Journal: Physiological Reports

Article Title: Differential regulation of K Ca 2.1 ( KCNN1 ) K + channel expression by histone deacetylases in atrial fibrillation with concomitant heart failure

doi: 10.14814/phy2.14835

Figure Lengend Snippet: Remodeling of K Ca 2.1 channels associated with AF in HF patients and a porcine AF model. K Ca 2.1 ( KCNN1 ) channel transcript levels were assessed in patients with paroxysmal AF (pAF; n = 10, a, b) or chronic AF (cAF; n = 10, c, d) and are displayed for left atrium (LA; a, c) and right atrium (RA; b, d) in comparison to sinus rhythm patients (SR; n = 10). (e) Mean threshold cycle (CT) levels of the housekeeping gene glyceraldehyde 3‐phosphate dehydrogenase ( GAPDH ) in human left (LA) and right atria (RA) ( n = 10 each). (f–i) K Ca 2.1 ( KCNN1 ) mRNA levels obtained from AF pigs after 7 days ( n = 5, f, g) or 14 days of atrial burst pacing ( n = 5, h, i) are provided compared to respective SR controls (SR; n = 5 each). (f, g) LA, left atrium. (h, i) RA, right atrium. (j) Mean porcine GAPDH CT levels in LA and RA tissue ( n = 5 each). Data are provided as box plots with underlying dots indicating original data; * p < 0.05, *** p < 0.001 versus SR controls

Article Snippet: Membranes were blocked with 5% milk in PBS‐T for 2 h at room temperature and developed using primary antibodies directed against K Ca 2.1 (1:1,000; APC‐039, Alomone Labs).

Techniques: Comparison, Tissue, Real-time Polymerase Chain Reaction

Journal: Physiological Reports

Article Title: Differential regulation of K Ca 2.1 ( KCNN1 ) K + channel expression by histone deacetylases in atrial fibrillation with concomitant heart failure

doi: 10.14814/phy2.14835

Figure Lengend Snippet: Expression of K Ca 2.1 channel protein in a porcine atrial fibrillation (AF)/heart failure model. K Ca 2.1 protein levels assessed in AF pigs after 14 days of atrial burst pacing (14 days AF; n = 5) are provided compared to SR controls (SR; n = 5). Data are expressed as box plots with underlying dots indicating original data relative to SR controls. (a) LA, left atrium. (b) RA, right atrium. For reference, full blots incubated with anti‐K Ca 2.1 antibodies (c, d) and anti‐GAPDH antibodies (e, f) are shown, corresponding to cropped pictures displayed in panels (a and b)

Article Snippet: Membranes were blocked with 5% milk in PBS‐T for 2 h at room temperature and developed using primary antibodies directed against K Ca 2.1 (1:1,000; APC‐039, Alomone Labs).

Techniques: Expressing, Incubation, Western Blot

Journal: Cancer Communications

Article Title: Targeting SPHK1 in macrophages remodels the tumor microenvironment and enhances anti‐PD‐1 immunotherapy efficacy in colorectal cancer liver metastasis

doi: 10.1002/cac2.70047

Figure Lengend Snippet: SPHK1 was selectively expressed in TAMs from CRLM and predicted poor adverse prognosis in CRC patients . (A) UMAP plots show clusters of main immune cell types and SPHK1 gene expression in CRLM using scRNA‐seq data from datasets GSE164522 and GSE178318 . (B) Violin plots depict the relative expression of SPHK1 in TAMs from human CRC and CRLM using data from datasets GSE164522 and GSE178318 . (C) Proportions of SPHK1 + TAMs among total TAMs in CRC and CRLM samples were analyzed using data from datasets GSE164522 and GSE178318 . (D) Representative IF images of co‐staining for SPHK1 (red) and CD68 (green) in human normal colon mucosa, primary CRC, normal liver, and CRLM samples from 11 CRC and 9 CRLM patients. (E) Representative images of IHC staining for SPHK1 in a human CRC tissue microarray (up). Overall survival of 155 CRC patients was stratified by the infiltration level of SPHK1 + cells (down). The mean number of SPHK1 + infiltrating cells was used as the cut‐off value. (F) Overall survival of different CD68 expression CRC cohorts was stratified by SPHK1 expression in tumor tissues. The median expression levels of SPHK1 and CD68 serve as the cut‐off values. Data from GSE17538 . (G) Disease‐specific survival of different CD68 expression CRC cohorts was stratified by SPHK1 expression in tumor tissues. The median expression levels of SPHK1 and CD68 serve as the cut‐off values. Data from GSE17538 . Data in (B) are represented as mean ± SD by unpaired Student's t ‐test. Data in (C) were obtained via Chi‐square test. Abbreviations: B, B cell; CRC, colorectal cancer; CRLM, colorectal cancer liver metastasis; DC, dendritic cell; DAPI, 4',6‐diamidino‐2‐phenylindole; IF, immunofluorescence; IHC, immunohistochemistry; Mast, mast cell; Mono, monocyte; NK, natural killer cell; Plasma, plasma cell; scRNA‐seq, single‐cell RNA sequencing; SPHK1, sphingosine kinase 1; T, T cell; TAM, tumor‐associated macrophage; TIDE, tumor immune dysfunction and exclusion; UMAP, uniform manifold approximation and projection.

Article Snippet: To inhibit SPHK1 activity, C57BL/6 and BALB/c mice with an intrasplenic tumoral injection were given SPHK1 inhibitor PF543 (5 mg/kg; MedChemExpress, Monmouth Junction, NJ, USA) or SKI II (50 mg/kg; MedChemExpress) at day 4 by intraperitoneal injection every three days.

