Tumor-associated macrophages respond to chemotherapy by detrimental transcriptional reprogramming and suppressing stabilin-1 mediated clearance of EGF

Abstract

Introduction: Tumor resistance to chemotherapy and metastatic relapse account for more than 90% of cancer specific mortality. Tumor-associated macrophages (TAMs) can process chemotherapeutic agents and impair their action. Little is known about the direct effects of chemotherapy on TAMs.

Methods: The effect of chemotherapeutic platinum agent cisplatin was assessed in the model system of human ex vivo TAMs. Whole-transcriptome sequencing for paired TAMs stimulated and not stimulated by cisplatin was analysed by NGS. Endocytic uptake of EGF was quantified by flow cytometry. Confocal microscopy was used to visualize stabilin-1-mediated internalization and endocytic trafficking of EGF in CHO cells expressing ectopically recombinant stabilin-1 and in stabilin-1+ TAMs. In cohort of patients with breast cancer, the effect of platinum therapy on the transcriptome of TAMs was validated, and differential expression of regulators of endocytosis was identified.

Results: Here we show that chemotherapeutic agent cisplatin can initiate detrimental transcriptional and functional programs in TAMs, without significant impairment of their viability. We focused on the clearance function of TAMs that controls composition of tumor microenvironment. For the first time we demonstrated that TAMs' scavenger receptor stabilin-1 is responsible for the clearance of epidermal growth factor (EGF), a potent stimulator of tumor growth. Cisplatin suppressed both overall and EGF-specific endocytosis in TAMs by bidirectional mode: suppression of positive regulators and stimulation of negative regulators of endocytosis, with strongest effect on synaptotagmin-11 (SYT11), confirmed in patients with breast cancer.

Conclusion: Our data demonstrate that synergistic action of cytostatic agents and innovative immunomodulators is required to overcome cancer therapy resistance.

Keywords: EGF; breast cancer; cisplatin; clearance; endocytosis; stabilin-1; tumor-associated macrophage (TAM).

Figure 1

Cisplatin does not affect TAM viability and M2 phenotype. (A) IHC analysis of CD68, stabilin-1 and CD206 expression in human breast cancer tissue treated and not treated with cisplatin-based therapy. (B) Quantitative analysis of CD68, stabilin-1 and CD206 expression. Tumor tissue was obtained after surgery for all patients. (C) Schematic description of ex vivo TAM generation which were stimulated with MCF-7 supernatants and treated by cisplatin. (D) Immunofluorescent staining/confocal microscopy of CD206 and stabilin-1 expression in CD68+ ex vivo TAMs. Scale bars are equal to 11µM. (E) Viability assay for ex vivo TAMs treated with cisplatin. Viability was estimated on day 6 before cisplatin treatment and day 9 after cisplatin-treatment (n=6 independent experiments). Data information: In (B), the level of protein expression is presented as Me (Q1;Q3). In (E), OD means defined in the percentage of NS control are presented as mean ± SD. The Manna-Whitney test was applied to compare two independent groups.

Figure 2

Transcriptional reprogramming of TAMs by cisplatin. (A) Effect of cisplatin on major inflammatory, scavenging and angiogenic factors analyzed by qPCR in ex vivo TAMs. Untreated – red, cisplatin-treated – blue (n=12 donors). (B) Results of RNA sequencing of ex vivo TAMs treated by cisplatin. Heatmap on the left depicts hierarchical clustering of upregulated and downregulated genes in ex vivo TAMs treated by cisplatin (p-value<0,05, log2 fold-change>0,58). On the right - heatmap with top 20 most DEGs induced by cisplatin. (C) Volcano plot shows significance and log2 fold-change value for DEGs in cisplatin-treated and untreated TAMs. (D) Bar plot with GSEA results demonstrates representative endocytosis pathways among other valuable pathways deregulated by cisplatin in ex vivo TAMs. The x-axis indicates NES (FDR<0,25, endocytosis pathways are highlighted in light-red). Data information: In (A), the level of gene expression is presented as Me (Q1;Q3). The Manna-Whitney test was applied to compare two independent groups. **p ≤ 0.01.

