ABSTRACT
To better understand intrinsic resistance to immune checkpoint blockade (ICB), we established a comprehensive view of the cellular architecture of the treatment-naive melanoma ecosystem and studied its evolution under ICB. Using single-cell, spatial multi-omics, we showed that the tumor microenvironment promotes the emergence of a complex melanoma transcriptomic landscape. Melanoma cells harboring a mesenchymal-like (MES) state, a population known to confer resistance to targeted therapy, were significantly enriched in early on-treatment biopsies from non-responders to ICB. TCF4 serves as the hub of this landscape by being a master regulator of the MES signature and a suppressor of the melanocytic and antigen presentation transcriptional programs. Targeting TCF4 genetically or pharmacologically, using a bromodomain inhibitor, increased immunogenicity and sensitivity of MES cells to ICB and targeted therapy. We thereby uncovered a TCF4-dependent regulatory network that orchestrates multiple transcriptional programs and contributes to resistance to both targeted therapy and ICB in melanoma.
Subject(s)
Melanoma , Humans , Gene Regulatory Networks , Immunotherapy , Melanocytes , Melanoma/drug therapy , Melanoma/genetics , Transcription Factor 4/genetics , Tumor MicroenvironmentABSTRACT
Intra-tumor heterogeneity (ITH) is a mechanism of therapeutic resistance and therefore an important clinical challenge. However, the extent, origin, and drivers of ITH across cancer types are poorly understood. To address this, we extensively characterize ITH across whole-genome sequences of 2,658 cancer samples spanning 38 cancer types. Nearly all informative samples (95.1%) contain evidence of distinct subclonal expansions with frequent branching relationships between subclones. We observe positive selection of subclonal driver mutations across most cancer types and identify cancer type-specific subclonal patterns of driver gene mutations, fusions, structural variants, and copy number alterations as well as dynamic changes in mutational processes between subclonal expansions. Our results underline the importance of ITH and its drivers in tumor evolution and provide a pan-cancer resource of comprehensively annotated subclonal events from whole-genome sequencing data.
Subject(s)
Genetic Heterogeneity , Neoplasms/genetics , DNA Copy Number Variations , DNA, Neoplasm/chemistry , DNA, Neoplasm/metabolism , Databases, Genetic , Drug Resistance, Neoplasm/genetics , Humans , Neoplasms/pathology , Polymorphism, Single Nucleotide , Whole Genome SequencingABSTRACT
Although clonal neo-antigen burden is associated with improved response to immune therapy, the functional basis for this remains unclear. Here we study this question in a novel controlled mouse melanoma model that enables us to explore the effects of intra-tumor heterogeneity (ITH) on tumor aggressiveness and immunity independent of tumor mutational burden. Induction of UVB-derived mutations yields highly aggressive tumors with decreased anti-tumor activity. However, single-cell-derived tumors with reduced ITH are swiftly rejected. Their rejection is accompanied by increased T cell reactivity and a less suppressive microenvironment. Using phylogenetic analyses and mixing experiments of single-cell clones, we dissect two characteristics of ITH: the number of clones forming the tumor and their clonal diversity. Our analysis of melanoma patient tumor data recapitulates our results in terms of overall survival and response to immune checkpoint therapy. These findings highlight the importance of clonal mutations in robust immune surveillance and the need to quantify patient ITH to determine the response to checkpoint blockade.
