hyperion helios imaging mass cytometry platform  (fluidigm)

Journal: Nature cancer

Article Title: DUSP6 mediates resistance to JAK2 inhibition and drives leukemic progression

doi: 10.1038/s43018-022-00486-8

Figure Lengend Snippet: a) Heatmap of top differentially expressed genes from microarray analysis in lin- CD34 + cells from sAML patients (n = 14) compared to MF patients (n = 6), and reference expression in healthy donor bone marrow (NBM; n = 5). b) DUSP6 expression from CD34 + cells from NBM (n = 31 patients), AML bone marrow CD34 + subfraction (n = 46 patients), and AML bone marrow CD34- subfraction (n = 44 patients) from GSE30029. DUSP6 values represent quartile normalized, log-transformed values. Statistics were assessed by two-tailed Student’s t test. Data are presented as mean values + /− s.d. c) Immunofluorescence of bone marrow from additional MF and sAML patients, and healthy donors. White arrows denote DUSP6-positive cell staining. IF image acquired from one section. Scale bar: 50 μM. d) Imaging mass cytometry analysis of PBMC cell pellets from normal donor peripheral blood (LRS2), MF (MF20), or sAML (sAML1) patients. Individual images show overlap of indicated channels as denoted, acquired from one section. Scale bar = 16 μM.

Article Snippet: Mass cytometry signals were recorded with the Hyperion imaging mass cytometry platform (Fluidigm).

Techniques: Expressing, Microarray, Transformation Assay, Two Tailed Test, Immunofluorescence, Staining, Imaging, Mass Cytometry

Journal: Nature cancer

Article Title: DUSP6 mediates resistance to JAK2 inhibition and drives leukemic progression

doi: 10.1038/s43018-022-00486-8

Figure Lengend Snippet: a) Enrichment plots of top upregulated Hallmark pathways in DUSP6high vs DUSP6low patients by GSEA. b) Relative mRNA expression of DUSP family genes across 35 AML cell lines from the Cancer Cell Line Encyclopedia. Boxplots represent min to max ranges with median, 25th, and 75th percentiles. c) Relative mRNA expression of DUSP family genes in HEL cells from the Cancer Cell Line Encyclopedia. d) Cell viability curves of HEL cells treated with BCI or trametinib across multiple drug doses. Cells were treated for 72 hours and viability was normalized to control. N = 6 independently treated cell cultures pooled from two independent experiments at each drug dose. Mean and standard deviation presented. e) Immunoblot of HEL cells treated with increasing doses of BCI or the MEK inhibitor trametinib. Cells were treated at their indicated drug dose for 24 hours. Immunoblot representative of two experiments. f) Phospho-STAT3 and phospho-STAT5 flow cytometry of HEL cells treated with 1 μM of BCI or control for 24 hours. n = 1 independently treated cell culture. g) Immunoblot profiling of different signaling pathways altered by BCI and trametinib. HEL cells were treated with 1 μM BCI or 1 μM trametinib for 24 hours. Immunoblot representative of 2 independent experiments. h) Hallmark gene set enrichment analysis showing top altered pathways by normalized enrichment score (NES) and enrichment plots of E2F targets and G2M checkpoint from RNA-seq of HEL cells treated with 1 μM of BCI, or DMSO control for 24 hours. i) Immunblot of HEL and UKE-1 cells treated with 1 μM BCI for 24 hours. Immunoblot representative of 3 independent experiments. j) Immunoblot of HEL cells ectopically expressing DUSP6 or GFP control vector. Immunoblot representative of 3 independent experiments. k) Cell viability assay of HEL cells ectopically expressing DUSP6 or GFP control vector. n = 6 independently treated cell cultures pooled from two independent experiments for each condition and grown for 96 hours with viability normalized to the control vector. Mean and standard deviation presented. Statistics were assessed by two-tailed Student’s t test. l) Cell viability assay of HEL cells ectopically expressing DUSP6 or GFP control vector treated with 300 nM BCI. Cells were plated at n = 6 independently treated cell cultures pooled from two independent experiments grown for 96 hours with viability from normalized to control treatment from each group. Mean and standard deviation presented. Statistics were assessed by two-tailed Student’s t test. m) Representative images of lin- CD34 + colonies grown in MethoCult H4034 Optimum for 12 days in 0.5 μM BCI or RPMI control. Samples plated in duplicate. Images acquired from one field of view representative of two plates/condition. Scale bar: 1000 μM.

Article Snippet: Mass cytometry signals were recorded with the Hyperion imaging mass cytometry platform (Fluidigm).

