Malignancy can be suppressed when malignant cells are fused with certain non-malignant ones. The hybrid cells derived from such fusions give rise to segregants in which a loss of chromosomes is associated with reversion to malignancy. The expression of histo-compatibility antigens can also be suppressed when cells bearing these antigens are fused with others that express them poorly.

Suppression of Malignancy by Cell Fusion

In the accompanying paper (Luo et al., Cancer Res., 58: 2652-2660, 1998), we demonstrated that vascular endothelial growth factor (VEGF), also designated vascular permeability factor (VPF), significantly accumulated in all mouse malignant ascites tested, suggesting its fundamental role in ascites tumors. Removal of VEGF may inhibit the development of ascites tumors. In this study, using a goat antimouse VEGF-neutralizing antibody, we tested this hypothesis with two well-defined syngeneic mouse ascites tumors: MM2 breast adenocarcinoma and OG/Gardner lymphoma 6C3HED (expressing moderate and low levels of VEGF, respectively). This antibody significantly inhibited MM2 and OG cell-free ascites fluid-induced hyperpermeability of mouse peritoneal microvessels and in vitro endothelial cell growth. Mice bearing tumors were administered i.p. daily with the antibody or normal goat IgG as controls for 8 days, at doses of 20-fold (for MM2-bearing mice) or 40-fold (for OG-bearing mice) the estimated amounts of VEGF that kinetically accumulated in the ascites fluid after the tumor inoculation. The average volume of ascites fluid, number of tumor cells and leaked RBCs, and the peritoneal microvessel permeability in MM2-bearing mice that received the antibody treatment were significantly lower than those in the matched controls (P < 0.01). Unexpectedly, OG-bearing mice did not show satisfactory response to the anti-VEGF treatment. This discrepancy was not likely due to inadequate doses or different host immune responses, but it was quite possibly to the different characteristics of MM2 carcinoma and OG lymphoma tumors, the latter being strongly invasive, and/or the existence of an inflammatory mediator(s), such as bradykinin or cytokine(s) other than VEGF. In summary, our results directly demonstrated, for the first time, differential roles for VEGF in ascites tumors in vivo and suggest the potential of VEGF inhibition as a specific therapy for ascites tumors of carcinoma origin, which are the major cause of the malignant ascites in adult humans.

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Differential inhibition of fluid accumulation and tumor growth in two mouse ascites tumors by an antivascular endothelial growth factor/permeability factor neutralizing antibody.

Studies were made on the effects of inducers on the leukemogenicity of sensitive mouse myeloid leukemia cells (M1) that could be induced to undergo cell differentiation into mature granulocytes and macrophages in vitro by incubation with inducers (certain proteins, bacterial lipopolysaccharides, or glucocorticoids) and of resistant M1 cells that could not be induced to differentiate into mature cells. Inducers of cell differentiation significantly enhanced the survival times of mice inoculated with sensitive cells but scarcely affected the survival times of mice inoculated with resistant cells. Some mice inoculated with the sensitive cells and treated with lipopolysaccharide did not develop leukemia. The sensitive and resistant clone cells contained similar common tumor-related surface antigens. Treatment with lipopolysaccharide was also effective in athymic nude mice inoculated with the sensitive M1 cells. Lipopolysaccharide or glucocorticoid significantly stimulated differentiation of the sensitive cells cultured in a diffusion chamber in vivo but had little effect on differentiation of resistant cells. These results suggest the possibility of treating, with partial success, leukemia in vivo with differentiation inducers.

https://cancerres.aacrjournals.org/content/canres/39/8/3167.full.pdf

Prolongation of Survival Time of Mice Inoculated with Myeloid Leukemia Cells by Inducers of Normal Differentiation

By means of a modified immune adherence (IA) technique, sera from melanoma patients were tested for the presence of antimelanoma antibodies. In total 13/73 sera tested showed a positive IA reaction of which 4/6 sera showed a positive reaction in the autologous situation. Sera from 33 patients with other tumors, 7 patients with non-neoplastic diseases and 50 healthy individuals did not show any IA reactivity towards melanoma cells. The reaction seemed to be selectively directed against tumor-associated antigens (TAA) on melanoma cells. No correlation with the stage of the disease could be found. Longitudinal studies indicated that conversions in antibody activity did not correlate with the clinical state of the patients. There was also no correlation with the corresponding in vitro data obtained in cell-mediated immunity tests. Cell lines and short-term cultures originating from tumors from different melanoma patients shared a common antigenicity. The expression of TAA on cells from a melanoma cell line fluctuated significantly during prolonged culture. The expression of TAA was influenced by the culture conditions and the growth state of the cells. A relation between TAA-expression and cell cycle phase was demonstrated.

