respectively.Results The weight of xenografts in the control group,emodin group,5AzA-cdR group,and combination group were (0.28 ±0.01),(0.17 ± 0.01),(0.12 ± 0.02),(0.08 ± 0.01)g,respectively.The tumor volume was (517 ±0.02),(382 ± 0.01),(232 ± 0.03),(169 ± 0.01) mm3.The methylation levels of p16 were 1.00 ± 0.00,0.89 ± 0.02,0.63 ± 0.02,and 0.19 ± 0.01;the methylation levels of RASSF1A were 1.00 ± 0.00,0.88 ± 0.02,0.51 ± 0.01,and 0.32 ± 0.01;the methylation degree of ppENK was 1.00 ± 0.00,0.92 ± 0.02,0.77 ± 0.02 and 0.31 ± 0.01,respectively.The expression of p16 mRNA was 1.00 ± 0.00,1.71 ±0.02,2.67 ± 0.02,3.81 ± 0.01.The expression of RASSF1A mRNA was 1.00 ± 0.00,1.92 ±0.02,2.73 ± 0.03,3.77 ± 0.01.The expression of ppENK mRNA was 1.00 ± 0.00,1.69 ± 0.03,2.17 ± 0.02 and 4.28 ± 0.01.The expression of p16 protein was 1.00 ± 0.00,1.71 ± 0.02,2.67 ± 0.02,3.81 ± 0.01;the expression of RASSF1A protein was 1.00 ± 0.00,1.92 ± 0.02,2.73 ± 0.03.3.77 ± 0.01;ppENK protein expression levels were 1.00 ±0.00,1.69 ±0.03,2.17 ±0.02,4.28 ±0.01.The weight and volume of xenografts in the three treatment groups were significantly smaller than those in the control group.The methylation of three tumor suppressor genes was lower than that of the control group,and the expression of tumor suppressor mRNA and protein was all significantly higher than the control group,which the combination drug group was also significantly stronger than that in emodin group and 5AzA-cdR group,and the differences were statistically significant (P ＜ 0.05 or ＜ 0.01).Conclusions The combination of emodin and 5AzA-cdR can enhance the demethylation effect of 5A6A-cdR on the tumor suppressor genes p16,RASSF1A and ppENK in the tumor tissue of pancreatic cancer xenograft model.

Modeling guidelines and latest applications of patient-derived xenograft models / 国际外科学杂志

Tumor models are required to keep the most original characteristics of the primary tumor in precision treatment.Patient-derived xenograft models are established when cancerous cells or tissues directly from patients'primary tumors are transplanted into immunodeficient mice to mimic human tumor biology in vivo,which have been widely used in cancer research.In this review,we initially summarize the methodology and its progress to create patient-derived xenograft models from three aspects including grafts,hosts and grafting regions,and then go over recent applications of patient-derived xenograft models in basic cancer research on the areas of tumorigenesis,metastasis and drug resistance and in translational medical research of tumor,such as exploring cancer biomarkers,screening anti-cancer drugs and personalized therapy for neoplasm.Finally,we propose the problems of patientderived xenograft,which must be solved urgently.

Muscarinic cholinergic receptor antagonist inhibits the growth and angiogenesis of small cell lung cancer in vivo / 肿瘤

