In situ chemoimmunotherapy hydrogel elicits immunogenic cell death and evokes efficient antitumor immune response

Abstract

Background: Chemoimmunotherapy has shown promising advantages of eliciting immunogenic cell death and activating anti-tumor immune responses. However, the systemic toxicity of chemotherapy and tumor immunosuppressive microenvironment limit the clinical application.

Methods: Here, an injectable sodium alginate hydrogel (ALG) loaded with nanoparticle albumin-bound-paclitaxel (Nab-PTX) and an immunostimulating agent R837 was developed for local administration. Two murine hepatocellular carcinoma and breast cancer models were established. The tumor-bearing mice received the peritumoral injection of R837/Nab-PTX/ALG once a week for two weeks. The antitumor efficacy, the immune response, and the tumor microenvironment were investigated.

Results: This chemoimmunotherapy hydrogel with sustained-release character was proven to have significant effects on killing tumor cells and inhibiting tumor growth. Peritumoral injection of our hydrogel caused little harm to normal organs and triggered a potent antitumor immune response against both hepatocellular carcinoma and breast cancer. In the tumor microenvironment, enhanced immunogenic cell death induced by the combination of Nab-PTX and R837 resulted in 3.30-fold infiltration of effector memory T cells and upregulation of 20 biological processes related to immune responses.

Conclusions: Our strategy provides a novel insight into the combination of chemotherapy and immunotherapy and has the potential for clinical translation.

Keywords: Chemoimmunotherapy; Hydrogel; In situ vaccine; Nab-PTX; TLR7 agonist.

Fig. 1

Schematic diagram of a chemoimmunotherapy hydrogel to play an anti-tumor role by reprogramming the tumor microenvironment. 1) In vitro, CaCl₂ (1.8 × 10^–3 mol L⁻¹) is added to sodium alginate solution (ALG, 20 mg mL⁻¹) mixed with nanoparticle albumin-bound-paclitaxel (Nab-PTX, 1 mg mL⁻¹) and an immunostimulating agent R837 (0.5 mg mL^–1), to form our chemoimmunotherapy hydrogel at room temperature. 2) ALG mixed with Nab-PTX and R837, will be cross-linked with the cations present in vivo to form hydrogel near the tumor. Then, some dying tumor cells release antigens due to immunogenic cell death induced by Nab-PTX. Immature DCs are activated by immunogenic tumor antigens accompanied by R837, and migrate to lymph nodes to induce sufficient effector memory T cells (T_EM), eventually increasing the proportion of T_EM in tumor microenvironment

Fig. 2

Preparation and characterization of the chemoimmunotherapy hydrogel. A Pictures of hydrogel containing sodium alginate (ALG) solution (20 mg mL⁻¹), CaCl₂ (1.8 × 10^–3 mol L⁻¹) and Cy5 (0.1 mg mL⁻¹) formed in vitro at room temperature. B Pictures of hydrogels containing sodium ALG solution (20 mg mL⁻¹) formed in vivo. C Scanning electron microscope images of blank ALG hydrogel (the scale bar is 100 µm). D Near-infrared (NIR) images of tumor-bearing ICR mice were taken on 1 h, 4 h, 12 h, 48 h, 72 h and 120 h, after peritumoral injection with pure Cy5 dye, or ALG hydrogel loaded with Cy5 (n = 3). E Relative total radiant efficiency of ICR mice after peritumoral injection with pure Cy5 dye, or ALG hydrogel loaded with Cy5 (n = 3). The error bars represented mean ± SEM

Fig. 3

In vitro function of the chemoimmunotherapy hydrogel. A Representative flow cytometry images of mature DCs (mDCs, CD11c⁺CD80⁺CD86⁺) after co-incubation with R837 (5 µg mL⁻¹), normal saline (NS, negative control), or LPS (1 µg mL⁻¹, positive control) in vitro for 48 h. B The percentage of mDCs after co-incubation with R837, NS or LPS in vitro for 48 h (n = 3). The error bars represented mean ± SEM. P-values were calculated by two-tailed unpaired Student’s t-tests. ***P = 0.0004 (R837 vs NS) 0.0001 (LPS vs NS). C Cumulative release of R837 and nanoparticle albumin-bound-paclitaxel (Nab-PTX) from sodium alginate solution (ALG) based hydrogel in vitro at 37 ℃. D Cell viability of human renal epithelial cells 293 T after co-incubation with NS, Nab-PTX (10 mg mL⁻¹), or ALG loaded with Nab-PTX (Nab-PTX/ALG) in vitro for 24 h, 48 h or 72 h respectively (n = 5). The error bars represented mean ± SEM. p-values were calculated by two-tailed unpaired Student’s t-tests. ***P = 0.0005, ****P < 0.0001. E Cell viability of mouse breast cancer cells 4T1 after co-incubation with NS, Nab-PTX (10 mg mL⁻.¹), or ALG loaded with Nab-PTX (Nab-PTX/ALG) in vitro for 24 h, 48 h or 72 h respectively (n = 5). The error bars represented mean ± SEM. p-values were calculated by two-tailed unpaired Student’s t-tests. *P = 0.0312, **P = 0.004, ***P = 0.0017

