Tumor Associated Macrophages Express High-Levels of FLT3 Ligand, Which Induces Activation of FLT3 Signaling That Promotes Survival of Neoplastic Cells in B-Cell Acute Lymphoblastic Leukemia
Authors: Guo Yao , Huang Junbin , Zhang Dengyang , Zhang Liqin , Zhao Yuming , Yu Liuting , Chang Zhiguang , Pei Han Zhong , Chen Chun , Xue Hongman , Xu Xiaojun , Zhao Zhizhuang Joe , Chen Yun 
Published: February 2021

B-cell acute lymphoblastic leukemia (B-ALL) is a malignancy of B lymphocytes blocked at an early stage of differentiation, which accounts for approximately 30% of childhood cancers. Almost all B-ALL blasts aberrantly express FMS-like tyrosine kinase 3 (FLT3), a receptor tyrosine kinase involved in the development of dendritic cells and B lymphocytes. In B-ALL, the high expression of FLT3 in blasts significantly increases the odds of relapse/death in patients, indicating a pathological role of FLT3 in B-ALL. Note that gain-of-function mutations of FLT3 are relatively rare (&lt;5%) in patients with B-ALL, suggesting an essential function of the ligand activating FLT3 in the initiation and progression of the disease. However, the presence, origin and function of FLT3 ligand (FLT3L) in B-ALL are not fully uncovered. In the present study, we observed high levels of FLT3L (234.7 ± 47.6 pg/ml) in plasma from 28 patients with B-ALL, compared with age-matched healthy donors (41.2 ± 15.5 pg/ml) by using ELISA. Among these samples, 12 were from patients achieved remission with negative minimal residue disease (MRD) but maintained high levels FLT3L (286.9 ± 71.2 pg/ml), suggesting cell-source(s) of FLT3L other than neoplastic B-ALL blasts. We hypothesized that tumor-associated macrophages (TAMs) from the microenvironment of B-ALL express FLT3L, because studies show that activated macrophages from patients with autoimmune disease express high levels of FLT3L to promote inflammation and destruction of local articular components. Previously, we cultured nurse-like cells, which are TAMs with a M2 phenotype from peripheral blood mononuclear cells (PBMCs) of patients with chronic lymphocytic leukemia (CLL) (Chen Y, et al. Blood. 2019; 134:1084-1094.). These cells are key components of microenvironment of CLL cells to support survival and migration of neoplastic B cells. In the present study, we cultured PBMCs from B-ALL patients and obtained non-neoplastic TAMs with a similar morphology to nurse-like cells. These cells were stained positive for CD68 and CD163, which are markers for M2 macrophages by immune fluorescent microscopy. We isolated RNA from purified B-ALL blasts or TAMs of the same patient for real-time PCR to measure the levels of FLT3L transcripts. FLT3L transcripts were abundant in the RNA isolated from TAMs, but were negligible in RNA isolated from B-ALL cells in each patient tested (N = 5). We also evaluated for secreted FLT3L protein via ELISA in the culture-medium of isolated TAMs and B-ALL cells with an equal cell number from 18 different patients. The cultured medium of TAMs had significantly higher concentration of FLT3L than B-ALL cells. Furthermore, we performed co-culture studies to assess the involvement of FL/FLT3 signaling in the survival of B-ALL cells cultured with TAMs. B-ALL cells co-cultured with TAMs had significantly more live cells detected by flow cytometry with annexin V and PI staining, compared with B-ALL cells cultured alone. Immunoblot showed that B-ALL cells co-cultured with TAMs had high levels of phosphorylated FLT3 and ERK, indicating the activated FLT3 signaling cascade in these cells. We speculate that the elevated level of FLT3L noted in the plasma of patients with B-ALL is produced primarily by TAMs, which reside in the microenvironment of neoplastic cells. Such high-level FLT3L may provide chemoresistance of B-ALL cells within the microenvironment. Treatment with FLT3 tyrosine kinase inhibitors may mitigate such effects, potentially enhancing the clearance of leukemia cells when used in combination with chemotherapy.

Disclosures
No relevant conflicts of interest to declare.