Subject(s)
Genetic Heterogeneity/radiation effects , Melanoma/genetics , Melanoma/immunology , Skin Neoplasms/genetics , Skin Neoplasms/immunology , Ultraviolet Rays/adverse effects , Animals , Carcinogenesis/genetics , Cell Line, Tumor , Cohort Studies , Disease Models, Animal , Female , Humans , Lymphocytes, Tumor-Infiltrating , Melanoma/mortality , Mice , Mice, Inbred C57BL , Mice, Inbred NOD , Mice, Knockout , Mutation/radiation effects , Phylogeny , Skin Neoplasms/mortality , Survival Rate , T-Lymphocytes/immunology , Tumor Microenvironment/immunology , Tumor Microenvironment/radiation effectsABSTRACT
Single-cell DNA sequencing (scDNA-seq) has been an effective means to unscramble intra-tumor heterogeneity, while joint inference of tumor clones and their respective copy number profiles remains a challenging task due to the noisy nature of scDNA-seq data. We introduce a new bioinformatics method called CoT for deciphering clonal copy number substructure. The backbone of CoT is a Copy number Transformer autoencoder that leverages multi-head attention mechanism to explore correlations between different genomic regions, and thus capture global features to create latent embeddings for the cells. CoT makes it convenient to first infer cell subpopulations based on the learned embeddings, and then estimate single-cell copy numbers through joint analysis of read counts data for the cells belonging to the same cluster. This exploitation of clonal substructure information in copy number analysis helps to alleviate the effect of read counts non-uniformity, and yield robust estimations of the tumor copy numbers. Performance evaluation on synthetic and real datasets showcases that CoT outperforms the state of the arts, and is highly useful for deciphering clonal copy number substructure.
Subject(s)
Computational Biology , DNA Copy Number Variations , Neoplasms , Single-Cell Analysis , Humans , Neoplasms/genetics , Single-Cell Analysis/methods , Computational Biology/methods , Sequence Analysis, DNA/methods , AlgorithmsABSTRACT
BACKGROUND: Accurately deciphering clonal copy number substructure can provide insights into the evolutionary mechanism of cancer, and clustering single-cell copy number profiles has become an effective means to unmask intra-tumor heterogeneity (ITH). However, copy numbers inferred from single-cell DNA sequencing (scDNA-seq) data are error-prone due to technically confounding factors such as amplification bias and allele-dropout, and this makes it difficult to precisely identify the ITH. RESULTS: We introduce a hybrid model called scGAL to infer clonal copy number substructure. It combines an autoencoder with a generative adversarial network to jointly analyze independent single-cell copy number profiles and gene expression data from same cell line. Under an adversarial learning framework, scGAL exploits complementary information from gene expression data to relieve the effects of noise in copy number data, and learns latent representations of scDNA-seq cells for accurate inference of the ITH. Evaluation results on three real cancer datasets suggest scGAL is able to accurately infer clonal architecture and surpasses other similar methods. In addition, assessment of scGAL on various simulated datasets demonstrates its high robustness against the changes of data size and distribution. scGAL can be accessed at: https://github.com/zhyu-lab/scgal . CONCLUSIONS: Joint analysis of independent single-cell copy number and gene expression data from a same cell line can effectively exploit complementary information from individual omics, and thus gives more refined indication of clonal copy number substructure.
Subject(s)
DNA Copy Number Variations , Neoplasms , Single-Cell Analysis , Single-Cell Analysis/methods , Humans , Neoplasms/genetics , Neoplasms/pathology , Algorithms , Cell Line, Tumor , Single-Cell Gene Expression AnalysisABSTRACT
The rapid development of single-cell DNA sequencing (scDNA-seq) technology has greatly enhanced the resolution of tumor cell profiling, providing an unprecedented perspective in characterizing intra-tumoral heterogeneity and understanding tumor progression and metastasis. However, prominent algorithms for constructing tumor phylogeny based on scDNA-seq data usually only take single nucleotide variations (SNVs) as markers, failing to consider the effect caused by copy number alterations (CNAs). Here, we propose BiTSC$^2$, Bayesian inference of Tumor clonal Tree by joint analysis of Single-Cell SNV and CNA data. BiTSC$^2$ takes raw reads from scDNA-seq as input, accounts for the overlapping of CNA and SNV, models allelic dropout rate, sequencing errors and missing rate, as well as assigns single cells into subclones. By applying Markov Chain Monte Carlo sampling, BiTSC$^2$ can simultaneously estimate the subclonal scCNA and scSNV genotype matrices, subclonal assignments and tumor subclonal evolutionary tree. In comparison with existing methods on synthetic and real tumor data, BiTSC$^2$ shows high accuracy in genotype recovery, subclonal assignment and tree reconstruction. BiTSC$^2$ also performs robustly in dealing with scDNA-seq data with low sequencing depth and variant missing rate. BiTSC$^2$ software is available at https://github.com/ucasdp/BiTSC2.
