Specificity of the immune response leading to protection or enhancement by regressive and progressive variants of a rat colon carcinoma

Two cell clones, K12/TRb (PROb) and K12/TSb (REGb), have been isolated from the same serially transplantable tumor, DHD, established from a colon carcinoma chemically induced in the rat. Inoculation of REGb cells gives a tumor which regresses within 4 to 8 weeks and generates immune protection against subsequent injection of the progressive tumor cells, PROb. Inoculation of PROb cells gives a progressive tumor and generates tolerance allowing progressive growth of contralaterally injected REGb cells. Inoculation of REGb cells fully protects the host against growth of a DHD tumor graft, the tumor from which REGb and PROb cells were originally obtained. On the other hand, inoculation of REGb cells does not confer any protection against growth of 4 other syngeneic tumor grafts, DHA, DHB, DHC and DHE. These tumors were obtained from other colonic tumors induced as DHD by 1.2 dimethylhydrazine (DMH). Progressive growth of the tumor induced by inoculation of REGb cells is observed in animals bearing a contralateral DHD tumor, but not in animals bearing tumor from other transplantable lines, DHA, DHB, DHC and DHE. Our results show that immune enhancement of a regressive tumor and the immune protection that it confers constitute specific responses to a tumor-specific transplantation antigen present on a single transplantable colon tumor.     

In vivo and in vitro reactivity of rat spleen cells against regressor and progressor colon-cancer cell variants

Previous reports demonstrated that progressor and regressor tumor-cell variants isolated from the same colon carcinoma chemically induced in a BD-IX rat differed in their capacity to induce an immune response. The present study was aimed at analyzing the characteristics of the responses to the regressor REGb and progressor PROb clones. Spleen cells from rats bearing early REGb tumors neutralized PROb cell tumorigenicity in a Winn-type local transfer assay, but responded occasionally to REGb and PROb cells in vitro. However, spleen cells from rats immunized by several injections of REGb and PROb cells strongly proliferated when cultured with PROb or REGb cells. This response was selective for the cell lines generated from the individual tumor at the origin of PROb and REGb lines, was dependent on CD4⁺ spleen cells, and was partially inhibited by an antibody against major histocompatibility complex class-II molecules. Although PROb cells shared tumor-rejection antigen(s) with REGb cells, splenocytes from PROb tumor-bearing rats did not neutralize PROb-cell tumorigenicity in vivo, nor did they proliferate when cultured with PROb or REGb cells in vitro. The unresponsiveness of spleen cells from PROb tumor-bearing rats was not due to the presence of immune suppressive cells, and a defect of antigen-presenting cells was shown to be unlikely. This unresponsiveness was limited to a lymphocyte subpopulation, since spleen cells from tumor-bearing rats responded normally to stimulation by PHA or allogeneic lymphocytes. These results strongly suggest that unresponsiveness of spleen cells from tumor-bearing rats is due to a tumor-specific anergy of the lymphocyte clones able to respond to tumorassociated antigens.     

Microglial cells induce cytotoxic effects toward colon carcinoma cells: Measurement of tumor cytotoxicity with a γ-glutamyl transpeptidase assay

Activated macrophages have been shown to exert cytostatic and cytotoxic effects toward tumor cells via nitric oxide (NO) release. In the CNS, microglial cells are considered to be the main resident population of immune effector cells. In this study, cytotoxic activity of N11, an immortalized murine microglial cell line, toward rat progressive DHD/PROb and regressive DHD/REGb colon carcinoma cells was examined in parallel with NO production. Cytotoxicity was evaluated using a novel method, the γ-glutamyl transpeptidase (γ-GTP) assay, based on the fact that DHD tumor cells expressed high levels of γ-GTP activity, while no γ-GTP activity was found in cells of the monocyte/macrophage lineage. Results showed that activation of N11 cells by interferon-γ plus either lipopolysaccharide or tumor necrosis factor-α induced high amounts of NO release and cytotoxic effects toward DHD/PROb as well as DHD/REGb cells. NO release by activated N11 cells was augmented by addition of tumor cell-conditioned medium. Both NO release by N11 cells and cytotoxicity were blocked by addition of N^G-monomethyl-L-arginine (L-NMA), an inhibitor of NO synthase, suggesting that cytotoxicity was mediated by N11-derived NO. However, in the presence of L-NMA an increased production of interleukin-6 was also observed. In conclusion, in opposition to information obtained with brain-derived endothelial cells, brain-derived microglial cells did not differentiate between progressive and regressive clones of colon carcinoma cells. Our results point to a specific role for both endothelial and microglial cell types in the context of brain metastasis. Microglial cells can be cytotoxic for tumor cells, and this cytotoxicity is mediated by NO. Int. J. Cancer, 70:169–174, 1997. © 1997 Wiley-Liss, Inc.     

