Inducible CCR2+ nonclassical monocytes mediate the regression of cancer metastasis.

A major limitation of immunotherapy is the development of resistance resulting from cancer-mediated inhibition of host lymphocytes. Cancer cells release CCL2 to recruit classical monocytes expressing its receptor CCR2 for the promotion of metastasis and resistance to immunosurveillance. In the circulation, some CCR2-expressing classical monocytes lose CCR2 and differentiate into intravascular nonclassical monocytes that have anticancer properties but are unable to access extravascular tumor sites.

Multiorgan transplant for therapy-associated lung and liver failure in a patient with stage III lung cancer.

Immunotherapy can significantly improve efficacy of cancer treatments. For locally advanced stage III lung cancers, chemoimmunotherapy in the neoadjuvant setting can achieve complete pathological response in about 40% of cases. However, optimal cancer response in patients receiving immunotherapy is sometimes associated with potentially fatal bystander injury to lung and liver.

Anaplastic transformation in thyroid cancer revealed by single-cell transcriptomics.

The deadliest anaplastic thyroid cancer (ATC) often transforms from indolent differentiated thyroid cancer (DTC); however, the complex intratumor transformation process is poorly understood. We investigated an anaplastic transformation model by dissecting both cell lineage and cell fate transitions using single-cell transcriptomic and genetic alteration data from patients with different subtypes of thyroid cancer. The resulting spectrum of ATC transformation included stress-responsive DTC cells, inflammatory ATC cells (iATCs), and mitotic-defective ATC cells and extended all the way to mesenchymal ATC cells (mATCs).

Delineating genotypes and phenotypes of individual cells from long-read single cell transcriptomes.

Single-cell nanopore sequencing of full-length mRNAs (scNanoRNAseq) is transforming singlecell multi-omics studies. However, challenges include computational complexity and dependence on short-read curation. To address this, we developed a comprehensive toolkit, scNanoGPS to calculate same-cell genotypes-phenotypes without short-read guidance.

Single-cell analysis of human glioma and immune cells identifies S100A4 as an immunotherapy target.

A major rate-limiting step in developing more effective immunotherapies for GBM is our inadequate understanding of the cellular complexity and the molecular heterogeneity of immune infiltrates in gliomas. Here, we report an integrated analysis of 201,986 human glioma, immune, and other stromal cells at the single cell level. In doing so, we discover extensive spatial and molecular heterogeneity in immune infiltrates.

Wnt/ß-catenin-mediated p53 suppression is indispensable for osteogenesis of mesenchymal progenitor cells.

The developmental origins of mesenchymal progenitor cells (MPCs) and molecular machineries regulating their fate and differentiation are far from defined owing to their complexity. Osteoblasts and adipocytes are descended from common MPCs. Their fates are collectively determined by an orchestra of pathways in response to physiological and external cues.

MEDALT: single-cell copy number lineage tracing enabling gene discovery.

We present a Minimal Event Distance Aneuploidy Lineage Tree (MEDALT) algorithm that infers the evolution history of a cell population based on single-cell copy number (SCCN) profiles, and a statistical routine named lineage speciation analysis (LSA), whichty facilitates discovery of fitness-associated alterations and genes from SCCN lineage trees. MEDALT appears more accurate than phylogenetics approaches in reconstructing copy number lineage. From data from 20 triple-negative breast cancer patients, our approaches effectively prioritize genes that are essential for breast cancer cell fitness and predict patient survival, including those implicating convergent evolution.

Delineating copy number and clonal substructure in human tumors from single-cell transcriptomes.

Single-cell transcriptomic analysis is widely used to study human tumors. However, it remains challenging to distinguish normal cell types in the tumor microenvironment from malignant cells and to resolve clonal substructure within the tumor. To address these challenges, we developed an integrative Bayesian segmentation approach called copy number karyotyping of aneuploid tumors (CopyKAT) to estimate genomic copy number profiles at an average genomic resolution of 5 Mb from read depth in high-throughput single-cell RNA sequencing (scRNA-seq) data.

