Immunostimulatory nanomedicines synergize with checkpoint blockade immunotherapy to eradicate colorectal tumors

Abstract

Nanoparticles can potentially stimulate tumour microenvironments to elicit antitumour immunity. Herein, we demonstrate effective immunotherapy of colorectal cancer via systemic delivery of an immunostimulatory chemotherapeutic combination in nanoscale coordination polymer (NCP) core-shell particles. Oxaliplatin and dihydroartemesinin have contrasting physicochemical properties but strong synergy in reactive oxygen species (ROS) generation and anticancer activity. The combined ROS generation is harnessed for immune activation to synergize with an anti-PD-L1 antibody for the treatment of murine colorectal cancer tumours. The favourable biodistribution and tumour uptake of NCPs and the absence of peripheral neuropathy allow for repeated dosing to afford 100% tumour eradication. The involvement of innate and adaptive immune systems elicit strong and long lasting antitumour immunity which prevents tumour formation when cured mice are challenged with cancer cells. The intrinsically biodegradable, well tolerated, and systemically available immunostimulatory NCP promises to enter clinical testing as an immunotherapy against colorectal cancer.

Fig. 1

Preparation and characterization of OxPt/DHA. a Schematic illustration showing layer-by-layer construction of the hybrid core-shell structure of OxPt/DHA. The OxPt/DHA consists of an OxPt prodrug coordinated to Zn²⁺ ions in the core and chol-DHA in the lipid shell. Compositions of the three NCPs investigated are also shown. b TEM image of OxPt/DHA. c Number-average diameter of OxPt/DHA characterized by DLS. d Stability test of OxPt/DHA at 37 °C in the presence of BSA (5 mg/mL). OxPt oxaliplatin, DHA dihydroartemisinin, TEM transmission electron microscopy, DLS dynamic light scattering

Fig. 2

Internalization and dissociation of NCP components in cells. a Uptake of chol-pyro-labelled NCP particles by CT26 cells at different incubation times. b The intracellular Pt in CT26 incubated with OxPt or OxPt/DHA as determined by ICP-MS. c CT26 cells incubated with fluorescent nanoparticles containing xylenol orange in the core, chol-pyro in the shell, and FITC-DOPE lipid to visualize OxPt in the core, chol-DHA in the shell, and the lipid layer of OxPt/DHA, respectively, for different times were observed under confocal laser scanning microscopy (CLSM). The experiments were done three times and data were expressed as mean ± SD in (a) and (b). The images in (c) were obtained without repetition. NCP nanoscale coordination polymer, OxPt oxaliplatin, DHA dihydroartemisinin, ICP-MS inductively coupled plasma-mass spectrometry

Fig. 3

Release of DHA and OxPt from OxPt/DHA. a Proposed DHA release via GSH-mediated disulphide cleavage and proton-catalysed hydrolysis. b Proposed release of OxPt via direct reduction by ascorbate or a two-step sequence of hydrolysis to generate OxPt-bc followed by reduction to OxPt. c Total Pt release from and chol-DHA remaining in OxPt/DHA particle when incubated in water at 37 °C with or without 0.5% Triton X-100 and 5 mM ascorbate. The data were obtained without repetition. OxPt oxaliplatin, DHA dihydroartemisinin, GSH glutathione

Fig. 4

Programmed cell death in colorectal cancer cells by ROS generation. a, b ROS generation in cells treated with OxPt/DHA, as indicated by the green fluorescence of 2′,7′-dichlorofluorescein (DCF) that was oxidized from 2′,7′-dichlorodihydrofluorescein diacetate (H₂DCFDA) by ROS. c, d Release of cytochrome c from mitochondria in cells incubated with OxPt/DHA. Mitochondria (red fluorescence) and cytochrome c (green fluorescence) were stained by MitoTracker Red CMXRos and anti-cytochrome c antibody, respectively. e, f Apoptosis induced by OxPt/DHA. After treatment, cells were stained by Alexa Fluor 488-labelled Annexin V and propidium iodide (PI) and analysed by flow cytometry. g, h Cell cycle arrest caused by OxPt/DHA. Treated cells were fixed with 70% ethanol overnight, treated with RNase A, stained by PI, and analysed by flow cytometry. Data are expressed as means ± SD, and one of three repetitions with similar results is shown here. *p < 0.05, **p < 0.01, ***p < 0.001 by Student’s two-tailed t test. OxPt oxaliplatin, DHA dihydroartemisinin, ROS reactive oxygen species

