WO2023215396A1

WO2023215396A1 - Devices, systems, and methods for classification of pancreatic cells

Info

Publication number: WO2023215396A1
Application number: PCT/US2023/020859
Authority: WO
Inventors: Vincent Jo Davisson
Original assignee: Amplified Sciences, Inc.
Priority date: 2022-05-03
Filing date: 2023-05-03
Publication date: 2023-11-09

Abstract

Devices, systems, and methods for classification of pancreatic cells. In a method for classifying pancreatic cysts of a patient, the method comprises the steps of preparing a bead suspension by binding magnetic beads with a gastricsin substrate and subsequently washing away any unbound substrate, activating a sample containing gastricin by lowering the pH of the sample, mixing the sample and the bead suspension to form a mixed sample, removing the magnetic beads from the mixed sample, resulting in a solution; and measuring the solution to determine gastricsin levels in the sample, wherein the gastricsin levels are indicative of a cyst type.

Description

DEVICES, SYSTEMS, AND METHODS FOR CLASSIFICATION OF PANCREATIC CELLS

PRIORITY

The present application is related to, and claims the priority benefit of, U.S. Provisional Patent Application Serial No. 63/337,637, filed May 3, 2022, the contents of which are incorporated herein directly and by reference in their entirety.

BACKGROUND

Occurrences of pancreatic cancer are projected to become the second leading cause of cancer-related death by 2030. Pancreatic cancer has a dismal 5-year survival rate of 11% and its incidence continues to increase. This grim outlook arises in part because the disease is most commonly diagnosed at late metastatic stages. Nevertheless, early diagnosis remains difficult because the early stages of the disease are poorly characterized and are largely asymptomatic.

The detection and classification of pancreatic cysts and cystic lesions is an important strategy towards early diagnosis of pancreatic cancer. Pancreatic cysts are incidentally detected in almost half of patients undergoing magnetic resonance imaging (MRI) and in 2.6% of patients undergoing computed tomography (CT) scans. Studies have shown that between 10 - 60 percent of cysts, particularly intraductal papillary mucinous neoplasms (IPMN) and mucinous cystic neoplasms (MCN), have significant potential to develop into malignant cancer. Patients presenting with likely mucinous cysts are candidates for surgical resection, which carries a 2-4% mortality risk, as well as a 21% risk of post-surgical new onset diabetes. Disappointingly, surgical interventions are often performed for cysts that are later found to be benign, while patients with non-mucinous cysts could (if accurately classified) be spared costly long-term surveillance. Accurate classification of pancreatic cysts as pre-cancerous mucinous cysts, IPMNs and MCNs, and non-mucinous benign cysts, such as serous cystic neoplasms (SCN), is essential to avoid misdiagnosis and unwarranted interventions.

Biomarker analysis of fine needle aspirate (FNA) fluid collected from pancreatic cysts offers great potential for high accuracy diagnosis of mucinous cysts. The FNA biomarker used most commonly in current clinical assessments is carcinoembryonic antigen (CEA) immunoassay, which has a reported pooled diagnostic sensitivity of 60.4% (38 - 73%) and specificity of 88.6% (65 - 96%) using a 192 ng/mL cutoff level. With room for diagnostic improvement, other biomarkers have also been studied for this purpose. The more intensive analysis of extracted genomic DNA for KRAS and GNAS mutations shows a pooled 94% sensitivity and specificity of 91% for IPMNs based upon a recent meta-analysis. A recent biomarker discovery effort of cyst fluids identified pepsinogen C (PGC) in mucinous pancreatic cysts. This discovery prompted development of a marker assay based upon the activation of PGC to gastricsin (also known as pepsin C) followed by catalytic turnover of a synthetic substrate tailored for detection by fluorescence resonance energy transfer (FRET). This assay format was used to discriminate mucinous versus non-mucinous cyst fluid samples from a small retrospective cohort with sensitivity 93%, specificity 100% and diagnostic accuracy of 95%.

The promising results of this previous gastricsin assay are an example of the potential clinical utility of protease activity in cancer diagnostics. As demonstrated for the gastricsin assay, an internally quenched fluorescent substrate offers a general approach to fluorescent signals upon cleavage by target proteases in homogenous solutions such as ADAMTS13. A number of additional protease assay platforms have been reported, each with potential utility for clinical applications. Examples including quantum dot-based quenched-fluorescent systems have been successfully used for multiplex protease assays. Nanoparticle bound fluorescent peptide substrates have been used to measure metalloprotease activity in vivo towards diagnosis of colorectal cancer. Similarly, substrate-masked antibodies were used to profile metalloprotease activity in cancerous tissues. High sensitivity detection technologies have also been applied to protease activities, including mass spectrometry and surface- enhanced Raman spectroscopy (SERS).

BRIEF SUMMARY

This disclosure describes a gastricsin assay using a magnetic bead-based platform, with both fluorescent and SERS detection modes. The method incorporates a single ultrasensitive dye to enable the detection of low turnover numbers and a highly selective peptide substrate for discrimination of gastricsin activity over other proteases in samples. These features have enabled rapid 7 minute measurements without significant matrix effects in complex clinical samples, including cyst fluid. Importantly, the concentration of released proteolytic product can easily be assessed, allowing for quantitative activity analysis rather than the previous FRET cutoff-based readout. The disclosure also describes exemplary embodiments capable of differentiating between mucinous and non-mucinous pancreatic cysts while consuming only 1 pL of cyst fluid. Gastricin are markers indicative of mucinous and non-mucinous cysts. The embodiments described provide for method and device of distinguishing between mucinous and non-mucinous cysts and therefore identifying potentially pre-cancerous cysts, and assisting in directing patient care.

