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PMC9878562
ACKR3 promotes CXCL12/CXCR4-mediated cell-to-cell-induced lymphoma migration through LTB4 production
Moreover, in the mixture, the CFP VAL-ko cells migrated with a similar efficiency as VAL-wt cells ( Figures 2A, B ).
PMC9878562
ACKR3 promotes CXCL12/CXCR4-mediated cell-to-cell-induced lymphoma migration through LTB4 production
Next, we addressed the role of the C-terminus of ACKR3 in promoting the migration of VAL-ko.
PMC9878562
ACKR3 promotes CXCL12/CXCR4-mediated cell-to-cell-induced lymphoma migration through LTB4 production
VAL-ACKR3-ΔC, barely supported VAL-ko migration towards CXCL12 in a 1:1 mixture, highlighting the importance the C-terminus for scavenging ( Figure S1C ), migration ( Figure S1Eii ) and stimulation in trans ( Figure 2B ).
PMC9878562
ACKR3 promotes CXCL12/CXCR4-mediated cell-to-cell-induced lymphoma migration through LTB4 production
It is conceivable that the scavenging activity is essential for chemotaxis of ACKR3 expressing cells and to support migration of VAL-ko, however it cannot be excluded that the C-terminus mediates additional intracellular signaling unrelated to receptor trafficking.
PMC9878562
ACKR3 promotes CXCL12/CXCR4-mediated cell-to-cell-induced lymphoma migration through LTB4 production
The later assumption is in line with previous findings (9) where it was shown that phosphorylation-deficient (C-terminus) ACKR3 does not scavenge chemokines but rescues the lethal phenotype of receptor-deficient mice.
PMC9878562
ACKR3 promotes CXCL12/CXCR4-mediated cell-to-cell-induced lymphoma migration through LTB4 production
VAL-wt cells induce the migration of VAL-ko cells. (
PMC9878562
ACKR3 promotes CXCL12/CXCR4-mediated cell-to-cell-induced lymphoma migration through LTB4 production
Transendothelial migration of VAL cells (as in Figure 1 ) towards different concentrations of CXCL12 (1, 10, 30 nM).
PMC9878562
ACKR3 promotes CXCL12/CXCR4-mediated cell-to-cell-induced lymphoma migration through LTB4 production
Cyan symbols and cyan lines (VAL-ko) displays % migrated VAL-ko in the absence of VAL-wt.
PMC9878562
ACKR3 promotes CXCL12/CXCR4-mediated cell-to-cell-induced lymphoma migration through LTB4 production
The % migrated VAL-ko (gated on CFP) in a 1:1 mixture with VAL-wt was calculated from the input of VAL-ko in the mixture (VAL-ko mixed, cyan filled symbols and black lines).
PMC9878562
ACKR3 promotes CXCL12/CXCR4-mediated cell-to-cell-induced lymphoma migration through LTB4 production
Black symbols: % migrated VAL-wt calculated from the respective input.
PMC9878562
ACKR3 promotes CXCL12/CXCR4-mediated cell-to-cell-induced lymphoma migration through LTB4 production
Calculation by flow cytometry as in Figure 1 .
PMC9878562
ACKR3 promotes CXCL12/CXCR4-mediated cell-to-cell-induced lymphoma migration through LTB4 production
Cumulative data of three independent experiments measured in triplicates (means ± SD, Dunn’s multiple comparisons ONE WAY ANOVA, *p ≤ 0.05, ***p ≤ 0.001) (B) Transendothelial migration of VAL cells towards 10 nM CXCL12 or medium.
PMC9878562
ACKR3 promotes CXCL12/CXCR4-mediated cell-to-cell-induced lymphoma migration through LTB4 production
VAL-ko were mixed 1:1 with VAL-wt (dark cyan) or VAL-ΔC (green) and the % migrated VAL-ko was calculated from the input of VAL-ko in the mixture.
PMC9878562
ACKR3 promotes CXCL12/CXCR4-mediated cell-to-cell-induced lymphoma migration through LTB4 production
Quantification was performed as described in Figure 1 .
