Induction of lymphomas on implantation of human oral squamous cell carcinomas in nude mice

Cancer cells from five oral cancer patients and pleomorphic adenoma cells from one individual were inoculated as single cell suspension into subcutis of 30 Swiss nude mice and tail vein of additional 30 mice. Further, tumor tissue pieces from three oral cancer patients were xenografted s.c. in 18 nude mice, and 10 mice were kept as controls. In animals implanted with tumor pieces, 7/18 (39%) mice, developed squamous cell carcinoma at the site of inoculation within 8-15 days, while tumors were not observed in mice inoculated with single cell suspension, up to 60/90 days. In 8/68 (12%) mice, white foci were observed in several tissues, with hepatomegaly and splenomegaly noted in 27/68 (39%) mice. Histopathological examination of various tissues revealed presence of large cell lymphoma in several organs in 14/68 (21%) mice. No regional or distant metastasis of the implanted oral tumor cells was detected. Mice injected with cells from pleomorphic adenoma, also demonstrated large cell lymphoma in 2/10 (20%) mice, whereas none of the 10 control animals showed any gross abnormalities or microscopic abnormalities in several organs. 2/16 (12%) lymphomas exhibited positive reaction with mouse B cell antibodies illustrating the murine origin of the lymphomas, and these were immunophenotyed as B cell lymphomas. The lymphomas were also examined with mouse T cell antibodies and none reacted positively with the mouse T cell antibodies. The lymphomas also failed to react with human T cell, B cell and human Leucocyte common antigen (LCA) antibodies, indicating that the induced lymphomas were not of human origin. The tumor specimens from seven of eight oral cancer patients and the pleomorphic adenoma patient induced lymphomas in nude mice. Thus it appears that xenografting oral tumor cells into nude mice may cause induction of the murine lymphomas, and this needs further investigation.     

Characterization of casein kinase II in human colonic carcinomas after heterotransplantation into nude mice

Casein kinase II (CKII) activity in colorectal tumours was compared before and after heterotransplantation onto nude mice. The test revealed that the enzyme activity was about two-fold enhanced in the tumours isolated from the nude mice when compared to the respective primary tumours from which they were derived. Immunoblots using a polyclonal CKII-specific antibody showed that the increase of activity was due to a higher expression of the enzyme. Immunohistochemical studies on cross sections of nude mouse tumours showed that most of the CKII molecules were located at the peripheral part of the tumour; the central part did not show intense CKII-specific staining.     

Chemo-adoptive immunotherapy of nude mice implanted with human colorectal carcinoma and melanoma cell lines

Summary The antitumor effects of chemotherapy, recombinant human interleukin-2 (IL-2), recombinant human interferon A/D (IFN), allogeneic human lymphokine-activated killer (LAK) cells, and antitumor monoclonal antibody (mAb), administered alone and in various combinations, were tested in athymic nude mice carrying human tumor xenografts. Treatment began 6–18 days after i.v. or i.p. inoculation of colorectal carcinoma or melanoma cell lines, when macroscopic growths were evident. Chemotherapy consisted of two or three courses of 5-fluorouracil (5-FU) or dacarbazine. IL-2 and/or IFN were administered three to five times weekly for 1–3 weeks, usually starting 2–5 days after chemotherapy. Human LAK cells were infused once or twice weekly for 2 or 3 weeks concurrently with IL-2. In some experiments, murine anticolorectal carcinoma mAb (SF25) was administered. In both tumor systems, chemotherapy alone or immunotherapy alone (IL-2, IL-2 + LAK cells, IFN, IL-2 + IFN ± LAK cells) had little or no therapeutic effects. Additive effects were obtained by combining chemotherapy with IL-2 and LAK cells or with IL-2 and IFN. In the majority of the experiments, the most effective combination was chemotherapy + IL-2 + IFN + LAK cells. Treatment with mAb was beneficial in the colorectal carcinoma system when combined with 5-FU + IL-2 or 5-FU + IL-2 + IFN. Homing experiments with radiolabeled human and mouse LAK cells injected i.v. showed increased early accumulation in the liver and lungs, whereas freshly explanted mouse splenocytes localized mostly in the spleen and liver. The tissue distribution pattern of human LAK cells was similar in normal and tumor-bearing mice (with lung metastases). These findings suggest that combination of chemotherapy with cytokines and LAK cells can be partially effective for advanced solid human tumors even in the absence of the host's T-cell immune response. Preliminary experiments showed that tumor-specific, anti-melanoma T-cell clones were effective in local (s.c.) tumor growth inhibition (Winn assay) following coinjection with the autologous tumor cells.     

