Synergistic effects of active specific immunotherapy and chemotherapy in guinea pigs with disseminated cancer

Sewall Wright strain 2 guinea pigs bearing pulmonary metastases of the syngeneic line 10 (L10) hepatocarcinoma were treated with a vaccine composed of 10(7) bacillus Calmette-Guérin admixed with 10(7) x-irradiated L10 tumor cells beginning 10 days after tumor inoculation. Although this treatment failed to cure most of the guinea pigs of their metastatic disease, histologic examination of the pulmonary tumors in the vaccinated guinea pigs provided evidence of a cell-mediated hypersensitivity response that disrupted the normally compact architecture seen in control tumors. When a monoclonal antibody against the L10 tumor was injected i.v. to evaluate the vascular permeability of the tumors, significantly more antibody localized in tumors of vaccinated guinea pigs than in tumors of untreated controls. These results suggested that blood-borne substances could be delivered more efficiently to L10 metastases after the tumor-bearing guinea pigs had been treated with vaccine. To determine whether such increased vascular permeability would enhance the antitumor effects of chemotherapeutic agents, combined immunotherapy and chemotherapy studies were performed. Although cyclophosphamide treatment by itself did not cure L10-bearing guinea pigs, cyclophosphamide used in conjunction with prior immunotherapy increased the survival rate of animals to more than twice that of animals treated with immunotherapy alone (74 vs 33%). These results suggest that one mechanism by which active specific immunotherapy enhances chemotherapy of disseminated tumors is by rendering tumor foci more permeable to subsequently administered cytotoxic drugs.     

Restriction endonuclease DNA analysis of antigenic variants of leptospires selected by monoclonal antibodies

The genome of antigenic variant CV (CT3)-1 derived from Leptospira interrogans serovar canicola was compared by cleavage with restriction endonucleases with the parent and serovar bafani, to which the variant was serologically most closely related. No differences were observed between the parent and variant in DNA restriction endonuclease patterns using eight restriction endonucleases. Serovar bafani was different in the patterns from the parent and antigenic variant CV (CT3)-1. The two antigenic variants derived from serovar hebdomadis, HV (H16)-1 and HV (H19)-1 which belonged serologically to serovars jules and hebdomadis, respectively, were compared by restriction endonuclease DNA analysis with the parent and serovar jules. No differences were observed between the parent and variants in DNA restriction endonuclease patterns using the same enzymes. But some differences were observed in DNA restriction endonuclease patterns between HV (H16)-1 and serovar jules. Thus, the antigenic variant selected from the parent by the anti-parent monoclonal antibody and serologically different from the parent, being identified either as a new serovar or as a known one, was found to be similar to the parent by the restriction endonuclease DNA analysis.     

Cell surface sialylation and tumor metastasis. Metastatic potential of B16 melanoma variants correlates with their relative numbers of specific penultimate oligosaccharide structures