PMC9878562
ACKR3 promotes CXCL12/CXCR4-mediated cell-to-cell-induced lymphoma migration through LTB4 production
Cumulative data of three independent experiments measured in triplicates (means ± SD, Turkey’s multiple comparison, ONE WAY ANOVA, ***p ≤ 0.001). (
PMC9878562
ACKR3 promotes CXCL12/CXCR4-mediated cell-to-cell-induced lymphoma migration through LTB4 production
Migration of DOHH2 cells.
PMC9878562
ACKR3 promotes CXCL12/CXCR4-mediated cell-to-cell-induced lymphoma migration through LTB4 production
DOHH2 cells were mixed 1:1 with VAL-ko and the mixture allowed to migrate for 8 h towards 10 nM CXCL12 or medium.
PMC9878562
ACKR3 promotes CXCL12/CXCR4-mediated cell-to-cell-induced lymphoma migration through LTB4 production
The % migrated DOHH2 (grey bars) and of VAL-ko (cyan bars) were calculated from the respective inputs in the mixture.
PMC9878562
ACKR3 promotes CXCL12/CXCR4-mediated cell-to-cell-induced lymphoma migration through LTB4 production
Experiments performed in triplicates, representative example of three independent experiments (means ± SD, Turkey’s multiple comparison, ONE WAY ANOVA ***p ≤ 0.001).
PMC9878562
ACKR3 promotes CXCL12/CXCR4-mediated cell-to-cell-induced lymphoma migration through LTB4 production
To extend the observation of DLBCL migration we measured mLEC transwell chemotaxis of DOHH2 cells in response to CXCL12.
PMC9878562
ACKR3 promotes CXCL12/CXCR4-mediated cell-to-cell-induced lymphoma migration through LTB4 production
DOHH2 cells migrated more efficiently than VAL-wt, but also displayed enhanced chemokinesis.
PMC9878562
ACKR3 promotes CXCL12/CXCR4-mediated cell-to-cell-induced lymphoma migration through LTB4 production
When DOHH2 cells were mixed with VAL-ko cells they induced chemokinesis of VAL-ko cells and barely supported chemotaxis towards CXCL12 ( Figure 2C ).
PMC9878562
ACKR3 promotes CXCL12/CXCR4-mediated cell-to-cell-induced lymphoma migration through LTB4 production
Taken together, the data suggest that expression of full-length ACKR3 on leader cells is needed to induce the CXCL12-dependent cell-to-cell-induced migration of CXCR4 VAL-ko.
PMC9878562
ACKR3 promotes CXCL12/CXCR4-mediated cell-to-cell-induced lymphoma migration through LTB4 production
We used a previously reported localized tumor xenograft model in NOD/SCID mice (32) to investigate lymphoma cell migration in vivo.
PMC9878562
ACKR3 promotes CXCL12/CXCR4-mediated cell-to-cell-induced lymphoma migration through LTB4 production
We injected 10 lymphoma cells subcutaneously in the flanks of NOD/SCID mice.
PMC9878562
ACKR3 promotes CXCL12/CXCR4-mediated cell-to-cell-induced lymphoma migration through LTB4 production
All animals showed a comparable increase in body weight and tumor size ( Figure 2SA ).
PMC9878562
ACKR3 promotes CXCL12/CXCR4-mediated cell-to-cell-induced lymphoma migration through LTB4 production
After 20-21 days, animals were sacrificed and the mass of the primary tumor was measured.
PMC9878562
ACKR3 promotes CXCL12/CXCR4-mediated cell-to-cell-induced lymphoma migration through LTB4 production
No significant differences in mass were observed between tumors grown from wild type DLBCL, VAL-wt cells and DOHH2, VAL-ko and the respective mixtures of wild type and VAL-ko ( Figure 3A ).
PMC9878562
ACKR3 promotes CXCL12/CXCR4-mediated cell-to-cell-induced lymphoma migration through LTB4 production
NOD/SCID mice barely developed sizable lymph nodes due to the lack of endogenous lymphocytes.
