Simultaneous Detection Research Articles

Assay for the simultaneous detection of Raillietina spp. (R. echinobothrida, R. tetragona, and R. cesticillus) and Ascaridia galli infection in chickens using duplex loop-mediated isothermal amplification integrated with a lateral flow dipstick assay

Raillietina species and Ascaridia galli are two of the significant intestinal parasites that affect chickens in a free-range system production. They destroy the intestinal mucosa layer, leading to several clinical symptoms such as weight loss, a slowed growth rate, and economic value loss. Thus, the objective of this study was to develop an assay for simultaneously detecting Raillietina spp. (R. echinobothrida, R. tetragona, and R. cesticillus) and A. galli in a single reaction using duplex loop-mediated isothermal amplification (dLAMP) coupled with a lateral flow dipstick (LFD) assay. The analytical specificity of the dLAMP-LFD assay showed a high specific amplification of Raillietina spp. and A. galli without non-target amplification. Regarding the analytical sensitivity, this approach was capable of simultaneously detecting concentrations as low as 5 pg/μL of mixed-targets. To evaluate the efficiency of the dLAMP assay, 30 faecal samples of chickens were verified and compared through microscopic examination. The dLAMP-LFD assay and microscopic examination results showed kappa values of Raillietina spp. and A. galli with moderate (K= 0.615) to high (K= 1) agreements, respectively, while the McNemar’s test indicated that the efficiency between assays was not significantly different. Therefore, the developed dLAMP-LFD assay can be used as an alternative screening method to the existing classical method for epidemiological investigation, epidemic control, and farm management, as well as for addressing poultry health problems.

Viscoelastic parameters derived from multifrequency MR elastography for depicting hepatic fibrosis and inflammation in chronic viral hepatitis

ObjectivesThe capability of MR elastography (MRE) to differentiate fibrosis and inflammation, and to provide precise diagnoses is crucial, whereas the coexistence of fibrosis and inflammation may obscure the diagnostic accuracy.MethodsIn this retrospective study, from June 2020 to December 2022, chronic viral hepatitis patients who underwent multifrequency MRE (mMRE) were included in, and further divided into, training and validation cohorts. The hepatic viscoelastic parameters [shear wave speed (c) and loss angle (φ) of the complex shear modulus] were obtained from mMRE. The logistic regression and receiver operating characteristic (ROC) curves were generated to evaluate performance of viscoelastic parameters for fibrosis and inflammation.ResultsA total of 233 patients were assigned to training cohort and validation cohorts (mean age, 52 years ± 13 (SD); 51 women; training cohort, n = 170 (73%), and validation cohort, n = 63 (27%)). Liver c exhibited superior performance in detecting fibrosis with ROC (95% confidence interval) of ≥ S1 (0.96 (0.92–0.99)), ≥ S2 (0.86 (0.78–0.92)), ≥ S3 (0.89 (0.84–0.95)), and S4 (0.88 (0.83–0.93)). Similarly, φ was effective in diagnosing inflammation with ROC values of ≥ G2 (0.72 (0.63–0.81)), ≥ G3 (0.88 (0.83–0.94)), and G4 (0.92 (0.87–0.98)). And great predictive discrimination for fibrosis and inflammation were shown in validation cohort (all AUCs > 0.75).ConclusionThe viscoelastic parameters derived from multifrequency MRE could realize simultaneous detection of hepatic fibrosis and inflammation.Critical relevance statementFibrosis and inflammation coexist in chronic liver disease which obscures the diagnostic performance of MR elastography, whereas the viscoelastic parameters derived from multifrequency MR elastography could realize simultaneous detection of hepatic fibrosis and inflammation.Key points• Hepatic biomechanical parameters derived from multifrequency MR elastography could effectively detect fibrosis and inflammation.• Liver stiffness is useful for detecting fibrosis independent of inflammatory activity.• Fibrosis could affect the diagnostic efficacy of liver viscosity in inflammation, especially in early-grade of inflammation.Graphical

Screen-printed electrode containing bismuth/graphene oxide hybrid for simultaneous detection of cadmium and lead ions

The heavy metal ions pollution of the soil and water ecosystem resulted in thorny environmental issues throughout the world. Herein, a screen-printed electrode (SPE) with carbon ink mixed with bismuth/graphene oxide hybrid (Bi/GO-SPE) was successfully prepared to simultaneously determine Cd2+ and Pb2+. The detection range of Cd2+ is from 5 to 50 μg/L with the limit of detection (LOD) of 1.55 μg/L. The detection range of Pb2+ is from 5 to 50 μg/L with LOD of 1.31 μg/L. When the Bi/GO-SPE detects Cd2+ and Pb2+ simultaneously, the linear detection range is from 5 to 50 μg/L, with LOD of 2.43 μg/L for Cd2+ and 2.09 μg/L for Pb2+ respectively. The Bi/GO-SPE exhibited favorable conductivity, exceptional stability, and robust anti-interference. The Bi/GO-SPE holds great potential in the detection of heavy metal ions in real water samples.

