Isolation Of Cells Research Articles

Abstract LB094: Assessment of minimal residual disease in lung cancer patients treated with osimertinib using liquid biopsy

Abstract Purpose: Drug tolerance has emerged as one of the major mechanisms driving resistance to targeted therapies (TT), but a comprehensive understanding of the dynamics and molecular heterogeneity underlying the adaptive drug response in patients is still lacking. Here, we used iterative liquid biopsies from osimertinib-treated lung cancer patients to monitor and characterize minimal residual disease (MRD) using both circulating tumor DNA (ctDNA) and circulating tumor cells (CTC). Experimental design: LUNG-RESIST (NCT04222335) is a prospective research study enrolling EGFR-mutant adenocarcinoma patients treated by osimertinib frontline. We collected blood samples at baseline, 1 month and every 3 months until progression. CtDNA was used to monitor the initial EGFR mutation (EGFRm) by digital PCR, and to identify new potential biomarkers of resistance by next-generation sequencing (NGS). CTC were enriched and isolated based on their size, absence of leucocyte staining and viability, to perform single-cell transcriptomic and genomic analyses. Results: 40 patients have been enrolled with a median progression-free survival (mPFS) of 14.5 months (95%CI, 8.5-24.5 months). 28/40 (70%) patients had detectable EGFRm in ctDNA at baseline, and decreased ctDNA level could be measured in 25/26 (96%) patients after 1 month of treatment, with a complete clearance in 15/26 (58%) patients. 7/11 (64%) patients without clearance at 1 month relapsed within 9 months. Clinical relapse could be anticipated 3 months ahead based on ctDNA increase for 9/20 (45%) patients. CTC-like cells have been collected at baseline and MRD (between 1 and 3 months) from respectively 31/40 (78%) and 29/40 (73%) patients, and at clinical progression for 12/22 (54%) patients who relapsed, for a total number of 1,510 cells. The number of CTC-like cells decreased after 1 month of treatment in 18/29 (62%) patients, and increased again in 7/12 (58%) patients at the time of clinical relapse. The results of NGS analyses on ctDNA and single-cell transcriptomic and genomic analyses on CTC will be presented at the congress, and should provide a better insight into the dynamics of molecular events occurring throughout the adaptive response in patients. Conclusion: We report the first molecular profiling of both ctDNA and CTC during the adaptive response to osimertinib in lung cancer patients. Monitoring the response and predicting the clinical outcome for EGFR-mutant lung cancer patients undergoing TT treatment can be done using ctDNA level as a biomarker. The isolation of CTC-like cells represents a non-invasive approach to study drug tolerance and ultimately develop new combinatory treatments to prevent relapse. Citation Format: Celia Delahaye, Anne Casanova, Estelle Clermont-Tarenchon, Aurelia Doussine, Juan-Pablo Cerapio, Anaïs Anton, Emma Devienne, Vincent Dongay, Julie Milia, Gilles Favre, Olivier Calvayrac, Anne Pradines, Julien Mazieres. Assessment of minimal residual disease in lung cancer patients treated with osimertinib using liquid biopsy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 2 (Late-Breaking, Clinical Trial, and Invited Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(7_Suppl):Abstract nr LB094.

Research progress on optimization of in vitro isolation, cultivation and preservation methods of dental pulp stem cells for clinical application.

Due to high proliferative capacity, multipotent differentiation, immunomodulatory abilities, and lack of ethical concerns, dental pulp stem cells (DPSCs) are promising candidates for clinical application. Currently, clinical research on DPSCs is in its early stages. The reason for the failure to obtain clinically effective results may be problems with the production process of DPSCs. Due to the different preparation methods and reagent formulations of DPSCs, cell characteristics may be affected and lead to inconsistent experimental results. Preparation of clinical-grade DPSCs is far from ready. To achieve clinical application, it is essential to transit the manufacturing of stem cells from laboratory grade to clinical grade. This review compares and analyzes experimental data on optimizing the preparation methods of DPSCs from extraction to resuscitation, including research articles, invention patents and clinical trials. The advantages and disadvantages of various methods and potential clinical applications are discussed, and factors that could improve the quality of DPSCs for clinical application are proposed. The aim is to summarize the current manufacture of DPSCs in the establishment of a standardized, reliable, safe, and economic method for future preparation of clinical-grade cell products.

Turning Trash Into Treasure: A Simple Protocol for Human Mesenchymal Stromal Cell Isolation From Used Bone Marrow Collection Kits.

