Biomimetic Nanocarriers Guide Extracellular ATP Homeostasis to Remodel Energy Metabolism for Activating Innate and Adaptive Immunity System

Abstract Metabolic interventions via targeting intratumoral dysregulated metabolism pathways have shown promise in reinvigorating antitumor immunity. However, approved small molecule immunomodulators often suffer from ineffective response rates and severe off‐target toxicity. ATP occupies a crucial role in energy metabolism of components that form the tumor microenvironment (TME) and influences cancer immunosurveillance. Here, a nanocarrier‐assisted immunometabolic therapy strategy that targets the ATP‐adenosine axis for metabolic reprogramming of TME is reported. An ecto‐enzyme (CD39) antagonist POM1 and AMP‐activated protein kinase (AMPK) agonist metformin are both encapsulated into cancer cell‐derived exosomes and used as nanocarriers for tumor targeting delivery. This method increases the level of pro‐inflammatory extracellular ATP (eATP) while preventing the accumulation of immunosuppressive adenosine and alleviating hypoxia. Elevated eATP triggers the activation of P2X7‐NLRP3‐inflammasome to drive macrophage pyroptosis, potentiates the maturation and antigen capacity of dendritic cells (DCs) to enhance the cytotoxic function of T cells and natural killer (NK) cells. As a result, synergistic antitumor immune responses are initiated to suppress tumor progress, inhibit tumor distant metastases, provide long‐term immune memory that offers protection against tumor recurrence and overcome anti‐PD1 resistance. Overall, this study provides an innovative strategy to advance eATP‐driven antitumor immunity in cancer therapy.


Materials
All chemicals were purchased from Sigma-Aldrich unless otherwise specified.

Cell culture
The murine melanoma cell lines B16F10 and macrophages RAW264.7, and the human colon cancer cells HCT116 were purchased from the Cell Bank of Shanghai, Chinese Academy of Sciences. B16F10-Luc cell lines were established by transfection of B16F10 with vectors carrying luciferase and puromycin resistance gene. B16F10, B16F10-Luc and RAW264.7 cells were cultured in DMEM medium supplemented with 10% FBS and 1% antibiotics (penicillin/streptomycin). HCT 116 cells were maintained in McCoy's 5A medium containing 10% FBS and 1% antibiotics (penicillin/streptomycin). Bone marrow-derived macrophages (BMDMs) and bone marrow-derived dendritic cells (BMDCs) were prepared following the steps blow. C57BL/6 mice were sacrificed and bone marrow cells were isolated from leg bones. To generate BMDMs, the isolated bone marrow cells were resuspended and maintained in complete RPMI medium supplemented with 10% FBS, 1% antibiotics (penicillin/streptomycin) and M-CSF (20 ng/mL) for 7 days. To generate BMDCs, the isolated bone marrow cells were resuspended and cultured in complete RPMI medium containing 10% FBS, 1% antibiotics (penicillin/streptomycin) and GM-CSF (20 ng/mL) for 7 days. All the cells were maintained in a normal oxygen incubator at 37 ℃ with 5% CO 2 . Hypoxia condition was performed in a hypoxic cell incubator with 2% O 2 , 5% CO 2 and balanced nitrogen.

Animals
Male C57BL/6 mice (6 to 7 weeks of age) were obtained from the Chinese Academy of Medical Science (Beijing, China). All animal handling and experimental procedures were approved by the Center for Animal Experiments/Animal Biosafety Level 3 Laboratory of Wuhan University.

Preparation and characterization of exosome/C-PMet
The exosomes harvested from B16F10 tumor cells were prepared according to the manufacturer's protocol of Exosome Isolation Reagent (RIBOBIO biotechnology co. LTD, China). To load POM1 and Met into the exosomes, the exosomes (0.5 mg) and drugs (50 μg in 10 μL DMSO) were mixed in 250 μL PBS in 0.4 cm cuvette (Bio-Rad). Electroporation was then carried out at 250 V and 350 μF on a Bio-Rad Gene Pulser X-cell Electroporation System. After electroporation, the mixture was incubated at 37 °C for 30 min to allow the recovery of the membrane of the electroporated exosomes. The obtained solution was centrifuged at 8000 g for 5min to remove the precipitate (redundant POM1 and Met) and the C-PMet in supernatant were collected by using Exosome Isolation Reagent again and stored at 4 ℃ for further use. The size and size distribution of C-PMet were measured by dynamic light scattering. The morphology structures of C-NV and C-PMet were observed by the TEM (JEOL-2100). The amount of Met loaded into C-NV and release from C-PMet curves followed by quantification via an HPLC system.

Western blot analysis
C-NV and C-PMet were prepared in SDS sample buffer and loaded into 10-12% SDS polyacrylamide gel. The proteins electrophoresed were then transferred onto polyacrylamide fluoride (PVDF) membranes. The membranes were further blocked with 5% skim milk for 1 h and incubated with primary antibodies against CD9 (ab223052, Abcam) and CD63 (ab217345, Abcam) at 4 °C over night. The PVDF membranes were then incubated with second antibody and detected using a Clarity TM Western ECL Substrate (Bio-Rad).
In vitro cancer targeting study B16F10 and RAW 264.7 cells were seeded in 24-well plates and cultured for 12 h.
Then 10 μL of C-PMet (FITC) were added into the culture medium. The cells were incubated at 37 °C, 5% CO 2 for 2 h, and washed with PBS for three times. Afterwards, the cells were further fixed with PFA for 30 min at room temperature, and then stained with DAPI and imaged by using a confocal laser scanning microscope (CLSM; IX81, Olympus, Japan).

