The CXCL12/CXCR7 signaling axis, isoforms, circadian rhythms, and tumor cellular composition dictate gradients in tissue

<div><p>Chemokine CXCL12 gradients drive chemotaxis in a CXCR4-dependent mechanism and have been implicated in cancer metastasis. While CXCL12 gradients are typically studied in organized, defined environments, the tumor microenvironment is disorganized. <i>In vivo</i>, CXCL12 gradients depend on many factors: the number and arrangement of cells secreting and degrading CXCL12, isoform-dependent binding to the extracellular matrix, diffusion, and circadian fluctuations. We developed a computational model of the tumor microenvironment to simulate CXCL12 gradient dynamics in disorganized tissue. There are four major findings from the model. First, CXCL12-β and -γ form higher magnitude (steeper) gradients compared to CXCL12-α. Second, endothelial CXCR7+ cells regulate CXCL12 gradient direction by controlling concentrations near but not far from the vasculature. Third, the magnitude and direction of CXCL12 gradients are dependent on the local composition of secreting and scavenging cells within the tumor. We theorize that “micro-regions” of cellular heterogeneity within the tumor are responsible for forming strong gradients directed into the blood. Fourth, CXCL12 circadian fluctuations influence gradient magnitude but not direction. Our simulations provide predictions for future experiments in animal models. Understanding the generation of CXCL12 gradients is crucial to inhibiting cancer metastasis.</p></div

CXCL12-secreting and -scavenging cell numbers in the tumor microenvironment influence gradient direction and magnitude.

<p><b>A</b>, Maximum CXCL12-α (left), -β (center), and -γ (right) gradients over a 24-h period illustrate that gradients are dependent upon cellular makeup. The * and ~ show the strongest gradients into the blood and into the tissue, respectively, and correspond to the same symbols in B. Gradient magnitudes were averaged over 5 runs to account for potential variation in the randomization of cell placement in the model. In all simulations, 200 endothelial CXCR7+ cells were present. White squares designate a gradient of exactly 0. <b>B</b>, Time-series of CXCL12 gradients demonstrate that gradients fluctuate according to circadian rhythm. Dotted lines represent the minimum gradient (0.002 nM/μm) that we determined to be necessary to trigger significant cell migration in a previous work [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0187357#pone.0187357.ref021" target="_blank">21</a>]. CT 24 corresponds to 9 pm while CT 12 corresponds to 9 am. Positive gradients correspond to higher CXCL12 tissue concentrations while negative gradients correspond to higher CXCL12 concentrations in the blood. <b>C</b>, Circadian fluctuations of all combinations of CXCL12-secreting and -scavenging cells shown in A.</p

Model setups.

<p><b>A</b>, In Setup 1, we capture the cell-derived CXCL12 gradient along the red line formed in the presence of clusters of CXCL12-secreting cells and CXCR7+ cells to examine the characteristics of isoform-specific gradients. <b>B</b>, In Setup 2, we simulate a section of a tumor and calculate the blood-tissue and endothelial-tissue gradients. All variables shown in figure are detailed in Appendix 2. <b>C,</b> A cross-sectional slice of the grid used for Setup 2.</p

CXCL12 circadian fluctuations in CXCL12 concentration occur independent of endothelial CXCR7.

<p><b>A</b>, Blood (left), femur (center), and tibia (right) CXCL12 levels vary 2-fold between 5 am and 8 pm. Error bars represent SEM of 5 repeated measurements per group of mice. <b>B</b>, CXCL12 isoform-specific human blood variations that we use in our model (Eq (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0187357#pone.0187357.e001" target="_blank">1</a>)). CT 24 corresponds to 9 pm while CT 12 corresponds to 9 am. For humans, we offset the time at which the maximum and minimum blood levels occur in the mice by 12 hours because mice are nocturnal. Mice and humans exhibit CXCL12 blood level maxima at night and in the morning, respectively.</p

Cell-derived CXCL12 gradients are isoform-specific with regards to the time to steady state, gradient magnitude, and amount of CXCL12 in the proximate environment.

<p><b>A</b>, Higher ECM binding affinity and higher secretion rate correspond to a higher gradient magnitude, but a longer time to form it. Error bars represent SEM of 5 simulations. Both clusters contain 100 cells. Cell clusters are placed 100 μm apart. <b>B</b>, CXCL12 gradients and total concentrations in the tissue vary with each isoform. Cell numbers were the same as in A. <b>C</b>, CXCL12-β (center) and -γ (right) create microenvironments with higher CXCL12 concentrations than CXCL12-α (left). On each plot, the x- and y-axes correspond to the number of cells in each cluster: 25, 50, 75, 100, 125, 150, 175, 200. <b>D</b>, the number of CXCL12-secreting cells in a cluster influences gradients, while the effect of the number of CXCR7+ cells is less pronounced. The x- and y-axes are the same as in C. For C and D, free and ECM-bound CXCL12 was summed. For all simulations in this figure, an average over 5 simulations is reported.</p

Endothelial CXCR7 influences CXCL12 gradient direction by controlling CXCL12 concentrations near but not far from the vasculature.

