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Page 6 of 22 García-Pardo et al. J Cancer Metastasis Treat 2021;7:62 https://dx.doi.org/10.20517/2394-4722.2021.103

analyses, our group demonstrated that binding of MMP-9 to CLL cells is mediated by a docking complex
[75]
consisting of α4β1 integrin and a 190 kDa CD44v isoform . Interestingly, silencing either α4β1 integrin or
190 kDa CD44 with specific siRNAs reduced the amount of cell surface-bound MMP-9 and increased the
levels of secreted MMP-9, indicating that there is a dynamic traffic between soluble and cell-bound MMP-9
in CLL. Perhaps this could explain why we could not find in our studies a clear association between the
expression of membrane-bound MMP-9 and the clinical stage of the disease [76-79] . The ability to bind and
retain MMP-9 at their surface appears to be characteristic of CLL cells as it was not observed in normal B-
[75]
cells .
α4β1 integrin is present in approximately 40% of CLL cases, and there is now extensive evidence showing
that the α subunit of this integrin, also known as CD49d, is a strong independent prognostic marker in CLL,
associated with an aggressive disease [10,80] . Our studies have shown that, as a receptor for MMP-9, α4β1
integrin contributes to disease progression by regulating CLL cell migration and survival [77-79,81] . This
[82]
contribution was recently confirmed based on a bimodal pattern of expression of CD49d in CLL cells .
CD44 (standard and variant forms) is expressed at high levels in CLL cells and has also been proposed as a
prognostic marker, particularly due to its role in CLL cell migration and organ localization .
[17]

REGULATION OF ANGIOGENIC FACTORS IN CLL
Regulation by conditions of the microenvironment - hypoxia
The constitutive production of angiogenic molecules and their receptors by CLL cells is subjected to
regulation by several stimuli. Because progression of CLL is characterized by the migration and localization
of malignant cells in lymphoid tissues [18,30] , these stimuli are mainly provided by the microenvironment of
these tissues. Hypoxia, a common condition in human bone marrow and other tissues, is a key regulator of
VEGF in physiological and pathological conditions [20,47,49] . Indeed, hypoxia stabilizes the hypoxia-inducible
factor-1α (HIF-1α), a major transcription factor for VEGF, allowing its binding to specific elements in the
VEGF promoter and increasing VEGF synthesis and secretion [47,49] . Several authors have shown that, upon a
hypoxic stimulus, cultured CLL cells increase their VEGF production, at both mRNA and protein
levels [36,38] . Moreover, we and others demonstrated by immunohistochemical analyses that VEGF is present
in the bone marrow and lymph nodes of CLL patients, and at higher expression than in normal tissues [38,65] .
Kay et al. found that culturing CLL cells under hypoxic conditions also reduced the levels of TSP-1
[36]
produced by these cells, thus favoring an angiogenic switch. The levels of PDGF and Ang-2 in CLL cells
were also shown to be upregulated by hypoxia [36,52,83] .

Regulation by stromal cells - induction of a proangiogenic phenotype on CLL cells
Upon migration and localization in lymphoid tissues, CLL cells interact with surrounding stromal cells,
establishing an active crosstalk that provides survival and proliferation signals to the malignant cells. The
molecular bases of these interactions have been recapitulated in excellent previous reviews [30,84] and involve
direct cell-cell contact via adhesion molecules, release of soluble factors such as chemokines, or material
exchange via extracellular vesicles [Figure 1]. One important consequence of these cellular interactions is
the modification of the gene expression profile of CLL cells, mainly resulting in the upregulation of anti-
apoptotic molecules (Bcl-2, Bcl-xL, Mcl-1, and XIAP) and the activation of survival signaling pathways
(PI3-K/NF-κB and Notch) . Additionally, the balance of pro- and antiangiogenic factors on CLL cells is
[30]
also affected by the contact with stromal cells. Kay et al. showed that culturing CLL cells on primary bone
[31]
marrow-derived stromal cells dramatically increased the secretion of bFGF and reduced the levels of the
antiangiogenic molecule TSP-1. In another study, Edelmann et al. performed gene microarray analyses on
[32]
CLL cells that had been co-cultured with the murine bone marrow fibroblast cell line M2-10B4, either in
direct cell-cell contact or separated by Transwell inserts. They found that direct contact with the fibroblastic
cells significantly increased VEGF expression at both gene and protein levels and decreased TSP-1