Inhibition of metastasis in rats immunized with xenogenized autologous tumor cells after excision of the primary tumor.

Subcutaneously transplanted 3-methylcholanthrene-induced KMT-17 tumor in WKA rats yielded not only a local solid tumor but also frequent metastases in regional lymph nodes. Neither active immunization with xenogenized identical tumor cells nor surgical excision of the solid tumor prohibited the metastases when each treatment was given 4 days after tumor transplantation. However, immunization combined with surgery significantly decreased the metastases and prolonged survival.

Change of antigenic expression on rat tumor cells after their transplantation.

Antigenic expression of 3-methylcholanthrene-induced transplantable fibrosarcoma KMT-17 cells was investigated in relation to days after ip transplantation. Cytotoxicity tests with antiserum against tumor-associated surface antigen of KMT-17 cells revealed that cytotoxic sensitivity and absorbing capacity decreased after transplantation, but they increased when other normal rats were given transplants of tumor cells. A decrease in the sensitivity was observed when immunosuppressively irradiated rats were given tumor transplants. Tumor cell density in the abdominal cavities of rats directly and absorbing capacity of KMT-17 cells to antiserum against the histocompatibility antigen did not change after transplantation. The possible mechanisms of antigenic change of KMT-17 cells were discussed.

Correlation between concanavalin A agglutinability and cytotoxic sensitivity to antiserum against tumor-associated antigen in rat fibrosarcoma cells.

An ip transplantation of 3-methylcholanthrene-induced, transplanted fibrosarcoma KMT-17 cells (1 X 10(8)) grew rapidly and killed syngeneic WKA rats in 3-4 days. Agglutinability induced by concanavalin A (Con A) and antigenic expression of KMT-17 cells were investigated in relation to days after ip transplantation. Agglutinability was highest in 1-day-old cells and lowest in 3-day-old cells. The agglutinability of 3-day-old cells increased again when these cells were transplanted into normal rats. The cytotoxic sensitivity of tumor cells to antiserum against tumor-associated surface antigen (TASA) changed simultaneously with the degree of Con A agglutinability. This phenomenon disappeared after artificial infection of tumor cells with Friend murine leukemia virus. The result of the quantitative absorption test at 4 degrees C overnight was that 1- and 3-day-old cells did not differ in their absorbing capacities to anti-TASA sera. However, when the absorption test was done at 37 degrees C for 60 minutes, 1-day-old cells had approximately 16 times more absorbing capacity than 3-day-old cells. However, the cytotoxic sensitivity to antiserum against histocompatibility antigen did not change, regardless of the number of days after ip transplantation. Analysis based on the quantitative absorption test revealed no difference in antibody-absorbing capacities between 1- and 3-day-old cells at both 4 degrees C and 37 degrees C. The relationship between Con A agglutinability and cytotoxic sensitivity to anti-TASA serum is discussed from the viewpoint of "lateral receptor mobility" on the cell surface.

Regression of established tumors in rats by injection of diethylaminoethyl-dextran and Friend murine leukemia virus.

Subcutaneously established tumors in WKA rats were treated with polycation DEAE-dextran (DEAE-D) and Friend murine leukemia virus (F-MuLV). This idea was based on "xenogenization of tumors," which is defined as the immunologic regression of transplanted tumors in syngeneic rats after artificial infection of tumors with murine leukemia viruses. Regressions of subcutaneously established tumors were induced in 13 of 40 (33%) rats by injection of DEAE-D and F-MuLV. Intratumor injections of DEAE-D and F-MuLV increased the regression of tumors in 7 of 12 (58%) rats as compared to that of tumors in 6 of 28 (21%) rats treated with DEAE-D and F-MuLV by other injection routes. Electron-microscopic and immunofluorescence examinations revealed that tumor cells were infected with F-MuLV and acquired F-MuLV-related surface antigen on the cell surfaces. Therefore, the regression in rats of subcutaneously established tumors by the injection of DEAE-D and F-MuLV may have been due to an immunologic mechanism that may have been the same as the xenogenization of transplanted tumors previously infected with murine leukemia viruses.

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.

Effect of combination treatment with cyclophosphamide and nonspecific passive immunization on a transplantable tumor in WKA rats.