The therapeutic potential of mesenchymal stromal cells (MSCs) has been extensively investigated in both preclinical and clinical settings. Recent years have witnessed the emergence of numerous isolation protocols and culture techniques, ranging from the selection of subpopulations to preserve stemness to preconditioning strategies aimed at enhancing therapeutic efficacy, tailored to the specific tissue source. In this protocol, we present a straightforward and cost-effective method for isolating human MSCs (hMSCs) from discarded bone marrow collection kits (comprising bag and filter systems) originally intended for removing impurities and unwanted cellular debris from the collected bone marrow aspirate, ensuring the purity of the stem cell population during stem cell transplantation. Utilizing basic laboratory equipment, we demonstrate the isolation of hMSCs, highlighting the expression of specific surface antigens, and multilineage differentiation into adipogenic, osteogenic, and chondrogenic lineages in vitro. This sustainable and resource-efficient approach not only contributes to reducing medical waste but also holds promise for advancing regenerative medicine applications. © 2024 Wiley Periodicals LLC. Basic Protocol 1: Isolation of human mesenchymal stromal cells from bone marrow collection kits Basic Protocol 2: Culture of human mesenchymal stromal cells Basic Protocol 3: Characterization of human mesenchymal stromal cells with flow cytometry analysis Basic Protocol 4: Characterization of human mesenchymal stromal cells with multilineage differentiation under in vitro conditions.

Gross as well as microscopic anatomy and physiological functions of fetal placenta in Jaffrabadi buffaloes

The present study was conducted to know the gross morphology and histo-morphological structure of fetal placenta in Jaffrabadi buffaloes. Parameters like calf weight, placental weight, numbers, and size of cotyledons were observed in Jaffrabadi buffaloes and morphologically, fetal cotyledons were convex and non-pendunculated in Jaffrabadi buffaloes were found. Histological studies of small and large cotyledons showed extensive branching of secondary and tertiary villi that were longer, slender, and well developed in Jaffrabadi buffalo. A less developed basal lamina was seen in small cotyledon whereas developed basal lamina with numerous capillaries and connective tissue were observed in the large cotyledon. The diameter of trophoblast giant cells (TGC) in larger cotyledons were significantly (P<0.05) than the small cotyledons in expelled placenta at full term in Jaffrabadi buffaloes. A distinct distribution of carbohydrate and lipids in cotyledons were observed between large and small cotyledons as evident by acid mucopolysaccharides, neutral polysaccharides, and sudanophilic staining. Specific staining for calcium with Alizarin red stain showed that calcium is not present in a noticeable amount in small and large cotyledons. Isolation and culture of Jaffrabadi placental cells in M-199 medium with antibiotics and 2% FBS results in the efficient production of progesterone, estrogen, and testosterone. This study has shown that trophoblast cells are the actual sites for steroid hormone production. These cultured placental cells (1x106 cells/ ml) produce Progesterone, Estradiol-17β and Testosterone in the range of 1.72 to 2.12, 16.03 to 19.51 and 0.51 to 0.58 ng/ml, respectively in Jaffrabadi buffalo.

450 Developing Methods for High-Resolution Characterization of Plasma Cells

OBJECTIVES/GOALS: Antibodies play an important role in the pathogenesis of a wide range of diseases, including cancer, autoimmune diseases, and infections. There are currently no reliable methods to isolate and study specific plasma cell subpopulations as antibody production sources. We aim to develop methods to study plasma cells in high resolution. METHODS/STUDY POPULATION: We will use molecular cloning to engineer fusion proteins that would bind plasma cell proteins to study these cells based on their surface features. The first phase of our study consists of assessing the efficacy of this plasma cell isolation method in established cell lines (e.g., RPMI 8226) and also antibody-secreting cell lines that we are establishing as a part of this study. In the second phase of the study, we will assess the efficacy of this method by studying antigen-specific plasma cell populations in the bone marrow aspiration samples of 20 healthy volunteers using various assays, including ELISPOT, flow cytometry, and fluorescent microscopy. RESULTS/ANTICIPATED RESULTS: We have designed the constructs and have completed the cloning. The final plasmids have been verified using various restriction enzymes and Sanger sequencing. Following the transfection of Freestyle HEK 293F cells and isolation of respective proteins, we expect to be able to utilize these engineered proteins to differentiate various antibody-secreting plasma cells. We will use cell lines for proof-of-concept experiments and will subsequently move this method to human bone marrow samples. We expect to be able to visualize multiple specific antibody-secreting plasma cell populations using fluorescent microscopy and utilize this method to isolate them by cell sorting via flow cytometry. DISCUSSION/SIGNIFICANCE: We expect to be able to use this method to target specific plasma cell clones in the advancement of precision medicine regarding the treatment of plasma cell disorders (e.g., multiple myeloma) and also expand its use in other areas, such as antibody discovery and the assessment of the humoral immune responses in infectious diseases.

