Metal-enriched HSP90 nanoinhibitor overcomes heat resistance in hyperthermic intraperitoneal chemotherapy used for peritoneal metastases

Abstract

Clinical hyperthermic intraperitoneal chemotherapy (HIPEC) is regarded as a potential treatment that can prolong survival of patients with peritoneal metastases after cytoreductive surgery. However, treated tumor cells are prone to becoming heat resistant to HIPEC therapy through high expression of heat shock proteins (HSPs). Here, a carrier-free bifunctional nanoinhibitor was developed for HIPEC therapy in the management of peritoneal metastases. Self-assembly of the nanoinhibitor was formed by mixing Mn ion and epigallocatechin gallate (EGCG) in a controllable manner. Such nanoinhibitor directly inhibited HSP90 and impaired the HSP90 chaperone cycle by reduced intracellular ATP level. Additionally, heat and Mn ion synergistically induced oxidative stress and expression of caspase 1, which activated GSDMD by proteolysis and caused pyroptosis in tumor cells, triggering immunogenic inflammatory cell death and induced maturation of dendritic cells through the release of tumor antigens. This strategy to inhibit heat resistance in HIPEC presented an unprecedented paradigm for converting "cold" tumors into "hot" ones, thus significantly eradicating disseminated tumors located deep in the abdominal cavity and stimulating immune response in peritoneal metastases of a mouse model. Collectively, the nanoinhibitor effectively induced pyroptosis of colon tumor cells under heat conditions by inhibiting heat stress resistance and increasing oxidative stress, which may provide a new strategy for treatment of colorectal peritoneal metastases.

Keywords: Epigallocatechin gallate; Heat shock protein 90 inhibitor; Heat stress resistance; Hyperthermic intraperitoneal chemotherapy; Pyroptosis.

Fig. 1

Design and characterization of MnEGCG nanoparticles. A Schematic illustration of the strategy for synthesis of MnEGCG nanoparticles. B TEM images of MnEGCG nanoparticles; the inset is the particle size distribution. C, Carbon. Mn, Manganese. O, Oxygen. C XRD, (D) FTIR and (E) XPS spectrum of MnEGCG nanoparticles. F Degradation of the nanoparticles in PBS at different pH values

Fig. 2

Cellular uptake and antiproliferation effect of nanoinhibitor in vitro. A Confocal fluorescent images showing cellular uptake of 5 FAM-nanoinhibitor in CT26 cells at 4, 6, 9, and 12 h. Green fluorescence represents 5-FAM conjugated to nanoinhibitor, and blue fluorescence indicates nucleus (DAPI). B AM/PI staining images of CT26 cells after treated with the nanoinhibitor at the concentrations of 12.50, 25.00, 50.00, and 100.00 mg/L for 24 h. Green fluorescence represents living cells dyed by acetoxymethyl ester (AM), and red fluorescence represents dead cells dyed by propidium iodide (PI). Cell viabilities after different treatments in CT26 cells for 24 h (C), HCT116 cells for 24 h (D), MGC803 cells for 24 h (E), CT26 cells for 48 h (F), HCT116 cells for 48 h (G), and MGC803 cells for 48 h (H). *, EGCG 37 °C vs EGCG 43 °C, ****p < 0.0001. #, Nano 37 °C vs Nano 43 °C, ####p < 0.0001. Nano, nanoinhibitor

Fig. 3

Antiproliferation mechanism of nanoinhibitor. A Brightfield images of tumor cells after treatment with the non-lethal high temperature (39 °C) once, twice, or thrice, respectively. Each heat session lasted for 30 min. B Cell viabilities after treatment with non-lethal high temperature (39 °C) corresponding to (A). C Western blot analysis of the effect of high temperature on the expression of heat shock proteins. D Docking models of EGCG bound to human HSP90α, and the structural view of EGCG in the HSP90 C-terminal domain. E Western blot analysis of HSP90 level in tumor cells treated with EGCG 37 °C (50 mg/L), nanoinhibitor 37 °C (50 mg/L), EGCG 43 °C (50 mg/L), or nanoinhibitor 43 °C (50 mg/L) at different time points. HSP90¹, HSP90⁶, and HSP90.²⁴, stand for HSP90 expression for 1, 6, 24 h after treatment, respectively. F Heat map of differentially expressed genes related to endoplasmic reticulum stress. G, H GSEA plots of differentially expressed genes enriched in (G) the protein processing in endoplasmic reticulum, and (H) oxidative phosphorylation. I ROS production after different treatments in CT26 tumor cells. *p < 0.05, **p < 0.01, and ****p < 0.0001

