Cyclophosphamide loaded thermo-responsive hydrogel system synergize with a hydrogel cancer vaccine to amplify cancer immunotherapy in a prime-boost manner

Abstract

Although neoantigen-based cancer vaccines show great potential in cancer immunotherapy due to their ability to induce effective and long-lasting anti-tumor immunity, their development is hindered by the limitations of neoantigens identification, low immunogenicity, and weak immune response. Cyclophosphamide (CTX) not only directly kills tumors but also causes immunogenic cell death, providing a promising source of antigens for cancer vaccines. Herein, a combined immunotherapy strategy based on temperature-sensitive PLEL hydrogel is designed. First, CTX-loaded hydrogel is injected intratumorally into CT26 bearing mice to prime anti-tumor immunity, and then 3 days later, PLEL hydrogels loaded with CpG and tumor lysates are subcutaneously injected into both groins to further promote anti-tumor immune responses. The results confirm that this combined strategy reduces the toxicity of CTX, and produces the cytotoxic T lymphocyte response to effectively inhibit tumor growth, prolong survival, and significantly improve the tumor cure rate. Moreover, a long-lasting immune memory response is observed in the mice. About 90% of the cured mice survive for at least 60 days after being re-inoculated with tumors, and the distant tumor growth is also well inhibited. Hence, this PLEL-based combination therapy may provide a promising reference for the clinical promotion of chemotherapy combined with cancer vaccines.

Keywords: Cancer vaccine; Cyclophosphamide; Immunogenic cell death; Immunotherapy; Thermo-responsive hydrogels.

Graphical abstract

Scheme 1

Schematic illustration of the PLEL-based combination strategy to amplify cancer immunotherapy. a) the preparation process of CTX@PLEL and CpG&TL@PLEL hydrogels. b) In vivo immune mechanism of PLEL hydrogel-based combination therapy strategy (Step 1: CTX@PLEL intratumoral injection to prime antitumor immune response; step 2: CpG&TL@PLEL subcutaneous vaccination to boost tumor-specific immunity).

Fig. 1

Efficacy of free cyclophosphamide combined with the hydrogel cancer vaccine. a) Photographs of tumors in different groups. b) Bodyweight curves of CT26 tumor-bearing mice over time, the black arrows represent CTX administration and the red arrows represent hydrogel vaccine administration. c) Tumor weight in all groups. d) Tumor growth curves of different treatment groups. e) Serum cytokine levels in CT26 tumor-bearing mice (n = 3–4). *p < 0.05, **p < 0.01 and ***p < 0.001.

Fig. 2

Immune cell activation of free cyclophosphamide combined with the hydrogel cancer vaccine. a) Representative flow cytometric analysis images. b-d) Relative quantification of CD3⁺, CD4⁺, and CD8⁺ T cells in inguinal lymph nodes. e-g) Relative quantification of CD11c+, CD86⁺, and MHC-I + DCs in inguinal lymph nodes analyzed by flow cytometry. *p < 0.05, **p < 0.01 and ***p < 0.001.

Fig. 3

The therapeutic effect of PLEL hydrogel loaded with different concentrations of cyclophosphamide combined with hydrogel vaccine. a) Reversible sol-gel phase transition process of CTX-loaded thermosensitive PLEL hydrogel. b) Change in storage (Gʹ) and loss (Gʹʹ) modulus for blank hydrogel and CTX-loaded hydrogel with increasing temperature. c) Cumulative release of CTX from the hydrogel (n = 3). d) Bodyweight curves of CT26 tumor-bearing mice over time, the black arrows represent CTX administration and the red arrows represent hydrogel vaccine administration. e) Relative body weight after administrating CTX. f) Lowest relative body weight after administrating CTX and hydrogel vaccine. g) Tumor growth curves (when one mouse in each group was sacrificed, the tumor growth curve of that group was stopped.) and h) Survival rate of different treatment groups. i) Representative photographs of CT26 tumor-bearing mice on day 23 (The letters in red represent low, medium, and high concentrations of CTX, L: 50 mg kg⁻¹, M: 100 mg kg⁻¹, H: 200 mg kg⁻¹). 1: Saline, 2: CpG&TL@PLEL, 3: L-CTX + CpG&TL@PLEL, 4: L-CTX@PLEL + CpG&TL@PLEL, 5: M-CTX + CpG&TL@PLEL, 6: M-CTX@PLEL + CpG&TL@PLEL, 7: H-CTX + CpG&TL@PLEL, 8: H-CTX@PLEL + CpG&TL@PLEL, 9: H-CTX, 10: H-CTX@PLEL. ****p < 0.0001.

Fig. 4

Immune cell population in mouse lymph nodes after different treatment. a) Representative flow cytometric analysis images of DCs. b-d) Quantification of expression levels of CD11c, CD86 and MHC-Ⅰ on the surface of DCs. e) Representative flow cytometric analysis images of T cells, and f-i) corresponding quantification of CD3⁺, CD4⁺ and CD8⁺ T cells. (The letters in red represent low, medium, and high concentrations of CTX, L: 50 mg kg⁻¹, M: 100 mg kg⁻¹, H: 200 mg kg⁻¹). *P < 0.05, **P < 0.01, ***P < 0.001, ****p < 0.0001.

Fig. 5

CTL anti-tumor immune response caused by combination therapy. Representative flow cytometric analysis images of DCs a) and T cells b). c-e) Quantification of surface markers on DCs. f-h) Quantification of surface markers on T cells. i) Tumor growth curves over time.

Fig. 6

Suppressor Treg cells of spleen and serum cytokine analysis. a) Representative flow cytometric analysis images of Treg cells. b) Quantification of the CTL to Treg cells ratio. c) Serum cytokine levels of mice in each group (n = 3–4).

Fig. 7

Anti-tumor immune memory response after CTX@PLEL + CpG&TL@PLEL treatment. a) Schematic diagram of evaluating immune memory response mediated by PLEL-based combination therapy strategy. b) Representative flow cytometric analysis images and d) percentage of effector and central memory T cells (n = 4). e, f) Tumor growth and c) Survival rate curves of control (n = 5) and the cured mice (n = 9). The cured: CT26 tumor-bearing mice survived for 60 days after CTX@PLEL + CpG&TL@PLEL treatment.

Fig. 8

The PLEL-based combined therapy for treating distant tumors. a) Schematic illustration to evaluate the inhibition efficacy to distant tumors generated by PLEL-based combination therapy strategy. b) Representative photographs of mice. c) Photographs of tumors in each group. d) Untreated and e) Treated tumor weight of mice. f) Untreated and g) Treated tumor growth curves of mice.