Numerous investigations suggest that cell surface glycoconjugates, and in particular sialic acids, are directly involved in determining the metastatic phenotype. To further evaluate this hypothesis, we have used a variety of techniques to probe the cell surfaces of several metastatic variants of the murine B16 melanoma that were selected for experimental lung-colonizing ability (Fidler, I. (1973) Nature 242, 148-149) or for their ability to spontaneously metastasize from the site of a subcutaneous injection (Stackpole, C. W., Alterman, A. L., and Fornabaio, D. M. (1985) Invasion & Metastasis 5, 125-142). Using a highly sensitive high performance liquid chromatography sialic acid assay in conjunction with Vibrio cholerae sialidase, we find that none of these metastatic variants differ significantly in their overall levels of cell surface sialic acid. Using highly purified, linkage-specific sialyltransferases, in conjunction with specific glycosidases, to probe the cell surface saccharide topography of specific penultimate oligosaccharides, we also find no significant differences between the efficient lung-colonizing variant, B16-F10 and the poorly-colonizing B16-F1 or B16-Flr variants. In contrast, the spontaneously metastatic variants examined contain substantially different levels of specific penultimate sialylation sites. The tumorigenic but nonmetastatic B16-LM3/G3.26 variant contains 4-fold more penultimate Gal beta 1-3GalNAc sialylation sites than the tumorigenic and highly metastatic B16-LM3/G3.12 variant when CMP[3H]NeuAc and the alpha 2-3Gal beta 1-3GalNAc sialyltransferase are used to probe the melanoma cell surfaces. Several prominent glycoconjugates of apparent Mr 43,000, 40,000, and 30,000 are especially evident upon sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the nonmetastatic cells. The nonmetastatic variant also contains 2-fold more Gal beta 1-4GlcNAc sialylation sites than the metastatic variant when the alpha 2-6Gal beta 1-4GlcNAc sialyltransferase is used as a cell surface probe. In this case, glycoconjugates of apparent Mr 74,000, 45,000, and 43,000 are more prominently observed on the cell surfaces of the nonmetastatic variant. These data indicate that the differences in lung-colonizing abilities of B16 melanoma metastatic variants do not correlate with the numbers or sialylation states of specific penultimate oligosaccharide structures on their surfaces. However, the relative levels of specific penultimate saccharide structures do correlate with the ability of the cells to undergo spontaneous metastasis from a subcutaneous tumor.     

Immunologic enhancement of experimental metastasis in the rat

Summary Using a series of immunologically cross-reactive metastatic tumor variants, we demonstrate that serum from animals bearing pulmonary tumor colonies possesses enhancing properties in the experimental metastasis (lung colony) assay. Enhancement is produced by chronic serum administration and promotes the growth of tumor cells arrested in the lungs which would not otherwise proliferate to form grossly detectable lung nodules. Tumor-bearer serum from animals with lung colonies derived from the most highly metastatic variant examined is shown to possess enhancing properties in both BD-IX(H-1^d) and BD-IV(H-1^d) rat strains, while tumor-bearer serum from animals with lung colonies derived from the less metastatic parent tumor cell line possesses enhancing properties in the BD-IX rat strain only. Removal of immunoglobulin from enhancing serum by affinity column chromatography simultaneously removes the enhancing factor(s), and enhancing activity correlates with the presence of increased levels of Clq-binding immune complexes in the serum. Serum levels of immune complexes are shown to be more elevated in serum from animals bearing lung colonies derived from the most highly metastatic variant. The enhancing moieties are shown to bind to concanavalin A, but not to staphylococcal protein A, and the active fraction elutes from concanavalin A-Sepharose with -methyl-mannoside. Consideration of immunoprecipitation studies on whole and fractionated enhancing sera, along with studies on affinity purified isotype fractions reveals that the activity resides with antibodies of IgG_2b subclass. Abbreviations used: NK, natural killer cell; CIC, circulating immune complex; RhC, rheumatoid-Clq protein complex; Ig, immunoglobulin     

A 6-thioguanine-resistant variant of the rat mammary adenocarcinoma 13762 that is more immunogenic

Summary A 6-thioguanine-resistant (TgR) variant of the metastatic mammary tumor 13762 was found to be very immunogenic. This TgR variant was nontumorigenic and nonmetastatic, whereas the parent 13762 cell line is very tumorigenic and metastatic in normal syngeneic animals. The TgR variant was tumorigenic in irradiated animals. The mechanism of the hosts' immune rejection of this TgR variant was investigated. A ⁵¹Cr-release cytotoxic cell assay was used to assess lymphocyte cell-mediated cytotoxicity (CMC) of tumor-draining lymph nodes and spleens from animals injected with tumor cells. In a secondary CMC response of splenic T cells from animals injected with TgR cells, there was a much stronger response as compared to animals injected with 13762 cells. This strong cytotoxic T cell response was short-term and correlated to the host rejection of TgR cells. Previously, we selected revertant cell lines (TgRrev, TgRrevM) from the TgR variant line that were more metastatic and tumorigenic. The revertant cell lines induced a lower CMC response than the TgR line, but a higher response compared to the parent 13762 line. The poor CMC response from 13762 tumorbearing animals was investigated and appeared to be due to a suppressor T cell response.     