Fig. 4

Tumor cell death and immunostimulation induced by nanoinhibitor in vitro. A Pyroptosis induced by different treatments. The top-right images are shown at twofold magnification. B Western blot analysis of pyroptosis-related proteins (the canonical pathway, GSDMD, Caspase 1) level. C Heat map showing differentially expressed genes related to pyroptosis. D-F GSEA plots of differentially expressed genes enriched in nucleotide oligomerization domain (NOD)-like receptor signaling pathway (D), myeloid dendritic cell activation (E), chemokine signaling pathway (F). G Illustration of pyroptosis by combined therapy of nanoinhibitor and 43 °C heating. EGCG, epigallocatechin gallate. HSPs, heat shock proteins. GSDM, gasdermin. DC, dendritic cell. H, I ELISA for IL-1β (H) and LDH (I) release of tumor cells after different treatments. J Extracellular ATP level of tumor cells after different treatment. K Flow cytometry of in vitro DCs maturation (CD80⁺CD86⁺, gated on CD11c.⁺) proportion (%) after incubation with CT26 cells with different treatments. Casp1_p20, cleaved caspase 1. LPS is regarded as the positive control group. LPS, Lipopolysaccharide. **p < 0.01, ****p < 0.0001

Fig. 5

The biodistribution of the nanoinhibitor in tumor-bearing mice. Images of bioluminescence (Luc) for tumor and fluorescence (Fluo) for DiR in CT26-Luc tumor-bearing mice after intraperitoneal treatment with (A) nanoinhibitor-DiR, (C) free DiR at different time points. Images of bioluminescence (Luc) and fluorescence (Fluo) for tumors and main organs in (B) nanoinhibitor-DiR group, (D) free DiR group at 168 h intraperitoneal injection. E, F The quantitative fluorescence analyses of tumors and major organs in the two groups of mice at 72 h and 168 h intraperitoneal injection, respectively. Tu, tumor. Ki, kidney. Lu, lung. Sp, spleen. Li, liver. He, heart. Avg, average. *p < 0.05 and **p < 0.01

Fig. 6

Antitumor effect of nanoinhibitor in vivo. A Schematic illustration of the experimental protocol in CT26-Luc tumor-bearing Balb/c mice. B Schematic illustration of the hyperthermic intraperitoneal perfusion procedure. C Bioluminescence images of tumor-bearing mice individually treated with PBS, Nano 37 °C (50 mg/L), PBS 43 °C, Nano 43 °C (50 mg/L) at day 0, 5, 10, 20, 30, 40, and 50. D Representative images of tumor tissues harvested from intraperitoneal at day 17. E Tumor immunohistochemical staining of HSP90 and cleaved GSDMD in different treatment groups. GSD-N, GSDMD-N fragment. F Bioluminescence intensity of tumors in different groups of mice individually corresponding to (C). G Survival curves of mice receiving the indicated treatment. i.p., intraperitoneal injection. Indi, individual

Fig. 7

In vivo immune-stimulation induced by nanoinhibitor. A Representative photographs of abdominal cavity and mesenteries of mice after different treatments. H&E staining and CD3 immunostained images of tumors in different groups. B Schematic illustration of modified PCI score derived from human PCI score. C IFN-γ, TNF-α, and IL-6 concentrations in ascites compared with control group post-treatment. D Modified PCI score of mice individually in different groups. E IFN-γ, TNF-α, and IL-6 concentrations in serum compared with control group post-treatment. F Illustration of proposed antitumor mechanism of the nanoinhibitor. ****p < 0.0001

Fig. 8

Translational research of nanoinhibitor in organoid. A Western blot analysis of heat shock protein 90 level in organoids from a CRC tumor patient after treatment with PBS, EGCG 37 °C (50 mg/L), Nano 37 °C (50 mg/L), PBS 43 °C, EGCG 43 °C (50 mg/L), Nano 43 °C (50 mg/L). B Cell viabilities of organoid after treatment with EGCG 37 °C, Nano 37 °C, EGCG 43 °C and Nano 43 °C at the drug concentrations of 3.12, 6.25, 12.50, 25.00, 50.00, and 100.00 mg/L. C Microscopical images of organoids after treatment with different concentrations of nanoinhibitor. D AM/PI staining images of organoids after treatment with different concentrations of the nanoinhibitor. CC, Colorectal cancer. *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001. NS, not significant

Fig. 9

Biosafety evaluation of nanomedicine. A Cell viabilities of GSE1 cells treated with Nano 37 °C, Nano 43 °C for 24 h. B, C Biochemical markers of liver function (ALT, AST) of mice with different treatments. D Cell viabilities of HMrSV5 cells treated for 24 h. E, F Biochemical markers of kidney function (UREA, CRE) of mice with different treatments. G H&E staining images of main organs of mice with different treatments