Interplay between monocyte subsets and leukemic blasts shapes the immune microenvironment and sustains minimal residual disease in pediatric B-cell acute lymphoblastic leukemia.

Objective: Persistent minimal residual disease (MRD) significantly contributes to chemotherapy resistance and relapse in pediatric B-cell acute lymphoblastic leukemia (B-ALL), with conventional clinical risk factors insufficiently explaining the variability in patient prognoses. This study sought to explore potential mechanisms linked to sustained MRD by integrating analyses of clinical cohorts with single-cell transcriptomics, aiming to identify reliable biomarkers for risk stratification and targeted therapy. Methods: Ninety-seven children with newly diagnosed B-ALL were enrolled and stratified according to their end-of-induction (EOI)-MRD status. Survival analysis and regression models were employed to identify independent risk factors influencing clinical outcomes and MRD development. Single-cell sequencing and CellChat analysis were conducted on paired diagnostic and EOI bone marrow samples to characterize leukemic heterogeneity and the interactions between monocyte subsets and leukemic blasts. Results: Clinical survival analysis showed that pediatric B-ALL patients with positive EOI-MRD had significantly shorter event-free survival. Multivariate regression analysis identified patient age and conventional clinical risk stratification as independent prognostic factors for long-term clinical outcomes. Moreover, age, baseline risk grouping, and the elevated proportion of non-classical monocytes served as independent predictors of positive EOI-MRD status. Single-cell transcriptomic profiling revealed remarkable intratumoral heterogeneity in newly diagnosed pediatric B-ALL cases. Interferon (IFN)-responsive leukemic blasts constituted candidate drug-resistant clones associated with chemotherapy resistance and persistent MRD. This blast subset exhibited inherent malignant characteristics, including blocked B-cell differentiation, cell cycle dormancy, and adaptive IFN tolerance. These malignant clones displayed transcriptional signatures consistent with evasion of IFN-dependent anti-leukemic immune clearance and IFN signaling reprogramming under chemotherapy stress to support cell survival, which may correlate with the formation of residual leukemic lesions. Immune microenvironment analysis confirmed disrupted bone marrow immune homeostasis in MRD-positive patients, which was characterized by impaired T-cell immune surveillance and aberrant accumulation of non-classical monocytes. Global cell-cell communication analysis uncovered bidirectional crosstalk between leukemic blasts and monocytes in the B-ALL immune microenvironment. Leukemic blasts exerted predominant unidirectional regulation on monocytes, while monocytes reciprocally remodeled the malignant phenotypes of leukemic cells through multiple ligand-receptor interactions. Functional heterogeneity existed across different monocyte subsets in the leukemic niche. Classical and intermediate monocytes were the primary sources of CCL signaling, mediating chemotactic signals to leukemic blasts via the CCL axis. Galectin signaling was universally activated to support basal cell adhesion and niche crosstalk in the bone marrow microenvironment. In comparison, non-classical monocytes exhibited transcriptional signatures suggestive of pro-tumor activities via activation of TNF/TNFSF13B-dependent pro-inflammatory signaling and downstream VEGF-related vascular remodeling pathways, which may correlate with the persistence and progression of residual leukemia. Notably, despite intensive inflammatory crosstalk between non-classical monocytes and IFN-responsive residual leukemic blasts, non-classical monocytes only displayed compensatory upregulation of HLA-DRB5 without full transcriptional activation of inflammatory programs. This incomplete immune response may be unable to eradicate residual leukemic cells and favor the formation of a tumor-permissive bone marrow niche, potentially correlating with persistent MRD in pediatric B-ALL. In conclusion, clinical high-risk factors, intrinsic leukemic heterogeneity, and monocyte-associated immune disturbances are jointly correlated with MRD progression. Aberrant crosstalk between non-classical monocytes and leukemic cells is linked to chemotherapy resistance, providing candidate immune biomarkers for prognostic evaluation and personalized targeted therapy in pediatric B-ALL.