Immunomagnetic Selection Research Articles

Мутации в генах KRAS/NRAS и эффективность индукционной терапии по трехкомпонентным схемам на основе бортезомиба у пациентов с впервые диагностированной множественной миеломой

AIM. To identify the KRAS and NRAS gene mutations in patients with newly diagnosed multiple myeloma (ММ) and to classify them according to the depth of antitumor response to bortezomib-based triplet induction therapy. MATERIALS & METHODS. The trial enrolled 89 patients with newly diagnosed MM prior to chemotherapy. Among them, there were 45 women and 44 men aged 30–82 years (median 58.5 years). ММ was diagnosed according to IMWG criteria (2014). Bone marrow (BM) plasma cells were isolated from the aspirate using gradient method with subsequent immunomagnetic CD138 marker selection. The KRAS and NRAS gene mutations in BM CD138+ cells were identified with Sanger sequencing method. The proteomic programs MutationTaster, Polyphen2, and FATHMM-XF were used for mutation analysis in the KRAS and NRAS genes. All patients received bortezomib-based triplet chemotherapy as first-line treatment. The response depth was assessed after completing 6 cycles of PAD and VCD regimens. Antitumor response was evaluated according to IMWG (2016) criteria. RESULTS. The mutation rate in the gene family RAS was 42 % (37/89). The analysis focused on the data from 33 patients with mutations detected and response identified after 6 cycles of treatment. In 22 out of 33 patients, deep response was not achieved, whereas 11 patients showed complete remission (CR) + very good partial remission (VGPR). In the group of patients without mutations in the gene family RAS, the response to therapy meeting the CR + VGPR criteria was 64 % (27/42). The differences appeared to be significant (p = 0.008). The clinical data and the evaluation of primary treatment outcomes provided the basis for distinguishing a group of 9 prognostically unfavorable mutations: NRAS Gly13Asp, Gln61His; KRAS Gly12Ala, Gly12Asp, Gly12Val, Gly13Asp, Gln61Arg, Gln61His, and Ala146Val. CONCLUSION. The mutations in KRAS and NRAS belonging to the gene family RAS had a negative effect on the efficacy of the bortezomib-based triplet induction therapy. Mutation variants in the RAS family genes differed in prognostic significance. The analysis results helped to identify the mutation variants associated with the worse response to therapy: NRAS Gly13Asp, Gln61His; KRAS Gly12Ala, Gly12Asp, Gly12Val, Gly13Asp, Gln61Arg, Gln61His, and Ala146Val.

Update: Induction of Inflammatory Bowel Disease in Immunodeficient Mice by Injection of Naïve CD4+ T cells (T Cell Transfer Model of Colitis).

The intestinal inflammation induced by injection of naïve CD4+ T cells into lymphocyte-deficient hosts (more commonly known as the T cell transfer model of colitis) shares many features of idiopathic inflammatory bowel disease (IBD) in humans, such as epithelial cell hyperplasia, crypt abscess formation, and dense lamina propria lymphocyte infiltration. As such, it provides a useful tool for studying mucosal immune regulation as it relates to the pathogenesis and treatment of IBD in humans. In the IBD model described here, colitis is induced in Rag (recombination-activating gene)-deficient mice by reconstitution of these mice with naïve CD4+CD45RBhi T cells through adoptive T cell transfer. Although different recipient hosts of cell transfer can be used, Rag-deficient mice are the best characterized and support studies that are both flexible and reproduceable. As described in the Basic Protocol, in most studies the transferred cells consist of naïve CD4+ T cells (CD45RBhi T cells) derived by fluorescence-activated cell sorting from total CD4+ T cells previously purified using immunomagnetic negative selection beads. In a Support Protocol, methods to characterize colonic disease progression are described, including the monitoring of weight loss and diarrhea and the histological assessment of colon pathology. © 2024 The Author(s). Current Protocols published by Wiley Periodicals LLC. Basic Protocol: Induction of IBD in Rag-deficient mice by the transfer of naïve CD4+CD45RBhi T cells Support Protocol: Monitoring development of colitis.

Abstract PO1-16-05: Matched multi-analyte and multi-omic liquid biopsy of cell-free DNA, circulating tumor cells and extracellular vesicles in HER2-positive metastatic breast cancer patients