FAS(CD95) ligand expression by tumor cell variants can be unrelated to their capacity to induce tolerance or immune rejection.

According to the results of in vitro experiments, Fas(CD95) ligand expression by cancer cells might induce apoptosis of activated T cells and contribute to immune tolerance. However, Fas ligand expression had never been explored in vivo in tumor cell models yielding either immune response or tolerance. In the present study, we analyzed the expression and function of Fas ligand in 2 clones of tumor cells originating from the same rat colon carcinoma. REGb cells were immunogenic and yielded tumors that regressed in immunecompetent syngeneic hosts, whereas PROb cells induced active tolerance and yielded progressive tumors. Fas ligand was expressed on the plasma membrane of both REGb and PROb cells, and its cDNA sequencing showed no mutation. However, neither REGb nor PROb cells induced apoptosis of co-cultured Fas-sensitive target cells. Our results show that surface expression of Fas ligand by tumor cells does not always induce killing of adjoining Fas-sensitive cells and that tumor cells may induce a protective immune response or an active tolerance independently of Fas ligand expression.     

Characterization of autoantigen (p105) in a model of colonic adenocarcinoma in the rat]

Sera from BDIX rats bearing the syngeneic colon tumors PROb or REGb were analysed by Western blotting in order to detect a possible humoral response against the grafted tumor. The PROb clone grows progressively in syngeneic hosts and metastasizes, whereas the REGb clone grows slowly and then is rejected. This model was developed by F Martin and his group in Dijon, France. We observed that rats bearing PROb tumors only develop an antibody response against a water-soluble protein of 105 kDa (p105) which is expressed by both tumor clones. This antibody response has never been detected in rats bearing REGb tumors. The antigen p105 was also expressed by normal adult colon as well as some other foetal or adult tissues. It is also present in extracts from several tumor cell lines including human colorectal carcinoma cell lines. Moreover, the titer of detected antibody at day 30 was inversely correlated with the survival of rats after tumor inoculation, suggesting a possible facilitating role of this antibody response.     

Identification and characterization of a rat protein (p 105) auto-antigenic in rats bearing a progressive syngeneic colon carcinoma.

Sera from BDIX rats inoculated with 2 tumor clones derived from a single syngeneic colon carcinoma were assayed by Western blotting for the presence of antibodies against the grafted tumor. The PROb clone is progressive and produces metastases. We observed that rats bearing this tumor developed antibodies against an unglycosylated water-soluble protein of 105 kDa. The magnitude of this humoral response, as assessed by the intensity of the signal on immunoblots, was inversely correlated with survival of the rats. Furthermore, rats inoculated with the REGb clone, which is immunologically rejected, never developed detectable antibodies against the tumor. Antisera from rats injected with PROb tumor detected p105 antigen in cellular extracts from the REGb clone and from a series of rat and human cell lines. This protein was also detected in variable amounts in some normal adult and fetal tissues. Treatment of PROb or REGb cells by either interferon-gamma or heat shock did not significantly alter the expression of the p105 auto-antigen.     

In vitro proliferative responses of spleen lymphocytes from rats bearing progressive or regressive tumors induced by cell variants of a syngeneic colon carcinoma