MCF-7 human breast carcinomas in nude mice as a model for evaluating aromatase inhibitors

While hormone-dependent, mammary tumors induced with carcinogens (DMBA or NMU) in intact rats have been used extensively for studying aromatase inhibitors, there is currently no suitable model to investigate their effects in human breast cancers in vivo. While hormone responsive tumors can be formed in the athymic mouse using human breast carcinoma MCF-7 cells, due to the low ovarian estrogen production, tumor growth is induced with estradiol supplementation. Thus, this model is unsuitable for studies of aromatase inhibitors. We have induced tumors without the need for estrogen supplementation by co-inoculating MCF-7 cells with Matrigel, a basement membrane preparation, into intact athymic mice. In one experiment, 45 days after inocubation, mice were assigned to the control group or 4-hydroxyandrostenedione (4-OHA) (1 mg/day s.c.) treatment for 52 days. Tumor volumes in the control mice increased 672%, whereas tumor volumes in the treated mice did not change significantly (178.9 ± 16.2 to 336.6 ± 120 mm³). In the second experiment, 55 days after inoculation, groups of mice were treated with the antiestrogen, tamoxifen (5 μg/day s.c.) or vehicle (controls). Tumor volumes in the control mice increased 325% in 58 days, whereas there was no significant change in tumor volume in the tamoxifen treated group (338.8 ± 55.3 to 330.6 ± 84.9 mm³). The results suggest that (1) the tumors resulting from MCF-7 cells co-inoculated with Matrigel are estrogen-dependent and (2) tamoxifen and 4-OHA were effective in suppressing growth of these tumors. The results suggest that this model should be useful for evaluating the effects of aromatase inhibitors and for comparing breast cancer treatments.     

Ultrastructural and ultracytochemical investigation of human gastric carcinomas

Summary We describe the ultrastructure of various types of gastric carcinoma cells as well as their histochemical properties as visualized at the electron-microscope level. The histochemical properties of tumour cells were compared with those of homologous normal epithelial cells. The localization and activity of ATPase, IDPase, acidic phosphatase and alkaline phosphatase as well as of the oxidoreductases (cytochrome oxidase, succinate dehydrogenase and NADH-dehydrogenase) were studied. Our findings demonstrated that, in tumour cells, a complicated process of structural-functional restructuring takes place. It seems that a number of ultracytochemical properties may be preserved or may disappear altogether, also, such properties may become enhanced or weaker. This heterogeneity of the histochemical properties of tumour cells is discussed with regard to the role of the stem (polypotent) cell in the process of the histogenesis (cytogenesis) of human gastric carcinomas.In honour of Prof. P. van Duijn     

Ultrastructural ultracytochemical investigation of human gastric carcinomas

We describe the ultrastructure of various types of gastric carcinoma cells as well as their histochemical properties as visualized at the electron-microscope level. The histochemical properties of tumour cells were compared with those of homologous normal epithelial cells. The localization and activity of ATPase, IDPase, acidic phosphatase and alkaline phosphatase as well as of the oxidoreductases (cytochrome oxidase, succinate dehydrogenase and NADH-dehydrogenase) were studied. Our findings demonstrated that, in tumour cells, a complicated process of structural-functional restructuring takes place. It seems that a number of ultracytochemical properties may be preserved or may disappear altogether; also, such properties may become enhanced or weaker. This heterogeneity of the histochemical properties of tumour cells is discussed with regard to the role of the stem (polypotent) cell in the process of the histogenesis (cytogenesis) of human gastric carcinomas.     

A novel immunohistochemical technique for the detection of human oral carcinomas transplanted in nude mice using mouse monoclonal antibodies

 We have developed a new immunostaining technique specifically for the detection of human tumors transplanted into nude mice using mouse monoclonal antibodies (MoAbs) produced in our laboratory. The formation of a molecular complex consisting of three components (mouse MoAb or hybridoma supernatant, biotin-labeled anti-mouse immunoglobulins, and normal nude mouse serum) markedly reduced background staining and enhanced specific reaction with the transplanted tumors in nude mice. When MoAb production by electrofusion was screened with this new method, the incidence of hybridoma supernatant reactive with sections of transplanted tumor was 2.3 per 100 wells immunostained. These results suggest that production of MoAbs using transplanted tumors is immunohistochemically possible and that this method may provide a new means for developing useful tumor markers. Accepted: 30 September 1996     

