Although genetic mutations are required for cancer development, reversible non-genetic alterations also play a pivotal role in cancer progression. Failure of well-orchestrated gene regulation by chromatin states and master transcription factors can be one such non-genetic etiology for cancer development. Master transcription factor-mediated cellular reprogramming of human cancer cells allows us to model cancer progression. Here I cover the history and recent advances in reprogramming cancer cells, followed by lessons from cellular reprogramming of normal cells that may apply to cancer. Lastly, I share my perspective on cellular reprogramming for studying epigenetic alterations that have occurred in tumorigenesis, discuss the current limitations, and propose ways to overcome the obstacles in the reprogramming of cancer. However, evidence now shows that epigenetic changes—both intrinsic and extrinsic to cells—may drive malignancy, supporting the possibility of cancer reversibility. The first evidence for cancer reversibility was shown in the pioneering work of Dr. Braun with crown-gall tumors of plants  in the 1In this review, I give a concise overview of the past, present, and future of cellular reprogramming to understand and model human cancer. I first summarize the historical evidence for cancer reversibility in mammalian cells by blastocyst injection, cell fusion, and nuclear transplantation experiments. I then briefly describe the basic concept of cellular reprogramming in normal somatic cells and discuss the up-to-date advances on cellular reprogramming of various cancers. I compare similar and distinctive aspects of cancer development and cellular reprogramming, and lastly discuss the prospects of cellular reprogramming for neoplastic disease along with the challenges associated with iPSC-based approaches in cancer.2The altered interplay between genetic and epigenetic networks contributes to tumorigenesis . Yet, inEarly attempts to control the cancerous phenotype in mammals were made in murine teratocarcinoma cells by blastocyst injection in the 1970s . Dr. BriTo test the developmental consequences of genetic variations occurring in malignant carcinoma cells, Dr. Mintz injected single euploid teratocarcinoma cells (derived from the core of embryoid bodies grown as an ascites tumor) into blastocysts bearing many genetic markers ; 44% of While these early chimeric studies indicated that the embryonic environment could maintain control of the aberrant proliferation of teratocarcinoma cells, the studies used euploid teratocarcinoma cells, which are pluripotent, and efficiency was very low. Thus, it was not clear whether somatic cancer of aneuploidy could be reprogrammed. The first experiment of reprogramming of aneuploid tumors was performed in frogs  in the 1In the early 2000s, several studies showed examples of both reprogramming of the cancer genome into pluripotency and predisposition of cloned embryos or animals to malignancy using NT in a subset of murine tumors .+/-Ptc1 mice into enucleated eggs  cells , an embr-/-p53 breast cancer and +/Ink4a/Arf-/-Ras fibroblasts developed more efficiently. Blastocysts derived from one RAS-inducible melanoma cell line (+/Ink4a/Arf-/-Ras) were further able to generate ES cells, which were capable of differentiation into multiple somatic cell types in vivo  Oocyte cytoplasm can, to some extent, reset accumulated epigenetic modifications associated with genetic mutations and cell lineages, and keep malignant phenotypes under control until the blastocyst stage, regardless of cancer type (despite variable cloning efficiency caused by unknown factors); b) During late cell specification, irreversible genetic alterations inherited by the donor nucleus contribute to reactivation of tumorigenic potential within the context of specific cell lineages where mutations can be expressed to oncogenic pathways. These observations suggest that nuclear reprogramming strategies can be harnessed to model cancer progression. However, so far, no human cancers have been successfully reprogrammed by NT.Several studies in the 1970s and 80s also showed malignancy suppression by fusion with healthy somatic cells without reprogramming cancer cells into pluripotency. Yet, normal cells' ability to reverse tumorigenicity in malignant cells depended on gene dosage and cell types . For exa3A defined set of master transcription factors (TFs), namely OCT4, SOX2, KLF4, and MYC , can reprogram somatic cells into induced pluripotent stem cells (iPSCs) and allow the study of dynamic events that occur during cell fate decisions . Since TCancer modeling using iPSC methodology is most advanced for blood cancers, including human  or murin3.1V600EBRAF or NRAS mutations with TetO-inducible OSK  did not show a definitive increase in gene expression . This suES cells . ReprogrIn summary, Zhang et al. showed that partially reprogrammed sarcoma cancer cell lines (pre-iPS) exhibit reduced tumorigenicity, resemble mesenchymal stem cells (MSC), and can differentiate into lineages that were blocked in their parental cell lines . They alTES, and CDKN1C in GBM. To study the functional consequences of resetting GBM-associated DNA methylation defects, Stricker et al. reprogrammed glioblastoma (GBM)-derived neural stem cells (GNS) into iPSCs (“GiPSC”), using piggyback transposon-mediated OCT4 and KLF4  were hypermethylated in cancer . Half  alleles (NF1 (-/-) iPSC clones exhibited, on average, a 10%-15% increase in cell proliferation. The NF1 (-/-) iPSCs were then differentiated into neural crest (NC) lineages from which SCs developmentally originate. NC cells from NF1 (-/-) iPSCs were not noticeably distinct compared to NC cells from NF1 (+/+) iPSCs. NC cells give rise to multiple cell lineages, such as peripheral neurons, melanocytes, as well as SC, from which PNFs develop. NF1 (-/-) iPSC-NC were able to differentiate into peripheral neurons and melanocytes. However, NF1 (-/-) iPSC-NC showed uncontrolled proliferation capacity even after day 14 of differentiation into SC, whereas control NF1 (+/+) iPSC-NC arrested their proliferation. NF1 (-/-) iPSC-NC also exhibited a lack of myelination capacity when co-cultured with dorsal root ganglion neurons in myelination medium .KRAS and TP53 genes, a heterozygous deletion of CDKN2A, homozygous deletions of SMAD4, as well as chromosomal rearrangements of the original tumor epithelial cells. The 10-22 cells acquired pluripotency to some extent, yet preferentially generated PanIN2/3-like ductal lesions after three months in immunocompromised mice. The lesions progressed to the invasive stage by 6-9 months. Thus, cancer iPS-like cells replicated the natural course of human PDAC progression , and have been reviewed in detail elsewhere . Here, I4.1Successful reprogramming requires both erasure of the somatic cell identity and activation of the pluripotency gene program. The first phase of reprogramming involves rapid proliferation , suppres4.2Despite the low efficiency of reprogramming and attempts to replace OSKM factors with alternatives, enforced OSKM expression provides the most robust reprogramming efficiency thus far. What is the role of these reprogramming factors in cellular reprogramming?It is clear that OSKM induction triggers rapid genome-wide changes in chromatin before gene activation, and such chromatin remodeling is dynamic throughout the entire process . For exaDoes ectopic OSKM expression change chromatin structure directly? Various studies showed the causative effect of ectopic OCT4 expression on the reorganization of chromatin structure. One study showed that ectopic OCT4 competed with relatively unstable nucleosomes and subsequently established nucleosome-depleted regions in the regulatory regions of target genes in hypomethylated clones derived from the colon cancer line HCT 116 . AnotherHow does ectopic OSKM expression initiate a new gene program? OCT4, SOX2, and KLF4 are widely known to function as pioneer factors , especially in human fibroblasts . In contAdditionally, OSKM expression has been shown to erase the original cell identity directly or indirectly . For exaIn summary, OSKM induction is accompanied by highly dynamic chromatin remodeling to initiate cellular reprogramming of fibroblasts to iPSCs. These chromatin dynamics translate into distinct phenotypes at the very early and late stages, as well as in reprogramming intermediates that may follow diverse paths through transient states.55.1Kras in mice  displayed teratomas  in reprogrammed cells vs. their parental fibroblast cells (R-DMR) and in cancer cells vs. their matched normal cell types (C-DMR) . HypometNotably, all three factors  were required to reprogram cancer cells and reduce their tumorigenicity in many studies . OverexpIn summary, although initial cellular reprogramming is reminiscent of cancer development, pluripotent stem cells have an epigenetic landscape that is distinct and reciprocal to that of cancer cells. Moreover, several cancer iPSC studies have provided evidence of the rewiring of part of the cancer epigenome as a result of reprogramming. This raises exciting questions: Can cellular reprogramming offer a powerful tool to deconstruct and reconstruct aberrant cancer epigenomes? Does it provide a new way to tackle cancer as well as serve as a cancer progression model?66.1We now know that OSKM can cooperatively repress somatic enhancers, which at least in part, erases the original cellular identity at the beginning of fibroblast reprogramming (see section 3). The OSKM factors can also reorganize chromatin structure before the activation of pluripotency. It remains to be determined if these mechanisms occur during the reprogramming of cancer cells. A growing number of studies show the potential of cellular reprogramming to model cancers. Yet, it remains unclear how the OSKM TFs engage the cancer genome and begin erasing the cancer epigenome to guide the reversion of cellular identity back to a pluripotent state. Common epigenetic factors can differentially elicit tumor formation or reprogramming to pluripotency. Why is it necessary to study early reprogramming events in cancer?First, reprogramming TFs can change the chromatin structure of the original cells with pioneering functions as well as by recruiting other chromatin co-factors. OCT4/SOX2 can redistribute somatic TFs to the transiently accessible regions by OCT4/SOX2  or direcSecond, malignancy can be manifested in cells in which oncogenic pathways can be active. Not all cell types harboring the same mutations can develop cancer. Reprogramming to pluripotency or lineage-transdifferentiation by master