Subject(s)
Neoplasms , Algorithms , Bayes Theorem , DNA Copy Number Variations , Humans , Neoplasms/genetics , Sequence Analysis, DNA , SoftwareABSTRACT
BACKGROUND: The tumor microenvironment is profoundly heterogeneous particularly when comparing sites of metastases. Establishing the extent of this heterogeneity may provide guidance on how best to design lipid-based drug delivery systems to treat metastatic disease. Building on our previous research, the current study employs a murine model of metastatic cancer to explore the distribution of ~ 100 nm liposomes. METHODS: Female NCr nude mice were inoculated with a fluorescently labeled, Her2/neu-positive, trastuzumab-resistant breast cancer cell line, JIMT-1mkate, either in the mammary fat pad to create an orthotopic tumor (OT), or via intracardiac injection (IC) to establish tumors throughout the body. Animals were dosed with fluorescent and radio-labeled liposomes. In vivo and ex vivo fluorescent imaging was used to track liposome distribution over a period of 48 h. Liposome distribution in orthotopic tumors was compared to sites of tumor growth that arose following IC injection. RESULTS: A significant amount of inter-vessel heterogeneity for DiR distribution was observed, with most tumor blood vessels showing little to no presence of the DiR-labelled liposomes. Further, there was limited extravascular distribution of DiR liposomes in the perivascular regions around DiR-positive vessels. While all OT tumors contained at least some DiR-positive vessels, many metastases had very little or none. Despite the apparent limited distribution of liposomes within metastases, two liposomal drug formulations, Irinophore C and Doxil, showed similar efficacy for both the OT and IC JIMT-1mkate models. CONCLUSION: These findings suggest that liposomal formulations achieve therapeutic benefits through mechanisms that extend beyond the enhanced permeability and retention effect.
Subject(s)
Antineoplastic Agents , Liposomes , Mice, Nude , Neoplasm Metastasis , Animals , Cell Line, Tumor , Female , Antineoplastic Agents/pharmacology , Antineoplastic Agents/therapeutic use , Antineoplastic Agents/pharmacokinetics , Antineoplastic Agents/administration & dosage , Humans , Treatment Outcome , MiceABSTRACT
Gastric cancers are highly heterogeneous malignant tumors. To reveal the relationship between differentiation status of cancer cells and tumor immune microenvironments in gastric cancer, single-cell RNA-sequencing was performed on normal mucosa tissue, differentiated gastric cancer (DGC) tissue, poorly differentiated gastric cancer (PDGC) tissue and neuroendocrine carcinoma (NEC) tissue sampled from surgically resected gastric cancer specimens. We identified the signature genes for both DGC and PDGC, and found that signature genes of PDGC strongly enriched in the epithelial-mesenchymal transition (EMT) program. Furthermore, we found that DGC tends to be immune-rich type whereas PDGC tends to be immune-poor type defined according to the density of tumor-infiltrating CD8+ T cells. Additionally, interferon alpha and gamma responding genes were specifically expressed in the immune-rich malignant cells compared with immune-poor malignant cells. Through analyzing the mixed adenoneuroendocrine carcinoma, we identified intermediate state malignant cells during the trans-differentiation process from DGC to NEC, which showed double-negative expressions of both DGC marker genes and NEC marker genes. Interferon-related pathways were gradually downregulated along the DGC to NEC trans-differentiation path, which was accompanied by reduced CD8+ cytotoxic T-cell infiltration. In summary, molecular features of both malignant cells and immune microenvironment cells of DGC, PDGC and NEC were systematically revealed, which may partially explain the strong tumor heterogeneities of gastric cancer. Especially along the DGC to NEC trans-differentiation path, immune-evasion was gradually enhanced with the decreasing activities of interferon pathway responses in malignant cells.
