Downstream Molecular Analysis Research Articles

Optimizing Ewing Sarcoma and Osteosarcoma Biopsy Acquisition: A Children's Oncology Group Bone Tumor Committee Consensus Statement.

Trends in diagnostic biopsy sample collection approaches for primary bone sarcomas have shifted in the past 2 decades. Although open/incisional biopsies used to be the predominant approach to obtain diagnostic material for Ewing sarcoma and osteosarcoma, image-guided core needle biopsies have increased in frequency and are safe for patients. These procedures are less invasive and reduce recovery times but have potential limitations. The quantity and quality of tissue obtained through these procedures vary between institutions. Acquired viable tissue volumes can be low, limiting the conduct of downstream expanded clinical workup, molecular analyses, and research. Patients with advanced Ewing sarcoma and osteosarcoma continue to have overall poor outcomes despite dose-intensive cytotoxic chemotherapy. The biology of treatment resistance is not currently well understood, partly due to limited availability of relevant tissue to study. There is a need for access to quality tumor specimens for molecular and other analyses to identify high-risk tumor subsets and drive discovery to improve patient outcomes. Given broad variability in bone tumor tissue procurement and processing across member institutions, the Children's Oncology Group Bone Tumor Committee convened a multidisciplinary group of experts to outline the current and near-future tissue needs for optimal clinical care and access to research platforms. The goal of this working group was to provide high-level guidance on biopsy practices that safely meet these evolving needs. Harmonizing tissue collection practices is paramount to improving the care of children, adolescents, and young adults diagnosed with Ewing sarcoma and osteosarcoma.

Tumour purity assessment with deep learning in colorectal cancer and impact on molecular analysis.

Tumour content plays a pivotal role in directing the bioinformatic analysis of molecular profiles such as copy number variation (CNV). In clinical application, tumour purity estimation (TPE) is achieved either through visual pathological review [conventional pathology (CP)] or the deconvolution of molecular data. While CP provides a direct measurement, it demonstrates modest reproducibility and lacks standardisation. Conversely, deconvolution methods offer an indirect assessment with uncertain accuracy, underscoring the necessity for innovative approaches. SoftCTM is an open-source, multiorgan deep-learning (DL) model for the detection of tumour and non-tumour cells in H&E-stained slides, developed within the Overlapped Cell on Tissue Dataset for Histopathology (OCELOT) Challenge 2023. Here, using three large multicentre colorectal cancer (CRC) cohorts (N = 1,097 patients) with digital pathology and multi-omic data, we compare the utility and accuracy of TPE with SoftCTM versus CP and bioinformatic deconvolution methods (RNA expression, DNA methylation) for downstream molecular analysis, including CNV profiling. SoftCTM showed technical repeatability when applied twice on the same slide (r = 1.0) and excellent correlations in paired H&E slides (r > 0.9). TPEs profiled by SoftCTM correlated highly with RNA expression (r = 0.59) and DNA methylation (r = 0.40), while TPEs by CP showed a lower correlation with RNA expression (r = 0.41) and DNA methylation (r = 0.29). We show that CP and deconvolution methods respectively underestimate and overestimate tumour content compared to SoftCTM, resulting in 6-13% differing CNV calls. In summary, TPE with SoftCTM enables reproducibility, automation, and standardisation at single-cell resolution. SoftCTM estimates (M = 58.9%, SD ±16.3%) reconcile the overestimation by molecular data extrapolation (RNA expression: M = 79.2%, SD ±10.5, DNA methylation: M = 62.7%, SD ±11.8%) and underestimation by CP (M = 35.9%, SD ±13.1%), providing a more reliable middle ground. A fully integrated computational pathology solution could therefore be used to improve downstream molecular analyses for research and clinics. © 2024 The Author(s). The Journal of Pathology published by John Wiley & Sons Ltd on behalf of The Pathological Society of Great Britain and Ireland.

Development of a Microphysiological Cartilage-on-Chip Platform for Dynamic Biomechanical Stimulation of Three-Dimensional Encapsulated Chondrocytes in Agarose Hydrogels.

