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

Coordinated Cellular Neighborhoods Orchestrate Antitumoral Immunity at the Colorectal Cancer Invasive Front.

Antitumoral immunity requires organized, spatially nuanced interactions between components of the immune tumor microenvironment (iTME). Understanding this coordinated behavior in effective versus ineffective tumor control will advance immunotherapies. We re-engineered co-detection by indexing (CODEX) for paraffin-embedded tissue microarrays, enabling simultaneous profiling of 140 tissue regions from 35 advanced-stage colorectal cancer (CRC) patients with 56 protein markers. We identified nine conserved, distinct cellular neighborhoods (CNs)-a collection of components characteristic of the CRC iTME. Enrichment of PD-1+CD4+ T cells only within a granulocyte CN positively correlated with survival in a high-risk patient subset. Coupling of tumor and immune CNs, fragmentation of T cell and macrophage CNs, and disruption of inter-CN communication was associated with inferior outcomes. This study provides a framework for interrogating how complex biological processes, such as antitumoral immunity, occur through concerted actions of cells and spatial domains

GateFinder: projection-based gating strategy optimization for flow and mass cytometry

Motivation: High-parameter single-cell technologies can reveal novel cell populations of interest, but studying or validating these populations using lower-parameter methods remains challenging.Results: Here, we present GateFinder, an algorithm that enriches high-dimensional cell types with simple, stepwise polygon gates requiring only two markers at a time. A series of case studies of complex cell types illustrates how simplified enrichment strategies can enable more efficient assays, reveal novel biomarkers and clarify underlying biology

Comparative genomics analysis reveals the <i>de novo</i> origin of the <i>PBOV1</i> protein-coding sequence.

<p><b>A</b>: The evolutionary tree of 34 mammals with available genomic sequences. The values next to species names show fractions of CDS of human <i>PBOV1</i> that could be aligned with the respective genome and fractions of encoded proteins (assuming that they exist) that could be aligned with the human PBOV1 protein. For selected taxons, the most probable values of those fractions in the last common ancestor (LCA) are given. The genome of LCA of <i>Boreoeutheria</i> most likely contained the start codon of <i>PBOV1</i>, 97% of respective genomic sequence (as the maximum of 97% of human sequence could be aligned to the genomes of horse and megabat) and 7% of the putative protein sequence. However, in rodents and <i>Lagomorpha</i> the frame was lost due to a mutation in the ATG codon. <i>Laurasiatheria</i> retain up to 97% of the genomic sequence homologous to <i>PBOV1</i> CDS, but the protein homology is below 3% due to a synapomorphic frame-shift deletion. All higher primates contain at least 99% of human genomic sequence, but the protein homology is only 20%. An important evolutionary event along the human lineage was the A→T substitution at the position 90 in the last common ancestor of <i>Hominidae</i> which removed the stop codon. However, all <i>Hominidae</i> genomes lack an in-frame stop codon over the span of the human transcript, which could make the transcript in this species a target of the non-stop decay <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0056162#pone.0056162-Vasudevan1" target="_blank">[24]</a>. Finally, a single nucleotide deletion that occurred after the divergence from chimp led to a frame-shift that finally shaped the modern human PBOV1 protein sequence. <b>B</b>: Multiple alignments of human <i>PBOV1</i> CDS with orthologous loci from selected mammalian species. The stretches of genomes that contribute to the putative protein homology to human PBOV1 are highlighted in yellow, followed by the features that disrupt protein homology (frame-shifts and stop codons). For the sake of representation, the exact sequences of species-specific insertions are omitted from the alignment.</p

PBOV1 Is a Human <em>De Novo</em> Gene with Tumor-Specific Expression That Is Associated with a Positive Clinical Outcome of Cancer

