Ultrastructure of giant cells in tumours induced in golden hamsters by murine sarcoma virus-Harvey

Tumours induced in golden hamsters by murine sarcoma virus-Harvey were studied in four situations: in subcutaneous tissue, on paws or distal ends of limbs, in muscle and in association with bone destruction. Most of the tumours contained multinucleate giant cells. In subcutaneous tumours, a “filament ous” type of giant cell was seen with long filamentous cell processes and bizarre-shaped nuclei. Also present was an “interlocking” type which had short cell processes that interlocked with those of surrounding uninuclear cells. The groups of cells formed in this way resembled a granuloma. Degenerating multinucleate sarcolemmal cells were seen at the edge of muscle tumours and “vegetative” muscle cells were also present. A “pleomorphic” type of giant cell was seen in paw, muscle and bone-eroding tumours. This was a multinucleate cell differentiated to respond to cellular damage caused by tumour infiltration. Groups of cells resembling those seen in granulomas were also present in these tumours.     

Histochemistry of giant cells in tumours induced in golden hamsters by murine sarscoma virus-Harvey

Tumours induced by murine sarcoma virus-Harvey (MSV-H) in golden hamsters are characterized by the presence of multinucleate giant cells. A histochemical study was undertaken to determine the relationship of the giant cells to each other and to the surrounding tumour. It was shown that the giant cells were not a homogeneous cell population. Those found in subcutaneous tumours showed no substrate-specific phosphatases and had marked histochemical similarity to cells of the adjacent tumour. In paw, muscle and bone-eroding tumours, most of the giant cells showed strong substrate-specific phosphatase activity and no histochemical resemblance to cells of the adjacent tumour. Giant cells probably related to skeletal muscle were seen only where muscle was infiltrated by tumour. It was concluded that the giant cells were possibly related to the tumour cells only in the case of the subcutaneous tumours and that in the other tumours they were probably related to macrophages.     

Proliferative patterns of lymphocytes in lymph nodes during tumour development: involvement of T and B cell areas

DNA-synthetizing lymphocytes were identified in the lymph nodes regional and more distal to the site of developing P-815 tumours by incorporation of [3H]-thymidine followed by autoradiography of lymph node sections. It appeared that not only T but also B cell areas of draining and to a lesser extent of distal lymph nodes were stimulated by the growing tumour. This result was unexpected since neither humoral nor tumour cell-bound antibody could be identified so far as a functional correlate of B cell stimulation. In general the proliferative response of lymphocytes followed a biphasic pattern with an early peak of reactivity on days 2-3 and a second peak around day 12-15 after tumour cell inoculation. In the draining (axillary) lymph node the second peak of reactivity was suppressed, possibly as a consequence of metastatic tumour cells in this node when tumour cells were inoculated in the flank. The pattern of lymphocyte stimulation revealed larger individual variations after tumour cell inoculation in the flank than the foot pad. These results were associated with a slower and less regular drainage of carbon particles from the flank to the axillary and exceptionally the brachial lymph node than from the foot pad to the popliteal node after injection of India ink.     

Quantitative and functional study of breast cancer axillary lymph nodes and those draining other human malignant tumors

Lymphocyte functional activity from lymph nodes draining human malignancies reflects the host immune response against tumour. Breast cancer is the neoplasia with the greatest amount of identified antigens but a weak inducer of a host efficient immune response. In our study we compared the mitogen stimulated-proliferative response of cells isolated from metastases-free lymph nodes draining breast cancer (Group 1), other malignant tumours (Group 2), and those obtained from patients without malignancies (Control group). A significant decrease of the proliferative response in cells isolated from lymph nodes draining breast cancer was observed comparing it to the other groups. Quantitative analysis of B and T cells showed a higher number of B cells than T cells in Groups 1 and 2. Moreover, Group 1 presented a two fold increase of T cells compared with Group 2. Our results suggest that the immunosuppression observed in lymph nodes draining breast cancer is higher than the inmunosuppression presented in other malignant tumours and that impaired function is not correlated with the increased number of T cells.     