Techniques: Functional Assay, Expressing, Control, Standard Deviation, Western Blot, Flow Cytometry, Cell Culture, Protein-Protein interactions, RNA Sequencing, Plasmid Preparation, Viability Assay, Two Tailed Test

Journal: Nature cancer

Article Title: DUSP6 mediates resistance to JAK2 inhibition and drives leukemic progression

doi: 10.1038/s43018-022-00486-8

Figure Lengend Snippet: a) Percentage of myeloid cells from hCD45+ PB (left) and CD71 + CD235a + from hCD45- BM (right) from NSGS mice transplanted with CD34 + cells ectopically expressing control (n = 9 mice) and DUSP6 (n = 10 mice) at endpoint. Both statistics assessed by two-tailed non-parametric Mann-Whitney U test after testing for normality by Shapiro-Wilk test. b) DUSP6 overexpression PDX with CD34 + cells from a second MF patient (MF106). Plots show percentage of human CD45 (hCD45) in peripheral blood and bone marrow of transplanted mice ectopically expressing control (n = 5) or DUSP6 (n = 5) across multiple timepoints, and spleen and liver weights of mice at endpoint normalized by mouse weight. %hCD45 in PB statistics assessed by two-way ANOVA incorporating weeks 4–8 post transplant. %hCD45 in BM, and normalized spleen and liver weights statistics were assessed by two-tailed Student’s t test. Data are presented as mean values + /− s.d. c) Kaplan-Meier survival analysis of mice from control or DUSP6 cohorts assessed by log-rank test. d) Colony assay of CD34 + cells from sAML15 after transduction with shRNAs targeting DUSP6 or control vector. Sorted cells were grown in MethoCult H4034 Optimum for 12 days. Samples were plated in triplicate (n = 3 replicates). Statistics were assessed by Two-tailed Student’s t test. Data are presented as mean values + /− s.d. e) CD34 + healthy donor normal bone marrow (NBM) PDX model. Cells were transduced with 2 independent shRNAs targeting DUSP6 or control and transplanted into NSGS mice. Plots show percentage of human CD45 (hCD45) in peripheral blood and bone marrow of transplanted mice treated ectopically expressing control (n = 5), shDUSP6 #1 (n = 5), or shDUSP6 #2 (n = 5) across multiple timepoints, and spleen and liver weights of mice at endpoint normalized by mouse weight. %hCD45 in PB statistics assessed by two-way ANOVA with Dunnett’s multiple comparisons test with control. %hCD45 in BM, and normalized spleen and liver weights statistics were assessed by two-tailed Student’s t test with Dunnett’s multiple comparisons test with control. f) Normalized spleen and liver weights from mice at end-point from sAML14 CD34 + PDX. Mice were treated with vehicle (n = 7), 25 mg/kg BCI (n = 8), 90 mg/kg ruxolitinib (n = 7), or combination (n = 7). Data are presented as mean values + /− s.d. g) tSNE dimensional reduction clustering of mouse and human CD45 + cells from bone marrow of sAML14 PDX mice. h) sAML PDX14 mass cytometry analysis showing percentage of CD123 + CD33 + leukemic cells gated from hCD45+ cells from 3 mice in each treatment group. Statistics were assessed by one-way ANOVA with Dunnett’s multiple comparison test. i) Erythroblast progenitors gated from hCD45+ cells. Statistics were assessed by one-way ANOVA with Dunnett’s multiple comparison test. j) SPADE tree cluster algorithm showing CD123 + and CD71 + populations from vehicle and BCI treated groups. k) UMAP clustering showing CD123 + and CD71 + populations. l) Schematic of the CD34 + healthy donor PDX model. CD34 + cells were isolated from BMMCs from normal donors, pooled, and transplanted into NSGS mice. Mice were treated with vehicle (n = 6), 25 mg/kg BCI (n = 6), 90 mg/kg ruxolitinib (n = 6), or combination (n = 6) following weekly schedule of 5 days on, 2 days off treatment starting on day 38. m) Plots show % hCD45 in peripheral blood and bone marrow of transplanted mice treated with vehicle or BCI across multiple timepoints and spleen weights of mice at endpoint normalized by mouse weight %hCD45 in PB statistics were assessed by two-way ANOVA comparing vehicle vs each individual treatment group with Dunnett’s multiple comparison test. %hCD45 in BM, and spleen and liver weights statistics were assessed by one-way ANOVA with Dunnett’s multiple comparison test. Data are presented as mean values + /− s.d.

