Establishment of an orthotopic pancreatic cancer mouse model: Cells suspended and injected in Matrigel

AIM: To establish an orthotopic mouse model of pancreatic cancer that mimics the pathological features of exocrine pancreatic adenocarcinoma.

Promising xenograft animal model recapitulating the features of human pancreatic cancer

BACKGROUND Multiple sites of metastasis and desmoplastic reactions in the stroma are key features of human pancreatic cancer(PC).There are currently no simple and reliable animal models that can mimic these features for accurate disease modeling.AIM To create a new xenograft animal model that can faithfully recapitulate the features of human PC.METHODS Interleukin 2 receptor subunit gamma(IL2RG)gene knockout Syrian hamster was created and characterized.A panel of human PC cell lines were transplanted into IL2RG knockout Syrian hamsters and severe immune-deficient mice subcutaneously or orthotopically.Tumor growth,local invasion,remote organ metastasis,histopathology,and molecular alterations of tumor cells and stroma were compared over time.RESULTS The Syrian hamster with IL2RG gene knockout(named ZZU001)demonstrated an immune-deficient phenotype and function.ZZU001 hamsters faithfully recapitulated most features of human PC,in particular,they developed metastasis at multiple sites.PC tissues derived from ZZU001 hamsters displayed desmoplastic reactions in the stroma and epithelial to mesenchymal transition phenotypes,whereas PC tissues derived from immune-deficient mice did not present such features.CONCLUSION ZZU001 hamsters engrafted with human PC cells are a superior animal model compared to immune-deficient mice.ZZU001 hamsters can be a valuable animal model for better understanding the molecular mechanism of tumorigenesis and metastasis and the evaluation of new drugs targeting human PC.

Histological analysis of human pancreatic carcinoma following irreversible electroporation in a nude mouse model

AIM To determine changes in the morphology and function of pancreatic cancer cells after irreversible electroporation(IRE) treatment, and to explore the clinical significance of IRE treatment for pancreatic cancer providing an experimental basis for the clinical application of IRE treatment. METHODS IRE was carried out in an athymic nude mouse model of pancreatic carcinoma generated with human pancreatic cancer cells 1. In therapy groups, IRE electrodes were inserted with 90 pulses per second at 800 V/cm applied to ablate the targeted tumor tissues. Histological assessment of the affected tissue was performed by hematoxylin and eosin staining(HE). Quantification of cell proliferation and apoptosis was performed by evaluating Ki67 and caspase-3 levels, respectively. Flow cytometry was used to assess cell apoptosis. Ultrasound imaging was carried out to evaluate IRE treatment results. Pathological correlation studies showed IRE is effective for the targeted ablation of pancreatic tumors in an orthotopic mouse model.RESULTS IRE was efficacious in removing tumors in the orthotopic mouse model. The IRE-ablated zone displays characteristics of nude mouse models at different time-points as assessed by hematoxylin and eosin staining. Immunohistochemical analysis of samples from the pancreatic cancer models showed significantly enhanced caspase-3 cleavage and Ki67. Flow cytometry data corroborated the above findings that apoptosis in tumor cells was observed immediately on the first postoperative day, and with time the middle and late stages of apoptosis were observed. For ultrasound imaging studies, the IRE ablation zone became a hyperechoic area due to increasing inflammatory and immunologic cellular contents. CONCLUSION IRE is a promising new approach for pancreatic cancer, with many potential advantages over conventional ablation techniques.

Functional magnetic resonance imaging in an animal model of pancreatic cancer

AIM: To test the hypotheses that diffusion weighed (DW)and transcatheter intraarterial perfusion (TRIP)magnetic resonance imaging (MRI) can each be used to assess regional differences in tumor function in an animal pancreatic cancer model.METHODS: VX2 tumors were implanted in pancreata of 6 rabbits. MRI and digital subtraction angiography (DSA) were performed 3 wk following implantation. With a 2-French catheter secured in the rabbit's gastroduodenal artery, each rabbit was transferred to an adjacent 1.5T MRI scanner. DWand TRIP-MRI were performed to determine if necrotic tumor core could be differentiated from viable tumor periphery. For each, we compared mean differences between tumor core/ periphery using a 2-tailed paired t-test (α = 0.05). Imaging was correlated with histopathology. RESULTS: Tumors were successfully grown in all rabbits, confirmed by necropsy. On DW-MRI, mean apparent diffusion coeffi cient (ADC) value was higher in necrotic tumor core (2.1 ± 0.3 mm2/s) than in viable tumor periphery (1.4 ± 0.5 mm2/s) (P < 0.05). On TRIP-MRI, mean perfusion values was higher in tumor periphery (110 ± 47 relative units) than in tumor core (66 ± 31 relative units) (P < 0.001). CONCLUSION: Functional MRI can be used to differentiate necrotic from viable tumor cells in an animal pancreatic cancer model using ADC (DW-MRI) and perfusion (TRIP-MRI) values.

