Liposome-facilitated enhancement of in vitro immunity to human colon cancer.

Liposome-antigens, in the presence of dialyzable leukocyte extracts (DLE)(1), were tested for their ability to enhance in vitro blastogenic responses to colon tumor antigens. Liposomes made with 6:4:1 molar ratios of phosphatidylcholine, cholesterol, and phosphatidic acid and bearing human colon cancer cell antigens were shown to be immunogenic in vitro, producing specific blastogenic responses. Addition of nonimmune DLE (500 micrograms/5 X 10(5) lymphocytes) in the in vitro culture system enhanced the blastogenic response 3x over liposome-antigen alone (p less than 0.001). Immune DLE (immune to keyhole limpet hemocyanin (KLH) and tuberculin (PPD) antigens) were suppressive (p less than 0.05) in the same study. Blastogenic reactivity to KLH was also generated in nonimmune lymphocytes and enhanced by liposomes with DLE specific to KLH (p less than 0.01). Nonspecific DLE (e.g., to PPD) caused suppression of KLH-induced blastogenesis (p less than 0.05). These findings confirm and extend prior reports noting that DLEs contain both specific and nonspecific lymphocyte blastogenic factors and suggest the use of this liposome-adjuvant system for boosting tumor immune responses.

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 facilitated xenogeneic approach for studying human colon cancer immunity: carrier and adjuvant effect of liposomes.

Liposomes prepared with human LS174T colon tumour cell membranes induce specific primary xenogeneic immune responses in BALB/c splenocytes in vitro. Characterization of the adjuvant role of these liposomes was accomplished by determining the effect on immune induction of several modifications on the liposomal carrier. The results showed that the carrier effect of liposomes was mediated primarily by tumour antigens exposed on the outer surface. Trypsin treatment of the liposomes eliminated 95% of the surface protein and significantly (P less than 0.05) reduced the ability of liposomes to induce cytotoxic splenocytes. The generation of cytolytic activity with liposomes was dose-dependent, with a 10 micrograms protein threshold and a maximal response at 100 micrograms. 'Rigid' liposomes were shown to be significantly (P less than 0.05) more efficacious than fluid liposomes in inducing cytotoxicity. In addition, the data indicate that the xenogeneic cell-mediated immunity exhibits identical classes of effector cells as found in murine-murine reactions. Lymphocytes bearing the THY-1, Lyt-1 and Lyt-2 surface markers were necessary for immune induction. The role of Lyt-123 subpopulation was suggested by the inability to achieve normal cytolytic levels by reconstitution with Lyt-1 plus Lyt-2 cells. Adherent cells were, as expected, necessary for the generation of primary immunity. Indeed, the interaction of I-A+ adherent cells with liposomes for at least 8 h was required to generate subsequent maximal T cell cytotoxic activity. The phenotype of the cytotoxic effector cell was Thy-1+, Lyt-2+, and I-Ad-. If this were an allo-or syngeneic, and not a xenogeneic system, this study would be of less interest. However, when coupled with the known molecular homologies between murine and human lymphocyte antigens, these results suggest that the concept of cross species major histocompatibility complex (MHC) restriction is tenable. Thus the liposome is not only an effective antigen carrier, but also a functional adjuvant for in vitro induced cell-mediated immunity.

Characterization of a human colonic adenocarcinoma cell line, LS123.

An epithelial cell line, LS123, was established in 1974 from the second in a series of three primary colonic tumors resected from a Caucasian female. The cell line is aneuploid, releases low concentrations of carcinoembryonic antigen (CEA), fails to grow progressively in nude mice, and forms colonies only in enriched semisolid medium developed for tumor stem cells. LS123 cells grow on confluent cell monolayers and in either low serum or serum-free medium. In the chick embryonic skin assay, LS123 cells grew as a well-differentiated abnormal colonic epithelium with little mitotic activity but with some indication of invasion. On floating collagen gels LS123 cells formed a one to three-cell-layer-thick undifferentiated epithelial sheet. The apparent low invasiveness of the cells of this line is supported by the patient's history of three primary colon tumors without systemic metastases during the past 30 yr. Therefore, although LS123 cells possess several properties associated with neoplasia, they have little invasive potential. Thus, LS123 cells may represent an important model for the study of human colon cancer.

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.

A new approach for the immunogenic presentation of membrane-bound human colon tumor antigens.