444 Deciphering the role of IL-4 in post-colitis repair

OBJECTIVES/GOALS: Incomplete mucosal healingand dysbiosis prevent long-term remission after colitis. IL4 may restore colon homeostasis through its action on immune cells and the microbiome. We will demonstrate this mechanism using genetically modified mice and molecular tools. This may result in target therapies that prolong remission in patients with IBD. METHODS/STUDY POPULATION: Mice were treated with 3% dextran sulfate sodium (DSS) in drinking water for 5 days to induce colitis. Mice were monitored daily for changes in body weight, and to monitor colitis severity. At each endpoint, mice were sacrificed and colon length was measured. For disease severity assessment, mouse colons were prepared in paraffin sections by the 'swiss-rolling' method. For flow cytometry, lamina propria mononuclear cell isolation was performed and cellular populations were stained with fluorophore-conjugated antibodies. IL4-eGFP-expressing (4get) mice were used to analyze the cellular expression of IL4 after colitis. Cell-specific IL4 deletion mice were generated using the cre-lox system. RESULTS/ANTICIPATED RESULTS: IL4-deficient mice had worse colitis compared with wild-type controls. Flow cytometry of lamina propria cells from 4get mice showed that most IL4-producing cells after colitis are eosinophils (CD11b+SiglecF+). Flow cytometry of C57bl6 mice showed an influx of IL4Ra+ monocytes (CD11b+Ly6C+) and macrophages (CD11b+F480+). IL4-stimulated bone marrow-derived macrophages demonstrated an increase in HB-EGF mRNA transcription. Myeloid-specific IL4R deleted mice had no difference in colitis severity compared with controls. Neutrophil-specific IL4R-deleted mice had increased colitis severity and mortality. Co-housing of littermate mice rescued recovery after DSS in IL4 deficient mice. DISCUSSION/SIGNIFICANCE: IL4 appears to play a role in restoring homeostasis after colitis. The mechanism depends on eosinophil-derived IL4, and action through neutrophils. However, the reparative function of IL4 can be shared with deficient mice through the microbiome. I will study the cellular and microbial mechanism by which IL4 restores homeostasis after colitis.

Investigation of Peste des petits ruminants virus circulation in Uttarakhand, India: a step towards global eradication of PPR by 2030.

This study was conducted to estimate the seroprevalence of Peste des petits ruminants virus (PPRV) and to determine the virus distribution in unvaccinated goats in the Pantnagar region of Uttarakhand state, India. A total of 212 serum samples from goats were collected randomly from various villages in three districts (Udhamsingh Nagar, Nainital, and Almora) of Uttarakhand. Serum samples were tested for anti-PPRV antibodies by a commercially available kit. RNA was extracted from the clinical samples and it was subjected to one-step RT-PCR, followed by virus isolation from positive samples. A total of 41 animals from various villages were found to be seropositive with a prevalence rate of 19.33%. PPR outbreaks were also reported from the Tarai region of Uttarakhand, and detection by PCR confirmed PPRV in 8 goats. Two representative swab samples were subjected to virus isolation in Vero cells and both samples showed typical cytopathic effects. The present study shows that PPRV is circulating in the Tarai region of Uttarakhand and mass vaccination for PPR must be followed in this region to increase herd immunity to a protective level. To the best of our knowledge, this is the first investigation of PPRV seroprevalence in unvaccinated goats of Uttarakhand, India.

The Influence of Lysosomal Stress on Dental Pulp Stem Cell-Derived Schwann Cells.

Dysregulation of the endo-lysosomal-autophagy pathway has been identified as a critical factor in the pathology of various demyelinating neurodegenerative diseases, including peripheral neuropathies. This pathway plays a crucial role in transporting newly synthesized myelin proteins to the plasma membrane in myelinating Schwann cells, making these cells susceptible to lysosome-related dysfunctions. Nevertheless, the specific impact of lysosomal dysfunction in Schwann cells and its contribution to neurodegeneration remain poorly understood. We aim to mimic lysosomal dysfunction in Schwann cells using chloroquine, a lysosomal dysfunction inducer, and to monitor lysosomal leakiness, Schwann cell viability, and apoptosis over time. Additionally, due to the ethical and experimental issues associated with cell isolation and the culturing of human Schwann cells, we use human dental pulp stem cell-derived Schwann cells (DPSC-SCs) as a model in our study. Chloroquine incubation boosts lysosomal presence as demonstrated by an increased Lysotracker signal. Further in-depth lysosomal analysis demonstrated an increased lysosomal size and permeability as illustrated by a TEM analysis and GAL3-LAMP1 staining. Moreover, an Alamar blue assay and Caspase-3 staining demonstrates a reduced viability and increased apoptosis, respectively. Our data indicate that prolonged lysosomal dysfunction leads to lysosomal permeability, reduced viability, and eventually apoptosis in human DPSC-SCs.

