Development and characterization of a human colon adenocarcinoma xenograft deficient in thymidine salvage

In order to determine the contribution of thymidine (dThd) salvage to intrinsic resistance to antimetabolites (5-fluoropyrimidines, antifolates) in the human colon adenocarcinoma xenograft, H X GC3, a subline deficient in thymidine kinase has been developed. A cell line (GC3/M) was established in continuous culture that demonstrated a karyotype identical to that of the stem line of H X GC3 (46,X, - Y + 12). After inoculation of GC3/M cells into immune-deprived CBA/CaJ mice, the H X GC3/M xenografts retained histological, histochemical, and growth characteristics of the H X GC3 xenograft. To develop a line deficient in dThd salvage, GC3/M cells were selected with BrdUrd (100 micrograms/ml). Three clones characterized were unable to proliferate in HAT medium, and were deficient (less than 10% control) in the cytosolic form of thymidine kinase. Activities of dThd phosphorylase and dTMP synthase were unchanged from parental GC3/M cells. Of the three clones inoculated into mice, GC3/M TK- 100 C3 was tumorigenic, the xenografts demonstrating histological and growth characteristics similar to H X GC3. The in vivo activity of the cytosolic form of dThd kinase was 3.5% of that in H X GC3 xenografts. Incorporation in vivo of [methyl-3H]dThd into acid insoluble material was 14% of that in H X GC3 tumors. Autoradiographs prepared from these tumors demonstrated that incorporation of radiolabel into nuclei occurred only in stromal cells derived from the host. It is anticipated that H X GC3/M TK- 100 C3 will be a line valuable for determining the role of dThd salvage in intrinsic resistance to 5-fluoropyrimidines or antifolates in human colon adenocarcinomas growing as xenografts and also the relevance of dTMp synthase as a target for antimetabolites in this histiotype.     

Development and characterization of a tamoxifen-resistant breast carcinoma xenograft

A human tamoxifen-resistant mammary carcinoma, MaCa 3366/TAM, originating from a sensitive parental xenograft 3366 was successfully established by treatment of tumour-bearing nude mice with 1-50 mg kg(-1) tamoxifen for 3 years during routine passaging. Both tumours did not differ significantly in OR- and PR-positivity, however, when compared with the sensitive tumour line, the mean OR content of the TAM-resistant subline is slightly lower. An OR-upregulation following withdrawal of oestradiol treatment was observed in the parental tumours but not in the resistant xenografts. Following long-term treatment with tamoxifen, the histological pattern of the breast carcinoma changed. The more differentiated structures being apparent after treatment with 17beta-oestradiol in the original 3366 tumour were not induced in the resistant line. Tamoxifen failed to induce a tumour growth inhibition in comparison to the tamoxifen-sensitive line. The pure anti-oestrogen, ICI 182 780, revealed cross-resistance. Sequence analysis of the hormone-binding domain of the OR of both lines showed no differences, suggesting that either mutations in other regions of the OR are involved in the TAM-resistance phenotype or that mechanisms outside of this protein induced this phenotype. Oestrogen and anti-oestrogen regulate pS2 and cathepsin D expression in 3366 tumours as in the human breast cancer cell line MCF-7. The resistant 3366/TAM tumours have lost this regulation. The established breast cancer xenografts 3366 and 3366/TAM offer the possibility of investigating mechanisms of anti-oestrogen resistance in an in vivo situation. They can be used to test novel approaches to prevent, or to overcome, this resistance in a clinically related manner.     

Establishment and characterization of novel xenograft models of human biliary tract carcinomas

