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. 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 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 SYT11, confirmed in patients with breast cancer. Our data demonstrate that synergistic action of cytostatic agents and innovative immunomodulators is required to overcome cancer therapy resistance.
Overall design: Human monocytes were obtained from the German Red Cross Blood Service Baden-Württemberg– Hessen (Mannheim, Germany). Three donors of German cohort were used for preparation of ex vivo TAMs and whole-transcriptome sequencing. Monocytes were isolated from the buffy coats of healthy donors by density gradients followed by positive magnetic selection using CD14+ MACS beads (no. 130-050-201, Miltenyi Biotech, Germany), resulting to 90–98% monocyte purity as confirmed by flow cytometry. Monocytes were cultured at the concentration of 106 cells/ml in serum-free X-VIVO medium (Lonza, Germany) supplemented with 10 ng/ml of macrophage colony-stimulating factor (M-CSF) (no. 300-25, Peprotech, Germany), 10−8 M of dexamethasone (no. D2915, Sigma-Aldrich, Germany) and 10 ng/ml of IL-4 (no. 200-04, Peprotech, Germany). Supernatants from MCF-7 cells were added to freshly isolated human primary monocytes at a final volume of 20% of a cultivation medium. Monocytes were differentiated to TAMs for 6 days in the presence of 7.5% CO2. Cisplatin (Cisplatin-Teva, Teva Pharmaceutical Industries, Ltd. L01XA01 injection solution) was used for chemotherapeutic treatment of TAMs in vitro. For ex vivo TAMs, cisplatin was used at the selected concentrations (20 μM, 40 μM, and 80 μM) to determine the optimal concentration for further analysis. The 20 μМ concentration of cisplatin was used for further analysis. Cisplatin was added on day 6 of macrophages differentiation and TAMs were incubated with cisplatin for 3 days. Whole-transcriptome sequencing was performed on 3 untreated TAMs and 3 TAMs treated with cisplatin. Prepared libraries were then pooled and sequenced on an Illumina NextSeq 500 instrument (Illumina, USA) with NextSeq 500/550 High-Output v2.5 Kit (75 cycles) (cat #20024906).
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