Journal: STAR Protocols

Article Title: Simplified mass cytometry protocol for in-plate staining, barcoding, and cryopreservation of human PBMC samples in clinical trials

doi: 10.1016/j.xpro.2022.101362

Figure Lengend Snippet: Surface antibody master mix, titrated for staining of up to 3 M cells per condition

Article Snippet: Anti-Human CD279/PD-1 (EH12.2H7)-174Yb—100 Tests (1:100) , Fluidigm , Cat#3174020B.

Techniques: Staining

Journal: STAR Protocols

Article Title: Simplified mass cytometry protocol for in-plate staining, barcoding, and cryopreservation of human PBMC samples in clinical trials

doi: 10.1016/j.xpro.2022.101362

Figure Lengend Snippet:

Article Snippet: Anti-Human CD279/PD-1 (EH12.2H7)-174Yb—100 Tests (1:100) , Fluidigm , Cat#3174020B.

Techniques: Recombinant, Saline, Staining, Blocking Assay, Isolation, Software, Cell Counting, Cytometry

Journal: Cell Reports Methods

Article Title: Optimization and validation of CAR transduction into human primary NK cells using CRISPR and AAV

doi: 10.1016/j.crmeth.2022.100236

Figure Lengend Snippet:

Article Snippet: 155Gd_PD-1 , Fluidigm , Cat# 3155009B, RRID: AB_2811087.

Techniques: Virus, Recombinant, Electroporation, Antibody Labeling, Labeling, Software

Journal: bioRxiv

Article Title: A class act: HDAC1- Malat1 regulates MDSC apoptosis and cell cycling to decrease suppression of T cells

doi: 10.64898/2026.03.23.713743

Figure Lengend Snippet: (A) UMAP visualization of major cell clusters identified from single cell RNA sequencing (scRNA-seq) of NT2.5-LM breast-to-lung metastatic tumors after 3 weeks of treatment with: vehicle (V), Entinostat (E), anti-CTLA-4 + anti-PD-1 immune checkpoint inhibitors (PC), and Entinostat + anti-CTLA-4 + anti-PD-1 combination (EPC). Subclusters of MDSCs identified: G-MDSCs and M-MDSCs. (B) Iterative logistic regression analyses conducted on the major MDSC cluster comparing the Vehicle (V) and Entinostat + ICIs combination treatment (EPC). (C) Expression of Malat1 / MALAT1 in G- and M-MDSCs isolated from lung metastases of NT2.5-LM mice treated with vehicle vs. Entinostat for 3 weeks (left, n=2 from 5 pooled mice per treatment group), J774M murine MDSC-like cell line treated with Entinostat for 24 hours (middle, n=3), and human PBMC-derived MDSCs (hMDSCs) treated with Entinostat for 24 hours (right, n=3). (D) Median Fluorescence Intensity expression of Malat1 in CD45 + CD11b + MHC-II - F4/80 - Ly6G - Ly6C hi cells from lung metastases in 4T1 mice, treated with vehicle (V), Entinostat (E), and Entinostat + ICIs (EPC) for 3 weeks. (E) Expression of Malat1 in J774M cell line treated with various concentrations of various HDAC inhibitors, with corresponding targeted class and specific HDACs. One-way ANOVA for (C, E: all statistically significant with p<0.05, unless indicated as non-significant (ns)), Kruskal-Wallis test with Dunn’s correction for (D). * p<0.05, *** p< 0.001, **** p<0.0001. ENT = Entinostat; PAN = Panobinostat; BEL = Belinostat; VOR = Vorinostat; TSA = Trichostatin A; DOM = Domatinostat; CHL = Chlopynostat; PYR = Pyroxamide; SCA = Santacruzamate A; RGF = RGFP966; TMP = TMP269; SIS = SIS17.

Article Snippet: Immune checkpoint inhibitors (ICIs) used in this study: anti-CTLA-4 (BioXCell cat. #BE0131) and anti-PD-1 (BioXCell cat. #BE0146).

