Selective apoptosis of tumor-associated platelets boosts the anti-metastatic potency of PD-1 blockade therapy

Abstract

Despite the transformative impact of programmed cell death protein-1 (PD-1) blockade therapy on metastatic/advanced solid tumor treatment, its efficacy is hindered by a limited response rate. Platelets play a pivotal role in tumor metastasis by shielding circulating tumor cells and secreting immunosuppressive factors. We here demonstrate that selectively inducing apoptosis in tumor-associated platelets (TAPs) using ABT-737-loaded nanoparticles (cyclic arginine-glycine-aspartate containing peptide-modified ABT-737-loaded nanoparticles [cRGD-NP@A]) enhances the anti-metastatic efficacy of the anti-PD-1 antibody (aPD-1). cRGD-NP@A specifically binds to TAPs, disrupting platelet-tumor cell interactions and exposing tumor cells to immune surveillance in vivo. Combined with aPD-1, cRGD-NP@A substantially augments immune activation and reduces TAP-derived immunosuppressive factors, notably transforming growth factor β1 (TGF-β1), consequently improving anti-metastatic outcomes across multiple metastasis-bearing animal models without observable adverse effects. Our study underscores the importance of depleting TAPs to enhance PD-1 blockade therapy, presenting a promising strategy to improve response rates and clinical outcomes for patients with metastatic cancer.

Keywords: PD-1 blockade therapy; PD-1 response improvement; platelet modulation; platelet-mediated immunosuppression; polymeric nanomedicine; tumor-associated platelets.

Graphical abstract

Figure 1

Preparation and characterization of the cRGD-NP@A nanoparticles (A) Schematic illustration of the preparation of cRGD-NP@A. (B) Representative TEM image of cRGD-NP@A. Scale bar, 100 nm. (C) Size distribution of cRGD-NP@A analyzed using dynamic light scattering. (D) In vitro drug release profile of ABT-737 from cRGD-NP@A at pH 7.4 and pH 5.0 within 48 h. Data are presented as mean ± SD, n = 3 biological replicates. See also Figures S1–S5.

Figure 2

cRGD-NP@A binding to TAPs triggers their apoptosis in vitro (A) Flow cytometry analysis of Cy5.5 fluorescence signals of resting or activated platelets after incubation with Cy5.5-labeled PEG-NP@A or cRGD-NP@A for 30 min, respectively. (B and C) Measurement of mitochondrial membrane potential (ΔΨm) depolarization (B) and PS externalization (C, presented as annexin V fluorescence fold change normalized to resting platelets without cRGD-NP@A treatment) in resting or ADP-activated platelets following treatment with cRGD-NP@A for 2 h. (D) Representative confocal microscopy images of B16-F10-stimulated or unstimulated platelets after incubation with Cy5.5-labeled PEG-NP@A or cRGD-NP@A for 10 min, respectively. Scale bar, 10 μm. (E and F) Measurement of ΔΨm depolarization (E) and PS externalization (F) in resting or B16-F10-stimulated platelets after treatment with free ABT-737, PEG-NP@A, or cRGD-NP@A, respectively. Data are presented as relative apoptotic rates of B16-F10-stimulated platelets normalized to unstimulated controls after treatment with the same concentration of ABT-737 in different formulations. Values are mean ± SD, n = 3 biological replicates. Statistical significance was analyzed using two-way ANOVA followed by Dunnett’s test. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001, ∗∗∗∗p < 0.0001. See also Figures S6–S8.

Figure 3

cRGD-NP@A accumulates in TAPs to inhibit platelet-tumor cell interaction in vivo (A and B) Ex vivo fluorescence imaging (A) and average Cy5 fluorescence intensity (B) of major organs harvested from C57BL/6N mice bearing B16-F10 pulmonary metastasis at 24 h after intravenous injection of free Cy5, Cy5-labeled PEG-NP@A, or cRGD-NP@A (n = 3 mice per group). (C) Immunofluorescence analysis of cRGD-NP@A distribution within metastatic foci 2 h post injection of Cy5-labeled PEG-NP@A or cRGD-NP@A. CD41 staining was used to visualize platelets. Scale bar, 100 μm. (D) Analysis of platelet adherence to B16-F10 tumor cells in vivo. Lungs from healthy C57BL/6N mice treated with various formulations, following co-injection of CellMask Orange-labeled B16-F10 cells and FITC-anti-CD41 antibody, were harvested for frozen section analysis. Scale bar, 50 μm. (E) Determination of circulating platelet activation level in B16-F10 lung metastasis-bearing mice after three treatments with saline, cRGD-NP, free ABT-737, and cRGD-NP@A, respectively, at an interval of three days (n = 5 mice per group). Data are presented as mean ± SD. Statistical significance was analyzed using two-way (B) or one-way (E) ANOVA with Dunnett’s test. ∗∗∗p < 0.001, ∗∗∗∗p < 0.0001; ns, not significant. See also Figures S9–S12.

