Anticancer drug response and expression of molecular markers in early-passage xenotransplanted colon carcinomas.

Despite some success in the treatment of colorectal carcinomas, novel rational therapies targeting specific cancer-related molecules are under development and urgently needed. These approaches need careful preclinical evaluation in models that closely mirror the clinical situation. Therefore, we established a panel of 15 xenotransplantable tumours directly from fresh surgical material. We showed that both the histology and expression of tumour-associated markers (Epithelial Cell Adhesion molecule (EpCAM), E-cadherin, carcinoembryonic antigen (CEA)) could be maintained during passaging in nude mice. Xenotransplanted tumours were characterised for chemosensitivity and revealed a response rate of 5/15 (33%) for 5-fluorouracil (5-FU), 15/15 (100%) for irinotecan and 8/14 (57%) for oxaliplatin. 5 patients out of 15 were treated with cytostatics because of synchronous metastases. The response to chemotherapy in these patients coincided very closely with the response of the individual xenografts. All of the xenografts expressed the proliferation marker Ki67 and the nuclear enzyme, Topoisomerase IIalpha (Topo IIalpha) at the protein level. Most of the xenografts also expressed the tumour suppressor, p53 (9/14) and the nuclear enzyme Topoisomerase Ialpha (Topo Ialpha) (13/14) at the protein level. Interestingly, the presence of a K-ras mutation in codon 12 (5/15 xenografts) coincided with a low response rate towards oxaliplatin. This observation needs further confirmation using a larger number of tumours. In conclusion, we were able to establish transplantable xenografts suitable to mimic the clinical situation. These well characterised models are useful tools for the preclinical development of novel therapeutic approaches and for investigating translational research aspects.

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 characteristics of two new human mammary carcinoma lines in nude mice with special reference to the estradiol receptor status and the importance of stroma for in vivo and in vitro growth.

Two new human mammary carcinoma lines originating from surgical material were established in nude mice. According to the adopted criteria, the tumor 4049 has been classified as estradiol receptor positive and mammary carcinoma 4296 as estradiol receptor negative. Both tumors proved to be c-erbB-2 protein positive and EGF-receptor negative. In contrast to carcinoma 4296, the in vitro growth and the take rate of mammary carcinoma 4049 in nude mice seems to be dependent on stromal components. Pretreatment of mice with estradiol/peanut oil before tumor engraftment was an essential precondition for the growth of the primary tumor in nude mice. After successful establishment the tumor growth was significantly stimulated by estradiol. The growth rate of mammary carcinoma 4296 was independent of any supplementation of estradiol. The two breast tumors were characterized with regard to their growth behaviour, histology, and sensitivity to cytostatics and antihormones. They are considered suitable tumor models for the testing of antineoplastic substances and for biological experiments.

Wild-type p53 is not involved in reversion of the tumorigenic phenotype of breast-cancer cells after transfer of normal chromosome-17.

A number of different candidate tumor suppressor genes involved in human breast cancer are presumed to be located on chromosome 17. To verify the relevance of chromosome 17 abnormalities in breast cancer cells, a normal human chromosome 17 was transferred by microcell fusion to R30 tumor cells derived from an infiltrating ductal mammary carcinoma. The tumorigenicity of the microcell hybrids in nude mice was examined. The tumor volume obtained with different clones was reduced by up to 94% of the value corresponding to the parental tumor cells. This effect was accompanied by a reduction of anchorage-independent growth, as well as cell growth rates on plastic plates. These effects were independent of the continued presence of a transferred 17q arm and could not be attributed to the action of the normal p53 gene. The results support the assumption that in addition to p53 a further tumor suppressor gene is located on 17p which is involved in breast cancer.

[Fine structure studies and identification of lectin receptors on the viral envelope of two HIV-isolates]. / Feinstrukturuntersuchungen und Identifizierung von Lektinrezeptoren auf der Virushülle zweier HIV-Isolate.

The electron microscopic particle findings were compared with the levels of revertase in corresponding samples over a longer period of time, and a good correlation was found. Comparative investigations of the fine-structure of two HIV isolates did not reveal any morphological differences. It can be assumed, on the basis of the comparative studies on lectin receptors using Helix pomatia lectin, that the viral envelopes of the two isolates are equipped similarly with N-acetyl-d-galactosamine. The differences are not significant with mature particles.

Recombinant plasmid DNA variation of Clostridium oncolyticum--model experiments of cancerostatic gene transfer.

The specific germinating capacity of spores of Clostridium oncolyticum in tumours in vivo, and various reports on concomitant partial oncolysis (tumour lysis) have prompted us to propose a concept of equipping C. oncolyticum with genes of other organisms producing cancerostatics. As an example we used Colicin E3, whose structural genes lies in the E. coli plasmid pCo1E3-CA38. After in vitro recombination of restrictase EcoRI-fragmented pCo1E3-CA38 DNA with an uncharacterized plasmid fraction from C. concolyticum a plasmid-free strain of C. oncolyticum was infected with recombinant DNA. A special microbiological selection system allows the identification of C. oncolyticum clones with Colicin E3-similar formation of cleared haloes.

Cardiotoxicity of free and liposomally encapsulated rubomycin (daunorubicin) in mice.