In order to develop new therapeutic regimens for biliary tract cancers, which carry dismal prognoses, the establishment of a human biliary tract cancer xenograft model is essential. Herein, we report the successful establishment and characterization of two xenograft models of human biliary tract cancers. An adenosquamous gallbladder cancer cell line (TGBC-44) and a bile duct adenocarcinoma cell line (TGBC-47) were obtained from fresh surgical specimens in our department and subcutaneously inoculated into nude mice. The overall tumor take rate was 100% and solid tumors grew measurable after 5 and 7 days for TGBC-44 and TGBC-47, respectively. Tumor doubling time was 3.9+/-1.1 and 4.1+/-0.5 days in the exponential growth phase in TGBC-44 and TGBC-47 xenografts, respectively. Isozyme test and karyotype analysis confirmed the human origin. Histopathology analysis revealed that the TGBC-44 xenograft retained both the squamous and the adenocarcinoma components, and the TGBC-47 xenograft exhibited poorly differentiated adenocarcinoma as in the corresponding original tumors. Immunohistochemistry and Western blotting studies revealed positive and similar expression of platelet derived endothelial growth factor/thymidine phosphorylase (PDGF/TP), thymidylate synthase (TS), and cyclooxygenase-2 (COX-2) in both original tumors and xenograft models. No macroscopic metastases were found at the time of sacrifice. We have successfully established two models of human biliary tract cancer, gallbladder and bile duct cancer. Models retained the morphological and biochemical characteristics of the original tumor and demonstrated constant biological behavior in all transplanted mice. These models could be useful tools for developing new diagnostic and therapeutic strategies against biliary tract cancers.     

Development of a new metastatic human breast carcinoma xenograft line.

Xenografts originated from human tumours offer the most appropriate research material for in vivo experimental research. However, primary human breast carcinomas are difficult to grow when transplanted in athymic mice: tumour take is less than 15%. Recently, we have achieved 60% tumour take by injecting tumour cell suspensions mixed with Matrigel. Human breast xenografts originated from primary breast carcinoma also frequently show the potential to metastasize spontaneously. In the present study, we generated a human breast carcinoma xenograft line (UISO-BCA-NMT-18) that shows 100% tumorigenicity and 80-100% lung metastasis when transplanted s.c. in athymic mice. We have studied in detail the characteristics of the xenograft and the patient''s tumour from which the xenograft line originated. Both the xenograft and the patient''s tumour showed intense staining for mutant p53 nuclear protein, and high expression of U-PA, PAI and u-PAR. In vivo growth of the xenograft is stimulated by exogenous supplementation of oestrogen. This xenograft is continuously growing in mice and has shown 80-100% metastasis for the last three successive in vivo passages. This well-characterized, oestrogen-responsive, metastatic breast carcinoma xenograft line will provide excellent research material for metastasis-related research.     

The disease relevance of human hepatocellular xenograft models: Molecular characterization and review of the literature

In recent years a number of new therapeutics has been developed that were not general toxins and inhibitors of cell division like classical chemotherapeutics, but were designed to target a specific pathway. A prerequisite for this development was the comprehensive characterization of molecular alterations occurring in human hepatocellular carcinoma (HCC). However, while much knowledge of the molecular pathogenesis of human HCC has been gained, the model systems used to test the functional relevance of these alterations and applied for preclinical evaluation of drug candidates are still poorly characterized. In this paper, we reviewed the literature about several commonly used HCC cell lines and xenotransplantation models and present our own data on the molecular characterization of these. Results obtained demonstrate that it is important to have a sound knowledge of the specific molecular constitution of the experimental model and to carefully evaluate the functional status of the pathway of interest. For this reason, we make the gene expression profiles publicly available to help researchers making an informed decision about which model to use.     

Development of a large-animal human brain tumor xenograft model in immunosuppressed cats

A large-animal model was developed to facilitate the noninvasive investigation of the effect on the human glioma-derived D-54 MG (glioblastoma multiforme) continuous cell line of a variety of therapeutic regimens. Twenty random-bred male cats were inoculated intracerebrally with 1 x 10(7) D-54 MG tumor cells after being initiated on one of three preparatory regimens of cyclosporin A p.o. Reproducible success of D-54 MG xenotransplantation (100%, 6 of 6 cats) was achieved only after pretreatment with 120 mg cyclosporin A p.o. (24-30 mg/kg) daily for greater than or equal to 10 days prior to tumor implantation. High-performance liquid chromatography-derived whole blood cyclosporin A 12-h trough levels of greater than or equal to 640 ng/ml were seen in successful implants. Lesions ranging from 2 to 20 mm in diameter were seen in cats sacrificed 27-44 days after implantation with no growth seen in control animals. Histopathological examination revealed the tumors to be well-circumscribed anaplastic intracerebral tumors with some invasion into surrounding host parenchyma. Perivascular lymphocytic cuffing was observed, but intratumoral lymphocytic infiltration was minimal. Gadolinium-EDTA-enhanced nuclear magnetic resonance imaging provided accurate tumor localization in T1-weighted images (TE 26 ms; TR 600 ms). Biochemical tests of kidney, liver, and hematological function were within normal limits, although 10% (2 of 20) of the animals developed gingival hyperplasia, and 5% (1 of 20) developed intussusception. The reproducible growth of the D-54 MG human glioblastoma cell line in a large-animal model eliminates many of the limitations associated with the standard nude mouse/rat model, thereby providing a novel test bed for a variety of imaging modalities as well as for drug immunoconjugate localization and toxicity studies.     

