Inhibition of Oxidative Phosphorylation Reverses Bone Marrow Hypoxia Visualized in Imageable Syngeneic B-ALL Mouse Model

Abstract

Abnormally low level of interstitial oxygen, or hypoxia, is a hallmark of tumor microenvironment and a known promoter of cancer chemoresistance. Inside a solid tumor mass, the hypoxia stems largely from inadequate supply of oxygenated blood through sparse or misshapen tumor vasculature whilst oxygen utilization rates are low in typical tumor's glycolytic metabolism. In acute leukemias, however, markers of intracellular hypoxia such as increased pimonidazole adduct staining and HIF-1α stabilization are observed in advanced leukemic bone marrows (BM) despite an increase in BM vasculogenesis. We utilized intravital fast scanning two-photon phosphorescence lifetime imaging microscopy (FaST-PLIM) in a BCR-ABL B-ALL mouse model to image the extracellular oxygen concentrations (pO₂) in leukemic BM, and we related the extracellular oxygen levels to intracellular hypoxia, vascular markers and local leukemia burden. We observed a transient increase in BM pO₂ in initial disease stages with intermediate leukemia BM burden, which correlated with an expansion of blood-carrying vascular network in the BM. Yet, we also observed increased formation of intracellular pimonidazole adducts in leukemic BM at the same time. This intermediate stage was followed by a significant decrease of extracellular pO₂ and further increase of intracellular hypoxia as leukemia cellularity overwhelmed BM in disease end-stage. Remarkably, treatment of leukemic mice with IACS-010759, a pharmacological inhibitor of mitochondrial Complex I, substantially increased pO₂ in the BM with advanced B-ALL, and it alleviated intracellular hypoxia reported by pimonidazole staining. High rates of oxygen consumption by B-ALL cells were confirmed by Seahorse assay including in ex vivo cells. Our results suggest that B-ALL expansion in BM is associated with intense oxidative phosphorylation (OxPhos) leading to the onset of metabolic BM hypoxia despite increased BM vascularization. Targeting mitochondrial respiration may be a novel approach to counteract BM hypoxia in B-ALL and, possibly, tumor hypoxia in other OxPhos-reliant malignancies.

Keywords: acute lymphobastic leukemia; bone marrow; hypoxia; leukemia; oxidative phosphorylation; oxygen; vascularity.

Figure 1

B-ALL engraftment and progression in immunocompetent recipient mice. (A) 2-photon intravital images of the calvarial bone marrow on day 1, 3, and 4 post-injection (p.i.) of 2.5 × 10⁵ B-ALL in C57BL6 mice. The blood was visualized using i.v. injection of BSA-AF647. The white arrows point to individual leukemia cells on day 1 p.i. (B) Leukemia tissue invasion index at different time points post-injection. B-ALL cells were injected in C57BL6 mice then tissues were collected and imaged (3 mice per time point day 5, 7, and 13 p.i.).

Figure 2

B-ALL cells localize and expand in the close proximity of blood vessels and osteoblasts (OB). The OB-GFP mice were infused iv with B-ALL-tdTomato cells followed by intravital 2-photon microscopy. (A) day 3 p.i.; (B) day 9 p.i.; (C) day 14 p.i. Red: B-ALL; blue: blood; green: GFP. Scale bars represent 100 μm.

Figure 3

Evaluation of BM oxygenation using pimonidazole (Pimo) IHC. Mice were euthanized on specified day after injecting the leukemia cells and 3 h after ip injection of pimonidazole (3 mice per group). (A–C) Images of pimonidazole IHC in representative histological sections of formalin fixed paraffin embedded femurs (FFPE, 200x magnification). (A) C57BL6 mouse without leukemia. (B) Intermediate stage of leukemia (day 10+ p.i.). (C) Advanced stage of leukemia (day 14+ p.i.). (D–F) Corresponding IHC intensity and cell/tissue segmentation. Brown +3: high intensity of pimonidazole staining; Orange +2: medium intensity of pimonidazole staining; Yellow +1: low intensity of pimonidazole staining; Blue 0: negative/no intensity of pimonidazole staining. (G) Semi quantitative analysis (Histo-score, H-score) of the intensity of pimonidazole IHC in FFPE femur sections of leukemic and normal mice (Healthy): intermediate (Intermediate) and advanced (Advanced) stages of disease progression (n = 3 mice per time point). (H) H-score analysis of the pimonidazole signal intensity in FFPE skull sections in different stages of disease progression (3 mice per time point). ****p < 0.0001.

