The Bone Marrow-Targeted Conditioning Stress Test Demonstrates Vertebrae Niche Being More Radio-Tolerant Than Femur, Indicating Skeleton Resilience and Global Heterogeneity

Introduction:

Myeloablative conditioning prior to Hematopoietic cell transplant, the curative treatment for several hematopoietic disorders, is essential for successful donor cell homing and engraftment. Despite the anatomical classifications, it has been challenging to grasp the complexity of large skeletal systems' global and local bone marrow (BM) environments, which may play a crucial role in donor homing, engraftment, and treatment response. Moreover, due to a lack of comprehensive in vivo studies, there is sparse information about radio-sensitivity on BM niches in local (i.e, endosteal and sinusoidal) and regional skeletal systems (femur, spine etc.,). This may be due to a lack of technology to deliver targeted radiation and therefore using systemic whole-body radiation which resulted in lethality at higher doses due to the composite effect of hematopoietic failure and organ toxicity. Hence, we developed a preclinical image-guided total marrow irradiation (TMI) model, which allows BM specific increase in radiation dose while sculping dose to vital organs, therefore providing an opportunity to evaluate the radio-sensitivity of the BM niche. Here, using TMI dose escalation, we determined the radiation sensitivity of BM niches and its effect on donor homing.

Methods:

TMI treatment delivery was developed as described previously (Zuro et al., 2022). Different radiation doses (12, 16, 20 and 24 Gy) were delivered to the host (CD45.1 B6 mouse) skeleton in two fractions and was transplanted with 10 million Donor BM cells (CD45.2 B6). The femur and L Spine were harvested for these treated mice at 20+ h, post BMT. The central marrow (CM) cells were harvested by flushing/gently crushing the bone with Collagenase I and Dispase I enzymatic solution, while endosteal marrow (EM) was harvested by digesting the bone fragments with Collagenase IV enzyme, for 30 minutes at 150-200 rpm at 37° C. The isolated cells from endosteal and Central marrow were stained with standard verified markers for Hematopoietic stem cells, Mesenchymal Stem cells and Endothelial cells (18 antibody Panel) and data was acquired in Aurora spectral flow cytometer (Cytek) and was analyzed using FloJo software.

Results:

The CM cells were more sensitive to radiation, ~≤10% cell survived compared to untreated controls (20h post BMT) while EM cells were more radio tolerant both in Femur (~≥30% survived) and L spine (≥80% survived), however the endosteal cells from l-spine were more radio tolerant than Femur (~≥ 80% vs 16-28%) ( Fig 1 A-B). Further, in femur it was dose dependent i.e., cell number reduced with increasing doses (~28% (12 Gy) to ~16% (24 Gy), unlike l-spine where cell numbers were similar across treated doses. Also, in femur, the Donor cells preferred EM niche over CM, however at higher dose (24 Gy) this spatial preference was lost ( Fig 1C), unlike l-spine which was similar in CM and EM at all doses. The Sca1 ⁺ MSC was significantly higher in EM than CM both in the femur and l-spine, however it decreased in femur at higher doses (24 Gy), while remained unchanged in l-Spine ( Fig 1D-E). The arteriolar endothelial cells (AECs) were predominantly present in the EM both in femur and spine, and while sinusoidal endothelial cells (SEC) were higher in EM than CM in Femur, however Spine did not show this pattern. Although, at higher doses (24 Gy), the AECs were significantly reduced even in the EM but were still higher than CM ( Fig 1F). The donor HSPCs (Lineage- cKit+ cells) were significantly higher in the central marrow while the HSCs (Lineage-, cKit+ Sca1+, CD150+) were higher in endosteal marrow. Only in femur, at higher doses AECs, SECs, and Sca1+ MSCs were reduced, resulting in lower CD150+ HSC homing, which was not affected in the l-spine ( Fig 1G-H).

Conclusion:

In summary, skeletal niche tolerates much higher radiation than previously performed using conventional myeloablative studies and support donor homing. Increasing radiation doses reduced the BM niche constituent cells like AECs, SECs and Sca1+ MSCs, preferentially in femur and reducing homing, surprisingly though the spine BM was more resistant and did not show any reduction in above mentioned cellular composition. This preferential radiotolerance between skeletal regions warrants more studies to understand the' BM stromal cells radio sensitivity with respect to regional and spatial skeletal niche.