"Sontaneous" neoplastic transformation in vitro: a form of foreign body (smooth surface) tumorigenesis.

Explants of subcutaneous connective tissue from adult BALB/c mice into plastic petri dishes were serially subcultured and tested for tumorigenicity in two ways: by the subcutaneous implantation of cells attached to plastic plates (1 by 5 by 10 millimeters), and by the subcutaneous injection of cells suspended in saline. Cells grown in vitro for 18 or more days before being implanted attached to a plastic plate (2.4 x 10(4) to 3.4 x 10(5) cells per plate) formed tumors after 24 to 79 weeks. The latent period before tumor appearance correlated inversely with the time spent by the cells in tissue culture. Cells inoculated in saline suspension (10 to 100 times the above number per plate) did not form tumors until after 84 days in vitro; plates alone did not induce tumor formation within more than 1 1/2 years of implantation. The tumors arising from the plate-attached cells were transplantable without plates and histologically appeared to be undifferentiated sarcomas. It is well established that smooth-surfaced foreign bodies, regardless of their chemical composition, will produce sarcomas when transplanted subcutaneously in rodents. We interpret our data, particularly the decrease in tumor latent period with time spent in tissue culture, as indicating that a smooth surface was acting as a carcinogen first in vitro (the surface of the tissue culture dish) and then in vivo (the surface of the plastic plate).

Spontaneous hepatic copper accumulation in Long-Evans Cinnamon rats with hereditary hepatitis. A model of Wilson's disease.

Long-Evans Cinnamon (LEC) rats, an inbred strain of a mutant rat isolated from Long-Evans rats, develop hereditary hepatitis. To elucidate the role of copper metabolism in the development of the hepatitis in LEC rats, we examined the copper concentration in the tissues and serum levels of copper and ceruloplasmin. Copper concentration in the liver of LEC rats was over 40 times that of normal Long-Evans Agouti (LEA) rats, while the serum ceruloplasmin and copper concentrations in LEC rats decreased significantly. The hepatocytes of LEC rats show steatosis in cytoplasm and pleomorphism of mitochondria, resembling the histologic features of the liver in Wilson's disease. These findings suggest that the hereditary hepatitis in LEC rats is closely associated with copper toxicity, and may be dealing with a rat form of Wilson's disease. Thus the LEC rats will provide a unique and useful animal model for clarifying the mechanism and for developing treatment strategies for Wilson's disease and other abnormal copper metabolism in humans.

Local adoptive transfer of the antitumor cellular immune response in syngeneic and allogeneic mice studied with a rapid radioisotopic footpad assay.

The immunologic nature of the cellular immune response against tumor cells inoculated in the footpad of mice was studied with a rapid, quantitative, and specific assay. The results indicate: a) The antitumor cellular immune response could be transferred adoptively in syngeneic and allogeneic mice with specific immune thymus (T) lymphocytes isolated on nylon columns; b) T-independent cells of host origin were necessary for the manifestation of the antitumor footpad reaction; and c) there was a close correlation between immune responses detected by the footpad assay and those detected by transplantation techniques. The footpad reaction consisted of several nonspecific and specific components. Nonspecific factors disturbing the specific footpad reaction in syngeneic and allogeneic recipients were discussed.

Local adoptive transfer of antitumor cellular immunity to xenogeneic animals studied with a rapid radioisotopic footpad assay.

The antitumor cellular immune response to Gross virus-induced rat tumor cells in F344 rats, as measured by a sensitive radioisotopic footpad assay, was adoptively transferred to syngeneic rats and to xenogeneic irradiated BALB/c mice. Xenogeneic transfer was accomplished by the injection of a mixture of rat tumor cells and syngeneic spleen cells, peritoneal exudate cells, or blood lymphocytes from specific immune rats into the footpads of mice. Peritoneal exudate cells produced the strongest footpad reaction in xenogeneic recipients. Use of the xenogeneic adoptive transfer system in a bioassay for human antitumor immunity appeared feasible.

Development of runting syndrome in Friend and Gross virus-induced doubly tolerant rats.