Kanglaite combined Gemcitabine inhibits growth of nude mouse subcutaneous transplantation tumor of human PC-3 pancreatic cancer cell

Objective:To study the mechanisms of pancreatic cancer treatment with Kanglaite combined Gemcitabine by investigating the relationship between the apoptosis and the expression of bcl-2, Bax and VEGF in pancreatic cancer cells.Methods:Nude mouse subcutaneous transplantation tumor model of Human PC-3 pancreatic cancer was established; the expressions of bcl-2, Bax and VEGF of transplantation tumor cell were determined; the earlier apoptosis rate of pancreatic cancer cell and the gross tumor volume were determined. Results:Kanglaite combined Gemcitabine remarkably decreased the protein expression of bcl-2,raised the expression of Bax,increased the apoptosis rate of the pancreatic cancer and contract the gross tumor volume. Kanglaite greatly decreased the protein expression of VEGF of the tumor cell. Conclusion:Therapeutic efficacy of Kanglaite combined Gemcitabine is far better than separate use of the two medicines in the pancreatic cancer transplantation tumor treatment.

THE ESTABLISHMENT OF A NEW ANIMAL MODEL FOR GASTRIC CANCER STUDY BY ORTHOTO PIC IMPLANTATION OF GASTRIC CANCER CELLS INTO ATHYMIC NUDE MICE

An animal model mimicking human gastric cancer by gastric wall implantation technique in athymic nude mice was reported. Two human gastric cancer cell lines. MKN-45 and MKN-28, were used in this study. All animals with gastric wall implantation of cancer cells of these two cell lines developed grossly visible gastric tumors after 3-4 weeks of implantation. Histopathological examination showed that tumors prirnarily grew at serosal side of stomach, and progressively invaded the gastric mucosa, but none showed metastasis in this study. All tumor-bearing animals died within 5-8 weeks after implantation. These results indicated that gastric wall of nude mice provided a good soil for growth and propagation of human gastric cancer cells. The model was useful for in vivo study on biological behavior of various types of human gastric cancers.

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.

In vivo comparison of transduction efficiency with recombinant adenovirus-mediated p53 in a human colon cancer mouse model by different delivery routes

Objective： To evaluate transduction efficiency with recombinant adenovirus-mediated p53 （rAd/p53） therapy in a human colon cancer mouse model by intra-tumoral injection and intra-arterial delivery. Methods： The tumor pieces of human colon cancer SW480 were implanted in the livers of 45 nude mice. These mice were administrated with rAd/p53 by intratumoral injection and intra-artedal delivery. After 24 h, 48 h and 72 h tAd/p53 administration, 5 mice each group were killed with over anesthesia and their livers were removed. P53 expression and apoptosis of tumor and liver were assessed. Results： P53 expression and apoptosis of intratumoral administration group was higher than tail vein group and control group. Apoptosis and p53 expression of livers in three groups had no significant difference. Conclusion： p53 gene transducUon efficiency and anticancer effect of rAd/p53 is much better by intra-tumoral injection than intra-arterial delivery,

Evidence-Based Dampness-Heat ZHENG(Syndrome) in Cancer: Current Progress toward Establishing Relevant Animal Model with Pancreatic Tumor

