Journal: bioRxiv

Article Title: The Aryl Hydrocarbon Receptor Controls IFNγ-Induced Immune Checkpoints PD-L1 and IDO via the JAK/STAT Pathway in Lung Adenocarcinoma

doi: 10.1101/2024.08.12.607602

Figure Lengend Snippet: A) Expression of Cd274 , Ido1 , Ido2 , Cyp1a1 , and Cyp1b1 mRNA was quantified by RT-qPCR in control and CMT167 AhR-KO cells (left two bars in each plot) or after 72 hours of treatment with 10 µM benzo(a)pyrene (B(a)P)(right two bars in each plot). Data from three independent experiments, each in duplicate or triplicate are presented as Gapdh -normalized means + SE. B) Protein extracted from cells treated as in ( A ) was probed by western immunoblotting for PD-L1 and, as a loading control, β-actin. One of three representative western blots is shown on the left and β-actin-normalized protein band densities are on the right. Band density data are presented as means from three experiments, each in triplicate, + SE. C) The percentage of Kyn + CMT167 WT or CMT167 AhR-KO cells treated with vehicle or B(a)P as in ( A ) was quantified by flow cytometry. Data from two experiments, each in triplicate, are presented as means + SE from. *p<0.05, **p<0.01, ****p<0.0001 (Student’s t-test, equal variance).

Article Snippet: The following TaqMan assays were purchased from Thermo Fisher Scientific: Cd274 (Mm03048248_m1), Ido1 (Mm00492590_m1), Ido2 (Mm00524210_m1), Cyp 1a1 (Mm00487218_m1), Cyp1b1 (Mm00487229_m1), Jak2 (Mm01208489_m1), Stat1 (Mm012 57286_m1), gapdh (Mm99999915_g1), CD274 (Hs00204257_m1), IDO1 (Hs00984148_m1), J AK2 (Hs01078136_m1), STAT1 Hs01013996_m1), STAT3( Hs00374280_m1 ), Muc1 ( Mm00449604_m1 ), Col5a1 ( Mm00489299_m1 ), Thbs1 ( Mm01335418_m1 ), Egfr ( Mm01187858_m1 ), Itgb2 ( Mm00434513_m1 ), Cd109 ( Mm00462151_m1 ), Ccl2 ( Mm00441242_m1 ), Ccl5 ( Mm01302427_m1 ), GAPDH (Hs99999905_m1).

Techniques: Expressing, Quantitative RT-PCR, Control, Western Blot, Flow Cytometry

Journal: bioRxiv

Article Title: The Aryl Hydrocarbon Receptor Controls IFNγ-Induced Immune Checkpoints PD-L1 and IDO via the JAK/STAT Pathway in Lung Adenocarcinoma

doi: 10.1101/2024.08.12.607602

Figure Lengend Snippet: A) CMT167 Ctrl or CMT167 AhR-KO cells were untreated or treated with 100 ng/ml IFNγ. Cd274 mRNA was quantified by RT-qPCR 24h later. Ido1 , Ido2 , Cyp1a1 , and Cyp1b1 mRNA was quantified 72h later. Data are from three independent experiments, each in duplicate or triplicate, are expressed as fold change of Gapdh -normalized means + SE. B) CMT167 Ctrl or CMT167 AhR-KO cells were left untreated or treated for 24h with 100 ng/ml IFNγ and PD-L1 protein expression assayed by western immunoblotting. A representative immunoblot is on the left and β-actin normalized band densities, averaged from three independent experiments, is on the right. (Bands from IFNγ-treated cells reached saturation prior to bands from untreated cells becoming visible). C) CMT167 Ctrl or CMT167 AhR-KO cells were treated for 24h with IFNγ and IDO1 protein expression assayed by western immunoblotting. A representative immunoblot is on the left and β-actin normalized band densities, averaged from three independent experiments, are on the right. D) CMT167 Ctrl or CMT167 AhR-KO cells were treated with 1-1000 ng/ml IFNγ for 24h and Kyn released into the media quantified via colorimetric assay. Data are averaged from two independent experiments each in quadruplicate + SE. E) CMT167 Ctrl or CMT167 AhR-KO cells were treated with 100 ng/ml IFNγ and Jak2 and Stat1 mRNA quantified 24h later. RT-qPCR data are from three independent experiments, each in triplicate, and presented as Gapdh -normalized means + SE. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001 (Student’s t-test, equal variance).

