In vitro expression of suppressogenic and enhancing activities in human colon cancer cells.

The presence of immunoregulatory factors in a human colon cancer cell line, LS174T, was exhibited in mitogenic and immunogenic assays. The simultaneous presence of stimulatory and suppressive factors in the spent media of LS174T colon cancer cells was shown. Opposing suppressive and stimulatory activities were extractable from viable tumor cells and found in 3 M KCl extracts fractionated by isoelectric focusing. Suppressogenic activity was demonstrated in concanavalin A and phytohemagglutinin mitogenesis in both human and murine lymphocytes; the blastogenesis of mixed lymphocyte culture reactions was suppressed, while responses of immune cells were not. Naive lymphocytes were stimulated or not affected by the same spent media. The factors appeared to require cell viability for optimal expression and were not species specific. These results further illustrate the complexity of tumor cell-host cell interactions and emphasize the need for in vitro assays to measure, characterize, and exploit the factors.

Liposome uptake into human colon adenocarcinoma cells in monolayer, spinner, and trypsinized cultures.

The purpose of this study was to begin investigating the nature of liposome interactions with colon tumor cells. Thus, experiments were performed to study the uptake and incorporation of multilamellar and of reverse-phase evaporation liposomes of neutral charge into monolayers, suspended spinner cultures, and trypsinized cells of a human colon adenocarcinoma cell line, LS174T. The results showed that the same tumor cells cultured under each condition exhibited a distinct pattern of vesicle uptake as determined at 0, 15, 30, 60, and 120 min. In monolayer cultures of LS174T cells, the uptake of liposomes bearing [3H]actinomycin D in the lipid bilayers was linear throughout the incubation period. In contrast, in trypsinized and spinner suspension cultures, uptake of liposomes was biphasic. There was a proportional uptake of both liposome (labeled with [3H]phosphatidylcholine or [14C]cholesterol) and of actinomycin D (trace labeled with 3H) into the cells under all culture conditions, indicating quantitative delivery of the drug with the intact lipid vesicle. Although the amount of actinomycin D presented to tumor cells by the two liposomes was equivalent, reverse-phase evaporation liposomes were more effective than multilamellar vesicles in inhibiting uridine uptake. In the presence of excess liposomes (10 times the uptake studies), saturation of the tumor cell surface occurred by 120 min. However, the liposomes remained accessible to enzymatic removal for 60 min. Liposome-saturated tumor cells remained refractory to further binding of liposomes for at least 2 hr. The results thus revealed that differences in cell uptake were due to the state of the target cells and not the liposome types, or their differential leakage of labels.

Noncytolytic extraction of murine tumor-specific transplantation antigens with the nonionic detergent octyl-beta-D-glucopyranoside.

The nonionic detergent octyl-beta-D-glucopyranoside (C8Glu) was used to extract immunogenic tumor-specific transplantation antigen (TSTA) from intact cells and purified plasma membranes of the 3-methylcholanthrene-induced fibrosarcoma MCA-F. Pretreatment of syngeneic C3H/HeJ mice with 100 micrograms of C8Glu extracts induced specific immunoprotection such that mice resisted the outgrowth of MCA-F but not the antigenically distinct tumors MCA-D or MCA-2A. Incubation of intact cells with 7 mM (0.2%) C8Glu for 30 minutes at 23 degrees C was judged to be noncytolytic because extracted cells excluded trypan blue. Preparative isoelectric focusing partially purified the MCA-F-specific antigen from crude C8Glu extracts into the pH 6.5-6.8 region of the gradient. Electrofocusing yielded 60% of the applied antigen activity and a fourfold to fivefold increase in specific activity. In addition, immunoprotective activity was obtained in 2% C8Glu extracts of MCA-F plasma membranes, confirming the membrane localization of the MCA-FTSTA. Three properties of C8Glu rendered it an attractive agent for the preparation of cell surface proteins: a nonionic character, large critical micellar concentration, and its capacity to extract antigens without complete membrane solubilization or cell disruption.

