Study of CXCL4 and CXCL4L1 isoforms in colon cancer: modulation of angiogenesis, monocytic stromal cell differentiation and tumor cell progression

Apart from their indispensable role in immune cell migration, chemokines are nowadays appreciated as multifunctional cytokines in several physiological and pathological circumstances. For instance, by modulating angiogenesis and shaping the tumor microenvironment, chemokines are recognized as essential players in tumor biology. This doctoral study mainly focused on the platelet-derived chemokines CXCL4 and CXCL4L1. These non-allelic variants differ only in three amino acids situated near the carboxy-terminal end. These two similar proteins mediate their biological effect by binding to the G protein-coupled CXCR3 receptor, which exists as 2 alternative splice forms: CXCR3A and CXCR3B. The CXCR3A splice variant mediates pro-migratory and proliferative signaling, whereas CXCR3B initiates opposite signaling. By inhibiting endothelial cell proliferation, CXCL4 and CXCL4L1 in particular, are known to shift the angiogenic/angiostatic balance in favor of angiostasis. As a result of their angiostatic action and the attraction of anti-tumural lymphocytes, CXCL4 and CXCL4L1 have been attributed anti-tumoral activity. In this doctoral project, we first aimed to identify the different NH2-terminal isoforms of CXCL4L1. Therefore, blood platelets of healthy volunteers were isolated and stimulated with thrombin. After purification of the supernatant, the samples were analyzed using a mass spectrometric approach. Besides the classical isoform CXCL4L1(1-70) and the amino-terminally extended isoform CXCL4L1(-4-70), which probably arises through alternative signal peptide removal, we also found two carboxy-terminally truncated isoforms CXCL4L1(1-69) and CXCL4L1(-4-69). The classical isoform was the most abundant, whereas the extended isoform CXCL4L1(-4-70) was only detected in 50% of the cases. In the past, the laboratory of Molecular Immunology and other groups already demonstrated that small alterations at the amino-terminal side of chemokines can have drastic biological consequences. Therefore, we wanted to investigate the impact of the extension of CXCL4L1 with four amino acids on its biological activity. Our in vitro and in vivo data indicated that both CXCL4L1 isoforms had a similar angiostatic potential. Furthermore, both isoforms were able to induce the chemotactic migration of CXCR3A-transfected cells to the same extent and had equal affinity for heparin. In addition, the modulatory effect of CXCL4L1 on monocyte-derived macrophages and immature monocyte-derived dendritic cells (iMDDC) was compared to that of CXCL4. Indeed, besides macrophage-colony stimulating factor (M-CSF), CXCL4 has been shown to prevent monocyte apoptosis and to induce the differentiation into an M4 specific macrophage subtype. Differently to M-CSF and CXCL4, CXCL4L1 is not a survival factor for monocytes. Additionally, CXCL4L1-stimulated monocytes acquired a more pro-inflammatory phenotype compared to CXCL4-differentiated monocytes. Indeed, CXCL4L1-stimulated monocytes were characterized by an increased expression of the chemokine receptors CCR2, CCR5 and CXCR3 and decreased expression of the IL-1 receptor antagonist. Furthermore, CXCL4L1-treated monocytes released higher amounts of the inflammatory chemokines CCL2 and CXCL8, whereas the production of CCL22 was lower compared to CXCL4-differentiated monocytes. For iMDDC generated in the presence of CXCL4, we detected an increased phagocytic capacity and a downregulation of matrix metalloproteinases compared to CXCL4L1. Our findings suggest a different modulatory effect of CXCL4 and CXCL4L1 on distinct cell types. Despite their well-described angiostatic and, consequently indirect, anti-tumoral effect, little is known about the direct effect of CXCL4 and CXCL4L1 on tumor cells. Finally, we evaluated the effect of both platelet-derived chemokines on colorectal cancer cells. Interestingly, CXCL4L1 increased proliferation and migration of HCT 116 cells. Of note, in the presence of CXCL4L1, we found a downregulation of the CXCR3B isoform in this cell line. On the other hand, stimulation with CXCL4 and CXCL10 led to a decreased proliferation rate of HCT 116 cells. However, CXCL4, CXCL4L1 and CXCL10 did not affect the growth of HT 29 cells, which were characterized by expression of both CXCR3 isoforms. In summary, we showed that a small alteration at the amino-terminal end of CXCL4L1 does not affect its biological activity. Additionally, we showed a different modulatory effect of CXCL4 and CXCL4L1 on monocytes, iMDDC and colon cancer cells. Despite its indirect anti-tumoral properties, CXCL4L1 stimulates the migration and proliferation of HCT 116 colon cancer cells via modulation of the receptor expression profile and might therefore promote cancer progression of CXCL4L1-expressing colon tumors.nrpages: 115status: publishe