Journal: Scientific reports

Article Title: A novel immune score model predicting the prognosis and immunotherapy response of breast cancer.

doi: 10.1038/s41598-023-31153-2

Figure Lengend Snippet: Figure 4. Weighted gene co-expression network analysis (WGCNA) and identification of candidate immune-related hub genes. (A) Analysis of network topology for various soft-thresholding powers. (B) Gene dendrogram and module colours. (C) Immune-related genes (IRGs) in the brown, green, and yellow modules were significantly correlated with BC progression. (D) Venn diagram results confirmed that IRGs ANO6, NPR3, CCL24, FLT3, and ULBP2 were also recruited to construct our prognostic risk model. (E) Module membership (MM) vs. gene significance (GS) analysis of 7 modules showed that MM was significantly correlated with GS in brown, yellow, blue, and red modules. WGCNA weighted gene co-expression network analysis, IRG immune- related gene, MM, module membership, GS gene significance.

Article Snippet: The mammary sections were incubated overnight at 4 °C with a cocktail of primary antibodies against human NPR3 (1:100; Proteintech, China), followed by incubation with a cocktail of secondary antibodies (Life Technologies, CA, USA) for 1 h at room temperature on the next day.

Techniques: Expressing, Construct

Journal: Scientific reports

Article Title: A novel immune score model predicting the prognosis and immunotherapy response of breast cancer.

doi: 10.1038/s41598-023-31153-2

Figure Lengend Snippet: Figure 7. Genetic alteration of immune-related genes (IRGs). In this study, we explored Tumour mutation burden (TMB), somatic mutation, and copy number variations (CNVs) between high- and low-risk groups using the “maftools” R package. Moreover, the cBio Cancer Genomics Portal database (cBioPortal) was performed to assess mutations and CNVs in breast cancer (BC) tissues. (A) Analysis of the mutation status of the 7 IRGs in our risk model. (B) The most frequent genetic alterations of NPR3 were missense mutation and amplification. (C) TMB comparison between the high- and low-risk groups. (D) The different survival probabilities for high-risk score BC patients with high TMB and BC patients with both low-risk score and low TMB. (E) The mutation frequency and classification of the top 20 gene mutations in the high-risk group. (F) The mutation frequency and classification of the top 20 gene mutations in the low-risk group. IRG immune-related gene, TMB tumour mutation burden, CNV copy number variation, cBioPortal cBio Cancer Genomics Portal database, BC breast cancer.

Article Snippet: The mammary sections were incubated overnight at 4 °C with a cocktail of primary antibodies against human NPR3 (1:100; Proteintech, China), followed by incubation with a cocktail of secondary antibodies (Life Technologies, CA, USA) for 1 h at room temperature on the next day.

Techniques: Mutagenesis, Amplification, Comparison

Journal: Scientific reports

Article Title: A novel immune score model predicting the prognosis and immunotherapy response of breast cancer.

doi: 10.1038/s41598-023-31153-2

Figure Lengend Snippet: Figure 8. Patients with high- and low-risk scores had different immune statuses. Comparison of the single- sample gene set enrichment analysis (ssGSEA) scores of 22 types of immune cells (A) and 13 immune-related pathways (B) between low- (blue box) and high-risk (red box) groups in The Cancer Genome Atlas (TCGA) project. (C) Immune-related gene prognostic index (IRGPI) differed among subtypes in the high and low-risk groups. (D) The tumour stemness of breast cancer (BC) patients between the high- and low-risk groups. (E) The relationship between copy number variations (CNVs) of 4 immune-related genes (IRGs) (ANO6, CCL24, FLT3, and NPR3) and the level of infiltration of B cells, T cells, and macrophages in BC. *P < 0.05; **P < 0.01; ***P < 0.001. ssGSEA single-sample gene set enrichment analysis, TCGA The Cancer Genome Atlas, IRGPI immune-related gene prognostic index, BC breast cancer, CNV copy number variation, IRG immune-related gene.

Article Snippet: The mammary sections were incubated overnight at 4 °C with a cocktail of primary antibodies against human NPR3 (1:100; Proteintech, China), followed by incubation with a cocktail of secondary antibodies (Life Technologies, CA, USA) for 1 h at room temperature on the next day.

Techniques: Comparison

Journal: Scientific reports

Article Title: A novel immune score model predicting the prognosis and immunotherapy response of breast cancer.

doi: 10.1038/s41598-023-31153-2

Figure Lengend Snippet: Figure 11. Validation of the predictive ability of the risk model in an external cohort. (A) The relative expression of the 7 immune-related genes (IRGs) in breast cancer (BC) samples through quantitative reverse transcription polymerase chain reaction (qRT‒PCR) (n = 30). * indicated P < 0.05, ** indicated P < 0.01, P value based on t-test. (B) The expression levels of PD-1, PD-L1, and CTLA-4 were decreased in the high-risk group, as evaluated by qRT‒PCR (n = 30). *** indicated P < 0.001, P value based on t-test. The immunohistochemistry (IHC) results revealed that the expression of NPR3 (C) was increased but that of PD-1 (D), PD-L1 (E), and CTLA-4 (F) was decreased in the high-risk group. (G) The immunofluorescence (IF) assay demonstrated that M2 macrophages were more abundant in high-risk patients. IRG immune-related gene, BC breast cancer, qRT‒PCR quantitative reverse transcription polymerase chain reaction, IHC immunohistochemistry, IF immunofluorescence.

Article Snippet: The mammary sections were incubated overnight at 4 °C with a cocktail of primary antibodies against human NPR3 (1:100; Proteintech, China), followed by incubation with a cocktail of secondary antibodies (Life Technologies, CA, USA) for 1 h at room temperature on the next day.

Techniques: Biomarker Discovery, Expressing, Reverse Transcription, Polymerase Chain Reaction, Immunohistochemistry, Immunofluorescence

Journal: Scientific reports

Article Title: A novel immune score model predicting the prognosis and immunotherapy response of breast cancer.

doi: 10.1038/s41598-023-31153-2

Figure Lengend Snippet: Figure 12. The effect of NPR3 on the proliferation, migration, and apoptosis of breast cancer (BC) cells. The results of quantitative reverse transcription polymerase chain reaction (qRT‒PCR) showed that si-NPR3-1 successfully knocked down the expression of NPR3 in MDA-MB-231 (A) and MCF-7 (B) cells (n = 3). * indicated P < 0.05, ** indicated P < 0.01, ns indicated no significance, P value based on t-test. The depletion of NPR3 observably suppressed the proliferation ability in both MDA-MB-231 (C) and MCF-7 (D) cells compared with the si-NC group (n = 3). *** indicated P < 0.001, P value based on t-test. The knockdown of NPR3 significantly decreased the migration of MDA-MB-231 (E,G) and MCF-7 (F, H) cells (n = 3). ** indicated P < 0.01, *** indicated P < 0.001, P value based on t-test. (I) Flow cytometry indicated that NPR3 deletion could increase apoptosis in MDA-MB-231 and MCF-7 cells (n = 3). ** indicated P < 0.01, *** indicated P < 0.001, P value based on t-test. All experiments were implemented separately in triplicate. BC breast cancer, qRT‒PCR quantitative reverse transcription polymerase chain reaction.

Article Snippet: The mammary sections were incubated overnight at 4 °C with a cocktail of primary antibodies against human NPR3 (1:100; Proteintech, China), followed by incubation with a cocktail of secondary antibodies (Life Technologies, CA, USA) for 1 h at room temperature on the next day.

Techniques: Migration, Reverse Transcription, Polymerase Chain Reaction, Expressing, Knockdown, Flow Cytometry