Liposomes bearing human tumor membrane vesicles were effective immunogenic complexes for inducing antibodies in rabbits. Multilamellar liposomes (MLV) at 7:4:1 molar ratios of phosphatidylcholine, cholesterol and phosphatidic acid were prepared with sonicated membrane (MN) isolated from LS174T colon tumor cells. MLV liposomes prepared together with MN (i.e., MN-MLV antigens) or MN added to preformed MLV (i.e., MN+MLV antigens), and MN antigens alone were used as immunogens. Rabbits were immunized i.v. with 100 g protein of each antigen group. Boosters (i.v.) were at days 13 and 29. Binding assays were performed by indirect radioimmunoassay on viable tumor cell targets. The MN+MLV groups were distinguished by earlier reactivity and greater specificity to the colon tumor antigens.

Immunosuppression of experimental allergic encephalomyelitis in guinea pigs by antibrain and antithymocyte heteroantisera.

Experimental allergic encephalomyelitis (EAE), induced by central nervous system (CNS) myelin basic protein (MBP) in adjuvant, is considered a thymus dependent autoimmune disease. Brain contains the thymic antigen, thy 1. The possibility that brain associated anti thy 1 immunoglobulin may be provoked in certain pathologic conditions of the CNS suggested a comparative evaluation of brain and thymocyte antisera on the development of EAE. Antisera produced in rabbits against brain from guinea pigs, rats and mice or fetal guinea pig thymus were highly reactive against thy 1 containing cells when assessed by indirect immunofluorescent staining or complement-mediated cell lysis. Treatment of guinea pigs with heteroantisera to guinea pig and mouse, but not to rat brain, for 3 days around the time of MBP sensitization markedly reduced physical signs of disease, particularly paralysis, but had little effect on the development of inflammatory lesions in the CNS. Anti-guinea pig thymocyte sera eliminated all physical signs of EAE with only residual pathology. These results establish the relative immunosuppressive effect of brain and thymocyte antisera in EAE and corroborate the thymus-dependent nature of EAE in guinea pigs.

Active specific immunotherapy potential for the treatment of large bowel cancer.

Active specific immunotherapy, harnessing the strength and specificity of the host immune response to destroy neoplastic cells, may offer an ideal surgical adjuvant treatment modality for human colon cancer. Unfortunately, achievement of this goal has been obscured by 1) the effect of excess residual disease to interfere with the host's destructive response, 2) the weak nature of tumor resistance, 3) the potential adverse effect of concomitant treatments such as chemotherapy, and 4) the present limitation of poorly defined immunogens to induce, as well as insensitive assay systems to detect, host sensitizaion. Recent immunologic and chemical research revealing distinctive surface membrane structures on colon cancer cells suggests that a controlled trial of irradiated, autochtonous cell vaccines (without mycobacterial adjuvants) may provide a new therapeutic tool for Dukes B2 and C stages of human colon cancer.

Human colon adenocarcinoma cells. II. Tumorigenic and organoid expression in vivo and in vitro.

A human colon epithelial tumor cell line (LS174T) recultured in vitro following passage through hamsters displayed differences in its cell doubling time and synthesis of carcinoembryonic antigen when compared with the cells grown solely in vitro. These animal-passaged cells more closely resembled the parent tumor cell line (LS180) derived from the primary tumor than LS174T, the trypsinized variant of LS180. Analysis of lactate dehydrogenase isoenzymes indicated that the tumor cells recovered from the hamsters were free of xenogeneic host tissue. Furthermore, LS174T grafted to athymic (nude) mice grew as a mucinous adenocarcinoma microscopically resembling the original tumor. The altered growth potential of LS174T was also demonstrated on confluent feeder monolayers of normal cells and by uninhibited multiplication in vitro. These results suggest that, at least in this one case, short-term passage of long-term cultured cells into xenogeneic hosts may effect a phenotypic reversion such that the cells regain properties observed in the primary tumor and the initial in vitro explant.

Cell surface alterations on colon adenocarcinoma cells.

