Clinical samples

This study included 20 fresh tumor tissues (10 each in stages I/II and III) and adjacent-normal tissues from OSCC patients admitted to Shanghai Ninth People’s Hospital. Human OSCC tissue microarrays were used for measuring USP30 levels by immunohistochemistry (IHC). The protocols for cancer specimen retrieval had the approval from the medical ethics commission of Shanghai Ninth People’s Hospital (no. SH9H-2022-TK84-1, date: 11 February 2021).

Immunohistochemistry (IHC)

OSCC tissues were fixed in 10% formaldehyde, embedded in paraffin, and sliced into 4 μm sections. The sections were baked in a 60 °C incubator for 1 h, conventionally dewaxed by xylene, dehydrated with gradient alcohol, incubated in 3% H2O2 (Sigma–Aldrich Chemical Company, St Louis, MO, USA) at 37 °C for 30 min, and washed with phosphate-buffered saline (PBS; Gibco, Invitrogen, Paisley, UK). The sections were then boiled in 10 mM citric acid buffer at 95 °C for 20 min, cooled to room temperature, and washed with PBS. Sections were incubated with anti-USP30 antibody (Abcam; ab235299), followed by HRP-conjugated anti-IgG antibody (Long Island Biotech, China, D-3004) using the standard protocol. IHC results were analyzed by two pathologists and scored based on percentage of positively stained cells. OSCC patients were divided into the low expression (H-score < 50%) group and the high expression (H-score > 50%) group.

Cell culture

Human oral epithelial cell HOEC (Procell Life Science & Technology Co., Ltd., China; CP-H203) and human OSCC cell lines SCC4 (CRL-1624), SCC9 (CRL-1629), SCC15 (CRL-1623), CAL27 (CRL-2095; all from ATCC, USA), and HSC4 (RIKEN Cell Bank, Tukuba, Japan; RCB1902) were maintained in DMEM with 10% FBS at 37 °C.

USP30 silencing or overexpression

shRNA targeting USP30 (shUSP30-1, 5′-GCTGCTTGTTGGATGTCTT-3′; shUSP30-2, 5′-CCAGAGTCCTGTTCGATTT-3′) were inserted into pLKO.1. USP30 cDNA was inserted into pLVX-puro. The recombinant plasmids were transfected and amplified in 293 T cells (ATCC; CRL-3216) along with the packaging plasmids psPAX2 and pMD2G to generate lentiviral particles. Transfection was performed using Lipofectamine 2000 (Invitrogen, Carlsbad, CA, USA) according to the manufacturer’s manual. The lentiviral particles were collected 48 h after transfection and used to transduce OSCC cell lines. Blank pLVX-Puro (vector) and pLKO.1-scrambled shRNA (shNC) were used as negative controls for overexpression and knockdown studies, respectively. C-terminally flag-tagged human USP30 DUB-domain cDNA (wild type or the C77S mutant) expression vectors were constructed as previously described [18].

Preparation of MF-094@ZIF-8

MF-094@ZIF-8 was synthesized as follows: 180 mg 2-methylimidazole (2-MIM) and 20 mg Zn(NO3)2 6H2O were dispersed in 1 mL methanol. Then, 0.5 mL MF-094 and 2-MIM was mixed. Next, Zn(NO3)2 6H2O was quickly injected and mixed for 1.5 h. The product was centrifuged at 7000 rpm for 5 min and washed with methanol. MF-094@ZIF-8 was collected.

Preparation of MF-094@ZIF-8-PDA

The MF-094@ZIF-8 (50 mg) and 3-hydroxytyramine hydrochloride (10 mg) were poured into 50 mL pH 8.5 Tris–HCl buffer and stirred for 3 h. The mixed solution was spun at 10,000 rpm to get MF-094@ZIF-8-PDA nanoparticles (NPs) and washed with deionized water three times and freeze dried.