Techniques: Gene Expression, Expressing, Staining, Immunohistochemistry, Microarray, Immunofluorescence, Clinical Proteomics, Single Cell, RNA Sequencing

Journal: Cancer Communications

Article Title: Targeting SPHK1 in macrophages remodels the tumor microenvironment and enhances anti‐PD‐1 immunotherapy efficacy in colorectal cancer liver metastasis

doi: 10.1002/cac2.70047

Figure Lengend Snippet: Pharmacologic and genetic inhibition of SPHK1 in TAMs prevented CRLM . (A) Schematic describing the treatment of mice with SPHK1 inhibitors after an intrasplenic tumor cell inoculation. (B) Representative images and quantification of liver lesions in MC38‐injected C57BL/6 mice treated with or without PF543. (C) Representative images and quantification of liver lesions in MC38‐injected C57BL/6 mice treated with or without SKI II. (D) Representative IF images and quantification of co‐staining of p‐SPHK1 (red) and F4/80 (green) in liver tumor tissues from C57BL/6 mice with or without SPHK1 inhibitor. (E) The relative mRNA expression of SPHK1 in indicated immune cells derived from the spleens of WT and SPHK1 −/− C57BL/6 mice was detected by qPCR. (F) FACS analysis of proportions of different immune cell types derived from the spleens of WT and SPHK1 −/− C57BL/6 mice was performed. (G) Schematic for establishing experimental liver metastasis via orthotopic or intrasplenic tumor inoculation in WT and SPHK1 −/− C57BL/6 mice. (H) Representative images and quantification of liver lesions in WT and SPHK1 −/− C57BL/6 mice intrasplenically injected with MC38 cells. (I) Representative images and quantification of liver lesions in WT and SPHK1 −/− C57BL/6 mice orthotopically injected with MC38 cells. (J) Levels of S1P in MC38 liver tumors with or without SPHK1 deficiency or SPHK1 inhibition were measured by LC‐MS/MS. Data are represented as mean ± SD by unpaired Student's t ‐test (B‐F and H‐J). Abbreviations: DC, dendritic cell; FACS, fluorescence activated cell sorting; IF, immunofluorescence; LC‐MS/MS, liquid chromatography‐tandem mass spectrometry; Mø, macrophage; Mono, monocyte; Neut, neutrophil; NK, natural killer cell; p‐SPHK1, phosphorylated‐SPHK1; S1P, sphingosine‐1‐phosphate; SPHK1, sphingosine kinase 1; SPHK1 −/− , SPHK1 knockout; WT, wild‐type.

Article Snippet: To inhibit SPHK1 activity, C57BL/6 and BALB/c mice with an intrasplenic tumoral injection were given SPHK1 inhibitor PF543 (5 mg/kg; MedChemExpress, Monmouth Junction, NJ, USA) or SKI II (50 mg/kg; MedChemExpress) at day 4 by intraperitoneal injection every three days.

Techniques: Inhibition, Injection, Staining, Expressing, Derivative Assay, Liquid Chromatography with Mass Spectroscopy, Fluorescence, FACS, Immunofluorescence, Liquid Chromatography, Mass Spectrometry, Knock-Out

Journal: Cancer Communications

Article Title: Targeting SPHK1 in macrophages remodels the tumor microenvironment and enhances anti‐PD‐1 immunotherapy efficacy in colorectal cancer liver metastasis

doi: 10.1002/cac2.70047

Figure Lengend Snippet: SPHK1 deficiency in TAMs reversed the immunosuppressive TME of CRLM . (A) t‐SNE plot of CyTOF showing the population of all CD45 + cells in liver metastatic tissues from MC38‐injected WT and SPHK1 −/− C57BL/6 mice; n = 3 per group. (B) t‐SNE plots of CyTOF showing each immune cell population in MC38 liver metastases from WT and SPHK1 −/− C57BL/6 mice; n = 3 per group. Each cell cluster was annotated into a specific immune cell type according to the known marker expression and indicated by different color. (C) Proportions of each cell cluster among CD45 + cells in liver metastatic tissues collected from MC38‐injected WT and SPHK1 −/− C57BL/6 mice. (D) Representative flow plots and frequencies of CD11b + F4/80 + TAMs in MC38 liver tumors from WT and SPHK1 −/− C57BL/6 mice. (E) Representative flow plots and frequencies of CD8 + T cells in MC38 liver tumors from WT and SPHK1 −/− C57BL/6 mice. (F) Representative flow plots and frequencies of exhausted (PD‐1 + ) CD8 + T cells in liver lesions from the indicated mice. (G) Representative flow plots and frequencies of effector (IFN‐γ + ) CD8 + T cells in liver lesions from the indicated mice. (H) Relative expression of exhaustion marker (PD‐1) and activation markers (CD44, CD25, and T‐bet) in CD8 + T cells between WT and SPHK1 −/− C57BL/6 mice revealed by CyTOF analysis. (I) Schematic for establishing a co‐culture system with MC38 cells, BMDMs, and mouse CD8 + T cells at a 1:1:1 ratio. (J) Schematic for establishing a co‐culture system with HCT116 cells, PMA‐differentiated THP‐1 cells, and human CD8 + T cells at a 1:1:1 ratio. (K) Representative flow plots and the expression of PD‐1 and IFN‐γ in mouse CD8 + T cells between different co‐culture groups. (L) WT and SPHK1 −/− C57BL/6 mice bearing MC38 liver tumors were treated with PBS liposomes plus IgG antibody (Ctrl), clodronate liposomes plus anti‐CSF1R, or anti‐CD8. Representative images and quantification of liver lesions between groups are shown; n = 5 per group. Data are represented as mean ± SD by unpaired Student's t ‐test (C‐H) and one‐way ANOVA (K and L). Abbreviations: ANOVA, analysis of variance; BMC, bone marrow cell; BMDM, bone marrow‐derived macrophage; cDC, conventional dendritic cell; BW, body weight; CL, clodronate liposome; CSF1R, colony stimulating factor 1 receptor; CyTOF, cytometry by time‐of‐flight; IFN‐γ, interferon gamma; LM, liver weight; Mono, monocyte; Neut, neutrophil; NK, natural killer cell; PD‐1, programmed cell death 1; pDCs, plasmacytoid dendritic cell; PMA, phorbol 12‐myristate 13‐acetate; SPHK1 −/− , SPHK1 knockout; t‐SNE, t‐distributed stochastic neighbor embedding; WT, wild‐type; γδ T, gamma‐delta T cells.

