Single Cancer Cells Research Articles

Single-Cell Analysis of circRNA Using ddPCR.

Droplet digital polymerase chain reaction (ddPCR) is a new quantitative PCR method based on water-oil emulsion droplet technology. ddPCR enables highly sensitive and accurate quantification of nucleic acid molecules, especially when their copy numbers are low. In ddPCR, a sample is fractionated into ~20,000 droplets, and every nanoliter-sized droplet undergoes PCR amplification of the target molecule. The fluorescence signals of droplets are then recorded by an automated droplet reader. Circular RNAs (circRNAs) are single-stranded, covalently closed RNA molecules that are ubiquitously expressed in animals and plants. CircRNAs are promising as biomarkers for cancer diagnosis and prognosis and as therapeutic targets or agents to inhibit oncogenic microRNAs or proteins (Kristensen LS, Jakobsen T, Hager H, Kjems J, Nat Rev Clin Oncol 19:188-206, 2022). In this chapter, the procedures for the quantitation of a circRNA in single pancreatic cancer cells using ddPCR are described.

Effect of capillary fluid flow on single cancer cell cycle dynamics, motility, volume and morphology.

From primary tumours and disseminating to secondary organs, cancer cells experience a wide variety of fluid flow profiles when passing through blood vessels or the lymphatic system before extravasation. Sinusoidal capillaries are a common site for extravasation. Therefore, we aim to investigate how metastatic cancer cells react to a biophysical cue such as capillary fluid flow by quantifying its effect on metastatic cell cycle progression, motility, cell and nuclear volume, and morphology. We use MDA-MB-231 breast cancer cells genetically modified with fluorescent ubiquitination-based cell cycle indicator 2 (FUCCI2) as a model system. Single cells are trapped using a microfluidic device and exposed to different laminar flows. Quantitative time-lapse imaging in both 2D epifluorescence and 3D confocal microscopy is performed using in-house software FUCCItrack. In addition, 3D time-lapse with cell and nuclear segmentation is performed with a deep learning approach to streamline the image processing of big datasets. We show that at a single cell level, faster fluid flow leads to a shorter S/G2/M phase and an overall shorter cell cycle, as well as increase in cell motility irrespective of the flow direction. 3D time-lapse confocal imaging of MDA-FUCCI2 single cells reveals the evolution of cell and nuclear volume and morphology as a function of a specific cell cycle phase. Both cell and nuclear volume increase linearly over time. Cell morphology elongates more strongly during the S/G2/M phase, whereas the nuclear shape remains constant. Under the highest flow conditions, only during the S/G2/M phase can we observe a more elongated nucleus, while the cell sphericity remains similar to the control. Collectively, this data, together with the deep learning approach, provides new insights into the potential impact of fluid flow at a single cell level.

Virtual-freezing fluorescence imaging flow cytometry with 5-aminolevulinic acid stimulation and antibody labeling for detecting all forms of circulating tumor cells.

Circulating tumor cells (CTCs) are precursors to cancer metastasis. In blood circulation, they take various forms such as single CTCs, CTC clusters, and CTC-leukocyte clusters, all of which have unique characteristics in terms of physiological function and have been a subject of extensive research in the last several years. Unfortunately, conventional methods are limited in accurately analysing the highly heterogeneous nature of CTCs. Here we present an effective strategy for simultaneously analysing all forms of CTCs in blood by virtual-freezing fluorescence imaging (VIFFI) flow cytometry with 5-aminolevulinic acid (5-ALA) stimulation and antibody labeling. VIFFI is an optomechanical imaging method that virtually freezes the motion of fast-flowing cells on an image sensor to enable high-throughput yet sensitive imaging of every single event. 5-ALA stimulates cancer cells to induce the accumulation of protoporphyrin (PpIX), a red fluorescent substance, making it possible to detect all cancer cells even if they show no expression of the epithelial cell adhesion molecule, a typical CTC biomarker. Although PpIX signals are generally weak, VIFFI flow cytometry can detect them by virtue of its high sensitivity. As a proof-of-principle demonstration of the strategy, we applied cancer cells spiked in blood to the strategy to demonstrate image-based detection and accurate classification of single cancer cells, clusters of cancer cells, and clusters of a cancer cell(s) and a leukocyte(s). To show the clinical utility of our method, we used it to evaluate blood samples of four breast cancer patients and four healthy donors and identified EpCAM-positive PpIX-positive cells in one of the patient samples. Our work paves the way toward the determination of cancer prognosis, the guidance and monitoring of treatment, and the design of antitumor strategies for cancer patients.