Cancer is one of the deadliest diseases affecting the health of human beings. With limited therapeutic options available, complementary and alternative medicine has been widely adopted in cancer management and is increasingly becoming accepted by both patients and healthcare workers alike. Chinese medicine characterized by its unique diagnostic and treatment system is the most widely applied complementary and alternative medicine. It emphasizes symptoms and ZHENG(syndrome)-based treatment combined with contemporary disease diagnosis and further stratifies patients into individualized medicine subgroups. As a representative cancer with the highest degree of malignancy, pancreatic cancer is traditionally classified into the "amassment and accumulation". Emerging perspectives define the core pathogenesis of pancreatic cancer as "dampness-heat" and the respective treatment "clearing heat and resolving dampness" has been demonstrated to prolong survival in pancreatic cancer patients, as has been observed in many other cancers. This clinical advantage encourages an exploration of the essence of dampness-heat ZHENG(DHZ) in cancer and investigation into underlying mechanisms of action of herbal formulations against dampness-heat. However, at present, there is a lack of understanding of the molecular characteristics of DHZ in cancer and no standardized and widely accepted animal model to study this core syndrome in vivo. The shortage of animal models limits the ability to uncover the antitumor mechanisms of herbal medicines and to assess the safety profile of the natural products derived from them. This review summarizes the current research on DHZ in cancer in terms of the clinical aspects, molecular landscape, and animal models. This study aims to provide comprehensive insight that can be used for the establishment of a future standardized ZHENG-based cancer animal model.

Synergetic anticancer effect of combined gemcitabine and photodynamic therapy on pancreatic cancer in vivo

AIM： To investigate the anti-tumor effects of combined cytotoxic drug （gemcitabine） and photodynamic therapy （PDT） on human pancreatic cancer xenograft in nude mice.METHODS： Human pancreatic cancer cell line SW1990 was used in the investigation of the in vivo effect of combined gemcitabine and PDT on human pancreatic cancer xenograft in mice. Sixty mice were randomly allocated into a control group （without treatment）, photosensitizer treatment group （2 mg/kg photosan, without illumination）, chemotherapy group （50 mg/kg gemcitabine i.p.）, PDT group （2 mg/kg photosan ＋ laser irradiation） and combined treatment group （photosan ＋ chemotherapy）, with 12 mice in each group. Tumor size was measured twice every week. Anti-tumor activity in different groups was evaluated by tumor growth inhibition （TGI）RESULTS： No significant anti-tumor effect was observed either in photosensitizer treatment group or in chemotherapy group. PDT led to necrosis in cancer lesions and significantly reduced tumor volume compared with photosensitizer on day 6 and at the following time points after initialization of therapy （0.24 ± 0.15-0.49 ± 0.08 vs 0.43 ± 0.18-1.25± 0.09, P 〈 0.05）. PDT significantly reduced tumor volume in combined treatment group compared with photosensitizer treatment group （0.12 ± 0.07-0.28 ± 0.22 vs 0.39 ± 0.15-2.20 ± 0.12, P 〈 0.05）, small dose chemotherapy group （0.12 ± 0.07-0.28 ± 0.12 vs 0.32 ± 0.14-1.16 ± 0.08, P 〈 0.05） and control group （0.12 ± 0.07-0.28 ± 0.12 vs 0.43 ± 0.18-1.25 ± 0.09, P 〈 0.05）. TGI was higher in the combined treatment group （82.42%） than in the PDT group （58.18%）.CONCLUSION： PDT has a significant anti-tumor effect, which is maintained for a short time and can be significantly enhanced by small doses of gemcitabine.

Topology impacts TRAIL therapy: Differences in primary cancer growth and liver metastasis between orthotopic and subcutaneous xenotransplants of pancreatic ductal adenocarcinoma cells

Background: To study novel treatment modalities for pancreatic ductal adenocarcinoma(PDAC), we need to transfer the knowledge from in vitro to in vivo. It is important to mirror the clinical characteristics of the typically local invasive growth of pancreatic cancer and the distant spread resulting in liver metastasis. Notably, for xenotransplant studies using human specimen, two models, i.e. subcutaneous(s.c.) and orthotopic(o.t.) transplantation are widely used. Methods: The subcutaneously and orthotopically inoculated Colo357 Bcl-x L cell-derived tumors were directly compared with and without TNF-related apoptosis inducing ligand(TRAIL) treatment. The size of primary tumors, number of liver metastasis and the histologic markers Ki67, M30, TNF-α and CD31 were assessed. Results: Upon TRAIL treatment, the primary tumors did not change their size, neither in the s.c. nor in the o.t. approaches. But when s.c. was compared to o.t., the size of the s.c. tumors was more than twofold bigger than that of the o.t. tumors( P<0.01). However, mice with orthotopically inoculated PDAC cells developed liver metastasis upon TRAIL treatment much more frequently( n=13/17) than mice with subcutaneously inoculated PDAC cells( n=1/11)( P<0.01). As a likely driving force for this increased metastasis, a higher TNF-α staining intensity in the o.t. tumors was observed by immunohistochemistry. Conclusions: These data from a direct side-by-side comparison underline the importance of the proper inoculation site of the PDAC cells. Local invasion and liver metastases are a hallmark of PDAC in the clinic;the o.t. model is clearly superior in reflecting this setting. Moreover, a serious side-effect of a possible new therapeutic compound became obvious only in the o.t. model.

