Extracellular Silica Nanomatrices Promote In Vitro Maturation of Anti-tumor Dendritic Cells via Activation of Focal Adhesion Kinase

Abstract

The efficacy of dendritic cell (DC)-based cancer vaccines is critically determined by the functionalities of in vitro maturated DCs. The maturation of DCs typically relies on chemicals that are cytotoxic or hinder the ability of DCs to efficiently activate the antigen-specific cytotoxic T-lymphocytes (CTLs) against tumors. Herein, the maturation chemicals are replaced with extracellular silica nanomatrices, fabricated by glancing angle deposition, to promote in vitro maturation of murine bone marrow-derived DCs (mBMDCs). The extracellular nanomatrices composed of silica nanozigzags (NZs) enable the generation of mature mBMDCs with upregulated levels of co-stimulatory molecules, C-C chemokine receptor type-7, X-C motif chemokine recetpor-1, DC-specific ICAM-3 grabbing nonintegrin, and enhanced endocytic capacity. The in vitro maturation is partially governed by focal adhesion kinase (FAK) that is mechanically activated in the curved cell adhesions formed at the DC-NZ interfaces. The NZ-maturated mBMDCs can prime the antigen-specific CTLs into programmed cell death protein-1 (PD-1)^lowCD44^high memory phenotypes in vitro and suppress the growth of tumors in vivo. Meanwhile, the NZ-mediated beneficial effects are also observed in human monocyte-derived DCs. This work demonstrates that the silica NZs promote the anti-tumor capacity of in vitro maturated DCs via the mechanoactivation of FAK, supporting the potential of silica NZs being a promising biomaterial for cancer immunotherapy.

Keywords: cancer immunotherapy; dendritic cells; extracellular nanomatrices; focal adhesion kinase; glancing angle deposition.

Figure 1

Schematic illustration of the effects of silica nanozigzags (NZs) on enhancing the in vitro maturation of anti‐tumor dendritic cells (DCs) via the activation of focal adhesion kinase (FAK). Silica NZs (with a pitch (P) of 245 nm and the number pitch (N) of 3, i.e., P245‐N3) promote the maturation of DCs into a phenotype that can potentially enhance their uptake of antigens, activation of cytotoxic T‐lymphocytes (CTLs) and homing to lymph nodes, via the mechanoactivation of FAK initiated by the formation of curved cell adhesions at the DC‐NZ interfaces. The NZ‐maturated murine bone marrow‐derived DCs (mBMDCs) can activate the antigen‐specific CTLs and program them into the PD‐1^lowCD44^high effector memory CTLs in vitro. Injection of the NZ‐maturated mBMDCs suppresses the growth of tumors in vivo. The silica NZs are also applicable to the in vitro maturation of human monocyte‐derived DCs (Hu‐moDCs).

Figure 2

The NZ‐mediated maturation of mBMDCs is modulated with an engineering of NZ nanostructures. A) Schematics of experimental timeline to characterize the properties of mBMDCs maturated with the silica nanomatrices composed of different nanostructures. The mBMDCs attached to the tissue culture polystyrene (TCPS), without any maturation treatment, served as the negative control group. B) Schematic illustration of three‐pitch silica NZs composed of controllable pitches (with a P of 165 and 245 nm, i.e., NZs‐P165‐N3 and NZs‐P245‐N3). C) Representative histograms, and the geometric mean fluorescence intensity (MFI) of D) CD86, E) DC‐SIGN, and F) XCR1 in the CD11c⁺ MHC‐II⁺ mBMDCs maturated with the three‐pitch silica NZs made of different P (with the number of independent trials (n) of 4). G) Schematic illustration of silica NZs composed of P of 245 nm and the controllable number of pitch (N of 2, 3, 4, and 5). H) Representative histograms, and I) the geometric MFI of CD86, J) DC‐SIGN, and K) XCR1 in the CD11c⁺ MHC‐II⁺ mBMDCs maturated with the silica NZs (P of 245 nm) with N of 2–5 (n = 4). The lines on histograms indicate the peaks in histograms of TCPS‐attached mBMDCs. The error bars represent the standard error of the mean. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001, one‐way ANOVA with Tukey's HSD post‐hoc test.

