Atypical Chemokine Receptor CCRL2 Shapes Tumor Spheroid Structure and Immune Signaling in Melanoma

Abstract

C-C motif chemokine receptor-like 2 (CCRL2) is an atypical chemokine receptor (ACKR) that binds chemerin with high affinity but lacks classical G protein-coupled signaling. Instead, it functions as a non-signaling presenter of chemerin to CMKLR1-expressing cells, modulating antitumor immunity. CCRL2 is highly expressed in the tumor microenvironment and various human cancers, and its expression has been linked to delayed tumor growth in mouse models, primarily through the chemerin/CMKLR1 axis. While CCRL2's role in immune surveillance is well established, its tumor cell-intrinsic functions remain less clear. Here, we investigated the impact of CCRL2 overexpression and knockout on tumor cell behavior in vitro. Although CCRL2 did not affect proliferation, migration, or clonogenicity in B16F0 melanoma and LLC cells, it significantly influenced spheroid morphology in B16F0 cells. Transcriptomic analysis revealed that CCRL2 modulates innate immune signaling pathways, including TLR4 and IFN-γ/STAT1, with context-dependent downstream effects. These findings suggest that CCRL2 shapes tumor architecture by rewiring inflammatory signaling networks in a cell-intrinsic manner. Further studies in other cancer types and cell models are needed to determine whether CCRL2's regulatory role is broadly conserved and to explore its potential as a therapeutic target in solid tumors.

Keywords: CCRL2; atypical chemokine receptor; melanoma; tumor spheroids.

Figure 1

Effect of CCRL2 expression on the migratory capacity of B16 and LLC tumor cell lines. (A,C) Representative images of wound healing assays performed on B16 melanoma cells (A) and LLC cells (C) either overexpressing CCRL2 (B16/LLC-CCRL2^OE) or knocked out for Ccrl2 (B16/LLC-CCRL2^KO), compared to the controls. Standardized wound areas were generated using culture inserts, and migration was monitored over time. Images include overlays highlighting the remaining cell-free space. (B,D) Quantification of the remaining cell-free space (%) at each time point, calculated as (Cell-free area/Cell-free area at T = 0 h) × 100%. Data are presented as mean ± SEM from three independent experiments (ns: not significant). Migration was analyzed using ImageJ software. Scale bars: 100 μm.

Figure 2

Impact of CCRL2 expression on clonogenic potential and colony architecture in B16 cells. (A) Representative images of crystal violet-stained colonies formed by control B16 cells, CCRL2-overexpressing (B16-CCRL2^OE), and CCRL2-knockout (B16-CCRL2^KO) cells after plating 500 cells and culturing for one week. The rightmost column shows image analysis overlays generated using ImageJ software for colony counting. (B) Quantification of colony formation efficiency calculated as (Number of colonies formed/Number of cells seeded) × 100%. Data are presented as mean ± SD from six independent experiments. No statistically significant differences were observed among the groups (ns: not significant). (C) Quantification of average colony size (µm²) on day 7. Data represent mean ± SD from four independent experiments. One-way ANOVA with Tukey’s post hoc test (** p < 0.01, **** p < 0.0001).

Figure 3

Effect of CCRL2 expression on 3D spheroid formation and morphology in LLC and B16 tumor cell lines under different culture conditions. (A) Representative images of spheroids formed by LLC and B16 cells at day 6 (control, CCRL2-overexpressing, and CCRL2-knockout) cultured in RPMI medium supplemented with 10% FBS. (B,C) B16 spheroids grown in RPMI medium containing 1% (B) or 5% (C) FBS. (D,E) B16 spheroids cultured in RPMI medium with either 10% (D) or 5% (E) FBS supplemented with 2 mg/mL methylcellulose (MC), used to enhance aggregation. Magnified insets highlight spheroid structure and compactness. Scale bars: 100 μm. Images are representative of at least three independent experiments.