Figure 3

Stabilin-1 mediated endocytic uptake of EGF in CHO cells. (A) Flow cytometry analysis of acLDL and EGF uptake in CHO-Stab1 and CHO-vector control cells (CHO-V3a) (n=3). (B) Immunofluorescence/confocal microscopy of EGF-Alexa488 and acLDL-Alexa488 internalization in CHO-stabilin-1 and CHO-V3a. Yellow indicates co-localization of fluorescently labeled ligands and stabilin-1. White indicates co-localization of EGF-Alexa488 or acLDL-Alexa488, stabilin-1, and EEA-1. EGF-Alexa488 and acLDL-Alexa488 were internalized only in CHO-stabilin-1 cells. Scale bars: 10 µm. In (A), MFI index is presented as mean ± SD. *** - p<0,001. T-test was applied.

Figure 4

Cisplatin inhibits EGF clearance by human TAMs. (A) 3D reconstruction of Z-stack confocal microcopy images for EGF and stabilin-1 co-localization in EEA1+ endosomes in TAMs (B) Confocal microscopy analysis of EGF transfer to Lamp1+ lysosomes after 30 min of endocytosis in TAMs. (C) Flow cytometry analysis of endocytosis of EGF and acLDL in ex vivo TAMs treated with cisplatin (n=4 donors). (D) Representative flow cytometry diagrams of ligand uptake. The fluorescent intensity is significantly lower in TAMs, treated with cisplatin. (E) Immunofluorescent/confocal analysis of stabilin-1 mediated internalization and trafficking of EGF and acLDL in TAMs without and with cisplatin treatment. White indicates co-localization of fluorescently labeled ligand, stabilin-1, and EEA-1 (n=4 donors). Scale bars: 10µM. Data information: In (C), MFI index is presented as Me (Q1;Q3), * - p<0,05, ** - p<0,01. The Manna-Whitney test was applied to compare two independent groups.

Figure 5

Cisplatin induces imbalance in the endocytic machinery of TAMs. (A) Gene set from endocytosis pathways (RT-PCR validated genes are highlighted in light-red). Results are obtained with RNAseq. (B) GSEA enrichment plots illustrating GO endocytosis pathways. The y-axis indicates enrichment score, the x-axis represents gene ranks (FDR<0,1, FDR<0,25, respectively). (C) Cisplatin suppresses expression of genes, that positively regulate endocytosis and vesicular transport, and stimulates expression of negative regulator of endocytosis (SYT11). DNM3, DENND1A, STX8 and SYT11 were analyzed by qPCR in ex vivo TAMs (n=12). (D) Differential SYT11 and DENND1A expression in patients` breast cancer samples identified by microarray analysis. Breast cancer tissues before and after cisplatin-based NACT were compared. PR – partial regression, SD – stable disease, FC – Fold Change. FC defines the differences in the pre- and post-treatment levels of gene expression. Data information: In (C), the level of gene expression is presented as Me (Q1;Q3), * - p<0,05, **p ≤ 0.01, *** - p<0,001. Untreated – red, cisplatin-treated – blue. Manna-Whitney test was applied to compare two independent groups.

Figure 6

Schematic presentation of detrimental effect of cisplatin on TAMs. M2 scavenger receptor stabilin-1 remains to be expressed after cisplatin treatment. Stabilin-1 is able to clear EGF from breast cancer TME specifically. Cisplatin suppresses endocytic vesicular machinery by dual effect. The first effect is suppression of the expression of positive regulators of endocytic membrane biogenesis (STX8, DENND1A, DNM3). The second effect is stimulation of the expression of negative regulator of endocytosis (SYT11). Combination of such cisplatin effects results in significantly impaired receptor-mediated endocytic clearance of soluble simulators of tumor growth.