Ganglioside patterns and phenotypic characteristics in a normal variant and a transformed back variant of a simian virus 40-induced hamster tumor cell line.

Ganglioside patterns of a cloned Simian virus 40- (SV40) induced hamster tumor cell (Cl(2)TSV(5)-S), its normal variant (Cl(2)TSV(5)-R) which are Cl(2)TSV(5)-S gradually adapted to grow in the presence of 2 microg/ml actinomycin D and exhibit certain normal phenotypic characteristics, and its back variant (Cl(2)TSV(5)-RR), which are Cl(2)TSV(5)-R cells grown in the absence of actinomycin D for more than 60 passages and which exhibit greater phenotypic similarity to Cl(2)TSV(5)-S cells, have been analyzed. All three cell lines contain N(acetylneuraminyl) galactosylglycosyl ceramide (hematoside, GM(3)), N-acetylgalactosaminyl (N-acetylneuraminyl) galactosylglucosyl ceramide (GM(2)), and a higher ganglioside tentatively identified as disialohematoside. However, Cl(2)TSV(5)-R have more GM(2) than Cl(2)TSV(5)-S whereas Cl(2)TSV(5)-RR contain an intermediate amount of GM(2). The amount of GM(2) is correlative with the activity of UDP-N-acetylgalactosamine: hematoside N-acetylgalactosaminyl transferase in the extract of the three cell lines and with their agglutination by wheat germ agglutinin.

Growth of normal and transformed rat embryo fibroblasts: effects of glycolipids from Salmonella minnesota R mutants.

Addition of glycolipids obtained from Salmonella minnesota R mutants to normal, spontaneously transformed, and SV40-transformed rat embryo fibroblasts in culture results in an inhibition of growth of transformed cells but not of normal cells. In the presence of the glycolipid with the smallest carbohydrate chain length, spontaneously transformed cells stop growing when they reach confluency. Inhibition of growth of transformed cells is inversely related to the chain length of the core sugars. Glycolipid mR595 is shown to bind with the cell membrane of transformed cells and elicits an augmentation in the intracellular level of cyclic AMP. Normal cells bind relatively less glycolipid mR595 and show a lower percent of increase in cyclic AMP due to glycolipid mR595 than do transformed cells.

Effect of core lipopolysaccharides from Salmonella minnesota R mutants on the survival times of mice bearing Ehrlich tumor.

Injection of a homologous series of bacterial core lipopolysaccharides obtained from Salmonella minnesota R mutants to Ehrlich solid tumor-beating mice results in an increase of survival times of treated animals. Lower chain length favors greater antitumor activity. Oligosaccharides and polysaccharides derived from lipopolysaccharides were found to be ineffective in increasing survial time of tumore-bearing mice. Smaller-sugar-chain-length core lipopolysaccharides were found to be better adjuvant than were those with longer sugar chains. Implication of adjuvant action of lipopolysaccharides in the elicitation of antitumor activity is suggested.

Maltose tetrapalmitate, a nontoxic immunopotentiator with antitumor activity.

The attempt to synthesize a lipid A-like component (the active portion of lipopolysaccharides, but lacking its endotoxic activity) resulted in the production of fatty acyl sugars of which maltose tetrapalmitate was seen to yield the most promising results. It shows no endotoxic activity and elicits an antitumor response in tumor-transplanted animals as shown by (a) an enhancement of the host's capacity to reject a large number of tumor cells, (b) retardation of growth in tumor size, and (c) induction of hemorrhagic necrosis in certain tumors. Experiments with mammary ascites carcinoma show maltose tetrapalmitate to be as effective as is bacterial glycolipid mR595 in its antitumor activity. The degree of sensitivity to maltose tetrapalmitate varies with the tumor-host system: mammary ascites carcinoma less than NH = Cl2TSV5S = B16 less than L26. The mode of action of maltose tetrapalmitate appears to be via its modulation of the immune system. It is itself noncytotoxic to tumor cells in vitro. It is seen to stimulate the spleen cells of certain animals mitogenically, although it causes tumor rejection in all the types of animals tested. Also, it activates peritoneal exudate macrophages in tumor-bearing animals; whether specifically or nonspecifically has not yet been established.

