B-cell acute lymphoblastic leukemia (B-ALL) is the most common cancer among children and characterized by the overproduction of immature B-cell blasts within bone marrow (BM). Despite the significant progress achieved over the last decades with multi-drug chemotherapy regimens, allogeneic hematopoietic stem cell (HSC) transplantation and, most recently, CD19-targeted CAR (chimeric antigen receptor) T-cell immunotherapy, relapse is common after initial treatment and the leading cause of death for pediatric B-ALL patients. A clearer understanding of the microenvironmental evolution during leukemia pathogenesis and the heterogeneity of acquired chemo-resistance mechanisms from distinct B-ALL subtypes may provide novel therapeutic targets for optimized therapy for refractory and relapsed B-ALL patients. To dissect the BM niche mechanisms associated with treatment resistance and disease relapse, there is a critical demand for both an accurate, real-time, and modular methodology and reliable clinical biomarkers to identify and screen promising therapy targets for patients with refractory and relapsed B-ALL diseases. However, the ability to study these heterogeneous interactions among distinct B-ALL subtypes and their BM niches is limited by current in vivo methods.Herein we report an in vitro biomimetic 3D ‘Leukemia-on-a-Chip’ model to dissect the evolving heterogeneity of the BM niche in regulating B-ALL chemotherapy resistance. The 3D microfluidics-based organotypic ‘Leukemia-on-a-Chip’ device is composed of three distinct functional regions: a central sinus region vascularized by endothelial cells (ECs), an inner ring region serves as an interface of leukemia blasts (B-ALL cells) and niche cells (ECs and MSCs) interactions, and the outer ring for cell culture media supplies and waste removal. The system was characterized by time-lapse imaging, single-cell mRNA sequencing (scRNA-Seq) and fluorescence immunostaining, as well as drug testing. Using the model, we monitored dynamic behaviors of B-ALL blasts with or without niche cells and found that B-ALL blasts without niche continuously migrated during culture, whereas those with niche cells became less motile after 2-day culture. To understand the heterogeneity of distinct human B-ALL types, such as favorable REH and unfavorable SUP, we mapped the two niches with scRNA-Seq and found that B-ALL blasts in both niches were variously activated in TNFA signaling via NF-κB. We confirmed this finding with on-chip immunostaining NF-κB and found that NF-κB was significantly enhanced in those with niche cells. In addition, we interrogated the spatial heterogeneity in the leukemic niche and found increased dormancy in B-ALL cells located at the endosteal niche relative to those in the perivascular niche, suggesting that BM sub-niches may differentially regulate leukemia progression, similar to the observances of its healthy counterpart. Finally, we demonstrated the preclinical use of our platform to screen niche-co-targeting regimens.In this study, we demonstrated a unique organotypic Leukemia-on-a-Chip microphysiological system and comparatively dissected the underlying heterogeneous mechanisms regulated by niche cells to drive leukemia progression and chemo-resistance. We anticipate this platform can be applied to many other hematological malignancies, presenting a powerful tool for drug development under a more physiologically relevant context.

Leukemia-on-a-Chip for Modeling and Decoding Chemotherapy Resistance

Paper Proceedings: https://asmedigitalcollection.asme.org/IMECE/IMECE2021/volume/85543

ASME 2021 International Mechanical Engineering Congress and Exposition (IMECE2021)

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