A spleen weight assay for Murine Sarcoma Virus Harvey (MSV-H)

A spleen weight assay has been developed for titrating murine sarcoma virus-Harvey (MSV-H) 3 weeks after intraperitoneal infection of newborn BALB/c and random-bred albino mice. Induction of splenomegaly corresponded in time with development of sarcomas and anaemia. The assay was also used to evaluate sera for neutralizing antibodies to MSV-H. Weanling mice were much less susceptible to erythroblastic splenomegaly and tumour induction than newborns. Random-bred mice were found more satisfactory for the assays than BALB/c mice. Newborn Sprague-Dawley rats also responded to MSV-H inoculation with a dose-dependent erythroblastic splenomegaly.     

Effect of di-isopropanolnitrosamine in European hamsters

The carcinogenic effects of di-isopropanolnitrosamine (DIPN) were tested in hibernating and non-hibernating European hamsters. The results obtained were compared with those produced by the same substance in Syrian golden hamsters and Sprague-Dawley rats. In European hamsters, tumours were produced in the nasal cavity, trachea, lung, liver and pancreas. The main target organs were the anterior part of the nasal cavity and liver. Only cholangiomas and cholangiocarcinomas were found in the liver. Early changes in the intrahepatic bile ducts and duct epithelium of the pancreas were seen 4 weeks after treatment was started. Fourteen out of 144 treated hamsters developed pancreatic-duct tumours, 2 of which were malignant. The tumorigenic response in the target organs was lower in hibernating than in non-hibernating animals.     

Species differences in sodium o-phenylphenate induction of urinary bladder lesions

The effects of sodium o-phenylphenate (Na-OPP) treatment on urinary bladder epithelium were examined in male F344 rats, B6C3F1 mice, Syrian golden hamsters and Hartley guinea pigs. Na-OPP was incorporated into diet at a dose of 2% and administered for 4, 8, 12, 24, 36 or 48 weeks. Simple and papillary or nodular (PN) hyperplasias were evident on light microscopy and pleomorphic microvilli demonstrated by scanning electron microscopy were only observed in rats, the lesions becoming more advanced with continued chemical feeding. In mice, hamsters and guinea pigs, proliferative lesions relating to Na-OPP administration were not observed. No significant differences in urinary pH, osmolality or crystal formation were apparent between the various animal species. Since carcinogenicity has been demonstrated for Na-OPP in rats but not in mice, the present findings suggest that Na-OPP might not exert urinary bladder carcinogenic potential in hamsters and guinea pigs.     

Further studies of the relationship between lymphatic dissemination and lymphnodal metastasis in non-immunogenic murine tumours.

In all 6 different murine tumours of spontaneous origin, a high proportion (22-95%) of the regional lympgh nodes draining small intradermal tumours gave rise to tumours after their isogeneic transplantation as whole nodes. In separate experiments with 4 of these tumours, equivalent tumour-bearing mice had their tumours surgically excised and were observed for the development of regional nodal corresponding frequency of tumour formation by transplanted nodes. After high-dose radiotherapy of intradermal carcinomas, there was a progressive fall in the incidence of positive regional node transplants from 48 to 96 h after irradiation. It is concluded that continual lymphatic dissemination of viable cancer cells is characteristic of malignant tumours, but that there is a relatively small chance of such cells giving rise to nodal metastatic growth. Related studies showed that the ability of a small number of cancer cells to give rise to tumours was very much greater if they were incorporated in a lymph node at transplantation than if they were transplanted directly as a suspension.     

The effect of cyclophosphamide on MSV-H oncogenesis.

