Co‐delivery Nano System of MS‐275 and V‐9302 Induces Pyroptosis and Enhances Anti‐Tumor Immunity Against Uveal Melanoma

Abstract In the treatment of uveal melanoma (UVM), histone deacetylase inhibitors (HDACi) have emerged as a promising epigenetic therapy. However, their clinical efficacy is hindered by the suboptimal pharmacokinetics and the strong self‐rescue of tumor cells. To overcome these limitations, reactive oxygen species (ROS)‐responsive nanoparticles (NPs) are designed that encapsulate HDACi MS‐275 and the glutamine metabolism inhibitor V‐9302. Upon reaching the tumor microenvironment, these NPs can disintegrate, thereby releasing MS‐275 to increase the level of ROS and V‐9302 to reduce the production of glutathione (GSH) related to self‐rescue. These synergistic effects lead to a lethal ROS storm and induce cell pyroptosis. When combined with programmed cell death protein 1 monoclonal antibodies (α‐PD‐1), these NPs facilitate immune cell infiltration, improving anti‐tumor immunity, converting “immune‐cold” tumors into “immune‐hot” tumors, and enhancing immune memory in mice. The findings present a nano‐delivery strategy for the co‐delivery of epigenetic therapeutics and metabolic inhibitors, which induces pyroptosis in tumors cells and improves the effectiveness of chemotherapy and immunotherapy.


Instrumentation and methods:
The size distribution of nanoparticles was measured by dynamic light scattering (DLS, Malvern Zetasizer, UK).The morphology and size of nanoparticles were characterized by transmission electron microscope (TEM, Hitachi HT7700, Japan).
Localization of nanoparticles and all the immunofluorescence slides were imaged using a confocal laser scanning microscope (CLSM, LSM 800, ZEISS, Germany).MTT assay was conducted using a microplate reader (SpectraMax, USA).In vivo imaging was conducted by In Vivo Imaging System (IVIS, Perkin Elmer, USA).Fluorescence intensity in cells was carried out using a CytoFLEX Flow Cytometry (Beckman Coulter, USA).

In vitro cellular uptake of NPs by CLSM and flow cytometry (FCM)
A cover slide was placed in the bottom of each well of a 24-well plate.Cells (1×10 5 ) in 1 mL medium were added to each well and incubated at 37 °C for 12 h.Then, the cells were incubated with NPs-Cy5.5 diluted in cell media for various time intervals (1 h, 4 h, 7 h).Next, the cells were washed with phosphate-buffered saline (PBS) and further incubated with the nucleus specific stain DAPI (ab285390, Abcam) and the cytoskeleton specific stain Alexa-488 (1:500, Beyotime) for 0.5 h.Subsequently, the cellular uptake was assessed by CLSM (DAPI, λex = 405 nm, λem = 460 nm, Cy5.5, λex = 673 nm, λem = 692 nm).The Zen software was used to analysis and draw the pictures.
To perform flow cytometry, cells were seeded into 12-well plate at 3×10 5 cells/well and incubated at 37 °C for 12 h.The NPs-Cy5.5 were added to each well in different time points (1 h, 4 h, 7 h), and the wells without any treatment were performed as negative control.Afterwards, the cells were harvested and quantified using FCM.The flowjo software was used to analysis.

In vitro cytotoxicity study
MTT assay was used to examine the cytotoxicity.Cells were seeded into 96-well plates (8×10 3 cells/well) and then incubated at 37 ℃ overnight.Then, the cells were treated with MS-275, V-9302, MS-275+V-9302, NPs for 24/48 h, respectively.MTT reagent in DMEM (10 μL) was added into each well and allowed to incubate for 4 h.10% SDS (100 μL) was added to replace the media.After gentle agitation (5 mins), the absorbance of each well was recorded on a microplate reader (Spectra Max) at 570 nm (peak absorbance) and 650 nm (peak background).
After 24 h, cells were detected by flow cytometry (Becton Dickinson and Company, USA), using Cell Cycle and Apoptosis Analysis Kit (Beyotime, C1052).

