Single Cancer Cells Research Articles

Repeated FRAP of the actin-binding protein CapG in the cell nucleus—a functional assay for EGF signaling in the single live breast cancer cell

Compartmentalization and differential distribution of proteins within a cell maintain cellular function and viability. CapG is a gelsolin-related actin-binding protein that distributes in steady state diffusively throughout cytoplasm and cell nucleus. To detect changes in CapG’s nucleocytoplasmic shuttling in response to external stimuli on the single cell level, we established repeated FRAP experiments of one and the same breast cancer cell. With this experimental set up, we found that ATP-depletion reversibly decreased CapG’s shuttling into the cell nucleus. The addition of epidermal growth factor (EGF) increased CapG’s nuclear shuttling within minutes. Serum-starvation doubled the number of breast cancer cells from 40 to 80% displaying increased CapG shuttling in response to EGF. Testing five different potential CapG phosphorylation sites, we found that serine 70 mediates the increase in CapG’s nuclear shuttling triggered by EGF. Thus, repeated FRAP of CapG in the cell nucleus can be used as functional readout of signaling cascades in the same single live breast cancer cell.

Construction of a Bifunctional Nanoelectrode for Intracellular Natural Product Delivery and Monitoring Anticancer Efficiency in Single Cells.

Natural products play a significant role in new drug discovery and anticancer therapy, making the evaluation of their anticancer efficiency crucial for clinical application. However, delivering natural products to single cells and in situ monitoring of induced signaling molecule fluctuation to evaluate anticancer efficiency remain significant challenges. Hence, we proposed a universal and straightforward strategy to construct a bifunctional nanoelectrode that integrates drug loading and monitoring of signal molecule fluctuations at the single-cell level. Platinum (Pt) nanoparticles/reduced graphene oxide (rGO) composites were first electrochemically deposited on the carbon fiber nanoelectrode (CFNE@Pt/rGO) to serve as electrocatalytic materials for the monitoring of natural-product-induced reactive oxygen species (ROS) generation. The GO/natural product complex, formed by π-π stacking and hydrophobic interactions, was further electrochemically reduced on the surface of CFNE@Pt/rGO to enable the CFNE drug-loading function. Using this bifunctional functional nanoelectrode, a series of natural products (such as capsaicin, curcumin, and chrysin) were delivered into single cancer cells, and their anticancer efficiency was evaluated by measuring ROS generation. The results showed that intracellular ROS production induced by chrysin was 1.5-fold greater than that of curcumin and 2.1-fold greater than that of capsaicin. This work proposes an effective tool to evaluate the anticancer efficiency of various natural products. Additionally, this nanotool can be expanded to monitor the fluctuation of other biomolecules (such as RNS, GSH, NADH, etc.) by replacing Pt nanoparticles with other electrocatalytic materials, which is significant for comprehensively exploring the anticancer efficiency of new drugs and for the clinical treatment of various diseases.

Kinetic Trajectories of Glucose Uptake in Single Cancer Cells Reveal a Drug-Induced Cell-State Change Within Hours of Drug Treatment.

The development of drug resistance is a nearly universal phenomenon in patients with glioblastoma multiforme (GBM) brain tumors. Upon treatment, GBM cancer cells may initially undergo a drug-induced cell-state change to a drug-tolerant, slow-cycling state. The kinetics of that process are not well understood, in part due to the heterogeneity of GBM tumors and tumor models, which can confound the interpretation of kinetic data. Here, we resolve drug-adaptation kinetics in a patient-derived in vitro GBM tumor model characterized by the epithelial growth factor receptor (EGFR) variant(v)III oncogene treated with an EGFR inhibitor. We use radiolabeled 18F-fluorodeoxyglucose (FDG) to monitor the glucose uptake trajectories of single GBM cancer cells over a 12 h period of drug treatment. Autocorrelation analysis of the single-cell glucose uptake trajectories reveals evidence of a drug-induced cell-state change from a high- to low-glycolytic phenotype after 5-7 h of drug treatment. Information theoretic analysis of a bulk transcriptome kinetic series of the GBM tumor model delineated the underlying molecular mechanisms driving the cellular state change, including a shift from a stem-like mesenchymal state to a more differentiated, slow-cycling astrocyte-like state. Our results demonstrate that complex drug-induced cancer cell-state changes of cancer cells can be captured via measurements of single cell metabolic trajectories and reveal the extremely facile nature of drug adaptation.