Antitumour activity of a sterically blocked ricin immunotoxin on a human colorectal adenocarcinoma grafted subcutaneously in nude mice

Summary We prepared a ricin-antibody conjugate, lacking the ability to bind the galactosidic residues of Sepharose 6B, a so-called blocked immunotoxin. The monoclonal antibody AR-3 was cross-linked to ricin through a thioether bond. Further studies showed that the immunoconjugate suppressed the tumour growth of HT-29 cells in intraperitoneally grafted nude mice, without showing any undesirable ricin toxicity.In this work, to demonstrate the therapeutic activity of the AR-3—ricin conjugate injected into mice bearing subcutaneous tumour, we first evaluated its pharmacokinetic behaviour and biodistribution. The behaviour of the immunoconjugate injected intravenously was almost intermediate between that of the antibody and ricin. Moreover, when the immunotoxin was intravenously administered to nude mice bearing subcutaneous tumour, no therapeutic effects appeared, in accordance with the relatively low permeability of the immunotoxin from the blood to the skin. In contrast, peritumoral treatment produced a strong reduction of the neoplastic nodules without substantial regrowth of the malignant cells. This result was also achieved when the immunotoxin treatment was performed on a well-established tumour. This finding was strictly related to the specificity of the immunoconjugate, since the analogous treatment with an irrelevant immunotoxin showed therapeutic failure.     

Proteomic identification of differentially-expressed genes in human gastric carcinomas

Although genetic alterations in proto-oncogenes, tumor-suppressor genes, cell cycle regulators, and cell growth factors have been implicated in the process of human gastric carcinogenesis, the principle carcinogenic mechanisms are not fully understood. In this study, we used a proteomic approach to search for genes that may be involved in gastric carcinogenesis and that might serve as diagnostic markers. We identified nine proteins with increased expression and 13 proteins with decreased expression in gastric carcinomas. The two most notable groups included proteins involved in mitotic checkpoint (MAD1L1 and EB1) and mitochondrial functions (CLPP, COX5A, and ECH1). This suggested that there are links between dysfunctions in these processes and gastric carcinogenesis. We also observed the differential expression of HSP27 and CYR61 proteins in gastric carcinoma, whose expression is known to be altered in other types of tumors. Furthermore, the study identified proteins whose function in gastric carcinomas was previously unsuspected and that may serve as new molecular markers for gastric carcinomas. Importantly, immunohistochemical analyses confirmed that the levels of expression of MAD1L1, HSP27, and CYR61 were altered in gastric carcinoma tissues. Therefore, our study suggested not only that the proteins identified in this study can be useful diagnostic markers but also that a proteomics-based approach is useful for developing a more complete picture of the pathogenesis and function of gastric carcinomas.     

Stability and localization of biotin-labeled murine monoclonal antibody to human gastric cancer in normal mice and nude mice-human tumor models

We examined the tissue localization of biotin-labeled murine monoclonal antibody (MAb) S202 directed against the human scirrhous gastric carcinoma cell line MK-01 in normal and tumor-bearing mice after intravenous (IV) administration. The biotin-labeled MAb proved to be stable in vivo under normal conditions, antibody titer being 1:256 at 4 hr after IV injection. At 24 hr after injection, the tumor was stained by the avidin-biotin-peroxidase complex (ABC) method. Biotin-labeled MAb was found to be suitable for detection of the xenografted tumor of nude mice. This study provides new information concerning the dynamics of the distribution of biotin-labeled MAb in vivo.     

Impaired local natural killer cell activity in human colorectal carcinomas

Summary The present study was undertaken to study natural killer (NK) cell activity in patients with colorectal cancer at peripheral and local levels. Mononuclear cells were isolated from uninvolved colorectal mucosa, tumor tissue and peripheral blood, and tested against the colon carcinoma cell line CaCo-2 and the erythroleukemia cell line K-562. Peripheral blood NK cell activity from the patients showed similar levels compared with healthy controls, whereas, mononuclear cells of tumor tissue were found to have a significantly decreased NK cell activity compared to the normal intestinal mucosa (P<0.01). No relation was found between the NK cell activity and the advancement of the disease according to the Duke's stage. Interferon- (IFN-) stimulated the NK cell activity of the mononuclear cells from blood, mucosa and tumor. However, the increase of NK cell activity after IFN- stimulation was lower in the tumor compared to the mucosa (P<0.02). The lectin, phytohaemagglutinin, increased the cytotoxicity of mononuclear cells from blood, mucosa and tumor to a similar level. These results suggest that patients with colorectal tumors exhibit a normal NK cell activity in peripheral blood and intestinal mucosa; however, a diminished NK cell activity exists at the tumor level. Although mononuclear cells isolated from the tumor have a normal response to lectin stimulation they show hyporesponsiveness to IFN- stimulation with regard to their NK cell activity.     

