CCR1 and CCR5 mediate cancer-induced myelopoiesis and differentiation of myeloid cells in the tumor

Background Cancer-induced ‘emergency’ myelopoiesis plays a key role in tumor progression by inducing the accumulation of myeloid cells with a suppressive phenotype peripherally and in the tumor. Chemokine receptors (CCRs) and, in particular, CCR1, CCR2, CCR5, and CCR7 are emerging as key regulators of myeloid cell trafficking and function but their precise role has not been completely clarified yet because of the signal redundancy, integration, and promiscuity of chemokines and of the expression of these CCRs on other leukocyte subsets. Methods We used the 4PD nanoparticle for the in vivo targeted silencing of CCR1, CCR2, CCR5, and/or CCR7 in the myeloid cells of tumor bearing mice to evaluate the effect of treatments on tumor growth, myeloid cell trafficking and polarization. We used flow and image cytometry and functional assays to monitor changes in the tumor microenvironment and depletion experiments and immune deficient mice to determine the role of Ly6G+cells during tumor progression. We further evaluated in vitro the impact of chemokine receptor inhibition and tumor derived factors on myeloid cell differentiation from mouse and human hematopoietic stem and precursors cells (HSPCs) using flow cytometry, transcriptome analysis, cytokines beads arrays, functional assays, and mice deficient for CCR1 or CCR5. Results 4PD-mediated in vivo silencing of CCR1 and CCR5 on myeloid cells and myeloid precursors was necessary and sufficient to inhibit tumor progression. Functional studies indicated that this antitumor effect was not mediated by alteration of myeloid cell chemotaxes but rather by the repolarization of polymorphonuclear myeloid-derived suppressor cells (MDSCs) into tumoricidal neutrophils. Transcriptome functional and cytokine analysis indicated that tumor derived factors induced CCL3 and CCL4 in HSPCs that, through the autocrine engagement of CCR1 and CCR5, induced HSPCs differentiation in MDSCs. These finding were confirmed across mice with different genetic backgrounds and using HSPCs from umbilical cord blood and peripheral blood of patients with cancer. Conclusions Our data support the notion that CCR1 and CCR5 and their ligands are a master immunological hub activated by several tumor derived factors. Activation of this pathway is necessary for the differentiation of MDSCs and protumoral macrophages.

the relevant peptide (1 µM) in the presence of 10 6 syngeneic splenocytes and syngeneic CD11b + cells for 3 days in 96 well flat bottom plates. Proliferation was evaluated by flow cytometry on the viable CD3 + CD8 + population.
Tumor-Myeloid cell co-culture assay. Magnetically isolated CD11b + cells (purity >90% by flow cytometry) or FACS sorted Ly6G or Ly6C cells were cultured at different ratio with 0.2x10 5 4T1-luciferase cells, in complete medium for 18h at 37°C. 4T1-luciferase cells were enumerated using the Li-COR system by luciferase assay after a 5' incubation with luciferin at 37ºC using a freshly diluted known number of 4T1-luciferase cells as standard curve.
Inhibitors targeting the main neutrophil tumoricidal pathway were chosen through a literature search, used at optimal reported concentration, and added to the cultures.

Analysis of microarray gene expression data
Twenty-four hours after culture with RPMI, 4T1 TCM with Bx471 and Maraviroc, or 4T1 TCM with vehicle, BM cells were washed with PBS and RNA extracted by Trizol (Invitrogen) and cleaned with RNeasy columns (Qiagen). For each chip, 2.5 g of total RNA was amplified to biotinylated complementary RNA (cRNA) as described in the Affymetrix GeneChip® Expression Analysis Technical Manual. Pre-hybridization quality controls were performed with the Agilent 2100 bioanalyzer (Agilent Technologies). RNA from 3 biological replicates was then hybridized on Affymetrix MG-U74Av2 arrays. Microarray probe fluorescence signals were converted to log2 expression values using the Robust Multiarray Average procedure of the affy Bioconductor package in R. Briefly, fluorescence intensities were background-adjusted and normalized using quantile normalization, and expression values were calculated using median polish summarization and a custom chip definition file for the Mouse Gene To identify differentially expressed genes, we compared the expression levels of BM cells cultured in 4T1-TCM and in complete media (RPMI) using the Significance Analysis of Microarray (SAM) algorithm coded in the samr R package. 1 In SAM, we estimated the percentage of false positive predictions (i.e. False Discovery Rate, FDR) with 100 permutations and selected those gene IDs with FDR ≤ 5% and absolute fold change larger than a selected threshold (e.g. ≥ 2). The volcano plot, showing the most significantly differentially expressed genes in BMJ Publishing Group Limited (BMJ) disclaims all liability and responsibility arising from any reliance Supplemental material placed on this supplemental material which has been supplied by the author(s) the comparison of BM CD11b + cells from 4T1-TCM and complete media (RPMI), was generated using the ggplot function of the ggplot2 R package. P-values were derived from SAM q-values using the function samr.pvalues.from.perms of the samr R package.
The gene expression levels of CD11b + cells from BM cells cultured with RPMI, 4T1-TCM with vehicle and 4T1-TCM with Bx471 and Maraviroc have been merged with publicly available gene expression data of CD11b + cells. in complete media (RPMI). Gene sets were considered significantly enriched at FDR <5% when using Signal2Noise as metric and 1,000 permutations of gene sets. Except for the over-representation analysis, all data analyses were performed in R version 3.5.1.
BMJ Publishing Group Limited (BMJ) disclaims all liability and responsibility arising from any reliance Supplemental material placed on this supplemental material which has been supplied by the author(s)  Mice (n=9-10) challenged with the indicated tumors were treated with 4PD conjugated with shRNAs specific for CCR1 and CCR5 or scrambled shRNAs once the tumors become palpable. Data derived from two independent experiments.  4T1 tumors from mice treated with scrambled shRNAs or CCR1 and CCR5 shRNAs were stained with DAPI and antibodies against Ly6G, RB1, and LAMP2. B) Scanned images from each channel of whole tumor sections were sliced in 500x500 pixel images using totalImageSlicer (https://www.coolutils.com/) and fed into cellprofiler as 8bit gray images. Primary objects (nuclei) were identified using the DAPI channel with a nuclei diameter set between 2 and 8 pixels, using the three classes Otsu Adaptive threshold method with a correction factor of 1 and the lower and upper bounds on threshold 0.1-1.0. Clumped objects were distinguished by shape and the size of the smoothing filter and minimum allowed distance between local maxima were automatically calculated. Secondary objects were identified using the autofluorescence and fluorescence of the merged image from the 3 channels acquired using the nuclei propagation method with three-classes Otzu Adaptive threshold method, 0.9 as threshold correction factor (0.0-1.0 range) and 0.02 as regularization factor. The cytoplasm as tertiary object was define as cell (secondary object) area minus the nuclei (primary object) area. For each cell, the integrated intensity mean of the DAPI channel in the nuclei and the integrated intensity mean of the Ly6G, RB1, and Ly6G channels in the cells were exported as .cpout file and analyzed using FCS Express PLUS vs7. A "tumor" gate/ROI was drawn to delineate the tumor area identified in H&E serial sections and RB1 and LAMP2 expression was evaluated within the intratumoral Ly6G + and Ly6Gcells. Data were normalized to the background (all cells gate) median intensity.