Journal: Journal of Experimental & Clinical Cancer Research : CR

Article Title: Pre-therapeutic bone marrow-resident leukemic cells in acute myeloid leukemia exhibit a distinct dysregulated calcium signature and stem-like profile reflecting minimal residual disease precursors

doi: 10.1186/s13046-025-03634-x

Figure Lengend Snippet: Identification and characterization of rare human BM-resident leukemic cells (BMresLC) in a xenograft model. A Experimental workflow: primary human AML cells were intravenously injected into immunodeficient mice. Peripheral blood was sampled twice monthly and analyzed by flow cytometry for human CD45 pos cells. At endpoint (≥ 180 days), mice were sacrificed, and BM was collected for RT-qPCR, flow cytometry, and 3D imaging after 3DISCO tissue clearing. B Representative 3D-IMARIS reconstructions of cleared tibial/femoral BM, stained for β2-microglobulin (human MHC class I subunit, red) and Ki67 (proliferation marker, green), showing human B2M pos and Ki67 pos cells or their negative counterparts. Images illustrate the localization and proliferative state of hBMresLC (e.g., B2M pos /Ki67 pos and B2M pos /Ki67 neg subpopulations) in different AML samples. Scale bar:150 µm. C Immunofluorescence staining of 3D-cleared BM tissue with anti-human CD33-PeCy7 (myeloid marker, red) and anti-Ki67-FITC (proliferation marker, green) showing spatial localization of hCD33 pos /Ki67 pos and hCD33 pos /Ki67 neg cell subpopulations. Scale bar: 300 µm. D Flow cytometry-based quantification of human leukemic cells in the BM mouse model, based on B2M, CD33, and Ki67 expression level (Ki67 neg : quiescent cells). E Flow cytometry-based quantification of human CD33 pos cells (blue) in diagnosis and mice BM, and the proportion of CD33 pos cells lacking Ki67 expression (CD33 pos /Ki67 neg quiescent cells) (red) in diagnosis and BMresLC samples. Statistical analysis was performed using Student’s t test (**** p < 0.0001, non-significant (ns)). F Violin plots illustrating mRNA expression levels of Ki67 across distinct clinical subgroups. RNA-seq data were generated from samples at AML diagnosis ( n = 445) and MRD ( n = 130) [ , , ]. RNA-Seq data were obtained from public domain AML datasets ( https://www.cbioportal.org ) and extracted as described in . Each violin represents the distribution of log₂ RPKM expression values. The central dotted marker (larger dot) indicates the median, while the upper and lower dotted markers (smaller dots) represent the interquartile range (IQR). Statistical significance between groups was assessed using the Student t -test. (** p < 0.01). G Quantification according to patient prognosis at diagnosis (adverse vs favorable) and among BMresLC from BM mouse model according to the AML prognosis (adverse vs favorable). Statistical analysis was performed using Student’s t test (** p < 0.01, non-significant (ns)). H Violin plots showing Ki67 mRNA expression levels in patient samples stratified according to adverse versus favorable prognosis (public datasets , cBioportal). Each violin represents the distribution of log₂ RPKM expression values; The central dotted marker (larger dot) indicates the median, while the upper and lower dotted markers (smaller dots) represent the interquartile range (IQR), and the shape reflects data density. Statistical significance between groups was assessed using the Student t -test (** p < 0.01, non-significant (ns))

Article Snippet: Whole-bone clearing and 3D-Imaris imaging enabled spatial localization of rare leukemic cells.

Techniques: Injection, Flow Cytometry, Quantitative RT-PCR, Imaging, Staining, Marker, Immunofluorescence, Expressing, Biomarker Discovery, RNA Sequencing, Generated