Tumor initiating cells in pancreatic cancer: A critical view

Emerging evidence points to the existence of pan-creatic cancer stem cells (CSC) as the culprit in the initiation, maintenance, metastasis, and treatment resistance of pancreatic cancer. The existence of such a cell population would have an important im-pact on the design of novel therapies against this devastating disease. However, no in vivo validation or rebuttal of the pancreatic CSC hypothesis exists. Major backlashes in the discussion on CSC are firstly, the confusion between the terms CSC and cell of origin of pancreatic ductal adenocarcinoma (PDAC), secondly the ambiguity of the cell of origin itself and thirdly, the fact that the CSC hypothesis is based on cell sorting and xenografting experiments; the latter of which often precludes solid conclusions because of the lack of a natural microenvironment and differences in drug delivery. Nonetheless, recent studies in other cancers partially support the CSC hypothesis by demonstrating a link between epithelial-to-mesenchymal transdifferentiation/transition (EMT) and CSC properties. Such a link is again open to dispute as EMT is a reversible process which is highly dependent on major oncogenic pathways in PDAC [e.g. K-Ras, transforming growth factor-β (TGF-β)] rather than on presumed cancer stem cell pathways. Hence, the available evidence does not robustly support the CSC concept in PDAC and a thorough validation of this hypothesis in well-defined genetically engineered mouse models of pancreatic cancer is required.

Radioimmunodetection of human choriocarcinoma xenograft in nude mouse

Objective: To study the efficiency of radioimmuno-detection in locating the xenograft of human chorio-carcinoma in nude mouse. Methods: Radioimmuno-detection was performed using cocktail antibodies of 131I-labeled mouse anti-human chorionic gonadotropin monoclonal antibodies to locate the xenograft of human choriocarcinoma in nude mouse. Radioactivity in different tissues was measured and the tumor/non-tumor ratio was calculated. Normal mouse IgG was used as control IgG. Results: The accumulation of radioactivity in the xenograft area could be recognized as early as 24 h after the injection of the radiolabelled antibodies. 72-96 h after the injection, the xenograft could be clearly shown. The minimal shown xenograft was 0.8 cm in diameter. The tumor/non-tumor ratio increased with the time and was obviously higher than that in control group. Conclusion: Radioimmunodetection can efficiently locate human choriocarcinoma xenograft in nude mouse.

Chronic pancreatitis,pancreatic adenocarcinoma and the black box in-between

Pancreatic cancer is a challenging disease for patients,doctors and researchers who for decades have searched for a cure for this deadly malignancy.Although existing mouse models of pancreatic cancer have shed light on the mecha-nistic basis of the neoplastic conversion of the pancreas,their impact in terms of offering new diagnostics and therapeu-tic modalities remains limited.Chronic pancreatitis is an inflammatory disease of the pancreas that is associated with a gradual damage of the organ and an increased risk of developing neoplastic lesions.In this review,we propose that detailed studies of chronic inflammatory processes in the pancreas will provide insights into the evolution of pancreatic cancer.This information may prove useful in the design of effective therapeutic strategies to battle the disease.

Intravoxel incoherent motion diffusion-weighted imaging for monitoring chemotherapeutic efficacy in gastric cancer