Abstract Background: Liquid biopsy, specifically cell-free DNA (cfDNA) mutation analysis, has recently entered clinical practice for treatment decision-making in hormone receptor (HR)-positive (+)/HER2-negative (-) metastatic breast cancer (mBC) patients. However, we showed that the analysis of multiple liquid biopsy analytes in the course of treatment in this BC subgroup has additive value. Since no multi-analyte studies are currently available for the subgroup of HER2+ mBC patients, we here studied multiple analytes [cfDNA, circulating tumor cells (CTCs) and extracellular vesicles (EVs)] and utilized our multi-omics perspective (CpG island methylation, mutations and mRNA expression) to get insight into their utility in therapy management of HER2+ mBC. Methods: 2x 9 ml blood was drawn from 21 HER2+ (HR+ n=16 and HR- n=5) mBC patients at the time of disease progression and at two consecutive staging time points. CTCs and their mRNA were isolated from 2x5 ml blood by positive immunomagnetic selection targeting EpCAM, EGFR and HER2 (AdnaTest EMT2/StemCell Select/Detect). Plasma of CTC-depleted blood was used for cfDNA isolation, while mRNA from EVs was isolated from 4 ml pre-filtered plasma by affinity-based binding to a spin column (exoRNeasy) using the remaining blood. The mRNA purified from CTCs and EVs by Oligo-dT beads was reverse transcribed, pre-amplified and analyzed by a multi-marker (18 genes) qPCR panel. qPCR data was normalized to CD45 (for CTC analysis) and data of 20 healthy female donor controls to identify BC specific overexpression signals with a specificity of >90% for all targets. Variants in the cfDNA were analyzed with a customized QIAseq Targeted DNA Panel with unique molecular indices (UMIs) and high coverage (mean 22.000x), while CpG island methylation in the cfDNA was analyzed with a customized QIAseq Targeted Methyl Panel with UMIs covering 9786 CpG sites (mean 900x). Consumables: QIAGEN, Germany. Results: In general, substantial differences occurred between the CTC and EV mRNA profiles. Transcripts involved in the PI3K pathway (mTOR, AKT2, PIK3CA) as well as ERBB2 signals were significantly more prevalent in CTCs compared to EVs and mTOR, ERCC1, SRC, AKT2 und PIK3CA CTC overexpression signals were found in >50% of these patients. In contrast, PARP1, AURKA, SRC und ERCC1 signals were significantly more prominent in the EVs and detected in >50% of the patients. ERCC1 signals were present in EVs with highest prevalence whereas ERBB2 signals were only present in CTCs. ERBB3+ CTCs were only detected in patients showing disease progression. Currently, cfDNA mutations as well as cfDNA CpG island methylation are under evaluation and the complete multi-analyte and multi-omic data set as well as their relation to clinical outcome will have been finalized to be presented at the meeting. Conclusion: We successfully established a workflow for parallel isolation of multiple liquid biopsy analytes from a minimized blood volume in HER2+ mBC. Preliminary mRNA profile results for CTCs and EVs show the complementary nature of these two liquid biopsy analytes. Besides the multi-analyte nature of this workflow, the pending results for genomic and epigenomic information will show which one of the analytes, alone or in combination, will help to optimize therapy management in HER2+ mBC patients. Citation Format: Sabine Kasimir-Bauer, Markus Storbeck, Ioanna Andreou, Siegfried hauch, Oliver Hoffmann, Rainer Kimmig, Corinna Keup. Matched multi-analyte and multi-omic liquid biopsy of cell-free DNA, circulating tumor cells and extracellular vesicles in HER2-positive metastatic breast cancer patients [abstract]. In: Proceedings of the 2023 San Antonio Breast Cancer Symposium; 2023 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2024;84(9 Suppl):Abstract nr PO1-16-05.

Analysis of maternal Fc gamma receptor IIIb isoantibodies using immunomagnetic negative selected neutrophils.

The isolation of neutrophils and subsequent detection of anti-human neutrophil antigens (HNA) antibodies are crucial in clinical medicine for the diagnosis of autoimmune neutropenia, neonatal alloimmune neutropenia (NAIN) and transfusion-related acute lung injury (TRALI). This study reports two cases of maternal anti-Fc-gamma-receptor-IIIb (FcγRIIIb) isoimmunization without NAIN symptoms and compares the efficiency of immunomagnetic negative selection (IMNS) with traditional dextran/Ficoll for neutrophil isolation in HNA serological assays. Investigating two cases of maternal anti-FcγRIIIb isoimmunization, neutrophils from three donors were isolated from 8 mL of whole blood using IMNS and dextran/Ficoll. Serological assays included the granulocyte agglutination and immunofluorescence test, monoclonal antibody immobilization of granulocyte antigens and the LABScreen Multi (One Lambda). IMNS and dextran/Ficoll were compared in terms of cell yield, viability, time, cost and purity. Maternal anti-FcγRIIIb isoantibodies with FCGR3B gene deletion were detected in both cases. Newborns and fathers exhibited specific gene combinations: FCGR3B*02/FCGR3B*02 (Case 1) and FCGR3B*02/FCGR3B*03 (Case 2). IMNS outperformed dextran/Ficoll, yielding four times more neutrophils (average neutrophil counts: 18.5 × 103/μL vs. 4.5 × 103/μL), efficiently removing non-neutrophil cells and reducing processing time (30-40 min vs. 70-90 min), although it incurred a higher cost (2.7 times). Two cases of maternal anti-FcγRIIIb isoantibodies, unrelated to NAIN, were identified. Although neutropenia has not been described in these cases, we emphasize the importance of identifying asymptomatic cases with the potential for severe neutropenia. Additionally, IMNS is introduced as a rapid, high-yield, high-purity neutrophil isolation technique, beneficial for serological assays detecting anti-HNA antibodies.

