Archival single-cell genomics reveals persistent subclones during DCIS progression.

Ductal carcinoma in situ (DCIS) is a common precursor of invasive breast cancer. Our understanding of its genomic progression to recurrent disease remains poor, partly due to challenges associated with the genomic profiling of formalin-fixed paraffin-embedded (FFPE) materials. Here, we developed Arc-well, a high-throughput single-cell DNA-sequencing method that is compatible with FFPE materials. We validated our method by profiling 40,330 single cells from cell lines, a frozen tissue, and 27 FFPE samples from breast, lung, and prostate tumors stored for 3-31 years. Analysis of 10 patients with matched DCIS and cancers that recurred 2-16 years later show that many primary DCIS had already undergone whole-genome doubling and clonal diversification and that they shared genomic lineages with persistent subclones in the recurrences. Evolutionary analysis suggests that most DCIS cases in our cohort underwent an evolutionary bottleneck, and further identified chromosome aberrations in the persistent subclones that were associated with recurrence.

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. We applied TSCS to 1,293 single cells from 10 synchronous patients with both DCIS and IDC regions in addition to exome sequencing. Our data reveal a direct genomic lineage between in situ and invasive tumor subpopulations and further show that most mutations and copy number aberrations evolved within the ducts prior to invasion. These results support a multiclonal invasion model, in which one or more clones escape the ducts and migrate into the adjacent tissues to establish the invasive carcinomas.

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). Deep-exome sequencing identified 10 patients in which NAC led to clonal extinction and 10 patients in which clones persisted after treatment. In 8 patients, we performed a more detailed study using single-cell DNA sequencing to analyze 900 cells and single-cell RNA sequencing to analyze 6,862 cells. Our data showed that resistant genotypes were pre-existing and adaptively selected by NAC, while transcriptional profiles were acquired by reprogramming in response to chemotherapy in TNBC patients.

Simple oligonucleotide-based multiplexing of single-cell chromatin accessibility.

Microdroplet single-cell ATAC-seq is widely used to measure chromatin accessibility, however, highly scalable and simple sample multiplexing procedures are not available. Here, we present a transposome-assisted single nucleus barcoding approach for ATAC-seq (SNuBar-ATAC) that utilizes a single oligonucleotide adaptor for multiplexing samples during the existing tagmentation step and does not require a pre-labeling procedure. The accuracy and scalability of SNuBar-ATAC was evaluated using cell line mixture experiments. We applied SNuBar-ATAC to investigate treatment-induced chromatin accessibility dynamics by multiplexing 28 mice with lung tumors that received different combinations of chemo, radiation, and targeted immunotherapy. We also applied SNuBar-ATAC to study spatial epigenetic heterogeneity by multiplexing 32 regions from a human breast tissue. Additionally, we show that SNuBar can multiplex single cell ATAC and RNA multiomic assays in cell lines and human breast tissue samples. Our data show that SNuBar is a highly accurate, easy-to-use, and scalable system for multiplexing scATAC-seq and scATAC and RNA co-assay experiments.

Breast tumours maintain a reservoir of subclonal diversity during expansion.

Our knowledge of copy number evolution during the expansion of primary breast tumours is limited1,2. Here, to investigate this process, we developed a single-cell, single-molecule DNA-sequencing method and performed copy number analysis of 16,178 single cells from 8 human triple-negative breast cancers and 4 cell lines. The results show that breast tumours and cell lines comprise a large milieu of subclones (7-22) that are organized into a few (3-5) major superclones. Evolutionary analysis suggests that after clonal TP53 mutations, multiple loss-of-heterozygosity events and genome doubling, there was a period of transient genomic instability followed by ongoing copy number evolution during the primary tumour expansion. By subcloning single daughter cells in culture, we show that tumour cells rediversify their genomes and do not retain isogenic properties. These data show that triple-negative breast cancers continue to evolve chromosome aberrations and maintain a reservoir of subclonal diversity during primary tumour growth.

Dissociation protocols used for sarcoma tissues bias the transcriptome observed in single-cell and single-nucleus RNA sequencing.