<p>200 endothelial CXCR7 cells (83% coverage of the blood vessel), 200 CXCL12-secreting cells, and 200 CXCR7+ cells in tissue were placed in the simulation. <b>A</b>, Locally, endothelial CXCR7+ cells scavenge CXCL12 and influence the endothelial-tissue gradient direction. We arbitrarily define positive gradients as those that point from the vasculature to the tissue, and negative gradients point from the tissue to the vasculature. Maximum gradients over a 24-hour simulation are shown. <b>B</b>, CXCL12 concentration profiles (corresponding to the maximum gradients shown in A) indicate endothelial CXCR7 significantly decreases concentrations near the blood vessel and has a much lower effect far away. Curves begin at 10μm because this designates the first grid compartment next to the vasculature (Setup 2, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0187357#pone.0187357.g001" target="_blank">Fig 1B and 1C</a>).</p

Cancer cells within “micro-regions” of the tumor containing either high or low ratios of CXCL12-secreting to non-endothelial CXCR7+ cells are more migratory than the tumor bulk.

<p>We assume tumors are mostly composed of immobile or weakly mobile cells due to a balance of CXCL12-secreting and CXCR7+ cells and a weak CXCL12 gradient. We predict that cancer cells located in environments containing a high number of CXCR7+ cells and few CXCL12-secreting cells will likely migrate towards vasculature, whereas cancer cells in environments with high secreting cell numbers and few CXCR7+ cells will likely migrate deeper into the tissue.</p

CXCL12-CXCR4 signaling pathway in ovarian cancer cells.

<p>A) HeyA8-CXCR4-CBRN/Ar-CBC and parental HeyA8 cells were treated with increasing concentrations of CXCL12 for 10 minutes. Western blot of total cell lysates shows phosphorylated and total AKT, respectively. We used GAPDH as a loading control. Graph shows relative band intensities for phosphorylated AKT in each cell line normalized to total AKT and GAPDH. B) HeyA8-CXCR4-CBRN/Ar-CBC cells were treated with 100 ng/ml CXCL12-α for 0, 5, 15, or 30 minutes in the absence or presence of 1 µM AMD3100. Total cell lysates were probed for phosphorylated and total ERK1/2, respectively. Lysates also were analyzed by Western blot for expression of β-arrestin 2-CBC and endogenous β-arrestin 1 and 2. GAPDH is shown as a loading control. C) Flow cytometry of CXCR4 expression in various HeyA8 cell lines used in this study. Dark line and dashed lines in histogram plots denote isotype control and staining with CXCR4 antibody.</p

Combination therapy with AMD3100 and cisplatin decreases tumor burden.

<p>A) Area under the curve analysis of imaging data for ratios of click beetle red luciferase complementation for CXCR4 and β-arrestin 2 normalized to eqFP650 fluorescence (tumor burden) in mice implanted with HeyA8-CXCR4-CBRN/Ar-CBC and HeyA8-CXCL12-GL cells. Data are shown for groups treated with vehicle control, AMD3100, cisplatin, or both AMD3100 and cisplatin for two weeks beginning one week after implanting cells. Graph shows mean values+SEM (n = 7 mice per group). *, p<0.05. B) Area under the curve analysis of fluorescence from eqFP650 produced by implanted ovarian cancer cells over the course of the experiment. Graph shows mean values+SEM. *, p<0.05, **, p<0.01. C) Kaplan-Meier curves for survival of mice treated with vehicle, AMD3100, cisplatin, or AMD3100 and cisplatin. All treatment groups differ from vehicle control (p<0.05) but not from each other.</p

Imaging association of CXCR4 and β-arrestin 2 in living mice.

<p>A) Representative images of mice with intraperitoneal implants of HeyA8-CXCR4-CBRN/Ar-CBC and HeyA8-CXCL12-GL cells. Images were obtained before and following 5 days of treatment with osmotic pumps containing AMD3100 or vehicle control. Scale bar depicts ranges of photon flux values displayed on pseudocolor images. B) Fluorescence from ovarian cancer cells was measured in living mice treated with AMD3100 or vehicle control. Graph shows mean values+SEM for fluorescence relative to values measured on day 0 before beginning treatment. Arrows show the period when osmotic pumps were in place. C) Quantified photon flux data for click beetle red complementation in mice treated with AMD3100 or vehicle control, respectively. Data were normalized to tumor fluorescence for each mouse and graphed as mean values+SEM (n = 7 mice per group). *, p<0.05.</p