Combination of cyclophosphamide (CY) and passive immunization with lymphoid cells sensitized to allogeneic tumor was studied in the treatment of a methylcholanthrene-induced transplantable fibrosarcoma in WKA rats. For determination of the most effective timing of combination treatment, rats given an injection of CY on Day 3 received passive transfer of the sensitized lymphoid cells on Day 0, 1, 2, 3, 4, or 6. A remarkable therapeutic effect was observed only when the passive transfer was combined on Day 4 with CY on Day 3. Rats inoculated with tumor succumbed in all cases without any treatment. After i.v. injection of CY on Day 3, 2 of 28 rats were cured (7.1%). Passive immunization with the sensitized lymphoid cells on Day 4 resulted in no therapeutic effect. After combination of CY on Day 3 and transfer of nonsensitized normal lymphoid cells on Day 4, 2 of 15 rats survived (13.3%). However, combination of CY and passive transfer of the nonspecifically sensitized lymphoid cells resulted in 23 survivors of 29 rats (79.3%).

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.

Immunochemotherapy of transplanted KMT-17 tumor in WKA rats by combination of cyclophosphamide and immunostimulatory protein-bound polysaccharide isolated from basidiomycetes.

Protein-bound polysaccharide Kureha (PS-K) isolated from Basidiomycetes was used in combination with cyclophosphamide (CY) for the treatment of a 3-methylcholanthrene- induced KMT-17 fibrosarcoma in WKA/Mk rats. A single administration of PS-K exhibited no inhibitory effect on the growth of s.c.-inoculated KMT-17 tumor at any timing and dose. However, PS-K exhibited a marked antitumor effect when it was combined with CY. The effect of PS-K dependend on the combination timing of PS-K and CY; a marked antitumor effect was observed when PS-K was administered before CY but not if it was given after CY or before tumor inoculation. When PS-K was administered on Day 1 followed by CY on Day 3, the highest survival rate of 78.5% (11 of 14) was obtained. Delayed hypersensitivity response of rats to KMT-17 was investigat ed by radioisotopic footpad assay. On Day 12, the hypersensitivity response in rats treated with PS-K on Day 1 and CY on Day 3 was significantly higher than that in nontreated rats, indicating an enhanced specific immunity to KMT-17 possibly resulting in a marked antitumor effect.

Effect of normal allogeneic lymphoid cell transfer in combination with chemotherapy on a transplantable tumor in rats.

In combination with chemotherapy [ftorafur (FT)], allogeneic lymphoid cells were transferred to inhibit the growth of a 3-methylcholanthrene-induced transplantable KMT-17 fibrosarcoma in Wistar-King-Aptekman/Hok rats. The transference of allogeneic lymphoid cells 4 days after tumor inoculation did not prove effective in inhibiting the growth of the tumor; administration of FT (300 mg/kg) on Day 3 resulted in a 16.7% survival rate. However, a combination of the transfer of cells and administration of FT resulted in an improved effect of 55.0%. A low dose of FT (100 mg/kg) also increased the therapeutic effect in combination with allogeneic lymphoid cell transfer (28.0%). However, a high dose of FT (500 mg/kg) did not increase the therapeutic effect (30.0%), even if combined with allogeneic lymphoid cell transfer on Day 2, 4, 6, or 9, although a high dose of FT administered alone on Day 4 showed a direct antitumor effect (32.0%). This effect was rather diminished when the transfer was combined on Day 2. (7.1%). The differences in the combination effect of doses of FT seem to depend on the immune status of the host induced by treatment with FT. In order to investigate the mechanism of combination timing of FT and allogeneic lymphoid cell transfer, the specific delayed hypersensitivity reaction to KMT-17 tumor was assayed by the radioisotopic footpad method. The specific delayed hypersensitivity reaction was strengthened by an injection of FT (300 mg/kg).

Nonspecific inhibition of tumor growth in vivo by admixed allogeneic tumor-sensitized lymphoid cells and identical inactivated allogeneic tumor cells.