Combined cyclophosphamide chemotherapy and maltose tetrapalmitate immunotherapy in the treatment of transplanted bladder and prostate carcinoma of the rat.

Chemoimmunotherapy in two animal models for urological cancers was studied. The models were Dunning R3327A prostatic carcinoma transplanted s.c. in Fischer X Copenhagen F1 hybrids and a well-differentiated bladder carcinoma transplanted orthotopically in the bladder submucosa of female Fischer rats. Cyclophosphamide, cis-platinum, and Adriamycin were initially used as anticancer chemotherapeutic agents, and the most effective ones were used in combination with maltose tetrapalmitate (MTP), which was used as an immunopotentiator. In the case of prostatic carcinoma, cyclophosphamide was the most effective among the anticancer agents in controlling tumor growth after inoculation of either 10(4) or 10(5) tumor cells. Combination of cyclophosphamide with i.p. MTP delayed tumor take and controlled tumor size more effectively than did either of the treatments given alone. Similar results were obtained in the case of bladder tumor. A combination of cis-platinum with MTP significantly controlled bladder tumor size, and a combination of cyclophosphamide with MTP cured 75% of the rats. The remaining 25% of this group had a small tumor that did not increase in size during the subsequent 2 weeks of observation without treatment. The incidence of metastasis of bladder tumor to lymph nodes and lung was reduced by MTP and cis-platinum and eliminated by cyclophosphamide alone and in combination with MTP. Nonspecific immunity as measured by phytohemagglutinin stimulation of spleen lymphocytes and antitumor immunity as measured by cytotoxicity and macrophage migration inhibition assays were highest in rats subjected to cyclophosphamide and MTP combined therapies.

Orthotopic implantation of primary N-[4-(5-Nitro-2-furyl)-2-thiazolyl]formamide-induced bladder cancer in bladder submucosa: an animal model for bladder cancer study.

Primary bladder tumors induced in Fischer 344 inbred rats by N-[4-(5-nitro-2-furyl)-2-thiazolyl]formamide were transplanted in syngeneic rats by the intravesical, s.c., i.v., and orthotopic routes. Attempts were made to establish bladder cancer cell lines in vitro. No success was achieved in transplantation by either the s.c., i.v., or intravesical routes when primary tumor cells were transplanted as cell suspensions. Cell suspensions of primary tumors also failed to grow in culture. However, orthotopic implantation into the bladder submucosa gave 45% success. Tumor fragments obtained from either the primary tumor or its lung metastases resulted in 10.6 and 36% tumor takes, respectively, when implanted s.c. However, after one orthotopic passage in the bladder submucosa, the tumor cells injected as cell suspension grew s.c. in 14% and orthotopically in 79% of the animals. Tumor fragments obtained from orthotopic tumors and implanted s.c. resulted in 15% tumor takes. After the second orthotopic passage, tumor cells could be grown in cultures and orthotopically in 100% of animals. The technique of orthotopic implantation as well as the usefulness of this tumor model for bladder cancer studies are described.

Systemic, bladder wall, and bladder tumor concentration of misonidazole following intravesical administration in the rat.

The hypoxic-cell radiosensitizer misonidazole was administered intravesically to normal and to bladder tumor-bearing female Fischer rats. Drug concentration was measured in the bladder wall, the tumor and in the serum using high pressure liquid chromatography at different times following administration. The data shows that misonidazole is readily adsorbed by the bladder wall and the tumor with tissue levels reaching up to 10 times those measured in the serum.

Combination of 60Co gamma-radiation, misonidazole, and maltose tetrapalmitate in the treatment of Dunning prostatic tumor in the rat.

Maltose tetrapalmitate (MTP), a synthetic nontoxic immunoadjuvant, the radiosensitizer misonidazole (MISO), and 60Co gamma-radiation, alone or in combination, were used in the management of Dunning prostatic tumor in the rat. Nine groups of 10 rats each were used to assess the efficacy of various therapeutic modalities. Tumor growth rates and animal survival times were determined for each group. Radiation was more effective when combined with MTP, but the adjuvant must be present when radiation is given for synergism to occur. MISO was as effective as MTP when used with radiation, but combining them cancels out their individual effects. In a clinical situation it would be advantageous to use separately the synergisms existing between MISO and radiation on the one hand and MTP and radiation on the other hand.