Article Snippet: The following TaqMan assays were purchased from Thermo Fisher Scientific: Cd274 (Mm03048248_m1), Ido1 (Mm00492590_m1), Ido2 (Mm00524210_m1), Cyp 1a1 (Mm00487218_m1), Cyp1b1 (Mm00487229_m1), Jak2 (Mm01208489_m1), Stat1 (Mm012 57286_m1), gapdh (Mm99999915_g1), CD274 (Hs00204257_m1), IDO1 (Hs00984148_m1), J AK2 (Hs01078136_m1), STAT1 Hs01013996_m1), STAT3( Hs00374280_m1 ), Muc1 ( Mm00449604_m1 ), Col5a1 ( Mm00489299_m1 ), Thbs1 ( Mm01335418_m1 ), Egfr ( Mm01187858_m1 ), Itgb2 ( Mm00434513_m1 ), Cd109 ( Mm00462151_m1 ), Ccl2 ( Mm00441242_m1 ), Ccl5 ( Mm01302427_m1 ), GAPDH (Hs99999905_m1).

Techniques: Quantitative RT-PCR, Expressing, Western Blot, Colorimetric Assay

Journal: bioRxiv

Article Title: The Aryl Hydrocarbon Receptor Controls IFNγ-Induced Immune Checkpoints PD-L1 and IDO via the JAK/STAT Pathway in Lung Adenocarcinoma

doi: 10.1101/2024.08.12.607602

Figure Lengend Snippet: A) A549 Ctrl or A549 AhR-KO cells were untreated or treated with 100 ng/ml IFNγ for 24h and CD274 , IDO1, and CYP1B1 expression quantified by RT-qPCR. Data from four experiments, each in triplicate, are represented as fold change of GAPDH -normalized means + SE. B) The percent positive PD-L1 + cells treated as in ( A ) was quantified by flow cytometry. Data from three experiments, each in triplicate, are presented as mean percent PD-L1 + + SE. C) The baseline percent of Kyn + A549 ctrl and A549 AhR- KO cells in two experiments, each in triplicate, was determined by flow cytometry. D) A549 Ctrl or A549 AhR-KO cells were treated with 0-1000 ng/ml IFNγ for 24h and Kyn release quantified by the Kyn-specific colorimetric assay using a standard Kyn curve. Data from two experiments, each in quadruplicate, are presented as average μM Kyn + SE. E) A549 Ctrl or A549 AhR-KO cells were left untreated or treated with 100 ng/ml IFNγ for 24h and baseline or 100 ng/ml IFNγ-induced JAK2 , STAT1, and STAT3 expression quantified by RT-qPCR. Data from four experiments, each in triplicate, are presented as average fold change of Gapdh -normalized means + SE. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001 (Student’s t-test, equal variance).

Article Snippet: The following TaqMan assays were purchased from Thermo Fisher Scientific: Cd274 (Mm03048248_m1), Ido1 (Mm00492590_m1), Ido2 (Mm00524210_m1), Cyp 1a1 (Mm00487218_m1), Cyp1b1 (Mm00487229_m1), Jak2 (Mm01208489_m1), Stat1 (Mm012 57286_m1), gapdh (Mm99999915_g1), CD274 (Hs00204257_m1), IDO1 (Hs00984148_m1), J AK2 (Hs01078136_m1), STAT1 Hs01013996_m1), STAT3( Hs00374280_m1 ), Muc1 ( Mm00449604_m1 ), Col5a1 ( Mm00489299_m1 ), Thbs1 ( Mm01335418_m1 ), Egfr ( Mm01187858_m1 ), Itgb2 ( Mm00434513_m1 ), Cd109 ( Mm00462151_m1 ), Ccl2 ( Mm00441242_m1 ), Ccl5 ( Mm01302427_m1 ), GAPDH (Hs99999905_m1).

