The neutrophil protein CD177 is a novel PDPN receptor that regulates human cancer-associated fibroblast physiology

Abstract

The cancer-associated fibroblast (CAF) marker podoplanin (PDPN) is generally correlated with poor clinical outcomes in cancer patients and thus represents a promising therapeutic target. Despite its biomedical relevance, basic aspects of PDPN biology such as its cellular functions and cell surface ligands remain poorly uncharacterized, thus challenging drug development. Here, we utilize a high throughput platform to elucidate the PDPN cell surface interactome, and uncover the neutrophil protein CD177 as a new binding partner. Quantitative proteomics analysis of the CAF phosphoproteome reveals a role for PDPN in cell signaling, growth and actomyosin contractility, among other processes. Moreover, cellular assays demonstrate that CD177 is a functional antagonist, recapitulating the phenotype observed in PDPN-deficient CAFs. In sum, starting from the unbiased elucidation of the PDPN co-receptome, our work provides insights into PDPN functions and reveals the PDPN/CD177 axis as a possible modulator of fibroblast physiology in the tumor microenvironment.

Fig 1. PDPN⁺ CAFs are associated with poor survival outcomes in CRC patients.

Two publicly available CRC microarray gene expression datasets (GSE33113 and GSE39582) were analyzed to determine whether PDPN expression levels were significant prognostic factors in recurrence-free survival (RFS). (A, B) Kaplan-Meier curves for the survival probability of patients in GSE33113 (A) or GSE39582 (B) split into tertiles by levels of PDPN expression. Throughout the Fig., log-rank p-values are associated with Kaplan-Meier curves while Cox proportional hazard (CoxPH) p-values are associated with the univariate models detailed in Table 2. (C) The correlation between PDPN expression and tumor content. (D) The correlation between PDPN expression and a signature of activated fibroblasts. (E, F) Fresh human CRC tumors were digested and PDPN expression on major cell populations was analyzed by FACS. Bar graph indicating the percentage of each cell type that are PDPN⁺ (E) and a histogram of PDPN expression on each cell type (F). (G, H) Kaplan-Meier curves for the survival probability of patients split into tertiles by expression levels of the activated fibroblast signature. See also S1 Fig and Table 2.

Fig 2. The PDPN cell surface interactome identifies the neutrophil marker CD177 as a novel binding partner.

(A) Schematic representation of the Conditioned Media AlphaScreen platform for receptor-ligand discovery. A library consisting of 1,200 unique STM proteins as ectodomain-Fc fusions was expressed in the conditioned media of human cells. Conditioned media enriched in individual STM proteins was captured on acceptor protein A-coated beads. Site-directed biotinylated PDPN ectodomain was captured on streptavidin-coated donor beads, and subsequently incubated with the library of STM proteins captured on acceptor beads. Putative PDPN binding partners are detected by measuring the chemiluminescence signal. (B) PDPN screens identify CD177 as a specific, high-scoring hit. Hits shown in grey circles have been empirically determined as non-specific binders in previous screens. Analysis of the new interactions identified using SPR. Recombinant (C) CLEC-2 or (D) CD177 ectodomains were immobilized on sensor chips and purified PDPN, expressed as a Fc-tagged ectodomain, was injected at the concentrations indicated. Dissociation constant (K_D) values for each interaction, measured using recombinant PDPN expressed as a monomeric ectodomain, are indicated below each plot.

Fig 3. CLEC-2 and CD177 have a profound effect on the CAF phosphoproteome.

(A) Schematic detailing the experimental workflow of multiplexed phospho- and global proteome profiling in parallel with TMT and fractionation for deep coverage. (B-C) Volcano plots depicting significant changes in CD177- (circle) and CLEC-2- (triangle) treated CAFs relative to untreated cells after (B) 2 min, or (C) 30 min of stimulation. Phosphosites that changed significantly (|Log2FC| > 1 and p < 0.05) in at least one treatment are represented. (D) Plot summarizing the enriched (q<0.05) GO pathways comprising proteins with significantly altered phosphosites compared to untreated cells. Numbers indicate the phosphosite groups associated with each curated GO term. (E) Heatmap representing the relative change across treatment conditions at 30 min of stimulation (relative to untreated cells) for selected phosphorylation sites on selected proteins.

Fig 4. PDPN controls primary human CRC CAFs growth and contractility.

(A) Histogram showing the expression of PDPN on WT CAFs and PDPN^-/- CAFs compared to an isotype control analyzed by FACS. (B) Graph depicting the percent confluency of WT and PDPN^-/- CAFs over time. Each point represents the mean of 16 different fields of view from 4 wells per condition, and the plot is representative of 4–6 independent experiments. ****p < 0.0001, ANOVA. (C) Left: Morphology index (perimeter²/4**area) of WT and PDPN^-/- CAFs seeded into 3D gels. Each dot represents an average of one well containing >50 cells and the plot is representative of 3 independent experiments. Right: Representative images of CAFs in 3D gels with staining for actin (red) and nuclei (blue). (D) Graph representing the relative contraction of PDPN^-/- cells compared with WT cells. Each dot represents the average of 3–4 wells each from 4 independent experiments. *p < 0.05, **p < 0.01, Mann Whitney.

Fig 5. CD177 and CLEC-2 inhibit PDPN-mediated contractility in primary human CRC CAFs.

(A) Graph showing morphology index of WT CAFs treated with isotype control, recombinant human CLEC-2, or recombinant human CD177. Dots represent the average of >50 cells in four fields of view per experiment, and six independent experiments. Data are plotted relative to the isotype control for each experiment. (B) Representative images of cells with various treatments in the 3D gels. Scale bar, 20 μm. (C) Contraction of WT CAFs treated with an isotype control, CLEC-2, or CD177 proteins relative with untreated CAFs. Dots represent the average of 2–3 wells per condition and graph comprises data from 4 independent experiments. (D) Morphology index of three different primary human fibroblasts from healthy tissues (bladder, colon, and ovary) treated with an isotype control, CLEC-2, or CD177 recombinant protein. Dots represent the average of 4 fields of view per experiment and graph represents data from three independent experiments. *p < 0.05, **p < 0.01, Kruskal-Wallis with Dunn’s multiple comparisons test. (E) Histogram showing representative staining for PDPN expression on the different fibroblasts compared with isotype control.