Calorie restriction-induced leptin reduction and T-lymphocyte activation in blood and adipose tissue in men with overweight and obesity

BackgroundT-Lymphocyte activation is modulated by the adipokine leptin and serum concentrations of this hormone can be reduced with short-term calorie restriction. The aim of this study was to understand whether leptin per se is important in determining levels of T-lymphocyte activation in humans, by investigating whether the reduction in leptin concentration following calorie restriction is associated with a decrease in T-Lymphocyte activation in blood and adipose tissue.MethodsTwelve men with overweight and obesity (age 35–55 years, waist circumference 95–115 cm) reduced their calorie intake by 50% for 3 consecutive days. Blood and subcutaneous adipose tissue were obtained for isolation of immune cells and cytokine analysis. CD4+ and CD8 + T-Lymphocytes were identified and characterised according to their expression of activation markers CD25 and CD69 by flow cytometry.ResultsSerum leptin was reduced by (mean ± SEM) 31 ± 16% (p < 0.001) following calorie restriction. The percentage of blood CD4 + CD25 + T-lymphocytes and level of CD25 expression on these lymphocytes were significantly reduced by 8 ± 10% (p = 0.016) and 8 ± 4% (p = 0.058), respectively. After calorie restriction, ex vivo leptin secretion from abdominal subcutaneous adipose tissue explants was not changed, and this corresponded with a lack of change in adipose tissue resident T-Lymphocyte activation.ConclusionsSerum leptin was reduced after calorie restriction and this was temporally associated with a reduction in activation of blood CD4 + CD25 + T-Lymphocytes. In abdominal subcutaneous adipose tissue, however, leptin secretion was unaltered, and there were no observed changes in adipose resident T-Lymphocyte activation.

Isolation and characterization of mammary epithelial cells derived from Göttingen Minipigs: A comparative study versus hybrid pig cells from the IMI-ConcePTION Project

The value of pig as “large animal model” is a well-known tool for translational medicine, but it can also be beneficial in studying animal health in a one-health vision. The ConcePTION Project aims to provide new information about the risks associated with medication use during breastfeeding, as this information is not available for most commonly used drugs. In the IMI-Conception context, Göttingen Minipigs have been preferred to hybrid pigs for their genetic stability and microbiological control. For the first time, in the present research, three primary cell cultures of mammary epithelial cells were isolated and characterized from Göttingen Minipigs (mpMECs), including their ability to create the epithelial barrier. In addition, a comparative analysis between Göttingen Minipigs and commercial hybrid pig mammary epithelial cells (pMECs) was conducted. Epithelial markers: CKs, CK18, E-CAD, ZO-1 and OCL, were expressed in both mpMECs and pMECs. RT2 Profiler PCR Array Pig Drug Transporters showed a similar profile in mRNA drug transporters. No difference in energy production under basal metabolic condition was evidenced, while under stressed state, a different metabolic behaviour was shown between mpMECs vs pMECs. TEER measurement and sodium fluorescein transport, indicated that mpMECs were able to create an epithelial barrier, although, this turned out to be less compact than pMECs. By comparing mpMECs with mammary epithelial cells isolated from Hybrid pigs (pMECs), although both cell lines have morphological and phenotypic characteristics that make them both useful in barrier studies, some specific differences exist and must be considered in a translational perspective.