Article Snippet: Mass cytometry signals were recorded with the Hyperion imaging mass cytometry platform (Fluidigm).

Techniques: Expressing, Control, Two Tailed Test, MANN-WHITNEY, Over Expression, Colony Assay, Transduction, Plasmid Preparation, Mass Cytometry, Comparison, Isolation

Journal: Nature cancer

Article Title: DUSP6 mediates resistance to JAK2 inhibition and drives leukemic progression

doi: 10.1038/s43018-022-00486-8

Figure Lengend Snippet: a) Heatmap of inhibitors of upstream regulators of S6 activity and the pearson correlation of their area under curve (AUC) with DUSP6 expression in AML cell lines. b) CRISPR dropout screen showing RPS6KA1 as an essential gene in AML. Candidates were identified if meeting criteria of FDR < 10% and whose inhibition affected # of AML lines but neither of non-AML lines. Data retrieved from Tzelepis et al. c) RPS6KA1 expression across 10,071 patient samples representing 31 distinct cancer subtypes from the TCGA Pan-Cancer cohort. Expression values provided as log2 (value +1). See additional information in methods. Boxplots represent min to max ranges with median, 25th, and 75th percentiles. d) Immunoblot analysis of RPS6KA1 knockdown by shRNA or control vector in HEL cells. Immunoblot representative of 5 independent experiments. e) Cell viability assay of HEL cells after RPS6KA1 knockdown relative to control vector. Cells were grown for 96 hours and viability was normalized to the pLKO control vector. n = 6 independently treated cell cultures pooled from two independent experiments per construct. Mean and standard deviation presented. Statistics were assessed by two-tailed Student’s t test. f) Heatmap of altered signaling pathways of lin- CD34 + cells from unique normal bone marrow donors and peripheral blood of MF patients by mass cytometry. Patient samples were treated with 5 μM BI-D1870 for 4 hours, 20 ng/mL TPO for 1 hour, or combination. Signals were normalized to the control treatment of each individual patient sample and reported as 90th percentile Arcsinh ratio. g) Ridge plot of RPS6KA1 expression from CD34 + scRNA-seq of N34, N39, and 381812 at MF and sAML stages. h) RPS6KA1 expression from CD34 + cells from NBM (n = 5), MF (n = 14), and sAML (n = 6) patient samples. RPS6KA1 values represent RMA from microarray. Statistics were assessed by two-tailed Student’s t test. i) Downregulation of HES1 expression from RNA sequencing of HEL cells treated for 4 hours with 1 μM BCI vs DMSO control. n = 2 independently treated cell cultures. j) qRT-PCR of HES1 in HEL cells treated with 1 μM BCI for 24 hours. HES1 mRNA expression normalized to ACTB. n = 3 independent experiments. Statistics were assessed by two-tailed Student’s t test. Data are presented as mean values + /− s.d. k) Immunoblot of HES1 in HEL cells after DUSP6 knockdown by shRNA or control vector. Immunoblot representative of 3 independent experiments. l) qRT-PCR of HES1 in HEL cells after DUSP6 knockdown by shRNA or control vector. HES1 mRNA expression normalized to ACTB for each group and then normalized to pLKO vector. n = 3 independent experiments. Statistics were assessed by two-tailed Student’s t test. Data are presented as mean values + /− s.d. m) Immunoblot of HEL cells after HES1 knockdown by shRNA or control vector. Immunoblot representative of 2 independent experiments. n) Cell viability curve of HEL cells after ectopic expression of RPS6KA1 or GFP control treated with increasing concentrations of BI-D1870. Cells were treated for 96 hours and viability was normalized to the control treatment from each group. n = 6 independently treated cell cultures pooled from two independent experiments per construct. Mean and standard deviation presented. o) Cell viability assay of HEL cells treated with 1 μM BI-D1870, 300 μM BCI or combination, and UKE-1 cells treated with 2 μM BI-D1870, 200 μM BCI, or combination. Cells were treated for 72 hours and viability was normalized to the control treatment. n = 6 independently treated cell cultures pooled from two independent experiments at each drug dose. Mean and standard deviation presented. p) Hallmark gene set enrichment analysis showing top altered pathways from RNA-seq of HEL cells treated for 24 hours with 10 μM BI-D1870 + 1 μM BCI compared to DMSO control (left) and 10 μM BI-D1870 + 1 μM BCI compared to 10 μM BI-D1870 alone (right). q) Dot plot of mass cytometry analysis of lin- CD34 + cells from MF103 treated with 1 μM BCI for 4 hours, 5 μM BI-D1870 for 4 hours, 20 ng/mL TPO for 1 hour, or combination. Signals of key phosphorylated proteins were normalized to the control treatment and reported as 90th percentile Arcsinh ratio.