TFs provides a way to change cellular identity. Reprogramming has been shown to disrupt oncogenic addiction and result in resistance to therapy. Likewise, transdifferentiation also results in decreased malignancy in a subset of tumors. For example, transdifferentiation of B lymphomas into macrophage-like cells impaired tumorigenicity in xenograft hosts . Reintro6.2The evolution of cancer has not been well characterized in many solid tumors, in part due to the lack of appropriate models. Current models for the study of solid human tumors recapitulate advanced stages of tumors and do not reflect the events that occur early in the disease's progression. Reversible epigenetic alterations can play a crucial role in the progression of cancers. Such epigenetic changes that accumulate in cancer cells can be reset to some extent during reprogramming to pluripotency by NT in murine tumors  or by iPIn embryogenesis, each cell of the blastocyst's inner cell mass must remain in the ground state of pluripotency until implantation . OncogenHowever, upon release from pluripotency, the rewired epigenetic landscapes can be re-established in the presence of the genetic mutations and cellular specifications that originally contributed to the cancer transformation. While NT-reprogrammed cancer cells differentiated into multiple early developmental cell types of the embryo, pediatric tumor-derived embryos died partly through organogenesis . In cont7To answer these provocative questions, we need to overcome a few challenges in the next few years. The biggest obstacles in cancer reprogramming mainly arise from the fact that tumors are highly heterogeneous, yet only a subset of cells are reprogrammed. Genetic mutations could be barriers impeding the reprogramming process. Most of the reprogramming mechanistic studies have been done in normal fibroblasts or blood cells, and the cumulative information from such cell types may not apply to cancer cells.7.1KRAS mutant alleles (unpublished data). Considering that the efficiency of reprogramming is extremely low and the reprogramming process is a stochastic event, it is not feasible to select cancer iPSC clones harboring the given genetic alteration. We were only able to identify one iPS-like cell line harboring PDAC classical mutations after multiple attempts. In the case of blood cancers, this may be less of a challenge as blood cells can easily be separated from stroma, and indeed the most significant advances in the cancer iPSC field have been made in blood cancer. Nonetheless, blood cancer samples still contain non-neoplastic cells, thus it is always crucial to confirm the origin of iPSCs.Solid tumors consist of cancer cells mixed with non-neoplastic cells. When it comes to human PDAC, this is a more severe problem since PDAC epithelial cells are tightly surrounded by stromal cells. We found that 90% of tumor epithelial cells did not harbor KRAS alleles by pyrosequencing and then confirmed the origin of iPSCs by comparative genome hybridization (CGH)  . Today, on (CGH)  also all7.2Intratumoral heterogeneity can give rise to subclones, which may differ in reprogramming efficiency. A stochastic model of reprogramming posits that cells have equal potential to give rise to iPSCs with variable latency at the single-cell level . HoweverSince there is significant variability among patients, generating iPSCs from multiple patients is needed. It is also essential to validate the significant results obtained from cancer iPSCs in more patient samples by other methods. Our study validated a subset of biomarkers generated from a single-iPS-like cell line in multiple patient cohorts . Intertu7.3Both NT and iPSC studies show the low reprogramming efficiency of cancer cells. In contrast to most myeloid leukemia iPSC studies that documented complete removal of somatic identity in iPSCs, solid cancer iPSC studies show persisting epigenetic changes that prevent cells from being fully reprogrammed pluripotency . ParadoxCells with certain mutations are resistant to reprogramming , and rea8Despite the caveats and challenges to overcome, a handful of proof-of-principle cancer reprogramming studies have provided new insights into disease progression, cancer epigenetics, and cancer therapy. By better understanding cellular reprogramming of cancer cells, we may offer new opportunities to model and understand neoplastic diseases.The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.


The adult mammalian heart is incapable of regeneration following cardiac injury, leading to a decline in function and eventually heart failure. One of the most evident barriers limiting cardiac regeneration is the inability of cardiomyocytes to divide. It has recently become clear that the mammalian heart undergoes limited cardiomyocyte self-renewal throughout life and is even capable of modest regeneration early after birth. These exciting findings have awakened the goal to promote cardiomyogenesis of the human heart to repair cardiac injury or treat heart failure. We are still far from understanding why adult mammalian cardiomyocytes possess only a limited capacity to proliferate. Identifying the key regulators may help to progress towards such revolutionary therapy. Specific noncoding RNAs control cardiomyocyte division, including well explored microRNAs and more recently emerged long noncoding RNAs. Elucidating their function and molecular mechanisms during cardiomyogenesis is a prerequisite to advance towards therapeutic options for cardiac regeneration. In this review, we present an overview of the molecular basis of cardiac regeneration and describe current evidence implicating microRNAs and long noncoding RNAs in this process. Current limitations and future opportunities regarding how these regulatory mechanisms can be harnessed to study myocardial regeneration will be addressed. The heart is one of the most vital organs of the human body but at the same time forms one of the least adaptive organs in terms of regenerative capacity. The human adult myocardium, like that of most mammalians, is incapable of adequate regeneration following cardiac injury. This failure to repair severe cardiomyocyte loss is a leading cause of heart failure and death worldwide . One of An important group of regulators are the noncoding RNAs (ncRNAs). These molecules are involved in the regulation of practically all biological processes, particularly during development and disease . DiffereCardiac regenerative potential has been compared among vertebrate species with the aim to find a molecular explanation for the limitations in human cardiomyogenesis. In the following paragraphs, we describe the regenerative capacity of the newt, zebrafish, mouse, and human heart and describe similarities and differences between them.Lower vertebrates such as the newt ,12 and zIn contrast to lower vertebrates, mammalian species such as mice have a limited capacity for cardiomyocyte division. Studies using multi-isotope imaging mass spectrometry to study cardiomyocyte turnover during normal ageing show that cardiomyocyte renewal occurs at a slow annual rate of 0.76% in mice . ImportaAlkass and colleagues  monitoreHuman cardiomyocyte renewal is proven to persist throughout life albeit at a low rate. Bergmann et al.  reportedEmbryonic mammalian cardiomyocytes are able to divide and share physiological conditions comparable to lower vertebrates . Fish heSurprisingly, despite the discrepancies in myocardial regenerative capacity between lower vertebrates and mammals, the cellular mechanisms controlling cardiomyocyte division seem to be evolutionary conserved. Cell cycle regulators, growth factors, and signaling through the Meis homeobox 1 (Meis1)-, Hippo/Yes-associated protein (YAP)-, switch/sucrose nonfermentable (SWI/SNF)-, neuregulin 1 (NRG1)-, and p38-pathway are proven to exert a fundamental role in cardiomyogenesis during postnatal development ,48,49. FOverall, comparative analyses of cardiac repair and regeneration in different animals provide important insights into the evolutionary conservation of key processes driving heart regeneration. Although the molecular regulators of cardiomyocyte division remain poorly understood, the similarity in cardiac regeneration capability between lower vertebrates and neonatal mammals is promising as it suggests that humans are able to overcome severe cardiac injury. Increased understanding of the regulators defining the decline in cardiomyocyte proliferation in mammals is required. Non-coding RNA (ncRNA) genes such as tRNA and rRNA genes have long been recognized to play vital roles in the cell, but ncRNA with unknown functionality were initially considered ‘junk DNA’ upon their first identification in 1972 . NowadayMiRNAs are a class of evolutionary conserved small (18–24 nucleotides) ncRNAs, and up to date 2654 microRNAs have been annotated in humans . MiRNAs Intergenic lncRNAs—located between two protein-coding genes. The majority of lncRNAs are included within this classification.Intronic lncRNAs—located within introns of protein-coding genes.Bidirectional lncRNAs—transcribed within 1 kb of promoters in the opposite direction from the protein-coding transcript.Enhancer lncRNAs—located within close proximity (<2 kb) and transcribed from enhancer regions of the genome.Sense lncRNAs—transcribed from the sense strand of protein-coding genes and can overlap introns and part or all of the exon.Antisense lncRNAs—transcribed from the antisense strand of protein-coding genes and can overlap an exon of the coding gene in the sense strand, an intron, or both.Long noncoding RNAs (lncRNA) are genes that do not code for proteins and are defined by an arbitrary criterion of length (>200 nucleotides) . AccordiAlthough the mechanisms how lncRNAs exert their functions are not yet fully elucidated, a striking difference between miRNAs and lncRNAs is that the latter can either repress or activate gene expression. LncRNAs are mostly localized in the nucleus, where they regulate gene expression at the epigenetic level. LncRNAs make use of very diverse mechanisms to control gene expression, which can be categorized into ,75:Signal lncRNAs—which can regulate gene expression in a time- and space-dependent manner. Decoy lncRNAs—which can titrate transcription factors and other proteins away from chromatin or titrate miRNAs out from their target . Guide lncRNAs—which can recruit chromatin modifying enzymes to target genes, either in cis (near the site of lncRNA production) or in trans to distant target genes. Scaffold lncRNAs—which can facilitate the assembly of multiple proteins to form ribonucleoprotein (RNP) complexes, affecting histone modifications on chromatin. Specific microRNAs (miRNAs) and long noncoding RNAs (lncRNAs) have been reported to engage in cardiac regeneration and cardiomyogenesis. Here we review current knowledge concerning the regulatory function of miRNAs and lncRNAs during cardiac regeneration through control of cardiomyogenesis. Understanding and utilizing these master regulators may form the solution to overcome the limited cardiomyogenesis potential of the adult mammalian heart.MiRNAs modulate numerous signaling pathways and cellular processes, including cardiac regeneration and cardiomyogenesis. Eulalio et al.  performeProfiling studies during postnatal cardiac development identified upregulation of multiple members of the miR-15 family when comparing the onset of cardiomyocyte cell cycle arrest and binucleation  with highly proliferative state  . These fAn additional miR-15 family member identified to be induced during postnatal development and contributing to cardiomyocyte cell cycle arrest is miR-128. In neonatal mice, cardiac-specific overexpression of miR-128 impaired cardiomyocyte proliferation and cardiac function, while miR-128 deletion extended proliferation of postnatal cardiomyocytes . FurtherMiRNA genes can be located within close genomic proximity, so-called miRNA clusters. Evolutionary highly conserved miRNAs are more frequently organized in clusters . It is pAmong the most abundantly expressed miRNAs in the myocardium is the highly conserved miR-1/miR-133 cluster, which has been shown to play a fundamental role in cardiomyogenesis and cardiac development by different groups. Both miRNAs exhibit cardiac- and skeletal muscle-enriched expression ,82. MiR-Similarly, miR-133 negatively regulates cardiomyocyte proliferation by inhibiting Cyclin D2 and Serum Response Factor . ExpressThe miR-302-367 cluster has been shown to be essential for cardiomyocyte proliferation and cardiac repair in mice. Expression of the miR-302-367 cluster is present in the embryonic mouse heart and diminishes during maturation . Re-exprThe miR-17-92 is among the best-studied miRNA clusters and was initially identified as a human oncogene that promotes cell proliferation of different types of tumors through phosphatase and tensin homolog (PTEN) targeting . DeletioAguirre et al.  identifiIn addition to the well-studied miRNA clusters and families, multiple other miRNAs have been reported to contribute to cardiac repair through regulating cardiomyogenesis, including miR-222 , miR-31aIn addition to the well-studied miRNA clusters and families, multiple other miRNAs have been reported to contribute to cardiac repair through regulating cardiomyogenesis, including miR-222 , miR-31aTaken together, evidence accumulating over the past two decades marks the crucial regulatory role of various miRNA clusters and families in cardiac regeneration through controlling cardiomyogenesis . MiRNA-bLncRNAs in general are considered to be expressed at a relatively low level . HoweverOunzain et al.  characteWithin the human genome, a crucial cell cycle-modulating genomic locus spans approximately 42 kbs on human chromosome 9p21. This region contains three important tumor suppressors: p15, p16, and p14 ,106. AntKlattenhoff and colleagues  identifiGrote et al.  identifiThe expression of lncRNA H19 is regulated during development and implicated in cancer . H19 harThe murine Delta-like homolog 1 (Dlk1)- type III iodothyronine deiodinase (Dio3) ncRNA locus is a mega-cluster (~1.000 kb) consisting of 3 protein coding genes, 61 miRNAs, and 3 lncRNAs ,117. TheThus, over the past few years an increasing number of profiling studies identified lncRNAs to be cardiac-enriched and/or differentially expressed during myocardial development or disease . AlthougUnraveling the cellular process of cardiomyocyte proliferation and the regulatory networks involving ncRNAs has yielded promising candidates to trigger cardiac regeneration and repair. Although huge progress has been made, major challenges have to be overcome in order to increase our understanding and to ultimately utilize this knowledge to boost repair of the adult mammalian heart.First and most importantly, cardiomyocyte division in the adult mammalian heart is an extremely rare event. A tremendous number of cardiomyocytes are lost during MI. Repairing the heart by generating a sufficient number of cardiomyocytes to compensate for the tissue loss and to preserve function is challenging. To date, ncRNA-based therapeutic approaches that are successful in promoting cardiomyogenesis are predominantly performed in small animal models, with only few studies making use of large animals ,132. WheSecond, measuring true cardiomyocyte division remains a technical challenge because of biological and technical factors. Cellular count of cardiomyocytes isolated via tissue dissociation are often subject to inconsistent results and selection bias due to variation in digestion efficiency. Similarly, whole heart nuclear fraction isolation followed by detection of cardiomyocyte specific nuclei using PCM-1 has been performed . HoweverMonitoring of various cell cycle activity markers in cardiomyocytes is therefore more commonly used as indicator of proliferation. Determination of these indirect surrogate markers implicated in proliferation can be done in a ‘static’ or ‘dynamic’ approach. The latter is applied when detecting cardiomyocyte DNA synthesis over time by nucleotide analog (EdU or BrdU) incorporation in cultured cardiomyocytes or in vivo. Static evaluation of cardiomyocyte proliferation markers is often performed by quantification of the percentage of cardiomyocytes positive for the cell cycle activity marker Ki67, the mitotic marker phosphohistone H3, or Aurora kinase B, which is expressed during binucleation and division. Although these markers are active during all or specific cell cycle phases, their expression does not necessarily indicate definitive cardiomyocyte division. Therefore, Milliron et al.  very recLarge scale identification of ncRNAs associated with cardiomyogenesis improved significantly with the advancements in genome-wide and RNA deep-sequencing techniques. Various groups have performed miRNA and/or lncRNA profiling of models associated with increased cardiomyogenesis, including the postnatal switch from proliferation to terminal differentiation ,7,8. AltGenome-wide association studies (GWAS) are used to identify genomic regions associated with a disease phenotype by scanning along the genome for known single-nucleotide polymorphisms. Indeed, GWAS have associated a plethora of SNPs among the ~20000–21000 protein-coding genes with cardiac disease ,140. SNPAnother hinderance to overcome when embarking on finding therapeutic strategies for cardiac regeneration is to efficiently modulate ncRNA function, in a time-, dose-, and cell type-specific manner. Following identification of a cardiomyogenesis-associated ncRNA, gain- and loss-of-function studies need to determine its functional involvement. Transgenic animal models can be generated to cell- and/or time-specifically modulate the expression of a ncRNA in an all-or-nothing approach, but this can be costly and time-consuming. Sophisticated tools to better control ncRNA function have been well established for miRNAs, although the therapeutic options for lncRNAs are more limited due to their greater length . NcRNA kThe recent advancements in AAV-mediated gene therapy allow for prolonged cardiomyocyte-enriched delivery of transcripts (with a maximum length of ~4.7kb) at a specific moment during development or disease . GeFinally, once ncRNAs associated with cardiac regeneration have been identified, the complex network of ncRNAs and their interaction partners has to be elucidated. A commonly used tool to study interaction between miRNAs and their targets is a luciferase assay. This assay investigates the interaction between miRNAs and their targets, but often only (part of) the target gene’s 3’UTR is incorporated. As a result, the assay may suffer from a lack of preservation of the tertiary structure of the studied RNA molecules. Moreover, luciferase assays performed in non-cardiomyocyte cells may not offer a representative intracellular transcriptome and therefore fail to identify relevant transcripts ,154. RNA and DNA immunoprecipitation assays give a more biologically relevant representation of interactions between miRNAs, lncRNAs, mRNAs, and proteins. Recent developments in immunoprecipitation methods using Argonaute 2—the main interacting protein that provides miRNAs their inhibitory function- enables largescale identification of miRNA-mRNA networks in a cardiac-relevant in vitro  and in vRegeneration of the adult mammalian heart is limited, but recent findings have marked its capacity to recover from severe injury early after birth, and to renew cardiomyocytes throughout life. Monitoring the regenerative capacity of the heart along evolution has taught us that several many underlying cellular mechanisms may be shared between lower vertebrates and neonatal mammals. This holds promise as it suggests that cardiomyogenesis-regulating ncRNAs identified in animal models may act in a similar manner in humans.Accumulating evidence has made it clear that both short and long ncRNAs are important orchestrators of gene regulatory networks in this process. A remarkable amount of evolutionary conserved miRNA clusters and families have been proven to control cardiomyocyte division, including the miRNA cluster of miR-99/100 and Let-7 families. Similarly, an increasing number of studies demonstrate that cardiomyocyte proliferation is under epigenetic control of the more recently emerged class of lncRNAs, such as ANRIL. MiRNA research kicked off more than two decades ago and is therefore several steps ahead in comparison with the field of other classes of ncRNAs. This may explain the superior body of knowledge of miRNAs in cardiac regeneration over lncRNAs. At the same time, this also provides hope for future developments since significant advancements have been achieved by studying 2654 miRNAs, while the 96,308 lncRNA genes forms an immense, unexplored source to explain the limitations of cardiac regeneration. Recent advancements in sequencing and gene therapy technologies improved largescale identification and precise in vivo modulation of ncRNAs to further elucidate the regulatory networks involved in cell cycle arrest and activation. Understanding and employing these critical regulators of cardiomyocyte proliferation, especially the miRNAs and lncRNAs, will aid our search to remove the breaks on myocardial regeneration.