Neonatal injections of Friend virus (FV) or Gross virus (GV) into rats produced immunological tolerance to the virus-induced tumors. The inoculation of specific immune lymphoid cells into the FV-induced tolerant rats brought about the runting syndrome, whereas the GV-induced tolerance was completely abrogated by the same procedure. To investigate the mechanism of the runting syndrome, rats were made doubly tolerant by neonatal injections of a mixture of FV and GV. The adoptive transfer of lymphoid cells from rats immunized with the FV-induced lymphomas into the doubly tolerant rats produced the runting syndrome. On the other hand, adoptive transfer of lymphoid cells from rats immunized with GV lymphomas into the doubly tolerant rats did not produce the runting syndrome and broke down the GV-induced tolerance but not the FV-induced tolerance. By the use of a complement-dependent cytotoxicity test, FV-infected cells were homogeneously detected in thymus, spleen, and bone marrow of the doubly tolerant rats, whereas GV-infected cells were detected only in the thymus. Studies with a rosette formation test as a rat thymus marker showed that none of the FV lymphomas formed rosettes with guinea pig erythrocytes, whereas GV lymphomas formed rosettes. These results suggest that FV and GV have different target cells for infection and transformation and that the development of the runting syndrome is closely associated with infection of bone marrow and spleen cells with FV.

Treatment of runting syndrome and prevention of primary lymphomas in friend virus-tolerant rats.

A series of experiments showed that the inoculation of spleen and lymph node cells from rats immunized with Friend lymphoma (WFT-13) CELLS INTO Friend virus-tolerant rats induced the runting syndrome in nearly all cases, and immunological tolerance to WFT-13 was not broken in any survivors. However, th inoculation of specific immune spleen and lymph node cells admixed with normal bone marrow cells suppressed the runting death. In addition, in these animals the primary lymphomas that ordinarily occur about 200 days after neonatal inoculation of Friend virus did not appear. The mixture of immune spleen and lymph node cells and normal spleen and lymph node cells or normal thymus cells was ineffective in preventing the runting death or the incidence of primary lymphoma. Spleen and lymph node cells from normal rats or rats immunized with antigenically different AH-66 cells were also without effect. Spleen and lymph node cells from rats immunized with sheep red blood cells had a relatively high incidence of the runting syndrome; a few survivors rejected the WFT-13 transplants and also did not develop primary lymphomas. These results suggest that a supplement of hematopoietic stem cells from bone marrow eill not only prevent the runting death of Friend virus-tolerant rats produced by inoculating immune lymphoid cells but will also prevent the expected occurrence of primary lymphomas.

A thymus cell marker in murine leukemia virus-induced lymphomas of rats.

A specific marker for an immature population of thymus cells in the rat was shown by the rosette formation between thymus cells and guinea pig erythrocytes. This method was used to classify murine leukemia virus-induced rat lymphomas. Eight of nine Gross virus-induced rat lymphoma lines, which originated in the thymus, formed rosettes; whereas Friend, Rauscher, or Moloney virus-induced rat lymphoma lines, which originated in either the thymus, spleen, or mesenteric lymph nodes, did not form rosettes. The percentage of the total cells which formed rosettes in the Gross lymphoma lines decreased with in vivo passages. If the tumor cells were exposed to trypsin treatment, then the tumor cells would form rosettes. Lymphoma lines which lacked rosette-forming cells did not show rosette formation after trypsin treatment. An immunofluorescence test showed that none of the lymphoma lines induced by Gross, Friend, Rauscher, or Moloney viruses carried the surface immunoglobulin characteristic of B-cells. These results suggest that Gross lymphomas may be derived from the thymic cortex and that Friend, Rauscher, or Moloney lymphomas may be derived from either mature thymus cells (non-rosette-forming cells) or from a subpopulation of the B-cell series which does not have the surface immunoglobulin G receptor.

Age-related changes of natural antitumor resistance in spontaneously hypertensive rats with T-cell depression.

