Multiparameter Flow Research Articles

Early dynamic changes to monocytes following major surgery are associated with subsequent infections.

Post-operative infections are a common cause of morbidity following major surgery. Little is understood about how major surgery perturbs immune function leading to heightened risk of subsequent infection. Through analysis of paired blood samples obtained immediately before and 24 h following surgery, we evaluated changes in circulating immune cell phenotype and function across the first 24 h, to identify early immune changes associated with subsequent infection. We conducted a prospective observational study of adult patients undergoing major elective gastrointestinal, gynecological, or maxillofacial surgery requiring planned admission to the post-anesthetic care unit. Patients were followed up to hospital discharge or death. Outcome data collected included mortality, length of stay, unplanned intensive care unit admission, and post-operative infections (using the standardized endpoints in perioperative medicine-core outcome measures for perioperative and anesthetic care criteria). Peripheral blood mononuclear cells were isolated prior to and 24 h following surgery from which cellular immune traits including activation and functional status were assessed by multi-parameter flow cytometry and serum immune analytes compared by enzyme-linked immunosorbent assay (ELISA). Forty-eight patients were recruited, 26 (54%) of whom developed a post-operative infection. We observed reduced baseline pre- and post-operative monocyte CXCR4 and CD80 expression (chemokine receptors and co-stimulation markers, respectively) in patients who subsequently developed an infection as well as a profound and selective post-operative increase in CD4+ lymphocyte IL-7 receptor expression in the infection group only. Higher post-operative monocyte count was significantly associated with the development of post-operative infection (false discovery rate < 1%; adjusted p-value = 0.001) with an area under the receiver operating characteristic curve of 0.84 (p < 0.0001). Lower monocyte chemotaxis markers, higher post-operative circulating monocyte counts, and reduced co-stimulatory signals are associated with subsequent post-operative infections. Identifying the underlying mechanisms and therapeutics to reverse defects in immune cell function requires further exploration.

Loss of hematopoietic progenitors heterogeneity is an adverse prognostic factor in lower-risk myelodysplastic neoplasms

Myelodysplastic neoplasms (MDS) are characterized by clonal evolution starting from the compartment of hematopoietic stem and progenitors cells (HSPCs), leading in some cases to leukemic transformation. We hypothesized that deciphering the diversity of the HSPCs compartment may allow for the early detection of an emergent sub-clone that drives disease progression. Deep analysis of HSPCs repartition by multiparametric flow cytometry revealed a strong disorder of the hematopoietic branching system in most patients at diagnosis with different phenotypic signatures closely related to specific MDS features. In two independent cohorts of 131 and 584 MDS, the HSPCs heterogeneity quantified through entropy calculation was decreased in 47% and 46% of cases, reflecting a more advanced state of the disease with deeper cytopenias, higher IPSS-R risk and accumulation of somatic mutations. We demonstrated that patients with lower-risk MDS and low CD34 + CD38+HSPCs entropy had an adverse outcome and that this parameter is as an independent predictive biomarker for progression free survival, leukemia free survival and overall survival. Analysis of HSPCs repartition at diagnosis represents therefore a very powerful tool to identify lower-risk MDS patients with a worse outcome and valuable for clinical decision-making, which could be fully integrated in the MDS diagnostic workflow.

Water‐Soluble Fluorescent Polymer Dyes with Tunable Emission Spectra for Flow Cytometry Applications

AbstractThe application of spectrally unique, bright, and water‐soluble fluorescent dyes is indispensable for the analysis of biological systems. Multiparameter flow cytometry is a powerful tool for characterization of mixed cell populations. To discriminate the different cell populations, they are typically stained by a set of fluorescent reagents, e.g., antibody‐fluorophore conjugates. The number of parameters which can be studied simultaneously strongly depends on the availability of reagents which can be differentiated by their spectral properties. In this study a series of fluorescent polymer dyes was developed, that can be excited with a single violet laser (405 nm) but distinguished by their unique emission spectra. The polyfluorene‐based polymers can be used on their own, or in combination with covalently bound small‐molecule dyes to generate energy transfer constructs to red‐shift the emission wavelength based on Förster resonance energy transfer (FRET). The polymer dyes were utilized in a biological flow cytometry assay by conjugating several of them to antibodies, demonstrating their effectiveness as reagents. This report represents the first systematic investigation of structure–property relationships for this type of fluorescent dyes.