Multiple Types of Cancer Classification Using Histopathological Images Based on Learning Without Forgetting Powered Deep Learning Models

Cancer detection represents a paramount challenge in modern medicine, where the ability to accurately identify malignancies across various anatomical regions can profoundly impact patient outcomes. In this study, we introduce an innovative approach for the simultaneous detection of brain, lung, and breast cancers using histopathological images, which are rich in cellular information critical for diagnosis. Through the utilization of Convolutional Neural Networks (CNNs), our system achieves remarkable accuracy in distinguishing cancerous tissues amidst complex histological backgrounds. The seamless integration of CNN models with Flask for backend deployment and HTML, CSS, and Python for frontend web development ensures a user-friendly interface conducive to both healthcare professionals and patients. Extensive validation of our methodology, conducted on diverse datasets encompassing a spectrum of cancer types and stages, underscores its robustness and reliability. The results obtained showcase not only the high accuracy of our system in detecting individual cancer types but also its versatility in concurrently predicting multiple malignancies. Furthermore, our approach demonstrates promising generalization capabilities, indicating its potential applicability across various healthcare settings and patient populations. By harnessing the power of advanced machine learning techniques, our research represents a significant leap forward in the realm of cancer diagnostics. The implementation of our methodology has the potential to revolutionize clinical practice by enabling earlier detection, personalized treatment planning, and improved patient outcomes.

Shigella Detection and Molecular Serotyping With a Customized TaqMan Array Card in the Enterics for Global Health (EFGH): Shigella Surveillance Study.

Quantitative polymerase chain reaction (qPCR) targeting ipaH has been proven to be highly efficient in detecting Shigella in clinical samples compared to culture-based methods, which underestimate Shigella burden by 2- to 3-fold. qPCR assays have also been developed for Shigella speciation and serotyping, which is critical for both vaccine development and evaluation. The Enterics for Global Health (EFGH) Shigella surveillance study will utilize a customized real-time PCR-based TaqMan Array Card (TAC) interrogating 82 targets, for the detection and differentiation of Shigella spp, Shigella sonnei, Shigella flexneri serotypes, other diarrhea-associated enteropathogens, and antimicrobial resistance (AMR) genes. Total nucleic acid will be extracted from rectal swabs or stool samples, and assayed on TAC. Quantitative analysis will be performed to determine the likely attribution of Shigella and other particular etiologies of diarrhea using the quantification cycle cutoffs derived from previous studies. The qPCR results will be compared to conventional culture, serotyping, and phenotypic susceptibility approaches in EFGH. TAC enables simultaneous detection of diarrheal etiologies, the principal pathogen subtypes, and AMR genes. The high sensitivity of the assay enables more accurate estimation of Shigella-attributed disease burden, which is critical to informing policy and in the design of future clinical trials.

Dual-Encoded Affinity Microbead Signature Combinatorial Profiling for Acute Myocardial Infarction High-Sensitivity Diagnosis.

The early diagnosis of acute myocardial infarction (AMI) is dependent on the combined feedback of multiple cardiac biomarkers. However, it remains challenging to precisely detect multicardiac biomarkers in complex blood early due to the lack of sensitive and specific diagnostic indicators and the low abundance and small size of associated biomarkers with high specificity (such as microRNAs). To make matters worse, spectral overlap significantly limits the multiplex analysis of cardiac biomarkers by fluorescent probes, leading to bias in the diagnosis of myocardial infarction. Herein, we developed a method for simultaneous detection of miRNAs and protein biomarkers using size- and color-coded microbeads that carry signature for target capture. We also constructed a microfluidic chip with different spacer arrays that segregate these microbeads in different chip regions according to their size to produce signature signals, indicating the level of different biomarkers. The signals on the microbeads were hugely amplified by catalytic hairpin assembly and rolling circle amplification. Notably, this strategy enables the simultaneous and in situ sensitive profiling of six kinds of biomarkers via adding two different fluorescent labels, removing the limitations of spectral overlap. We envision that the strategy has great potential for application in clinical diagnosis for AMI.