A Novel Method for the Early Detection of Single Circulating, Metastatic and Self-Seeding Cancer Cells in Orthotopic Breast Cancer Mouse Models.

Metastasis is the main cause of cancer-related deaths, but efficient targeted therapies against metastasis are still missing. Major gaps exist in our understanding of the metastatic cascade, as existing methods cannot combine sensitivity, robustness, and practicality to dissect cancer progression. Addressing this issue requires improved strategies to distinguish early metastatic colonization from metastatic outgrowth. Luciferase-labelled MDA-MB-231, MCF7, and 4T1 breast cancer cells were spiked into samples from tumour-naïve mice to establish the limit of detection for disseminated tumour cells. Luciferase-labelled breast cancer cells (±unlabelled cancer-associated fibroblasts; CAFs) were orthotopically implanted in immunocompromised mice. An ex vivo luciferase assay was used to quantify tumour cell dissemination. In vitro luciferase assay confirmed a linear and positive correlation between cancer cell numbers and the bioluminescence detected at single cell level in blood, brain, lung, liver, and mammary fat pad samples. Remarkably, single luciferase-labelled cancer cells were detectable in all of these sites, as the bioluminescence quantified in the analysed samples was substantially higher than background levels. Ex vivo, circulating tumour cells, metastasis, and tumour self-seeding were detected in all samples from animals implanted with highly metastatic luciferase-labelled MDA-MB-231 cells. In turn, detection of poorly metastatic luciferase-labelled MCF7 cells was scarce but significantly enhanced upon co-implantation with CAFs as early as 20 days after the experiment was initiated. These results demonstrate the feasibility of using an ultrasensitive luciferase-based method to dissect the mechanisms of early metastatic colonization to improving the development of antimetastatic therapies.

Deep Learning-Based Automated Analysis of NK Cell Cytotoxicity in Single Cancer Cell Arrays

The cytotoxicity assay of immune cells based on live cell imaging offers comprehensive information at the single cell-level information, but the data acquisition and analysis are labor-intensive. To overcome this limitation, we previously developed single cancer cell arrays that immobilize cancer cells in microwells as single cell arrays, thus allow high-throughput data acquisition. In this study, we utilize deep learning to automatically analyze NK cell cytotoxicity in the context of single cancer cell arrays. Defined cancer cell position and the separation of NK cells and cancer cells along distinct optical planes facilitate segmentation and classification by deep learning. Various deep learning models are evaluated to determine the most appropriate model. The results of the deep learning-based automated data analysis are consistent with those of the previous manual analysis. The integration of the microwell platform and deep learning would present new opportunities for the analysis of cell–cell interactions.

Multistage Nanocarrier Based on an Oil Core-Graphene Oxide Shell.

Potent synthetic drugs, as well as biomolecules extracted from plants, have been investigated for their selectivity toward cancer cells. The main limitation in cancer treatment is the ability to bring such molecules within each single cancer cell, which requires accumulation in the peritumoral region followed by homogeneous spreading within the entire tissue. In the last decades, nanotechnology has emerged as a powerful tool due to its ability to protect the drug during blood circulation and allow enhanced accumulation around the leaky regions of the tumor vasculature. However, the ideal size for accumulation of around 100 nm is too large for effective penetration into the dense collagen matrix. Therefore, we propose a multistage system based on graphene oxide nanosheet-based quantum dots (GOQDs) with dimensions that are 12 nm, functionalized with hyaluronic acid (GOQDs-HA), and deposited using the layer-by-layer technique onto an oil-in-water nanoemulsion (O/W NE) template that is around 100 nm in size, previously stabilized by a biodegradable polymer, chitosan. The choice of a biodegradable core for the nanocarrier is to degrade once inside the tumor, thus promoting the release of smaller compounds, GOQDs-HA, carrying the adsorbed anticancer compound, which in this work is represented by curcumin as a model bioactive anticancer molecule. Additionally, modification with HA aims to promote active targeting of stromal and cancer cells. Cell uptake experiments and preliminary penetration experiments in three-dimensional microtissues were performed to assess the proposed multistage nanocarrier.

