Human cells transformed in vitro by human cytomegalovirus: tumorigenicity in athymic nude mice.

Athymic nude mice were inoculated with human embryo lung cells transformed in vitro by human cytomegalovirus (CMV). Of the inoculated animals, 62% developed tumors after an average latent period of 19 days. The tumors were composed of small, polygonal cells with large nulei and scanty cytoplasm embedded in an abundant collagenous matrix. The cells were poorly differentiated but may have been of epithelial origin. Adjacent structures were rarely invaded. CMV-related intracellular and membrane antigens were detected by indirect and anticomplement immunofluorescence techniques in cells cultured in vitro from the tumors.     

Enhancement of tumorigenicity of human breast adenocarcinoma cells in nude mice by matrigel and fibroblasts.

The failure of MCF7 cells to induce the formation of tumours after sub-cutaneous inoculation into athymic nude mice can be obviated by the simultaneous injection of an extract of basement membrane proteins (matrigel). Tumour growth is promoted and the latency period is low (2 to 4 weeks). In the absence of matrigel, the simultaneous inoculation of fibroblasts and MCF7 cells also resulted in the development of tumours, but with a longer latency period (about 2 months). The tumorigenic synergy between matrigel and fibroblasts was evidenced by co-inoculating MCF7 cells MDA-MB 231 cells with fibroblasts and matrigel. This co-inoculation decreased the delay of appearance of the tumours and/or accelerated the tumour growth, depending upon the number of fibroblasts injected. Repeated injections of fibroblasts conditioned medium, at the site of inoculum of tumour cells also enhanced tumour growth, suggesting the involvement of soluble factors secreted by fibroblasts. Histologically, tumours induced by co-inoculation of tumour cells and fibroblasts contained more stromal structures including vimentin-positive cells, fibronectin and interstitial collagens. These data suggest that human tumours may be reconstituted and grown in athymic nude mice using basement membrane components and fibroblasts as inductors.     

Two-step chromosomal control of tumorigenicity of chinese hamster cells in nude mice

A simple method for microinjecting isolated chromosomes into a single living cell under an inverted microscope has been developed. Of the 368 injected cells, 85 were able to form a colony and could be cloned. Clones of Chinese hamster V79 cells microinjected with chromosomes isolated from murine D56 cells [V79 (D56) cells] were tested for tumorigenicity in immunodeficient nude mice and for colony-forming ability in soft agar. Untreated recipient V79 cells were highly tumorigenic and had a high colony-forming ability in soft agar. In contrast, two out of the 21 microinjected clones tested were non-tumorigenic in nude mice and had only weak colony-forming ability in soft agar. The chromosome banding pattern was analyzed in microinjected clones and tumors derived from cells of these clones. In cells of the two non-tumorigenic clones, a telocentric chromosome 1 (t1) was specifically involved in translocations with other chromosomes or chromosome fragments. In all tumor cells obtained from nude mice, a supernumerary piece or a whole biarmed chromosome 14 (b14) was specifically found. The results suggest that the t1 chromosome bears the gene which controls in vitro transformation and that the additional genetic change, i.e. the extra piece of b14 chromosome, was required for tumor formation in vivo.     

Diamine oxidase activity in human melanoma cell lines with different tumorigenicity in nude mice.

The activity of diamine oxidase (DO, EC 1.4.3.6.) which converts putrescine into gamma-aminobutyraldehyde in the degradative pathway of polyamine, was studied in 4 human melanoma cell lines, 2 of which produce tumours in greater than 80% of nude mice (M3Dau, M4Beu), whereas the other 2 induce tumours in less than 25% (M1Dor, M2GeB). The activity of DO in these cells varies with the growth rate: 24 h after seeding there is an initial increase in DO activity, followed by a steep decline during exponential growth. At 96 h, when cells reach saturation density, the activity of DO is significantly greater in the highly tumorigenic cell lines than in the poorly tumorigenic cell lines. Kinetic studies show that for the highly tumorigenic lines apparent Km values are 10.6 X 10(-6)M +/- 0.2 (M3Dau) and 14.2 X 10(-6) M +/- 0.6 (M4Beu), whereas for the poorly tumorigenic lines the values are 4.5 X 10(-6) M +/- 0.3. After transplantation into nude mice, the M1Dor cell line, which exhibits a low Km (app.) for DO of which had high Km (app.) value. Km (app.) determination of DO could be an approach for characterizing human melanoma cells differing in their tumorigenic potential in nude mice.     

