Minimally Invasive Injectable Gel for Local Immunotherapy of Liver and Gastric Cancer

Abstract

Local immunotherapy represents a promising solution for preventing tumor recurrence and metastasis post tumor surgical resection by eliminating residue tumor cells as well as eliciting tumor-specific immune responses. Minimally invasive surgery has become a mainstream surgical method worldwide due to its advantages of aesthetics and rapid postoperative recovery. Unfortunately, the currently reported local immunotherapy strategies are mostly designed to be used after open laparotomy, which go against the current surgical philosophy of minimally invasive therapy and is not suitable for clinical translation. Aiming at this problem, a minimally invasive injectable gel (MIGel) is herein reported loaded with immunotherapeutic agents for gastric and liver cancer postoperative treatment. The MIGel is formed by crosslinking between oxidized dextran (ODEX) and 4-arm polyethylene glycol hydroxylamine (4-arm PEG-ONH₂) through oxime bonds, which can be injected through a clinic-used minimally invasive drainage tube and adhered tightly to the tissue. The loaded oxaliplatin (OxP) and resiquimod (R848) can be released constantly over two weeks and resulted in over 75% cure rate in orthotopic mouse gastric and liver cancer model. Collectively, a concept of minimally invasive local immunotherapy is proposed and MIGel is designed for local intraperitoneal cancer immunotherapy through minimally invasive surgery, with good clinical translation potential.

Keywords: cancer immunotherapy; gastric cancer; hydrogels; liver cancer; minimally invasive injection.

Scheme 1

Schematic illustration of the preparation and mechanism of MIGel for orthotopic liver and gastric tumor therapy. The MIGel is cross‐linked by 4‐arm PEG‐ONH₂ and ODEX. With the assistance of laparoscopy, the MIGel was minimally invasive injected through microcatheter. After the injection of MIGel, instantaneous adhesion to the surface of tissues was occurred due to the interaction between the aldehyde group of the material and the amino group on the surface of the tissue. The entire surgical process is being monitored in real‐time. The released OxP could induce tumor cell death thus promoting the release of antigens. Subsequently, the antigens were captured by immature DCs (iDCs) and presented to naïve T cells. Meanwhile, effector T cells mobilize to the tumor bed and kill tumor cells with the help of the separately injected aPD‐1 antibody.

Figure 1

Construction and characterization of MIGel. A) Schematic illustration of the MIGel construction process. B) The gelation time of MIGels at different concentrations at 4 °C and 37 °C. C,D) Rheology properties of gels formed at different temperatures, and mass ratios. (E) The optical images of gels via a tube inverted test at 37 °C; The MIGel can pass through the needle (29 G), and then CIAC was drawn. F) SEM images of lyophilized MIGel. Scale bar: 10 µm. G) Viscosity of the MIGel at the strain of 1% to 500%. H) Shear stress sweep tests of the 6% MIGel at 37 °C. I) Self‐healing ability of the 6% MIGel evaluated with an alternating strain of 1% and 500% at room temperature. J) Tensile strength of MIGel between mouse skins and MIGel. K) Adhesion to various tissues including stomach, spleen, and kidney. L) The images of MIGel adhered to full and empty stomach.

Figure 2

The degradation, injectability and drug release, and safety evaluation of MIGel. A) In vitro degadation of MIGel under physiological conditions (PBS, pH 7.4). The white circles represent the remaining hydrogels during degradation period. B) In vivo degradation of MIGel after injecting on the surface of gastric. C) The images of MIGel injected on the liver and stomach of rats though the syringe with 29 G needle. D) The images of MIGel injected on the liver and stomach of rabbits though laparoscopic procedure. E) The schematic for the preparation of MIGel@OxP/R848. F) The accumulated release profiles of OxP and R848 in vitro. G) In vivo pharmacokinetic profiles following the intraperitoneal injection of free OxP/R848 or MIGel@OxP/R848 injected on the gastric of rats. H) The complete blood count after injection of MIGel on the stomach at day 3 and day 7. I) The IL‐6 concentration in the serum after injection of MIGel on the stomach at day 3 and day 7. Data are the mean ± standard deviation (n = 3). ns, not significant.