Article Snippet: To inhibit SPHK1 activity, C57BL/6 and BALB/c mice with an intrasplenic tumoral injection were given SPHK1 inhibitor PF543 (5 mg/kg; MedChemExpress, Monmouth Junction, NJ, USA) or SKI II (50 mg/kg; MedChemExpress) at day 4 by intraperitoneal injection every three days.

Techniques: Injection, Marker, Expressing, Activation Assay, Co-Culture Assay, Liposomes, Derivative Assay, Cytometry, Knock-Out

Journal: Cancer Communications

Article Title: Targeting SPHK1 in macrophages remodels the tumor microenvironment and enhances anti‐PD‐1 immunotherapy efficacy in colorectal cancer liver metastasis

doi: 10.1002/cac2.70047

Figure Lengend Snippet: The SPHK1‐S1P axis promoted IL‐1β secretion through NLRP3 inflammasome in TAMs . (A) RNA‐seq analysis was applied for TAMs extracted from the liver metastases of WT and SPHK1 −/− C57BL/6 mice and BMDM‐derived TAMs with or without S1P treatment (10 µmol/L for 12 h). The results showed that 110 DEGs were downregulated in SPHK1 −/− TAMs compared with WT TAMs, and 142 DEGs were upregulated by S1P treatment in TAMs. Seventeen common DEGs included NLRP3 and IL‐1β. (B) GSEA results showing the inflammasome complex pathway and NLRP3 inflammasome complex assembly pathway enriched in WT TAMs. (C) Relative mRNA expression of inflammasome genes in TAMs from liver tumors with or without PF543 treatment were determined by qPCR. (D) Representative images and relative levels of IL‐1β and CXCL9 in Ctrl‐ and PF543‐treated liver tumors determined by a mouse inflammatory cytokine array. (E) Representative IF images of co‐staining for IL‐1β (red) and F4/80 (green) in liver lesions between groups (left); quantification of the number of IL‐1β + TAMs per HPF (right). (F) Representative IF images of co‐staining for SPHK1 (white), IL‐1β (red), and CD68 (green) in human CRC (n = 7) and CRLM tissues (n = 7). (G) TAMs derived from BMDMs or THP‐1 cells were treated with S1P (10 µmol/L) for 24 h, followed by using Western blotting to detect the expression of NLRP3, IL‐1β, and Caspase1. β‐actin was used as a loading control. (H) Relative protein expression of inflammasome genes was determined in shNC and sh SPHK‐1 THP‐1 cells by Western blotting. β‐actin was used as a loading control. (I) TAMs were pretreated with 10 µmol/L S1P for 12 h and then primed with LPS (500 ng/mL) for 3 h, followed by stimulation with ATP (5 mmol/L, 30 min). Levels of IL‐1β in the supernatant were determined by ELISA. (J) Western blotting showing the protein expression of p‐SPHK1 (Ser225) and inflammasome genes in BMDMs after co‐cultured with the indicated cells for 48 h. β‐actin was used as a loading control. (K) BMDMs were co‐cultured with MC38 and mouse CD8 + T cells for 48 h, followed by measuring the level of IL‐1β in the supernatant by ELISA. (L) Representative images and quantification of liver lesions in BALB/c mice intrasplenically injected with CT26 following AAV‐ SPHK1 or anakinra treatment. Data are represented as mean ± SD by unpaired Student's t ‐test (C, E, F, and I) and one‐way ANOVA (K and L). Abbreviations: AAV, adeno‐associated virus; ANOVA, analysis of variance; ATP, adenosine tri‐phosphate; BMDM, bone marrow‐derived macrophage; BSA, bovine serum albumin; CASP1, Caspase 1; CCL2, C‐C motif chemokine ligand 2; CD30L, lymphocyte adhesion molecule 30L; CRC, colorectal cancer; CRLM, colorectal cancer liver metastasis; CXCL9, C‐X‐C motif chemokine ligand 9; DAPI, 4',6‐diamidino‐2‐phenylindole; GSEA, gene set enrichment analysis; IF, immunofluorescence; IL13, interleukin‐13; IL‐1β, interleukin 1 beta; LPS, lipopolysaccharide; MIP‐1γ, macrophage inflammatory protein‐1 gamma; NLRP3, NLR family pyrin domain containing 3; p‐SPHK1, phosphorylated‐SPHK1; RNA‐seq, RNA sequencing; S1P, sphingosine‐1‐phosphate; sh SPHK1 , short hairpin RNA of SPHK1 ; SPHK1 −/− , SPHK1 knockout; sTNF RII, soluble tumor necrosis factor receptor II; TAMs, tumor‐associated macrophages; WT, wild‐type.

Article Snippet: To inhibit SPHK1 activity, C57BL/6 and BALB/c mice with an intrasplenic tumoral injection were given SPHK1 inhibitor PF543 (5 mg/kg; MedChemExpress, Monmouth Junction, NJ, USA) or SKI II (50 mg/kg; MedChemExpress) at day 4 by intraperitoneal injection every three days.