Techniques: Expressing, Quantitative RT-PCR, Flow Cytometry, Colorimetric Assay

Journal: bioRxiv

Article Title: Sialidases derived from Gardnerella vaginalis remodel the sperm glycocalyx and impair sperm function

doi: 10.1101/2025.02.01.636076

Figure Lengend Snippet: A) Schematic describing lectin flow cytometry and representative glycan epitopes. B, C, D) Sperm were exposed to sialidases (AUS 20 units or GvNanH2 160 units) for 1 hour, fixed in formalin, and stained with MAL-II-biotin/Neutravidin-Alexa Fluor 488, SNA-Cy5, or PSA-FITC respectively. Each point represents the median fluorescence intensity from one donor. Statistics represent a mixed model comparison with post-hoc Holm-Šidák tests. E) Schematic representing change in zeta potential after removal of negatively charged sialic acid on sperm surface. F) Sperm were treated with sialidase at various doses for 1 hour, and zeta potential was measured via dynamic light scattering. Each measurement represents the average of seven technical replicates, and statistics represent a mixed model comparison with post hoc Holm-Šidák tests.

Article Snippet: Biotinylated Maackia Amurensis Lectin II (MAL II, Cat #:B-1265-1) and Cy5-conjugated Sambucus Nigra Lectin (SNA, Cat #: CL-1305-1) were purchased from Vector Labs. Human Contraception Antibody (HCA), was a gift from ZabBio.

Techniques: Flow Cytometry, Staining, Fluorescence, Comparison, Zeta Potential Analyzer

Journal: bioRxiv

Article Title: Single Particle Tracking of Genetically Encoded Nanoparticles: Optimizing Expression for Cytoplasmic Diffusion Studies

doi: 10.1101/2024.11.17.623896

Figure Lengend Snippet: A) Characteristic log-log plots of MSD vs. lag time (τ). Slope α indicates diffusion type: Brownian (α=1), subdiffusive (α<1), superdiffusive (α>1). Plateau indicates confinement. B) Log-log plot of ensemble- and time-averaged MSD ( ate ) for particles longer than 10 frames (50 ms), corrected for static error and fitted to a power law with different lag time Δt values of 5, 10, 15, or 20 ms (τ = nΔt), the corresponding alpha values are given in the legend; C) image of U2OS with lower (fluorescent intensity = 16 r.u.) and higher GEM expression (fluorescent intensity = 147 r.u.). Sample size was 60 cells. D) Density probability function of collected D α , E) α,and F) D eff collected after fit with power-law relationship with σ/Δt = 0.5; G) Plot of D eff vs. fluorescence intensity in cells throughout the U2OS colony. The red line indicates the division between cells with “High GEM expression” (30 cells) and “Low GEM expression” (30 cells); H) Corresponding plot of α vs. fluorescence intensity.

Article Snippet: U2OS cells expressing pCMV-pfv-Sapphire-Ires-DsRed (Addgene #116934) or pCMV-pfv-paGFP were sorted by flow cytometry using a Cell Sorter SH800S, while U2OS cells expressing TRE-pfv-Sapphire were selected with 2 μg/mL puromycin.

Techniques: Diffusion-based Assay, Expressing, Fluorescence

Journal: bioRxiv

Article Title: DDX41 dissolves G-quadruplexes to maintain erythroid genome integrity and prevent cGAS-mediated cell death

doi: 10.1101/2024.10.14.617891

Figure Lengend Snippet: (A) Ter119 negative cells and Ter119 positive erythroid cells were purified from wild-type mouse bone marrow cells. G4 levels were tested by flow cytometry using the BG4 antibody that specifically recognizes G4. Quantification is on the right. (B) Bone marrow lineage-negative cells were cultured in Epo medium for 2 days. G4 levels were tested on different days using flow cytometry by the BG4 antibody. Quantification is on the right. (C) CD34+ human HSPCs were cultured in Epo medium for 21 days. The levels of G4 were measured by flow cytometry as in B at the indicated time. Cells at day 7, 14, and 21 represent proerythroblasts, polychromatic to orthochromatic erythroblasts, and orthochromatic to mature red blood cells, respectively. (D) Flow cytometric assays of G4 levels in the indicated bone marrow lineage cells purified from wild-type mice. (E) Quantification of D. (F) Gating strategy of various erythroblasts. Populations I to VI represent proerythroblasts, basophilic erythroblasts, polychromatic erythroblasts, orthochromatic erythroblasts, late orthochromatic to reticulocytes, and mature red blood cells, respectively. (G-H) Flow cytometric assay of G4 level in bone marrow erythroid populations I (G) and V (H) from the indicated mice. Quantification is on the right. (I) Bone marrow lineage negative cells from the indicated mice were cultured in Epo medium for 2 days. G4 levels on different days were measured by flow cytometry using BG4 antibody. Quantification is below the histogram. (J) CD34+ cells were transduced with lentiviral vectors expressing indicated sgRNAs and Cas9. Cells were then harvested for Western blotting of the indicated proteins at day 9 in culture. (K) Quantitative analyses of G4 levels in cells from J using flow cytometric assays. (L) Quantitative analyses of cell death in cells from J using flow cytometric assays. The dead cells are defined as propidium iodide and annexin V double positive. (M) Quantitative analyses of G4 levels in bone marrow mononuclear cells from the patient with DDX41 mutated MDS. All the error bars represent the SEM of the mean. The comparison between two groups was evaluated with 2 tailed t tests, and the comparison among multiple groups was evaluated with 1-way ANOVA tests. * p<0.05, **p<0.01, ***p<0.001, and ****p<0.0001. ns: not significant.

