Guinea Pig Hepatoma Research Articles

Endobronchial Intratumoral Chemotherapy

Endobronchial Intratumoral Chemotherapy

Tumor cell detection method using complement-mediated cytolytic reaction and imaging sensor system.

A novel tumor-detection system consisting of complement-mediated cytolytic reaction and an image processing system was developed for the simple and rapid determination of tumor cells. The present system consists of a CCD image sensor, image memory board, personal computer, and microscope. When monoclonal antibody 3C4, which is specific to the guinea pig hepatoma L-10, was added to cell suspension, only L-10 cytolysis occurred. Cytolysis caused a decrease in brightness of the cells observed by phase-contrast microscopy. The phase contrast image of the cells before cytolysis was converted to a digitalized signal and stored in computer memory. After cytolysis, a brightness threshold above that of lysed cells was subtracted from the digitalized signal and compared to the signal stored before reaction. L-10 cells in mixed cell suspension were determined specifically by the system. Measurement time was only 2 sec and overall time, including reaction time, was approximately 30 min. Since this method does not require a cell washing process, automation of the whole system is possible.

Enhancement of macrophage invasion of tumors by administration of chemotactic factor-antitumor antibody conjugates

The numbers of macrophages in peritoneal guinea pig hepatomas were significantly ( P < 0.005) elevated by the intraperitoneal injection of a covalent conjugate of the chemotactic peptide, formylmethionylleucylphenylalanine (fMLP), and IgG reactive with surface antigens on the hepatoma cells. These conjugates, which were previously shown to be chemotactic for guinea pig peritoneal exudate macrophages in vitro, increased the numbers of macrophages in the tumors approximately twofold when injected either in a single dose or in five doses. Although five injections of unconjugated fMLP were nearly as effective as the IgG-fMLP conjugates, free fMLP did not enhance the numbers of macrophages in tumors when injected as a single dose. Unconjugated IgG had no effect. The mean tumor weights were decreased in those groups of guinea pigs which received IgG-fMLP but statistical significance was not achieved due to tumor weight variability in all groups.

In vitro effects of antitumor antibody-chemotactic factor complexes

Antitumor antibodies with in vitro chemotactic activity for guinea pig peritoneal exudate macrophages and polymorphonuclear leukocytes were obtained by covalent linkage of the chemotactic peptide, formyl-methionyl-leucyl-phenylalanine (fMLP) to rabbit IgG reactive with a strain 13 guinea pig hepatoma. In addition to its chemotactic properties, the fMLP coupled to antibody also retained its ability to stimulate the production of superoxide anions by macrophages. Neither the IgG nor the IgG treated with the carbodiimide coupling reagent exhibited any of these biological activities. The antibodies, which were adsorbed with sheep erythrocytes and normal strain 13 liver cells prior to the coupling procedure, reacted with surface antigens present on the hepatoma cells but not with those on fibrosarcoma cells or fibroblasts as determined by complement-dependent cytotoxicity. This complement-mediated function was, however, greatly diminished by coupling the IgG to fMLP although indirect immunofluorescent staining patterns established binding of the complexes to hepatoma cells but not to normal liver cells. When bound to tumor cells the antibody-peptide conjugates were also chemotactic, implying that the conjugated antibodies, conjugated antibody-tumor antigen complexes, or fMLP may be released and establish an effective chemotactic gradient for peritoneal exudate cells in vitro.

Effect of lipids, structural precursors of lipids and fatty acids on complement-mediated killing of antibody-sensitized nucleated cells

