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, 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: eBioMedicine

Article Title: Characterising the effect of circulating sphingolipids on metastatic prostate cancer cells

doi: 10.1016/j.ebiom.2025.106058

Figure Lengend Snippet: Sphingolipid metabolism drives response to 3LS plasma. a) Relative viability of PC3 cells incubated with 3LS negative (blue, n = 6) or 3LS positive (red, n = 6) and treated with either DMSO or 20 μM PF543. Viability is shown relative to the median viability of PC3 cells treated with 3LS negative plasma and DMSO. b) Summary statistics from two-factor ANOVA comparison of PC3 viability data. c) Relative viability of C4–2B cells incubated with 3LS negative (blue, n = 7) or 3LS positive (red, n = 6) and treated with either DMSO or 20 μM PF543. Viability is shown relative to the median viability of C4–2B cells treated with 3LS negative plasma and DMSO. d) Summary statistics from two-factor ANOVA comparison of C4–2B viability data. ∗p < 0.05, ns = not significant, significant values shown in red in summary statistics.

Article Snippet: Cells were incubated with plasma at 37 °C, 5% CO 2 for 48 h. For drug treatments, cells were treated as above with the addition of either 20 μM PF543 (MedChemExpress, Monmouth Junction, NJ, USA) in DMSO (final concentration of DMSO <0.1%) or an equivalent concentration of DMSO alone.

Techniques: Clinical Proteomics, Incubation, Comparison

Journal: Frontiers in Cell and Developmental Biology

Article Title: Ceramide Kinase Inhibition Blocks IGF-1-Mediated Survival of Otic Neurosensory Progenitors by Impairing AKT Phosphorylation

doi: 10.3389/fcell.2021.678760

Figure Lengend Snippet: Inhibition of endogenous SPHK1 has no effect on early inner ear development. (A) Morphological aspect of organotypic cultures after 20 h in serum-free medium (SFM), 1 μM PF-543, 10 nM IGF-1, IGF-1 plus PF-543. Immunostaining with TUNEL (cyan) and PH3 (red) of at least 5 otic vesicle per condition. Scale bar: 150 μm. (B) Quantification of otic vesicle area, (C) TUNEL-positive or (D) PH3-labeled cells in the presence or absence of PF-543 (0.25 and 1 μM) and IGF-1 (10 nM). (E) SOX2 intensity (F) and Tuj-1 intensity as neurogenesis markers normalized to data for SFM. Data are represented as mean ± SEM from at least n = 4 otic vesicles per condition. Statistical significance was calculated by one-way ANOVA followed by Bonferroni’s (B,E) or Dunnett’s T3 post hoc test (C) . * p < 0.05, ** p < 0.01, *** p < 0.001 vs control.

Article Snippet: Culture treatments included serum-free medium (SFM, control condition), 10 nM IGF-1 (recombinant IGF-I; Roche Molecular Biochemicals, Basel, Switzerland), the CERK inhibitor adamantane-1-carboxylic acid (2-benzoylamino-benzothiazol-6-yl)amide (NVP-231, hereafter called CKi) (Tocris Bioscience, Bristol, United Kingdom) prepared in DMSO, and the sphingosine kinase 1 (SPHK1) inhibitor PF-543 hydrochloride (Tocris Bioscience).

Techniques: Inhibition, Immunostaining, TUNEL Assay, Labeling, Control

Journal: bioRxiv

Article Title: Regulatory role of sphingolipid metabolites, sphingosine-1-phosphate and C16:0 ceramide, in immunogenic cell death of colon cancer cells induced by Bak/Bax-activation

