Plasticity and heterogeneity of tumor cell morphology and motility behavior.

<p><b>A–B</b>. Bright field images of MDA-MB-231 cells embedded in 3D collagen matrices within the microfluidic channel at t = 0 and 8 h. Here, t = 0 is defined as the time when buffer and 100 nM SDF-1α solution were introduced into the two side channels. Cells were pre-incubated for 24 hours after seeding before the introduction of the gradients. <b>C.</b> Graphical description of cell speed , cell velocity along the gradient direction , persistence length , and gradient-directed persistence length . <b>D.</b> Graphical description of aspect ratio. Distribution of cell aspect ratios at t = 0 and 8 h. <b>E.</b> Distribution of cell speed of elongated cells (aspect ratio larger than 3) and amoeboid-like cells (aspect ratio less than 3).</p

Chemoinvasive and chemokinetic behavior of tumor cells to linear SDF-1α gradients.

<p><b>A.</b> Average cell velocity along the SDF-1α gradient as a function of the SDF-1α gradient . Solid line is a fit to the ligand – receptor binding kinetics . <b>B.</b> Average cell speed as a function of the SDF-1α concentration gradient. <b>C.</b> Average persistence length along the gradient direction as a function of SDF-1α concentration gradient. <b>D.</b> Average persistence length as a function of SDF-1α concentration gradient. The stars were obtained using a nonparametric <i>t</i>-test compared to the control group (Mann-Whitney test with * for 0.01<<i>p</i><0.05, ** for 0.001<<i>p</i><0.01, and *** for <i>p</i><0.001).</p

Cooperative roles of EGF and SDF-1α in tumor cell chemoinvasion.

<p>Average cell velocity (<b>A</b>) and speed (<b>B</b>) in the presence of a SDF-1α gradient of 111 nM/mm and a uniform EGF concentration of 0, 0.25 or 8.33 nM. Control conditions were without SDF-1α and EGF. <b>C.</b> Average cell speed under indicated conditions. The stars were obtained using a nonparametric <i>t</i>-test compared to the control group (Mann-Whitney test with * for 0.01<<i>p</i><0.05, ** for 0.001<<i>p</i><0.01, and *** for <i>p</i><0.001).</p

Live immunostaining detects only extracellular basement membrane-bound CCL21, while staining on fixed tissue reveals intracellular as well as extracellular CCL21.

<p>(<b>a–b</b>) In the live ear dermis CCL21 accumulated in patches (arrowheads) and along continuous lymphatic segments (arrows), and co-localized with (<b>a</b>) perlecan and (<b>b</b>) collagen IV, both basement membrane components of the collecting lymphatic vessels. (<b>c</b>) Occasionally observed CCL21-positive (green) initial lymphatic vessels (iLy) stained more weakly for collagen IV (red). (<b>d</b>) Extracellular CCL21 deposits (green) were seen around lymphatic vessels in the exposed ear skin did not correlate with injured blood vessel, as determined by i.v. injection of TRITC-dextran (cyan) that leaked from areas of injured vessels (arrowheads). The tissue was pre-stained for perlecan and CCL21 before dextran injection. Scale bars in a, b and d (left), 400 µm; c and d (right), 100 µm.</p

Tumor cells display no chemoinvasion but mild chemokinesis in linear EGF gradients.

<p>Average cell velocity along the EGF gradient (<b>A</b>), average cell speed (<b>B</b>), average persistence length along the EGF concentration gradient (<b>C</b>) and average persistence length (<b>D</b>) as a function of EGF gradients. The stars were obtained using a nonparametric <i>t</i>-test compared to the control group (Mann-Whitney test with * for 0.01<<i>p</i><0.05, ** for 0.001<<i>p</i><0.01, and *** for <i>p</i><0.001).</p

Microfluidic device setup and data acquisition.

