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PMC9878562
ACKR3 promotes CXCL12/CXCR4-mediated cell-to-cell-induced lymphoma migration through LTB4 production
In neoplasms, the chemokine system plays a promiscuous role depending on the type of cancer, the cellular and environmental context, and the involved chemokine axis.
PMC9878562
ACKR3 promotes CXCL12/CXCR4-mediated cell-to-cell-induced lymphoma migration through LTB4 production
The chemokine system can influence disease progression in a beneficial or detrimental way (14–21).
PMC9878562
ACKR3 promotes CXCL12/CXCR4-mediated cell-to-cell-induced lymphoma migration through LTB4 production
In many solid cancers altered expression levels of ACKR3 correlate with poor prognosis and tumor metastasis (22–29).
PMC9878562
ACKR3 promotes CXCL12/CXCR4-mediated cell-to-cell-induced lymphoma migration through LTB4 production
In addition, the CXCL12/ACKR3/CXCR4 axis was proposed to be involved in lymphoma (30–32).
PMC9878562
ACKR3 promotes CXCL12/CXCR4-mediated cell-to-cell-induced lymphoma migration through LTB4 production
Diffuse large B cell lymphoma (DLBCL) arise in the germinal center when B cells undergo somatic hypermutation and class-switch recombination.
PMC9878562
ACKR3 promotes CXCL12/CXCR4-mediated cell-to-cell-induced lymphoma migration through LTB4 production
During these events, genetic alterations can cause malignant tumor transformation leading to GC-derived B cell lymphoma (33).
PMC9878562
ACKR3 promotes CXCL12/CXCR4-mediated cell-to-cell-induced lymphoma migration through LTB4 production
We have previously shown that GC derived DLBCL VAL cells, require the functional expression of ACKR3 to disseminate into distant organs in NOD/SCID mice (32).
PMC9878562
ACKR3 promotes CXCL12/CXCR4-mediated cell-to-cell-induced lymphoma migration through LTB4 production
Tumor spreading is a multistage process where cancer cells undergo transformation, leave the primary tumor and migrate into adjacent and distant tissues, where they proliferate (34–38).
PMC9878562
ACKR3 promotes CXCL12/CXCR4-mediated cell-to-cell-induced lymphoma migration through LTB4 production
It was previously assumed that cancers metastasize through single tumor cells escaping from the primary site arriving at distant tissues and propagating there.
PMC9878562
ACKR3 promotes CXCL12/CXCR4-mediated cell-to-cell-induced lymphoma migration through LTB4 production
However, in the last decade, emerging evidence suggests that metastases can disseminate with a more complex mechanism called collective cell migration (39).
PMC9878562
ACKR3 promotes CXCL12/CXCR4-mediated cell-to-cell-induced lymphoma migration through LTB4 production
This process describes the presence of leading cells that trace the way into adjacent tissues for the migration of follower cells (40) or induce the movement of cohesive clusters (39).
PMC9878562
ACKR3 promotes CXCL12/CXCR4-mediated cell-to-cell-induced lymphoma migration through LTB4 production
Collective migration has been extensively studied to describe the metastasis of solid tumors but is seldom used to describe the movement of hematological cancers in a contact independent manner.
PMC9878562
ACKR3 promotes CXCL12/CXCR4-mediated cell-to-cell-induced lymphoma migration through LTB4 production
Through chemokine sequestration expression of scavenger receptors can contribute to the formation of self-generated gradients (40, 41).
PMC9878562
ACKR3 promotes CXCL12/CXCR4-mediated cell-to-cell-induced lymphoma migration through LTB4 production
The mechanisms by which ACKR3, CXCR4 and CXCL12 induce the dissemination of VAL cells in a synergetic manner have not been addressed.
PMC9878562
ACKR3 promotes CXCL12/CXCR4-mediated cell-to-cell-induced lymphoma migration through LTB4 production
Here, we demonstrate that in the presence of CXCL12 wild type VAL cells (VAL-wt), which can express ACKR3 on the cell surface, create cues that in vitro can be sensed by CXCR4 follower VAL-wt.