We investigated the relationship between age-related changes in natural resistance and antitumor effects using a spontaneously hypertensive rat (SHR rat) strain which shows a progressive decline of the number of T-cells and their functions as a result of aging. The growth of a weakly antigenic mammary adenocarcinoma SST-2 was significantly suppressed in SHR rats ages 2 and 3 months, whereas in SHR rats ages 1 or 8 months no suppression of the tumor growth was observed. Splenic natural killer cell activity among the SHR rats was still low at 1 month, when the T-cell function is relatively intact; it reached a maximum level at 3 months and thereafter rapidly decreased. On the other hand, the cytostatic activity of peritoneal macrophages, which is also low at 1 month and becomes high at 3 months, thereafter remained at high levels until 8 months of age. That is, the kinetics of natural killer cell activity during the aging processes runs parallel to the function of suppressing tumor growth. Treatment with anti-asialomonoganglioside antiserum abrogated the suppressive activity of SST-2 tumor growth in 3-month-old SHR rats. Treatment with double stranded RNA polyinosinate-polycytidylate, an interferon inducer, produced significant suppression of the tumor growth in SHR rats ages 3 and 8 months. These results suggest that the participation of natural killer cells is a principal effector mechanism in the suppression of SST-2 tumor growth in SHR rats ages 2 and 3 months.

Metastatic capacity and intercellular communication between normal cells and metastatic cell clones derived from a rat mammary carcinoma.

Three highly metastatic clones and two weakly metastatic clones were obtained from a spontaneously arising mammary carcinoma in an SHR rat. The difference in their capacity to generate metastatic ability was recognized when the tumor cells were implanted s.c. but not when they were implanted i.v. This evidence possibly indicates that the difference in the metastatic capacity of these clones is caused by different potential for detachment from the primary site and for intravasation during the various steps of metastasis. There are no differences between highly and weakly metastatic clones with regard to their in vitro growth characteristics (doubling time, saturation density, plating efficiency, etc.), homotypic aggregation, and adhesiveness to plastic matrices and fibroblast monolayers. Therefore, we used a dye transfer method to examine the relationship between the metastatic capacity of tumor cells and the capacity of tumor cells to make junctional communication with normal fibroblasts. We found that the incidence of intercellular communication between weakly metastatic clone cells and fibroblasts (derived from normal s.c. tissues and tumor tissues) was significantly higher than that between highly metastatic clone cells and fibroblasts. These results suggest that junctional communication between tumor cells and normal fibroblasts may play a part in the early stage of cancer metastasis.

Low incidence of gross leukemia virus-induced lymphomas in spontaneously hypertensive rats with thymic dysfunction.

We compared the incidence of lymphomas induced by Gross leukemia virus (GLV) between spontaneously hypertensive rats (SHR) with a congenital T-cell depression related to thymic dysfunction and normal Wistar rats, the original strain of SHR. Of 20 SHR given neonatal injections of GLV, only 3 (15%) died with thymic lymphomas about 100 days after the virus infection. In contrast, 27 of 28 Wistar rats (96%) developed lymphomas of mostly thymic origin. The 3 lymphomas derived from the SHR bore only a Thy 1.1 antigen, whereas most of the lymphomas derived from Wistar rats carried not only a Thy 1.1 antigen but also a guinea pig red blood cell rosette receptor and a T (W3/13) antigen. Grafts of 1-week-old male Wistar thymus into the neonatal female SHR promoted a differentiation of thymocytes and markedly increased the incidence of the lymphomas which were positive for a guinea pig red blood cell rosette receptor and a T-antigen; grafts of 1-week-old SHR thymus, however, failed to do this. These results suggest that the low incidence of GLV-induced lymphomas in SHR may correlate closely with the absence or decreased numbers of the rosette-forming thymocytes which are presumably the target cells for GLV.

Restoration of T-cell function and induction of antitumor immune response in T-cell-depressed spontaneously hypertensive rats by treatment with thymosin fraction 5.