Development of a multiplex polymerase chain reaction assay for detection of hepatitis C virus, hepatitis B virus, and human immunodeficiency virus 1.

Hepatitis C virus (HCV), hepatitis B virus (HBV), and human immunodeficiency virus 1 (HIV-1) are the most epidemic blood-borne viruses, posing threats to human health and causing economic losses to nations for combating the infection transmission. The diagnostic methodologies that depend on the detection of viral nucleic acids are much more expensive, but they are more accurate than serological testing. To develop a rapid, cost-effective, and accurate diagnostic multiplex polymerase chain reaction (PCR) assay for simultaneous detection of HCV, HBV, and HIV-1. The design of the proposed PCR assay targets the amplification of a short conserved region featured with a distinguishable melting profile and electrophoretic molecular weight inside each viral genome. Therefore, this diagnostic method will be appropriate for application in both conventional (combined with electrophoresis) and real-time PCR facilities. Confirmatory in silico investigations were conducted to prove the capability of the approached PCR assay to detect variants of each virus. Then, Egyptian isolates of each virus were subjected to the wet lab examination using the given diagnostic assay. The in silico investigations confirmed that the PCR primers can match many viral variants in a multiplex PCR assay. The wet lab experiment proved the efficiency of the assay in distinguishing each viral type through high-resolution melting analysis. Compared to related published assays, the proposed assay in the current study is more sensitive and competitive with many expensive PCR assays. This study provides a simple, cost-effective, and sensitive diagnostic PCR assay facilitating the detection of the most epidemic blood-borne viruses; this makes the proposed assay promising to be substitutive for the mistakable and cheap serological-based assays.

Selected Ion Monitoring for Orbitrap-Based Metabolomics.

Orbitrap mass spectrometry in full scan mode enables the simultaneous detection of hundreds of metabolites and their isotope-labeled forms. Yet, sensitivity remains limiting for many metabolites, including low-concentration species, poor ionizers, and low-fractional-abundance isotope-labeled forms in isotope-tracing studies. Here, we explore selected ion monitoring (SIM) as a means of sensitivity enhancement. The analytes of interest are enriched in the orbitrap analyzer by using the quadrupole as a mass filter to select particular ions. In tissue extracts, SIM significantly enhances the detection of ions of low intensity, as indicated by improved signal-to-noise (S/N) ratios and measurement precision. In addition, SIM improves the accuracy of isotope-ratio measurements. SIM, however, must be deployed with care, as excessive accumulation in the orbitrap of similar m/z ions can lead, via space-charge effects, to decreased performance (signal loss, mass shift, and ion coalescence). Ion accumulation can be controlled by adjusting settings including injection time and target ion quantity. Overall, we suggest using a full scan to ensure broad metabolic coverage, in tandem with SIM, for the accurate quantitation of targeted low-intensity ions, and provide methods deploying this approach to enhance metabolome coverage.

Color- and background-free Raman-encoded lateral flow immunoassay for simultaneous detection of carbendazim and imidacloprid in a single test line

Multiple detection based on lateral flow immunoassay (LFA) holds significant research importance across various fields. Recently, the use of multi-color surface-enhanced Raman scattering (SERS) nanotags has enabled simultaneous detection of multiple targets within a single test line (T-line) in LFA, greatly enhancing detection efficiency, reliability, and application flexibility. However, challenges regarding signal overlap in the Raman-fingerprint region (400–1800 cm−1) and the difficulty in determining specific targets based on typical color analysis have persisted. In this study, we developed two types of nanoprobes that encode both color and Raman information to address these challenges in simultaneous multiple detection on a single T-line in SERS-LFA. The nanoprobes were encoded with distinguishable blue and red colors, respectively, enabling colorimetric study in multiplex LFA for qualitative analysis. Additionally, they were encoded with distinctive and non-overlapping Raman signals in the Raman-silent region (1800–2800 cm−1), ensuring accurate and sensitive detection by effectively avoiding interference from each other as well as background signals in the sample matrix. Using carbendazim (CBZ) and imidacloprid (IMI) as model targets, the color- and Raman-encoded SERS-LFA demonstrated promising potential for the simultaneous detection of CBZ and IMI in both standard and real samples, whether in a colorimetric or a SERS mode.

