Tumor-Derived Factors Differentially Affect the Recruitment and Plasticity of Neutrophils

SIMPLE SUMMARY: Neutrophils are the most abundant type of circulating leukocytes and play an important role in tumor biology. In the context of cancer, the origin of tumor-associated neutrophils (TANs) as well as the interplay between circulating neutrophil subpopulations (NDN and LDN) are still not clear. Our results show for the first time that TANs can originate from both NDN and LDN, with LDN infiltrating at a higher extent than NDN. CXCL1 and CXCL2 chemokines strongly affect NDN and LDN migration, and tumor-conditioned media differently impact their chemotaxis. Moreover, NDN display tumor-induced phenotypical plasticity, transitioning towards a low-density state (LD-NDN). We found that tumor-conditioned media and CXCL1 promote LD-NDN formation, suggesting that multiple factors mediate neutrophils’ plasticity. Newly formed LD-NDN present similar characteristics to LDN in terms of morphology, functional activity, and surface receptor expression, indicating that a portion of LDN could originate from NDN undergoing phenotypical changes. ABSTRACT: Neutrophils play a key role in cancer biology. In contrast to circulating normal-density neutrophils (NDN), the amount of low-density neutrophils (LDN) significantly increases with tumor progression. The correlation between these neutrophil subpopulations and intratumoral neutrophils (TANs) is still under debate. Using 4T1 (breast) and AB12 (mesothelioma) tumor models, we aimed to elucidate the source of TANs and to assess the mechanisms driving neutrophils’ plasticity in cancer. Both NDN and LDN were found to migrate in response to CXCL1 and CXCL2 exposure, and co-infiltrate the tumor site ex vivo and in vivo, although LDN migration into the tumor was higher than NDN. Tumor-derived factors and chemokines, particularly CXCL1, were found to drive neutrophil phenotypical plasticity, inducing NDN to transition towards a low-density state (LD-NDN). LD-NDN appeared to differ from NDN by displaying a phenotypical profile similar to LDN in terms of nuclear morphology, surface receptor markers, decreased phagocytic abilities, and increased ROS production. Interestingly, all three subpopulations displayed comparable cytotoxic abilities towards tumor cells. Our data suggest that TANs originate from both LDN and NDN, and that a portion of LDN derives from NDN undergoing phenotypical changes. NDN plasticity resulted in a change in surface marker expression and functional activity, gaining characteristics of LDN.