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A new soluble CSF1R-dimeric mutein with enhanced trapping of both CSF-1 and IL-34 reduces suppressive tumor-associated macrophages in mesothelioma.

ABSTRACT: Background: Signalling through the colony stimulating factor-1 receptor (CSF-1R) upon binding of its ligands CSF-1 and IL-34 has tumorigenic effects in several cancers through both induction of suppressive macrophages, and survival/proliferation of tumor cells. In addition, IL-34 tumorigenic effect can also be mediated by signalling through its other receptors, protein-tyrosine phosphatase zeta (PTPzeta), Syndecan-1 (CD138) and triggering receptor expressed on myeloid cells 2 (TREM2). Inhibition of CSF-1R signalling with small tyrosine kinase inhibitors or neutralization of CSF-1 and/or IL-34 using antibodies have been proposed to be used in cancer but their effect is limited, they have drawbacks since they only partially inhibit CSF-1 or IL-34 activity and in the case of small tyrosine kinase inhibitors lead to secondary effects due to lack of specificity. Thus, there is a need for a more specific and yet integrative approach. Methods: A human mutated form of the extracellular portion of CSF-1R was in silico modelized to trap both IL-34 and CSF-1 with higher affinity than the wild-type CSF-1R by replacing the methionine residue at position 149 by a Lysine (M149K). The extracellular portion of the mutated CSF-1R M149K was dimerized using the immunoglobulin Fc sequence of a silent human IgG1 (sCSF-1RM149K-Fc). Signalling through CSF-1R, survival of monocytes and differentiation of suppressive macrophages were analyzed using mesothelioma patient’s samples and mesothelioma/macrophage spheroids in the presence of sCSF-1RM149K-Fc or sCSF-1R-Fc wild type control (sCSF-1RWT-Fc). Results: We defined that the D1 to D5 domains of the extracellular portion of CSF-1R were required for efficient binding to IL-34 and CSF-1. The mutein sCSF-1RM149K-Fc trapped with higher affinity than sCSF-1RWT-Fc both recombinant CSF-1 and IL-34 added in culture and naturally produced in mesothelioma pleural effusions. This was shown by inhibition of CSF-1R signalling, survival and differentiation of human suppressive macrophage in vitro and in vivo induced by mesothelioma cells. Neutralization of IL-34 and CSF-1 resulted in higher killing of mesothelioma cells by a tumor-specific CD8+ T cell clone in mesothelioma/macrophage spheroids. Conclusions: sCSF-1RM149K-Fc efficiently inhibited CSF-1R signalling, monocyte survival and suppressive macrophage differentiation induced by mesothelioma cells producing CSF-1 and IL-34 as well as restored cytotoxic T cell responses. sCSF-1RM149K-Fc efficiently and simultaneously trap both CSF-1 and IL-34 with high therapeutic potential versus other therapies under development targeting single components of this complex cytokine pathway involved in cancer.

ORGANISM(S): Homo sapiens

PROVIDER: GSE282239 | GEO | 2025/03/26

REPOSITORIES: GEO

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Project description:Macrophage polarization between the M2 (repair, pro-tumorigenic) and M1 (inflammatory) phenotypes is seen as a continuum of states. The detailed transcriptional events and signals downstream of CSF-1R that contribute to amplification of the M2 phenotype and suppression of the M1 phenotype are largely unknown. Macrophage CSF-1R pTyr-721 signaling promotes cell motility and enhancement of tumor cell invasion in vitro. Combining analysis of cellular systems for CSF-1R gain-of-function and loss-of-function with bioinformatic analysis of the macrophage CSF-1R pTyr-721-regulated transcriptome, we uncovered miR-21 as a downstream molecular switch controlling macrophage activation and identified ERK1/2 and NF-M-NM-:B as CSF-1R pTyr-721-regulated signaling nodes. We show that CSF-1R pTyr-721 signaling suppresses the proinflammatory phenotype, predominantly by induction of miR-21. Profiling of the miR-21-regulated mRNAs revealed that 80% of the CSF-1-regulated canonical miR-21 targets are pro-inflammatory molecules. Additionally, miR-21 positively regulates M2 marker expression. Moreover, miR-21 feeds back to positively regulate its own expression and to limit CSF-1R-mediated activation of ERK1/2 and NF-M-NM-:B. Consistent with an anti-inflammatory role of miRNA-21, intraperitoneal injection of mice with a miRNA-21 inhibitor increases the recruitment of inflammatory monocytes and enhances the peritoneal monocyte/macrophage response to lipopolysaccharide (LPS). M-bM-^@M-^C These results identify the macrophage miR-21 network as a novel target for controlling macrophage polarization. We performed microarray-based analysis on four mouse macrophage cell lines, two CSF-1R pTyr-721-expressing cell lines (M-/-.WT and M-/-.3ABY721) and two CSF-1R Tyr-721-deficient lines (M-/-.Y721F and M-/-.3AB). Total RNA (two biological replicates) was extracted from CSF-1-starved cells (UR) or from cells constitutively grown in CSF-1 (CONST). Additionally, CSF-1-starved M-/-.WT and M-/-.Y721F cell lines were stimulated with CSF-1 for 0min, 20 min, 60 min and 180 min and used for total RNA extraction. Total RNA preparations with Ribosomal Integrity Numbers (RIN) > 9.5 were used for microarray analysis. A total of 100 ug/cell line/replicate was used for gene expresion analysis on the Affymetrix Mouse Gene ST1.0 chips at the Genomics Core at Einstein, according to manufacturerM-bM-^@M-^Ys instructions. Differential expression analysis was performed using the M-bM-^@M-^XlimmaM-bM-^@M-^Y package of R/Bioconductor to identify significantly differentially expressed mRNAs over time, in response to CSF-1 treatment and to the genotype. CSF1-regulated genes were identified according to the cutoff with both expression folder change > 1.5 and p-value < 0.05.

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A new soluble CSF1R-dimeric mutein with enhanced trapping of both CSF-1 and IL-34 reduces suppressive tumor-associated macrophages in mesothelioma.

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