Correlation between quantitative expression of H-2K, H-2D and MuLV antigens on spontaneous AKR lymphomas

Thymocytes of leukemic AKR mice were analysed with a fluorescence-activated cell sorter (FACS) using monoclonal antibodies (MAbs) reactive with H-2Kk and H-2Dk and conventional hetero-antibodies against MuLV gp-70 antigens. Comparison was made with thymocytes of the low-leukemia H-2k strain C3H, as well as with thymocytes of young AKR mice, late preleukemic AKR mice (6-9 months old) and mitogen-stimulated thymocytes of young AKR mice. Expression of H-2 antigens increased on cells of the majority of tumors, on thymocytes of some late preleukemic mice and on mitogen-stimulated thymocytes. An increase in H-2K and H-2D antigens was noted: imbalance of the K/D ratio in favor of H-2D was observed mostly in tumors with relatively lower total amounts of H-2K and D antigens; ratio shifts in favor of H-2K were also found, mostly in tumors with relatively higher amounts of H-2K and D antigens. Increased expression of MuLV antigens was found on cells of all tumors and on thymocytes of several late preleukemic mice. We show that in primary spontaneous AKR leukemias, in spite of large individual differences, the expression of high amounts of MuLV gp70 is not random, but is associated with high expression of H-2K and H-2D antigens. The same phenomenon is seen in thymocytes of preleukemic mice, but in mitogen-stimulated thymus cells increase of H-2 expression is not accompanied by increase of MuLV gp70.     

Analysis of the expression of H-2 and H-2-linked antigens on mammary tumor cells.

The products of the recently discovered H-2L locus were expressed on BALB/c mammary tumor cells and behaved as histocompatibility antigens, in contrast to the products of H-2 linked loci (Qa-loci) that did not influence the acceptance or rejection of tumor transplants.     

Reduction of metastatic rate by immunotherapy: a comparison of the immunogenic properties of metastasizing tumor cells versus tumor cells in the primary mass.

The vasculature of a poorly immunogenic, highly metastatic transplantable fibrosarcoma (T-241) maintained in the femoral muscle of C57BL/6J mice was perfused. This permitted collection of tumor cells which had invaded into the tumor vascular channels (ie, metastasizing tumor cells). Also collected as a separate population were tumor cells from the primary tumor mass. Immunization was carried out with these cell populations in conjunction with BCG and the effect on the growth of primary tumor and metastatic rate was evaluated following rechallenge with unfractionated tumor cells. The rate of tumor growth at the primary site was not affected by any of the immunization schedules. However, immunization with venous effluent cells (metastasizing tumor cells) and BCG was two times more effective in reducing the number of pulmonary metastases than immunization using tumor cells isolated from the primary tumor mass. Passively transferred spleen cells from donors immunized with the cell populations listed above had exactly the same effect, that is, no effect on the growth of the primary tumor, but a dramatic reduction in the metastatic rate when effluent tumor cells were used to immunize cell donors. The data point to an antigenic heterogeneity with this particular transplantable tumor.     

Expression of H-2 antigens and inducibility of antitumor immune responses in various tumor cell clones established from methylcholanthrene-induced fibrosarcomas

A large number of fibrosarcoma cell lines was established in vitro from a tumor mass induced freshly by inoculating 3-methylcholanthrene (MCA) subcutaneously (sc) into C3H/HeN mice, and more than five clones were isolated from each cell line by the limiting dilution technique. The present study investigated a) qualitative and quantitative comparison of the immunogenicity [tumor-associated transplantation antigen (TATA) activity] of different tumor clones and b) the relationship between such immunogenicity and the expression of H-2 class I antigens. When TATA were compared between different clones from the same tumor, these TATA were revealed to be cross-reactive to each other. On the other hand, the comparison of TATA between clones from different tumors demonstrated the existence of individually unique TATA in these clones. In addition to qualitative heterogeneity of TATA from different tumors, the magnitude of immunogenicity was also heterogeneous in the individual clones established. Whether or not such quantitative heterogeneity of immunogenic strength was related to the expression of H-2 (class I) antigens was examined by flow microfluorometry studies using anti-H-2k antibodies. The results demonstrated that there was no correlation between TATA activity capable of inducing in vivo tumor resistance and the expression of H-2 antigens. This contrasted with parallelism between the expression of H-2 antigens and inducibility of cytotoxic T lymphocytes (CTL) or lysability of tumor cell clones by CTL. These results are discussed in the context of the cellular mechanism of tumor cell eradication in vivo and the regulation of cell surface H-2 expression in vitro and in vivo.     

