CCL9/CCR1 axis-driven chemotactic nanovesicles for attenuating metastasis of SMAD4-deficient colorectal cancer by trapping TGF- β

Abstract

SMAD4 deficiency in colorectal cancer (CRC) is highly correlated with liver metastasis and high mortality, yet there are few effective precision therapies available. Here, we show that CCR1⁺-granulocytic myeloid-derived suppressor cells (G-MDSCs) are highly infiltrated in SMAD4-deficient CRC via CCL15/CCR1 and CCL9/CCR1 axis in clinical specimens and mouse models, respectively. The excessive TGF-β, secreted by tumor-infiltrated CCR1⁺-G-MDSCs, suppresses the immune response of cytotoxic T lymphocytes (CTLs), thus facilitating metastasis. Hereby, we develop engineered nanovesicles displaying CCR1 and TGFBR2 molecules (C/T-NVs) to chemotactically target the tumor driven by CCL9/CCR1 axis and trap TGF-β through TGF-β-TGFBR2 specific binding. Chemotactic C/T-NVs counteract CCR1⁺-G-MDSC infiltration through competitive responding CCL9/CCR1 axis. C/T-NVs-induced intratumoral TGF-β exhaustion alleviates the TGF-β-suppressed immune response of CTLs. Collectively, C/T-NVs attenuate liver metastasis of SMAD4-deficient CRC. In further exploration, high expression of programmed cell death ligand-1 (PD-L1) is observed in clinical specimens of SMAD4-deficient CRC. Combining C/T-NVs with anti-PD-L1 antibody (aPD-L1) induces tertiary lymphoid structure formation with sustained activation of CTLs, CXCL13⁺-CD4⁺ T, CXCR5⁺-CD20⁺ B cells, and enhanced secretion of cytotoxic cytokine interleukin-21 and IFN-γ around tumors, thus eradicating metastatic foci. Our strategy elicits pleiotropic antimetastatic immunity, paving the way for nanovesicle-mediated precision immunotherapy in SMAD4-deficient CRC.

Keywords: CCR1; Chemotactic nanovesicles; G-MDSCs; Metastasis; SMAD4-deficient CRC; TGF-β; TGFBR2; Tertiary lymphoid structures.

Graphical abstract

Scheme 1

Schematic illustration of chemotactic C/T-NVs as a pleiotropic therapeutic strategy to eliminate metastasis SMAD4-deficient colorectal cancer. (A) SMAD4-deficient colorectal cancer cells ectopically express chemokine CCL9 and recruit CCR1⁺-G-MDSCs to metastatic foci via the CCL9/CCR1 axis in the mouse model. CCR1⁺-G-MDSCs produce excessive abundant TGF-β which directly inhibits the activity of CTLs and promotes metastasis. (B) C/T-NVs chemotactically target metastatic foci driven by the CCL9/CCR1 axis and counteract the accumulation of CCR1⁺-G-MDSCs, thus suppressing CCR1⁺-G-MDSCs-derived TGF-β. Tumor-infiltrated C/T-NVs trap excessive and essential TGF-β, thus leading to the activation of CTLs and attenuating metastasis of SMAD4-deficient CRC. (C) The combination of C/T-NVs and aPD-L1 facilitates the formation of TLSs around metastatic foci, contributing to the sustained activation of CTLs and release of cytotoxic IFN-γ and GzmB, and eradicates SMAD4-deficient CRC.