<p><b>A.</b> An image of the microfluidic device on the microscope stage. A penny is placed on the side for scale. <b>B.</b> Schematic illustration of the microfluidic device. Three parallel channels are patterned on a 1-mm thick agarose gel membrane. A stable linear gradient is generated across the center channel by flowing solutions of chemokine and buffer through the source and the sink channels respectively. A mixture of cells (1 million cells/ml) embedded in type I collagen (1.5 mg/ml) is seeded in the center channel. All three channels are 400 µm wide and 250 µm deep, and the ridges between the channels are 250 µm wide. <b>C.</b> 3D reconstruction of a z-stack of 65 images (5 µm each) of the cell-embedded collagen matrix viewed from x-y plane (top view) and the x-z plane (side view); scale bar, 50 µm. <b>D.</b> Cell trajectory plots (60 cells each) from the four conditions indicated. In the last panel, the uniform 0.25 nM EGF is generated by supplying 0.25 nM EGF solutions along the two side channels. Each colored line represents one cell trajectory tracked in 16 h.</p

Intravital immunofluorescence reveals different migratory behavior between leukocyte subsets in the skin.

<p>(<b>a</b>) Bone marrow-derived dendritic cells (BMDCs), isolated from the EGFP mouse and matured with LPS, were overlaid onto the Lyve1-α-rabbit Alexa 488 (red) stained ear dermis, and after 30 min the tissue was imaged for 3 hours. BMDCs were tracked (example tracks shown in white); (i) and (ii) show sequential images of two indicated areas where BMDCs (arrows, with yellow tracks shown at right) were observed entering Lyve1⁺ initial lymphatic vessels. (<b>b</b>) After staining for collagen IV (streptavidin-Alexa Pacific Blue, red) and CD45 (Alexa 596, green), the ear skin was stimulated with CVF-treated serum (anaphylatoxins) and imaged. Left, arrowhead points to pericyte imprint, indicative of collecting lymphatic vessels. Migrating CD45⁺ cells, most likely dermal dendritic cells (DDCs), were tracked (white). Insets (i) and (ii) show DDCs (arrows) entering collecting lymphatic vessels, with their migratory tracks (yellow) shown at right. (<b>c</b>) <i>Ex vivo</i> isolated peritoneal macrophages were stimulated with LPS and stained for CD11b-Alexa 594 before overlaying onto PECAM-1-Alexa 488 stained dermis. After 30 min, the tissue was imaged for 2 hours. Inset area (white box, left; sequential images, right) highlights the slow speed of migration of these cells compared to the DCs. (<b>d</b>) Quantification of cell migration speeds in (a)–(c). BMDCs and DDCs migrated with roughly the same speeds while macrophages remained immotile; since BMDCs were 5 times larger than intrinsic DDCs, their relative speed of migration (speed divided by cell diameter) was 5 times slower (p<0.0001). Scale bars, 50 µm.</p

Leukocyte migration and extravasation events in anaphylatoxin-stimulated tissue can be imaged with intravital immunofluorescence (IF).