PMC9878562
ACKR3 promotes CXCL12/CXCR4-mediated cell-to-cell-induced lymphoma migration through LTB4 production
Injection of wild type DLBCL cells led to measurable mass of the draining lymph node.
PMC9878562
ACKR3 promotes CXCL12/CXCR4-mediated cell-to-cell-induced lymphoma migration through LTB4 production
By contrast, the draining lymph nodes of tumors from VAL-ko remained hardly detectable ( Figure 3A ).
PMC9878562
ACKR3 promotes CXCL12/CXCR4-mediated cell-to-cell-induced lymphoma migration through LTB4 production
However, an increase in lymph node mass was observed, when 1:1 mixtures of wild type DLBCL and VAL-ko were injected ( Figure 3A ).
PMC9878562
ACKR3 promotes CXCL12/CXCR4-mediated cell-to-cell-induced lymphoma migration through LTB4 production
To test whether under these conditions VAL-ko contributed to the increase in mass of the draining lymph node, we used flow cytometry to measure the presence of CFP VAL-ko.
PMC9878562
ACKR3 promotes CXCL12/CXCR4-mediated cell-to-cell-induced lymphoma migration through LTB4 production
While VAL-ko alone did not migrate to the axillary lymph node, in the presence of VAL-wt or DOHH2 the CFP VAL-ko could readily be detected ( Figure 3B ).
PMC9878562
ACKR3 promotes CXCL12/CXCR4-mediated cell-to-cell-induced lymphoma migration through LTB4 production
Hence, like in vitro, also in vivo wild type lymphoma cells appeared to promote the migration of ACKR3 deficient VAL-ko.
PMC9878562
ACKR3 promotes CXCL12/CXCR4-mediated cell-to-cell-induced lymphoma migration through LTB4 production
In the mixture preferentially ACKR3 positive VAL-wt migrated to the draining lymph node along with VAL-wt, which do not express ACKR3 at the surface and CFP VAL-ko ( Figure 3C ).
PMC9878562
ACKR3 promotes CXCL12/CXCR4-mediated cell-to-cell-induced lymphoma migration through LTB4 production
Consistent with our in vitro observations ( Figure 1D ) the proportion of VAL-wt expressing ACKR3 on the surface was markedly increased from 18% to over 50% in the draining lymph node.
PMC9878562
ACKR3 promotes CXCL12/CXCR4-mediated cell-to-cell-induced lymphoma migration through LTB4 production
By contrast, the expression of CCR7 was unaltered on VAL-wt and remained essentially absent on CFP VAL-ko, suggesting that the receptor may not contribute to draining lymph node infiltration ( Figure S2B ).
PMC9878562
ACKR3 promotes CXCL12/CXCR4-mediated cell-to-cell-induced lymphoma migration through LTB4 production
Migration of VAL-ko from the primary tumor to the draining lymph node is enhanced in the presence of ACKR3 expressing cells. (
PMC9878562
ACKR3 promotes CXCL12/CXCR4-mediated cell-to-cell-induced lymphoma migration through LTB4 production
Tumor (left) and lymph node (right) weights from NOD/SCID common γ-chain mice injected with 10 VAL-wt, VAL-ko and DOHH2 cells alone and as 1:1 mixture VAL-wt:VAL-ko and DOHH2:VAL-ko.
PMC9878562
ACKR3 promotes CXCL12/CXCR4-mediated cell-to-cell-induced lymphoma migration through LTB4 production
Weights are reported as means ± SD from three independent xenograft experiments with 4-5 animals per group.
PMC9878562
ACKR3 promotes CXCL12/CXCR4-mediated cell-to-cell-induced lymphoma migration through LTB4 production
Tumor and lymph node t-test single comparison: ns, p≥0.05, *p≤ 0.05, **p ≤ 0.01. (
PMC9878562
ACKR3 promotes CXCL12/CXCR4-mediated cell-to-cell-induced lymphoma migration through LTB4 production
Total number of CFP VAL-ko present in the draining axillary lymph node from the same experiments.
