Journal: bioRxiv

Article Title: Single-cell profiling guided combinatorial immunotherapy for fast-evolving CDK4/6 inhibitor resistant HER2-positive breast cancer

doi: 10.1101/671198

Figure Lengend Snippet: ( A ) Waterfall plots showing percent change of tumor volume with 14-day’s treatment in MMTV-Neu mice (n=8,6,6 and 9 for Ctrl, Ab, Pal and Ab+Pal). Ctrl, vehicle treated; Ab, anti-Her2/Neu antibody; Pal, CDK4/6 inhibitor Palbociclib. ( B ) Tumor volume curves showing tumors rebounded during sustained Ab+Pal combination treatment (n=12 for Ctrl and n=10 for Ab+Pal). ( C ) Schematic for sample processing, enrichment of tumor cells and Drop-seq based single-cell RNA sequencing. ( D ) t-distributed stochastic neighbor embedding (t-SNE) plots colored by treatment groups and clustering of 4711 tumor cells derived from Ctrl, Ab or Pal alone, responsive/residual tumors (APP) and resistant tumors (APR) with Ab+Pal treatment. Each point represents a single cell. ( E ) Abundance of each cell cluster in tumors with indicated treatment as presented and classified in panel D. ( F ) Volcano plots comparing ssGSEA enrichment score of 1053 canonical pathways/gene sets of the C2 collection of Molecular Signatures Database between APR and Ctrl single cell RNA-Seq data. Each point represents one pathway/gene set. X-axis, mean difference of single cell ssGSEA enrichment score; Y-axis, −log10 (P-value by t-test). ( G ) ssGSEA enrichment score violin plots for single cells in each group for indicated signatures. ( H ) Volumes of Ab+Pal resistant tumors after treatment combining immune checkpoint blockades (n=7 for Ab+Pal and n=6 for Ab+Pal+ICB). ICB, anti-CTLA4 and anti-PD-1 antibody cocktail. P-value by two-tailed Student’s t-test.

Article Snippet: The following pre-conjugated antibodies purchased from Fluidigm were used in this study: CD45-089Y (3089005B, 30-F11); Ly-6G-141Pr (3141008B, 1A8,); CD11c-142Nd (3142003B, N418); CD45R-144Nd (3144011B, RA3-6B2); CD4-145Nd (3145002B, RM4-5); CD11b-148Nd (3148003B, M1/70); CD44-150Nd (3150018B, IM7); CD25-151Eu (3151007B, 3C7); CD3e-152Sm (3152004B, 145-2C11); PD-L1-153Eu (3153016B, 10F.9G2); CTLA-4-154Sm (3154008B, UC10-4B9); PD-1-159Tb (3159024B, 29F.1A12); Ly-6C-162Dy (3162014B, HK1.4); CX3CR1-164Dy (3164023B, SA011F11); NK1.1-165Ho (3165018B, PK136); c-Kit-166Er (3166004B, 2B8); CD8a-168Er (3168003B, 53-6.7); CD86-172Yb (3172016B, GL1); I-A/I-E-209Bi (3209006B, M5/114.15.2).

Techniques: RNA Sequencing Assay, Derivative Assay, Two Tailed Test

Journal: bioRxiv

Article Title: Single-cell profiling guided combinatorial immunotherapy for fast-evolving CDK4/6 inhibitor resistant HER2-positive breast cancer