Article Snippet: The sgRNAs targeting DDX41 or scrambled sgRNA were cloned into the lentiviral vector lentiCRISPR v2 (Addgene, #52961, encoding Cas9) using the previously reported protocol .

Techniques: Purification, Flow Cytometry, Cell Culture, Transduction, Expressing, Western Blot, Comparison

Journal: bioRxiv

Article Title: DDX41 dissolves G-quadruplexes to maintain erythroid genome integrity and prevent cGAS-mediated cell death

doi: 10.1101/2024.10.14.617891

Figure Lengend Snippet: (A) Epo medium-cultured mouse bone marrow lineage negative HSPCs were treated with 1 μM PDS for the indicated time. Immunofluorescence assays of γ-H2AX were performed, and representative images of the erythroid cells were presented. Scale bar: 5 μm. (B) Flow cytometry assay of the cells in A. (C) Statistical quantification of γH2AX signals in B. (D) Epo medium-cultured mouse bone marrow lineage negative HSPCs were cultured for 1 day, followed by the treatment of 1 μM PDS for 6 hours. Quantitative RT-PCR analyses of indicated ribosome RNAs were performed using different primer sets. (E) Western blotting assays of indicated in cells from D. Actin was used as a loading control. (F) Same as D except that bone marrow lineage negative HSPCs from HBBCre:Ddx41 fl/fl mouse were cultured for 1 day before the quantitative RT-PCR assays. (G) Western blotting assays of the indicated proteins in F. Cells from both day 1 and day 2 cultured cells were analyzed. (H) CD34+ cells were transduced with lentiviral vectors expressing indicated sgRNAs and Cas9. Cells were then harvested for Western blotting of the indicated proteins at day 9 in culture. (I) Immunohistochemical stains of p53 in bone marrow core biopsies from the patient in normal individual. Scale bar: 100 μm. (J) Quantification of γ-H2AX in bone marrow mononuclear cells from the patient in I and 2 control individuals. All the error bars represent the SEM of the mean. The comparison between two groups was evaluated with 2 tailed t tests, and the comparison among multiple groups was evaluated with 1-way ANOVA tests. * p<0.05, **p<0.01, ns: not significant.

Article Snippet: The sgRNAs targeting DDX41 or scrambled sgRNA were cloned into the lentiviral vector lentiCRISPR v2 (Addgene, #52961, encoding Cas9) using the previously reported protocol .

Techniques: Cell Culture, Immunofluorescence, Flow Cytometry, Quantitative RT-PCR, Western Blot, Control, Transduction, Expressing, Immunohistochemical staining, Comparison

Journal: bioRxiv

Article Title: DDX41 dissolves G-quadruplexes to maintain erythroid genome integrity and prevent cGAS-mediated cell death