The inhibition of lymphocyte stimulation by autologous human metastatic melanoma cells correlates with the expression of HLA-DR antigens on the tumor cells

Previous studies indicated that peripheral blood lymphocytes from patients (Pt-PBL) with lymph node metastatic melanomas proliferated in vitro and developed into tumor-restricted cytotoxic lymphocytes in response to alloantigens or interleukin 2 (IL-2). However, Pt-PBL were not stimulated by irradiated autologous metastatic melanoma (Auto-Me) cells. In the present study we report that the lack of stimulatory activity of Auto-Me cells may be due to a suppressive effect exerted by Auto-Me cells on the responder lymphocytes. In fact, we found that in 62% of cases examined, the addition of 5-10% Auto-Me cells to Pt-PBL cultures strongly inhibited both proliferation and the generation of tumor cytotoxic lymphocytes induced by alloantigens or IL-2. The inhibition was dose-dependent and tumor-restricted, and was not due either to toxicity, medium depletion or IL-2 absorption by Auto-Me cells. Normal fibroblasts, K562 cells and autologous E- lymphocytes were not suppressive. Auto-Me cells were able to inhibit Pt-PBL responses only when added during the first 24 h of culture and not later. Phenotypic analysis of Auto-Me cells using monoclonal antibodies directed against HLA-A,B,C, HLA-DR and melanoma-associated antigens revealed that the expression of high levels of DR antigens on Auto-Me cells was associated with an elevated suppressive activity. Conversely, Auto-Me cells with low or undetectable levels of DR antigens were not inhibitory. Furthermore, the increased expression of DR antigens on Auto-Me cells obtained by in vitro treatment with human interferon gamma (IFN-γ) also resulted in an increased suppressive activity. We conclude that HLA-DR+ metastatic melanoma cells can interfere with the generation of an anti-tumor immune response, thus potentially favoring the escape of the tumor from the host's control mechanism.     

Upregulated phospholipase D2 expression and activity is related to the metastatic properties of melanoma

The incidence rates of melanoma have increased steadily in recent decades and nearly 25% of the patients diagnosed with early-stage melanoma will eventually develop metastasis, for which there is currently no fully effective treatment. The link between phospholipases and tumors has been studied extensively, particularly in breast and colon cancers. With the aim of finding new biomarkers and therapeutic options for melanoma, the expression of different phospholipases was assessed in 17 distinct cell lines in the present study, demonstrating that phospholipase D2 (PLD2) is upregulated in metastatic melanoma as compared to normal skin melanocytes. These results were corroborated by immunofluorescence and lipase activity assays. Upregulation of PLD2 expression and increased lipase activity were observed in metastatic melanoma relative to normal skin melanocytes. So far, the implication of PLD2 activity in melanoma malignancies has remained elusive. To the best of our knowledge, the present study was the first to demonstrate that the overexpression of PLD2 enhances lipase activity, and its effect to increase the proliferation, migration and invasion capacity of melanoma cells was assessed with XTT and Transwell assays. In addition, silencing of PLD2 in melanoma cells reduced the metastatic potential of these cells. The present study provided evidence that PLD2 is involved in melanoma malignancy and in particular, in its metastatic potential, and established a basis for future studies evaluating PLD2 blockade as a therapeutic strategy to manage this condition.     

