K1735 Mouse Melanoma Research Articles

Abstract 5031: Salmonella-mediated suppression of Akt/mTOR signaling inhibits metastasis via downregulation of Snail expression in melanoma

Abstract The expression of EMT inducer Snail in many cancer types has been implicated in high-grade malignancies and correlated with poor prognosis. Tumor-intrinsic Snail not only has direct effects on metastasis but also impacts tumor microenvironment by altering secretomes. Our previous reports demonstrated that systemic administration of Salmonella significantly reduced tumor growth and metastatic nodules of tumor-bearing mice, in part, by coaxing changes in the tumor microenvironment. These led us to hypothesize that Salmonella targets metastasis inducers. Here, we use B16F10 and K1735 mouse melanoma cells to investigate the anti-metastatic potentials of Salmonella against melanoma. Immunoblotting revealed that the expression of Snail in both B16F10 and K1735 melanoma cell lines was significantly downregulated following Salmonella treatment in a dose-dependent manner. Likewise, the phosphorylation of Akt and mTOR was significantly reduced after Salmonella treatment. Our findings further revealed that in vitro metastatic activities of Salmonella-treated melanoma cells were significantly inhibited as demonstrated in the wound-healing and Transwell assays. When transfected with constitutively active AKT plasmid, B16F10 and K1735 markedly increased the phosphorylation of Akt and mTOR accompanied by an elevated Snail expression. Interestingly, Salmonella treatment reversed the Akt/mTOR and Snail upregulation of cells overexpressing Akt. Our data therefore suggest that Salmonella significantly reduces melanoma metastasis by downregulating Snail. Citation Format: Christian R. Pangilinan, Che-Hsin Lee. Salmonella-mediated suppression of Akt/mTOR signaling inhibits metastasis via downregulation of Snail expression in melanoma [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 5031.

Abstract A46: Antibody-mediated phosphatidylserine blockade significantly enhances the efficacy of downstream immune checkpoint inhibition in K1735 and B16 mouse melanoma

Abstract Background: The underlying cause for the failure of immune checkpoint blockade is the overwhelming, persistent and multifocal immune suppression in the tumor microenvironment. This is due to the absence of pre-existing antitumor Teff because of the action of important upstream immune checkpoints that recruit tumor infiltrating myeloid-derived suppressor cells (MDSCs), regulatory T cells (Tregs) and M2 macrophages, which also produce potent immunosuppressive cytokines (e.g., TGF-beta and IL-10). Phosphatidylserine (PS) is an upstream immune checkpoint that drives global immunosuppression. Previous work has shown that PS targeting agents can override PS-driven immunosuppression and re-program the tumor microenvironment from immunosuppressive to immunosupportive, break tumor immune tolerance, and elicit potent de novo antitumor T-cell immunity. Methods: In the present study, the antitumor effect of the combination of a PS-targeting antibody with antibodies that inhibit the immune checkpoints programmed death receptor-1 (PD-1) or cytotoxic T-lymphocyte associated antigen (CTLA-4) in the K1735 mouse melanoma model was examined. Tumor-bearing mice were treated with each antibody alone or the combination at 5 to 10 mg/kg, twice a week. Tumor growth, survival, as well as intra-tumoral and peripheral immune cell profiles after treatment were assessed by FACS and IHC. Results: Here we demonstrate that combination of PS-targeting antibody (mch1N11) with anti-PD-1 or anti-CTLA-4 antibodies significantly improved therapeutic efficacy in mice bearing K1735 melanoma compared to single agent treatment. Similar results were seen in the B16 mouse melanoma model. Importantly, we found that the level of MDSCs in the spleen was significantly reduced by combination treatment, 9.9% in the combination treated group, as compared with 18.27% and 14.69% in anti-PD-1 (p<0.0005) or control C44 (p<0.05) treated group, respectively. In addition, combination therapy had the highest ratio of tumor-infiltrating immune effector to suppressor cells and the most IL-2 and IFNγ-secreting T cells in the spleen in response to non-specific stimulation. The percentage of IFNγ-secreting CD8+ T cells in the combination group was 5.85%, as compared with 4.1% and 4.0% in anti-PD-1 or control C44 group, respectively (p<0.05). The percentage of IL-2-secreting CD8+ T cells in the combination group was 2.4%, as compared with 1.29% and 1.17% in anti-PD-1 or control C44 group, respectively (p<0.05). The percentage of IL-2-secreting CD4+ T cells in the combination group was 6.5%, as compared with 2.9% and 3.2% in anti-PD-1 or control C44 group, respectively (p<0.05). Finally, combination of PS-targeting with downstream immune checkpoint blockade did not induce any observable toxicity following multiple treatment doses. Conclusions: Our findings indicate that the combination of PS-blockade with blockade of downstream immune checkpoints (e.g., PD-1 and CTLA-4) represents a promising strategy to enhance the efficacy of cancer immunotherapy. Citation Format: Bruce Freimark, Jian Gong, Dan Ye, Van Nguyen, Shen Yin, Rich Archer, Chris Hughes, Jeff Hutchins, Jeff Hutchins, Alan Schroit, Rolf Brekken, Xianming Huang. Antibody-mediated phosphatidylserine blockade significantly enhances the efficacy of downstream immune checkpoint inhibition in K1735 and B16 mouse melanoma. [abstract]. In: Proceedings of the AACR Special Conference: Tumor Immunology and Immunotherapy: A New Chapter; December 1-4, 2014; Orlando, FL. Philadelphia (PA): AACR; Cancer Immunol Res 2015;3(10 Suppl):Abstract nr A46.

