Journal: Journal for Immunotherapy of Cancer

Article Title: Preventing surgery-induced natural killer cell suppression and metastases by inhibiting PI3K-gamma signaling in myeloid-derived suppressor cells

doi: 10.1136/jitc-2025-013304

Figure Lengend Snippet: Large expansion of phenotypically defined sx-MDSCs comprised mainly of M-MDSC and PMN-MDSCs immediately after surgery (POD1). ( A ) Representative flow plots showing CD33 versus Lineage (CD3/CD56/CD19) for baseline (left) and POD1 (right) PBMCs. ( B ) Representative flow plots gated down on CD33 + Lin − cells showing CD15 versus CD14 expression. ( C ) Healthy controls (n=15), Baseline, and POD1 patients (n=44) proportion of CD33 + Lin − cells (left) and MDSC subsets (right). ( D ) The MFI of HLA-DR gated on M-MDSCs (left) and the proportion of M-MDSCs that are HLA-DR lo (right) in healthy controls, baseline and POD1 patients with cancer. ( E ) Representative histograms of common M-MDSC and PMN-MDSC markers, before and after surgery. Statistical analysis was performed using Wilcoxon matched pairs signed-rank test or Kruskal-Wallis with Dunn’s post test. **** p≤0.0001. FMO, fluorescence minus one control; HLA-DR, human leukocyte antigen - DR isotype; E-MDSC, early stage-MDSC; M-MDSC, monocytic-MDSC; MDSC, myeloid derived suppressor cell; sx-MDSC, surgery-induced MDSC, MFI; mean fluorescence instensity; PBMC, peripheral blood mononuclear cell; PMN-MDSC, polymorphonuclear-MDSC; POD1, postoperative day 1.

Article Snippet: Sx-MDSCs were isolated using the MDSC Isolation Kit (Miltenyi #130-094-538) from surgically stressed mice on POD1.

Techniques: Expressing, Fluorescence, Control, Derivative Assay

Journal: Journal for Immunotherapy of Cancer

Article Title: Preventing surgery-induced natural killer cell suppression and metastases by inhibiting PI3K-gamma signaling in myeloid-derived suppressor cells

doi: 10.1136/jitc-2025-013304

Figure Lengend Snippet: Sx-MDSCs suppress NK92-MI cytotoxicity. ( A ) Schematic of MDSC-NK92 co-culture suppression assay with CP450-labeled K562 target cells. ( B ) NK92-MIs were co-cultured at increasing ratios with CD33+ cells for 6 or 24 hours (in the absence of K562s) to assess CD33+ cell death by NK92-MI (left). Sx-MDSCs from POD1 patient samples (n=10) were co-cultured with NK92-MI cells and NK cytotoxicity was measured as % dead K562 (right). ( C ) % suppression was calculated as the reduction of NK cytotoxicity, normalized to NK cells alone (ratio=0). CD33+ cells isolated from baseline and patient-matched POD1 blood were co-cultured with NK92-MI and K562 targets to measure their suppressive capacity (n=5). ( D ) Representative images of sorted cells stained with Giemsa Wright. ( E ) Bulk Sx-MDSCs, M-MDSCs, PMN-MDSCs, and HDN from POD1 patients (n=3) co-cultured with NK92-MI to measure effect on NK cytotoxicity. The NK92-MI alone control group is indicated by the broken horizontal line. Statistical analysis used Kruskal-Wallis with Dunn’s post-test. ****p≤0.0001. HDN, high-density neutrophils; M-MDSC, monocytic-MDSC; MDSC, myeloid-derived suppressor cell; NK, natural killer; sx-MDSC, surgery-induced MDSC, PMN-MDSC, polymorphonuclear-MDSC; POD1, postoperative day 1.

Article Snippet: Sx-MDSCs were isolated using the MDSC Isolation Kit (Miltenyi #130-094-538) from surgically stressed mice on POD1.