PMC9878562
ACKR3 promotes CXCL12/CXCR4-mediated cell-to-cell-induced lymphoma migration through LTB4 production
Moreover, VAL-wt support in vitro chemotaxis of VAL cells in which ACKR3 was genetically deleted (VAL-ko).
PMC9878562
ACKR3 promotes CXCL12/CXCR4-mediated cell-to-cell-induced lymphoma migration through LTB4 production
The latter by themselves are not able to migrate in response to CXCL12.
PMC9878562
ACKR3 promotes CXCL12/CXCR4-mediated cell-to-cell-induced lymphoma migration through LTB4 production
In vivo VAL-wt can induce the migration of CXCR4 VAL-ko, contributing to the infiltration of draining lymph nodes.
PMC9878562
ACKR3 promotes CXCL12/CXCR4-mediated cell-to-cell-induced lymphoma migration through LTB4 production
Using a novel computational analysis of 3D migration, we show that in the presence of CXCL12 VAL-wt stimulate cell-to-cell-induced migration presumably through the release of leukotriene B4 (LTB4).
PMC9878562
ACKR3 promotes CXCL12/CXCR4-mediated cell-to-cell-induced lymphoma migration through LTB4 production
Moreover, the release of LTB4 from VAL cells induces cell polarization and chemotaxis in an autocrine and paracrine fashion.
PMC9878562
ACKR3 promotes CXCL12/CXCR4-mediated cell-to-cell-induced lymphoma migration through LTB4 production
We propose a novel mechanism by which ACKR3 sustains the movement of otherwise migration incompetent cells and partially reveal the molecular components of this pathway.
PMC9878562
ACKR3 promotes CXCL12/CXCR4-mediated cell-to-cell-induced lymphoma migration through LTB4 production
The atypical chemokine receptor ACKR3 is known to be upregulated on both solid cancers and blood neoplasms (22, 24, 30, 31).
PMC9878562
ACKR3 promotes CXCL12/CXCR4-mediated cell-to-cell-induced lymphoma migration through LTB4 production
Here we investigated its expression and functionality on two DLBCL lines, VAL and DOHH2.
PMC9878562
ACKR3 promotes CXCL12/CXCR4-mediated cell-to-cell-induced lymphoma migration through LTB4 production
Flow cytometry was performed on wild type DLBCL cells and VAL cells in which ACKR3 was genetically ablated (VAL-ko).
PMC9878562
ACKR3 promotes CXCL12/CXCR4-mediated cell-to-cell-induced lymphoma migration through LTB4 production
In the cells with deleted ACKR3 cyan fluorescent protein (CFP) was introduced under constitutively active spleen focus-forming virus (SFFV) promoter, which allows to easily identify cells in tissues and mixed cultures (32).
PMC9878562
ACKR3 promotes CXCL12/CXCR4-mediated cell-to-cell-induced lymphoma migration through LTB4 production
We measured surface expression on these cells of ACKR3, CXCR4 and CCR7.
PMC9878562
ACKR3 promotes CXCL12/CXCR4-mediated cell-to-cell-induced lymphoma migration through LTB4 production
All cells expressed similar levels of CXCR4 on the plasma membrane ( Figure 1A ).
PMC9878562
ACKR3 promotes CXCL12/CXCR4-mediated cell-to-cell-induced lymphoma migration through LTB4 production
Conversely, ACKR3 was expressed only on a fraction of VAL-wt (10-18%), despite their clonal nature and continuous mRNA levels of the receptor (32).
PMC9878562
ACKR3 promotes CXCL12/CXCR4-mediated cell-to-cell-induced lymphoma migration through LTB4 production
When cells were sorted for ACKR3 surface expression and cultured, the mixed phenotype reestablished rapidly within 2-3 weeks.