The present disclosure includes disclosure of a method for classifying pancreatic cysts of a patient, comprising the steps of preparing a bead suspension by binding magnetic beads with a gastricsin substrate and subsequently washing away any unbound substrate, activating a sample containing gastricin by lowering the pH of the sample, mixing the sample and the bead suspension to form a mixed sample, removing the magnetic beads from the mixed sample, resulting in a solution; and measuring the solution to determine gastricsin levels in the sample, wherein the gastricsin levels are indicative of a cyst type.

The present disclosure includes disclosure of a method for classifying pancreatic cysts of a patient, wherein the gastricin substrate comprises a biotinylated lysine residue at a C- terminus of the gastricin substrate using a diethylene glycol spacer.

The present disclosure includes disclosure of a method for classifying pancreatic cysts of a patient, wherein the gastricin substrate comprises a fluorescent dye at an N-terminus of the gastricin substrate.

The present disclosure includes disclosure of a method for classifying pancreatic cysts of a patient, wherein the step of mixing the sample and the bead suspension to form the mixed sample is performed to allow the gastricin in the sample to cleave the gastricin substrate having the fluorescent dye.

The present disclosure includes disclosure of a method for classifying pancreatic cysts of a patient, wherein the gastricsin substrate is a peptide that is selectively cleaved at low pH by gastricsin without interference from any other proteins found in cyst fluid.

The present disclosure includes disclosure of a method for classifying pancreatic cysts of a patient, wherein the gastricin substrate comprises a reporter that is fluorescent.

The present disclosure includes disclosure of a method for classifying pancreatic cysts of a patient, wherein the gastricin substrate comprises a reporter reactive to surface enhanced Raman spectroscopy (SERS).

The present disclosure includes disclosure of a method for classifying pancreatic cysts of a patient, wherein the patient sample contains pepsin.

The present disclosure includes disclosure of a method for classifying pancreatic cysts of a patient, further comprising the step of adding pepstatin to the solution.

The present disclosure includes disclosure of a method for classifying pancreatic cysts of a patient, wherein the sample is a patient sample containing pepsinogen C. The present disclosure includes disclosure of a method for classifying pancreatic cysts of a patient, wherein the sample is a patient sample containing blood.

The present disclosure includes disclosure of a method for classifying pancreatic cysts of a patient, wherein the sample is less than 5 pL.

The present disclosure includes disclosure of a method for classifying pancreatic cysts of a patient, wherein the step of preparing the bead suspension by binding the magnetic beads with the gastricsin substrate is performed using streptavidin or amine binding or click chemistry.

The present disclosure includes disclosure of a method for classifying pancreatic cysts of a patient, wherein the step of measuring the solution is performed utilizing surface enhanced Raman spectroscopy (SERS).

The present disclosure includes disclosure of a method for classifying pancreatic cysts of a patient, wherein the step of measuring the solution is performed utilizing fluorescence.

The present disclosure includes disclosure of a method for classifying pancreatic cysts of a patient, further comprising the step of adding silver nanoparticles to the solution.

The present disclosure includes disclosure of a method for classifying pancreatic cysts of a patient, wherein the step of measuring the resulting solution is performed utilizing surface enhanced Raman spectroscopy (SERS).

The present disclosure includes disclosure of a method for classifying pancreatic cysts of a patient, wherein the step of utilizing SERS is performed at a wavelength selected from the group consisting of 538nm, 638nm, and 785 nm.

The present disclosure includes disclosure of a method for classifying pancreatic cysts of a patient, wherein silver nanoparticles are added to the resulting solution such that the ratio of the silver nanoparticles is greater than 1 : 1 by volume, weight, or density.

The present disclosure includes disclosure of a method for classifying pancreatic cysts of a patient, wherein the step of preparing the bead suspension by binding magnetic beads with a gastricsin substrate is performed by preparing the bead suspension by binding magnetic beads with a gastricsin substrate having a fluorescent dye coupled thereto.

The present disclosure includes disclosure of a method for classifying pancreatic cysts of a patient, wherein the step of measuring the solution is performed to determine the gastricin levels as being indicative of a mucinous cyst or a non-mucinous cyst.

The present disclosure includes disclosure of a method for classifying pancreatic cysts of a patient, wherein if the step of measuring the solution is performed and the gastricin levels are indicative of a mucinous cyst, the method further comprises the step of determining that the sample contains cancerous cells.

The present disclosure includes disclosure of a method of quantitating enzymatic activity for the purposes of assessing a subject’s disease state, comprising diluting a minimal volume sample to a working concentration range, immobilizing a selective peptide substrate via conjugation to a magnetic particle and removing an unreacted substrate via magnetic separation after a time period has elapsed, detecting a cleaved product using fluorescence and/or surface enhanced Raman spectroscopy (SERS), quantifying product generation or turnover by comparing to a standard curve of product detected using the fluorescence and/or SERS, and using turnover to determine patient diagnosis status.

The present disclosure includes disclosure of a method of quantitating enzymatic activity in the presence of biological matrices using surface enhanced Raman spectroscopy (SERS), comprising lowering the pH of a biological sample solution to below pH 5 by adding pH 2 assay buffer, aggregating a rhodamine-dimer dye with an excess of silver nanoparticles, and exciting the aggregated dye with a laser and detecting the aggregated dye using a Raman spectrometer.