Fibroblast-dependent tumorigenicity of cells in nude mice: implication for implantation of metastases

The inadequate nature of the microenvironment is one of several factors considered in the failure of tumor engraftment in athymic mice; in the present work, we have tried to more adequately reconstitute it by injecting tumor cells together with fibroblasts. We have demonstrated that the s.c. co-inoculation of fibroblasts with different kinds of tumor cells of animal origin [rat rhabdomyosarcoma (RMS) 9-4/0, rat hepato-carcinoma FAO] or human origin (colonic adenocarcinoma HT29, Ewing's sarcoma pleural metastasis EW-S1) is necessary for tumor take and growth when the number of tumor cells alone is below the tumorigenic dose. We have shown that the s.c. coinoculation of 10(6) fibroblasts and 10(2) RMS 9-4/0 tumor cells induced a tumor take in all the recipient mice, while 10(2) tumor cells alone never gave any tumor. With a tumorigenic number of RMS 9-4/0 tumor cells (10(4), addition of 10(6) fibroblasts decreased the delay between cell injection and tumor appearance, thereby increasing tumor take and growth rate. These results were observed not only in nude animals (mice and rats) used as recipient animals but also in normal WAG rats receiving the syngeneic RMS 9-4/0 tumor cells, and they were independent of the nature or origin of the different fibroblasts cells. This helper effect has also been observed in the normal WAG rats. I.v. injection of tumor cells from a poorly metastatic 9-4/8 subline, derived from the RMS 9-4/0 line and mixed with 10(6) fibroblasts, gave a high number of lung colonies. Addition of 10(6) irradiated 9-4/8 tumor cells instead of fibroblasts did not increase the lung colonizing potential. Fibroblast-conditioned medium mixed with tumor cells instead of fibroblasts also enhanced tumor take and size but to a lesser extent than did the fibroblasts themselves. Only endothelial cells cultured from porcine aorta had a similar helper effect among the cells tested. It is argued in the discussion that the proliferating state of cultivated fibroblasts is a determinant factor conferring upon them the ability to promote tumor cell growth, while fibroblasts very numerous at the implantation site but quiescent might not be efficient in cooperation. Changes in fibroblast morphology and physiology may be necessary in order for tumor cells to express their tumorigenicity.     

Intracranial heterotransplantation of human hematopoietic cells in nude mice.

A panel of established cell lines and many primary cell specimens from lymphomas and leukemias as well as from normal lymphatic tissues were tested for tumorigenicity by intracranial heterotransplantation in nude mice. Not only lymphoma and leukemia cell lines, but also lymphoblastoid cell lines, lacking markers of malignancy, were tumorigenic in the brains of nude mice. These findings indicate that tumorigenicity following intracranial heterotransplantation in nude mice cannot be used as proof for the malignant nature of established cell lines. Heterotraplantation of primary cell specimens yielded only a few tumor takes. When primary cells were infected with exogenous Epstein-Barr virus prior to the transplantation procedure, tumorigenicity could be significantly increased. Cytogenetic evaluation of tumors growing after intracranial transplantation of human hematopoietic cells showed, in some cases, a selection of cytogenetically aberrant cell clones.     

