Journal: bioRxiv

Article Title: Antibody-Based Targeting of the SPP1-CD44 Axis in Pediatric High-Grade Glioma through Single-Cell and Structural Bioinformatics

doi: 10.1101/2025.05.01.651763

Figure Lengend Snippet: Cellchat cell-cell communication analysis results. a) Outgoing signal heatmap. The intensity of color represents higher communication probability. b) incoming signal heatmap. The intensity of color represents higher communication probability. c) All outgoing signaling network from MGD TAMs. e) Network topology heatmap of SPP1. f) Violin plots of SPP1 transcript abundance across all clusters.

Article Snippet: Adding anti-SPP1 antibody i.e. bioXcell Clone 100D3 and clone MPIIIB10 showed reversal of immunosuppression TME and enhanced CAR T cell antitumor responses [ ].

Techniques:

Journal: bioRxiv

Article Title: Antibody-Based Targeting of the SPP1-CD44 Axis in Pediatric High-Grade Glioma through Single-Cell and Structural Bioinformatics

doi: 10.1101/2025.05.01.651763

Figure Lengend Snippet: pySCENIC results highlight that no one TF dominates SPP1 gene expression regulation in MGD TAM. a) TF Regulon activity profile of SPP1 transcription factors, here size of the dot shows regulon activity and color shows gene expression correlation of the corresponding TF with SPP1 gene expression. b) TF gene expression dot plot. The size of the dot represents number of cells expressing corresponding TF in each cell type population, and the color represents average expression of the gene within the cell type population, c) MAFB gene expression feature plot showing expression of this gene across all cell types. d) MAFB violin plot showing expression of this gene across all cell types.

Article Snippet: Adding anti-SPP1 antibody i.e. bioXcell Clone 100D3 and clone MPIIIB10 showed reversal of immunosuppression TME and enhanced CAR T cell antitumor responses [ ].

Techniques: Gene Expression, Activity Assay, Expressing

Journal: bioRxiv

Article Title: Antibody-Based Targeting of the SPP1-CD44 Axis in Pediatric High-Grade Glioma through Single-Cell and Structural Bioinformatics

doi: 10.1101/2025.05.01.651763

Figure Lengend Snippet: SPP1 protein structure and sequence analysis results. a) Root mean squared fluctuation of SPP1 protein residues under replica exchange molecular dynamics simulation starting at temperatures 283.15K (black), 303.15K (red), 333.15K (green), and 353.15K (blue). 8, b) Energy landscape-based conformation sampling from four replicate exchange runs. Here y-axis represents radius of gyration values across all 4 replicates and x axis represents RMSD values across all 4 replicates. c) Most stable SPP1 conformation across all 4 replica exchange trajectories. CD44 binding motif (residue 121-140) is shown in orange, 23C3 binding motif is shown in blue, and 2K1 and C2K1 binding motif is shown in cyan. and known SPP1 antibody binding interface shown in blue, and cyan, d) SPP1 protein residue phosphorylation score heatmap. Arrow highlights the top computationally predicted likely phosphorylation site 169 with score > 0.8, e) Sequence alignment of SPP1 across mammals. Orange bar highlights SPP1-CD44 binding motif, blue bar highlights 23C3 binding motif, cyan bar highlights 2K1 and C2K1 binding motif, and red arrow represent phosphorylation site 169 on SPP1 protein sequence. f) Root mean squared fluctuation quadratic mean of normal SPP1 (black) and residue 169 phosphorylated SPP1 (red) across all three independent MD simulation replicates, showing increase in stability in SPP1-CD44 binding interface upon phosphorylation.

Article Snippet: Adding anti-SPP1 antibody i.e. bioXcell Clone 100D3 and clone MPIIIB10 showed reversal of immunosuppression TME and enhanced CAR T cell antitumor responses [ ].

