Ultralow-dose irradiation enables engraftment and intravital tracking of disease initiating niches in clonal hematopoiesis

Abstract

Recent advances in imaging suggested that spatial organization of hematopoietic cells in their bone marrow microenvironment (niche) regulates cell expansion, governing progression, and leukemic transformation of hematological clonal disorders. However, our ability to interrogate the niche in pre-malignant conditions has been limited, as standard murine models of these diseases rely largely on transplantation of the mutant clones into conditioned mice where the marrow microenvironment is compromised. Here, we leveraged live-animal microscopy and ultralow dose whole body or focal irradiation to capture single cells and early expansion of benign/pre-malignant clones in the functionally preserved microenvironment. 0.5 Gy whole body irradiation (WBI) allowed steady engraftment of cells beyond 30 weeks compared to non-conditioned controls. In-vivo tracking and functional analyses of the microenvironment showed no change in vessel integrity, cell viability, and HSC-supportive functions of the stromal cells, suggesting minimal inflammation after the radiation insult. The approach enabled in vivo imaging of Tet2^+/- and its healthy counterpart, showing preferential localization within a shared microenvironment while forming discrete micro-niches. Notably, stationary association with the niche only occurred in a subset of cells and would not be identified without live imaging. This strategy may be broadly applied to study clonal disorders in a spatial context.

Keywords: Cancer microenvironment; Clonal hematopoiesis; Fluorescence imaging; Multiphoton microscopy; Myelodysplastic syndrome; TET2; Time-lapse imaging.

Fig. 1

Low-dose (0.5 Gy) irradiation enables engraftment and tracking of healthy hematopoietic cells in vivo. (a) Timelines of the transplantation, imaging and peripheral blood analyses. (b) Maximum intensity projection of a montage of tile-scanned z-stacks showed single cells (yellow arrows) and cells that underwent expansion (white arrows) at 1 and 4 weeks after transplantation into 0.5 Gy whole body irradiated recipient mice, compared to the non-conditioned control (Green, UBI-GFP cells; Magenta, Rhodamine dextran; representative images from N = 3—7 mice). (c) Strategies of small-animal radiation research platform (SARRP) local irradiation. Ventral view of the animal and placement of calvarial-targeted isocenter (red) and radiation field (10 × 10 mm, purple square). The dose volume histogram shows 100% of 0.5 Gy dose targeted to only the radiation field. (d) Maximum intensity projection of a montage of tile-scanned z-stacks showed cell engraftment in the non-irradiated site, at 1 week after transplantation into 0.5 Gy local irradiated recipient mice (Green, UBI-GFP cells; Magenta, Rhodamine dextran; representative images from N = 3 mice). (e,f) Engraftment analyses (GFP⁺ cells in the peripheral blood) after transplantation of 2 × 10⁶ GFP⁺ whole bone marrow cells in 0.5 Gy whole body irradiated (WBI), non-conditioned, and 0.5 Gy locally irradiated recipients (N = 3–8 mice per experimental group). Each dot represents an individual mouse. Data from all mice are shown. Two-sided Mann–Whitney test. Data shows mean ± s.d.

Fig. 2

Vascular integrity and cell viability are preserved after 0.5 Gy whole body irradiation. (a) Heat maps showing average intensity projection from the first 8 seconds of rhodamine—dextran leakage after the dye was administered retro-orbitally. Bright color indicates higher pixel intensity (s: sinusoidal vessels with diameter > 15 μm; a: arterioles with diameter < 12 μm; H: high permeability zone; L: low permeability zone). (b,c) Average vascular permeability and diameter measured in whole body irradiated (WBI), non-irradiated (Ctrl), and locally irradiated mice. (Each data point represents measurements from individual vessel segments. n = 33–73 segments. N = 3 mice from 0.5 or 4.5 Gy WBI and Local groups, N = 2 mice from Ctrl). (d) Vascular density characterized in fraction of dextran⁺ voxels field of view between 0.5 Gy WBI and non-irradiated (Ctrl) groups 1 week after 0.5 Gy irradiation, Two-sided Mann–Whitney test. Data shows mean ± s.d. (e) Maximum intensity projection of representative z-stacks of bone marrow cavities. Images were taken from day 1 after 0.5 Gy, 4.5 Gy WBI or from a non-conditioned mouse (Red: propidium iodide; Green: Fluorescein-dextran). (f) Quantifications of PI⁺ cells per unit volume from the measured cavities (n = 6–9 cavities, N = 3 mice). Osteocytes (determined by the lacuna space from the bone channel) are excluded throughout the analyses as all osteocytes are labeled with propidium iodide in both groups. Two-sided Mann–Whitney test. Data shows mean ± s.d. (g,h) FACs analysis of endothelial and stromal cell populations 1 week after 0.5 Gy WBI (n = 3 vials cell suspension pooled from N = 5 mice, two-sided unpaired t-test).

Fig. 3

Tri-lineage differentiation of MSCs and the hematopoietic support are maintained after 0.5 Gy irradiation. (a) Representative images from osteogenic (Alizarin Red staining), adipogenic (Oil Red staining) and chondrogenic (Alcian Blue staining) assays. (b) Quantifications of tri-lineage differentiation capacity based on the number of labeled cells out of total cells. (Each data point represents measurements from replicates. n = 3 replicates per mouse. N = 3 mice per group). (c) CXCLl12 ELISA performed on total bone marrow interstitial fluid taken from non-conditioned mice, 0.5 Gy WBI mice and 4.5 Gy WBI mouse (n = 2 replicates per mouse, N = 7 mice per group, Two-sided unpaired t-test). (d) Engraftment analyses (GFP⁺ cells in the peripheral blood) of LSK cells transplanted to lethally irradiated mice. LSK cells were co-cultured with MSCs undergoing 0.5 Gy WBI (green) vs the control group (red). Each dot represents a replicate. (n = 2–3 replicates per mouse. N = 3 mice per group, Two-sided Mann–Whitney test. Data shows mean ± s.d.).

Fig. 4

Intravital imaging revealed a high frequency of Tet2^+/- and WT (Tet2^+/+) cells cohabitating the same bone marrow cavity while a cell subset formed discrete micro-niches. (a) A sagittal section of bone marrow cavities (yellow and cyan dashed lines) containing transplanted WT (red) and Tet2^+/- (green) cells. (b) The frequency of a single population (Tet2^+/- or WT alone) or both populations found in the same bone marrow cavity. Data are analyzed from cavities where cells were present, from calvaria and freshly isolated tibia metaphysis. (n = 47 cavities from calvaria, 23 cavities from long bones, N = 3 mice per group) (c) Representative maximum intensity projected images showing growth advantages from the WT or the Tet2^+/− clones at 1–2 weeks after transplantation. (d) The minimal inter-cellular distance between the same clone (Green-to-Green distance or Red-to-Red distance) or between Tet2^+/− and the WT clones (Green-to-Red distance) in the same bone marrow cavity. Each data points were measured based on 3D coordinates between a cell and its closest cell (n = 180, 180, 205 data points for G–G, G–R, R–R, respectively. N = 3 mice, Two-sided Mann–Whitney test. Data shows full range, median, the first and third quartiles.) (e) Time-lapse acquisition showing stationary vs. motile cell population with their trajectories. Images were displayed in 2D by maximum intensity projection. (f) The fraction of stationary vs. motile cell population. Quantifications was based on Tet2^+/- cell displacement greater than 10 μm in 3D over an 1 h with 1-min time interval (n = 19 cells, N = 4 mice).