By the use of five independent techniques, cell surface alterations distinctive of malignant as compared to normal colon cells were detected on in vivo surgical specimens and on cultured cell lines established in our laboratory. The findings, which were distinctive of the malignant as compared to the normal cell included: (a) polymorphism of surface microvilli on scan electron microscopy; (b) decreased susceptibility to infection with vaccinia and reovirus, but not to herpes, adeno- or echovirus: (c) production of large quantities of carcinoembryonic antigen; (d) presence of specific membrane proteins on sodium dodecyl sulfate-polyacrylamide gel analysis of plasma membranes purified from cell homogenates by ultracentrifugation in polyethylene glycol-dextran partitions; and (e) reaction with specific, cytotoxic, rabbit heteroantisera. Solubilized extracts of the malignant cells formed precipitin lines with the heteroantisera, suggesting that the distinctive antigens could be released from the cell surface. These results suggest that human colon carcinomas bear tumor-distinctive proteins and offer the prospect of specific immunodiagnostic reagents and immunotherapeutic tools.

Human colonic adenocarcinoma cells. I. Establishment and description of a new line.

A series of human colonic epithelial cell lines have been cultured from a single patient: LS-180 the original adenocarcinoma, LS-174T a trypsinized variant, and normal colonic tissue. The malignant cells, 20 to 40, mum in diameter and oval to polygonal, exhibited characteristics of normal colonic mucosal cells, namely, abundant microvilli prominent in secretory cells, and the presence of intracytoplasmic mucin vacuoles. The cultured adenocarcinoma cells, but not normal, demonstrated neoplastic properties by producing high levels of carcinoembryonic antigen (CEA) and by the ability to be propagated in hamster cheek pouches and in immunodeprived mice. The CEA production by the newly established line LS-180 released 900 times more CEA per cell into the culture medium and bore 30 times more cell-associated material than the established line, HT-29. These cell lines may permit detection of distinctive chemical, physiological, pharmacologic, and immunologic characteristics of neoplastic colonic cells.

Purification of tumor-specific antigens. An overview of the relevance to human colon carcinoma.

Methods which dissociate intramolecular noncovalent bonds have been used to prepare soluble derivatives of cell-surface antigens. Applications of these techniques to human colon carcinoma are underway. Continuous tissue-culture strains derived from primary lesions were developed and shown to be composed of malignant epithelial elements. Parallel data on the preparation and activity of soluble materials in a murine model methylcholanthrene system reveal that although cultured cells are a satisfactory source for antigen extraction, they are poor targets of the immune response. The development of methods to quantitate the biologic activity of colon-specific, soluble materials may provide indicator systems to define the antigenic determinants, to permit purification, and to serve as assays of the efficacy of immunotherapy.

The leukocyte aggregation test: immunodiagnostic applications and immunotherapeutic implications for clinical renal transplantation.

The leukocyte aggregation test (LAT) detects the in vitro adhesion of sensitized, but not nonimmune, recipient leukocytes onto donor kidney cell monolayers. The test specifically detects cell-mediated homograft immunity up to 15 days prior to the appearance of clinical signs or alteration of chemical indexes. The presence of a positive reaction always signified incipient homograft rejection, which was usually controlled by intravenously administered, high-dose methylprednisolone sodium succinate (Solu-Medrol) therapy. There was no instance in which methyl-prednisolone treatment effectively reversed rejection in the presence of a negative leukocyte aggregation test. One common form of homograft rejection may be characterized by positive LAT results, a cellular infiltrate on the renal biopsy specimen, and sensitivity to methylprednisolone therapy.

Complement-dependent anaphylactic reactions.

The concept of elicitation of reactions of anaphylactic type by non-tissue-fixing antibody, through activation of complement and release of anaphylatoxins by antigen-antibody complexes in vivo, is not clearly defined by published evidence. Experimental data are presented to demonstrate that guinea pig immunoglobulin G2 (noncytotropic) complexed with antigen in vitro elicits dermal reactions in guinea pigs, and that pretreatment of animals with complement-inactivating cobra venom factor diminishes such reactions. The various pathways through which immediate hypersensitive reactions may occur are discussed.

Characteristics of the leucocyte-aggregation assay for cell-mediated immunity.

The leucocyte aggregation assay detects cell-mediated immunity by the specific adherence of sensitized lymphocytes onto target cell monolayers. Leucocyte aggregates appear to develop by instruction of non-immune cells by sensitized T lymphocytes. B cells may function in a secondary capacity to amplify aggregate formation. The reactions are sensitive to low temperatures and to metabolic inhibitors of protein and RNA synthesis and function. Serum factors alternatively enhanced or blocked the phenomenon, depending upon the immune status of the patient. This assay may prove useful for the dissection of allograft rejection and tumor resistance due to its brevity, reflection of T-cell immunity, and sensitivity to host humoral factors.