Techniques: Co-Culture Assay, Suppression Assay, Labeling, Cell Culture, Isolation, Staining, Control, Derivative Assay

Journal: Journal for Immunotherapy of Cancer

Article Title: Preventing surgery-induced natural killer cell suppression and metastases by inhibiting PI3K-gamma signaling in myeloid-derived suppressor cells

doi: 10.1136/jitc-2025-013304

Figure Lengend Snippet: scRNA-seq of cryopreserved PBMCs before and after surgery reveals drastically altered monocyte/myeloid cell expression profiles on POD1. ( A ) Schematic showing PBMCs (cryopreserved) from six matched baseline and POD1 patients were processed for multiplexed scRNA-seq with the 10x Genomics Chromium platform. ( B ) Dot plot displaying the relative expression of the top three marker genes (x-axis) of each cluster (y-axis). ( C ) UMAP plot of scRNA-seq data. Each point corresponds to a single cell and is colored by cluster. ( D ) Identical UMAP embedding as in ( C ), with Baseline and POD1 cells labeled. ( E ) UMAP plot of the CD14+ and CD16+ monocyte population at baseline and POD1. ( F ) GSEA showing the NES of the top upregulated and downregulated pathways. All gene sets are significantly enriched (FDR<0.05). ( G ) UMAP plot showing activity of an M-MDSC gene set from Alshetaiwai et al ; in monocyte populations in Baseline and POD1 samples. ( H ) NMF plots of gene expression programs for NMF 1, 2, 3 and 4 (see for all NMF programs). ( I ) A heatmap of selected genes driving the various NMF gene expression programs (z-score transformed; ranked according to NMF1). FDR, false discovery rate; GSEA, gene set enrichment analysis; M-MDSC, monocytic-myeloid-derived suppressor cell; NES, normalized enrichment scores; NMF, non-negative matrix factorization; PBMC, peripheral blood mononuclear cell; POD1, postoperative day 1; scRNA-seq, single-cell RNA sequencing; UMAP, uniform manifold approximation and projection.

Article Snippet: Sx-MDSCs were isolated using the MDSC Isolation Kit (Miltenyi #130-094-538) from surgically stressed mice on POD1.

Techniques: Expressing, Marker, Labeling, Activity Assay, Gene Expression, Transformation Assay, Derivative Assay, RNA Sequencing

Journal: Journal for Immunotherapy of Cancer

Article Title: Preventing surgery-induced natural killer cell suppression and metastases by inhibiting PI3K-gamma signaling in myeloid-derived suppressor cells

doi: 10.1136/jitc-2025-013304

Figure Lengend Snippet: PI3K inhibitors reverse the suppressive effects of sx-MDSCs on NK cells. ( A ) A library of 147 small molecules covering the major cellular signaling pathways was screened (n=4 experiments) at 1 µM in the MDSC-NK suppression assay. Compounds that improved NK cell cytotoxicity (blue, left y-axis) by >50% from DMSO control (black dotted line), without impacting NK cell viability (red, right y-axis), were considered hits. LY294002 (purple circle) improved cytotoxicity in all screens and was the top hit in 3/4 screens. ( B ) LY294002 improves NKC (n=6). ( C ) Dose response of LY294002 and the effect on NKC and K562 viability. ( D ) pan-PI3K and ( E ) PI3K-γ specific inhibitors (IPI-549, TG100-115) in NK92—MDSC suppression assays. ( F ) pan-PI3K and ( G ) PI3K-γ specific inhibitors (IPI-549, TG100-115) in MDSC suppression assays with primary healthy donor NK cells. Statistical analysis used one-way ANOVA with Dunnett’s multiple comparisons test. ***p<0.0001. ANOVA, analysis of variance; DMSO, dimethyl sulfoxide; MDSC, myeloid-derived suppressor cell; NK, natural killer; sx-MDSC, surgery-induced MDSC.

Article Snippet: Sx-MDSCs were isolated using the MDSC Isolation Kit (Miltenyi #130-094-538) from surgically stressed mice on POD1.

Techniques: Protein-Protein interactions, Suppression Assay, Control, Derivative Assay

Journal: Journal for Immunotherapy of Cancer

Article Title: Preventing surgery-induced natural killer cell suppression and metastases by inhibiting PI3K-gamma signaling in myeloid-derived suppressor cells