Abstract 7495: Validation of microfluidic isolation of circulating pancreatic tumor cells from blood

Abstract Background. Pancreatic ductal adenocarcinoma (PDAC) has a poor prognosis and is the fourth leading cause of cancer-related deaths in the US. PDAC is diagnosed in the late stages, which limits treatment options and yields poor clinical outcomes. Recurrence is a significant issue for patients undergoing surgery for PDAC. Circulating tumor cells (CTCs) are thought to be a potential mediator of recurrence, facilitating the formation of secondary tumor sites after surgery. Thus, there is a need to isolate CTCs to improve understanding of disease recurrence, to ultimately improve outcomes. Herein, we aim to validate our inertial microfluidic (iMF) system for isolating pancreatic CTCs from blood. We also benchmark our iMF system to a commercially available immunomagnetic negative selection system, EasySep™, and quantify target cell recovery and cell enrichment. Methods. Our iMF system employs the differential cell sizes of CTCs from blood cells and inertial migration of such cells in a straight microchannel (150 µm × 50 µm × 24 mm) to enrich CTCs from blood. The iMF system sample flow rate used is 100 µL/min, with a 200 µL/min PBS buffer flow rate. The EasySep™ Direct Human CTC Enrichment kit targets CD2, CD14, CD16, CD19, CD45, CD61, CD66b, and Glycophorin to remove blood cells from the sample using magnetic particles, thereby separating them from CTCs. We spiked low cell concentrations of 50, 100, and 500 Hoechst-stained pancreatic carcinoma cells, PANC1, in 1 mL of healthy blood then captured using either EasySep™ or iMF. Cell enumeration was performed after centrifugation or Cytospin. Results. PANC1 cells and WBCs have average sizes of 18 µm and 8.4 µm, respectively. We characterized our iMF device to capture cells ≥ 12 µm, which yields capture of most PANC1 cells. Indeed, our iMF system achieved 82.8±19.2%, 72.5±16.3%, and 59.3±3.8% recovery for 50, 100, and 500 cell-spikes in lysed blood, respectively. For the same 50, 100, and 500 cell-spikes, the total iMF CTC yields after Cytospin were 46.3±11.8, 44.3±9.1, and 32.7±2.1%, while the total CTC yields from EasySep™ were 4.6±3, 5.3±2.1, 8.3±1%, respectively. When we processed whole blood directly, EasySep™ had lower cell recovery, 3.8±0.2, 4.6±1, and 3.3±0.4%, respectively. Our system also demonstrated 8 × higher target cell enrichment compared to EasySep™. Blood lysis before iMF separation did not drastically affect the recovery of target cells. Conclusions. We determined that our system has improved performance over the commercial isolation system, EasySep™, thereby validating the effectiveness of microfluidic isolation for PDAC. In future studies, we can then use our iMF system in CTC isolation from routine blood draws from PDAC patients undergoing surgical resection. We may then isolate real patient samples and study relevant phenotypes of PDAC CTCs which may therefore be used for identifying certain mechanisms of PDAC progression. Citation Format: Celine Macaraniag, Ifra Khan, Jian Zhou, Pier Cristoforo Giulianotti, Alain Borgeat, Gina Votta-Velis, Ian Papautsky. Validation of microfluidic isolation of circulating pancreatic tumor cells from blood [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 7495.

Abstract 1491: The enhanced formation of neutrophil extracellular traps (NETs) may contribute to the exacerbation of peritoneal metastasis in a diabetic host

Abstract Background: Diabetes is associated with increased risk of cancer development and progression. Recent studies have shown that neutrophils in diabetic patients are prone to forming neutrophil extracellular traps (NETs) which have been reported to capture circulating cancer cells, leading to the promotion of liver and lung metastasis. This study aims to clarify the causative relationship between NETs and the peritoneal metastasis (PM) in diabetic condition. Methods: Outcome of the diabetic patients who received curative gastrectomy for gastric cancer from 2010 to 2021 in our institute was comparatively investigated with those without diabetes. Neutrophils were isolated from the bone marrow from C57BL/JHamSlc-ob/ob mouse (type 2 diabetes model) and wild type mouse using Ly6G-based positive immunomagnetic selection. The neutrophils were stimulated with 100nM of PMA for 4 hours and NETs formation was evaluated by the addition of Sytox Green under fluorescent microscope. A mouse gastric cancer cell, YTN16P (5 × 105 cells) was intraperitoneally (IP) injected, the number of metastatic nodules on mesentery was comparatively evaluated two weeks later. Results: Disease-free survival (p=0.004, HR=1.65) and overall survival (p=0.035, HR=1.46) of 203 diabetic patients were significantly worse those of 975 non-diabetic patients. Neutrophils isolated from ob/ob mice produced markedly increased NETs than that from wild-type mice. NETs produced by neutrophils obtained from the peritoneal cavity of ob/ob mice after IP injection of a 9% casein solution effectively captured many YTN16P in vitro. The concentration of double strand DNA (dsDNA) in plasma in ob/ob mice compared with their counterparts (676ng/mL vs 590ng/mL, p=0.036, n=3). The number of PM in ob/ob mice was significantly more than that in wild-type controls (39.1 vs 15.5, p=0.027, n=10). Conclusion: Enhanced NETs formation in diabetic conditions may be associated with an increased risk of PM in gastric cancer. Citation Format: Rei Takahashi, Hideyuki Ohzawa, Yuki Kaneko, Misaki Matsumiya, Yurie Futoh, Kohei Tamura, Kazuya Takahashi, Yuki Kimura, Akira Saito, Hideyo Miyato, Naohiro Sata, Joji Kitayama. The enhanced formation of neutrophil extracellular traps (NETs) may contribute to the exacerbation of peritoneal metastasis in a diabetic host [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 1491.

Reduced miR-99a-3p levels in systemic lupus erythematosus may promote B cell proliferation via NCAPG and the PI3K/AKT signaling pathway.