Intracellular ROS generation
A cover slide was placed in the bottom of each well of a 24-well plate.Cells (1×10 5 ) in 1 mL complete media were added to each well and incubated at 37 °C for 12 h.Afterward, the cells were treated with MS-275(10 μM), V-9302 (2 μM), MS-275(10 μM) + V-9302 (2 μM), NPs (10 μM) at the photosensitive unit for 7 h respectively.Subsequently, the culture medium of the cells was replaced with a serum-free medium and then incubated with ROS indicator DCFH-DA (10 μM) for 20 mins.Then the cover slide of each well was placed on the microslide, and the cell nuclei were stained with DAPI.Subsequently, images were collected with CLSM.
Furthermore, the intracellular ROS level was further detective and quantify by FCM.First, cells were seeded in 12-well plate at a density of 3 ×10 5 per well and incubated at 37 °C for 12 h.Afterward, the cells were treated with the same conditions as the above CLSM analysis.Finally, the cells were harvested to examine the intracellular DCF by FCM.
PD-L1: The above cells were washed with PBS and further incubated with PD-L1 Rabbit Polyclonal Antibody for 2 h at 37 ℃.Subsequently, the cells were incubated with Goat Anti-Rabbit IgG H&L (Alexa Fluor ® 555) for 1 h at 37 ℃.The expression of PD-L1 inside the cells was observed by CLSM.MitoSox: The above cells were washed with PBS and further incubated with MitoSox for 2 h at 37 ℃.Then the cover slide of each well was placed on the microslide, and the cell nuclei were stained with DAPI.Subsequently, images were collected with CLSM.

Western blot
OCM-1 cells were seeded in 6-well plates (6×10 5 cells/well) and allowed to adhere overnight.The cells were treated with MS-275(10 μM), V-9302 (2 μM), MS-275(10 μM) + V-9302 (2 μM), NPs (10 μM) for 24 h.Cells were washed three times with cold PBS and maintained in medium for 0.5 h.RIPA lysis buffer with protease and phosphatase inhibitors was added into well.The proteins of cells were extracted through centrifuge at a speed of 12000 rpm for 5 mins.Protein content quantification was carried out by the BCA protein assay kit (Beyotime, P0011).Then, the electrophoreses process was conducted through SDS-PAGE by a gel-electrophoretic apparatus (Bio-Rad mini, USA), and the proteins were transferred to the PVDF films and incubated with the antibodies against various proteins overnight on a shaker at 4℃.Subsequently, the PVDF films were washed 5 times and incubated with HRP conjugated antibodies for 1 h.The Western blot images were obtained by Amersham Imager 600 (AI600, General Electric Co., Ltd., USA) with 300 μL of ECL chemiluminescent reagent (Beyotime biotechnology Co., Ltd., P0018AS) added on the top of the membrane.

In vivo biodistribution analysis
The biodistribution of nanoparticles was investigated by in vivo imaging system (IVIS, Spectrum CT，PerkinElmer).OCM-1 cells (5×10 6 cells) were injected into right buttock subcutaneously of BALB/c nude mice.When the tumor volumes reached about 80 mm 3 , the mice were injected with NPs-Cy7.5 intravenously.After injection, the fluorescence imaging was performed by IVIS spectrum imaging system (Ex/Em=745 nm/840 nm) at various time points.Mice were sacrificed at 48 h post injection for fluorescence imaging of the major organs (heart, liver, spleen, lung, intestine, and kidney) and tumor tissues ex vivo.

Establishment subcutaneous tumor model of OCM-1 and therapeutic effect evaluation
To investigate the antitumor effect of nanoparticles, OCM-1 cells (5×10 6 cells) were injected into right buttock subcutaneously of BALB/c nude mice.When the tumor volumes reached about 80 mm 3 , the mice were injected with Saline, Cisplatin, MS-275+V-9302, and NPs at the dose of 4 mg/kg intravenously.The tumor volume was monitored at a time interval of 2 days.Tumor volume was calculated by the following formula: Volume = (Length * Width 2 ) / 2.