The effect of cyclophosphamide on MSV-H oncogensis and the immune response of young mice has been investigated. A single, sublethal dose (100 and 50 mg/kg of cyclophosphamide) in 8-day-old mice given 24 h before or after MSV-H infection led to an earlier and lower incidence of tumours in comparison with controls infected only with MSV-H. The protective effect of cyclophosphamide, and the mechanism of action of both cyclophosphamide and MSV-H on the target cells, mesenchymal cells in rapid replication, as well the immunological implications of the findings are discussed.     

The cellular events associated with regression and progression of murine (Moloney) sarcomas

Tumors were induced in adult and neonatal mice by intramuscular injections of either 10⁴ or 10⁶ cells from a cultured murine (Moloney) sarcoma line. Neoplasms that progressed were induced in neonates by either dose, and in adults only by the larger dose; adult mice receiving 10⁴ cells usually developed tumors that regressed. Light microscopic examinations were performed at 2–3 day intervals throughout the course of tumor development and subsequent regression or progression. Initially all tumors became infiltrated with polymorphonuclear leukocytes—mainly neutrophils—and edema was extensive. By the end of the second week post inoculation, this acute inflammatory infiltrate had been replaced in adult mice by one consisting of mononuclear cells; neonatal mice never developed significant numbers of these inflammatory cells in their tumors. Of particular significance, since mononuclear inflammatory cells were associated intimately with tumors during the process of regression, was the disappearance of these cells 12–14 days post inoculation from tumors destined to progress in adult mice. Hyperplastic changes were found in the cortices and medullae of regional lymph nodes draining both progressing and regressing sarcomas. Secondary neoplasms developed commonly, and the distribution of these lesions was related to the ages of mice at the time of inoculation.     

Inhibition of dendritic cell migration by transforming growth factor-β1 increases tumor-draining lymph node metastasis

Background

Transforming growth factor (TGF)-β is known to be produced by progressor tumors and to immobilize dendritic cells (DCs) within those tumors. Moreover, although TGF-β1 has been shown to promote tumor progression, there is still no direct, in vivo evidence as to whether TGF-β1 is able to directly induce distant metastasis.

Methods

To address that issue and investigate the mechanism by which TGF-β1 suppresses DC activity, we subdermally inoculated mouse ears with squamous cell carcinoma cells stably expressing TGF-β1 or empty vector (mock).

Results

The numbers of DCs within lymph nodes draining the resultant TGF-β1-expressing tumors was significantly lower than within nodes draining tumors not expressing TGF-β1. We then injected fluorescently labeled bone marrow-derived dendritic cells into the tumors, and subsequent analysis confirmed that the tumors were the source of the DCs within the tumor-draining lymph nodes, and that there were significantly fewer immature DCs within the nodes draining TGF-β1-expressing tumors than within nodes draining tumors not expressing TGF-β1. In addition, 14 days after tumor cell inoculation, lymph node metastasis occurred more frequently in mice inoculated with TGF-β1 transfectants than in those inoculated with the mock transfectants.

Conclusions

These findings provide new evidence that tumor-derived TGF-β1 inhibits migration of DCs from tumors to their draining lymph nodes, and this immunosuppressive effect of TGF-β1 increases the likelihood of metastasis in the affected nodes.     

Tumorigenicity of inorganic arsenic compounds following intratracheal instillations to the lungs of hamsters

The tumorigenicity of arsenic trioxide (As2O3), calcium arsenate (Ca3(AsO4)2) and arsenic trisulfide (As2S3) was studied in male Syrian golden hamsters which received arsenic compounds containing a total dose of 3.75 mg arsenic by means of intratracheal instillations once a week for 15 weeks. As controls, some hamsters were treated with only the vehicle, phosphate buffer solution. During the animals' total life span, one lung adenocarcinoma was seen in the 17 hamsters in the As2O3 group, one lung adenocarcinoma and 6 lung adenomas in the 25 hamsters in the Ca3(AsO4)2 group, one lung adenoma in the 22 hamsters in the As2S3 group and one lung adenosquamous carcinoma in the 21 hamsters in the control group. From these results, we conclude that calcium arsenate is tumorigenic to the lungs of Syrian golden hamsters. The results concerning the other 2 arsenic compounds were not conclusive.     