SCOPIT: sample size calculations for single-cell sequencing experiments.

Background: In single cell DNA and RNA sequencing experiments, the number of cells to sequence must be decided before running an experiment, and afterwards, it is necessary to decide whether sufficient cells were sampled. These questions can be addressed by calculating the probability of sampling at least a defined number of cells from each subpopulation (cell type or cancer clone).

Results: We developed an interactive web application called SCOPIT (Single-Cell One-sided Probability Interactive Tool), which calculates the required probabilities using a multinomial distribution (www.

MYB-activated models for testing therapeutic agents in adenoid cystic carcinoma.

Objective: There are no effective systemic therapies for adenoid cystic cancer (ACC) and lack of tumor lines and mouse models have hindered drug development.We aim to develop MYB-activated models for testing new therapeutic agents.

Materials And Methods: We studied new ACC patient-derived xenograft (PDX) models and generated a matched cell line from one patient.

GSK3β regulates epithelial-mesenchymal transition and cancer stem cell properties in triple-negative breast cancer.

Background: Triple-negative breast cancers (TNBCs), which lack receptors for estrogen, progesterone, and amplification of epidermal growth factor receptor 2, are highly aggressive. Consequently, patients diagnosed with TNBCs have reduced overall and disease-free survival rates compared to patients with other subtypes of breast cancer. TNBCs are characterized by the presence of cancer cells with mesenchymal properties, indicating that the epithelial to mesenchymal transition (EMT) plays a major role in the progression of this disease.

Restructuring of the Gut Microbiome by Intermittent Fasting Prevents Retinopathy and Prolongs Survival in Mice.

Intermittent fasting (IF) protects against the development of metabolic diseases and cancer, but whether it can prevent diabetic microvascular complications is not known. In mice, we examined the impact of long-term IF on diabetic retinopathy (DR). Despite no change in glycated hemoglobin, mice on the IF regimen displayed significantly longer survival and a reduction in DR end points, including acellular capillaries and leukocyte infiltration.

Chemoresistance Evolution in Triple-Negative Breast Cancer Delineated by Single-Cell Sequencing.

Triple-negative breast cancer (TNBC) is an aggressive subtype that frequently develops resistance to chemotherapy. An unresolved question is whether resistance is caused by the selection of rare pre-existing clones or alternatively through the acquisition of new genomic aberrations. To investigate this question, we applied single-cell DNA and RNA sequencing in addition to bulk exome sequencing to profile longitudinal samples from 20 TNBC patients during neoadjuvant chemotherapy (NAC).

Multiclonal Invasion in Breast Tumors Identified by Topographic Single Cell Sequencing.

Ductal carcinoma in situ (DCIS) is an early-stage breast cancer that infrequently progresses to invasive ductal carcinoma (IDC). Genomic evolution has been difficult to delineate during invasion due to intratumor heterogeneity and the low number of tumor cells in the ducts. To overcome these challenges, we developed Topographic Single Cell Sequencing (TSCS) to measure genomic copy number profiles of single tumor cells while preserving their spatial context in tissue sections.

Integrative genome analysis of somatic p53 mutant osteosarcomas identifies Ets2-dependent regulation of small nucleolar RNAs by mutant p53 protein.

is the most frequently mutated gene in human cancer. Many mutant p53 proteins exert oncogenic gain-of-function (GOF) properties that contribute to metastasis, but the mechanisms mediating these functions remain poorly defined in vivo. To elucidate how mutant p53 GOF drives metastasis, we developed a traceable somatic osteosarcoma mouse model that is initiated with either a single mutation (p53R172H) or loss in osteoblasts.

Nanogrid single-nucleus RNA sequencing reveals phenotypic diversity in breast cancer.