Figure 4

2-photon PLIM imaging of oxygen in the calvarial BM of mice with and without B-ALL. (A) Oxygen images showing pO₂ levels represented in the color scale. (B) Mean pO₂ levels. **p < 0.01, ***p < 0.001.

Figure 5

BM immunofluorescence microscopy of the vascular niche in leukemic OB-GFP mice at consecutive stages of leukemia progression. (A) Representative immunofluorescence images of frozen femur sections. Red: B-ALL (tdTomato fluorescence); yellow: VE-cadherin-Alexa647; green: osteoblasts (GFP); blue: DNA (DAPI) (representative of 3 mice per group). The early, intermediate and advanced stages of leukemia are defined in Results. (B) Quantification of VE-cadherin+ blood vessels in the bone marrow of mice with early vs. advanced B-ALL. ****p < 0.0001.

Figure 6

Quantitative imaging of BM vasculature. (A) Representative 2-photon intravital images were maximum intensity projected and evaluated using AngioTool and the output was overlaid. (B) AngioTool numbers of junctions; (C) AngioTool average vessels length; (D) AngioTool total vessels length. *Indicates Student t-test p < 0.05.

Figure 7

Inhibition of OxPhos decreases oxygen consumption by B-ALL cells. (A) Mitochondrial respiration (OCR) of ex-vivo B-ALL cells compared to mouse normal B cells. (B) Mitochondrial respiration of B-ALL cells treated with OxPhos_i (IACS-010759). (C) Extracellular acidification rate (ECAR) of B-ALL cells ex-vivo compared to mouse normal B cells. (D) Representative graph of mitochondrial respiration for BM cells from a mouse that received three doses of IACS-010759 (OxPhos_i, 7.5 mg/kg) on day 12, 13, and 14 p.i. and control leukemic mouse BM cell measured by day 14 p.i. (E) Average basal mitochondrial respiration (BM cells) for 3 mice per group derived from mice treated with vehicle or IACS-010759 respectively, (day 14 p.i.). *p-value = 0.05. (F) Representative graph of extracellular acidification rate for BM cells from a mouse that received three doses of IACS-010759 (7.5 mg/kg) on day 12, 13, and 14 p.i. and control leukemic mouse BM cell measured on day 14 p.i. ***p < 0.001, ****p < 0.0001.

Figure 8

Complex I inhibition counteracts the hypoxia in the BM with advanced B-ALL. C57BL6 mice with B-ALL were treated with vehicle or Complex I inhibitor IACS-010759 (OxPhos_i, 7.5 mg/kg for 3 consecutive days and 5 mg/kg for 3 more days). On indicated days, mice were infused i.v. with dextran-TRITC (blue) and PtP-C343 oxygen probe, anesthetized, and calvaria BM was imaged through intact skull by fast scanning two-photon phosphorescence lifetime imaging microscopy (FaST-PLIM) followed by quantification of pO₂ levels. (A) Intravital oxygen images of skull BM on day 15 p.i. (B) Corresponding fluorescence images. (C) Local oxygen level probability distribution. (D) Mean oxygen tension in BM of B-ALL leukemic mice without treatment or with IACS-010759 treatment. *p-value < 0.05.

Figure 9

Evaluation of the effect of Complex I inhibitor (OxPhos_i) on BM hypoxia using pimonidazole IHC. (A,C) representative IHC of skull section of untreated leukemic mouse and (B,D) IHC of skull section of IACS-010759 treated mouse; (E,F) H&E on corresponding adjacent sections. (G) Semi quantitative analysis of pimonidazole IHC intensity by Histo-score (H-score) for skull section of individual leukemic mice on day 15 p.i. (H) Average pimonidazole IHC intensity for 3 mice in each group day 15 p.i. ****p < 0.0001.