Techniques: Sequencing, Sampling, Binding Assay, Residue, Phospho-proteomics

Journal: bioRxiv

Article Title: Antibody-Based Targeting of the SPP1-CD44 Axis in Pediatric High-Grade Glioma through Single-Cell and Structural Bioinformatics

doi: 10.1101/2025.05.01.651763

Figure Lengend Snippet: Antibody directed evolution results obtained from RosettaAntibodyDesign tool. a) UMAP showing 2500 new antibodies shown as colored dots and the parental 23C3 antibody shown as blast star. Color of the dots highlight energy required to break interaction between SPP1 and the corresponding antibody variant. UMAP is calculated using cosine distance between amino acid sequence esm2 embeddings of CDR regions of two antibody variants. b) Hex plot showing 2500 antibody variants. Here energy required to break SPP1 binding with the corresponding antibody variant is shown as x axis, and cosine distance of amino acid sequence esm2 embeddings of CDR regions of an antibody variant with parental 23C3 amino acid sequence esm2 embeddings of CDR regions. c) Most stable variant (23C3-v1) protein structure (grey) bonded to SPP1 protein structure (wheat). Blue spheres are the residue changes in 23C3-v1 as compared to 23C3 amino acid sequence. d) 23C3-v1 heavy chain amino acid sequence aligned to 10 humanized antibody heavy chain amino acid sequence. Class I and class II epitope sequence region is highlighted using black line and corresponding residue location, e) 23C3-v1 light chain amino acid sequence aligned to 10 humanized antibody light chain amino acid sequence. Class II epitope sequence region is highlighted using black line and corresponding residue location, f) Heavy chain (light green) mutations (red spheres), and light chain (cyan) mutations (blue spheres) induced in 23C3-v1 to neutralize class I and class II epitope. Heavy chain residues 62-68 are shown as green spheres, g) Three independent MD simulation replicates RMSF quadratic mean of 23C3-v1 (black) and Hu23C3-v1 (red) heavy chain, showing no significant change across SPP1 binding region of heavy chain. A sudden increase in fluctuation was observed in residue 62-68, which is distant and upward from SPP1 binding region (shown as green spheres in ), h) Three independent MD simulation replicates RMSF quadratic mean of 23C3-v1 (black) and humanized 23C3-v1 (Hu23C3-v1) (red) light chain, showing no significant change across SPP1 binding region of light chain, i) Box-whiskers plot of 23C3-v1 and Hu23C3-v1 heavy and light chain binding affinity with SPP1 across all three independent MD simulation replicates calculated using gmx_MMPBSA, shows no significant change in Hu23C3-v1 as compared to 23C3-v1 upon epitope neutralization.

Article Snippet: Adding anti-SPP1 antibody i.e. bioXcell Clone 100D3 and clone MPIIIB10 showed reversal of immunosuppression TME and enhanced CAR T cell antitumor responses [ ].

Techniques: Variant Assay, Sequencing, Binding Assay, Residue, Neutralization

Journal: bioRxiv

Article Title: Antibody-Based Targeting of the SPP1-CD44 Axis in Pediatric High-Grade Glioma through Single-Cell and Structural Bioinformatics

doi: 10.1101/2025.05.01.651763

Figure Lengend Snippet: Cellchat cell-cell communication analysis results. a) Outgoing signal heatmap. The intensity of color represents higher communication probability. b) incoming signal heatmap. The intensity of color represents higher communication probability. c) All outgoing signaling network from MGD TAMs. e) Network topology heatmap of SPP1. f) Violin plots of SPP1 transcript abundance across all clusters.

Article Snippet: Recent work showed that using an anti-SPP1 antibody (bioXcell Clone: 100D3) and (clone MPIIIB10) in combination with mIL13Rα2 CAR T cell therapy shows enhanced CAR T cell antitumor response [ ].

Techniques:

Journal: bioRxiv

Article Title: Antibody-Based Targeting of the SPP1-CD44 Axis in Pediatric High-Grade Glioma through Single-Cell and Structural Bioinformatics

doi: 10.1101/2025.05.01.651763

Figure Lengend Snippet: pySCENIC results highlight that no one TF dominates SPP1 gene expression regulation in MGD TAM. a) TF Regulon activity profile of SPP1 transcription factors, here size of the dot shows regulon activity and color shows gene expression correlation of the corresponding TF with SPP1 gene expression. b) TF gene expression dot plot. The size of the dot represents number of cells expressing corresponding TF in each cell type population, and the color represents average expression of the gene within the cell type population, c) MAFB gene expression feature plot showing expression of this gene across all cell types. d) MAFB violin plot showing expression of this gene across all cell types.

Article Snippet: Recent work showed that using an anti-SPP1 antibody (bioXcell Clone: 100D3) and (clone MPIIIB10) in combination with mIL13Rα2 CAR T cell therapy shows enhanced CAR T cell antitumor response [ ].