Article Snippet: Mass cytometry signals were recorded with the Hyperion imaging mass cytometry platform (Fluidigm).

Techniques: Functional Assay, Activity Assay, Expressing, CRISPR, Inhibition, Western Blot, Knockdown, shRNA, Control, Plasmid Preparation, Viability Assay, Construct, Standard Deviation, Two Tailed Test, Protein-Protein interactions, Mass Cytometry, Microarray, RNA Sequencing, Quantitative RT-PCR

Journal: Nature cancer

Article Title: DUSP6 mediates resistance to JAK2 inhibition and drives leukemic progression

doi: 10.1038/s43018-022-00486-8

Figure Lengend Snippet: a) tSNE dimensional reduction clustering of distinct subpopulations from sAML4 and altered signaling upon BCI, TPO induction, or combination treatment post mass cytometry analysis. Samples were treated with 1 μM BCI for 4 hours, 20 ng/mL TPO for 1 hour, or combination. tSNE plots of sAML4 representative of plots from 2 MF, 3 sAML, and 2 normal patient samples. b) TPO-induced signaling across different subpopulations from sAML4. Patient samples were treated with 20 ng/mL TPO for 1 hour. Signals were normalized to the control treatment and reported as median Arcsinh ratio. tSNE plots of sAML4 representative of plots from 3 sAML patients. c) TPO-induced cytokine production across different subpopulations from sAML5. Patient samples were treated with 20 ng/mL TPO for 4 hour. Signals were normalized to the control treatment and reported as 90 percentile Arcsinh ratio. tSNE plots of sAML5 representative of plots from 3 sAML patients. d) Heatmap and dot plots of altered cytokine production of CD14 + monocytes from bone marrow (NBM40) and peripheral blood (NPB LRS2) of healthy donors, and PBMCs from MF and sAML patients by mass cytometry. Unique patient samples were treated with 1 μM BCI for 4 hours, 20 ng/mL TPO for 4 hour, or combination. Signals were normalized to the control treatment of each individual patient sample and reported as 90th percentile Arcsinh ratio. Basal cytokine expression in CD14 + cells from MF and sAML are also presented (left panel) and are normalized to the NBM/NPB within each individual CyTOF run to control for batch effect: run 1 - sAML4 and sAML6 normalized to NBM40; run 2 - MF20 and MF102 normalized to NPB LRS2; run 3- MF40 and sAML5 normalized to NBM40. e) Dot plot of MIP-1β/CCL4 in CD123 + and CD16 + monocyte populations from sAML5. Samples were treated with 1 μM BCI for 4 hours, 20 ng/mL TPO for 4 hours, or combination. Signals were normalized to the control treatment and reported as 90 percentile Arcsinh ratio. n = 1 independent experiment with sample sAML5.

Article Snippet: Mass cytometry signals were recorded with the Hyperion imaging mass cytometry platform (Fluidigm).

Techniques: Mass Cytometry, Control, Expressing

Journal: Nature cancer

Article Title: DUSP6 mediates resistance to JAK2 inhibition and drives leukemic progression

doi: 10.1038/s43018-022-00486-8

Figure Lengend Snippet: a) WBC subpopulation counts, platelet counts, and body weight from Jak2 transplanted mice treated with vehicle (n = 9) or BCI (n = 10) across multiple timepoints. Statistics were assessed by two-way ANOVA comparing vehicle to BCI. Data are presented as mean values + /− s.d.. b) Representative gross spleen of Jak2 mice treated with vehicle or BCI at endpoint. c) Hematocrit, white blood cell (WBC) counts and differentials, and platelets counts of wildtype primary mice treated with vehicle (n = 4) or 25 mg/kg BCI (n = 5) following weekly schedule of 5 days on, 2 days off treatment across multiple timepoints. Liver, spleen, and body weights collected at endpoint. Two-way ANOVA and two-tailed Student’s t test statistical analysis resulted in non-significant values across all comparisons between vehicle and BCI treatment. Data are presented as mean values + /− s.d. d) Representative flow cytometry analysis of peripheral blood from CD45.1 mice showing engraftment of MPL W515 GFP + CD45.2 cells. e) WBC subpopulation counts, platelet counts, and normalized spleen weight from MPL W515 MF model of transplanted mice treated with vehicle (n = 8) or BCI (n = 8) across multiple timepoints. Statistics were assessed by two-way ANOVA for white count differential comparisons between vehicle and BCI and two-tailed Student’s t test for normalized spleen weight. Data are presented as mean values + /− s.d.