PMC9878562
ACKR3 promotes CXCL12/CXCR4-mediated cell-to-cell-induced lymphoma migration through LTB4 production
Lymph node cells were analyzed by flow cytometry and gated for hCD19 (HIB19 or SJ25C1), mCD45 (30-F11) and CFP expression (means ± SD, t-test single comparison, ns, p≥0.05 **P ≤ 0.01). (
PMC9878562
ACKR3 promotes CXCL12/CXCR4-mediated cell-to-cell-induced lymphoma migration through LTB4 production
Surface expression of ACKR3 on VAL-wt cells and CFP expression in VAL-ko extracted from a representative localized tumor (upper panel) and from the corresponding lymph node (lower panel).
PMC9878562
ACKR3 promotes CXCL12/CXCR4-mediated cell-to-cell-induced lymphoma migration through LTB4 production
Representative data from one experiment shown in A (n= 4-5). (
PMC9878562
ACKR3 promotes CXCL12/CXCR4-mediated cell-to-cell-induced lymphoma migration through LTB4 production
Confocal images of sections of a tumor generated by injecting a 1:1 mixture of VAL-wt and VAL-ko (CFP).
PMC9878562
ACKR3 promotes CXCL12/CXCR4-mediated cell-to-cell-induced lymphoma migration through LTB4 production
Tumors were fixed for 48h in a solution of 4% paraformaldehyde and Vibratome sections stained overnight with anti-hCD19 (mAb HIB19) anti-mPDPN (mAb 8.1.1) and anti-mCD31 (mAb MEC13.3). (
PMC9878562
ACKR3 promotes CXCL12/CXCR4-mediated cell-to-cell-induced lymphoma migration through LTB4 production
Confocal images of sections of a draining lymph node from a tumor generated by injecting a 1:1 mixture of VAL-wt and VAL-ko (CFP).
PMC9878562
ACKR3 promotes CXCL12/CXCR4-mediated cell-to-cell-induced lymphoma migration through LTB4 production
Lymph nodes were fixed for 5h in a solution 4% paraformaldehyde and Vibratome sections were stained overnight with anti-hCD19 Ab (mAb HIB19), anti-mPDPN (mAb 8.1.1).
PMC9878562
ACKR3 promotes CXCL12/CXCR4-mediated cell-to-cell-induced lymphoma migration through LTB4 production
Immunofluorescence (IF) analysis of sections from tumors of VAL-wt:VAL-ko mixtures by confocal microscopy revealed that VAL-wt and VAL-ko are randomly distributed in the tumor mass.
PMC9878562
ACKR3 promotes CXCL12/CXCR4-mediated cell-to-cell-induced lymphoma migration through LTB4 production
Using podoplanin (PDPN) and CD31 staining for mouse lymphatic and endothelial vessels we found a similar localization for both cell populations ( Figure 3D ).
PMC9878562
ACKR3 promotes CXCL12/CXCR4-mediated cell-to-cell-induced lymphoma migration through LTB4 production
By contrast, IF analysis of axillary lymph node sections from mice bearing VAL-wt:Val-ko tumors revealed striking differences in the distribution of the two cell populations.
PMC9878562
ACKR3 promotes CXCL12/CXCR4-mediated cell-to-cell-induced lymphoma migration through LTB4 production
Human CD19 VAL-wt almost completely populated the entire draining lymph node.
PMC9878562
ACKR3 promotes CXCL12/CXCR4-mediated cell-to-cell-induced lymphoma migration through LTB4 production
By contrast, CFP VAL-ko were found at the edge of the lymph node, close to the sub-capsular sinus in clusters surrounded by VAL-wt cells ( Figure 3E ).
PMC9878562
ACKR3 promotes CXCL12/CXCR4-mediated cell-to-cell-induced lymphoma migration through LTB4 production
Taken together, the data indicate that like in vitro lymphoma cells can also promote the migration of VAL-ko in vivo.
PMC9878562
ACKR3 promotes CXCL12/CXCR4-mediated cell-to-cell-induced lymphoma migration through LTB4 production
To investigate further the migration and the cooperation between cells, we used a 3D migration and quantitative computational (45) analysis system.
