PSME2 offers value as a biomarker of M1 macrophage infiltration in pan-cancer and inhibits osteosarcoma malignant phenotypes

A growing number of studies have revealed an association between proteasome activator complex subunit 2 (PSME2) and the progression of various forms of cancer. However, the effect of PSME2 on osteosarcoma progression is unknown. Pan-cancer analyses focused on the immunological activity and prognostic relevance of PSME2 have yet to be conducted. The Cancer Genome Atlas and Genome-Tissue Expression databases were leveraged to evaluate PSME2 expression and activity across 33 cancer types. Significant PSME2 dysregulation was noted in a wide range of cancer types and this gene was found to offer significant diagnostic and prognostic utility in most analyzed cancers. From a mechanistic perspective, PSME2 expression levels were correlated with DNA methylation, DNA repair, genomic instability, and TME scores in multiple cancer types. PSME2 was subsequently established as a pan-cancer biomarker of M1 macrophage infiltration based on a combination of bulk, single-cell, and spatial transcriptomic data and confirmatory fluorescent staining results. In osteosarcoma cells, overexpressing PSME2 significantly suppressed tumor proliferative, migratory, and invasive activity. Screening efforts also successfully identified the PSME2-activating drug irinotecan, which can synergistically promote the death of osteosarcoma cells when combined with the chemotherapeutic drug paclitaxel. As a biomarker of M1 macrophage infiltration, PSME2 expression levels may offer insight into tumor development and progression for a wide range of cancers including osteosarcoma, emphasizing its potential utility as a prognostic and therapeutic target worthy of further study.


Supplemental Figures For: PSME2 offers value as a biomarker of M1 macrophage infiltration in pan-cancer and inhibits malignant phenotypes of osteosarcoma
LGG, LUAD, HNSC, LUSC, and STAD tumors.

Multiple Fluorescence Staining of Pan-Cancer Tissue Chip
Multiple fluorescence staining was performed on pan-cancer paraffin sections to validate the M1 macrophage biomarker potential of PSME2.The sections in this study contained 10 cancer types.
These sections were deparaffinized and blocked with 5% bovine serum albumin (BSA).

Cell lines and culture
Human osteosarcoma cell lines (HOS and U2OS) were purchased from the American Type Culture Collection (ATCC).Cells were maintained in DMEM/F12 containing 10% fetal bovine serum and 1% penicillin/ streptomycin (Invitrogen, USA) in a humidified incubator at 37 °C with 5% CO2.

Cell transfection
Cells were cultured in 6-well plates until 40% confluent after which they were transduced with lentiviral (Genechem) for 24 h and selected with puromycin (3 mg/mL) for 24 hours.
Transfections with lentivirus were performed with a multiplicity of infection (MOI)=20 in HOS cell line and a MOI=10 in U2OS cell line.Stability of expression was determined.

RNA isolation and qRT-PCR
Total RNA was extracted from transfected cells with TRIzol (Thermo Fisher Scientific) according to the provided directions.Gene expression relative to 18S rRNA was measured by qRT-PCR using an IQ5 Multicolour Real-Time PCR Detection system (Bio-Rad Laboratories, CA, USA).

Western blotting
Proteins were extracted from cells in RIPA buffer with a protease and phosphatase inhibitor cocktail (Keygen, Nanjing, China).After determination of concentrations using BCA, the proteins were separated on SDS-PAGE and transferred to nitrocellulose.The blots were treated with anti-PSME2 (1:3000) (ab183727, Abcam, UK) and anti-GAPDH (1:5000) (ab181602, Abcam, UK) antibodies and the bands were visualized using enhanced chemiluminescence.The loading control was GAPDH.

Cell proliferation assays
CCK-8 assays were performed by seeding and maintaining 6000 cells in 96-well plates for 1, 2 or 3 days.Fresh medium was replaced with 100µL, and CCK-8 (10µL, Sigma) was added.
Cells were then incubated for 2 hours for OD 450 measurement.Cells were fixed and permeabilized after EdU was added to culture medium at 10 μM and incubated for 2h.Click-iT reaction was performed following the manufacturer's manual (ThermoFisher Scientific).As a final step, nuclear staining was carried out using DAPI, and images were obtained using a fluorescence microscope (ThermoFishr Scientific).In colony formation assays, 600 cells were seeded and cultured for 10 days in 6-well plates.Next, they were washed with PBS and fixed with 4% paraformaldehyde for 30 minutes.Cells were stained with 0.5% crystal violet for 1 hr at room temperature, and colonies were counted with clone-counter program.
The viability of irinotecan alone or in combination with paclitaxel in osteosarcoma cells was analysed by the CCK8 assay.Briefly, HOS or U2OS cells underwent seeding process in a final volume of 100 μL in a 96 well plate at 3000 cells/well density and cultured for 24 hours.After adding different concentrations of irinotecan and/or paclitaxel, the cells were further cultured for another 48 hours.Then, we dropped CCK-8 regent (10µL, Sigma) in every well, and the culture plate underwent incubation as shielded from light for 2 hours at 37°C.We ascertained optical density (OD) value at a wavelength of 450 nm.Using the median-effect model of Chou-Talalay and with CompuSyn software, we ascertained the combination index (CI) 2 .CI < 1 indicates synergism.Each experiment was carried out in triplicate.
Wound-healing assay 10 6 cells were seeded per well in a six-well plate after being trypsinized.Micropipettes were scraped with sterile tips after overnight incubation.We photographed the initial gap width and the residual gap width 24 hours after scratching.