Bioprobe-RNA-seq-microRaman system for deep tracking of the live single-cell metabolic pathway chemometrics

The integration between RNA-sequencing and micro-spectroscopic techniques has recently profiled the advanced transcriptomic discoveries on the cellular level. In the current study, by combining the sensation approach (including bio-molecules structural evaluation, high throughput next-generation sequencing (HT-NGS), and confocal Raman microscopy) the functionality on the single live cancer cells’ ferroptosis and apoptosis signaling pathways is visualized. Our study reveals a hydrophobic tunnel by phycocyanin-isoprene molecule (PC-SIM) electrostatic charge within hepatoma cells (HepG2) that activates the ferritin light chain (FTL) and caspase-8 associate protein (CASP8AP2) ferroptosis responsible genes. Moreover, this research proves that PC-vanillin (VAN) stimulation induces the actin-binding factor profilin-1 (PFN1), subsequently in situ tracking its expression at 1139.75 cm−1 microRaman wavenumber. While PC-thymol (THY) induces the lysophospholipase-2 (LYPLA2) (p-value = 0.009) and acetylneuraminate-9-O-acetyltransferase (CASD1) (p-value = 0.022) at 1143.19 cm−1. Our findings establish a new concept to promote the cross-disciplinary use of instant cellular-based detection technology for intermediary evaluating the signaling cellular transcriptome.

Acetic acid is a superior ion pairing modifier for sub-nanogram and single cell proteomics.

A recent study demonstrated a substantial increase in peptide signal and corresponding proteome coverage when employing 0.5% acetic acid (AA) as the ion pairing modifier in place of the 0.1% formic acid traditionally used in shotgun proteomics. In this study, we investigated the effect of modifier in the context of sub-nanogram and single cell proteomics (SCP). We first evaluated a tryptic digest standard down to 20 picograms total load on column on a TIMSTOF SCP system. In line with the previous results, we observed a signal increase when using AA, leading to increased proteome coverage at every peptide load assessed. Relative improvements were more apparent at lower concentrations, with a 20 picogram peptide digest demonstrating a striking 1.8-fold increase to over 2,000 protein groups identified in a 30 minute analysis. Furthermore, we find that this increase in signal can be leveraged to reduce ramp times, leading to 1.7x more scans across each peak and improvements in quantification as measured by %CVs. When evaluating single cancer cells, approximately 13% more peptide groups were identified on average when employing AA in the place of FA. All vendor raw and processed data are available through ProteomeXchange as PXD046002 and PXD051590.

Chain electrospray ionization mass spectrometry for ultra-low volume sample analysis

In this work, chain electrospray ionization (chain-ESI) was developed to efficiently ionize trace samples for mass spectrometry analysis. The primary ion source was found to have the ability to induce secondary electrospray ionization with an extraordinarily low sample consumption rate in the picoliters per minute (pLs/min). This allows low volume sample to generate substantial tandem mass spectrum (MS2) data for metabolite annotations. Notably, chain-ESI can effectively prevent the electro-redox reaction in the process of electrospray, so as to reflect the native state of the analytes. Furthermore, from a single Broussonetia papyrifera (B. papyrifera) trichome and a single A549 cancer cell, 1426 and 617 metabolites were detected respectively. All of those observations demonstrated that chain-ESI offers the advantages of direct, rapid analysis with extreme-low volumes and high coverage, enabling the measurement of bio-information in low volume samples.

Designing SERS nanotags for profiling overexpressed surface markers on single cancer cells: A review