Osteoarthritis (OA) is one of the most prevalent joint diseases globally, characterized by the progressive breakdown of articular cartilage, resulting in chronic pain, stiffness, and loss of joint function. Despite its significant socioeconomic impact, therapeutic options remain limited, largely due to an incomplete understanding of the molecular mechanisms driving cartilage degradation and OA pathogenesis. Recent advances in in vitro modeling have revolutionized joint tissue research, transitioning from simplistic two-dimensional cell cultures to sophisticated three-dimensional (3D) constructs that more accurately mimic the physiological microenvironment of native cartilage. Over the last decade, organ-on-chip technologies have emerged as transformative tools in tissue engineering, offering microphysiological platforms with precise control over biomechanical and biochemical stimuli. These platforms are providing novel insights into tissue responses and disease progression and are increasingly integrated into the early stages of drug screening and development. In this article, we present a detailed experimental protocol for constructing a cartilage-on-chip system capable of delivering controlled dynamic biomechanical stimulation to 3D-encapsulated chondrocytes in an agarose hydrogel matrix. Our protocol, optimized for both bovine and human chondrocytes, begins with Basic Protocol 1, detailing the preparation and injection of cell-laden hydrogels into the microdevice. Basic Protocol 2 describes the application of dynamic mechanical loading using a calibrated pressurized pump. Finally, Basic Protocols 3 and 4 focus on the retrieval of the hydrogel and RNA extraction for downstream molecular analyses. This platform represents a critical advancement for in vitro studies of cartilage biology, enabling more precise modeling of OA pathophysiology and evaluation of experimental therapeutics. © 2024 The Author(s). Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1: Cartilage-on-chip injection Basic Protocol 2: Cartilage-on-chip actuation Basic Protocol 3: Cartilage-on-chip agarose hydrogel removal Basic Protocol 4: Preparation of cartilage-on-chip for RNA extraction.

Functional genetics reveals modulators of antimicrotubule drug sensitivity.

Microtubules play essential roles in diverse cellular processes and are important pharmacological targets for treating human disease. Here, we sought to identify cellular factors that modulate the sensitivity of cells to antimicrotubule drugs. We conducted a genome-wide CRISPR/Cas9-based functional genetics screen in human cells treated with the microtubule-destabilizing drug nocodazole or the microtubule-stabilizing drug paclitaxel. We further conducted a focused secondary screen to test drug sensitivity for ∼1,400 gene targets across two distinct human cell lines and to additionally test sensitivity to the KIF11 inhibitor, STLC. These screens defined gene targets whose loss enhances or suppresses sensitivity to antimicrotubule drugs. In addition to gene targets whose loss sensitized cells to multiple compounds, we observed cases of differential sensitivity to specific compounds and differing requirements between cell lines. Our downstream molecular analysis further revealed additional roles for established microtubule-associated proteins and identified new players in microtubule function.

Abstract A036: Next gen liquid biopsy: Comprehensive analysis from a single tube of blood

Abstract Introduction There remains a largely untapped potential for utilizing liquid biopsy as a non- invasive, real-time window into tumor biology. In this study, we report on the use of the high- multiplex protein quantitation capability of the OrionTM spatial biology platform (RareCyte®, Inc) and cell picking capability of the CyteFinder® rare cell platform, to perform a comprehensive liquid biopsy analysis of whole blood that provides a deeper understanding of samples. This novel technology was used to characterize circulating tumor cells (CTCs), perform immune profiling of white blood cells (WBCs), and perform targeted mutation profiling of CTCs and cell-free DNA (cfDNA). All of these analytes can be measured from one 7.5 mL draw of blood. This array of tests was applied to cancer samples to demonstrate clinical feasibility. MethodsBlood samples were collected into AccuCyte® blood collection tubes. Buffy coats were processed to microscope slides using AccuCyte® blood processing kits. Slides were fixed and stained with an 18-plex immuno-oncology panel and imaged using the Orion Spatial Biology platform to evaluate protein biomarker expression on CTCs and to profile WBCs. Individual CTCs and WBCs from clinical samples were isolated using the CytePicker® for downstream molecular analysis. cfDNA was isolated from plasma collected during the AccuCyte process. Picked CTCs, WBCs, and cfDNA were sequenced using the CleanPlex® OncoZoom® Cancer Hotspot Kit. ResultsCancer patient blood was tested with a high-plex immunofluorescence assay using the Orion spatial biology system. CTCs were identified and phenotypically characterized in multiple cancer types. On the same slides, immunophenotyping of WBCs was performed. T- cell, B-cell, and macrophage populations were quantified. Additional sub-populations of T-cells (regulatory and memory) and proliferating cells (Ki67+) were enumerated. Expression of checkpoint-specific markers PD-L1 and PD-1 was measured on CTCs and WBCs. Sequencing results from individual picked CTCs showed evidence of minor clones representative of tumor heterogeneity that were not detectable in the cfDNA analysis. Conclusions This novel application of a high-plex spatial biology imaging system to liquid biopsies, in conjunction with the proven capabilities of rare cell detection and genomic analysis of single cells and cfDNA, has the potential to revolutionize liquid biopsy testing. From a single blood sample, this approach allows one to ask important questions about the complex interactions between the immune system and the tumor and to follow these interactions longitudinally in real time over the course of the disease. The goal is to leverage this unprecedented depth of analysis to improve patient outcomes and better understand the biological complexity of disease. Future directions of this work are to study rare types of WBCs that indicate immune activation and response to the tumor, and the detection and longitudinal monitoring of cell therapies such as CAR T cells after infusion. Citation Format: Arturo B Ramirez, Jon Ladd, Erin Bayer, Brock Bartels, Rachel Ponting, Arista Tischner. Next gen liquid biopsy: Comprehensive analysis from a single tube of blood [abstract]. In: Proceedings of the AACR Special Conference: Liquid Biopsy: From Discovery to Clinical Implementation; 2024 Nov 13-16; San Diego, CA. Philadelphia (PA): AACR; Clin Cancer Res 2024;30(21_Suppl):Abstract nr A036.