Transwell assays
Migration assays were carried out in Transwell chambers 8 mm in diameter, Corning.For the invasion assay, growth factor-reduced Matrigel (354234; Corning) was precoated on the chambers before the migration assays.A suspension of osteosarcoma cells in 200 μL serum-free DMEM medium was added to the upper chamber, while 10% fetal bovine serum was added to the bottom chamber.After 24 hours of cultivation, cells in the chamber were fixed with 4% paraformaldehyde for 30 minutes and stained with 0.1% crystal violet.We viewed cells under an inverted light microscope in three random fields of view that had migrated or invaded to the lower surface.

In vivo growth
Animal experiments were carried out in accordance with the Guide for the Care and Use of Laboratory Animals (Ministry of Science and Technology of China, 2006), and were approved by the animal ethics committee of Shanxi Medical University (approval number DW2022054).
Five-week-old BALB/c nude mice were obtained from Charles River (China).Randomly selected mice received subcutaneous injections of 10 6 lentivirus-transfected HOS cells in 200 μL Matrigel (n = 6 per group).Tumor growth was assessed each day.The mice were euthanized after five weeks, and the tumors were removed and measured 3 .

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Figure S3 Pan-cancer diagnostic ROC curves.

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Figure S4 Kaplan-Meier curves representing associations between PSME2 levels and the OS, DSS, DFI, and PFI of patients with the indicated cancer types.

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Figure S5 Pan-cancer analyses of the association between PSME2 levels and promoter methylation.

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Figure S6 Correlations between PSME2 methylation and prognostic outcomes in different cancers.(A-D) Kaplan-Meier curves highlighting associations between the degree of PSME2 methylation and OS (B), DSS (C), DFI (D), and PFI (E) for the indicated cancer types.(E) Scatter plots representing relationships between levels of PSME2 methylation and markers of CTLs in specific cancer types.

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Figure S7 (A) PSME2_ AA_26864 PSI values in pan-cancer, adjacent, and normal tissues.Colored labels correspond to cancers and matching adjacent tissues.(B) Differences in PSI values between tumors and adjacent/normal tissues and the association between PSME2_AA_26864 events and prognostic outcomes.(C) A network of experimentally validated protein-protein interactions for PSME2.(D) Enriched GO pathways of the top 100 genes co-expressed with PSME2.

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Figure S8Correlations between PSME2 levels and oncogenic pathway signatures.

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Figure S9 Associations between PSME2 expression and pan-cancer immunity.(A) Scatter plots for the 6 tumor types with the strongest correlations between PSME2 expression levels and Immune score values.(B) PSME2 expression levels in the indicated immune subtypes of BRCA,

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Figure S11 (A) Box plots representing PSME2 expression before and after cytokine treatment in the indicated tumor cell lines as analyzed with the TISMO tool.(B) Box plots representing the expression of PSME2 before and after treatment with ICIs (anti-PD1, anti-PDL1, and anti-CTLA4) as analyzed with the TISMO tool.*P < 0.05, **P < 0.01, ***P < 0.001.

Figure S12
Figure S12 Correlation between PSME2 expression and the infiltration of immune cells.(A-D) Heatmap showing the association between PSME2 expression and the infiltration of immune cells in pan-cancer generated by using MCPCOUNTER algorithm (A), Xcell algorithm (B), ssGSEA algorithm from the ImmuCellAI database (C), and CIBERSORT algorithm (D).

Figure
Figure S13 (A) TIMER2.0 was used to calculate the association between PSME2 expression and the infiltration of CD8+ T-cell infiltration in different cancers using multiple algorithms.(B) The association between PSME2 promoter methylation and M1 macrophage infiltration.
Eschenbach AC, Giavazzi R, Fidler IJ.Growth and metastasis of tumor cells isolated from a human renal cell carcinoma implanted into different organs of nude mice.
1.Livak KJ, Schmittgen TD.Analysis of relative gene expression data using real-time