The cytotoxic activity of limiting concentrations of human complement against antibody-sensitized line-10 guinea-pig hepatoma cells can be inhibited and/or enhanced by pretreatment of the complement with lipids and/or fatty acids. Cardiolipin (CL), phosphatidylglycerol (PG), phosphatidylcholine (PC), phosphatidylserine (PS), sphingomyelin (SPHMY), cholesterol (CHOL), cholesteryl oleate (CHOL-O), phosphatidic acid (PA) or mammalian phosphatidylethanolamine (PE m) were effective in inhibiting C activity. Bacterial phosphatidylethanolamine (PE b) had no effect. Several cholesteryl esters and triglycerides of known fatty acid composition were tested. CHOL-O and glyceryl-1, 2-stearate-3-oleate were effective, but cholesteryl palmitate, stearate, arachidonate and glyceryl-1, 3-stearate-2-oleate were not effective in inhibiting HuC action. Structural precursors of phosphoJipids effective in inhibiting HuC activity (e.g. l-α-glycerophosphoryl esters of choline, serine and ethanolamine) did not modify the action of HuC on antibody-sensitized line-10 cells under any conditions tested. In contrast to the larger mol. wt lipid, conditions were found in which fatty acid could inhibit or enhance the cytotoxic action of C. The modification of C activity was dependent upon the specificity of the antibody used to sensitize the cells, the chain length, and the concentration of the fatty acid. The results suggest that the composition and possible subtle structural characteristics of lipids may play a role in influencing the functional activity of complement.

Xenogeneic immunotherapy of transplantable guinea pig hepatoma with primate leukocyte lysates

Injection of leukocyte lysates prepared from rhesus monkeys xenografted with syngeneic strain-2 guinea pig line 10 hepatoma cells had the ability to increase survival times in guinea pigs receiving lethal, metastasizing doses of the tumor cells. Immunotherapy of the line 10 hepatoma with these xenogeneic anti-line 10 leukocyte lysates resulted in 5 out of 11, or 45.4%, of the animals surviving tumor challenge. Substantially increased survival times were observed in groups of animals treated with lysates from normal or anti-line 1 hepatoma sensitized rhesus monkeys as well. All survivors possessed in vivo skin reactivity and in vitro positive cell-migration inhibition against the line 10 antigen and purified protein derivative. The survivors exhibited negative or marginal immune reactivity against line 1 antigen, strain-2 salt-extracted liver antigen, and a nonspecific antigen, keyhole limpet hemocyanin (KLH). Intact leukocyte lysates from line 10 xenografted baboons, rhesus monkey lysate dialysates, RNA extracts of leukocyte lysates, or lysates of nonlymphoid liver cells obtained from line 10 sensitized rhesus monkeys all failed to extend survival times significantly in strain-2 guinea pigs bearing the line 10 tumor.

Anchorage-dependent culture of line 10 guinea-pig hepatoma cells.

A simple method for the anchorage-dependent culture of line 10 guinea-pig hepatoma cells is described.

Immunotherapeutic effectiveness of BCG inactivated by various modalities

Factors responsible for the limited effectiveness of Bacillus Calmette-Guérin (BCG) immunotherapy are completely understood. One limitation is that although the effect is dose-related, high-dose administration increases the risk of BCG toxicity, possibly the result of disseminated BCG infection. In the present study, we compared the relative effectiveness of Tice lyophilized BCG which was inactivated by heat, sonication, irradiation, streptomycin, or isoniazid (INH). The model systems were the 13762A rat mammary adenocarcinoma and the line 10 guinea pig hepatoma. In the 13762A system, tumor were injected on day 7 with living or killed BCG preparations, or with Corynebacterium parvum as a positive control. Tumors were excised on day 20. Rats treated with surgery alone usually died within 40--50 days with extensive metastases to lymph nodes, lungs, and viscera. Guinea pig line 10 hepatoma was treated with vaccine containing irradiated tumor cells and BCG. In both the rat and guinea pig models, BCG inactivated by means of irradiation was effective as viable BCG and heat-killed BCG also had a strong effect. Streptomycin treatment diminished the efficacy of the BCG and sonication destroyed BCG effectiveness even though the organisms were not all killed. The INH treatment of tumor-bearing rats did not alter the benefits of single or repeated injections of high-dose viable BCG, irradiated BCG, or C parvum. We conclude that inacativation of BCG with heat, irradiation, or INH host treatment preserves but does not improve the immunotherapeutic benefits of BCG.