Antibodies and antigen expression in human melanoma detected by the immune adherence test.

In studies on antitumor antibody:drug conjugates as potential antitumor agents, methotrexate (MTX) was conjugated with a murine monoclonal antibody (aMM46) to an antigen on ascitic mouse mammary tumor MM46 cells (MM antigen) with human serum albumin (HSA) as an intermediary. MTX was linked to HSA which had been conditioned to have about 1 mol of thiol group per mol of HSA by dithiothreitol treatment followed by oxidation on standing at 4°C. The MTX linking was performed, without protection of the thiol group of HSA, by using MTX N -succinimidyl ester prepared via MTX intramolecular anhydride. The resulting HSA:MTX was reacted with the immunoglobulin with the maleimide group introduced. The aMM46:HSA:MTX obtained retained both antibody binding and drug activities. The cytotoxicity of aMM46:HSA:MTX against MM antigen-positive MM46 cells was greater than that of control 96.5 (anti-human melanoma-associated antigen, p97):HSA:MTX and was inhibited by unconjugated aMM46. No different cytotoxicity of aMM46:HSA:MTX compared with that of 96.5:HSA:MTX was observed against MM antigen-negative mouse mammary tumor MM48 cells. The presence of ammonium chloride or leupeptin abrogated the selective cytotoxicity against MM46 cells of aMM46 conjugate but did not affect the nonspecific cytotoxicity of 96.5:HSA:MTX. These results support the idea that the selective cytotoxicity of aMM46:HSA:MTX is antibody directed and exhibited through lysosomal degradation of the conjugate.

https://cancerres.aacrjournals.org/content/canres/47/4/1076.full.pdf

In vitro cytotoxicity of a human serum albumin-mediated conjugate of methotrexate with anti-MM46 monoclonal antibody.

Tumor-specific antibodies reacted with an isografted ascitic form of mouse mammary tumor, MM2, with a higher sensitivity in an immune adherence test than in an immune cytotoxicity test.



Immune adherence and immune adherence inhibition tests were applied for the detection of surface antigens of MM2 tumor reacting with tumor-specific antibodies. After treatment with ether, ethanol, methanol, ethanol-ether mixture, acetone, chloroform-methanol mixture, deoxycholate, triton × 100, snake venoms, phospholipase A, or heating at 100° C for 15 minutes, the MM2 cells did not react with syngeneic antibodies in immune adherence. The reactivity of the cells was not suppressed by heating at 62° C for 15 minutes after treatment with deoxyribonuclease, ribonuclease, proteinases, neuraminidase, ultraviolet irradiation or freezing and thawing.



In the immune adherence inhibition test, deoxycholate extract or triton × 100 extract of MM2 cells inhibited the immune adherence reactivity of syngeneic antibodies with MM2 cells, while the organic solvent extracts did not. This indicates that antigenic material was extractable in deoxycholate or triton × 100.



After removal of surface antigens with 0.1% deoxycholate, fractions were prepared from treated MM2 cells. Fractions precipitated at 105,000 × g (microsomal fractions) as well as fractions precipitated at 11,000 × g (mitochondrial fractions) inhibited the immune adherence reactivity of tumor-specific antibodies with MM2, indicating that antigens were also present in these fractions.

Immunological studies on mouse mammary tumors. Iii. Surface antigens reacting with tumor-specific antibodies in immune adherence.