Preparation of MF-094@ZIF-8-PDA-PEGTK NPs

The MF-094@ZIF-8-PDA NPs (50 mg/mL) solution and NH2-PEG-NH2 (50 mg/mL) aqueous solution were mixed in deionized water with 1-ethyl-3-(-3-dimethyl aminopropyl) carbodiimide hydrochloride (EDC, 50 mg/mL) and N-hydroxysuccinimide (NHS, 50 mg/mL) and stirred for another 24 h. The mixture was spun at 10,000 rpm to get MF-094@ZIF-8-PDA-PEGTK NPs and washed with deionized water three times.

NP characterization

NPs were observed by a transmission electron microscopy (TEM). The Brunauer–Emmett–Teller (BET) method was used for evaluating surface areas. DynaPro NanoStar (Wyatt, USA) was used to measure the size distribution of NPs. Thermogravimetric analysis (TGA) was used for evaluating the loading capacity of NPs. The chemical composition of NPs was examined with Fourier transform infrared (FT-IR).

Cellular uptake behavior

We used confocal laser scanning microscopy (CLSM) to evaluate the cellular uptake ability of MF-094@ZIF-8-PDA-PEGTK by HSC4 cells. Briefly, HSC4 cells were seeded in a 24-well plate at a density of 3 × 104 cells/well and incubated with 50 μg/mL of Cy7-labeled MF-094@ZIF-8-PDA-PEGTK. After incubation for 6 h, the nuclei, cytoskeleton, and MF-094@ZIF-8-PDA-PEGTK was stained with 4′,6-diamidino-2-phenylindole (DAPI; Beyotime Biotechnology, Shanghai, China), fluorescein isothiocyanate (FITC)-phalloidin (Abcam), and Cy7 (MedChemExpress) for 40 min, respectively, and observed via CLSM at 405, 562, and 488 nm laser excitation. TEM detection for samples fixed with 4% glutaraldehyde was performed as previously described [19].

Quantitative RT–PCR (qRT–PCR)

RNA was isolated using TRIzol (Invitrogen, Carlsbad, CA, USA; 15596018) and reverse transcribed into cDNA using the RevertAid First Strand cDNA Synthesis Kit (Thermo Fisher Scientific, Waltham, MA, USA; K1622). The qRT–PCR was performed using a Maxima SYBR Green/ROX qPCR Master Mix (Thermo Fisher Scientific, Waltham, MA, USA; K0223). The primers sequences were as follows: USP30, 5′-CCCAGACAGAACAGAAACAG-3′ and 5′-AAGAGCAGCAGCAATTCC-3′; c-Myc, 5′-AGGCTGCGGCTTGGATTAAC-3′ and 5′-CAAACCCTCTCCCTTTCTC-3′; GAPDH, 5′-AATCCCATCACCATCTTC-3′ and 5′-AGGCTGTTGTCATACTTC-3′. Reaction conditions were: 95 °C for 10 min, followed by 40 cycles of 95 °C for 15 s, 60 °C for 45 s. The fold change was calculated by the 2−△△CT method.

Immunoblotting

Proteins were isolated with RIPA buffer and the protein concentrations were quantified using a BCA Protein Assay Kit (Beyotime Biotechnology, Shanghai, China; P0012S). Proteins were separated with SDS-PAGE, electro-blotted to membranes, blocked, and probed with anti-USP30 (ab235299), anti-c-Myc (ab32072), anti-GLS1 (ab131554), anti-SLC1A5 (ab187692), and anti-β-actin antibodies (ab8227; all from Abcam) in accordance with the manufacturer’s instruction. After incubating with primary antibodies, the membranes were incubated with HRP-conjugated secondary antibodies (Beyotime Biotechnology, Shanghai, China; A0208, A0216). Chemiluminescence (Merck Chemicals, Co., Ltd, Shanghai, China; WBKLS0100) was used for detecting antibody binding. Image J analysis software was used to quantify the gray levels of each band for protein study. β-Actin served as a loading control.