Techniques: Single Cell, RNA Sequencing, Expressing, Isolation, Derivative Assay, Fluorescence

Journal: bioRxiv

Article Title: A multivalent peptide-polymer conjugate material mimics STING to therapeutically activate innate immune signaling

doi: 10.64898/2026.03.24.712780

Figure Lengend Snippet: ( A ) Mice were inoculated with 3×10 6 BPPNM cells IP and dosed with 20 μg of STING or Scr conjugate delivered by LNP IP at 10, 13, 16, and 19 days after inoculation. Omental tumor was collected on day 20 for analysis by flow cytometry. Groups included N = 5 mice. ( B ) Cell populations in tumor after treatment, displaying percentage of CD45 + cells made up by T cells (CD3 + ), CD8 + T cells, CD4 + T cells, NK cells (CD3 - NK1.1 + ), B cells (CD19 + ), Macrophages (F4/80 + ), DCs (CD11c + MHCII + ), and MDSCs (CD11b + Gr-1 hi ). Percentage of CD45 - cells made up by BPPNM cancer cells (GFP + ) are also displayed. ( C-D ) Polarization of Macrophages (F4/80 + ), showing MFI and representative distributions of ( C ) CD86 and ( D ) CD206. ( E ) Activation of DCs (CD11c + MHCII + ), showing MFI and representative distributions of CD86. ( F ) Expression of PD-1 on CD8 + T cells, showing percentage of cells that are PD-1 + and representative distributions of PD-1. P values computed with a one-way ANOVA followed by Tukey’s post-hoc test are displayed above each figure. All flow data is represented as mean ± SD. ( G ) Mice were inoculated with 3×10 6 BPPNM cells IP and dosed with 20 μg of STING or Scr conjugate delivered by LNP IP at 10, 13, 16, 19, and 22 days after inoculation. A subset of groups were additionally treated with 100 μg αPD-1 antibody at 11 and 17 days after inoculation. Groups included N = 6 (PBS, STING LNP, Scr LNP), N = 5 (STING LNP + αPD-1), or N = 4 (PBS + αPD-1, Scr LNP + αPD-1) mice. ( H ) Survival plot with P values determined by log(rank) (Mantel–Cox) test. ( I-K ) Tumor burden measured by IVIS, displaying ( I ) geometric mean ± SD bioluminescent intensity over the treatment period, ( J ) an image of the mouse with the median bioluminescent intensity from each group at the measurement following the end of treatment (day 24), and ( K ) individual mouse bioluminescent intensity values.

Article Snippet: In groups specified in figure captions, mice were also administered a 100 μg dose of anti-mouse PD-1 antibody (Bio X Cell #BE0273 InVivoMAb, Clone 29F.1A12) in 200 μL of 1× PBS on days 11 and 17 after tumor inoculation.

Techniques: Flow Cytometry, Activation Assay, Expressing

Journal: Cancer Communications

Article Title: NAT10 regulates tumor progression and immune microenvironment in pancreatic ductal adenocarcinoma via the N4‐acetylated LAMB3‐mediated FAK/ERK pathway

