Genetic and epigenetic variation, together with transcriptional plasticity, contribute to intratumour heterogeneity1. The interplay of these biological processes and their respective contributions to tumour evolution remain unknown. Here we show that intratumour genetic ancestry only infrequently affects gene expression traits and subclonal evolution in colorectal cancer (CRC). Using spatially resolved paired whole-genome and transcriptome sequencing, we find that the majority of intratumour variation in gene expression is not strongly heritable but rather ‘plastic’. Somatic expression quantitative trait loci analysis identified a number of putative genetic controls of expression by cis-acting coding and non-coding mutations, the majority of which were clonal within a tumour, alongside frequent structural alterations. Consistently, computational inference on the spatial patterning of tumour phylogenies finds that a considerable proportion of CRCs did not show evidence of subclonal selection, with only a subset of putative genetic drivers associated with subclone expansions. Spatial intermixing of clones is common, with some tumours growing exponentially and others only at the periphery. Together, our data suggest that most genetic intratumour variation in CRC has no major phenotypic consequence and that transcriptional plasticity is, instead, widespread within a tumour.

Molecular clocks that record cell ancestry mutate too slowly to measure the short-timescale dynamics of cell renewal in adult tissues. Here, we show that fluctuating DNA methylation marks can be used as clocks in cells where ongoing methylation and demethylation cause repeated ‘flip–flops’ between methylated and unmethylated states. We identify endogenous fluctuating CpG (fCpG) sites using standard methylation arrays and develop a mathematical model to quantitatively measure human adult stem cell dynamics from these data. Small intestinal crypts were inferred to contain slightly more stem cells than the colon, with slower stem cell replacement in the small intestine. Germline APC mutation increased the number of replacements per crypt. In blood, we measured rapid expansion of acute leukemia and slower growth of chronic disease. Thus, the patterns of human somatic cell birth and death are measurable with fluctuating methylation clocks (FMCs).

ObjectiveThe crypt population in the human intestine is dynamic: crypts can divide to produce two new daughter crypts through a process termed crypt fission, but whether this is balanced by a second process to remove crypts, as recently shown in mouse models, is uncertain. We examined whether crypt fusion (the process of two neighbouring crypts fusing into a single daughter crypt) occurs in the human colon.DesignWe used somatic alterations in the gene cytochrome c oxidase (CCO) as lineage tracing markers to assess the clonality of bifurcating colon crypts (n=309 bifurcating crypts from 13 patients). Mathematical modelling was used to determine whether the existence of crypt fusion can explain the experimental data, and how the process of fusion influences the rate of crypt fission.ResultsIn 55% (21/38) of bifurcating crypts in which clonality could be assessed, we observed perfect segregation of clonal lineages to the respective crypt arms. Mathematical modelling showed that this frequency of perfect segregation could not be explained by fission alone (p<10−20). With the rates of fission and fusion taken to be approximately equal, we then used the distribution of CCO-deficient patch size to estimate the rate of crypt fission, finding a value of around 0.011 divisions/crypt/year.ConclusionsWe have provided the evidence that human colonic crypts undergo fusion, a potential homeostatic process to regulate total crypt number. The existence of crypt fusion in the human colon adds a new facet to our understanding of the highly dynamic and plastic phenotype of the colonic epithelium.

Cell lipid membranes are the primary site of irreversible injury during freezing/thawing and cryopreservation of cells, but the underlying causes remain unknown. Here, we probe the effect of cooling from 20 °C to 0 °C on the structure and mechanical properties of 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) bilayers using atomic force microscopy (AFM) imaging and AFM-based nanoindentation in a liquid environment. The Young’s modulus of elasticity (E) at each temperature for DPPC was obtained at different ionic strengths. Both at 20 mM and 150 mM NaCl, E of DPPC bilayers increases exponentially –as expected–as the temperature is lowered between 20 °C and 5 °C, but at 0 °C E drops from the values measured at 5 °C. Our results support the hypothesis that mechanical weakening of the bilayer at 0 °C  is produced by  structural changes at the lipid-fluid interface.