Methods for Selective Gene Expression Profiling in Single Tertiary Lymphoid Structure Using Laser Capture Microdissection.

Tertiary lymphoid structures (TLS) are de novo lymphoid formations that are induced within tissues during inflammatory episodes. TLS have been reported at various anatomic sites and in many different contexts like cancer, infections, autoimmunity, graft rejection, and idiopathic diseases. These inducible, ectopic, and transient lymphoid structures exhibit the prototypical architecture found within secondary lymphoid organs (SLO) and have been increasingly recognized as a major driver of local adaptive immune reaction. As TLS emerge within tissues, the isolation in situ and the molecular characterization of these structures are challenging to perform. Laser capture microdissection (LCM) is a powerful tool to isolate selective structural components and cells from frozen or paraffin-embedded tissues. We and other groups previously applied LCM to decipher the molecular network within TLS and uncover their intrinsic connection with the local microenvironment. In this chapter, we describe a detailed LCM method for selecting and isolating TLS in situ to perform comprehensive downstream molecular analyses.

Be bold, start cold! cold formalin fixation of colorectal cancer specimens granted superior DNA and RNA quality for downstream molecular analysis

The use of cold formalin fixation (CFF; i.e., fixating tissue samples with 4 °C precooled formalin) recently attracted further attention owing to its putative improved ability to preserve nucleic acid compared with standard room temperature formalin (SFF). In this study, we aimed to assess the effect of four formalin-based fixation protocols (SFF, CFF, delayed formalin fixation-DFF, and cold formalin hyperfixation; CFH) on both DNA and RNA quality. We collected 97 colorectal cancer (CRC) and analyzed 23 metrics of nucleic acid quantity and quality yield using a multiplatform approach by combining spectrophotometric, fluorimetric, electrophoretic, and polymerase chain reaction (PCR) assays. Following confirmation of fixation-protocol-related different effects via clustering analysis, CFF presented best metrics compared with all protocols, specifically positive coefficients of DV1000-60000, DV2/DV1, DNA λ ratio 260/230, and ABL gene expression absolute copies, and negative coefficient of DV150-1000. The SFF subgroup presented a positive coefficient of DV150-1000 and negative coefficients for DV1000-60000, DV2/DV1, RNA λ ratio 260/230, RNA QuBit concentration, DV100/200, RNA electrophoresis concentration and absolute quantity, and ABL copies. Overall, we confirmed the superior yield performances of CFF preservation for both DNA and RNA compared with the other protocols in our series of CRC samples. Pending further validations and clarification of the specific mechanisms behind these findings, our study supports the implementation of CFF in the pathology unit routine specimen management for tumor tissue molecular profiling.

Programmable Framework Nucleic Acid-Modified Nanomagnetic Beads for Efficient Isolation of Exosomes and Exosomal Proteomics Analysis.

