Functional integration of acute myeloid leukemia into the vascular niche

Abstract

Vascular endothelial cells are a critical component of the hematopoietic microenvironment that regulates blood cell production. Recent studies suggest the existence of functional cross-talk between hematologic malignancies and vascular endothelium. Here we show that human acute myeloid leukemia (AML) localizes to the vasculature in both patients and in a xenograft model. A significant number of vascular tissue-associated AML cells (V-AML) integrate into vasculature in vivo and can fuse with endothelial cells. V-AML cells acquire several endothelial cell-like characteristics, including the upregulation of CD105, a receptor associated with activated endothelium. Remarkably, endothelial-integrated V-AML shows an almost fourfold reduction in proliferative activity compared with non-vascular-associated AML. Primary AML cells can be induced to downregulate the expression of their hematopoietic markers in vitro and differentiate into phenotypically and functionally defined endothelial-like cells. After transplantation, these leukemia-derived endothelial cells are capable of giving rise to AML. These novel functional interactions between AML cells and normal endothelium along with the reversible endothelial cell potential of AML suggest that vascular endothelium may serve as a previously unrecognized reservoir for AML.

Figure 1. AML localizes to vascular endothelium in vivo.

(A) Transplantation schema. (B) Engraftment analysis of primary AML in NSG mice. Representative flow cytometry data showing the frequency of AML cells from one donor in the peripheral blood (PB) and bone marrow (BM) of xenografted animals. Each diamond in the scatter plot on the right represents an individual mouse. (C) AML cells in the bone marrow of an NSG recipient femur. Tissue sections were stained with antibodies to mouse CD31 (red), human CD45 (green). Arrowheads indicate sinusoids, which are compressed in regions with high levels of human cell engraftment. Nuclei are stained with DAPI (blue). (D) Infiltrates of primary human AML cells (outlined with dashed lines) are found immediately adjacent to portal veins (PV) in the livers of NSG recipient mice (H&E stain). (E) Human CD45⁺ AML cells (hCD45; green) localize next to mouse CD31⁺ portal vein endothelial cells (mCD31⁺, red). Nuclei are blue (DAPI). (F-H) Perivascular accumulation of AML cells around the portal vessels in human liver. (F) H&E stained section. (G) Extensive infiltration of CD33⁺ cells (brown) in a portal triad (BD: bile duct; A: artery) is shown. (H) CD45⁺ AML cells (blue) surround a CD31⁺ (brown) portal vessel (PV). Scale bars for all images are 20 microns.

Figure 2. Subpopulations of AML cells tightly adhere to vascular endothelium in vivo.

(A) An example of a single V-AML cell (in box; hCD45⁺, green) with a region of cell membrane that is so adherent to mouse CD31 (mCD31+, red) labeled portal vein (PV) endothelium that the mouse and human markers overlap. Arrowheads indicate V-AML cells adjacent to the endothelium but not tightly associated with mCD31+ portal ECs. (B) Z-stack analysis of the boxed cell shown in panel A. L indicates the PV lumen. A representative Z plane is shown. (C) Quantitation of AML integration into PV endothelium in individual primary (black diamonds) and secondary (white diamonds) NSG recipient mice (n=11). A minimum of 140 nucleated endothelial cells from non-adjacent tissue sections were scored for each mouse. The bar indicates the mean. (D) Strategy for isolating V-AML from the livers of AML recipient mice. Representative FACS sorting gates are shown for mouse endothelial cells (EC), AML and V-AML. (E) Genomic PCR analysis of FLT3 in thrice sorted, single V-AML cells. The FLT3-ITD was detected in 96% of V-AML cells analyzed, confirming their leukemic origin. An example of a rare single cell containing only wild type FLT3 is shown (Lane 8). FLT3-ITD PCR data from pooled mouse cells, bulk input AML cells, and normal peripheral human peripheral blood cells (WT) are shown. nt: no template control. (F) IF analysis of sorted V-AML revealed the presence of mouse CD31⁺ membranes on AML cells. Scale bars: (A): 20 microns; (B): 10 microns; (E): 5 microns.

