Probe Library Research Articles

Biarsenical-based fluorescent labeling of metallothioneins as a method for ultrasensitive quantification of poly-Cys targets.

Mammalian metallothioneins (MTs) play a crucial role in maintaining Zn(II) and Cu(I) homeostasis, as well as regulating the cellular redox potential. They are involved in cancer resistance to cisplatin-related drugs and the sequestration of toxic metal ions. To investigate their participation in specific physiological and pathological processes, it is imperative to develop an analytical method for measuring changes in protein concentration both in vitro and in vivo. Over the years, numerous methods have been developed for this purpose; however, they often suffer from issues such as low sensitivity, lengthy sample preparation, or variability in antibody batches. In this study, we present, for the first time, the application of the fluorescent biarsenical probe CrAsH-EDT2 for the specific and ultrasensitive detection of human MTs. The interaction between the small library of biarsenical probes and 11 human MT isoforms (MT1-MT4), isolated MT2 domains, snail LlMT, and plant phytochelatin PC4 was investigated using spectrofluorimetry and mass spectrometry. A key advantage of our method is that the probe excess does not need to be removed before measurement as unbound is virtually non-fluorescent. We established protocols for MT detection in various assays, including cuvette-based, SDS-PAGE, and capillary electrophoresis. A detection limit (LOD) of 100fg for human MT2 was achieved. Finally, we observed increased metallothionein concentration in lysates of HeLa cells treated with ZnSO4. In conclusion, our results demonstrate that the established method is easy to apply, versatile, and can be adapted to detect metallothioneins and other poly-Cys targets in different assays and applications.

P0151 Multi-modal single cell sequencing identifies CCR9 expression in lamina propria with highest protein expression on CD4 tissue resident memory cells in the inflamed ileum in Crohn’s disease

Abstract Background Crohn’s Disease (CD) is a chronic condition that can impact any part of the gastrointestinal tract. The sustained inflammation is driven by the continuous influx of inflammatory leukocytes into the gut mucosa, which is regulated through chemokine gradients and adhesion molecules, including integrins. In the gut, the migration of inflammatory cells to specific segments is thought to be mediated by selected integrins and/or chemokine axes. The CCL25/CCR9 axis has been identified as a key pathway for immune cell migration into small bowel segments, such as the ileum. In this study, CITE-seq and spatial transcriptomic analysis was used to integrate proteomic and transcriptomic profiles to characterise CCR9+ immune cells in the ileum compared to the colon. Methods Biopsies were obtained via ileocolonoscopy from macroscopically inflamed and normal mucosa with appropriate consent from healthy volunteers and IBD patients visiting the Department of Gastroenterology at the Sahlgrenska Hospital. Biopsies were frozen in Cryostor CS10 or formalin fixed and paraffin embedded. Cells from frozen biopsies were sorted, CD45+ cells stained with CCR9 and Totalseq-C human universal antibody cocktail, followed by amplification, separation of antibody-derived DNA from mRNA derived cDNA, library construction and sequencing of both libraries. For spatial transcriptomics, tissue sections were deparaffinised, stained with H&E and decrosslinked before human whole transcriptome probe hybridisation, ligation, extension, library construction and sequencing. Cells were annotated using a transfer learning approach combining several reference single cell datasets including detailed CD4+ and CD8+ T cell datasets and utilising both antibody data and mRNA transcriptomic data. Results CCR9 positive cells appeared scattered throughout the lamina propria and expression at mRNA level was enriched in the ileum compared to the colon with about 10% of CD4 and 17% of CD8 T cells expressing CCR9. Interestingly at protein level, expression was more restricted with the highest expression on cells that exhibited proteomic and transcriptomic features associated with resident CD4 memory T cells (Trm). This population almost exclusively (>90%) consisted of CCR9 positive cells and their proteomic and transcriptomic profile consistent with the recently described effector and innate-like phenotype of Trm cells that is thought to contribute to Crohn’s disease pathology. Conclusion These data may point to CCR9 positive cells, and in particular Trm cells, in the ileum as important pathological cells in Crohn’s disease and provide further support for depleting CCR9 cells as a potential therapeutic target for the treatment of ileal Crohn’s disease.