Antigens present in mouse mammary tumor MM2 reacting with tumor-specific antibodies were extracted with 0.2% deoxycholate. After the removal of deoxycholate by dialysis, most antigens were precipitated by centrifugation at 105,000 × g for 60 min. Hyperimmunization of C3H/He mice with these antigens induced heightened resistance against MM2. Antibodies appeared in the serum after a booster injection of deoxycholate extract of tumor cells was given. Antigens were solubilized from deoxycholate extract with distilled water. Purified soluble antigen inhibited the neutralization activity of the tumor-specific antibody to MM2 tumor. From these observations, it is concluded that transplantation antigens of isografted mammary tumors in C3H/He mice are present in deoxycholate extracts of MM2 tumor.

Immunological studies on mouse mammary tumors. IV. Extraction and solubilization of transplantation antigen of mouse mammary tumor.

An isografted tumor MM2, originating from a spontaneous mammary tumor in a C3H/He/mouse, killed 4- to 5-week-old mice within 30 days through intraperitoneal injection of 2 × 104 cells per mouse. It was shown that resistance to the isograft was induced by injection of 1.5 × 105 or 2.0 × 105 tumor cells sensitized with serum (5 μg antibody N) of rabbits immunized with the tumor. Four weeks after injection of the sensitized MM2 cells, 173 C3H/He mice were challenged intraperitoneally with fresh unsensitized MM2 in doses of 5 × 105 to 1 × 106 cells per mouse. Of these, 119 mice survived without any sign of tumor growth while all untreated mice died. The mean survival time for untreated mice was 18.1 days (± 1.7) when inoculated with 5 × 105 cells and 16.2 days (± 1.8) when inoculated with 1 × 106 cells. After the second challenge with the same number of fresh unsensitized cells, 117 out of 119 mice survived without any sign of tumor growth. All of 15 mice randomly selected from these resistant mice were tested for acceptance of skin grafts from normal C3H/He mice. The grafts showed no rejection even 150 days after the second graft. After hyperimmunization of these resistant mice, the serum of the spleen extract taken from the mice inhibited growth of tumors when the tumors were premixed in vitro and injected intraperitoneally into C3H/He mice.

Immunological studies on mouse mammary tumors. I. Induction of resistance to tumor isograft in C3H/He mice.

After injection of isografted mammary tumor MM2 sensitized with rabbit antiserum, resistance was induced in C3H/He mice after repeated challenges with MM2. The serum taken from these mice was found to be cytotoxic against MM2 cells. The serum also inhibited the outgrowth of transplant of primary tissue culture cells of spontaneous mammary tumor of C3H/He mice. A series of transplanted mammary tumors recently converted into ascitic form in our laboratory (MM3, MM4-1, MM4-2, MM4-3, MM6, MM8, MM9, MM11, MM12 and MM13), and Ehrlich ascites tumor were found to be susceptible to this serum, as tested by trypan blue uptake in vitro. Outgrowth of these tumors was also inhibited when tumor premixed with this serum was injected.



No cytotoxic effect was observed against normal mouse mammary gland cells of a C3H/He mouse.



Sera obtained from hyperimmunized syngeneic C3H/He mice were able to fix complement with MM2 tumors. They were partially inactivated by heating at 56° C and by treatment with 2-mercaptoethanol. After gel filtration through Sephadex G-200, complement fixing and cytotoxic activity were found in both 19S and 7S fractions. The 7S fractions, after DEAE cellulose column chromatography, gave a precipititin line at the IgG position in immunoelectrophoresis.



From the above evidence, it is concluded that the target cells of the cytotoxic factors of this serum are primary cultured cells or isografted cells of mammary tumors of C3H/He mice. The cytotoxic factors present in the serum are considered to be antibodies against isografts of mammary tumors in C3H/He mice.

Shoshichi Takeuchi

Papers

Immunological studies on mouse mammary tumors. Iii. Surface antigens reacting with tumor-specific antibodies in immune adherence.

Immunological studies on mouse mammary tumors. IV. Extraction and solubilization of transplantation antigen of mouse mammary tumor.

Immunological studies on mouse mammary tumors. I. Induction of resistance to tumor isograft in C3H/He mice.

Immunological studies on mouse mammary tumors. II. Characterization of tumor‐specific antibodies against mouse mammary tumors