Modulation by IFN-gamma of the metastatic ability of murine, human, and H-2-transfected tumor cells

Interferon-gamma (IFN-gamma) can enhance the experimental metastatic ability of B16 melanoma. The in vitro treatment with IFN-gamma of four clones derived from the murine mammary adenocarcinoma TS/A increased the number of lung colonies observed after intravenous injection in syngeneic mice. The spontaneous metastatic ability of these clones was not altered by the IFN-gamma pretreatment nor by daily intratumor injection of low-dose IFN-gamma. The experimental metastatic ability in nude mice of the human rhabdomyosarcoma cell line RD was decreased by in vitro pretreatment with IFN-gamma. To study the role played by major histocompatibility complex gene products in the IFN-gamma-mediated enhancement of B16 experimental metastasis, a mutant B16 clone, B78H1, was transfected with the H-2Kb gene. B78H1 cells are not capable of expressing H-2b even after treatment with IFN-gamma; IFN-gamma readily induced high levels of H-2Kb in a set of transfected clones, but did not enhance their experimental metastatic ability.     

Resistance to NK and metastatic potential of fibrosarcoma cells is associated with products encoded by the H-2D region.

We have used the murine 3-methylcholanthrene induced T10 fibrosarcoma tumor cell system originating in (C3II/en x C57BL/6)F1 mice (H-2b x H-2k) to elucidate the possible correlation between metastatic potential, expression of individual H-2 antigens and susceptibility to NK cells. Transfection of the non metastatic and NK sensitive IC9 cells (Db+, Kk, Kb, Kk-) with the H-2Dk gene, altered the metastatic phenotype of the parental cells, yet had no effect on the susceptibility of these tumor cells to lysis by NK and did not elicit a specific CTL response in syngeneic hosts. Variants of the metastatic and NK resistant IE7 clone (Db+, Kk-, Kb-, Kk-), lacking H-2Dk, were selected by treatment with monoclonal anti H-2Dk antibodies and complement. These variants were sensitive to NK and poorly or non metastatic. Transfection of Dk negative variants with the H-2Dk gene, resulted in the isolation of several clones which expressed a wide range of metastatic phenotypes but maintained sensitivity to NK. In addition, by cloning the cDNA of the H-2Dk gene of the metastatic T10-IE7 variant cells and analyzing its nucleotide sequence, we found four single nucleotide changes. Two of them are not expected to alter the encoded amino acids, whereas the others should result in two amino acid substitutions in the alpha-2 domain of the class I H-2Kd protein product. These changes might account, at least partially, for the failure of the transfection of H-2Dk to restore resistance to NK.(ABSTRACT TRUNCATED AT 250 WORDS)     

Induction of a tumor with greatly increased metastatic growth potential by injection of cells from a low-metastatic H-2 heterozygous tumor cell line into an H-2 incompatible parental strain

An H-2 heterozygous sarcoma, MDAY, originally induced with methylcholanthrene in an (A × DBA/2)F₁ (H-2^a × H-2^d) hybrid host was selected for growth in the H-2^d homozygous parental DBA/2 strain by serial intraperitoneal transplantation of ascites tumor cells. An apparent variant, designated MDAY-D2, was obtained which showed the expected loss of the private and public H-2K^k haplotype antigens normally associated with the A strain parent and the original MDAY tumor. Comparison of the original and variant lines revealed a wide variety of cell surface antigen and receptor differences. Both tumors were found to be highly anaplastic and histologically unclassifiable. Examination of the two tumor lines growing in vivo revealed a remarkable difference in their metastatic growth potential. The original MDAY line showed little propensity to spread to any organ site, with the occasional exception of liver, after subcutaneous inoculation of (A × DBA/2)F₁ mice. In striking contrast, there was a rapid and massive spread of MDAY-D2 to liver, spleen, lungs and kidneys within 12–16 days: liver and spleen could be totally replaced by tumor within 2–3 weeks. These characteristics were observed in both (A × DBA/2)F₁ and DBA/2 mice. The tendency to metastasize, as well as loss of the H-2K^k haplotype, appeared stable and irreversible. Although the precise origin of MDAY-D2 is not clear, its metastasizing properties are unique, making it a useful and desirable model to study the biology of metastasis.     