Subject(s)
Stomach Neoplasms , Humans , Stomach Neoplasms/pathology , CD8-Positive T-Lymphocytes/metabolism , Single-Cell Gene Expression Analysis , Cell Differentiation/genetics , Interferons/genetics , Tumor Microenvironment/geneticsABSTRACT
Cancer is an evolutionary disease. Intra-tumor heterogeneity (ITH), which describes the diversity within individual tumors, sets the foundation for evolution. The fitness of tumor cells is determined by their microenvironment, which exerts intense selection pressure that generally favors cells with survival and proliferation advantages. It has been revealed that host immunity dramatically influences the evolutionary trajectory of cancer. As technologies advance, a refined map of the immune system's involvement in cancer evolution has gradually come to our knowledge. Here we specifically view cancer through the lens of evolutionary immunological biology. We will cover the neoplastic evolution under immunosurveillance, including how the host immunity shapes the tumor evolutionary trajectory and how progressive tumors modulate the host immunity to survive. A comprehensive understanding of the interplay between cancer evolution and cancer immunity provides clues to combating cancer strategically.
Subject(s)
Neoplasms , Humans , Neoplasms/genetics , Tumor Microenvironment/geneticsABSTRACT
Tumors are genetically heterogeneous and many mutations are actually present in subclonal populations. The clonal status of mutations is valuable for accurate prognosis, clinical management. The aim of this study was to identify the clonal status of somatic mutations and systematically evaluate their prognostic values across various cancer types. We totally identified 227 clonal and 432 subclonal mutations contributed to prognosis and demonstrated the importance of clonal status in improving mutation-related clinical guidance. We further developed a customized multi-step approach to identify gene-specific prognostic patterns of clonal status at pan-cancer level and found some cancer-specific prognostic patterns. The 'subclonal-dependent risk' subpattern was one of the most common subpatterns, it usually accompanied by high genomic in-stability and high extent of intra-tumor heterogeneity and could be used to improve the accuracy of prognostic analysis. Our results revealed the importance of clonal status, especially subclonal mutation in clinical survival.
Subject(s)
Neoplasms , Clonal Evolution , Genomics , Humans , Mutation , Neoplasms/genetics , Neoplasms/pathology , PrognosisABSTRACT
Gingivobuccal oral squamous cell carcinoma (OSCC-GB) occurs among persons who excessively chew smokeless tobacco in India. To understand the role of cancer stem cells (CSCs) in the disease, we have performed transcriptomics analysis on RNA-seq data from OSCC-GB primary tumors. The mutational signature analysis of the identified novel and Catalogue of Somatic Mutations in Cancer (COSMIC) variants reveals DNA damage associated etiology based on identified COSMIC signatures showing a higher prevalence of C > T mutations and 1 bp T/(A) nucleotide insertions, pointing to the role of smokeless tobacco carcinogens. The differential somatic mutational, functional impact predictions, and survival analysis reveals the role of DNA damage response-related genes, with the CREBBP gene as a major player. The new CSC somatic variants identified in the study may play a crucial role in cancer metastasis, local-regional recurrence, chemo- and/or radioresistance that contributes to high mortality of the Indian OSCC-GB patients.