doi: 10.1101/2021.09.16.460650

Figure Lengend Snippet: A. In DLD-1 cells, ABT and AZD alone or in combination had no effect on the cell surface exposure of ectoCRT. PF-543 alone also had no effect on ectoCRT exposure as high as 20 μM. In combination with ABT/AZD, PF-543 dose dependently induce cell surface exposure of ectoCRT (*=p<0.05). B. To determine whether ABT/AZD±PF-543 activated Caspase 8, we examined levels of Pro-Caspase 8 (Full length) and cleavage of the Casp8 substrate Bap31. ABT/AZD and MTX alone induce Casp8 activity and Bap31 cleavage. PF-543 enhanced these effects for both ABT/AZD and MTX (n=3). C. ABT/AZD and MTX induce the formation of dimeric CRT (non-reducing conditions –βME ) and PF-543 enhances these effects (n=3). D. Monomeric CRT migrates as two distinct species (Monomer and Monomer+). The Monomer+ form is arginylated at the N-terminus as determined by antibodies specific for the N-term Arg-CRT form (Millipore ABS1671). Dimeric CRT is predominantly comprised of the N-term Arg-CRT form denoted by Dimer+ suggesting that homodimerization of CRT occurs from a cytosolic form of CRT (n=3). E. Activation of Bak/Bax induces pore formation allowing for ER luminal Ca 2+ leakage to the cytosol. The activity of SERCA can attenuate cytosolic Ca 2+ levels by replenishing the ER. Inhibition of SERCA with Thapsigargin ( TH ) enhances ABT/AZD induced ectoCRT exposure (*=p<0.05). F. Inhibition of SERCA activity enhances ABT/AZD induced dimerization of Arg-CRT (n=3). Statistical analysis was performed by one-way ANOVA followed by Tukey’s multiple comparison test using GraphPad PRISM.

Article Snippet: As we observed previously, the SphK inhibitor PF-543 (Pfizer) alone has no effect on production of ectoCRT.

Techniques: Activity Assay, Activation Assay, Inhibition

Journal: bioRxiv

Article Title: Regulatory role of sphingolipid metabolites, sphingosine-1-phosphate and C16:0 ceramide, in immunogenic cell death of colon cancer cells induced by Bak/Bax-activation

doi: 10.1101/2021.09.16.460650

Figure Lengend Snippet: DLD-1 cells were treated with ABT/AZD (AA; 0.5/0.25μM, respectively) ± PF-543 (5μM) for 16h. AAPF induced depletion of c-FLIP L/S and induced activation of Caspase 8 as determined by cleavage of the Casp8 substrates Bap31 and BID. Inhibition of Casp8 (z-IETD-fmk; 10 μM) blocked Bap31 and BID cleavage but c-FLIP L/S depletion was independent of Casp8 activity suggesting that SphK generated S1P stabilizes c-FLIP under conditions of ER stress (n=3). B. ABT/AZD+PF-543 induced dimerization of Arg-CRT was also blocked by Casp8 inhibition ( D+, D dimer and M+ M monomeric forms of CRT are indicated). C. Casp8 inhibition dose-dependently reduced ABT/AZD alone and ABT/AZD+PF-543 induced ectoCRT exposure. D. Inhibition of Cer synthesis using fumonisin B1 (FB1; 70 μM) had no effect on ABT/AZD+PF-543 induced depletion of c-FLIP and cleavage of the Casp8 substrate, Bap31. E. FB1 treatment enhanced dimerization of cell associated CRT in response to ABT/AZD+PF-543 treatment. F. FB1 significantly reduced cell surface exposure of ectoCRT induced by ABT/AZD+PF-543 treatment (*=p<0.0001). Statistical analysis was performed by one-way ANOVA followed by Tukey’s multiple comparison test using GraphPad PRISM.

Article Snippet: As we observed previously, the SphK inhibitor PF-543 (Pfizer) alone has no effect on production of ectoCRT.

Techniques: Activation Assay, Inhibition, Activity Assay, Generated

Journal: bioRxiv

Article Title: Regulatory role of sphingolipid metabolites, sphingosine-1-phosphate and C16:0 ceramide, in immunogenic cell death of colon cancer cells induced by Bak/Bax-activation

doi: 10.1101/2021.09.16.460650

Figure Lengend Snippet: Sphingolipidomic analysis was conducted on DLD-1 cell samples treated with ABT/AZD low (0.5μM/0.25μM) alone and in combination with PF-543 (5μM) or ABT/AZD high (1.0μM/0.5μM) for 24h, (n=3). Values are presented as pmoles of sphingolipid/μg of total cellular protein.