Evolution of the expression of fetal acinar antigens during carcinogenesis of the pancreas in hamsters: individual follow-up by open biopsy

Individual lesions in the pancreas and the presence of fetal acinar antigens along with carcinogenesis induced by N-nitrosobis(2-oxopropyl)amine (CAS: 60599-38-4) were studied by open biopsy in 16 Syrian golden hamsters 13, 22, and 40 weeks after initiation of treatment. At 13 weeks, cystadenoma and regular ductal hyperplasia were noted in 3 animals and 1 animal, respectively. Staining for fetal acinar antigens in the pancreas was found in 69% of the hamsters. At 22 weeks, cystadenoma and hyperplastic ducts were common (60 and 53%), and 3 hamsters developed pancreatic adenocarcinoma. Fetal acinar antigens persisted in the acini and extended to irregular hyperplastic ducts and tumor cells. At 40 weeks, ductal proliferation was the main lesion in all pancreatic tissue, and 9 animals had adenocarcinoma. Acinar antigens were found in the remaining acini, in irregular hyperplastic ducts, and in tumor cells. Thus, once reexpressed, fetal acinar antigens persist in pancreatic lesions and pancreatic carcinomas in the hamster.     

In vivo detection and partial characterization of effector and suppressor cell populations in spleens of mice with large metastatic fibrosarcomas

The MC-2 fibrosarcoma, which is a transplantable tumour syngeneic for BALB/c mice, metastasizes to lymph nodes draining subcutaneous inoculation sites, and also to the lungs. T cell-mediated immunity was detected in Winn assays using spleens from excision immunized mice. T cell-mediated anti-tumour immunity was also detected in spleens from mice with small tumours but disappeared as the tumour burden increased. Protective immune spleen cell activity in the Winn assay was inhibited by prior addition of spleen cells from mice with large tumours, causing increased tumour incidence. Splenic metastases occasionally occurred in the MC-2 model, but were not demonstrable by bioassay in any of the experiments detecting splenic suppressor cell activity. In vivo protective activity was restored to advanced-stage tumour-bearer spleens by whole-body ionizing irradiation (0.5 and 2.5 Gy) of donor mice 15 h before sampling. Spleen cells from mice with small tumours remained protective after 1.5, 2.5 and 4.0 Gy of irradiation in vivo. These results are consistent with the properties of radiosensitive suppressor T cells. In contrast to reports in other tumour models, suppressor cells were not detected until late in the course of MC-2 development. This is surprising in view of the aggressively metastatic nature of MC-2. It is postulated that modulation of the anti-tumour immune response by the suppressor cells is associated with metastasis in this tumour model. The late appearance of both suppressor cells and metastatic cells in the spleen may reflect similar processes occurring earlier in regional lymph nodes.     

Biological significance of isolated tumor cells and micrometastasis in lymph nodes evaluated using a green fluorescent protein-tagged human gastric cancer cell line.