Prophylactic antiangiogenic tumor treatment.

Complementary antitumor treatments are required to increase the cure rate achieved by surgery and/or radiotherapy by avoiding future recurrences and metastases. The growth of most solid tumors, particularly carcinomas, depends upon the simultaneous development of internal tumor vasculature to allow the proliferation of tumor cells. Inhibition of tumor vascularization is an indirect means of limiting tumor expansion. Daily administration of cortisone and maltose tetrapalmitate (MTP) abolished growth of implanted syngeneic C3HBA mammary tumor. Gross and macroscopic examination of these tumors revealed that tumor growth was prevented. Histological examination demonstrated lack of vascularization within the neoplastic tissue. We believe that this combination in an appropriate form could provide a prophylactic treatment regimen after conventional antitumor treatments in humans.

Increased survival of CD1 mice bearing dimethylhydrazine induced primary colon and anal cancers by difluoromethylornithine with concomitant increase in angiosarcoma incidence.

Sixty CDl mice received dimethylhydrazine 20 mg/kg s.c. once weekly for 26 weeks to induce colorectal cancer. At this time the animals harbored frank colorectal cancer and early epidermoid cancer. The animals were divided into six groups that were subjected to the following treatments: none, MTP immunotherapy (MTP) alone, radiotherapy (R) alone, difluoromethylornithine (DFMO) chemotherapy alone and combinations of R+DFMO and R+DFMO+MTP. Criteria of evaluation of treatment efficacy were: number of colorectal tumor lesion and their staging at death, the incidence and size of anal cancer at death and survival time. Radiotherapy alone was marginally effective and MTP treatment was moderately effective in preventing anal cancer and reducing the number of colorectal tumors as well as their size. DFMO was exceptional in preventing anal cancer in a majority of animals and increasing animal survival; the latter effect was due to its preventive action against pyelonephritis, the major cause for animal death. However, in DFMO treated animals, the incidence of angiosarcoma increased from 10-16% (in the absence of DFMO) to 35-50% (in the presence of DFMO). The most effective treatment of the colorectal tumor was the triple combination of R + DFMO + MTP.

Evaluation of cortisone-heparin and cortisone-maltose tetrapalmitate therapies against rodent tumors. II. Pathological studies.

Pathological studies were undertaken in three tumor-host models which were subjected to cortisone based treatments. The first model was Fisher 344 rats with established orthotopically implanted syngeneic bladder tumor. Cortisone-herapin and cortisone-maltose tetrapalmitate (MTP) treatments induced focal areas of tumor necrosis and necrobiosis, whereas cortisone alone caused necrobiosis. The second model was C3HBA mammary tumor fragments implanted subcutaneously in syngeneic MTP responder C3H/HeN and MTP non-responder C3H/HeJ female mice. Only cortisone-MTP treatment led to an absence of capillary extension from surrounding blood vessels into the scant tumor stroma. The third model, ethyl carbamate induced primary lung cancer in AJ mice, was tested only with cortisone-herapin combination. The treatment caused central zones of necrosis.

Effect of maltose tetrapalmitate on tumor vascularization and tumor growth.

C3HBA mammary tumor (H-2k) was implanted s.c. by trocar in MTP responder C3H/HeN (H-2k) and non-responder C3H/HeJ (H-2k) female mice. One half of the animals received MTP i.p. The size of the tumor was measured everyday. Tumor growth was slightly slower in the C3H/HeJ than in the C3H/HeN. MTP treatment was effective only for the tumor implanted in the C3H/HeN. Microscopic and microscopic visualization of the tumor 1, 2, 3, and 15 days after tumor implantation and MTP treatment revealed poor vascular development in the MTP-treated C3H/HeN compared to untreated controls at days 2 and 3.

Antitumor efficacies of maltose tetrapalmitate immunotherapy alone and in combinations with radiotherapy and with cyclophosphamide chemotherapy against dimethylhydrazine induced colon and anal cancers in CDI mice.