<p>(<b>a</b>) Sequential images of the ear dermis of a wild-type mouse, stained for collagen IV (red) and Lyve1 (white) and transfused with blood from an EGFP mouse. Tissue stimulated with cobra venom factor (CVF, an anaphylatoxin)-treated serum caused leukocytes (arrows) to roll within a venule (arrowhead), transmigrate across venules, and migrate within the tissue. (<b>b</b>) Sequential images show the extent to which stimulation with serum and CVF (anaphylatoxins, bottom row) drives massive extravasation of leukocytes from blood vessels, compared to that under control conditions (top row). (<b>c</b>) Quantification of tracked leukocytes does not only show 15 times increase in leukocytes extravasation after CVF treatment, but also that their migration speeds were significantly faster. (<b>d</b>) Sequential images from an EGFP-chimeric mouse showing number of leukocytes entering CCL21-zone with a single leukocyte potentially entering pre-lymphatic collecting vessels and then stopping at the basement membrane for 8 minutes (6 to 14 min), squeezing its cytoplasm at the level of the CCL21-positive pre-collecting lymphatic vessel (9 to 14 min), and subsequently gaining the rounded-cell morphology (17 min). Arrowheads point to front and tail of the potentially transmigrating cell. Arrows point to cells that are crawling over basement membrane of the collecting lymphatic vessel. Tissue was stimulated with anaphylatoxins from CVF treated serum. Squares in image at 0 min represent example regions of interests (ROIs) that were placed onto either a CCL21-positive zone (i) or in a control zone (ii, dashed square) and used to calculate the relative density of cells entering CCL21 and control zones. (<b>e</b>) Leukocytes preferentially migrated towards intrinsic CCL21 immunolabeled depots. Cross correlation intensity from 6 pairs of sequential images increases in CCL21-ROIs and it is significantly different from control-ROIs, with p-value declining from 0.13 (15 min), 0.03 (30 min), to 0.008 (45 min). Error bars represent standard deviation. In: b–e: collagen IV-streptavidin Alexa 647, CCL21-Alexa 488. A–c, blood from EGFP mouse was transfused to WT after the tissue was stained with antibodies as indicated; d and e, bone marrow from EGFP mouse was transplanted to WT mouse two months before the experiment. Detection reagents: collagen IV-streptavidin-Pacific Blue, CCL21-Alexa 594, white-Lyve1-Alexa 488. Scale bars, 100 µm.</p

Monocytes internalize nanoparticles via macropinocytosis while B and T cell associate externally.

<p>(<b>a</b>) Representative flow cytometry plots of <i>in vivo</i> NP-Dy649⁺ uptake kinetics after intradermal administration: monocytes (CD11b⁺GR1^midSSC^lowF4/80⁺) and B cells (B220⁺) in the spleen. (<b>b</b>) Characteristic flow cytometry plots of biotinylated nanoparticle (NP-biotin) association with splenic (B, CD4, and CD8 cells) and bone marrow (CD11b⁺Ly6c⁺ and CD11b⁺Ly6g⁺) cells after 12 h incubation <i>in vitro</i>. To distinguish surface-associated- from internalized-NPs, cells were incubated before permeabilization with streptavidin-A488 (for extracellular association) and after permeabilization with streptavidin-A647 (for intracellular uptake). (<b>c</b>) Percentage of fluorescently labeled NPs (NP-Dy649) taken up by bone marrow cells as a function of the PI3K inhibitor (Ly294002) concentration. Bone marrow cells were incubated with increasing concentrations of Ly294002 (maximum 50 µM) for 45′ prior to the addition of NP-Dy649 for 12 h. Cells were subsequently stained and analyzed by flow cytometry. Open circles: CD11b⁺Ly6c⁺, filled squares: CD11b⁺Ly6g⁺, continuous line: vehicle control (VH, DMSO) for CD11b⁺Ly6c⁺, dashed line: VH for CD11b⁺Ly6g⁺.</p

Nanoparticles are taken up by MDSCs in draining nodes, spleen and tumor.

<p>Mice were inoculated subcutaneously with 10⁶ E.G7-OVA thymoma cells underneath the left shoulder blade (dorsoanterior left lateral side). After tumors reached 100 mm³, mice were injected with Dy649-labeled nanoparticles (NPs). Flow cytometry plots illustrating targeting of (<b>a</b>) monocytic (MO) MDSCs and (<b>b</b>) polymorphonuclear (PMN) MDSCs in the tumor draining lymph node (TDLN), the spleen and the tumors. (<b>c</b>) Three-dimensional flow-cytometry representation of the MDSC compartment (MO and PMN) of the tumor. Comparison between different organs of interest of the (<b>d</b>) MO-MDSCs and (<b>e</b>) PMN-MDSCs subpopulation accumulating NPs. One-way ANOVA followed by Bonferroni post test. n = 3 *<i>p</i>≤0.05, **<i>p</i>≤0.01. Tu: Tumor; Sp: Spleen.</p