doi: 10.1101/671198

Figure Lengend Snippet: (A) Enrichment analysis of cabozantinib target genes across single TILs grouped and plotted by different phenotypes (left) or by different immune cell types (right) as annotated in . P -value by Student’s t-test (two-tailed). (B) Expression distribution of Kit and Met on t-SNE plot (left) and quantification of Kit and/or Met expressing cells among different immune celltypes as annotated (right). P -value by three-sample Chi-square test. (C) Abundance of Kit and/or Met expressing IMCs among tumors with different phenotypes. P -value by three-sample Chi-square test. (D) Growth of Ab+Pal resistant tumors with Ab+Cabo treatment (n=7 for Ab+Pal, n=6 for Cabo and n=7 for Ab+Cabo). (E) Representative images and quantification of CD3 immunohistochemistry staining for tumors with Ab+Pal or Ab+Cabo treatment. Scale bar, 20 μm. P -value by Student’s t-test. (F) T-cell depletion during Ab+Cabo treatment against Ab+Pal resistant tumors. P -value by Student’s t-test. (G) Relative volumes of Ab+Pal resistant tumors after treatment with Cabo and ICB. Ab+Pal resistant tumors were transplanted to recipient MMTV-Neu mice and first treated with Ab+Pal to acquire the resistance phenotype (left), then treated with Ab+Pal (n=6), Ab+Pal+ICB (n=6), Ab+ICB (n=8), Ab+Cabo (n=5), or Ab+Cabo+ICB (n=7) for 2 weeks (right). (H) Growth of Ab+Pal resistant tumors after sequential treatment with Ab+Pal+ICB and Ab+Cabo+ICB (n=9). Ab+Pal resistant tumors were first treated with Ab+Pal+ICB for 1 week then switched to Ab+Cabo+ICB treatment for 3 weeks. (I) Survival time to doubled tumor volume from experiment in (D). P -value by log-rank (Mantel-Cox) test. Cabo, protein kinase inhibitor cabozantinib. ICB, immune checkpoint blockades cocktail with anti-CTLA4 and anti-PD-1 mAb.

Article Snippet: The following pre-conjugated antibodies purchased from Fluidigm were used in this study: CD45-089Y (3089005B, 30-F11); Ly-6G-141Pr (3141008B, 1A8,); CD11c-142Nd (3142003B, N418); CD45R-144Nd (3144011B, RA3-6B2); CD4-145Nd (3145002B, RM4-5); CD11b-148Nd (3148003B, M1/70); CD44-150Nd (3150018B, IM7); CD25-151Eu (3151007B, 3C7); CD3e-152Sm (3152004B, 145-2C11); PD-L1-153Eu (3153016B, 10F.9G2); CTLA-4-154Sm (3154008B, UC10-4B9); PD-1-159Tb (3159024B, 29F.1A12); Ly-6C-162Dy (3162014B, HK1.4); CX3CR1-164Dy (3164023B, SA011F11); NK1.1-165Ho (3165018B, PK136); c-Kit-166Er (3166004B, 2B8); CD8a-168Er (3168003B, 53-6.7); CD86-172Yb (3172016B, GL1); I-A/I-E-209Bi (3209006B, M5/114.15.2).

Techniques: Two Tailed Test, Expressing, Immunohistochemistry, Staining

Journal: bioRxiv

Article Title: Single-cell profiling guided combinatorial immunotherapy for fast-evolving CDK4/6 inhibitor resistant HER2-positive breast cancer

doi: 10.1101/671198

Figure Lengend Snippet: ( A ) Representative images of H&E staining for tumors with indicated treatment and quantification of hypocellularity. Scale bar, 100 μm. ( B ) Representative images and quantification of Ki67 immunohistochemistry staining tumors with indicated treatment. Scale bar, 20 μm. ( C ) Relative volumes of Ab+Pal resistant tumors after sequential treatment with Ab+Pal+ICB and Ab+Cabo+ICB (as in ). Ab+Pal resistant tumors were first treated with Ab+Pal+ICB for 1 week then switched to Ab+Cabo+ICB treatment for 3 weeks. Cabo, protein kinase inhibitor cabozantinib; ICB, immune checkpoint blockades, cocktail of anti-CTLA4 and anti-PD-1 antibodies. P -value by one-way ANOVA with Tukey’s test.