Diagnostic value of serum COMP and ADAMTS7 for intervertebral disc degeneration

ObjectiveIntervertebral disc degeneration (IVDD) is a major cause of morbidity and disability. Our study aimed to investigate the potential of cartilage oligomeric matrix protein (COMP) and ADAMTS7 (A disintegrin and metalloproteinases with thrombospondin motifs 7) as biomarkers for IVDD together with their functional relationship.MethodsIVD tissues and peripheral blood samples were collected from IVDD rabbit models over 1–4 weeks. Tissues and blood samples were also collected from clinical patients those were stratified into four equal groups according to Pfirrmann IVDD grading (I–V) with baseline data collected for each participant. COMP and ADAMTS7 expression were analyzed and biomarker characteristics were assessed using linear regression and receiver operating curve (ROC) analyses.ResultsCOMP and ADAMTS7 expression increased in tissues and serum during IVDD progression. Serum COMP (sCOMP) and serum ADAMTS7 (sADAMTS7) levels increased in a time-dependent manner following IVD damage in the rabbit model while significant positive correlations were detected between sCOMP and sADAMTS7 and Pfirrmann grade in human subjects. ROC analysis showed that combining sCOMP and sADAMTS7 assay results produced an improved diagnostic measure for IVDD compared to individual sCOMP or sADAMTS7 tests. In vitro assays conducted on human cell isolates revealed that COMP prevented extracellular matrix degradation and antagonized ADAMTS7 expression although this protective role was uncoupled under microenvironmental conditions mimicking IVDD.ConclusionsIncreases in circulating COMP and ADAMTS7 correlate with IVDD progression and may play regulatory roles. Assays for sCOMP and/or sADAMTS7 levels can discriminate between healthy subjects and IVDD patients, warranting further clinical assessment.

DNA Ploidy as a Potential Adjunct Prognostic Marker of Low-Risk Prostate Cancer Progression after Radical Prostatectomy.

Post prostatectomy PSA kinetics and General Grade Groups (GGG) are the strongest prognostic markers of biochemical recurrence (BCR) and prostate cancer (PCa)-specific mortality after radical prostatectomy. Despite having low-risk PCa, some patients will experience BCR, for some, clinically significant BCR. There is a need for an objective prognostic marker at the time of prostatectomy to improve risk stratification within this population. In this study, we investigated the prognostic potential of DNA ploidy. Prostatectomy samples from 97 patients with GGG1 and GGG2 with a low-risk CAPRA-S score were included in this study. PCa tissue with the worst Gleason pattern underwent tissue disaggregation, cell isolation and staining with a DNA stoichiometric stain. Using image cytometry, DNA ploidy was measured and a Ploidy Score (PS) was generated. Among the 97 patients, 79 had no BCR, 18 experienced BCR, of which 14 had a PSA doubling time (PSA-DT) >1 year (low-risk group) and 4 had a PSA-DT of <1 year (high-risk group). Using Logistic regression analysis, only pathological T stage (pT) and PS independently predicted BCR with PS being the most significant (p = 0.001). The number of aneuploid cells was significantly higher in the high-risk group compared to the other groups (p = 1.7x10-11). PS combined with GGG diagnosis further stratified risk groups of biochemical recurrence free survival within CAPRA-S low-risk cohort. DNA ploidy is an independent prognostic marker of BCR in low-risk PCa after radical prostatectomy, which could early on identify potentially aggressive PCa recurrences and introduce a more personalized approach to salvage treatments.

Ilheus Virus (ILHV) Resistance in Culex quinquefasciatus from the Northern Region of Brazil.

Orthoflavivirus ilheusense (ILHV) is a member of the Flaviviridae family. It was first isolated in 1944 from pools of Aedes serratus and Psorophora ferox mosquitoes; however, it has also been detected in species of the genus Culex, such as Cx. portesi and Cx. coronator. The objective of this study was to examine the vector competence of Cx. quinquefasciatus mosquitoes to ILHV infection and the subsequent transmission of the virus through their saliva during feeding on blood. F1 generation females of Cx. quinquefasciatus (Ananindeua/PA) were orally infected with goose blood infected with strain BeH7445, and body, head and saliva samples were analyzed at 7, 14, and 21 dpi using the techniques of virus isolation in cells and indirect immunofluorescence. The presence of ILHV was not detected in the body and head samples of Cx. quinquefasciatus females at any of the three dpi's analyzed, indicating that the lineage of mosquitoes analyzed was resistant to ILHV. According to the results obtained in this study, the species Cx. quinquefasciatus proved resistant to ILHV, regardless of the virus titers to which it was exposed, which suggests the possibility that this species does not act as a vector in the ILHV transmission cycle.