Thujone inhibits lung metastasis induced by B16F-10 melanoma cells in C57BL/6 mice

The antimetastatic potential of thujone, a naturally occurring monoterpene, was evaluated. Metastasis was induced in C57BL/6 mice by injecting highly metastatic B16F-10 melanoma cells through the lateral tail vein. Administration of thujone (1 mg·(kg body weight)(-1)), prophylactically and simultaneously with tumor induction, inhibited tumor nodule formation in the lungs by 59.45% and 57.54%, respectively, with an increase in the survival rate (33.67% and 32.16%) of the metastatic tumor bearing animals. These results correlated with biochemical parameters such as lung collagen hydroxyproline, hexosamine and uronic acid contents, serum sialic acid and γ-glutamyl transpeptidase levels, and histopathological analysis. Treatment with thujone downregulated the production of proinflammatory cytokines such as tumor necrosis factor-α, interleukin (IL)-1β, IL-6, and granulocyte-monocyte colony-stimulating factor. Thujone administration downregulated the expression of matrix metalloproteinase (MMP)-2, MMP-9, extracellular signal-regulated kinase (ERK)-1, ERK-2, and vascular endothelial growth factor (VEGF) and also upregulated the expression of nm-23, tissue inhibitor of metalloproteinase (TIMP)-1, and TIMP-2 in the lung tissue of metastasis-induced animals. Treatment with thujone inhibited the activity of MMP-2 and MMP-9 in gelatin zymographic analysis. Thujone treatment significantly inhibited the invasion of B16F-10 melanoma cells across the collagen matrix in a Boyden chamber. Thujone also inhibited the adhesion of tumor cells to collagen-coated microtire plate wells and the migration of B16F-10 melanoma cells across a polycarbonate filter in vitro. These results indicate that Thujone can inhibit the lung metastasis of B16F-10 cells through inhibition of tumor cell proliferation, adhesion, and invasion, as well as by regulating expression of MMPs, VEGF, ERK-1, ERK-2, TIMPs, nm23, and levels of proinflammatory cytokines and IL-2 in metastatic animals.     

Subtractive immunization yields monoclonal antibodies that specifically inhibit metastasis

Subtractive immunization allowed the isolation and characterization of monoclonal antibodies that specifically inhibit metastasis but not proliferation of highly metastatic human tumor cells. The tolerizing agent cyclophosphamide was used to suppress the immune system in mice to dominant immunodeterminants present on a non-metastatic variant (M-) of the human epidermoid carcinoma cell line (HEp3). Mice were then inoculated with a highly metastatic variant (M+) of HEp3 to enhance an immune response to antigenic determinants present on metastatic cells. Hybridomas were generated and screened by ELISA for differential reactivity to M+ HEp3 over M- HEp3 cells. This experimental approach, termed subtractive immunization (S.I.), was compared to a control immunization protocol, which eliminated the cyclophosphamide treatment. The S.I. protocol resulted in an eight-fold increase in the proportion of mAbs that react with molecules enriched on the surface of the M+ HEp3 cells. Two of the mAbs derived from the S.I. protocol, designated DM12-4 and 1A5, were purified and examined for their effect in a metastasis model system in which chick embryos are transplanted with primary HEp3 tumors. Purified mAbs DM12-4 and 1A5, inoculated i.v. into the embryos, inhibited spontaneous metastasis of HEp3 cells by 86 and 90%, respectively. The mAbs are specifically anti-metastatic in that they have no effect on the growth of HEp3 cells in vitro nor did they inhibit primary tumor growth in vivo. The mAbs recognize M+ HEp3 cell surface molecules of 55 kD and 29 kD, respectively. These data demonstrate that the S.I. protocol can be used for the development of unique mAbs that are reactive with antigenic determinants whose expression is elevated on metastatic human tumor cells and which function mechanistically in the metastatic cascade.     