Figure 4

Quantitative analysis of spheroid growth and morphology in LLC and B16 cells under different culture conditions. Spheroid area (µm²) and circularity (a measure of compactness, with values closer to 1.0 indicating a more spherical morphology) were quantified using ImageJ in parental (control), CCRL2-overexpressing (CCRL2^OE), and CCRL2-knockout (CCRL2^KO) variants of LLC and B16 melanoma cell lines. Cells were cultured under varying serum concentrations (panels A–C) or with methylcellulose supplementation (panels D,E). Quantifications were performed at days 3, 6, 9, or 10, depending on the condition. All corresponding statistical analyses are provided in Supplementary Table S1.

Figure 5

CCRL2 and E-cadherin expression in tumor spheroids derived from B16 and LLC cells. Flow cytometry analysis of CCRL2 (blue histograms) and E-cadherin (purple histograms) expression in (A) B16, B16-CCRL2^OE, and B16-CCRL2^KO spheroids and in (D) LLC, LLC-CCRL2^OE, and LLC-CCRL2^KO spheroids. Tumor spheroids were cultured in medium containing 10% FBS and harvested on day 6 for analysis. (B,E) Quantification of CCRL2 expression levels based on mean fluorescence intensity (MFI) in B16 and LLC spheroids, respectively. (C,F) Quantification of E-cadherin MFI in the same conditions. Data shown are representative of four independent experiments for B16 and two for LLC, with consistent results. Statistical significance was determined using one-way ANOVA followed by Tukey’s post hoc test (* p < 0.05, ** p < 0.01, **** p < 0.0001; ns: not significant).

Figure 6

CCRL2 expression modulates distinct transcriptional programs in B16 spheroids. (A) Principal component analysis (PCA) of bulk RNA sequencing data from B16 (green), B16-CCRL2^OE (red), and B16-CCRL2^KO (blue) spheroids cultured in 10% FBS and collected at day 6. Each point represents an individual biological replicate. (B,C) Volcano plots displaying differentially expressed genes between B16-CCRL2^OE vs. B16 (B) and B16-CCRL2^KO vs. B16 (C). Red dots represent significantly upregulated genes, and blue dots represent significantly downregulated genes (adjusted p < 0.05, |log₂FC| > 1). Vertical and horizontal dashed lines mark significance and fold change thresholds. (D) Venn diagrams showing the overlap between DEGs in the two comparisons for both upregulated and downregulated genes. (E) Gene Ontology (GO) enrichment analysis of DEGs performed using g:Profiler. Each dot represents an enriched GO term grouped by domain: Molecular Function (GO:MF, red), Biological Process (GO:BP, orange), and Cellular Component (GO:CC, green). The x-axis indicates GO categories, and the y-axis shows –log₁₀ of the adjusted p-value. The size of each dot reflects the size of the gene set associated with that term. (F) Bar graph showing the expression Z-scores of selected transmembrane receptors and G protein-coupled receptors (GPCRs) identified through Ingenuity Pathway Analysis (IPA). Receptors were selected based on both predicted upstream regulatory activity and the availability of a corresponding gene expression log ratio from RNA-seq data. (G) Schematic representation of CCRL2-related signaling interactions based on the published literature, illustrating crosstalk with TLR4, and IFNAR pathways. Downstream transcription factors such as STAT1, AP-1, NF-κB, and IRF3 are shown. The arrows indicate the stepwise progression of the signaling pathway. The question mark represents an unknown or independent pathway. Created with BioRender.com https://app.biorender.com/illustrations/67fcf56dd06583caecb642c5?slideId=79a3abef-dc31-4011-a16b-42bb183051d4 (accessed on 25 July 2025) (H) Z-score plot of downstream effectors and transcription factors identified through IPA downstream analysis as deregulated in B16-CCRL2^OE and B16-CCRL2^KO cells. Only molecules associated with TLR4 and IFNAR signaling pathways were included. (I) Heatmaps showing expression levels of downstream target genes regulated by STAT1, AP-1, NF-κB, and IRF3 transcription factors in B16-CCRL2^OE, and B16-CCRL2^KO cells compared to control. Each row represents a gene and each column a biological replicate.