The dynamical process of cell division that underpins homeostasis in the human body cannot be directly observed in vivo, but instead is measurable from the pattern of somatic genetic or epigenetic mutations that accrue in tissues over an individual's lifetime. Because somatic mutations are heritable, they serve as natural lineage tracing markers that delineate clonal expansions. Mathematical analysis of the distribution of somatic clone sizes gives a quantitative readout of the rates of cell birth, death, and replacement. In this review we explore the broad range of somatic mutation types that have been used for lineage tracing in human tissues, introduce the mathematical concepts used to infer dynamical information from these clone size data, and discuss the insights of this lineage tracing approach for our understanding of homeostasis and cancer development. We use the human colon as a particularly instructive exemplar tissue. There is a rich history of human somatic cell dynamics surreptitiously written into the cell genomes that is being uncovered by advances in sequencing and careful mathematical analysis lineage of tracing data.

Cancer genomic medicine relies on targeting driver genes. However, current catalogues of cancer drivers are mostly based on indirect measurements of mutation frequencies, positions or types, rather than their effect on clonal expansions in vivo. Moreover, nongenetic drivers are largely unknown, as are the epigenetic and transcriptomic effects of genetic drivers. Here we perform spatial computational inference on multiomic data with matched whole-genome sequencing, ATAC-seq and RNA-seq. Using 436 samples, we directly quantify the contribution, or lack thereof, of putative driver genes to subclonal expansions in vivo in 30 colorectal carcinomas (4-33 samples per patient, median=15). Although subclonal neutral evolution was widespread (13/26 cases with sufficient data), there were cases with clear evidence of subclonal selection (6/26) in which we measured epigenetic and transcriptomic differences between subclones in vivo. In 7/26 cases we could not distinguish between neutral or selective evolution with the available data. We identified expanding subclones that were not driven by known genetic alterations, and propose candidate epigenetic drivers. We identified the distinguishing patterns of genomic heterogeneity produced in fast, exponentially growing tumours (7/26) versus neoplasms growing only at the periphery (19/26), as well as identifying clonally intermixed (16/28 cases with sufficient data) versus segregated malignancies (10/28). Our model-based approach measures genetic and non-genetic subclonal selection, or lack thereof, in space and time and allows in vivo comparisons of the emergent phenotypic properties of subclones within human tumours.

Cancer evolution is driven by natural selection acting upon phenotypic trait variation. However, the extent to which phenotypic variation within a tumour is a consequence of intra-tumour genetic heterogeneity remains undetermined. Here we show that colorectal cancer cells frequently have highly plastic phenotypic traits in vivo in patient tumours. We measured the degree to which trait variation reflects genetic ancestry by quantifying the phylogenetic signal of gene expression across 297 samples with multi-region paired whole genome and transcriptome sequencing collected from 27 primary colorectal cancers. Within-tumour phylogenetic signal for genes and pathways was detected only infrequently, suggesting that the majority of intra-tumour variation in gene expression programmes was not strongly heritable. Expression quantitative trait loci analyses (eQTL) identified a small number of putative mechanisms of genetic control of gene expression due to the cis-acting coding, non-coding and structural genetic alteration, but most gene expression variation was not explained by our genetic analysis. Leveraging matched chromatin-accessibility sequencing data, enhancer mutations with cis regulatory effects on gene expression were associated with a change in chromatin accessibility, indicating that non-coding variation can have phenotypic consequence through modulation of the 3D architecture of the genome. This study maps the evolution of transcriptional variation during cancer evolution, highlighting that intra-tumour phenotypic plasticity is pervasive in colorectal malignancies, and may play key roles in further tumour evolution, from metastasis to therapy resistance.

31Small solid-state devices are candidates for accelerating biomedical assays/drug discovery, 32 however their potential remains unfulfilled. Here, we demonstrate that graphene-field 33 effect transistors (FET) can be used to successfully detect the key molecular events 34 underlying viral infections and the effect of antiviral drugs. Our device success is achieved 35 by bio-mimicking the host-cell surface during an influenza infection at the graphene 36 channel. In-situ AFM confirms the biological interactions at the sialic acid-functionalized 37 graphene: viral hemagglutinin (HA) binds to sialic acid, and neuraminidase (NA) reacts 38 with the sialic acid-HA complex. The graphene-FET detects HA binding to sialic acid, and 39 NA cleavage of sialic acid. The inhibitory effect of the drug "zanamivir" on NA-sialic 40 acid interactions is monitored in real-time; the reaction rate constant of NA-sialic acid 41 reaction was successfully determined. We demonstrate that graphene-FETs are powerful 42 platforms for measurement of biomolecular interactions and contribute to future 43 deployment of solid-state devices in drug discovery/biosensing. 44 45 46