This review focuses on the chemical design and the use of Surface-Enhanced Raman Scattering (SERS)-active nanotags for measuring surface markers that can be overexpressed at the surface of single cancer cells. Indeed, providing analytical tools with true single-cell measurements capabilities is capital, especially since cancer research is increasingly leaning toward single-cell analysis, either to guide treatment decisions or to understand complex tumor behaviour including the single-cell heterogeneity and the appearance of treatment resistance. Over the past two decades, SERS nanotags have triggered significant interest in the scientific community owing their advantages over fluorescent tags, mainly because SERS nanotags resist photobleaching and exhibit sharper signal bands, which reduces possible spectral overlap and enables the discrimination between the SERS signals and the autofluorescence background from the sample itself. The extensive efforts invested in harnessing SERS nanotags for biomedical purposes, particularly in cancer research, highlight their potential as the next generation of optical labels for single-cell studies. The review unfolds in two main parts. The first part focuses on the structure of SERS nanotags, detailing their chemical composition and the role of each building block of the tags. The second part explores applications in measuring overexpressed surface markers on single-cells. The latter encompasses studies using single nanotags, multiplexed measurements, quantitative information extraction, monitoring treatment responses, and integrating phenotype measurements with SERS nanotags on single cells isolated from complex biological matrices. This comprehensive review anticipates SERS nanotags to persist as a pivotal technology in advancing single-cell analytical methods, particularly in the context of cancer research and personalized medicine.

A collective strategy to promote the dissemination of single cancer cells.

The transition from collective to single-cell invasion in metastatic tumors has been regarded as the consequence of oncogenic drivers in concert with extracellular triggers received from the tumor microenvironment. In this issue, Yoon and colleagues (https://doi.org/10.1083/jcb.202308080) have identified an epigenetic program by which collective niches release laminin-332 and thereby cause the detachment and invasion of fully individualized tumor cells.

Abstract PO4-01-08: Co-expression of estrogen receptor (ER), progesterone receptor (PR), and Ki67 in a single breast cancer cell indicates a favorable prognosis in ER-positive breast cancer

Abstract Background: ER-positive breast cancer is biologically and clinically divided into two subtypes: luminal-A and luminal-B. This classification is mainly based on the status of cellular proliferation. At least two different pathways drive cellular proliferation in ER-positive breast cancer. One is a classical pathway where ER binds to estrogen responsive element (ERE), which leads to the expression of downstream molecules, including PR. The other is a non-classical pathway where a complex of ER and relevant factors bind to different sites than ERE. Growth factor signaling has been suggested to potentiate a non-classical pathway. We hypothesized that examining PR status in ER-positive proliferating cells could tell which pathway is more dominant in ER-positive breast cancer. Methods: To test the hypothesis, we used a newly developed triplex immunohistochemistry (IHC) that detects three molecules simultaneously under bright-field microscopy. Postmenopausal patients who were treated with neoadjuvant endocrine therapy with aromatase inhibitors from January 2007 to September 2016 at Saitama Cancer Center were included in this study. ER, PR, and Ki67 expressions were assessed in a single slide using the triplex IHC assay with anti-ER antibody (clone SP1), anti-PR antibody (clone 1E2), and anti-Ki67 antibody (clone 30-9). ER, PR and Ki67 expression was assessed in a single cell nucleus of cancer cells (567 to 4871 cells) from multiple areas in each case. An ER-positive proliferating cell was defined as an ER-positive and Ki67-positive cell. PR status in ER-positive proliferating cells was assessed. When PR was expressed in more than 50% of ER-positive proliferating cells in a clinical case, the tumor was categorized as the PR-positive group. Luminal A and luminal B breast cancers were defined based on the pre-treatment Ki67 labeling index with a cut-off of 14 %. Statistical analyses included the Mann-Whitney test, the log-rank test, and the Cox proportional hazard model. Results: Pre-treatment tissues from 55 patients were evaluated. The median age was 62 (range: 54 to 80) years. The patients were grouped into PR-positive and PR-negative groups. The two groups had no differences in age and pre-treatment T and N stages. The median pre-treatment Ki67 labeling index was 5.9 % in the PR-positive group and 9.9 % in the PR-negative group, which showed a statistically significant difference (P = 0.01). Clinical response to neoadjuvant endocrine therapy was compared, and no difference was observed. The median post-treatment Ki67 labeling index was 3.6 % in the PR-positive group and 13.1 % in the PR-negative group with a significant difference (P = 0.035). The survival was compared between the two groups. The PR-positive group showed a significantly more favorable disease-free survival (DFS) than the PR-negative group (P = 0.0079). To adjust for the background differences including Ki67 and PR status, a multivariate analysis was performed and showed that the PR-positive group had a significantly better DFS than the PR-negative group independent of clinical stage, Ki67 labeling index, and PR status (P = 0.042). Breast cancer-specific survival (BCSS) was also better in the PR-positive group than in the PR-negative group after adjusting for clinical stage, Ki67 labeling index, and PR status (P = 0.043). Interestingly, among patients with luminal A tumors, those in the PR-positive group showed a better DFS than those in the PR-negative group (P = 0.022). Conclusion: PR status in ER-positive proliferating cells was an independent prognostic factor in DFS and BCSS and divided patients with luminal A tumors further into two prognostic groups. Citation Format: Takayuki Ueno, Rie Horii, Hiroshi Matsumoto, Makiko Ono, Yurina Maeshima, Hiroaki Nitta. Co-expression of estrogen receptor (ER), progesterone receptor (PR), and Ki67 in a single breast cancer cell indicates a favorable prognosis in ER-positive breast cancer [abstract]. In: Proceedings of the 2023 San Antonio Breast Cancer Symposium; 2023 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2024;84(9 Suppl):Abstract nr PO4-01-08.