C2-Linked Arabinose-Functionalized Polystyrene Microbeads Selectively Target Staphylococcus aureus.

Carbohydrates play pivotal roles in the first stages of microbial infections and can be exploited as decoys to hijack the interactions between bacteria and the host cell. Multivalent glycan probes mimicking the natural presentation of glycans in living cells have been successfully employed to study fundamental carbohydrate/protein interactions in microbial systems; however, most pathogenic glycan receptors exhibit a shared specificity for commonly found sugars present in both healthy and pathogenic cells, posing a challenge for target selectivity. In this study, we report the synthesis of a small library of d-arabinose multivalent probes, a sugar absent in human physiology, and their evaluation in a bacteria agglutination assay using cluster analysis. Our findings reveal preferential binding to Staphylococcus aureus of C2-linked arabinose moieties over C1- or C5-linked probes, underscoring the importance of glycan presentation in targeting specificity. Furthermore, we demonstrate the selectivity of the C2-linked probe toward S. aureus across a panel of common bacterial pathogens. Additionally, these probes are able to disrupt biofilm formation in S. aureus SH1000, thereby further proving the cell surface interactions with S. aureus.

Identification of a pancreatic juice-specific fluorescent probe through 411 probes activated by aminopeptidases/proteases or phosphatases/phosphodiesterases

BackgroundThis study is a retrospective review aimed to identify pancreatic juice-specific fluorescent probes to visualize pancreatic juice using a library of 381 aminopeptidase/protease-activatable fluorescent probes and 30 phosphatase/phosphodiesterase probes. In 2013, we developed a fluorescence imaging technique using a chymotrypsin probe to visualize pancreatic juice, linked to postoperative pancreatic fistula (POPF). This probe required addition of trypsin to convert pancreatic chymotrypsinogen to chymotrypsin. Recently we accessed libraries of enzyme-activatable fluorescent probes to find probes that facilitated target-specific imaging. MethodsPancreatic juice and ascitic fluid samples were collected in eight patients undergoing pancreaticoduodenectomy. Reaction rates of pancreatic juice to background ascitic fluids were calculated for these 411 fluorescent probes. ResultsForty-four fluorescent probes were screened in terms of high reactivity with pancreatic juice. Only one candidate probe targeting ectonucleotide pyrophosphatase/phosphodiesterase (ENPP) 1 was selected for a pancreatic juice-specific fluorescent probe. Inhibitor experiments and Western blotting supported the presence of ENPP1 in the pancreatic juice. ConclusionENPP1-targeting fluorescent probe may have the potential to visualize pancreatic juice leakage during surgery. This finding may allow surgeons to suture leaking sites and decide the necessity of prophylactic abdominal drains; however, the role of ENPP1 in pancreatic juice remains to be clarified.

Encodable DNA Hairpin Probes for Nanopore Multiplexed Target Detection.

Owing to the co-occurrence of hazardous compounds, it is crucial to build multiple highly discriminative probe libraries for simultaneous determination. Drawing inspiration from nucleic acid barcodes, we developed a probe system that is exclusively based on the nucleic acid secondary structure's hairpin structure, which can be directly read by nanopores. The highly distinguishable hairpin probes were constructed, and a detailed explanation of the possible patterns in their design was provided. These probe-representative events measured through the α-hemolysin (α-HL) nanopores were both distinguished, either through visual observation or comparison of the nanopore parameters. Besides, the potential design pattern for probes with unique telegraphic switching between the two levels was also unveiled. Finally, these probes were utilized to realize simultaneous, ultrasensitive mycotoxin multiple-detection, and their prospective applications for the detection of proteins and microRNAs were presented, indicating their suitability for a wide range of sensing applications.

