In vivo imaging of specialized bone marrow endothelial microdomains for tumour engraftment

Abstract

The organization of cellular niches is known to have a key role in regulating normal stem cell differentiation and regeneration, but relatively little is known about the architecture of microenvironments that support malignant metastasis1,2. Using dynamic in vivo confocal imaging, here we show that murine bone marrow contains unique anatomic regions defined by specialized endothelium. This vasculature expresses the adhesion molecule E-selectin and the chemoattractant stromal-cell-derived factor 1 (SDF-1) in discrete, discontinuous areas that influence the homing of a variety of tumour cell lines. Disruption of the interactions between SDF-1 and its receptor CXCR4 inhibits the homing of Nalm-6 cells (an acute lymphoblastic leukaemia cell line) to these vessels. Further studies revealed that circulating leukaemic cells can engraft around these vessels, suggesting that this molecularly distinct vasculature demarcates a microenvironment for early metastatic tumour spread in bone marrow. Finally, purified haematopoietic stem/progenitor cells and lymphocytes also localize to the same microdomains, indicating that this vasculature might also function in benign states to demarcate specific portals for the entry of cells into the marrow space. Specialized vascular structures therefore appear to delineate a microenvironment with unique physiology that can be exploited by circulating malignant cells.

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Figure 1: Leukaemic cell homing and engraftment in mouse skull bone marrow in vivo.
Figure 2: In vivo immunofluorescence microscopy of vascular cell adhesion molecule expression in bone marrow.
Figure 3: Leukaemic cell homing to SDF-1 + E-selectin + microvascular domains is inhibited by SDF-1/CXCR4 blockade.
Figure 4: HSPCs home to SDF-1-positive vascular microdomains.

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Acknowledgements

We thank I. Alley, G. Adams, R. Klein and D. Worhunsky for help with stem cell harvesting and purification, and C. Pitsillides for assistance with T-cell purification techniques. We are grateful to D. Dombkowski for assistance with cell sorting. Special thanks to S. Harvey for assistance with illustrations and to A. Chenn for review of the manuscript. This work was supported by National Institute of Health (NIH) grants to C.P.L. and D.T.S., a NIH training grant (D.A.S.) and a Whitaker Foundation Graduate Fellowship (J.W.W.).

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Correspondence to Dorothy A. Sipkins or Charles P. Lin.

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Reprints and permissions information is available at npg.nature.com/reprintsandpermissions. The authors declare no competing financial interests.

Supplementary information

41586_2005_BFnature03703_MOESM1_ESM.mov

This video shows Nalm-6 leukemic cells rolling along but not adhering to skin vasculature. (MOV 2070 kb)

Supplementary Video S1

This video shows Nalm-6 leukemic cells rolling along but not adhering to skin vasculature. (MOV 2070 kb)

41586_2005_BFnature03703_MOESM2_ESM.mov

This movie shows Nalm-6 leukemic cells adhering to specific vascular domains in the bone marrow. (MOV 9159 kb)

Supplementary Video S2

This movie shows Nalm-6 leukemic cells adhering to specific vascular domains in the bone marrow. (MOV 9159 kb)

41586_2005_BFnature03703_MOESM3_ESM.mov

This movie shows another view of Nalm-6 leukemic homing interactions in a bone marrow parasagittal vascular bed. (MOV 9281 kb)

Supplementary Video S3

This movie shows another view of Nalm-6 leukemic homing interactions in a bone marrow parasagittal vascular bed. (MOV 9281 kb)

41586_2005_BFnature03703_MOESM4_ESM.mov

This movie shows footage of extensive Nalm-6 rolling and binding interactions in a large frontal vein possessing high blood flow-rate. (MOV 10144 kb)

Supplementary Video S4

This movie shows footage of extensive Nalm-6 rolling and binding interactions in a large frontal vein possessing high blood flow-rate. (MOV 10144 kb)

Supplementary Figure S1

This figure shows that MatLyLu (MLL) prostatic carcinoma cells home to the same vascular microdomains as Nalm-6 leukemic cells. (JPG 28 kb)

Supplementary Figure S2

This figure shows that Nalm-6 binding and SDF-1 expression occur in restricted regions of bone marrow. SDF-1+ regions are contrasted with SDF-1- areas. Corresponding Nalm-6 homing patterns are shown. (JPG 31 kb)

Supplementary Figure S3

This figure shows the restricted pattern of SDF-1 vascular expression in bone marrow by in vivo double-labeling of the vasculature with fluorescent SDF-1 and PECAM-1. (JPG 62 kb)

Supplementary Figure S4

This figure shows that Nalm-6 homing is inhibited after SDF-1-desensitization treatment. (JPG 18 kb)

Supplementary Figure S5

This figure shows that CXCR4 blockade causes a modest mobilization of engrafted Nalm-6 leukemic cells into the peripheral circulation. (JPG 19 kb)

Supplementary Figure S6

This figure shows in vitro studies that confirm the relative persistence of fluorescence signal in slowly dividing cells, as detected by flow cytometry. (JPG 23 kb)

Supplementary Figure S7

This figure shows that benign T lymphocytes home to the same SDF-1+ vascular microdomains as Nalm-6 leukemic cells. (JPG 17 kb)

Supplementary Figure S8

This figure shows that VLA-4/VCAM-1 interactions do not appear critical for initial homing of Nalm-6 pre-B ALL to bone marrow. (JPG 24 kb)

Supplementary Figure S9

This figure shows that Nalm-6 leukemic cells pre-treated with N-Cadherin blocking antibody show altered homing kinetics compared to control. (JPG 31 kb)

Supplementary Figure S10

This figure demonstrates that differences in signal intensity detection by in vivo immunoimaging appear to result from variations in optical properties of the surrounding tissue. (JPG 46 kb)

Supplementary Figures and Supplementary Videos Legends

Legends to accompany the above Supplementary Figures and Supplementary Videos (DOC 32 kb)

Supplementary Methods (DOC 35 kb)

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Sipkins, D., Wei, X., Wu, J. et al. In vivo imaging of specialized bone marrow endothelial microdomains for tumour engraftment. Nature 435, 969–973 (2005). https://doi.org/10.1038/nature03703

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