PURPOSE: The biological significance of isolated tumor cells and micrometastasis in lymph node defined by the International Union against Cancer remains essentially unknown because of the lack of appropriate animal models. In the present study, we developed a lymph node micrometastasis model featuring a human gastric cancer cell line tagged with green fluorescent protein gene (GCIY-EGFP), which allows visualization of even isolated tumor cells in the development of metastasis without histologic procedure. Using this model, we investigated the effect of surgery and chemotherapy on the growth of early-phase metastasis formation in the lymph nodes. EXPERIMENTAL DESIGN: The time course of spontaneous inguinal lymph node metastasis after s.c. inoculation of GCIY-EGFP cells into nude mice was examined with fluorescence dissecting microscopy. Then, the effects of surgical removal of the primary tumor with or without anti-asialo GM1 treatment or postoperative chemotherapy on the growth of isolated tumor cells and micrometastasis in the lymph nodes were examined. RESULTS: GCIY-EGFP cells were found to metastasize spontaneously to the inguinal lymph nodes to form isolated tumor cells, micrometastasis, and, finally, develop macroscopic metastasis at 1 to 2, 3 to 5, and 5 weeks postinjection, respectively. When the primary tumors were removed within 2 weeks of inoculation, isolated tumor cells, but not micrometastasis, in the lymph nodes regressed by 4 weeks after surgery in all the mice examined (five of five). This spontaneous regression of isolated tumor cells was completely reversed by anti-asialo GM1 treatment, which could deplete natural killer cells effectively in nude mice. Chemotherapy following resection of the primary tumor at an early stage partially eliminated the remaining micrometastasis in the lymph nodes. CONCLUSIONS: These results suggest that isolated tumor cells in the regional lymph nodes regressed by removal of the primary tumor mainly via natural killer cell-mediated antitumor activity and that micrometastasis in the lymph nodes could be effectively eliminated by the postoperative chemotherapy.     

Alterations of the basement membrane and connective tissue antigens in human metastatic lymph nodes

Antigens of the basement membrane (type-IV collagen and laminin) and the connective tissue (type-Ill collagen and fibronectin) were studied by immunofluorescence in 16 lymph nodes draining colorectal carcinomas and 6 lymph nodes draining breast carcinomas. A comparison was also made between 7 primary colorectal carcinomas and 9 lymph nodes draining these tumors. Anti-type-IV collagen and anti-laminin rarely stained the basement membrane of metastatic tumors. In contrast, we detected type-IV collagen in the peritumoral stroma, although similar images were rarely seen in primary tumors. When tumoral cells were in the vicinity of lymphoid cells, they were occasionaly separated by a barrier stained by the four antisera, or only by antifibronectin and anti-type-lll collagen. In other cases no barrier was observed between both types of cells which were in close contact. On the whole the above alterations were more marked in the lymph nodes draining breast carcinomas, in comparison to those draining colorectal carcinomas. Tumor cells were never stained by antitype-IV collagen or antilaminin serum. Some cells found either in the lymphoid or in the tumor area of metastatic lymph nodes were stained not only by these antisera, but also by a monoclonal antibody against Willebrand Factor, which is a marker of endothelial cells. Thus the labelled cells were characterized as being derived from the capillary wall.     

Interferon production in lymph nodes draining breast carcinoma and its augmentation by OK-432

In breast cancer patients on whom modified radical mastectomy is performed, relatively more of the regional lymph nodes draining the breast carcinoma remain in comparison with standard radical mastectomy. Therefore, investigation of the functions of lymph nodes draining breast carcinoma has become important. Lymphocyte subsets of 33 axillary lymph nodes from 19 breast cancer patients were analysed using flow cytometry. In axillary lymph nodes, both OKT-3(+) cells and OKT-8(+) cells were decreased in comparison with those in peripheral blood. However, the OKT 4/8 ratio was increased in axillary lymph nodes. These findings suggest that axillary lymph nodes are immunologically more functional against cancer spread than peripheral blood. OK-M1(+) cells, Leu-7(+) cells and Leu-11a(+) cells were decreased in axillary lymph nodes in comparison with peripheral blood. The ability of IFN production in axillary lymph nodes and peripheral blood was analysed using the cytopathic effect of VSV-sindbis virus. After 72 hours incubation, IFN production of axillary lymph nodes showed maximum titer. When lymph nodes were co-cultured with OK-432, IFN production of axillary lymph nodes was strongly augmented. IFN production of axillary lymph nodes draining breast carcinoma were increased in comparison with peripheral blood. Axillary lymph nodes draining breast carcinoma would thus seem to be important as cytokine-producing organs. IFN has been found to be an activator of NK cells, cytotoxic T cells and IL-2 production. Axillary lymph nodes may therefore play an important role against the spread of breast cancer.     