PRMT1 inhibition perturbs RNA metabolism and induces DNA damage in clear cell renal cell carcinoma

In addition to the ubiquitous loss of the VHL gene in clear cell renal cell carcinoma (ccRCC), co-deletions of chromatin-regulating genes are common drivers of tumorigenesis, suggesting potential vulnerability to epigenetic manipulation. A library of chemical probes targeting a spectrum of epigenetic regulators is screened using a panel of ccRCC models. MS023, a type I protein arginine methyltransferase (PRMT) inhibitor, is identified as an antitumorigenic agent. Individual knockdowns indicate PRMT1 as the specific critical dependency for cancer growth. Further analyses demonstrate impairments to cell cycle and DNA damage repair pathways upon MS023 treatment or PRMT1 knockdown. PRMT1-specific proteomics reveals an interactome rich in RNA binding proteins and further investigation indicates significant widespread disruptions in mRNA metabolism with both MS023 treatment and PRMT1 knockdown, resulting in R-loop accumulation and DNA damage over time. Our data supports PRMT1 as a target in ccRCC and informs a mechanism-based strategy for translational development.

Exploring karyotype by developing novel oligonucleotide probes for Leucanthemum paludosum and other plant species

Leucanthemum paludosum is a small, popular, perennial herbaceous flower that is widely used as a potted plant and in flower beds. It is highly adaptable and fast-growing, blooms from early to late spring, and can be maintained for two–three months, thus making it a potential model plant for cytological studies. However, it lacks applicable markers for identifying its chromosome numbers, and no karyotyping or cytological study about L. paludosum has been reported. In the present study, we aimed to analyze the morphology and chromosomal status of L. paludosum to provide a reference for its systematic utilization. The results showed that L. paludosum is diploid (2n = 2x = 18). The bivalent number was substantially higher than the monovalent bodies. L. paludosum also conducted a loosely patterned karyotype, with no fluorescence in situ hybridization (FISH) signal when using a conventional oligonucleotide (oligo) probe library. To address this problem, we developed a novel repetitive oligo probe ((ATC)10) using transcriptome sequences. Furthermore, we explored its feasibility in different plants and provided effective repetitive oligoprobes for species such as Avena sativa, Vicia faba, Allium sativum, Lilium davidii var. unicolor, and Antirrhinum majus. Our study provides new cytological data for L. paludosum and developed a novel method for designing oligo probes for karyotype detection.

Flaring Inflammation and ER Stress by an Organelle-Specific Fluorescent Cage.

The endoplasmic reticulum (ER) plays an important role in protein synthesis and its disruption can cause protein unfolding and misfolding. Accumulation of such proteins leads to ER stress, which ultimately promotes many diseases. Routine screening of ER activity in immune cells can flag serious conditions at early stages,but the current clinically used bio-probes have limitations. Herein, an ER-specific fluorophore based on abiocompatiblebenzothiadiazole-imine cage (BTD-cage) with excellent photophysical properties is developed. The cage outperforms commercially available ER stains in long-term live cell imaging with no fading or photobleaching over time.The cageisresponsive to different levels of ER stress whereitsfluorescence increases accordingly. Incorporating the bio-probe into an immune disorder model, a6-, 21-, and 48-foldincrease in intensity is shown in THP-1, Raw 246.7, and Jurkat cells, respectively (within 15min).Theseresults strongly support that this system can be used for rapid visual and selective detection of ER stress. It is envisaged that tailoring molecular interactions and molecular recognition usingsupramolecular improved fluorophorescan expand the library of biological probes for enhanced selectivity and targetability toward cellular organelles.

Dynamics of collagen oxidation and cross linking in regenerating and irreversibly infarcted myocardium

In mammalian hearts myocardial infarction produces a permanent collagen-rich scar. Conversely, in zebrafish a collagen-rich scar forms but is completely resorbed as the myocardium regenerates. The formation of cross-links in collagen hinders its degradation but cross-linking has not been well characterized in zebrafish hearts. Here, a library of fluorescent probes to quantify collagen oxidation, the first step in collagen cross-link (CCL) formation, was developed. Myocardial injury in mice or zebrafish resulted in similar dynamics of collagen oxidation in the myocardium in the first month after injury. However, during this time, mature CCLs such as pyridinoline and deoxypyridinoline developed in the murine infarcts but not in the zebrafish hearts. High levels of newly oxidized collagen were still seen in murine scars with mature CCLs. These data suggest that fibrogenesis remains dynamic, even in mature scars, and that the absence of mature CCLs in zebrafish hearts may facilitate their ability to regenerate.