Development of drug resistance in a human epidermoid lung carcinoma xenograft line

The development of resistance to vincristine, actinomycin D and cisplatin has been examined in a human epidermoid lung carcinoma xenograft line (HXL 55) growing in nude mice. Treatment of HXL 55 with 1 mg kg-1 vincristine or 0.5 mg kg-1 actinomycin D once in each in vivo passage resulted in a rapid reduction in tumour responsiveness to these drugs. A partial resistance was already acquired at the 2nd transplant generation. In contrast, a gradual decrease in therapeutic response was observed with 10 mg kg-1 cisplatin. Irradiation with a local dose of 10 Gy induced no resistance. The three induced drug-resistant sublines were characterized in terms of the time course of development of resistance, the degree of induced resistance, cross-resistance, growth rate and stability of the phenotype.     

Development and characterization of a bladder cancer xenograft model using patient‐derived tumor tissue

Most of the cancer xenograft models are derived from tumor cell lines, but they do not sufficiently represent clinical cancer characteristics. Our objective was to develop xenograft models of bladder cancer derived from human tumor tissue and characterize them molecularly as well as histologically. A total of 65 bladder cancer tissues were transplanted to immunodeficient mice. Passagable six cases with clinico‐pathologically heterogeneous bladder cancer were selected and their tumor tissues were collected (012T, 025T, 033T, 043T, 048T, and 052T). Xenografts were removed and processed for the following analyses: (i) histologic examination, (ii) short tandem repeat (STR) genotyping, (iii) mutational analysis, and (iv) array‐based comparative genomic hybridization (array‐CGH). The original tumor tissues (P 0) and xenografts of passage 2 or higher (≥P2) were analyzed and compared. As a result, hematoxylin and eosin staining revealed the same histologic architecture and degree of differentiation in the primary and xenograft tumors in all six cases. Xenograft models 043T_P2 and 048T_P2 had completely identical STR profiles to the original samples for all STR loci. The other models had nearly identical STR profiles. On mutational analysis, four out of six xenografts had mutations identical to the original samples for TP53, HRAS, BRAF, and CTNNB1. Array‐CGH analysis revealed that all six xenograft models had genomic alterations similar to the original tumor samples. In conclusion, our xenograft bladder cancer model derived from patient tumor tissue is expected to be useful for studying the heterogeneity of the tumor populations in bladder cancer and for evaluating new treatments.     

Development of transplantable human chordoma xenograft for preclinical assessment of novel therapeutic strategies

Background

Chordomas are rare and indolent bone tumors that arise in the skull base and mobile spine. Distant metastases occur in >20% of cases, but morbidity and mortality are mainly related to local relapses that affect the majority of patients. Standard chemotherapy has modest activity, whereas new targeted therapies (alone or in combination) have some activity in controlling disease progression. However, the scarcity of preclinical models capable of testing in vivo responses to these therapies hampers the development of new medical strategies.

Methods

In this study, 8 chordoma samples taken from 8 patients were implanted in nude mice. Four engrafted successfully and gave rise to tumor masses that were analyzed histologically, by means of fluorescence in situ hybridization and biochemical techniques. The data relating to each of the mouse tumors were compared with those obtained from the corresponding human tumor.

Results

All 4 engraftments retained the histological, genetic and biochemical features of the human tumors they came from. In one epidermal growth factor receptor(EGFR)-positive xenograft, responsiveness to lapatinib was evaluated by comparing the pre- and posttreatment findings. The treatment induced a low-level, heterogeneous switching off of EGFR and its downstream signaling effectors.

Conclusions

Overall, this model is very close to human chordoma and represents a new means of undertaking preclinical investigations and developing tailored therapies.     

Development of a metastatic human colon cancer xenograft model in the nude mouse

The objective of our experimental protocols was to develop a metastatic model for a human colon carcinoma xenograft in congenitally athymic nude mice. This model would be useful in evaluating the efficacy of radiolabeled monoclonal antibodies for detection and treatment of regional and distant metastases. The LS-174T human colon carcinoma line was used to establish primary subcutaneous tumors in nude mice. Mice were killed at varying time intervals to establish the incidence of spontaneous metastases. Only lung micrometastases were observed during the 2-month observation period. To increase the metastatic rate, the site of primary implantation was varied and/or surgical manipulations were performed. Excision of small primary tumors resulted in a low incidence of local recurrence and no distant metastases. However, with excision of large primary tumors, a high local recurrence rate was noted and over 30% of mice had gross metastases. Mice bearing hind footpad tumors underwent excision when tumors were at least 1 cm in size. There were no local recurrences, but by 8 weeks over 40% had large pulmonary metastases. The LS-174T tumor was also established as a primary implant in the spleen from which 10 to 15% of the mice developed liver or lung metastases. The LS-174T tumor can metastasize in the nude mice and the latter two models may prove very useful in imaging and therapy studies.     

5-杂氮-2′-脱氧胞苷对人鼻咽癌裸鼠移植瘤的抑制作用

背景与目的:5-杂氮-2′-脱氧胞苷是DNA甲基转移酶抑制剂,能使因甲基化而失活的抑癌基因重新激活,进而抑制肿瘤的生长。本文观察了5-杂氮-2′-脱氧胞苷对人鼻咽癌细胞及其裸鼠移植瘤的抑制作用,探讨其抗肿瘤效应及可能的机理,寻找鼻咽癌治疗的新靶点。方法:用5-杂氮-2′-脱氧胞苷处理人鼻咽癌细胞,观察其对细胞生长的抑制及死亡相关蛋白激酶基因(death-associated proteinkinase,DAPK)甲基化情况。建立人鼻咽癌移植瘤裸鼠模型,用5-杂氮-2′-脱氧胞苷处理裸鼠,观察移植瘤的生长情况,并用RT-PCR和免疫组织化学技术检测DAPK基因mRNA及蛋白表达的变化。结果:未经5-杂氮-2′-脱氧胞苷处理的CNE细胞中DAPKmRNA不表达。经5-杂氮-2′-脱氧胞苷处理后,不同药物浓度组的细胞中均有DAPKmRNA表达,且表达随浓度增高而增强。5-杂氮-2′-脱氧胞苷对人鼻咽癌细胞及其裸鼠移植瘤的生长均有明显的抑制作用,且能使失活的DAPK基因重新激活。药物作用4周后,用药组裸鼠体重(22.35±2.02)g与对照组(21.68±2.14)g之间无显著性差异(t=0.011,P>0.05),用药组肿瘤的体积(195.32±27.57)mm3较对照组(343.67±23.08)mm3明显减小,两者之间有显著性差异(t=10.11,P﹤0.01)。在鼻咽癌移植瘤中,对照组DAPK均不表达,而用药组出现表达。结论:5-杂氮-2′-脱氧胞苷对人鼻咽癌细胞及其裸鼠移植瘤的抑制作用可能是因为其使因甲基化而失活的抑癌基因DAPK再转录,诱导该基因的表达,从而抑制肿瘤细胞的生长和增殖。     

Development and characterization of 5 canine B-cell lymphoma cell lines

Canine and human lymphoma share similar characteristics in disease development and response to therapy. Translational research can be furthered using tools such as canine cell lines to model therapeutic compounds and strategies. We developed 5 B-cell lymphoma cell lines from dogs with confirmed large B-cell lymphoma. These cell lines were CD3, CD18, CD20, and CD90 positive with variable CD79a, CD1c and CD34 expression. All cell lines were tumorigenic in Nu/nu mice and were wild type for p53. Canine lymphoma cell lines serve as an important resource for translational lymphoma research.     

