Proliferation tracing with single-cell mass cytometry optimizes generation of stem cell memory-like T cells.

Selective differentiation of naive T cells into multipotent T cells is of great interest clinically for the generation of cell-based cancer immunotherapies. Cellular differentiation depends crucially on division state and time. Here we adapt a dye dilution assay for tracking cell proliferative history through mass cytometry and uncouple division, time and regulatory protein expression in single naive human T cells during their activation and expansion in a complex ex vivo milieu. Using 23 markers, we defined groups of proteins controlled predominantly by division state or time and found that undivided cells account for the majority of phenotypic diversity. We next built a map of cell state changes during naive T-cell expansion. By examining cell signaling on this map, we rationally selected ibrutinib, a BTK and ITK inhibitor, and administered it before T cell activation to direct differentiation toward a T stem cell memory (TSCM)-like phenotype. This method for tracing cell fate across division states and time can be broadly applied for directing cellular differentiation

Bile acid and inflammation activate gastric cardia stem cells in a mouse model of barrett-like metaplasia

Esophageal adenocarcinoma (EAC) arises from Barrett esophagus (BE), intestinal-like columnar metaplasia linked to reflux esophagitis. In a transgenic mouse model of BE, esophageal overexpression of interleukin-1β phenocopies human pathology with evolution of esophagitis, Barrett-like metaplasia and EAC. Histopathology and gene signatures closely resembled human BE, with upregulation of TFF2, Bmp4, Cdx2, Notch1, and IL-6. The development of BE and EAC was accelerated by exposure to bile acids and/or nitrosamines, and inhibited by IL-6 deficiency. Lgr5+ gastric cardia stem cells present in BE were able to lineage trace the early BE lesion. Our data suggest that BE and EAC arise from gastric progenitors due to a tumor-promoting IL-1β-IL-6 signaling cascade and Dll1-dependent Notch signaling. © 2012 Elsevier Inc

Proliferation tracing with single-cell mass cytometry optimizes generation of stem cell memory-like T cells.

Selective differentiation of naive T cells into multipotent T cells is of great interest clinically for the generation of cell-based cancer immunotherapies. Cellular differentiation depends crucially on division state and time. Here we adapt a dye dilution assay for tracking cell proliferative history through mass cytometry and uncouple division, time and regulatory protein expression in single naive human T cells during their activation and expansion in a complex ex vivo milieu. Using 23 markers, we defined groups of proteins controlled predominantly by division state or time and found that undivided cells account for the majority of phenotypic diversity. We next built a map of cell state changes during naive T-cell expansion. By examining cell signaling on this map, we rationally selected ibrutinib, a BTK and ITK inhibitor, and administered it before T cell activation to direct differentiation toward a T stem cell memory (TSCM)-like phenotype. This method for tracing cell fate across division states and time can be broadly applied for directing cellular differentiation

Biomarkers of Residual Disease, Disseminated Tumor Cells, and Metastases in the MMTV-PyMT Breast Cancer Model

<div><p>Cancer metastases arise in part from disseminated tumor cells originating from the primary tumor and from residual disease persisting after therapy. The identification of biomarkers on micro-metastases, disseminated tumors, and residual disease may yield novel tools for early detection and treatment of these disease states prior to their development into metastases and recurrent tumors. Here we describe the molecular profiling of disseminated tumor cells in lungs, lung metastases, and residual tumor cells in the MMTV-PyMT breast cancer model. MMTV-PyMT mice were bred with actin-GFP mice, and focal hyperplastic lesions from pubertal MMTV-PyMT;actin-GFP mice were orthotopically transplanted into FVB/n mice to track single tumor foci. Tumor-bearing mice were treated with TAC chemotherapy (docetaxel, doxorubicin, cyclophosphamide), and residual and relapsed tumor cells were sorted and profiled by mRNA microarray analysis. Data analysis revealed enrichment of the Jak/Stat pathway, Notch pathway, and epigenetic regulators in residual tumors. Stat1 was significantly up-regulated in a DNA-damage-resistant population of residual tumor cells, and a pre-existing Stat1 sub-population was identified in untreated tumors. Tumor cells from adenomas, carcinomas, lung disseminated tumor cells, and lung metastases were also sorted from MMTV-PyMT transplant mice and profiled by mRNA microarray. Whereas disseminated tumors cells appeared similar to carcinoma cells at the mRNA level, lung metastases were genotypically very different from disseminated cells and primary tumors. Lung metastases were enriched for a number of chromatin-modifying genes and stem cell-associated genes. Histone analysis of H3K4 and H3K9 suggested that lung metastases had been reprogrammed during malignant progression. These data identify novel biomarkers of residual tumor cells and disseminated tumor cells and implicate pathways that may mediate metastasis formation and tumor relapse after therapy.</p> </div

Up-regulation of chromatin modifying gene in MMTV-PyMT residual tumors.

<p>Fluidigm RT-PCR analysis of H3K4 methyltransferases (A), H3K9 methyltransferases (B) and other chromatin-modifying enzymes (C) in various tumor populations. GFP-positive tumor populations were FACS-sorted from indicated groups and mRNA was harvested for the analysis; mean ± s.e.m., n = 5 per group. Data were normalized to Gapdh expression.</p

Ifn-γ/Jak/Stat signaling in MMTV-PyMT residual tumor cells after chemotherapy.

<p>(A) Ingenuity pathway analysis of genes up-regulated in chemotherapy-treated residual tumors in the MMTV-PyMT transplant model. (B) Microarray expression values of Ifn-γ/Jak/Stat-associated genes in untreated (green), residual (red) and relapsed (blue) tumors; mean, n = 5 per group. (C) Fluidigm RT-PCR analysis of Ifn-γ/Jak/Stat-associated genes. GFP-positive tumor populations were FACS-sorted from the indicated groups and mRNA was harvested for the analysis; mean ± s.e.m., n = 5 per group. Data was normalized to Gapdh expression.</p

Histone-3 methylation marks in cancer progression.

<p>(A) Western blot of H3K4 tri-methyl, H3K9 tri-methyl, H3K27 tri-methyl and actin control in tumor lysates from adenomas (5 week outgrowth), carcinomas (18 week outgrowth), and lung metastasis; n = 3 per group. (B) Western blot of H3K4 tri-methyl, H3K4 di-methyl, H3K4 mono-methyl and Histone 3 control performed with serial dilutions of tumor lysates (1 ug, 5 ug, 10 ug total protein loaded per well). (C) Fluidigm RT-PCR analysis of Il-6, Il-6ra, and Prdm1 in the indicated tumor populations. GFP-positive tumor populations were FACS-sorted from the indicated groups and mRNA was harvested for the analysis; mean ± s.e.m., n = 5 per group. Data was normalized to Gapdh expression.</p