Analysis of the intralesional adriamycin-induced regression of primary and metastatic growth of line-10 guinea-pig hepatoma

A single intralesional injection of 2.4 mg adriamycin/kg into 7-day-old intradermal strain-2 guinea-pig hepatoma, line-10, caused regression of the tumor and prevented the growth of regional lymph node metastasis. Cured animals were resistant to challenge with the same tumor. Intratumoral injection of 20 mg DTIC/kg was not effective in causing tumor regression or preventing growth of regional lymph node metastasis. Comparative histological examinations performed on the tumor site and the draining lymph node showed that both chemotherapeutic drugs caused extensive necrosis at the injection site and that within 7 days only adriamycin eradicated tumor cells from the draining lymph nodes.

Demonstration of Natural Antibodies in Normal Rabbit Serum With Similar Specificity Pattern as Mouse Natural Killer Cells&lt;xref ref-type="fn" rid="FN2"&gt;2&lt;/xref&gt;&lt;xref ref-type="fn" rid="FN3"&gt;3&lt;/xref&gt;

Normal rabbit sera (NRS) obtained from noninbred animals were shown to contain antibody to YAC-1, a mouse lymphoma, in a complement-dependent 51Cr release assay. A positive correlation was found between the sensitivity to lysis by NRS antibody and the susceptibility to lysis by mouse natural killer (NK) cells when 7 mouse tumors and 1 guinea pig hepatoma were tested in both assay systems. This correlation was further established with a quantitative absorption assay in which NRS was absorbed with various mouse tumors and subsequently tested for cytotoxicity against YAC-1, the most NRS- and NK-sensitive tumor. A striking positive correlation appeared between the capacity to absorb the NRS anti-YAC-1 activity and the NK sensitivity of various mouse tumors to CBA spleen cells. Thymocytes from strain A/Sn mice less than 2 weeks old absorbed a substantial amount of the NRS anti-YAC-1 activity; these cells were also sensitive to mouse NK cytolysis, whereas thymocytes from older mice lacked both properties. These natural rabbit antibodies could not "arm" peripheral blood lymphocytes in the rabbit because 1) the peripheral blood lymphocytes from these animals used as serum donors showed only low cytotoxic activity in an NK assay against NK-sensitive mouse tumors, and 2) no positive correlation appeared between the levels of cytolytic activity and the levels of natural serum antibodies observed in the rabbits. The present data suggest that natural antibodies in NRS showed the same specificity pattern for mouse tumors displayed by mouse NK cells.

Physico-chemical properties of tumor cells that influence their susceptibility to humoral immune killing

Line-10 guinea pig hepatoma cells are susceptible to killing by antibody plus human complement, but resistant to killing by antibody plus guinea pig complement. Tumor cells treated with agents that reversibly increase (adriamycin), decrease (insulin or hydrocortisone), or have no effect (5-fluorouracil) on the susceptibility of the cells to antibody-complement killing were tested for their lipid and fatty acid composition. Hormone-treated (resistant) cells showed an increased total lipid content, an increased cholesterol: phospholipid ratio, and a depressed level of unsaturated fatty acids in the cellular neutral and phospholipids compared to control untreated cells. Adriamycin-treated (sensitive) cells showed exactly the opposite effects. The lipid composition of both hormone- and adriamycin-treated cells returned to control levels when the cells reverted to control levels of susceptibility to antibody-complement killing. 5-fluorouracil-treated cells were indistinguishable from untreated controls in their lipid and fatty acid composition. The chemical composition of the cell, and its effects on the physical properties of the cellular membranes, therefore appears to be fundamental for the ability of these tumor cells to resist humoral immune attack.

Immunotherapy of guinea pigs with a transplanted hepatoma: comparison of intralesionally administered killed BCG cells and BCG cell walls

Heat-killed whole BCG cells (KC) and BCG cell walls (CW) were each tested in emulsified form for their potency to cause regression of a transplanted guinea pig hepatoma. On a weight basis, KC were at least as effective as CW in causing tumor regression and elimination of microscopic lymph node metastasis, and they, as well as purified protein derivative of mycobacteria, provoked delayed cutaneous hypersensitivity reactions in animals immunized with CW or with KC. On a weight basis, KC were as active as CW in eliciting delayed cutaneous hypersensitivity in sensitized guinea pigs whether the animals were immunized with CW or with KC. In unimmunized animals the inflammatory response to intradermally administered KC was similar to that induced by CW. Because KC are easier to prepare than CW, it is suggested that whole killed BCG might be used instead of CW in clinical trials of cancer treatment requiring administration of nonliving mycobacteria.