BRIEF DESCRIPTION OF THE DRAWINGS

The disclosed embodiments and other features, advantages, and disclosures contained herein, and the matter of attaining them, will become apparent and the present disclosure will be better understood by reference to the following description of various exemplary embodiments of the present disclosure taken in conjunction with the accompanying drawings, wherein:

Figure 1 shows a diagram for a scheme for a conditional gastricsin activity assay, according to an exemplary embodiment of the present disclosure;

Figure 2 shows gastricsin activity assay in pH 2 buffer, with subsection A) showing the product formation calculated from fluorescence measurement at each concentration of gastricsin as indicated on the x-axis, subsection B) showing the product formation calculated from SERS measurements at each concentration of gastricsin, whereby the samples in subsection A) were used for these measurements after addition of silver, and whereby the error bars represent the standard deviation of triplicate measurements, subsection C) showing a correlation of product formation between SERS and fluorescence measurements from paired reaction samples, whereby the grey area is the 95% confidence interval from a linear regression analysis with a slope = 1.075 and R² = 0.914, according to exemplary embodiments of the present disclosure; Figure 3 shows the specificity of assay for gastricsin, with subsection A) showing the activity of VSOOf substrate loaded onto beads with proteolytic enzymes and bovine serum albumin (BSA), with concentrations for each analyte listed in parentheses, subsection B) showing the impact of 50 nM pepstatin on activity of 150 ng/mL gastricsin and/or 1 g/mL pepsin in assay as indicated in the table underneath where “+” indicates addition of analyte, and subsection C) showing the impact of the addition of pepstatin to assay buffer for clinical sample analysis, whereby controls are mock samples prepared with gastricsin at 150 ng/mL, and whereby the error bars represent standard deviation of triplicate measurements, according to exemplary embodiments of the present disclosure;

Figure 4 shows gastricsin activity assay in samples comprised of a mock mucus matrix containing pancreatic enzymes with a viscosity of 1.5 cP to simulate a pancreatic cyst fluid sample, with subsection A) showing measured product formation calculated from fluorescence measurement at each concentration of gastricsin as indicated on the x-axis, subsection B) showing measured product formation calculated from SERS measurement at each concentration of gastricsin as indicated on the x-axis, using the same samples used in subsection A), and whereby the error bars represent the standard deviation of triplicate measurements, subsection C) showing the correlation of product formation between SERS and fluorescence measurements from paired reaction samples, whereby the grey area is the 95% confidence interval from a linear regression analysis with a slope = 1.293 and R² = 0.836, according to exemplary embodiments of the present disclosure;

FIG. 5 shows Gastricsin activity assay in samples comprised of a mock mucus matrix containing pancreatic enzymes with a viscosity of 1.6 cP, while the assay buffer contained the indicated concentration of Hb to simulate a bloody pancreatic cyst fluid sample. A) Measured product formation calculated from fluorescence measurement at each concentration of gastricsin as indicated on the x-axis. B) Measured product formation calculated from SERS measurement at each concentration of gastricsin as indicated on the x-axis. The samples are the exact same from panel (A). Error bars represent the standard deviation of triplicate measurements. C) Correlation of product formation between SERS and fluorescence measurements from paired reaction samples. The grey area is the 95% confidence interval from a linear regression analysis with slope = 1.940 and R2 = 0.828, according to an exemplary embodiment of the present disclosure;

Figure 6 shows clinical sample results from a retrospective cohort of 69 pancreatic cyst fluid samples with known clinical diagnoses, with subsection A) showing the measured mass of pepsinogen C according to commercial ELISA kit, subsection B) showing a ROC curve of ELISA measurement of pepsinogen C mass to distinguish mucinous from non- mucinous pancreatic cysts (AUC = 0.873, 95% CI 0.794 - 0.900). subsection C) showing the measured product formation calculated from fluorescence measurement of clinical samples, subsection D) showing the measured product formation calculated from SERS measurement of clinical samples, whereby sample measurements are paired and both C) and D) are divided into respective diagnosis category as indicated on the x-axis, subsection E) showing a correlation of product formation between SERS and fluorescence measurements from paired reaction samples, whereby the grey area is the 95% confidence interval from a linear regression analysis with a slope = 0.348 and R² = 0.614, and whereby every measurement represents a single reaction and sample, and subsection F) showing ROC curves of clinical samples diagnosed according to the gastricsin activity assay using the product formation calculated from fluorescence (black, AUC = 0.936, 95% CI 0.882 - 0.990) or SERS (blue, AUC = 0.873, 95% CI 0.791 - 0.956), in comparison with clinical CEA measurement (red, AUC = 0.812, 95% CI 0.707 - 0.918) and a combination of all three (green, AUC = 0.950, 95% CI 0.900 - 0.999). **** = P < 0.0001 according to a Mann-Whitney test of significance, according to exemplary embodiments of the present disclosure;

Figure 7 shows the structure of peptide substrate, VS001, according to an exemplary embodiment of the present disclosure;

Figure 8 shows the structure of dye-labelled gastricsin product, according to an exemplary embodiment of the present disclosure;

Figure 9 shows standard curves using fluorescence intensity (subsection A)) or SERS AUC (subsection B)) measurements of the product of the enzyme reaction at the concentrations listed on the x-axis, whereby the samples were run in triplicate for fluorescence and a single well of each sample was run using SERS, and whereby the curves were fit with a linear regression and the equation and fit are shown in each graph, according to exemplary embodiments of the present disclosure;

Figure 10 shows a chromatogram and purity of VS001, according to an exemplary embodiment of the present disclosure; and

Figure 11 shows a chromatogram and purity of dye-labeled gastricsin product, according to an exemplary embodiment of the present disclosure.

An overview of the features, functions and/or configurations of the components depicted in the various figures will now be presented. It should be appreciated that not all of the features of the components of the figures are necessarily described. Some of these nondiscussed features, such as various couplers, etc., as well as discussed features are inherent from the figures themselves. Other non-discussed features may be inherent in component geometry and/or configuration.