Objective: To investigate the effects of muscarinic cholinergic receptor 3 (M3R) antagonist 4-diphenylacetoxy-N -methylpiperidine methiodide (4-DAMP) on the growth of small cell lung cancer and angiogenesis in nude mice. Methods: The mRNA and protein expressions of M3R in human small cell lung cancer SBC3 cells were detected by RT-PCR and Western blotting, respectively. The subcutaneous transplantation tumor model of SBC3 cells in nude mice was established. The model mice were randomly divided intofour groups, then intraperitoneally injected with 0.9% sodium chloride solution (as the control group) and different doses of 4-DAMP (0.5, 1 and 2 mg/kg), respectively (once a day, for 15 days). The size of xenograft tumor was measured every 2 days. After the nude mice were sacrificed, the tumor mass was measured, and the tumor inhibitory rate was calculated. The expressions of M3R and vascular endothelial growth factor (VEGF) in the tumor tissues were detected by real-time fluorescent quantitative PCR and Western blotting, respectively. The microvessel density (MVD) and VEGF expression were detected by immunohistochemistry. Results: Human small cell lung cancer SBC3 cells expressed M3R mRNA and protein. After the subcutaneous transplantation tumor model of SBC3 cells in nude mice was successfully established and treated with different doses (0.5-2 mg/kg) of 4-DAMP, the sizes of xenograft tumors were significantly decreased as compared with the control group (all P < 0.05), and the weights of tumor tissues were significantly reduced (all P < 0.01). The tumor growth inhibitory rates in 0.5, 1 and 2 mg/kg 4-DAMP-treated groups were 15.82%, 33.54% and 55.06%, respectively. Furthermore, the relative expression levels of M3R and VEGF as well as MVD in the tumor tissues of 0.5, 1 and 2 mg/kg 4-DAMP-treated groups were significantly down-regulated as compared with the control group (all P < 0.05). Conclusion: M3R antagonist 4-DAMP can inhibit the growth and angiogenesis of human small cell lung cancer in nude mice.

Down-regulation of microRNA-21 expression inhibits the growth of human colon carcinoma SW620 cells in nude mice / 肿瘤

Objective: To investigate the effect of microRNA-21 (miR-21) expression down-regulated by antisense oligonucleotides (ASOs) on the growth of human colon carcinoma SW620 cells in nude mice. Methods: Firstly, the nude mouse model of human colon carcinoma cell line SW620 was established. Then the recombinant plasmid p-miR-21-ASOs or the control plasmid p-Cont was locally injected into tumor tissues (once every 4 days), and the tumor growth was observed. On the 5th day after the last injection, all of mice were sacrificed, and the tumor tissues were removed and measured. Then the histomorphology of tumor tissues was observed by HE staining assay. The apoptosis of tumor cells was analyzed by TUNEL assay. The expression of Ki-67 proliferation nuclear antigen was measured by immunofluorescence assay. Furthermore, the expression levels of mature miR-21, as well as cyclin-dependent kinase (CDK) 2, CDK3, CDK4 and CDK6 were detected by real-time fluorescent quantitative PCR. And the expressions of phosphorylated protein kinase B (p-Akt) and phosphorylated extracellular regulated protein kinase (p-ERK) were analyzed by Western blotting. Results: Compared with p-Cont group, the xenograft tumors in p-miR-21-ASOs group grew slowly (P < 0.05), and the weight of tumor tissues was significantly reduced (P < 0.01). Meanwhile, there were large areas of necrosis in tumor tissues in p-miR-21- ASOs group. The expression level of miR-21 in tumor tissues was significantly decreased after p-miR-21-ASOs injection (P < 0.01). Moreover, the expression of Ki-67 in tumor tissues was significantly down-regulated (P < 0.01), and the relative number of apoptosis cells increased obviously (P < 0.05) in p-miR-21-ASOs group. Furthermore, the relative expression levels of CDK2, CDK3, CDK4, CDK6, p-Akt and p-ERK were significantly down-regulated in p-miR- 21-ASOs group as compared with p-Cont group (all P < 0.05). Conclusion: Down-regulation of miR-21 expression by ASOs can significantly inhibit the growth of human colon carcinoma SW620 cells in nude mice, indicating that miR-21 may be a potential target for gene therapy of colon cancer.

Baicalin induces cell cycle arrest and apoptosis of human colon cancer in vitro and in vivo / 肿瘤