Abstract PO1-06-07: EMSY enhances cancer stem cell self-renewal and tumorigenesis by reshaping methionine metabolism in triple-negative breast cancer

Abstract PO1-06-07: EMSY enhances cancer stem cell self-renewal and tumorigenesis by reshaping methionine metabolism in triple-negative breast cancer

Single-cell SERS imaging of dual cell membrane receptors expression influenced by extracellular matrix stiffness

Membrane receptors perform a diverse range of cellular functions, accounting for more than half of all drug targets. The mechanical microenvironment regulates cell behaviors and phenotype. However, conventional analysis methods of membrane receptors often ignore the effects of the extracellular matrix stiffness, failing to reveal the heterogeneity of cell membrane receptors expression. Herein, we developed an in-situ surface-enhanced Raman scattering (SERS) imaging method to visualize single-cell membrane receptors on substrates with different stiffness. Two SERS substrates, Au@4-mercaptobenzonitrile@Ag@Sgc8c and Au@4-pethynylaniline@Ag@SYL3c, were employed to specifically target protein tyrosine kinase-7 (PTK7) and epithelial cell adhesion molecule (EpCAM), respectively. The polyacrylamide (PA) gels with tunable stiffness (2.5–25 kPa) were constructed to mimic extracellular matrix. The simultaneous SERS imaging of dual membrane receptors on single cancer cells on substrates with different stiffness was achieved. Our findings reveal decreased expression of PTK7 and EpCAM on cells cultured on stiffer substrates and higher migration ability of the cells. The results elucidate the heterogeneity of membrane receptors expression of cells cultured on the substrates with different stiffness. This single-cell analysis method offers an in-situ platform for investigating the impacts of extracellular matrix stiffness on the expression of membrane receptors, providing insights into the role of cell membrane receptors in cancer metastasis.

Characterizing heterogeneous single-cell dose responses computationally and experimentally using threshold inhibition surfaces and dose-titration assays

Single cancer cells within a tumor exhibit variable levels of resistance to drugs, ultimately leading to treatment failures. While tumor heterogeneity is recognized as a major obstacle to cancer therapy, standard dose-response measurements for the potency of targeted kinase inhibitors aggregate populations of cells, obscuring intercellular variations in responses. In this work, we develop an analytical and experimental framework to quantify and model dose responses of individual cancer cells to drugs. We first explore the connection between population and single-cell dose responses using a computational model, revealing that multiple heterogeneous populations can yield nearly identical population dose responses. We demonstrate that a single-cell analysis method, which we term a threshold inhibition surface, can differentiate among these populations. To demonstrate the applicability of this method, we develop a dose-titration assay to measure dose responses in single cells. We apply this assay to breast cancer cells responding to phosphatidylinositol-3-kinase inhibition (PI3Ki), using clinically relevant PI3Kis on breast cancer cell lines expressing fluorescent biosensors for kinase activity. We demonstrate that MCF-7 breast cancer cells exhibit heterogeneous dose responses with some cells requiring over ten-fold higher concentrations than the population average to achieve inhibition. Our work reimagines dose-response relationships for cancer drugs in an emerging paradigm of single-cell tumor heterogeneity.

