Biodistribution and tumour localisation of a daunomycin-monoclonal antibody conjugate in nude mice with human tumour xenografts

Summary The blood kinetics and tissue distribution of a conjugate of daunomycin and a monoclonal antibody (791T/36) have been examined in mice, including nude mice with human tumour xenografts reactive with the antibody. For this the antibody moiety of the conjugate was labelled with ¹²⁵I and the drug moiety assayed by radioimmunoassay. After radioiodination, the preparation had an immunoreactive fraction in isotopic binding tests with 791T cells of 74%. Both drug and antibody moieties were precipitable with anti-mouse Ig anti-serum. Following i.v. injection, blood clearance of the two components of the conjugate was essentially identical, and with the serumborne conjugate both radiolabel and drug were co-precipitable. In mice with 791T xenografts, the tumour showed localisation of both drug and antibody moieties and at the time of analysis (3 days) tumour levels of drug were over 100 times those seen with free drug. In parallel studies with mice with antigen negative xenografts, there was no preferential localisation of antibody or drug moieties of the conjugate. These studies have shown in vivo stability of this conjugate, and site-specific targetting of an anti-tumour anthracycline.     

Imaging of human ovarian tumour xenografts in nude mice using a novel 111In-labelled monoclonal antibody (10B)

Monoclonal antibody (mAb) 10B, directed against the human ovarian adenocarcinoma cell line, HEY, was conjugated with cyclic DTPA anhydride and labelled with ¹¹¹In. The biodistribution of ¹¹¹In-DTPA-10B was determined in non-tumour bearing mice and mice bearing subcutaneous (s.c.) and intraperitoneal (i.p.) HEY tumours. The radiolabel was preferentially targeted to s.c. and i.p. tumours in comparison with a control mAb, ¹¹¹In-DTPA-2G3, which does not bind to HEY cells. Among normal organs, the predominant uptake of radiolabel was into liver and kidney. Subcutaneous tumours were successfully imaged using external gamma scintigraphy following i.v. injection of ¹¹¹In-DTPA-10B. The results suggest that ¹¹¹In-DTPA-10B may be a useful agent for the diagnostic imaging of tumour masses in patients with ovarian cancer.     

Chest roentgenographic techniques for demonstrating human lung tumour xenografts in nude rats.

Roentgenographic techniques were investigated for imaging orthotopic lung tumours in anaesthetized nude rats endobronchially implanted with human lung cancer cells. A conventional radiographic unit with a dual-screen, double-emulsion film mammographic receptor produced images preferable to those from a mammographic unit because of superior resolution. Typical exposure factors were 300 mA, 29 kVp, and 17 ms at a focus-film distance of 76 cm with a 2.11 by 2.41 mm effective focal spot and inherent filtration of 1.2 mm aluminium. Sensitivity for tumour detection was 0.93 for 59 animals with pathologically proved tumours and 0.96 for 54 animals with tumours larger than 4 mm or 50 mg. For 24 pathologically tumour-free animals, specificity was 1.00. For 55 animals radiographically judged to have tumours, positive predictive value was 1.00. For all 83 animals, accuracy was 0.95. This technique effectively demonstrates orthotopic human lung tumours in nude rats and should be useful for noninvasive monitoring of tumour presence, location, size, and changes in size.     

The immunohistochemical demonstration of thyroglobulin in human thyroid tumour xenografts using a monoclonal antibody

A monoclonal antibody (RBU/01) was raised against human thyroglobulin and its suitability for the immunohistochemical staining of thyroglobulin was determined on fixed, wax-embedded tissue, using the peroxidase anti-peroxidase (PAP) method. The antibody was then used to demonstrate the expression of human thyroglobulin in sections of a human follicular carcinoma of the thyroid which had been grown in immunodeficient mice. It is concluded that the immunohistochemical evaluation of the xenografts with the antibody provides useful information on this xenograft system as a potential model for thyroid carcinoma.     

The immunohistochemical demonstration of thyroglobulin in human thyroid tumour xenografts using a monoclonal antibody

Summary A monoclonal antibody (RBU/01) was raised against human thyroglobulin and its suitability for the immunohistochemical staining of thyroglobulin was determined on fixed, wax-embedded tissue, using the peroxidase anti-peroxidase (PAP) method. The antibody was then used to demonstrate the expression of human thyroglobulin in sections of a human follicular carcinoma of the thyroid which had been grown in immunodeficient mice. It is concluded that the immunohistochemical evaluation of the xenografts with the antibody provides useful information on this xenograft system as a potential model for thyroid carcinoma.     