Objective: To investigate the effects of baicalin on the cell cycle and apoptosis of human colon cancer in vitro and in vivo, and to further clarify its possible molecular mechanism. Methods: After treatment with different concentrations (0, 50, 100, 200, 400 and 800 μg/mL) of baicalin for 48 h, the morphology and viability of human normal colorectal mucosa FHC cells and human colon cancer HCT116 cells were detected by invert microscopy and MTT method, respectively. The changes of apoptosis rate and cell cycle distribution of HCT116 cells after baicalin treatment were detected by flow cytometry. The expression levels of apoptosis-related proteins [poly ADP-ribose polymerase-1 (Parp-1), caspase 3, X-linked inhibitor of apoptosis protein (XIAP), nuclear factor-κB (NF-κB), p53, Bcl-2 and Bax] and cell cycle-related proteins (cyclin D1 and cyclin B1) in HCT116 cells treated with baicalin were measured by Western blotting. After the orthotopic xenograft tumor model of colon cancer HCT116 cells in nude mice were constructed and treated with baicalin by gavage, the body weight of mice and the tumor size were checked, and the baicalin-induced apoptosis in xenograft tumors was also assayed using TUNEL methods. Results: As compared with baicalin-untreated control group, 50-800 μg/mL baicalin significantly suppressed the viability of colon cancer HCT116 cells (all P 0.05). Conclusion: Baicalin can inhibit the growth of colon cancer HCT116 cells in vivo and in vitro through inducing apoptosis and cell cycle arrest at G1 phase.

Inhibitory effect of 2,3',4,5'-tetramethoxystilbene on growth of endometrial carcinoma xenografts / 肿瘤

Objective: To investigate the effect of 2,3',4,5'-tetramethoxystilbene (TMS) on the growth of xenograft tumors of endometrial carcinoma Ishikawa cells, to explore its molecular mechanism, and to evaluate the adverse reaction of TMS. Methods: Human xenograft models were established in nude mice by inoculation with human endometrial carcinoma Ishikawa cells, and randomly divided into TMS group and control group by subcutaneously injection with 10 mgkg-1d-1 TMS and equal volume of olive oil, respectively. The volume and weight of xenograft tumors were calculated. The body weight of mice and the wet weight of tumor, heart, liver, spleen, lung, kidney, uterus and ovary in mice were measured. The solid organ damage was detected by HE staining. The expressions of cytochrome P4501B1 (CYP1B1), extracellular signal-regulated kinase (ERK) and phospho-ERK (p-ERK) proteins in xenograft tumors were detected by SP immunohistochemistry method. Results: After subcutaneous injection of TMS, the volume (P 0.05). No obvious lesion was found in various organs of nude mice in each group. The expressions of CYP1B1, ERK and p-ERK proteins were weakly positive in xenograft tumors. Conclusion: TMS can obviously inhibit the growth of human endometrial carcinoma xenografts in nude mice, and has no obvious toxic and side effects on tumor-bearing nude mice.

Establishing a colorectal cancer liver metastasis patient-derived tumor xenograft model for the evaluation of personalized chemotherapy

PURPOSE: In order to suggest optimal anticancer drugs for patient-tailored chemotherapy, we developed a colorectal cancer (CRC)-liver metastasis patient-derived tumor xenograft (PDTX) model. METHODS: Tissue obtained from a patient with CRC-liver metastasis (F0) was transplanted in a nonobese female mouse with diabetic/severe combined immune deficiency (F1) and the tumor tissue was retransplanted into nude mice (F2). When tumor volumes reached ~500 mm³, the F2 mice were randomly divided into 4 groups (n = 4/group) of doxorubicin, cisplatin, docetaxel, and nontreated control groups. The tumor tissues were investigated using H&E staining, terminal deoxynucleotidyl transferase dUTP nick end labeling assays, and immunohistochemistry. To determine where the mutant allele frequencies varied across the different passages, we isolated genomic DNA from the primary tumor, liver metastasis, and PDTX models (F1/F2). RESULTS: The physiological properties of the tumor were in accord with those of the patient's tumors. Anticancer drugs delayed tumor growth, inhibited proliferation, and caused apoptosis. Histological assessments revealed no observable heterogeneity among the intragenerational PDTX models. Target exon sequencing analysis without high-quality filter conditions revealed some genetic variations in the 83 cancer-related genes across the generations. However, when de novo mutations were defined as a total count of zero in F0 and ≥5 in F2, exactly prognostic impact of clone cancer profiling (EGFR, KRAS, BRAF, PIK3CA, NRAS, APC and TP53) were detected in the paired. CONCLUSION: A CRC liver metastasis PDTX model was established for the evaluation of chemotherapeutic efficacy. This model retained the physiological characters of the patient tumors and potentially provides a powerful means of assessing chemotherapeutic efficacy.