PMC9878562
ACKR3 promotes CXCL12/CXCR4-mediated cell-to-cell-induced lymphoma migration through LTB4 production
Cells were embedded in a collagen matrix and chemotaxis towards CXCL12 was followed by time-lapse imaging ( Figure 4A ).
PMC9878562
ACKR3 promotes CXCL12/CXCR4-mediated cell-to-cell-induced lymphoma migration through LTB4 production
Tracks of migrating cells were generated automatically by computational analysis and used to extrapolate data of statistical significance to calculate displacement, track length and velocity of single cells.
PMC9878562
ACKR3 promotes CXCL12/CXCR4-mediated cell-to-cell-induced lymphoma migration through LTB4 production
Single cell data were pooled into experimental groups for comparison ( Figure 4B ).
PMC9878562
ACKR3 promotes CXCL12/CXCR4-mediated cell-to-cell-induced lymphoma migration through LTB4 production
The analysis confirmed that VAL-ko are unable to migrate towards CXCL12 ( Figure S3A ) while the mixture of migrating VAL-ko:VAL-wt is mobile and migration competent ( Figure S3B ).
PMC9878562
ACKR3 promotes CXCL12/CXCR4-mediated cell-to-cell-induced lymphoma migration through LTB4 production
Displacement and track length of VAL-ko in the mixture is comparable to that of VAL-wt ( Figure 4B ).
PMC9878562
ACKR3 promotes CXCL12/CXCR4-mediated cell-to-cell-induced lymphoma migration through LTB4 production
However, cells in the mixture appeared to migrate with an intermediate velocity being faster than VAL-ko cells alone, but slower than VAL-wt cells ( Figure 4B ).
PMC9878562
ACKR3 promotes CXCL12/CXCR4-mediated cell-to-cell-induced lymphoma migration through LTB4 production
Similarly, plotting the tracks on common origin revealed that (i) VAL-wt in the mixture are highly motile, (ii) VAL-ko essentially do not move, while (iii) the migratory capacity of VAL-ko is partially restored in the presence of VAL-wt ( Figure 4C ).
PMC9878562
ACKR3 promotes CXCL12/CXCR4-mediated cell-to-cell-induced lymphoma migration through LTB4 production
The results are consistent with the transwell assays and the in vivo data, demonstrating that the presence of ACKR3 is essential to promote migration in cis and in trans.
PMC9878562
ACKR3 promotes CXCL12/CXCR4-mediated cell-to-cell-induced lymphoma migration through LTB4 production
CXCL12 stimulated VAL-wt increase the 3D motility and directional migration of VAL-ko. (
PMC9878562
ACKR3 promotes CXCL12/CXCR4-mediated cell-to-cell-induced lymphoma migration through LTB4 production
Schematic representation of 3D migration chamber showing a chemotactic gradient (CXCL12 diffusion) from left to right. (
PMC9878562
ACKR3 promotes CXCL12/CXCR4-mediated cell-to-cell-induced lymphoma migration through LTB4 production
Violin plots showing displacement, length and velocity of VAL-ko alone (light cyan), VAL-ko in a 1:1 mixture with VAL-wt (VAL-ko mixed, dark cyan) and VAL-wt in the same mixture (VAL-wt mixed, grey).
PMC9878562
ACKR3 promotes CXCL12/CXCR4-mediated cell-to-cell-induced lymphoma migration through LTB4 production
Tracking analyses were performed using Imaris software (means, Turkey’s multiple comparisons test, ONE-WAY ANOVA ns, p≥0.05, ****p ≤ 0.0001). (
PMC9878562
ACKR3 promotes CXCL12/CXCR4-mediated cell-to-cell-induced lymphoma migration through LTB4 production
Plots of tracks with a common origin. (
PMC9878562
ACKR3 promotes CXCL12/CXCR4-mediated cell-to-cell-induced lymphoma migration through LTB4 production
VAL-wt mixed, (ii) VAL-ko alone, (iii) VAL-ko mixed. (
PMC9878562
ACKR3 promotes CXCL12/CXCR4-mediated cell-to-cell-induced lymphoma migration through LTB4 production
Scatter plots of directionality vs arrest coefficients of 1000 VAL-wt mixed, VAL-ko alone, VAL-ko mixed.