Article Snippet: The following pre-conjugated antibodies purchased from Fluidigm were used in this study: CD45-089Y (3089005B, 30-F11); Ly-6G-141Pr (3141008B, 1A8,); CD11c-142Nd (3142003B, N418); CD45R-144Nd (3144011B, RA3-6B2); CD4-145Nd (3145002B, RM4-5); CD11b-148Nd (3148003B, M1/70); CD44-150Nd (3150018B, IM7); CD25-151Eu (3151007B, 3C7); CD3e-152Sm (3152004B, 145-2C11); PD-L1-153Eu (3153016B, 10F.9G2); CTLA-4-154Sm (3154008B, UC10-4B9); PD-1-159Tb (3159024B, 29F.1A12); Ly-6C-162Dy (3162014B, HK1.4); CX3CR1-164Dy (3164023B, SA011F11); NK1.1-165Ho (3165018B, PK136); c-Kit-166Er (3166004B, 2B8); CD8a-168Er (3168003B, 53-6.7); CD86-172Yb (3172016B, GL1); I-A/I-E-209Bi (3209006B, M5/114.15.2).

Techniques: Staining, Immunohistochemistry

Journal: International Journal of Molecular Sciences

Article Title: Multiple Immunostainings with Different Epitope Retrievals—The FOLGAS Protocol

doi: 10.3390/ijms23010223

Figure Lengend Snippet: Secondary antibodies, chromogens, fluorophores and metals. Except DAPI, the fluorophores not directly coupled to antibodies are linked to streptavidin for binding of biotinylated antibodies. Abbreviations: RTU: ready to use, AP: alkaline phosphatase, HRP: horseradish peroxidase. Tb: Terbium, Er: Erbium.

Article Snippet: , 159Tb (conjugated to CD68 clone KP1) , , - , Fluidigm (SKU 201300).

Techniques: Binding Assay

Journal: Methods in molecular biology (Clifton, N.J.)

Article Title: Flow Cytometry and Mass Cytometry for Measuring the Immune Cell Infiltrate in Atherosclerotic Arteries

doi: 10.1007/978-1-0716-1924-7_47

Figure Lengend Snippet: CyTOF panel as published in Winkels et al. [ 9 ]

Article Snippet: TCR-γ/S , 159Tb , GL3 , Fluidigm , 3159012B , Commercial.

Techniques: Conjugation Assay

Journal: Scientific Reports

Article Title: Salmonella enterica serovar Typhi exposure elicits ex vivo cell-type-specific epigenetic changes in human gut cells

doi: 10.1038/s41598-020-70492-2

Figure Lengend Snippet: Unsupervised clustering of epigenetic changes between and within the different mucosal cell subsets after wild-type S . Typhi infections. Cells isolated cells from healthy terminal ileum surgical tissues were exposed to S . Typhi strain Ty2 (Ty2) at 1:100 MOI. Cells cultured with media only were used as controls (media). After 3 h of incubation, cells were stained using a panel of 33 metal-labeled Abs, and the chromatin modifications were analyzed at the single-cell level by mass cytometry. To visualize the clustering of the 11 cell subsets ( i.e ., B-, CD3+ T-, CD4+ T-, CD8 + T-, NK, TCR-γδ, Mucosal associated invariant (MAIT) and NKT cells, as well as monocytes, macrophages, and epithelial cells), tSNE-defined population distributions and clustering were colored by meta-cluster. ( A ) t-SNE maps of chromatin modifications. Settings to run the t-SNE algorithm were set-up in Cytobank. ( B ) Color-coded key showing the location of epigenetic marks meta-clusters. Data are representative of one out of three experiments with terminal ileum segments from 2 different donors, one replicate each.