Techniques: RNA Sequencing, Derivative Assay, Expressing, Staining, Western Blot, Control, Enzyme-linked Immunosorbent Assay, Cell Culture, Injection, Virus, Immunofluorescence, shRNA, Knock-Out

Journal: Cancer Communications

Article Title: Targeting SPHK1 in macrophages remodels the tumor microenvironment and enhances anti‐PD‐1 immunotherapy efficacy in colorectal cancer liver metastasis

doi: 10.1002/cac2.70047

Figure Lengend Snippet: The S1P‐S1PR2 axis activated NLRP3 inflammasome via NF‐κB and HIF‐1α signaling . (A) TAMs differentiated from BMDMs were treated with or without S1P (10 µmol/L), and then subjected to RNA‐seq analysis. The DEGs upregulated in S1P‐treated group were utilized for KEGG enrichment analysis showing the HIF‐1 and NF‐κB signaling pathways significantly enriched. (B) TAMs derived from BMDMs or THP‐1 cells were treated with or without 10 µmol/L S1P for 24 h. Relative protein levels of HIF‐1α, p‐IKKα/β (Ser176/180), P65, and p‐P65 (Ser536) were determined by Western blotting. β‐actin was used as a loading control. (C) Western blotting of HIF‐1α, NLRP3, and IL‐1β of TAMs under hypoxia (1% O 2 ) or normal conditions (20% O 2 ) for 24 h was performed. β‐actin was used as a loading control. (D) TAMs were incubated under hypoxia (1% O 2 ) or normal conditions (20% O 2 ) for 24 h, followed by detecting the mRNA levels of inflammasome genes by qPCR. (E) BMDM‐derived TAMs were transfected with P65‐ or HIF‐1α‐specific siRNA for 48 h before incubation with 10 µmol/L S1P or left untreated for 24 h. qPCR analysis of NLRP3 and IL‐1β was performed. (F) Western blotting of P65, HIF‐1α, NLRP3, and IL‐1β in TAMs following the indicated treatments as in (E). β‐actin was used as a loading control. (G) UMAP plots show 3 TAM subpopulations and SPHK1 expression in human CRLM scRNA‐seq data from GSE164522 . (H) Violin plots show the relative expression of SPHK1, NLRP3 and IL‐1β among 3 TAM subpopulations in human CRLM specimens. Data from GSE164522 . (I) Representative IF images of co‐staining for SPHK1 (white), NLRP3 (red), and CD68 (green) in human CRLM tissues. (J) Relative mRNA levels of NLRP3 and IL‐1β in BMDM‐derived TAMs treated without or with S1PR2 agonist CYM5520 (2 µmol/L; 5 µmol/L) for 24 h. (K) Western blotting showing the protein expression of HIF‐1α, P65, and p‐P65 (Ser536) in TAMs treated without or with S1PR2 agonist CYM5520 (2 µmol/L; 5 µmol/L) for 24 h. β‐actin was used as a loading control. Data are represented as mean ± SD by unpaired Student's t ‐test (D) and one‐way ANOVA (E, H, and J). Abbreviations: ANOVA, analysis of variance; BMDM, bone marrow‐derived macrophage; BSA, bovine serum albumin; CASP1, Caspase 1; C1QC, complement C1q C chain; DMSO, dimethyl sulfoxide; HIF‐1α, hypoxia inducible factor 1 subunit alpha; IF, immunofluorescence; IKKα/β, IκB kinase alpha/beta; IL‐1β, interleukin 1 beta; KEGG, Kyoto encyclopedia of genes and genomes; NF‐κB, nuclear factor‐kappa B; NLRP3, NLR family pyrin domain containing 3; P65, RelA, a member of the NF‐κB transcription factor family; p‐P65, phosphorylated‐P65; S1P, sphingosine‐1‐phosphate; siHIF‐1α, small interfering RNA of HIF‐1α; siP65, small interfering RNA of P65; SPHK1, sphingosine kinase 1; SPP1, secreted phosphoprotein 1; TAMs, tumor‐associated macrophages; UMAP, uniform manifold approximation and projection.

Article Snippet: To inhibit SPHK1 activity, C57BL/6 and BALB/c mice with an intrasplenic tumoral injection were given SPHK1 inhibitor PF543 (5 mg/kg; MedChemExpress, Monmouth Junction, NJ, USA) or SKI II (50 mg/kg; MedChemExpress) at day 4 by intraperitoneal injection every three days.

Techniques: RNA Sequencing, Protein-Protein interactions, Derivative Assay, Western Blot, Control, Incubation, Transfection, Expressing, Staining, Immunofluorescence, Small Interfering RNA

Journal: Cancer Communications

Article Title: Targeting SPHK1 in macrophages remodels the tumor microenvironment and enhances anti‐PD‐1 immunotherapy efficacy in colorectal cancer liver metastasis

doi: 10.1002/cac2.70047

Figure Lengend Snippet: rIL‐1β‐treated CRC cells promoted TAM recruitment and CD8 + T cell dysfunction . (A) Heatmap showing the relative expression of the indicated inflammatory cytokines and chemokines in the co‐culture system of CD8 + T cells and HCT116 cells and shNC THP‐1 or sh SPHK1 THP‐1 cells. (B) BMDMs with or without SPHK1 deletion or S1P pretreatment, and THP‐1 cells with or without SPHK1 ‐knockdown were subjected to the co‐culture experiments with CD8 + T cells and CRC cells. Relative mRNA expression of CCL2 in TAMs isolated from the co‐culture system was determined by qPCR. (C) MC38 or HCT116 cells were treated with recombinant IL‐1β (50 nmol/L) for 24 h, followed by the detection of the transcription levels of CCL2, CCL7, CCL8, and CXCL12 by qPCR. (D) Representative transwell images showing the migrated macrophages with or without SPHK1 deficiency when co‐cultured with CRC cells. (E) Heatmap showing the relative expression of the secreted proteins from MC38 cells with or without rIL‐1β (50 nmol/L) pretreatment. (F) Representative flow plots and frequencies of CD62L + CD8 + T cells after incubation with rIL‐1β‐pretreated MC38 with or without ADAM17 ‐knockdown. CD8 + T cells were activated with anti‐CD3 (5 µg/mL) and anti‐CD28 (2 µg/mL) before incubation. (G) Statistical plots showing the proportion of CD62L + CD8 + T cells after treatment with a concentration gradient of mouse rADAM17. (H) Representative images of IHC staining for SPHK1 and ADAM17 in a human CRC tissue microarray (left). Overall survival in CRC patients was stratified by the infiltration level of SPHK1 + cells and tumor ADAM17 expression (right); n = 155 primary CRC. Data are represented as mean ± SD by unpaired Student's t ‐test (B‐D) and one‐way ANOVA (F and G). Abbreviations: ADAM17, ADAM metallopeptidase domain 17; ANOVA, analysis of variance; BMDM, bone marrow‐derived macrophage; CCL2, C‐C motif chemokine ligand 2; CCL7, C‐C motif chemokine ligand 7; CCL8, C‐C motif chemokine ligand 8; CD62L, lymphocyte adhesion molecule 62L; CXCL12, C‐X‐C motif chemokine ligand 12; IHC, immunohistochemistry; IL‐1β, interleukin 1 beta; IL‐2, interleukin 2; IL‐6, interleukin 6; rADAM17, recombinant ADAM17; rIL‐1β, recombinant IL‐1β; sh ADAM17 , short hairpin RNA of ADAM17 ; sh SPHK1 , short hairpin RNA of SPHK1 ; SPHK1 −/− , SPHK1 knockout; WT, wild‐type.

