Page 60 - Haematologica April 2020

M.G. Gorelashvili et al.
cular space with a predefined step size of 3 ± 2 μm (mean ± standard deviation). The time between two steps was set at 20 s. To analyze migration towards the vasculature, the number of steps until first contact with a BM vessel was assessed, assuming that the cell would subsequently migrate into the intraluminal space. To further character- ize the migration, the mean squared displacement (MSD) of cell trajectories was determined for different time scales. Simulations were performed with and without megakaryocytes in the BM to assess the influence of BM crowdedness on cell migration (Figure 4A-C; Online Supplementary Figure S7). We found that megakaryocytes dramatically reduced the motility of HSC and neutrophils. The number of steps (nsteps) to reach the vasculature increased in the presence of megakaryocytes (HSC: from nsteps = 513 ± 1107 to nsteps = 8057 ± 10310, neutrophils: from nsteps = 628 ± 1270 to nsteps = 7869 ± 10175) (Figure 4A) and the trajectories exhibited lower MSD values (HSC: from MSD20s = 9.52 ± 5.95 μm2 to MSD20s = 6.85 ± 1.27 μm2, neutrophils: from MSD20s =10.57 ± 5.06 μm2 to MSD20s = 6.96 ± 1.26 μm2) (Figure 4B, C; Online Supplementary Figure S7). Fitting the first 25% of the MSD trajectories to determine the apparent diffusion coefficient (Dapp)39 revealed reduced Dapp in the presence of megakary- ocytes (Online Supplementary Table S3). Likewise, satura- tion limits of the MSD curves were reduced in the pres- ence of megakaryocytes (HSC: from 171 ± 1.3 μm2 to 133 ± 0.8 μm2; neutrophils: from 182 ± 1.8 μm2 to 116 ± 0.5 μm2) (Online Supplementary Table S4). Collectively, these data suggest that megakaryocytes represent passive obsta- cles, and significantly hamper cell migration in the BM. Lowering cell velocity (step size of 2 ± 1 μm) further sup- pressed migration for both HSC and neutrophils (HSC: nsteps = 8100 ± 13162 and MSD20s = 3.73 ± 1.13 μm2, neu- trophil: nsteps = 19694 ± 13924 and MSD20s = 3.91 ± 1.02 μm2) (Figure 4A, D). Interestingly, there were no signifi- cant differences between the investigated cell types, despite their different size and shape.
Chemotaxis and weak cell-to-vessel adhesion reveal the impact of cell size on migration in silico
Cell migration in the BM can be guided by chemotactic processes. Thus, we introduced chemotaxis into our cell migration algorithm, with the vessel walls being assumed to be the source of the chemoattractant (Online Supplementary Figure S6). Furthermore, we extended the algorithm with an adjustable probability for entering the vessel (PEV) to reflect a highly physiological cell migration process. We found that the number of required steps to reach and enter the vessels decreased (Online Supplementary Table S2) as the chemotaxis increased with a stronger gradient guiding the cells towards nearby ves- sels (Figure 4E). At the same time the corresponding MSD values for both investigated PEV of 100% and 50% signifi- cantly increased (Online Supplementary Table S2 and S3, Figure 4F). Here, neutrophils appeared to enter the vascu- lature faster than HSC, which is in contrast to the simula- tions without chemotaxis. As expected, reducing PEV from 100% to 50% increased the time until entering the vascu- lature (Online Supplementary Table S2, Figure 4E), but did not change the MSD values (Online Supplementary Table S2, Figure 4C, G). Interestingly, for probability PEV=50% neutrophils reached the vasculature significantly faster than HSC even in the absence of chemoattractants. In other words, cell size matters for migration to the vascu-
lature, and this size effect can even be augmented by bio- physical parameters such as chemotaxis and cell-to-vessel adhesion probability.
Treatment known to deplete circulating platelets and increase megakaryocyte volume is associated with a reduction in neutrophil mobility in the bone marrow
Next, we assessed whether the data obtained from the computational simulations could be validated in vivo (Figure 5; Online Movies 1 and 2). A depletion of megakaryocytes would inevitably also remove the bio- chemical factors derived from megakaryocytes. Factors such as platelet factor-4 (PF4) and TGFβ1 have been shown to modulate HSC quiescence16,17 so it would be impossible to discriminate between the biochemical and biomechanical effects of megakaryocyte depletion. As an alternative approach we treated platelets with anti-GPIbα antibodies, which do not deplete megakaryocytes, but result in increased megakaryocyte volume of vessel-asso- ciated megakaryocytes on day 3 after platelet depletion.15 In our computational simulations the larger megakary- ocytes had a greater impact than steady-state megakary- ocytes on neutrophil mobility (not shown). Thus, we com- pared neutrophil mobility in naïve mice and mice on day 3 following platelet-depletion (Figure 5) with the param- eters detailed in the Online Supplementary Material (Online Supplementary Tables S3 and S4). As expected from our simulations neutrophil mobility was decreased in platelet-depleted mice (saturation limit from 120 ± 5.58 μm2 to 66.7 μm2 in platelet-depleted mice), supporting our hypothesis that megakaryocytes restrain the mobility of neutrophils.
Supporting information
We have uploaded two supporting videos - one exem- plary dataset of naïve and megakaryocyte-depleted mice – as well as the MatLab scripts used in the simulation and the Ilastik training file used in bone and BM segmentation on Zenodo under DOI: 10.5281/zenodo.3144732.
Discussion
Here, we provide a profound 3D image reconstruction and segmentation pipeline for different BM components and use these data for computational simulations by com- plex tailored cell localization and migration algorithms. Realistic simulation templates were deployed for migra- tion simulations of HSC and neutrophils. Our data clearly show that volumetric analysis of the number and localiza- tion of megakaryocytes provides additional information.
Furthermore, we performed computational 3D simula- tions of megakaryocyte distribution and BM cell migra- tion using the 3D segmented LSFM data. These simula- tions suggest that megakaryocytes play an important role in cell migration even if not migrating themselves. Instead, they represent passive obstacles, and thus significantly influence migration of other cells, such as HSC and neu- trophils, in the BM. We discovered this from realistic sim- ulations using templates with high physiological relevance derived from segmented cell and vessel objects in 3D LSFM images.
The image analysis pipeline is clearly superior to com- monly used strategies, and minimizes bias of crucial parameters such as cell number and volume. Our data
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