Techniques: Gene Expression, Activity Assay, Expressing

Journal: bioRxiv

Article Title: Antibody-Based Targeting of the SPP1-CD44 Axis in Pediatric High-Grade Glioma through Single-Cell and Structural Bioinformatics

doi: 10.1101/2025.05.01.651763

Figure Lengend Snippet: SPP1 protein structure and sequence analysis results. a) Root mean squared fluctuation of SPP1 protein residues under replica exchange molecular dynamics simulation starting at temperatures 283.15K (black), 303.15K (red), 333.15K (green), and 353.15K (blue). 8, b) Energy landscape-based conformation sampling from four replicate exchange runs. Here y-axis represents radius of gyration values across all 4 replicates and x axis represents RMSD values across all 4 replicates. c) Most stable SPP1 conformation across all 4 replica exchange trajectories. CD44 binding motif (residue 121-140) is shown in orange, 23C3 binding motif is shown in blue, and 2K1 and C2K1 binding motif is shown in cyan. and known SPP1 antibody binding interface shown in blue, and cyan, d) SPP1 protein residue phosphorylation score heatmap. Arrow highlights the top computationally predicted likely phosphorylation site 169 with score > 0.8, e) Sequence alignment of SPP1 across mammals. Orange bar highlights SPP1-CD44 binding motif, blue bar highlights 23C3 binding motif, cyan bar highlights 2K1 and C2K1 binding motif, and red arrow represent phosphorylation site 169 on SPP1 protein sequence. f) Root mean squared fluctuation quadratic mean of normal SPP1 (black) and residue 169 phosphorylated SPP1 (red) across all three independent MD simulation replicates, showing increase in stability in SPP1-CD44 binding interface upon phosphorylation.

Article Snippet: Recent work showed that using an anti-SPP1 antibody (bioXcell Clone: 100D3) and (clone MPIIIB10) in combination with mIL13Rα2 CAR T cell therapy shows enhanced CAR T cell antitumor response [ ].

Techniques: Sequencing, Sampling, Binding Assay, Residue, Phospho-proteomics

Journal: bioRxiv

Article Title: Antibody-Based Targeting of the SPP1-CD44 Axis in Pediatric High-Grade Glioma through Single-Cell and Structural Bioinformatics

doi: 10.1101/2025.05.01.651763

Figure Lengend Snippet: Antibody directed evolution results obtained from RosettaAntibodyDesign tool. a) UMAP showing 2500 new antibodies shown as colored dots and the parental 23C3 antibody shown as blast star. Color of the dots highlight energy required to break interaction between SPP1 and the corresponding antibody variant. UMAP is calculated using cosine distance between amino acid sequence esm2 embeddings of CDR regions of two antibody variants. b) Hex plot showing 2500 antibody variants. Here energy required to break SPP1 binding with the corresponding antibody variant is shown as x axis, and cosine distance of amino acid sequence esm2 embeddings of CDR regions of an antibody variant with parental 23C3 amino acid sequence esm2 embeddings of CDR regions. c) Most stable variant (23C3-v1) protein structure (grey) bonded to SPP1 protein structure (wheat). Blue spheres are the residue changes in 23C3-v1 as compared to 23C3 amino acid sequence. d) 23C3-v1 heavy chain amino acid sequence aligned to 10 humanized antibody heavy chain amino acid sequence. Class I and class II epitope sequence region is highlighted using black line and corresponding residue location, e) 23C3-v1 light chain amino acid sequence aligned to 10 humanized antibody light chain amino acid sequence. Class II epitope sequence region is highlighted using black line and corresponding residue location, f) Heavy chain (light green) mutations (red spheres), and light chain (cyan) mutations (blue spheres) induced in 23C3-v1 to neutralize class I and class II epitope. Heavy chain residues 62-68 are shown as green spheres, g) Three independent MD simulation replicates RMSF quadratic mean of 23C3-v1 (black) and Hu23C3-v1 (red) heavy chain, showing no significant change across SPP1 binding region of heavy chain. A sudden increase in fluctuation was observed in residue 62-68, which is distant and upward from SPP1 binding region (shown as green spheres in ), h) Three independent MD simulation replicates RMSF quadratic mean of 23C3-v1 (black) and humanized 23C3-v1 (Hu23C3-v1) (red) light chain, showing no significant change across SPP1 binding region of light chain, i) Box-whiskers plot of 23C3-v1 and Hu23C3-v1 heavy and light chain binding affinity with SPP1 across all three independent MD simulation replicates calculated using gmx_MMPBSA, shows no significant change in Hu23C3-v1 as compared to 23C3-v1 upon epitope neutralization.