Global profiling of functional histidines in live cells using small-molecule photosensitizer and chemical probe relay labelling.

Recent advances in chemical proteomics have focused on developing chemical probes that react with nucleophilic amino acid residues. Although histidine is an attractive candidate due to its importance in enzymatic catalysis, metal binding and protein-protein interaction, its moderate nucleophilicity poses challenges. Its modification is frequently influenced by cysteine and lysine, which results in poor selectivity and narrow proteome coverage. Here we report a singlet oxygen and chemical probe relay labelling method that achieves high selectivity towards histidine. Libraries of small-molecule photosensitizers and chemical probes were screened to optimize histidine labelling, enabling histidine profiling in live cells with around 7,200 unique sites. Using NMR spectroscopy and X-ray crystallography, we characterized the reaction mechanism and the structures of the resulting products. We then applied this method to discover unannotated histidine sites key to enzymatic activity and metal binding in select metalloproteins. This method also revealed the accessibility change of histidine mediated by protein-protein interaction that influences select protein subcellular localization, underscoring its capability in discovering functional histidines.

Chemogenic albumin-seeking NIR dyes for in vivo site-specific albumin tagging

In vivo receptor tagging with contrast agent is considered an effective approach to achieve targeted bioimaging and imaging-guided surgery in the near-infrared-II (NIR-II) region, yet there has been a lack of systematic investigation into their binding mechanism for efficient in vivo targeted imaging. In this study, we design and synthesize a wide range of chemogenic albumin-seeking dyes to systematically investigate relationship between dye structures and binding behavior in situ. The albumin-seeking dye with optimized structures can undergo supramolecular interaction followed by covalent binding with albumin to form stable dye@albumin. This dye@albumin acts as an artificial fluorescent protein, in which a dye chromophore is wrapped by a natural protein that exists abundantly in living body. We further explain the much-improved photostability of dye@albumin from the perspective of photooxidation owing to their similar structures with classical fluorescent proteins. Among the synthesized albumin-seeking dyes, the optimized dye IR-6B5 exhibits comparable brightness and photostability to the previously reported IR-780, but offers superior NIR-II targeting bioimaging for tumors. Our designed albumin-seeking dyes enrich the NIR probe library and open up new possibilities for in vivo imaging and tumor-targeted guided surgery.

Abstract 3658: Longitudinal tumor-informed ctDNA monitoring with the originally developed OTS-Assay system