Anti-β1 Integrin IgG Inhibits Pulmonary Macrometastasis and the Size of Micrometastases from a Murine Mammary Carcinoma

In the present report, we investigated the possible importance of β₁ integrins in the growth and metastasis of a murine mammary carcinoma, SP1, and a metastatic variant, SP1-3M in vivo. CBA/J female mice bearing SP1 tumor transplants were injected with anti-β₁ integrin IgG or control nonimmune IgG (200 μg per mouse; i.p.) every two days. Animals received anti-CD4 antibody (100 μg per mouse) at time zero to suppress immunity against rabbit IgG. Outgrowth of macroscopic metastases from SP1, but not from SP1-3M primary tumors, was markedly inhibited in animals receiving anti-β₁ integrin IgG but not nonimmune IgG. To assess the stage(s) in the metastatic cascade affected, we examined the number and diameter of micrometastatic nodules in treated and untreated groups. The diameter of micrometastases was significantly reduced in SP1-tumor-bearing mice treated with anti-β₁ integrin IgG compared to control IgG, although the number of nodules per cm² of lung sections examined remained unchanged. No change in the number or size of micrometastases in SP1-3M tumor-bearing mice was observed. No difference in the binding, or complement-mediated and antibody-dependent cell-mediated cytotoxicity of anti-β₁ integrin IgG with SP1 and SP1-3M cells was detected. The results suggest that under these conditions anti-β₁ integrin inhibits metastatic tumor growth in lung tissue, but has minimal effect on intravasation, adhesion to target organs and extravasation.     

Complete local tumor regression with antibody to fibrin Fragment E.

Rabbit antibody to fibrin fragment E (FFE) was used in an immunotherapy model for the treatment of the line-10 ascites variant of a diethylnitrosamine-induced hepatoma in strain 2 guinea pigs. When 0.75 or 1.0 mg of an IgG preparation containing anti-FFE antibody was injected s.c. 6 and 16 days after the injection of a uniformly lethal dose of line-10 tumor cells, complete regression of the i.d. growing tumor was observed in all 18 strain 2 guinea pigs treated. Thus, this therapy appears to be more effective than any BCG or other immunotherapeutic regimen thus far reported for this tumor. No significant anti-tumor effect was noted when normal rabbit IgG or smaller doses (0.25 or 0.50 mg) of the anti-FFE IgG preparation were used. The injection sites exhibited an inflammatory response for 7 to 10 days characterized by erythema and hemorrhage. Since all animals were treated after the metastatic progression of the tumor is known to frequently occur, the long-term tumor-free survival of these animals as well as their resistance to subsequent tumor challenge indicate that the anti-FFE antibody therapy led to systemic tumor immunity.     

Proportion of antigenic variants induced by in vitro UV irradiation differs in clones derived from a single tumor

The purpose of this study was to examine the capacity of different clones derived from the same tumor to generate highly antigenic cells after in vitro exposure to UV radiation. Cells from the metastatic murine melanoma K1735 and clones of K1735 differing in metastatic potential were exposed to UV radiation in vitro, cloned, and tested for antigenic properties in vivo. Approximately half of the clones isolated after UV irradiation of parental K1735 melanoma cells were highly antigenic (five of nine). Similar treatment of cells of a nonmetastatic clone of K1735 generated clones that were all antigenic (nine of nine). In contrast, only one of nine clones derived from UV-irradiated cells of a highly metastatic clone of K1735 were antigenic. Clones derived from unirradiated cultures were not antigenic variants. The increased antigenicity of cells derived from UV-irradiated cultures did not correlate with an increase in expression of cell surface class I major histocompatibility complex antigens. These results demonstrate that the frequency of antigenic variant production after UV irradiation is an intrinsic property of the particular cell line used, and that even cloned cell lines derived from a single tumor differ in their ability to generate antigenic variants after UV irradiation. In addition, they indicate that the increased antigenicity is not necessarily due to a UV-induced increase in expression of cell surface class I histocompatibility antigens.     