Multiplex SERS Phenotyping of Single Cancer Cells in Microdroplets

AbstractIn the context of personalized medicine, the analysis of single cells is key in order to understand the origin and evolution of cancer to provide an accurate prognosis. Microfluidics and microdroplets are increasingly used for the handling and understanding of the behavior of single cells, as they offer the perfect isolated environment. However, due to the small volumes handled, it is necessary to couple this technology with an ultrasensitive detection technique. Herein, surface‐enhanced Raman scattering (SERS) spectroscopy and droplet microfluidics are combined toward the multiplex phenotypic analysis of single cancer cells. For this, cancer cells are labeled with different SERS tags that recognize membrane proteins and encapsulated individually in microdroplets. Afterward, single cells within microdroplets are imaged by SERS spectroscopy. To the best of the authors’ knowledge, this is the first time that a multiplex phenotypic SERS analysis of single cells in microdroplets is shown. This integrated optofluidic platform paves the way toward the multiplex and automated characterization of cell populations in cancer patients.

Real-Time Detection of Circulating Tumor Cells in Bloodstream Using Plasmonic Fiber Sensors.

Circulating tumor cells (CTCs) are single cancer cells or cancer cell clusters that are present in the circulatory system. Assessing CTC levels in patients can aid in the early detection of cancer metastasis and is essential for the purposes of accurate cancer prognosis. However, current in vitro blood tests are limited by the insufficient blood samples and low concentration levels of CTCs, which presents a major challenge for practical biosensing devices. In this work, we propose the first surface plasmon resonance (SPR) fiber probe to work intravenously, which offers a real-time detection of CTCs in bloodstreams. By exposing the protein-functionalized fiber probe to circulating blood, a continuous capture of CTCs ensures a constant increase in enrichment and hence greatly enhances enumeration accuracy. The performance of our plasmonic fiber probe was demonstrated to specifically detect Michigan Cancer Foundation-7 (MCF-7) breast cancer cells in flowing whole mouse blood. Further, a detection limit of ~1.4 cells per microliter was achieved by using an epithelial cell adhesion molecule (EpCAM) antibody-based receptor layer and a 15 minute enrichment period. This pilot study validates real-time CTC detection directly in the bloodstream by using plasmonic fiber probes, which exhibit promising clinical potential for in vivo diagnostic tests involving low concentration biomarkers in circulating blood.

Cell-Taxi: Mesenchymal Cells Carry and Transport Clusters of Cancer Cells.

Cell clusters that collectively migrate from primary tumors appear to be far more potent in forming distant metastases than single cancer cells. A better understanding of the collective cell migration phenomenon and the involvement of various cell types during this process is needed. Here, an in vitro platform based on inverted-pyramidal microwells to follow and quantify the collective migration of hundreds of tumor cell clusters at once is developed. These results indicate that mesenchymal stromal cells (MSCs) or cancer-associated fibroblasts (CAFs) in the heterotypic tumor cell clusters may facilitate metastatic dissemination by transporting low-motile cancer cells in a Rac-dependent manner and that extracellular vesicles secreted by mesenchymal cells only play a minor role in this process. Furthermore, in vivo studies show that cancer cell spheroids containing MSCs or CAFs have faster spreading rates. These findings highlight the active role of co-traveling stromal cells in the collective migration of tumor cell clusters and may help in developing better-targeted therapies.