BACKGROUND: Single-cell RNA-seq has emerged as an innovative technology used to study complex tissues and characterize cell types, states, and lineages at a single-cell level. Classification of bulk tumors by their individual cellular constituents has also created new opportunities to generate single-cell atlases for many organs, cancers, and developmental models. Despite the tremendous promise of this technology, recent evidence studying epithelial tissues and diverse carcinomas suggests the methods used for tissue processing, cell disaggregation, and preservation can significantly bias gene expression and alter the observed cell types. To determine whether sarcomas - tumors of mesenchymal origin - are subject to the same technical artifacts, we profiled patient-derived tumor explants (PDXs) propagated from three aggressive subtypes: osteosarcoma (OS), Ewing sarcoma (ES), desmoplastic small round cell tumor (DSRCT). Given the rarity of these sarcoma subtypes, we explored whether single-nuclei RNA-seq from more widely available archival frozen specimens could accurately be identified by gene expression signatures linked to tissue phenotype or pathognomonic fusion proteins. RESULTS: We systematically assessed dissociation methods across different sarcoma subtypes. We compared gene expression from single-cell and single-nucleus RNA-sequencing of 125,831 whole-cells and nuclei from ES, DSRCT, and OS PDXs. We detected warm dissociation artifacts in single-cell samples and gene length bias in single-nucleus samples. Classic sarcoma gene signatures were observed regardless of the dissociation method. In addition, we showed that dissociation method biases could be computationally corrected. CONCLUSIONS: We highlighted transcriptional biases, including warm dissociation and gene-length biases, introduced by the dissociation method for various sarcoma subtypes. This work is the first to characterize how the dissociation methods used for sc/snRNA-seq may affect the interpretation of the molecular features in sarcoma PDXs.

Advances and applications of single-cell sequencing technologies.

Single-cell sequencing (SCS) has emerged as a powerful new set of technologies for studying rare cells and delineating complex populations. Over the past 5 years, SCS methods for DNA and RNA have had a broad impact on many diverse fields of biology, including microbiology, neurobiology, development, tissue mosaicism, immunology, and cancer research. In this review, we will discuss SCS technologies and applications, as well as translational applications in the clinic.

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. Single-cell copy number or mutational profiling was performed, in addition to bulk exome and targeted deep-sequencing. In the first patient, we observed monoclonal seeding, in which a single clone evolved a large number of mutations prior to migrating to the liver to establish the metastatic tumor. In the second patient, we observed polyclonal seeding, in which two independent clones seeded the metastatic liver tumor after having diverged at different time points from the primary tumor lineage. The single-cell data also revealed an unexpected independent tumor lineage that did not metastasize, and early progenitor clones with the "first hit" mutation in APC that subsequently gave rise to both the primary and metastatic tumors. Collectively, these data reveal a late-dissemination model of metastasis in two CRC patients and provide an unprecedented view of metastasis at single-cell genomic resolution.

Clonal evolution in breast cancer revealed by single nucleus genome sequencing.

Sequencing studies of breast tumour cohorts have identified many prevalent mutations, but provide limited insight into the genomic diversity within tumours. Here we developed a whole-genome and exome single cell sequencing approach called nuc-seq that uses G2/M nuclei to achieve 91% mean coverage breadth. We applied this method to sequence single normal and tumour nuclei from an oestrogen-receptor-positive (ER(+)) breast cancer and a triple-negative ductal carcinoma. In parallel, we performed single nuclei copy number profiling. Our data show that aneuploid rearrangements occurred early in tumour evolution and remained highly stable as the tumour masses clonally expanded. In contrast, point mutations evolved gradually, generating extensive clonal diversity. Using targeted single-molecule sequencing, many of the diverse mutations were shown to occur at low frequencies (<10%) in the tumour mass. Using mathematical modelling we found that the triple-negative tumour cells had an increased mutation rate (13.3×), whereas the ER(+) tumour cells did not. These findings have important implications for the diagnosis, therapeutic treatment and evolution of chemoresistance in breast cancer.

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.navinlab.com/SCOPIT). In addition, we created an R package called pmultinom for scripting these calculations. CONCLUSIONS: Our tool for fast multinomial calculations provide a simple and intuitive procedure for prospectively planning single-cell experiments or retrospectively evaluating if sufficient numbers of cells have been sequenced. The web application can be accessed at navinlab.com/SCOPIT.

The first five years of single-cell cancer genomics and beyond.

Single-cell sequencing (SCS) is a powerful new tool for investigating evolution and diversity in cancer and understanding the role of rare cells in tumor progression. These methods have begun to unravel key questions in cancer biology that have been difficult to address with bulk tumor measurements. Over the past five years, there has been extraordinary progress in technological developments and research applications, but these efforts represent only the tip of the iceberg. In the coming years, SCS will greatly improve our understanding of invasion, metastasis, and therapy resistance during cancer progression. These tools will also have direct translational applications in the clinic, in areas such as early detection, noninvasive monitoring, and guiding targeted therapy. In this perspective, I discuss the progress that has been made and the myriad of unexplored applications that still lie ahead in cancer research and medicine.

Genome evolution in ductal carcinoma in situ: invasion of the clones.