Simultaneous voltammetric detection of benzene, naphthalene and anthracene from water using boron-doped diamond electrode

In this study, a simple, fast and sensitive voltammetric detection using boron-doped diamond (BDD) electrode was proposed for simultaneous quantification of benzene (BZ), naphthalene (NF) and anthracene (AC) from priority organic pollutants class in real tap water. The electrochemical behaviors of individual and simultaneous BZ, NF and AC studied by cyclic voltammetry (CV) on BDD electrode showed a large separation between their oxidation potential, which allowed the development of simple simultaneous detection method. Differential-pulse voltammetry (DPV) technique operated at step potential of 5 mV and modulation amplitude of 200 mV enabled to reach the lowest limits of detection of 0.40 μM for BZ, 0.04 μM for NF and 0.70 nM for AC, which is appropriate for water quality control related to their environmental quality standards. No significant influence of chloride ions was found and the method was validated in real tap water and surface water spiked with known concentrations of BZ, NF and AC, which proved the practical utility of the method for water quality control.

Detection of Single Nucleotide Polymorphisms of Circulating Tumor DNA by Strand Displacement Amplification Coupled with Liquid Chromatography.

The detection of multiple single nucleotide polymorphisms (SNPs) of circulating tumor DNA (ctDNA) is still a great challenge. In this study, we designed enzyme-assisted nucleic acid strand displacement amplification combined with high-performance liquid chromatography (HPLC) for the simultaneous detection of three ctDNA SNPs. First, the trace ctDNA could be hybridized to the specially designed template strand, which initiated the strand displacement nucleic acid amplification process under the synergistic action of DNA polymerase and restriction endonuclease. Then, the targets would be replaced with G-quadruplex fluorescent probes with different tail lengths. Finally, the HPLC-fluorescence assay enabled the separation and quantification of multiple signals. Notably, this method can simultaneously detect both the wild type (WT) and mutant type (MT) of multiple ctDNA SNPs. Within a linear range of 0.1 fM-0.1 nM, the detection limits of BRAF V600E-WT, EGFR T790M-WT, and KRAS 134A-WT and BRAF V600E-MT, EGFR T790M-MT, and KRAS 134A-MT were 29, 31, and 11 aM and 22, 29, and 33 aM, respectively. By using this method, the mutation rates of multiple ctDNA SNPs in blood samples from patients with lung or breast cancer can be obtained in a simple way, providing a convenient and highly sensitive analytical assay for the early screening and monitoring of lung cancer.

A multiplex real-time RT-qPCR assay for simultaneous detection of porcine epidemic diarrhea virus, porcine deltacoronavirus, and swine acute diarrhea syndrome coronavirus.

Porcine epidemic diarrhea virus (PEDV), porcine deltacoronavirus (PDCoV), and swine acute diarrhea syndrome coronavirus (SADS-CoV) cause intestinal diseases with similar manifestations in suckling piglets. In this study, we developed a multiplex real-time PCR for differential diagnosis of PEDV, PDCoV, and SADS-CoV. The assay demonstrated high specificity with a detection limit of 5 copies/µl for each virus. The assay specifically detected PEDV, PDCoV, and SADS-CoV and excluded all other swine pathogens circulating in pigs. Furthermore, the assay exhibited satisfactory performance in analyzing clinical samples. The data indicate that the newly developed multiplex real-time PCR method can be applied for differential diagnosis of porcine enteric coronaviruses.

Simultaneous detection of testosterone, nandrolone, and boldenone esters in dried blood spots for doping control in sports by liquid chromatography-tandem mass spectrometry.

Testosterone, nandrolone, and boldenone, which are listed as doping substances on the World Anti-Doping Agency Prohibited List, are mostly available commercially in esterified forms. Isotope ratio mass spectrometry (IRMS) represents a key tool for identifying these substances, as they are hydrolyzed and discharged in the urine as pseudo-endogenous substances. However, IRMS, which comprises a complicated process, cannot achieve the direct detection of steroid esters in blood samples. These substances can be detected using dried blood spots (DBSs), reducing the impact of esterase hydrolysis. Here, a simultaneous liquid chromatography-tandem mass spectrometry method for detecting 28 steroid (13 testosterone, nine nandrolone, and six boldenone) esters was developed using three DBS types of samples, including a cellulose paper and polymer. The substances were first derivatized with methyloxime to increase their sensitivities (the limits of detection were <0.1-0.4, <0.1-0.9, and <0.1-0.9ng/mL for the testosterone, nandrolone, and boldenone esters, respectively). Further, the DBS absorbents were verified since the effect of interferences depended on it. Next, a study involving seven participants was conducted to detect intramuscularly administered testosterone enanthate (100 mg). Polymer and cellulose papers were used to collect blood from their upper arms and fingertips, respectively, and testosterone enanthate was identified and detectable at both blood-collection sites for up to 144 and 216 h, respectively. Furthermore, testosterone enanthate was detectable in the DBS samples stored under refrigeration after 6 months, indicating the stable nature of DBS.