From one colonic carcinoma chemically induced in the rat, 2 sublines of tumor cells have been cloned, one (PROb) inducing progressive tumors, the other (REGb) generating tumors that regress a few weeks after s.c. injection into syngeneic hosts. Our study was aimed at comparing cellular immunity between animals bearing PROb or REGb tumors. Spleen cells were first tested for in vitro proliferation in response to mitomycin-treated PROb or REGb cells. Only spleen cells from rats injected with REGb cells proliferated significantly when mixed with PROb or REGb cells. The proliferative response induced by REGb cells was considerably higher than the response to PROb cells. When spleen cells from rats bearing REGb tumors were cultured with a mixture of REGb and PROb cells at various PROb/REGb cell ratios, PROb cells significantly suppressed the strong proliferative response generated by the same number of REGb cells alone. REGb-immune spleen cells, after in vitro stimulation by PROb or REGb cells, were not cytotoxic for either cell variant. REGb-immune spleen cells did not differ in their content of T lymphocytes expressing CD4 or CD8 markers when they were stimulated by PROb or REGb cells in vitro, but REGb cells induced a larger number of activated lymphocytes expressing the IL-2 receptor. Our results indicate that, compared to REGb cells, PROb cells are poorer stimulators of proliferation of tumor-immune spleen cells, and that they are able to suppress the proliferative response induced by REGb cells.     

Correlation between cell surface oligosaccharides and tissue target-selective adhesion of two rat adenocarcinoma cell lines

We observed that two rat colon adenocarcinoma variants originating from a single parental cell line and differing by their progressive and metastatic capacities in syngeneic BDIX rats differed by their organ distribution after intravenous injections. The PROb cells accumulated in the lung, wherefrom the REGb cells were rapidly cleared. In order to explore the role of cell surface glycoconjugates in organ-specific metastasis, cytofluorometric and histochemical studies using labelled lectins were performed. This revealed that the metastatic variant PROb presented more alpha-L-Fuc(1----2) beta D-Gal-R structures than the regressive nonmetastatic variant REGb. At variance, REGb cells exposed more D-galactosyl and N-acetyl-D-galactosaminyl residues than PROb cells. Monosacharides inhibited specifically cell adhesions on frozen organ sections. L-Fuc and N-acetyl-D-galactosamine (D-GalNAc) most strongly inhibited the adhesion of PROb cells on lungs, whereas D-Gal and D-GalNAc most strongly inhibited that of REGb cells. On the liver, adhesions of both cell lines were inhibited by D-Gal and D-GalNAc. These observations support the involvement of sugar-lectin receptors in the adhesion of these cells to the lungs or liver. The possible involvement of previously described lectins is discussed.     

Relationship between sensitivity to natural killer cells and MHC class-I antigen expression in colon carcinoma cell lines.

The sensitivity of colorectal tumors to NK-cell-mediated cytotoxicity and their expression of major histocompatibility complex (MHC) class-I antigens were studied in an attempt to determine whether such antigens play a role in the susceptibility of colorectal tumors to NK-cell lysis. In a rat colon-carcinoma model, 2 clones differing in their sensitivity to NK-cell-mediated cytotoxicity were tested for class-I expression; it was seen that the more sensitive cells (REGb) expressed less class-I products than did the resistant cells (PROb). However, when MHC class-I antigen expression was increased by IFN-gamma treatment, no change in NK-cell lysis was found with the PROb cells, while an increase in cytotoxicity was obtained with the REGb cells. After in vivo or in vitro selection of NK-resistant REGb cells, we observed in the selected cells an important decrease in RT-I class-I antigen expression. Fifteen different human colorectal cell lines were also studied for HLA class-I expression and NK-cell susceptibility, and no quantitative correlation between these 2 features was seen. However, cell lines which were deficient in HLA class-I antigens were more sensitive than class-I-positive cells.     

Colon-cancer cell variants producing regressive tumors in syngeneic rats,unlike variants yielding progressive tumors,attach to interstitial collagens through integrin α2β1

Nine clones of tumor cells, derived from a single rat colon carcinoma, were analyzed for their adhesive properties and in vivo growth patterns. Four clones (denoted REG) gave rise to regressively growing tumors. Cells from the 4 REG clones attached significantly better to collagen types I and III than did cells from the 5 clones (denoted PRO) which grew progressively in vivo. In contrast, REG and PRO clones did not differ in their attachment to collagen type IV, laminin or fibronectin. The attachment of REG cells to collagen was dependent on Mg²⁺, but not Ca²⁺. Monospecific rabbit IgG to rat integrin β₁-chain inhibited REG cell attachment to collagen, demonstrating involvement of a β₁ integrin in this process. PRO and REG cells expressed an underglycosylated β₁ chain (M_r ∼ 105,000) that was somewhat smaller than β₁-chains described previously on rat fibroblasts and hepatocytes (M_r ∼ 115,000). Monoclonal IgG to rat integrin α₂β₁, but not to α₁β₁, readily inhibited REG cell attachment to collagen, demonstrating the involvement of integrin α₂β₁. However, β₁ and α₂ integrin subunits were found in purified glycoproteins from both PRO and REG cells. This suggests that α₂β₁ integrin is expressed by both cell variants, but is functional as a collagen receptor on REG cells only. In this system of tumor-cell variants, the clear-cut differences in attachment to interstitial collagens of the 9 clones suggest a possible relationship between this attachment and the capacity to induce progressive or regressive tumors. © 1996 Wiley-Liss, Inc.     