Systemic lupus erythematosus is an immunologically dysregulated disease characterized by the presence of multiple autoantibodies. In SLE, B lymphocytes contribute to the dysregulated production of autoantibodies and cytokines. Recently, we discovered that miR-99a-3p binds to both EIF4EBP1 and NCAPG mRNA and that lowering miR-99a-3p can promote B cell autophagy in SLE by increasing EIF4EBP1 expression. However, the functions of miR-99a-3p and NCAPG in SLE have not been extensively investigated. This work aims to evaluate the levels of miR-99a-3p and NCAPG expression in SLE B cells and to determine whether the aberrant expression of miR-99a-3p and NCAPG contributes to the pathological mechanisms in SLE. B lymphocytes were obtained through immunomagnetic negative selection. Using RT-qPCR, miR-99a-3p and NCAPG mRNA expressions in B lymphocytes and in the BALL-1 cell line were measured. To determine the relative abundance of NCAPG, PI3K, p-PI3K, AKT, and p-AKT, we normalize them to the level of β-actin using Western blotting. Evaluation of miR-99a-3p and NCAPG's impact on cell proliferation was done utilizing CCK-8 assay. Using flow cytometry, the cell cycle and apoptosis were both measured. Comparing SLE B cells to healthy controls, miR-99a-3p expression was significantly downregulated. Additionally, it was observed that SLE B cells had significantly higher NCAPG mRNA expression. Blocking miR-99a-3p expression in BALL-1 cells with an antagomir elevated NCAPG expression, facilitated PI3K/AKT pathway activation, improved cell proliferation, raised the fraction of S-phase cells, and prevented cell apoptosis. The opposite effects of upregulated miR-99a-3p levels on BALL-1 cells were observed by using an agomir. Furthermore, the effect of decreased miR-99a-3p expression on cell proliferation was partially mediated by elevating NCAPG levels and activating the PI3K/AKT pathway. Our research indicates that lower miR-99a-3p expression in SLE B cells appears to boost B cell number via the NCAPG and PI3K/AKT pathways.

Density Gradient Centrifugation-Independent Purification of Human Basophils.

Basophils represent the rarest type of granulocyte in human peripheral blood. Thus, researching basophils has historically been challenging and has often been reliant on enrichment protocols using density gradient centrifugation. This article describes a novel, fast, and cost-effective method to purify highly viable human basophils from peripheral blood through negative immunomagnetic selection, foregoing the density centrifugation step in the Basic Protocol. The technique is easy to use and consistently produces purities >96%. Furthermore, the Support Protocols describe procedures to determine basophil yield, purity, and viability, and how to investigate functional activity of the purified basophils through flow cytometry and visualize the basophils through microscopy. © 2024 The Authors. Current Protocols published by Wiley Periodicals LLC. Basic Protocol: Gradient centrifugation-independent basophil isolation Support Protocol 1: Flow cytometry staining to assess basophil yield, purity, and viability Support Protocol 2: Giemsa staining Support Protocol 3: Calcium flux analysis Support Protocol 4: Basophil activation test.

ULCERATIVE COLITIS-DERIVED INTESTINAL EPITHELIAL CELLS ARE PROTECTED FROM CELL DEATH BY THE IMMUNE MEDIATORS RELEASED FROM RESTING AND ACTIVATED SYSTEMIC T CELLS

Abstract Immune dysregulation is a major driver in the pathogenesis of ulcerative colitis (UC), but the specific mechanisms of disease initiated by the adaptive immune system remain unknown. While many studies have focused on the contribution of individual T cell subsets, disrupted communication between the epithelium and T cells in the underlying lamina propria is not fully understood in UC. This study examines the impact of T cells isolated from healthy participants on primary cultures of epithelial cells derived from UC donors. Blood derived human CD4+ and CD8+ T cells were isolated by immunomagnetic negative selection and activated by anti-CD3/CD28 cross-linking. Activated and resting T cells were then co-cultured with UC-derived colonic epithelial spheroids, consisting predominantly of stem cells, suspended in Matrigel. Spheroid damage and epithelial cell death were measured by histological scoring of brightfield microscopic images and zombie violet positive staining, detected using flow cytometry, respectively. Our earlier studies showed that activated CD4+ T cells co-cultured with spheroids from healthy donors stimulated significant spheroid damage and epithelial cell death in a dose- and time-dependent manner. In addition, we reported that the addition of conditioned media (CM) from activated CD4+ T cells to healthy spheroids also induces spheroid damage and epithelial cell death. Neither resting CD4+ nor CD8+ T cells nor activated CD8+ T cells stimulate the same response (Panel A). Our preliminary results indicate that UC-derived epithelial cells, isolated from spheroids, exhibit a higher base line of spontaneous lethal sectoring (42%), when compared to spheroids isolated from healthy colons (10%). In stark contrast to healthy spheroids, addition of CM from resting and activated CD4+ or CD8+ T cells protect the ulcerative colitis-derived spheroids from damage and isolated UC epithelial cells from spontaneous death. We have not yet identified which T cell subset are protecting UC-derived epithelial cells from damage and death. Thus, our results distinguish the healthy epithelial spheroid response to T cell CM from UC spheroids in three distinct ways: [1] CM is protective in UC, [2] Both CD4 and CD8 cells mediate this response in UC, and [3] CM from resting and activated T cells secrete the responsible immune mediators. Overall, these results signify that the biological response of UC-derived epithelial cells to immune mediators is different from that seen with healthy epithelial cells, indicating a metabolic and genetic difference in the epithelium from the UC patient.