Figure 3

Silica NZs‐P245‐N3 promotes the maturation of XCR1⁺DC‐SIGN⁺ mBMDCs, with an enhancement of endocytic capacity and production of pro‐inflammatory cytokines. A) Scanning electron microscopy (SEM) cross‐sectional image of the silica NZs (with a P of 245 nm and three pitches, i.e., NZs‐P245‐N3) perpendicularly protruding on a supporting substrate. Insets: (upper left) SEM image of an individual NZ (scale bar: 250 nm); (upper right) SEM top‐down image of the NZs. B) Schematics of experimental timeline to characterize the properties of mBMDCs maturated with the silica NZs or LPS. The mBMDCs attached to the TCPS, without any maturation treatment, served as the negative control group. C) Representative histograms, and D) the percentage of CD86⁺, E) CCR7⁺, F) XCR1⁺, G) DC‐SIGN⁺, and H) CD206⁺ cells in the CD11c⁺ MHC‐II⁺ mBMDC subset after the 20 h maturation period (D: n = 6, E‐G: n = 4, H: n = 3). I–L) Representative confocal images (I, K, yellow lines indicate the outlines of cells defined by the staining of F‐actin; scale bars: I, 20 µm; K, 10 µm), and J) the quantifications of fluorescence levels of intracellular dextran or L) microspheres in the maturated mBMDCs after a 15 min treatment of AF647‐conjugated dextran or dark‐red fluorescent microspheres. At least 200 cells were measured in each group per trial, and at least 1300 cells were measured in each group for five independent trials (n = 5). The error bars represent the standard error of the mean. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001, (D,E,G,H,J,L) one‐way ANOVA with Tukey's HSD post‐hoc test, (F) Welch's ANOVA with Dunnett's T3 post‐hoc test.

Figure 4

Characterization of cell adhesions at the DC‐NZ interfaces, with an activation of FAK. After being seeded on the silica NZs‐P245‐N3 for 20 h, the mBMDCs were fixed and visualized using SEM and confocal fluorescence microscopy. A) Representative SEM images show an attachment of mBMDCs on the silica NZs. B) An enlarged view of the area indicated with the black box in (A), where the cell membrane is shaded in a pale blue color. The white arrows show the locations where the silica NZs are in contact with the basal plasma membrane of mBMDC. The red dotted lines indicate the outline of a silica NZ. C) Representative maximum‐intensity projection (MIP) of deconvoluted confocal z‐stack shows the CellTracker Deep Red dye‐stained silica NZs (red) and wheat germ agglutinin‐stained plasma membrane (green) of NZ‐maturated mBMDCs. D) Orthogonal views in the XZ plane labeled with the dotted line in (C). E) Representative MIPs of deconvoluted confocal z‐stacks show the CellTracker Deep Red dye‐stained silica NZs (green), immunostaining of vinculin (magenta), and phalloidin‐stained F‐actin (blue) in the basal plasma membrane of NZ‐maturated mBMDCs. Inset: a schematic illustrates the formation of cell adhesions (magenta) at the interfaces between the DC (blue) and silica NZs (green). F) Fluorescence intensity profiles of the silica NZs, vinculin, and F‐actin across the white arrow labeled on the enlarged merge view in (E). G) Individual slices in the z‐stack of an enlarged view indicated by the orange box in (E). The white arrowheads mark the depth of a vertical cell adhesion formed at the DC‐NZ interfaces. H) Representative confocal images show the immunostaining results of vinculin (magenta) and p‐FAK (Y397, green) at the basal plasma membranes of mBMDCs attached to TCPS and silica NZs. The yellow arrows and arrowheads indicate the podosomes and focal adhesions, respectively, in the TCPS‐attached mBMDCs. I) Quantifications of the cell spreading area and J) the percentage of cell area covered with vinculin⁺ cell adhesions in the mBMDCs attached to TCPS or silica NZs. K) Fluorescence intensity profiles of vinculin and p‐FAK across the white arrows in (H). L) Quantification of fluorescence intensity of p‐FAK in the vinculin⁺ cell adhesions of mBMDCs attached to TCPS or silica NZs. The error bars represent the standard error of the mean. ****p < 0.0001, two‐tailed Welch's t‐test, n = 305 cells for the TCPS group and n = 313 cells for the NZ group, pooled from five independent experimental trials.

Figure 5

Silica NZs promote the maturation of mBMDCs via the activation of FAK. A) Schematics of the experimental timeline to examine the role of FAK in the NZs(‐P245‐N3)‐induced maturation of mBMDCs via the application of FAK inhibitor PF‐573228 (PF). The mBMDCs attached to the TCPS, with the treatment of 0.02% DMSO, served as the vehicle control group. B) Representative Western blots and C) quantification result of the level of Tyr‐397(Y397)‐phosphorylated FAK in the mBMDCs after the 20 h maturation period (n = 5). D) Representative histograms, and E) the percentage of CD86⁺, F) CCR7⁺, G) XCR1⁺, H) DC‐SIGN⁺ cells in the CD11c⁺ MHC‐II⁺ mBMDC subset after the 20 h maturation period (n = 4). I‐L) Representative confocal images (I, K, yellow lines indicate the outlines of cells defined by the staining of F‐actin; scale bars: 10 µm), and J) the quantifications of fluorescence levels of intracellular dextran or L) microspheres in the maturated mBMDCs after a 15 min treatment of AF647‐conjugated dextran or dark‐red fluorescent microspheres. At least 200 cells were measured in each group per trial, and at least 1000 cells were measured in each group for five independent trials (n = 5). The error bars represent the standard error of the mean. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001, one‐way ANOVA with Tukey's HSD post‐hoc test.