Abstract 35: In situ CAR T cell engineering using lipid nanoparticles for B cell lymphoma therapy

Abstract Chimeric antigen receptor (CAR)-T cell therapy represents a breakthrough in immunotherapy, offering promising outcomes in the management of hematological malignancies. Current CAR-T cell in vitro manufacturing routes involve T cell isolation, purification, activation, gene transduction and cell expansion. The process duration and the costs present significant barriers. Therefore, there is a need for innovative strategies to address these concerns. Herein, we developed lipid nanoparticles (LNPs) to deliver CAR-mRNA efficiently and safely to T cells in vivo, thereby enabling in situ CAR-T cell engineering. By optimizing their composition and introducing anti-CD5 as the targeting ligand, we were able to enhance mRNA delivery and T cell transfection efficiency while maintaining T cell function. Furthermore, to augment T cell responsiveness to exogenous genetic elements, we investigated the pretreatment of T cells with interleukin (IL-7), thereby modulating T cell homeostasis by enhancing survival and proliferation, and rendering the cells more receptive to exogenous genetic modifications. We tracked firefly luciferase (FLuc) in naïve Balb/c mice using in vivo and ex vivo bioluminescence imaging after intravenous injection of anti-CD5 FLuc-mRNA LNPs. We observed positive mRNA expression in lymphoid organs, particularly in the spleen. Additionally, we harvested the lymphoid organs, and performed immunophenotyping analysis using flow cytometry after the administration of anti-CD5 LNPs encapsulating mRNA encoded CD19-CAR with a flag tag. We showed enhanced LNP binding in 65.2% of T cells and CAR expression in 11.64% of T cells as compared with non-treated controls, anti-CD5 FLuc-mRNA LNPs, or IgG CAR-mRNA LNPs. In a CD19+ A20 tumor-bearing mouse model, treatment with CAR-LNPs resulted in a significant increase in chemoattractants (CCL2, CCL4, CCL5, CCL7 and CXCL10) and pro-inflammatory cytokines (TNF-α, IFN-γ, IL6, IL8 and IL15) in blood, and infiltration of T cells and other immune cells in tumors, compared to the non-treated control group over 48 hours post treatment. In vivo engineering allows simultaneous CAR T cell engineering and delivery, reducing the time between manufacturing and treatment, minimizing contamination risks, eliminating complex ex vivo processes, and reducing the need for high-dose preconditioning chemotherapy. In conclusion, the adoption of in situ CAR-T cell production holds promise for improving this therapeutic approach. The authors acknowledge the funding support from NIHR01CA253316 and Leukemia and Lymphoma Society (LLS, Grant ID: 8022-20) Citation Format: Nisi Zhang, Elise Robinson, Angelie Rivera-Rodriguez, James Wang, Yutong Guo, Bo Wu, Spencer K. Tumbale, Marina Raie, Mohammed Inayathullah, Debra K. Czerwinski, Ronald Levy, Katherine W. Ferrara. In situ CAR T cell engineering using lipid nanoparticles for B cell lymphoma therapy [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 35.

Abstract 995: Preservation of patient whole blood preserves cancer immune cell biology at single cell resolution

Abstract Blood is an easily accessible clinical sample type that helps inform treatments, investigate disease mechanisms, and monitor outcomes. Logistical challenges in the transportation and processing of blood samples impact data output from single cell RNA sequencing (scRNA-seq). Recent scRNA-seq studies have shown that significant changes in gene expression and immune cell populations occur in as little as a few hours post collection. Thus, there is a need for rapid preservation of blood upon collection before biological changes can occur. Existing preservation approaches compromise the cell membrane, making samples incompatible with single cell transcriptomic analyses. Here, we describe a novel workflow for fixation and long-term storage of whole blood, and isolation of peripheral blood mononuclear cells (PBMCs) from the fixed blood using a readily available, low-cost, high-throughput procedure. We then profile the gene expression of these cells using the fixation-compatible Single Cell Gene Expression Flex workflow. Importantly, this novel blood collection and preparation workflow eases blood transportation logistical constraints, allowing distributed sample collection and batched shipping of blood samples for scRNA-seq and potentially enabling large-scale translational research studies. We compared whole blood fixed immediately after collection using our workflow with clinical blood samples stored at ambient temperature. This revealed that immune cell populations and biological pathways shift significantly as early as five hours following sample collection, if not immediately stabilized. Across a time course of ambient temperature storage of whole blood without immediate stabilization, we observe significant upregulation of stress-related genes (FOS, JUN, CIRBP) in samples using Single Cell Gene Expression Flex profiling. Additionally, expression of a proto-oncogene TNFAIP3 was upregulated in whole blood stored at 20°C compared to samples stored at 4°C for the same lengths of time, unless samples were stabilized using the novel workflow shown here. This novel whole blood fixation method, coupled with automatable cell isolation methods and Single Cell Gene Expression Flex-enabled multiplexed single cell sequencing, will usher in a new age of economical, large-scale translational research studies that result in valuable clinical insights. Citation Format: Kelly Martin, Connor Kunihiro, Jawad Abousoud, Tingsheng Drennon, Yina Li, Sean Marrache, Nandhini Raman, Veronica Rodriguez, Paul Lund, Jens Durruthy Durruthy, Sarah Taylor, Andrew Kohlway, Peter Smibert, Dagmar Walter, Jill Herschleb. Preservation of patient whole blood preserves cancer immune cell biology at single cell resolution [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 995.