Altered mRNA expression of glycosyltransferases in human gastric carcinomas.

Biosynthesis of carbohydrate structures is tissue-specific and developmentally regulated by glycosyltransferases like fucosyl-, sialyl- and N-acetylglucosaminyltransferases. During carcinogenesis, aberrant glycosylation leads to the development of tumor subpopulations with different adhesion properties. The aim of this contribution was to directly compare mRNA expression of several glycosyltransferases in surgical specimens of gastric carcinomas. Carcinoma specimens were classified and characterized according to the WHO/UICC system. In each case, the expression of 12 glycosyltransferase enzymes was studied simultaneously by RT-PCR. For semi-quantitative analysis, amplification of the sample sequence was compared with that of beta-actin, co-amplified within the same tube. Expression of N-acetylglucosaminyltransferase V in gastric carcinomas was significantly enhanced compared to normal tissue. Also, expression of sialyltransferase ST3Gal-IV and fucosyltransferase FT-IV was significantly enhanced in carcinoma tissue. No significant differences in glycosyltransferase expression were found in samples positive for Helicobacter pylori or between the different gastric regions. Thus, carcinogenesis is characterized by specific alterations in mRNA expression of several glycosyltransferases. Future studies will show whether RT-PCR detection of the expression of these enzymes could be helpful for prognostic purposes.     

Expression of basic fibroblast growth factor in human gastric carcinomas

The expression of mRNA for the basic fibroblast growth factor (FGF) gene was examined in seven human gastric carcinoma cell lines and in tissue from 29 gastric carcinomas together with the adjacent normal mucosa. Among the seven gastric carcinoma cell lines, the MKN45 cell line expressed mRNA for the basic FGF gene. Basic FGF protein production was confirmed by flow cytometric analysis and immunohistochemistry. Among the surgical specimens, 16 (55%) of 29 gastric carcinomas showed higher levels of basic FGF mRNA than the normal mucosa. Interestingly, in scirr-hous gastric carcinomas characterized by their fibrous stroma and rapid growth, 9 (69%) of 13, samples examined revealed higher levels of basic FGF mRNA than normal mucosa, whereas only 3 (33%) of the 9 well differentiated adenocarcinomas studied produced similar results. Immunohistochemically, basic FGF protein was localized in tumor cells. These results suggest that basic FGF produced by tumor cells may play an important role in producing fibrosis and angiogenesis in gastric carcinomas.     

Effect of melphalan on growth curves and cell cycle distribution of four human small cell carcinomas of the lung grown in nude mice

Four human small cell carcinomas of the lung grown in nude mice were exposed to melphalan. Two of the tumors were derived from subpopulations isolated by in vitro cloning from the same tumor biopsy. The chemosensitivity of the tumors was determined by calculating the specific growth delay. Drug-induced changes in the cell cycle were detected by flow cytometric DNA analysis. The specific growth delay of the tumors was very different with the greatest differences between the two subpopulations originating from the same tumor. Melphalan induced a dose-related S phase accumulation in three sensitive tumors, whereas no changes were seen in a resistant tumor. Furthermore, the amount of S phase accumulation reflected the sensitivity to melphalan. The results suggest that heterogeneity in chemosensitivity is an important reason for chemotherapy failures.     

Establishment of human colon carcinoma lines in nude mice.

After subcutaneous inoculation into nude mice of 24 human colon adenocarcinomas, growth, defined as histopathologically confirmed tumor growth which has been passed, was observed in 13 cases (54%). Tumors from metastatic sites showed higher take rates (58%) than tumors from primary sites or recurrent tumors (50%). Nine continuous tumor lines were established (69% of growing tumors) with metastatic tumors establishing more readily (100% of growing tumors) than primary tumors (40%). The average period in primary transplant was shorter for metastasis (8.3 weeks), than for primary tumors (18.5 weeks); total material 10.6 weeks. Average periods between passages were shorter than primary transplant times; these periods were shorter for metastases (6.6 weeks) than for primary tumors (9.4 weeks); total material 7.4 weeks. Of four growing tumors not established as continuous lines, three were primary and one a recurrent tumor, and the loss of tumor growth occurred in very early passages, not later than passage 3. All nude mouse-grown colon tumors were moderately well differentiated.     