DETAILED DESCRIPTION

For the purposes of promoting an understanding of the principles of the present disclosure, reference will now be made to the embodiments illustrated in the drawings, and specific language will be used to describe the same. It will nevertheless be understood that no limitation of the scope of this disclosure is thereby intended. The exemplary embodiments and processes described herein may utilize particular methods materials, but it is understood that equivalents may be used to achieve the same resort.

Experimental method

Materials

Synthetic procedures for the dimeric rhodamine 6G dye used in the present study are reported previously.³³ This dye was coupled to the N-terminus of resin bound biotinylated peptides, purchased from GenScript. 2-(6-Chloro-lH-benzotriazole-l-yl)-l, 1,3,3- tetramethylaminium hexafluorophosphate (HCTU, NC0576737), dichloromethane (DCM, MK-4879-4), trifluoroacetic acid (TFA, AC139720025), diethyl ether (AC12399-0050), acetonitrile for HPLC (A998), and water for HPLC (600-30-13) were all purchased from Fisher Scientific. N,N -diisopropylethylamine (DIEA, D125806), N,N’ -dimethylformamide (DMF, 319937), and sodium chloride (NaCl, S9625-1KG) were purchased from Sigma Aldrich. Triisopropylsilane (TIPS, Al 87865) was purchased from Ambeed. Tween® 20 (97062-332) was purchased from VWR. A commercially available pH 2 buffer (Cat. No. LC122201) was purchased from LabChem. To prepare gastricsin/gastricsin standards, we purchased recombinant human pepsinogen c (Cat. No. 6186-AS-010) from R&D Systems. Mock cyst fluid samples were prepared using a proprietary artificial mucus matrix, supplemented with pancreatic enzymes and set to a viscosity of 1.5 centipoise (cP), prepared by Biochemazone (Alberta, Canada). Streptavidin-blocked sera-mag speed beads (Cat No. 21152104011150) were manufactured by Cytiva and purchased through VWR. Pepstatin A (S7381) was purchased from Selleck Chem. Trypsin (Research Products International Cat. No. T70010-1.0), pepsin (R&D Systems Cat. No. 6186-AS-010), thrombin (Cayman Chemical Company, Cat. No. 13188), hemoglobin (Hb, Sigma Aldrich, H7379), and bovine serum albumin (Sigma Aldrich, A7906-50G). 50 nm diameter citrate capped silver nanoparticles (1 mg/mL, Cat. No. AGCB50-5M) were purchased from nanoComposix. All reagents and proteins required for immunoassay of gastricsin were purchased as part of a kit (DY6186-05) sold by R&D systems. Pepstatin A was purchased from Selleck Chemicals (Cat. No. S7381). For running assays, 384 shallow well, black, flat bottom plate (Cat. No. 1230M76, Thomas Scientific) and 0.5 mL sterile centrifuge tubes (MTC Bio, Cat. No. C2007) were used. All assay samples were heated on an incubator (VWR Cat. No. 75838- 270), equipped with a 30-tube block (VWR Cat. No. 13259-000) and a temperature control probe (VWR Cat. No. 11301-112). All fluorescence readings were measured on a Biotek Synergy Hl Plate Reader. SERS spectra were obtained using a probe-based Raman spectrometer (Wasatch Photonics Inc., model WP-532-SR-IC) with a 532 nm laser source, 50 mW laser power, 25 pm slit width and standard lenses at 11 mm working distance using 20 - 30 ms integration time.

Preparation of Assay Buffer

Tween® 20 (0.1%) and NaCl (100 mM) were added to commercial pH 2 Buffer. Assay Buffer preparations were mixed by vortexing and prepared fresh before each assay. Clinical Samples

Clinical samples were obtained under an approved IRB from the UCSF Medical Center. Samples were banked in collaboration with several medical centers as part of a EDRN study. Briefly, cyst fluid was obtained at the time of operative resection and at the time of endoscopic ultrasound guided (EUS) aspiration. For operative cases, resected specimens underwent cyst aspiration within 30 minutes of resection. Aspiration was performed by a surgeon, pathologist, or technician. Cyst fluid samples were obtained with an 18-gauge to 21-gauge needle, divided into 100 pl aliquots, and stored at -70°C or colder. No additives or centrifugation were performed prior to freezing and total time between excision and freezing was < 60 minutes. For EUS obtained samples, aspiration was performed at the time of endoscopy. Cyst fluid was initially allocated for clinically needed tests for care of the patient and any remaining fluid was stored for research purposes. The remaining fluid samples were divided into 100 pl aliquots, and stored at -70°C. No additives or centrifugation were performed prior to freezing and specimens were frozen within 60 minutes of aspiration.

Magnetic Bead Preparation

Synthetic details and characterization of the peptide substrate, VS001 (Fig. 7), are given in the supplementary information. Streptavidin-coated magnetic beads were loaded with VS001 in bulk. First, an aliquot of beads was twice washed with pH 2 Assay Buffer, vortexed vigorously, and the beads were separated via magnetic separation with a magnetic rack. The washed beads were then incubated with a volume equal to the starting aliquot of 0.05 mM VS001 at room temperature for 15 minutes with occasional mixing via brief vortexing. After incubation, the beads were washed four times with 5x original aliquot volume of Assay Buffer and again isolated via magnetic separation after vigorous vortexing to remove unbound peptide. After the final wash, VS001 -loaded beads were resuspended in Assay Buffer to original volume to maintain 10 mg/mL bead concentration. pH 2 Assay Buffer Sample Preparation

One pL recombinant human PGC was diluted with 28.3 pL of pH 2 Assay Buffer and incubated at room temperature to activate for 10 minutes. After activation, the stock 15 pg/mL gastricsin solution was then further diluted to desired concentrations using pH 2 Assay Buffer prior to protease assay.

