Efficient derivation of purified metastasis-initiating cells from mouse mammary gland

Elucidation of the cellular and molecular mechanisms responsible for metastatic dissemination is of broad interest and is paramount to the design of more effective treatments for breast cancer. To date, occurrence of metastasis accounts for over 90% of breast cancer deaths, meaning that the invasion and development of metastasis are the most life threatening aspects of this disease. Only rare tumor cells shed into circulation have the capacity to colonize distal sites. It is possible that the colonizing ability is dependent on the developmental history of cells in a tumor. The developmental hierarchy of mammary epithelium in both mice and humans shows that even in the adult individuals, stem and progenitor cells are present in the breast. Substantial evidence indicates that this hierarchy plays an important role in regulating behavior of tumors arising in the breast, including their proclivity to metastasize. Experiments with mouse mammary cells harboring inducible oncogenic transgenes have established the existence of rare mammary cells in the untransformed breast that are able to seed and grow in the lungs in experimental metastasis protocols. In the absence of oncogene expression these cells give rise to the ectopic mammary outgrowths (micro-breast foci, MBF), and upon activation of potent oncogenes, such as Myc and Ras, or Her2/Neu, they give rise to ectopic breast tumors in the lung (micro-breast tumors, MBT). This suggests that cells that give rise to MBF (MBF cells) may contribute to late recurrences in breast cancer patients, as they may persist undetected for long periods of time, and revive as malignant ectopic tumors upon mutational stress. This possibility prompted us to look for a subpopulation of mammary epithelial cells (MECs) that may be responsible for MBF- and/or MBT-initiating properties in murine models.Using multiparameter cell sorting and modified experimental metastasis approach we now demonstrate the purification of a subset of adult mouse normal breast cells that are able to seed and persist at the ectopic site – the lung. This subpopulation, EpCAMIntCD29Hi, is enriched for MBF cells by intravenous injection. Only the cells within the EpCAMIntCD29Hi population are capable of forming ectopic new tumors in response to oncogene activation, property that defines them as tumor initiating cells. In support of a potential role for MBF cells in breast cancer metastasis, abundance of tumor cells with EpCAMIntCD29Hi phenotype is predictive of tumor ability to form experimental metastases in mice. Thus, our work supports the notion that metastatic colonization may depend on factors regulating normal stem cell behavior. In the future, we would like to investigate whether MBF-like normal cells may contribute to natural history of human breast cancer, for example by giving rise to late recurrences.Dérivation efficiente de cellules initiatrices de métastases purifiées provenant de glandes mammaires de sourisLa compréhension des mécanismes cellulaires et moléculaires responsables de la dissémination des métastases revêt un intérêt majeur, et est essentiel à l'élaboration de traitements plus efficaces contre le cancer du sein. À ce jour, l'occurrence de métastases dans les cancers du sein entraîne 90 % des décès, ce qui signifie que l'invasion et le développement des métastases représentent les aspects les plus menaçants de la maladie. Seules quelques rares cellules tumorales présentes dans la circulation sanguine ont la capacité de coloniser des sites ectopiques. La caractérisation du développement hiérarchique des cellules de l'épithélium mammaire (CEM), tant chez la souris que chez l'humain, démontre que même chez l'adulte, les cellules souches et les cellules pro- génitrices sont présentes dans le sein. Des preuves évidentes indiquent que cette hiérarchie joue un rôle important dans la régulation des tumeurs qui se développent dans la glande mammaire. Des expériences sur des cellules provenant de glandes mammaires de souris ayant des oncogènes transgéniques inductibles ont permis d'établir l'existence de rares cellules mammaires capables de coloniser les poumons lors d'études de modèles de métastases expérimentales. Ces cellules sont capables de générer des tumeurs mammaires ectopiques dans les poumons sur activation d'oncogènes potentiels tels que Myc et Ras, ou Her2/Neu. Ceci tend à démontrer que des cellules saines de poumons (CPP) peuvent contribuer à une récurrence tardive du cancer du sein chez les patients, puisqu'elles sont indétectables (en dormance) pendant de longues périodes et qu'elles forment des tumeurs malignes ectopiques causées par un stress mutationnel. Cette possibilité nous a amené à rechercher des sous-populations de CEM possiblement responsables des propriétés métastasiques chez les modèles murins.Grâce au trieur cellulaire multiparamétrique et à des essais de métastases expérimentales modifiés nous avons démontré la purification d'une rare sous-population de cellules mammaires adultes normales capables de coloniser et de persister dans un site ectopique. Cette sous-population (Lin-EpCAMIntCD29Hi) est fortement enrichie de cellules colonisatrices des poumons (CCP) par injection intraveineuse. Seules les cellules incluses dans EpCAMIntCD29Hi sont capables de former de nouvelles tumeurs ectopiques en réponse à l'activation des oncogènes, propriétés qui les définissent comme initiatrices de cellules tumorales. En appui an rôle potentiel des CPP dans les métastases du cancer du sein, l'abondance de cellules tumorales de phénotype Lin-EpCAMIntCD29Hi est précurseur de la capacité à former des métastases expérimentales chez la souris. Nos travaux nous permettent de soutenir que la colonisation métastasique dépend de facteurs régulant le comportement de cellules souches normales. Dans un avenir rapproché, nous souhaitons vérifier si la colonisation par des cellules normales peut contribuer à l'historique du cancer du sein chez l'humain, en prédisant, par exemple le risque de récurrence tardive