Abstract 540: Identification of therapy resistance mediating extracellular vesicles in single-cell level via a novel microfluidic co-culture device development

Abstract Once cancer has metastasized, it is incurable because tumors evolve resistance to all systemically administered anti-cancer therapies. Resistance to therapy has classically been attributed to the presence of a cancer stem cell or genetic tumor cell heterogeneity. Extracellular vesicles (EVs) are nano-sized (40-1000 nm) vesicles that are released by all cells into the extracellular space. EVs carry diverse cargo dependent on the cell of origin and have been studied as a source of novel cancer-specific liquid biopsy biomarkers and contributors to tumor progression and metastasis. However, the paracrine function of EVs in promoting therapy resistance is under-studied, and the impact of EVs released from therapy-resistant cancer cells on surrounding therapy-sensitive cancer cells is unknown. In this study, we develop an innovative microfluidic device, TREVCo, to enable simultaneous single-cell trap, EV detection, and co-culture modules to evaluate EVs-mediated transfer of cellular information between therapy-resistant cancer cells and adjacent cells, and to characterize EVs released from resistant cells and determine their impact on the resistance phenotype of adjacent therapy-sensitive cancer cells. We utilize resistant cells from multiple prostate (PCa) cell lines that have been selected for therapy resistance by culture with increasing dosages of cancer drugs such as cisplatin, docetaxel. We use the matched untreated therapy-sensitive parental cell lines as the sensitive cells. We hypothesize a novel model of therapy resistance in lethal PCa: EVs released from resistant cells contain pro-resistance factors that are transferred to adjacent sensitive cells upon EV uptake, thus directly enabling therapeutic resistance. We will assess the impact of resistant cell-EVs at a single cell level via a novel device, TREVCo, that enables comprehensive downstream analysis. This single-cell approach enables dissection of critical EV-associated therapy-resistant factors within the large heterogeneity of resistant PCa. Co-culture of a single resistant cell and a hundred adjacent sensitive cells mimicking intra-tumoral interactions is also critical to demonstrate evidence of resistant cell secreting factors inducing a pro-resistance phenotype. These studies will be the first-in-field to assess the impact of resistant PCa cell-derived EVs at single-cell resolution. An understanding of resistant cell-derived EVs and the impact of that EV cargo on adjacent sensitive cells will provide critical insight into the acquisition of therapy resistance in lethal PCa. The novel approach to apply resistant cell-derived EVs in co-culture assay with sensitive cells will enable the screening of transmittable resistance-associated factors and provides critical opportunities for accelerating precision medicine for advanced PCa patients. Citation Format: Chi-Ju Kim, Sarah Amend, Kenneth Pienta. Identification of therapy resistance mediating extracellular vesicles in single-cell level via a novel microfluidic co-culture device development [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 540.

Abstract 1539: A comprehensive analytical validation framework for antibodies used in the CellScape multiplex immunofluorescence assay