A Cross-Sectional Study of Tumor Budding in Correlation with Clinicopathological Parameters in Invasive Breast Carcinoma

Introduction: Breast carcinoma is the largest cause of mortality from cancer among women in India. Tumor budding is usually defined as isolated single cancer cells or clusters of up to four cancer cells located at the invasive tumor front. Tumor budding is associated with poor clinicopathological characteristics, such as Tumor size, tumor differentiation, lymph node invasion, lymphatic or vascular invasion etc. Aim: To study tumor budding and its association with known clinicopathological parameters and hormone receptor status in invasive breast carcinoma. Methods : A Cross-sectional study was conducted after approval from the ethics committee. The duration of study was 1.5 years (Jan 2022- June 2023). Results: A total of fifty cases of invasive breast carcinomas were included in this study. 70% (35cases) were >50yrs of age, 65% (33cases) were of T2 (tumor size) category. 82%(41cases) of Invasive duct carcinoma, No specific type, 12% (6 cases) of mixed carcinomas followed by three cases, one is invasive lobular carcinoma(6%), others are high grade papillary duct carcinoma(6%) and mucinous carcinoma (6%). 76%(38cases) showed presence of lymphovascular invasion, 74%(37cases) showed absence of necrosis, 54%(27 cases) are categorized as grade 2. High grade tumor budding was seen in 54% (27 cases). Conclusion: The following variables had a significant association with the tumor budding in present study: Tumor size, TNM staging, treatment status, lymphovascular invasion showing a p-value of <0.05. ER positive tumors are also associated with high grade tumor budding. Keywords: Breast Carcinoma, Clinicopathological Variables, Lymphovascular Invasion, Tumor Budding

Unveiling the Spatiotemporal and Dose Responses within a Single Live Cancer Cell to Photoswitchable Upconversion Nanoparticle Therapeutics Using Hybrid Hyperspectral Stimulated Raman Scattering and Transient Absorption Microscopy.

Photodynamic therapy (PDT) provides an alternative approach to targeted cancer treatment, but the therapeutic mechanism of advanced nanodrugs applied to live cells and tissue is still not well understood. Herein, we employ the hybrid hyperspectral stimulated Raman scattering (SRS) and transient absorption (TA) microscopy developed for real-time in vivo visualization of the dynamic interplay between the unique photoswichable lanthanide-doped upconversion nanoparticle-conjugated rose bengal and triphenylphosphonium (LD-UCNP@CS-Rb-TPP) probe synthesized and live cancer cells. The Langmuir pharmacokinetic model associated with SRS/TA imaging is built to quantitatively track the uptakes and pharmacokinetics of LD-UCNP@CS-Rb-TPP within cancer cells. Rapid SRS/TA imaging quantifies the endocytic internalization rates of the LD-UCNP@CS-Rb-TPP probe in individual HeLa cells, and the translocation of LD-UCNP@CS-Rb-TPP from mitochondria to cell nuclei monitored during PDT can be associated with mitochondria fragmentations and the increased nuclear membrane permeability, cascading the dual organelle ablations in cancer cells. The real-time SRS spectral changes of cellular components (e.g., proteins, lipids, and DNA) observed reflect the PDT-induced oxidative damage and the dose-dependent death pattern within a single live cancer cell, thereby facilitating the real-time screening of optimal light dose and illumination duration controls in PDT. This study provides new insights into the further understanding of drug delivery and therapeutic mechanisms of photoswitchable LD-UCNP nanomedicine in live cancer cells, which are critical in the optimization of nanodrug formulations and development of precision cancer treatment in PDT.

Abstract 3778: Comprehensive analysis of single disseminated cancer cells identifies a stem-like phenotype with strong prognostic impact