Hepadnaviviridae) are reverse-transcribing animal viruses that infect vertebrates. DNA sequences derived from ancient hepadnaviruses have been identified in the germline genome of numerous vertebrate species, and these ‘endogenous hepatitis B viruses’ (eHBVs) reveal aspects of the long-term coevolutionary relationship between hepadnaviruses and their vertebrate hosts. Here, we use a novel, data-oriented approach to recover and analyse the complete repertoire of eHBV elements in published animal genomes. We show that germline incorporation of hepadnaviruses is exclusive to a single vertebrate group (Sauria) and that the eHBVs contained in saurian genomes represent a far greater diversity of hepadnaviruses than previously recognized. Through in-depth characterization of eHBV elements, we establish the existence of four distinct subgroups within the genus Avihepadnavirus and trace their evolution through the Cenozoic Era. Furthermore, we provide a completely new perspective on hepadnavirus evolution by showing that the metahepadnaviruses (genus Metahepadnavirus) originated >300 million years ago in the Paleozoic Era and have historically infected a broad range of vertebrates. We also show that eHBVs have been intra-genomically amplified in some saurian lineages, and that eHBVs located at approximately equivalent genomic loci have been acquired in entirely distinct germline integration events. These findings indicate that selective forces have favoured the accumulation of hepadnaviral sequences at specific loci in the saurian germline. Our investigation provides a range of new insights into the long-term evolutionary history of reverse-transcribing DNA viruses and shows that germline incorporation of hepadnaviruses has played a role in shaping the evolution of saurian genomes.Hepadnaviruses (family  Hepadnaviridae) are reverse-transcribing DNA viruses that infect vertebrates. The type species—hepatitis B virus (HBV)—is estimated to infect ∼300 million people worldwide, causing substantial morbidity and mortality. Hepadnaviruses have enveloped, spherical virions and a small, circular DNA genome ∼3 kilobases (kb) in length. The genome is characterized by a highly streamlined organization incorporating extensive gene overlap—the open reading frame (ORF) encoding the viral polymerase (P) protein occupies most of the genome and typically overlaps at least one of the ORFs encoding the core (C), and surface (S) proteins.Hepadnaviruses , which infect amphibians and reptiles, as well as two highly distinct groups that infect fish—the metahepadnaviruses (genus Metahepadnavirus) and the parahepadnaviruses (genus Parahepadnavirus)  . Unexpecnavirus) .Whole genome sequencing has revealed the presence of DNA sequences derived from hepadnaviruses in some vertebrate genomes. These ‘endogenous hepatitis B viruses’ (eHBVs) are thought to have originated via ‘germline incorporation’ events in which hepadnavirus DNA sequences were integrated into chromosomal DNA of germline cells and subsequently inherited as novel host alleles. Most eHBV sequences that arise in this way will be quickly purged from the gene pool via drift and natural selection. Occasionally, however, some may persist long enough to become genetically fixed in the germline of ancestral species. Fixed eHBVs are expected to remain in the germline indefinitely unless removed by macrodeletion, but in the absence of selective pressure their sequences will gradually degrade via neutral mutation.Hepadnaviridae. eHBV sequences are in some ways equivalent to hepadnavirus ‘fossils’ in that they provide a source of retrospective information about the distant ancestors of modern hepadnaviruses. Before ancient eHBV sequences provided a means of calibrating the timeline of hepadnavirus evolution, the family was thought to have originated within the past 100,000 years. However, the discovery of ancient eHBV sequences exhibiting remarkable similarity to contemporary strains demonstrates that hepadnaviruses infected vertebrate ancestors millions of years ago, during the Mesozoic and Cenozoic Eras . Using GLUE, we implemented an automated process for constructing alignments of putatively orthologous sequences. These alignments were subsequently used to derive consensus sequences representing each set of orthologs. Consensus sequences were incorporated into multiple sequence alignments (MSAs) along with hepadnavirus genome sequence. We defined a set of ‘master’ reference sequences, each of which represents a distinct clade in the hepadnavirus phylogeny. We used these sequences to create a ‘constrained alignment tree’—a data structure, implemented in GLUE , that liWe used the database-integrated genome screening (DIGS) tool  to derivVia DIGS, we generated a database of genomic sequences disclosing similarity to hepadnaviruses. We extended the core schema of this database to incorporate additional tables representing the taxonomic classifications of viruses, eHBVs and host species included in our study. We used structured query language (SQL) to interrogate the database, filtering sequences based on their similarity to reference sequences, the taxonomic properties of the closest related reference sequence, and the taxonomic distribution of related sequences across hosts. Using this approach, we categorized sequences into: (1) putatively novel eHBV elements; (2) orthologs of previously characterized eHBVs (e.g. copies containing large indels); (3) non-viral sequences that cross-matched to hepadnavirus probes (e.g. retrotransposons). Sequences that did not match to previously reported eHBVs were further investigated by incorporating them into our genus-level, genome-length MSA along with all of our reference taxa and reconstructing maximum likelihood phylogenies using RAxML (version 8) .Where phylogenetic analysis supported the existence of a novel eHBV insertion, we also attempted to: (1) determine its genomic location relative to annotated genes in reference genomes; and (2) identify and align eHBV-host genome junctions and pre-integration insertion sites (see below). Where these investigations revealed new information (e.g. by confirming the presence of a previously uncharacterized eHBV insertion), we updated our reference library accordingly. This in turn allowed us to reclassify all of the putative eHBV loci in our database and group sequences more accurately into categories. By iterating this procedure, we progressively resolved the majority of eHBV sequences identified in our screen into groups of orthologous sequences derived from the same initial germline incorporation event . eHBV elhttps://www.genome.jp/tools/motif/) against motif libraries Pfam , we investigated the locus surrounding each putatively novel eHBV. We extracted DNA sequences flanking eHBV insertions and used BLASTn to identify the homologous insertion sites in species listed in the ENSEMBL genome browser. To assess potential presence of transposable elements (TE) around eHBVs of interest , we extrThe minimum ages of eHBV insertions reported in this study were inferred by identifying the most distantly related pair of host species sharing a particular eHBV and applying the estimated species divergence dates and confidence intervals (CI) as reported in TimeTree . A plot bona fide eHBV elements are only present in the genomes of saurian species. Furthermore, reconstruction of the phylogenetic relationships between eHBVs and contemporary hepadnaviruses revealed that saurian genomes contain a broader diversity of eHBV elements than previously recognized, with elements derived metahepadnaviruses being present, as well as elements derived from the Avihepadnavirus and Herpetohepadnavirus genera  for endogenous hepadnaviral elements (eHBVs). Via screening we identified 930  sequences genera . We estiVaranus komodoensis). Herpeto.6-Varanus was found to cluster robustly with skink hepatitis B virus (SkHBV)  .Paroedura pictus)—and the tuatara (Sphenodon punctatus). By contrast, herpetohepadnavirus-derived elements were only identified in reptiles, and avihepadnavirus-derived elements were only detected in birds.Notably, metahepadnavirus-like eHBV elements were identified in a wide range of saurian species, including birds, turtles, a lizard—the ocelot gecko . Notably, however, clade IV and clade II share a conserved, synapomorphic character: the insertion of a valine (V) or isoleucine (I) residue in the P protein, between positions 203 and 204 (Phylogenetic reconstructions demonstrate the presence of at least four distinct clades (I–IV) within the us genus . Clade Iunctions ), but wiunctions . The ord and 204 . This sh and 204 , Fig. 1bAvi.29-Psittacula) and in Anna’s hummingbird  as well as two distinct elements in songbirds (order Passeriformes). Among the two songbird elements, one was found only in warblers (Avi.37-Phylloscopus) while another (Avi.37-Passeriformes) was found in five distinct families within the superfamily Passeroidea, establishing that it integrated into the passeroid germline >38 Mya (CI: 16–43 Mya). We also identified clade I-derived elements in parrots that represent only fragments of a hepadnavirus genome. These elements, which appear to have been intra-genomically amplified (discussed below) and include some elements that are orthologous across all parrots (order Psittaciformes), indicate that germline incorporation occurred >49 Mya (CI: 29–71 Mya) prior to the divergence of the kea (Nestor notabilis) from other parrot lineages (Near-complete insertions derived from clade I were identified in rose-necked parakeets (lineages .Gavia stellata), and barn owls . The insertion in ducks was incorporated >30 Mya (CI 26–35 Mya), prior to the divergence of mallards (Anas platyrhynchos) and ruddy ducks (Oxyura jamaicensis). Notably, multiple, genome-length eHBV elements derived from this lineage were often identified in the same species or species group. For example, multiple, clade II-derived eHBVs were identified in both the Tyto (Avi.11 and Avi.22) and Gavia (Avi.14 and Avi15) germlines. However, in-depth analysis of these sequences shows that each derives from distinct germline incorporation events. Not only are they located in entirely distinct genomic loci (Clade II contains sequences derived from ducks (family Anatidae), red-throated divers , and in clade Australiaves, represented by the red-legged seriema (Cariama cristata). This broad distribution is consistent with the demonstrably ancient origins of this lineage.Clade III includes the ‘AHBV-FRY  and eZHBAHBV-FRY ), which Alauda arvensis) genome that shows a higher level of relatedness to modern hepadnaviruses than does any previously reported eHBV (eHBV-Avi.28-Alauda was the only avihepadnavirus-derived eHBV element found to exhibit similarity to modern avihepadnaviruses in the variable region of the genome (Buceros rhinoceros), within clade IV  sequences . This lineage, named dnavirus , and is equences , suggestequences ), or (2)Phylogenies indicate that the initial germline incorporation event that gave rise to this multi-copy eHBV lineage predates the diversification of the four cormorant species in which it was identified, as evidenced by the presence of multiple, multi-species sub-clusters in phylogenies see  and the We also identified apparently intra-genomically amplified, avihepadnavirus-derived eHBV elements in the genomes of parrots. In this case, the amplified elements appear to derive from an ancient clade I avihepadnavirus. Although the elevated eHBV copy number found in certain avian orders reflects intra-genomic amplification, it is nonetheless clear that the rate of germline incorporation is significantly higher in birds than in any other vertebrate group. We characterized eHBV loci in saurian genomes by identifying the nearest annotated genes upstream and downstream of EVE integration sites . ExcludiAvihepadnavirus genus. We also identified one case in which an avihepadnavirus-derived element found in the avian germline  is located at the same approximate genomic position as a herpetohepadnavirus-derived element (Herpeto.7) in snake genomes  in length. However, a group of orthologous, metahepadnavirus-derived eHBV elements identified in birds contained some copies that spanned a near-complete genome . Further∼282 Mya . Given t∼282 Mya , inserti∼282 Mya . Since D∼282 Mya , the equin ovo and this may be the case in other avian species —particularly as they relate to vertical transmission. Studies of avihepadnavirus infections in domestic ducks show that virus is normally transmitted via vertical transmission  species . ConceivAvihepadnavirus genus, each of which has a relatively broad distribution among avian species. So far, all exogenous virus species have only been identified in one of these clades. However, all appear to have circulated throughout a large part, if not most of the Cenozoic Era. However, due to lack of genome coverage across avian species, we were only able to obtain an approximate timeline of evolution for each of the four avihepadnaviral lineages. Conceivably, the existence of the four clades might reflect the historical compartmentalization of avian subpopulations (e.g. due to geographic isolation) during certain periods of their evolution. Currently, we do not have a sufficient level of precision to infer any association between the ancestral distribution of avihepadnavirus strains and the evolutionary history of specific bird lineages. However, the upcoming publication of data from the avian 10K genomes project  indicates that it was inserted into the germline an estimated 280 Mya (CI: 273–286 Mya) . Recent debate has focused on the relationship between short and long-term substitution rate estimates in virus genomes (eHBV-Meta.1-Sauria) provides an approximate estimate of 2.59 × 10−9 substitutions per site per year for the long-term rate of substitution rate in conserved regions of the pol gene. This matches closely with recently published estimates of long-term substitution rates inferred for polymerases and other highly conserved genes in several virus families, including hepadnaviruses  . This ma genomes . Dividin genomes  by 300 Maviruses .Metahepadnavirus genus groups as a relatively close sister taxon to the mammalian orthohepadnaviruses in phylogenies (Hepadnaviridae diverge into distinct ‘Meta-Ortho’ and ‘Herpeto-Avi’ lineages prior to the divergence of fish and tetrapods and then subsequently co-diverged (broadly speaking) with their host groups  activity—and a large number of these insertions have been fixed. We have previously reported a similar phenomenon for endogenous circoviral elements in carnivore genomes , and it Consistent with the idea that germline incorporation of hepadnavirus sequences might, in some cases, be favoured by selection at the level of the host, we identified multiple examples of loci containing multiple fixed eHBV elements, each derived from a distinct germline colonization event . In prinad hoc way that makes it difficult for other investigators to recapitulate or build on the work done by previous investigators. In this study, we sought to directly address these challenges by using a novel data-orientated approach. This allowed us to publish our findings in the form of an online resource that not only contains all of the data items associated with our investigation , but also represents the semantic relationships between these data items. Furthermore, via the GLUE engine (a platform-independent software environment)  , it provveab012_Supplementary_DataClick here for additional data file.