Abstract Background: Cancer Genome Profiling (CGP) tests are now widely available in daily practice. However, only a very small fraction of patients have the opportunity for genome-guided therapies. Somatic mutations from cancer specimens are unique to individual patients and hold utility as individual tumor markers in blood. We have developed an original circulating tumor DNA (ctDNA) monitoring system, called OTS (Off-The-Shelf)-Assay, at Iwate Medical University Hospital, Yahaba, Japan. Patients and Methods: Between April 2022 and October 2023, 79 patients who received the OTS-Assay were enrolled in the present analysis. The OTS-Assay includes three steps: (a) OTS-Scan (somatic mutation identification by either tissue or blood), (b) OTS-Select (somatic mutation selection), and (c) OTS-Monitor (periodical somatic mutation quantification as ctDNA). For the OTS-Select, we established an originally developed >90 stepwise selection algorithm. One of the determinants is the availability of appropriate digital PCR (dPCR) probes for individual patients. Here, we search the probes for individual patients from the dPCR probe library, OTS-1000ex, which consists of >1000 probes against frequently reported human somatic mutations (Quantdetect, Tokyo, Japan). Results: All 79 patients were subject to the analysis. The cancer stage of all the patients was advanced. More than 10 tumor types were enrolled, including esophagus (n=42), colorectal (n=12), breast (n=7), lung (n=4), pancreas (n=3), hematological (n=3), and others (n=8). Fifty patients received OTS-Scan, and 29 patients had received a CGP test before the OTS-Assay (i.e., OTS-Scan was not needed). Seventy-five patients received OTS-Select, meaning that four patients had somatic mutations that were designated for ctDNA monitoring. All 79 patients received at least one OTS-monitor. The number of OTS-Monitors received ranged from 1 to 11 during the 19-month period, and the average interval was 3.0 months. The average pretreatment variant allele frequency (VAF) was 4.96% and the majority of the follow-up VAF was less than 1%. More than 90% of the patients had at least one clinical validity defined by: (a) early relapse prediction, (b) treatment efficacy evaluation, or (c) no relapse corroboration. Conclusion: The OTS-Assay system provides stable longitudinal ctDNA monitoring at the range of less than 1% of VAF using the originally developed dPCR probe library. The sensitivity and affordability of the OTS-Assay system allows frequent monitoring, which leads to obtaining important clinical information in the context of advanced cancer therapy. Citation Format: Satoshi S. Nishizuka, Hayato Hiraki, Akiko Yashima-Abo, Takeshi Iwaya. Longitudinal tumor-informed ctDNA monitoring with the originally developed OTS-Assay 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 3658.

Abstract 1518 Development of a diverse activity-based probe library for studying serine proteases

Abstract 1518 Development of a diverse activity-based probe library for studying serine proteases

Development and application of whole-chromosome painting of chromosomes 7A and 8A of Arachis duranensis based on chromosome-specific single-copy oligonucleotides.

For peanut, the lack of stable cytological markers is a barrier to tracking specific chromosomes, elucidating the genetic relationships between genomes and identifying chromosomal variations. Chromosome mapping using single-copy oligonucleotide (oligo) probe libraries has unique advantages for identifying homologous chromosomes and chromosomal rearrangements. In this study, we developed two whole-chromosome single-copy oligo probe libraries, LS-7A and LS-8A, based on the reference genome sequences of chromosomes 7A and 8A of Arachis duranensis. Fluorescence in situ hybridization (FISH) analysis confirmed that the libraries could specifically paint chromosomes 7 and 8. In addition, sequential FISH and electronic localization of LS-7A and LS-8A in A. duranensis (AA) and A. ipaensis (BB) showed that chromosomes 7A and 8A contained translocations and inversions relative to chromosomes 7B and 8B. Analysis of the chromosomes of wild Arachis species using LS-8A confirmed that this library could accurately and effectively identify A genome species. Finally, LS-7A and LS-8A were used to paint the chromosomes of interspecific hybrids and their progenies, which verified the authenticity of the interspecific hybrids and identified a disomic addition line. This study provides a model for developing specific oligo probes to identify the structural variations of other chromosomes in Arachis and demonstrates the practical utility of LS-7A and LS-8A.

Selective labelling of GBA2 in cells with fluorescent β-d-arabinofuranosyl cyclitol aziridines.

GBA2, the non-lysosomal β-glucosylceramidase, is an enzyme involved in glucosylceramide metabolism. Pharmacological inhibition of GBA2 by N-alkyl iminosugars is well tolerated and benefits patients suffering from Sandhoff and Niemann-Pick type C diseases, and GBA2 inhibitors have been proposed as candidate-clinical drugs for the treatment of parkinsonism. With the ultimate goal to unravel the role of GBA2 in (patho)physiology, we sought to develop a GBA2-specific activity-based probe (ABP). A library of probes was tested for activity against GBA2 and the two other cellular retaining β-glucosidases, lysosomal GBA1 and cytosolic GBA3. We show that β-d-arabinofuranosyl cyclitol aziridine (β-d-Araf aziridine) reacts with the GBA2 active site nucleophile to form a covalent and irreversible bond. Fluorescent β-d-Araf aziridine probes potently and selectively label GBA2 both in vitro and in cellulo, allowing for visualization of the localization of overexpressed GBA2 using fluorescence microscopy. Co-staining with an antibody selective for the lysosomal β-glucosylceramidase GBA1, shows distinct subcellular localization of the two enzymes. We proffer our ABP technology for further delineating the role and functioning of GBA2 in disease and propose the β-d-Araf aziridine scaffold as a good starting point for the development of GBA2-specific inhibitors for clinical development.