Origin of cells in human oral mucosa heterotransplants in nude mice

Species identification of cells was performed in a model to study epithelial-mesenchymal interactions using combinations of human and murine tissue. The study comprised 34 successfully recovered transplants of human palatal mucosa with 15 human epithelial outgrowths formed on uncertainly species-identified connective tissue. Formalin-fixed and paraffin-embedded 5-micron sections were stained with bisbenzimide (0.8 microgram/ml) and examined in an epifluorescence microscope. The nuclear-staining pattern revealed limited peripheral invasion of the transplanted connective tissue by murine fibroblasts. In areas of epithelial outgrowth a small number of fibroblasts could not be identified but most subepithelial fibroblasts were murine. The study supports previous findings which indicate that the altered differentiation of the human epithelial outgrowths was caused by the murine connective tissue underlying the human epithelium.     

Effect of human lymphoblastoid interferon on human yolk sac tumors in nude mice

The effect of human lymphoblastoid interferon on the growth of human tumors heterotransplanted into nude mice was examined. The human tumor lines examined, named YST-1, YST-2 and YST-3, were derived from yolk sac tumors of the ovary. Daily intraperioneal injection of 3 X 10(4) U interferon per mouse for 14 days did not inhibit the growth of any of these three human tumor lines. A close correlation was observed between the tumor volume and the level of alpha-fetoprotein in sera of mice bearing the YST-1 tumor (r = 0.55) or YST-2 tumor (r = 0.70). No histological differences were detected between tumor cells of interferon-treated and control mice. Tumor-bearing mice treated with interferon showed no marked weight loss.     

Expression of basic fibroblast growth factor in human gastric carcinomas.

The expression of mRNA for the basic fibroblast growth factor (FGF) gene was examined in seven human gastric carcinoma cell lines and in tissue from 29 gastric carcinomas together with the adjacent normal mucosa. Among the seven gastric carcinoma cell lines, the MKN45 cell line expressed mRNA for the basic FGF gene. Basic FGF protein production was confirmed by flow cytometric analysis and immunohistochemistry. Among the surgical specimens, 16 (55%) of 29 gastric carcinomas showed higher levels of basic FGF mRNA than the normal mucosa. Interestingly, in scirrhous gastric carcinomas characterized by their fibrous stroma and rapid growth, 9 (69%) of 13, samples examined revealed higher levels of basic FGF mRNA than normal mucosa, whereas only 3 (33%) of the 9 well differentiated adenocarcinomas studied produced similar results. Immunohistochemically, basic FGF protein was localized in tumor cells. These results suggest that basic FGF produced by tumor cells may play an important role in producing fibrosis and angiogenesis in gastric carcinomas.     

An ascites form of human mammary carcinoma transplantable in nude mice

Mammary carcinoma cells from pleural effusion of a patient were inoculated into the peritoneal cavity of nude mice, and a large amount of ascites was produced about 120 days later. From the ascites, serial passages in the same form were successful in nude mice by intraperitoneal injection of 10(7) or more cells. The ascites cells retained the morphology almost similar to that of the patient tumor cells, whereas specific estrogen-binding proteins in the cytoplasm disappeared after growing in male nude mice. The results were compared with those of other established human cancer cell lines in nude mice.     

Collagenase inhibitors retarding invasion of a human tumor in nude mice

Tumor invasion has been correlated with the ability of tumor cells to produce collagenolytic enzymes which are capable of degrading normal host tissues. However, the human small cell carcinoma implanted subcutanouesly and growing progressively in athymic (nude) mice produced large quantities of collagenase but did not appear to significantly infultrate adjacent host tissue. In comparison, subcutaneously implanted murine Lewis lung tumors produced similar quantities of collagenase and were locally invasive. The human tumors were surrounded by a compact layer of fibroblast cells in a fibrous matrix. This fibrous sheath exhibited anticollagenase activity and indicated a mechanism of host tissue resistance to invasion via the formation of inhibitors to degradative enzymes produced by tumor cells.