PMC9878562
ACKR3 promotes CXCL12/CXCR4-mediated cell-to-cell-induced lymphoma migration through LTB4 production
However, cells negative for ACKR3 at sorting were more refractory for acquiring ACKR3 surface expression during this time ( Figure S1A ).
PMC9878562
ACKR3 promotes CXCL12/CXCR4-mediated cell-to-cell-induced lymphoma migration through LTB4 production
However, during prolonged culture (6 weeks) the cell established the initial phenotype.
PMC9878562
ACKR3 promotes CXCL12/CXCR4-mediated cell-to-cell-induced lymphoma migration through LTB4 production
Similarly, only a small fraction of DOHH2 expressed ACKR3 on the plasma membrane ( Figure 1A ).
PMC9878562
ACKR3 promotes CXCL12/CXCR4-mediated cell-to-cell-induced lymphoma migration through LTB4 production
Interestingly, CCR7 was predominantly expressed on the surface of wild type DLBCL (VAL-wt and DOHH2), which were negative for ACKR3 plasma membrane expression.
PMC9878562
ACKR3 promotes CXCL12/CXCR4-mediated cell-to-cell-induced lymphoma migration through LTB4 production
Genetic deletion of ACKR3 on VAL cells had no effect on CXCR4 expression.
PMC9878562
ACKR3 promotes CXCL12/CXCR4-mediated cell-to-cell-induced lymphoma migration through LTB4 production
Surprisingly, CCR7 transcripts were less frequent in VAL-ko (117 ± 23 vs. 898 ± 32, (n=5)) and surface expression was markedly reduced on VAL-ko ( Figure 1A ).
PMC9878562
ACKR3 promotes CXCL12/CXCR4-mediated cell-to-cell-induced lymphoma migration through LTB4 production
Chemokine receptor expression and function on DLBCL cells. (
PMC9878562
ACKR3 promotes CXCL12/CXCR4-mediated cell-to-cell-induced lymphoma migration through LTB4 production
ACKR3 (mAb 11G8), CXCR4 (mAb 12G5) and CCR7 (mAb G043H7) surface expression on VAL-wt, DOHH2, and VAL-ko was measured by flow cytometry.
PMC9878562
ACKR3 promotes CXCL12/CXCR4-mediated cell-to-cell-induced lymphoma migration through LTB4 production
Representative plots of three independent experiments. (
PMC9878562
ACKR3 promotes CXCL12/CXCR4-mediated cell-to-cell-induced lymphoma migration through LTB4 production
Uptake of the chimeric chemokine CXCL11_12 AF647 by DLBCL.
PMC9878562
ACKR3 promotes CXCL12/CXCR4-mediated cell-to-cell-induced lymphoma migration through LTB4 production
Mean fluorescence intensity (MFI) of DLBCL incubated with 50 nM of CXCL11_12 for 45 min at 37°C.
PMC9878562
ACKR3 promotes CXCL12/CXCR4-mediated cell-to-cell-induced lymphoma migration through LTB4 production
MFI (by flow cytometry) reports the mean ± SD (Dunnett’s multiple comparisons, ONE-WAY ANOVA **p ≤ 0.01, ****p ≤ 0.0001) of four independent experiments performed in triplicates. (
PMC9878562
ACKR3 promotes CXCL12/CXCR4-mediated cell-to-cell-induced lymphoma migration through LTB4 production
Transendothelial migration of lymphoma cells.
PMC9878562
ACKR3 promotes CXCL12/CXCR4-mediated cell-to-cell-induced lymphoma migration through LTB4 production
VAL cell migration through mLEC towards 10 nM CXCL12 was measured after 8h incubation.
PMC9878562
ACKR3 promotes CXCL12/CXCR4-mediated cell-to-cell-induced lymphoma migration through LTB4 production
Cell migration was calculated as percentage of the input (% migrated cells).