Article Snippet: Recent work showed that using an anti-SPP1 antibody (bioXcell Clone: 100D3) and (clone MPIIIB10) in combination with mIL13Rα2 CAR T cell therapy shows enhanced CAR T cell antitumor response [ ].

Techniques: Variant Assay, Sequencing, Binding Assay, Residue, Neutralization

Journal: Journal of Translational Medicine

Article Title: Spatial transcriptomics reveals unique metabolic profile and key oncogenic regulators of cervical squamous cell carcinoma

doi: 10.1186/s12967-024-06011-y

Figure Lengend Snippet: Differential analysis of immune characteristics in different tumor regions. ( A ) Volcano plots (P-value and fold change) comparing gene expression between the leading edge and tumor core. Upregulated (orange) and downregulated genes (cyan) are highlighted, and differentially expressed immune-related genes (dark red) or metabolic genes (red) are labelled. Horizontal dashed line indicates adjusted P-value of 0.05, while the vertical dashed lines represent log2FC = -1 and 1. ( B ) Volcano plots (P-value and fold change) comparing gene expression of macrophages between the leading edge and tumor core(left) or normal tissue(right). Upregulated (orange) and downregulated genes (cyan) are highlighted. Horizontal dashed line indicates adjusted P-value of 0.05, while the vertical dashed lines represent log2FC = -1 and 1. ( C ) Macrophages spatial location(left) and spatial expression of SPP1 in leading edge region(right). ( D ) Immunohistochemical images depicting endogenous protein expression levels (brown) of SPP1 in leading edge region of CSCC sample (left) and normal sample (right), with black arrows highlighting macrophages. ( E ) Spatial expression of selected immune checkpoint genes (PD-L1 and IDO1) in normal tissue, leading edge region, and tumor core region. ( F ) Immunohistochemical images depicting endogenous protein expression levels (brown) of PD-L1 and IDO1 immune checkpoint genes in normal tissue, leading edge region, and tumor core region samples. ( G ) GSEA showing enrichment of Oxidative phosphorylation signalling pathwa in cancer cells from the leading edge and tumor core regions

Article Snippet: The primary antibodies are as follows: (1) IDO1 (HUABIO; cat #HA721331; 1:200), (2) CD274/PD-L1 (Proteintech; cat #66248-1-Ig; 1:5000), (3) SPP1 (OriGene; cat #TA806784; 1:150).

Techniques: Expressing, Immunohistochemical staining

Journal: Current Issues in Molecular Biology

Article Title: SPP1 mRNA Expression Is Associated with M2 Macrophage Infiltration and Poor Prognosis in Triple-Negative Breast Cancer

doi: 10.3390/cimb46120806

Figure Lengend Snippet: GSEA results showing the Toll-like receptor signaling pathway is a differentially enriched pathway in the three TNBC. ( A ) KEGG pathway annotations of the Toll-like receptor signaling pathway. ( B ) NES (normalized enrichment score) of each Toll-like receptor signaling pathway-related genes. ( C ) Data for the positive association genes are visualized in a heat map. SPP1 (OPN) is marked with an asterisk.

Article Snippet: Afterward, the slides were rinsed under gently running tap water for 5 min and then placed in a PBS wash bath for 30 min. Immunohistochemistry (IHC) staining was performed using a primary mouse anti-human SPP1 monoclonal antibody (osteopontin/OPN/SPP1 antibody, AKm2A1: sc-21742; Santa Cruz Biotechnology, Inc. , Dallas, TX, USA) applied at a concentration of 1:200.