Article Snippet: To inhibit SPHK1 activity, C57BL/6 and BALB/c mice with an intrasplenic tumoral injection were given SPHK1 inhibitor PF543 (5 mg/kg; MedChemExpress, Monmouth Junction, NJ, USA) or SKI II (50 mg/kg; MedChemExpress) at day 4 by intraperitoneal injection every three days.

Techniques: Expressing, Co-Culture Assay, Knockdown, Isolation, Recombinant, Cell Culture, Incubation, Concentration Assay, Immunohistochemistry, Microarray, Derivative Assay, shRNA, Knock-Out

Journal: Cancer Communications

Article Title: Targeting SPHK1 in macrophages remodels the tumor microenvironment and enhances anti‐PD‐1 immunotherapy efficacy in colorectal cancer liver metastasis

doi: 10.1002/cac2.70047

Figure Lengend Snippet: SPHK1‐targeting treatment enhanced the efficacy of anti‐PD‐1 immunotherapy . (A) Heatmap showing the expression of the indicated markers in 4 subpopulations of CD8 + T cells with CyTOF analysis using the MC38 liver metastases from WT and SPHK1 −/− C57BL/6 mice. (B) Proportions of CD8 + T‐cell subclusters among CD45 + cells between WT and SPHK1 −/− liver tumors. (C) Schematic describing liver tumor‐bearing mice treated with anti‐PD‐1 antibody, SPHK1 inhibitor, or combination therapy. (D) Representative images and quantification of liver lesions in C57BL/6 mice receiving control, anti‐PD‐1, PF543, or combination treatment; n = 5 per group. (E) Survival of C57BL/6 mice administered with the indicated treatments; n = 8 per group. (F) Statistical plots of the proportion of PD‐1 + or IFN‐γ + CD8 + T cells in MC38 liver tumors with indicated treatment. (G) Representative IF images of co‐staining for p‐SPHK1 (red) and F4/80 (green) in MC38 liver tumors (left); quantification of the number of p‐SPHK1 + TAMs per HPF (right). (H) Schematic depicting the treatments applied to C57BL/6 mice. Mice bearing subcutaneous MC38 tumors were given IgG or anti‐PD‐1. Mice simultaneously bearing subcutaneous and liver tumors were treated with IgG, anti‐PD‐1, anti‐PD‐1 plus liver‐directed irradiation or anti‐PD‐1 plus liver‐directed irradiation plus PF543. (I) Representative images and quantification of liver lesions in C57BL/6 mice given the indicated treatments as described in (H); n = 5 per group. (J) Statistical analysis of IHC staining for F4/80 + TAMs and CD8 + T cells in liver tumors from different treatment groups. n = 9 random HPFs. (K) Representative images and statistical analysis of IHC staining for SPHK1 in CRC samples from patients prior to receiving anti‐PD‐1 treatment; n = 12. (L) Overall survival in patients with different solid tumors who received ICIs (data from PMID33020056) was stratified by the expression level of SPHK1. (M) Overall survival in mUC patients who received anti‐PD‐L1 therapy (data from IMvigor210) was stratified by the expression level of SPHK1. (N) Progression‐free survival in KIRC patients who received anti‐PD‐1 therapy (data from PMID32472114) was stratified by the expression level of SPHK1. Data are represented as mean ± SD by unpaired Student's t ‐test (B and K) and one‐way ANOVA (D, F, G, I, and J). Abbreviations: ANOVA, analysis of variance; BW, body weight; CRC, colorectal cancer; CyTOF, cytometry by time‐of‐flight; DAPI, 4',6‐diamidino‐2‐phenylindole; ICIs, immune checkpoint inhibitors; IF, immunofluorescence; IFN‐γ, interferon gamma; IHC, immunohistochemistry; int, intermediate; IR, irradiation; KIRC, kidney renal clear cell carcinoma; LM, liver weight; mUC, metastatic urothelial cancer; PD‐1, programmed cell death 1; PD‐L1, programmed cell death 1 ligand 1; p‐SPHK1, phosphorylated‐SPHK1; SPHK1 −/− , SPHK1 knockout; WT, wild‐type.

Article Snippet: To inhibit SPHK1 activity, C57BL/6 and BALB/c mice with an intrasplenic tumoral injection were given SPHK1 inhibitor PF543 (5 mg/kg; MedChemExpress, Monmouth Junction, NJ, USA) or SKI II (50 mg/kg; MedChemExpress) at day 4 by intraperitoneal injection every three days.