PMC9878562
ACKR3 promotes CXCL12/CXCR4-mediated cell-to-cell-induced lymphoma migration through LTB4 production
Cumulative data from three independent experiments measured in triplicates (means ± SD, Turkey’s multiple comparison ONE-WAY ANOVA ns (not significant), p≥0.05, ****p ≤ 0.0001). (
PMC9878562
ACKR3 promotes CXCL12/CXCR4-mediated cell-to-cell-induced lymphoma migration through LTB4 production
(Left) ACKR3 (mAb 11G8) surface expression on VAL-wt cells before (upper panel, before) and after transendothelial migration (lower panel, after) towards 10 nM CXCL12 as in (C).
PMC9878562
ACKR3 promotes CXCL12/CXCR4-mediated cell-to-cell-induced lymphoma migration through LTB4 production
Representative plots from ten independent experiments performed in duplicates. (
PMC9878562
ACKR3 promotes CXCL12/CXCR4-mediated cell-to-cell-induced lymphoma migration through LTB4 production
Percentage of cells expressing ACKR3 on the plasma membrane before and after migration.
PMC9878562
ACKR3 promotes CXCL12/CXCR4-mediated cell-to-cell-induced lymphoma migration through LTB4 production
Cumulative data from five different experiments measured in triplicates (means ± SD, unpaired t-test, ****p ≤ 0.0001). (
PMC9878562
ACKR3 promotes CXCL12/CXCR4-mediated cell-to-cell-induced lymphoma migration through LTB4 production
Left side: flow cytometry plot showing ACKR3 (mAb 11G8) and CXCR5 (mAb J252D4) surface expression on VAL-wt (left panel).
PMC9878562
ACKR3 promotes CXCL12/CXCR4-mediated cell-to-cell-induced lymphoma migration through LTB4 production
Transendothelial migration of VAL-wt towards increasing concentrations of CXCL13 (0.1, 0.3 and 1 µM) (bar chart).
PMC9878562
ACKR3 promotes CXCL12/CXCR4-mediated cell-to-cell-induced lymphoma migration through LTB4 production
Right side: flow cytometry plot showing ACKR3 (mAb 11G8) and CXCR5 (mAb J252D4) surface expression on VAL-ko (left panel).
PMC9878562
ACKR3 promotes CXCL12/CXCR4-mediated cell-to-cell-induced lymphoma migration through LTB4 production
Transendothelial migration of VAL-ko through mLEC towards increasing concentrations of CXCL13 (0.1, 0.3 and 1 µM) (bar chart).
PMC9878562
ACKR3 promotes CXCL12/CXCR4-mediated cell-to-cell-induced lymphoma migration through LTB4 production
Cell migration was calculated as described above.
PMC9878562
ACKR3 promotes CXCL12/CXCR4-mediated cell-to-cell-induced lymphoma migration through LTB4 production
Bar charts show cumulative data of three independent experiments performed in triplicates (means ± SD, Dunnett’s multiple comparisons, ONE WAY ANOVA, **p ≤ 0.01, ***p ≤ 0.001).
PMC9878562
ACKR3 promotes CXCL12/CXCR4-mediated cell-to-cell-induced lymphoma migration through LTB4 production
To assess the functionality of ACKR3 we took advantage of a previously developed specific chemokine uptake assay (42).
PMC9878562
ACKR3 promotes CXCL12/CXCR4-mediated cell-to-cell-induced lymphoma migration through LTB4 production
CXCL11_12 AF647 is a fluorescent chimeric chemokine side-specific labeled at the C-terminus, composed of the N-terminus of CXCL11 and the main body of CXCL12.
PMC9878562
ACKR3 promotes CXCL12/CXCR4-mediated cell-to-cell-induced lymphoma migration through LTB4 production
The chimera specifically binds ACKR3, but not CXCR3 or CXCR4, the respective cognate receptors for CXCL11 and CXCL12.
