Fig 2: Current model of the CXCL8-Calprotectin-CD147-neutrophil axis in penile tumor progression. (1) PeCa cells release CXCL8 and Calprotectin that promote chemotaxis and infiltration of neutrophils. (2) Infiltrating Calprotectin-positive TME-reprogrammed neutrophil-MDSCs supports tumor growth by releasing growth factors and matrix remodeling enzymes. (3) MDSCs synthesize Calprotectin, supporting an autocrine feedback loop that causes further accumulation of MDSCs. (4) Neutrophil-derived MDSCs have a notable T-cell suppressing activity associated with poor prognosis. (5) The expression of mCD147 impairs ADCC and, thus, the efficacy of immunotherapeutic approaches. (6) The Calprotectin-CD147 axis causes intracellular signaling that enhances the expression of pro-tumorigenic genes and thus promotes proliferation and dissemination of cancer cells. (7) ADAM proteases cleave mCD147 releasing sCD147 that fuels epithelial-to-mesenchymal transition of cancer cells, further boosting invasion and metastasis of cancer cells, and causes a positive feedback loop of sustained CD147-MMP production in fibroblasts. (8) These transform into cancer-associated fibroblasts that, in turn, further promote tumorigenesis by releasing MMP, recruiting further MDSCs and causing an immunosuppressive gene signature in myeloid cells. (9) Tumor-derived factors such as TNF-a, VEGF, and TGF-ß induced Calprotectin in distal organs initiating (10) the formation of Calprotectin-CD147-neutrophil MDSC-primed pre-metastatic niches that facilitate the colonization by tumor cells [modified by (53)].

Fig 3: CD147 surface expression correlates negatively with ADCC susceptibility. (A) Expression of CD147 was investigated by IHC on FFPE slides of 3D cultures from PeCa cell lines P2, L2, L3, and NFK on a matrix of rat collagen with embedded HFF using mouse anti-human CD147–specific antibody and the Vector goat-anti-rabbit-ImmPRESS DAB Kit. Pictures were recorded with a Leica DMI6000 and ×10 magnification. (B) Expression of sCD147 in conditioned media from (A) was determined by ELISA. (C) Surface expression of mCD147 was investigated by indirect immunofluorescence and flow cytometry using a mouse anti-human CD147–specific antibody and a PE-labeled mIgG-binding protein of the PeCa cell lines P2, L2, L3, and A431. Representative histograms of four independent experiments are shown. (D) Relative fluorescence intensity (RFI) of four independent experiments in (C) was summarized. (E) The capacity of EGFR-directed IgA antibodies (IgA isotype control, 225-IgA2.0, 10 µg/ml) to mediate ADCC of tumor cell lines within 3 h was tested by Calcein release assay using freshly isolated neutrophils (E:T ratio 40:1). A431 served as positive control (30). Calcein-loaded target cells in suspension were simultaneously treated with antibodies and neutrophils for 3 h and fluorescence measured in the supernatant. (F) Pearson correlation of mCD147 surface expression on tumor cells in C and specific lysis of the tumor cells in (D). Data are presented as box and whisker plot showing all data points and min and max as “CD147 in [pg/ml]” in (B), as “RFI” in (D), as “specific lysis %” as mean ± SEM in (E) of three independent experiment runs in triplicates. Significant differences are indicated by asterisks for p = 0.05 (*), p = 0.01 (**), p = 0.001 (***), and p = 0.0001 (****) as calculated by one-way ANOVA, in (E) with two-way ANOVA with Tukey’s multiple comparison test. NFK, normal foreskin keratinocytes; HFF, human foreskin fibroblasts.