Tumor metastases and cell-mediated immunity in a model system in DBA/2 mice. VIII. Expression and shedding of Fcγ receptors on metastatic tumor cell variants

The expression of receptors for the Fc portion of IgG immunoglobin molecules was studied on tumor cell lines with high and low metastatic capacity. Two tumor cell lines from DBA/2 mice that had high metastatic activity, ESb and MDAY-D2, contained a high percentage of Fc receptor positive cells, as detected in a rosette assay with IgG antibody-coated erythrocytes (EA). In contrast, the low metastatic parental line Eb, from which ESb was derived, contained only a low percentage of EA-rosette-forming cells. ESb ascites tumor cells adapted to tissue culture in the presence of 2-mercaptoethanol (2ME) had a high expression of Fc receptors, whereas a cell line adapted to tissue culture in the absence of 2ME had a low expression of Fc receptors. “Soluble” Fc receptors were detectable by their ability to bind to EA and to cause blocking of rosette formation. They were found to be present in fluids from tumor-bearing animals, such as serum and cell-free ascites. Even animals with an ascites tumor of the low-metastatic line Eb contained “soluble” Fc receptors. The results are discussed with regard to their possible significance for tumor metastasis.     

Tracking micrometastasis to multiple organs with lacZ-tagged CWR22R prostate carcinoma cells.

Metastasis to organs other than lung is rarely observed in animal model systems of human prostate carcinoma (PCA), with the exception of already metastatic isolates of human PCA cultured for long periods of time. To analyze more directly the evolution of metastatic variants from primary PCA tumor isolates, the lacZ histochemical marker gene was transfected into the CWR22Rv1 cell line isolated from the CWR22R xenograft (primary tumor). Three clones of varying lacZ-expression stability were analyzed for tumorigenicity and progression in athymic nude mice. Clones B and D were highly tumorigenic in the subcutis; however, lacZ expression was highly unstable. In contrast, clone H demonstrated highly stable lacZ expression for >25 passages in culture or in animals. Clone H, injected sc in a PBS vehicle, gave a 15-40% tumorigenic take. All primary tumor-bearing animals exhibited micrometastases in lung and other organs. Clone H injected in a Matrigel vehicle gave 100% tumorigenicity, with all animals displaying micrometastases in lung, liver, and/or bone (lower frequency in brain and kidney). Overall, the relative frequency of micrometastasis to multiple organs was lung>liver=bone>brain>kidney. Overt metastases were never observed in the lung or bone but were occasionally found in liver. lacZ-transfected clone H CWR22Rv1 cells represent a much more accurate model of metastasis of PCA to the organs normally involved in progression of the human disease. Use of marker gene-tagged cells and other high-resolution molecular techniques will now permit analyses of the earliest events in PCA progression and micrometastasis.     

Multiple variants of the human lymphocyte homing receptor CD44 generated by insertions at a single site in the extracellular domain.

The human CD44 cell-surface glycoprotein participates in a wide variety of cell-cell interactions including lymphocyte homing and tumor metastasis. The CD44 antigen is known to display extensive size heterogeneity when compared between different tissue sources although the structural basis for this variation is not yet clear. Recently, two further isotypes in addition to the basic hemopoietic form of the CD44 antigen have been cloned and sequenced and these have been found to contain all or part of a 200-400-base pair insert within the extracellular domain, suggesting that the characteristic heterogeneity in the molecule may be generated by a mechanism of alternative splicing. We have obtained further evidence for alternative splicing, and we report here the cloning and sequencing of six different CD44 sequence variants from a variety of cell lines using a combination of expression cloning and the polymerase chain reaction. Comparison of these variants indicates that each is probably assembled by the insertion of five different exon units in tandem into a discrete site within the membrane proximal region of the extracellular domain. One of the variants contains an exon that shares extensive amino acid sequence homology with a recently described rat CD44 variant that mediates tumor metastasis. Another variant contains a new exon that encodes a tandem repeat of the consensus sequence SG for covalent modification with chondroitin sulfate and is expressed predominantly on mammary tumors. We suggest that a mechanism of alternative exon splicing generates much of the observed structural heterogeneity of CD44 and that the particular set of CD44 variants expressed in a single cell may represent a precise postal code directing the final destination of migrating cells and metastatic tumors.     