Ductal carcinoma in situ (DCIS) is the most frequently diagnosed early-stage breast cancer. Only a subset of patients progress to invasive ductal carcinoma (IDC), and this presents a formidable clinical challenge for determining which patients to treat aggressively and which patients to monitor without therapeutic intervention. Understanding the molecular and genomic basis of invasion has been difficult to study in DCIS cancers due to several technical obstacles, including low tumour cellularity, lack of fresh-frozen tissues, and intratumour heterogeneity. In this review, we discuss the role of intratumour heterogeneity in the progression of DCIS to IDC in the context of three evolutionary models: independent lineages, evolutionary bottlenecks, and multiclonal invasion. We examine the evidence in support of these models and their relevance to the diagnosis and treatment of patients with DCIS. We also discuss how emerging technologies, such as single-cell sequencing, STAR-FISH, and imaging mass spectrometry, are likely to provide new insights into the evolution of this enigmatic disease. Copyright © 2016 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

Reconstructing mutational lineages in breast cancer by multi-patient-targeted single-cell DNA sequencing.

Single-cell DNA sequencing (scDNA-seq) methods are powerful tools for profiling mutations in cancer cells; however, most genomic regions sequenced in single cells are non-informative. To overcome this issue, we developed a multi-patient-targeted (MPT) scDNA-seq method. MPT involves first performing bulk exome sequencing across a cohort of cancer patients to identify somatic mutations, which are then pooled together to develop a single custom targeted panel for high-throughput scDNA-seq using a microfluidics platform. We applied MPT to profile 330 mutations across 23,500 cells from 5 patients with triple negative-breast cancer (TNBC), which showed that 3 tumors were monoclonal and 2 tumors were polyclonal. From these data, we reconstructed mutational lineages and identified early mutational and copy-number events, including early TP53 mutations that occurred in all five patients. Collectively, our data suggest that MPT can overcome a major technical obstacle for studying tumor evolution using scDNA-seq by profiling information-rich mutation sites.

Dimeric p53 Mutant Elicits Unique Tumor-Suppressive Activities through an Altered Metabolic Program.

Cancer-related alterations of the p53 tetramerization domain (TD) abrogate wild-type (WT) p53 function. They result in a protein that preferentially forms monomers or dimers, which are also normal p53 states under basal cellular conditions. However, their physiologic relevance is not well understood. We have established in vivo models for monomeric and dimeric p53, which model Li-Fraumeni syndrome patients with germline p53 TD alterations. p53 monomers are inactive forms of the protein. Unexpectedly, p53 dimers conferred some tumor suppression that is not mediated by canonical WT p53 activities. p53 dimers upregulate the PPAR pathway. These activities are associated with lower prevalence of thymic lymphomas and increased CD8+ T-cell differentiation. Lymphomas derived from dimeric p53 mice show cooperating alterations in the PPAR pathway, further implicating a role for these activities in tumor suppression. Our data reveal novel functions for p53 dimers and support the exploration of PPAR agonists as therapies. SIGNIFICANCE: New mouse models with TP53R342P (monomer) or TP53A347D (dimer) mutations mimic Li-Fraumeni syndrome. Although p53 monomers lack function, p53 dimers conferred noncanonical tumor-suppressive activities. We describe novel activities for p53 dimers facilitated by PPARs and propose these are "basal" p53 activities. See related commentary by Stieg et al., p. 1046. See related article by Choe et al., p. 1250. This article is highlighted in the In This Issue feature, p. 1027.

Targeting T cell checkpoints 41BB and LAG3 and myeloid cell CXCR1/CXCR2 results in antitumor immunity and durable response in pancreatic cancer.

Pancreatic ductal adenocarcinoma (PDAC) is considered non-immunogenic, with trials showing its recalcitrance to PD1 and CTLA4 immune checkpoint therapies (ICTs). Here, we sought to systematically characterize the mechanisms underlying de novo ICT resistance and to identify effective therapeutic options for PDAC. We report that agonist 41BB and antagonist LAG3 ICT alone and in combination, increased survival and antitumor immunity, characterized by modulating T cell subsets with antitumor activity, increased T cell clonality and diversification, decreased immunosuppressive myeloid cells and increased antigen presentation/decreased immunosuppressive capability of myeloid cells. Translational analyses confirmed the expression of 41BB and LAG3 in human PDAC. Since single and dual ICTs were not curative, T cell-activating ICTs were combined with a CXCR1/2 inhibitor targeting immunosuppressive myeloid cells. Triple therapy resulted in durable complete responses. Given similar profiles in human PDAC and the availability of these agents for clinical testing, our findings provide a testable hypothesis for this lethal disease.

Spatial charting of single-cell transcriptomes in tissues.

