Fibrosarcoma Clone Research Articles

T Lymphocytes Restrain Spontaneous Metastases in Permanent Dormancy

Tumor dormancy is a clinical phenomenon related to immune equilibrium during cancer immunoediting. The mechanisms involved in dormant metastases are poorly understood due to the lack of preclinical models. Here, we present a nontransgenic mouse model in which spontaneous metastases remain in permanent immunomediated dormancy with no additional antitumor treatment. After the injection of a GR9-B11 mouse fibrosarcoma clone into syngeneic BALB/c mice, all animals remained free of spontaneous metastases at the experimental endpoints (3-8 months) but also as long as 24 months after tumor cell injection. Strikingly, when tumor-bearing mice were immunodepleted of T lymphocytes or asialo GM1-positive cells, the restraint on dormant disseminated metastatic cells was relieved and lung metastases progressed. Immunostimulation was documented at both local and systemic levels, with results supporting the evidence that the immune system was able to restrain spontaneous metastases in permanent dormancy. Notably, the GR9-B11 tumor clone did not express MHC class I molecules on the cell surface, yet all metastases in immunodepleted mice were MHC class I-positive. This model system may be valuable for more in-depth analyses of metastatic dormancy, offering new opportunities for immunotherapeutic management of metastatic disease.

Precise Regulation of Porcupine Activity Is Required for Physiological Wnt Signaling

Gradients of diverse Wnt proteins regulate development, renewal, and differentiation. Porcupine (PORCN) is a membrane-bound O-acyltransferase that is required for post-translational modification of all Wnts to enable their transport, secretion, and activity. Mutations in PORCN are associated with focal dermal hypoplasia (FDH), whereas gene deletion causes embryonic lethality in mice. To study the protein in more detail, zinc finger nucleases were used to edit the PORCN genomic locus, establishing two HT1080 fibrosarcoma clones null for PORCN activity that facilitate the study of PORCN structure and function. We establish that PORCN is a key non-redundant node for the regulation of global Wnt signaling because PORCN null cells are completely incapable of autocrine Wnt signaling. The strength of Wnt signaling is exquisitely sensitive to PORCN expression, with a dynamic range of at least 3 orders of magnitude, suggesting that PORCN activity is a key modulator of all Wnt ligand activity. Consistent with this, we find that multiple FDH-associated mutants have only subtle alterations in enzyme activity yet are associated with a severe FDH phenotype. These studies support an essential regulatory role of PORCN in shaping Wnt signaling gradients.

New strategy for efficient selection of dendritic cell-tumor hybrids and clonal heterogeneity of resulting hybrids

Heterotypic hybrids created between dendritic cells (DC) and tumor cells represent an efficient approach for loading DC with tumor-associated antigens (TAA), and DC-tumor hybrid vaccines have shown promising outcomes in various preclinical and clinical studies. Conventional DC-tumor hybrid preparations, however, are unavoidably contaminated by DCtumor aggregates and DC loaded with tumor cell debris. Here we describe a new strategy for selecting genuine DC-tumor hybrids. A HAT-sensitive/zeocin-resistant DC clone (XS106-7 Zeo) was fused with a GFP-transduced fibrosarcoma clone (S1509a-GFP) by polyethylene glycol and heterotypic hybrid clones were established by limiting dilution in the presence of HAT and zeocin. CD45 (DC origin) and GFP (tumor origin) were both expressed in 91% (51/56 clones) of the resulting clones, indicating high efficiency of our strategy. Marked heterogeneity was observed among the hybrid clones, and only one clone exhibited characteristic features of DC (CD86 and I-A expression, dendritic morphology, T cellstimulatory capacity, and IL-1b, IL-6, and TNFa production), suggesting that only small fractions of DC-tumor hybrids acquire and maintain the properties of parental DC. Finally, vaccination with this hybrid clone protected mice from subsequent growth of S1509a tumor cells, documenting the in vivo activity of DC-tumor hybrids in the complete absence of exogenous TAA.

Real-time imaging of individual fluorescent-protein color-coded metastatic colonies in vivo.