Exosomes are increasingly being regarded as emerging and promising biomarkers for cancer screening, diagnosis, and therapy. The downstream molecular analyses of exosomes were greatly affected by the isolation efficiency from biosamples. Among the current exosome isolation strategies, affinity nanomaterials performed comparably better with selectivity and specificity. However, these techniques did not take the structure and size of exosomes into account, which may lead to a loss of isolation efficiency. In this article, a framework nucleic acid was employed to prepare a well-designed nanosized bead Fe3O4@pGMA@DNA TET@Ti4+ for enrichment of exosomes. The abundant phosphate groups in the framework nucleic acid provide binding sites to immobilize Ti4+, and its rigid three-dimensional skeleton makes them act as roadblocks to barricade exosomes and provide affinity interactions on a three-dimensional scale, resulting in the improvement of isolation efficiency. The model exosomes can be effectively isolated with 92% recovery in 5 min. From 100 μL of HeLa cell culture supernatant, 34 proteins out of the top 100 commonly identified exosomal proteins were identified from the isolated exosomes by the novel beads, which is obviously more than that by TiO2 (19 proteins), indicating higher isolation efficiency and exosome purity by Fe3O4@pGMA@DNA TET@Ti4+ beads. The nanobeads were finally applied for comparing exosomal proteomics analysis from real clinical serum samples. Twenty-five upregulated and 10 downregulated proteins were identified in the lung cancer patients group compared to the health donors group, indicating that the novel nanobeads have great potential in isolation of exosomes for exosomal proteomics analysis in cancer screening and diagnosis.

Non-Invasive Quality Control of Organoid Cultures Using Mesofluidic CSTR Bioreactors and High-Content Imaging.

Human brain organoids produce anatomically relevant cellular structures and recapitulate key aspects of in vivo brain function, which holds great potential to model neurological diseases and screen therapeutics. However, the long growth time of 3D systems complicates the culturing of brain organoids and results in heterogeneity across samples hampering their applications. We developed an integrated platform to enable robust and long-term culturing of 3D brain organoids. We designed a mesofluidic bioreactor device based on a reaction-diffusion scaling theory, which achieves robust media exchange for sufficient nutrient delivery in long-term culture. We integrated this device with longitudinal tracking and machine learning-based classification tools to enable non-invasive quality control of live organoids. This integrated platform allows for sample pre-selection for downstream molecular analysis. Transcriptome analyses of organoids revealed that our mesofluidic bioreactor promoted organoid development while reducing cell death. Our platform thus offers a generalizable tool to establish reproducible culture standards for 3D cellular systems for a variety of applications beyond brain organoids.

The impact of DNA extraction on the quantification of Legionella, with implications for ecological studies.

Monitoring the levels of opportunistic pathogens in drinking water is important to plan interventions and understand the ecological niches that allow them to proliferate. Quantitative PCR is an established alternative to culture methods that can provide a faster, higher-throughput, and more precise enumeration of the bacteria in water samples. However, PCR-based methods are still not routinely applied for Legionella monitoring, and techniques, such as DNA extraction, differ notably between laboratories. Here, we quantify the impact that DNA extraction methods had on downstream PCR quantification and community sequencing. Through a community science campaign, we collected 50 water samples and corresponding shower hoses, and compared two commonly used DNA extraction methodologies to the same biofilm and water phase samples. The two methods showed clearly different extraction efficacies, which were reflected in both the quantity of DNA extracted and the concentrations of Legionella enumerated in both the matrices. Notably, one method resulted in higher enumeration in nearly all samples by about one order of magnitude and detected Legionella in 21 samples that remained undetected by the other method. 16S rRNA amplicon sequencing revealed that the relative abundance of individual taxa, including sequence variants of Legionella, significantly varied depending on the extraction method employed. Given the implications of these findings, we advocate for improvement in documentation of the performance of DNA extraction methods used in drinking water to detect and quantify Legionella, and characterize the associated microbial community.IMPORTANCEMonitoring for the presence of the waterborne opportunistic pathogen Legionella is important to assess the risk of infection and plan remediation actions. While monitoring is traditionally carried on through cultivation, there is an ever-increasing demand for rapid and high-throughput molecular-based approaches for Legionella detection. This paper provides valuable insights on how DNA extraction affects downstream molecular analysis such as the quantification of Legionella through droplet digital PCR and the characterization of natural microbial communities through sequencing analysis. We analyze the results from a risk-assessment, legislative, and ecological perspective, showing how initial DNA processing is an important step to take into account when shifting to molecular-based routine monitoring and discuss the central role of consistent and detailed reporting of the methods used.