Article Snippet: As we observed previously, the SphK inhibitor PF-543 (Pfizer) alone has no effect on production of ectoCRT.

Techniques:

Journal: bioRxiv

Article Title: Regulatory role of sphingolipid metabolites, sphingosine-1-phosphate and C16:0 ceramide, in immunogenic cell death of colon cancer cells induced by Bak/Bax-activation

doi: 10.1101/2021.09.16.460650

Figure Lengend Snippet: A. Knock-out clones of DLD-1 cells were generated with Crispr/Cas9 gRNAs targeted to CerS6. Knock-out was confirmed by western blot analysis using anti-CerS6 antibodies. Clone #2 was chosen for further analysis. B. Sphingolipidomic analysis of CerS6 knock-out clone #2. Sphingolipid levels are expressed as fold change relative to wild-type DLD-1 cells. C. Knock-out clones of DLD-1 cells were generated with Crispr/Cas9 gRNAs targeted to CerS5. Knock-out was confirmed by western blot analysis using anti-CerS5 antibodies. Clone #3 was chosen for further analysis. D. CerS6 knock-out reduced and CerS5 knock-out enhanced cell surface exposure of ectoCRT determined by flow-cytometry (n=3). Representative results of at least 3 individual experiments. E. Western blot analysis of CerS6 expression in CerS5 and CerS6 knock-out clones normalized to GAPDH. F. Time course analysis. Wild-type DLD-1 and CerS6 knock-out cells were treated for the indicated times with vehicle ( V ), ABT/AZD low (0.5μM/0.25μM; L ), ABT/AZD low + PF-543 (5.0μM; C ) and ABT/AZD high (1.0μM/0.5 μM). Depletion of c-FLIP, cleavage of Bap31 and dimerization of CRT were determined by western blot analysis using appropriate antibodies.

Article Snippet: As we observed previously, the SphK inhibitor PF-543 (Pfizer) alone has no effect on production of ectoCRT.

Techniques: Knock-Out, Clone Assay, Generated, CRISPR, Western Blot, Flow Cytometry, Expressing

Journal: bioRxiv

Article Title: Regulatory role of sphingolipid metabolites, sphingosine-1-phosphate and C16:0 ceramide, in immunogenic cell death of colon cancer cells induced by Bak/Bax-activation

doi: 10.1101/2021.09.16.460650

Figure Lengend Snippet: According to our results, ER membrane localized Bak/Bax activation occurs upstream of Caspase 8 activation within the “canonical” ICD pathway. ABT/AZD at high concentrations can directly induce ER lumenal Ca 2+ depletion and activate Caspase 8 leading to ectoCRT exposure and ICD. At lower, non-cytotoxic concentrations of ABT/AZD, ER membrane localized Bak/Bax induced Caspase 8 activation is counter-balanced by Bak/Bax induced activation of IRE1α kinase activity. IRE1α phosphorylates SphK1 at Ser225 to enhance its catalytic activity above basal levels. SphK1, localized to the cytosolic face of the ER membrane, interacts with TRAF2 and produces S1P locally to activate TRAF2 K63 ubiquitin ligase activity to polyubiquitinate c-FLIP. K63-Ub c-FLIP serves as the substrate for M1 linear ubiquitination, by LUBAC, which stabilizes c-FLIP and inhibits Caspase 8 activity. Thus, induction of ectoCRT dimerization, cell surface exposure and ICD are inhibited. Inhibition of SphK1 activity using SKIs such as PF-543 (among others) blocks S1P production and TRAF2 activation leading to c-FLIP degradation/depletion. Caspase 8 auto-activates leading to ectoCRT dimerization/exposure and ICD.

Article Snippet: As we observed previously, the SphK inhibitor PF-543 (Pfizer) alone has no effect on production of ectoCRT.

