Figure 3 from Trabectedin Enhances the Antitumor Effects of IL-12 in Triple-Negative Breast Cancer

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posted on 2025-04-02, 07:24 authored by Emily Schwarz, Himanshu Savardekar, Sara Zelinskas, Abigail Mouse, Gabriella Lapurga, Justin Lyberger, Adithe Rivaldi, Emily M. Ringwalt, Katherine E. Miller, Lianbo Yu, Gregory K. Behbehani, Timothy P. Cripe, William E. Carson

Combination IL-12 and trabectedin selectively reduces immunosuppressive myeloid cells while increasing intratumoral CD8⁺ T cells. Mass cytometry analysis of splenic and tumor-infiltrating immune cells from 4T1 tumor–bearing mice treated with PBS control (n = 7), IL-12 (n = 6), trabectedin (n = 6), and IL-12 plus trabectedin (n = 4) for 15 days. A, Representative Uniform Manifold Approximation and Projection (UMAP) plots of immune cell populations at day 15 in each treatment group clustered in an unsupervised manner from live/CD45⁺ splenocytes. Percentages of (B) NK cells and (C) CD4⁺ cells, CD8⁺ cells, and Tregs of total live/CD45⁺ splenocytes. D, Percentage of total macrophages of live/CD45⁺ splenocytes and (E) the proportions of M1-like and M2-like macrophages of total macrophages. F, Percentage of cDC1 cells of live/CD45⁺ splenocytes. G, Representative UMAP plots of tumor-infiltrating immune cells from mice treated with PBS control (n = 3), IL-12 (n = 4), trabectedin (n = 3), and IL-12 plus trabectedin (n = 3) clustered in an unsupervised manner from live/CD45⁺ cells. H, Percentages of NK cells and (I) CD4⁺ cells, CD8⁺ cells, and Tregs of live/CD45⁺ tumor–infiltrating cells. J, Percentage of total macrophages of live/CD45⁺ tumor-infiltrating cells and (K) proportion of M1 and M2 macrophages of total macrophages. L, Percentage of cDC1 cells of live/CD45⁺ tumor-infiltrating cells. An ordinary one-way ANOVA with the Tukey multiple comparisons test was used for statistical analysis of bar graphs. Data represent mean ± SEM. *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001. Trab, trabectedin.

Funding

National Cancer Institute (NCI)

United States Department of Health and Human Services

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National Center for Advancing Translational Sciences (NCATS)

United States Department of Health and Human Services

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History

ARTICLE ABSTRACT

IL-12 is a potent NK cell–stimulating cytokine, but the presence of immunosuppressive myeloid cells such as myeloid-derived suppressor cells (MDSC) can inhibit IL-12–induced NK-cell cytotoxicity. Thus, we hypothesized that trabectedin, a myeloid cell–depleting agent, would improve the efficacy of IL-12 in triple-negative breast cancer (TNBC). In vitro treatment of healthy donor NK cells with trabectedin increased expression of the activation marker CD69 and mRNA expression of T-box transcription factor (Tbx21), the cytotoxic ligands TNF-related apoptosis–inducing ligand (TNFSF10), Fas ligand (FASLG), and the dendritic cell (DC)–recruiting chemokine lymphotactin (XCL1). The combination of IL-12 and trabectedin increased NK-cell cytotoxicity and activation and production of IFN-γ, TNF-α, and granzyme B in the presence of human TNBC cells. Treatment of 4T1 and EMT6 tumor–bearing mice with IL-12 and trabectedin led to a significant reduction in tumor burden compared with single-agent controls and the highest levels of plasma IFN-γ, intratumoral CD8+ T cells, and conventional type 1 DC. MDSC and M2-like macrophages were significantly decreased with combination therapy. NK-cell depletion abrogated the effects of combination therapy, as did the elimination of CD8+ T cells. NK-cell depletion led to lower levels of the NK cell–derived chemokine CCL5 and the DC-derived chemokine CXCL10, higher tumor burden, and decreased intratumoral CD8+ T cells. IL-12 and trabectedin also significantly enhanced the response of TNBC to anti–PD-L1 therapy. These data suggest that MDSC depletion augments the ability of IL-12–activated NK cells to drive the infiltration of DC and CD8+ T cells into TNBC for an antitumor effect.