doi: 10.1002/cac2.70045

Figure Lengend Snippet: NAT10 was associated with CD8 + T cell exhaustion in the PDAC TME. (A) Cell subset plot showing 10 subsets of T_NK cells in subcutaneous tumors collected from negative control (NC), Nat10 knockdown (sh Nat10 ‐1), and Nat10 overexpression ( Nat10 ‐OE) groups, as analyzed by the UMAP dimensionality reduction algorithm. (B) Histogram showing the proportions of 10 subsets of T_NK cells in sh Nat10 ‐1, NC, and Nat10 ‐OE groups. The red rectangle indicates the predominant abundance of CD8 + Tex (type I) subset in the Nat10‐ OE group. (C) Dot plot showing the mean expression and expression proportions of marker genes and functional signatures of the nine distinct T cell subsets. (D) Representative micrographs of immunofluorescence staining of CD8a (red) showing the proportion of CD8 + T cells in the subcutaneous tumor sections from sh Nat10 ‐1, NC, and Nat10 ‐OE groups ( n = 3 per group). Cell nuclei were counterstained with DAPI (blue). The quantification results are shown below. (E) Representative micrographs of multiplexed immunofluorescence staining of CD8a (red), PD‐1 (yellow), IFNg (orange), and Prf1 (green) showing the proportion of CD8 + Tex (type I) subset in the subcutaneous tumor sections from sh Nat10 ‐1, NC, and Nat10 ‐OE groups ( n = 3 per group). Cell nuclei were counterstained with DAPI (blue). The quantification results are shown below. Abbreviations: Bcl2, B‐cell lymphoma‐2; CD4 + Tex, exhausted CD4 + T cells; CD4 + Tn, naïve CD4 + T cells; CD8 + Tc, cytotoxic CD8 + T cells; CD8 + Tex, exhausted CD8 + T cells; CD8 + Tn, naïve CD8 + T cells; Ctla4, ctlantigen‐4; DAPI, 4',6‐Diamidino‐2‐phenylindole; γδT, gamma‐delta T cells; IFNg/Ifng, interferon gamma; Lag3, lymphocyte‐activation gene 3; NAT10, N‐acetyltransferase 10; NC, negative control; NK, natural killer cells; ns, not significant; OE, overexpression; PD‐1, programmed cell death 1; PDAC, pancreatic ductal adenocarcinoma; Pdcd1, programmed cell death 1; Prf1, perforin; Tgfb1, transforming growth factor beta 1; TME, tumor microenvironment; T_NK cells, T cells and NK cells; Treg, regulatory T cells; UMAP, Uniform Manifold Approximation and Projection.

Article Snippet: The cells were fixed in 4% PFA at room temperature for 15 minutes and incubated with recombinant human/mouse PD‐1 Fc chimeric protein (MedChemExpress) at room temperature for 1 hour, followed by incubation with Alexa Fluor TM 647‐conjugated secondary antibodies (Invitrogen) at room temperature for 1 hour.

Techniques: Negative Control, Knockdown, Over Expression, Expressing, Marker, Functional Assay, Immunofluorescence, Staining, Activation Assay

Journal: Cancer Communications

Article Title: NAT10 regulates tumor progression and immune microenvironment in pancreatic ductal adenocarcinoma via the N4‐acetylated LAMB3‐mediated FAK/ERK pathway

doi: 10.1002/cac2.70045

Figure Lengend Snippet: NAT10 may regulate CD8 + T cell function via PD‐1/PD‐L1 axis in PDAC. (A) Representative micrographs of immunofluorescence staining of PD‐1 (red) showing PD‐1/PD‐L1 binding on PDAC cells in negative control (NC) and NAT10 / Nat10 overexpression ( NAT10/Nat10 ‐OE) groups, with and without Naamidine J (10 µmol/L, 24 hours) treatment ( n = 3 per group). Cell nuclei were counterstained with DAPI (blue). The quantification results are shown on the right. (B) Representative results of Western blotting showing PD‐L1 expression in PDAC cells, including NC and NAT10 / Nat10 ‐OE groups, with and without Naamidine J (10 µmol/L, 24 hours) treatment ( n = 3 per group). GAPDH was used as a loading control. The numbers below the bands indicate the relative band density quantified by ImageJ, normalized to GAPDH. (C) Representative results of CD8 + T cell‐mediated tumor cell‐killing assay in NC and NAT10 ‐OE groups, with and without Naamidine J (10 µmol/L, 24 hours) treatment ( n = 3 per group). The quantification results are shown below. (D) Schematic showing the schedule of Naamidine J treatment in the animal models bearing subcutaneous tumors. PanO2 cells of negative control (NC) and Nat10 overexpression ( Nat10 ‐OE) were subcutaneously injected in the flanks of mice on day 0. Naamidine J was intraperitoneally injected into mice on days 28, 31, 34, 37, 40, 43, 46, 49, 52 (30 mg/kg, once every 3 days), and an equal volume of PBS was intraperitoneally injected accordingly as a control. The tumors were harvested on day 56 for analysis. (E) Growth curve of subcutaneous tumor size from the NC and Nat10 ‐OE groups, with PBS or Naamidine J treatment ( n = 5 per group). (F) Quantification results showing subcutaneous tumor size on day 56 in the NC and Nat10‐ OE groups, with PBS or Naamidine J treatment (n = 5 per group). (G) Quantification results showing the inhibitory efficacy of Naamidine J treatment on tumor size in the NC and Nat10‐ OE groups ( n = 5 per group). Inhibitory efficacy in the NC group = [tumor size NC + PBS – tumor size NC + Naamidine J ] / tumor size NC + PBS × 100%, and inhibitory efficacy in the Nat10 ‐OE group = [tumor size Nat10 ‐OE + PBS – tumor size Nat10 ‐OE + Naamidine J ] /tumor size Nat10 ‐OE + PBS × 100%. (H) Representative results of flow cytometry showing the proportion of infiltrating PD‐1 + IFNg + /PD‐1 − IFNg + CD8 + T cells from the subcutaneous tumors in the NC and Nat10‐ OE groups, with PBS or Naamidine J treatment ( n = 5 per group). The quantification results are shown below. (I) Representative results of flow cytometry showing the proportion of infiltrating PD‐1 + Prf1 + /PD‐1 − Prf1 + CD8 + T cells from the subcutaneous tumors in the NC and Nat10‐ OE groups, with PBS or Naamidine J treatment ( n = 5 per group). The quantification results are shown below. Abbreviations: CD8 + Tex, exhausted CD8 + T cells; CD8 + Tc, cytotoxic CD8 + T cells; DAPI, 4',6‐Diamidino‐2‐phenylindole; ‌‌GAPDH, glyceraldehyde 3‐phosphate dehydrogenase; IFNg, interferon gamma; PBS, phosphate buffered saline; PDAC, pancreatic ductal adenocarcinoma; PD‐1, programmed cell death 1; PD‐L1, programmed cell death ligand 1; Prf1, perforin; NAT10, N‐acetyltransferase 10; NC, negative control; ns, not significant; OE, overexpression.