Stress cardiomyopathy (Takotsubo cardiomyopathy) is very rare in the central nervous system (CNS) demyelinating disorders. Although this dysfunction of the heart-brain axis has been reported in several case series related to multiple sclerosis (MS), stress cardiomyopathy by neuromyelitis optica (NMO), which is rarer CNS demyelinating disorder than MS, is extremely rare. Herein, we report a case of stress cardiomyopathy associated with a medullary lesion as a presentation of NMO.A 30-year-old woman was treated by veno-arterial extracorporeal membrane oxygenation due to catastrophic cardiopulmonary dysfunction after prolonged and unexplained nausea, vomiting, and cough. Myoclonus on the limbs developed afterward. Taken with suspicion of area postrema syndrome (APS), the brain MRI showed a demyelinating lesion in the medulla oblongata. APS and severe heart failure by stress cardiomyopathy were completely resolved by ECMO and hydrocortisone therapy. However, the CNS demyelinating lesion recurred after 1 month. The patient was diagnosed with NMO evident by the presence of aquaporin-4 antibody; Steroid therapy improved her symptoms.NMO should be considered as one of the differential diagnoses in patients with APS preceding severe cardiopulmonary distress. A rare but relevant association between stress cardiomyopathy (Takotsubo cardiomyopathy) and multiple sclerosis (MS) has been reported in several case series –4. NeuroA 30-year-old woman without underlying disease suffered from unexplained nausea, vomiting, and cough without fever. After 1 month, she was admitted to the emergency department with suddenly deteriorated dyspnea and myoclonic jerks on all limbs. At admission, the chest X-ray showed multifocal patchy ground-glass opacity in the bilateral lungs. ST elevation of the inferior leads on the electrocardiography and increased cardiac markers  were observed. The echocardiography showed severe left ventricular (LV) systolic dysfunction  and regional wall motion abnormalities  image , ranging from the medulla to the thoracic spinal cord Fig. a,b,c. AqStress cardiomyopathy is an acute cardiac condition caused by intense emotional or physical stress leading to severe but reversible cardiac dysfunction . The casAlthough a catastrophic cardiopulmonary event like stress cardiomyopathy is very rare in CNS demyelinating diseases, several case series associated with MS have been reported –4. One cAlthough the pathogenesis of stress cardiomyopathy remains unclear, the predisposition of neurologic or psychiatric disorders was found very frequently in around 85% of stress cardiomyopathy . The comAPS is a condition with intractable nausea, vomiting, or hiccups associated with area postrema in the dorsal medulla. In this case, APS, as her initial presenting symptom, and the subsequent myoclonus of the limbs suggested a medullary lesion. APS is one of the core clinical characteristics for NMO and reported in up to 30% of the NMO patients during their illness. Most APS attacks precede inflammatory involvement of the optic nerves or spinal cord, making APS an important warning sign . UnexplaCatastrophic stress cardiomyopathy is very rare in NMO, but NMO should be considered as one of the differential diagnoses in patients with APS and a cardiopulmonary event.


Brain is one of the most common target organ of lung cancer metastasis, while descriptions of intraventricular carcinomatosis could hardly be found among previous cases. To date no cases from lung adenocarcinoma have been reported in the literature.We report here a case of multiple intraventricular metastases from lung adenocarcinoma with EGFR G719X mutation. This 64-year-old woman was referred to our hospital with complaints of dizziness and vomiting. Target therapy with afatinib was initiated and the lesions in both lung and brain achieved good partial responses.This case report revealed a phenomenon of rare intraventricular metastasis from lung cancer, which should be carefully distinguished from primary ventricular tumors. Compared to brain parenchyma metastasis, intraventricular lesions would cause more severe symptoms which may be rapidly progressive. Target therapy could become a potential option in such patients with non-drugresistant EGFR mutations. Up to 50% of pulmonary carcinoma patients would develop brain metastases throughout their clinical courses, and 10–25% of them have brain metastases at the time of initial diagnosis . AdenocaA sixty-four-year-old Chinese woman, with a ten-year history of diabetes and hypertension, presented with serious dizziness and vomiting in a month.Positron emission tomography (PET) performed in October 2017 indicated a nodular lesion in left upper lung with diffuse bilateral lung metastases . Afatinib, a second generation Epidermal Growth Factor Receptor- tyrosine kinase inhibitor (EGFR-TKI) was administered at a dose of 30 mg qd due to EGFR mutation in exon 18 (G719X). The symptoms of the patient improved significantly within one month and most of the daily activities were affordable. Reevaluation after three-month treatment showed partial remission in both lung and brain lesions Figs. b. RepeatIntraventricular neoplasms account for less than 1% of intracranial neoplasms , which wIt has been widely recognized that brain metastases frequently occur in the early course of lung cancer, especially in small cell lung cancer (SCLC) . Among aThe lateral ventricles are the most common sites of intraventricular metastases due to the abundant vascular supply of choroid plexus . It is dAfatinib, a second generation EGFR-TKI, was approved by the U.S. FDA for metastatic NSCLC with uncommon non-drugresistant EGFR mutations, such as L861Q, G719X, and S768I. Afatinib can achieve half maximal inhibitory concentration (IC50) against EGFR mutation in the cerebrospinal fluid  so that The described case adds pulmonary adenocarcinoma to the possible origin of an intraventricular cerebral metastasis. Furthermore, whether the patient has brain metastases or not, afatinib may be a potential option for patient with uncommon mutations of EGFR.


A 24-year-old pregnant female presented to the emergency department with lower abdominal cramping and vaginal bleeding. A point-of-care ultrasound demonstrated a calcified yolk sac.When identified, calcification of the yolk sac in the first trimester is a sign of fetal demise. It is important for an emergency physician to be aware of the various signs and findings on point-of-care ultrasound and be familiar with the management of these pathologies. A 24-year-old female presented to the emergency department (ED) with three days of lower abdominal cramping and vaginal spotting. Physical examination demonstrated suprapubic tenderness, scant blood in the vaginal vault, and closed cervix. The urine pregnancy test was positive. An ED point-of-care, pelvic ultrasound identified a fetal pole with an adjacent echogenic yolk sac . Fetal cCalcification of the yolk sac is likely due to dystrophic calcification, in which dead tissue undergoing necrosis accumulates calcium.4Calcified yolk sacs are usually indicative of a loss of fetal cardiac activity before 12 weeks of gestation. Greater than 80% of miscarriages occur in the first trimester, and calcification of the yolk sac may be seen at that time. However, the prevalence is currently unknown. Risk factors are the same as those that are commonly associated with miscarriage, including previous miscarriage, fetal chromosomal abnormalities, advanced maternal age, and diabetes. Signs and symptoms include abdominal pain or cramps and vaginal bleeding. Physical findings include abdominal tenderness and blood in the vaginal vault with an open or closed cervix. Treatment is expectant management and symptomatic therapy. Outpatient obstetrical follow-up with ultrasonography at 7–14 days to assess the pregnancy for viability is generally appropriate. A calcified yolk sac does not confer any higher risk of future miscarriage or infertility, similar to other first-trimester spontaneous abortions.5CPC-EM CapsuleWhat do we already know about this clinical entity?A calcified yolk sac is a sonographic sign associated with fetal demise and inevitable abortion.What is the major impact of the image(s)?This image can help physicians distinguish fetal demise from other signs of early pregnancy.How might this improve emergency medicine practice?Identification of a calcified yolk sac can help manage patient expectations and allow the emergency medicine clinician to counsel the patient, and arrange appropriate follow up.VideoTransvaginal sonogram demonstrating a fetal pole (solid arrowhead) with an adjacent echogenic yolk sac (hollow arrow).