Fig. 4

In vivo antitumor effect of the chemoimmunotherapy hydrogel. A Treatment schema of R837/Nab-PTX/ALG in tumor suppression experiment. 2 × 10⁶ murine hepatocellular carcinoma cells H22 in 100 µL PBS were injected subcutaneously on the lower right mammary gland of ICR mice to establish H22 tumor mouse models. B Average tumor-growth curves of ICR mice bearing H22 hepatocellular carcinoma with different treatments as indicated (n = 6). The mice were administered with R837 (50 µg) and/or Nab-PTX (100 µg) on day 6 and day 13, which were dissolved in normal saline or sodium ALG solution to a final volume of 100 µL per dose. The tumor size was measured every 2–3 days from the first day of administration until the average volume of the NS group reached 1000 mm³, which was considered to have reached the standard for euthanasia of mice. The error bars represented mean ± SEM and p-values were calculated by two-way ANOVA and Tukey post-test and correction. ns P > 0.05, ****P < 0.0001. C Tumor-growth curves of each mouse in different groups (n = 6). D Survival curves of ICR mice in different groups (n = 6). P-values were calculated by log-rank (Mantel–Cox) test, ns P > 0.05, *P = 0.0284, **P = 0.0013, ***P = 0.0006 (NS vs PTX/ALG, NS vs R837/Nab-PTX/ALG). E Average weight of different groups (n = 6). The error bars represent mean ± SEM and p-values were calculated by two-way ANOVA and Tukey post-test and correction, ns P > 0.05

Fig. 5

Immune responses and immunogenic cell death induced by the chemoimmunotherapy hydrogel. Representative flow cytometry images (A) and proportions (B) of mature DCs (mDCs, CD11c⁺CD80⁺CD86⁺) in tumor-draining lymph nodes (TDLNs) 1 week after the last treatment (n = 5). The error bars represented mean ± SEM. p-values were calculated by two-tailed unpaired Student’s t-tests. ns P > 0.05, **P = 0.0011 and ***P = 0.0003, ****P < 0.0001. Representative flow cytometry images (C) and proportions (D) of effector memory T cells (T_EM, CD3⁺CD8⁺CD44⁺CD62L^–) and central memory T cells (T_CM, CD3⁺CD8⁺CD44⁺CD62L⁺) in tumors 1 week after the last treatment (n = 5). The error bars represented mean ± SEM. p-values were calculated by two-tailed unpaired Student’s t-tests. *P = 0.0102 (T_EM) 0.0333 (T_CM), **P = 0.0032 (NS vs R837/ALG) 0.0049 (NS vs Nab-PTX/ALG) 0.0033 (Nab-PTX/ALG vs R837/Nab-PTX/ALG) and ***P = 0.0002. Representative flow cytometry images (E) and proportions (F) of cytotoxic lymphocytes (CTLs, CD3⁺CD8⁺) and helper T cells (Th, CD3⁺CD4.⁺) in spleens 1week after the last treatment (n = 5). The error bars represented mean ± SEM. p-values were calculated by two-tailed unpaired Student’s t-tests. *P = 0.0276 (CD4) 0.0145 (CD8), **P = 0.0063 (CD4), ***P = 0.0004 (NS vs R837/ALG) 0.005 (ALG vs R837/Nab-PTX/ALG) 0.0002 (NS vs R837/Nab-PTX/ALG). (G) Calreticulin (CRT) staining of tumors (brown) 1 week after the last treatment (the scale bar is 62.5 µm and 20 µm, respectively)

Fig. 6

RNA-sequencing analysis. A Volcano map of significantly differentiated expression between NS and R837/Nab-PTX/ALG groups. The red dot represents significant up-regulated gene and the blue dot represents significant down-regulated gene. B Biological process and molecular function of GO enrichment analysis. C Differential gene expressions in immune system process by GO enrichment analysis. D Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis. E, F Differential gene expressions in Toll-like receptor signaling pathway and TNF signaling pathway

Fig. 7

Antitumor effect and safety evaluation of the chemoimmunotherapy hydrogel. A Treatment schema of R837/Nab-PTX/ALG in tumor suppression experiment. 1 × 10⁶ murine breast cancer cells 4T1 in 100 µL PBS were injected subcutaneously on the lower right mammary gland of Balb/c mice to establish 4T1 tumor mouse models. B Average tumor-growth curves of Balb/c mice bearing 4T1 breast cancer with different treatments as indicated (n = 6). The mice were administered with R837 (50 µg) and/or Nab-PTX (100 µg) on day 6 and day 13, which were dissolved in normal saline or sodium ALG solution to a final volume of 100 µL per dose. The tumor size was measured every 2–3 days from the first day of administration until the average volume of the NS group reached 1000 mm³, which was considered to have reached the standard for euthanasia of mice. The error bars represented mean ± SEM and p-values were calculated by two-way ANOVA and Tukey post-test and correction. ***P = 0.0005, ****P < 0.0001. C Survival curves of Balb/c mice in different groups (n = 6). P-values were calculated by log-rank (Mantel–Cox) test, *P < 0.0194, **P < 0.0045, ***P = 0.0006 (NS vs R837/ALG, NS vs Nab-PTX/ALG, NS vs R837/Nab-PTX/ALG). D Hematoxylin–eosin staining of main organs, including heart, liver, spleen, lung and kidney. (the scale bar is 200 µm)