Complement-Dependent Cytotoxic Antitumor Antibody. II. Quantitative Determination of Cell-Bound Immunoglobulin M

A method for the quantitative determination of rabbit IgM bound to cell surfaces has been developed. This method was based on the ability of goat IgG specific for rabbit IgM heavy chains to bind 125I-labeled protein A when bound to the antigen. With the use of this technique the production of specific IgM antitumor antibodies in New Zealand White rabbits after immunization with guinea pig hepatoma cells line-1 and line-10 was followed. Differences in the production of IgM were observed between the different bleedings from rabbits immunized with line-1. No significant IgM antibody was produced following immunization of rabbits with line-10 tumor cells. This indirect method for determining IgM on the cell surfaces was objective, easy to perform, and detected complement-fixing and noncomplement-fixing antibodies. In addition, this technique could be applied to quantify other components on the cell surface for which a suitable specific antibody was available.

Some perspectives on the transfer of cell-mediated immunity by immune-RNA.

Ribonucleic acid extracts of lymphoid cells from immune hosts were used to transfer in vivo and in vitro cell-mediated immune reactivity to a variety of antigens. The in vivo immune responses transferred by RNA included the delayed cutaneous hypersensitivity reaction to fungal and chemically-defined antigens and the tumor-rejection reaction to guinea pig hepatoma antigens. The in vitro immune responses transferred by RNA included macrophage migration inhibition by fungal, chemically-defined, and tumor antigens. The transfer activity of RNA preparations was contained in the 8 s to 18 s species of RNA and was sensitive to RNase but not to DNase or trypsin. Antigen was not detectable in the RNA preparations and appeared to have no role in the transfer activity. Syngeneic, allogeneic, or xenogeneic sources of RNA could transfer immune reactivity. In each system tested, the transfer of cell-mediated reactivity by RNA was specific for the antigen used to sensitize the RNA donor. The potential use of RNA-mediated transfer of immunity is discussed.

Regression of a transplanted guinea pig hepatoma after intralesional injection of an emulsified mixture of endotoxin and mycobacterial sulfolipid

Mycobacterial sulfatides (sulfolipids) in admixture with endotoxin had tumor regressive activity comparable to trehalose-6,6'-dimycolate (TDM) and endotoxin in guinea pigs, but unlike TDM were neither toxic nor granulomagenic and had no antitumor activity in mice. The increased endotoxin lethality in mice pretreated with BCG was not observed in mice pretreated with sulfolipids or TDM instead of BCG.

Effect of hormones on the killing by antibody and complement of tumor cells treated with proteolytic enzymes

Line-10 guinea pig hepatoma cells are normally resistant to killing by antibody plus GPC but they are susceptible to killing by antibody plus HuC. Pretreatment of the cells with selected proteolytic enzymes renders the cells susceptible to killing by antibody plus GPC, whereas pretreatment with polypeptide, catecholamine, or steroid hormones renders the cells more resistant to killing by antibody plus HuC. Hormone pretreated tumor cells incubated with proteolytic enzymes remained resistant to antibody-GPC-mediated killing. Enzyme-pretreated cells incubated with hormones were either sensitive or resistant to antibody-GPC-mediated killing depending on the enzyme used to pretreat the cells, the enzyme concentration, the hormone class, and the specificity of the antibody used to sensitized the cells. The enzyme pretreated cells were able to bind amounts of hormone sufficient to render the cells resistant to humoral immune killing. These results suggest that the hormones exert their effects on certain areas of the tumor cell membrane rather than having a generalized effect on the cells. It is postulated that proteins, along with complex lipids, participate in the mechanism whereby tumor cells resist humoral immune killing.