Recent Advances in Single-Cell Metabolomics Based on Mass Spectrometry

Recent Advances in Single-Cell Metabolomics Based on Mass Spectrometry

Impact of Depth of Invasion Tumor Budding and Worst Pattern of Invasion on Clinical Outcome in Post-Operative Oral Cavity Squamous Cell Carcinoma

<h3>Purpose/Objective(s)</h3> Definitive treatment for oral cavity squamous cell carcinoma (OCSCC) is surgery followed by post-operative radiotherapy (PORT) with or without chemotherapy. Risk factors for PORT are T3/T4, node positive, lympho-vascular space invasion (LVI) or perineural invasion (PNI), close margin etc. Depth of invasion (DOI), tumor budding (TB) and worst pattern of invasion (WPOI) and tumor infiltrating lymphocytes (TILS) may have prognostic implications. We aim to report the incidence of these factors and its association with clinical outcome post-operative OCSCC patients treated at our institute. <h3>Materials/Methods</h3> 150 post-operative patients of OCSCC undergoing curative oncological resection were analyzed with median follow up time 18 months from year 2019 to 2021. WPOI (WPOI-5) was defined as tumor dispersion of ≥1mm between tumor satellites. High risk WPOI was defined as (WPOI 4/5). Tumor budding is defined as the presence of single cancer cell or cluster of <5 cancer cells at the invasive front. DOI and staging were used as per AJCC 8^th edition. Disease free survival (DFS) and overall survival (OS) were estimated with Kaplan- Meier method. <h3>Results</h3> Median age was 50 years with M:F ratio of 135:15. Tumor site was buccal mucosa: tongue:alveolus:others as 84 (56%):49 (32%):13 (8.6%):4 (2.6%). LVI:PNI: close/positive margin: extra nodal extension was seen in 57 (38%):52 (34%):35 (23%):33 (22%) patients respectively. DOI=>10, DOI<10, high risk WPOI, tumor budding and TILS were observed in 79(52.6%), 71(47.3%), 102(72.8%), 97(71.3%) and 30(21.5%) patients respectively. Association of these have been listed in Table 1. AJCC eighth stage was I:II:III:IV as 30 (20%):32 (21.3%):38 (25.3%):50 (33.3%). Median RT Dose was 60 Gray. Sixty-four patients received CTRT (Median 5 cycles). Median Overall Treatment Time (OTT) was 98 (95-120) days. 2-year OS and DFS for the entire cohort was 81% and 67% respectively. 2-year DFS for patient with TILS was 86% vs.64% without TILS (p=0.07). PNI (60% vs. 72%; p=0.06) and bone invasion (47% vs 84%; p=0.04) portended poorer DFS. TB, WPOI and DOI did not statistically impact DFS in our cohort. <h3>Conclusion</h3> TB, WPOI and DOI are often associated in post-operative OCSCC with other adverse risk features. Patients with TILS tend to have a better clinical outcome

Evolutionary Hypothesis in Cell Cycle of Breast Cancer Patients: Mosaic Phases in Single Cancer Cells

Introduction: Cell cycle shapes the initiation, progression and therapeutic approaches of neoplasms. An uncontrolled cell proliferation and growth are the key characteristics of either malignant or benign tumors. The programmed check points control the transition of phases through the related barriers. Therefore, balancing the carcinogenic processes may inhibit progression and facilitate a targeted-base therapy.&#x0D; Methods: The present study is performed in interphase. Detection of the Mosaic Phases (MPs) by Fluorescence In Situ Hybridization was confirmed by assaying the protein expression (PE) including immunofluorescence and flow cytometry.&#x0D; Results: The novel hypothesis reflects the presence of dual and/or multi-phases, as minor clones in single cells of breast cancer (BC) patients. This finding led to initiate a model with applicable ratio values and different MPs including G1/S, S/G2 and G1/S/G2, accompanied by normal phases (G1, S, G2). The remarkable harmonic behaviors between signal copy numbers and the corresponding PE, dual- and triple- co-expression between different cyclins combination including E/B1 and D1/E/B1 and the other involved proteins were observed. The ratio of gain to normal signals appeared to be a good prognosis for chromosome 1, but better survival was significantly obtained for this ratio in chromosome 3&#x0D; Conclusion: The predisposing-diagnostic-predictive-prognostic-preventive panels may lead to innovate the CDKs inhibitor-based therapy by considering the MPs Model; and may also be considered for clinical classification, in BC and other cancers.

Monitoring Angiotropic Extravascular Migratory Metastasis In Vitro.