Techniques: Expressing, Control, Staining, Irradiation, Immunohistochemistry, Cytometry, Immunofluorescence, Knock-Out

Journal: Frontiers in Pharmacology

Article Title: S1P/S1PR1 signaling is involved in the development of nociceptive pain

doi: 10.3389/fphar.2024.1407347

Figure Lengend Snippet: SPHK1 inhibitor reduced nociceptive pain. (A) The content of S1P in the serum, DRG and spinal cord of CFA mice on day 3 was analyzed by ELISA after administration of SK1-I (5 mg/kg, 10 mg/kg) (n = 3 biological repeats, 3 mice/repeat/group). Data are shown as the mean ± SEM. * p < 0.05 vs. Vehicle. (B–D) The PWF and PWL of CFA mice after intraperitoneal administration of the SPHK1 inhibitor SK1-I (n = 6 mice/group). Data are shown as the mean ± SEM. * p < 0.05, *** p < 0.001 vs. CFA + Vehicle. (E) The activation of astrocytes and microglial cells in the spinal dorsal horn was detected by immunofluorescence after SK1-I injection (n = 3–4 biological repeats). (F,G) The immunoreactivity levels of GFAP and IBA1 in the spinal dorsal horn after SK1-I administration (n = 3–4 biological repeats). Data are shown as the mean ± SEM. * p < 0.1 vs. CFA + Vehicle. The red arrow represents the time of model establishment. The green arrow represents the time of drug administration.

Article Snippet: Complete Freund’s adjuvant (F5881) was purchased from Sigma‒Aldrich Canada Ltd. S1P (HY-108496, 5 μL, 100 Um), SK1-I (HY-119016), FTY720 (HY-12005), siponimod (HY-12355), and KRP-203 (HY-13660) were purchased from MCE ( ).

Techniques: Enzyme-linked Immunosorbent Assay, Activation Assay, Immunofluorescence, Injection

Journal: bioRxiv

Article Title: Neuronal overexpression of potassium channel subunit Kcnn1 prolongs survival of SOD1-linked ALS and A53T alpha-synuclein mouse models

doi: 10.1101/2024.10.11.617887

Figure Lengend Snippet: Kaplan-Meier survival plots for G85R SOD1YFP homozygous mice of high copy number, 260-330, (panel A), and lower copy number, 199-245, (panel B), showing that survival (days to paralysis) is extended by Thy1.2-Kcnn1 transgenesis by 35% and 100%, respectively. Extended survival of Kcnn1 transgenic mice (red trace) is shown relative to both littermates lacking Kcnn1 (blue trace in A) and a cohort of homozygous G85R SOD1YFP mice (black trace). Note that Thy1.2-Kcnn1 is hemizygous in (A) and homozygous in (B), but that, in the case of G85R SOD1YFP, there was no significant difference in survival for copy number 260-330 between hemizygous and homozygous Kcnn1 (compare panel (A) with SF3).

Article Snippet: Antibodies used: anti-mouse Kcnn1 (Proteintech 17929-1-AP, 1:400), anti-ATF4 (Cell Signaling 11815, 1:100), anti-CHAT (Abcam ab178850, 1:100), anti-phospho(S757) ULK1 (Cell Signaling 6888, 1:100), anti-phospho(S51) eIF2α (Cell Signaling 3398, 1:100), anti-phospho(S129) α-synuclein (Abcam ab51253,1:800), anti-Ctip2 (Abcam ab18465, 1:100).

Techniques: Transgenic Assay

Journal: bioRxiv

Article Title: Neuronal overexpression of potassium channel subunit Kcnn1 prolongs survival of SOD1-linked ALS and A53T alpha-synuclein mouse models

doi: 10.1101/2024.10.11.617887

Figure Lengend Snippet: Kaplan-Meier survival plots for G93A SOD1/+ mice (cohort of 26 mice in both panels A and B) and G93A SOD1/+ mice homozygous for Kcnn 1-3 (totaling ∼8 copies by real time PCR measurement) and a separate hemizygous Kcnn1-6 line (with 6 copies by real time PCR) (red traces). Extension of time to paralysis was 54% in panel A and 34% in panel B.

Article Snippet: Antibodies used: anti-mouse Kcnn1 (Proteintech 17929-1-AP, 1:400), anti-ATF4 (Cell Signaling 11815, 1:100), anti-CHAT (Abcam ab178850, 1:100), anti-phospho(S757) ULK1 (Cell Signaling 6888, 1:100), anti-phospho(S51) eIF2α (Cell Signaling 3398, 1:100), anti-phospho(S129) α-synuclein (Abcam ab51253,1:800), anti-Ctip2 (Abcam ab18465, 1:100).

Techniques: Real-time Polymerase Chain Reaction

Journal: bioRxiv

Article Title: Neuronal overexpression of potassium channel subunit Kcnn1 prolongs survival of SOD1-linked ALS and A53T alpha-synuclein mouse models

doi: 10.1101/2024.10.11.617887

Figure Lengend Snippet: Expression of Kcnn1 RNA and protein in motor neurons in ventral horn region of Kcnn1-3/Kcnn1-3 homozygous mice and nontransgenic B6SJL. A, RNA fluorescence in situ hybridization (FISH) of Kcnn1 RNA present in ventral horn of cervical spinal cord section of a 7 month old Kcnn1-3/Kcnn1-3 mouse (left) and nontransgenic B6SJL mouse (right). B, Immunostaining with an anti-Kcnn1 antibody of the Kcnn1-3/Kcnn1-3 (left) and B6SJL (right). C, Immunostaining of a Kcnn1-6 mouse (left) and a B6SJL nontransgenic mouse (right). Scale bars are 20 μm.

Article Snippet: Antibodies used: anti-mouse Kcnn1 (Proteintech 17929-1-AP, 1:400), anti-ATF4 (Cell Signaling 11815, 1:100), anti-CHAT (Abcam ab178850, 1:100), anti-phospho(S757) ULK1 (Cell Signaling 6888, 1:100), anti-phospho(S51) eIF2α (Cell Signaling 3398, 1:100), anti-phospho(S129) α-synuclein (Abcam ab51253,1:800), anti-Ctip2 (Abcam ab18465, 1:100).

Techniques: Expressing, Fluorescence, In Situ Hybridization, Immunostaining

Journal: bioRxiv

Article Title: Neuronal overexpression of potassium channel subunit Kcnn1 prolongs survival of SOD1-linked ALS and A53T alpha-synuclein mouse models

doi: 10.1101/2024.10.11.617887

Figure Lengend Snippet: Comparison in a ventral horn neuron of Kcnn1 antibody staining pattern with G85R SOD1YFP homozygote fluorescence pattern. A 20 micron thick cervical spinal cord section from a 3 month old Kcnn1-3/ Kcnn1-3; G85R SOD1YFP homozygous mouse with copy number 298 was stained with anti-Kcnn1 antibody (top panel); the YFP fluorescence imaged (middle panel); and the two images merged (bottom panel). The Kcnn1 pattern was cytoplasmic and patchy, with variable intensity. The YFP fluorescence was more uniform with a rim of nuclear staining (see text), and the merge showed overlap of the patterns, but no distinct yellow color that would reflect precise colocalization. Scale bar is 10 µm.

Article Snippet: Antibodies used: anti-mouse Kcnn1 (Proteintech 17929-1-AP, 1:400), anti-ATF4 (Cell Signaling 11815, 1:100), anti-CHAT (Abcam ab178850, 1:100), anti-phospho(S757) ULK1 (Cell Signaling 6888, 1:100), anti-phospho(S51) eIF2α (Cell Signaling 3398, 1:100), anti-phospho(S129) α-synuclein (Abcam ab51253,1:800), anti-Ctip2 (Abcam ab18465, 1:100).

Techniques: Comparison, Staining, Fluorescence

Journal: bioRxiv

Article Title: Neuronal overexpression of potassium channel subunit Kcnn1 prolongs survival of SOD1-linked ALS and A53T alpha-synuclein mouse models

doi: 10.1101/2024.10.11.617887

Figure Lengend Snippet: Non-ocular cranial motor neurons innervating facial muscles (7N) and tongue (12N) are prevented from accretion of SOD1 YFP aggregates by overexpression of Kcnn1 in double homozygote transgenics G85R SOD1YFP/G85R SOD1YFP;Kcnn1-3/Kcnn1-3. Left panels, YFP fluorescence from G85R SOD1YFP protein; right panels, immunostaining with anti-Kcnn1 antibody. In the absence of Kcnn1, YFP fluorescent aggregates are present in as many as 50% of motor neurons in 7N (top left panel, insert), and in an even higher percentage in 12N, but in the presence of overexpressed Kcnn1, detected in the right panels where the Kcnn1-3 homozygous transgene is present, only a rare YFP fluorescent aggregate (or artifact) could be observed.

Article Snippet: Antibodies used: anti-mouse Kcnn1 (Proteintech 17929-1-AP, 1:400), anti-ATF4 (Cell Signaling 11815, 1:100), anti-CHAT (Abcam ab178850, 1:100), anti-phospho(S757) ULK1 (Cell Signaling 6888, 1:100), anti-phospho(S51) eIF2α (Cell Signaling 3398, 1:100), anti-phospho(S129) α-synuclein (Abcam ab51253,1:800), anti-Ctip2 (Abcam ab18465, 1:100).

Techniques: Muscles, Over Expression, Fluorescence, Immunostaining

Journal: bioRxiv

Article Title: Neuronal overexpression of potassium channel subunit Kcnn1 prolongs survival of SOD1-linked ALS and A53T alpha-synuclein mouse models

doi: 10.1101/2024.10.11.617887

Figure Lengend Snippet: Kaplan-Meier survival plots of G93A/+ cohort and G93A/+ mice that had been injected at P0 into both lateral cerebral ventricles with self-complementary recombinant AAV9 carrying a CMV promoter driving mouse Kcnn1 cDNA, at titer of 5 × 10E13 vg/ml. Median survival to paralysis was increased by 15%. Notably, an earlier test using a titer of 1 × 10E13 produced only a few days of extended survival. See also SF8 and SF9 for RNA and immunostaining analysis of spinal cord from virus-injected mice.

Article Snippet: Antibodies used: anti-mouse Kcnn1 (Proteintech 17929-1-AP, 1:400), anti-ATF4 (Cell Signaling 11815, 1:100), anti-CHAT (Abcam ab178850, 1:100), anti-phospho(S757) ULK1 (Cell Signaling 6888, 1:100), anti-phospho(S51) eIF2α (Cell Signaling 3398, 1:100), anti-phospho(S129) α-synuclein (Abcam ab51253,1:800), anti-Ctip2 (Abcam ab18465, 1:100).

Techniques: Injection, Recombinant, Produced, Immunostaining, Virus

Journal: bioRxiv

Article Title: Neuronal overexpression of potassium channel subunit Kcnn1 prolongs survival of SOD1-linked ALS and A53T alpha-synuclein mouse models

doi: 10.1101/2024.10.11.617887

Figure Lengend Snippet: Kaplan-Meier survival plots of Thy1.2-A53T alpha-synuclein transgenic mice in a B6SJL background. Survival time is measured as days to the time of reaching an endstage motor behavior in which mice were no longer able to ambulate and could not maintain an upright stance (see SF11). The presence of Kcnn1-3/+ extended survival time by 47%, while presence of Kcnn1-6 (higher copy number) in an ongoing experiment has extended survival time by more than 100%.

Article Snippet: Antibodies used: anti-mouse Kcnn1 (Proteintech 17929-1-AP, 1:400), anti-ATF4 (Cell Signaling 11815, 1:100), anti-CHAT (Abcam ab178850, 1:100), anti-phospho(S757) ULK1 (Cell Signaling 6888, 1:100), anti-phospho(S51) eIF2α (Cell Signaling 3398, 1:100), anti-phospho(S129) α-synuclein (Abcam ab51253,1:800), anti-Ctip2 (Abcam ab18465, 1:100).