Safety and efficacy of living BCG or BCG cell walls (CW) in the treatment of a guinea pig hepatoma

Guinea pigs with established intradermal tumors and microscopic lymph node metastases were treated by intralesional injection of graded doses of living BCG or BCG CW. The lowest dose of living BCG used produced a significant cure rate and no grossly evident toxicity. An intermediate and the highest dose of living BCG used cured some animals but others lost weight and a significant number died as a result of the treatment. Histologic examination of animals with significant weight loss showed fatty degeneration of the liver, granulomatous hepatitis and histiocytic infiltration of the spleen. None of the doses of BCG CW used was toxic and they were at least as effective as living BCG in intralesional treatment. In some experiments in which treatment was delayed it was found that the extent of disease required to render treatment ineffective was about the same for living BCG and for BCG CW.

Synthesis of Specific Lipids Associated with the Hormone-Induced Resistance of Tumor Cells to Humoral Immune Killing

Abstract Selected polypeptide (insulin), catecholamine (epinephrine), and glucocorticosteroid hormones (hydrocortisone and prednisolone) that increase the resistance of line-1 guinea pig hepatoma cells to humoral immune killing were studied for their effects on the ability of the cells to synthesize lipids. Line-1 tumor cells incubated for 1 hr at 37°C with 10-7 to 10-9 M concentrations of any of these hormones became more resistant to killing by antibody plus GPC and exhibited increased levels of fatty acid incorporation into lipids compared to untreated cells. Cells incubated with hormone for 4 hr at 37°C became as susceptible to antibody-GPC killing as untreated cells; lipid synthesis in these cells likewise reverted to control levels. When the reverted cells were reincubated with a different, but not the same, hormone, they were again rendered more resistant to antibody-GPC killing and their lipid synthesis was restimulated. Thin layer chromatography of lipid extracts from hormone-treated as well as from restimulated cells indicated that the increased resistance of the cells to killing correlated with increased synthesis of phosphatidyl choline, phosphatidyl serine, and, in some instances, triglycerides. The cells' reversion to the susceptible state during prolonged incubation with hormone or restimulation with the same hormone was accompanied by a reduction in the synthesis of these lipids along with an increase in cholesteryl ester and cardiolipin synthesis. The synthesis of specific phospholipids and neutral lipids, therefore, rather than lipid synthesis in general, appears to be associated with the mechanism whereby these tumor cells resist humoral immune killing.

Immunotherapy with an intralesionally administered synthetic cord factor analogue.

Injection of emulsified 6,6'-di-O-2-tetradecyl-3-hydroxyoctadecanoyl-a, a trehalose designated C76, a synthetic analogue of the mycobacterial glycolipid trehalose-6,6'-dimycolate (TDM), into transplants of an established, syngeneic murine fibrosarcoma induced complete regression of tumor in a number of animals. The number of animals in which tumor regressed completely dependent on the amount of oil in the emulsion. On a weight basis, C76 was at least as active as TDM. Intralesional injection of an emulsified mixture of C76 and endotoxin (ET) or of TDM and ET caused regression of an established transplant of a guinea-pig hepatoma in syngeneic animals. In mice, intravenously administered emulsions of C76 were less toxic and less granulomagenic than those made with TDM.

Immunotherapy of a guinea pig hepatoma with mycobacterial vaccines: comparison of BCG cell walls and cell wall skeletons

BCG cell wall skeletons (SK) derived from BCG cell walls (CW) by treatment with proteolytic enzymes and organic solvents were tested for their potency to cause regression of a transplanted guinea pig hepatoma. On a weight basic, SK were as effective as CW in causing tumor regression, and they, as well as purified protein derivative of mycobacteria, provoked delayed cutaneous hypersensitivity reactions in animals immunized with CW or with SK. On a weight basis, CW were more active than SK in eliciting delayed cutaneous hypersensitivity in sensitized guinea pigs whether the animals were immunized with CW or with SK. In unimmunized animals the inflammatory response to intradermally administered CW was greater than that evoked by SK. CW and SK provoked delayed cutaneous hypersensitivity reactions of similar strength in animals immunized with living BCG. This study provided no compelling reasons for using SK instead of CW in clinical trials of cancer treatment by mycobacterial vaccines.