The mechanism of cancer cell migration from the primary tumor toward secondary sites is not fully understood. In addition to intravascular cellular migration, angiotropic extravascular migratory metastasis (EVMM) has been recognized as a metastatic pathway involving tumor cells crawling along the abluminal vascular surface to distant sites. A very simple in vitro 3D assay is described here, which is based on a previous in vitro angiogenesis assay. The assay involves monitoring single fluorescence-tagged migrating cancer cells in the presence of vascular structures in real time. This coculture assay represents a quantitative approach for monitoring the migration processes of cancer cells along vessels, demonstrating phenotypic switching and migration dynamics. This protocol can be used for molecular analyses and can also be adapted for screening of therapeutic agents to block cancer metastasis.

Integrative In Situ Photodynamic Therapy-Induced Cell Death Measurement of 3D-Bioprinted MCF-7 Tumor Spheroids.

The development of new in vitro models that closely mimic the tumor microenvironment (TME) to evaluate the efficacy of anticancer drugs has received great attention. In this study, a three-dimensional (3D) bioprinted Michigan Cancer Foundation-7 (MCF-7) cancer spheroid-embedded hydrogel model was suggested for integrative in situ determination of the half-maximal inhibitory concentration (IC50) values of photosensitizers (PSs). The MCF-7 cell-laden alginate/gelatin hydrogel was printed for the fabrication of tumor spheroids. The hydrogel was used to mimic the extracellular matrix (ECM) surrounding the cancer cells in the TME. The fluorescence intensities corresponding to photodynamic therapy (PDT)-induced death of tumor spheroids probed by the laser showed a random distribution in the hydrogel, regardless of the focus of the laser and the vertical-axis direction in which the laser was passed. These results enabled integrative in situ measurement of all tumor spheroids probed by the laser without needing to separate the tumor spheroids in the hydrogel and measure them individually. When compared with two-dimensional (2D) monolayer cultures, very large IC50 values of the PSs, chlorin e6 (Ce6) and sulfonated tetraphenyl porphyrin (sTPP), were achieved in MCF-7 spheroid-embedded hydrogels mainly due to the drug resistance of the tumor spheroids. Additionally, the heterogenic PDT response of single MCF-7 cancer cells in a single tumor spheroid was observed through 3D imaging of irregular apoptosis in a single spheroid since single tumor spheroids showed a heterogenic PDT response. Furthermore, the laser-power-dependent IC50 values of PSs were obtained using the MCF-7 spheroid-embedded hydrogel model.

In vivo 3D profiling of site-specific human cancer cell morphotypes in zebrafish.

Tissue microenvironments affect the functional states of cancer cells, but determining these influences in vivo has remained a challenge. We present a quantitative high-resolution imaging assay of single cancer cells in zebrafish xenografts to probe functional adaptation to variable cell-extrinsic cues and molecular interventions. Using cell morphology as a surrogate readout of cell functional states, we examine environmental influences on the morphotype distribution of Ewing Sarcoma, a pediatric cancer associated with the oncogene EWSR1-FLI1 and whose plasticity is thought to determine disease outcome through non-genomic mechanisms. Computer vision analysis reveals systematic shifts in the distribution of 3D morphotypes as a function of cell type and seeding site, as well as tissue-specific cellular organizations that recapitulate those observed in human tumors. Reduced expression of the EWSR1-FLI1 protein product causes a shift to more protrusive cells and decreased tissue specificity of the morphotype distribution. Overall, this work establishes a framework for a statistically robust study of cancer cell plasticity in diverse tissue microenvironments.

ATAXIC: An Algorithm to Quantify Transcriptomic Perturbation Heterogeneity in Single Cancer Cells.

The single-cell RNA sequencing (scRNA-seq) has recently been widely utilized to quantify transcriptomic profiles in single cells of bulk tumors. The transcriptomic profiles in single cells facilitate the investigation of intratumor heterogeneity that is unlikely confounded by the nontumor components. We proposed an algorithm (ATAXIC) to quantify the heterogeneity of transcriptomic perturbations (TPs) in single cancer cells. ATAXIC calculated the TP heterogeneity level of a single cell based on the standard deviations of the absolute z-scored gene expression values for tens of thousands of genes, reflecting the asynchronous degree of transcriptomic alterations relative to the central (mean) tendency. By analyzing scRNA-seq datasets for eight cancer types, we revealed that ATAXIC scores were likely to correlate positively with the enrichment scores of various proliferation and oncogenic signatures, DNA damage repair, treatment resistance, and unfavorable phenotypes and outcomes in cancer. The ATAXIC scores varied among different cancer types, with lung cancer and melanoma having the lowest average scores and clear cell renal cell carcinoma having the highest average scores. The low TP heterogeneity in lung cancer and melanoma could bestow relatively higher response rates to immune checkpoint inhibitors on both cancer types. In conclusion, ATAXIC is a useful algorithm to quantify the TP heterogeneity in single cancer cells, as well as providing new insights into tumor biology.