Treatment of human colonic cancer in early stages when the process is still limited to the colonic wall is primarily surgery. We wished to see if maltose tetrapalmitate (MTP) immunotherapy alone or in combination with radiotherapy (R) and cyclophosphamide (C) chemotherapy would be effective against primary colon cancer in a fashion similar to that reported by us for primary liver cancer (Anticancer Research 6: 245-250, 1986). One hundred female CD1 mice were subjected to dimethylhydrazine (DMH) treatment once a week for 26 weeks, a period one week before which, colon cancer was histologically documented in each animal of a group that was sacrificed. Surprisingly, many of the animals harboured early anal cancer as well. At 28 weeks, 85 of the available animals were divided into 6 groups that received: Gr. 1, no treatment; Gr. 2, MTP alone (M); Gr. 3, radiotherapy alone (R); Gr. 4, cyclosphophamide alone (C); Gr. 5, R + C; Gr. 6, M + R + C. Criteria of treatment efficacy were: number, size and staging of colorectal tumors and the incidence and the size of anal tumors at death. Mean survival time was also determined although it remained a questionable criterium since most animals died due to complication (hepatic toxicity, pyelonephritis, thrombose) elicited by DMH, R and C toxicities and not as a result of colonic tumor size or metastases. As a single therapy, M appeared to be superior to either R or C alone. However, R + C combination was effective and was further improved upon by its association with M. With the triple combination, (M + R + C), lesions of both cancers decreased in size and/or number and the colon cancer histologically eclipsed from 46% of the treated animals.

An animal model of Kaposi's sarcoma. I. Immune status of CD1 mice undergoing dimethyl hydrazine treatment to induce angiosarcomas and other malignancies.

Dimethylhydrazine (DMH) induces colonic cancer and angiosarcomas in mice. In order to determine pertinence of mouse angiosarcoma as a model to AIDS associated Kaposi's sarcoma (KS), we investigated if immune dysfunction occurred during tumor development by DMH. Outbred CD1 male mice received once weekly DMH a 20 mg/kg body weight dose s.c. for 33 weeks. Every two weeks initially and then every week groups of DMH-treated and control animals were sacrificed to determine a) peripheral blood and splenic T cell subset ratio b) 4-day plaque forming cell (PFC) response to i.p. sheep red blood cells (SRBC) and c) mitogenic response of spleen cells to Concanavalin A (Con A) and lipopolysaccharide (LPS). No change in T helper/T suppressor + cytotoxic T cell (Th/Tsupp. + CTL) and mitogenic response to spleen cells to Con A was noted whereas PFC response of animals to SRBC and mitogenic response of spleen cells to LPS decreased. These data suggest that either infection with T cell depleting virus such as LP:BM5 or immunosuppressive drugs affecting T cell function, such as steroids may be required to bring the immune status of DMH treated animals closer to that of AIDS associated KS bearing human subjects.

An animal model of Kaposi's sarcoma. II. Pathogenesis of dimethyl hydrazine induced angiosarcoma and colorectal cancer in three mouse strains.

CDI, C57B1/6 and DBA2 mice were subjected to dimethyl hydrazine (DMH) carcinogenesis to determine incidences of various types of tumors developing internally in the three strains. The animals were also screened for skin angiosarcomas. Skin lesions histologically similar to cutaneous AIDS associated Kaposi's sarcoma were observed in CD1 mice. Angiosarcomas predominated over colorectal tumors in C57Bl/6 and DBA2 mice, whereas the reverse was true for CD1 mice. The visceral angiosarcomas had both histological similarities and differences with visceral AIDS associated Kaposi's sarcoma.

Evaluation of cortisone-heparin and cortisone-maltose tetrapalmitate therapies against rodent tumors. I. Biological studies.

The antitumor activity of either cortisone-heparin or cortisone-maltose tetrapalmitate combination or both was tested against two animal tumor models. The first model was orthotopically implanted bladder tumor established in syngeneic Fisher 344 rats. Shrinkage and growth arrest of the tumors were induced by cortisone and amplified by its combination with either heparin or maltose tetrapalmitate (MTP). The second model was trocar implanted C3HBA mammary tumor piece s.c. in syngeneic LPS and MTP responder C3H/HeN and non responder C3H/HeJ mice. The tumor was sensitive to growth inhibition by cortisone-MTP in the C3H/HeN but not by cortisone alone or cortisone-heparin. Tumor implanted in C3H/HeJ was much less sensitive to cortisone-MTP. Cortisone could be replaced by 17-alpha-hydroxyprogesterone, but not by cortexolone.