Subject(s)
Carcinoma, Squamous Cell , Head and Neck Neoplasms , Mouth Neoplasms , Carcinoma, Squamous Cell/genetics , Carcinoma, Squamous Cell/pathology , DNA Damage , Humans , Mouth Neoplasms/genetics , Mouth Neoplasms/pathology , Mutation , Squamous Cell Carcinoma of Head and Neck/geneticsABSTRACT
Objective: To analyze poly-guanine (poly-G) genotypes and construct the phylogenetic tree of colorectal cancer (CRC) and provide an efficient and convenient method for the study of intra-tumor heterogeneity and tumor metastasis pathway. Methods: The clinicopathological information of patients with primary colorectal cancer resection with regional lymph node metastases were retrospectively collected in the Department of General Surgery, General Hospital of Tianjin Medical University from January 2017 to December 2017. The paraffin sections of the paired tumor samples were performed consecutively, and multi-region microdissection was performed after histogene staining. The phenol-chloroform extraction and ethanol precipitation scheme was used to obtain DNA, and Poly-G multiplex PCR amplification and capillary electrophoresis detection were performed. The correlation between Poly-G mutation frequency and clinicopathological parameters was analyzed. Based on the difference of Poly-G genotypes between paired samples, the distance matrix was calculated, and the phylogenetic tree was constructed to clarify the tumor metastasis pathway. Results: A total of 237 paired samples were collected from 20 patients including 134 primary lesions, 66 lymph node metastases, 37 normal tissues, and Poly-G mutation was detected in 20 patients (100%). The mutation frequency of Poly-G in low and undifferentiated patients was (74.10±23.11)%, higher than that in high and medium differentiated patients [(31.36±12.04)%, P<0.001]. In microsatellite instability patients, the mutation frequency of Poly-G was (68.19±24.80)%, which was higher than that in microsatellite stable patients [(32.40±14.90)%, P=0.003]. The Poly-G mutation frequency was not correlated with age, gender, and pathological staging (all P>0.05). Based on Poly-G genotype difference of the paired samples, the phylogenetic trees of 20 patients were constructed, showing the evolution process of the tumor, especially the subclonal origins of lymph node metastasis. Conclusion: Poly-G mutations accumulate in the occurrence and development of CRC, and can be used as genetic markers to generate reliable maps of intratumor heterogeneity in large numbers of patients with minimal time and cost expenditure.
Subject(s)
Colorectal Neoplasms , Poly G , Humans , Lymphatic Metastasis , Retrospective Studies , Phylogeny , Mutation , Colorectal Neoplasms/genetics , Colorectal Neoplasms/pathology , Biomarkers, Tumor/geneticsABSTRACT
The cancer stem cell (CSC) concept emerged from the recognition of inherent tumor heterogeneity and suggests that within a given tumor, in analogy to normal tissues, there exists a cellular hierarchy composed of a minority of more primitive cells with enhanced longevity (ie, CSCs) that give rise to shorter-lived, more differentiated cells (ie, cancer bulk populations), which on their own are not capable of tumor perpetuation. CSCs can be responsible for cancer therapeutic resistance to conventional, targeted, and immunotherapeutic treatment modalities, and for cancer progression through CSC-intrinsic molecular mechanisms. The existence of CSCs in colorectal cancer (CRC) was first established through demonstration of enhanced clonogenicity and tumor-forming capacity of this cell subset in human-to-mouse tumor xenotransplantation experiments and subsequently confirmed through lineage-tracing studies in mice. Surface markers for CRC CSC identification and their prospective isolation are now established. Therefore, the application of single-cell omics technologies to CSC characterization, including whole-genome sequencing, RNA sequencing, and epigenetic analyses, opens unprecedented opportunities to discover novel targetable molecular pathways and hence to develop novel strategies for CRC eradication. We review recent advances in this field and discuss the potential implications of next-generation CSC analyses for currently approved and experimental targeted CRC therapies.