Suppression of Burkitt's lymphoma tumorigenicity in nude mice by co-inoculation of EBV-immortalized lymphoblastoid cells

EBV-immortalized B-lymphoblastoid cell lines (LCL) inoculated s.c. into T-cell-deficient nude mice regress completely after a short initial growth period. We tested whether the putative host response underlying this phenomenon might also be directed against progressively growing Burkitt's lymphoma (BL) tumors in nude mice. Outgrowth of BL tumors was suppressed when cells of the highly tumorigenic BL cell line BL 60 were mixed with cells of the autologous LCL IARC 277 before s.c. inoculation into nude mice. Even when the cells were inoculated separately and simultaneously into contralateral flanks of the mice, regression of initially growing BL tumors could be observed, albeit with reduced frequency and dependent on the dose of LCL cells. Tumor growth of BL 60 cells could also be suppressed by co-inoculation with the non-autologous LCL IARC 174 and IARC 277 cells could suppress growth of the non-autologous BL cell line Eli. Pronounced infiltration with murine (m)CD-11b-positive mouse macrophages and mCD-8a-positive mouse lymphoid cells, most probably natural killer cells, was seen in histological tissue sections of regressing BL 60 tumors when LCL cells were inoculated contralaterally. In regressing BL tumors, these mouse cells were present not only in necrotic areas but also in vital BL tissue, indicating that infiltration of mouse cells had taken place before the development of necrosis. Since tumor-infiltrating mouse cells can be activated at least by some human cytokines, we measured cytokine production of BL 60 and IARC 277. High amounts of IL 6 and IL 10 were produced by the LCL cells, whereas IL-6 and IL-10 production by the BL 60 cells was beyond or close to the detection threshold. In addition, IL 8 was secreted up to 5-fold more by the LCL than by the BL cells. The results presented here thus suggest a host response of the nude mouse, which is triggered by cytokines released from the LCL but, once induced, is directed also against BL cells.     

Telomerase activation during the linear evolution of human fibroblasts to tumorigenicity in nude mice

The ribonucleoprotein enzyme telomerase is active in most immortalcell lines, most tumors and all tumor-derived cell lines. Theenzyme is important because it prevents continual shorteningof telomeres and therefore plays a significant role in chromosomemaintenance. In man, telomerase is not active in most somaIcells with finite lifespans. Using the SV40 T antigen we immortalizedand transformed to fully tumorigenic a human fibroblast cellstrain. We wished to determine when telomerase was activatedduring this progression to tumorigenicity. Using the PCR-basedTRAP assay we found that eight of eight immortal cell linesthat were either not tumorigenic or rarely formed tumors weretelomerase positive at the time of inoculation. Additionally,10 of 11 newly immortal cell lines contained telomerase activitywithin the first 25–33 population doublings after crisis.None of the precrisis cells from which these immortal cellswere derived were positive for telomerase activity. Thus wefound that telomerase activation is not the final in vivo stepin the transformation of these cells and the window of activationis usually near the escape from crisis or M2. These resultsstrengthen the hypothesis that telomerase activation may allowthe rare cell to escape from crisis in those immortal cell populationsdependent on telomerase for telomere maintenance.     

S-adenosylmethionine decarboxylase overexpression reduces invasiveness and tumorigenicity in nude mice of MCF-7 breast cancer cells.

To elucidate the role of S-adenosylmethionine decarboxylase (SAMDC) in breast cancer biology, we have generated SAMDC overexpressing MCF-7 breast cancer cells. SAMDC overexpression did not alter in a major way growth properties of MCF-7 cells in soft agar, either under basal conditions or in response to estrogen and antiestrogen administration. SAMDC-MCF-7 cells, on the other hand, exhibited a markedly reduced invasive ability in matrigel (p=0.013). Furthermore, they were less tumorigenic in nude mice. The odds for control clones to form tumors were 3.13 (C.1.1.2-8.2, p=0.0184) higher than those for SAMDC clones. The odds ratio were identical in the absence and in the presence of estradiol. In addition, the growth rate of established tumors was slower for SAMDC than for control clones. Overall, our results are consistent with the notion that these phenotypic changes induced by SAMDC overexpression are primarily mediated by suppression of cellular putrescine (and, possibly, spermidine) levels.     

Interleukin 17, a T-cell-derived cytokine, promotes tumorigenicity of human cervical tumors in nude mice.