Figure 3. A subset of V-AML cells fuses with endothelium.

(A) Confocal, single Z-stack image of a mouse portal vessel (PV). A V-AML cell (CD45⁺, green) that also exhibits circumferential mCD31 staining (red) is shown within the boxed region. Panels on the right show single color and merged channels at a higher magnification. (B) Z-stack imaging of a single FACS sorted V-AML cell is shown. Circumferential, overlapping hCD45 and mCD31 expression is observed in the cell membrane, consistent with AML and EC syncytium formation. These syncytia typically represent ~1% of the total sorted mCD31⁺ V-AML population. (C) Interphase FISH analysis of sorted V-AML cells showing the presence of human (red) and mouse (green) centromeres. Scale bars: 10 microns.

Figure 4. V-AML cells up regulate CD105 expression and become quiescent.

(A) Input primary AML cells express hCD45 but not hCD105 by RT-PCR. By contrast, V-AML cells sorted from the liver of an NSG recipient show an induction of CD105 expression. Each lane shows ten sorted cells. (B) hCD105 expression in a subpopulation of V-AML that are tightly associated with the endothelium. Arrowheads indicate hCD105⁺ (red), isolectin⁺ (grey) HLA-ABC⁺ (green) V-AML cells. DNA is labeled with DAPI (blue). (C) Example of BrdU uptake in PV-adherent V-AML cells. In the top panel, two hCD45⁺ (green) V-AML cells (_*, arrowhead) that appear to share membrane with mouse endothelial cells are shown. The second panel shows mouse endothelial isolectin GS-IB4 expression (red). In the third panel, BrdU (pink) is detected in one of the two V-AML cells (*). Bottom panel is a merged image. L indicates the PV lumen. (D) Quantitation of BrdU uptake in mouse endothelial cells, AML and V-AML tightly associated to mouse CD31⁺ PV endothelium. VAML cells adherent to or incorporated into the endothelial layer of the PV proliferate significantly less than AML cells present throughout the rest of the liver. The mean ± SEM is shown for pooled samples. A minimum of 600 PV ECs, 75 PV integrated V-AML cells and 2500 AML cells were scored in non-adjacent sections from each liver. Scale bars: 20 microns

Figure 5. AML has endothelial cell potential in vitro.

(A) Leukemia-derived endothelial colony forming cells (L-ECFC) from the bone marrow of AML patients forms vascular tubules (B) when cultured in Matrigel. (C) Endothelial cell marker expression is upregulated in cultured L-ECFC by flow cytometry whereas hematopoietic cell marker expression is down regulated. Isotype controls are shown in grey. (D) FISH analysis of single L-ECFC cells shows the presence of the AML-specific mutations (green) including trisomy 9 and the MLL re-arrangement. Nuclei are stained blue (DAPI). Scale bars: (A,B) 100 microns; (D) 10 microns.

Figure 6. AML-derived endothelium can adopt a leukemic phenotype

(A) Experimental strategy to assess in vivo leukemic potential of AML-derived ECFCs. (B) Flow cytometry gating used for the purification of CD105⁺CD45^neg L-ECFC prior to transplant. RT-PCR analysis of sorted CD105⁺CD45^neg cells confirmed the absence of CD45 expression. (C) Analysis of bone marrow from CD105⁺CD45^neg L-ECFC recipient NSG mice. A significant population of human CD45⁺ cells is detected in mouse bone marrow by flow cytometry (left panel); and in tissue sections by immunohistochemistry (DAB brown). (D) Localization of hCD33⁺ expressing cells (brown) in the bone marrow of an L-ECFC engrafted NSG mouse. A representative merged brightfield and fluorescent image (DAPI, blue) is shown. (E) Multiple copies of human MLL gene (green, arrowheads) confirm the leukemic origin of the human donor cells in bone marrow from L-ECFC engrafted mice. All nuclei are stained with DAPI. (F) Detection of cytoplasmic NPM1 (blue, arrows) following transplant of L-ECFC derived from an NPM1 mutant donor patient. Scale bars: (C): 10 microns; (D-E): 5 microns; (F): 50 microns.