Subject(s)
Colorectal Neoplasms/drug therapy , Colorectal Neoplasms/metabolism , Computational Biology , Neoplastic Stem Cells , Animals , Antineoplastic Agents, Immunological/therapeutic use , Carcinogenesis , Colorectal Neoplasms/genetics , Colorectal Neoplasms/pathology , Computational Biology/methods , Drug Resistance, Neoplasm , Genomics , Humans , Immunotherapy , Molecular Targeted Therapy , Single-Cell AnalysisABSTRACT
BRCA1-associated protein-1 (BAP1) expression is commonly lost in several tumors including malignant pleural mesothelioma (MPM). Presence or absence of immunohistochemical BAP1 nuclear staining in tumor cells is currently used for differential diagnosis of MPM. In this study, a large cohort of 596 MPM tumors with available clinical data was analyzed to examine associations of BAP1 staining pattern with clinical and molecular features that may reflect the impact of BAP1 mutation on MPM biology. Cases were classified according to the BAP1 staining pattern of tumor cells. Exome and RNA-sequencing data were available for subsets of cases. Levels of mRNA encoding claudin 15 (CLDN15) and vimentin (VIM) were determined using RT-qPCR on 483 cases to estimate the relative proportions of epithelial-like and mesenchymal-like components in each tumor. Four BAP1 staining patterns were observed: single-pattern nuclear staining (36%), single-pattern cytoplasmic staining (25%), single-pattern absent staining (12%), and combinations of these staining patterns (27%). This study confirmed prior reports that nuclear BAP1 is more frequently associated with wild-type BAP1 and sarcomatoid histology. However, no associations between BAP1 staining pattern(s) and mutations in specific protein domains and/or mutation type were observed. BAP1 staining patterns were significantly associated (p < 0.001) with BAP1 gene expression, MPM histologic subtypes, molecular clusters, and markers of epithelial-to-mesenchymal transition. Frequent observation of combinations of BAP1 staining patterns in MPM tumors indicated intra-tumoral heterogeneity of BAP1 status. Cytoplasmic BAP1 staining was identified as a putative indicator of favorable prognosis in non-epithelioid MPM. In conclusion, novel significant associations among different BAP1 staining patterns and subgroups of MPM tumors were observed, suggesting that the role of BAP1 in tumor progression may be more complex than its presumed tumor suppressor function. Cytoplasmic staining was identified as a putative indicator of favorable prognosis in non-epithelioid MPM, potentially addressing a critical need in clinical decision-making in this disease. © 2020 The Authors. The Journal of Pathology published by John Wiley & Sons, Ltd. on behalf of The Pathological Society of Great Britain and Ireland.
Subject(s)
Biomarkers, Tumor/analysis , Mesothelioma, Malignant/chemistry , Pleural Neoplasms/chemistry , Tumor Suppressor Proteins/analysis , Ubiquitin Thiolesterase/analysis , Adolescent , Adult , Aged , Aged, 80 and over , Biomarkers, Tumor/genetics , Cell Nucleus/chemistry , DNA Mutational Analysis , Epithelial-Mesenchymal Transition , Female , Humans , Immunohistochemistry , Male , Mesothelioma, Malignant/genetics , Mesothelioma, Malignant/pathology , Mesothelioma, Malignant/therapy , Middle Aged , Mutation , Pleural Neoplasms/genetics , Pleural Neoplasms/pathology , Pleural Neoplasms/therapy , Prognosis , Tumor Suppressor Proteins/genetics , Ubiquitin Thiolesterase/genetics , Young AdultABSTRACT
Single-cell sequencing technologies are revolutionizing cancer research and are poised to become the standard for translational cancer studies. Rapidly decreasing costs and increasing throughput and resolution are paving the way for the adoption of single-cell technologies in clinical settings for personalized medicine applications. In this chapter, we review the state of the art of single-cell DNA and RNA sequencing technologies, the computational tools to analyze the data, and their potential application to precision oncology. We also discuss the advantages of single-cell over bulk sequencing for the dissection of intra-tumor heterogeneity and the characterization of subclonal cell populations, the implementation of targeted drug repurposing approaches, and describe advanced methodologies for multi-omics data integration and to assess cell signaling at single-cell resolution.