Antitumor effect of thermosensitive hydrogel co-loading paclitaxel and cisplatin on cervical cancer / 肿瘤

Objective: To develop a hydrogel-based drug delivery system coloading cisplatin (DDP) and paclitaxel (PTX), polyethylene glycolpolycaprolactone- polyethylene glycol (PECE)/DDP+methoxypolyethylene glycols-polycaprolactone (MPEG-PCL)/PTX (or PDMP in short), and to explore the therapeutic efficacy of PDMP hydrogel on cervical cancer in animal model and the possible mechanism. Methods: MPEG-PCL/PTX micelles were successfully synthesized by solid dispersion method. Then the PDMP gel-drug system was successfully developed after mixing MPEG-PCL/PTX with PECE hydrogel and DDP solution, and the physical and chemical properties of PDMP were verified by high performance liquid chromatography, rheometer and laser particle size analysis, respectively. The cervical cancer model of HeLa cells in nude mice was established and randomly divided into four groups: control group (0.9% NaCl solution), simple vector group (PECE+MPEG-PCL), free drug group (PTX+DDP) and PDMP gel drug group (12 mice in each group). The tumor-beared mice in the different groups were intratumorally administered with different drugs, then the tumor growth was observed. Half of the mice in each group (n = 6) were sacrificed after 10 days of treatment, and tumor tissues were harvested for immunohistochemistry and flow cytometry in order to evaluate Ki-67 expression, cell cycle distribution and apoptosis. The remaining tumor-beared mice in each group (n = 6) were cultured, and the survival time was observed. Results: PDMP gel drug system was successfully prepared. As compared with the other groups, PDMP inhibited tumor growth in nude mice and improved the survival time of tumor-bearing nude mice (all P<0.05). After PDMP treatment, the positive expression rate of Ki-67 in xenograft tumors was significantly reduced as compared with the other groups (all P<0.05), suggesting that the proliferation activity of tumor cells was decreased. PDMP caused the cell block at G1 phase, and increased the apoptosis rate as compared with the other groups (all P<0.05). Conclusion: PDMP may be a promising intratumoral drug delivery system to enhance antitumor efficacy for the treatment of cervical cancer in situ.

Inhibitory effect of ethanol extract of Forsythia suspensa root on growth of esophageal cancer xenograft in vivo / 肿瘤

Objective: To investigate the inhibitory effect of ethanol extract of Forsythia suspensa root (FSEER) on growth of esophageal cancer cells in nude mice. Methods: The inhibitory effect of FSEER on growth of esophageal cancer cell lines including TE-1, TE-13, Yes-2 and Eca-109 was measured by MTT method. The xenograft tumor models of TE-13 cells in nude mice were established and divided into two groups: treatment group [treated with FSEER (50 mg/kg)] and the control group (treated with equal volume of normal saline). Then the growth of xenograft tumors in nude mice was observed and their sizes and weights were measured. The apoptosis of tumor tissues in nude mice was deteced by TUNEL staining analysis. The expressions of Bcl-2 and Bax in tumor tissues were detected by immunohistochemical method. Morphological changes of lung and liver tissues in nude mice were observed by hematoxylin-eosin staining. Results: FSEER had obvious inhibitory effect on the growth of esophageal cancer cells in vitro (P < 0.01). Moreover, the growth of TE-13 cells was inhibited by FSEER in the dose-and timedependent manner (P < 0.01). The average volume and weight of tumor tissues in nude mice of FSEER treatment group were significantly smaller and lower, respectively, as compared with those of the control group (P < 0.05). Comparing to the control group, the number of apoptotic cells in FSEER treatment group was significantly increased (P < 0.05), while the expression level of Bcl-2 was decreased (P < 0.05), but Bax expression level was increased (P < 0.05). No obvious morphological changes were found in liver and lung tissues in FSEER treatment group and the control group. Conclusion: FSEER has inhibitory effect on the growth of xenograft tumor of esophageal cancer cells in nude mice, which may be related to inducing apoptosis.