Article Snippet: Cells were surface stained with anti-human mAbs to CD3 (clone UCHT1), CD4 (clone SK3), CD8 (clone RPA-T8), CD11b (clone ICRF44), CD16 (clone 3G8), CD19 (clone HIB19), CD38 (clone HIT2), CD45 (clone HI30), CD45RO (clone UCHL), CD56 (clone HCD56), CD57 (clone HCD57), CD69 (clone FN50), CD161 (clone HP-3G10), CD163 (clone GHI/61), CCR7 (clone G043H7), EpCAM (CD326, clone 9C4), HLA-DR (clone L243), TCR γδ (clone 11F2)(Fluidigm, Sunnyvale, CA), CD14 (clone 3C10) (Invitrogen, Carlsbad, CA), and TCR Vα7.2 (clone 3C10) (Biolegend, San Diego, CA).

Techniques: Isolation, Cell Culture, Incubation, Staining, Labeling, Mass Cytometry

Journal: Scientific Reports

Article Title: Salmonella enterica serovar Typhi exposure elicits ex vivo cell-type-specific epigenetic changes in human gut cells

doi: 10.1038/s41598-020-70492-2

Figure Lengend Snippet: Chromatin profiles of the epigenetic changes in TCR-γδ cells induced by S . Typhi. Cells isolated cells from healthy terminal ileum surgical tissues were exposed to S . Typhi strain Ty2 (Ty2) and cultured as described in Fig. . Cells cultured with media only were used as controls (media). FCOM data of the 28 combinations within the acceptability criteria for changes of the chromatin marks are shown. Bars represent the net difference ( S . Typhi-infected minus uninfected cultures). Bar graphs extend from the 25th to 75th percentiles; the line in the middle represents the median of the pooled data. The whiskers delineate the smallest to the largest value. Data are representative of three experiments with terminal ileum segments from 4 different donors, one replicate each. P values < 0.05 were considered significant (red-colored boxes).

Article Snippet: Cells were surface stained with anti-human mAbs to CD3 (clone UCHT1), CD4 (clone SK3), CD8 (clone RPA-T8), CD11b (clone ICRF44), CD16 (clone 3G8), CD19 (clone HIB19), CD38 (clone HIT2), CD45 (clone HI30), CD45RO (clone UCHL), CD56 (clone HCD56), CD57 (clone HCD57), CD69 (clone FN50), CD161 (clone HP-3G10), CD163 (clone GHI/61), CCR7 (clone G043H7), EpCAM (CD326, clone 9C4), HLA-DR (clone L243), TCR γδ (clone 11F2)(Fluidigm, Sunnyvale, CA), CD14 (clone 3C10) (Invitrogen, Carlsbad, CA), and TCR Vα7.2 (clone 3C10) (Biolegend, San Diego, CA).

Techniques: Isolation, Cell Culture, Infection

Journal: iScience

Article Title: Single cell characterization of blood and expanded regulatory T cells in autoimmune polyendocrine syndrome type 1

doi: 10.1016/j.isci.2024.109610

Figure Lengend Snippet:

Article Snippet: CD274 (PD-L1) , Fluidigm , 3159029B; RRID: AB_2861413.

Techniques: Isolation, Amplification, Hybridization, Cell Isolation, Staining, RNA Sequencing, Gene Expression, Mass Cytometry, Flow Cytometry, Software

Journal: iScience

Article Title: Defects in NK cell immunity of pediatric cancer patients revealed by deep immune profiling

doi: 10.1016/j.isci.2024.110837

Figure Lengend Snippet:

Article Snippet: Anti-CD161 (clone HP-3G10, conjugated to 159Tb) , Standard BioTools , Cat#3159004B.

Techniques: Purification, Clinical Proteomics, Recombinant, Blocking Assay, Staining, Saline, Mass Cytometry, Software, Cytometry

Journal: iScience

Article Title: Defects in NK cell immunity of pediatric cancer patients revealed by deep immune profiling

doi: 10.1016/j.isci.2024.110837

Figure Lengend Snippet:

Article Snippet: Anti-NKp30 (clone Z25, conjugated to 159Tb) , Standard BioTools , Cat#3159017B.

Techniques: Purification, Clinical Proteomics, Recombinant, Blocking Assay, Staining, Saline, Mass Cytometry, Software, Cytometry