Techniques:

Journal: Current Issues in Molecular Biology

Article Title: SPP1 mRNA Expression Is Associated with M2 Macrophage Infiltration and Poor Prognosis in Triple-Negative Breast Cancer

doi: 10.3390/cimb46120806

Figure Lengend Snippet: The mRNA expression of SPP1 across various cancer types and their corresponding normal tissues. * p < 0.05, ** p < 0.01, *** p < 0.001. Red boxes denote tumor tissues, while blue boxes represent normal tissues. The red highlights SPP1 expression in tumor tissues, and the blue indicates SPP1 expression in normal tissues. ACC: Adrenocortical carcinoma. BLCA: Bladder urothelial carcinoma. BRCA: Breast invasive carcinoma. CESC: Cervical squamous cell carcinoma and endocervical adenocarcinoma. CHOL: Cholangiocarcinoma. COAD: Colon adenocarcinoma. DLBC: Lymphoid neoplasm diffuse large B-cell lymphoma. ESCA: Esophageal carcinoma. GBM: Glioblastoma multiforme. HNSC: Head and neck squamous cell carcinoma. KICH: Kidney chromophobe. KIRC: Kidney renal clear cell carcinoma. KIRP: Kidney renal papillary cell carcinoma. LAML: Acute myeloid leukemia. LGG: Brain lower-grade glioma. LIHC: Liver hepatocellular carcinoma. LUAD: Lung adenocarcinoma. LUSC: Lung squamous cell carcinoma. MESO: Mesothelioma. OV: Ovarian serous cystadenocarcinoma. PAAD: Pancreatic adenocarcinoma. PCPG: Pheochromocytoma and paraganglioma. PRAD: Prostate adenocarcinoma. READ: Rectum adenocarcinoma. SARC: Sarcoma. SKCM: Skin cutaneous melanoma. STAD: Stomach adenocarcinoma. TGCT: Testicular germ cell tumors. THCA: Thyroid carcinoma. THYM: Thymoma. UCEC: Uterine corpus endometrial carcinoma. UCS: Uterine carcinosarcoma. UVM: Uveal melanoma.

Article Snippet: Afterward, the slides were rinsed under gently running tap water for 5 min and then placed in a PBS wash bath for 30 min. Immunohistochemistry (IHC) staining was performed using a primary mouse anti-human SPP1 monoclonal antibody (osteopontin/OPN/SPP1 antibody, AKm2A1: sc-21742; Santa Cruz Biotechnology, Inc. , Dallas, TX, USA) applied at a concentration of 1:200.

Techniques: Expressing

Journal: Current Issues in Molecular Biology

Article Title: SPP1 mRNA Expression Is Associated with M2 Macrophage Infiltration and Poor Prognosis in Triple-Negative Breast Cancer

doi: 10.3390/cimb46120806

Figure Lengend Snippet: Spearman correlations between the expression of SPP1 and the 32 Toll-like receptor signaling pathway genes in 191 TNBC patients.

Article Snippet: Afterward, the slides were rinsed under gently running tap water for 5 min and then placed in a PBS wash bath for 30 min. Immunohistochemistry (IHC) staining was performed using a primary mouse anti-human SPP1 monoclonal antibody (osteopontin/OPN/SPP1 antibody, AKm2A1: sc-21742; Santa Cruz Biotechnology, Inc. , Dallas, TX, USA) applied at a concentration of 1:200.

Techniques: Expressing

Journal: Current Issues in Molecular Biology

Article Title: SPP1 mRNA Expression Is Associated with M2 Macrophage Infiltration and Poor Prognosis in Triple-Negative Breast Cancer

doi: 10.3390/cimb46120806

Figure Lengend Snippet: Spearman correlation between SPP1 expression and BX795 sensitivity in TNBC cells.

Article Snippet: Afterward, the slides were rinsed under gently running tap water for 5 min and then placed in a PBS wash bath for 30 min. Immunohistochemistry (IHC) staining was performed using a primary mouse anti-human SPP1 monoclonal antibody (osteopontin/OPN/SPP1 antibody, AKm2A1: sc-21742; Santa Cruz Biotechnology, Inc. , Dallas, TX, USA) applied at a concentration of 1:200.

Techniques: Expressing

Journal: Current Issues in Molecular Biology

Article Title: SPP1 mRNA Expression Is Associated with M2 Macrophage Infiltration and Poor Prognosis in Triple-Negative Breast Cancer

doi: 10.3390/cimb46120806

Figure Lengend Snippet: Higher SPP1 mRNA expression had a poor prognosis in BRCA and TNBC. ( A ) Kaplan–Meier survival curve comparing the overall survival (OS) between the low and high SPP1 mRNA expression cohorts in BRCA patients. ( B ) Kaplan–Meier survival curve comparing the relapse-free survival (RFS) between the low and high SPP1 mRNA expression cohorts in BRCA patients. ( C ) Kaplan–Meier survival curve comparing the overall survival (OS) between the low and high SPP1 mRNA expression cohorts in TNBC patients. ( D ) Kaplan–Meier survival curve comparing the relapse-free survival (RFS) between the low and high SPP1 mRNA expression cohorts in TNBC patients.

