Nanoparticle delivery of miR-21-3p sensitizes melanoma to anti-PD-1 immunotherapy by promoting ferroptosis

Background Although anti-programmed cell death protein 1 (PD-1) immunotherapy is greatly effective in melanoma treatment, low response rate and treatment resistance significantly hinder its efficacy. Tumor cell ferroptosis triggered by interferon (IFN)-γ that is derived from tumor-infiltrating CD8+ T cells greatly contributes to the effect of immunotherapy. However, the molecular mechanism underlying IFN-γ-mediated ferroptosis and related potentially promising therapeutic strategy warrant further clarification. MicroRNAs (miRNAs) participate in ferroptosis execution and can be delivered systemically by multiple carriers, which have manifested obvious therapeutic effects on cancer. Methods MiRNAs expression profile in IFN-γ-driven ferroptosis was obtained by RNA sequencing. Biochemical assays were used to clarify the role of miR-21-3p in IFN-γ-driven ferroptosis and the underlying mechanism. MiR-21-3p-loaded gold nanoparticles were constructed and systemically applied to analyze the role of miR-21-3p in anti-PD-1 immunotherapy in preclinical transplanted tumor model. Results MiRNAs expression profile of melanoma cells in IFN-γ-driven ferroptosis was first obtained. Then, upregulated miR-21-3p was proved to facilitate IFN-γ-mediated ferroptosis by potentiating lipid peroxidation. miR-21-3p increased the ferroptosis sensitivity by directly targeting thioredoxin reductase 1 (TXNRD1) to enhance lipid reactive oxygen species (ROS) generation. Furthermore, miR-21-3p overexpression in tumor synergized with anti-PD-1 antibody by promoting tumor cell ferroptosis. More importantly, miR-21-3p-loaded gold nanoparticles were constructed, and the systemic delivery of them increased the efficacy of anti-PD-1 antibody without prominent side effects in preclinical mice model. Ultimately, ATF3 was found to promote miR-21-3p transcription in IFN-γ-driven ferroptosis. Conclusions MiR-21–3 p upregulation contributes to IFN-γ-driven ferroptosis and synergizes with anti-PD-1 antibody. Nanoparticle delivery of miR-21–3 p is a promising therapeutic approach to increase immunotherapy efficacy without obvious systemic side effects.

USA) overnight at 4°C. After being washed, the membranes were incubated with horseradish peroxidase-conjugated secondary antibodies (Goat Anti-Mouse IgG, 1:5000; Goat Anti-Rabbit IgG, 1:5000, Santa Cruz, CA, USA) for 2 h at room temperature. Bound antibodies were detected using the ECL western blotting detection system and Image J software (Bio-Rad, Hercules, CA, USA).

Tumor tissue microarray
Melanoma tissue microarray (DC-Mel21020) was purchased from Avilabio Inc. (Xi'an, China). Detailed information can be accessed via BMJ Publishing Group Limited (BMJ) disclaims all liability and responsibility arising from any reliance Supplemental material placed on this supplemental material which has been supplied by the author(s)

Malondialdehyde (MDA) detection
The MDA level in cell lysates was assessed using a lipid peroxidation assay kit (ab118970, Abcam, Cambridge, MA, USA) according to the manufacturer's instructions. After treatment, cells were washed with ice-cold PBS and homogenized on ice in 300 μl of the MDA lysis buffer. After being centrifuged at 13,000 g for 10 mins, 200 μl of the supernatant from each homogenized sample was transferred to a microcentrifuge tube. Then, 600 μl of the thiobarbituric acid (TBA) solution was added into each vial and incubated at 95°C for 1 h.
The MDA in the sample would react with TBA to generate an MDA-TBA adduct. After the samples were cooled to room temperature in an ice bath for 10 mins, 200 μl from each reaction mixture was added into a 96-well plate for analysis. The absorbance was then measured at 532 nm using a Model 680 Microplate Reader (Bio-Rad, Hercules, CA, USA).

Lipid ROS analysis
Lipid ROS level of cells was examined using flow cytometry. In brief, cells were seeded in 6-well plates (3 × 10 5 per well). After indicated treatment, the culture medium was discarded, and replaced with 5 μM of BODIPY 581/591 C11 (D3861, Invitrogen, Carlsbad, CA, USA) diluted in 1 mL fresh medium for each well, and then incubated at 37 ℃ for 30 mins. Cells were directly collected and washed with PBS, and then resuspended in 500 μl of PBS.
Oxidation of BODIPY-C11 resulted in a shift of the fluorescence emission peak from 590 nm to 510 nm proportional to lipid ROS generation. The fluorescence intensities were quantified

Colony formation assay
For colony formation assay, cells were collected, counted, and replated in appropriate dilutions in six-well plate after treatment. After 10-14 days of incubation, colonies were washed with PBS, fixed with 4% paraformaldehyde for 10-20 mins, and then stained with a mixture of 6 % glutaraldehyde and 0.5% crystal violet for 1-2 h. After removing the glutaraldehyde crystal violet mixture carefully and washing with PBS, the plates with colonies were leaved to dry naturally at room temperature. The number of clones was calculated and plating efficiency was determined for each sample.

Transwell invasion and migration assay
Melanoma cells with indicated transfection were incubated at 37 °C for 36 hours. Then, cells were starved in serum-free medium for 12 additional hours and then trypsinized for reseeding on the top chambers of 24-well transwell culture inserts (Corning). After 24 hours, cells were fixed in 4% paraformaldehyde at room temperature for 10 mins. For invasion assays, transwell chambers with 8 μm-pore size membrane filter inserts (Corning) coated with Matrigel (BD Biosciences) were used to determine cell invasion. The non-motile or non-invasive cells on the upper side of the filter were removed, while the motile or invasive cells on the lower side were stained with crystal violet. For the quantification of the invasive and migratory cells, we used the "Multi-point" tool in ImageJ software to count the stained cells. Five fields for each well were counted under the inverted system microscope (Ti-S, Nikon).

Iron assay
Intracellular ferrous iron (Fe 2+ ) level was accessed using the iron assay kit (cat. ab83366, Abcam, Cambridge, MA, USA) according to the manufacturer's recommendations. Briefly, cells after indicated treatment were collected and washed twice with ice-cold PBS. Cells were homogenized in 5× volumes of iron assay buffer on ice. The supernatant was collected and iron reducer was added to each sample before mixing, and then processed to incubation for 30 mins at 37 °C. Then, the iron probe was added to each sample before mixing, and then  Erastin was used at 10μM in both cell lines. RSL3 was used at 0.5μM in A375 and 1μM in A2058 cell line. Fer-1 was used at 2μM in both cell lines. IFN-γ was used at 50ng/ml in both cell lines. Data represent the mean ± SD of triplicates. P value was calculated by two-tailed Student's t-test. * P < 0.05, ** P < 0.01, *** P < 0.001. ns, non-significant. AgomiR-NC, agomiR-21-3p and antagomiR-21-3p were all used at 100nM in both cell lines.
Erastin was used at 10μM in both cell lines. RSL3 was used at 0.5μM in A375 and 1μM in A2058 cell line. Fer-1 was used at 2μM in both cell lines. IFN-γ was used at 50ng/ml except in the immunoblotting analysis. Data represent the mean ± SD of triplicates. P value was calculated by two-tailed Student's t-test. * P < 0.05, ** P < 0.01, *** P < 0.001. ns, non-significant.