Droplet Microfluidics-Based Fabrication of Monodisperse Poly(ethylene glycol)-Fibrinogen Breast Cancer Microspheres for Automated Drug Screening Applications.

Spheroidal cancer microtissues are highly advantageous for a wide range of biomedical applications, including high-throughput drug screening, multiplexed target validation, mechanistic investigation of tumor-extracellular matrix (ECM) interactions, among others. Current techniques for spheroidal tissue formation rely heavily on self-aggregation of single cancer cells and have substantial limitations in terms of cell-type-specific heterogeneities, uniformity, ease of production and handling, and most importantly, mimicking the complex native tumor microenvironmental conditions in simplistic models. These constraints can be overcome by using engineered tunable hydrogels that closely mimic the tumor ECM and elucidate pathologically relevant cell behavior, coupled with microfluidics-based high-throughput fabrication technologies to encapsulate cells and create cancer microtissues. In this study, we employ biosynthetic hybrid hydrogels composed of poly(ethylene glycol diacrylate) (PEGDA) covalently conjugated to natural protein (fibrinogen) (PEG-fibrinogen, PF) to create monodisperse microspheres encapsulating breast cancer cells for 3D culture and tumorigenic characterization. A previously developed droplet-based microfluidic system is used for rapid, facile, and reproducible fabrication of uniform cancer microspheres with either MCF7 or MDA-MB-231 (metastatic) breast cancer cells. Cancer cell-type-dependent variations in cell viability, metabolic activity, and 3D morphology, as well as microsphere stiffness, are quantified over time. Particularly, MCF7 cells grew as tight cellular clusters in the PF microspheres, characteristic of their epithelial morphology, while MDA-MB-231 cells displayed elongated and invasive morphology, characteristic of their mesenchymal and metastatic nature. Finally, the translational potential of the cancer microsphere platform toward high-throughput drug screening is also demonstrated. With high uniformity, scalability, and control over engineered microenvironments, the established cancer microsphere model can be potentially used for mechanistic studies, fabrication of modular cancer microtissues, and future drug-testing applications.

Microfluidic chip for precise trapping of single cells and temporal analysis of signaling dynamics

Microfluidic designs are versatile examples of technology miniaturisation that find their applications in various cell biology research, especially to investigate the influence of environmental signals on cellular response dynamics. Multicellular systems operate in intricate cellular microenvironments where environmental signals govern well-orchestrated and robust responses, the understanding of which can be realized with integrated microfluidic systems. In this study, we present a fully automated and integrated microfluidic chip that can deliver input signals to single and isolated suspension or adherent cells in a precisely controlled manner. In respective analyses of different single cell types, we observe, in real-time, the temporal dynamics of caspase 3 activation during DMSO-induced apoptosis in single cancer cells (K562) and the translocation of STAT-1 triggered by interferon γ (IFNγ) in single fibroblasts (NIH3T3). Our investigations establish the employment of our versatile microfluidic system in probing temporal single cell signaling networks where alternations in outputs uncover signal processing mechanisms.

The Emerging Impact of Tumor Budding in Oral Squamous Cell Carcinoma: Main Issues and Clinical Relevance of a New Prognostic Marker.