Microfluidic Interfaces for Chronic Bidirectional Access to the Brain.

Two-photon polymerization (TPP) is an additive manufacturing technique with micron-scale resolution that is rapidly gaining ground for a range of biomedical applications. TPP is particularly attractive for the creation of microscopic three-dimensional structures in biocompatible and noncytotoxic resins. Here, TPP is used to develop microfluidic interfaces which provide chronic fluidic access to the brain of preclinical research models. These microcatheters can be used for either convection-enhanced delivery (CED) or for the repeated collection of liquid biopsies. In a brain phantom, infusions with the micronozzle result in more localized distribution clouds and lower backflow compared to a control catheter. In mice, the delivery interface enables faster, more precise, and physiologically less disruptive fluid injections. A second microcatheter design enables repeated, longitudinal sampling of cerebrospinal fluid (CSF) over time periods as long as 250 days. Moreover, further in vivo studies demonstrate that the blood-CSF barrier is intact after chronic implantation of the sampling interface and that samples are suitable for downstream molecular analysis for the identification of nucleic acid- or peptide-based biomarkers. Ultimately, the versatility of this fabrication technique implies a great translational potential for simultaneous drug delivery and biomarker tracking in a range of human neurological diseases.

NGS method for parallel processing of high quality, damaged or fragmented input material using target enrichment.

Next-generation sequencing (NGS) has been increasingly popular in genomics studies over the last decade and is now commonly used in clinical applications for precision diagnostics. Many disease areas typically involve different kinds of sample specimens, sample qualities and quantities. The quality of the DNA can range from intact, high molecular weight molecules to degraded, damaged and very short molecules. The differences in quality and quantity pose challenges for downstream molecular analyses. To overcome the challenge with the need of different molecular methods for different types of samples, we have developed a joint procedure for preparing enriched DNA libraries from high molecular weight DNA and DNA from formalin-fixed, paraffin-embedded tissue, fresh frozen tissue material, as well as cell-free DNA.

Biochemical Separation of Cytoplasmic and Nuclear Fraction for Downstream Molecular Analysis.

Biochemical fractionation is a technique used to isolate and separate distinct cellular compartments, critical for dissecting cellular mechanisms and molecular pathways. Herein we outline a biochemical fraction methodology for isolation of ultra-pure nuclei and cytoplasm. This protocol utilizes hypotonic lysis buffer to suspend cells, coupled with a calibrated centrifugation strategy, for enhanced separation of cytoplasm from the nuclear fraction. Subsequent purification steps ensure the integrity of the isolated nuclear fraction. Overall, this method facilitates accurate protein localization, essential for functional studies, demonstrating its efficacy in separating cellular compartments. © 2024 The Authors. Current Protocols published by Wiley Periodicals LLC. Basic Protocol: Biochemical fractionation Support Protocol 1: Protein quantification using Bradford assay Support Protocol 2: SDS/PAGE and Western blotting.

Abstract 2771: Increased mtDNA copy number via EPOR signaling: A potential mechanism for metastatic dissemination in head and neck squamous cell carcinoma