The cell surface sulphydryl content of metastatic variants of B16 murine melanoma

The cell surface sulphydryl content of three metastatic variants of the B16 murine melanoma has been determined using isoelectric equilibrium techniques. The F1 variant, which has no ability for natural metastasis, and the F10 variant with moderate metastatic ability appeared to have no detectable surface thiol groups. The variant BL6, which shows a high degree of natural metastasis, possessed surface thiol groups. The variants were found to be heterogeneous in isoelectric distribution. Three subpopulations were identified based on isoelectric criteria. The size of the pI 5.0 subpopulation appeared to increase with metastatic ability. A proportion of this subpopulation, approximately 4% in the F10 and 10% in the BL6, was found to possess surface thiol groups. In the BL6 line, 10-20% of the pI 4.6 subpopulation possessed surface thiol groups. The surface negative charge density of the cell types showed no correlation with their natural metastatic ability.     

Inhibition of inflammatory angiogenesis by distant subcutaneous tumor in mice

We investigated angiogenesis, inflammatory cells accumulation and endogenous production of cytokines in sponge implants of tumor-bearing mice. Seven days after inoculation of Ehrlich tumor cells (2.5 x 10(6)), sponge discs were implanted subcutaneously in the dorsa of mice to induce the formation of fibrovascular tissue. The implants of tumor-bearing and non tumor-bearing animals were assessed for neovascularization and leukocyte accumulation, together with levels of relevant cytokines, vascular endothelial growth factor VEGF), tumor necrosis factor alpha (TNF-alpha), CXCL1-3/KC and CCL2/JE. In the implants of tumor-bearing animals angiogenesis (assessed by hemoglobin content and VEGF levels in the implants) and leukocyte accumulation (assessed by myeloperoxidase -MPO- and N- acetylglucosaminidase-NAG-enzyme activities) were all significantly less than those in the implants of non tumor-bearing animals. Although the chemokine CXCL1-3/KC was lower in the implants of tumor-bearing animals, the chemokine CCL2/JE was increased in this group. The production of TNF-alpha in the implants was not modified by the presence of the subcutaneous tumor. The combination of the methodologies used in this study has provided a novel approach to investigate the interaction between two distinct proliferating tissues that share common features (angiogenesis, cell recruitment, inflammation) and has shown that the predominant inhibitory effect of a tumor mass over repair process is associated with altered cytokine production.     

Immunological resistance to L1210 leukemia induced by viable L1210/DTIC cells

Summary Permanent, drug-induced antigenic alterations, not detectable in parental cells and transmissible after the withdrawal of treatment with the drug, have been obtained in mouse lymphoma. Viable L1210/DTIC cells, because they are rejected by syngeneic animals and carry L1210-associated TAA, can elicit host resistance to a subsequent inoculum of parental L1210. Mice challenged with viable L1210/DTIC cells, following rejection, were more resistant than mice immunized with inactivated parental cells. Resistance was specific and related to the immunogenicity of the TAA of the original tumor line employed.Active immunization was potentiated by adoptive transfer of immune lymphocytes, as evidenced by marked improvement in animal survival. Also, the treatment of tumor-bearing animals with anticancer compounds in conjunction with immunological alteration may result in an improved therapeutic response. BCNU administered to immunized animals 6 days after challenge with parental tumor cells resulted in augmented host survival, possibly attributable to partial resistance of a secondary immune response to the drug and a late nadir of immunosuppression, occurring after the completion of therapeutic action. Cyclophosphamide given before immunization enhanced host survival to a subsequent challenge of L1210 leukemia, conceivably as the result of preferential inhibition of T suppressor cells.     