Article Snippet: Afterward, the slides were rinsed under gently running tap water for 5 min and then placed in a PBS wash bath for 30 min. Immunohistochemistry (IHC) staining was performed using a primary mouse anti-human SPP1 monoclonal antibody (osteopontin/OPN/SPP1 antibody, AKm2A1: sc-21742; Santa Cruz Biotechnology, Inc. , Dallas, TX, USA) applied at a concentration of 1:200.

Techniques: Expressing

Journal: Current Issues in Molecular Biology

Article Title: SPP1 mRNA Expression Is Associated with M2 Macrophage Infiltration and Poor Prognosis in Triple-Negative Breast Cancer

doi: 10.3390/cimb46120806

Figure Lengend Snippet: Immunohistochemical analysis of normal and tumor cell expression of SPP1 from representative samples (100×). Panel shows the number of samples evaluated by the specific SPP1 antibody and that they were positive for SPP1 expression (+) or negative (−). ( A ) The left panel shows representative results of low SPP1 immunostaining in normal breast tissue, while the right panel illustrates high SPP1 immunostaining. ( B ) The left panel displays representative results of low SPP1 immunostaining in breast tumor tissue, while the right panel shows high SPP1 immunostaining. ( C ) SPP1 is not significantly expressed in BRCA tissue compared to its matched normal breast tissue. ( D ) SPP1 is downregulated in TNBC compared to its corresponding normal breast tissues.

Article Snippet: Afterward, the slides were rinsed under gently running tap water for 5 min and then placed in a PBS wash bath for 30 min. Immunohistochemistry (IHC) staining was performed using a primary mouse anti-human SPP1 monoclonal antibody (osteopontin/OPN/SPP1 antibody, AKm2A1: sc-21742; Santa Cruz Biotechnology, Inc. , Dallas, TX, USA) applied at a concentration of 1:200.

Techniques: Immunohistochemical staining, Expressing, Immunostaining

Journal: Current Issues in Molecular Biology

Article Title: SPP1 mRNA Expression Is Associated with M2 Macrophage Infiltration and Poor Prognosis in Triple-Negative Breast Cancer

doi: 10.3390/cimb46120806

Figure Lengend Snippet: SPP1 protein expression had no prognostic significance in BRCA and TNBC. ( A ) Kaplan–Meier plot of the overall survival (OS) was applied to the low and high SPP1 protein cohorts in BRCA patients. ( B ) Kaplan–Meier plot of the relapse-free survival (RFS) was applied to the low and high SPP1 protein cohorts in BRCA patients. ( C ) Kaplan–Meier plot of the overall survival (OS) was applied to the low and high SPP1 protein cohorts in TNBC patients. ( D ) Kaplan–Meier plot of the relapse-free survival (RFS) was applied to the low and high SPP1 protein cohorts in TNBC patients.

Article Snippet: Afterward, the slides were rinsed under gently running tap water for 5 min and then placed in a PBS wash bath for 30 min. Immunohistochemistry (IHC) staining was performed using a primary mouse anti-human SPP1 monoclonal antibody (osteopontin/OPN/SPP1 antibody, AKm2A1: sc-21742; Santa Cruz Biotechnology, Inc. , Dallas, TX, USA) applied at a concentration of 1:200.

Techniques: Expressing

Journal: Current Issues in Molecular Biology

Article Title: SPP1 mRNA Expression Is Associated with M2 Macrophage Infiltration and Poor Prognosis in Triple-Negative Breast Cancer

doi: 10.3390/cimb46120806

Figure Lengend Snippet: Associations between SPP1 mRNA expression and the infiltration of different macrophage subtypes in TNBC: ( A ) M0 macrophages, ( B ) M1 macrophages, and ( C ) M2 macrophages.

Article Snippet: Afterward, the slides were rinsed under gently running tap water for 5 min and then placed in a PBS wash bath for 30 min. Immunohistochemistry (IHC) staining was performed using a primary mouse anti-human SPP1 monoclonal antibody (osteopontin/OPN/SPP1 antibody, AKm2A1: sc-21742; Santa Cruz Biotechnology, Inc. , Dallas, TX, USA) applied at a concentration of 1:200.

Techniques: Expressing