Mock Sample Preparation

Mock samples were prepared by diluting 1 pL recombinant human PGC with 28.3 pL Artificial Mucus Matrix supplemented with pancreatic enzymes and a viscosity of 1.5 cP to create a 15 pg/mL stock solution. The stock mock sample was further diluted with the Artificial Mucus Matrix to desired concentrations, then 1 pL of each Mock Sample was activated by dilution with 99 pL of pH 2 Assay Buffer followed by gentle mixing and incubation at room temperature for 10 minutes prior to protease assay.

Hb Sample Preparation and Assay

Mock samples and pH 2 Assay Buffer were prepared as described previously. Hemoglobin was hydrated in distilled water before it was spiked to a final concentration of 1.5 mg/mL in an aliquot of the freshly prepared pH 2 Assay Buffer. Mock Samples were run after activation with either standard pH 2 Assay Buffer or with pH 2 Assay Buffer supplemented with 1.5 mg/mL of Hemoglobin. Whichever buffer was used for activation was used throughout the protease assay and data collection.

Clinical Sample Preparation

Clinical samples were thawed on ice and divided into 1 pL aliquots without any additives or centrifugation prior to assay. A single 1 pL aliquot of each sample was diluted with 99 pL of pH 2 Assay Buffer and gently mixed via pipetting. Samples were then activated for 10 minutes at room temperature.

General Protease Assay

The protease activity assay was performed in 0.5 mL sterile microtubes where 7.5 pL of VS001 loaded on magnetic beads were added to sample tubes containing 7.5 pL of activated gastricsin sample and gently vortexed before immediate placement on an incubator equipped with a 30-tube block heated to 37 °C controlled with a temperature probe. Reaction mixtures were covered with foil on the heat block and incubated for 7 minutes. After incubation they were promptly placed on a magnetic bead rack to stop the enzymatic reaction and isolate the resulting product solution. Each 15 pL reaction was divided into three 4 pL subsamples pipetted into individual wells of a 384 shallow well, black, flat bottom plate. Fluorescence intensity was measured in triplicate on a plate reader at Ex/Em of 16/557 nm and then sealed to prevent evaporation prior to SERS analysis.

Protease Comparison Assay

Proteases were hydrated in distilled water according to manufacturers’ specifications before dilution in the pH 2 Assay buffer. Proteases were activated and assayed according to the General Assay conditions. Reported concentrations are the final protease concentrations in the assay mixture.

Pepstatin Protease Assay

Two sets of pH 2 Assay Buffers were prepared as described previously wherein one buffer was spiked with 50 nM of pepstatin A while the other served as a control. Samples containing combinations of 150 ng/mL of gastricsin, and/or 30 nM pepsin in Artificial Mucus Matrix were run normally under General Assay conditions with either normal pH 2 Assay Buffer or with the pH 2 Assay Buffer supplemented with 50 nM pepstatin and analyzed concurrently.

Raman Spectroscopy

4 pL of a suspension of 50 nm diameter citrate capped silver nanoparticles (1 mg/mL, nanoComposix, Inc. Cat. No. AGCB50-5M) was added to 4 pL of protease assay sample in a 384-well plate. The fresh colloid aggregate samples were immediately subjected to Raman spectroscopy to obtain the SERS data. Spectral data were processed and the area under the curve (AUC) was analyzed using OPUS 8.2.28 software (Bruker Optics, Inc.). Graphs of the data were prepared using GraphPad Prism as described in the Data Analysis section.

Enzyme-linked Immunosorbent Assay (ELISA)

PGC mass was measured by ELISA according to the manufacturer’s specifications. The total mass of the PGC was measured in all clinical samples using a commercial sandwich ELISA assay for human Pepsinogen C/gastricsin (R&D Systems, Minneapolis, MN), following the manufacturer’s instructions. Optical density was measured with a Biotek Synergy HT plate reader at 450 nm using wavelength correction at 540 nm. Sample concentration was interpolated from a standard curve of recombinant gastricsin (R&D Systems) using a 4-parameter logistic regression.

Data Analysis

All data analysis and graph preparations were conducted using GraphPad Prism 9.3. Each sample assay raw fluorescence intensity or SERS area under curve (AUC) was converted to the corresponding product concentration according to the appropriate standard curve as shown in Fig. 9. Each sample was analyzed in triplicate and the mean and standard deviations were calculated and plotted for each point. For samples with both fluorescence and SERS measurements, paired sample results were plotted against each other to visualize the optical interferences resulting from the external milieu and the linear relationship between the two measurements was calculated. The grey bars in these graphs represent the 95% confidence interval, and the slope is proportional to optical interference of one or both measurements. Unblinded clinical results were plotted according to their diagnosis as either non-mucinous or mucinous, and receiver operating characteristic (ROC) curves were prepared with noted cutoffs, area under curve (AUC), sensitivities, and specificities. The significance of clinical sample discrimination was analyzed using the Mann- Whitney test for each measurement described, and the p-values are listed in the figure caption.

Results

The assay design is described in Fig. 1. Briefly, magnetic beads are loaded with gastricsin substrate, and any unbound material is washed away. Gastricsin samples (research or clinical) are activated by incubation at pH 2 at room temperature prior to mixing with substrate-loaded beads. Equal volumes of enzyme solution and bead suspension are mixed for a predetermined amount of time, and then the beads are removed with a magnetic tube rack. The resulting solution contains enzyme and reaction product at pH 2, which is quantified by either fluorescence or SERS measurements.