Interleukin (IL) 17 is a proinflammatory cytokine secreted mainly by activated human memory CD4 T cells that induces IL-6, IL-8, and nitric oxide. Because IL-6 and IL-8 have been implicated in the pathogenesis of cervical cancer, we investigated the action of IL-17 on human cervical tumor cell lines in vitro and in vivo. We showed that in vitro, IL-17 increases IL-6 and IL-8 secretion by cervical carcinoma cell lines at both protein and mRNA levels. No direct effect of IL-17 on in vitro proliferation of cervical tumor cell lines could be demonstrated. However, two cervical cell lines transfected with a cDNA encoding IL-17 exhibited a significant increase in tumor size as compared to the parent tumor when transplanted in nude mice. This enhanced tumor growth elicited by IL-17 was associated with increased expression of IL-6 and macrophage recruitment at the tumor site. A potential role of IL-17 in modulation of the human cervical tumor phenotype was also supported by its expression on the cervical tumor in patients with CD4 infiltration. IL-17 therefore behaves like a T-cell-specific cytokine with paradoxical tumor-promoting activity. This may partially explain previous reports concerning the deleterious effect of CD4 T cells in cancer.     

Chemokine gene transfection into tumour cells reduced tumorigenicity in nude mice in association with neutrophilic infiltration.

To study the effect of localised secretion of chemokines on tumour growth, the genes for human (hu) interleukin 8 (IL-8), hu-MCP-1 (MCAF), hu-MIP-1 alpha (LD78), murine (mu)-MCP-1 (JE), mu-MIP-1 alpha or mu-MIP-2 were introduced, via mammalian expression vectors, into Chinese hamster ovary (CHO) cells, and the ability of transfected cells to form tumours in vivo was evaluated. The production of hu-IL-8, hu-MIP-1 alpha or mu-MIP-1 alpha by transfected clones did not influence the growth rate in vitro, but drastically suppressed tumour growth when injected subcutaneously (s.c.) into nude mice. However, clones transfected with hu-MCP-1, mu-MCP-1 or mu-MIP-2 did not show any significant difference in growth rate in vivo compared with clones transfected with vector alone. Histological examination of the site of injection of CHO clones transfected with hu-IL-8, hu-MIP-1 alpha or mu-MIP-1 alpha showed predominantly neutrophilic infiltration. These results indicate that chemokines have potent anti-tumour activity when released, even at low doses, at the tumour site, which may be mediated by recruitment and targeting of neutrophilic granulocytes to chemokine-releasing cells. Our studies highlight the potential usefulness of localised chemokine secretion in inducing potent host anti-tumour defensive responses.     

Loss of tumorigenicity of human breast cancer cells engineered to produce IL-2, IL-4 or GM-CSF in nude mice.

Human breast cancer cells (OCUB-M), retrovirally transduced with granulocyte macrophage-colony stimulating factor (GM-CSF), interleukin-2 (IL-2) or IL-4 gene were examined for their antitumor activities in nude mice. Although cell proliferation rates in vitro of these cytokine-producing cells were not significantly different from that of wild-type cells, nude mice that were subcutaneously inoculated with cytokine-producing cells did not develop tumors in contrast to mice that were injected with wild-type cells. Injection of GM-CSF-producing cells into the vicinity of growing wild-type tumors retarded subsequent growth of wild-type tumors. Histological examination of tumors which received GM-CSF-producing cells revealed marked infiltration of mononuclear cells around the tumors. Irradiation of cytokine-producing cells diminished their proliferation capacity but production of cytokine(s) was retained. Therefore, inoculation of irradiated cytokine producer cells into growing tumors can be used as a therapeutic maneuver for breast cancer.     