In vitro toxicity of C-PMet
The toxicity was measured by MTT assay. B16F10 cells were seeded in 96-well plates at a density of 5 × 10 3 cells per well and incubated for 24 h. Afterwards, B16F10 cells were incubated for 4 different groups: (1) C-NV (2) C-Met (3) C-POM1 (4) C-PMet.
At the end of the incubation, 5 mg/mL MTT PBS solution was added, and the plate was incubated for another 4 h. Finally, the absorbance values of the cells were determined by using a microplate reader (Emax Precision, USA) at 570 nm. The background absorbance of the well plate was measured and subtracted. The cytotoxicity was calculated by dividing the optical density (OD) values of treated groups (T) by the OD values of the control (C) (T/C × 100%).

In vitro DC stimulation
For in vitro DC stimulation experiments, 1x10 5 B16F10 cells were cultured with 1x10 6 DC cells through a transwell co-culture system. After various treatments, DCs were stained with anti-CD45-APC/Cy7, anti-CD11c-FITC, anti-CD80-APC and anti-CD86-PE (Biolegend) antibodies, and then sorted by an FC500 flow cytometer (Beckman-Coulter, USA). The supernatants from the co-culture system were also collected to measure the levels of TNF-α and IL-12p70.

The priming and effector function assay of T cells
For in vitro T cells proliferation assay, CD8 + T lymphocytes from C57BL/6 mice spleen negatively selected by magnetic separation according to the protocol of CD8a + T Cell Isolation Kit (Miltenyi Biotec), were stained with the Cell Trace CFSE Cell Proliferation Kit (Invitrogen). CFSE-stained CD8 + T cells were then cultured with above treated DCs for 72 h, and the proliferation of splenic T cells was measured by flow cytometry. As a parallel experiment, the effector function of incubated CD8 + T cells was analyzed by measuring the expression of granzyme B (GZMB) and interferon-gamma (IFN-γ).

In vivo imaging
For in vivo fluorescence imaging, 2×10 5 B16F10 cells were injected into the right flank of the mice. When the tumor size reached around 100 mm 3 , the mice were intravenously injected with the Cy5.5 labeled C-PMet (C-PMet@Cy5.5). The tumor accumulation of C-PMet@Cy5.5 was recorded on an IVIS Spectrum at different time intervals (3, 6, 12 and 24 h after the injection).

In vivo anti-tumor effects
For the B16F10 primary tumor model, B16F10 (2×10 5 cells each) suspended in 100 μL of PBS were injected into the right flank of the mice. After 7 days of subcutaneously implantation, mice were randomly divided into seven groups and administered with one of treatments: PBS, POM1, C-POM1, Met, C-Met, PMet and C-PMet at the metformin dosage of 100 mg kg -1 and the POM1 dosage of 5 mg kg -1 , respectively. Afterwards, the growth of the tumors was carefully monitored. Tumor volume (V) was calculated by the formula: V = 0.5 x L x W 2 , where L and W represent the length and width of the tumor. Mice were sacrificed on day 23 after tumor inoculation. The tumor tissues, peripheral blood and the major organ of mice were collected for analysis. The remaining mice were used for the survival study.
Mice were sacrificed when the tumor burden exceeded 2 cm in diameter or if they lost up to 20% of their initial weight. Survival was evaluated from the first day of implantation until day 60.

In vivo toxicity evaluation
Blood samples were harvested from the B16F10 tumor-bearing mice to measure the levels of ALT (alanine aminotransferase), AST (aspartate aminotransferase), ALP (alkaline phosphatase), CRE (creatinine) and BUN (blood urea nitrogen). Major organs (hearts, livers, spleens, lungs and kidneys) were also collected and examined by H&E staining.

Transcriptomics study
For transcriptome analysis, 2× 10 5 B16F10 cells were injected into the right flank of the mice. When the tumor volume reached about 100 mm 3 , the mice were randomly divided into 2 groups and intravenously injected with PBS and C-PMet, respectively.
After 3 days, tumor tissues were harvested and total RNA were extracted. The transcriptome analysis was performed in the Beijing Genomics Institute. All data were analyzed online with BGIseq500 platform (BGI-Shenzhen, China).

In vivo anti-rechallenge study
To evaluate the long-term immune protection after the treatment with our strategy, tumor rechallenge model was established. Briefly, 2×10 5 B16F10 cells in 100 μL PBS were subcutaneously injected into the right flank of C57BL/6 mice (day -7). 7 days (day 0) later, the mice were randomly divided into four groups: Surgery (G1), C-PMet (G2), PD1 (G3) and C-PMet+PD1 (G4). For G2 and G4, the mice in G2 and G4 were iv injected with C-PMet three times for a week until the tumors were eliminated. For