Fig 2: Low-dose SNAP treatment induces alterations in macrophage subsets. (A) Subclustering of tumor-associated macrophages (TAMs) by UMAP and identifying the top three DEGs in each subset of TAMs. The top three DEGs were identified using the “FindAllMarker” function in the Seurat package. (B) The distribution of each TAM defined by gene expression patterns in the control or low-dose SNAP treatment groups. (C) The expression levels of M1-related and M2-related genes in the TAM subset. (D) Ridge plots determining the expression distributions of M1-related genes (Cxcl3) and M2-related genes (Arg1) in the TAM subsets. The X-axis represents log-normalized expression levels

Fig 3: Low-dose SNAP induces a subset of macrophages displaying an M1-related gene expression signature and Arg1 expression. (A) Subclustering of TAM1 (978 cells) and visualization of the subsets. UMAP embedding of single-cell RNA sequencing data revealed five clusters using the “FindCluster” function. Each dot represents one cell. (B) Average expression levels of the top 5 DEGs in each subset of TAM1 cells. (C) Distribution of each TAM1 subset in the control and SNAP treatment groups. (D) Bubble heatmap showing the expression levels of M1-related genes and M2-related genes in the subset of TAM1 cells. (E) Flow cytometry analysis of intratumoral TAM1-1 cells and (F) CD80+ Arg1+ macrophages between the control and low-dose SNAP treatment groups. (G) MSigDB hallmark gene set enrichment assay (GSEA) analysis of DEGs in the TAM1-0 (left) and TAM1-1 (right) clusters. TAM1-0 and TAM1-1 DEGs were determined using the “FindMarker” function in the Seurat package and are displayed in Supplementary Data 6. All pathways were significant (p-value < 0.05). Tumor tissues were collected Day 20 after tumor implantation and used for flow cytometry analysis

Fig 4: TRPC1-/- mice show no M1 phenotype development even upon LPS treatment(A) Ca2+ imaging performed on M1 cells from control (WT) and TRPC1-/- mice. Representative trace showing Thapsigargin (Tg)-evoked [Ca2+]i changes in naive and M1 cells pretreated with and without LPS (100 ng/mL) treatment for 6 h. Data shown are representative of three independent experiments with 110–140 cells each.(B) Bar diagram showing quantification of the fluorescence ratio (340/380) from an average of ~100 cells are shown underneath the traces. Bar graphs depict average ± SEM (Student's t test). Data shown are representative of three independent experiments with similar results.(C and D) Application of 2 µM Thapsigargin (Tg) in bath solution induced inward currents at -80 mV in control and LPS (100 ng/mL)-treated M1 cells. Respective IV curves of currents and quantitation (8–10 recordings) of current intensity at -80 mV are shown in (C). Bar graphs depict average ± SEM (Student's t test). Data shown are representative of three independent experiments with similar results.(E) Ca2+ imaging performed in M2 cells from control (WT) and TRPC1-/- mice. Representative trace showing Thapsigargin (Tg)-evoked [Ca2+]i changes in naive and M2 treated cells upon pretreatment with and without LPS (100 ng/mL) for 6 h. Data shown are representative of three independent experiments with similar results.(F) Bar diagram showing quantification of the fluorescence ratio (340/380) from an average of 90–120 cells are shown underneath the traces. Bar graphs depict average ±SEM, *p = 0.05 (Student's t test). Data shown are representative of three independent experiments with similar results.(G and H) Application of 2 µM Thapsigargin (Tg) in bath solution induced inward currents at -80 mV in control and LPS (100 ng/mL)-treated M2 cells. Respective IV curves of currents and quantitation (5–8 recordings) of current intensity at -80 mV are shown in (G). Bar graphs depict average ± SEM, *p = 0.05 (Student's t test). Data shown are representative of three independent experiments with similar results.(I) Heatmap showing Log2 Fold changes (N = 3) in selected M1-inflammatory and M2-anti-inflammatory genes in bone marrow-derived macrophages from WT mice treated with IFN? at 20 ng/mL over medium alone (NS), and corresponding cells from TRPC1-/- mice cultured in presence of IFN? are shown in columns 1 and 2, respectively.(J) A 2D scatter plot showing comparison as in (I). The x axis shows Log2 fold change values of IFN?-treated WT macrophages over unstimulated samples. The y axis shows Log2 fold change values of IFN?-treated TRPC1-/- over unstimulated TRPC1-/- samples. The color of dots represents statistical significance in differential gene expression. Genes with an adjusted p value < 0.05 were considered as differentially expressed. Red dots denote differentially expressed genes on both comparisons, whereas green dots represent genes that are significant in IFN? versus NS. Genes differentially expressed in TRPC1-/- (NS versus treated) are shown as purple dots and those non-significant in both conditions are represented by blue dots.(K) Box plots showing Log2 fold change of the expression of pro- and anti-inflammatory genes in IFN? treated over unstimulated WT and TRPC1-/- macrophages are shown in boxes 1 and 2, respectively from three independent experiments.(L) Flow cytometry was performed to measure the difference between wild type and TRPC1-/- of classic internal M1 markers (iNOS+) and M2 markers (Arg1+) and surface M1 (CD80+) and M2 (CD206+) markers. Data shown are representative of three independent experiments with similar results.(M) Macrophage (M0, M1, and M2) cells from TRPC1-/-mice cultured in 6-well plates (2 × 106 cells/well) and treated with or without LPS (100 ng/mL) for 6 h. The concentration of pro-inflammatory (IL-6 and IFN?) cytokines released in the media was performed by ELISA. Data shown are representative of three independent experiments with similar results. Bar graphs depict average ± SD for relative values, NS, non-significant (Student's t test).(N) Immunoblotting analysis showing the level of expression of wild-type mice versus TRPC1-/- M0, M1, and M2 BMDM polarization pathway genes pNF-?B, NF-?B, pJNK, JNK, pSTAT6, STAT6, pSTAT1, and STAT1 and ß-actin. Data shown are representative of three independent experiments with similar results.

Fig 5: Analysis of macrophages.(A) Subclustering of macrophages showing four subsets. The marker genes and composition for each subset are listed. (B) Marker genes for each macrophage/monocyte subset and their enriched gene sets in GO analysis. Expression of canonical M1 and M2 markers and other proposed markers is shown. (C) In vivo flow cytometry strategy using CD68 to identify monocytes and macrophages. ARG1+ macrophages expressing the surface markers MRC1 and NOS2 in the gated dataset represent anti-inflammatory macrophages (AIM1) and proinflammatory macrophages (PIM1), respectively. The ARG1+LY6C+ monocytes (Mono1) are also shown. (D) Pseudo-temporal ordering of macrophage/monocytes and the distribution of four subsets along the trajectory. (E) Correlation analysis of macrophage/monocyte subsets.