Effect of murine tumors upon delayed hypersensitivity to dinitrochlorobenzene. III. In vivo activity of the nonspecific suppressor cell.

Tumor-induced immunosuppression was investigated in an in vivo model of delayed hypersensitivity (DH) to the chemical sensitizer, dinitrochlorobenzene (DNCB). DH to DNCB as measured in a footpad assay was decreased in C3H/HeJ mice bearing MCA-F, a 3-methylcholanthrene-induced syngeneic fibrosarcoma. Suppressor cells from the spleens of tumor-bearing mice inhibited the induction of DH to DNCB in otherwise normal syngeneic C3H/HeJ recipients. Ten million spleen cells (SpC) harvested from mice bearing MCA-F for 10 days and adoptively transferred to tumor-free mice at the time of sensitization with DNCB suppressed the response to the sensitizer. The suppressor cells were macrophages, since they were adherent to plastic, removed by treatment with a magnet after phagocytosis of carbonyl iron, resistant to exposure to gamma radiation and to treatment with anti-Thy 1.2 serum and complement. Further, the nonspecific suppressor cells were activated by progressive tumor growth rather than by induction of tumor-specific immunity using irradiated tumor cells. Titration studies revealed that suppression of DH occurred with the transfer of as few as 10(6) SpC. Thus, nonspecific suppressor cells are effective at inhibiting in vivo DH to DNCB and suggest that nonspecific suppression in the intact host occurs through mechanisms different from those involved in suppression in vitro.

Immunobiological properties of 1-butanol-extracted cell surface antigens.

Extracts of viable 3-methylcholanthrene-induced murine sarcoma cells (MCA-F and MCA-2A) prepared using single-phase (2.5%) 1-butanol significantly retarded the outgrowth of the homotypic, but not the heterotypic, tumor of syngeneic C3H/HeJ mice. Butanol extracts specifically evoked a delayed hypersensitivity response in tumor-immune syngeneic mice, but not in alloimmune DBA/2J mice. Crude butanol extracts of MCA-F cells did not contain alloantigenic activity, as shown by their inability to block H-2 or Ia-specific antibodies in a complement-dependent cytotoxicity assay. Absorption of these same allospecific reagents with untreated or with butanol-extracted cells indicated that H-2 antigens remain associated with the cell surface during extraction. Thus, butanol appears to release tumor antigens, but not alloantigens, from the cell surface.

Delayed hypersensitivity to crude and partially purified murine tumor-specific transplantation antigens and alloantigens utilizing the footpad swelling assay.

Antigen preparations extracted from C3H/HeJ methylcholanthrene-induced fibrosarcomas by the 3M KCl extraction procedure were assessed for tumor-specific and allospecific antigenicity. Specificity of crude tumor antigen preparations and of fractions from preparative isoelectric focusing was investigated by evocation of footpad swelling (FPS) in syngeneic mice immunized with irradiated fibrosarcoma cells. Tumor immune mice displayed delayed hypersensitivity as positive FPS responses to challenge with 3M KCl extract and with fraction (Fr) 15 (pH 5.7 to 6.0) from preparative isoelectrically focused 3M KCl extract. Crude extracts and Fr 15 exhibited immunoprotective activity in vivo. Immune mice demonstrated a specific FPS response only to crude antigen preparations of Fr 15 from immunizing tumors, not to materials from a noncross-reactive neoplasm. DBA/2J mice immunized with C3H/HeJ spleen cells displayed FPS to challenge with crude antigen preparations, but not with the tumor-specific Fr 15. Alloantigen activity, however, was detected by a positive FPS response in C3H-immune DBA mice in fractions from the pH range 5.1 to 5.5. These experiments demonstrated that the FPS assay provides the setting for detection of specific delayed hypersensitivity responses to crude and fractionated tumor antigen preparations and for delineation of tumor-specific and histocompatibility antigen activities in fractions from crude extracts.