Abstract Background: Multiplex immunofluorescence (mIF) is a powerful tool that enables deep phenotyping of cells within the tumor microenvironment (TME). Careful optimization of multiplex antibody panels for specificity, sensitivity and reproducibility is crucial to achieving robust data and is a precondition for assay repeatability and standardization. We describe our validation procedure for VistaPlex™ multiplex assay panels. Methods: Experiments were performed using the CellScape™ platform for precise spatial multiplexing. The platform enables automated cyclic mIF for readily available fluorophore-conjugated antibodies compatible with fresh frozen or FFPE tissues. The use of high dynamic range imaging further facilitates quantitative phenotyping, while excellent imaging resolution provides single-cell discrimination with high accuracy. To design a ready-to-use panel for immune cell phenotyping, individual antibodies were first screened for specificity and then optimized for labeling sensitivity. Signal-to-noise, inter-assay reproducibility, and intra-assay precision were evaluated across serial sections in a multi-stage testing matrix to validate each antibody in isolation and assembled into a panel. Resulting from this effort, we introduce a 15-plex ready-to-use VistaPlex immune profiling panel for human FFPE tissues. Results: Image analysis of signal-to-noise ratios demonstrated robust performance of multiplexed antibodies across multiple tissue sections and tissue types. Measurements of both signal and quantitative immune phenotyping across technical replicates showed a high degree of intra-assay precision and inter-assay reproducibility. The 15-plex panel was validated on human tonsil, lung, breast, and colon tissues. Conclusions: The CellScape platform enables simultaneous detection of multiple protein biomarkers on a single tissue section for deep immune cell profiling in the TME. Rigorously validated VistaPlex panels further streamline this spatial biology workflow and will aid in the generation of consistent and reliable results for end users. Citation Format: Charles E. Jackson, Arne Christians, Jannik Boog, Matthew H. Ingalls, Xenia Meshik, Adam Northcutt, Kayla E. Cashion, J Spencer Schwarz, Oliver Braubach. A comprehensive analytical validation framework for antibodies used in the CellScape multiplex immunofluorescence assay [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 1539.

Abstract 1470: Advancing cancer research: A novel PanCancer digital PCR tool for simultaneous detection of multiple hallmark mutations in BRAF and EGFR

Abstract The paradigm shift towards precision medicine in oncology emphasizes the importance of identifying specific genetic mutations driving cancer. Some hallmark mutations in cancer-associated genes, such as BRAF and EGFR, are important in analyzing many cancer types. With the emergence of digital PCR (dPCR) technology, high-sensitivity and quantitative mutation analysis can be performed, lending valuable support to cancer research. In this work, we introduce a new and innovative dPCR method, the dPCR PanCancer assays (RUO), designed for concurrently detecting multiple hallmark mutations in BRAF and EGFR. This work includes two novel PanCancer assays for BRAF and EGFR, each tailored to target a spectrum of mutations associated with these genes, facilitating a comprehensive mutation analysis. The dPCR assay for BRAF targets multiple V600 hallmark mutations, and the dPCR assay for EGFR targets various important deletions on exon 19. This also includes a reference gene as a control for PCR efficiency in the duplex reaction. Here, we present our initial data from various sample types, including blood, plasma and FFPE samples. Through a meticulously optimized dPCR setup, we achieved exceptional sensitivity and specificity, enabling the detection of multiple mutations in a single channel at allelic frequencies below 1%. Both dPCR PanCancer Kits (RUO) have potential use in research for pre-screening samples (e.g., prior to next-generation sequencing) or monitoring cancer cells. They simultaneously assess the mutations, reducing time and costs and saving sample material. Additionally, our technology is adaptable to other cancer-associated genes, where similar assays can potentially be developed. Overall, we have demonstrated that our dPCR PanCancer Kits (RUO) provide a robust, fast and efficient technology to identify critical mutations, ultimately enhancing our understanding of BRAF and EGFR-driven cancers. The dPCR PanCancer Kit is for research use only. Not for the diagnosis, prevention, or treatment of a disease. Citation Format: Claudia Kappmeier, Sherina Edward, Corinna Hochstein, Ellen Inga Bruske, Francesca Di Pasquale, Ronny Kellner. Advancing cancer research: A novel PanCancer digital PCR tool for simultaneous detection of multiple hallmark mutations in BRAF and EGFR [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 1470.

Abstract 2180: Modulation of immune infiltration patterns in inflammation-associated colorectal cancer through rice bran-based dietary strategies