The substrate peptide sequence used is based on previous work. This peptide sequence, selected by multiplex substrate profiling, is shown to be selectively cleaved at low pH by gastricsin without interference from any other proteins found in cyst fluid. To the C-terminus of this peptide, we have appended a biotinylated lysine residue separated from the substrate sequence by a diethylene glycol spacer. At the N-terminus, we have attached a dimeric rhodamine 6G (R6G)-based dye, which we developed as an ultrasensitive and stable reporter for SERS detection. The resulting substrate was immobilized on magnetic beads coated with streptavidin and used for assaying gastricsin activity. Briefly, magnetic beads are loaded with gastricsin substrate, and any unbound material is washed away. Gastricsin samples (standard or clinical) are activated by incubation at pH 2 at room temperature prior to mixing with substrate-loaded beads. Equal volumes of enzyme solution and bead suspension are mixed and incubated at 37°C for 7 minutes, then the beads are removed with a magnetic tube rack. The resulting solution contains enzyme and reaction product at pH 2, which is quantified by either fluorescence or SERS measurements. These assays were responsive to varying amounts of gastricsin (Fig. 2). Fluorescence measurements were performed using a 384-well plate in a plate reader. SERS measurements were performed in the same plate after addition of silver nanoparticles. Signals in both detection modes showed a linear dependence with gastricsin concentration in our assays, allowing for direct quantitation of enzymatic activity. Furthermore, results calculated from each of the detection methods correlated well with each other, as evidenced by the linear slope of 1.075 in a plot of SERS vs. Fluorescence results, suggesting that both methods are equally well-suited for analysis of gastricsin turnover (Fig. 2, subsection C). Gastricsin is known to have a broad substrate specificity but cleaves the designed peptide between the alanine and tryptophan residues, as demonstrated previously. Using standard curves (Fig. 9) made from varying concentrations of a pre-synthesized proteolysis product (Fig. 8), the assay was standardized for the product production. As such, we are able to calculate turnover number and activity in product per unit time.

To ensure the selectivity of our assay for gastricsin, we next studied a panel of common proteins and proteases using a fluorescence readout (Fig. 3). While the assay was very selective in this protease panel, some activity was observed with high levels of the structurally similar pepsin. For the application of classifying pancreatic cysts, this interference is not of concern because pepsin is not found in cyst fluid to the best of our knowledge. However, in matrices containing both gastricsin and pepsin this assay will present limitations. A remedy was to evaluate the gastricsin in the presence and absence of the selective pepsin inhibitor pepstatin. These results showed that gastricsin was not inhibited by pepstatin, while the activity of pepsin was completely abrogated (Fig. 3, subsection B). Therefore, in matrices where pepsin is suspected to be present, pepstatin can be used to determine the gastricsin activity. We further confirmed this principle by testing several clinical samples activated with and without pepstatin in the pH 2 Assay Buffer. Despite some differences in the positive controls, the clinical samples themselves did not show a change in signal intensity which suggests that these samples did not contain pepsin as expected.

Prior to studies of patient samples, a mock pancreatic cyst fluid preparation was prepared for assay testing and validation (Fig. 4). This mixture consisted of an artificial mucus matrix product to which pancreatic enzyme extracts had been added. The viscosity of this matrix was adjusted to 1.5 cP, based on previous rheology studies of pancreatic cyst fluid.³⁷ Recombinant pepsinogen C was diluted with this matrix at varied concentrations and used for gastricsin activity assays. While fluorescence measurements decreased in intensity, SERS analyses showed no matrix effects (Fig. 4, subsection B). Furthermore, the relationship between product calculated using fluorescence and SERS is skewed, as indicated by the linear slope of 1.293 in Fig. 4, subsection C. This suggests that, due to decreased fluorescence related to optical interference from the mock matrix, SERS measurements can provide a more accurate analysis of enzyme activity and prove to be more reliable in clinical applications.

Previous reports of cyst fluid sample contamination with blood during fine needle aspiration are of considerable importance to activity assay performance. To anticipate possible optical interferences from hemoglobin in blood-contaminated samples, assays were performed in buffer containing 1.5 mg/mL hemoglobin (Fig. 5). The concentration was chosen to model that found in hemolyzed blood. Indeed, fluorescence measurements were decreased in the presence of hemoglobin. Impressively, however, the assay results using SERS based detection was not compromised. Combined with the studies using mock pancreatic cyst fluid, these data suggest that the SERS-based detection can be used when matrix effects are significant in fluorescence-based activity assays and supported by the steep slope of 1.940 shown in Fig. 5, subsection C.

Finally, a study of a retrospective cohort of 69 cyst fluid samples with known assignments as mucinous or non-mucinous classification was conducted (Table 1, Fig. 6). Both fluorescence (AUC = 0.936, 95% CI 0.882 - 0.990, Sensitivity = 85%, Specificity = 93% at cutoff of 5.79 pmol product) and SERS (AUC = 0.873, 95% CI 0.781 - 0.954, Sensitivity = 80%, Specificity = 90% at cutoff of 15.14 pmol product) assays were able to differentiate between these two classifications. The assay classifies cysts with better accuracy than the most commonly used biomarker, CEA (AUC = 0.812, 95% CI 0.707 - 0.918, Sensitivity = 69%, Specificity = 89% at cutoff of 192 ng/mL), as reported in the clinical records for each sample. The accuracy of the activity-based assay is also significantly superior to a protein mass-based pepsinogen C immunoassay (AUC = 0.873, 95% CI 0.784 - 0.961, Sensitivity = 77%, Specificity = 93% at cutoff of log2 = 16.51 pg/mL). The ROC curve’s AUC is further improved when all assays are combined (AUC = 0.941. 95% CI 0.916 - 1.000, Sensitivity = 87%, Specificity = 93%), suggesting potentially improved diagnostic power when combining biomarkers in diagnosis of cysts. Furthermore, the results for the entire 69 sample cohort were collected and analyzed in a single day, outpacing currently available clinical diagnostics that often require between 1 - 21 days. Table 1 - n = 69 clinical sample cohort characteristics. Values are as listed in the row heading.