Subject(s)
Neoplasms , Single-Cell Analysis , Humans , Medical Oncology , Neoplasms/drug therapy , Neoplasms/genetics , Precision Medicine , Sequence Analysis, RNA , Single-Cell Analysis/methodsABSTRACT
BACKGROUND: Intra-tumor heterogeneity (ITH) encompasses cellular differences in tumors and is related to clinical outcomes such as drug resistance. However, little is known about the dynamics of ITH, owing to the lack of time-series analysis at the single-cell level. Mouse models that recapitulate cancer development are useful for controlled serial time sampling. RESULTS: We performed single-cell exome and transcriptome sequencing of 200 cells to investigate how ITH is generated in a mouse colorectal cancer model. In the model, a single normal intestinal cell is grown into organoids that mimic the intestinal crypt structure. Upon RNAi-mediated downregulation of a tumor suppressor gene APC, the transduced organoids were serially transplanted into mice to allow exposure to in vivo microenvironments, which play relevant roles in cancer development. The ITH of the transcriptome increased after the transplantation, while that of the exome decreased. Mutations generated during organoid culture did not greatly change at the bulk-cell level upon the transplantation. The RNA ITH increase was due to the emergence of new transcriptional subpopulations. In contrast to the initial cells expressing mesenchymal-marker genes, new subpopulations repressed these genes after the transplantation. Analyses of colorectal cancer data from The Cancer Genome Atlas revealed a high proportion of metastatic cases in human subjects with expression patterns similar to the new cell subpopulations in mouse. These results suggest that the birth of transcriptional subpopulations may be a key for adaptation to drastic micro-environmental changes when cancer cells have sufficient genetic alterations at later tumor stages. CONCLUSIONS: This study revealed an evolutionary dynamics of single-cell RNA and DNA heterogeneity in tumor progression, giving insights into the mesenchymal-epithelial transformation of tumor cells at metastasis in colorectal cancer.
Subject(s)
Colorectal Neoplasms , Animals , Colorectal Neoplasms/genetics , DNA , Exome/genetics , Genetic Heterogeneity , Mice , RNA , Sequence Analysis, RNA , Tumor MicroenvironmentABSTRACT
Metastatic disease represents the primary cause of breast cancer (BC) mortality, yet it is still one of the most enigmatic processes in the biology of this tumor. Metastatic progression includes distinct phases: invasion, intravasation, hematogenous dissemination, extravasation and seeding at distant sites, micro-metastasis formation and metastatic outgrowth. Whole-genome sequencing analyses of primary BC and metastases revealed that BC metastatization is a non-genetically selected trait, rather the result of transcriptional and metabolic adaptation to the unfavorable microenvironmental conditions which cancer cells are exposed to (e.g., hypoxia, low nutrients, endoplasmic reticulum stress and chemotherapy administration). In this regard, the latest multi-omics analyses unveiled intra-tumor phenotypic heterogeneity, which determines the polyclonal nature of breast tumors and constitutes a challenge for clinicians, correlating with patient poor prognosis. The present work reviews BC classification and epidemiology, focusing on the impact of metastatic disease on patient prognosis and survival, while describing general principles and current in vitro/in vivo models of the BC metastatic cascade. The authors address here both genetic and phenotypic intrinsic heterogeneity of breast tumors, reporting the latest studies that support the role of the latter in metastatic spreading. Finally, the review illustrates the mechanisms underlying adaptive stress responses during BC metastatic progression.