PMC9878562
ACKR3 promotes CXCL12/CXCR4-mediated cell-to-cell-induced lymphoma migration through LTB4 production
Gates with differently colored dots indicate four different motility patterns. (
PMC9878562
ACKR3 promotes CXCL12/CXCR4-mediated cell-to-cell-induced lymphoma migration through LTB4 production
Violin plots of changes in velocity of VAL-ko and VAL-wt, relative to the number of motile (0, 1, 2) VAL-wt in a 50 µm radius (means, Mann-Whitney t-test ns, p≥0.05, ****p ≤ 0.0001). (
PMC9878562
ACKR3 promotes CXCL12/CXCR4-mediated cell-to-cell-induced lymphoma migration through LTB4 production
Directionality changes over time of a VAL-ko (red thick line) with respect to directionality changes of two VAL-wt in a 50 µm radius (orange and red thin lines, upper plot).
PMC9878562
ACKR3 promotes CXCL12/CXCR4-mediated cell-to-cell-induced lymphoma migration through LTB4 production
The lower plot shows the same directionality changes of a single VAL-ko (red thick line), compared to the average of directionality value of VAL cells in the 50 µm radius.
PMC9878562
ACKR3 promotes CXCL12/CXCR4-mediated cell-to-cell-induced lymphoma migration through LTB4 production
To better characterize differences in motility, we analyzed the behavior of individual cells over time.
PMC9878562
ACKR3 promotes CXCL12/CXCR4-mediated cell-to-cell-induced lymphoma migration through LTB4 production
We applied an algorithm using mobility values to generate plots that distinguish four populations according to their migratory behavior.
PMC9878562
ACKR3 promotes CXCL12/CXCR4-mediated cell-to-cell-induced lymphoma migration through LTB4 production
We quantified instantaneous motility measures over time, by dividing each track into fragments (tracklets), and performed a tracklet-based action analysis as previously described (46).
PMC9878562
ACKR3 promotes CXCL12/CXCR4-mediated cell-to-cell-induced lymphoma migration through LTB4 production
From the motility measures, we computed the continuous arrest coefficient (46), that is the proportion of time during which a cell does not move.
PMC9878562
ACKR3 promotes CXCL12/CXCR4-mediated cell-to-cell-induced lymphoma migration through LTB4 production
Cells with low motility are associated with high values of arrest coefficient.
PMC9878562
ACKR3 promotes CXCL12/CXCR4-mediated cell-to-cell-induced lymphoma migration through LTB4 production
Moreover, we computed the instantaneous directionality (46), which was associated with high values for polarized cells migrating along straight trajectories.
PMC9878562
ACKR3 promotes CXCL12/CXCR4-mediated cell-to-cell-induced lymphoma migration through LTB4 production
We assigned these coefficients to 1000 tracklets of each group: VAL-wt and VAL-ko in the mixture (separately analyzing CFP and CFP cells) and VAL-ko alone ( Figure 4D ).
PMC9878562
ACKR3 promotes CXCL12/CXCR4-mediated cell-to-cell-induced lymphoma migration through LTB4 production
For VAL-wt, in a 1:1 mixture with VAL-ko, 233 tracklets displayed low arrest coefficients and low directionality, which indicates that these cells perform chemokinesis (green gate), whereas 511 tracklets were associated with immobile cells, characterized by tracklets with a high arrest coefficient and low directionality (blue gate).
PMC9878562
ACKR3 promotes CXCL12/CXCR4-mediated cell-to-cell-induced lymphoma migration through LTB4 production
Tracklets of cells, which protruded lamellipods, hence visibly polarized, but showed slow or no migration (red gate 58 tracklets), were defined by a high arrest coefficient and high directionality (deduced from the polarity).
PMC9878562
ACKR3 promotes CXCL12/CXCR4-mediated cell-to-cell-induced lymphoma migration through LTB4 production
Finally, 198 tracklets (orange gate) displayed low arrest coefficients and high directionality and were associated with cells that performed a pronounced directional migration.