Figure 3

In vivo therapeutic efficacy on subcutaneous gastric tumor resection model. A) Treatment scheme for subcutaneous gastric tumor model (90% tumor resection) and immune analysis (50% tumor resection). B) Average tumor growth curves after operation. n = 8. C) Individual gastric tumor growth curves after surgical operation. D) Survival curves of mice in various treatment groups. n = 8. (E) The body weight changes of the mice during the treatment period. n = 8. (F) The analysis of activated DCs (CD11c⁺MHCII⁺ and CD11c⁺CD80⁺), CD4⁺ T cells, and CD8⁺ T cells in the tumor at day 19. n = 4. Data are presented as mean ± standard error on the mean (S.D.) *p < 0.05, **p < 0.01, and ***p < 0.001. ns, not significant.

Figure 4

In vivo therapeutic efficacy on orthotopic gastric cancer model and immune analysis after various treatments. A) Treatment scheme. B) The representative images of the excised stomachs after various treatments (day 24). C) The stomach weights after various treatments. n = 6. D) The proportions of activated DCs in tumors after various treatments at day 24. n = 5. E) Tumoral CD4⁺ and CD8⁺ T cells after various treatments at day 24. n = 5. F) The proportions of NK (CD49b⁺) cells in tumors after various treatments at day 24. n = 5. G) The level of IFN‐β, IFN‐γ, and IL‐12 cytokines in tumors after various treatments. n = 5. H) Survival curves of mice after various treatments. n = 8. I) Rechallenge tumor growth curves of naïve mice and the survival mice after 60 days of MIGel@OxP/R848+aPD‐1 treatment. n = 6. *p < 0.05, **p < 0.01, and ***p < 0.001. ns, not significant.

Figure 5

The whole genome RNA‐seq for gastric tumors after various treatments. A) Venn interaction of control (G1), MIGel@OxP (G2), MIGel@OxP/R848 (G3) and MIGel@OxP/R848+aPD‐1 (G4). n = 3. B) The volcano plots illustrating differentially regulated gene expression from RNA‐seq analysis between the control and MIGel@OxP, MIGel@OxP and MIGel@OxP/R848, MIGel@OxP/R848 and MIGel@OxP/R848+aPD‐1. Upregulated and downregulated genes are shown in red and green, respectively. n = 3. C) KEGG pathway analysis of changed genes between the control and MIGel@OxP, MIGel@OxP and MIGel@OxP/R848, MIGel@OxP/R848 and MIGel@OxP/R848+aPD‐1. n = 3. D) KEGG functional clustering of genes that were changed for biological processes. E) Heatmaps of differentially expressed genes related to T cells activation, antigen processing, and presentation. n = 3. (*p < 0.05, **p < 0.01, and ***p < 0.001).

Figure 6

In vivo therapeutic efficacy on liver metastasis model and orthotopic liver cancer model. A) Treatment scheme for liver metastasis model. B) The liver weights after various treatments. n = 5. C) Survival curves of mice after various treatments. n = 7. D) Tumoral CD4⁺ and CD8⁺ T cells after various treatments at day 24. n = 4‐5. E) The proportions of CD4⁺, CD8⁺, and IFN‐γ⁺ T cells in blood after various treatments at day 24. n = 4‐5. F) Treatment scheme for orthotopic liver model. G) The representative images of the excised livers after various treatments (day 24). The white circles represent the orthotopic liver tumors. H) The liver weights after various treatments. n = 5. I) The nuclear magnetic resonance images (T1) of the livers in various treatment groups. The white circles represent the orthotopic liver tumors. *p < 0.05, **p < 0.01, and ***p < 0.001. ns, not significant.