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. 2022 May 20;6(10):3126–3141. doi: 10.1182/bloodadvances.2021004321

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Figure 4. — IVM identifies subsets of AML cells affected by MMP inhibition. (A) Selected frames from representative time-lapse IVM of BM calvarium with intermediate infiltration. Red: mTomato⁺ healthy hematopoietic cells; green: YFP⁺ AML cells. White and yellow arrows: examples of explorative and nonexplorative cells, respectively. Boxed areas are shown at higher magnification in A’ and A”, where nonexplorative and explorative cells’ morphology is highlighted by dashed lines. Scale bars represent 80 µm in A and 25 µm in A’ and A”. (B) Rose plots showing tracks of nonexplorative and explorative AML cells. n = 26 nonexplorative and n = 27 explorative AML cells. (C) Quantification of displacement (track length), Euclidean distance (distance from track start to end points), velocity, linear progression, and (D) circularity and perimeter variance of nonexplorative and explorative cells. n = 27 explorative cells and n = 26 nonexplorative cells pooled from 8 fields of view from 3 mice in 2 independent experiments. (E) Diffusion coefficient of explorative (blue lines) and nonexplorative (orange lines) cells represented as TAMSD. Red lines are the fit by ordinary least square of the mean of all trajectories (black dots). (F) Frequency of explorative cells observed per field of view. n = 10 FoWs from 4 mice from 3 independent experiments. (G) Selected, representative timeframe of time-lapse IVM showing mTomato+ AML blasts (red) and in vivo immunostaining for CX3CR1 (turquoise). Scale bar represents 50 µm. (H) Boxed areas in (G) shown at higher resolution are examples of CX3CR1⁺ explorative and CX3CR1^- nonexplorative AML cells (scale bar = 10 µm). (I) Percentage of CX3CR1⁺ explorative and nonexplorative AML cells per field of view analyzed. n = 8 fields of view pooled from 3 mice. (J) Simulation results when each field of view is initially seeded by 2 adjacent cells, and progeny is generated according to available proliferation rates. Left: all cells are explorative. Middle: all cells are nonexplorative. Right: 10% of cells are explorative. (K) Quantification of explorative and nonexplorative AML cells in vehicle- and prinomastat-treated leukemic mice in the calvarium BM. n = 3 mice per condition. (L) Average velocity of AML blasts in calvarium BM of PBS- and prinomastat-treated AML-burdened mice. n = 338 and n = 293 cells per group, pooled from n = 3 mice per group from 2 independent experiments. All data are mean ± SEM. *P < .05; **P < .001; ***P < .0001; ns, not significant. P values determined by Student t tests. TAMSD, time-averaged mean square displacement.

Figure 4. — IVM identifies subsets of AML cells affected by MMP inhibition. (A) Selected frames from representative time-lapse IVM of BM calvarium with intermediate infiltration. Red: mTomato⁺ healthy hematopoietic cells; green: YFP⁺ AML cells. White and yellow arrows: examples of explorative and nonexplorative cells, respectively. Boxed areas are shown at higher magnification in A’ and A”, where nonexplorative and explorative cells’ morphology is highlighted by dashed lines. Scale bars represent 80 µm in A and 25 µm in A’ and A”. (B) Rose plots showing tracks of nonexplorative and explorative AML cells. n = 26 nonexplorative and n = 27 explorative AML cells. (C) Quantification of displacement (track length), Euclidean distance (distance from track start to end points), velocity, linear progression, and (D) circularity and perimeter variance of nonexplorative and explorative cells. n = 27 explorative cells and n = 26 nonexplorative cells pooled from 8 fields of view from 3 mice in 2 independent experiments. (E) Diffusion coefficient of explorative (blue lines) and nonexplorative (orange lines) cells represented as TAMSD. Red lines are the fit by ordinary least square of the mean of all trajectories (black dots). (F) Frequency of explorative cells observed per field of view. n = 10 FoWs from 4 mice from 3 independent experiments. (G) Selected, representative timeframe of time-lapse IVM showing mTomato+ AML blasts (red) and in vivo immunostaining for CX3CR1 (turquoise). Scale bar represents 50 µm. (H) Boxed areas in (G) shown at higher resolution are examples of CX3CR1⁺ explorative and CX3CR1^- nonexplorative AML cells (scale bar = 10 µm). (I) Percentage of CX3CR1⁺ explorative and nonexplorative AML cells per field of view analyzed. n = 8 fields of view pooled from 3 mice. (J) Simulation results when each field of view is initially seeded by 2 adjacent cells, and progeny is generated according to available proliferation rates. Left: all cells are explorative. Middle: all cells are nonexplorative. Right: 10% of cells are explorative. (K) Quantification of explorative and nonexplorative AML cells in vehicle- and prinomastat-treated leukemic mice in the calvarium BM. n = 3 mice per condition. (L) Average velocity of AML blasts in calvarium BM of PBS- and prinomastat-treated AML-burdened mice. n = 338 and n = 293 cells per group, pooled from n = 3 mice per group from 2 independent experiments. All data are mean ± SEM. *P < .05; **P < .001; ***P < .0001; ns, not significant. P values determined by Student t tests. TAMSD, time-averaged mean square displacement.