Simple SummaryThe results of tumor budding are an independent prognostic factor of locoregional recurrences in many solid cancers, including colorectal, nasopharyngeal, esophageal, and lung cancer. Regarding oral squamous cell carcinoma, tumor budding predicts poor survival outcomes regardless of the tumor subsite and the pathological stage. Several detection methods for tumor budding have been proposed in the literature in order to reach an agreement on a standardized scoring system. However, the lack of a standardized assessment method prevents its evaluation in a multidisciplinary setting. In this context, this study aims to critically review the literature data regarding the prognostic role of tumor budding in oral squamous cell carcinoma. The validation of a tumor budding detection method based on tumor stage and site could be relevant to improving risk stratification and planning the clinical management of patients. Moreover, tumor budding could be included in daily pathological practice to better predict the patient’s outcomes.Tumor Budding (TB) represents a single cancer cell or a small cluster of less than five cancer cells on the infiltrative tumor front. Accumulating evidence suggests TB is an independent prognostic factor in oral squamous cell carcinoma (OSCC). However, its exact role is not yet elucidated, and a standardized scoring system is still necessary. The study aims to extensively review the literature data regarding the prognostic role of TB in OSCC. The results of TB are an independent prognostic factor of poor survival outcomes in OSCC. To date, the manual detection of hematoxylin and eosin-staining or pancytokeratin-immunostaining sections are the most commonly used methods. Between the several cut-offs, the two-tier system with five buds/field cut-offs provides better risk stratification. The prognostic role of the BD model in predicting survival outcomes was extensively validated; however, the inclusion of DOI, which is already a staging parameter, encouraged other authors to propose other models, integrating TB count with other adverse risk factors, such as the tumor–stroma ratio and tumor-infiltrated lymphocytes. The prognostic relevance of TB in OSCC highlights its evaluation in daily pathological practice. Therefore, the TB detection method and the TB scoring system should be validated based on tumor stage and site.

2D MOF Nanosensor-Integrated Digital Droplet Microfluidic Flow Cytometry for In Situ Detection of Multiple miRNAs in Single CTC Cells.

Current circulating tumor cells (CTCs) detection strategies based on surface epithelial markers suffer from low specificity in distinguishing between CTCs and epithelial cells in hematopoietic cell population. Tumor-associated miRNAs within CTCs are emerging as new biomarkers due to their high correlation with tumor development and progress. However, in-situ simultaneous analysis of multiple miRNAs in single CTC cell is still challenging. To overcome this limitation, a digital droplet microfluidic flow cytometry based on biofunctionalized 2D metal-organic framework nanosensor (Nano-DMFC) is developed for in situ detection of dual miRNAs simultaneously in single living breast cancer cells. Here, 2D MOF-based fluorescent resonance energy transfer (FRET) nanosensors are established by conjugating dual-color fluorescence dye-labeled DNA probes on MOF nanosheet surface. In the Nano-DMFC, 2D MOF-based nanoprobes are precisely microinjected into each single-cell encapsulated droplets to achieve dual miRNA characterization in single cancer cell. This Nano-DMFC platform successfully detects dual miRNAs at single-cell resolution in 10 mixed positive MCF-7 cells out of 10 000 negative epithelial cells in serum biomimic samples. Moreover, this Nano-DMFC platform shows good reproductivity in the recovery experiment of spiked blood samples, which demonstrate the high potential for CTC-based cancer early diagnosis and prognosis.

Anticancer activity of D-LAK-120A, an antimicrobial peptide, in non-small cell lung cancer (NSCLC)

Non-small cell lung cancer (NSCLC) is a major cause of global cancer mortalities and accounts for approximately 80–85% of reported lung cancer cases. Conventional chemotherapeutics show limited application because of poor tumor selectivity and acquired drug resistance. Antimicrobial peptides (AMPs) have gained much attention as potential anticancer therapeutics owing to their high potency and high target selectivity and specificity with limited scope for drug resistance. In this study, D-LAK (D-LAK-120A), a cationic AMP, was evaluated for its anticancer efficacy in various NSCLC cell lines. D-LAK peptide demonstrated enhanced cytotoxicity in A549, H358, H1975, and HCC827 cell lines with inhibitory concentrations between 4.0 and 5.5 μM. An increase in the lactate dehydrogenase (LDH) levels and propidium iodide (PI) uptake across compromised membrane suggested membranolytic activity as an inhibition pathway. In addition, we found D-LAK induced lung cancer cell apoptosis and arrested cells in the S phase (DNA synthesis) of cell cycle. Moreover, a decreased mitochondrial membrane potential and elevated ROS levels were observed after D-LAK treatment, suggesting induction of mitochondria-mediated apoptosis. Additionally, D-LAK inhibited single cell proliferation and cancer cell migration in vitro. The tumor reduction observed in the 3D spheroid assay further suggests the potential use of D-LAK as an anticancer agent for NSCLC treatment. Our results postulate innovative insights on the anticancer mechanism of D-LAK, which may contribute to its further development into preclinical studies and a potential therapeutic.

