Allogenic bone-marrow transfusion suppresses development of lung metastases in osteogenic sarcoma patients after radical surgery.

On the basis of experimental data obtained in Syrian hamsters, demonstrating the highly efficient suppression of experimental and spontaneous metastases of highly-metastatic sarcoma cells by the use of allogeneic normal bone-marrow cells (BMC), a clinical protocol for the suppression of lung metastases of osteogenic sarcoma was started in 1984 in the Cancer Research Center, Moscow. From this time onwards, 24 osteogenic sarcoma patients, at stages 2A and 2B were treated with a combination of radical surgery and a single transfusion of normal (non-activated) allogeneic BMC (blood-group and Rhesus compatible). The first results of this ongoing study are now presented. Metastases appeared in 11 out of the 24 patients, generally very early during the first 3-9 months after treatment and in no case after 2 years. More than 50% of the BMC-treated patients were free of lung metastases after 2 or more years of observation; 8 out of 15 are still metastasis-free after 3-4 or more years of observation following treatment. The differences in the frequency of metastasis and duration of survival without metastasis of treated patients compared with a group of 41 osteogenic sarcoma patients at stages 2A and B, treated with radical surgery only (controls) reached significant levels 12 months after treatment and thereafter. Rapid recovery of NK cytotoxic activity has been observed in nearly all successfully BMC-treated patients.

In vitro bioassay for type E prostaglandins based on their NK immunodepressing activity.

A biological assay for PGE in commercial preparations, or secreted by tumor cells in culture fluid was developed on the basis of the immunosuppressive effect of PGE on the cytotoxic activity of NK cells. The assay is simple, rapid and convenient for detecting PGE cell secretion, either spontaneous or induced by various signals. The sensitivity of the bioassay is limited by the sensitivity of NK cells to the immunosuppressive activity of PGE (i.e., about 10(-8) M).

Clustering of discrete cell properties essential for tumorigenicity and metastasis. III. Dissociation of the properties in N-ras-transfected RSV-SR-transformed cells.

We have previously shown that RSV-SR-transformed hamster cells acquire high resistance to H2O2, i.e. the cytotoxic product of activated macrophages (H2O2R) and that they begin to secrete PGE (PGES), thus inactivating the CTA of NK cells. Among normal cells, the same phenotype is expressed in activated macrophages. In all our RSV-transformed cells these 2 properties were jointly expressed and correlated with high tumorigenicity and experimental metastasizing of these cells. We now show that transfection of 3 RSV-SR-transformed cell strains with activated N-ras leads either to complete inhibition of the H2O2R + PGES phenotype in all clones of one strain, or to inhibition of PGES only in the majority of clones of 2 other strains. Unexpectedly, the complete or partial inhibition of this phenotype did not alter the high tumorigenicity of 2 strains of these cells, but lower tumorigenicity was evident in almost all clones of the third strain (as well as in some gene-neo-transfected clones of these strains). The loss of PGES made these cells susceptible to the CTA of NK cells, while the loss of H2O2R did not alter their resistance to the CTA of macrophages. Expression of the H2O2R + PGES phenotype was retained in all cloned variants of control, gene-neo-transfected cells. The possible relation of the N-ras gene to regulation of src gene activities in RSV-SR-transformed cells is discussed.

Clustering of discrete cell properties essential for tumorigenicity and metastasis. II. Studies of Syrian hamster embryo fibroblasts transformed by Rous sarcoma virus.

Tumorigenic and metastasizing activities (TGA; MA) and susceptibility, or resistance to H2O2 and PGE-releasing activity (H2O2R + PGEs+ phenotype) have been examined in 6 Syrian hamster embryo cell strains transformed in vitro with Rous sarcoma viruses (Schmidt-Ruppin and Prague strains). Early observations of extremely high level of TGA and even MA of RSV-SR-transformants never selected in vivo have been confirmed. The correspondence of these properties with a high level of expression of H2O2R + PGEs+ phenotype and its clustering character were demonstrated in 4 RSV-SR transformants, while significantly lower expression of all these characteristics, including TGA, was observed in 2 RSV-Prague transformants. High level of spontaneous MA was noticed in some RSV-SR transformants. A tumor cell line induced in vivo by RSV-SR did not differ from the cell strain transformed in vitro by RSV-SR. Inhibition of H2O2R + PGEs+ phenotype in one of RSV-SR transformants was obtained with non-toxic doses of BCNU and indomethacin, leading to a marked decrease of TGA.