Actigraphy has become a popular, non-invasive means of continuously monitoring physical activity and sleep. One optional setting, the low frequency extension (LFE) filter, reduces the movement threshold to capture low acceleration activity that is common in older adults. This filter significantly alters physical activity outcomes , but it is unclear if this has implications for sleep interval calculations that rely upon accurate differentiation between physical activity and sleep. We investigated the effects of the LFE filter on wrist-worn sleep estimates in older adults. Participants were 9 older adults who wore the ActiGraph GT9X on their non-dominant wrist for 7 days in a free-living environment. Raw data was processed with and without the LFE filter enabled, and sleep intervals were calculated by a proprietary ActiGraph algorithm. Paired samples t-tests demonstrated that the LFE filter generated significantly later bedtimes, fewer minutes spent in bed, shorter sleep duration, and fewer awakenings during the night compared to when the filter was disabled . Use of the LFE filter did not lead to differences in arise time, sleep latency, efficiency, or wake after sleep onset . While the LFE filter was designed to improve accuracy of physical activity estimates in more sedentary populations, these findings suggest that the LFE filter also has the potential to impact sleep estimates of older adults. Researchers using ActiGraph-calculated sleep would benefit from careful consideration of this software-dependent impact.


Cancer immunotherapy has shown remarkable progress in recent years. Nanocarriers, such as liposomes, have favorable advantages with the potential to further improve cancer immunotherapy and even stronger immune responses by improving cell type-specific delivery and enhancing drug efficacy. Liposomes can offer solutions to common problems faced by several cancer immunotherapies, including the following: (1) Vaccination: Liposomes can improve the delivery of antigens and other stimulatory molecules to antigen-presenting cells or T cells; (2) Tumor normalization: Liposomes can deliver drugs selectively to the tumor microenvironment to overcome the immune-suppressive state; (3) Rewiring of tumor signaling: Liposomes can be used for the delivery of specific drugs to specific cell types to correct or modulate pathways to facilitate better anti-tumor immune responses; (4) Combinational therapy: Liposomes are ideal vehicles for the simultaneous delivery of drugs to be combined with other therapies, including chemotherapy, radiotherapy, and phototherapy. In this review, different liposomal systems specifically developed for immunomodulation in cancer are summarized and discussed. Cancer immunotherapy has been widely explored because of its durable and robust effects . Tumors The generation of clinically effective antitumor responses normally requires the successful execution of several immune processes . FirstlyNext, productive T cell responses are generated in lymphoid organs . During Once activated, effector T cells must migrate to the tumor site and infiltrate the TME to perform their killing job. Here, negative regulatory signals that dampen T cell activation or induce anergy and exhaustion must be avoided as much as possible . TypicalImmunotherapy refers to the approach to treat cancer through generating or regulating an immune response against it . Recentl®).The increasing research on the applicability of nanotechnology in cancer therapy is based on its unique hallmarks from the fields of drug delivery, diagnosis, and imaging . NanocarLiposomes are lipid-based nanoparticles with high potential to improve cancer immunotherapies, since they can incorporate and/or associate a high variety of cancer drug molecules  ,31. LipoLiposomes, in particular polyethylene glycol (PEG)ylated liposomes, tend to passively accumulate in tumor tissue via the enhanced permeability and retention (EPR) effect ,35,36. CIn spite of improved biodistribution and tumor accumulation, there are also certain issues that need to be addressed to get the maximum benefit from current liposomal platforms. As the complexity of liposomes increases, so do the expenses and difficulties associated with their preparation and quality control. The physicochemical properties of liposomes, including their size, charge, polarity, and any modifications, may also have a negative impact on the ability of the liposomes to reach the tumor via the EPR effect ,46. For It has been established that liposomes with immunostimulatory properties can induce potent immune responses . GeneralHence, the efficient and targeted delivery of these stimulatory molecules to the cells of interest is one of the important keys for successful cancer immunotherapy. Compared to injecting “free” drugs directly, liposomes can prevent cargos from degrading in the surrounding biological environment, improve their biodistribution, and promote their delivery to target cells. There are two well-known targets to improve immunotherapy through liposomes .The efficient activation and maturation of DCs is a prerequisite to induce proper anti-tumor immune responses. The most critical elements to achieve sufficient efficacy are immunopotentiators (enhance immune responses) and delivery systems (minimize the toxicity and enhance the efficacy). Liposomes can efficiently protect cargoes from rapid degradation and co-deliver antigens with immune adjuvants to induce powerful immune responses . In addiCpG ODN has been widely reported to enhance immune responses . Various280-288 peptide and MPLA induced significantly higher uptake by DCs and CD8 T cell responses compared to the co-administration of “free” MPLA [MPLA is another type of effective immune adjuvant that can be encapsulated in liposomes, which is among the first generation of Toll-like receptors agonists approved for human application . Mechaniee” MPLA . In thisIn addition to their delivery capability, liposomes themselves can act as adjuvants to stimulate immune responses intrinsically ,78,79,80Unlike peptides or protein, nucleic acids are inherently immunogenic, and there is a lesser need to use adjuvant simultaneously. The propensity of liposomes to accumulate in reticuloendothelial organs such as the liver and spleen, where antigen-presenting cells are abundant, can be exploited for the efficient delivery of antigen-coding RNA. Once foreign RNA is introduced into cells, it can be recognized by pathogen recognition receptors , which further incites the production of type I interferon to induce potent immune responses ,92. Fusogenic liposomes are regarded as ideal candidates for RNA delivery vehicles because delivery via fusogenic liposomes is one of the early proposed solutions to the problem of endocytic sequestration and the subsequent lysosomal degradation of RNA . SynthetRNA lipoplexes also represent a promising delivery system for DC targeting. They can protect RNA from extracellular ribonucleases and enable the systemic delivery to lymphoid tissues to induce the selective expression of their RNA cargo in resident APCs. Salomon et al. reported RNA lipoplexes encoding CD4 T cell-recognizable neoantigens and established potent adaptive T cell responses by boosting in situ CD8 T cell immunity . WhereasTernary complexes comprising of cationic liposomes, cationic polymers, DNA or RNA lipopolyplexes were studied thoroughly by Huang and collaborators ,102. LipIntensifying the effector phase of immune responses by the direct activation of T cells or by using genetically engineered T cells is also a promising strategy to improve immunotherapy . DCs canIn the last decade, harnessing the power of the immune system against cancer has become an increasingly effective therapeutic option that can result in potent and durable responses in multiple cancer types. However, the activated tumor specific T cells do not often correlate with tumor regression in patients, which goes in accordance with the discovery and characterization of immune escape mechanisms ,114. ImmDuring early T cell activation, CTLA-4 is expressed and then competitively inhibits CD28 binding to CD80 and CD86 on DCs, thus leading to decreased T cell survival and expansion ,125,126.The PD-1/PD-L1 pathway impairs T cell responses and induces T cell anergy, exhaustion, or apoptosis upon engagement. PD-1 is expressed on a large proportion of tumor-infiltrating lymphocytes in many different tumor types, and increased PD-1 expression is linked to tolerance to avoid auto-immunity ,132. PD-64Cu) and doxorubicin to simultaneously treat and track therapy progression in a breast cancer model [To assist anti-PD-1 therapy, various effective approaches that employed liposomes have been developed. Lang et al. reported an effective liposomal system that incorporated the PD-1 inhibitor HY19991 and thioridazine into the double-layer structure of liposomes . The liper model . The accPD-L1, expressed on the surface of APCs and malignant cancer and tumor-associated cells, can be upregulated to a high level in various cell types by proinflammatory cytokines, particularly IFN-γ . Merino There are still several other immune checkpoints under exploration as potential therapeutic targets, such as the inhibitory receptors lymphocyte activation gene 3 protein , T cell In addition to cell interaction that contributes to a suppressive microenvironment, there are some soluble mediators with complex mechanisms that can also inhibit anti-tumor immunity. Genetic alterations in tumors facilitate their growth and invasion into the surrounding tissue and also orchestrate the persistence of chronic inflammatory mediators . These mThe inhibition of IDO presents a promising approach to relieve the immunosuppressive state of the TME. In the TME, IDO is generated by cancer cells, tumor-associated macrophages, myeloid-derived suppressor cells, and others. IDO catalyzes the degradation of tryptophan and production of tryptophan metabolites that limits T cell function . A recen2A. A2A is a receptor that is expressed on the surface of CD8 tumor-infiltrating T cells, myeloid-derived suppressor cells, and natural killer (NK) cells to regulate immune response during (chronic) inflammation. Emerging studies have shown that blocking the adenosine-A2A pathway through pharmacologic inhibition or genetic silencing could significantly reverse the immune suppression in the TME by improving the function of T cells and NK cells in multiple tumor models [2A pathway antagonists, and it cannot be applied widely in the clinic due to its poor pharmacokinetic profile and hydrophobic nature [2A blockade. However, switching off adenosine–A2A pathway directly can also induce undesired effects such as increased tissue inflammation and damage [Adenosine is also an important ribonucleoside and metabolite regulating immune function that can be detected in the TME. The hypoxia in the TME can induce the upregulation of extracellular adenosine signals through the adenosine receptor Ar models . SCH famc nature . Liposomc nature . In thisd damage . An alted damage . Allard d damage . Based oThe intervention in the TGF-β pathway provides a good opportunity to augment the anti-tumor efficacy using liposomes. In a pancreatic ductal adenocarcinoma model, Meng et al. developed PEGylated liposomes with TGF-β inhibitors and observed