Selective localization of tumor-immune spleen cells at the tumor challenge site after adoptive transfer of line 10 tumor immunity in strain 2 guinea pigs

In this study, the distribution of immune spleen cells was investigated after adoptive transfer of immunity in inbred strain 2 guinea pigs. Spleen cells obtained from line 10 immune donor animals became specifically restimulated in vitro with 3 M KCl-extracted line 10 soluble proteins, but not with 3 M KCl-extracted line 1 or liver proteins. After 4 days culture in vitro, these specifically restimulated immune spleen cells retained their antitumor activity in vivo after adoptive transfer. The specifically restimulated immune spleen cells were radiolabeled with [3H]thymidine, 1 X 10(8) viable cells were adoptively transferred in tumor-bearing guinea pigs, and their distribution was investigated. As controls for the specific localization of the immune cells at the line 10 tumor, the presence of labeled cells was studied in the contralateral transplanted line 1 hepatoma as well as in cellular inflammatory reactions elicited by injection with incomplete Freund's adjuvant (IFA) and complete Freund's adjuvant (CFA). A significantly higher localization of the labeled immune spleen cells in the line 10 tumor and the first and second draining lymph nodes of the line 10 challenge site were found when compared to the influx of these cells in the line 1 tumor and the nontumor antigen-related inflammatory reactions. Because our immune donor animals were immunized with a mixture of line 10 cells and BCG, these animals are immune to both. Line 10 immune spleen cells were restimulated in vitro with PPD and were radiolabeled. These PPD-restimulated immune spleen cells showed no preferential localization at the line 10 tumor challenge site but, as expected, a tendency for localization at the CFA (H37Ra) injection site. Furthermore, PPD-reactive spleen cells from BCG-immunized guinea pigs showed a significantly higher accumulation at the CFA injection site compared to the IFA injection site and the line 10 and line 1 tumor challenge site. From the results, it is concluded that line 10 tumor-immune and BCG-immune spleen cells are two distinct cell populations, and that the existence of cross-reacting antigens between BCG and the line 10 hepatocarcinoma are of no importance for the rejection of the line 10 tumor by immune spleen cells.     

Homologous recombination in variants of the B16 murine melanoma with reference to their metastatic potential

Genomic instability has been accepted as providing a phenotypic variety of malignant cells within a developing tumour. Defects in genetic recombination can often lead to phenotypic differences; therefore, it is possible that metastatic variant cell lines exhibit their particular phenotype as a result of an altered ability to catalyse homologous recombination. We have investigated recombination efficiency in B16 melanoma metastatic variants, using a plasmid, pDR, as a recombination substrate. The plasmid contains two truncated, nontandem but overlapping segments of the neomycin resistance gene (neo 1 and neo 2), separated by the functional gpt gene unit. Only a successful recombination of the two neo segments will generate a functionally intact neomycin gene. Extrachromosomal recombination here was a transient measure of the cells to recombine the neo fragments in an intra- or intermolecular manner. Extrachromosomal recombination frequencies were higher in the high metastasis variants (BL6, ML8) compared with the low metastatic F1 cells. On the other hand, the frequency of chromosomal recombination (after plasmid integration) was higher for the low metastasis (F1) cell line compared with the highly metastatic variants, BL6 and ML8. Since the recombination assay measures only successful recombination events, we have interpreted the observed higher incidence of chromosomal recombination in the low metastatic variant line as indicative of a more stable genome. Similarly, a higher inherent instability in the genome of the high metastasis variants would render these less efficient at producing and maintaining successful recombination events, and this was found to be true by Southern analysis. The results presented show that frequency of recombination may be adduced as evidence for implicating genomic instability in the generation of variant cell populations during metastatic spread. Such an interpretation is also compatible with the Nowell hypothesis for tumour progression. © 1996 Wiley-Liss, Inc.     