Simple and automatic monitoring of cancer cell invasion into an epithelial monolayer using label-free holographic microscopy

The invasiveness of cancer cells describes the metastasizing capability of a primary tumor. The straightforward detection and quantification of cancer cell invasion are important to predict the survival rate of a cancer patient and to test how anti-cancer compounds influence cancer progression. Digital holographic microscopy based M4 Holomonitor (HM) is a technique that allows the label-free monitoring of cell morphological and kinetical parameters in real-time. Here, a fully confluent epithelial monolayer derived from the African green monkey kidney (Vero) on a gelatin-coated surface was established, then HeLa cells were seeded on top of the monolayer, and their behavior was monitored for 24 h using HM. Several cancer cells showing invasiveness were detected during this period, while other HeLa cells did not show any signs of aggressivity. It was demonstrated that the invasion of single cancer cells is soundly observable and also quantifiable through monitoring parameters such as phase shift, optical volume, area, and motility, which parameters can easily be obtained and processed automatically. Based on the experimental data, the invasion speed of cancer cells entering the epithelial layer can be defined as the shrinking of detected single-cell volume per unit time. The invasion speed and its correlation with cell migration parameters were analyzed in depth. A clear linear relationship between migration and invasion speed was found, cancer cells with stronger migration have slower invasion speed. These results not only describe the effect of how cancer cells invade the underlying monolayer in contrast to non-invasive HeLa cells, but could help in future research to optimize drugs affecting cell invasibility in a fully automated, label-free and high-throughput manner.

Abstract 1693: Dissecting intercellular extrachromosomal circular DNA heterogeneity in single cancer cells with scEC&amp;T-seq

Abstract Extrachromosomal DNA circularization is a common event in cancer cells and frequently serves as a vehicle for cancer oncogene amplification. Random segregation of oncogene-containing extrachromosomal circular DNA promotes rapid intercellular heterogeneity, conferring tumors the ability to rapidly evolve and escape therapy. Smaller, copy-number neutral extrachromosomal circular DNAs are also abundantly identified in both healthy and malignant tissues, but their function in cancer is still unknown. Understanding how extrachromosomal circular DNAs contribute to intercellular heterogeneity in cancer cells remains crucial, however methods for an unbiased characterization of extrachromosomal circular DNAs in single cells are lacking. We introduce scEC&amp;T-seq (single cell extrachromosomal circular DNA and transcriptomic sequencing), a method for parallel detection of extrachromosomal circular DNAs and full-length mRNA in single cells. We demonstrate the ability of our method to isolate and detect extrachromosomal circular DNAs genome-wide from all range of sizes in single cells. We observed that whereas large oncogene-containing circular DNAs are clonally present in most cancer cells, only a very small fraction of small circular DNAs are recurrently identified in single cells, indicating yet unknown prerequisites for maintenance and propagation. Our method was able to capture and recapitulate the structural complexity of oncogene-containing extrachromosomal circular DNAs in single cells, and the matching transcriptomic data allowed us to identify fusion transcripts resulting from the rearranged extrachromosomal structures. In addition, we observed that whereas the main structure of extrachromosomal circular DNAs is mostly stable in single cells, intercellular differences in extrachromosomal circular DNAs’ content can drive differences in oncogene transcription levels in single cells. We envision that by integrating extrachromosomal circular DNA and mRNA sequencing, our method will not only be useful to investigate the impact of intercellular heterogeneity in extrachromosomal circular DNA in tumor evolution, but also to interrogate its function in other biological and pathological processes. Citation Format: Rocio Chamorro Gonzalez, Thomas Conrad, Robin Xu, Madalina Giurgiu, Maja Cwikla, Katharina Kasack, Lotte Brückner, Eric van Leen, Elias Rodriguez-Fos, Konstantin Helmsauer, Heathcliff Dorado Garcia, Yi Bei, Karin Schmelz, Sascha Sauer, Angelika Eggert, Johannes H. Schulte, Roland F. Schwarz, Kerstin Haase, Richard P. Koche, Anton G. Henssen. Dissecting intercellular extrachromosomal circular DNA heterogeneity in single cancer cells with scEC&amp;T-seq [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 1693.