PMC9878562
ACKR3 promotes CXCL12/CXCR4-mediated cell-to-cell-induced lymphoma migration through LTB4 production
VAL-wt and DOHH2 readily internalized the chimera ( Figure 1B ).
PMC9878562
ACKR3 promotes CXCL12/CXCR4-mediated cell-to-cell-induced lymphoma migration through LTB4 production
Hence, despite the limited surface expression of ACKR3 ( Figure 1A ), the receptor appears functional on both DLBCL lines.
PMC9878562
ACKR3 promotes CXCL12/CXCR4-mediated cell-to-cell-induced lymphoma migration through LTB4 production
The observation is in agreement with the notion that the receptor cycles between the plasma membrane and endocytic compartments (6).
PMC9878562
ACKR3 promotes CXCL12/CXCR4-mediated cell-to-cell-induced lymphoma migration through LTB4 production
As expected, VAL-ko did not scavenge the chimera and displayed low unspecific binding ( Figure 1B and Figure S1B ).
PMC9878562
ACKR3 promotes CXCL12/CXCR4-mediated cell-to-cell-induced lymphoma migration through LTB4 production
Chemokine scavenging and receptor internalization requires the intact C-terminus of ACKR3 (6).
PMC9878562
ACKR3 promotes CXCL12/CXCR4-mediated cell-to-cell-induced lymphoma migration through LTB4 production
We reintroduced into VAL-ko ACKR3 with a truncated C-terminus by stable transfection resulting in VAL-ACKR3-ΔC cells (VAL-ACKR3-ΔC).
PMC9878562
ACKR3 promotes CXCL12/CXCR4-mediated cell-to-cell-induced lymphoma migration through LTB4 production
VAL-ACKR3-ΔC poorly scavenged CXCL11_12 AF647 indicating that the C-terminus of ACKR3 is required for scavenging and receptor internalization ( Figures S1B, C ), consistent with previous findings (6, 43).
PMC9878562
ACKR3 promotes CXCL12/CXCR4-mediated cell-to-cell-induced lymphoma migration through LTB4 production
Next, we assessed the chemotactic response towards CXCL12.
PMC9878562
ACKR3 promotes CXCL12/CXCR4-mediated cell-to-cell-induced lymphoma migration through LTB4 production
Chemotaxis assays were performed as previously described with transwell plates coated with a monolayer of endothelial cells grown on a collagen-based extracellular matrix (32).
PMC9878562
ACKR3 promotes CXCL12/CXCR4-mediated cell-to-cell-induced lymphoma migration through LTB4 production
The presence of endothelial cells optimizes the migration of lymphoma cells.
PMC9878562
ACKR3 promotes CXCL12/CXCR4-mediated cell-to-cell-induced lymphoma migration through LTB4 production
VAL cells were allowed to migrate through a layer of mouse lymphatic endothelial cells (mLEC) for 8h in the absence and presence of an optimal concentration of CXCL12 (10 nM) in the lower compartment.
PMC9878562
ACKR3 promotes CXCL12/CXCR4-mediated cell-to-cell-induced lymphoma migration through LTB4 production
In agreement with our previous findings (32), under these conditions about 20% of VAL-wt migrated into the lower compartment in the presence of chemokine, which accounts for a 3-fold increase compared to unstimulated chemokinesis.
PMC9878562
ACKR3 promotes CXCL12/CXCR4-mediated cell-to-cell-induced lymphoma migration through LTB4 production
By contrast, VAL-ko, which express equal surface levels of CXCR4 showed no response to CXCL12, but also no spontaneous migration ( Figure 1C ).
PMC9878562
ACKR3 promotes CXCL12/CXCR4-mediated cell-to-cell-induced lymphoma migration through LTB4 production
The observation was confirmed with genetically different clones to exclude off-target effects on cell migration through editing of ACKR3 by CRISPR/Cas9.