Inhibitory effect of sanguinarine on growth of xenograft tumors of gastric cancer GTL-16 cells in nude mice and its related mechanism / 肿瘤

Objective: To investigate the antitumor effect of sanguinarine (SAN) in vivo and its possible mechanism. Methods: The tumor-bearing BALB/c male nude mouse models were established by subcutaneous inoculation of gastric cancer GTL-1 6 cells. The mouse models were divided into three groups [14 mice in each group were intragastrically administered with 0.9% NaCl solution (as the control group), 2.5 mg/kg SAN and 5.0 mg/kg SAN every other day for 7 times, respectively]. The general status of the mice was observed, and the tumor volume was measured. After 28 days, the mice were sacrificed, then the tumor volume and weight were recorded. The histopathologic examination of tumor tissues was performed using hematoxylin-eosin (HE) staining. Then the protein expression of epidermal growth factor receptor (EGFR) in tumor tissues was detected by Western blotting. Results: As compared with the control group, the tumors grew slowly in SAN treatment groups. At autopsy, the volume and weight of tumors in SAN treatment groups were all smaller than those in the control group (P < 0.01), and the tumor volume and weight were smallest in 5.0 mg/kg SAN group (P < 0.01). The result of histopathologic examination revealed that SAN treatment could reduce the accumulation of tumor cells and epidermal invasion of tumor cells. The expression levels of EGFR were significantly lower in SAN treatment groups than that in the control group (P < 0.05). Conclusion: SAN inhibits the growth of tumor in nude mice inoculated with gastric cancer GTL-16 cells, and which may be related to the downregulation of EGFR expression.

Tumor Growth Suppression and Enhanced Radioresponse by an Exogenous Epidermal Growth Factor in Mouse Xenograft Models with A431 Cells / Journal of the Korean Cancer Association, 대한암학회지

PURPOSE: The purpose of this study was to evaluate whether an exogenous epidermal growth factor (EGF) could induce anti-tumor and radiosensitizing effects in vivo. MATERIALS AND METHODS: BALB/c-nu mice that were inoculated with A431 (human squamous cell carcinoma) cells in the right hind legs were divided into five groups: I (no treatment), II (EGF for 6 days), III (EGF for 20 days), IV (radiotherapy [RT]), and V (RT plus concomitant EGF). EGF was administered intraperitoneally (5 mg/kg) once a day and the RT dose was 30 Gy in six fractions. Hematoxylin and eosin (H&E) stained sections of tumor, liver, lung, and kidney tissues were investigated. Additionally, tumors were subjected to immunohistochemistry staining with caspase-3. RESULTS: EGF for 6 days decreased tumor volume, but it approached the level of the control group at the end of follow-up (p=0.550). The duration of tumor shrinkage was prolonged in group V while the slope of tumor re-growth phase was steeper in group IV (p=0.034). EGF for 20 days decreased tumor volume until the end of the observation period (p < 0.001). Immunohistochemistry revealed that mice in group V showed stronger intensity than those in group IV. There were no abnormal histological findings upon H&E staining of the normal organs. CONCLUSION: EGF-induced anti-tumor effect was ascertained in the xenograft mouse models with A431 cells. Concomitant use of EGF has the potential role as a radiosensitizer in the design of fractionated irradiation.

Research progress of patient-derived xenograft model / 国际外科学杂志

Tumor cell line culture technologies emerged as a strong impetus to the development of tumor biology research.However,cell line tumor model presented limited predictive value and instable properties in the clinical translational research of new cancer therapies.Recently Patient-derived Xenograft Model (PDX) has aroused researcher's attention and been widely applied to new cancer therapy research.This new model could highly retain the stroma heterogeneity、histological characteristics and molecular diversity of the original tumor,and is expected to bring new breakthroughs in individualized treatment of tumor patients.