Plasminogen receptors on rat colon carcinoma cells.

Cells from rat carcinoma cell lines PROb (giving progressive tumours) and REGb (giving regressive tumours) have cell surface receptors which bind specifically rat plasminogen and plasmin. Affinity for Pg was found to be higher in PROb (Kd = 10(-7) M) than in REGb cells (Kd = 5.10(-7) M) but with a concomitant decrease in the number of binding sites, 0.9 x 10(6)/cell (range from 0.6 to 1.2 x 10(6)) in PROb vs 3.6 x 10(6)/cell (range 1.2 to 6 x 10(6)) in REGb cells. The number and the affinity of binding sites varied in an opposite way in PROb and REGb cells. The difference in affinity parameters was unrelated to the degree of invasiveness of tumour cells in syngenetic rats. Bound plasmin retained its enzymatic activity, which indicates that its binding does not involve the catalytic active site. In cell solubilisates plasminogen receptor appeared as one major band situated in the area of 50-60 kDa.     

Cinchonine, a potent efflux inhibitor to circumvent anthracycline resistance in vivo.

Circumvention of multidrug resistance is a new field of investigation in cancer chemotherapy, and safe and potent multidrug resistance inhibitors are needed for clinical use. We investigated several analogues of quinine for their ability to increase anthracycline uptake in resistant cancer cells. Cinchonine was the most potent inhibitor of anthracycline resistance in vitro, and its activity was little altered by serum proteins. Serum from rats treated with i.v. cinchonine produced greater uptake of doxorubicin in cancer cells (DHD/K12/PROb rat colon cells and K562/ADM human leukemic cells) than did serum from quinine-treated rats (ex vivo assay). Cinchonine was more effective than quinine in reducing tumor mass and increasing the survival of rats inoculated i.p. with DHD/K12/PROb cells and treated i.p. with deoxydoxorubicin. Moreover, the acute toxicity of cinchonine in rats and mice was lower than that of other quinine-related compounds. The lower toxicity and greater potentiation of in vivo anthracycline activity produced by cinchonine are favorable characteristics for its use as an anti-multidrug resistance agent in future clinical trials.     

In vivo and in vitro invasiveness of a rat colon-cancer cell line maintaining E-cadherin expression: An enhancing role of tumor-associated myofibroblasts

In various cell systems, an inverse relationship was found between expression of E-cadherin, a molecule involved in the Ca²⁺-dependent homophylic cell-to-cell attachment of epithelial cells, and the capacity to invade extracellular matrix gels or normal tissues in vitro., DHD/K12/TRb (PROb) cells, maintained as a cell line derived from a rat colon carcinoma, homogeneously expressed. in vitro immunoreactive E-cadherin, which was functional as shown in cell dissociation-reassociation assays. PROb cells were found to be non-invasive in 3 different assays in vitro., However, tumors resulting from a s.c. injection of PROb cells into syngeneic BD-IX rats were invasive, although PROb cells maintained E-cadherin expression in the tumors. Cells from a freshly dissociated PROb tumor showed, not only PROb cells but also tumor-associated myofibrobfasts and were able to cross a Matrigel-coated filter. PROb tumors were indeed infiltrated by numerous myofibroblasts, mainly located at the invasive edge of the tumor. Cells from an established culture of tumor-infiltrating myofibroblasts were able to confer upon PROb cells invasiveness through Matrigel-coated filter or into chick-heart fragments. PROb cells maintained their capacity to express E-cadherin after myofibroblast-enhanced Matrigel invasion. Tumor-associated myofibroblasts, but not PROb cells, secreted a 72-kDa collagenase that could play a role in tumor-cell invasion. These results strongly suggest that cells from the tumor stroma, and more specifically myofibroblasts, may be involved in the invasiveness of epithelial tumor cells in vivo, even when E-cadherin expression prevents tumor-cell invasiveness in different in vitro assays.     