We have established stable, bright green fluorescent protein (GFP)- or red fluorescent protein (RFP)-expressing HT-1080 human fibrosarcoma clones. These cell lines showed similar cell proliferation rates and high-frequency experimental lung metastasis. The HT-1080-GFP and -RFP clones enable simultaneous real-time dual-color imaging in the live animal. HT-1080 cells were transduced with retroviral vectors containing GFP or RFP and the neomycin resistance gene. Stable transformants were selected stepwise with G418 up to 800 microl/ml. Subsequently, high GFP- or RFP-expressing clones, HT-1080-GFP or HT-1080-RFP, respectively, were selected. 3 x 10(6) cells from each clone were mixed and injected into the tail vein of SCID mice. The cells seeded the lung at high frequency with subsequent formation of pure green and pure red colonies as well as mixed yellow colonies with different patterns visualized directly on excised lungs. The lung metastases were also visualized by external fluorescence imaging in live animals through skin-flap windows over the chest wall. Lung metastases were observed on the lung surface of all mice. SCID mice well tolerated multiple surgical procedures for direct-view imaging via skin-flap windows. Real-time metastatic growth of the two different colored clones in the same lung was externally imaged with resolution and quantification of green, red, or yellow colonies in live animals. The color coding enabled determination of whether the colonies grew clonally or were seeded as a mixture with one cell type eventually dominating, or whether the colonies grew as a mixture. The simultaneous real-time dual-color imaging of metastatic colonies described in this report gives rise to the possibility of color-coded imaging of clones of cancer cells carrying various forms of gene of interest.

Cell Cycle Control with Minimal Participation of Cdk2 in a Murine Fibrosarcoma Clone Cultured in Protein-free Medium

The differences in the protein expression of cyclins, cyclin-dependent kinases (cdks), and their inhibitors and cdk kinase activities were examined in serum dependent (SD) and independent (PF) clones of the murine fibrosarcoma cell line, Gc-4. The expression of cyclin A in SD was minimal in contrast to PF. Furthermore, cdk2 kinase activity in PF was remarkably lower than that in SD, yet the G1/S transition in PF appeared normal. PF was also resistant against the selective inhibitor of cdk2, butyrolactone I. These findings suggest that tumor cell proliferation and tumor progression can be promoted by the activation of a molecule(s) downstream of cdk2.

Tumor responsiveness to the metastasis-stimulatory effects of prostaglandin E2 is restricted by protein phosphatases.

Prostaglandin E2 (PGE2) is secreted by both tumor and host cells, and can enhance tumor dissemination (1,2). However, the amount of PGE2 that tumor cells secrete does not necessarily reflect their metastatic capacity (1). We have shown that cloned metastatic Lewis lung carcinoma cells (LLC-LN7) secrete less PGE2 than do cloned nonmetastatic LLC (LLC-C8) cells, but are more responsive to PGE2 in their in vitro motility and in vivo metastasis (1,3,4). Such differences in responsiveness to PGE2 have also been demonstrated for fibrosarcoma clones (1).

Apoptosis of a fibrosarcoma induced by protein-free culture involves DNA cleavage to large fragments but not internucleosomal fragmentation.

A murine fibrosarcoma clone, Gc-4 SD, grows depending on fetal calf serum. In MTT assay, protein-free cultivation resulted in a reduction of the viable cell number time-dependently. Electron-microscopic and flow-cytometric analyses revealed that the reduction in growth was accompanied by the appearance of apoptotic cells. However, no internucleosomal fragmentation was observed even after SI-nuclease treatment. On the other hand, pulse field gel electrophoresis revealed that cleavage of DNA into high-molecular-weight fragments estimated as 50 to 150 kilobase pairs (kbp), with a peak of 100 kbp, was found in the serum-deprived cells. Large fragments disappeared from the DNA extracts when the smaller cells with high blue fluorescence with Hoechst 33342 were removed by flow cytometry, suggesting direct correlation between the large DNA fragmentation and apoptosis. The addition of aurintricarboxylic acid neither abolished the large DNA fragmentation nor inhibited the reduction in the number of viable cells. Both cycloheximide and actinomycin D enhanced the reduction in the number of viable cells as well as the large DNA fragmentation. These results suggest that apoptosis of a fibrosarcoma induced by protein-free culture involves a specific endogenous endonuclease, which may be distinct from and independent of the ATA-sensitive endonuclease producing internucleosomal DNA fragmentation.

In vivo activation of NK cells induces inhibition of lung colonization of H-2 positive and H-2 negative fibrosarcoma tumor clones.

The role of different tilorone analogs in the abrogation of the metastatic spread of H-2 positive and H-2 negative tumor clones was studied. Pre-treatment of BALB/c mice with RMI 10,874DA compound completely abolished lung colonization of an H-2 negative (GR9.B9) MCA-induced fibrosarcoma clone in an experimental metastasis assay. This effect was also evident when clones were treated with other tilorone analogs (R11,567DA or R11,513DA). Other H-2 positive and H-2 negative chemically induced fibrosarcoma clones were also tested. The effect was not due to direct toxicity of the tilorone analog on tumor cells, but instead was dependent on NK cells; this was suggested by the finding that treatment of mice with anti-asialo GM1 abrogated the effect of the tilorone analog (RMI 10,874DA compound). Interestingly, the inhibition of lung colonization after intravenous injection was again observed regardless of the H-2 phenotype of the tumor clones, and H-2+ and H-2- clones were similarly inhibited. In vitro assays of NK sensitivity of tumor clones showed that lysis varied depending on the H-2 phenotype of tumor clones, indicating an absence of correlation between in vivo and in vitro results.