AIM: To assess intravoxel incoherent motion diffusion-weighted imaging (IVIM-DWI) for monitoring early efficacy of chemotherapy in a human gastric cancer mouse model.METHODS: IVIM-DWI was performed with 12 b-values (0-800 s/mm2) in 25 human gastric cancer-bearing nude mice at baseline (day 0), and then they were randomly divided into control and 1-, 3-, 5- and 7-d treatment groups (n = 5 per group). The control group underwent longitudinal MRI scans at days 1, 3, 5 and 7, and the treatment groups underwent subsequent MRI scans after a specified 5-fluorouracil/calcium folinate treatment. Together with tumor volumes (TV), the apparent diffusion coefficient (ADC) and IVIM parameters [true water molecular diffusion coefficient (D), perfusion fraction (f) and pseudo-related diffusion coefficient (D*)] were measured. The differences in those parameters from baseline to each measurement (ΔTV%, ΔADC%, ΔD%, Δf% and ΔD*%) were calculated. After image acquisition, tumor necrosis, microvessel density (MVD) and cellular apoptosis were evaluated by hematoxylin-eosin (HE), CD31 and terminal-deoxynucleotidyl transferase mediated nick end labeling (TUNEL) staining respectively, to confirm the imaging findings. Mann-Whitney test and Spearman’s correlation coefficient analysis were performed.RESULTS: The observed relative volume increase (ΔTV%) in the treatment group were significantly smaller than those in the control group at day 5 (ΔTVtreatment% = 19.63% ± 3.01% and ΔTVcontrol% = 83.60% ± 14.87%, P = 0.008) and day 7 (ΔTVtreatment% = 29.07% ± 10.01% and ΔTVcontrol% = 177.06% ± 63.00%, P = 0.008). The difference in ΔTV% between the treatment and the control groups was not significant at days 1 and 3 after a short duration of treatment. Increases in ADC in the treatment group (ΔADC%treatment, median, 30.10% ± 18.32%, 36.11% ± 21.82%, 45.22% ± 24.36%) were significantly higher compared with the control group (ΔADC%control, median, 4.98% ± 3.39%, 6.26% ± 3.08%, 9.24% ± 6.33%) at days 3, 5 and 7 (P = 0.008, P = 0.016, P = 0.008, respectively). Increases in D in the treatment group (ΔD%treatment, median 17.12% ± 8.20%, 24.16% ± 16.87%, 38.54% ± 19.36%) were higher than those in the control group (ΔD%control, median -0.13% ± 4.23%, 5.89% ± 4.56%, 5.54% ± 4.44%) at days 1, 3, and 5 (P = 0.032, P = 0.008, P = 0.016, respectively). Relative changes in f were significantly lower in the treatment group compared with the control group at days 1, 3, 5 and 7 follow-up (median, -34.13% ± 16.61% vs 1.68% ± 3.40%, P = 0.016; -50.64% ± 6.82% vs 3.01% ± 6.50%, P = 0.008; -49.93% ± 6.05% vs 0.97% ± 4.38%, P = 0.008, and -46.22% ± 7.75% vs 8.14% ± 6.75%, P = 0.008, respectively). D* in the treatment group decreased significantly compared to those in the control group at all time points (median, -32.10% ± 12.22% vs 1.85% ± 5.54%, P = 0.008; -44.14% ± 14.83% vs 2.29% ± 10.38%, P = 0.008; -59.06% ± 19.10% vs 3.86% ± 5.10%, P = 0.008 and -47.20% ± 20.48% vs 7.13% ± 9.88%, P = 0.016, respectively). Furthermore, histopathologic findings showed positive correlations with ADC and D and tumor necrosis (rs = 0.720, P < 0.001; rs = 0.522, P = 0.007, respectively). The cellular apoptosis of the tumor also showed positive correlations with ADC and D (rs = 0.626, P = 0.001; rs = 0.542, P = 0.005, respectively). Perfusion-related parameters (f and D*) were positively correlated to MVD (rs = 0.618, P = 0.001; rs = 0.538, P = 0.006, respectively), and negatively correlated to cellular apoptosis of the tumor (rs = -0.550, P = 0.004; rs = -0.692, P < 0.001, respectively).CONCLUSION: IVIM-DWI is potentially useful for predicting the early efficacy of chemotherapy in a human gastric cancer mouse model.

The 150 most important questions in cancer research and clinical oncology series:questions 76-85

Since the beginning of 2017, Chinese Journal of Cancer has published a series of important questions in cancer research and clinical oncology to promote cancer research and accelerate collaborations. In this article, 10 questions are presented as followed. Question 76. How to develop effective therapeutics for cancer cachexia? Question 77.How can we develop preclinical animal models to recapitulate clinical situations of cancer patients for more effective anti-cancer drug development? Question 78. How can we develop novel effective therapeutics for pancreatic cancer and hepatocellular carcinoma? Question 79. What are the true beneficial mechanisms of antiangiogenic therapy in cancer patients? Question 80. How to approach the complex mechanisms of interplay among various cellular and molecular components in the tumor microenvironment? Question 81. Can tissue oxygenation improve the efficacy of conventional chemotherapy on cancer? Question 82. Can tissue oxygenation improve the efficacy of radiotherapy on digestive system tumors including liver cancer? Question 83. Can we integrate metabolic priming into multimodal management of liver cancer? Question 84. Has the limit of anti-androgen strategy in prostate cancer treatment been reached by the new generation of anti-androgen drugs? Question 85. Can we identify individuals with early-stage cancers via analyzing their clinical and non-clinical information collected from social media, shopping history, and clinical, pathological, and molecular traces?