Dynamics of single cell genomes and transcriptomes in response to chemotherapies

Cancer is an ecosystem of genetically diverse evolving clones, which emerge in time and space as a result of genomic and non-genomic instability. Consequently, clonal evolution provides a basis for fluctuating cell fitness, which impacts etiology and drug resistance. However, progress in defining clonal fitness by copy number alterations (CNA), has been impeded by a lack of perturbation experiments with timeseries sampling. This dissertation is focused on understanding and measuring clonal fitness determined by copy number genomic instability in breast cancer to single cell resolution. First, I established a series of transplantable human breast cancers in immunodeficient mice strains that were tolerant to DNA damaging chemotherapy. I selected four PDXs (patient derived xenografts) for detailed analysis of clonal fitness under no treatment and after treatment with DNA damaging agents, cisplatin and CX5461. Subsequently, I optimized methods of tumor dissociations, and determined the effects of digestion time and temperature on single cell gene expression. Next, I established that single cell whole genome sequencing of CNA in PDX passaged over time resulted in positive fitness over clones that gradually sweep the population. I established a mixture re-transplant paradigm to demonstrate that the fitness predictions of a Wright-Fisher population genetics model applied to the data, were reproducible experimentally. I described that fitness landscapes of all TNBC tumors were inverted under drug because low fitness clones under no treatment gave rise to drug resistant clones. Drug holiday experiments showed that cisplatin resistance had a fitness cost. I demonstrated using CX5461, that drug resistance could arise in a common background, suggesting common drug resistant states. Finally, I examined the impact of CNA on transcriptional phenotypes. By making assignments of single cells RNA-seq, to CNA defined clones, I observed that in cis, CNA mediated clonal gene expression impacts between 5-50% of the transcriptome. Time series analysis of expression revealed reversible, but time dependent expression reprogramming, thus defining the non-CNA dependence of clonal transcriptomes. Pathway analysis revealed common sets of pathways associated with late drug resistance to platinum in TNBC. These comprehensive measurements and analysis of clonal structure will advance interpretation of polyclonal resistance to therapy.Medicine, Faculty ofPathology and Laboratory Medicine, Department ofGraduat

Identification of transcriptional programs using dense vector representations defined by mutual information with GeneVector

Abstract Deciphering individual cell phenotypes from cell-specific transcriptional processes requires high dimensional single cell RNA sequencing. However, current dimensionality reduction methods aggregate sparse gene information across cells, without directly measuring the relationships that exist between genes. By performing dimensionality reduction with respect to gene co-expression, low-dimensional features can model these gene-specific relationships and leverage shared signal to overcome sparsity. We describe GeneVector, a scalable framework for dimensionality reduction implemented as a vector space model using mutual information between gene expression. Unlike other methods, including principal component analysis and variational autoencoders, GeneVector uses latent space arithmetic in a lower dimensional gene embedding to identify transcriptional programs and classify cell types. In this work, we show in four single cell RNA-seq datasets that GeneVector was able to capture phenotype-specific pathways, perform batch effect correction, interactively annotate cell types, and identify pathway variation with treatment over time

clonealign: statistical integration of independent single-cell RNA and DNA sequencing data from human cancers

Measuring gene expression of tumor clones at single-cell resolution links functional consequences to somatic alterations. Without scalable methods to simultaneously assay DNA and RNA from the same single cell, parallel single-cell DNA and RNA measurements from independent cell populations must be mapped for genome-transcriptome association. We present clonealign, which assigns gene expression states to cancer clones using single-cell RNA and DNA sequencing independently sampled from a heterogeneous population. We apply clonealign to triple-negative breast cancer patient-derived xenografts and high-grade serous ovarian cancer cell lines and discover clone-specific dysregulated biological pathways not visible using either sequencing method alone.Graduate and Postdoctoral StudiesMedicine, Faculty ofScience, Faculty ofOther UBCNon UBCPathology and Laboratory Medicine, Department ofStatistics, Department ofReviewedFacult

Epiclomal: Probabilistic clustering of sparse single-cell DNA methylation data.