Abstract P1185: Spatial Transcriptome Profiling In The Mouse Heart With Single-Cell Resolution

Background: The spatial organization of the diverse cells in the heart shapes biological function. For instance, during the inflammatory and fibrotic processes that are central to heart disease, many cells act mainly within their local microenvironment. Recent studies using single-cell RNA sequencing (scRNA-seq) have provided a detailed list of the cell types and states involved in development, homeostasis and in cardiovascular diseases. However, scRNA-seq requires tissue dissociation, which removes spatial information and thereby limits our ability to determine how cells interact with each other to maintain health or cause disease. As a consequence, the precise spatial organization of cell types and states in the mammalian heart remains unknown. We sought to address this knowledge gap by using multiplexed error-robust fluorescence in situ hybridization (MERFISH), a method that enables simultaneous identification and localization of single transcripts from hundreds of different genes in intact tissue sections. Methods/Results: We adapted MERFISH and developed a new single-cell image segmentation method to creating a single-cell atlas of the mouse heart. First, we designed and constructed a 128-gene MERFISH probe library including cell type marker genes and also genes covering a broad range of cardiac signaling pathways. We then optimized sample preparation, including cell boundary staining and tissue clearing. Once optimized, we used this approach to image 10 μm-thick sections from healthy mouse heart, resolving tens of thousands of cells and millions of transcripts. Our data thus constitute the first single-cell spatial transcriptome atlas of the mouse heart. Conclusion: Through our newly developed cardiac MERFISH platform, we identified tens of thousands of cells and labeled millions of individual RNA transcripts in intact sections of mouse heart. Our cellular transcriptome profile maps enable new insights into how the organization of the heart shapes its biological function.

A protease activity-based machine-learning approach as a complementary tool for conventional diagnosis of diarrhea-predominant irritable bowel syndrome.

Irritable bowel syndrome (IBS) has no clinically accepted biomarkers even though it affects a large number of individuals worldwide. To address this lack of understanding, we evaluated peptidase activity in fecal samples from 35 patients with diarrheal IBS without symptom exacerbation (IBS-n) and 35 healthy subjects using a library of 384 fluorescent enzymatic substrate probes. IBS-n patients had high trypsin-like peptidase activity for cleavage of C-terminal lysine and arginine residues and low elastase-like activity for cleavage of C-terminal serine and glycine residues. These fluorescent probe library data, together with diagnostic machine-learning techniques, were able to accurately predict IBS-n. This approach can be used to diagnose diseases where no clinically accepted biomarkers exist, in which fecal enzyme activity is altered and also suggests that the development of new therapies targeting enzyme activities is possible.

Protein@cyanine-based NIR-II lymphography enables the supersensitive visualization of lymphedema and tumor lymphatic metastasis.

Visualization of the lymphatic system is clinically indispensable for the diagnosis and/or treatment of lymphatic diseases. Although indocyanine green (ICG) lymphography becomes an alternate imaging modality compared to traditional lymphoscintigraphy, it's still far from ideal due to the insufficient detection depth and low spatiotemporal resolution. Herein, w e rationally develop protein@cyanine probes to solve the limitations of the current NIR-I lymphography. The protein@cyanine probes are synthesized following a chlorine-containing dye-labeling strategy based on structure-selectivity (facile covalent binding between the dye and protein with a 1:1 molar ratio). As expected, ou r probes display exceptional NIR-II imaging ability with much-improved imaging contrast/resolution and controllable pharmacokinetics, superior to the clinical ICG. Ou r protein@cyanine probes locate lymph nodes (LNs) and delineate lymphatic vessels (LVs) with super-high sensitivity and signal-to-background ratio, enabling real-time diagnosing lymphatic diseases such as lymphedema and tumor lymphatic metastasis. In particular, the NIR-II lymphography provides us an opportunity to discover the disparate morbidity rate of primary lymphedema in different types of mice. Given the fact of lacking clinically transferable NIR-II probes, this work not only provides a promising strategy for enriching of the current library of NIR-II probes, but also promotes the clinical translation of NIR-II lymphography technology. This article is protected by copyright. All rights reserved.

