A tumor-to-lymph procedure navigated versatile gel system for combinatorial therapy against tumor recurrence and metastasis

Abstract

Application of cancer vaccines is limited due to their systemic immunotoxicity and inability to satisfy all the steps, including loading of tumor antigens, draining of antigens to lymph nodes (LNs), internalization of antigens by dendritic cells (DCs), DC maturation, and cross-presentation of antigens for T cell activation. Here, we present a combinatorial therapy, based on a α-cyclodextrin (CD)-based gel system, DOX/ICG/CpG-P-ss-M/CD, fabricated by encapsulating doxorubicin (DOX) and the photothermal agent indocyanine green (ICG). Upon irradiation, the gel system exhibited heat-responsive release of DOX and vaccine-like nanoparticles, CpG-P-ss-M, along with chemotherapy- and phototherapy-generated abundant tumor-specific antigen storage in situ. The released CpG-P-ss-M acted as a carrier adsorbed and delivered antigens to LNs, promoting the uptake of antigens by DCs and DC maturation. Notably, combined with PD-L1 blocking, the therapy effectively inhibited primary tumor growth and induced tumor-specific immune response against tumor recurrence and metastasis.

Fig. 1. LDIMP process.

(A) Fabrication of the integrated regimen and the release process of CpG-P-ss-M. (B) Simplified mechanism of CpG-P-ss-M–mediated DC maturation for cancer immunotherapy. Letters LDIMP in orange frame represent loading tumor-specific antigens by DDS, draining to LNs, internalization by DCs, DC maturation for costimulatory molecule expression, and presenting peptide–MHC-I complexes to T cells, respectively.

Fig. 2. CpG-P-ss-M elicits antigen-specific CD8⁺ CTL response by improving the LDIMP process.

(A to C) TEM images and DLS analysis of CpG-PP (A), CpG-PM (B), and CpG-P-ss-M (C). (D) Photograph of gelation process after ultrasonication. (E) Scanning electron microscopy images of PP/CD gel. (F) Frequency-dependent rheological properties of gels. (G and H) Cumulative release rate of DOX (G) and nanoparticles (NPs) (H) upon irradiation. (I) Quantification of proteins captured by nanoparticles. (J) Live images of melanoma-bearing mice after intratumoral administration. Red circles, location of subiliac LNs. (K) Ex vivo imaging of popliteal and axillary LNs collected at different times after administration. (L) Semiquantitative data of fluorescence signal from popliteal, sciatic, axillary, and accessory axillary LNs. (M and N) Percentages of CD86⁺ (M) and CD80⁺ (N) BMDCs gated by CD11c⁺. (O) Mean fluorecent intensity (MFI) of 25D1.16 signal gated by CD11c⁺CD8⁺ and (P) percentages of CD80⁺CD86⁺ gated by CD11c⁺ in BMDCs/B16-OVA multicellular spheroid cocultured system. (Q to S) Percentages of CD8⁺CTLs (Q and R) and CFSE dilution (S) in CD8⁺ T/BMDCs/B16-OVA multicellular spheroid cocultured system. *P < 0.05, **P < 0.01, ****P < 0.0001. ns, not significant. Error bars represent mean ± SD; n = 3. Photo credit (D): Lin Qin, West China School of Pharmacy, Sichuan University.

Fig. 3. Antitumor effects in the bilateral B16F10 tumor model.

(A) Schematic depicting the experimental approach. (B) Body weight of B16F10 tumor–bearing mice (n = 6). (C and D) Representative images of primary (C) and distant tumors (D) after treatment. (E and F) Primary (E) and distant (F) tumor size curves (n = 6). (G and H) Weight of primary (G) and distant tumors (H). (I) TUNEL staining of primary and distant tumors; scale bar, 50 μm. *P < 0.05, **P < 0.01, ***P < 0.001. Error bars represent mean ± SD. Photo credit (C and D): Lin Qin, West China School of Pharmacy, Sichuan University.

Fig. 4. Combinatorial therapy promoted DC maturation and induced potent CD8⁺ T cell response in vivo.

(A to C) Percentages of CD80⁺ cells, CD86⁺ cells (A and B), and CD83⁺ cells (C) gated on CD11c⁺ cells in spleens. (D) Percentages of CD11c⁺ cells in the spleen. (E and F) Percentages of CD83⁺ (E) and CD86⁺ cells (F) gated on CD11c⁺ cells in LNs. (G and H) Percentages of CD3⁺CD8⁺ T cells in the primary (G) and distant tumors (H). (I and J) Percentages of T_regs (CD3⁺CD4⁺Foxp3⁺) in the primary (I) and distant tumors (J). (K) Ratio of CD8⁺ T cells:Foxp3⁺ T_regs in primary and distant tumors. (L and M) IFN-γ (L) and TNF-α (M) levels in serum, determined by enzyme-linked immunosorbent assay (ELISA). (N and O) IFN-γ production by antigen-specific CD8⁺ CTL cells in the spleens (N) and LNs (O). *P < 0.05, **P < 0.01, ***P < 0.001. Error bars represent mean ± SD (n = 3 to 4).

Fig. 5. Combinatorial therapy suppresses postoperative tumor relapse.

(A) Schematic diagram of the postoperative model and the administration schedule. i.p. intraperitoneal; i.v., intravenous. (B) Survival curve of each group using the log-rank (Mantel-Cox) test (n = 8). (C to E) Percentages of CD8⁺MHC-I⁺ cells (C and D) and CD8⁺MHC-II⁺ cells (E) in total CD11c⁺ DCs in spleens (n = 3). (F to H) The percentage of CD8⁺ 25D1.16⁺ gated on CD11c⁺ in LNs (F and G) and spleens (H) was detected by flow cytometry (n = 3). (I and J) Percentages of CD8⁺IFN-γ⁺ cells in spleen (I) and LN (J) (n = 3). (K) IFN-γ and TNF-α levels in serum measured by ELISA kit (n = 4). (L and M) In vivo CTL response (n = 3). *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001. Error bars represent mean ± SD.

Fig. 6. Long-term immune response induced by combinatorial therapy.

(A) Male C57BL/6 mice bearing melanoma received resection after two treatments. All mice were randomly divided into three parts. Memory study in the first cohort of mice. (B and C) Macroscopic tumor nodules on the lung surface (n = 6). (D) Weight of lung tissues (n = 6). (E to G) T_EM (CD44⁺CD62L⁻ gated by CD3⁺CD8⁺) in spleens were detected by flow cytometry (n = 5). (H) Detection of tumor antigen–specific CTLs by intracellular IFN-γ staining in LNs (n = 5). (I) IFN-γ and TNF-α levels in the serum (n = 5). (J) Survival curve of (i) phosphate-buffered saline (PBS) and (ii) ICG/DOX/CpG-P-ss-M/CD + Laser + anti–PD-L1 groups (n = 12) in the second cohort. (K to O) Rechallenge study in the last cohort. (K) Survival curve of (i) naïve mice, as age-matched control + rechallenge, and (ii) ICG/DOX/CpG-P-ss-M/CD + Laser + anti–PD-L1 + rechallenge (n = 7). (L and M) Individual contralateral tumor growth curves of two groups. (N and O) Frequencies of CD3⁺CD8⁺ T cells and T_EM in the LNs (n = 4). Data in (J) and (K) were analyzed using the log-rank (Mantel-Cox) test. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001. Error bars represent mean ± SD. Photo credit (B): Lin Qin, West China School of Pharmacy, Sichuan University.