We studied the effect of treatment with Thymosin Fraction 5 (Fr-5) on the restoration of T-cell functions and the induction of antitumor immunity in spontaneously hypertensive rats (SHR) with congenital T-cell depression. SHR showed a reduced number of rosette-forming thymocytes early in their lives, and in vitro incubation of SHR thymocytes with Fr-5 restored the numbers of rosette-forming T-cells to the normal level for Wistar/HMK rats, the original strain of SHR. In vivo treatment of SHR with various doses of Fr-5 (0.5, 1.0, or 2.0 mg/kg, 6 times every other day) also increased significantly the blastogenic responses of their spleen cells to phytohemagglutinin but failed to promote plaque-forming cell responses to sheep red blood cells. These immunological restorative effects by Fr-5 were dose dependent. In contrast, treatment with lower doses of Fr-5 (0.25 or 0.50 mg/kg) showed greater curative effects on a high antigenic fibrosarcoma (SMT-6) than did treatment with higher doses of Fr-5 (1.0 or 2.0 mg/kg). This was confirmed by the fact that treatment with a 0.5-mg/kg dose of Fr-5 caused a significant suppressive effect on the growth of a weakly antigenic and highly metastatic adenocarcinoma (SST-2) in SHR with a consequent prolongation of survival days, whereas treatment with a 1.0- or 2.0-mg/kg dose of Fr-5 was without any effect. In order to clarify this mechanism, we studied the effect of pretreatment with cyclophosphamide (CY) on the development of antitumor delayed-type hypersensitivity (DTH) reaction in the SMT-6-bearing SHR treated with Fr-5 (0.5 or 2.0 mg/kg). Treatment with 0.5 mg of Fr-5 per kg significantly increased the DTH reaction to SMT-6 cells in both CY-pretreated and untreated SHR. In contrast, treatment with 2.0 mg of Fr-5 per kg produced a significant antitumor DTH reaction in SHR pretreated with CY but failed to induce the DTH reaction in SHR untreated with CY. These results suggest that higher doses of Fr-5 may induce preferentially suppressor T-cells rather than killer T-cells in tumor-bearing SHR with congenital T-cell depression.

Accelerated regeneration of trypsin-treated surface antigens of simian virus 40-transformed BALB/3T3 cells induced by X-irradiation.

The antigens of SV40-transformed BALB/3T3 cells measured by a radioisotopic footpad assay after removal by trypsin treatment regenerated in vitro in 3 to 6 hr. After X-irradiation with 3000 R, however, the antigens were regenerated to normal levels within 1 h. X-ray doses of between 1000 and 5000 R accelerated the regeneration of cell surface antigens, while X-irradiation with the larger dose of 8000 R did not. X-irradiation of nontrypsinized tumor cells was without effect. Possible mechanisms of this phenomenon are discussed.

Vasoformative sarcomas arising from BALB/3T3 cells attached to solid substrates.

The BALB/3T3 mouse embryo cell line, noted for its marked postconfluence inhibition of proliferation, anchorage dependence, and high serum requirement, and frequently studied as a prototype nontumorigenic "fibroblast" line that is compared with tumorigenic sublines transformed with various agents, produced tumors within 2 to 3 months when an average of 3 X 10(4) cells were implanted s.c. attached to 1- X 5- X 10-mm polycarbonate platelets. Plastic platelets alone produced no tumors after 1 year of observation. The tumors, as well as others arising from implants of BALB/3T3 cells attached to 3-mm glass beads, were given the histological diagnosis of "vasoformative saroma" because the tumor cells frequently formed vascular channels. The vasoformative pattern and the results of specific staining for reticulin and collagen support the likelihood that BALB/3T3 cells originated from endothelial cells rather than from fibroblasts. That the tumors were derived from BALB/3T3 cells and not host cells was proved when tumors arising in BALB/c X C57BL/6 F1 hybrids were shown to be transplantable to BALB/c but not to C57BL/6 mice. The cultured tumor cells showed loss of both postconfluence inhibition of proliferation and anchorage dependence. Evidence of the induction of endogenous oncornaviruses was obtained in only one of four tumors tested. These tumors also exhibited tumor-unique transplantation rejection antigens. We conclude that BALB/3T3 cells are preneoplastic and give rise to different spontaneously transformed clones bearing unique tumor rejection antigens when implanted in vivo attached to a solid substrate.

Karyotypes of vasoformative sarcomas arising from BALB/3T3 cells attached to polycarbonate plates.