Parallelized Immunomagnetic Isolation of Basophils Directly from Whole Blood.

Basophils are the rarest circulating white blood cells (WBCs), but they play important roles in allergic disorders and other diseases. To enhance diagnostic capabilities, it would be desirable to isolate and analyze basophils efficiently from small blood samples. In 100 μL of whole blood, there are typically ~103 basophils, outnumbered by ~105 WBCs and ~108 red blood cells (RBCs). Basophils' low abundance has therefore presented a significant challenge in their isolation from whole blood. Conventional in-bulk basophil isolation methods require lengthy processing steps and cannot work with small volumes of blood. Here we report a parallelized integrated basophil isolation device (pi-BID) for the negative immunomagnetic selection of basophils directly from 4 samples of 100 μL of whole blood, in parallel, within 14 minutes including sample preparation time. The pi-BID interfaces directly with standard sample tubes, and uses a single pressure source to drive the flow in parallel microfluidic channels. Compared with conventional in-bulk basophil isolation, the pi-BID is >3× faster, and has higher purity (~93%) and similar recovery (~67%). Compared with other microfluidic devices for the immunomagnetic isolation of WBC sub-types, our pi-BID achieves 10× higher enrichment of target cells from whole blood, with no prior removal of RBCs necessary.

MYC Pathway Upregulation Identified in Non-Responders to Hypomethylating Agents and Presents a Therapeutic Target in Acute Myeloid Leukemia

Background: In the current era of acute myeloid leukemia (AML) management, hypomethylating agents (HMAs) remain as the backbone of combination regimens for front-line treatment of elderly or unfit patients (pts) and/or as salvage therapy in relapsed/refractory pts. Data on biological predictors of HMA response are limited. Our previous work explored cytidine deaminase (CDA), known to inactivate HMAs, and nucleophosmin 1 (NPM1), determined by core pathway analysis to indirectly influence CDA expression. No clear correlation was identified in pt samples between CDA protein expression and NPM1 status or response, and pharmacogenomic analysis showed no CDA single nucleotide polymorphisms were predictive of response to HMAs. We aimed to expand prior findings using RNA sequencing (RNA seq) and gene set enrichment analysis (GSEA) to identify gene signatures predictive of HMA response and propose potential therapeutic targets. Methods: AML pts with banked samples who received frontline, bridging, or salvage HMA-based therapy between January 2014 to December 2018 were reviewed. Responses following at least 2 cycles of HMA were categorized as complete response (CR), CRi (CR with incomplete hematologic recovery), morphologic leukemia-free state (MLFS), complete hematologic response (CHR), or refractory. Pts were categorized as responders (CR, CRi, MLFS, CHR) or non-responders (refractory). Tumor cells were purified using immunomagnetic selection from bone marrow aspirates collected at diagnosis (dx). RNA seq was performed on 20 available pt tumor samples. Unsupervised clustering was performed and GSEA was completed to compare identified clusters using a false discovery rate (FDR) cut-off of less than 25%. GSEA-derived gene pathway scores were assessed by Wilcoxon rank-sum test. To evaluate AML cell lines to explore the non-responder phenotype, viability of AML cell lines was assessed following treatment with HMA azacitidine (Aza) and MS177 (a proteolysis targeting chimeric EZH2 degrader, also known to degrade MYC). Immunoblotting was used to assay proteolysis targeting chimeric (PROTAC)-induced MYC degradation. Results: Unsupervised clustering of RNA seq from 20 tumor samples at dx, labeled for response and NPM1 status, revealed 3 distinct groups: a non-responder group, a responder group, and a mixed response group ( Figure 1A). GSEA comparing the non-responder group to the responder group revealed several enriched genes in the non-responder group, including MYC targets (FDR <0.001), E2F targets (FDR <0.001), and G2M checkpoint (FDR=0.244). Upregulation of MYC targets was confirmed by enrichment profiling. Application of enrichment pathway scores revealed significant differences between non-responders and responders for E2F targets (p=0.026), G2M checkpoint (p=0.026), and MYC targets (p=0.0022). To explore MYC as a therapeutic target, we assayed MYC degradation in leukemia cells treated with two PROTAC degraders. The PROTAC MS177 induced marked MYC degradation in HL-60 cells ( Figure 1B). Cytotoxicity assays showed that in Kasumi-1 cells, the 50% inhibitory concentrations (IC50) for Aza and MS177 were 2.9 µM (responder phenotype) and 1.1 µM, respectively ( Figure 1C); in HL-60 cells the IC50 for Aza and MS177 were 10.9 µM (non-responder phenotype) and 0.06 µM, respectively ( Figure 1D). MS177 also showed efficacy in cytotoxicity assays using pt derived tumor samples. Conclusions: Using tumor samples from AML pts receiving HMAs, we identified a non-responder gene phenotype enriched with MYC upregulation. GSEA-derived pathway scoring differentiated non-responders from responders in our dataset. To further evaluate whether the enrichment pathway score is predictive for response across other AML cohorts, we are applying enrichment scores to publicly available AML datasets. With MYC upregulation identified as a potential driver of HMA failure, MYC degradation or inhibitory targets are recognized as potential therapeutic targets for pts with a non-responder gene signature. We established the HL-60 AML cell line, found to be less sensitive to Aza than the Kasumi-1 cell line, demonstrated greater sensitivity to MYC degradation compared to Kasumi-1 cells and may reflect the non-responder phenotype. Further in vitro efforts are ongoing to evaluate MYC targets for the HMA non-responder phenotype.