<div><p><em>PBOV1</em> is a known human protein-coding gene with an uncharacterized function. We have previously found that <em>PBOV1</em> lacks orthologs in non-primate genomes and is expressed in a wide range of tumor types. Here we report that <em>PBOV1</em> protein-coding sequence is human-specific and has originated <em>de novo</em> in the primate evolution through a series of frame-shift and stop codon mutations. We profiled <em>PBOV1</em> expression in multiple cancer and normal tissue samples and found that it was expressed in 19 out of 34 tumors of various origins but completely lacked expression in any of the normal adult or fetal human tissues. We found that, unlike the cancer/testis antigens that are typically controlled by CpG island-containing promoters, <em>PBOV1</em> was expressed from a GC-poor TATA-containing promoter which was not influenced by CpG demethylation and was inactive in testis. Our analysis of public microarray data suggests that <em>PBOV1</em> activation in tumors could be dependent on the Hedgehog signaling pathway. Despite the recent <em>de novo</em> origin and the lack of identifiable functional signatures, a missense SNP in the <em>PBOV1</em> coding sequence has been previously associated with an increased risk of breast cancer. Using publicly available microarray datasets, we found that high levels of <em>PBOV1</em> expression in breast cancer and glioma samples were significantly associated with a positive outcome of the cancer disease. We also found that <em>PBOV1</em> was highly expressed in primary but not in recurrent high-grade gliomas, suggesting the presence of a negative selection against <em>PBOV1</em>-expressing cancer cells. Our findings could contribute to the understanding of the mechanisms behind <em>de novo</em> gene origin and the possible role of tumors in this process.</p> </div

List of mammalian genomes used in the comparative genomics study.

<p>List of mammalian genomes used in the comparative genomics study.</p

<i>PBOV1</i> expression in pancreas cancer xenografts is downregulated by HhAntag treatment (data from GSE11981 dataset).

<p>The data comes from a study that profiled the gene expression response of human pancreatic cancer xenografts in mice to the treatment with HhAntag, a potent inhibitor of Hedgehog signaling and a prospective anti-cancer drug <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0056162#pone.0056162-Yauch1" target="_blank">[44]</a>. In three out of four replicates <i>PBOV1</i> expression was downregulated by more than 75%.</p

<i>PBOV1</i> expression profiling by PCR in cDNA panels from human tumors shows that <i>PBOV1</i> is expressed in multiple tumor types.

<p><b>A</b>. Tumor cDNA Panel (BioChain Institute, USA): 1 – Brain medulloblastoma, with glioma, 2 – Lung squamous cell carcinoma, 3 – Kidney granular cell carcinoma, 4 – Kidney clear cell carcinoma, 5 – Liver cholangiocellular carcinoma, 6 – Hepatocellular carcinoma, 7 – Gallbladder adenocarcinoma, 8 – Esophagus squamous cell carcinoma, 9 – Stomach signet ring cell carcinoma, 10 – Small Intestine adenocarcinoma, 11 – Colon papillary adenocarcinoma, 12 – Rectum adenocarcinoma, 13 – Breast fibroadenoma, 14 – Ovary serous cystoadenocarcinoma, 15 – Fallopian tube medullary carcinoma, 16 – Uterus adenocarcinoma, 17 – Ureter papillary transitional cell carcinoma, 18 – Bladder transitional cell carcinoma, 19 – Testis seminoma, 20 – Prostate adenocarcinoma, 21 – Malignant melanoma, 22 – Skeletal Muscle malignancy fibrous histocytoma, 23 – Adrenal pheochromocytoma, 24 – Non-Hodgkin's lymphoma, 25 – Thyroid papillary adenocarcinoma, 26 – Parotid mixed tumor, 27 – Pancreas adenocarcinoma, 28 – Thymus seminoma, 29 – Spleen serous adenocarcinoma, 30 – Hodgkin's lymphoma, 31 – T cell Hodgkin's lymphoma, 32 – Malignant lymphoma. NC – PCR with no template, PC – PCR with human DNA. DNA contamination was controlled using gDNA-CTR primers. Full-sized images of gels are presented on Figure S7 and Figure S8 in File S1. <b>B</b>. PBOV1 expression in clinical tumor samples (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0056162#s4" target="_blank">Materials and Methods</a> for full description of samples). PBOV1 is expressed in breast cancer (9–250), ovary cancer (1, 6), cervical cancer (2, 13), endometrial cancer (156, 270), lung cancer (12, 14, 17), seminoma (7), meningioma (63), non-Hodgkin lymphomas (67, 82, 92, 102, 113) Full-sized images of gels are presented on Figure S9 and Figure S10 in File S1.</p