Different serum enzyme levels (creatine kinase, CK; lactate dehydrogenase, LDH; alanine amino-transferase, ALAT; aspartate aminotransferase, ASAT) were determined after treatment of mice with rubomycin in free or liposomally encapsulated form. Especially increase in CK activity, measure for cardiac toxicity, was significantly altered in animals that were treated with rubomycin-containing liposomes. Organ distribution studies showed a significantly lower uptake of encapsulated drug in heart tissues. Electron microscopic studies confirmed these results by indicating only small anthracycline-induced cardiac lesions using the liposomal form.

Reconstitution of the liver microsomal monooxygenase system in liposomes from dimyristoylphosphatidylcholine.

Different techniques to incorporate the essential isolated and purified enzymes of the hepatic endoplasmic reticulum into monolamellar dimyristoylphosphatidylcholine vesicles are compared with respect to structural and functional parameters of the reconstituted system. By use of gel penetrating chromatography on Sepharose 4B, dynamic light scattering and electron microscopy the structural properties of the reconstituted system were proved comparing the reduction of P-450 LM2 via the NADPH dependent reductase and by dithionite as well with the microsomal reduction rates and by studying the binding of benzphetamine to P-450 LM2 in soluble and liposomal form.

[Preparation, properties and therapeutic effect of Daunorubicin-containing liposomes]. / Darstellung, Eigenschaften und therapeutische Wirksamkeit von daunorubicinhaltigen Liposomen.

Preparation and analytic characterization of uni- and multilamellar lipid vesicles with different lipid composition, charge and size, containing Daunorubicin are described. A critical review is given for the methods of preparation of lipids and other amphiphilic compounds. Variations of the lipid composition, charge and size of the liposomes do not alter the therapeutic effectiveness.

Biological characterization of cells derived from a human breast carcinoma. I. Some characteristics of cells cultivated in vitro prior to and after transplantation into nude mice.

Single cells from a patient (B. E., 65 years) have been isolated by collagenase treatment and cultivated in vitro. The human tumour of the mammary gland showed predominantly simple undifferentiated and also tubular structures. The 5th in vitro passage of these cells was characterized by DNA-distribution pattern, cell doubling time, chromosome number, ultrastructure, hormone and drug sensitivity. Cells of the 5th in vitro passage were i.p. transplanted into nude mice. The characteristics of in vitro cultivated cells (5th in vitro passage) were compared with both ascitic cells (1703/A) and cells of solid tumour material (1703/S) grown in nude mice for several passages (0, 10, 21). DNA-distribution patterns, chromosome numbers and cell doubling times are in good correlation. The number of polyploid cells is increased in ascitic cells. These malignant cells are best able to proliferate in vitro after transplantation into nude mice. Ultrastructure examination of the 21st passage has shown similarity between cultured cells, ascitic cells and cells of solid tumour material grown in nude mice. Virus particles could be observed in cells of solid only. Estrogen binding could be observed in the original tumour material only. All other cell or tissue preparations contained no receptors. Drug sensitivity was changed in the case of Vinblastin, Daunoblastin and Sarkolysin treatment of cells of solid tumour material more than in ascitic cells grown in nude mice. The environment dependence of cells and biological differences between in vivo-in vitro tumours and human neoplasms has to be taken into account when using cells as in vivo or in vitro model systems in experimental and clinical cancer research.

[Germination of Clostridium oncolyticum in spheroid cell cultures as an in vitro tumor model]. / Auskeimung von Clostridium oncolyticum in Sphäroid-Zellkulturen als In-vitro-Tumormodell.

An aggregation technique is used for construction of multicellular spheroids from trypsinized cell cultures of several normal and cancerous cell lines. This technique is applicable also in the presence of 1 X 10(9)/ml spores of Clostridium oncolyticum, and spores and vegetative bacterial cells can be counted by a soft agar colonization technique after trypsinization of spheroids. 24 hours after aerobic cultivation of spore-containing spheroids, germination and multiplication of Clostridium is detected by microscopy, electron-microscopy, and by bacterial cultivation technique. In this response there was no considerable difference between spheroids from tumour or from fresh cell lines. By electron microscopy many spores and vegetatively multiplying Clostridia became visible in the central necrotic part of spheroids. In the peripheral aerobic rim of spheroids, however, also few vegetative Clostridia were detectable.

Cocultivation of Clostridium oncolyticum with normal and tumour cell lines.

During incubation of monolayer cell cultures with clostridia spores in the presence of 1.5 per cent of oxygen vegetative bacterial rods become visible and cell necrosis occurs preferentially in tumour cell lines. After such cocultivation tumour cells show a higher amount of clostridia not elutable by washing than finite cells. Electron microscopy of associated clostridia shows vegetative rods in contact to and inside of tumour cells.

Therapeutic evaluation of liposome-encapsulated Daunoblastin in murine tumor models.

Daunoblastin in free and liposome-encapsulated form was tested in the L1210 murine leukemia and the intramuscularly transplanted 276A sarcoma. Both therapeutic (% ILS, tumor volume inhibition) and toxicologic (leukocytes, body weight difference) parameters were evaluated. The liposome preparations showed similar therapeutic effects as the free substance but caused a lower toxicity with a lower mortality rate, higher leukocyte values and smaller body weight reduction. Longer sonication time with the output of more smaller unilamellar vesicles had no influence on the parameters in the solid model, but resulted in shorter ILS values in the L1210 model. Administration of empty liposomes immediately before liposomal Daunoblastin did not result in better antineoplastic activity but yielded higher leukocyte values.