Effects of acitretin combined with clarithromycin on tumor growth and angiogenesis in human oral epidermoid carcinoma xenografts in nude mice / 中华皮肤科杂志

Objective To evaluate the effects of acitretin combined with clarithromycin on tumor growth in human oral epidermoid carcinoma xenografts in nude mice,and to investigate their antitumor mechanisms.Methods A cell line of human oral epidermoid carcinoma was subcutaneously inoculated into 31 Balb/c nude mice to establish a xenograft model of human skin tumor.Then,the nude mice were randomly classified into 6 groups according to a double blind protocol:control group (n =6) remaining untreated,placebo group (n =5) treated with wheat flour,acitretin group (n =5) treated with acitretin 7.2 mg/kg per day,clarithromycin group (n =5) treated with clarithromycin 100 mg/kg per day,acitretin + placebo group (n =5) treated with both acitretin (7.2 mg/kg per day) and wheat flour,and acitretin + clarithromycin group (n =5) treated with acitretin (7.2 mg/kg per day) and clarithromycin 100 mg/kg per day.All the drugs were intragastrically administrated once daily.After three weeks of treatment,mice were sacrificed and xenografts were removed.Then,the size and weight of xenografts were measured,and pathological analysis was conducted.Real time-PCR was performed to quantify the mRNA expressions of vascular endothelial growth factor (VEGF) and nuclear factor (NF)-κB,and immunohistochemistry was carried out to observe the expression of VEGF as well as to determine microvessel density (MVD) and Ki-67 proliferation index.By using the software SPSS 19.0,analysis of variance was performed for comparison of measurement data,and least significant difference (LSD) test for paired comparisons.Results Both the size and weight of xenografts in the acitretin + clarithromycin group were significantly lower than those in the other groups (all P ＜ 0.05).Real-time fluorescence-based PCR revealed weaker mRNA expressions of VEGF and NF-κB in the acitretin + clarithromycin group compared with the control group,clarithromycin group and acitretin group (all P ＜ 0.05).As immunohistochemistry showed,the acitretin + clarithromycin group displayed a decrease in the expression rate (all P ＜ 0.01) and staining intensity of VEGF,MVD (all P ＜ 0.01) with a sparse distribution of microvessels,Ki-67 proliferation index (all P ＜ 0.05) and proliferative activity of tumor cells compared with the control group,clarithromycin group and acitretin group.Conclusion Acitretin combined with clarithromycin can synergistically inhibit the growth of human oral epidermoid carcinoma xenografts in nude mice,downregulate VEGF expression,and suppress angiogenesis and tumor proliferation.

New preclinical mouse model: Patient-derived xenografts / 肿瘤

The mouse models of nude mice bearing subcutaneous xenografts derived from tumor cell lines provide a preclinical testing system for exploring novel anticancer therapies. However, due to the lack of heterogeneity and an ability of spontaneous distant metastasis, these traditional models are difficult to accurately simulate the clinical condition of the patient. Recently, some researchers have developed better preclinical models by using patient-derived xenografts (PDXs) in mice. It has been shown that transplanting a variety of patient-derived tumor tissues into an appropriate anatomical site of immunocompromised or transgenic mice can authentically mimic the primary tumor in patients, especially with distant metastatic ability. PDXs can not only faithfully preserve the molecular phenotypes and genomic alterations of the primary tumors in patients, but also reproduce the heterogeneity of primary tumors; therefore, PDXs have been used in investigation of mechanism of drug resistance. This review summarizes the methodology of establishing PDXs, verification of similarity in original tumor and the corresponding xenograft, and the value and limitations of PDXs used in the development of new drugs. Copyright © 2014 by TUMOR.

Effects of tissue factor pathway inhibitor-2 on the growth and angiogenesis of subcutaneous tumor xenografts in nude mice established from human liver cancer cells / 肿瘤