Abstract The correlation between gut microbiota dysbiosis and inflammation-associated colorectal cancer (CRC) has generated considerable interest in dietary interventions targeting immune modulation. Our prior study demonstrated the colon cancer protective effects of rice bran (RB) and ex vivo Bifidobacterium longum fermented rice bran (FRB) diet intervention against azoxymethane/dextran sodium sulfate (AOM/DSS)-induced CRC in gut microbiota-intact specific pathogen-free mice (SPF). RB showed higher levels of protection compared to FRB against CRC, as evidenced by diminished neoplastic lesion size and restored colonic epithelial integrity. The major objective of the present study was to leverage cutting-edge multispectral imaging technique for simultaneous detection of multiple immune cells markers and for a comprehensive analysis of distinct immune landscapes. We also performed spatial correlations within the tumor microenvironment of AOM/DSS-SPF mice undergoing RB and FRB dietary interventions. The primary focus was on T cell subtypes including cytotoxic T cells, helper T cells, regulatory T (Treg) cells, natural killer (NK) cells, NKT cells, B cells and macrophages (MΦ) subtypes (M1 MΦ, and M2 MΦ) to uncover the intricacies of the immune landscape and the interplay for CRC prevention through dietary intervention. The results indicated that RB intervention for 15 weeks led to reductions in overall T cell subtypes in the colonic tissue, particularly Treg cells, which are associated with protecting tumors against immune surveillance in CRC. On the other hand, FRB intervention also resulted in reductions in overall T cell subtypes but exhibited higher infiltration of NK cells and proinflammatory M1 macrophages in the colonic tissue after 15 weeks. While an increased presence of NK cells might be beneficial in countering CRC, the infiltration of M1 macrophages could potentially lessen the impact of FRB compared to RB intervention in CRC protection. The outcomes suggest that the better protective efficacy of non-fermented RB diets is linked to the modulation of Treg cell infiltration, whereas the efficacy of the FRB diet is to some extent limited by the presence of M1 macrophages. Overall, this study provides valuable insights into the complex immune landscape influenced by RB and FRB interventions in a murine model of inflammation-induced CRC, and a novel mechanism for the immunomodulatory effects of dietary RB interactions with gut microbiota. In the future, observing immune modulation throughout diet intervention would enhance our understanding of the role played by these immune cells. Citation Format: Robin Kumar, Lakshmi Sai Pratyusha Bugata, Md. Imtiazul Kabir, Rajesh Agarwal, Elizabeth P. Ryan, Komal Raina. Modulation of immune infiltration patterns in inflammation-associated colorectal cancer through rice bran-based dietary strategies [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 2180.

Abstract 3706: Single cell and cell-free nucleic acid analyses of liquid biopsy specimens from a blood collection tube using a new droplet digital PCR system

Abstract Introduction: Liquid biopsy-based testing has increasingly been adopted by oncologists for biomarker-driven diagnostics, therapy selection, and patient monitoring. Unlike tissue-based approaches, the target analytes in blood specimens are often rare events that must be discriminated from a background of non-tumor derived material. Droplet digital PCR (ddPCR) is an ideal platform for detection of rare nucleic acid markers in the clinic, because it is highly sensitive and specific, robust, and quantitative. Additionally, the workflow is simple to adopt and execute. The QX600 System is a new ddPCR instrument designed for advanced multiplexing which enables simultaneous detection of up to 6 unique fluorophores in a single well. This capability can decrease the sub-sampling needed for detection of multiple analytes. The objective of our study was to develop multiplexed ddPCR assays for detection of cell-based and cell-free circulating biomarkers from a single tube of whole blood. Methods: Tumor cells were enriched from the buffy coat fraction of whole blood collected in fixative-containing cfDNA BCT based on size and morphology using a microfluidics-based system. Enriched tumor cells were enumerated by immunofluorescence (DAPI+/Pan-CK+/CD45-), and gene expression was measured using the QX600 for a panel of cytokeratins (CK) and PD-L1 in CTC negative, NSCLC donor specimens (n=8). Results were compared to CTC positive, contrived specimens (whole blood spiked with breast tumor cell line MCF7 cells; n=5). For ctDNA analysis of the plasma fraction, a multiplexed ddPCR assay was designed to test for five actionable mutations prevalent in NSCLC, including EGFR del19, L858R, T790M, KRAS G12C, BRAF V600E, and wildtype EGFR. To evaluate performance of the multiplexed assay, concordance evaluation was conducted using cfDNA from 8 mutation-positive NSCLC donors on the QX600 and compared to single assays run on the QX200 system. Results: CK8 and CK19 were differentially expressed in the cells enriched from the CTC negative NSCLC cohort as compared to the contrived positives (p=0.0058, and p=0.0049, respectively). All the NSCLC cfDNA samples were positive for the reference variant, and linear regression analysis of the minor variant frequency for ctDNA biomarkers showed good concordance between QX systems (R2=0.8847). Conclusions: These studies demonstrate proof of concept for single cell and cell-free analysis from a single blood draw into a cfDNA BCT using the QX600 system. Liquid biopsy analysis of CTCs and ctDNA can enable minimally invasive, comprehensive decision making in the diagnostic workup of patients. Future studies will focus on further optimizing the cell enrichment methodology from cfDNA BCT and development of additional multiplexed ddPCR assays for NSCLC-relevant biomarkers on the QX600. Citation Format: Leisa Jackson, Colin Cochran, Claire Gould, Sarah Kreston, Jazmin Bello, Brittany D’Alessio, Janice Riley, Alisa Tubbs, Gary A. Pestano. Single cell and cell-free nucleic acid analyses of liquid biopsy specimens from a blood collection tube using a new droplet digital PCR system [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 3706.

