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. 2021 Aug 4;11(1):15840.
doi: 10.1038/s41598-021-95039-x.

Co-culture model of B-cell acute lymphoblastic leukemia recapitulates a transcription signature of chemotherapy-refractory minimal residual disease

Stephanie L Rellick #  1   2 , Gangqing Hu #  1   3   4 , Debra Piktel  1   2 , Karen H Martin  1   2 , Werner J Geldenhuys  5 , Rajesh R Nair  1   2 , Laura F Gibson  6   7
Affiliations

Co-culture model of B-cell acute lymphoblastic leukemia recapitulates a transcription signature of chemotherapy-refractory minimal residual disease

Stephanie L Rellick et al. Sci Rep. .

Abstract

B-cell acute lymphoblastic leukemia (ALL) is characterized by accumulation of immature hematopoietic cells in the bone marrow, a well-established sanctuary site for leukemic cell survival during treatment. While standard of care treatment results in remission in most patients, a small population of patients will relapse, due to the presence of minimal residual disease (MRD) consisting of dormant, chemotherapy-resistant tumor cells. To interrogate this clinically relevant population of treatment refractory cells, we developed an in vitro cell model in which human ALL cells are grown in co-culture with human derived bone marrow stromal cells or osteoblasts. Within this co-culture, tumor cells are found in suspension, lightly attached to the top of the adherent cells, or buried under the adherent cells in a population that is phase dim (PD) by light microscopy. PD cells are dormant and chemotherapy-resistant, consistent with the population of cells that underlies MRD. In the current study, we characterized the transcriptional signature of PD cells by RNA-Seq, and these data were compared to a published expression data set derived from human MRD B-cell ALL patients. Our comparative analyses revealed that the PD cell population is markedly similar to the MRD expression patterns from the primary cells isolated from patients. We further identified genes and key signaling pathways that are common between the PD tumor cells from co-culture and patient derived MRD cells as potential therapeutic targets for future studies.

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Conflict of interest statement

The authors declare no competing interests.

Figures

Figure 1
Figure 1
Schematic of in vitro co-culture model. (a) A representation of the three cell populations in the co-culture with the suspension and phase bright cells (light grey circles), phase dim cells (PD-dark grey circles), and BMSC or HOB (adherent cells-green). (b) To isolate the PD cells, after removing the media and rinsing to remove cells attached to the top of the adherent cells, the adherent cells and PD cells are collected by trypsinization and purified by size exclusion using Sephadex G-10 (yellow circles).
Figure 2
Figure 2
HOB co-culture up-regulated various environment-sensing signaling pathways and down-regulated cell cycle genes. (a) Volcano plot for expressed genes between control cells (LTMC) and ALL co-cultured with HOB. Red: up-regulated genes in ALL co-cultured with HOB; Black: down-regulated genes; Blue background: all expressed genes. (b) Smoothed scatter plot for the expression changes induced by co-culture of ALL with HOB or with BMSC. r: Pearson Coefficient. (c) DAVID gene-ontology enrichment analysis on biological processes (BP3), cellular components (CC3), and molecular functions (MF3) for genes down-regulated in ALL co-cultured with HOB. (d) DAVID gene-ontology enrichment analysis on biological processes (BP3), cellular components (CC3), and molecular functions (MF3) for genes up-regulated in ALL co-cultured with HOB. (e) Bar graphs for the normalized enrichment scores of Gene Set Enrichment Analysis (GSEA) of expressed genes, ranked by the FC of expression (ALL co-cultured/LTMC), against the MSigDB HALLMARK gene sets (FDR q-val < 0.05 and enrichment > 1.5). (f) Gene Set Enrichment Analysis (GSEA) of expressed genes, ranked by the FC of expression (ALL co-cultured/LTMC), against the MSigDB gene set “OSWALD HEMATOPOIETIC STEM CELL IN COLLAGEN GEL UP” (top panel), which includes genes up-regulated in hematopoietic stem cells cultured with collagen gel compared to in suspension, gene set “WONG ADULT TISSUE STEM MODULE” (middle panel), which includes genes up-regulated in adult tissue stem cells, and gene set “LIM MAMMARY STEM CELL UP” (bottom panel), which includes genes up-regulated in mammary stem cells.
Figure 3
Figure 3
HOB co-culture up-regulated signature genes associated with minimal residual disease of ALL. (a) Bright-field microscopy images showing REH co-cultured on HOB (left) and ALL primary patient cells grown on HOB. Arrow heads indicate ALL cell buried under HOB and therefore are referred to as phase-dim (PD). (b) Gene Set Enrichment Analysis (GSEA) of expressed genes, ranked by the FC of expression (ALL co-cultured/LTMC), against genes that are up-regulated (left panel) or down-regulated (right panel) in ALL cells corresponding to MRD as compared to ALL cells collected at time of diagnosis and before disease treatment in patients. (c) Heat map visualization of expression values in ALL co-cultured with HOB and in suspension (LTMC) for genes differentially expressed between “MRD” and “diagnosis”, sorted by fold-change.
Figure 4
Figure 4
The PD cells are distinctive from the PB cells and are related to MRD. (a) MA plot for the average expression and expression fold-changes between PD and PB cells co-cultured with HOB. Red: genes up-regulated from PB to PD; Black: genes down-regulated; blue background: all expressed genes. (b) Gene ontology enrichment analysis for genes up-regulated from PB to PD on biological processes (BP3). (c) Gene Set Enrichment Analysis (GSEA) of expressed genes, ranked by the FC of expression (PD/PB), against KEGG gene set “Cell cycle”. (d) Smoothed scatter plot for the expression changes of PD vs PB (x-axis) and PD vs LTMC (y-axis). r: Pearson Coefficient. (e) Gene Set Enrichment Analysis (GSEA) of expressed genes, ranked by the FC of expression (PD/PB), against genes that are up-regulated (left panel) or down-regulated (right panel) in ALL cells corresponding to MRD vs control ALL cells collected at time of diagnosis.
Figure 5
Figure 5
Expression of MRD signature genes in LTMC, PB, and PD cells. (a) PCA analysis based the expression of all genes as measured by RNA-Seq analysis for LTMC, PB, and PD cells. (b) Heat map visualization of expression values in ALL co-cultured with HOB (PB and PD cells) and in suspension (LTMC) for genes differentially expressed between “MRD” and “diagnosis”, sorted by fold-change. (c) Similar to panel b but for genes that are differentially expressed between PD and PB cells.
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