Four vasoformative sarcoma in vivo tumor lines arising from the s.c. implantation of 3 X 10(4) BALB/3T3 cells attached to a 1- X 5- X 10-mm polycarbonate platelet were shown to be quite similar in chromosome constitution to the parental 3T3 line. All tumors contained the same marker chromosomes that were present in the parent BALB/3T3 cells. The findings conclusively showed that the tumors had derived from BALB/3T3 cells and not from host cells or from the in vivo fusion of the inoculated 3T3 cells with host cells. Each tumor line had a unique karyotype that was markedly more homogeneous than that of the parental BALB/3T3 cells, strongly suggesting that each tumor represented a separate clone. An M1 marker chromosome was consistently present in each of the four tumor lines but was present in only about 50% of the parent BALB/3T3 cells; it therefore appeared to be a distinct and stable feature of the tumors.

Enhancement of antitumor transplantation resistance in rats by appropriately timed administration of busulfan.

Enhancement of specific transplantation resistance to a syngeneic tumor (KMT-17) was observed in WKA rats by treatment with the antileukemia drug busulfan (BU) (15 mg/kg) 5 days before and 5 days after immunization with X-irradiated KMT-17 tumor cells. Rats immunized with X-irradiated KMT-17 cells and then treated with BU showed specific transplantation resistance only against KMT-17 tumor. Carrageenan administration after BU treatment had no effect on enhancement by BU, which indicated that macrophages were not playing a major role in the observed enhancement. With the Winn assay, it was found that spleen cells from rats immunized with X-irradiated tumor cells followed by BU inhibited the growth of admixed tumor cells more strongly than did spleen cells from rats only immunized or only BU treated and that the tumor-neutralizing activity of spleen cells from rats treated by immunization followed by BU was abrogated by treatment with anti-T-serum and complement. It was suggested that the enhanced antitumor transplantation resistance caused by BU was due to enhanced T-cell immune responses to tumor cells. Enhancement of anti-tumor transplantation resistance by BU was significantly abrogated by adoptive transfer with thymus cells and was slightly abrogated with spleen cells from rats immunized with X-irradiated KMT-17 cells 1 day before tumor challenge but receiving no other treatment. Transfer of sera from the immunized rats had no effect on enhancement by BU. These results, taken together, suggest that the mechanism of the enhancement by BU involved a selective elimination of the immunosuppressor cells from the immunized hosts.

Characterization of immunosuppressor cells in rats immunized with solubilized tumor-associated antigens prepared from a methylcholanthrene-induced fibrosarcoma.

Antiimmune responses in rats previously immunized with soluble tumor antigens prepared by sodium deoxycholate (DOC-STA) from chemically induced fibrosarcoma KMT-17 were measured by the Winn assay. Enhancement of tumor growth was demonstrated at a tumor:effector ratio of 1:500 with DOC-STA-immune spleen cells, although inhibition of tumor growth was demonstrated at a tumor:effector ratio of 1:100. The tumor-neutralizing ability of KMT-17-immune spleen cells was abrogated when DOC-STA-immune spleen cells were added to a mixture of KMT-17 cells and KMT-17-immune spleen cells. This suppressor activity of the spleen cells was diminished by the treatment with rabbit anti-rat T-cell serum and immune complement. The suppressor activity of DOC-STA-immune spleen cells was also shown in 51Cr release assay and was specific for the tumor line used. After fractionation of spleen cells from DOC-STA-immune rats by the Ficoll density gradient, the cells in the light layer showed an enhancing effect on tumor growth detected by the Winn assay, whereas the cells in the heavier region of the gradient had an inhibiting effect.

Histopathology of regression of tumor metastasis in the lymph nodes.