Abstract Introduction: Recent studies on disseminated cancer cells (DCCs) in Non-Small-Cell Lung Cancer (NSCLC) patients demonstrated their clinical relevance even in early tumor stages. However, their molecular and phylogenetic link to the primary tumor is currently unclear. Certain genetic alterations, such as TP53 or KRAS mutations, have been proposed to be clonal events and drivers of dissemination and metastasis. In this study, we analyzed the genome and transcriptome of single DCCs to identify the characteristics of early DCCs and their translational clinical value. Materials and methods: From 2011 to 2019 we prospectively collected and analyzed bone marrow aspirates and lymph node samples from 296 newly diagnosed NSCLC patients undergoing tumor resection with curative intention. Presence of DCC was examined by i) immunocytology (IC) for Cytokeratin (clone A45-B/B3, bone marrow, BM-DCCs) or EpCAM (clone BerEp4, lymph node, LN-DCCs) and ii) immunofluorescent (IF) staining for EpCAM in a live cell suspension (clone HEA-125) from BM or LN. Single cells were isolated using micromanipulation and subjected to Whole Genome Amplification or combined Whole Genome/Transcriptome Amplification for IC and IF, respectively. Sanger sequencing for known hotspot mutations in TP53 and KRAS was performed on single DCCs. Gene expression profiles of DCCs were analyzed by single cell RNA sequencing and the results were compared with clinical data with a mean follow-up of 2.93 years (range 0.03-7.22). Results: LN-DCCs were found in 44.8% and 59.6% by IC and IF, respectively. CKIC pos BM-DCCs were found in 45.2%, while EpCAMIF pos BM-DCCs were present in 52.8%. LN-DCCs correlated with reduced tumor-specific survival (TSS, p=0.009) and progression-free survival (PFS, p=0.025). EpCAMIF pos, but not CKIC pos BM-DCCs correlated with reduced TSS (p=0.003) and PFS (p=0.083). Genetic analysis of single DCCs revealed that TP53 or KRAS mutations could be found in 3.6% and 2.8% of analyzed BM-DCCs and 5.0% and 29.3% of LN-DCCs for IF and IC, respectively. Single cell RNA-seq showed distinct transcriptomic profiles for LN- and BM-DCCs, and identified a DCC phenotype with high stemness score in the bone marrow. Further analysis of potential transcriptome-based biomarkers identified a novel candidate marker for activated EpCAMpos BM-DCCs that strongly correlates with PFS and TSS (p<0.001). Conclusions: DCCs in early-stage NSCLC have high prognostic value and predict reduced PFS and TSS. While genetic alterations in the TP53 and KRAS gene were not detected in the majority of analyzed DCCs, transcriptomic phenotypes provided relevant information and identified a subset of activated DCCs that correlate strongly with relapse and poor outcome. Citation Format: Tobias Mederer, Daniel Spitzl, Felix Elsner, Bernhard Polzer, Michael Ried, Reiner Neu, Tobias Robold, Hans-Stefan Hofmann, Christoph A. Klein. Comprehensive analysis of single disseminated cancer cells identifies a stem-like phenotype with strong prognostic impact [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 3778.

Abstract 3704: Whole genome and targeted proteome analysis of single cancer cells

Abstract The simultaneous genome and proteome analysis of very rare single cells would enable addressing important biological and diagnostic questions particularly in cancer. Here, we introduce a novel genome and proteome (G&P) analytic workflow, a method allowing comprehensive analysis of single-cell DNA and targeted profiling of protein expression in single cells. As a proof of concept, we demonstrate the capability of the method by quantifying copy number of the ERBB2 gene alongside with protein expression levels of HER2 and phospho-HER2 in single cells. The G&P workflow utilizes antibodies conjugated with DNA oligonucleotides designed to enable their amplification alongside the single-cell genome facilitating parallel genome and proteome profiling by either quantitative PCR (qPCR) or sequencing. We utilized antibodies targeting cytokeratin 8/18/19, HER2 and phospho-HER (Tyr1248). Performance of each DNA-conjugated antibody was assessed by immunofluorescence staining in a variety of breast cancer cell lines (MDA-MB231, MDA-MB453, BT474 and MCF7) and sample types. Stained cells were isolated by manual micromanipulation and subjected to whole genome amplification (WGA). Protein expression levels, derived from quantification of the amplified DNA tags, were measured by qPCR. Shallow whole genome sequencing was employed to facilitate genome-wide profiling of copy number variations (CNVs), including the ERBB2 locus. We successfully developed and tested a G&P workflow allowing parallel genome-wide CNV profiling and quantification of three target proteins. G&P measurement yielded concordant results to immunofluorescence quantification for all tested markers. Furthermore, the workflow allowed direct correlation of copy numbers of the ERBB2 gene with HER2 expression in single cancer cells. Notably, the method enabled measurements of cellular and molecular responses at the single-cell level to lapatinib treatment, i.e. monitoring of the HER2 pathway activation level. Our novel multiplex G&P technique is capable of generating multiparametric measurements linking DNA copy numbers and expression levels of selected genes of interest. The method is optimized for analysis of single cells from rare cell populations and is readily translatable to study circulating tumor cells (CTCs) and disseminated cancer cells (DCCs), enabling for the first time pathway analysis via phosphoprotein analyses of these cells. Citation Format: Atieh Alyasin, Kathrin Weidele, Catherine Botteron, Christoph A. Klein, Zbigniew T. Czyż. Whole genome and targeted proteome analysis of single cancer cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 3704.