Subject(s)
Breast Neoplasms , Breast Neoplasms/metabolism , Female , Humans , Neoplasm MetastasisABSTRACT
BACKGROUND: Tumors are composed by a number of cancer cell subpopulations (subclones), characterized by a distinguishable set of mutations. This phenomenon, known as intra-tumor heterogeneity (ITH), may be studied using Copy Number Aberrations (CNAs). Nowadays ITH can be assessed at the highest possible resolution using single-cell DNA (scDNA) sequencing technology. Additionally, single-cell CNA (scCNA) profiles from multiple samples of the same tumor can in principle be exploited to study the spatial distribution of subclones within a tumor mass. However, since the technology required to generate large scDNA sequencing datasets is relatively recent, dedicated analytical approaches are still lacking. RESULTS: We present PhyliCS, the first tool which exploits scCNA data from multiple samples from the same tumor to estimate whether the different clones of a tumor are well mixed or spatially separated. Starting from the CNA data produced with third party instruments, it computes a score, the Spatial Heterogeneity score, aimed at distinguishing spatially intermixed cell populations from spatially segregated ones. Additionally, it provides functionalities to facilitate scDNA analysis, such as feature selection and dimensionality reduction methods, visualization tools and a flexible clustering module. CONCLUSIONS: PhyliCS represents a valuable instrument to explore the extent of spatial heterogeneity in multi-regional tumour sampling, exploiting the potential of scCNA data.
Subject(s)
DNA Copy Number Variations , Neoplasms , Cluster Analysis , Genetic Heterogeneity , Humans , Sequence Analysis, DNA , Single-Cell AnalysisABSTRACT
Pancreatic ductal adenocarcinoma (PDAC) is composed of stromal, immune, and cancerous epithelial cells. Transcriptomic analysis of the epithelial compartment allows classification into different phenotypic subtypes as classical and basal-like. However, little is known about the intra-tumor heterogeneity particularly in the epithelial compartment. Growing evidences suggest that this phenotypic segregation is not so precise and different cancerous cell types may coexist in a single tumor. To test this hypothesis, we performed single-cell transcriptomic analyses using combinational barcoding exclusively on epithelial cells from six different classical PDAC patients obtained by Endoscopic Ultrasound (EUS) with Fine Needle Aspiration (FNA). To purify the epithelial compartment, PDAC were grown as biopsy-derived pancreatic cancer organoids. Single-cell transcriptomic analysis allowed the identification of four main cell clusters present in different proportions in all tumors. Remarkably, although all these tumors were classified as classical, one cluster present in all corresponded to a basal-like phenotype. These results reveal an unanticipated high heterogeneity of pancreatic cancers and demonstrate that basal-like cells, which have a highly aggressive phenotype, are more widespread than expected.
Subject(s)
Carcinoma, Pancreatic Ductal/pathology , Organoids/pathology , Pancreatic Neoplasms/pathology , Single-Cell Analysis/methods , Biopsy , Humans , RNA-Seq , Signal Transduction/physiologyABSTRACT
BACKGROUND: A number of simulators have been developed for emulating next-generation sequencing data by incorporating known errors such as base substitutions and indels. However, their practicality may be degraded by functional and runtime limitations. Particularly, the positional and genomic contextual information is not effectively utilized for reliably characterizing base substitution patterns, as well as the positional and contextual difference of Phred quality scores is not fully investigated. Thus, a more effective and efficient bioinformatics tool is sorely required. RESULTS: Here, we introduce a novel tool, SimuSCoP, to reliably emulate complex DNA sequencing data. The base substitution patterns and the statistical behavior of quality scores in Illumina sequencing data are fully explored and integrated into the simulation model for reliably emulating datasets for different applications. In addition, an integrated and easy-to-use pipeline is employed in SimuSCoP to facilitate end-to-end simulation of complex samples, and high runtime efficiency is achieved by implementing the tool to run in multithreading with low memory consumption. These features enable SimuSCoP to gets substantial improvements in reliability, functionality, practicality and runtime efficiency. The tool is comprehensively evaluated in multiple aspects including consistency of profiles, simulation of genomic variations and complex tumor samples, and the results demonstrate the advantages of SimuSCoP over existing tools. CONCLUSIONS: SimuSCoP, a new bioinformatics tool is developed to learn informative profiles from real sequencing data and reliably mimic complex data by introducing various genomic variations. We believe that the presented work will catalyse new development of downstream bioinformatics methods for analyzing sequencing data.