We present Epiclomal, a probabilistic clustering method arising from a hierarchical mixture model to simultaneously cluster sparse single-cell DNA methylation data and impute missing values. Using synthetic and published single-cell CpG datasets, we show that Epiclomal outperforms non-probabilistic methods and can handle the inherent missing data characteristic that dominates single-cell CpG genome sequences. Using newly generated single-cell 5mCpG sequencing data, we show that Epiclomal discovers sub-clonal methylation patterns in aneuploid tumour genomes, thus defining epiclones that can match or transcend copy number-determined clonal lineages and opening up an important form of clonal analysis in cancer. Epiclomal is written in R and Python and is available at https://github.com/shahcompbio/Epiclomal

Clonal fitness inferred from time-series modelling of single-cell cancer genomes

Progress in defining genomic fitness landscapes in cancer, especially those defined by copy number alterations (CNAs), has been impeded by lack of time-series single-cell sampling of polyclonal populations and temporal statistical models1-7. Here we generated 42,000 genomes from multi-year time-series single-cell whole-genome sequencing of breast epithelium and primary triple-negative breast cancer (TNBC) patient-derived xenografts (PDXs), revealing the nature of CNA-defined clonal fitness dynamics induced by TP53 mutation and cisplatin chemotherapy. Using a new Wright-Fisher population genetics model8,9 to infer clonal fitness, we found that TP53 mutation alters the fitness landscape, reproducibly distributing fitness over a larger number of clones associated with distinct CNAs. Furthermore, in TNBC PDX models with mutated TP53, inferred fitness coefficients from CNA-based genotypes accurately forecast experimentally enforced clonal competition dynamics. Drug treatment in three long-term serially passaged TNBC PDXs resulted in cisplatin-resistant clones emerging from low-fitness phylogenetic lineages in the untreated setting. Conversely, high-fitness clones from treatment-naive controls were eradicated, signalling an inversion of the fitness landscape. Finally, upon release of drug, selection pressure dynamics were reversed, indicating a fitness cost of treatment resistance. Together, our findings define clonal fitness linked to both CNA and therapeutic resistance in polyclonal tumours.ISSN:0028-0836ISSN:1476-468

Dissociation of solid tumor tissues with cold active protease for single-cell RNA-seq minimizes conserved collagenase-associated stress responses

Background: Single-cell RNA sequencing (scRNA-seq) is a powerful tool for studying complex biological systems, such as tumor heterogeneity and tissue microenvironments. However, the sources of technical and biological variation in primary solid tumor tissues and patient-derived mouse xenografts for scRNA-seq are not well understood. Results: We use low temperature (6 °C) protease and collagenase (37 °C) to identify the transcriptional signatures associated with tissue dissociation across a diverse scRNA-seq dataset comprising 155,165 cells from patient cancer tissues, patient-derived breast cancer xenografts, and cancer cell lines. We observe substantial variation in standard quality control metrics of cell viability across conditions and tissues. From the contrast between tissue protease dissociation at 37 °C or 6 °C, we observe that collagenase digestion results in a stress response. We derive a core gene set of 512 heat shock and stress response genes, including FOS and JUN, induced by collagenase (37 °C), which are minimized by dissociation with a cold active protease (6 °C). While induction of these genes was highly conserved across all cell types, cell type-specific responses to collagenase digestion were observed in patient tissues. Conclusions: The method and conditions of tumor dissociation influence cell yield and transcriptome state and are both tissue- and cell-type dependent. Interpretation of stress pathway expression differences in cancer single-cell studies, including components of surface immune recognition such as MHC class I, may be especially confounded. We define a core set of 512 genes that can assist with the identification of such effects in dissociated scRNA-seq experiments.Medicine, Faculty ofScience, Faculty ofOther UBCNon UBCObstetrics and Gynaecology, Department ofPathology and Laboratory Medicine, Department ofStatistics, Department ofReviewedFacult

Single-cell genomic variation induced by mutational processes in cancer

How cell-to-cell copy number alterations that underpin genomic instability1 in human cancers drive genomic and phenotypic variation, and consequently the evolution of cancer2, remains understudied. Here, by applying scaled single-cell whole-genome sequencing3 to wild-type, TP53-deficient and TP53-deficient;BRCA1-deficient or TP53-deficient;BRCA2-deficient mammary epithelial cells (13,818 genomes), and to primary triple-negative breast cancer (TNBC) and high-grade serous ovarian cancer (HGSC) cells (22,057 genomes), we identify three distinct ‘foreground’ mutational patterns that are defined by cell-to-cell structural variation. Cell- and clone-specific high-level amplifications, parallel haplotype-specific copy number alterations and copy number segment length variation (serrate structural variations) had measurable phenotypic and evolutionary consequences. In TNBC and HGSC, clone-specific high-level amplifications in known oncogenes were highly prevalent in tumours bearing fold-back inversions, relative to tumours with homologous recombination deficiency, and were associated with increased clone-to-clone phenotypic variation. Parallel haplotype-specific alterations were also commonly observed, leading to phylogenetic evolutionary diversity and clone-specific mono-allelic expression. Serrate variants were increased in tumours with fold-back inversions and were highly correlated with increased genomic diversity of cellular populations. Together, our findings show that cell-to-cell structural variation contributes to the origins of phenotypic and evolutionary diversity in TNBC and HGSC, and provide insight into the genomic and mutational states of individual cancer cells.ISSN:0028-0836ISSN:1476-468