Cell viability

Cell viability was measured using cell counting kits (CCK)-8 (Dojindo Molecular Technologies, Kumamoto, Japan; CK04). Briefly, cells were grown to the logarithmic phase, harvested, and seeded into 96-well plates at a cell density of 3 × 103 cells per well. At 12, 24, and 48 h after treatment, 10 μL CCK-8 solution was added to each well and maintained for the reaction time of 1 h. Absorbance was measured at 450 nm using a microplate reader and used for the calculation of cell viability.

Apoptosis assay

Cells were kept in a 6-well plate (3 × 105/ well) until 50% confluence. After treatment, the cells were collected and incubated with 5 µL of recombinant annexin V labeled with fluorescein isothiocyanate (Annexin V-FITC) for 15 min in the dark at 4 °C, and then with 5 µL Propidium iodide (PI) (Beyotime Institute of Biotechnology) for another 15 min. Apoptosis was examined using a FACScan flow cytometer (BD, Franklin Lakes, NJ, USA).

Glutamine assay

After treatment, the levels of glutamine in cells were analyzed with a glutamine assay kit (Abcam; ab197011) following the manufacturers’ instructions. According to the principle of glutamine conversion into glutamic acid and ammonia, the amount of glutamine was calculated by measuring the amount of ammonia. The relative glutamine uptake was normalized by the protein amount of each group.

Co-immunoprecipitation (Co-IP)

Cell lysates were prepared with lysis buffer (1% Triton X-100, 150 mM NaCl, 20 mM Tris pH7.5, and 1 mM EDTA) supplemented with protease inhibitor cocktail (Sigma-Aldrich), incubated with normal immunoglobulin (Ig)G (Santa Cruz Biotechnology; sc-2027), anti-USP30 (Invitrogen; PA5-113053), or anti-c-Myc (Abcam; ab168727) antibodies, followed by further incubation with Protein A/G PLUS-Agarose beads (Santa Cruz Biotechnology; sc-2003) at 4 °C for 2 h. Then, the immunocomplex was washed three times by the lysis buffer for Western blot analysis.

Half-life of c-Myc

HSC4 cells were infected with lentivirus expressing shUSP30 or control shRNA (shNC) for 24 h, then exposed to cycloheximide (CHX, 20 mM, Sigma-Aldrich). Cells were collected at 0, 1, 4, or 8 h after exposure and subjected to immunoblotting.

Animal model

Male nude mice (4 weeks old) were subcutaneously injected with 1 × 106 SCC4 cells. Ten days after inoculation, MF-094 or Cy7-labeled NPs loaded with or without MF-094 were injected into the mice via the tail vein, at a dose of 1 mg/kg/day for 4 weeks. Herein, four experiment groups were established (n = 10 mice per group), including mice bearing SCC4 cells as control (group 1), mice bearing SCC4 cells injected with MF-094 (group 2), and mice bearing SCC4 cells injected with NPs loaded with (group 3) or without MF-094 (group 4). NP fluorescent images were obtained using the IVIS 200 system (PerkinElmer, Waltham, MA, USA) at 6, 12, and 24 h to verify the NP biodistribution. In vivo surviving SCC4 cells were detected using bioluminescence at 0, 1, 2, 3, and 4 weeks to verify the in vivo targeting efficacy of NPs. Tumor volume was recorded every 4 days. Mice were euthanized on day 38, and their key organs (liver, heart, lungs, kidneys, and spleen) and xenograft tumors were collected, washed, fixed with 4% paraformaldehyde (PFA), and processed to produce paraffin-embedded sections. To evaluate toxicity and apoptosis, sections were stained with H&E or TUNEL. Animal experiments were performed in concordance with the NIH Guidelines and approved protocols of Animal Care and Use Committee of Shanghai Rat@Mouse Biotech Co., Ltd., China (no. 2020-0213, date: 15 March 2020).

Statistical analysis

Data are presented as the mean ± standard deviation (SD) from at least three independent experiments. Statistical analysis was performed using GraphPad Prism 8.4.2 (San Diego, CA). Student’s t-test and ANOVA were applied for comparisons. P-values < 0.05 were defined significant.