doi: 10.1101/2024.10.14.617891

Figure Lengend Snippet: (A) Representative wide-field picture and H&E stains of bone marrow organoid in culture. (B) Whole-mount 3D imaging of the organoids. Imaris was used for cell surface rendering. Organoids were stained with indicated antibodies and subsequently imaged using a laser scanning confocal platform. (C) Confocal immunofluorescence assays of erythroid islands in the iPSC-derived bone marrow organoids (left) and a primary human bone marrow biopsy (right). CD71 was labeled with green for organoids and magenta for primary bone marrow. DAPI: blue. (D) Flow cytometry assays of the organoids using indicated antibodies for various lineages. (E) 10,000 CellVue-labeled donor CD34+ HSPCs were co-incubated with iPSC-derived bone marrow organoids for 3 days in each well of a 96-well plate, followed by an immunofluorescence assay. Representative pictures show the engraftment of donor hematopoietic cells into the organoid. Green, red, and blue represent CD71, CellVue, and DAPI-positive nuclei, respectively. The arrow points to an engrafted CellVue positive cell expressing CD71. (F) Flow cytometry of the organoids using indicated antibodies for various lineages of the engrafted cells in organoids from E. (G) Same as E, except the donor CD34+ cells were transduced with lentiviral vectors expressing Cas9 and indicated sgRNAs before co-incubation. After 3 days, the cells were collected for flow cytometric assays of erythroid and myeloid differentiation of CellVue-positive donor hematopoietic cells and negative iPSC-derived hematopoietic cells. Each data point represents cells combined from 10 organoids. The comparison was evaluated with 1-way ANOVA tests. * p<0.05, **p<0.01. (H) Schematic model of the function of DDX41 during erythropoiesis. The diagram is generated through BioRender.

Article Snippet: The sgRNAs targeting DDX41 or scrambled sgRNA were cloned into the lentiviral vector lentiCRISPR v2 (Addgene, #52961, encoding Cas9) using the previously reported protocol .

Techniques: Imaging, Staining, Immunofluorescence, Derivative Assay, Labeling, Flow Cytometry, Incubation, Expressing, Transduction, Comparison, Generated

Journal: bioRxiv

Article Title: Elimination of senescent cells with senolytic host-directed therapy reduces tuberculosis progression in mice

doi: 10.1101/2025.03.28.645957

Figure Lengend Snippet: (a) Schematics of the experimental strategy to measure senescence markers (p21, p16 and SA-βGal activity) and SASP cytokines levels in mice lungs at 10 and 20 days p.i. Day 1 CFU= 55. (b) %p21+p16+ and (c) %SA-βGal+ (CellEvent Senescence green+) cells out of all live lung (uninfected and Mtb- infected mice) and Spleen ( Mtb- infected mice) cells at 10 days p.i. as determined by multicolor flow cytometry. (d) %p21+p16+ and (e) %SA-βGal+ cells out of different lung-cell types at 10 days p.i., as determined by multicolor flow cytometry. Gating for p16⁺, p21⁺, and SA-βGal⁺ events for each cell type were established using reference cells from Mtb -infected WT B6 mice (set at 1 %). (AMs: Alveolar macrophages, IMs: Interstitial macrophages) (f) Normalized concentration of SASP-cytokines in lung homogenates at 20 dpi and 4 wpi as measured by a LEGENDplex™ Mouse Inflammation Panel (13-plex). The data are means ± SEM. Each data point represents a mouse (n=11-12). (two-way ANOVA with Tukey’s (b-e) or Dunnett’s (f) multiple comparisons test).

Article Snippet: The PVDF membranes were blocked using 5% Blotting-grade blocker (Bio-Rad; 1706404) in TBST buffer-Tris-buffered saline (Quality Biological; 351086101)+ 0.1% Tween 20 (Sigma; P2287) for 1-2 hour and incubated overnight at 4°C with the primary antibodies from Mouse Reactive Senescence Marker Antibody Sampler Kit (Cell Signaling Technology-78551)-Phospho-Histone H2A.X (Ser139) (20E3) Rabbit mAb (1: 1000 dilution), Lamin B1 (E6M5T) Rabbit mAb (1: 1000 dilution), HMGB1 (D3E5) Rabbit mAb (1: 1000 dilution), p16 INK4A (E5F3Y) Rabbit mAb (1: 1000 dilution) and β-Actin (D6A8) Rabbit mAb (Cell Signaling Technology; 8457) (1:5000 dilution).