Abstract LB-262: Phosphatidylserine-targeting antibody synergizes with anti-PD-1 antibody to inhibit tumor growth in K1735 mouse melanoma model

Abstract A large body of evidence indicates that PS is a major player in tumor-associated immunosuppression, including the induction and expansion of immune suppressor cells, such as MDSCs and M2 macrophages, and the production of immunosuppressive cytokines, such as TGF-beta and IL-10. Furthermore, PS also inhibits DCs maturation and antigen presentation. Thus, PS is a critical target for cancer immunotherapy. We have previously shown that anti-PS antibody can override PS-driven immunosuppression and re-program the tumor microenvironment from immunosuppressive to immunosupportive, breaks tumor immune tolerance and elicits potent de novo antitumor T-cell immunity. In the present study, we examined the combination of anti-PD-1 with anti-PS antibody in K1735 mouse melanoma model. Tumor-bearing mice were treated with each antibody alone or the combination at 2.5 mg/kg, twice a week. The combination treatment showed significantly superior tumor growth inhibition than with either antibody alone, animals treated with the combination survived significantly longer than with either antibody alone. Flow cytometry analysis showed the combination has the highest ratio of tumor-infiltrating immune effector to suppressor cells. Finally, no toxicity was observed in all treatment groups following multiple treatment doses. These data suggest that combination of PS blockade with immune checkpoint blockade represents a promising combinatorial strategy for cancer immunotherapy. Citation Format: Xianming Huang, dan Ye, Rolf Brekken, Yi Yin. Phosphatidylserine-targeting antibody synergizes with anti-PD-1 antibody to inhibit tumor growth in K1735 mouse melanoma model. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr LB-262. doi:10.1158/1538-7445.AM2014-LB-262

Fluorescent-tilmanocept for tumor margin analysis in the mouse model

BackgroundDendritic cells (DC) are localized in close proximity to cancer cells in many well-known tumors, and thus maybe a useful target for tumor margin assessment. Materials and methods[99mTc]- cyanine 7 (Cy7)-tilmanocept was synthesized and in vitro binding assays to bone marrow-derived DC were performed. Fifteen mice, implanted with either 4T1 mouse mammary or K1735 mouse melanoma tumors, were administered 1.0 nmol of [99mTc]-Cy7-tilmanocept via tail vein injection. After fluorescence imaging 1 or 2 h after injection, the tumor, muscle, and blood were assayed for radioactivity to calculate percent-injected dose. Digital images of the tumors after immunohistochemical staining for DC were analyzed to determine DC density. ResultsIn vitro binding demonstrated subnanomolar affinity of [99mTc]-Cy7-tilmanocept to DC (KA = 0.31 ± 0.11 nM). After administration of [99mTc]-Cy7-tilmanocept, fluorescence imaging showed a 5.5-fold increase in tumor signal as compared with preinjection images and a 3.3-fold difference in fluorescence activity when comparing the tumor with the surgical bed after tumor excision. Immunohistochemical staining analysis demonstrated that DC density positively correlated with tumor percent of injected dose per gram (r = 0.672, P = 0.03), and higher DC density was observed at the periphery versus center of the tumor (186 ± 54 K versus 64 ± 16 K arbitrary units, P = 0.001). Conclusions[99mTc]-Cy7-tilmanocept exhibits in vitro and in vivo tumor-specific binding to DC and maybe useful as a tumor margin targeting agent.