With the in vivo tumor neutralization test (Winn test), growth of a transplanted (KMT-17) from Wistar-King-Aptekman rats was inhibited by allogeneic tumor (AH-66 from Donryu rats)-sensitized syngeneic lymphoid cells admixed with mitomycin C (MMC)-treated AH-66 cells. The observed tumor inhibition may be immunologically nonspecific, since no cross-antigens were detected by membrane immunofluorescence on the surfaces of KMT-17 and AH-66 cells. Close contact among KMT-17, AH-66-sensitized lymphoid cells and MMC-treated AH-66 cells was required for the inhibition of KMT-17 growth. AH-66 cells pretreated with formalin or ultrasonication lost tumor inhibitory activity when they were admixed with AH-66-sensitized lymphoid cells, and only MMC-treatment effectively preserved the tumor inhibitory activity of AH-66 cells. The sensitized spleen cells, draining lymph node, or peripheral blood cells inhibited tumor growth when they were admixed with MMC-treated AH-66 cells, whereas nucleated cells from bone marrow, thymus, or distal lymph node did not. Growths of KMT-17 were inhibited by admixed sensitized spleen cells and MMC-treated AH-66 even when pre-irradiated rats were used as recipients.

Prevention of tissue factor generation in mononuclear cells by agents known to increase intracellular cyclic AMP.

Agents known to increase intracellular levels of cyclic 3', 5'-adenosine monophosphate (cyclic AMP) were examined for their effect on tissue factor generation by mononuclear cells cultured with E. coli endotoxin. Aminophylline, an inhibitor of phosphodiesterase, and epinephrine, a beta-adrenergic agent, showed an inhibitory effect, and these effects were reversible. Moreover, dibutyryl cyclic AMP also exhibited the effect. Dibutyryl cyclic GMP, however, did not enhance the tissue factor generation by mononuclear cells. On the basis of these observations, it was concluded that the phenomenon of tissue factor generation by mononuclear cells is a biological event, and that intracellular cyclic AMP has a possible role in modulating this phenomenon.

Effect of x-irradiation of recipients on in vivo neutralization of syngeneic tumors by spleen cells nonspecificially immunized with allogeneic tumors.

Spleen cells from WKA rats immunized with allogeneic Donryu tumor (AH66), when examined by the Winn test, inhibited lethal growth of admixed antigenically unrelated syngeneic tumor (KMT-17) only when a mixture of spleen cells and tumor cells was injected into pre-irradiated recipients. On the other hand, spleen cells specificially sensitized to KMT-17 inhibited growth of admixed KMT-17 cells either in irradiated or non-irradiated rats.

Immunological characteristics of leukemia and lymphoma in allogeneic cell immunity: growth of syngeneic tumors in rats immunized with allogeneic cells.

The growth of transplanted tumors was strongly inhibited in syngeneic Wistar King Aptekman (WKA) rats immunized with allogeneic tumor cells from Donryu rats. This phenomenon of non-specific immunity against tumors is referred to as "allogeneic cell immunity". However, an exception to the "allogeneic cell immunity" was observed in leukemias and lymphomas. Four transplanted leukemia or lymphoma lines were not inhibited in syngeneic rats immunized with allogenic tumor cells. Furthermore, immunization with allogeneic leukemic cells had only a relatively weak inhibitory effect upon a syngeneic fibrosarcoma and no inhibitory effect upon leukemias. In WKA rats immunized with allogeneic lymphoid cells from Donryu rats, the growth of fibrosarcoma, but not of lymphoma, was inhibited. Using transplantation experiments, both fibrosarcoma and lymphoma were defined as antigenic tumors in WKA rats. Transplantation of mixtures of syngeneic tumor cells and allogeneic tumor cells in WKA rats confirmed the above findings. These results revealed that leukemia and lymphoma differ from non-leukemic tumors in regard to "allogeneic cell immunity".

Inhibition of syngeneic tumor growth in rats immunized with allogeneic skin grafts.

The growth of a transplantable tumor (KMT-17) in syngeneic Wistar King Aptekman/Mk (WKA) rats was inhibited by preimmunization with allogeneic normal cells from Donryu strain rats. The phenomenon is referred to as allogeneic cell immunity. A slight inhibition was observed in rats immunized with normal liver, spleen, kidney, embryonal cells, whole blood and white blood cells from allogeneic Donryu rats. A strong inhibition was observed in animals which had rejected allogeneic skin grafts, particularly from the Donryu and Kyoto rats. The inhibition was comparatively weak after immunization with skin grafts from the Long Evans, ACI, Buffalo, Ficsher, Sprague Dawley or Tokyo allogeneic rat strains. This immunizing effect was dependent on the viability of the grafts and was easily abrogated by low-dose irradiation. The mechanism of allogeneic cell immunity is considered to be a non-specific stimulation of the immunity against antigenic tumors in the syngeneic host