Figure 4

cRGD-NP@A enhances anti-metastatic effects of aPD-1 therapy (A) Schematic illustration of the action mechanisms of cRGD-NP@A. Intravenous administration of cRGD-NP@A specifically targets TAPs in the circulation and metastatic sites. cRGD-NP@A then induces apoptosis in TAPs upon endocytosis, thereby exposing tumor cells to immune cell recognition and killing. This intervention simultaneously suppresses the secretion of TGF-β1 by TAPs, further activating immune response. Ultimately, these actions potentiate the anti-metastatic effects of concurrent aPD-1 therapy. (B) Image of lungs collected from B16-F10 metastasis-bearing mice after different treatments. (C) H&E (upper) analysis and CD41 immunofluorescence staining (lower) of lung tissue sections from mice bearing B16-F10 pulmonary metastasis. Scale bar, 2 mm for the upper panel, 50 μm for the lower panel. (D and E) Number of surface nodules (D) and metastatic area quantification (E, metastasis region/total region) of lungs harvested from B16-F10 metastasis-bearing mice (n = 5 mice per group). (F) Plasma level of TGF-β1 of B16-F10 lung metastasis-bearing mice after different treatments (n = 4 mice per group). (G) Treatment schedule for anti-metastasis evaluation in F344 rats bearing MADB106 pulmonary metastasis. (H) Representative images (upper), H&E analysis (middle), and CD41 staining (lower) of lung sections from rats bearing MADB106 metastasis. Scale bars, 5 mm for the middle panel, 100 μm for the lower panel. (I and J) Number of surface metastatic nodules (I) and metastatic area quantification (J) of lungs from MADB106 metastasis-bearing rats after different treatments. n = 4 rats for each group. Values are mean ± SD. Statistical significance was determined by one-way ANOVA with Dunnett’s test. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001, ∗∗∗∗p < 0.0001. See also Figures S13–S15.

Figure 5

cRGD-NP@A augments cytotoxic T cell response of aPD-1 therapy (A) Schematic illustration of cRGD-NP@A enhancing CTL response to aPD-1 therapy by depleting TAPs for blocking TGF-β1-mediated immunosuppression. (B) Immunofluorescence analysis of CD8⁺ T cell infiltration in B16-F10 pulmonary metastatic foci. Scale bar, 50 μm. (C and D) Flow cytometry analysis of granzyme B (C) and IFN-γ (D) secretion by CD3⁺CD8⁺ T cells in peripheral blood from B16-F10 metastasis-bearing mice at the end of treatment. n = 5 mice for each group. (E–H) Proportions of CD3⁺CD8⁺ T cells in lymphocytes (E), and Ki67⁺ (F), IFN-γ⁺ (G), and granzyme B⁺ (H) CD3⁺CD8⁺ T cells in the spleen from B16-F10 metastasis-bearing mice following various treatments. n = 5 mice for each group. (I–L) Serum levels of major pro-inflammatory cytokines in B16-F10 lung metastasis-bearing mice after different treatments. n = 4 mice for each group. Data are presented as mean ± SD. Statistical significance was determined by one-way ANOVA with Dunnett’s test. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001, ∗∗∗∗p < 0.0001. See also Figures S16 and S17.

Figure 6

Reduction of TGF-β1 level mediates the immune-enhancing effects of cRGD-NP@A on aPD-1 therapy (A) Image of lungs collected from B16-F10 metastasis-bearing mice after different treatments. (B) Number of surface nodules of lungs harvested from B16-F10 metastasis-bearing mice after different treatments. (C) Flow cytometry analysis of the proportion of CD4⁺FOXP3⁺ Tregs in peripheral blood from B16-F10 metastasis-bearing mice at the end of the experiment. (D and E) Flow cytometry analysis of NKG2D and CD107a expression on NK cells (D) and CD80 and CD86 expression on DCs (E) in peripheral blood after various treatments. (F and G) Flow cytometry analysis of IFN-γ and granzyme B secretion by CD3⁺CD8⁺ T cells in the peripheral blood (F) and spleen (G) at the end of treatment. Data are presented as mean ± SD, n = 5 mice for each group. Statistical significance was analyzed by one-way ANOVA with Dunnett’s test. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001, ∗∗∗∗p < 0.0001.