PMC9878562
ACKR3 promotes CXCL12/CXCR4-mediated cell-to-cell-induced lymphoma migration through LTB4 production
VAL-ko when placed alone in the chamber were found to be mostly immobile (956 tracklets in the blue gate).
PMC9878562
ACKR3 promotes CXCL12/CXCR4-mediated cell-to-cell-induced lymphoma migration through LTB4 production
However, VAL-ko in the mixture displayed similar motility as VAL-wt with slightly less directional migration ability (103 vs. 198 tracklets).
PMC9878562
ACKR3 promotes CXCL12/CXCR4-mediated cell-to-cell-induced lymphoma migration through LTB4 production
Our algorithm applied to a large number of tracklets confirmed the motility of VAL-wt and their ability to promote the migration of VAL-ko.
PMC9878562
ACKR3 promotes CXCL12/CXCR4-mediated cell-to-cell-induced lymphoma migration through LTB4 production
We further analyzed whether the velocity of VAL-ko and VAL-wt migration depends on a motile VAL-wt in the direct neighborhood.
PMC9878562
ACKR3 promotes CXCL12/CXCR4-mediated cell-to-cell-induced lymphoma migration through LTB4 production
Figure 4E shows that a VAL-ko in the presence of at least one motile VAL-wt within a radius of 50 µm migrated with significantly higher velocity compared to VAL-ko where no motile VAL-wt were in close proximity.
PMC9878562
ACKR3 promotes CXCL12/CXCR4-mediated cell-to-cell-induced lymphoma migration through LTB4 production
We observed that the increase in velocity of migration of VAL-ko was similar whether one or two motile VAL-wt were in the neighborhood, suggesting no additive effect.
PMC9878562
ACKR3 promotes CXCL12/CXCR4-mediated cell-to-cell-induced lymphoma migration through LTB4 production
The same analysis performed for the velocity of VAL-wt revealed that the motility of the cells was also influenced by the presence of a motile VAL-wt within a radius of 50 µm.
PMC9878562
ACKR3 promotes CXCL12/CXCR4-mediated cell-to-cell-induced lymphoma migration through LTB4 production
The finding suggested that VAL-ko and VAL-wt might perform a secondary migration, which could be triggered either by microgradients of CXCL12 created by ACKR3 VAL-wt or by another stimulus that is released during the migration of VAL-wt along a CXCL12 gradient.
PMC9878562
ACKR3 promotes CXCL12/CXCR4-mediated cell-to-cell-induced lymphoma migration through LTB4 production
By analyzing in depth the tracks of VAL-ko we saw that the cells often migrate to a spot where a VAL-wt had briefly paused and left (Movies S1-3).
PMC9878562
ACKR3 promotes CXCL12/CXCR4-mediated cell-to-cell-induced lymphoma migration through LTB4 production
In addition, we measured the changes in directionality of VAL-ko and VAL-wt in close proximity and plotted the data over time ( Figure 4F ).
PMC9878562
ACKR3 promotes CXCL12/CXCR4-mediated cell-to-cell-induced lymphoma migration through LTB4 production
We found that the changes in directionality of VAL-ko were in most cases preceded by a change in VAL-wt directionality ( Figure 4F upper panel).
PMC9878562
ACKR3 promotes CXCL12/CXCR4-mediated cell-to-cell-induced lymphoma migration through LTB4 production
Moreover, the migration of a VAL-ko was similar to the overall movement of motile cells in a radius of 50 µm ( Figure 4F , lower panel).
PMC9878562
ACKR3 promotes CXCL12/CXCR4-mediated cell-to-cell-induced lymphoma migration through LTB4 production
The observations support the assumption that the migration of VAL-ko depends on the movement of VAL-wt, in line with the hypothesis that DLBCL invade tissues through cell-to-cell-induced migration as a heterogeneous population of cells.
PMC9878562
ACKR3 promotes CXCL12/CXCR4-mediated cell-to-cell-induced lymphoma migration through LTB4 production
Pertussis toxin (PTX) treatment inhibits the transmigration of VAL-wt towards CXCL12 but has no effect on the VAL cell chemokinesis ( Figure S4A ).