Antibody-mediated phosphatidylserine blockade significantly enhances the efficacy of downstream immune checkpoint inhibition in K1735 mouse melanoma

Phosphatidylserine (PS) is an upstream immune checkpoint that drives global immunosuppression. Previous work has shown that PS targeting agents can override PS-driven immunosuppression and re-program the tumor microenvironment from immunosuppressive to immunosupportive, break tumor immune tolerance, and elicit potent de novo antitumor T-cell immunity. In the present study, the antitumor effect of the combination of a PS-targeting antibody with antibodies that inhibit the downstream immune checkpoints PD-1 or CTLA-4 antibody in the K1735 mouse melanoma model was examined. Tumor-bearing mice were treated with each antibody alone or the combination at 5 to 10 mg/kg, twice a week. Combination therapy potently suppressed tumor growth and improved overall survival compared to single agent treatment. Flow cytometry revealed that combination therapy induced the highest ratio of tumor-infiltrating immune effector to suppressor cells. Importantly, combination treatment also significantly decreased the levels of myeloid-derived suppressor cells (MDSC) in the spleen. In addition, inhibition of PS and PD-1 or CTLA-4 resulted in significantly more IL-2 and IFNg-secreting splenic CD4+ and CD8+ T cells than any single agent treatment. Finally, combined immune checkpoint blockade did not induce any observable toxicity following multiple treatment doses. In summary, our findings demonstrate that the combination of antibody-mediated PS blockade with an inhibition of established immune checkpoints (e.g., PD-1 and CTLA-4) represents a promising strategy for cancer immunotherapy.

Immunotherapy for murine K1735 melanoma: combinatorial use of recombinant adenovirus expressing CD40L and other immunomodulators.

We have constructed and tested five recombinant adenoviruses (Ads) that express a variety of immunomodulators, including CD40 ligand (CD40L), a potent costimulator of several components of the immune system. We demonstrate that CD40L expressed from Ad in K1735 mouse melanoma cells leads to a strong reduction in tumorigenicity and to efficient protective immunity in a vaccination setting. Subsequently, using a therapeutic approach, we found that local, intratumoral coinjection of CD40L- and IL-2-expressing Ads was superior to any other agents tested and resulted in an at least 1.9-fold increase in mean survival time, in contrast to systemic application of recombinant CD40L or GM-CSF proteins, which had no significant effects. When using vaccination as a therapeutic approach, the combinations of CD40L plus IL-2 or GM-CSF plus IL-2 from Ad gave rise to an extended (2.8-fold) increase in mean survival time. A detailed analysis of immune cells present within regressing tumors indicated that mainly CD4(+) and CD8(+) T cells, and to a lesser extent dendritic cells, infiltrated the tumor mass, but not NK cells, macrophages, or granulocytes. These results propose that a combination of CD40L plus IL-2 has an improved efficacy over the use of single agents when applied for direct in situ therapy or vaccination therapy.

Fractal dimension of K1735 mouse melanoma clones and spheroid invasion in vitro.

An in vitro tumour-host confrontation method to investigate the invasion behaviour of cancer has been applied to K1735 mouse melanomas. Fluorescently labelled spheroids of cancer cells and host cells were confronted and the temporal course of cancer invasion into the host was investigated using confocal laser scanning microscopy. To improve the quantitative data of this method, the boundary images of the fluorescently labelled confrontation pairs were treated as fractals. The physical and mathematical framework for determination of the fractal capacity dimension is widely used in biology and medicine and has proved to be a very useful tool for describing the cancer invasion process. The fractal capacity dimension determination was carried out by dilation of the binary boundaries of the objects, which were treated as an estimate of the Minkowski-Bouligand dimension. The fractal dimension correlated well with the degree of invasion of the K1735-M2 clone. Control experiments, with host-host confrontations and various K1735 clones with reduced invasiveness, support these results.