Clinical and Molecular Characterization of Patients with Therapy Related Myeloid Neoplasms (TRMN)

Acknowledgements Chemotherapy and/or radiotherapy in cancer patients compose a future risk of developing a therapy related myeloid neoplasm (TRMN). However, the presence of a germline landscape might contribute and modify the overall risk. It has been suggested that around 18.5% of TRMN patients have a germline cancer mutation. In particular, cancer predisposing genes or syndromes involving alterations in the DNA repair mechanism such as CHEK2, BRCA1, BRCA2, MLH1, MSH2, MSH6 or PMS2 might be enriched in patients who develop a primary tumor (PT) and a subsequent secondary myeloid neoplasm. The role of the alterations in cancer susceptibility genes in association with the treatment for the PT in the development of TRMN is still unclear. To evaluate the clinical and genetic landscape of TRMN patients to understand the role of different risk factors and provide tools for differential diagnosis and clinical management. Clinical evaluation of 96 patients with TRMN and 12 patients with a secondary myeloid neoplasm after a non-treated with chemotherapy primary tumor (nTMN) was performed. In addition, germline landscape and somatic alterations were studied in the TRMN series by tNGS (N=49) and WES (N=47). Tumoral sample was obtained from bone marrow. CD3+ T lymphocytes from peripheral blood (PB) were enriched by immunomagnetic selection and used as germline/control sample. A minimum of 98% purity in CD3+ was selected to prevent myeloid contamination. Variant frequency (VAF) <30% found in tumor but not in CD3+ sample was annotated as somatic alteration. Only pathogenic/likely pathogenic variants were considered. The median age of onset was 62.3 and 71.1 years for the PT and the TRMN, respectively. 32.5% had complex karyotype. Similar positive correlation was found between the age of onset of the PT and the age of onset of the TRMN and nTNM. A negative correlation was found between the age of onset of the PT and the latency until the TRMN and nTMN diagnosis, which suggests that elderly age condition is more relevant than the cytotoxic treatment (Fig.1). No differences in latencies were found between MN subtypes, despite ring sideroblasts and del(5q) myeloid subtypes correlated with shorter and higher latencies, respectively. Different latencies were neither found when the PT was solid or hematologic, but interestingly, TRMN patients with previous colorectal cancer had shorter latencies. On the other hand, 91.3% of the TRMN patients presented a molecular alteration (considering both genetic or cytogenetic alterations), including 60.9% and 75% of patients with germline and acquired alterations, respectively. Germline mutations included 52.2% and 47.8% annotated in cancer predisposition genes of the DNA repair mechanisms and myeloid genes, respectively. Only 39.1% of TRMN patients were found with pathogenic somatic variants in myeloid genes while 68.75% were found with an abnormal karyotype. Finally, higher number of germline and somatic mutations was found in TRMN patients with an abnormal karyotype. However, a higher percentage of germline mutations in myeloid genes was found in TRMN with a normal karyotype (Fig.2). Higher percentage of molecular alterations (both germline and somatic) in myeloid genes was found in TRMN patients with a previous solid PT. However, higher percentage of mutations in cancer predisposition genes was found when the PT was hematologic. Our results highlight the role of mutations in cancer predisposition genes in the development of TRMN. Similar latency associations were found for TRMN and nTMN patients, which suggest that other risk factors different than treatment might contribute to the TRMN development. Altered karyotypes in TRMN patients are associated with acquired mutations in myeloid genes but not with germline mutations, which suggests two different entities with different prognosis. Molecular alterations in myeloid genes are more relevant in TRMN patients with solid PT, while molecular alterations in cancer predisposition genes are associated with TRMN patients with hematologic PT. This work was supported in part by a grant from the Instituto de Salud Carlos III, Ministerio de Economia y Competitividad, Spain (PI 20/00531)(Co-funded by European Regional Development Fund. ERDF, a way to build Europe). 2021 SGR 00560 (GRC) Generalitat de Catalunya; economical support from CERCA Programme and Fundació Adey.

Isolation methods determine human neutrophil responses after stimulation.

Studying neutrophils is challenging due to their limited lifespan, inability to proliferate, and resistance to genetic manipulation. Neutrophils can sense various cues, making them susceptible to activation by blood collection techniques, storage conditions, RBC lysis, and the isolation procedure itself. Here we assessed the impact of the five most used methods for neutrophil isolation on neutrophil yield, purity, activation status and responsiveness. We monitored surface markers, reactive oxygen species production, and DNA release as a surrogate for neutrophil extracellular trap (NET) formation. Our results show that neutrophils isolated by negative immunomagnetic selection and density gradient methods, without RBC lysis, resembled untouched neutrophils in whole blood. They were also less activated and more responsive to milder stimuli in functional assays compared to neutrophils obtained using density gradients requiring RBC lysis. Our study highlights the importance of selecting the appropriate method for studying neutrophils, and underscores the need for standardizing isolation protocols to facilitate neutrophil subset characterization and inter-study comparisons.