Generation and control of metastasis in experimental tumor systems; inhibition of experimental metastases by a tilorone analogue.

The role of the chemical compound RMI 10,874DA (3,6-bis[2-(dimethylamino)-ethoxyl]-9H-xanthene-9-one dihydrochloride) in the abrogation of the metastatic spread of tumor cells was studied. Pre-treatment of BALB/c mice with the RMI 10,874DA compound (referred to below as tilorone analogue) completely eliminated lung colonization of an H-2-negative (GR9.B9) MCA-induced fibrosarcoma clone in an experimental metastasis assay. Other murine tumors, including H-2-positive and H-2-negative chemically induced fibrosarcoma clones and B16 melanoma, were also sensitive to the treatment; orally administered tilorone analogue given one day before the i.v. injection of tumor cells markedly inhibited lung colonization. The effect was not due to direct toxicity of tilorone analogue on tumor cells, but instead it was dependent on NK cells; this was suggested by the finding that anti-asialo GM, treatment of mice abrogated the effect of tilorone analogue. Kinetic studies of splenic NK activity in tilorone-treated mice showed a rapid boosting of NK-cell activity, the greatest stimulation occurring the day before removal of splenocytes for 51Cr-release assay against YAC-I target cells. These kinetics correlated with the inhibition of in vivo lung colonization after tilorone analogue treatment. Inhibition of experimental tumor metastasis was dose-dependent and was observed when animals were treated the day before or the day after tumor-cell injection. Furthermore, repeated treatment of mice with this tilorone analogue significantly reduced lung colonization.

Augmented expression of a type IV collagen-binding protein in a highly metastatic murine fibrosarcoma clone.

The adhesive properties of highly and weakly metastatic murine sarcoma (Meth A) clones were investigated. A highly metastatic clone, MH‐02, preferentially adhered to type IV collagen‐coated plastic dishes and to bovine pulmonary arterial endothelial cell‐coated plastic dishes as compared to a weakly metastatic clone, ML‐01. Pretreatment of MH‐02 and ML‐01 cells with antisera against MH‐02 cells resulted in almost equivalent adhesiveness to type IV collagen. Preincubation of 125I‐radiolabeled tumor cells with the antisera against MH‐02 significantly reduced the arrest of MH‐02 cells in the lung, but ML‐01 cells were not affected. The number of pulmonary metastatic nodules of MH‐02 cells was reduced to the same level as that of ML‐01 cells by preincubation of the tumor cells with the antisera in an experimental metastasis experiment. These results indicated that the high metastatic ability of MH‐02 can be attributed to its preferential adhesiveness to type IV collagen. The type IV collagen‐binding proteins of MH‐02 and ML‐01 were analyzed by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS‐PAGE) and autoradiography. Among several proteins which bound to type IV collagen, expression of a protein with a molecular weight of 29 kD was significantly greater in MH‐02 than in ML‐01. These results suggest that the greater adhesion of highly metastatic MH‐02 cells to type IV collagen is due to enhanced expression of the type IV collagen‐binding 29 kD protein.

Murine fibrosarcoma clone established in defined medium

Murine fibrosarcoma clone established in defined medium

Potentiation of tnf‐mediated cell killing by VP‐16: Relationship to DNA single‐strand break formation

Interaction between tumor necrosis factor (TNF) and the DNA topoisomerase II inhibitor, etoposide VP-16, in cell killing has been studied. To accurately investigate the nature of DNA damage during the cell killing process, experiments were assessed using the highly TNF-sensitive WEHI164.13 murine fibrosarcoma clone and DNA filter elution methodology. Concomitant treatment of cells with combination of TNF/VP-16 resulted in marked enhancement of cell lysis. Using the alkaline elution technique, we show that TNF fails to induce DNA single-strand breaks as compared to those generated by VP-16. In addition, the potentiating effect of VP-16 on TNF-mediated WEHI164.13 cell killing was not associated with an increase in its intrinsic activity with respect to DNA single-strand break formation. While the 2 phospholipase A2 inhibitors, quinacrine and dexamethasone, were efficient in inhibiting TNF-mediated cell lysis, only quinacrine was efficient in selectively abrogating the TNF/VP-16 cell killing pathway. The inhibitory effect of quinacrine on VP-16/TNF-mediated cell lysis was accompanied by a marked decrease in VP-16-mediated DNA single-strand break generation. Taken together, our findings suggest that TNF and TNF/VP-16 treatments may involve different events during cell killing and support the hypothesis that 2 signals are required for optimal induction of cell lysis by the combination of VP-16/TNF: one signal provided by VP-16 resulting in topoisomerase II inhibition and subsequent DNA single-strand break generation, and a second signal involving TNF.