Single cell RNA sequencing has emerged as a powerful tool for resolving transcriptional diversity in tumors, but is limited by throughput, cost and the ability to process archival frozen tissue samples. Here we develop a high-throughput 3' single-nucleus RNA sequencing approach that combines nanogrid technology, automated imaging, and cell selection to sequence up to ~1800 single nuclei in parallel. We compare the transcriptomes of 485 single nuclei to 424 single cells in a breast cancer cell line, which shows a high concordance (93.

Immunodeficiency in Pancreatic Adenocarcinoma with Diabetes Revealed by Comparative Genomics.

Pancreatic adenocarcinomas (PAAD) often are not diagnosed until their late stages, leaving no effective treatments. Currently, immunotherapy provides a promising treatment option against this malignancy. However, a set of immunotherapy agents benefit patients with many types of cancer, but not PAAD.

Single-cell DNA sequencing reveals a late-dissemination model in metastatic colorectal cancer.

Metastasis is a complex biological process that has been difficult to delineate in human colorectal cancer (CRC) patients. A major obstacle in understanding metastatic lineages is the extensive intra-tumor heterogeneity at the primary and metastatic tumor sites. To address this problem, we developed a highly multiplexed single-cell DNA sequencing approach to trace the metastatic lineages of two CRC patients with matched liver metastases.

Tumor evolution: Linear, branching, neutral or punctuated?

Intratumor heterogeneity has been widely reported in human cancers, but our knowledge of how this genetic diversity emerges over time remains limited. A central challenge in studying tumor evolution is the difficulty in collecting longitudinal samples from cancer patients. Consequently, most studies have inferred tumor evolution from single time-point samples, providing very indirect information.

Punctuated copy number evolution and clonal stasis in triple-negative breast cancer.

Aneuploidy is a hallmark of breast cancer; however, knowledge of how these complex genomic rearrangements evolve during tumorigenesis is limited. In this study, we developed a highly multiplexed single-nucleus sequencing method to investigate copy number evolution in patients with triple-negative breast cancer. We sequenced 1,000 single cells from tumors in 12 patients and identified 1-3 major clonal subpopulations in each tumor that shared a common evolutionary lineage.

Highly multiplexed targeted DNA sequencing from single nuclei.

Single-cell DNA sequencing methods are challenged by poor physical coverage, high technical error rates and low throughput. To address these issues, we developed a single-cell DNA sequencing protocol that combines flow-sorting of single nuclei, time-limited multiple-displacement amplification (MDA), low-input library preparation, DNA barcoding, targeted capture and next-generation sequencing (NGS). This approach represents a major improvement over our previous single nucleus sequencing (SNS) Nature Protocols paper in terms of generating higher-coverage data (>90%), thereby enabling the detection of genome-wide variants in single mammalian cells at base-pair resolution.

A unifying gene signature for adenoid cystic cancer identifies parallel MYB-dependent and MYB-independent therapeutic targets.

MYB activation is proposed to underlie development of adenoid cystic cancer (ACC), an aggressive salivary gland tumor with no effective systemic treatments. To discover druggable targets for ACC, we performed global mRNA/miRNA analyses of 12 ACC with matched normal tissues, and compared these data with 14 mucoepidermoid carcinomas (MEC) and 11 salivary adenocarcinomas (ADC). We detected a unique ACC gene signature of 1160 mRNAs and 22 miRNAs.

Role of LKB1-CRTC1 on glycosylated COX-2 and response to COX-2 inhibition in lung cancer.

Background: Cyclooxygenase-2 (COX-2) directs the synthesis of prostaglandins including PGE-2 linking inflammation with mitogenic signaling. COX-2 is also an anticancer target, however, treatment strategies have been limited by unreliable expression assays and by inconsistent tumor responses to COX-2 inhibition.

Methods: We analyzed the TCGA and Director's Challenge lung cancer datasets (n = 188) and also generated an LKB1-null lung cancer gene signature (n = 53) to search the Broad Institute/Connectivity-MAP (C-MAP) dataset.