Involvement of histo-blood-group antigens in the susceptibility of colon carcinoma cells to natural killer-mediated cytotoxicity.

The susceptibility to natural-killer-cell lysis and expression of histo-blood-group antigens of 2 clones from a rat colon adenocarcinoma, of variants derived from them and of 17 human colon carcinoma cell lines were assessed in an attempt to determine if the major glycosidic tissue antigens of epithelial cells could influence the NK susceptibility of tumor target cells of epithelial origin. The rat REGb clone, which is relatively NK-sensitive, expressed higher levels of precursor structures T and Tn and lower levels of H antigenic determinants than the PROb clone, which displays higher resistance to NK-cell lysis. Cell variants were obtained from these 2 clones; it appeared that whether the cell variants were selected on the basis of expression of a blood-group antigenic determinant or on the basis of altered susceptibility to NK-cell lysis, there was a link between increased resistance and higher expression of cell-surface A and H histo-blood-group antigens, or conversely, between increased sensitivity and higher expression of precursor structures. Similar conclusions were obtained upon study of the human cell lines, since a significant correlation was found between the level of expression of T or Tn antigens and sensitivity to NK-cell lysis. A significant relationship was found between the expression of Lewis antigens and increased resistance to NK-cell-mediated cytotoxicity.     

Possible involvement of TGF beta 1 in the distinct tumorigenic properties of two rat colon carcinoma clones.

The presence in tumors of numerous cytokines suggests that they potentially modulate tumor cell activities and host tissue remodelling. To investigate the possible involvement of transforming growth factor type beta (TGF beta) in the metastatic process of cancer development, we have studied the effect of this factor on two rat colon carcinoma cell lines. These cell clones had been previously tested and selected for their ability to develop metastases in syngenic animals or lack of it. The two cell lines were characterized for their production of TGF beta. Production of active and latent forms of TGF beta 1 in the medium conditioned by the rat colon cancer cells were quantified using a bioassay. The presence of active TGF beta 1 was demonstrated in conditioned medium from the progressive tumor (PROb) cells and significant expression of latent forms of TGF beta 1 were found in the conditioned media from both cell clones. TGF beta 1 slightly inhibited proliferation of PROb cells which had been previously described as moderately differentiated, and significantly stimulated proliferation of the regressive (REGb) cells, described as poorly differentiated. On the basis of our observations, we suggest that this endogenous factor could be involved in autocrine regulation of tumor cell activities and in paracrine regulation of stroma cell and immune responses. Active and/or latent expression of TGF beta 1 by the two rat colon carcinoma cell lines, and their variable responses to the growth factor, strongly suggest that this polypeptide is involved in the regulation of tumorigenic expression of adenocarcinoma cells.     

Epinephrine enhances penetration and anti-cancer activity of local cisplatin on rat sub-cutaneous and peritoneal tumors.

Despite the theoretical advantages of a high local concentration of anti-cancer drugs, local chemotherapy often fails to produce complete and lasting responses in experimental and human solid tumors. Experiments using Patent Blue dye showed that fluids diffused poorly into tumor mass when injected inside or around s.c. rat tumors in rats. In the same way, Patent Blue dye distributed poorly from the peritoneal cavity into the tumor nodules of rats with peritoneal carcinomatosis. The potent vasoconstrictor, epinephrine (1 mg/kg of body weight) was shown to facilitate the penetration of Patent Blue dye into s.c. and peritoneal rat tumors. Platinum concentration evaluated by micro-PIXE in s.c. DHD/K12/ PROb colon tumors or by atomic absorption spectrometry in DHD/K12/PROb peritoneal tumors was 4- to 12-fold higher when epinephrine was added to local cisplatin. Peri-tumoral or intra-tumoral injection of cisplatin (2 mg/kg) alone does not cure s.c. DHD/K12/PROb colon tumors or GV1A1 glioma tumors in BD IX rats. By contrast, a complete and lasting cure of s.c. tumors was achieved regularly and without skin necrosis when epinephrine was added to intra-tumoral or peri-tumoral cisplatin. Rats with peritoneal-tumor nodules 1 to 2 mm in diameter, and insensitive to i.p. cisplatin alone, were cured when the anti-cancer drug was combined with epinephrine. These experimental results could justify clinical trials using a combination of cisplatin and epinephrine in the treatment of locally growing solid tumors.     