Article Snippet: The cells were fixed in 4% PFA at room temperature for 15 minutes and incubated with recombinant human/mouse PD‐1 Fc chimeric protein (MedChemExpress) at room temperature for 1 hour, followed by incubation with Alexa Fluor TM 647‐conjugated secondary antibodies (Invitrogen) at room temperature for 1 hour.

Techniques: Cell Function Assay, Immunofluorescence, Staining, Binding Assay, Negative Control, Over Expression, Western Blot, Expressing, Control, Injection, Flow Cytometry, Saline

Journal: Cancer Communications

Article Title: NAT10 regulates tumor progression and immune microenvironment in pancreatic ductal adenocarcinoma via the N4‐acetylated LAMB3‐mediated FAK/ERK pathway

doi: 10.1002/cac2.70045

Figure Lengend Snippet: Graphic illustration on the role and mechanism of NAT10 in regulating tumor progression and immune microenvironment in PDAC. NAT10 promotes the malignant progression of PDAC cells by enhancing LAMB3 mRNA stability via ac4C modification, thereby activating the FAK/ERK pathway. In addition, NAT10 upregulates PD‐L1 expression in tumor cells through the LAMB3/FAK/ERK pathway and is associated with the abundance of intermediate exhausted CD8 + T cells in the PDAC TME. Blocking of PD‐1/PD‐L1 axis with Naamidine J increases the proportion of cytotoxic CD8 + T cells and decreases the proportion of intermediate exhausted CD8 + T cells in the PDAC TME, thereby inhibiting tumor malignant progression. Abbreviations: ac4C, N4‐acetylcytidine; ERK, extracellular regulated protein kinases; FAK, focal adhesion kinase; IFNg, interferon gamma; NAT10, N‐acetyltransferase 10; PDAC, pancreatic ductal adenocarcinoma; PD‐1, programmed cell death 1; PD‐L1, programmed cell death ligand 1; p‐ERK, phosphorylated extracellular regulated protein kinases; p‐FAK, phosphorylated focal adhesion kinase; Prf1, perforin; p‐Src, phosphorylated sarcoma; Src, sarcoma; TME, tumor microenvironment.

Article Snippet: The cells were fixed in 4% PFA at room temperature for 15 minutes and incubated with recombinant human/mouse PD‐1 Fc chimeric protein (MedChemExpress) at room temperature for 1 hour, followed by incubation with Alexa Fluor TM 647‐conjugated secondary antibodies (Invitrogen) at room temperature for 1 hour.

Techniques: Modification, Expressing, Blocking Assay