ic808p  (R&D Systems)

Journal: Genes & Diseases

Article Title: SPP1 + macrophages in colorectal cancer: Markers of malignancy and promising therapeutic targets

doi: 10.1016/j.gendis.2024.101340

Figure Lengend Snippet: Tissue distribution and potential mechanisms of SPP1 + macrophages. (A) Bar graphs of the ratio of SPP1 + macrophages to all macrophages in the six scRNA-seq datasets (NC vs . CRC). The sample size is shown above the bar. Mean ± standard error of the mean; ∗ P < 0.05, ∗∗ P < 0.01, ∗∗∗ P < 0.001, ∗∗∗∗ P < 0.0001; ns, not significant; Mann–Whitney U test. (B) Bar graphs of the ratio of SPP1 + macrophages to all macrophages in the three scRNA-seq datasets (CRC vs . LM). (C) Bar graphs of the ratio of SPP1 + macrophages to all macrophages in different tissue types (CRC, NC, LM, NL, and HCC) in the GSE164522 and GSE156625 datasets. (D) Pie charts of the proportions of four macrophage subsets in the CRC-Mix dataset. (E–G) The expression of (E) EMT, (F) glycolysis, and (G) hypoxia signatures in the three major macrophage subsets in the CRC-Mix dataset. (H) Heatmap of cellular interactions among the four macrophage subsets and six major cell types (GSE178341). (I) Receptor‒ligand interactions among four macrophage subsets and six major cell types (GSE178341). P values are indicated by circle size; colors indicate the average expression levels of ligands and receptors or molecules in the corresponding cell types. SPP1, secreted phosphoprotein 1; CRC, colorectal cancer; NC, normal colorectum (adjacent colorectum); LM, liver metastases; NL, normal liver (adjacent); HCC, hepatocellular carcinoma; EMT, epithelial–mesenchymal transition.

Article Snippet: The primary antibodies used for validating SPP1 + macrophages were as follows: mouse anti-human CD68 (Proteintech, Cat# 66231-2-Ig, 1:2000) and mouse anti-human SPP1 (Santa Cruz Biotechnology, Cat# sc-21742, 1:1000).

Techniques: MANN-WHITNEY, Expressing

Journal: Genes & Diseases

Article Title: SPP1 + macrophages in colorectal cancer: Markers of malignancy and promising therapeutic targets

doi: 10.1016/j.gendis.2024.101340

Figure Lengend Snippet: SPP1 is a novel macrophage marker in CRC. (A) Heatmap of the proportion of M0, M1, and M2 macrophages (CIBERSORT), immune score (ESTIMATE), expression of markers ( CD45 , CD68 , CD86 , iNOS , CD163 , CD206 , and SPP1 ), CD68/CD45 , and SPP1/CD68 in 10 bulk RNA-seq datasets (NC vs . CRC). The color gradient of each tile signifies changes in the marker expression level, where red represents an increase in CRC, blue represents a decrease, and the depth of the color reflects the statistical significance. Additional information provided includes the analysis method (right), dataset source (top), and the sample sizes of NC and CRC for each dataset (bottom). (B) Quantitative real-time PCR analysis of SPP1 mRNA expression between 50 paired NC and CRC samples. Wilcoxon test; ∗∗∗ P < 0.001. (C) Violin plots of the expression of SPP1 in CD45 − cells, other immune cells, other myeloid cells, and SPP1 + macrophages (GSE178341). (D) UMAP plot of the distribution of CD45 − cells, other immune cells, other myeloid cells, and SPP1 + macrophages (left). UMAP of iterative subsets of cells from the global level to immune cells to myeloid cells shows the enrichment of SPP1 expression levels in different cells (right). (E) Images of immunohistochemical staining showing SPP1 protein expression in NC, CRC, NL, and HCC samples. Data are from The Human Protein Atlas. Scale bar, 200 μm. (F) Significantly enriched GO BP (Gene Ontology Biological Process) terms in genes coexpressed with SPP1 in the TCGA-CRC cohort. SPP1, secreted phosphoprotein 1; CRC, colorectal cancer; NC, normal colorectum (adjacent colorectum); NL, normal liver (adjacent); HCC, hepatocellular carcinoma; iNOS, inducible nitric oxide synthase; FDR, false discovery rate.

Article Snippet: The primary antibodies used for validating SPP1 + macrophages were as follows: mouse anti-human CD68 (Proteintech, Cat# 66231-2-Ig, 1:2000) and mouse anti-human SPP1 (Santa Cruz Biotechnology, Cat# sc-21742, 1:1000).