Single-cell RNA sequencing methods can profile the transcriptomes of single cells but cannot preserve spatial information. Conversely, spatial transcriptomics assays can profile spatial regions in tissue sections, but do not have single-cell resolution. Here, we developed a computational method called CellTrek that combines these two datasets to achieve single-cell spatial mapping through coembedding and metric learning approaches. We benchmarked CellTrek using simulation and in situ hybridization datasets, which demonstrated its accuracy and robustness. We then applied CellTrek to existing mouse brain and kidney datasets and showed that CellTrek can detect topological patterns of different cell types and cell states. We performed single-cell RNA sequencing and spatial transcriptomics experiments on two ductal carcinoma in situ tissues and applied CellTrek to identify tumor subclones that were restricted to different ducts, and specific T cell states adjacent to the tumor areas. Our data show that CellTrek can accurately map single cells in diverse tissue types to resolve their spatial organization.

Learning to distinguish progressive and non-progressive ductal carcinoma in situ.

Ductal carcinoma in situ (DCIS) is a non-invasive breast neoplasia that accounts for 25% of all screen-detected breast cancers diagnosed annually. Neoplastic cells in DCIS are confined to the ductal system of the breast, although they can escape and progress to invasive breast cancer in a subset of patients. A key concern of DCIS is overtreatment, as most patients screened for DCIS and in whom DCIS is diagnosed will not go on to exhibit symptoms or die of breast cancer, even if left untreated. However, differentiating low-risk, indolent DCIS from potentially progressive DCIS remains challenging. In this Review, we summarize our current knowledge of DCIS and explore open questions about the basic biology of DCIS, including those regarding how genomic events in neoplastic cells and the surrounding microenvironment contribute to the progression of DCIS to invasive breast cancer. Further, we discuss what information will be needed to prevent overtreatment of indolent DCIS lesions without compromising adequate treatment for high-risk patients.

A Decade's Experience in Pediatric Chromosomal Microarray Reveals Distinct Characteristics Across Ordering Specialties.

Chromosomal microarray (CMA) is a testing modality frequently used in pediatric patients; however, published data on its utilization are limited to the genetic setting. We performed a database search for all CMA testing performed from 2010 to 2020, and delineated the diagnostic yield based on patient characteristics, including sex, age, clinical specialty of providers, indication of testing, and pathogenic finding. The indications for testing were further categorized into Human Phenotype Ontology categories for analysis. This study included a cohort of 14,541 patients from 29 different medical specialties, of whom 30% were from the genetics clinic. The clinical indications for testing suggested that neonatology patients demonstrated the greatest involvement of multiorgan systems, involving the most Human Phenotype Ontology categories, compared with developmental behavioral pediatrics and neurology patients being the least. The top pathogenic findings for each specialty differed, likely due to the varying clinical features and indications for testing. Deletions involving the 22q11.21 locus were the top pathogenic findings for patients presenting to genetics, neonatology, cardiology, and surgery. Our data represent the largest pediatric cohort published to date. This study is the first to demonstrate the diagnostic utility of this assay for patients seen in the setting of different specialties, and it provides normative data of CMA results among a general pediatric population referred for testing because of variable clinical presentations.

Single-Cell Sequencing Reveals Trajectory of Tumor-Infiltrating Lymphocyte States in Pancreatic Cancer.

Pancreatic ductal adenocarcinoma (PDAC) has few effective treatments. Immunotherapy, an attractive alternative strategy, remains challenging with the lack of knowledge on the tumor-infiltrating lymphocyte (TIL) landscape in PDAC. To generate a reference of T-cell subpopulations, we profiled 80,000 T cells from 57 PDAC samples, 22 uninvolved/normal samples, and cultured TIL using single-cell transcriptomic and T-cell receptor analysis. These data revealed 20 cell states and heterogeneous distributions of TIL populations. The CD8+ TIL contained a putative transitional GZMK+ population based on T-cell receptor clonotype sharing, and cell-state trajectory analysis showed similarity to a GZMB+PRF1+ cytotoxic and a CXCL13+ dysfunctional population. Statistical analysis suggested that certain TIL states, such as dysfunctional and inhibitory populations, often occurred together. Finally, analysis of cultured TIL revealed that high-frequency clones from effector populations were preferentially expanded. These data provide a framework for understanding the PDAC TIL landscape for future TIL use in immunotherapy for PDAC. SIGNIFICANCE: To improve the efficacy of immunotherapy in PDAC, there is a great need to understand the PDAC TIL landscape. This study represents a reference of PDAC TIL subpopulations and their relationships and provides a foundation upon which to base future immunotherapeutic efforts. This article is highlighted in the In This Issue feature, p. 2221.