Flow cytometric DNA ploidy in salivary gland tumours

This study on 279 tumours of the salivary glands was conducted to analyse whether the assessment of DNA ploidy by flow cytometry may assist histopathology in discriminating benign from malignant types of tumours. The group of benign tumours included 164 pleomorphic adenomas, 51 Warthin's tumours, 7 basal cell adenomas, 2 lipomas as well as 5 other different tumours. All of the 229 benign tumours were diploid. The malignant tumours consisted of 18 adenoid cystic adenomas, 10 mucoepidermoid carcinomas, 5 acinic cell carcinomas, 5 carcinoma in pleomorphic adenoma as well as of 12 other malignancies belonging to 7 different tumour entities. Twelve of 50 malignant salivary gland tumours were aneuploid. There was no significant relationship between the DNA ploidy status and histopathological grading, lymph node metastasis and local recurrence development, respectively. In three cases which initially were taken for pleomorphic adenomas by routine histological examination, aneuploid cell populations exposed by DNA flow cytometric analysis gave rise to a closer inspection of the suspect lesions. Examination of consecutive slides actually revealed small assemblies of carcinoma cells that required a final diagnosis as non-invasive carcinoma in pleomorphic adenoma. The most obvious value of DNA flow cytometry in salivary gland tumours is thus its contribution to assist histopathology in identifying potentially malignant lesions.     

Growth and spread in nude mice of Epstein-Barr virus transformed B-cells from a chronic lymphocytic leukemia patient

Subcutaneous inoculation of Epstein-Barr virus (EBV) transformed peripheral blood B-lymphocytes (PBL) from an untreated chronic lymphocytic leukemia (CLL) patient produced progressively growing lethal tumors in 4 of 11 whole body irradiated (440 rads) nude mice. In one tumor bearing mouse there was splenomegaly and generalized enlargement of lymph nodes. Chromosomal analysis and membrane immunofluorescence revealed that cells in all the 4 s.c. tumors and a proportion of cells in the enlarged spleen and lymph nodes had human chromosomes and contained human kappa or lambda chains demonstrating that these were polyclonal human B-cells. Epstein-Barr virus associated nuclear antigen could be detected in 100% of cells in all the 4 EBV transformed B-cell lines in vitro and aliquots of cells from several s.c. tumors and metastatic lesions examined. Successful serial transplantation into irradiated nude mice was possible for at least 3 generations with one of the 4 s.c. tumors. During serial transplantation, spread of tumor cells to the spleen and lymph nodes could be detected in all the 3 passage mice investigated; however, there was no evidence in any mouse of dissemination of tumor cells into the bloodstream or into any organ other than lymph nodes and spleen. s.c. tumors also developed in a proportion of irradiated nude mice after inoculation of cells from two other s.c. tumors and the metastatic spleen and lymph nodes, but all these tumors regressed during the first or second transplant passage. Two % of PBL from the untreated patient and 4% of EBV transformed PBL maintained in vitro were found to have trisomy of chromosome 12 which is the most frequently reported anomaly associated with human CLL B-cells. It is highly probable that the cells with trisomy were derived from the leukemic clone of this patient. Cells with this trisomy predominated in most metastatic sites compared to the parent s.c. tumors. Inoculation of irradiated nude mice with EBV transformed PBL from this patient after chlorambucil therapy (100% metaphase plates with 46,XY,11q+ karyotype) or with EBV transformed PBL from 2 normal adults failed to produce any progressively growing tumor in a total of 12 irradiated animals observed greater than 300 days. Although there are several reports of EBV induced immortalization of CLL B-cells in vitro, we have not seen any previous report on the successful serial transplantation and dissemination of EBV transformed CLL B-cells in nude mice.