Lurbinectedin reduces tumour-associated macrophages and the inflammatory tumour microenvironment in preclinical models

Abstract

Background: Lurbinectedin is a novel anticancer agent currently undergoing late-stage (Phase II /III) clinical evaluation in platinum-resistant ovarian, BRCA1/2-mutated breast and small-cell lung cancer. Lurbinectedin is structurally related to trabectedin and it inhibits active transcription and the DNA repair machinery in tumour cells.

Methods: In this study we investigated whether lurbinectedin has the ability to modulate the inflammatory microenvironment and the viability of myeloid cells in tumour-bearing mice.

Results: Administration of lurbinectedin significantly and selectively decreased the number of circulating monocytes and, in tumour tissues, that of macrophages and vessels. Similar findings were observed when a lurbinectedin-resistant tumour variant was used, indicating a direct effect of lurbinectedin on the tumour microenviroment. In vitro, lurbinectedin induced caspase-8-dependent apoptosis of human purified monocytes, whereas at low doses it significantly inhibited the production of inflammatory/growth factors (CCL2, CXCL8 and VEGF) and dramatically impaired monocyte adhesion and migration ability. These findings were supported by the strong inhibition of genes of the Rho-GTPase family in lurbinectedin-treated monocytes.

Conclusions: The results illustrate that lurbinectedin affects at multiple levels the inflammatory microenvironment by acting on the viability and functional activity of mononuclear phagocytes. These peculiar effects, combined with its intrinsic activity against cancer cells, make lurbinectedin a compound of particular interest in oncology.

Figure 1

Effects of lurbinectedin on human purified monocytes in vitro. (A) Cell viability of monocytes treated with lurbinectedin or trabectedin at two different doses (5 and 10 nM) for up to 48 h, evaluated with Annexin/PI staining by flow cytometry; results are shown as % of live cells compared with untreated cells (mean±s.e. of three experiments). (B) Caspase-8 activation in treated monocytes evaluated by flow cytometry with anti-cleaved CASP8 antibody. Caspase-8 activation peaks at 12 h and remains at plateau at 18 h. (C) Modulation of cytokine production (ELISA) by lurbinectedin and trabectedin in human LPS-stimulated monocytes. Monocytes were treated with drugs (5 nM) for 1 h, washed and cultured with fresh medium for 24 h. Both drugs significantly and similarly inhibited the production of CCL2, CXCL8 and VEGF. (D) Migration assay (left) and adhesion assay (right) of human monocytes pre-treated with lurbinectedin and trabectedin (5 nM), in response to CCL2 (100 ng ml⁻¹). Statistical analysis: *P<0.05, **P<0.01 and ***P<0.001 (Student’s t-test).

Figure 2

Modulation of gene expression by lurbinectedin in human LPS-stimulated monocytes. Monocytes were treated with lurbinectedin (10 nM), trabectedin (10 nM) or doxorubicin (1 μM) for 6 h (mean±s.e. of five donors). (A) Dendogram plot showing the similarity of gene modulation for lurbinectedin (PM) and trabectedin (ET), in contrast with doxorubicin. (B) Modulation of Rho GTPase genes in human LPS-stimulated monocytes, in the myeloid cell line THP1, primary IL-2-activated human T lymphocytes and the murine fibrosarcoma MN/MCA1. Both lurbinectedin and trabectedin inhibit Rho GTPase genes selectively in myeloid cells. Statistical analysis: *P<0.05, **P<0.01 and ***P<0.001 (Student’s t-test). Data were analysed by the DDCT method and expressed as fold change (arbitrary unit) compared with untreated control (set as 1). Values below 0.5 or above 2 (red dashed lines) are genes differentially expressed.

Figure 3

In vivo anti-tumour activity of lurbinectedin in mice. The syngenic fibrosarcoma MN/MCA1 (A) and its resistant variant RES-MN/MCA1 (B) were used. Mice were treated with lurbinectedin or trabectedin i.v. q7d × 3, respectively, at the indicated doses (see text). A representative experiment of two performed with similar results is shown. Lurbinectedin and trabectedin have similar anti-tumour efficacy. Statistical analysis: ***P<0.001 (two-way ANOVA; Bonferroni post hoc test).

Figure 4

Effects of lurbinectedin on the tumour microenvironment of the fibrosarcoma MN/MCA1. (A) Flow cytometry analysis of circulating leukocytes of mice treated with lurbinectedin or trabectedin as detailed in the legend of Figure 3. Results are shown after 1 cycle of treatment. Both drugs selectively decrease the number of total monocytes (CD11b+CD115+), especially the Ly6C^high monocyte subset. (B) Flow cytometry analysis of splenic leukocytes in mice treated after three cycles. Both drugs selectively decrease the number of macrophages (CD11b+ F4/80+Ly6C^high). Statistical analysis: *P<0.05, **P<0.01 and ***P<0.001 (two-way ANOVA; Bonferroni T-test. (C) Representative immunohistochemistry pictures of tumour sections after treatment (three cycles), stained for macrophages (F4/80) or blood vessels (CD31). (D) Quantification of immunohistochemistry. Both macrophages and vessels were significantly reduced in tumours treated with lurbinectedin and trabectedin. The immunoreactive areas are calculated as mean from five microscopic fields for each sample, five mice per group. Images were analysed using Image-Pro Analyzer software. Original magnification, × 20. Statistical analysis: *P<0.05, **P<0.01 and ***P<0.001 (Student’s t-test).

Figure 5

Effects of lurbinectedin on the tumour microenvironment of the fibrosarcoma resistant variant RES-MN/MCA1. (A) Flow cytometry analysis of circulating leukocytes of mice treated with lurbinectedin or trabectedin as detailed in the legend of Figure 3. Results are shown after one cycle of treatment. Both drugs selectively decrease the number of total monocytes (CD11b+CD115+), especially the Ly6C^high monocyte subset. (B) Flow cytometry analysis of splenic leukocytes in mice treated after three cycles. Both drugs selectively decrease the number of macrophages (CD11b+ F4/80+Ly6C^high). Statistical analysis: *P<0.05, **P<0.01 and ***P<0.001 (two-way ANOVA; Bonferroni T-test. (C) Representative immunohistochemistry pictures of tumour sections after treatment (three cycles), stained for macrophages (F4/80) or blood vessels (CD31). (D) Quantification of immunohistochemistry. Both macrophages and vessels were significantly reduced in tumours treated with lurbinectedin and trabectedin. The immunoreactive areas are calculated as mean from five microscopic fields for each sample, five mice per group. Images were analysed using Image-Pro Analyzer software. Original magnification, × 20. Statistical analysis: *P<0.05, **P<0.01 and ***P<0.001 (Student’s t-test).

Figure 6

Schematic representation of the mechanisms of action of lurbinectedin on the tumour microenvironment. Lurbinectedin induces the apoptotic death of monocytes; at sub-cytotoxic concentrations lurbinectedin inhibits the cell migration in response to chemotactic signals (CCL2) and the production of inflammatory mediators produced in the tumour microenvironment (CCL2 and CXCL8) and the angiogenic factor VEGF.