Figure 1

CCR1⁺-G-MDSCs accumulate to SMAD4-deficient CRC tissues via the CCL15/CCR1 axis and CCL9/CCR1 axis in clinical specimens and mouse models. (A) Clinical specimens of human CRC were examined using immunohistochemistry staining for SMAD4, CCR1 and CD33, and (B) representative images of indicated specimens. Scale bar = 100 μm. (C) Immunohistochemistry analysis of human CRC specimens showing the correlations of SMAD4 and CCR1 expression. Pearson correlation analysis was used to indicate correlation. (D) Fluorescence Activated Cell Sorter (FACS) analysis of the percent of MDSCs in PBMC from healthy subjects and clinical patients with CRC. (E) Representative FACS plots of CD33⁺ CCR1⁺ in CD11b⁺ HLA-DR^– cells. (F) Quantification of CCL15 concentration in plasma from healthy subjects and clinical patients with CRC. (G) Representative FACS plots and (H) quantification of the percent of CD14^– CD15⁺ (G-MDSCs) in MDSCs, respectively. G: G-MDSCs, M: MDSCs. (I) MC38 or CT26 cells were injected into spleens of C57BL/6 or BALB/c mice respectively. Mice were reared for 14 days and sacrificed for analysis of liver metastasis, n = 4. (J) Representative FACS plots of MDSCs in CD11b⁺, CCR1⁺ in MDSCs from MC38 and CT26 metastasis tissues, respectively. (K) Liver metastasis from mice was stained for CD11b (green) and Ly6G/Ly6C (red). Inset shows invasion front of liver metastasis. Scale bar = 100 μm, scale bar for zoom out = 30 μm. (L) Quantification of the percent of G-MDSCs in CD11b⁺, n = 4, from MC38 and CT26 metastasis tissues, respectively. (M) Immunohistochemistry staining for CCL9 and CCR1 from MC38 and CT26 metastasis tissues, Scale bar = 100 μm, scale bar for zoom out = 20 μm. (N) Quantification of CCL9 concentration in tumors, n = 4. (O) Schematic summary showing SMAD4-deficient CRC recruited CCR1⁺-G-MDSC via the CCL9/CCR1 axis. Data are presented as mean ± SD of at least two independent experiments. Two-tailed unpaired Student's t-test (H, L, N), and Brown-Forsythe and Welch ANOVA tests were used (D, F) for statistical analysis with calculated P values shown.

Figure 2

G-MDSCs-derived excessive TGF-β promotes malignant liver metastasis of SMAD4-deficient CRC. (A) MC38 or CT26 cells were injected into spleens of C57BL/6 or BALB/c mice, respectively. Mice received intraperitoneal (i.p.) injection of anti-Gr1 antibody, lgG2b isotype control antibody (200 μg/dose) or vehicle control. Mice were reared for 14 days and sacrificed for analysis of liver metastasis, n = 3. (B) Quantification of liver weight, n = 3. (C) FACS analysis and quantification of the percent of MDSCs in live cells from MC38 and CT26 metastasis tissues, n = 3. (D) Representative FACS plots of MDSCs in CD11b⁺. (E) Quantification of TGF-β1 concentration in tumors from MC38 and CT26 metastasis tissues, n = 3. (F) Immunohistochemistry staining for TGF-β1 in metastatic tissues. Scale bar = 100 μm, scale bar for zoom out = 30 μm. (G) Representative FACS plots of sorted G-MDSCs, M-MDSCs and ctr-cells. (H) Relative Tgfb1 mRNA level in indicated sorted cells, n = 3. (I) MC38 cells were injected into the spleens of C57BL/6 mice. Mice received galunisertib (i.g.,75 mg/kg) or vehicle control daily for 7 days. Mice were reared for 7 days and sacrificed for analysis of liver metastasis, n = 3. (J) Quantification of liver weight, n = 3. Gal: galunisertib. (K) Immunohistochemistry staining for TGF-β1, p-SMAD2 and CD3 of liver metastasis tissues. Scale bar = 100 μm. (L) FACS analysis and quantification of the percent of CD3⁺ T cells in live cells, (M) CD69⁺ in CD8⁺ and (N) granzyme B (GzmB⁺) in CD8⁺ T cells from liver metastatic tissues, respectively, n = 3. (O) Schematic summary of CCR1⁺-G-MDSC-derived TGF-β promoting malignant liver metastasis of SMAD4-deficient CRC. Data are presented as mean ± SD of at least two independent experiments. Two-tailed unpaired Student's t-test (J, L, M, N) and Ordinary one-way ANOVA with a Tukey's multiple comparisons test (B, C, E, H) were used for statistical analysis with calculated P values shown.