Abstract Introduction: Head and Neck Squamous cell carcinoma (HNSCC) is characterized by high degree of invasion, locoregional metastasis, relapse which leads to overall poor prognosis. The molecular mechanism responsible for aggressiveness is poorly understood. A better understanding of molecular pathways involved shall precede the identification of newer diagnostic tools and therapeutic targets. We explored EPOR signaling pathway in Circulating tumor cells (CTCs) and HNSCC cell lines. Methods and Results: By using a validated in-house CTC isolation protocol, which addresses various drawbacks of currently available techniques and isolates CTCs suitable for various downstream molecular analysis, we isolated CTCs from HNSCC patients. The whole transcriptome analysis was performed from CTCs using ultra-low cell number RNA sequencing and corresponding primary tumors in 24 paired samples. We found a significant transcriptomic heterogeneity in CTCs, which are associated with overall disease outcome in HNSCC patients. To explore the plausible molecular pathways, we also identified 521 differentially expressed genes in CTCs with respect to primary tumors and various canonical pathways including oxidative phosphorylation, TCA cycle, cytokine-cytokine receptor interaction signaling, and ECM receptor interaction were enriched in CTC samples. Overexpression of erythropoietin receptor (EPOR) along with various genes encoded by mitochondrial genome and genes involved in maintenance of mtDNA in CTCs as compared to primary tumors was an important finding from our data. When analyzed in depth, there was a significant positive correlation between various genes involved in mtDNA maintenance (ABAT, NT5M) and encoded by mitochondrial genome (MT-ND1, MT-ND2, MT-ND4, MT-ND5, MT-ND6, MT-CO1, MT-CO2, MT-ATP6, MT-CYTB) with EPOR expression in our RNA seq data which is also substantiated by analyzing TCGA data. We confirmed these findings experimentally in HNSCC cell lines Cal 27 and FaDu. It was also observed that metastatic cell line FaDu has significantly higher mtDNA content as compared to non-metastatic cell line Cal 27, which was correlated with EPOR expression. Treatment with low dose Erythropoietin (0.1IU/mL) which a natural ligand for EPOR, enhanced mtDNA content in both the cell lines at low doses and boosted the metastatic capability while minimal effect on cell proliferation was observed. Conclusion: EPOR signaling might promote the metastatic ability of cancer cells and may act as a potential target for cancer dissemination and metastasis. Citation Format: Anshika Chauhan, Geeta S. Boora, Sahana Ghosh, Subrata K. Patra, Roshan K. Verma, Jaimanti Bakshi, Debajyoti Chatterjee, Radhika Srinivasan, Sushmita Ghoshal, Arindam Maitra, Arnab Pal. Increased mtDNA copy number via EPOR signaling: A potential mechanism for metastatic dissemination in head and neck squamous cell carcinoma [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 2771.

Abstract 6934: Integrating deep learning morphology profiling, cell sorting, and scRNASeq reveals sample diversity and enriched sub-populations

Abstract Advances in single cell technologies have ushered in a new multi-omics era that has provided more granular information on cell types and functions at unprecedented resolution. Morpholomics (the information encoded by cell morphology) has traditionally been used as a readout of cell identity, state, and function; but scalability, interpretation, and sorting based on morphology has remained challenging. The Deepcell® platform, REM-I, performs high-dimensional morphology analysis of unlabeled, single cells using deep-learning on high-resolution bright-field images captured in microfluidic flow to profile and sort target cells in real-time and at scale. Morphology analysis of single cells is achieved by a self-supervised deep learning foundation model, ‘Human Foundation Model’ (HFM), which combines deep learning features with morphometrics. HFM determines cell identities using morphological features associated with distinct processes characterized by granules, vesicles, cell size, pigmentation. and others. To demonstrate the ability of HFM modeling to distinguish and characterize a diversity of cell types representative of tumor microenvironments, we processed various sample types on the REM-I platform. Human melanoma, immune cells, stromal cells, and dissociated biopsies were combined to recapitulate heterogeneous tumor samples. HFM analysis enabled morphology-based exploration of the resulting bright-field images, revealing the presence of various morphologically distinct populations within each sample. Tumor cells were morphologically distinct and clustered separately from non-tumor cells. Immune cells exhibited subtle but separable morphologies, including T cells of distinct activation states. To demonstrate the morphology-based sorting capabilities of REM-I, we applied it to a ‘reference sample’ consisting of several fixed cell lines, at proportions ranging from 1% to >90%. Using only brightfield images of single cells analyzed through HFM, we used the platform to enrich each cell line from the ‘reference sample’ with high yield and purity, and identified cell types constituting a small percentage (~1%) of the total population. We next showed that unlabeled cells enriched on REM-I that are characterized as unperturbed and viable are amenable to downstream molecular analyses. These cell populations were sorted and the retrieved live cells were analyzed by scRNA-Seq (10x Chromium), demonstrating enrichment of various cell types of the tumor microenvironment in each cell population relative to the pre-sorted sample. These results illustrate: 1) the ability to perform downstream molecular analyses on sorted cells from REM-I, 2) the platform can be used to parse the tumor microenvironment, and 3) the link between morphology, cell identity, and molecular signatures. Citation Format: Andreja Jovic, Christian Corona, Kiran Saini, Tiffine Pham, Ryan Carelli, Vivian Lu, Senzeyu Zhang, Stephane C. Boutet, Maddison Masaeli. Integrating deep learning morphology profiling, cell sorting, and scRNASeq reveals sample diversity and enriched sub-populations [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 6934.