Techniques: Transgenic Assay

Journal: bioRxiv

Article Title: Neuronal overexpression of potassium channel subunit Kcnn1 prolongs survival of SOD1-linked ALS and A53T alpha-synuclein mouse models

doi: 10.1101/2024.10.11.617887

Figure Lengend Snippet: Kcnn1 overexpression in Kcnn1-6/+ spinal cord ventral horns does not affect the transcriptional expression of G93A human SOD1. Approximately 100 ventral horns of 20 micron thickness were laser capture-microdissected from each of B6SJL, G93A/+, and Kcnn1-6/+;G93A/+. RNA was prepared, and qRT- PCR performed on ∼10 ng of each RNA sample. The level of each indicated RNA was taken relative to an internal GAPDH reference and expression then measured as the fold difference with, in each sample, the B6SJL measurement as the control. Thus, for B6SJL, the values for Kcnn1 and mouse SOD1 were taken as 1. The presence of mouse Kcnn1-6 thus produced an ∼175-fold increase of Kcnn1 over endogenous mouse Kcnn1. For human SOD1 in G93A/+ and in Kcnn1-6;G93A/+, the ratio had to be taken relative to mouse SOD1 (since human SOD1 is absent from mouse). This ratio resulted in a value of ∼75 regardless of the presence of Kcnn1 transgene, indicating that expression of Kcnn1 does not impair the transcription of the pathogenic G93A transgene.

Article Snippet: Antibodies used: anti-mouse Kcnn1 (Proteintech 17929-1-AP, 1:400), anti-ATF4 (Cell Signaling 11815, 1:100), anti-CHAT (Abcam ab178850, 1:100), anti-phospho(S757) ULK1 (Cell Signaling 6888, 1:100), anti-phospho(S51) eIF2α (Cell Signaling 3398, 1:100), anti-phospho(S129) α-synuclein (Abcam ab51253,1:800), anti-Ctip2 (Abcam ab18465, 1:100).

Techniques: Over Expression, Expressing, Quantitative RT-PCR, Control, Produced

Journal: bioRxiv

Article Title: Neuronal overexpression of potassium channel subunit Kcnn1 prolongs survival of SOD1-linked ALS and A53T alpha-synuclein mouse models

doi: 10.1101/2024.10.11.617887

Figure Lengend Snippet: Expanded ER in spinal cord motor neuron of a 4 month old Kcnn1-6 mouse. Ultrathin sections were prepared following PFA perfusion, dissection of the spinal cord, PFA/glutaraldehyde post-fixation, epon embedding, and microtome sectioning. Note the whorl patterns of stacked ER membranes that tails off in a downward leftward direction. Darkly stained structures are lipofuscin granules, some containing typical lipid droplets. More lightly staining round structures are likely to be lysosomes. The nucleus is positioned at the bottom aspect of the image and surrounding Golgi has a dispersed appearance. Scale bar, 2 microns.

Article Snippet: Antibodies used: anti-mouse Kcnn1 (Proteintech 17929-1-AP, 1:400), anti-ATF4 (Cell Signaling 11815, 1:100), anti-CHAT (Abcam ab178850, 1:100), anti-phospho(S757) ULK1 (Cell Signaling 6888, 1:100), anti-phospho(S51) eIF2α (Cell Signaling 3398, 1:100), anti-phospho(S129) α-synuclein (Abcam ab51253,1:800), anti-Ctip2 (Abcam ab18465, 1:100).

Techniques: Dissection, Staining

Journal: bioRxiv

Article Title: Neuronal overexpression of potassium channel subunit Kcnn1 prolongs survival of SOD1-linked ALS and A53T alpha-synuclein mouse models

doi: 10.1101/2024.10.11.617887

Figure Lengend Snippet: Heat map showing gene ontology analysis of RNAseq data comparing laser captured spinal cord motor neurons from B6SJL with those from Kcnn1-6 transgenic mice. Of the 20 most significantly enriched categories in the gene ontology analysis, only two showed strongly upregulated transcription in Kcnn1-6 transgenic mice, both involving the ER. (see text).

Article Snippet: Antibodies used: anti-mouse Kcnn1 (Proteintech 17929-1-AP, 1:400), anti-ATF4 (Cell Signaling 11815, 1:100), anti-CHAT (Abcam ab178850, 1:100), anti-phospho(S757) ULK1 (Cell Signaling 6888, 1:100), anti-phospho(S51) eIF2α (Cell Signaling 3398, 1:100), anti-phospho(S129) α-synuclein (Abcam ab51253,1:800), anti-Ctip2 (Abcam ab18465, 1:100).

Techniques: Transgenic Assay

Journal: bioRxiv

Article Title: Neuronal overexpression of potassium channel subunit Kcnn1 prolongs survival of SOD1-linked ALS and A53T alpha-synuclein mouse models

doi: 10.1101/2024.10.11.617887

Figure Lengend Snippet: Presence of an integrated stress response (ISR) in Kcnn1-6 transgenic mice. Immunostaining with anti-phosphorylated-S51-eIF2alpha (A) and anti-ATF4 (B), comparing spinal cord sections of a Kcnn1-6 mouse with those of a nontransgenic B6SJL mouse. (A) shows an at least several-fold increase of anti-phospho-S51-eIF2alpha signal in Kcnn1-6 motor neurons (left) compared with that in those of the B6SJL mouse (right), reflecting activation of the ISR. (B) shows three representative ventral horn sections from a Kcnn1-6 mouse and three from a B6SJL mouse. Motor neurons are identified by anti-ChAT antibody staining (middle row of images). The Kcnn1-6 sections (upper left images, first and third with 3 and 2 motor neurons, respectively) show a variably intense anti-ATF4 signal that is localized to the nucleus. This compares to a relatively weak or absent signal that is localized to the cytoplasm in the nontransgenic cord (upper right images). This reflects the selective translation that occurs as a feature of the ISR. Scale bar 20 μm in (A), 5 μm in (B).

Article Snippet: Antibodies used: anti-mouse Kcnn1 (Proteintech 17929-1-AP, 1:400), anti-ATF4 (Cell Signaling 11815, 1:100), anti-CHAT (Abcam ab178850, 1:100), anti-phospho(S757) ULK1 (Cell Signaling 6888, 1:100), anti-phospho(S51) eIF2α (Cell Signaling 3398, 1:100), anti-phospho(S129) α-synuclein (Abcam ab51253,1:800), anti-Ctip2 (Abcam ab18465, 1:100).

Techniques: Transgenic Assay, Immunostaining, Activation Assay, Staining