Objective: To investigate the effects of TFPI-2 (tissue factor pathway inhibitor 2) on the growth and angiogenesis of subcutaneous tumor xenografts in nude mice established from human liver cancer cells. Methods: The Hep3B cells stably expressing TFPI-2 (Hep3B-TFPI-2 group) and the Hep3B cells transfected with empty vector PCDNA3.1 (Hep3B-V group) or without transfection (Hep3B-P group) were subcutaneously transplanted into nude mice respectively to generate subcutaneous tumor xenografts. The volume of tumor xenograft was measured every three days, and the growth curve of tumor xenograft was drawn when the subcutaneous tumor xenograft was visible. The nude mice were killed three weeks after transplant, the volume of tumor xenograft was measured, and the total RNAs and proteins in tumor xenografts were extracted. The mRNA and protein expressions of TFPI-2 and VEGF (vascular endothelial growth factor) in tumor xenografts were analyzed by RFQ-PCR (real-time fluorescence quantitative PCR) and Western blotting, respectively. The expression of TFPI-2 protein and the MVD (microvessel density) in tumor xenografts were observed by immunohistochemistry. Results: The eventual tumor volume of tumor xenografts in Hep3B-TFPI-2 group was apparently smaller than those in Hep3B-V group and Hep3B-P group (both P < 0.05). The expression of mRNA and abundance of protein of TFPI-2 in Hep3B-TFPI-2 group were significantly higher than those in the other two groups (P < 0.05); while the expression of mRNA and abundance of protein of VEGF in Hep3B-TFPI-2 group were apparently lower than those in the other two groups. Compared with Hep3B-V group and Hep3B-P group, the inhibitory rates of VEGF protein expression in Hep3B-TFPI-2 group were 19.8% and 23.5%, respectively (P < 0.05). The MVD in Hep3B-TFPI-2 group was apparently lower than those in the other two groups (P < 0.05). Conclusion: TFPI-2 can significantly inhibit the growth and angiogenesis of subcutaneous tumor xenografts in nude mice established from hepatocarcinoma Hep3B cells. Copyright © 2013 by TUMOR.

Effect and mechanism of gene therapy of lentivirus mediated RhoA shRNA on ovarian cancer xenograft in vivo / 中华妇产科杂志

Objective To investigate treatment effects of lentivirus mediated RhoA short hairpin RNA(shRNA) on xenograft tumor of ovarian cancer in nude mice in vivo and the underlying mechanism.Methods Human ovarian cancer cell line HO8910 were inoculated to establish subcutaneous xenograft model of human ovarian cancer.Tumor-bearing nude mice were assigned randomizely to three groups:LentiRhoA-sh group,Lenti-negative control (NC) group and phosphate buffered saline (PBS) group.lentivirus mediated RhoA shRNA,negative control lentivirus and PBS were respectively injected in the three groups.Effects of treatment were observed by tumor growth curve,tumor volume,tumor weight,and tumor inhibition rate.Xenograft tissues and liver,spleen,lung,and renal tissues were examined by hematoxylin and eosin (HE) staining or were detected by streptavidin-perosidase (SP)immunochemical method.The changes of RhoA gene expression in xenograft tissues after lentivirus mediated RhoA shRNA treated were also detected by real-time qPCR,immunochemistry and Western blot assay.Cell apoptosis in xenograft tissues were examined by terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling(TUNEL) method and apoptotic index (AI) were counted.Results Compared with Lenti-NC group and PBS group,the growth speed of xenograft in Lenti-RhoA-sh group delayed significantly after injection 9 days(P ＜ 0.01).Tumor volume (338 ± 114) mm3 decreased significantly in the Lenti-RhoA-sh group when compared with those in Lenti-NC group(1190 ± 332)mm3 and PBS group (1101 ± 396) mm3 (P ＜ 0.01).Tumor weight (0.23±0.11)g decreased significantly in the Lenti-RhoA-sh group when compared with Lenti-NC group (0.79 ± 0.19)g and PBS group (0.74 ± 0.17)g (P ＜ 0.01).Real-time qPCR result shown that the expression of RhoA mRNA (0.30 ± 0.05) decreased significantly in the Lenti-RhoA-sh group compared with Lenti-NC group (0.95 ±0.06) and PBS group(1.00 ±0.11 ; P ＜0.01).Western blot result showed that the expression level of RhoA protein decreased significantly in the Lenti-RhoA-sh group (0.14 ± 0.06) compared with those in Lenti-NC group(0.78 ± 0.14) and PBS group (0.75 ± 0.13;P ＜ 0.01).TUNEL staining displayed that AI significantly increased in the Lenti-RhoA-sh group (20.9 ± 3.4) ％ compared with those in Lenti-NC group(5.2 ±±2.0)％ and PBS group(6.0 ±2.1)％ (P ＜0.01).Conclusion Lentivirus mediated RhoA shRNA may be effectively down-regulate of the expression of RhoA,inhibit the growth of subcutaneous xenograft tumor of ovarian cancer in nude mice by increasing the cell apoptosis.