Abstract 5054: Optimization and evaluation of an FFPE dual extraction protocol for next-generation sequencing applications

Abstract Formalin-fixed paraffin-embedded (FFPE) biopsies are highly valuable and widely used tissue specimens for clinical diagnostics. However, obtaining sufficient and high-quality nucleic acid material from limited FFPE samples presents a challenge for downstream molecular analysis, such as next-generation sequencing (NGS). We present an optimized sequential extraction method that generates high-quality DNA and RNA from a single set of input tissues that is automatable and operation-friendly. This workflow performs well with reduced FFPE tissue input and efficiently supports various high-throughput clinical NGS applications. The DNA/RNA yield, quality, purity, and impacts on NGS assay performances were on par or better than an existing validated comparator extraction method. With comparable FFPE input, the new method demonstrated a superior extraction performance with significantly higher yield, quality, and purity. For DNA, NGS libraries were made with two different library preparation methods: a TA-ligation-based method and a single-strand-based method, followed by hybrid capturing and sequencing. The DNA from the new extraction method demonstrated a superior library conversion rate and improved target enrichment uniformity with both chemistries. This provides the potential of reduced input requirements, allowing very limited tissue, such as fine needle aspirate or core needle biopsy, for clinical NGS testing. The sequencing results were highly concordant between the existing and new extraction methods when the extracted DNA was subjected to a validated comprehensive genomic profiling (CGP) clinical test, demonstrating the quality and robustness of the extraction. For RNA, NGS libraries were made following the validated CGP clinical test with hybrid capturing, and the results were comparable between the two extraction methods. In conclusion, an FFPE extraction method was optimized to allow more clinical biopsy samples to be tested with different NGS workflows, providing a better diagnostic value for patient care. Citation Format: Rebecca Hong, Jieying Chu, Steven Hsiao, Daniel Whang, Steven P. Rivera, Heng Xie, Jiannan Guo. Optimization and evaluation of an FFPE dual extraction protocol for next-generation sequencing applications [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 5054.

A FACS-based novel isolation technique identifies heterogeneous CTCs in oral squamous cell carcinoma.

Isolating circulating tumour cells (CTCs) from the blood is challenging due to their low abundance and heterogeneity. Limitations of conventional CTC detection methods highlight the need for improved strategies to detect and isolate CTCs. Currently, the Food and Drug Administration (FDA)-approved CellSearch™ and other RUO techniques are not available in India. Therefore, we wanted to develop a flexible CTC detection/isolation technique that addresses the limitation(s) of currently available techniques and is suitable for various downstream applications. We developed a novel, efficient, user-friendly CTC isolation strategy combining density gradient centrifugation and immuno-magnetic hematogenous cell depletion with fluorescence-activated cell sorting (FACS)-based positive selection using multiple CTC-specific cell-surface markers. For FACS, a stringent gating strategy was optimised to exclude debris and doublets by side scatter/forward scatter (SSC/FSC) discriminator, remove dead cells by 4',6-diamidino-2-phenylindole (DAPI) staining, and eliminate non-specific fluorescence using a "dump" channel. APC-labelled anti-CD45mAB was used to gate remaining hematogenous cells, while multiple epithelial markers (EpCAM, EGFR, and Pan-Cytokeratin) and an epithelial-mesenchymal transition (EMT) marker (Vimentin) labelled with fluorescein isothiocyanate (FITC) were used to sort cancer cells. The technique was initially developed by spiking Cal 27 cancer cells into the blood of healthy donors and then validated in 95 biopsy-proven oral squamous cell carcinoma (OSCC) patients. CTCs isolated from patients were reconfirmed by Giemsa staining, immuno-staining, and whole transcriptome amplification (WTA), followed by qRT-PCR. In vitro culture and RNA sequencing (RNA-Seq) were also performed to confirm their suitability for various downstream applications. The mean detection efficiency for the Cal 27 tongue cancer cells spiked in the whole blood of healthy donors was 32.82% ± 12.71%. While ~75% of our patients (71/95) had detectable CTCs, the CTC positivity was independent of the TNM staging. The isolated potential cancer cells from OSCC patients were heterogeneous in size. They expressed different CTC-specific markers in various combinations as identified by qRT-PCR after WTA in different patients. Isolated CTCs were also found to be suitable for downstream applications like short-term CTC culture and RNA-Seq. We developed a sensitive, specific, flexible, and affordable CTC detection/isolation technique, which is scalable to larger patient cohorts, provides a snapshot of CTC heterogeneity, isolates live CTCs ready for downstream molecular analysis, and, most importantly, is suitable for developing countries.