Evaluation of Circulating Endothelial Cells (CECs) As Marker of Endothelial Damage in Allo-Transplanted Patients at High Risk of Hepatic Veno-Occlusive Disease/Sinusoidal Obstruction Syndrome (VOD/SOS): The Cecinvod Study

INTRODUCTION Sinusoidal obstruction syndrome (SOS), also known as veno-occlusive disease (VOD), is a potentially fatal complication after allogeneic stem cell transplantation (alloSCT). Identifying a predictive biomarker for VOD has been challenging. Since endothelial injury is considered one of the main pathogenic factors for VOD onset, with the CECinVOD prospective study we aimed to evaluate Circulating Endothelial Cells (CEC) in allo-transplanted patients (pts) at higher risk to develop VOD. METHODS From October 2020 to November 2022, 150 pts have been enrolled in the CECinVOD study from 11 Italian Bone Marrow Transplantation Units. All pts must be older than 18 years and undergoing myeloablative alloSCT. CECs were detected using the CellSearch system, the FDA-approved immunomagnetic selection approach incorporating ferrofluid nanoparticles (anti CD146) and fluorophore-labelled antibodies (anti CD105, CD45 and DAPI). CEC were defined as CD146+, CD105+, DAPI+ and CD45-. CEC were collected at the following timepoints: before conditioning regimen (T0), at the end of conditioning regimen and before alloSCT (T1), at the time of neutrophils engraftment (T2), and 7-10 days after engraftment (T3). In pts who developed VOD, additional timepoints were collected as follows: at any time of suspected or proven VOD onset (T4), and then weekly during Defibrotide treatment (T5-T8). SOS/VOD was defined according to the 2016 European Group for Blood and Marrow Transplantation criteria. RESULTS Pts' main characteristics are summarized in Table 1. Six out of 150 pts (4%) developed VOD during the follow up (4 “severe”, and 2 “very severe”). All pts were treated with Defibrotide, obtaining a complete remission in 5 of them, while 1 pt died due to VOD complications. Pts receiving TBI-based regimen were more likely to develop VOD compared to those receiving Treosulfan (10 to 14 g/m2) or Busulfan ev (9.6 to 12.8 mg/kg) (p 0.08). Similarly, higher baseline levels of bilirubin were associated with a higher incidence of VOD (p 0.08). Considering the CECs analysis, 615 samples were evaluated. At the enrollment, CECs levels were not related with any of clinical characteristics analyzed, except for the number of previous treatments. Indeed, those pts with 2 or more previous lines of treatments had higher levels of CECs (OR=0.53; p<0.001). Considering the different timepoints, conditioning regimen and alloSCT result in higher levels of CECs. Thus, CEC were higher at T1 than at T0 (p 0.02), as well as they were even higher at T2 than at T1 (p<0.0001) (Fig.1). Conversely, no significant differences have been observed between T3 and T2. No other clinical characteristic was correlated with CEC counts at the different timepoints. Pts who developed VOD had higher median levels of CEC at all the timepoints analyzed, but this difference has not achieved statistical significance, probably due to the low number of pts in the VOD group. At VOD onset, the pts always had an increase in CEC levels compared with the previous timepoint. After defibrotide treatment, the CEC levels increased in the first week, while they progressively decreased during the VOD treatment (T6 and T7, -50,7% and -71,5%, respectively). Recently, another endothelial activation marker has been considered: the Easix score. We didn't find any relationship between the Easix score at transplant and the VOD onset, even if a trend may be observed. We also investigated whether the CEC levels may be related with Easix score, but we didn't find any relationship at each timepoint. Interestingly, in a subgroup of pts CECs have been observed as a cluster of multiple cells. This data is firstly described in allo-SCT. Its functional significate remains unclear, but we found a relationship between CECs cluster and the number of CECs (p < 0.001). CONCLUSIONS The incidence of VOD in our prospective study was low with respect to the one reported in the literature. This can be explained by the changes in alloSCT from the past (better selection of the pts, lower use of TBI, higher use of reduced intensity regimen) and the retrospective nature of most of the previous reports. We show that CECs can be considered reliable marker of endothelial damage in alloSCT pts, highlighting the impact of previous treatments, the conditioning regimen, and allo-SCT itself. Increased CEC level may be helpful to confirm VOD diagnosis, as well as their monitoring may be useful to evaluate the response to the treatment for VOD.

Isolation of Mouse Neutrophils.

Neutrophils represent the first line of defense against bacterial and fungal pathogens. Indeed, patients with inherited or acquired qualitative and quantitative neutrophil defects are at high risk for developing bacterial and fungal infections and suffering adverse outcomes from these infections. Therefore, research aiming at defining the molecular factors that modulate neutrophil effector function under homeostatic conditions and during infection is essential for devising strategies to augment neutrophil function and improve the outcomes of infected individuals. This article describes reproducible density-gradient-centrifugation-based as well as positive and negative immunomagnetic selection protocols that can be applied in any laboratory to harvest large numbers of highly enriched and highly viable neutrophils from the bone marrow of mice. In another protocol, we also present a method that combines gentle enzymatic tissue digestion with a positive immunomagnetic selection technique or fluorescence-activated cell sorting (FACS) to harvest highly pure and highly viable preparations of neutrophils directly from mouse tissues such as the kidney, the liver, or the spleen. Mouse neutrophils isolated by these protocols can be used to examine several aspects of cellular function ex vivo, including pathogen binding, phagocytosis, and killing, neutrophil chemotaxis, oxidative burst, degranulation, and cytokine production, and for performing neutrophil adoptive transfer experiments. © 2023 Wiley Periodicals LLC. This article has been contributed to by U.S. Government employees and their work is in the public domain in the USA. Basic Protocol 1: Isolation of Neutrophils from Mouse Bone Marrow Using Positive Immunomagnetic Separation Alternate Protocol 1: Purification of Neutrophils from Bone Marrow Using Negative Immunomagnetic Separation Alternate Protocol 2: Purification of Neutrophils from Bone Marrow Using Histopaque-Based Density Gradient Centrifugation Basic Protocol 2: Isolation of Neutrophils from Mouse Tissues Using Positive Immunomagnetic Separation Alternate Protocol 3: Isolation of Neutrophils from Mouse Tissues Using FACS.