Abstract 4675: Reduction in NSCLC tumorigenesis by targeting sphingosine kinase 2

Abstract Lung cancer will account for 21% of US cancer deaths in 2023 with 238,340 cases and 127,070 deaths. Treatment with EGFR-TKI (epidermal growth factor receptor tyrosine kinase) inhibitors initially decrease tumor growth, but often leads to TKI resistance. According to studies, non-small cell lung cancer (NSCLC) tumors express more SphK2 (Sphingosine kinase 2) than normal lung tissue and patients with overexpression have worse survival chances. However, SphK2's role in TKI resistance is unclear. We hypothesize that inhibiting/downregulating SphK2 could reduce tumorigenicity and overcome ER (Erlotinib) and OR (Osimertinib) resistance. We studied the expression of SphK2 in both drug-resistant (OR/ER) and drug sensitive (parental) H358, H2170, H3255, and PC9 NSCLC cell lines using Western blotting, qRT-PCR, and immunofluorescence. H3255OR cells were also bioprinted with GelMA hydrogel using an R-Gen 200 bioprinter to mimic the physiological tumor environment and treated with SphK2 siRNA or ABC294640 (SphK2i). Immunoblotting was done to analyze changes in the expression of SphK2 in H358 (WT-EGFR) and H3255 (mutant EGFR- L858R) parental and OR-resistant cell lines. Results indicated a 1.5-1.8-fold upregulation of SphK2. Increased SphK2 gene expression was observed using qPCR in TKI-resistant H3255OR (4.3-fold), PC9ER/OR with mutant EGFR (E746-A750del) (1.8-3.3-fold), and H2170ER/OR with WT EGFR (1.3-1.5-fold) compared to parental cells. Immunofluorescence studies showed increased fluorescence of SphK2 in H3255OR (2.6-fold), H358 ER (2.4-fold) and OR cells (2.3-fold) compared to parental cells. Spheroids formed from single cancer cells can "re-create" the growth of a tumor. The spheroid count of H3255OR was reduced by 73.98% and 56%, respectively, when SphK2i was added to the 3D-cell culture with Matrigel, compared to the diluent and OR alone (p≤0.01). We used 3D bioprinting to precisely stack cells on biological scaffolds to recreate lung tumors from patients and boost the therapeutic efficacy of these findings. When bioprinted spheroids of H3255OR were treated with SphK2i in combination with OR, the number of spheroids per microscopic field were reduced by 87.3% compared to OR alone. Bioprinted H3255OR spheroids treated with SphK2 siRNA and OR revealed a 75.8% and 84% reduction of spheroid count, respectively, compared to treatments with siRNA alone or OR alone (p≤0.05). This data supports the hypothesis that SphK2 suppression or downregulation can assist in mitigating oncogenesis. We also used H3255OR transwell migration assays to examine how SphK2 inhibition affects cell migration. Combining OR with SphK2i resulted in a 94.7% and 91.4% decrease in cell migration compared to diluent and OR alone (p≤0.01). In H358OR-resistant cells, combining OR with SphK2i resulted in a 94.97% inhibition compared to diluent (p≤0.01). These results suggest that targeting SphK2 could be a valuable approach for treating lung cancer. Citation Format: Ayesha Khan, Bamby Dieng, Namrata Dube, Mohammad Fazle Alam, Neelu Puri. Reduction in NSCLC tumorigenesis by targeting sphingosine kinase 2 [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 4675.