doi: 10.1136/jitc-2025-013304

Figure Lengend Snippet: PI3K pathway activation in postoperative MDSCs and functional impact of PI3K inhibition. ( A ) Cohort of differentially expressed genes from baseline versus POD1 MDSCs. ( B ) Box plot inference of PI3K pathway activity in baseline versus POD1 MDSCs. ( C ) Pathway diagram of PI3K signaling and Akt phosphorylation. Phosphorylation status of AKT at the T308 (left) and S473 residue (right) (n=5). ( D ) Effect of PI3K inhibitors on pAKT (T308) MFI (top), MDSC-NK suppression (middle), and NKC (bottom). ( E ) Effect of IPI-549 on POD1 expression of anti-inflammatory versus pro-inflammatory mRNA transcripts, normalized to baseline. Statistical analysis used Wilcoxon matched pairs signed-rank tests and one-way ANOVA with Dunnett’s multiple comparisons test (vs DMSO control). ****p>0.0001. ANOVA, analysis of variance; ARG1, arginase-1; CCL, CC chemokine ligand; DMSO, dimethyl sulfoxide; IL, interleukin; MDSC, myeloid-derived suppressor cell; mRNA, messenger RNA; MFI, mean fluorescence intensity; NK, natural killer; POD1, postoperative day 1; TGF, transforming growth factor; TNF, tumor necrosis factor.

Article Snippet: Sx-MDSCs were isolated using the MDSC Isolation Kit (Miltenyi #130-094-538) from surgically stressed mice on POD1.

Techniques: Activation Assay, Functional Assay, Inhibition, Activity Assay, Phospho-proteomics, Residue, Expressing, Control, Derivative Assay, Fluorescence

Journal: Journal for Immunotherapy of Cancer

Article Title: Preventing surgery-induced natural killer cell suppression and metastases by inhibiting PI3K-gamma signaling in myeloid-derived suppressor cells

doi: 10.1136/jitc-2025-013304

Figure Lengend Snippet: Blockade of PI3Kγ signaling in sx-MDSCs reduces NK cell suppression and metastatic disease in mouse models of surgical stress. ( A ) Ex vivo effect of PI3K inhibitors on pAKT (T308) phosphorylation in splenic MDSCs from C57Bl/6 mice (n=4). ( B ) Effect of preoperative in vivo administration of PI3K inhibitors on pAKT (T308) phosphorylation measured on POD1 in splenic MDSCs (n=4). ( C ) Ex vivo effect of PI3K inhibitors on MDSC-NK %suppression (n=3). ( D ) Effect of preoperative in vivo administration of PI3K-γ inhibitors on sx-MDSC suppressive capacity. ( E ) Adoptive transfer of representative images of lungs is shown. One-way ANOVA with Holm-Sidak or two-way ANOVA with Dunnett’s multiple comparisons tests were performed. ****p<0.0001. ANOVA, analysis of variance; M-MDSC, monocytic-MDSC; MDSC, myeloid-derived suppressor cell; MFI, mean fluorescence intensity; NK, natural killer; sx-MDSC, surgery-induced MDSC, PMN-MDSC, polymorphonuclear-MDSC; POD1, postoperative day 1.

Article Snippet: Sx-MDSCs were isolated using the MDSC Isolation Kit (Miltenyi #130-094-538) from surgically stressed mice on POD1.

Techniques: Ex Vivo, Phospho-proteomics, In Vivo, Adoptive Transfer Assay, Derivative Assay, Fluorescence

Journal: Acta neuropathologica communications

Article Title: BDNF augmentation reverses cranial radiation therapy-induced cognitive decline and neurodegenerative consequences.

doi: 10.1186/s40478-024-01906-9

Figure Lengend Snippet: Fig. 2 Riluzole treatment restores hippocampal BDNF in the irradiated hippocampus. 10–12 weeks old WT male mice received cranial radiation therapy (RT) followed by riluzole (RZ) treatment (13 mg/kg) in drinking water for 6–7 weeks. An ELISA-based quantification of BDNF from the micro-dissected hippocampus showed RT-induced reductions in the RT + Vehicle group. Importantly, RZ treatment in the cranially irradiated mice showed significant restoration of BDNF levels. Data are presented as mean ± SEM (N = 6–10 mice per group). P values were derived from two-way ANOVA and Bonferroni’s multiple comparisons test

Article Snippet: BDNF levels were quantified using a commercially available ELISA kit (E-EL-M0203, Elabscience Biotechnology) and uncoated ELISA plates (Nunc MaxiSorp, Biolegend).