Techniques: Activation Assay, Activity Assay, Inhibition

Journal: Cells

Article Title: S1P Signalling Axis Is Necessary for Adiponectin-Directed Regulation of Electrophysiological Properties and Oxidative Metabolism in C2C12 Myotubes

doi: 10.3390/cells11040713

Figure Lengend Snippet: Role of S1P biosynthesis in the effect of Adn on the passive electrophysiological properties of C2C12 myotubes. ( a ) Representative photographs of Ctrl patched myotubes and of Adn, PF543, PF543 + Adn, ABC294640 and ABC294640 + Adn treated myotubes (Magnification 40×; scale bar 20 µm). C2C12 myotubes were pre-treated with specific SK1 inhibitor PF543 (10 µM) or SK2 inhibitor ABC294640 (1 µM) for 1 h before being challenged with 1 µg/mL Adn for 24 h. ( b ) Resting membrane potential (RMP) evaluated in current-clamp mode (Ctrl, n = 8; Adn, n = 21; PF543, n = 6; PF543 + Adn, n = 10; ABC294640 n = 7; ABC294640 + Adn, n = 8). ( c ) Membrane resistance (Rm) (Ctrl, n = 19; Adn, n = 39; PF543, n = 16; PF543 + Adn, n = 13; ABC294640, n = 10; ABC294640 + Adn, n = 14). ( d ) Membrane capacitance (C m ) estimated in voltage-clamp (Ctrl, n = 8; Adn, n = 29; PF543, n = 12; PF543 + Adn, n = 19; ABC294640, n = 6; ABC294640 + Adn, n = 8). In all the panels values are means ± SD. Data are from three independent experiments, each performed in duplicate, n is the total number of analyzed myotubes. One-way ANOVA with Bonferroni’s post hoc test, * indicates p < 0.05 vs. Ctrl; # p < 0.05 vs. PF453; ns, not significant. The pharmacological blockade of SK1 (10 µM PF543) or SK2 (1 μM ABC294640) affects Adn-induced changes in electrophysiological passive properties in a statistically significant way by two-way ANOVA followed by Bonferroni’s post hoc test, § p < 0.05.

Article Snippet: All biochemicals, TRI reagent, cell culture reagents, Dulbecco’s Modified Eagle Medium (DMEM), foetal bovine serum (FBS), horse serum (HS), protease inhibitor cocktail, bovine serum albumin (BSA), D-erythro-MAPP, specific anti-Spns2 (N-TERM) antibodies and the specific SK1 inhibitor PF543 were purchased from Merck Life Sciences (Burlington, MA, USA).

Techniques:

Journal: Cells

Article Title: S1P Signalling Axis Is Necessary for Adiponectin-Directed Regulation of Electrophysiological Properties and Oxidative Metabolism in C2C12 Myotubes

doi: 10.3390/cells11040713

Figure Lengend Snippet: Role of S1P biosynthesis in the effect of Adn on ionic currents in C2C12 myotubes. C2C12 myotubes were pre-treated with specific SK1 inhibitor PF543 (10 µM) or SK2 inhibitor ABC294640 (1 µM) for 1 h before being challenged with 1 µg/mL Adn for 24 h. Overall I–V plots related to K + currents measured in the presence of PF543 ( a ) or ABC294640 ( b ) are shown: Ctrl, filled triangles, n = 7; Adn, filled squares, n = 7; PF543, filled circles, n = 5; PF543 + Adn, filled diamonds, n = 3; ABC294640, open circles, n = 3; ABC294640 + Adn, open diamonds, n = 4). Adn increases K + current compared to Ctrl ( a , b ), * p < 0.05 vs. Ctrl, § p < 0.05 vs. Adn (two-way ANOVA and Bonferroni’s post hoc test). Overall I–V plots related to Ca 2+ currents measured following the pre-treatment with SK1 inhibitor PF543 10 µM ( c ) or SK2 inhibitor ABC294640 (1 µM) ( d ): Ctrl, filled triangles, n = 7; Adn, filled squares, n = 5; PF543, filled circles, n = 2; PF543 + Adn, filled diamonds, n = 4; ABC294640, open circles, n = 3; ABC294640 + Adn, open diamonds, n = 3). Adn increases Ca 2+ inflow compared to Ctrl cells ( c , d ), * p < 0.05 vs. Ctrl, § p < 0.05 vs. Adn (two-way ANOVA and Bonferroni’s post hoc test). Data points are mean ± SD. Data are from three independent experiments, each performed in duplicate, n is the total number of analyzed myotubes. Error bars are shown when their size is bigger than the symbol.

