Research ArticleIMMUNOLOGY

Targeting pulmonary tumor microenvironment with CXCR4-inhibiting nanocomplex to enhance anti–PD-L1 immunotherapy

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Science Advances  15 May 2020:
Vol. 6, no. 20, eaaz9240
DOI: 10.1126/sciadv.aaz9240
  • Fig. 1 Preparation and immune activation mechanism of FX/siPD-L1@HP.

    (A) Preparation of FX/siPD-L1@HP. PTX was encapsulated in HSA to obtain HP as a core of the nanocomplex and FX was wrapped on the outer layer of HP forming FX@HP. The final preparation FX/siPD-L1@HP is formed by electrostatic adsorption of siRNA. (B) FX/siPD-L1@HP was administered by pulmonary delivery and modulates TME to enhance anti–PD-L1 immunotherapy through three pathways. First, FX/siPD-L1@HP can decrease α-SMA and collagen expression to attenuate fibrosis in tumors, which will facilitate T cell infiltration. Second, low dosage of PTX can expose the CRT proteins on the surface of tumor cells, thus promoting the maturation and antigen presentation of DCs. Third, the released PTX and CXCR4 antagonism effect can reduce the MDSCs and Tregs, thereby relieving the immunosuppressive state in the TME.

  • Fig. 2 Physicochemical characterization of the nanoparticles.

    (A) TEM image of FX/siRNA@HP. (B) Hydrodynamic size and (C) zeta potential of the nanocomplex. (D) siRNA condensation by the nanocomplex and release of siRNA from the nanocomplex treated with GSH. (E) In vitro cumulative release profiles of PTX released from the nanocomplex. (F) Cell uptake determined by flow cytometry at 4 hours after incubation with the FAM-siRNA nanocomplex in LLC cells.

  • Fig. 3 In vitro immune activation.

    (A) CXCR4 antagonism ability of the nanocomplex relative to AMD3100. Data were analyzed with unpaired t test (****P < 0.0001 versus PEI). (B) PD-L1 gene silence by the nanocomplex (100 nM siPD-L1) in vitro. (C) Expression of CRT on LLC cells treated with PBS, free PTX, PEI/siPD-L1, FX/siScr@HP, FX/siPD-L1, and FX/siPD-L1@HP (100 nM PTX and 100 nM siPD-L1). (D) Flow cytometry assay of CRT expression on LLC cells after different treatments. Expression of (E) CD86 and (F) CD40 of CD11c+ BMDCs after coculturing with LLC cells with different treatments.

  • Fig. 4 Biodistribution and antitumor efficacy of the nanocomplex in orthotopic LLC in vivo.

    (A) In vivo fluorescence imaging at different times after pulmonary delivery of the nanocomplex. (B) Ex vivo fluorescence images of major organs at 1, 12, and 24 hours. From left to right, heart, liver, spleen, lung, kidney, and intestines. (C) Organ distribution of siRNA-Cy5 based on ex vivo fluorescence intensity. (D) CXCR4 expression in LLC on day 8. (E) Schematic illustrating the in vivo treatments of the LLC-bearing mice. (F) H&E assay of the lung tumor after different treatments on day 16. (G) Mice were treated with the nanocomplex on days 8, 10, 12, 14, 16, and 18, and survivals were monitored to day 60 (n = 8). Data were analyzed with log-rank test: FX/siPD-L1 versus PEI/siPD-L1 (P = 0.0040); FX/siPD-L1@HP versus PEI/siPD-L1 (P < 0.0001).

  • Fig. 5 Antitumor efficacy of the nanocomplex in LMBC in vivo.

    (A) Schematic illustrating the in vivo treatments of the LMBC-bearing mice. (B) In vivo bioluminescence imaging of the mice bearing LMBC after different treatments. (C) H&E assay of the harvested lungs on day 16 (scale bar, 1 mm). (D) Representative photographs of the lungs after different treatments on day 16. (1) Untreated, (2) PEI/siPD-L1, (3) FX/siScr@HP, (4) FX/siPD-L1, and (5) FX/siPD-L1@HP. (E) Survival curves after different treatments (n = 8). Data were analyzed with log-rank test. Median survival: untreated (20 days), PEI/siPD-L1 (27.5 days), FX/siScr@HP (31 days), FX/siPD-L1 (43.5 days), and FX/siPD-L1@HP (53 days).

  • Fig. 6 Antitumor mechanism in vivo.

    (A) After implantation of LLC cells, the treatments were given on days 8, 10, 12, and 14, and the mice were euthanized to collect the tumors on day 16 for further analysis. (B) CRT exposure of LLC cells. (C and D) Percentages and representative flow cytometry plots of CD4+ and CD8+ T cells in tumors. (E) α-SMA immunofluorescence staining and picrosirius red staining of the collected tumors after different treatments (scale bars, 100 μm). (F and G) Percentages and representative flow cytometry plots of Tregs in tumors. (H and I) Percentages and representative flow cytometry plots of MDSCs in tumors. (J) IFN-γ and (K) TNFα expression detected by ELISA. (L) IL-10 and (M) TGF-β expression analyzed by immunofluorescence staining. The IL-10 expression and TGF-β expression of the untreated group were set as 100%. Data were analyzed with unpaired t test, *P < 0.05, **P < 0.01, ***P < 0.001, ns. not significant.

Supplementary Materials

  • Supplementary Materials

    Targeting pulmonary tumor microenvironment with CXCR4-inhibiting nanocomplex to enhance anti–PD-L1 immunotherapy

    Zhaoting Li, Yixin Wang, Yuexin Shen, Chenggen Qian, David Oupicky, Minjie Sun

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