A transplanted rat lymphosarcoma (SGT-4), induced by Gross virus and inoculated s.c. into the foot of a normal syngeneic rat, initially grew but ultimately regressed. The tumor cells metastasized to the regional popliteal, lumbar, and inguinal lymph nodes and formed massive metastatic foci there. These lymph node metastases also regressed spontaneously. However, in Gross-tolerant rats inoculated with Gross virus at birth, no regression was observed. Histopathologically, infiltration and proliferation of lymphoid cells, reticulum cells, and bifrocytes occurred in the regressing metastatic tumor in lymph nodes as well as in the regressing transplanted tumor in the foot. Only in lymph nodes of normal rats, in which tumor metastasis regressed, was the characteristic "starry sky" appearance observed. Our results suggest that regression of metastatic tumor in lymph nodes, as well as of transplanted tumor in syngeneic rats, was due to an immunological reaction by the host and that an immunological factor may be responsible for the "starry sky" picture.

Temperature-dependent alteration in immunogenicity of tumor-associated transplantation antigen monitored via paraformaldehyde fixation.

Paraformaldehyde-fixed mKSA tumor cells of BALB/c mice were shown to retain tumor-associated transplantation antigen (TATA) activity to a degree comparable to that of X-ray-inactivated tumor cells under the optimal conditions of 1% paraformaldehyde for 30 min at 37 degrees. Unexpectedly, the TATA activity of cells fixed below 10 degrees was greatly reduced. This temperature effect was reversible. TATA activity was restored if the cells were returned to 37 degrees before fixation. Fixation at all temperatures for longer than 2 hr or at paraformaldehyde concentrations greater than 1% also caused a decrease in immunogenicity. Spleen cells from mice immunized with tumor cells fixed at 37 degrees were able to more effectively neutralize tumor growth in the Winn assay compared with those from mice immunized with cells fixed at 0 degrees. Immunization with paraformaldehyde-fixed tumor cells was completely specific. Mice immunized with an antigenically unrelated tumor were not rendered immune to tumor challenge. Fixed tumor cells could be stored for at least 1 month without loss of TATA activity.

Augmented immunogenicity of tumor cell membranes produced by infection with influenza virus as compared to Moloney sarcoma virus.

The tumor-associated transplantation antigens (TATA) of crude membrane extracts from SV40-transformed BALB/3T3 tumor cells lytically infected with influenza virus were markedly more immunogenic than were extracts from uninfected cells measured either by the ability to induce heightened resistance to tumorgraft challenge or by heightened lymphocyte-mediated cytotoxicity against tumor cells in vitro. When intact tumor cells (as opposed to membrane extracts) were productively infected with Moloney sarcoma virus, they were made so immunogenic that they would only grow in X-irradiated syngeneic animals. Yet crude membrane extracts from the Moloney sarcoma virus-infected tumor cells showed no increase in TATA activity analogous to that seen after infection with influenza virus. Thus, influenza virus augmentation of tumor membrane TATA may operate by a different mechanism than does the oncornavirus augmentation of intact tumor cell TATA reported by others. It appears that Moloney sarcoma virus and possibly other oncornaviruses cannot be used to augment the TATA activity of tumor cell membranes in the same way that other surface-budding viruses can.

Adoptive immunotherapy of a Gross virus producing lymphoma and a methylcholanthrene-induced fibrosarcoma in tolerant rats.

Immunological tolerance to Gross virus-specific transplantation antigens in rats given neonatae transfer of donor lymphoid cells beneath the kidney capsule of syngeneic recipient rats. Immune or normal donor cells invariably developed a cell-mediated immune reaction in kidneys of GV-tolerant recipients, presumably against GV antigens present on the surface of recipient lymphoid cells in the kidney. Spleen and lymph node cells from tolerant rats failed to develop a reaction in tolerant recipients, but developed a strong reaction to histoincompatible antigens in the kidneys of semisyngeneic tolerant rats. The immunologically tolerant state in the rats could be broken by adoptive transfer of spleen and lymph node cells from syngeneic rats immunized with GV-induced lymphoma cells. Immunotherapy of a GV-induced and also a GV-infected methylcholanthrene-induced fibrosarcoma growing in tolerant rats was successful when immune spleen and lymph node cells were administered i.p. 3 days after s.c. inoculation of 2 X 10(7) tumor cells in the case of the lymphoma, and 1 day after inoculation of 5 X 10(6) tumor cells in the case of the fibrosarcoma.