Toxicity of anticancer agents, growth and chemosensitivity of human tumour xenografts in a segregating stock of AF nude mice.

An investigation of the usefulness of a segregating stock of nude mice [AF nude mice (AF-nu)] for screening anticancer agents was undertaken. The toxicity of anticancer agents, takes and growth rates of human tumour xenografts and chemosensitivities of xenografts in AF-nu were studied and compared with those in BALB/cA nude mice (BALB/cA-nu). The results showed differences in the pattern of mortalities of AF-nu and BALB/cA-nu administered a range of anticancer agents. Body weight changes in the two nude mouse strains differed in the case of 5-fluorouracil, but not for nimustine, adriamycin and vincristine. All tumours transplanted in AF-nu grew as in BALB/cA-nu. Growth rates of 2 xenografts (gastric cancer and glioblastoma) were not significantly different between the 2 nude mouse strains, but those of 2 lung tumour xenografts were significantly greater in AF-nu than those in BALB/cA-nu. There were no significant differences in chemosensitivities of human tumours in AF-nu and BALB/cA-nu (consistency rate as evaluated by our criteria was 88%). From these results, it is suggested that AF-nu are more suitable for anticancer agent screening and experimental chemotherapy of human tumour xenografts than BALB/cA-nu because of lower costs and high reproductive rate. Although they are genetically heterogeneous, sets of experimental animals sharing the same gene pool can be produced routinely.     

A monoclonal antibody (Po66) directed against human lung squamous cell carcinoma immunolocalization of tumour xenografts in nude mice

Summary Po66, a mouse IgG1 monoclonal antibody, was produced by immunization against a patient lung squamous cell carcinoma. The tissue reactivity of the antibody was measured by a radioimmunological assay with enzymatically dissociated cells, by an immunofluorescence test on frozen tissue sections and by peroxidase-staining of paraffin sections. The antibody bound to lung squamous cell carcinoma, oesophagous carcinoma and, inconsistantly to lung adenocarcinoma but not to the other tumours tested. Some normal tissues also reacted positively, in particular bronchial serous glands, oesophagus epithelium and renal distal and collecting tubules. In normal and malignant tissues showing epithelioid differentiation, Po66 bound to the intermediate maturation area. The antigen immunoprecipitated by Po66 from lung squamous cell carcinoma appeared as a single band with a molecular weight 47000 to 50000 daltons. Purified monoclonal antibody Po66 and an unrelated IgG1 immunoglobulin were labelled with radioactive iodine and injected i. v. into nude mice bearing subcutaneous xenografts of human lung squamous cell carcinoma. The localization index in the tumour was 3.3. Antibody labelled with ¹³¹I allowed gamma-scintigraphic imaging of the xenografts which were clearly outlined by days 9 to 11.This work was supported by Association pour la Recherche sur le Cancer     

Cell proliferation in human tumour xenografts: measurement using antibody labelling against bromodeoxyuridine and Ki-67

Cell proliferation was investigated in human tumour xenografts using bromodeoxyuridine (BrdUrd) labelling, evaluated either by flow cytometry or in tissue sections, and also using the proliferation marker Ki-67. BrdUrd labelling was found to increase when cryostat tumour sections were digested with an enzymic solution. This yielded a labelling index up to four times higher than that obtained using the flow cytometer. Ki-67 indices were found to be higher than those reported for human tumour biopsies, as may be expected due to the enhanced growth rate of the xenografts. Significant heterogeneity was observed in the results for cervix, breast and bladder tumours, and the results of the three methods were poorly correlated. However, three of the four tumour types showed that the tumour with the lowest Ki-67 index also had the longest potential doubling time. Since the measurement of Ki-67 index was found technically easier to perform, and also adequately reflects relative tumour cell proliferation, it is preferred over the other techniques.     

Acoustical imaging of a model of a human hand using pulsed microwave irradiation

Pulsed 5.66-GHz microwave energy irradiated a model of a human hand that was positioned above a submerged planar array of 400 hydrophones. Hydrophone response data were analyzed by a computer that graphically reproduced the image.     

Origin of laminin in the extracellular matrix of human tumor xenografts in nude mice

Monoclonal antibodies reacting exclusively with laminin of human origin and a polyclonal antibody reacting with both murine and human laminin were used to immunohistochemically study the extracellular matrix of four human tumors grown as xenografts in nude mice: a lung carcinoma and a yolk sac carcinoma because they produced cell associated laminin in vitro; and two hepatocellular carcinomas which did not produce cell associated laminin in vitro. The extracellular matrix of the xenografts of the lung carcinoma and the yolk sac carcinoma contained laminin of both human and murine origin. Xenografts of liver carcinoma contained only laminin of mouse origin. This shows that the malignant cells capable of laminin production in vitro contribute this glycoprotein to the extracellular matrix of the solid tumor formed by them in vivo.     

The circulatory bed of the human uterine tube

By means of combined morphological methods blood vessels have been studied in 54 uterine tubes of child-birth women. The main pathways for carrying and distribution of blood to corresponding parts of the tube are sector arteries. They are situated in the subserous tela along the anterior and posterior semicircles of the organ. The microcirculatory bed (MCB) of the uterine tube is presented by serous, subserous, muscular and mucosal plexuses. The MCB of the serous tunic is characterized by vascular compositions--modules. Angioarchitectonics of the mucosal tunic is determined with differences in vascularization of complex and simple folds. Organospecific for small arteries and veins of the tubes is presence of vascular mechanisms, regulating the blood stream (intimal cushions, muscular-elastic constrictors, valves and others). Blood capillaries of the mucosal tunic possess a number of ultrastructural peculiarities: thickened peripheral part of endotheliocyte cytoplasm, that contains fenestrae; wide continuous basal membrane with pericytes in its duplication; three types of pericytic-endothelial contacts etc.     

抗肝癌hdsFv-hEDN重组免疫毒素对荷人肝癌裸鼠皮下移植瘤生长的抑制作用

目的:研究抗肝癌hdsFv-hEDN重组免疫毒素对荷人肝癌裸鼠皮下移植瘤生长的抑制作用,评价其作用为导向治疗药物的临床应用价值.方法:将体外培养的人肝癌细胞系SMMC-7721细胞接种于裸鼠皮下,建立荷人肝癌裸鼠皮下移植瘤动物模型,随机分为hdsFv-hEDN治疗组和对照组,分别给予尾静脉注射hdsFv-hEDN和生理盐水,1次/日,共2周.比较各组裸鼠皮下移植瘤生长速度、肿瘤体积和重量,并计算肿瘤的抑制率.取各组裸鼠肿瘤组织,心,肺,肝,肾组织HE染色,光学显微镜下观察.结果:抗肝癌hdsFv-hEDN治疗组裸鼠皮下移植瘤生长抑制作用显著,肿瘤生长速度减慢,肿瘤体积从42.62±0.57 mm3增加到74.28±2.59 mm3、瘤重为155.82±14.43 mg,而对照组肿瘤体积从41.94±0.91 mm3增加到127.42±4.81 mm3、瘤重为283.28±15.21 mg,两组比较差异非常显著.肿瘤体积抑瘤率和瘤重抑瘤率分别达到41.59±0.02%和45.51±0.09%.组织学观察抗肝癌hdsFv-hEDN组肿瘤组织出现大片坏死,凋亡明显增加,心、肝、肺、肾等重要器官未见明显异常.结论:抗肝癌hdsFv-hEDN重组免疫毒素对荷人肝癌裸鼠皮下移植瘤生长具有良好的抑制作用.     

Establishment of a primary human sarcoma model in athymic nude mice

Improvement of soft tissue sarcoma patient outcome requires well-characterized animal models in which to evaluate novel therapeutic options. Xenograft sarcoma models are frequently used, but commonly with established cell lines rather than with primary human sarcoma cells. The objective of the present study was to establish a reproducible xenograft model of primary human soft tissue sarcoma in athymic nude mice. Primary soft tissue sarcoma cells from four resected human sarcomas were isolated, cultured until the third passage and injected subcutaneously into athymic nude mice. The sarcoma xenograft was further analyzed by histological and immunohistochemical staining. In two out of four sarcomas tumor growth could successfully be established leading to solid tumors of up to 540 mm³ volume. Histological and immunohistochemical staining confirmed the mouse xenograft as identical sarcoma compared with the original patient’s tissue. In the present study a reproducible xenograft model of primary human soft tissue sarcoma in athymic nude mice was established. This animal model is of great interest for the study of sarcomogenesis and therapy.     