Article Snippet: All biochemicals, TRI reagent, cell culture reagents, Dulbecco’s Modified Eagle Medium (DMEM), foetal bovine serum (FBS), horse serum (HS), protease inhibitor cocktail, bovine serum albumin (BSA), D-erythro-MAPP, specific anti-Spns2 (N-TERM) antibodies and the specific SK1 inhibitor PF543 were purchased from Merck Life Sciences (Burlington, MA, USA).

Techniques:

Journal: Cells

Article Title: S1P Signalling Axis Is Necessary for Adiponectin-Directed Regulation of Electrophysiological Properties and Oxidative Metabolism in C2C12 Myotubes

doi: 10.3390/cells11040713

Figure Lengend Snippet: Role of S1P biosynthesis in the oxidative metabolism of Adn in C2C12 myotubes. C2C12 myotubes were pre-treated with specific SK1 inhibitor PF543 (10 µM) or SK2 inhibitor ABC294640 (1 µM) for 1 h before being challenged with 1 µg/mL Adn for 24 h. The percentage of oxygen consumption rate (OCR) values were measured in cell lysates using a Clark-type O 2 electrode from Hansatech Instruments as described in . Histogram reports the percentages of mean values ± SD with respect to controls. Data are obtained from at least three independent experiments, each performed in duplicate. The statistical analysis was carried out using one-way ANOVA test followed by Tukey’s multiple comparisons test using GraphPad Prism software v 6.0 (*** p < 0.001 vs. Ctrl; # p < 0.05 vs. PF543, § p < 0.01 vs. Adn, ns, not significant).

Article Snippet: All biochemicals, TRI reagent, cell culture reagents, Dulbecco’s Modified Eagle Medium (DMEM), foetal bovine serum (FBS), horse serum (HS), protease inhibitor cocktail, bovine serum albumin (BSA), D-erythro-MAPP, specific anti-Spns2 (N-TERM) antibodies and the specific SK1 inhibitor PF543 were purchased from Merck Life Sciences (Burlington, MA, USA).

Techniques: Software

Journal: Cells

Article Title: S1P Signalling Axis Is Necessary for Adiponectin-Directed Regulation of Electrophysiological Properties and Oxidative Metabolism in C2C12 Myotubes

doi: 10.3390/cells11040713

Figure Lengend Snippet: Role of S1P biosynthesis in the metabolome of Adn-treated C2C12 myotubes. C2C12 myotubes were pre-treated with specific SK1 inhibitor PF543 (10 µM) or SK2 inhibitor ABC294640 (1 µM) for 1 h before being challenged with 1 µg/mL Adn for 24 h. Adn-dependent metabolomic changes in cell lysates (endo-metabolome) ( a ) and conditioned media (exo-metabolome) ( b ) were analysed in four experiments. Boxplots of the significant metabolites: changes in metabolite levels caused by Adn were statistically significant by paired Wilcoxon test, (* p < 0.05 vs. Ctrl, § p < 0.05 vs. Adn, ° p < 0.05 vs. ABC294640, ns, not significant).

Article Snippet: All biochemicals, TRI reagent, cell culture reagents, Dulbecco’s Modified Eagle Medium (DMEM), foetal bovine serum (FBS), horse serum (HS), protease inhibitor cocktail, bovine serum albumin (BSA), D-erythro-MAPP, specific anti-Spns2 (N-TERM) antibodies and the specific SK1 inhibitor PF543 were purchased from Merck Life Sciences (Burlington, MA, USA).

Techniques: