Comparison Of Single Cells Research Articles

Single-Cell Atlas of Lineage States, Tumor Microenvironment, and Subtype-Specific Expression Programs in Gastric Cancer.

We profiled gastric malignancies at single-cell resolution and identified increased plasma cell proportions as a novel feature of diffuse-type tumors. We also uncovered distinct cancer-associated fibroblast subtypes with INHBA-FAP-high cell populations as predictors of poor clinical prognosis. Our findings highlight potential origins of deregulated cell states in the gastric tumor ecosystem. This article is highlighted in the In This Issue feature, p. 587.

P-061: Single-cell whole-exome DNA sequencing traces clonal trajectory in paired evolution of MGUS to multiple myeloma

<h3>Background</h3> Accurate measurement of heterogeneity and reconstruction of evolutionary pathways are key to decipher clonal dynamics in tumour origins. Although bulk population genetic sequencing data has been used extensively to understand the life history of tumours, sequencing single cell DNA is likely to provide a much higher resolution of clonal dynamics and yield new insights into competing cellular populations with different genotypes. This is of significant importance when clonal evolution from a benign state to malignant disease is being considered, such as in the clonal evolution of MGUS to multiple myeloma (MM). <h3>Methods</h3> To this end, we further refined a multiple displacement amplification-based single cell whole-exome sequencing (scWES) protocol and applied it to examine MM cells (2 cases), and in one of these cases, probed evolution from a pre-existing MGUS stage in a paired setting. Single tumour cells were isolated from CD138+CD38+ fluorescence-activated cell sorted populations and CD3+ for T cells as germline controls. We developed a novel computational pipeline to accurately call somatic SNVs/indels and copy number aberration (CNA) events in the form of loss-of-heterozygosity (LOH) and gains in each single cell, identified drivers at the genome-wide level and reconstructed the tumour phylogeny while estimating and accounting for allelic dropout (N=176 single cells, 25× each cell). <h3>Results</h3> By focusing on the patient that allowed comparison of single cells at pre- and post-malignancy states, we show that pre-malignant subclones can be readily identified, and strikingly that malignant cells arise and expand from one of the MGUS subclones in the combined tumour phylogeny. <h3>Conclusions</h3> These results suggest that this scWES based method is a pragmatic approach to investigate clonal dynamics by allowing: 1) the analysis of genetic heterogeneity at the genome-wide level (much higher resolution than targeted panels), and 2) at low sequencing costs across large set of cells when compared with whole genome sequencing-based methods.

Abstract P5-02-03: Highly multiplexed tissue-based cyclic immunofluorescence in breast cancer for precision oncology

Abstract Background: Tissue-based cyclic immunofluorescence (t-CyCIF) is a recently described technique for highly multiplexed immunofluorescence microscopy of formalin-fixed, paraffin-embedded (FFPE) specimens. Via an iterative process, successive four-channel images are collected from the same sample and then registered to each other to generate a high-dimensional representation that is used for visualization and analysis. This technique can be used to capture up to 60 different antigens on a single FFPE tumor section and permits quantification of cell lineage and state markers, intracellular signaling proteins, drug targets and immune cell antigens, thereby promoting biomarker discovery efforts that are fundamental to precision oncology. As with most technologies that utilize immunostaining, proper antibody validation is key to reliable performance. In this study we used t-CyCIF to evaluate multiple antibodies directed against proteins commonly used to characterize breast carcinomas and their associated microenvironment. Our goal was to validate these antibodies on the t-CyCIF platform prior to the more widespread use of this technology to propel novel discoveries on breast cancer initiation, progression and treatment. Methods: To choose the optimal antibody candidate for each biomarker in t-CyCIF, we compared multiple fluorophore-conjugated antibodies for each of the following three proteins routinely evaluated in breast carcinomas: estrogen receptor (ER), progesterone receptor (PR) and HER2. For each of these, a single antibody commonly used in clinical practice was used as a reference. Analyses were performed at the level of pixels, cells and tissue cores. In addition, inter-assay analyses were performed comparing: (1) t-CyCIF vs. immunohistochemistry (IHC), the latter assessed both by digital pathology and by two independent pathologists; and (2) t-CyCIF vs. fluorescence in situ hybridization (FISH) for HER2. Following validation of these antibodies, we evaluated the expression of CD45, CD68, PD-L1, p53, Ki67 and androgen receptor along with ER, PR and HER2, to better understand both the tumor microenvironment and the cell identities/states in breast carcinomas. Results: A total of 948 tissue cores were included in the study. In the first phase, 13 different antibodies were analyzed: three raised against ER and five each against PR and HER2. The pixel-to-pixel evaluation resulted in r scores using Pearson correlation equal to 0.86 for both ER markers tested; ranging from 0.88 to 0.93 for PR; and from 0.56 to 0.94 for HER2. The correlation scores in single-cell comparisons ranged from 0.76 to 0.88 for ER, 0.54 to 0.81 for PR, and from 0.56 to 0.76 for HER2. Comparisons were then performed at the tissue core level. In light of the data generated through these multiple levels of analyses, we identified fluorophore-conjugated candidates for use in t-CyCIF. Correlation scores on the tissue core level were high in the inter-assay analyses, i.e. t-CyCIF vs. IHC (e.g. r scores up to 0.91 for HER2 on t-CyCIF vs. IHC) and t-CyCIF vs. HER2 FISH (r scores up to 0.71). In the second phase, we characterized the tumor microenvironment and cell identities present in 260 breast carcinomas. With a qualified panel of antibodies, we performed single-cell analyses of 589,343 cells and identified unexpected patterns of PD-L1 expression in distinct populations of tumor cells. Conclusion: This study is the first to evaluate the performance of breast cancer-specific antibodies in a highly multiplexed imaging platform such as t-CyCIF. This work demonstrates a step-by-step approach for qualifying reagents to be used in a multiplexed, spatially resolved tissue imaging modality. This validation study will facilitate the use of t-CyCIF for additional studies in breast cancer to evaluate both tumor elements and components of the microenvironment. Citation Format: Ricardo G Pastorello, Jia-Ren Lin, Ziming Du, Shaolin Mei, Krishan Taneja, Deborah A Dillon, Stuart J Schnitt, Peter K Sorger, Elizabeth A Mittendorf, Sandro Santagata, Jennifer L Guerriero. Highly multiplexed tissue-based cyclic immunofluorescence in breast cancer for precision oncology [abstract]. In: Proceedings of the 2019 San Antonio Breast Cancer Symposium; 2019 Dec 10-14; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2020;80(4 Suppl):Abstract nr P5-02-03.

A new workflow for whole-genome sequencing of single human cells.

Unbiased amplification of the whole-genome amplification (WGA) of single cells is crucial to study cancer evolution and genetic heterogeneity, but is challenging due to the high complexity of the human genome. Here, we present a new workflow combining an efficient adapter-linker PCR-based WGA method with second-generation sequencing. This approach allows comparison of single cells at base pair resolution. Amplification recovered up to 74% of the human genome. Copy-number variants and loss of heterozygosity detected in single cell genomes showed concordance of up to 99% to pooled genomic DNA. Allele frequencies of mutations could be determined accurately due to an allele dropout rate of only 2%, clearly demonstrating the low bias of our PCR-based WGA approach. Sequencing with paired-end reads allowed genome-wide analysis of structural variants. By direct comparison to other WGA methods, we further endorse its suitability to analyze genetic heterogeneity.

Feedforward Origins of Response Variability Underlying Contrast Invariant Orientation Tuning in Cat Visual Cortex

Contrast invariant orientation tuning in simple cells of the visual cortex depends critically on contrast dependent trial-to-trial variability in their membrane potential responses. This observation raises the question of whether this variability originates from within the cortical circuit or the feedforward inputs from the lateral geniculate nucleus (LGN). To distinguish between these two sources of variability, we first measured membrane potential responses while inactivating the surrounding cortex, and found that response variability was nearly unaffected. We then studied variability in the LGN, including contrast dependence, and the trial-to-trial correlation in responses between nearby neurons. Variability decreased significantly with contrast, whereas correlation changed little. When these experimentally measured parameters of variability were applied to a feedforward model of simple cells that included realistic mechanisms of synaptic integration, contrast-dependent, orientation independent variability emerged in the membrane potential responses. Analogous mechanisms might contribute to the stimulus dependence and propagation of variability throughout the neocortex.

Cellular Mechanisms Underlying Stimulus-Dependent Gain Modulation in Primary Visual Cortex Neurons In Vivo

Gain modulation is a widespread neuronal phenomenon that modifies response amplitude without changing selectivity. Computational and in vitro studies have proposed cellular mechanisms of gain modulation based on the postsynaptic effects of background synaptic activation, but these mechanisms have not been studied in vivo. Here, we used intracellular recordings from cat primary visual cortex to measure neuronal gain while changing background synaptic activity with visual stimulation. We found that increases in the membrane fluctuations associated with increases in synaptic input do not obligatorily result in gain modulation in vivo. However, visual stimuli that evoked sustained changes in resting membrane potential, input resistance, and membrane fluctuations robustly modulated neuronal gain. The magnitude of gain modulation depended critically on the spatiotemporal properties of the visual stimulus. Gain modulation in vivo may thus be determined on a moment-to-moment basis by sensory context and the consequent dynamics of synaptic activation.

The Emergence of Contrast-Invariant Orientation Tuning in Simple Cells of Cat Visual Cortex

Simple cells in primary visual cortex exhibit contrast-invariant orientation tuning, in seeming contradiction to feed-forward models that rely on lateral geniculate nucleus (LGN) input alone. Contrast invariance has therefore been thought to depend on the presence of intracortical lateral inhibition. In vivo intracellular recordings instead suggest that contrast invariance can be explained by three properties of the excitatory pathway. (1) Depolarizations evoked by orthogonal stimuli are determined by the amount of excitation a cell receives from the LGN, relative to the excitation it receives from other cortical cells. (2) Depolarizations evoked by preferred stimuli saturate at lower contrasts than the spike output of LGN relay cells. (3) Visual stimuli evoke contrast-dependent changes in trial-to-trial variability, which lead to contrast-dependent changes in the relationship between membrane potential and spike rate. Thus, high-contrast, orthogonally oriented stimuli that evoke significant depolarizations evoke few spikes. Together these mechanisms, without lateral inhibition, can account for contrast-invariant stimulus selectivity.

The effects of contrast on visual orientation and spatial frequency discrimination: a comparison of single cells and behavior.

We have compared the effects of contrast on human psychophysical orientation and spatial frequency discrimination thresholds and on the responses of individual neurons in the cat's striate cortex. Contrast has similar effects on orientation and spatial frequency discrimination: as contrast is increased above detection threshold, orientation and spatial frequency discrimination performance improves but reaches maximum levels at quite low contrasts. Further increases in contrast produce no further improvements in discrimination. We measured the effects of contrast on response amplitude, orientation and spatial frequency selectivity, and response variance of neurons in the cat's striate cortex. Orientation and spatial frequency selectivity vary little with contrast. Also, the ratio of response variance to response mean is unaffected by contrast. Although, in many cells, response amplitude increases approximately linearly with log contrast over most of the visible range, some cells show complete or partial saturation of response amplitude at medium contrasts. Therefore, some cells show a clear increase in slope of the orientation and spatial frequency tuning functions with increasing contrast, whereas in others the slopes reach maximum values at medium contrasts. Using receiver operating characteristic analysis, we estimated the minimum orientation and spatial frequency differences that can be signaled reliably as a response change by an individual cell. This analysis shows that, on average, the discrimination of orientation or spatial frequency improves with contrast at low contrasts more than at higher contrasts. Using the optimal stimulus for each cell, we estimated the contrast threshold of 48 neurons. Most cells had contrast thresholds below 5%. Thresholds were only slightly higher for nonoptimal stimuli. Therefore, increasing the contrast of sinusoidal gratings above approximately 10% will not produce large increases in the number of responding cells. The observed effects of contrast on the response characteristics of nonsaturating cortical cells do not appear consistent with the psychophysical results. Cells that reach their maximum response at low-to-medium contrasts may account for the contrast independence of psychophysical orientation and spatial frequency discrimination thresholds at medium and high contrasts.