胡桃醌不同给药途径对人肝癌细胞BEL-7402裸鼠皮下移植瘤生长的影响

目的采用裸鼠皮下移植瘤模型,通过不同给药途径对胡桃醌抗肿瘤活性和毒性进行评价。方法建立人肝癌BEL-7402细胞裸鼠皮下移植瘤模型,通过腹腔注射和局部注射两个给药途径观察胡桃醌抑制肿瘤生长的效果。结果①以600、300和150μg/kg胡桃醌腹腔注射于人肝癌BEL-7402细胞裸鼠皮下移植瘤模型,发现该剂量胡桃醌对肿瘤生长没有明显的影响;NK细胞活性检测发现,600、300μg/kg胡桃醌对裸鼠免疫功能有影响(P均<0.01),150μg/kg胡桃醌则没有影响(P>0.05);与阳性对照组(5-Fu)相比,600μg/kg胡桃醌组NK细胞活性差异无显著性(P>0.05),300和150μg/kg胡桃醌组NK细胞活性差异有显著性(P<0.05,P<0.01),结果提示胡桃醌对小鼠免疫系统有一定的损伤作用。②以4.5、3和1.5 mg/kg胡桃醌腹腔注射于人肝癌BEL-7402细胞裸鼠皮下移植瘤模型,抑瘤率分别为为78.24%、66.57%、48.94%;4.5、3 mg/kg胡桃醌的抑瘤作用可与阳性对照组比拟(P均>0.05)。但4.5 mg/kg胡桃醌组裸鼠出现明显的皮下脂肪减少、消瘦,并有死亡现象。③以pH 7.4和pH 4.0的600、300和150μg/kg胡桃醌人肝癌BEL-7402细胞裸鼠皮下移植瘤模型局部给药,结果发现不同pH(pH 7.4或4.0)600、300μg/kg的胡桃醌局部注射抑瘤作用与阳性对照组(5-Fu)组差异无显著性(P>0.05),而不同pH的150μg/kg胡桃醌抑瘤作用不明显。同一浓度不同pH药物的抑瘤作用差异无显著性(P均>0.05),但pH 4.0的胡桃醌组肿瘤细胞肝转移较少。结论胡桃醌不同给药途径均可抑制人肝癌BEL-7402细胞裸鼠皮下移植瘤的生长,但有一定的毒副作用,药物安全范围较小。     

The suitability of immunosuppressed mice kept in a standard animal unit as recipients of human tumour xenografts

CBA/Lac mice were immunosuppressed by thymectomy and whole body irradiation with 250 kVp X-rays following pretreatment with cytosine arabinoside. The optimum radiation dose for immunosuppression with prolonged survival was 7.35 Gy. The animals were kept in a standard animal unit with an overall survival rate of 83%. They were found to be suitable for large scale, long-term, xenotransplantation experiments at 20% of the cost of nude mice.     

Overexpression of P-glycoprotein in human lung carcinoma xenografts after fractionated irradiation in vivo.

In vivo exposure of a human epidermoid lung carcinoma xenograft to seven irradiation treatments of 10 Gy in consecutive passages resulted in expression of resistance to vincristine. This about threefold drug resistance was detectable with a single dose of 1 mg/kg vincristine. Characterization of the radiation-pretreated subline showed that overexpression of P-glycoprotein, as determined by immunofluorescence and Mabs C219 and 265/F4, occurred in this tumor. After six X-ray fractions, only single positive cells were observed, whereas seven fractions produced an intense immunofluorescent reaction with both antibodies. Southern blot analyses indicated that no gene amplification had occurred. This result shows that irradiation can influence expression of P-glycoprotein and in this way influences drug resistance.     

Tumour localization of monoclonal antibody against a rat mammary carcinoma and suppression of tumour growth with adriamycin-antibody conjugates

Summary Monoclonal antibody to the rat mammary carcinoma Sp4 isolated from hybridoma supernatants and labelled with ¹²⁵I showed preferential in vivo localization into subcutaneous growths of Sp4 compared with normal tissues and a range of other transplanted tumours. No specific localization was seen with ¹²⁵I-labelled normal rat immunoglobulin. Adriamycin conjugated to monoclonal antibody significantly retarded Sp4 tumour growth at 1/25th of the effective dose of the free drug, and in some cases brought about total tumour regression. Normal rat immunoglobulin-adriamycin conjugates were relatively ineffective. These studies indicate that monoclonal antibody directed against tumour cell surface antigens may be highly effective for tumour targeting of therapeutic agents.