Figure 6

The NZ‐maturated mBMDCs promote in vitro activation of antigen‐specific CTLs into the PD‐1^lowCD44^high memory phenotypes. A) Schematics of the experimental timeline to analyze the properties of antigen‐specific CTLs primed by the mBMDCs maturated with the silica NZs(‐P245‐N3) or LPS. The CTLs primed by the TCPS‐attached mBMDCs served as the negative control group. B) Representative histograms and C) the geometric MFI of IFNγ in the antigen‐specific H‐2k^b‐OVA_257‐264‐tetramer⁺ CD8⁺ CTL subsets after 4 days of DC‐CTL co‐culture and the treatment with GolgiPlug on the day of harvest (n = 4). D,F,H) Representative histograms, E) the normalized percentage of CFSE^low divided cells, G) the percentage of CD44^high cells, and I) the geometric MFI of PD‐1 in the antigen‐specific H‐2k^b‐OVA_257‐264‐tetramer⁺ CD8⁺ CTL subsets after 4 days of DC‐CTL co‐culture (E: n = 5, G, I: n = 6). The percentage of CFSE^low CTLs is normalized by mean. The lines on histograms indicate the gating for CFSE^low (D) and CD44^high (F) cells. The error bars represent the standard error of the mean. *p<0.05, **p<0.01, ***p<0.001, two‐way ANOVA with Tukey's HSD post‐hoc test.

Figure 7

The NZ‐maturated mBMDCs suppress the growth rate of tumors in vivo. A) Schematics of the experimental timeline to examine the in vivo effect of the OVA_257‐264‐pulsed mBMDCs maturated with silica NZs(‐P245‐N3) or LPS on the mice inoculated with B16‐OVA melanoma cells. The tumor‐bearing mice injected with the vehicle (HBSS) without any mBMDCs served as the sham control group. B) The Kaplan‐Meier survival curves of tumor‐bearing mice after the injection of mBMDCs (log‐rank test, p = 0.0493, sham control versus NZ‐maturated mBMDCs). C) The growth rate of tumors in individual mice. D) The summarized tumor volume of each experimental group (^*sham control versus NZ‐maturated mBMDCs, day 8: p = 0.0507, day 10: p = 0.0004, day 12: p<0.0001; ^#LPS‐maturated mBMDCs versus NZ‐maturated mBMDCs, day 10: p = 0.0673, day 12: p = 0.0123; ^†sham control versus LPS‐maturated mBMDCs, day 8: p = 0.0626, two‐way ANOVA with Tukey's HSD post‐hoc test). The error bars represent the standard error of the mean. n = 9 for the sham control group, n = 10 for the NZ‐maturated mBMDCs and LPS‐maturated mBMDCs, pooled from three independent trials.

Figure 8

Silica NZs promote the maturation of human monocyte‐derived dendritic cells (hu‐moDCs). A) Schematics of the experimental timeline to examine the properties of hu‐moDCs maturated with silica NZs(‐P245‐N3). The hu‐moDCs attached to the TCPS, without any maturation treatment, served as the negative control group. The TCPS‐attached hu‐moDCs maturated with 100 ng mL⁻¹ LPS for 20 h were used as the positive control for the validation of the assay method. B–J) Representative histograms (B,E) and C) the geometric MFI of MHC‐I, D) MHC‐II, F) CD80, G) CD86, H) CCR7, I) XCR1 and J) DC‐SIGN in the CD11c⁺ MHC‐II⁺ hu‐moDC subset after the 20 h maturation period (C, F: n = 6, D: n = 8, G: n = 5, H‐J: n = 4). The lines on histograms mark the peaks in histograms of the unpulsed TCPS‐attached hu‐moDCs. The error bars represent the standard error of the mean. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001, (TCPS‐attached versus NZ‐maturated hu‐moDCs, with or without CEA pulsing) two‐way ANOVA with Šídák's multiple comparisons test, (comparison among the hu‐moDCs without CEA pulsing) Welch's ANOVA with Dunnett's T3 post‐hoc test (C‐D, F, H), one‐way ANOVA with Tukey's HSD post‐hoc test (G, I‐J).