PMC9878562
ACKR3 promotes CXCL12/CXCR4-mediated cell-to-cell-induced lymphoma migration through LTB4 production
Chemokinesis of VAL cells through mLEC was not inhibited in the presence of the CXCR4 antagonist AMD3100, suggesting a basal migratory activity that was not mediated by CXCR4 ( Figure S1D ).
PMC9878562
ACKR3 promotes CXCL12/CXCR4-mediated cell-to-cell-induced lymphoma migration through LTB4 production
Similarly, VAL-ACKR3-ΔC, which express comparable CXCR4 levels on the surface ( Figure S1Ei ) did not perform CXCL12-dependent migration ( Figure S1Eii ).
PMC9878562
ACKR3 promotes CXCL12/CXCR4-mediated cell-to-cell-induced lymphoma migration through LTB4 production
The observation suggests that the C-terminus is necessary for ACKR3 activity during VAL cell migration.
PMC9878562
ACKR3 promotes CXCL12/CXCR4-mediated cell-to-cell-induced lymphoma migration through LTB4 production
We next compared the surface expression of ACKR3 on VAL-wt before and after migration towards CXCL12.
PMC9878562
ACKR3 promotes CXCL12/CXCR4-mediated cell-to-cell-induced lymphoma migration through LTB4 production
The fraction of cells, which expressed ACKR3 at the plasma membrane increased from about 17% to 40-50% after chemotaxis ( Figure 1D ).
PMC9878562
ACKR3 promotes CXCL12/CXCR4-mediated cell-to-cell-induced lymphoma migration through LTB4 production
The observation could indicate that cells, which express ACKR3 at the plasma membrane, migrate more efficiently.
PMC9878562
ACKR3 promotes CXCL12/CXCR4-mediated cell-to-cell-induced lymphoma migration through LTB4 production
However, it cannot be excluded that cells upregulate surface expression of the receptor during migration.
PMC9878562
ACKR3 promotes CXCL12/CXCR4-mediated cell-to-cell-induced lymphoma migration through LTB4 production
To get a better insight we sorted ACKR3 and ACKR3 VAL cells and analyzed their migratory properties in chemotaxis assays.
PMC9878562
ACKR3 promotes CXCL12/CXCR4-mediated cell-to-cell-induced lymphoma migration through LTB4 production
The efficiency of cells expressing ACKR3 at the surface was notably higher compared to cells that did not present ACKR3 at the plasma membrane.
PMC9878562
ACKR3 promotes CXCL12/CXCR4-mediated cell-to-cell-induced lymphoma migration through LTB4 production
Moreover, VAL-wt lacking ACKR3 surface expression did not upregulate the receptor during migration ( Figure S1F ).
PMC9878562
ACKR3 promotes CXCL12/CXCR4-mediated cell-to-cell-induced lymphoma migration through LTB4 production
Nevertheless, in ACKR3 negative VAL-wt the mRNA of the receptor is not absent compared to the genetically modified VAL-ko (32).
PMC9878562
ACKR3 promotes CXCL12/CXCR4-mediated cell-to-cell-induced lymphoma migration through LTB4 production
Hence, it cannot be excluded that ACKR3 expressed on endosomal structures somehow contributes to the migration.
PMC9878562
ACKR3 promotes CXCL12/CXCR4-mediated cell-to-cell-induced lymphoma migration through LTB4 production
All together, these findings suggest that functional ACKR3 plasma membrane expression is necessary to support efficiently CXCR4-dependent cell migration.
PMC9878562
ACKR3 promotes CXCL12/CXCR4-mediated cell-to-cell-induced lymphoma migration through LTB4 production
In order to exclude a general inhibitory effect on VAL cell migration by ACKR3 deletion we examined the chemotaxis towards CXCL13, the ligand for CXCR5, which is expressed on both, VAL-wt and VAL-ko ( Figure 1E ).