Techniques: Irradiation, Enzyme-linked Immunosorbent Assay, Derivative Assay

Journal: Cancer research

Article Title: Inhibition of BCL2 family members increases the efficacy of copper chelation in BRAF V600E -driven melanoma

doi: 10.1158/0008-5472.CAN-19-1784

Figure Lengend Snippet: A, Schematic diagram of high throughput screen of 2123 Selleckchem bioactive compound library at 100nM alone or in combination with the IC20 of TTM (dashed red line) in A375 or WM88. B,C, Normalized Percent Inhibition (NPI) of TTM + Compound versus NPI Compound graph for indicated cells treated with Selleckchem bioactive compound library alone or in combination with IC20 of TTM. Hits (blue circles) are defined as NPI TTM + Compound ≥ 20 (dashed red line). D,E, Observed effect versus expected effect graph for indicated cells of Hits from B and C. Hits (blue circles) are defined as Bliss Index ≥ 1.5 (dashed red line). F,G, Graphical representation of Hits defined as NPI TTM + Compound ≥ 20 and Bliss Index ≥ 1.5 in indicated cells. H, Venn diagram relationship between Compound Hits. I, NPI of 5 overlapping Hit Compounds from H alone or in combination with IC20 of TTM (dashed red line). J,K, Scatter dot plot of %ATP normalized to VEH ± s.e.m. of indicated cells treated with vehicle or indicated concentrations of drugs. L,M,N,O, Scatter dot plot of %ATP normalized to VEH ± s.e.m. of indicated cells treated with VEH or indicated concentrations of drugs. Results were compared using a one-way ANOVA followed by a Tukey’s multi-comparisons test. One asterisk, P<0.05, Two asterisks, P<0.01, Three asterisks, P<0.001, Four asterisks, P<0.0001. n=3.

Article Snippet: 293T/17 (ATCC, catalog #CRL-11268), A375 (ATCC, catalog #CRL-1619), WM88 (Rockland, catalog #WM88-01-0001), WM3311 (Rockland, catalog #WM3311-01-0001), WM3743 (Rockland, catalog #WM3743-01-0001) cells were purchased from the indicated companies and maintained in Dulbecco’s Modified Eagle Media (DMEM, Gibco) supplemented with 10% v/v fetal bovine serum (FBS, GE Lifesciences) and 1% penicillin-streptomycin (P/S, Gibco).

Techniques: High Throughput Screening Assay, Drug discovery, Inhibition

Journal: Cancer research

Article Title: Inhibition of BCL2 family members increases the efficacy of copper chelation in BRAF V600E -driven melanoma

doi: 10.1158/0008-5472.CAN-19-1784

Figure Lengend Snippet: A,C, Relative CellTiter-Glo® cell viability ± s.e.m. of indicated cells stably expressing two independent Dox-inducible shRNAs (#1 and #2) against indicated genes treated with indicated concentrations of TTM without or with Dox n=3. B,D, Scatter dot plot of TTM IC50 ± s.e.m in indicated cells cells stably expressing two independent Dox-inducible shRNAs against indicated genes treated without or with Dox. Results were compared using a two-way ANOVA followed by a Sidak’s multi-comparisons test. Three asterisks, P<0.001, Four asterisks, P<0.0001. n=3. E,G, Relative CellTiter-Glo® cell viability ± s.e.m. of indicated cells treated without or with indicated concentrations of TTM and increasing concentrations of indicated BH3 mimetics. n=3. F,H, Scatter dot plot of BH3 mimetic IC50 ± s.e.m in indicated cells treated without or with indicated concentrations of TTM and increasing concentrations of indicated BH3 mimetics. n=3. Results were compared using a two-way ANOVA followed by a Tukey’s multi-comparisons test. n=3. I,J, Graphical representation of Bliss Index (observed effect versus expected effect) from A375 (E) and WM88 (G) at the indicated drug combinations. Synergistic combinations are indicated by Bliss Index values >1. Two asterisks, P<0.01, Three asterisks, P<0.001, Four asterisks, P<0.0001.

Article Snippet: 293T/17 (ATCC, catalog #CRL-11268), A375 (ATCC, catalog #CRL-1619), WM88 (Rockland, catalog #WM88-01-0001), WM3311 (Rockland, catalog #WM3311-01-0001), WM3743 (Rockland, catalog #WM3743-01-0001) cells were purchased from the indicated companies and maintained in Dulbecco’s Modified Eagle Media (DMEM, Gibco) supplemented with 10% v/v fetal bovine serum (FBS, GE Lifesciences) and 1% penicillin-streptomycin (P/S, Gibco).

Techniques: Stable Transfection, Expressing