High expression of shMDG1 gene is associated with low metastatic potential of tumor cells

Metastasis is the primary cause of mortality associated with cancer. Molecular mechanisms leading to metastatic spread are poorly studied. To get a better understanding of this process, we compared the gene expression pattern of two isogenic cell lines, HET-SR and HET-SR1 (Rous Sarcoma Virus-transformed embryo hamster fibroblasts) with different metastatic activity using the differential display technique. A novel cDNA of hamster gene shMDG1 (Syrian hamster homologue of microvascular differentiation gene 1), which had 94% homology with rat MDG1 gene, was identified. Expression of shMDG1 was increased in low metastatic HET-SR cell line in comparison to high metastatic HET-SR1. Sequence analysis of the ORF of shMDG1 gene showed that it belongs to the DnaJ/heat-shock proteins of 40 kDa (HSP40) chaperones family, considered to function as a cochaperone of HSP70 family. In order to confirm involvement of shMDG1 in metastasis, we injected parental and shMDG1 overexpressed cells into animals. We showed that overexpression of the shMDG1 gene significantly diminished the metastatic activity of both HET-SR and HET-SR1 cells. The shMDG1-induced repression of metastasis was not connected with alterations in cell proliferation and motility in vitro, but correlated well with a decrease in content of the Asn-linked beta1-6 branched oligosaccharides on cell surface.     

The differential staurosporine-mediated G1 arrest in normal versus tumor cells is dependent on the retinoblastoma protein

Previously, we reported that breast cancer cells with retinoblastoma (pRb) pathway-defective checkpoints can be specifically targeted with chemotherapeutic agents, following staurosporine-mediated reversible growth inhibition in normal cells. Here we set out to determine if the kinetics of staurosporine-mediated growth inhibition is specifically targeted to the G(1) phase of cells, and if such G(1) arrest requires the activity of wild-type pRb. Normal human mammary epithelial and immortalized cells with intact pRb treated with low concentrations of staurosporine arrested in the G(1) phase of the cell cycle, whereas pRb-defective cells showed no response. The duration of G(1) and transition from G(1) to S phase entry were modulated by staurosporine in Rb-intact cells. In pRb(+) cells, but not in Rb(-) cells, low concentrations of staurosporine also resulted in a significant decrease in cyclin-dependent kinase 4 (CDK4) expression and activity. To directly assess the role of pRb in staurosporine-mediated G(1) arrest, we subjected wild-type (Rb(+/+)) and pRb(-/-) mouse embryo fibroblasts (MEFs) to staurosporine treatments. Our results show that whereas Rb(+/+) MEFs were particularly sensitive to G(1) arrest mediated by staurosporine, pRb(-/-) cells were refractory to such treatment. Additionally, CDK4 expression was also inhibited in response to staurosporine only in Rb(+/+) MEFs. These results were recapitulated in breast cancer cells treated with siRNA to pRb to down-regulate the pRb expression. Collectively, our data suggest that treatment of cells with nanomolar concentrations of staurosporine resulted in down-regulation of CDK4, which ultimately leads to G(1) arrest in normal human mammary epithelial and immortalized cells with an intact pRb pathway, but not in pRb-null/defective cells.     

Abolishment of metastasis formation by murine tumor cells transfected with "foreign" H-2K genes

High metastatic, low immunogenic Lewis lung carcinoma clones express low levels of H-2Kb major histocompatibility complex antigens. These cells metastasize spontaneously in mice with C57BL/6 genetic background possessing the H-2Db locus. Transfection of different H-2K genes abrogates metastasis in H-2K, H-2D compatible mice and in C57BL/6 recipients. The transfected cells are potent inducers of H-2K-restricted and alloreactive cytotoxic lymphocytes that kill H-2K-positive cells and cross-react with parental nontransfected cells.     