Fig. 5

Immunostimulatory effects in colorectal cancer cells. a, b CRT cell surface expression upon treatment with OxPt/DHA, determined by flow cytometry (a) and CLSM (b). c HMGB-1 release from tumour cells treated with OxPt/DHA, detected by ELISA. d–f Uptake of treated MC38 cells by bone-marrow-derived dendritic cells (d) and macrophages (e). f Priming of T-cell responses triggered by OxPt/DHA. MC38 tumour cells were treated with OxPt/DHA, and injected into the right footpads of C57BL/6 mice to determine the capacity of draining lymph node cells to produce IFN-γ in response to MC38 lysates. g, h In vivo anticancer vaccination of OxPt/DHA in immunocompetent C57BL/6 mice (g) and immunodeficient Rag^2−/− mice (h). The antitumour response was measured by immunizing mice with OxPt/DHA-treated tumour cells in one flank and challenging mice with untreated, live tumour cells in the opposite flank 7 days later. Data are expressed as means ± SD. One of three repetitions with similar results is shown here for (a)−(e). The result was obtained without repetition for (f)−(h) (n = 6). *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001 by Student’s two-tailed t test. CRT calreticulin, OxPt oxaliplatin, DHA dihydroartemisinin, CLSM confocal laser scanning microscopy

Fig. 6

Enhanced anti-PD-L1 immunotherapy on colorectal cancers. a Experimental design for the treatment and challenge of CT26 tumour-bearing mice. Tumours were allowed to grow for 12 days before treatment to form more immunosuppressive tumours. Then, tumour-bearing mice were intraperitoneally injected with OxPt/DHA combined with α-PD-L1 every 3 days for 12 total doses. Three months after all tumours had disappeared, mice were challenged with CT26 cells, followed by rechallenge with 4T1 1 month later. b Growth curves of CT26 tumours after treatment with OxPt/DHA combined with α-PD-L1 and challenge with CT26 cells (red arrow). c Experimental design for surgery control of CT26 tumour-bearing mice. Percentage tumour-free mice (d, f) and tumour growth curve (e, g) after challenge with CT26 cells (d, e) or rechallenge with unrelated 4T1 tumour cells (f, g) in naïve mice or OxPt/DHA and α-PD-L1-treated mice. h Growth curves of MC38 tumour on C57BL/6 after treatment with OxPt/DHA (8 mg/kg OxPt) combined with α-PD-L1. i, j Therapeutic effect of OxPt/DHA plus α-PD-L1 on C57BL/6 (i) at the dose of 16 mg/kg OxPt and Rag2^−/− mice (j) at the dose of 8 mg/kg OxPt. Data are each pooled from two independent experiments and expressed as means ± SD (n = 6 except for (j); n = 5 for (j)). OxPt oxaliplatin, DHA dihydroartemisinin

Fig. 7

Tumour infiltration of innate immune cells. a Immunofluorescence analysis showing the infiltration of dendritic cells and macrophages 2 days after treatment. b, c The densities of CD11c⁺ (b) and F4/80⁺ (c) cells in the whole tumours 2 days after treatment, from the confocal images of immunofluorescence staining (n = 3). d, e The percentages of dendritic cells (d), total macrophages (e) and M1 macrophages (f) by flow cytometry of cell-surface staining 12 days after the first treatment. Data are each pooled from three independent experiments for (d) and (e). Data were obtained without repetition for (f). *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001 by Student’s two-tailed t test

Fig. 8

OxPt/DHA promotes tumour-specific T cell response. a Immunofluorescence analysis showing the infiltration of CD8⁺ T cells 12 days after the first treatment. b The density of CD8⁺ T cells in the tumour sections, analysed from the confocal images of immunofluorescence staining (n = 3). c CD8⁺ T cells in tumours by flow cytometry of cell-surface staining 12 days after the first treatment. d KSPWFTTL antigen-specific IFN-γ producing T cells detected by ELISPOT assay 12 days after the first treatment. e The percentage of effector memory T cells in total CD8⁺ T cells in spleens after treatment with OxPt/DHA plus α-PD-L1. Data are each pooled from two independent experiments for (c)−(e). *p < 0.05, **p < 0.01, ***p < 0.001 and ****p < 0.0001 by Student’s two-tailed t test. OxPt oxaliplatin, DHA dihydroartemisinin, ELISPOT Enzyme-Linked ImmunoSpot

Fig. 9

Anticancer and immune mechanisms of OxPt/DHA. left, The disruption of the lipid bilayer of OxPt/DHA upon endocytosis exposes the chol-DHA and OxPt prodrugs to triggered release by hydrolysis and/or reduction. The resultant parent drugs exhibit the expected mechanisms of action of DNA adduct formation and/or ROS generation. right, Systemically delivered OxPt/DHA can accumulate in the tumours and release the drug payload, as shown on the left, for immunogenic cell death of the cancer cells. Cell-surface CRT expression and release of DAMPs, such as HMGB1, lead to phagocytosis by macrophages and/or dendritic cells, which travel to the regional lymph node to prime T cells. Macrophages can release the antigens for DC uptake and subsequent T cell activation. The tumour-specific T cells proliferate and infiltrate into the tumour, where they can exert their cytotoxic effects. The α-PD-L1 prevents the binding of tumour PD-L1 and T cell PD-1, thereby inhibiting deactivation of the T cells