Non-Mucinous Mucinous

Samples (n) 15 14 13 27

Age 54.2 + 18.5 60.1 + 9.6 53.2 + 17.3 66.8 + 11.6

(Years + SD)

, Female 7 8 10 13

^Gcndcr Male 7 6 3 14

Pittsburgh 4 0 2 4

, Indiana 6 9 6 13

Institution Stanford 4 3 5 10

UCSF 0 2 0 0

Collection Surgery 6 10 13 24

Method** EUS-FNA 8 4 0 2

Cyst Size*** 66.6 + 33.6 60.2 + 39.7 54.5 + 38.1 47.8 + 32.8

(mm + SD)

CEA 31 + 43 7354 + 10773 + 5398 + 16290

(ng/mL + SD) 27413 25538

Gastricsin Mass 220 + 775 10231 + 6972 + 10630 +

(ng/mL + SD) 12485 10449 19431

Gastricsin Fluorescence 4.7 + 1.2 3.9 + 0.6 56 + 54 46 + 47

Activity (pmol SERS 16 + 5.7 13 + 3.5 36 + 22 34 + 21 prod + SD)

*One pseudocyst sample without patient characteristic information

** One pseudocyst without sample collection information, one IPMN collected via endoscopic retrograde cholangiopancreatography (ERCP)

***Five pseudocyst and two IPMN samples without cyst size data

Discussion

These studies demonstrate a highly accurate magnetic bead-based protease assay platform with both fluorescence and SERS readouts. The work applies the newly developed assay platform to measure gastricsin activity, which has been found enriched in mucinous pancreatic cyst fluids collected through fine needle aspiration, either pre- or post-surgical resection as indicated in Table 1. The gastricsin and CEA analyses can assist clinical decisions regarding the potential risk of cysts to develop into pancreatic cancer. The first step for clinical decision-making is to address whether they are dealing with a benign, non- mucinous, or potentially cancerous, mucinous cyst. Mucinous cysts are significantly more likely to become cancerous but surgical pancreatic resection can prevent this near-universal deadly outcome. As such, accurate diagnosis, aided by the assays, will be useful in the determining the risk of the patient developing a pancreatic cancer. The assay uses a peptide substrate that is selectively cleaved by gastricsin which aims to prevent false negatives. Notably, the presence of pepsin, which is structurally similar to gastricsin, can give a false response in the assays. This situation can be remedied by addition of pepstatin, which selectively inhibits pepsin. While pepsin is not a major concern in cyst fluid applications, this adaptation may be important in other biological matrices. Matrix interference with fluorescence readout in assays are common as also encountered with the gastricsin activity assay in the presence of simulated mucus or hemoglobin. These matrix effects were not a factor in the SERS based readouts. The 69 retrospective cyst fluid samples were quantified using two orthogonal detection methods in the same assay conditions. Impressively, these methods successfully differentiated mucinous and non-mucinous samples.

An important future direction is to study the performance of the assay using larger cohorts of cyst fluid samples since 69 has limited statistical power. The current assay workflow and detection methods offer a robust platform to pursue larger scale clinical studies. Current estimates of sample turnaround times for 100 clinical samples are less than 24 hours, even using manual execution. This situation could be greatly improved by adaptation to automated clinical analyzer systems. Technology improvement efforts will seek to incorporate this assay platform onto a system capable of magnetic bead purification and fluorescence readout. Ideally, a system would utilize SERS to remain agnostic to matrix effects. Finally, ultrasensitive SERS-based detection significantly reduces required clinical fluids to nano-sized volumes. Future directions could incorporate nanoliter liquid handling equipment to improve variations. The assay approach offers a suitable platform for improved early detection of pancreatic cancer in conjunction with current clinical standards.

Synthesis and Characterization of Dye-labelled Peptide Substrate, VS001

To 13 pmol of resin-bound peptide substrates, the preactivated mixture of dimeric rhodamine 6G dye (1.1 eq, 14.8 pmol, 16 mg), HCTU (1.1 eq., 14.8 pmol, 6 mg), and DIEA (3.3 eq., 44.4 pmol, 8 pL), in 2 mL of DMF, were was added. This mixture was incubated at room temperature for 18 hours with mechanical shaking. The resin was then filtered and washed with (6x) DMF (6x) and DCM (3x) sequentially. The peptide substrate, VS001, was then cleaved from the resin by shaking with 10 mL of TFA:TIPS: Water (95:2.5:2.5) mixture for 2 hours. The resin was removed by filtration and the crude dye labelled peptide (VS001) concentrated in vacuo, precipitated with diethyl ether, and purified by high performance liquid chromatography (HPLC). For semi-preparative separations, an Alltech Econosil Cl 8 was used. The compound was purified by eluting with water and acetonitrile, each containing 0.01% trifluoroacetic acid (solvent A and solvent B respectively). A gradient elution, with a constant flow rate of 3 mL, was performed: 5% solvent B in A for the first 5 minutes, followed by a linear increase from 5% solvent B to 95% solvent B from until 20 min., a wash period of 95% solvent B from 20 to 25 min., and finally a linear decrease from 95% to 5% solvent B in A from 25 to 30 min. All peaks containing an absorption at 520 nm were collected, combined, and dried. The product was confirmed by electrospray mass spectrometry, performed using an Advion Expression^L spectrometer (M²⁺: Calc: 1244.64, Exp: 1244.4; MH³⁺: Calc: 830.10, Exp: 830.1).