Immune response analysis in vivo
To examine immune response in vivo, B16-F10 cells (2 × 10 6 cells) were injected into right buttock subcutaneously of C57BL/6 mice, the mice bearing the subcutaneous tumor model of B16-F10 were treated with various formulations.The tumors, tumor draining lymph nodes (TDLNs) and spleens were harvested after treatment.

Statistical Analysis
GraphPad Prism 9 (GraphPad, La Jolla, CA, USA) was used for statistical analysis.
Data were presented as mean ± SD from at least 3 independent experiments of biological replicates, if not stated in the figure legend.Data were analyzed using twosided Student's t-test when two groups were being compared.One-way or two-way analysis of variance (ANOVA) was used when more than two groups were compared (multiple comparisons).The difference was regarded as significant when the p value was less than or equal to 0.05.*p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001, ns, not significant.Data were from three independent experiments.Datas were presented as mean ± SD.
Statistical significance between all groups was calculated via one-way ANOVA.****p < 0.0001, ns, not significant.n = 5 mice per group.In the heart sections of each group, the cardiomyocytes were neatly arranged, the distribution of nuclei was centered and clear, and the morphology was complete.In liver tissue sections of each group, the structure of the liver lobules was clear, and the hepatocytes were arranged in a radial pattern centered on the central vein to form the liver plate.In the spleen sections of each group, the structure of the germinal center was complete, and the cells were neatly arranged.In the lung tissue sections of each group, the shape of alveoli was intact, no obvious thickening of alveolar wall was seen, and a few red blood cells were seen in the alveoli.In the kidney tissue sections of each group, the glomerular structure was intact, and no obvious degeneration or edema was seen in the renal interstitium.

Figure S6 .Figure S7 .
Figure S6.Relative cell viabilities of OCM-1 with treatment of NPs for 24 h, NPs for 48 h, MS+V for 24 h, MS+V for 48 h via MTT assay, respectively.

Figure S8 .
Figure S8.Relative expression of proteins in the mTOR pathway.a) Relative expression of Akt.b) Relative expression of P-Akt.c) Relative expression of mTOR.d) Relative expression of P-mTOR.e) Relative expression of P-70S6.e) Relative expression of P-S6.β-Actin was used as the internal reference protein.Data were from three independent experiments.Datas were presented as mean ± SD.Statistical significance between all groups was calculated via one-way ANOVA.*p < 0.05, **p < 0. 01, ***p < 0.001, ns, not significant.

Figure
Figure S9.a) CLSM images of ROS in cells after various treatments.(blue, DAPI; green, ROS).b) Flow cytometric profiles and c) Quantification of ROS in OCM-1 cells for 24 h treatment of MS-275, V-9302, MS-275+V-9302 and NPs.Data were from three independent experiments.Datas were presented as mean ± SD.Statistical significance between all groups was calculated via one-way ANOVA.****p < 0.0001.

Figure S11 .
Figure S11.Original western blot images of a, b, c) full length of GSDMD (FL-GSDMD), N-terminal of GSDMD (NT-GSDMD) and d, e, f) β-Actin after different treatments for 24 h in OCM-1 cells.Figures S11a and S11e are used in Figure 4h in the main text.

Figure S12 .
Figure S12.H&E staining of major organs (heart, liver, spleen, lung, and kidney) of BALB/c nude mice treated with saline, MS+V, cisplatin, or NPs at 4 mg/kg body weight.

Figure S16 .
Figure S16.The percentage of mature DCs populations (CD80 + CD86 + ) within the TDLNs in each group are presented as histograms.Statistical significance between all groups was calculated via one-way ANOVA.****p < 0.0001.

Table S2 .
IC50 (95%CI) of OCM-1 with treatment of NPs for 24 h, NPs for 48 h, MS+V for 24 h and MS+V for 48 h.