Isolement et caractérisation de neutrophiles avec des propriétés anti-tumorales

Neutrophils were initially characterized as the innate immune cells which serve as first line defense against invading microorganisms. Today it is known that neutrophils have more far-reaching functions, being involved in mounting adaptive immune responses against foreign antigens^1,2, regulating hematopoiesis³, angiogenesis⁴ and wound healing⁵. In addition, neutrophils may affect tumor growth and metastatic progression by virtue of their pro- and anti-tumor activities^6,7. Neutrophils are characterized by a polymorphic segmented nucleus (hence termed polymorphonuclear (PMN) leukocytes) and contain at least three distinct subclasses of granules as well as secretory vesicles⁸ (Figure 1A-C).

Neutrophils possess high phagocytic capacity and high NADPH oxidase activity critical for microbial elimination, and secrete a wide range of chemokines important for attraction of additional neutrophils and other immune cells to the site of inflammation^8,9. Neutrophils are characterized by the expression of a large amount of surface receptors including Toll-like receptors (TLRs), C-type Lectin Receptors (CLRs), complement receptor 3 (CD11b/CD18) and other adhesion molecules (e.g., L-selectin, LFA-1, VLA-4 and carcinoembryonic antigen-related cell adhesion molecule 3 (CEACAM3/CD66b)), chemokine receptors (e.g., CXCR1, CXCR2, CCR1, CCR2), chemoattractant receptors (e.g., PAFR, LTB₄R and C5aR), cytokine receptors (e.g., G-CSFR, IL-1R, IL-4R, IL-12R, IL-18R, TNFR), formyl-peptide receptors (e.g., FPR1-3), and Fc receptors (e.g., CD16 (FcγRIII), CD32 (FcγRII), and CD64 (FcγRI)¹⁰. In mice, neutrophils are usually identified as CD11b⁺Ly6G⁺, whereas human neutrophils are identified using the CD11b, CD15, CD16 and CD66b leukocyte markers. It is also generally accepted to stain for the granule proteins myeloperoxidase (MPO) and neutrophil elastase (NE) for detection of neutrophils in tissues.

It is still unclear whether the diverse functions of neutrophils are mediated by the same cell or by distinct cell sub-populations. Accumulating data suggest for the presence of a heterogenic neutrophil population that exhibits a high degree of plasticity affected by pro-inflammatory stimuli and the microenvironment^11,12. Fridlender et al.¹³ have grossly divided the neutrophils in cancer into two major sub-populations termed N1 with anti-tumor properties and N2 with pro-tumor properties. In cancer, as well as in chronic inflammation, there is an additional sub-population composed of granulocytic myeloid-derived suppressor cells (G-MDSCs) that suppress T cell responses¹⁴. G-MDSCs are considered to be immature myeloid cells characterized by a CD11b⁺Ly6C^lowLy6G^hi phenotype in mice¹⁵, while having a CD15⁺/CD16^low phenotype in human¹⁶. G-MDSCs express higher levels of arginase and myeloperoxidase, while lower levels of cytokines and chemokines than normal circulating neutrophils. They are less phagocytic and migratory, but produce higher levels of ROS^15,17,18. In the present paper we will describe some basic methodologies for isolation and characterization of neutrophils with anti-tumor properties.

While neutrophils constitute the largest population of all white blood cells in the human circulation (45 – 70%; 1,800 – 6,000/μl), in mice, under normal conditions, they are rather sparse (10 – 15%; 300 – 500/μl). The neutrophil count increases steadily upon inflammation and occasionally in cancer, which represents a state of chronic inflammation⁷. Neutrophils develop from multipotent common myeloid precursor (CMP) cells in the bone marrow, through a differentiation process passing the stages of myeloblasts (MB), promyelocytes (PM), myelocytes (MC), metamyelocytes (MM) and band cells (BC)⁸. The mature, post-mitotic neutrophils may remain within the bone marrow for 4 – 7 days before they are released to the circulation⁸. Neutrophil turnover in the blood is usually rapid with an average half-life of 6 – 12 hrs, which may be prolonged under inflammatory conditions. Unstimulated neutrophils have limited anti-tumorigenic activity, a feature that can be acquired by exposing the naïve neutrophils to the chemokines IL-8 (CXCL2), CCL2, CCL5 and CXCL5^6,19 or artificially, by exposing them to the phorbol ester phorbol 12-myristate 13-acetate (PMA)⁶.

The short half-life of blood neutrophils together with the low number of neutrophils (~ 3 – 5 x 10⁵) achieved from 1 ml blood of a naïve 6 – 8 week old mouse, have made it difficult to explore the function of circulating mouse neutrophil in vitro. To overcome this difficulty, other sources have been used. For instance, large numbers of neutrophils may be obtained from the bone marrow²⁰ or the peritoneum following the induction of sterile inflammation (e.g., after intraperitoneal injection of thioglycollate broth or Zymosan A). It should be noted that neutrophils obtained from the peritoneal cavity do not exert any anti-tumorigenic activity (unpublished observation).

Granot et al.⁶ observed that BALB/c mice inoculated orthotopically with the mouse 4T1 breast carcinoma cell line develop neutrophilia which aggravates with tumor progression⁶ (Figure 2A), such that 20 – 40 million blood neutrophils can be easily isolated from 1 ml blood 3 – 4 weeks post-tumor inoculation. These neutrophils have acquired anti-tumor activities, and have accordingly been coined tumor-entrained neutrophils (TEN), in order to distinguish them from naïve neutrophils⁶ (Figure 2B). While high-density neutrophils (HDN, Figure 1A) are highly anti-tumorigenic, low-density neutrophils (LDN, Figure 1B) generated in the context of cancer are not²¹. Also, high-density neutrophils from the bone marrow and spleen of tumor-bearing mice have anti-tumor activity (unpublished data). It should be noted that with tumor progression the spleen becomes gradually enlarged (splenomegaly), with increasing amounts of neutrophils.

It should be noted that TEN are also generated in other models of cancer including both spontaneous (MMTV-PyMT and MMTV-Wnt1 mammary tumors and k-Ras driven lung tumors) and injected (AT-3 (MMTV-PyMT) and E0771 breast carcinoma cells, LLC Lewis lung carcinoma cells and B16-F10 melanoma cells). However, the extent of neutrophil mobilization in these tumor models is far less than of 4T1-inoculated mice, reaching 5 – 10 x 10⁶ neutrophils in 1 ml blood after 3 weeks.