Techniques: Activity Assay, Infection, Flow Cytometry, Concentration Assay

Journal: bioRxiv

Article Title: Elimination of senescent cells with senolytic host-directed therapy reduces tuberculosis progression in mice

doi: 10.1101/2025.03.28.645957

Figure Lengend Snippet: (a) Representative H&E-stained images of Mtb -infected mice lungs treated with Vehicle or drugs, and respective ImageJ-based quantification. Yellow arrow indicates necrotic granuloma in Vehicle treated B6.Sst1S mice at 5 wpi. The data are means ± SEM. Each data point represents a mouse. Statistical analysis between two groups was done by unpaired two-tailed Student’s t test. Square represents necrotic granuloma formation in the lung. (b) Representative γH2A.X-Immunohistochemistry images of mice after lungs vehicle/ drugs treatment, and respective Violin plots to show ImageJ quantification of γH2A.X-stained area. Each data point represents a mouse. Statistical analysis between two groups was done by unpaired two-tailed Student’s t test. (c) %p21+βGal+ and %p16+βGal+ subpopulation observed in all live lung cells of Mtb -infected mice (n= 5-8). The data are means ± SEM. Each data point represents a mouse. Statistical analysis was calculated by two-way ANOVA with Dunnett’s multiple comparisons test. Bubble plot to show %p21+βGal+ and %p16+βGal+ subpopulation in different lung cell types in Mtb -infected (d) B6.Sst1S mice (n= 8) and (e) WT B6 old mice (n= 5) at indicated time point and treatment groups. The size of the bubble indicates %subpopulation out of all of particular cell types and color is adjusted p values relative to Veh group. Statistics are calculated by one-way ANOVA with Tukey’s multiple comparisons test ( p > 0.15: ns).

Article Snippet: The PVDF membranes were blocked using 5% Blotting-grade blocker (Bio-Rad; 1706404) in TBST buffer-Tris-buffered saline (Quality Biological; 351086101)+ 0.1% Tween 20 (Sigma; P2287) for 1-2 hour and incubated overnight at 4°C with the primary antibodies from Mouse Reactive Senescence Marker Antibody Sampler Kit (Cell Signaling Technology-78551)-Phospho-Histone H2A.X (Ser139) (20E3) Rabbit mAb (1: 1000 dilution), Lamin B1 (E6M5T) Rabbit mAb (1: 1000 dilution), HMGB1 (D3E5) Rabbit mAb (1: 1000 dilution), p16 INK4A (E5F3Y) Rabbit mAb (1: 1000 dilution) and β-Actin (D6A8) Rabbit mAb (Cell Signaling Technology; 8457) (1:5000 dilution).

Techniques: Staining, Infection, Two Tailed Test, Immunohistochemistry

Journal: bioRxiv

Article Title: Targeting CD74-positive macrophages improves neoadjuvant therapy in cervical cancer as revealed by single-cell transcriptomics analysis

doi: 10.1101/2023.11.09.566505

Figure Lengend Snippet: (A) Venn diagram of the 18 candidate genes shared by the Macro 1, Strong, and Positive subgroups. (B) Violin plot of the CD74, GRN, CYBA, and CTSH expression in the Strong, Moderate, and Weak subgroups. (C) The normalized CD74 expression level after pre-treatment, NACT, and anti-PD-1 Ab combination therapy (NACT+anti-PD-1 Ab) in T2, T3, and T4 patients. The patient T3 samples received two rounds of NACT and anti-PD-1 Ab combination therapy were analyzed. (D) UMAP projection of the CD74 expression in macrophage subgroups. The expression plot was split based on the Strong, Moderate, and Weak subgroups. (E) The unsupervised transcriptional trajectory of macrophages from Monocle (version 3), colored by the pseudo-time. The circle labeled 1 indicated the start of the trajectory. (F) UMAP projection of the normalized expression of CD74, GRN, NPC2, CYBA, CTSH, and LAPTM5 in macrophage subgroups. The gray curve indicates the pseudo-time trajectory. (G) The CD74 and GRN expression dynamic change along with the pseudo-time, colored by the Strong, Moderate, and Weak subgroups. (H) The immunofluorescence assay showed the CD74 (red) and CD68 (green) expression in the tissues of patient T4 receiving NACT and anti-PD-1 Ab combination therapy. The green boxes represent CD74 negative macrophages, and the white boxes represent macrophages expressing CD74. (I) The CD74 mRNA level in HeLa or CaSki cells cocultured with macrophages derived from THP-1 cells. Anti-PD-1 Ab and CDDP were used to establish a model of NACT combined with immunotherapy in vitro. (J) The percentage of CD74-positive macrophages cocultured with CaSki cells was detected by flow cytometry. (K) The immunofluorescence assay showed the CD74 (red) and CD68 (green) expression of subcutaneous tumors treated with CDDP and/or anti-PD-1 Ab. (L) Flow cytometry revealed the number of CD74-positive macrophages in single-cell suspensions isolated from mice subcutaneous tumors. The p-value was obtained by a two-tailed unpaired Student’s t-test, and the results are presented as the mean ±SD.

