Neutrophil infiltration and whole-cell vaccine elicited by N-dihydrogalactochitosan combined with NIR phototherapy to enhance antitumor immune response and T cell immune memory

Abstract

Melanoma is one of the deadliest malignancies with a high risk of relapse and metastasis. Long-term, tumor-specific, and systemic immunity induced by local intervention is ideal for personalized cancer therapy. Laser immunotherapy (LIT), a combination of local irradiation of laser and local administration of an immunostimulant, was developed to achieve such an immunity. Although LIT showed promising efficacy on tumors, its immunological mechanism is still not understood, especially its spatio-temporal dynamics. Methods: In this study, we investigated LIT-induced immunological responses using a 980-nm laser and a novel immunostimulant, N-dihydrogalactochitosan (GC). Then we followed the functions of key immune cells spatially and temporally using intravital imaging and immunological assays. Results: Immediately after LIT, GC induced a rapid infiltration of neutrophils which ingested most GC in tumors. The cytokines released to the serum peaked at 12 h after LIT. Laser irradiations produced photothermal effects to ablate the tumor, release damage-associated molecular patterns, and generate whole-cell tumor vaccines. LIT-treated tumor-bearing mice efficiently resisted the rechallenged tumor and prevented lung metastasis. Intravital imaging of tumor at rechallenging sites in LIT-treated mice revealed that the infiltration of tumor-infiltrating lymphocytes (TILs) increased with highly active motility. Half of TILs with arrest and confined movements indicated that they had long-time interactions with tumor cells. Furthermore, LIT has synergistic effect with checkpoint blockade to improve antitumor efficacy. Conclusion: Our research revealed the important role of LIT-induced neutrophil infiltration on the in situ whole-cell vaccine-elicited antitumor immune response and long-term T cell immune memory.

Keywords: immune memory; intravital imaging; laser immunotherapy; whole-cell vaccine.

Figure 1

The treatments of subcutaneous B16 and CFP-B16 tumors. (A) Infrared (IR) thermal images of mice bearing CFP-B16 tumors under different treatments (GC + PTT, PBS + PTT, GC, or PBS). (B) Tumor temperature changes based on IR thermal imaging data in (A). Data are presented as mean ± SD (n = 3 mice). (C) Volume of B16 tumors in the mice of different treatment groups. Data are presented as mean ± SD (n = 9-10 mice, two independent experiments, PBS versus GC + PTT, *** P < 0.001, and GC versus GC + PTT, *** P < 0.001). (D) Survival rates of mice bearing B16 tumors after various treatments (9-10 mice per group). (E) Volume of CFP-B16 tumors in the mice of different treatment groups. Data are presented as mean ± SD (n = 10 mice, two independent experiments, GC + PTT versus PBS, *** P < 0.001, and GC + PTT versus GC, *** P < 0.001). (F) Survival rates of mice bearing CFP-B16 tumors after various treatments (10 mice per group). Statistical analysis was performed using the Kruskal-Wallis test followed by Dunn's multiple comparison tests and the log-rank Mantel-Cox test.

Figure 2

Immunological responses induced by LIT in the tumors, serum, and TDLNs. (A) Schematics of the procedures and timeline of GC + PTT treatment for CFP-B16 and analysis of LIT-induced antitumor immune response. (B) Proportions of neutrophils in immune cells in the treated primary tumors after various treatments at different times. (C) Proportions of neutrophils in immune cells with GC-RB (gated by CD45⁺ and RB⁺) in the tumors treated with GC-RB + PTT at different times. Data are presented as mean ± SD (n = 3-5 mice, two independent experiments). (D-F) Cytokine levels in serum (TNF-α, IL-6, and IL-1β) from mice at different times after various treatments. Data are presented as mean ± SD (n = 3-4 mice, two independent experiments). (G) HSP70 protein expression in the treated primary tumors 24 h after various treatments was analyzed using WB. (H) HSP70 and HMGB1 expressions in TDLNs at 48 h after different treatments were analyzed using WB (n = 3, two independent experiments). (I, J) The frequency of CD69⁺ in the CD4⁺ (I) and CD8⁺ (J) T cells of TDLNs 24 h after different treatments. (K) The frequency of mature DCs (CD11c⁺CD80⁺CD86⁺) in TDLNs 72 h after various treatments. Data are presented as mean ± SD (n = 4-7 mice, three independent experiments). Statistical analysis was performed using the unpaired t-test, and the one-way ANOVA test followed by the Bonferroni post-test. * P < 0.05, ** P < 0.01, *** P < 0.001, ns: not significant.