A wearable microfluidic system for efficient sweat collection and real-time detection

Sweat is an important biofluid with rich physiological information that can evaluate human health condition. Wearable sweat sensors have received widespread attention in recent years due to the benefits of non-invasive, continuous, and real-time monitoring. Currently, an efficient device integrating sweat collection and detection is still needed. Here, a wearable sweat microfluidic system was fabricated for real-time collection and analysis of sweat. The fabricated microfluidic system consisted of four layers, including a skin adhesive layer, a microfluidic layer, an electrode layer, and a capping layer. The sweat collection rate was around 0.79 μL/min, which demonstrated efficient sweat sampling, storage, and refreshing capabilities. Simultaneous detection of multiple sweat biomarkers was achieved with a screen-printed sweat sensing array, which could realize high-precision detection of Na+, K+, and glucose. Moreover, the sensing array also showed good repeatability and stability, with a relative standard deviation of sensitivity of less than 5%. Additionally, human testing was conducted to demonstrate that this microfluidic system can continuously monitor Na+, K+, and glucose in subjects' sweat during exercise, which showed high potential for non-invasive human health monitoring.

Abstract 94: Interrogation of STING induced cytokines in the tumor microenvironment using an Integrated MultiOmyx-RNAscope panel for spatial and quantitative profiling

Abstract The STING (Stimulator of Interferon Genes) pathway is a pivotal player in the innate immune response, responsible for detecting cytosolic accumulation of endogenous DNA and initiating a cascade of events leading to the production of pro-inflammatory type I interferons, which promote an antitumor environment through activation of T cells, dendritic cells, and natural killer cells. Therefore, STING agonists are currently being explored as a potential therapeutic for cancer treatment, often in combination with immune checkpoint inhibitors. While the STING pathway can be stimulated in multiple cell types, activation in antigen presenting cells, such as dendritic cells (DC), is crucial for antitumor activity in the tumor microenvironment (TME). Additionally, the type 1 interferon family consists of multiple subtypes, including IFNb and IFNa, which can produce differential biological responses. Although the STING pathway is known to induce expression of multiple cytokines, combined spatial characterization of STING and type I interferons in the TME has not been performed. To characterize STING-interferon expression in the TME, we use the Integrated MultiOmyx-RNAscope platform. MultiOmyxTM (NeoGenomics Laboratories, Inc) is a proprietary multiplex immunofluorescence (mIF) platform for the visualization and characterization of up to 60 protein biomarkers in a single formalin-fixed paraffin-embedded (FFPE) section and offers high-resolution spatial and quantitative analysis of protein expression in tissue samples. RNAscopeTM (Bio-Techne) Multiplex is a highly sensitive fluorescent in-situ hybridization (ISH) assay that can detect up to 3 RNA markers in a single FFPE section. The Integrated MultiOmyx-RNAscope assay allows for simultaneous detection of both protein and RNA markers in a single sample. Herein we report the design and use of a novel panel of commercially-available antibodies and ISH probes broad enough to characterize various immune subpopulations and cytokine expressing cells, including DCs and interferons, in the TME of a variety of tumor indications including melanoma and head and neck squamous cell carcinoma. Quantification and analysis will be performed using NeoLYTXTM, the proprietary MultiOmyx Analytics pipeline, to examine the spatial distribution and expression levels of key STING pathway induced cytokines to elucidate the dynamic communication of signaling molecules and their localization within specific cell populations. Understanding of the variety and phenotype of STING/cytokine expressing cells in the TME is crucial to define the populations being targeted by therapies for cancer treatment. Citation Format: Sandra Lam, Courtney Todorov, Jiong Fei, Harry Nunns, Eric Leones, Marianne Thio, Flora Sahafi, Erinn Parnell, Qingyan Au. Interrogation of STING induced cytokines in the tumor microenvironment using an Integrated MultiOmyx-RNAscope panel for spatial and quantitative profiling [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 94.