Immuno-selection in vivo of H-2D phenotypic variants from a metastatic clone of sarcoma cells results in cell lines of altered metastatic competence

To find out whether manipulation of H-2 expression on metastatic cells could alter their metastatic properties, we immunoselected in vivo H-2 antigen variants from a metastatic clone of the T10 sarcoma [originating in a (C57BL/6J X C3H.eB)FI mouse] and tested their metastatic capacity. The unselected metastatic cells (IE7) were previously found to express H-2Db and H-2Dk antigens, but they did not express the H-2K antigens of either parental haplotype. Transplantation of IE7 cells into C57BL/6J irradiated mice resulted in loss of H-2Dk expression and a reduction in H-2Db antigen density. Further transplantation of these cells into non-irradiated C57BL/6J mice led to a total loss of H-2 expression. The cells concomitantly lost their metastatic potency. Immunoselection of IE7 cells in C3H.eB irradiated and non-irradiated mice resulted in cells which were H-2Dk-positive but H-2Db-negative. Cells of these selected variants not only retained their metastatic potential, but in fact were far more metastatic than the unselected IE7 cells. Thus, changes in H-2 expression on tumor cells may alter their metastatic potential. In the case of T10 cells, H-2Dk expression seems to be directly involved in their metastatic capacity.     

Expression of metastatic potential of tumor cells in young nude mice is correlated with low levels of natural killer cell-mediated cytotoxicity

Of 6- and 3-week-old nude mice given intravenous injections of murine tumor cells with well-defined metastatic properties, only the 3-week-old mice developed lung tumor colonies in significant numbers. The quantitative differences in metastatic potential among tumor cell lines injected into syngeneic recipients were also maintained following intravenous injection into young nude mice. Successful metastasis in 3-week-old nude mice is correlated with the low levels of natural killer cell activity detected in these young recipients. Boosting of the natural killer cell activity of 3-week-old nude mice by the administration of bacterial adjuvants and interferon inducers significantly inhibited metastasis formation. The differences in metastasis development could not be attributed to differences in the initial arrest of tumor cells in the pulmonary vascular bed, but rather to a better survival of the arrested cells in the lungs of 3-week-old nude mice as compared with 6-week-old counterparts. We conclude that low levels of NK cell activity are associated with increased incidence of experimental metastasis.     

Increased plasminogen binding is associated with metastatic breast cancer cells: differential expression of plasminogen binding proteins.

Overexpression of urokinase-type plasminogen activator and its receptor correlates with metastatic capacity in breast cancer. In this study we show that the urokinase/urokinase receptor-overexpressing, metastatic human breast cancer cell line MDA-MB-231 (1) bound significantly more cell-surface plasminogen in a lysine-dependent manner and (2) was capable of generating large amounts of plasmin compared with the non-metastatic cell lines MCF-7 and T-47D. In addition, distinct plasminogen binding proteins were detected in the plasma membranes of the cell lines, suggesting heterogeneity of binding proteins. Plasminogen binding was analysed using a combination of dual-colour fluorescence flow cytometry and ligand histochemistry (for comparative and cellular localization of ligand binding), and fluorimetry (for Scatchard analysis). Apart from revealing the greater plasminogen binding capacity of MDA-MB-231 cells, flow cytometry and histochemistry also revealed that, in all three cell lines, non-viable or permeabilized cells bound significantly more plasminogen in a lysine-dependent manner than viable or non-permeabilized cells. Viable MDA-MB-231 cells bound plasminogen with moderate affinity and high capacity (Kd = 1.8 microM, receptor sites per cell 5.0 x 10(7). Our results indicate that differences in cell surface-specific plasminogen binding capacity between cell lines may not be detectable with binding techniques that cannot distinguish between viable and non-viable cells.     

Identification of known and novel immunogenic T-cell epitopes from tumor antigens recognized by peripheral blood T cells from patients responding to IL-2-based treatment