Expression of the Elm1 gene, a novel gene of the CCN (connective tissue growth factor, Cyr61/Cef10, and neuroblastoma overexpressed gene) family, suppresses In vivo tumor growth and metastasis of K-1735 murine melanoma cells.

We previously isolated a partial cDNA fragment of a novel gene, Elm1 (expressed in low-metastatic cells), that is expressed in low-metastatic but not in high-metastatic K-1735 mouse melanoma cells. Here we determined the full-length cDNA structure of Elm1 and investigated the effect of Elm1 expression on growth and metastatic potential of K-1735 cells. The Elm1 gene encodes a predicted protein of 367 amino acids showing approximately 40% amino acid identity with the CCN (connective tissue growth factor [CTGF], Cyr61/Cef10, neuroblastoma overexpressed gene [Nov]) family proteins, which consist of secreted cysteine-rich proteins with growth regulatory functions. Elm1 is also a cysteine-rich protein and contains a signal peptide and four domains conserved in the CCN family proteins. Elm1 was highly conserved, expressed ubiquitously in diverse organs, and mapped to mouse chromosome 15. High-metastatic K-1735 M-2 cells, which did not express Elm1, were transfected with an Elm1 expression vector, and several stable clones with Elm1 expression were established. The in vivo growth rates of cells expressing a high level of Elm1 were remarkably slower than those of cells expressing a low level of Elm1. Metastatic potential of transfectants was reduced in proportion to the level of Elm1 expression. Thus, Elm1 is a novel gene of CCN family that can suppress the in vivo growth and metastatic potential of K-1735 mouse melanoma cells.

Isolation of a NovelSry-Related Gene That Is Expressed in High-Metastatic K-1735 Murine Melanoma Cells

To identify genes differentially expressed in association with metastatic potential of K-1735 mouse melanoma cells, the mRNA differential display method was applied to compare mRNAs from high- and low-metastatic K-1735 cells. A novel gene was identified as being expressed in high-metastatic cells but not in low-metastatic cells. Sequence analysis revealed that this gene had an open reading frame of 538 amino acid residues containing aSryhigh-mobility group (HMG) box domain, known as a DNA binding motif, particularly in theSoxfamily genes. This gene showed the highest homology to the humanSOX9gene, which is the responsible gene for an inherited disease, campomelic dysplasia. Thus, this gene was designated theSox21gene (the 21stSoxfamily gene). TheSox21gene was mapped to mouse Chromosome 15, to which neither genes containing the HMG box region nor the loci of hereditary diseases similar to campomelic dysplasia have been previously mapped. This gene was highly conserved and specifically expressed in the brain. These results suggest that expression of theSox21gene is involved in the development of nerve systems and may function to enhance the metastatic potential of K-1735 mouse melanoma cells of nerve cell origin.

Transfected mouse melanoma lines that express various levels of human melanoma-associated antigen p97.

The gene for the human melanoma-associated antigen p97 has been introduced by cDNA transfection into cells from clone M2 of the K1735 mouse melanoma, which metastasizes to the lung when injected iv into syngeneic C3H/HeN mice. Tumor clones were established from the transfected cells and found to differ in the level of p97 expression. Their outgrowth in immunocompetent syngeneic mice was shown to inversely correlate with p97 antigen expression, and lines that express higher p97 levels elicited a stronger delayed-type hypersensitivity response when injected into the footpads of mice immune to p97. Five clones which expressed very high levels of p97 failed to grow in immunocompetent C3H/HeN mice while they formed tumors in nude (nu/nu) mice. The highest expressing clone, 2A, grew slightly faster than any of the other clones when cultured in vitro. Since several of the transfected clones were found to express a stable level of p97 and have consistent in vivo growth behavior, they provide a useful model for various forms of antigen-specific active and passive immunotherapy with the same agents as those intended for human application.