Techniques: Marker, Expressing, RNA Sequencing, Real-time Polymerase Chain Reaction, Immunohistochemical staining, Staining

Journal: Genes & Diseases

Article Title: SPP1 + macrophages in colorectal cancer: Markers of malignancy and promising therapeutic targets

doi: 10.1016/j.gendis.2024.101340

Figure Lengend Snippet: Role of SPP1 + macrophages in CRC progression, metastasis, and prognosis. (A–E) Heatmaps of the immune score (ESTIMATE) and expression of markers ( CD45 , CD68 , CD86 , iNOS , CD163 , CD206 , and SPP1 ) in bulk RNA-seq CRC datasets, comparing stage I/II vs . stage III/IV (A), T 1/2 vs . T 3/4 (B), N0 vs . N1-3 (C), M0 vs . M1 (D), and CRC vs . LM (E). Top, dataset source; bottom, sample size. ∗GSE14333 uses the Duke stage. (F) Univariate Cox analysis of macrophage markers in four CRC datasets (TCGA, GSE17536, GSE39582, and GSE41258). (G) The top 10 enriched hallmark terms in the H- SPP1/CD68 group according to GSEA. SPP1, secreted phosphoprotein 1; CI, confidence interval; GSEA, Gene Set Enrichment Analysis; HR, hazard ratio; iNOS, inducible nitric oxide synthase.

Article Snippet: The primary antibodies used for validating SPP1 + macrophages were as follows: mouse anti-human CD68 (Proteintech, Cat# 66231-2-Ig, 1:2000) and mouse anti-human SPP1 (Santa Cruz Biotechnology, Cat# sc-21742, 1:1000).

Techniques: Expressing, RNA Sequencing

Journal: Genes & Diseases

Article Title: SPP1 + macrophages in colorectal cancer: Markers of malignancy and promising therapeutic targets

doi: 10.1016/j.gendis.2024.101340

Figure Lengend Snippet: Comparison of clinical parameters between the L- SPP1 / CD68 and H- SPP1 / CD6 8 CRC groups.

Article Snippet: The primary antibodies used for validating SPP1 + macrophages were as follows: mouse anti-human CD68 (Proteintech, Cat# 66231-2-Ig, 1:2000) and mouse anti-human SPP1 (Santa Cruz Biotechnology, Cat# sc-21742, 1:1000).

Techniques: Comparison, Standard Deviation, Mutagenesis

Journal: Genes & Diseases

Article Title: SPP1 + macrophages in colorectal cancer: Markers of malignancy and promising therapeutic targets

doi: 10.1016/j.gendis.2024.101340

Figure Lengend Snippet: Spatial distribution characteristics of SPP1 + macrophages and their potential impact on the CRC TME. (A) Spatial plots of hematoxylin-eosin staining (column 1), cell count (column 2), region (column 3), and the expression of three macrophage marker genes (column 4–6) in NC (top), CRC (middle), and LM (bottom) samples. (B) Stacked plots of the percentage of spots expressing SPP1 in each region in NC, CRC, and LM samples. (C) MIA map of scRNA-seq-identified immune cell types and spatial transcriptomics (ST)-defined regions in CRC (upper). Red indicates enrichment and blue indicates depletion. The violin plot shows the expression of nine macrophage marker genes in ST-defined regions in CRC (lower). (D) MIA map of scRNA-seq-identified immune cell types and ST-defined regions in LM samples (upper). The violin plot shows the expression of nine macrophage marker genes in ST-defined regions in LM (lower). (E) Schematic diagram of the spatial distribution of macrophages. The fibroblast-enriched area at the tumor border had the highest macrophage density, while the tumor area had the highest proportion of SPP1 + macrophages. (F) Immunofluorescence staining of human CRC tissue and paired NC tissue. CD68 (red), SPP1 (green), and DAPI (blue) in individual and merged channels are shown. CRC, colorectal cancer; NC, normal colorectum (adjacent colorectum); LM, liver metastases; MIA, multimodal intersection analysis; SPP1, secreted phosphoprotein 1; TME, tumor microenvironment.

Article Snippet: The primary antibodies used for validating SPP1 + macrophages were as follows: mouse anti-human CD68 (Proteintech, Cat# 66231-2-Ig, 1:2000) and mouse anti-human SPP1 (Santa Cruz Biotechnology, Cat# sc-21742, 1:1000).