Article Snippet: Mass cytometry signals were recorded with the Hyperion imaging mass cytometry platform (Fluidigm).

Techniques: Two Tailed Test, Flow Cytometry

Journal: Nature cancer

Article Title: DUSP6 mediates resistance to JAK2 inhibition and drives leukemic progression

doi: 10.1038/s43018-022-00486-8

Figure Lengend Snippet: a, Schematic of the CD34+ sAML PDX mouse models. CD34+ cells were isolated from patients sAML11 and sAML14 and transplanted into NSGS mice. For the sAML11 PDX, mice were treated with vehicle (n = 6) or 25 mg per kg BCI (n = 7) daily for 5 d followed by 2 d off treatment starting on day 24. For the sAML14 PDX, mice were treated with vehicle (n = 7), 25 mg per kg BCI (n = 8), 90 mg per kg ruxolitinib (n = 7) or the combination (n = 7) following a weekly schedule of 5 d on treatment and 2 d off treatment starting on day 31. b, Efficacy of BCI in the sAML11 PDX model. Plots show percentage of hCD45+ cells in PB and BM of transplanted mice treated with vehicle or BCI across multiple timepoints and spleen and liver weights of mice at the endpoint normalized by mouse weight. Percentages of hCD45+ cells in PB were assessed by two-way ANOVA comparing vehicle to BCI. Percentages of hCD45+ cells in BM and normalized spleen and liver weight statistics were assessed by two-tailed Student’s t-test. Data are presented as mean values ± s.d. c, Efficacy of treatment in the sAML14 PDX model. Plots show percentages of hCD45+ cells in PB and BM of transplanted mice treated with vehicle, BCI, ruxolitinib or the combination across multiple timepoints. Percentages of hCD45+ cells in PB were assessed by two-way ANOVA comparing vehicle versus each individual treatment group with Dunnett’s multiple-comparison test and by two-way ANOVA for ruxolitinib versus the combination. Percentages of hCD45+ cells in BM were assessed by one-way ANOVA with Dunnett’s multiple-comparison test. Right, mass cytometry of cells isolated from the BM of three sAML14 PDX mice from each treatment group at the endpoint. Heatmap denotes row z score calculated from the mean signal intensity of each marker. Data are presented as mean values ± s.d. mCD45, mouse CD45.

Article Snippet: Mass cytometry signals were recorded with the Hyperion imaging mass cytometry platform (Fluidigm).

Techniques: Isolation, Two Tailed Test, Comparison, Mass Cytometry, Marker

Journal: Science Advances

Article Title: Spatiotemporal architecture of immune cells and cancer-associated fibroblasts in high-grade serous ovarian carcinoma

doi: 10.1126/sciadv.adk8805

Figure Lengend Snippet: ( A ) Patient-matched primary, synchronous metastasis, and recurrent metastasis HGSOC samples from 42 patients were assembled into a TMA. The Kaplan-Meier survival curve below shows the time to relapse, with patients classified as early or late relapse (<15 or >15 months to relapse, respectively). Scale bars, 4 mm. ( B ) Imaging mass cytometry was performed by staining the tissue with metal ion–tagged antibodies, ablating the tissue, and performing image data analysis. ( C ) Three major cell types and their protein expression patterns are shown in sample IMC images and a heatmap: immune cells, fibroblasts, and epithelial (cancer) cells. Scale bar, 25 μm. ( D ) Cell composition of (regions of interest) ROIs was approximately 50 to 55% epithelial cancer cells, 25 to 30% fibroblasts, and 15 to 20% immune cells in primary, synchronous metastasis, and recurrence tumor types. ( E ) Lymph node metastases contained elevated levels of immune cells, P = 1.1 × 10 −5 , analysis of variance (ANOVA). ( F ) Early and late relapse conditions were associated with similar proportions of immune cells, fibroblasts, and epithelial cells. Stacked bar plots are shown to illustrate the summation to 1, error bars denote SD, and the error bar is single-sided to reduce visual clutter. Cell proportion differences are not statistically significant by ANOVA unless noted with *.

Article Snippet: Data were acquired on the Hyperion/Helios Imaging Mass Cytometry platform (Fluidigm/Standard BioTools) at the Cedars Sinai Spatial Molecular Profiling Shared Resource.

Techniques: Imaging, Mass Cytometry, Staining, Expressing