Clustering of discrete cell properties essential for tumorigenicity and metastasis. I. Studies of Syrian hamster embryo fibroblasts spontaneously transformed in vitro.

The expression of two discrete cell properties related to the host natural effector mechanisms, i.e., resistance to damage by H2O2, a cytotoxic product of activated macrophages, and the ability to secrete PGE, which inhibits NK-cell cytotoxicity, has been examined in parental Syrian hamster embryo cells spontaneously transformed in vitro (STHE strain) and in 18 in vivo selected sublines. In all cell variants, resistance to H2O2 and PGE-releasing activity were either both expressed, or not expressed at all. Parental STHE cells and 5 variants selected in vivo, which were equally highly susceptible to H2O2-induced damage, did not release any detectable amount of PGE upon contact with NK cells. In contrast, 13 other STHE variants selected in vivo and characterized by their resistance to H2O2, all released PGE upon contact with NK cells. Thus, these two biochemically unrelated cell phenotypic characteristics are likely to be either simultaneously selected in vivo, or united in cluster which pre-exist or appear in rare cell variants of the parental cell population in the conditions of in vivo natural selection pressure.

Inhibition of experimental and spontaneous lung metastases of highly metastatic Syrian hamster sarcoma cells by non-activated bone marrow and peritoneal exudate cells.

The possibility of inhibiting local tumor growth (experimental and spontaneous lung metastases) of the selected highly-metastatic Syrian hamster sarcoma of STHE-LM8 subline by means of non-activated syngeneic and allogeneic spleen, bone marrow (BMC) and peritoneal exudate (PEC) cells was studied. Retroorbital inoculation of Syrian hamsters with native, or lethally irradiated allogeneic BMC and PEC, but not spleen cells, or hamster embryo cells effectively inhibited the development of experimental and spontaneous lung metastases induced by STHE-LM8 cells in the animals. Spontaneous lung metastases were effectively inhibited in about 50% of STHE-LM8 tumor-bearing animals (with or without tumor excision) inoculated with BMC five times at 5- to 7-day intervals beginning from 1-10 days after subcutaneous palpable tumor appearance. Experimental lung metastases were inhibited in 20-80% of animals inoculated with BMC or PEC once 5-7 days before the tumor cells, simultaneously with them, or during the 5-7 days following the inoculation of tumor cells, thus demonstrating that BMC metastasis-inhibiting activity was expressed during 10-14 days against single tumor cells, or small clusters of tumor cells, and was not effective at the stage of micro-, or macrometastasis formation. BMC and PEC of normal allogeneic donors were significantly more active in metastasis inhibition than the same cells of tumor-bearing animals. BMC of inbred normal Syrian hamsters of the ICV line were significantly less active, or did not inhibit experimental lung metastases either in syngeneic or in random-bred allogeneic hamsters, thus apparently demonstrating an unknown genetic defect of their NR system.

Correlation between the metastasizing activity of in vitro spontaneously transformed hamster cells and their natural resistance-depressing activity.

In our previous experiments in Syrian hamsters the in vivo semiquantitative method of determination of the natural resistance-depressing (NRD) activity of tumor cells was described. It was demonstrated that inactivated cells of different tissue culture cell lines derived from in vivo tumors in contrast to normal, or in vitro spontaneously transformed hamster embryo (STHE) cells, bear the NRD factor. It was suggested that NRD activity of tumor cells might be essential for the development of primary tumors and metastases (Deichman et al., 1979). Therefore, in this study we compared the NRD activity and metastatic activity (MA) of the parental STHE cells never passaged in vivo and STHE in vivo sublines, obtained from subcutaneous tumor nodules and from individual spontaneous lung metastases (LM) after s.c. transplantation of STHE cells. Five LM sublines of STHE strain (out of eight investigated) were NRD active as well as the cells of one of two s.c. tumor nodules. Study of MA, i.e. the relation between the number of cells inoculated retroorbitally in the bloodstream of hamsters and the number of experimental metastases developed in the lungs of inoculated animals, demonstrated that the highest MA was observed with the cells of LM sublines possessing the highest NRD activity. Parental STHE cells did not depress natural anti-tumor resistance and their MA was the lowest. The data presented demonstrate the heterogeneity of the population of constantly cultured in vitro STHE cells in relation to their NRD activity and the selection of NRD active cells during the development of lung metastases.