Article Snippet: Anti-mouse PD-1 (CD279)-InVivo (10 mg/kg, every other day, Selleck, China) and Cisplatin (7 mg/kg, every four days, MedChemExpress, China) were injected into the abdominal cavity of mice to build a NACT combined with PD-1 therapy model. Milatuzumab (anti-CD74) (15 mg/kg, every other day, MedChemExpress, China) was used to block the expression of CD74.

Techniques: Expressing, Labeling, Immunofluorescence, Derivative Assay, In Vitro, Flow Cytometry, Isolation, Two Tailed Test

Journal: bioRxiv

Article Title: Targeting CD74-positive macrophages improves neoadjuvant therapy in cervical cancer as revealed by single-cell transcriptomics analysis

doi: 10.1101/2023.11.09.566505

Figure Lengend Snippet: (A, B) Phagocytosis ability detected by flow cytometry. Macrophages derived from THP-1 cells were co-cultured with SiHa cells knockdown (A) or overexpressing (B) CD74. Phagocytosis efficiency was quantified by the percentage of double fluorophore-positive macrophages in the upper right quadrant. (C) The phagocytic function of macrophages co-cultured with SiHa cells treated with anti-CD74 was measured by flow cytometry. (D) Density plot of the M1 and M2 ssGSEA score in the Strong, Moderate, and Weak subgroups. (E) M1/M2 ssGSEA ratio of the macrophages in the pretreatment, NACT, and anti-PD-1 Ab combination group. (F, G) UMAP projection of the normalized density of CD74 expression (F), M1 score, and M2 score (G) in the macrophage subgroups. (H, I) CD86 (H) and CD206 (I) expression of THP-1-derived macrophages after CD74 overexpression. CD86 is the surface marker of M1 macrophages, while CD206 is the surface marker of M2 macrophages. (J, K) CD86 (J) and CD206 (K) expression of THP-1-derived macrophages after CD74 knockdown. (L) Phagocytosis efficiency of THP-1-derived macrophages cocultured with SiHa cells. CDDP-induced phagocytic function loss of macrophages was rescued by knocking down CD74. (M) Flow cytometry detected the differences in phagocytic function among the control, the CDDP, and the anti-CD74 Ab combined with the CDDP group. THP-1-derived macrophages were cocultured with SiHa cells. (N) The representative image of the subcutaneous tumor model. Mouse cervical cancer cells TC-1 were subcutaneously inoculated into immunocompetent CD74 humanized BALB/c-hCD74 mice. Each group consisted of 6 mice receiving CDDP or CDDP combined with intraperitoneal injected anti-CD74 Ab. Tumors of four mice in the drug-combination group became nonpalpable on Day 4 after administration. (O) Growth curve of subcutaneous tumors in mice. (P) Phagocytosis assay was performed using macrophages isolated from the mice’s spleen and mouse cervical cancer cell line TC-1. Phagocytosis efficiency was quantified by the percentage of Dil and Dio double fluorophore-positive THP-1-derived macrophages. The p-value was obtained by a two-tailed unpaired Student’s t-test, and the results are presented as the mean ±SD. *p<0.05, **p < 0.01, ***p<0.001

Article Snippet: Anti-mouse PD-1 (CD279)-InVivo (10 mg/kg, every other day, Selleck, China) and Cisplatin (7 mg/kg, every four days, MedChemExpress, China) were injected into the abdominal cavity of mice to build a NACT combined with PD-1 therapy model. Milatuzumab (anti-CD74) (15 mg/kg, every other day, MedChemExpress, China) was used to block the expression of CD74.

Techniques: Flow Cytometry, Derivative Assay, Cell Culture, Knockdown, Expressing, Over Expression, Marker, Control, Injection, Phagocytosis Assay, Isolation, Two Tailed Test