Phorbol 12-myristate 13-acetate enhances nm23 gene expression in murine melanocytes but not in syngeneic B16-Bl6 melanoma variants

The nm23 gene has been described as a potential metastasis suppressor gene in certain rodent and human tumors. We previously demonstrated that tyrosine and phenylalanine restriction suppresses metastatic heterogeneity of B16-BL6 murine melanoma and selects for tumor variants with decreased metastatic potential. In this study, we investigated nm23 expression in the highly metastatic B16-BL6 (ND) melanoma, its nutritionally derived poorly metastatic (LT) variant, and the syngeneic non-tumorigenic Mel-ab melanocytes. No differences in nm23 expression were observed between ND and LT cells, and nm23 expression varied between different isolates. Previously, we showed that metastatic potential of 1-ND cells decreases and is not altered in 1-LT cells after prolonged in vitro cell passage; however, nm23 expression is equivalently increased by 2-fold. In 2-ND and 2-LT cells, expression of nm23 is not different at higher in vitro cell passage. Expression of nm23 decreased about 2-fold when phorbol 12-myristate 13-acetate (PMA) was removed from Mel-ab cells, which induces these cells to become quiescent. Although membrane-associated protein kinase C (PKC) activity decreased after prolonged PMA treatment in all cells, neither nm23 expression nor proliferation of ND and LT cells was affected by PMA. These data indicate that nm23 expression is related to proliferative activity rather than to the suppression of metastatic potential. © 1996 Wiley-Liss, Inc.     

Passive transfer of tumor immunity with spleen cells from guinea pigs cured of hepatocarcinoma by nonspecific immunotherapy

Summary The purpose of this study was to evaluate cell-mediated tumor immunity in strain-2 guinea pigs cured of line-10 hepatocarcinoma by oil-in-water emulsions containing phenol-water extracts from either BCG or the Re mutant of Salmonella typhimurium (Re ET) admixed with mycobacteria glycolipid (P3). Treatment with these emulsions produced complete regression of established tumor nodules and prevented the growth of lymph node metastases in 25 of the 28 animals inoculated intradermally (ID) with 10⁶ line-10 cells and given intralesional immunotherapy 6 days later. No tumor regression was observed in animals given phenol-water extracts alone. Spleen cells, taken from guinea pigs cured of line-10 by BCG extract + P3 or Re ET + P3, were tested for their influence on tumor growth by means of an in vivo adoptive neutralization test (Winn test). Cell transfer was accomplished by the subcutanous injection of various concentrations of spleen cells admixed with 10⁵ viable line-10 cells. The results showed that as few as 10⁷ immune spleen cells completely inhibited the growth of 10⁵ tumor cells in 46–54% of the animals. The best tumor growth inhibition (77–85%) was observed in animals given 5 × 10⁷ immune cells admixed with 10⁵ tumor cells. The onset of transferrable tumor immunity was earlier in animals treated with the BCG extract + P3 than in those given the Re ET + P3. However, the duration of detectable tumor immunity was longer in the latter group. In contrast, no inhibition of tumor growth was observed in animals given spleen cells from normal or tumor-bearing guinea pigs. Moreover, spleen cells obtained from guinea pigs immunized with BCG extract + P3 or Re ET + P3 emulsions only and admixed with line-10 cells failed to transfer tumor immunity to normal animals. Thus, results from this study clearly demonstrated that cell-mediated tumor immunity was elicited in animals cured of line-10 tumor with combinations of P3 and phenol-water extracts of either BCG or Re mutant of S. typhimurium and that sensitized spleen cells effectively transferred systemic tumor immunity to normal recipients.