Intravesical Instillation in Pure Line LEW Rats and Nude Mice

In order to study bladder intravesical instillation methods in pure line LEW rats and nude mice, female LEW rats and nude mice aged 2 to 4 weeks were sacrificed. Their urethra and bladder were observed under anatomical microscopy. A trochar was prepared according to the outline and an- gle of the urethra. Ink was poured into female rats and nude mice bladder though urethra. Filling and staining of bladder were observed and evaluated under anatomical microscopy. Status and urethral injury of rats and mice were observed. The results showed that urethra anatomic structure of rats and nude mice was different from that of human urethra. When bladder was filled with ink and became blue, liquid was not seen to leak out. The success rate of intubation was high (100%). Living activi- ties of animals weren’t influenced by intravesical instillation. It was concluded that bladder irrigation might be a kind of valid and utilizable method in pure line rat and nude mouse empirical study. The model may be a more effective tool for study of bladder tumor.

白细胞介素-6对胰腺癌小鼠癌组织的细胞增殖及上皮间质转化的影响

目的 探讨白细胞介素-6(IL-6)在胰腺癌裸鼠模型癌组织增殖与上皮间质化中的作用,并分析其分子机制。方法 将18只成功构建胰腺癌细胞荷瘤的BALB/c裸鼠模型随机分为模型对照组、IL-6过表达组和免疫球蛋白G(IgG)抗体组,每组6只。模型对照组于肿瘤部位注射生理盐水进行干预,IL-6过表达组于肿瘤部位注射IL-6过表达质粒进行干预,IgG抗体组予肿瘤部位注射IgG抗体进行干预。3组均于末次给药24 h后处死小鼠,并收集胰腺肿瘤组织。对各组肿瘤组织进行离体称重并计算抑瘤率。通过苏木精-伊红(HE)染色观察胰腺组织的病理学改变。采用酶联免疫吸附试验(ELISA)检测各组裸鼠肿瘤组织中IL-6、p-STAT3、NF-κB p65和VEGF蛋白的表达水平。采用蛋白质印迹法(Western blot)检测各组肿瘤组织中E-cadherin、Vimentin、MMP-9蛋白的表达水平。采用实时荧光定量聚合酶链反应(qRT-PCR)检测各组IL-6、p-STAT3、SOCS3基因的表达水平。结果 3组裸鼠胰腺肿瘤组织的平均瘤重及抑瘤率比较,差异均无统计学意义(P>0.05)。HE染色结果显示,模型对照组肿瘤组织细胞体积增大,癌细胞排列呈巢状;IL-6过表达组可见肿瘤组织轻度纤维化,少量炎症浸润;IgG抗体组可见核分裂象,大量肿瘤细胞坏死,局灶组织纤维化。IL-6过表达组E-cadherin蛋白、SOCS3基因表达水平低于模型对照组和IgG抗体组,而Vimentin、MMP-9、IL-6、p-STAT3、NF-κB p65、VEGF蛋白的表达水平及IL-6、NF-κB p65基因的表达水平高于模型对照组和IgG抗体组,差异均有统计学意义(P<0.05);IgG抗体组Vimentin、MMP-9、IL-6、p-STAT3、NF-κB p65、VEGF蛋白的表达水平及IL-6、NF-κB p65基因的表达水平低于模型对照组,而E-cadherin和SOCS3基因水平高于模型对照组,差异均有统计学意义(P<0.05)。结论 IL-6可以通过p-STAT3和(或)NF-κB p65信号通路调节胰腺癌裸鼠模型肿瘤组织中的相关细胞因子,影响肿瘤组织的增殖及上皮间质化。