A novel model of distal colon cancer in athymic mice / Novo modelo de câncer de cólon distal em camundongos atímicos

PURPOSE: The present a novel adenocarcinoma model in athymic mice. METHODS: Seven athymic mice were used. Colon diversion and distal fistula were made. Adenocarcinoma cells were inoculated in the submucosa of fistula. Tumor growth was monitored daily. Scintigraphy with 99mTc-MIBI was performed to identify the tumor. RESULTS: The model of distal colon cancer is feasible. Tumor detection was possible by both, macroscopically and molecular imaging. All resections demonstrated poorly differentiated tumors. Colon obstruction occurred in one case, similarly to evolution in human tumors of distal colon. CONCLUSION: The proposed model of distal colon cancer is feasible, allows for easy monitoring of tumoral growth by both, macroscopically and molecular imaging, and is suitable for studying the evolution of tumor with implementation of cytotoxic therapy in vivo.

OBJETIVO: Apresentar novo modelo de adenocarcinoma distal em camundongos atímicos. MÉTODOS: Foram utilizados sete camundongos atímicos. Desvio do cólon distal e fístula foram feitas. Células de adenocarcinoma foram inoculadas na submucosa da fístula. O crescimento do tumor foi monitorado diariamente. Cintilografia com 99mTc-MIBI foi realizada para identificar o tumor. RESULTADOS: O modelo de câncer de cólon distal é viável. Detecção do tumor foi possível macroscopicamente e por imagem molecular. Todas as ressecções demonstraram tumores pouco diferenciados. Obstrução do cólon ocorreu em um caso, de forma semelhante à evolução em tumores humanos do cólon distal. CONCLUSÃO: O modelo de câncer do cólon distal proposto é viável, permite a monitorização fácil do crescimento tumoral macroscopicamente e por imagem molecular, sendo adequado para o estudo da evolução de tumor com aplicação de terapia citotóxica in vivo.

Changes of moleculars involved in PI3K/AKT pathway in emodin-induced apoptosis of human leukemia K562 cells in nude mice / 肿瘤

Objective: To observe emodin-induced molecular changes in PI3K/AKT signaling pathway in human leukemia K562 cells transplanted into BALB/c nude mice, and to explore whether emodin induces the apoptosis of K562 cells through PI3K/AKT signaling pathway. Methods: The subcutaneously transplanted tumor model of human K562 cells in nude mice was established. After continuously intraperitoneal injection with different doses of emodin for 12 d, the mice were sacrificed. Then the tumor weight and volume were measured, and the tumor inhibition rate was calculated. Emodininduced apoptotic morphological changes of K562 cells were detected by HE stain and scanning electron microscopy. RT-PCR and Western blotting were used to detect the expressions of PI3K, AKT and FoxO 3a mRNAs and proteins, respectively. Results: The relative tumor volumes (V/V0) were significantly smaller in the low-, moderate- and high-dose emodin-treated groups (8.90±0.24, 5.62±0.17 and 2.06± 0.31, respectively) than that in the untreated group (11.83±0.47; P <0.01). Significant apoptosis of K562 cells was found in emodin-treated groups under a light microscope and an electron microscope. RT-PCR revealed down-regulation of PI3K and AKT mRNAs expression and up-regulation of FoxO 3a mRNA expression induced by different concentrations of emodin in a dose-dependent manner. Western blotting analysis showed that the expression levels of PI3K and AKT proteins were markedly decreased and the expression level of FoxO 3a protein was significantly elevated in xenografted tumors treated with emodin. Conclusion: Emodin can significantly inhibit the growth of K562 cell xenografts in nude mice. The underlying mechanism may be associated with inhibition of PI3K/AKT signaling pathway. Copyright© 2011 by TUMOR.