Abstract A26: Age and Dose Related Changes to the Bone Marrow Microenvironment after Cytotoxic Conditioning with Busulfan

Abstract

Hematopoietic Cell Transplantation (HCT) is a frequently used treatment for hematologic malignancies such as acute myeloid leukemia, multiple myeloma, lymphoma and non-malignant diseases. Preparative regimens before HCT damage the Bone Marrow (BM) niche, but it is not fully known how the cytotoxic preconditioning, whether High or Low intensity, impacts bone and BM remodeling, regeneration, and subsequent hematopoietic recovery over time. In addition, the effect of recipient age on these factors has not been completely described. In this study, we sought to longitudinally investigate bone and BM remodeling after Low and High intensity Busulfan (BU) conditioning with the aim of understanding the role that BM niche alterations play in the recovery of the hematopoietic system after transplantation. Using two-photon intravital microscopy, we visualized bone and BM changes in young and adult mice on days 2, 5, and 42 post-transplantation. Both Low and High intensity conditioning were administered with injections of 40 mg/kg and 80 mg/kg busulfan, respectively. Busulfan is a DNA alkylating drug that in combination with cyclophosphamide is being clinically used to treat leukemia. Mice were then transplanted with 1 × 106 whole BM cells from a C57BL/6-Tg(UBC-GFP) mouse. During live imaging, functionality of the vascular system and hematopoietic recovery were studied. For ex vivo imaging, vascular labeling fluorescent antibodies (Alexafluor 647 conjugated to anti-CD31, CD144, and Sca-1) and calcium binding dyes (dye1; Calcein, Dye2; Alizarin) were administered before intracardiac perfusion. Then long bones were harvested, frozen, and the cortical layer was shaved to enable visualization of the BM. Vascular analysis indicated increased leakage in both Low and High intensity conditioning even after 42 days suggesting delayed endothelial recovery following conditioning. Morphological evaluation of the vascular network revealed a decrease in the frequency and increase in the size and density of the vessels at early timepoints that was partially restored by day 42 in young mice only. Bone remodeling in young mice showed a reduction in the dye1/dye2 ratio at day 5 and a more significant decrease by day 42. In the adult mice, a low ratio was observed in BU-conditioned mice only at day 42 post treatment. To further investigate, we classified the metaphyseal and epiphyseal long bone cavities as deposition type, mixed type, and resorption type based on the ratio of the two dyes. The classification analysis indicates that the reduction in dye1/dye2 ratio in BU-conditioned mice over time is primarily due to an increase in resorption type bone cavities in BU-conditioned mice compared to the control. Although donor HSC engraftment is known to be age and dose dependent, our results indicate that long-term microenvironmental changes in the bone and bone marrow may further impact hematopoietic recovery. Overall, our results demonstrate new aspects of bone remodeling and bone marrow regeneration that may influence hematopoietic recovery and warrant further investigation. Citation Format: Joel A Spencer, Christian Burns, Ruth Verrinder, Farhad Ghazali, Nastaran Abbasizadeh. Age and Dose Related Changes to the Bone Marrow Microenvironment after Cytotoxic Conditioning with Busulfan [abstract]. In: Proceedings of the AACR Special Conference: Acute Myeloid Leukemia and Myelodysplastic Syndrome; 2023 Jan 23-25; Austin, TX. Philadelphia (PA): AACR; Blood Cancer Discov 2023;4(3_Suppl):Abstract nr A26.