重组腺病毒Ad5F35-SH2-DED对K562细胞裸鼠致瘤能力的影响

目的:研究融合蛋白SH2-DED(SD)对白血病K562细胞裸鼠皮下移植瘤的影响.方法:建立K562细胞裸鼠皮下移植瘤的重组腺病毒预防和治疗模型.预防模型采用皮下注射经重组腺病毒Ad5F35-SD或Ad5F35-SmD预处理的K562细胞;治疗模型采用皮下注射K562细胞建立皮下移植瘤后,进行Ad5F35-SD或Ad5F35-SmD的瘤体内多点注射.观察裸鼠移植瘤的生长情况以及移植瘤肿瘤细胞的形态学变化.应用TUNEL法和电子显微镜观察移植瘤肿瘤细胞的凋亡情况.结果:治疗模型组中,Ad5F35-SD处理组移植瘤体积明显缩小,肿瘤细胞核浓缩,细胞质染色加深.TUNEL法和电子显微镜检测结果提示,肿瘤细胞发生凋亡,且凋亡相关蛋白caspase-3和caspase-8表达上调.Ad5F35-SD在预防模型组中同样具有抑制移植瘤生长的作用.结论:重组腺病毒Ad5F35-SD能够抑制K562细胞在裸鼠体内的致瘤能力以及裸鼠皮下移植瘤的生长,并促进肿瘤细胞凋亡.     

Over-expression of hsp70 confers tumorigenicity to mouse fibrosarcoma cells

Over-expression of the major heat-shock protein hsp70 in WEHI-S tumor cells renders them resistant to the cytotoxic effects of tumor necrosis factor (TNF). To study the significance of this resistance in vivo, the tumorigenic potential of WEHI-S cells transfected with human hsp70 in sense and anti-sense orientation was investigated in athymic and in normal syngenic mice. A striking correlation was observed between the level of hsp70 expression and tumorigenicity in athymic mice. Hsp70 expression rendered WEHI cells tumorigenic also in normal mice, but higher numbers of cells were required for tumor formation than in athymic mice. Over-expression of hsp70 in WEHI-S cells did not enhance their anchorage-dependent growth in vitro or their ability to form colonies in soft agar. The hsp70-transfected cells exhibited greatly increased resistance against killing by murine natural cytotoxic cells and macrophages in vitro. A similar tumorigenic phenotype could also be induced independently of hsp70 by prolonged culture of WEHI-S cells with TNF. These results suggest that over-expression of hsp70 increases the tumorigenic potential of WEHI-S cells in mice, by allowing these cells to escape from the early TNF-mediated anti-tumor immune surveillance.     

Hyperthermia enhances mitoxantrone cytotoxicity on human breast carcinoma and sarcoma xenografts in nude mice.

In this preclinical in vivo study, we measured antitumor response, local side effects and systemic toxicity of locally applied water-bath hyperthermia given alone or simultaneously with mitoxantrone (3 mg/kg b.w. i.v.; LD 10) on a human derived breast carcinoma (MX 1) or a human sarcoma (S 117) transplanted to female athymic (nude) mice. A single hyperthermia treatment at a tumor temperature up to 43 degrees C for 1 hr caused in both tumor lines only minor tumor regressions and transient tumor growth delay. However, the antitumor effect of mitoxantrone was significantly enhanced by local hyperthermia at 42 degrees C and particularly at 43 degrees C. In both tumor lines complete tumor regressions were achieved only if mitoxantrone was combined with hyperthermia. Undesired side effects and drug toxicity were not enhanced by hyperthermia. According to in vitro data and the results of the present in vivo study mitoxantrone seems to be a good candidate for clinical trials in combination with locoregional hyperthermia.     

Effects of epidermal growth factor receptor concentration on tumorigenicity of A431 cells in nude mice

To test the relationship between the concentration of epidermal growth factor (EGF) receptors and tumor growth in vivo, we measured the rate of growth of several independently isolated A431 cell lines in athymic mice. This series of A431 clonal variants with differing extents of EGF receptor gene amplification and protein expression were implanted into athymic mice and the time to solid tumor formation and rate of growth were measured. Results of these experiments indicate that the degree of gene amplification and concentration of EGF receptors are directly correlated with the growth of these cells as solid tumors in host animals. Complementary DNA hybridization analysis revealed no change in the extent of gene amplification and expression in implanted cells versus excised tumors nor any evidence of further gene rearrangement in vivo. A high concentration of EGF receptors appears to facilitate the growth of tumor cells in vivo and in vitro.