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Functional effects on macrophages
Macrophages are also important components of tissue microenvironments. In CLL, the specific population
of leukemia-associated macrophages, known as nurse-like cells, plays a fundamental role in inducing and
maintaining CLL cell proliferation and survival [29,100,101] . The important function of tumor-associated
macrophages in angiogenesis is also well documented [102,103] . Macrophages can be activated in vitro by
[104]
specific stimuli towards two different functional phenotypes: M1 and M2 . M1 macrophages are pro-
inflammatory and induce strong antitumor immune responses by producing relevant specific molecules and
cytotoxic factors. In contrast, M2 macrophages are anti-inflammatory and secrete chemokines/cytokines
that suppress immune responses and support tumor expansion [105,106] . In vivo evidence indicates that tumor-
associated macrophages are mostly polarized towards the M2-like phenotype and that tumor cells
contribute to this polarization . In the case of CLL, Audrito et al. showed that activated CLL cells
[107]
[108]
induced monocyte differentiation to M2 macrophages, and this was mediated by the nicotinamide
phosphoribosyltransferase enzyme produced by CLL cells [Figure 1]. These M2 macrophages sustained CLL
cell survival and reduced T-cell proliferation, thus favoring disease progression . In another study, the
[108]
high-mobility group protein B-1 produced by CLL cells was shown to differentiate nurse-like cells to M2-
polarized macrophages, concomitant with a STAT3 and NF-κB activation on both cell types . The hypoxic
[109]
conditions found in CLL niches also indirectly induced M2 macrophage polarization via increase
production of adenosine .
[29]

Polarization of macrophages towards the M2 subtype also has important consequences for angiogenesis.
Zajac et al. showed that M2 macrophages have higher proangiogenic capacity than the M1 subtype, due
[110]
to the increased production of MMP-9 free of tissue inhibitor of metalloproteinases-1 (TIMP-1), the specific
MMP-9 inhibitor. Indeed, these authors showed that M2 macrophages shutdown their TIMP-1 gene
expression, a fact that was also demonstrated with murine bone marrow-derived macrophages. The
importance of MMP-9 for M2 macrophage angiogenic function was further demonstrated by showing that
MMP-9-null M2 macrophages were not angiogenic, despite their downregulation of the TIMP-1 gene .
[110]
Moreover, the same group showed that neutrophils, which do not produce TIMP-1 , are the major source
[111]
of proangiogenic MMP-9 in tumor tissues . MMP-9 is present in CLL tissues, where it is produced by the
[112]
mentioned stromal cellular components as well as by CLL cells. Our group demonstrated that the CM of
24 h CLL cell cultures induced a proangiogenic profile in both M1 and M2 macrophages after 7 days of
culture, as determined by the increased expression of MMP-9 in these cells, at both mRNA and protein
[79]
levels . Although we did not test whether the MMP-9 produced by M2 macrophages was more angiogenic
than that produced by M1 macrophages, this study constitutes another example of how CLL cells modulate
the angiogenic profile of microenvironmental cells in CLL tissues.

Functional effects on neutrophils
The role of neutrophils in tumor initiation and angiogenesis is well recognized . In solid tumors, such as
[113]
melanoma, hepatocellular carcinoma, and breast carcinoma, the recruitment of proangiogenic neutrophils
triggers STAT3 activation and the production of angiogenic molecules, including VEGF and MMP-9 .
[114]
There is also a proangiogenic subpopulation of neutrophils, which express high levels of α4β1 integrin,
CXCR4, and MMP-9 and are recruited to hypoxic tumor sites . Indeed, as mentioned above,
[115]
[112]
inflammatory neutrophils are the major producers of angiogenic MMP-9 in tumor tissues . In the case of
CLL, neutrophils were shown to be activated, expressing high levels of CD54 and presenting some
functional defects . Podaza et al. showed that CLL cells induced neutrophil survival and their
[117]
[116]
reprogramming to an immunosuppressive phenotype. In turn, neutrophils were shown to affect CLL cell
activation and survival through the induction of extracellular traps , thus establishing a reciprocal cellular
[117]
interaction that supports the proangiogenic function of neutrophils in CLL niches.