Abstract 1161: Deep spatial proteomics: A new approach for obtaining insight into the glioblastoma microenvironment

Abstract Glioblastoma is the most frequent and malignant brain tumor, with a median survival of approximately 14 months. The tumor microenvironment is composed of different cell types and is known to support the glioblastoma cells and contribute to poor patient prognosis. Since accumulating studies report discordance between mRNA and protein abundances both on population and single-cell levels, and since proteins are the effector molecules of the cells and the actual target molecules of most drugs, our aim is to dissect the tumor microenvironment by spatial LC-MS based proteomics. Glioblastoma formalin-fixed paraffin-embedded (FFPE) tissue was sectioned and placed on membrane glass slides suitable for laser capture microdissection (LCM), followed by precise isolation of single cells from the heterogeneous tumor microenvironment. We used the Zeiss Palm MicroBeam laser microdissection instrument, combined with an Orbitrap Eclipse Tribrid Mass Spectrometer running our in-house sensitivity tailored Data Independent Acquisition method (WISH-DIA) to analyze and quantify the proteomes. We obtained up to 3000 proteins out of only 100 cells and were able to demonstrate the feasibility of collecting and analyzing various cell types at the single-cell level. The proteins identified belonged to many different classes/families of proteins and were able to distinguish the different cell types. In conclusion, this on-going study demonstrates our ability to isolate single cells from the highly heterogeneous tumor microenvironment of glioblastoma with ultra-high sensitivity, thereby allowing the characterization of the proteome of the glioblastoma microenvironment followed by identification of potential therapeutic and/or diagnostic targets. Citation Format: Kartikey Saxena, Rune Daucke, Vilde Pedersen, Pia Helene Klausen, Erwin Schoof, Bjarne Winther Kristensen. Deep spatial proteomics: A new approach for obtaining insight into the glioblastoma microenvironment [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 1161.

Dielectric properties of human macrophages are altered by Mycobacterium tuberculosis infection.

The analysis of cell electrophysiology for pathogenic samples at BSL3 can be problematic. It is virtually impossible to isolate infected from uninfected without a label, for example green fluorescent protein, which can potentially alter the cell electrical properties. Furthermore, the measurement of highly pathogenic organismsoften requires equipment dedicated only for use with these organisms due to safety considerations. To address this, we have used dielectrophoresis to study the electrical properties of the human THP-1 cell line and monocyte-derived macrophages before and after infection with non-labelled Mycobacterium tuberculosis. Infection with these highly pathogenic bacilli resulted in changes including a raised surface conductance (associated with reduced zeta potential) and increased capacitance, suggesting an increase in surface roughness. We have also investigated the effect of fixation on THP-1 cells as a means to enable study on fixed samples in BSL1 or 2 laboratories, which suggests that the properties of these cells are largely unaffected by the fixation process. This advance results in a novel technique enabling the isolation of infected and non-infected cells in a sample without labelling.

Size effect of dynamic bioactive magnetic particles on regulated isolation of tumor cells

Circulating tumor cells (CTCs) are critical biomarkers in cancer metastasis, yet their isolation from the bloodstream remains a challenging task due to their low abundance. This study introduces a method for the efficient isolation of CTCs, utilizing dynamic biointerface-based magnetic particles (DBMPs), with a particular emphasis on the role of particle size. We have developed three distinct sizes DBMPs specifically tailored for effective CTCs interaction, leveraging the unique properties of polydopamine (PDA) coatings and phenylboronic acid (PBA)-modified capture peptides through reversible catechol-boronate interactions. Comparative analysis of DBMPs of varying sizes was assessed under uniform experimental conditions for their capture and release efficiencies. The findings reveal a clear distinction: smaller particles (DBMP1 and DBMP2) achieved greater efficiency in capturing CTCs, whereas the larger particles (DBMP3) were more effective in releasing them. This difference is ascribed to the varied interaction dynamics between the particles and CTCs. Furthermore, we have fine-tuned DBMP3 to optimize CTC capture while still maintaining a high efficiency in cell release. The integration of these DBMPs into a magnetic isolation framework is poised to significantly enhance CTCs isolation methods, marking a pivotal advancement in cancer diagnostics and management.

