A role for macrophages in remodeling the bone marrow microenvironment in B-ALL

B-cell acute lymphoblastic leukemia (B-ALL) is characterized by the accumulation of malignant B-lymphocytes in the bone marrow (BM), and evidence indicates that B-ALL blasts create a favorable microenvironment by disrupting the BM hematopoietic stem cell niche.¹ Data also suggests that macrophages may play a pivotal role in malignant cell growth, not only in solid tumors, but also in hematologic malignancies.¹

Erica Dander and colleagues sought to evaluate the role of monocyte/macrophage compartments in B-ALL and the involvement of chemotactic signaling pathways that may regulate their recruitment into the leukemic BM.¹ Their findings have been published in the British Journal of Hematology and are summarized here.

Patient samples

• BM plasma samples were collected from pediatric patients with B-ALL at diagnosis (n = 160) or relapse (n = 39), and from pediatric healthy donors (n = 53) who donated BM cells for sibling allografts.
• BM biopsies were collected for immunohistochemical analysis from eight adult patients with B-ALL and six non-leukemic controls.
• For flow-cytometric analysis of monocyte subsets, peripheral blood samples were collected from 13 patients with B-ALL at disease diagnosis and from eight healthy donors.
• Primary B-ALL cells for co-culture experiments were isolated from 11 pediatric BM aspirates.

Key observations

Bone marrow infiltration of CD68⁺ macrophages

Immunohistochemical assessment of BM biopsies revealed an increase in CD68⁺ macrophages in six out of eight patients with B-ALL, two of whom had a vast infiltration of these cells, compared with non-leukemic controls. In all eight patients, there was strong and widespread CD163 and CD206 expression, indicating that the infiltrating macrophages had an M2-like signature.

Bone marrow upregulation of CCL2 and CX3CL1

A role for the monocyte/macrophage chemoattractant chemokine ligand 2 (CCL2) in the leukemic microenvironment was indicated by significantly increased CCL2 levels in the BM plasma of pediatric patients with B-ALL, both at diagnosis (n = 77; median, 146.4 pg/mL; p < 0.0001), and at disease relapse (n = 27; median, 139.0 pg/mL; p = 0.0003), compared with pediatric healthy donors (n = 19; median, 53.6 pg/mL). Investigations into the mechanism of increased CCL2 levels revealed:

• Increased levels of CCL2 in supernatants of BM-derived mesenchymal stromal cells (MSCs) from healthy donors cocultured with B-ALL blasts from patients at diagnosis.
• Upregulation of CCL2 production from vascular cells (human umbilical vein endothelial cells [HUVEC]) when cultured with leukemic blasts; and
• Increased production of CCL2 by MSCs and HUVEC cells when stimulated with IL-6, TNF-α, and IL-1β.

Taken together these results suggest a CCL2-mediated interaction of leukemic blasts with MSCs and vascular cells in the BM, stimulated by inflammatory cytokines that are known to be abundant in the leukemic BM.

The following observations also suggest alteration of the CX3CL1/CX3CR1 monocyte recruitment pathway in patients with B-ALL:

• Significantly increased CX3CL1 levels in the BM plasma of 90 patients with B-ALL at diagnosis (median, 3.14 ng/mL; p < 0.05) compared with healthy donors (n = 33; median, 2.19 ng/mL).
• Significantly increased CX3CL1 levels in HUVEC cell cultures following stimulation with inflammatory cytokines IL-6, TNF-α, and IL-1β (from undetectable levels to a median of 35.68 ng/mL; p < 0.01).

Increased proportion of non-classical monocytes in peripheral blood

Flow cytometric analysis of peripheral blood revealed an increased proportion of non-classical (NC; CD14⁺CD16⁺⁺) monocytes at diagnosis in patients with B-ALL (58.11% vs 2.76% in healthy donor samples). These NC monocytes had increased expression of the M2-like marker CD163 and expressed higher levels of the CX3CL1 receptor, CX3CR1, compared with classical monocytes, possibly responding to the high levels of CX3CL1 observed in the B-ALL BM.

Deregulation of the C5a/PTX3 axis

Prior literature has indicated a role for the complement fragment C5a in the motility of leukemic cells. Here, a 3.5-fold increase in C5a levels in the BM plasma of patients with B-ALL at diagnosis compared with healthy donors (median, 11,113 pg/mL vs 3,686 pg/mL; p < 0.0001) supports a role for C5a in promoting monocyte/macrophage recruitment in B-ALL.

Overexpression of C5a in the B-ALL BM may be partially influenced by the complement regulator pentraxin3 (PTX3), supported by the following observations:

• Decreased levels of PTX3 in the BM plasma of patients with B-ALL both at diagnosis (n = 117; median, 1,597 pg/mL; p < 0.0001) and relapse (n = 37; median, 1,946 pg/mL; p < 0.0001), compared with healthy donors (n = 23; median, 3,275 pg/mL).
• Reduced production of PTX3 by BM MSCs cocultured with B-ALL cell line cells and primary B-ALL blasts.

Summary

Based on these results, the authors concluded that B-ALL cells profoundly influence the BM microenvironment, hypothesizing that NC monocytes are recruited to the leukemic BM by migratory signals, including CX3CL1, to an environment that is abundant in M2 polarizing factors, such as CCL2 and C5a.

Further studies of the B-ALL monocyte/macrophage compartment will be an important aid to the development of dual-impact therapeutic strategies, targeting both the leukemic blasts and tumor microenvironment.