Establishment and characterization of human gastric and colonic xenograft lines resistant to CPT-11 (A new derivative of camptothecin)

CPT-11-resistant human gastric and colonic xenograft lines were established by direct intratumoral injection of CPT-11 into subcutaneous SC-1-NU and CC-2-NU tumors in nude mice once a week for 10 months. The resistance of these xenograft lines to CPT-11 was confirmed by growth inhibition rate, to be 36.3% and 45.4%, respectively, compared to each parent cell line. DNA topoisomerase I activity of the nuclear extracts of SC-1-NU/CPT-11 and CC-2-NU/CPT-11, as assayed by relaxation of supercoiled DNA Col-E1, was significantly less than those of the parent lines. The cellular levels of topoisomerase I in those resistant lines measured by Western blot analysis were 0.57- and 0.79-fold lower than those of the parental lines, respectively. However, the activity of DNA topoisomerase II of those resistant cell lines assayed by decatenation of kinetoplast DNA was higher than that of the parental lines and the cellular levels of topoisomerase II in the resistant lines measured by Western blot analysis were 10.8- and 8.1-fold higher than those of the parent lines. Intracellular accumulation of CPT-11 in CPT-11-resistant tumors was not changed as compared to that of the parental lines, but hydrolysis of CPT-11 to more active SN-38 was reduced in the resistant tumors.     

A new temperature-sensitive liposome for use with mild hyperthermia: characterization and testing in a human tumor xenograft model

The single biggest challenge now facing drug delivery (for liposomes and indeed other carriers) is to initiate and produce release of the encapsulated drug only at the diseased site and at controllable rates. Our efforts have focused on developing a new thermal-sensitive drug delivery system, specifically for the local control of solid tumors. We describe here a new lipid formulation containing doxorubicin that has been optimized for both mild hyperthermic temperatures (39 degrees C to 40 degrees C) that are readily achievable in the clinic and rapid release times of drug (tens of seconds). This new liposome, in combination with mild hyperthermia, was found to be significantly more effective than free drug or current liposome formulations at reducing tumor growth in a human squamous cell carcinoma xenograft line (FaDu), producing 11 of 11 complete regressions lasting up to 60 days posttreatment.     

Radioimmunodetection of human choriocarcinoma xenograft in nude mouse

n order to collect data for further clinical study, we investigated the efficiency of radioimmunodetection (RID) in locating the xenograft of human chorio- carcinoma in nude mouse using 131I labeled mouse anti-human chorionic gonadotropin (hCG) monoclonal antibody (MAb).Materials and methodsPreparation of AntibodiesHybridoma ascites containing three strains of mouse anti-hCG MAbs was provided by the Monoclonal Antibody Lab of the Department of Endocrinology of Peking Union Medical Coll…     

Intratracheally administered 5-azacytidine is effective against orthotopic human lung cancer xenograft models and devoid of important systemic toxicity

Introduction: Hypermethylation of key tumor suppressor genes plays an important role in lung carcinogenesis. The purpose of this study is to explore the therapeutic potential of regional administration (via the airways) of the demethylating agent 5-azacytidine (5-Aza) for the treatment of early lung cancer.Patients and MethodsWe administered 5-Aza solution directly into the trachea in imprinting control region (ICR) mice (to study its toxicity) and in nude mice bearing orthotopic human lung cancer xenografts (to assess its antitumor activity).ResultsIn vitro, 5-Aza inhibited the growth of human lung cancer cell lines H226, H358, and H460 in a dose-dependent manner. The concentrations to inhibit cell growth by 50% (IC₅₀) were about 0.6-4.9 μg/mL. 5-Azacytidine reversed hypermethylation in the promoter of tumor suppressor gene RASSF1a in the H226 cells at a 6000-fold lower concentration than its IC₅₀. In animal studies, intratracheal (I.T.) administration of 90 mg/kg 5-Aza (the maximum tolerated dose of 5-Aza intravenous injection [I.V.]) resulted in moderate pulmonary toxicity and 5-fold reduced myelosuppression compared with the same dose of I.V. 5-Aza. Using an optimized multiple dose schedule, I.T. 5-Aza was about 3-fold more effective than I.V. 5-Aza in prolonging the survival of mice bearing orthotopic H460 and H358 xenografts, and did not cause any detectable toxicity.Conclusion5-Azacytidine can reverse the hypermethylation in the human lung cancer cell lines at a nontoxic dose. Regional administration to the airways enhances the therapeutic index of 5-Aza by 75-fold. The potential of regional administration of 5-Aza (including by aerosolization) for the treatment of advanced bronchial premalignancy deserves further investigation.