Abstract 7498: Acoustophoresis enriches tumor cell clusters from patients with metastatic prostate cancer

Abstract Purpose: A subset of aggressive prostate cancers (PCa) progress to invade surrounding tissue to form distant metastases by shed of circulating tumor cells (CTCs). However, due to the scarcity of CTCs among the millions of nucleated blood cells, there is an unmet need to improve CTC isolation assays. Our technique Acoustophoresis uses standing ultrasound waves in a flow-through microfluidic chamber to discriminate cells based on their size, density, and compressibility. Method: We analyzed the effectiveness of acoustophoretic circulating tumor cell (CTC) isolation from 6 mL PFA fixed red blood cell lysed blood from 12 metastatic prostate cancer patients and 20 healthy control subjects and compared it to the golden standard CellSearch. For acoustic cell separation, cells were diluted in 12 mL PBS based buffer and separated by acoustophoresis and analyzed by imaging flow cytometry using a standardized antibody panel (EpCAM, cytokeratin, CD45, CD66b, DAPI). Generally, CTCs, and clusters migrate faster than white blood cells (WBCs) when exposed to ultrasound in the microchannel during acoustophoresis and can therefore be separated from nucleated blood cells. Major findings: Our pilot study shows that acoustophoresis finds both singlets and clusters of CTCs in patients as well as aggregates of CTCs and WBCs. In total, 67 cancer cell containing cell clusters were identified. Most identified cell clusters were small, containing less than 4 cells. In addition, acoustophoresis is a label-free separation technique, and could therefore identify additional subclasses of CTCs among the detected CTCs with e.g., EpCAM low/- expression. Further, we provide extensive data for the effect of storage time on acoustophoretic separation performance for fixed prostate cancer cell line cells and WBCs. Demonstrating that sample processing can be done up to 72 hours after blood sampling. We believe this is to date the most comprehensive study of acoustic separation of CTCs in patients and control subjects. Conclusion: Label-free acoustophoresis is a potential method for future non-invasive molecular interrogation of metastatic cancers. We believe Acoustophoresis is a promising technology for CTC enrichment with the possibility to detect new subclasses of CTCs as well as cell clusters not detectable with CellSearch. CTC-clusters has previously been proposed as a promising and potent predictor to monitor progressive disease and is associated with earlier onset of metastatic disease and poor prognosis, which makes acoustophoresis ability to identify clusters of high interest. Citation Format: Cecilia Magnusson, Per Augustsson, Andreas Lenshof, Andreas Josefsson, Anders Bjartell, Yvonne Ceder, Hans Lilja, Thomas Laurell. Acoustophoresis enriches tumor cell clusters from patients with metastatic prostate cancer [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 7498.

Abstract 7497: Advancing multi-biomarker CTC assay and CDx development through the automated GenoCTC system

Abstract Introduction: The successful outcomes observed in clinical trials emphasize the effectiveness of therapeutic approaches relying on biomarker tests, particularly in the development of cancer drugs. The parallel development of a drug and its associated predictive biomarker is considered an ideal strategy for achieving rapid and successful drug development in precision medicine. In this context, we employ an automated circulating tumor cell (CTC) isolation platform, known as GenoCTC, to facilitate the development of biomarker-driven CTC assays, with the ultimate goal of developing companion diagnostic (CDx) tests. Method: This study employed the 5th generation GenoCTC device, a fully automated system designed for marker-based CTC enrichment using immune magnetophoresis. We confirmed the isolation of CTCs through spiking tests, utilizing cancer biomarkers such as cMET, Mesothelin (MSLN), and Claudin 3 (CLDN3). cMET is a biomarker extensively studied in various cancers, particularly non-small cell lung cancer. We previously showed the prognostic significance of cMET expressing CTCs in HR+ and HER2- metastatic breast cancer patients. MSLN and CLDN3 are tumor-associated proteins showing overexpression across diverse cancer types, with a potential for the development of antibody-based cytotoxic drugs. The GenoCTC device allows efficient separation of CTCs expressing these biomarkers, ensuring high accuracy, precision, specificity, and sensitivity. Simultaneously, therapeutic drugs directed at these markers are progressing through phase trials or preclinical studies. Conclusion: Our results show that cMET, MSLN, and CLDN3 are potential markers for the development of biomarker tests, that can improve the patient selection process for personalized therapy targeting these biomarkers. Citation Format: Chaithanya Chelakkot, Jieun Park, Chae Rin Kim, Yeonu Lee, Yu Rim Lee, Saehyung Lee, Jun Young Choi, Young Ho Moon, Yoon-La Choi, Young Kee Shin, Hun Seok Lee. Advancing multi-biomarker CTC assay and CDx development through the automated GenoCTC 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 7497.