Figure 3

Long-term tumor resistance induced by LIT. (A) Schematics of the procedures and timeline of the CFP-B16 tumor rechallenge of successfully treated tumor-bearing mice and immunological assays. (B) Tumors collected from mice 24 days after tumor rechallenge. Healthy, age marched C57BL/6 mice were used as control. (C-E) Tumor growth curves for mice in different treatment groups (n = 10 mice, two independent experiments) after tumor rechallenge. (F) Frequency of effector memory T cells (T_EM) in the spleens were analyzed (CD8⁺CD62L^-CD44⁺) on day 40 after tumor treatments. Data are presented as mean ± SD (n = 7-8 mice, three independent experiments). (G) IFN-γ secretion by CD8⁺ T cells in TDLNs collected from mice 11 days after tumor rechallenge. Data are presented as mean ± SD (n = 5 mice, two independent experiments). Statistical analysis was performed using the one-way ANOVA test followed by the Bonferroni post-test. * P < 0.05, ** P < 0.01, *** P < 0.001, ns: not significant.

Figure 4

Migration of endogenous TILs in the tumor microenvironment of CXCR6-GFP mice with CFP-B16 rechallenge. (A) Schematics of the procedures and timeline of intravital imaging of endogenous TILs in the tumor microenvironment. (B) In vivo time-lapse images of endogenous GFP⁺ TILs in the CFP-B16 tumor area. Scale bar: 70 μm. (C) Quantification of the density of endogenous GFP⁺ TILs on day 11 after CFP-B16 tumor cell implantation. Data are presented as mean ± SD (n = 11-13 fields, from 3-5 mice per group). (D) Trajectories of GFP⁺ T cells in different groups, following the alignment of their starting positions. (E-G) Scatter plots of the mean velocity (E) confinement ratio (F) and arrest coefficient (G) of GFP⁺ TILs in tumor areas. Each data point represents a single cell, and the red bars indicate mean values. (H) Histograms representing the relative fraction of the different classes of interactions between TILs and tumor cells. The data from 5-7 mice, 3 independent experiments were pooled. Statistical analysis was performed using the one-way ANOVA test followed by the Bonferroni post-test, and Kruskal-Wallis test followed by Dunn's multiple comparison tests. * P < 0.05, ** P < 0.01, ***P < 0.001, ns: not significant.

Figure 5

Inhibition of lung metastasis by LIT. (A) Schematics of the procedures and timeline of LIT in the inhibition of CFP-B16 tumor lung metastasis. (B) Tumor nodules in the lungs. Lungs collected from mice of different groups 21 days after 2×10⁵ CFP-B16 tumor cells were injected through tail vein. (C) Number of metastases in the lungs of different mice groups. Data are presented as mean ± SD (n = 9-12 mice, two independent experiments). (D) H&E staining of lung tissues of different mice groups. Scale bar: 500 μm. (E-J) Cytokine levels (IFN-γ, IL-1 β, GM-CSF, IL-6, TNF-α, and IL-4) in the lungs collected from mice in different groups, and were analyzed using ELISA. Data are presented as mean ± SD (n = 3-5 mice, two independent experiments). Statistical analysis was performed using the one-way ANOVA test followed by the Bonferroni post-test. * P < 0.05, ** P < 0.01, *** P < 0.001, ns: not significant.

Figure 6

Synergistic inhibitory effect of LIT combined anti-PD-1 for distant secondary B16 tumors. (A) Schematics of the procedures and timeline of LIT combined with anti-PD-1 to treat primary and secondary B16 tumors. (B) Tumor growth curves (both flanks) in different groups. Data are presented as mean ± SD (n = 6-7 mice). (C) Percentage of PD-L1⁺ cells in the secondary tumor cells 13 days after different treatments of the first tumors. (D) Percentage of CD3⁺ T cells (TILs) in the CD45⁺ immune cells in the secondary tumors after various treatments. (E) Percentage of CD4⁺ and CD8⁺ T cells in TILs from (D). (F) Percentage of TIM3⁺ and PD-1⁺ cells in CD8⁺ T cells from (E). Data are presented as mean ± SD (n = 3-4 mice). Statistical analysis was performed using the unpaired t-test, or Mann-Whitney test (nonparametric). * P < 0.05, ** P < 0.01, *** P < 0.001, ns: not significant.

Figure 7

Schematics of LIT mediated antitumor immune response for primary, secondary, and re-challenged and metastatic melanoma. (A) Schematic illustration of laser immunotherapy (LIT) for subcutaneous primary tumor and the timelines of the immune response in the tumor, serum and TDLNs. (B) Schematic of the treatment of secondary tumors by the combination of LIT and the anti-PD-1 antibody. (C) Schematic of the antitumor mechanism of LIT in the subcutaneous re-challenged tumors and lung metastasis.