Techniques: Staining, Cell Counting, Expressing, Marker, Immunofluorescence

Journal: Genes & Diseases

Article Title: SPP1 + macrophages in colorectal cancer: Markers of malignancy and promising therapeutic targets

doi: 10.1016/j.gendis.2024.101340

Figure Lengend Snippet: Implications of SPP1 + macrophages for CRC immunotherapy. (A) The Kaplan–Meier survival curve showed the high expression of SPP1/CD6 8 was associated with worse overall survival (OS) in TCGA-CRC cohorts ( n = 593). Log-rank test. (B, C) Box plots of the effect of preoperative chemotherapy on SPP1 expression in macrophages in the scRNA-seq cohorts of (B) GSE178318 and (C) Wu et al. R represents responders and NR represents non-responders. Mann–Whitney U test. (D) SubMap analysis in TCGA-CRC showed that the H- SPP1/CD68 group was more sensitive to CTLA4 inhibitors (Bonferroni-corrected P = 0.023) and PD-1 inhibitors (Bonferroni-corrected P = 0.021) than the L- SPP1/CD68 group. (E) SubMap analysis in GSE39582 showed that the H- SPP1 group was more sensitive to the CTLA4 inhibitor (Bonferroni-corrected P = 0.023) and PD-1 inhibitor (Bonferroni-corrected P = 0.024). (F) The expression of CSF1R in the three major macrophage subsets (CRC-Mix). (G) Spatial feature plots of the expression of SPP1 (column 2), C1QC (column 3), and CSF1R (column 4) in the CRC sample. (H) Stacked plots of the percentage of spots expressing CSF1R in regions of all spots ( n = 3138), SPP1 + spots, and C1QC + spots in the CRC sample. SPP1, secreted phosphoprotein 1; CRC, colorectal cancer; CTLA4, cytotoxic T-lymphocyte associated protein 4; PD-1, programmed death-1; CSF1R, colony-stimulating factor 1 receptor; C1QC, complement C1q C chain.

Article Snippet: The primary antibodies used for validating SPP1 + macrophages were as follows: mouse anti-human CD68 (Proteintech, Cat# 66231-2-Ig, 1:2000) and mouse anti-human SPP1 (Santa Cruz Biotechnology, Cat# sc-21742, 1:1000).

Techniques: Expressing, MANN-WHITNEY

Journal: Genes & Diseases

Article Title: SPP1 + macrophages in colorectal cancer: Markers of malignancy and promising therapeutic targets

doi: 10.1016/j.gendis.2024.101340

Figure Lengend Snippet: Schematic diagram of the SPP1 + macrophage model in CRC. (A) Classification of macrophage subsets in CRC, including FCN1 + , C1QC + , SPP1 + , and MKI67 + macrophages, defined by their core features of inflammation, phagocytosis, malignancy, and proliferation, respectively. (B) Model of the developmental trajectory of monocyte/macrophage lineages in different sample types from CRLM and HCC patients. This model shows the cell origin and tissue distribution of SPP1 + macrophages. (C) The number and proportion of SPP1 + macrophages increased during CRC occurrence, progression, and metastasis, making them a marker of CRC malignancy. Blue, yellow, and red dots represent the numbers of all immune cells, all macrophages, and SPP1 + macrophages in the sample, respectively. (D) SPP1 + macrophages had distinct signatures compared with other macrophages (left). Red: signature specifically elevated in SPP1 + macrophages. Yellow: signature elevated in SPP1 + macrophages compared with C1QC + macrophages. Blue: signature decreased in SPP1 + macrophages compared with C1QC + macrophages. The right shows the crosstalk between SPP1 + macrophages and other cell subsets through the SPP1 protein. (E) Patients with a high proportion of SPP1 + macrophages have a worse prognosis, along with signatures related to genome instability and mutations. Immunotherapy has the potential to improve outcomes in patients with a high proportion of SPP1 + macrophages, whereas targeting CSF1R is less effective. SPP1, secreted phosphoprotein 1; CSF1R, colony-stimulating factor 1 receptor; CRC, colorectal cancer; CRLM, colorectal cancer liver metastases; HCC, hepatocellular carcinoma; C1QC, complement C1q C chain.

Article Snippet: The primary antibodies used for validating SPP1 + macrophages were as follows: mouse anti-human CD68 (Proteintech, Cat# 66231-2-Ig, 1:2000) and mouse anti-human SPP1 (Santa Cruz Biotechnology, Cat# sc-21742, 1:1000).

Techniques: Marker