PMC9878562
ACKR3 promotes CXCL12/CXCR4-mediated cell-to-cell-induced lymphoma migration through LTB4 production
With increasing concentrations of CXCL13 we observed a typical bell-shaped migration response by both cell types ( Figure 1E ), indicating that VAL-ko retain the capability to sense a chemokine gradient, polarize and migrate.
PMC9878562
ACKR3 promotes CXCL12/CXCR4-mediated cell-to-cell-induced lymphoma migration through LTB4 production
In addition, we explored the ability of VAL-wt and VAL-ko to migrate in response to stimulation of CCR7 with CCL19.
PMC9878562
ACKR3 promotes CXCL12/CXCR4-mediated cell-to-cell-induced lymphoma migration through LTB4 production
VAL-wt, which heterogeneously express CCR7 ( Figure 1A ), were sorted for CCR7 surface expression and tested separately.
PMC9878562
ACKR3 promotes CXCL12/CXCR4-mediated cell-to-cell-induced lymphoma migration through LTB4 production
CCR7 VAL-wt, which do not express ACKR3 on the surface ( Figure 1A ), migrated more efficiently than the CCR7 ACKR3 sorted cells ( Figure S1G ).
PMC9878562
ACKR3 promotes CXCL12/CXCR4-mediated cell-to-cell-induced lymphoma migration through LTB4 production
The data indicate that the moderate chemotaxis towards CCL19 correlates with CCR7 surface expression.
PMC9878562
ACKR3 promotes CXCL12/CXCR4-mediated cell-to-cell-induced lymphoma migration through LTB4 production
In line with this, VAL-ko cells, which do not express CCR7 ( Figure 1A ) did not migrate towards CCL19 ( Figure S1G ).
PMC9878562
ACKR3 promotes CXCL12/CXCR4-mediated cell-to-cell-induced lymphoma migration through LTB4 production
The observation that VAL-wt, which do not express ACKR3 on the surface, can migrate towards CXCL12 in the presence of cells exposing the receptor on the plasma membrane was intriguing ( Figure 1D ).
PMC9878562
ACKR3 promotes CXCL12/CXCR4-mediated cell-to-cell-induced lymphoma migration through LTB4 production
Previous data showed that in zebrafish ACKR3 expressed in trans on stromal cells can support the CXCL12-mediated migration of primordial germ cells (44).
PMC9878562
ACKR3 promotes CXCL12/CXCR4-mediated cell-to-cell-induced lymphoma migration through LTB4 production
It is plausible that ACKR3 positive VAL-wt, when locally scavenging CXCL12, shape micro-gradients that are sensed by CXCR4 VAL cells that do not express ACKR3 on the surface.
PMC9878562
ACKR3 promotes CXCL12/CXCR4-mediated cell-to-cell-induced lymphoma migration through LTB4 production
The hypothesis implies that ACKR3 scavenging activity is necessary for VAL cell migration.
PMC9878562
ACKR3 promotes CXCL12/CXCR4-mediated cell-to-cell-induced lymphoma migration through LTB4 production
To address the assumption, we used mLEC transwell assays to measure CXCL12-induced chemotaxis of VAL-ko in the presence of VAL-wt.
PMC9878562
ACKR3 promotes CXCL12/CXCR4-mediated cell-to-cell-induced lymphoma migration through LTB4 production
We mixed VAL-wt and CFP VAL-ko in a 1:1 ratio, which enables the identification of the two cell types by flow cytometry.
PMC9878562
ACKR3 promotes CXCL12/CXCR4-mediated cell-to-cell-induced lymphoma migration through LTB4 production
The mixture was allowed to migrate towards different concentrations of CXCL12 ( Figure 2A ).
PMC9878562
ACKR3 promotes CXCL12/CXCR4-mediated cell-to-cell-induced lymphoma migration through LTB4 production
CFP VAL-ko in the 1:1 mixture with VAL-wt showed a typical bell-shaped migratory response towards CXCL12, whereas VAL-ko cells alone poorly responded ( Figure 2A ).