a decrease in pericyte coverage of the tumor vasculature, which allowed higher access of liposomes to the tumor site . As a plMotivated by such findings, several small molecule inhibitors targeting TGF-β ligands or receptors have started undergoing clinical trials . HoweverIncreasing evidence indicate that immunotherapies are effective in multiple cancer types, and therefore, the combination of immunotherapy with other therapies may be beneficial in a broad range of tumors. Liposomes integrated with different therapies and loaded with multiple drugs can not only facilitate anti-tumor effects but also modulate the tumor immune environment efficiently compared to monotherapy only. The choice of therapeutic agents and timing of these combinations is critical, because different agents normally show different mechanisms and target sites. Combinatorial therapy can be a double-edged sword, as it can elicit potent anti-tumor effects and also increase the risk of systemic toxicity. In this regard, liposomes show great potential to overcome the aforementioned limitations, as they can simultaneously deliver agents with different physicochemical properties and mitigate adverse effects. A good liposome-based delivery system needs to meet the following requirements: (1) co-load different molecules in sufficient concentrations; (2) overcome biologic barriers without losing its bioactivity; (3) release cargos at the desire site and time; (4) have the ability to target specific tumor or cell type; (5) exhibit synergistic or additive effects; and (6) must utilize economic, efficient, and safe preparation methods.Immunochemotherapy might be one of the typical strategies with intriguing results that indicate that chemotherapy can enhance the therapeutic outcome of immunotherapy and further reverse chemoresistance ,169,170.Sonodynamic therapy involves the combination of low-intensity ultrasound with sonosensitizers for high tissue-penetrating capability to generate reactive oxygen species to induce cell death and subsequent immune responses against the released TAAs ,177. YuePhototherapy  can also be enhanced with rationally designed liposomes for the targeted delivery of photosensitizers and by a process similar to that of sonodynamic therapy. It produces reactive oxygen species to destroy cancer cells and stimulate the immune system through the release of TAAs ,180,181.Shi et al. devised a liposome-based immune stimulation strategy to immune regulate the TME and to significantly augment tumor growth inhibition when combined with photodynamic therapy . Kim et Recently, several studies have reported that the combination of nanocarriers with photothermal therapy and immunotherapy could generate higher antitumor immunological effects than without nanocarriers ,188,189.Many great achievements of liposome-based drug delivery systems for cancer immunotherapy in recent years have been reported, and there is little doubt that liposomes provide a promising platform to improve cancer treatments via different immunotherapeutic mechanisms. However, further optimization of liposomes is required tuned to their specific purposes. It is crucial to design liposomes to precisely target the specific sites in the TME and to reduce off-target effects, which lead to poor outcomes that can be more unpredictable than traditional therapies. Areas for further improvement are (1) improving the pharmacokinetics of liposomes to reduce biodistribution to achieve the least possible toxicity, and (2) adjusting the liposomes according to the purpose of the specific immunomodulator and its target. In summary, liposomes exhibit distinctive advantages for cancer immunotherapy, such as high safety, efficient delivery for multiple drugs, as well as inducing immune activation by themselves; therefore, they are likely to play an important role in future immunotherapeutic cancer strategies.


TP53, MYC and PTEN are enriched in hypoxic tumours, and mutations in PTEN interact with hypoxia to direct tumour evolutionary trajectories. Overall, hypoxia plays a critical role in shaping the genomic and evolutionary landscapes of cancer.Many primary tumours have low levels of molecular oxygen (hypoxia), and hypoxic tumours respond poorly to therapy. Pan-cancer molecular hallmarks of tumour hypoxia remain poorly understood, with limited comprehension of its associations with specific mutational processes, non-coding driver genes and evolutionary features. Here, as part of the ICGC/TCGA Pan-Cancer Analysis of Whole Genomes (PCAWG) Consortium, which aggregated whole genome sequencing data from 2658 cancers across 38 tumour types, we quantify hypoxia in 1188 tumours spanning 27 cancer types. Elevated hypoxia associates with increased mutational load across cancer types, irrespective of underlying mutational class. The proportion of mutations attributed to several mutational signatures of unknown aetiology directly associates with the level of hypoxia, suggesting underlying mutational processes for these signatures. At the gene level, driver mutations in  Many tumours exhibit hypoxia (low oxygen) and hypoxic tumours often respond poorly to therapy. Here, the authors quantify hypoxia in 1188 tumours from 27 cancer types, showing elevated hypoxia links to increased mutational load, directing evolutionary trajectories. Sub-regions of hypoxia can result from disrupted oxygen supply: irregular and disorganized tumour vasculature can reduce oxygen availability5. Hypoxia can also be caused by changes in oxygen demand: altered tumour metabolism7 can increase intra-cellular demand for oxygen, potentially extending hypoxia signalling to liquid tumours. The adaptation of tumour cells to this imbalance in oxygen supply and demand is associated with poor clinical prognosis in several cancer types, attributed at least in part to hypoxia-associated genomic instability and clonal selection16.Approximately half of all solid tumours are characterized by low levels of molecular oxygen (hypoxia)TP53 mutations and for those that are apoptosis-deficient18. Indeed mutations in TP53 occur at a higher frequency in hypoxic primary tumours of at least 9 types16. The abundance of proteins involved in homologous recombination (e.g. RAD51) and non-homologous end joining (e.g. Ku70) are reduced under hypoxia, and these changes can persist for 2 days after reoxygenation21. Genes central to efficient mismatch repair (e.g. MLH1 and MSH2) are also downregulated under hypoxia23. Further, co-presence of tumour hypoxia and high genomic instability15, specific cellular morphologies like intraductal and cribriform carcinoma24 or specific mutations like loss of PTEN16, synergistically predict for rapid relapse after definitive local therapy in some tumour types, particularly prostate cancer. These data underscore the relationship between hypoxia and DNA repair defects, and suggest the tumour microenvironment applies a selective pressure leading to the development of specific genomic profiles.Previous work has provided insight into the molecular origins and consequences of tumour hypoxia and genomic instability. Dynamic cycling of hypoxia can select for cells with 16. However, the influence of tumour hypoxia on pan-cancer driver alterations, mutational signatures, and subclonal architectures remains unclear. To fill this gap, we calculated tumour hypoxia scores for 1188 tumours with whole-genome sequencing (WGS) and RNA sequencing, spanning 27 cancer types. Genome sequencing data was aggregated by the Pan-Cancer Analysis of Whole Genomes (PCAWG) consortium and generated by the ICGC and TCGA projects. These sequencing data were re-analyzed with standardized, high-accuracy pipelines to align to the human genome (reference build hs37d5) and identify germline variants and somatic mutations, as described previously25. This sequencing data together with our high-quality hypoxia quantitation represents a powerful hypothesis-generating mechanism to suggest useful back-translational in vitro experiments and better define the hypoxia-associated mutator phenotype across cancers. We associated hypoxia with key driver alterations in coding and non-coding regions of the genome, and find hypoxia is associated with specific mutational signatures of unknown aetiology. We illustrate the joint impact of PTEN and the tumour microenvironment in influencing the evolutionary trajectory of tumours. Overall, these data highlight the genomic changes through which hypoxia drives aggressive cancers.We previously evaluated the exomic and copy-number alteration (CNA) consequences of tumour hypoxia across 19 cancer types25 and RNA-sequencing26 analyses were systematically carried out by centralized teams with consistent and high-accuracy bioinformatics pipelines. Normal reference samples had a mean WGS coverage of 39 reads per base-pair while coverage for tumour samples had a bimodal distribution with modes at 38 and 60 reads per base-pair25. All samples underwent an extensive and systematic quality assurance process25.We compiled a cohort of 1188 tumours from 27 cancer types via the PCAWG Consortium. All samples had matched tumour and reference normal WGS and tumour RNA sequencing data generated by the ICGC and TCGA projects. WGS28. Cancer type was further incorporated as a random effect in every model, allowing us to consider a different baseline value for the feature of interest for each cancer type. As a measure of effect size we report conditional R2 values, denoted as 29. We also report marginal R2 values, denoted as 29.We used linear mixed-effect models to associate hypoxia with features of interest across cancers while adjusting for tumour purity, age, and sex30, Winter31 and Ragnum32 , mature B-cell lymphomas , lung adenocarcinomas  and breast adenocarcinomas . This was in contrast to chronic lymphocytic leukaemias  and prostate adenocarcinomas  where little inter-tumoural variability in hypoxia was observed. The variability in hypoxia score was not significantly associated with the median hypoxia score within cancer types , an engineered feature that is a surrogate for genomic instability and is associated with hypoxia across several tumour types16 . Overall, hypoxia is associated with increased numbers of most types of somatic mutations.We then considered the association of hypoxia scores with 14 other metrics of the mutation density of CNAs, structural variants (SVs) and single nucleotide variants (SNVs) using linear mixed-effect models Fig. , 3. The 25, with CNA, SV and SNV data available for 1096 patients. In cases where a patient had multiple mutations in the same gene (e.g. a CNA and an SNV) we denoted these as compound events. We again used linear-mixed effect models to associate hypoxia with each driver feature across cancers  signatures tested, nine showed differential activity in hypoxic tumours compared to non-hypoxic ones, while controlling for cancer type, tumour purity, age and sex  signatures illustrated a similar story. Of the 17 ID signatures analyzed, the activity of 5 were associated with tumour hypoxia scores while controlling for cancer type, tumour purity, age and sex . This supports previous in vitro work that demonstrated the contextual synthetic lethality of PARP inhibition in cells with defective DNA repair due to hypoxia43. While we have not specifically included in vitro experimental validation