Analysis of Clonal Evolution in a Tumor Consisting of pSV2neo‐transfected Mouse Fibrosarcoma Clones

The process of clonal evolution was analyzed in a line of methylcholanthrene‐induced mouse fibrosarcomas. The tumor cells were transfected with pSV2neo gene and 22 clones were randomly isolated. Genetically tagged clones were mixed and inoculated into syngeneic mice. Southern blot analysis revealed that one of the clones, no. 11, dominated both in tumors in situ and in lung metastatic nodules. No. 11 clone and other clones were similar in growth rates in vitro and in vivo, in spontaneous and experimental metastatic abilities, in immunogenicity, and in the capacity of intercellular communication in vitro. Although no. 11 clone overgrew other clones in vivo, this was not the case when clones were mixed and maintained in vitro. We conclude that clonal interactions in vivo may be responsible for the dominance of no. 11 clone in the tumor. It is likely that the preferential metastasis of no. 11 clone to the lung may be a simple reflection of the proliferative advantage of the dominant clone in the tumor in situ.

NK sensitivity and lung clearance of MHC‐class‐I‐deficient cells within a heterogeneous fibrosarcoma

The present study examines clonal variations in NK sensitivity in a methylcholanthrene-induced fibrosarcoma. Previous studies of clones from this tumor have shown considerable heterogeneity in H-2 expression, and an association between deleted or low levels of class-I products and increased tumorigenicity after subcutaneous implantation in immunocompetent syngeneic mice. Here, fibrosarcoma clones with no or low expression of MHC-class-I products were found to be sensitive to NK-mediated lysis, while clones with high levels of MHC-class-I expression were relatively resistant. One H-2+ (G2) and one H-2- (B9) clone were chosen for more detailed studies. Cold-target competition assays and conjugate cytotoxicity assays in agarose showed that splenic effector cells bound equally well to the H-2+ and H-2- tumor clone, although only the latter was sensitive to NK cell lysis. Treatment with 50 U/ml of rIFN-gamma for 48 hr increased the levels of H-2 expression and made both clones more resistant to NK-mediated lysis. In vivo studies with radiolabelled tumor cells showed that cells from the H-2+ clone survived better than cells from the H-2- clone in the pulmonary capillary bed after i.v. inoculation. This difference disappeared in mice treated with anti-asialo GM1 serum, known to deplete NK cell activity.

Inhibition of tumor cell-induced platelet aggregation and experimental tumor metastasis by the synthetic Gly-Arg-Gly-Asp-Ser peptide.

The mechanism by which the murine fibrosarcoma clone PAK 17.15 induces platelet aggregation [tumor cell-induced platelet aggregation (TCIPA)] was studied because platelet activation by this clone is necessary for metastasis to the lungs. PAK 17.15 TCIPA was completely inhibited by ADP-clearing enzymes, such as apyrase, or a mixture of creatine phosphate and creatine phosphokinase. Thrombin and collagen were not involved in PAK 17.15 TCIPA. Further studies showed that ADP is most likely secreted from activated platelets and that membrane protein(s) on PAK 17.15 cells are responsible for platelet activation. Inasmuch as ADP-dependent platelet aggregation requires fibrinogen and can be inhibited by the Gly-Arg-Gly-Asp-Ser (GRGDS) synthetic peptide, the effect of this peptide on PAK 17.15 TCIPA was studied. PAK 17.15 TCIPA was completely inhibited by the GRGDS peptide (0.4 mM) but not by a control peptide, Gly-Arg-Gly-Glu-Ser (0.8 mM). In addition, the GRGDS peptide inhibited adhesion of PAK 17.15 cells to immobilized fibronectin. As expected, the GRGDS peptide almost completely inhibited lung colonization by iv injected PAK 17.15 cells in C57BL/6 mice. Our results indicate that GRGDS may inhibit pulmonary metastases by interfering with TCIPA as well as with tumor cell adhesion to extra-cellular matrix components in the host.

Correlation between bizarre colony morphology and metastatic potential of tumor cells

The morphology of colonies developing from cells of UV-2237 fibrosarcoma clones grown in an unattached state correlated with their experimental metastatic potential in vivo. The frequency of bizarre-shaped (non-symmetrical) colonies was increased in colonies that developed from cells of highly metastatic clones growing in an overlay of media on agar, in 1.3% Methocel, or in 0.3% Noble agar. These bizarre-shaped colonies rarely developed from cells of clones with low metastatic frequency. In addition, when tested for in vivo experimental metastasis, the cells from the bizarre colonies were highly metastatic.