Figure 3

Characterization of CCR1/TGFBR2-engineered NVs. (A) Schematic illustration of the construction of CCR1/TGFBR2-engineered nanovesicles (C/T-NVs). (B) Immunofluorescence staining for CCR1 and TGFBR2 in indicated MC38 stable cell lines. MC38-C: MC38-CCR1 stable cells, MC38-T: MC38-TGFBR2 stable cells, MC38-C/T: MC38-CCR1/TGFBR2 stable cells. Scale bar = 10 μm. (C) Western blot analysis of CCR1 or TGFBR2 in indicated MC38 stable cells. (D) Western blot analysis of stable cells and derived NVs. 1: MC38-ctr and derived ctr-NVs. 2: MC38-T and derived T-NVs. 3: MC38-C and derived C-NVs. 4: MC38-C/T and derived C/T-NVs. (E) Representative nanoparticle tracking analysis (NTA) of size distributions for indicated NVs sample and (F) quantification of corresponding size, n = 3. (G) Zeta potential of the engineered NVs, n = 3. (H) Representative TEM images of indicated NVs. Scale bar = 200 nm. (I) Representative TEM images of immunogold labeling of CCR1 and TGFBR2 molecules (10 nm nanogold particles) in indicated NVs. Scale bar = 200 nm. (J) The ratio of the CCR1 and (K) TGFBR2 on engineered NVs, n = 3. The protein level of CCR1 in ctr-NVs and T-NVs was below the detection limit of the ELISA kit, so it was not shown. Data are presented as mean ± SD.

Figure 4

C/T-NVs chemotactically target SMAD4-deficient tumor cells and trap TGF-β molecules. (A) Transwell to investigate chemotactic migration of CCR1⁺ cells to CCL9. (B) Migratory cells on the slide were stained and photographed. CS, cultured supernatant. (C) Transwell to investigate the chemotaxis of CCR1⁺ NVs to SMAD4-deficient cells. (D) DiO-NVs (green) and MC38 cells were stained with DAPI (blue) for CLSM image, Scale bar = 10 μm. (E) Quantification of the integrated density of DiO in each image analyzed by Image J, n = 5. (F) The ex vivo biodistribution of free DiR and indicated DiR-NVs in spontaneous metastasis model at 4 and 24 h post intravenous injection. He: heart, Li: liver, Sp: spleen, Lu: lung, Ki: kidney, T: tumor. (G) Quantification of average DiR fluorescent signal in isolated liver metastasis sections and splenic tumors, n = 4. (H) Immunofluorescence image of PKH26-NVs biodistribution in tumor tissue at 24 h post intravenous injection. Scale bar = 100 μm. (I) Transwell to investigate the counteracting effect of CCR1⁺ NVs on the chemotactic migration of CCR1⁺ cells. (J) Migratory CCR1⁺ cells and (K) sorted intratumoral CCR1⁺ MDSCs, respectively were stained and photographed. (L) Schematic diagram of TGFBR2⁺ NVs trapping TGF-β1 molecules. (M) Representative TEM images of trapped TGF-β1 molecular (10 nm immunogold labeling) on indicated NVs. Scale bar = 200 nm. (N) Quantification of residual TGF-β1 in supernatant, n = 3. (O) Western blot analysis of MC38 cells treated with SB431542 or 5E09 count NVs. 1: ctr-NVs. 2: T-NVs. 3: C-NVs. 4: C/T-NVs. Data are presented as mean ± SD of at least two independent experiments. Ordinary one-way ANOVA with a Tukey's multiple comparisons test (E, G, N) was used for statistical analysis with calculated P values shown.