data in this report, we and others have previously validated associations first revealed by analysis of mRNA-based hypoxia signatures. For example, our group previously described microRNA-133a-3p as a hypoxia-associated miRNA in prostate cancer based on mRNA signature-based associations across multiple independent datasets16. We went on to validate that microRNA-133a-3p was indeed induced under hypoxia in multiple prostate cancer cell lines and confirmed its capacity to modulate cell proliferation and invasion. Similarly, Ye et al. applied hypoxia signatures to 10 independent datasets of cell lines and primary tumour fragments under hypoxia and normoxia44. These 10 datasets represented seven cancer types and within each dataset samples under hypoxia showed higher hypoxia scores compared to normoxic samples. Further, they generated predictions of drugs that would be more or less potent under hypoxia and validated four drug–hypoxia interactions in vitro. These data illustrate that hypoxia signatures applied to large cohorts of primary tumours can generate reliable hypotheses, many of which have been validated in controlled systems. However, some aerobic cancer cells may also mimic the biological state of hypoxia  and this may affect signature-derived hypoxia estimates. Further, while pimonidazole (which was used to develop the Ragnum hypoxia signature32) reflects oxygen tensions below 10 mmHg (1.3% O2), it is difficult to directly relate hypoxia signature scores with oxygen tension45. Overall, hypoxia signalling can be distinct from microenvironmental hypoxia and this remains a critical caveat of this study.Our study cannot conclusively say whether hypoxia exerts a selective pressure that enriches for specific genomic alterations or if these genomic changes directly result in hypoxia. Experimental studies of single genes support that both effects may contribute to the associations we describeDiving into the mutational processes related to hypoxia, we confirmed that several SBS and small indel signatures related to impaired DNA repair were associated with hypoxia. This raises the potential confounder that because hypoxic tumours have more mutations, we have more power to detect related mutational signatures. However, we demonstrated that hypoxia is indeed strongly associated with many mutational signatures with unknown aetiology, particularly SBS5, which is found in nearly all cancer types. Modelling these associations in vitro is particularly difficult and these data provide a high confidence measure of the mutational signatures that may be directly or indirectly driven by tumour oxygen levels. It is difficult to disentangle the timing of these events: whether a specific driver mutation gives rise to a specific mutational signature or if these are separate processes. Better mapping of the evolutionary timing of hypoxia will be particularly important in addressing this question and the advent of hypoxia signatures may facilitate future studies in this area.16, and other models that link hypoxia to genomic instability and downstream clonal selection42. Previous work has also demonstrated that patients with allelic loss of PTEN and elevated hypoxia rapidly relapse after definitive treatment for localized prostate cancer16. Here, we showed that tumours with alterations in PTEN and elevated hypoxia are enriched for a polyclonal tumour architecture. This illustrates the joint influence of the tumour mutational landscape and microenvironment in guiding evolutionary trajectories across cancers. Further, these data suggest that increased subclonal diversification may be a novel route via which PTEN drives aggressive tumour phenotypes, in concert with tumour hypoxia, and this can be better defined with future back-translational in vitro experiments. The PCAWG dataset is the largest publicly available pan-cancer dataset to date and this limits our ability to validate our discoveries in independent datasets. Ultimately, it will be necessary to validate our findings in large, independent cohorts. Hypotheses generated here, particularly those around hypoxia and tumour evolution, will require long term, systematic in vitro modelling and will be the subject of future studies. Overall, this work shows that a hypoxic tumour microenvironment is associated with specific mutational processes and distinct somatic mutational profiles, and may direct the subclonal architecture of cancers.We observed a significant association between elevated hypoxia and the number of clonal mutations. This supports the idea that hypoxia is an early event in cancer, as we have suggested previously31, Buffa et al. 30 and Ragnum et al. 32, as described previously16  using mRNA-abundance-based signatures of tumour hypoxia developed previously by Winter et al.16 TCGA data between comparable cancer groups. The groups compared are as follows (PCAWG cancer type and TCGA cancer type): Bladder-TCC and BLCA; Breast-AdenoCA and BRCA; Cervix-SCC and CESC; CNS-GBM and GBM; ColoRect-AdenoCA and COADREAD; Head-SCC and HNSC; Kidney-RCC and KIRC; Liver-HCC and LIHC; Lung-AdenoCA and LUAD; Lung-SCC and LUSC; Ovary-AdenoCA and OV; Panc-AdenoCA and PAAD; Prost-AdenoCA and PRAD; Skin-Melanoma and SKCM; Thy-AdenoCA and THCA; Uterus-Adeno and UCEC. Of the 27 cancer types in PCAWG, 11  did not have hypoxia data from comparable cancers in TCGA and were not used for the comparison16. For Spearman’s correlations, p-values were calculated using algorithm AS89.To compare hypoxia scores generated by the different signatures, the median hypoxia score was calculated for each of the PCAWG cancer types based on each signature. The median hypoxia scores from each signature were then scaled from +1 to −1 using the plotrix package (v3.7). Scaled median hypoxia values for the PCAWG cancer types were also compared to scaled median hypoxia values from previously published16. Hypoxia scores were then calculated for all normal tissue samples related to the 19 cancer types with hypoxia scores . Tumour hypoxia scores were compared to normal tissue hypoxia scores for each tissue type where at least 15 normal tissue samples were available (Mann–Whitney U-test). A total of 5649 independent tumours and 625 independent normal tissue samples were evaluated in the comparisons.Previously calculated pan-cancer tumour hypoxia scores were gathered for 7791 independent tumours from 19 cancer types based on the Buffa hypoxia signatureTP53 mutational status, etc.) across cancers using the lme4 package (v1.1-17). For each feature of interest, we compared a full model  to a null model (i.e. a model without the feature of interest) using an ANOVA to determine if hypoxia was significantly associated with the feature of interest across cancers. A generic example of this is shown below with Eqs.  which uses a simulation-based approach to create standardized residuals33. We used linear mixed-effect models to associate each feature with hypoxia score across cancers and compared each full model with a null model. Cancer type was incorporated as a random effect in each model while tumour purity, age and sex were incorporated as fixed effects. Tumours belonging to cancer types with fewer than 15 samples were excluded from the analysis. A Bonferroni p-value adjustment was applied to the p-values from linear mixed-effect modelling since fewer than 20 tests were conducted. All models were adjusted for tumour purity based on previously published purity data33. The full model for evaluating PGA is shown below as an example as follows:Previously published data for 15 mutational density and summary features were downloaded for 1188 tumoursTo assess if SNVs per megabase were independently associated with hypoxia after adjusting for the total number of deletions we created two linear mixed-effect models. The full model associated hypoxia with SNVs per megabase while adjusting for cancer type, age, sex, tumour purity and the number of deletions (Eq. ). For co25. For each of the 653 driver features, we summarized if a patient had an SNV, CNA or SV. Some tumours had more than one type of event in a gene (e.g. a CNA and an SNV) and these events were classified as compound events. We then used linear mixed-effect models to associate the mutational status of each gene with hypoxia score and compared each full model with a null model. Cancer type was incorporated as a random effect in each model while tumour purity, age and sex were incorporated as fixed effects. The driver mutation analysis did not specifically consider the type of mutation in the gene and only considered if the gene had a mutation or was wildtype. Tumours belonging to cancer types with fewer than 15 samples were excluded from the analysis. An FDR adjustment was applied to the p-values from linear mixed-effect modelling. The full model for evaluating PTEN is shown below as an example as follows:Data for driver mutations was first summarized at the gene level for 1096 tumours with previously published driver mutation data35. For each tumour, we calculated the proportion of total mutations attributed to each mutational signature. The proportion of mutations attributed to each signature were calculated by dividing the number of mutations attributed to each signature by the total number of mutations in the tumour. We used linear mixed-effect models to associate the proportion of mutations attributed to each signature with hypoxia score and compared each full model with a null model. Cancer type was incorporated as a random effect in each model while purity, age and sex were incorporated as fixed effects. Tumours belonging to cancer types with fewer than 15 samples were excluded from the analysis. An FDR adjustment was applied to the p-values from linear mixed-effect modelling. The full model for SBS1 is shown below as an example as follows:Previously published data for mutations attributed to various specific signatures was downloaded for 1188 tumours38 subclonal reconstruction data was used to summarize the number of clonal and subclonal mutations in all 1188 tumours. We used linear mixed-effect models to associate the number of these timed mutations with hypoxia score and compared each full model with a null model. Cancer type was incorporated as a random effect in each model while purity, age and sex were incorporated as fixed effects. Tumours belonging to cancer types with fewer than 15 samples were excluded from the analysis. A Bonferroni adjustment was applied to the p-values from linear mixed-effect modelling since fewer than 20 tests were conducted.Previously reported37. Hypoxia scores were median dichotomized to classify patients as hypoxic or normoxic. To test for an interaction between tumour hypoxia and PTEN mutational status in selecting for a particular subclonal architecture, we used linear mixed-effect models together with an ANOVA. An interaction model was first created where the relationship between the hypoxia scores and PTEN mutational status was modelled as an interaction :9\docum47 (v5.9.1). Figures were compiled using Inkscape (v0.91).All data analysis was performed in the R statistical environment (v3.4.3). Data visualization was performed using the BPG packageFurther information on research design is available in the Supplementary InformationDescription of Additional Supplementary FilesSupplementary Data 1Reporting Summary