An enzyme-free method for isolating testicular macrophages from rodent models

Macrophages are the major type of immune cell in the testis of both humans and rodents. Testicular macrophages (TMs) play critical roles in maintaining the testicular microenvironment, such as Leydig cell-dependent hormone production, spermatogenesis, and immune balance. A substantial number of studies have used rodent models to investigate the functions of TMs with various methods and harvest macrophages from the testis. Studies have demonstrated that enzyme digestion, an essential part of these methods, can improve the number and purity of TMs while unavoidably altering the immunoprofile of macrophages, which is detrimental for further study in terms of immune investigation. Here, we modified the existing method of microglia isolation and set up a novel method without the enzyme digestion step to isolate TMs. According to the characteristics of testicular tissue looseness and the physical and biological characteristics of macrophages, by combining mechanical separation, gradient centrifugation, and immuno-magnetic bead selection, we can effectively avoid the enzymatic digestion of testis tissue and maintain the immune characteristics of macrophages. Additionally, we verified the purity of TM with flow cytometry (FC) at approximately 91–95%, and the production of tumor necrosis factor alpha (TNF-α) and interleukin-6 (IL-6) was lower than that isolated with enzyme digestion. In contrast to the traditional method, this novel protocol can assist those who have no convenient access to fluorescence-activated cell sorting (FACS) to isolate a sufficient number of TMs and, most importantly, avoid altering the immunoprofile of TMs without enzyme digestion.

Isolation of Monocytes from Whole Blood by Immunomagnetic Negative Selection

Isolation of Monocytes from Whole Blood by Immunomagnetic Negative Selection

Isolation of Monocytes from Whole Blood by Immunomagnetic Negative Selection

Isolation of Monocytes from Whole Blood by Immunomagnetic Negative Selection

The β2-adrenergic receptor agonist terbutaline upregulates T helper-17 cells in a protein kinase A-dependent manner

BackgroundT helper 17 (Th17) cells produce IL-17A cytokine and can exacerbate autoimmune diseases and asthma. The β2 adrenergic receptor is a g protein-coupled receptor that induces cAMP second messenger pathways. We tested the hypothesis that terbutaline, a β2-adrenergic receptor-specific agonist, promotes IL-17 secretion by memory Th17 cells in a cAMP and PKA-dependent manner. MethodsVenous peripheral blood mononuclear cells (PBMC) from healthy human participants were activated with anti-CD3 and anti-CD28 antibodies. Secreted IL-17A was measured by enzyme linked immunosorbent assay, intracellular IL-17A, and RORγ were measured using flow cytometry, and RORC by qPCR. Memory CD3+CD4+CD45RA-CD45RO+ T cells were obtained by immunomagnetic negative selection and activated with tri-antibody complex CD3/CD28/CD2. Secreted IL-17A, intracellular IL-17A, RORC were measured, and phosphorylated-serine133-CREB was measured by western blotting memory Th cells. ResultsTerbutaline increased IL-17A (p < 0.001), IL-17A+ cells (p < 0.05), and RORC in activated PBMC and memory Th cells. The PKA inhibitors H89 (p < 0.001) and Rp-cAMP (p < 0.01) abrogated the effects of terbutaline on IL-17A secretion in PBMC and memory T cells. Rolipram increased IL-17A (p < 0.01) to a similar extent as terbutaline. P-Ser133-CREB was increased by terbutaline (p < 0.05) in memory T cells. ConclusionTerbutaline augments memory Th17 cells in lymphocytes from healthy participants. This could exacerbate autoimmune diseases or asthma, in cases where Th17 cells are considered to be pro-inflammatory.

Integrative Magneto-Microfluidic Separation of Immune Cells Facilitates Clinical Functional Assays.

Accurately analyzing the functional activities of natural killer (NK) cells in clinical diagnosis remains challenging due to their coupling with other immune effectors. To address this, an integrated immune cell separator is required, which necessitates a streamlined sample preparation workflow including immunological cell isolation, removal of excess red blood cells (RBCs), and buffer exchange for downstream analysis. Here, a self-powered integrated magneto-microfluidic cell separation (SMS) chip is presented, which outputs high-purity target immune cells by simply inputting whole blood. The SMS chip intensifies the magnetic field gradient using an iron sphere-filled inlet reservoir for high-performance immuno-magnetic cell selection and separates target cells size-selectively using a microfluidic lattice for RBC removal and buffer exchange. In addition, the chip incorporates self-powered microfluidic pumping through a degassed polydimethylsiloxane chip, enabling the rapid isolation of NK cells at the place of blood collection within 40min. This chip is used to isolate NK cells from whole blood samples of hepatocellular cancer patients and healthy volunteers and examined their functional activities to identify potential abnormalities in NK cell function. The SMS chip is simple to use, rapid to sort, and requires small blood volumes, thus facilitating the use of immune cell subtypes for cell-based diagnosis.