Figure 5

C/T-NVs attenuate liver metastasis of SMAD4-deficient CRC. (A) MC38-Luc cells were injected into the spleens of C57BL/6 mice. Mice received 5E11 count of indicated NVs and HBSS vehicle (i.v.), respectively. In vivo bioluminescence images of MC38-Luc metastases in indicated groups at indicated time point and (B) quantification of liver photon flux of each mouse, n = 4. (C) MC38 cells were injected into the spleens of C57BL/6 mice. Mice received 5E11 count of indicated NVs and HBSS vehicle (i.v.), respectively. Mice were reared for 14 days and sacrificed for analysis of liver metastasis, n = 4. (D) Quantification of the liver weight, n = 4. (E) Overall survival time of mice in indicated groups, n = 12. (F) Quantification of CCL9 concentration in tumors with indicated NVs treatment, n = 3. (G) FACS analysis and quantification of the percent of MDSCs in CD11b⁺, (H) G-MDSCs in MDSCs in metastatic tumors, respectively, n = 3. (I) Representative FACS plots of G and H. (J) Quantification of TGF-β1 concentration in tumors with indicated NVs treatment, n = 3. (K) FACS analysis and quantification of the percent of CD3⁺ in live cells in liver metastasis, n = 3. (L) Immunohistochemistry staining for TGF-β1 and CD3 of liver metastasis. Inset shows invasion front of liver metastasis. Scale bar = 100 μm, scale bar for zoom out = 30 μm. (M) FACS analysis and quantification of the percent of CD69⁺ and (N) granzyme B (GzmB⁺) in CD8⁺ cells in liver metastasis, respectively, n = 3. Data are presented as mean ± SD of at least two independent experiments. Ordinary one-way ANOVA with a Tukey's multiple comparisons test (D, F, G, H, J, K, M, N) was used for statistical analysis with calculated P values shown.

Figure 6

The combination of C/T-NVs and aPD-L1 facilitates the formation of tertiary lymphoid structures around tumors and eradicates metastatic foci. (A) Immunohistochemistry staining for SMAD4, TGF-β1 and PD-L1 of clinical specimens of human primary CRC with SMAD4 deficiency or proficiency. Scale bar = 100 μm. (B) Pearson correlation between the SMAD4 intensity and PD-L1 intensity in immunohistochemistry image of human CRC. (C) MC38 cells were injected into the spleens of C57BL/6 mice. Mice were treated with HBSS vehicle, ctr-NVs, ctr-NVs + aPD-L1, C/T-NVs, and C/T-NVs + aPD-L1. Mice were reared for 14 days and sacrificed for analysis of liver metastasis, n = 4. (D) Illustration of indicated groups for the following figures and quantification of liver weight, n = 4. (E) Representative FACS plots of the percent of MDSCs and G-MDSCs in liver metastasis. (F) Quantification of TGF-β1 concentration in tumors with indicated treatment, n = 3. (G) FACS analysis and quantification of the percent of CD3⁺ in live cells, (H) CD8⁺ in live cells, granzyme B (GzmB⁺) in CD8⁺, (I) CD4⁺ in live cells, CD69⁺ in CD4⁺, (J) B220⁺ B in live cells and MHC-II⁺, CD80⁺ in B220⁺, respectively, n = 3. (K) Immunohistochemistry staining for CD3 and CD20 of liver metastasis. Black insets show invasion front of liver metastasis. Red insets and arrows show the necrosis area. Scale bar = 100 μm. (L) Relative mRNA level of Cxcl13, (M) Cxcr5 in liver metastasis with indicated treatment, respectively, n = 3. (N) Representative images of immunohistochemistry staining for CD3, CD20, CD8, CD4, Ki67, IFN-γ and immunofluorescence staining for CXCR5, CXCL13 for analysis of the formation of TLS. Red arrows show TLSs. Scale bar = 100 μm. (O) Relative mRNA level of ll21, (P) lfng in liver metastasis with indicated treatment, respectively, n = 3. Data are presented as mean ± SD of at least two independent experiments. Ordinary one-way ANOVA with a Tukey's multiple comparisons test (D, F, G, H, I, J, I, M, O, P) was used for statistical analysis with calculated P values shown.