PH-sensing supramolecular fluorescent probes discovered by library screening

A new design concept for pH-sensing supramolecular fluorescent probes is reported. Supramolecular fluorescent pH probes based on pro-guest are designed and prepared. Pro-guests are designed to degrade under acidic condition and convert to competitive guests to displace encapsulated dyes, which leads to a significant enhancement in fluorescence intensity. A library of potential fluorescent pH probes is generated and screened to discover workable probes. These probes are capable of detecting the acidic pH in solution phase. We confirm that these supramolecular probes could detect the acidic environment in endosomal compartments in live cells.

Abstract 5599: An introduction of a highly sensitive circulating tumor DNA monitoring system for minimal residual disease detection using a library of 1000 digital PCR probes

Abstract Background: Many patients undergo a next generation sequencing (NGS)-based cancer genome profiling (CGP) test, but it often does not aid in choosing an optimal therapeutic regimen. Therefore, the genomic information should be used for minimal residual disease (MRD) detection, which directly leads to improved post-treatment cancer patient management. Circulating tumor DNA (ctDNA) is a small fraction of DNA present in blood that carries somatic mutations in cancer patients. Our previous studies demonstrated the clinical validity of ctDNA as a tumor marker for MRD detection. However, the variant allele frequency (VAF; reflecting the fraction of somatic mutations) of ctDNA is less than 1%, at which stable detection is difficult for NGS. Instead of NGS, we have established a highly sensitive ctDNA monitoring system for MRD detection using an originally developed library of >1000 digital PCR (dPCR) probes. Patients and Methods: Using a Clinical Institutional Review Board approval from the Iwate Medical University Hospital, we initiated a dPCR assay system, called off-the-shelf (OTS)-Assay, in April 2022 under a partnership with Quantdetect, Inc (Tokyo, Japan). The OTS-Assay has three main components: OTS-Scan, OTS-Select, and OTS-Monitor. The OTS-Scan can provide an original CGP if a patient comes with no CGP results. The OTS-Select chooses matched somatic mutations from our dPCR probe library. The OTS-Monitor quantifies VAFs of ctDNA periodically in the patient plasma with a sensitivity of 0.05% VAF. There is no strict patient eligibility criteria other than the requirement of previous therapy for treatment of an advanced cancer. Results: Between April and November 2022, 26 patients visited Iwate Medical University Hospital for the OTS-Assay. Twenty-five patients were referred from other Departments of Iwate Medical University Hospital. The primary cancer types include esophagus (n=21), colorectal (n=2), pancreas (n=2), and breast (n=1). Twenty patients had CGP results at their first visit. OTS-Select were used for 13 patients and picked an average of 11.4 mutations per patient. Among those, each of eight patients were immediately able to start ctDNA monitoring with one of the 1000 dPCR probes. As of November 2022, there are four patients who received OTS-Monitor more than one time. Any unexpected VAF increase of ctDNA has not been noted, although a substantial decrease of ctDNA VAF was found in one patient who received one cycle of chemotherapy. Conclusion: The OTS-Assay is a practical introduction of our previous clinical research products based on a core technology, which uses an originally developed library of >1000 dPCR probes. The potential of early and accurate MRD detection will improve post-treatment cancer patient management. Citation Format: Satoshi S. Nishizuka, Hayato Hiraki, Masakazu Abe, Akiko Yashima-Abo, Takeshi Iwaya. An introduction of a highly sensitive circulating tumor DNA monitoring system for minimal residual disease detection using a library of 1000 digital PCR probes. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 5599.