For purity analysis, a Vydac Protein and Peptides Cl 8 column was used. A gradient elution, with a constant flow rate of 1 mL, was performed: 5% solvent B in A for the first 5 minutes, followed by a linear increase from 5% solvent B to 95% solvent B from until 20 min., a wash period of 95% solvent B from 20 to 25 min., and finally a linear decrease from 95% to 5% solvent B in A from 25 to 30 min. The compound eluted at 16.09 as a peak containing a shoulder feature, observed at multiple wavelengths using a Hitachi L7450 detector. When this shoulder was collected and analyzed again, the same peak with a shoulder feature was observed, indicating that this behavior was not due to an overlapping impurity. Detection at 220 nm showed an impurity at 15.07 min., which was not observed at 520 nm. Analysis of peak areas determined that VS001 was 96% pure.

Synthesis and Characterization of Dye labelled Gastricsin Product

Synthesized gastricsin product was prepared analogously to VS001 (see previous procedure). The resin-bound peptide sequence was ordered from Genscript, labelled with dimeric rhodamine dye, cleaved from resin, purified using semi-preparative HPLC on an Alltech Econosil Column (using the same method as described for VS001), and concentrated in vacuo. The peptide was identified using electrospray ionization mass spectrometry, performed using an Advion Expression^L spectrometer (M²⁺: Calc: 763.88, Exp: 764.0).

For purity analysis, a Vydac Protein and Peptides Cl 8 column was used. A gradient elution, with a constant flow rate of 1 mL, was performed: 5% solvent B in A for the first 5 minutes, followed by a linear increase from 5% solvent B to 95% solvent B from until 20 min., a wash period of 95% solvent B from 20 to 25 min., and finally a linear decrease from 95% to 5% solvent B in A from 25 to 30 min. The compound eluted at 17.27 min. as a single sharp peak, by detection using UV- visible absorption at 220 nm (using a Hitachi L7400 detector). Because no other peaks were observed, the product was deemed to be greater than 99% pure.

While various embodiments have been described in considerable detail herein, the embodiments are merely offered as non-limiting examples of the disclosure described herein. It will therefore be understood that various changes and modifications may be made, and equivalents may be substituted for elements thereof, without departing from the scope of the present disclosure. The present disclosure is not intended to be exhaustive or limiting with respect to the content thereof.

Further, in describing representative embodiments, the present disclosure may have presented a method and/or a process as a particular sequence of steps. However, to the extent that the method or process does not rely on the particular order of steps set forth therein, the method or process should not be limited to the particular sequence of steps described, as other sequences of steps may be possible. Therefore, the particular order of the steps disclosed herein should not be construed as limitations of the present disclosure. In addition, disclosure directed to a method and/or process should not be limited to the performance of their steps in the order written. Such sequences may be varied and still remain within the scope of the present disclosure.

Claims

1. A method for classifying pancreatic cysts of a patient, comprising the steps of: preparing a bead suspension by binding magnetic beads with a gastricsin substrate and subsequently washing away any unbound substrate; activating a sample containing gastricin by lowering the pH of the sample; mixing the sample and the bead suspension to form a mixed sample; removing the magnetic beads from the mixed sample, resulting in a solution; and measuring the solution to determine gastricsin levels in the sample, wherein the gastricsin levels are indicative of a cyst type.

2. The method of claim 1, wherein the gastricin substrate comprises a biotinylated lysine residue at a C-terminus of the gastricin substrate using a diethylene glycol spacer.

3. The method of claim 1, wherein the gastricin substrate comprises a fluorescent dye at an N-terminus of the gastricin substrate.

4. The method of claim 3, wherein the step of mixing the sample and the bead suspension to form the mixed sample is performed to allow the gastricin in the sample to cleave the gastricin substrate having the fluorescent dye.

5. The method of claim 1, wherein the gastricsin substrate is a peptide that is selectively cleaved at low pH by gastricsin without interference from any other proteins found in cyst fluid.

6. The method of claim 1, wherein the gastricin substrate comprises a reporter that is fluorescent.

7. The method of claim 1, wherein the gastricin substrate comprises a reporter reactive to surface enhanced Raman spectroscopy (SERS).

8. The method of claim 1, wherein the patient sample contains pepsin.

9. The method of claim 8, further comprising the step of adding pepstatin to the solution.

10. The method of claim 1, wherein the sample is a patient sample containing pepsinogen C.

11. The method of claim 1, wherein the sample is a patient sample containing blood.

12. The method of claim 1, wherein the sample is less than 5 pL.

13. The method of claim 1, wherein the step of preparing the bead suspension by binding the magnetic beads with the gastricsin substrate is performed using streptavidin or amine binding or click chemistry.

14. The method of claim 1, wherein the step of measuring the solution is performed utilizing surface enhanced Raman spectroscopy (SERS).

15. The method of claim 3, wherein the step of measuring the solution is performed utilizing fluorescence.

16. The method of claim 3, further comprising the step of adding silver nanoparticles to the solution.

17. The method of claim 16, wherein the step of measuring the resulting solution is performed utilizing surface enhanced Raman spectroscopy (SERS).

18. The method of claim 17, wherein the step of utilizing SERS is performed at a wavelength selected from the group consisting of 538nm, 638nm, and 785 nm.

19. The method of claim 16, wherein silver nanoparticles are added to the resulting solution such that the ratio of the silver nanoparticles is greater than 1 : 1 by volume, weight, or density.

20. The method of claim 1, wherein the step of preparing the bead suspension by binding magnetic beads with a gastricsin substrate is performed by preparing the bead suspension by binding magnetic beads with a gastricsin substrate having a fluorescent dye coupled thereto.

21. The method of claim 1, wherein the step of measuring the solution is performed to determine the gastricin levels as being indicative of a mucinous cyst or a non- mucinous cyst.

22. The method of claim 21, wherein if the step of measuring the solution is performed and the gastricin levels are indicative of a mucinous cyst, the method further comprises the step of: determining that the sample contains cancerous cells.