Effects of a single injection of anti-asialo GM1 serum on natural cytotoxicity and the growth of a regressive colonic tumor in syngeneic rats

The REGb tumor cell line is a cloned variant of the DHD-K12 cell line, established from a colon carcinoma chemically induced in the rat. Unlike the parent DHD-K12 cell line, or other clones, which give progressive tumors when inoculated to the syngeneic rat, REGb cells produce tumors which regress in 3 to 5 weeks and never cause metastasis. In order to explore the role of natural killer (NK) cells in REGb tumor regression, each rat was given one injection of anti-asialoGM1 (anti-asGM1) serum, a known inhibitor of NK activity. This injection was done 24 hr before REGb cell challenge. This injection significantly depressed the in vitro cytotoxicity of peripheral blood lymphocytes on REGb cells for 2 weeks. REGb tumors grew larger and regressed later in the treated animals than those in the controls. Furthermore, a progressive or recurrent tumor was observed in 4 out of 10 treated rats, giving lung and/or lymph-node metastases in 2 cases. Immuno-histological study of the cells infiltrating the REGb tumors in control and treated animals showed a decrease number of asGM1+ and OX8+ lymphocytes, presumably NK cells, after anti-asGM1 treatment. An increase in number of macrophages was demonstrated in the progressive tumors of treated animals. These results suggest that NK cells play an important role in the initial stage of the regression TSb tumors in untreated syngeneic rats.     

Failure of expression of class I major histocompatibility antigens to alter tumor immunogenicity of a spontaneous murine carcinoma

We have shown previously that clonal immunogenic variants of murine mammary adenocarcinoma 10.1 can be isolated after treatment in vitro with the DNA-hypomethylating agent 5-azacytidine (5-aza). Such immunogenic variants frequently express elevated class I major histocompatibility complex antigens relative to the level of expression in the parent tumor and are rejected in syngeneic mice by a T-cell-dependent process. To ascertain whether elevated immunogenicity is a function of increased class I antigen expression, we isolated high class I antigen expressors from 5-aza-treated 10.1 cells by using the fluorescence-activated cell sorter. Clonal variants displaying any increase in class I antigen expression were more efficient stimulators of allo-class I antigen-specific cytolytic T-cell precursors. However, these variants displayed unaltered tumorigenicity in immunocompetent syngeneic mice. Thus, phenotypic changes other than, or in addition to, elevated class I antigen expression cause the reduced tumorigenicity of immunogenic clones of 10.1 cells isolated after 5-aza treatment.     

A cell type-specific and gap junction-independent mechanism for the herpes simplex virus-1 thymidine kinase gene/ganciclovir-mediated bystander effect.

Tumor cells expressing the herpes simplex virus type 1 thymidine kinase (HSV-tk) gene are killed by nucleoside analogues such as ganciclovir (GCV). GCV affects not only the cells expressing HSV-tk but also neighboring cells that do not express the gene; this phenomenon commonly is called "bystander effect." GCV metabolites transfer via gap junctional intercellular communication (GJIC) accounts for the bystander effect in different cell lines, but other mechanisms have also been described. In this study, we analyzed the mechanisms of the bystander effect in two cell lines exhibiting different capacities of communication (DHD/K12 and 9L). The 9L cells exhibited a very good bystander effect, which was completely blocked by a long-term inhibitor of GJIC, 18 alpha-glycyrrhetinic acid. DHD/K12 cells exhibited a moderate bystander effect that was not abolished by 18 alpha-glycyrrhetinic acid or 1-octanol, another strong inhibitor of GJIC. Interestingly, we also observed a bystander effect in cultures where HSV-tk-expressing DHD/K12 cells were physically separated from their untransfected counterparts but grown in the same medium. Moreover, the transfer of filtered conditioned medium from GCV-treated HSV-tk-expressing DHD/K12 cells to DHD/K12 parental cells induced a decrease of survival in a concentration-dependent manner, suggesting that the bystander effect in this cell line was mediated by a soluble factor.