In previous studies CD8+ T cells specific for melanocyte antigens have been frequently found in melanoma patients responding to interleukin-2 (IL-2)-based therapies. In our study we analyzed the suitability of using circulating T cells from melanoma patients with clinical response after IL-2-based therapy to identify novel T-cell epitopes from defined tumor antigens. Using unstimulated peripheral blood mononuclear cells and the interferon-gamma (IFN-gamma) ELISPOT assay, we studied CD8(+) T-cell responses against 5 peptides from the tumor antigen tyrosinase (Tyr) selected by epitope prediction using an HLA-A1-binding computer algorithm. T cells specifically secreting IFN-gamma in response to 3 of these 5 peptides, namely, Tyr (454-463), Tyr (146-156) and Tyr (243-251), could be detected in 4 of 4 HLA-A1-positive patients with clinical response. In contrast, no T-cell responses against these peptides were seen in 6 HLA-A1-positive melanoma patients with progressive disease and in 8 healthy subjects. We could generate specific cytotoxic T lymphocytes (CTL) against Tyr (454-463) using peptide-pulsed autologous dendritic cells as antigen-presenting cells. The induced CTLs efficiently killed melanoma cells that express HLA-A1 and tyrosinase. The peptides Tyr (146-156) and Tyr (243-251) had recently been identified as CTL epitopes by other groups. Further ex vivo characterization of the T cells reactive against the novel epitope Tyr (454-463) in 1 patient by multicolor flow cytometry showed specific CD3+/CD8+/IFN-gamma+ T cells with frequencies of up to 0.41% of the CD3+/CD8+ T-cell population. Most of this T-cell population also expressed granzyme B. Our data confirm that in patients with tumor regressions induced by immunotherapy or chemoimmunotherapy circulating T cells reactive with tyrosinase epitopes can frequently be detected. Peripheral blood T cells from such patients are a valuable source for screening peptides selected by epitope prediction This strategy facilitates the rapid identification of immunogenic T-cell epitopes that are probable targets of immune-mediated tumor rejection.     

Platelet aggregation induced by adenosine diphosphate released from cloned murine fibrosarcoma cells is positively correlated with the experimental metastatic potential of the cells.

We established five clones (ML-01, ML-02, MH-01, MH-02, MH-03) from murine 3-methylcholanthrene-induced fibrosarcoma A (Meth A), and investigated their experimental metastatic potentials in relation to their platelet-aggregating activities. A clone with a high metastatic potential (MH-02) showed a characteristic biphasic pattern of platelet aggregation, of which the first peak was not present in the aggregation patterns of the clone with low metastatic potential (ML-01). The first peak was eliminated by treatment of the cells with apyrase, indicating that adenosine diphosphate (ADP) was the causative substance of this particular peak. The metastatic potential of clones correlated well with the ADP concentration of the culture media. These results suggest that the increased ADP production and consequential enhancement of platelet-aggregating activity are closely related to the increment of pulmonary metastatic potential of MH-02 clone.     

Growth of rat mammary adenocarcinoma cells in semisolid clonogenic medium not correlated with spontaneous metastatic behavior: heterogeneity in the metastatic, antigenic, enzymatic, and drug sensitivity properties of cells from different sized colonies

Using rat 13762NF mammary tumor cell clones of varying spontaneous metastatic potentials and biochemical properties and known phenotypic stabilities we studied the relationship between cell colony growth in a clonogenic assay and the biological and biochemical properties of cells derived from different cell colonies. The spontaneous metastatic potential of in vivo or in vitro grown 13762NF tumor cells was not related to their in vitro cloning efficiencies; cells of both low and high metastatic potential formed colonies of various sizes and shapes during 14 days of growth in 0.3% or 0.6% semisolid agarose. A highly metastatic cell clone of relatively low growth potential in agarose was examined further. Individual tumor cell colonies derived from this cell clone were removed from agarose and their properties determined. Cells from small (less than 100-microns-diameter) or large (greater than 500-microns-diameter) agarose colonies had similar self-renewal capacities in agarose and formed variously sized cell colonies when replated in agarose medium. Metastatic potential, drug sensitivity parameters, and expression of a high Mr mucin-like glycoprotein antigen and type IV collagenolytic activity known to be associated with spontaneous metastasis of 13762NF tumor cells were dissimilar in cells from different colonies, and these characteristics were independent of original tumor cell colony size in agarose. In contrast, the expression of cell surface proteins of Mr less than 300,000 were similar among cells derived from different agarose colonies. The data indicate that heterogeneity exists in the ability of 13762NF adenocarcinoma cells of different biochemical and metastatic potentials and drug sensitivities to grow in semisolid agarose. In addition, the cells that grow in agarose to form detectable colonies (greater than 50 cells) are not necessarily those with a high potential of metastasizing spontaneously to distant sites.