Assessment of Strategies for Preserving Swine Viral RNA Targets in Diagnostic Specimens.

Successful downstream molecular analyses of viral ribonucleic acid (RNA) in diagnostic laboratories, e.g., reverse transcription-quantitative polymerase chain reaction (RT-qPCR) or next-generation sequencing, are dependent on the quality of the RNA in the specimen. In swine specimens, preserving the integrity of RNA requires proper sample handling at the time the sample is collected on the farm, during transport, and in the laboratory until RNA extraction is performed. Options for proper handling are limited to maintaining the cold chain or using commercial specimen storage matrices. Herein, we reviewed the refereed literature for evidence that commercial specimen storage matrices can play a role in preserving swine viral RNA in clinical specimens. Refereed publications were included if they compared RNA detection in matrix-treated vs. untreated samples. At present, the small number of refereed studies and the inconsistency in reported results preclude the routine use of commercial specimen storage matrices. For example, specimen storage matrices may be useful under specific circumstances, e.g., where it is mandatory to render the virus inactive. In a broader view, statistically sound side-by-side comparisons between specimens, viral RNA targets, and storage conditions are needed to establish if, when, and how commercial specimen storage matrices could be used in diagnostic medicine.

Extracting cell-free extracellular DNA from wastewater: Evaluation of extraction protocols for use in complex matrices

Cell-free extracellular DNA (exDNA) plays important roles in DNA-based surveillance of wastewater treatment processes and reclaimed water quality. Several techniques have been applied to extract cell-free exDNA in wastewater. However, the applicability and feasibility of these techniques has not been evaluated. Five different cell-free exDNA extraction techniques were evaluated in this study. By spiking multiple DNA standards in buffer system, it was shown that recovery efficiencies for the five extraction methods were dependent on the size of the DNA. ExDNA recovery was also influenced differently among the five methods by the water quality parameters including conductivity, pH and dissolved organic carbon. By sequencing exDNA samples, it was shown that extraction techniques affect the downstream PCR and functional diversity derived from exDNA. Overall, anion exchange method presents the most optimal performance to extract cell-free exDNA in wastewater due to highest recovery efficiency, reproducibility and success rate of PCR for downstream molecular analysis among the five evaluated methods.

A universal protocol for high-quality DNA and RNA isolation from diverse plant species

Next-generation sequencing demands high-quality nucleic acid, yet isolating DNA and RNA is often challenging, particularly from plant tissues. Despite advances in developing various kits and reagents, these products are tailored to isolation of nucleic acid from model plant tissues. Here we introduce a universal lysis buffer to separate nucleic acid from various plant species, including recalcitrant plants, to facilitate molecular analyses, such as quantitative PCR (qPCR), transcriptomics, and whole-genome sequencing (WGS). The protocol is a modification of the original CTAB methods, which leads to nucleic acid isolation from many plant species, including monocots and eudicots. The lysis buffer consists of hexadecyltrimethylammonium bromide (CTAB), sodium chloride (NaCl), Tris base, ethylenediaminetetraacetic acid (EDTA) and β-mercaptoethanol (βME). The modified CTAB method enables the isolation of nucleic acid from small amounts of plant tissues (e.g., 15–100 mg) in a timely manner, which is well-suited for a large number of samples and also when adequate sample collection is a limiting factor. The protocol isolates not only DNA from various plant species but also RNA. This makes it highly effective for molecular analyses compared to previously described CTAB methods optimised for DNA isolation. The appropriate concentration of the components enables high-quality DNA and RNA isolation from plant tissues simultaneously. Additionally, this protocol is compatible with commercially available columns. For DNA and RNA to be qualified for next-generation sequencing platforms, the protocol is supplemented with columns to purify either DNA or RNA from the same tissue to meet high standards for sequencing analyses. This protocol provides an ideal approach to overcome potential obstacles in isolating high-quality DNA or RNA from a wide range of plant species for downstream molecular analysis.