Reproducibility and relation to specific and non-specific anti-tumor resistance of the tumor "sneaking through" phenomenon.

The reproducibility and immunological specificity of the tumor "sneaking through" phenomenon and enhancement of tumor growth were studied in syngeneic and random-bred Syrian hamsters by means of a quantitative modification of the transplantation test. After primary challenge the phenomenon was neither observed in normal animals nor in animals effectively immunized against tumor. However, it was regularly observed in some "immune" animals after secondary challenge. In primary challenge of animals "sneaking through" phenomenon was most often observed in animals pretreated with large doses of heat-inactivated tumor cells. This characteristic could not be transferred with serum of pretreated animals. In contrast to specific tumor immunity, the "sneaking through" pbenomenon appeared to be immunologically non-specific. This was observed in cross-transplantation tests with tumor cells bearing different TSTAs. Thus, TSTA is not an inducer and apparently not a target for a response leading to enhancement of tumor growth in pretreated hamsters. Experiments demonstrating enhanced tumor growth in pretreated animals at the same time demonstrate two other possibly more essential findings: (1) normal animals are naturally resistant to transplantation of 1 to about 1 x 10(3) (or more) tumor cells; and (2) this resistance can be totally abrogated by the pretreatment of normal animals with tumor cell preparations. The preliminary data demonstrate that abrogation of natural anti-tumor resistance in adult hamsters subsequently inoculated with SV40 leads to rapid development of primary tumors in such animals. The development of specific anti-tumor immune response in animals treated with inactivated tumor cell preparations was also studied. Significant non-specific inhibition of sponse in Syrian hamsters treated with inactivated syngeneic tumor cells was observed. The data obtained are considered to demonstrate two anti-tumor defense systems in the animal, i.e., non-specific natural resistance and specific anti-tumor immunity. The first seems to be responsible for elimination of low numbers of tumor cells in the normal organism and also to be esseitial for effective induction and functioning of the specific anti-tumor immunity.

Sensitivity of TSTA and species-specific cell membrane antigens of tumor cells to glutaraldehyde treatment.

In vivo immunogenicity and in vitro species-specific membrane antigens in tumor cells treated or untreated with glutaraldehyde (GA) were studied. Two different syngeneic Syrian hamster transplantable tumor cell lines (spontaneous liver cancer and SV40-induced sarcoma) not only lost immunogenicity after GA treatment but were responsible for enhancement of test-tumor growth in immunized animals. In vitro mixed hemadsorption test used for determination of species-specific membrane antigens in Syrian hamster, green monkey and interspecies hybrid cells revealed drastic alteration of antigens on the membrane of cells treated with GA.

In vivo and in vitro TSTA-inducing activity of temperature-sensitive (ts) mutants of SV40.

In vivo TSTA induction in Syrian hamsters was studied with the use of SV40 ts mutants (A, B, C, BC and D). The ts A30, TS A239 and possibly also the ts BC210 mutants were defective in resistance-inducing activity in hamsters in contrast to wild type SV40 and other ts mutnats. At the permissive temperature ts A30 and ts A239 did not induce TSTA in hamster cells during abortive infection in vitro, while they did so in green monkey cells at both permissive and non-permissive temperatures. In hamster cells transformed by ts A30, ts A239 and ts A209 mutants none at all or very little TSTA was detected by in vivo transplantation immunological tests. Thus, expression of TSTA induced by these three SV40 ts A mutants was found to be dependent from the species of infected cells and was being a temperature independent viral function.

Use of the blocking procedure in mixed hemadsorption tests for the analysis of surface antigens in liver tissue culture cells.

For detection of surface species-specific and specific tumor antigens in the various tissue culture cell lines, including interspecies hybrid cell line, the blocking procedure was included in a microhemadsorption test (MHT). Use of the appropriate high-titered blocking antisera unadequate to the indicatory system of MHT permits the detection of different species-specific and tumor antigens on the surface of the liver tissue culture cells with the use of heterologous unabsorbed immune sera.