Combined radiotherapy, chemotherapy, and maltose tetrapalmitate immunotherapy in the treatment of 4'dimethylaminoazobenzene-induced liver cancer.

Therapeutic effects of radiotherapy (R), chemotherapy, and maltose tetrapalmitate (MTP) immunotherapy alone and in combinations were tried against 4' dimethylaminoazobenzene (DAB) induced primary liver cancer in Wistar rats in three separate protocols. Rats were fed a low protein synthetic diet containing 0.06% DAB for 90-120 days. Around 90 days, liver cancers developed in all the animals. In the first protocol, animals were either left untreated or treated with cyclophosphamide (Cy), MTP (i.p. or oral) and Cy plus oral MTP. Rats in the MTP (i.p.) group maintained a steady liver weight but neither Cy nor Cy + MTP influenced the survival time or liver weight. In the second protocol, R as well as a 3-drug combination at 2 dose levels were tried alone and with MTP before or soon after cessation of DAB feeding. Survival times were decreased by R and chemotherapy due to combined toxicities of DAB and treatments and were partially restored by MTP. In the third protocol, MTP, R, and Cy were each tried alone and in combinations, 21 days after cessation of 100-day DAB feeding. Increase in survivals were obtained by each treatment, although tumor weight was best controlled by triple R+ Cy + MTP combination.

Adjuvant immunotherapy of N-[4(5-nitro-2-furyl)-2-thiazolyl]-formamide-induced bladder tumors.

The prophylactic effect of maltose tetrapalmitate (MTP), a newly developed non specific immunoadjuvant in preventing or delaying bladder cancer induction by N-[4(5-nitro-2-furyl)-2-thiazolyl] formamide (FANFT) or in reducing the growth rate of the induced tumor was compared to other well-known immunoadjuvants (BCG, C. parvum, levamisole and pyran copolymer). The evaluation of the prophylactic role of immunoadjuvants demonstrated that MTP, levamisole and C. parvum were the most effective in prolonging animal survival. MTP was found superior to either of them in reducing the tumor size. The development of lung metastasis was lower in the group receiving MTP or C. parvum. Next, MTP was studied for its therapeutic effect against primary FANFT induced tumor. The subcutaneous (s.c.) and oral routes of MTP administration and their combination with intravesical route were tried. Combinations of intravesical with s.c. or oral MTP were most effective in reducing tumor size, obtaining lower metastases along with greater mononuclear lymphocyte infiltration in the primary tumor.

Effects of single and combined maltose tetrapalmitate immunotherapy, cyclophosphamide chemotherapy and radiotherapy on ethyl carbamate accelerated primary lung cancer in A/J mice.

A/J mice were given ethyl carbamate to accelerate and to raise to 100 percent the incidence of lung tumours at 34 weeks (day 237) of age. The animals were then divided into groups which received the following treatments: group 1, no treatment; group 2, MTP alone; group 3, radiotherapy alone; group 4, cyclophosphamide alone; group 5, radiotherapy + MTP; group 6, MTP + cyclophosphamide; group 7, radiotherapy followed by cyclophosphamide and group 8, MTP and radiotherapy together followed by MTP and cyclophosphamide. Except for radiotherapy, which was given for 5 consecutive days, MTP and cyclophosphamide were continued till the death of the animals. The treatment efficacies were evaluated by the number and size of tumour nodules, taking into consideration the survival time of the animal. Animals in groups receiving cyclophosphamide died earlier (between days 290 and 315) due to its toxic effects, and half of the radiotherapy-MTP were sacrificed at day 314 for comparison. Although cyclophosphamide alone and radiotherapy plus cyclophosphamide demonstrated antitumour activity, the number of tumour nodules and the nodule diameter were reduced most effectively in group 8 (receiving MTP, radiotherapy and cyclophosphamide). Among the animals in the non-cyclophosphamide group, radiotherapy alone was ineffective. MTP given before and after radiotherapy (group 5) kept tumour volume in control although this group died suddenly. The animals receiving only MTP died between day 430 and 470. The number of tumour nodules and the nodule diameter in the MTP group were, however, significantly reduced when compared to controls or radiotherapy group animals dying at or near the same time.