RAD51C表达下调对小鼠卵巢癌体内成瘤和VEGF、NRP-2表达的调控

目的探讨RAD51旁系同源基因C(RAD51C)表达下调对小鼠卵巢癌体内成瘤和血管内皮生长因子(VEGF)、神经纤毛蛋白-2(NRP-2)表达调控的影响。方法使用A2780卵巢癌细胞,通过培养检测细胞中RAD51C的表达,构建3种RAD51C干扰载体(SiRNA-47、SiRNA-183和SiRNA-285),并包装成慢病毒,转染细胞,逆转录实时定量聚合酶链式反应(RT-qPCR)验证转染效果,进行稳筛,构建稳转细胞株。使用Balb/c雌性裸鼠建立细胞系来源的异体移植肿瘤模型(CDX)模型,将18只建模成功的裸鼠分为3组:A2780细胞组(Control组)、A2780细胞+空载体对照组(NC组)、A2780细胞+RAD51C干扰组(Si-RAD51C组),每组各6只。Control组注射生理盐水,NC组注射空载慢病毒50μL,Si-RAD51C组注射RAD51C干扰慢病毒50μL。观察各组成瘤情况,取肿瘤组织,采用蛋白质印迹法(WB)、免疫组织化学检测RAD51C、VEGF、NRP-2蛋白表达情况。结果RT-qPCR验证RAD51C干扰慢病毒转染效果以SiRAN-285最明显(P<0.05)。Si-RAD51C组与Control组、NC组比较瘤体的体积最小、重量也最轻,且RAD51C、NRP-2及VEGF蛋白的表达显著降低(P<0.05)。结论RAD51C干扰慢病毒可抑制小鼠A2780卵巢癌细胞肿瘤的形成,且对RAD51C、NRP-2及VEGF蛋白的表达均有抑制作用。

预免疫策略结合mVenus-p27K-系统构建休眠肿瘤小鼠模型

目的·通过融合预免疫策略与mVenus-p27K-细胞G0期指示系统、DTR-HSV/TK自杀基因系统以及Luc2-tdTomato示踪系统,构建一个无明显转移灶的肿瘤休眠小鼠模型。方法·在KPC1199小鼠胰腺癌细胞系中,依次引入mVenusp27K-细胞G0期指示系统、DTR-HSV/TK自杀基因系统及Luc2-tdTomato示踪系统,构建KPC1199-PDL稳定表达细胞株。KPC1199-PDL细胞在无血清条件下培养后,通过流式细胞术分选为mVenus(+)细胞及mVenus(−)细胞,采用实时荧光定量PCR(qPCR)验证G0期相关基因的表达。采用CCK-8细胞增殖实验评估KPC1199-PDL细胞对白喉毒素(DTX)和更昔洛韦(GCV)的敏感性。在野生型C57BL/6小鼠中构建经脾-门静脉-肝转移模型,利用免疫荧光技术验证KPC1199-PDL细胞在体内的功能。在C57BL/6小鼠皮下注射KPC1199-PDL细胞,5 d后原位注射DTX和GCV消融皮下瘤,获得预免疫小鼠,并在此基础上构建经脾-门静脉-肝转移模型。采用生物发光成像评估皮下瘤消融和肝转移情况,利用免疫荧光染色检测预免疫小鼠肝脏中肿瘤细胞的分布及休眠状态。结果·KPC1199-PDL细胞稳定表达3种工具系统,且其增殖能力未受影响。在无血清条件培养下,部分KPC1199-PDL细胞表达mVenus蛋白,即进入G0期;经流式细胞术分选后得到的mVenus(+)细胞G0期相关基因较mVenus(−)细胞显著高表达(均P<0.05),而增殖相关基因显著低表达(P<0.05)。CCK-8实验显示KPC1199-PDL细胞对DTX和GCV高度敏感。体内实验证实KPC1199-PDL细胞可通过表达tdTomato蛋白有效示踪,以及表达mVenus蛋白提示细胞进入G0期。经皮下种瘤和药物消融后成功获得预免疫小鼠,在此基础上构建的经脾-门静脉-肝转移模型,生物发光成像未在肝脏观察到转移信号,但肝脏组织切片经免疫荧光检测发现存在单个或小簇状同时表达mVenus和tdTomato,但不表达增殖标志物Ki67的G0期肿瘤细胞。结论·胰腺癌预免疫小鼠模型结合mVenus-p27 K-指示系统、DTR-HSV/TK自杀基因系统及Luc2-tdTomato示踪系统,成功得到可识别、可示踪的休眠肿瘤动物模型。