Water-Soluble Fluorescent Polymer Dyes with Tunable Emission Spectra for Flow Cytometry Applications.

The application of spectrally unique, bright, and water-soluble fluorescent dyes is indispensable for the analysis of biological systems. Multiparameter flow cytometry is a powerful tool for characterization of mixed cell populations. To discriminate the different cell populations, they are typically stained by a set of fluorescent reagents, e.g., antibody-fluorophore conjugates. The number of parameters which can be studied simultaneously strongly depends on the availability of reagents which can be differentiated by their spectral properties. In this study a series of fluorescent polymer dyes was developed, that can be excited with a single violet laser (405 nm) but distinguished by their unique emission spectra. The polyfluorene-based polymers can be used on their own, or in combination with covalently bound small-molecule dyes to generate energy transfer constructs to red-shift the emission wavelength based on Förster resonance energy transfer (FRET). The polymer dyes were utilized in a biological flow cytometry assay by conjugating several of them to antibodies, demonstrating their effectiveness as reagents. This report represents the first systematic investigation of structure-property relationships for this type of fluorescent dyes.

Sodium fluorescein uptake by the tumor microenvironment in human gliomas and brain metastases.

Intravenous sodium fluorescein (SF) is increasingly used during surgery of gliomas and brain metastases to improve tumor resection. Currently, SF is believed to permeate the brain regions where the blood-brain barrier (BBB) is damaged and to accumulate in the extracellular space but not in tumor or healthy cells, making it possible to demarcate tumor margins to guide resection. By evaluating the immune contexture of a number of freshly resected gliomas and brain metastases from patients undergoing SF-guided surgery, the authors recurrently observed fluorescence-positive cells. Therefore, the aim of this study was to determine if SF accumulates inside the cells of the tumor microenvironment (TME), and if so, in which type of cells, and whether incorporation can also be observed in the leukocytes of peripheral blood. Freshly resected tumor specimens were dissociated to single cells and analyzed by multiparametric flow cytometry. Peripheral blood leukocytes, macrophages, and a glioma cell line were treated with SF in vitro, and their cell uptake was assessed by multiparametric and imaging flow cytometry and by confocal microscopy. The ex vivo and in vitro analyses revealed that SF accumulates intracellularly in leukocytes as well as in tumor cells, but with a high variability of incorporation in the different cell subsets analyzed. Myeloid cells showed the highest level of fluorescence. In vitro uptake experiments showed that SF accumulation increases over time. The imaging analyses confirmed the internalization of the compound inside the cells. SF is not just a marker of BBB damage, but its intracellular detection suggests that it selectively accumulates intracellularly. Future efforts should target the mechanisms of its differential uptake by the different TME cell types in depth.

Placental circulating T cells: a novel, allogeneic CAR-T cell platform with preserved T-cell stemness, more favorable cytokine profile, and durable efficacy compared to adult PBMC-derived CAR-T

BackgroundChimeric antigen receptor (CAR)-T cell quality and stemness are associated with responsiveness, durability, and memory formation, which benefit clinical responses. Autologous T cell starting material across patients with cancer is...

Novel B cell subsets as potential biomarkers in Idiopathic Inflammatory Myopathies: insights into disease pathogenesis and disease activity.

Idiopathic inflammatory myopathies are a heterogeneous group of rare autoimmune disorders characterized by progressive muscle weakness and the histopathologic findings of inflammatory infiltrates in muscle tissue. Although their pathogenesis remains indefinite, the association of autoantibodies with clinical manifestations and the evidence of high effectiveness of depleting therapies suggest that B cells could be implicated. Therefore, we explored the landscape of peripheral B cells in this disease by multiparametric flow cytometry, finding significant numerical decreases in memory and double negative subsets, as well as an expansion of the naïve compartment relative to healthy controls, that contribute to defining disease-associated B cell subset signatures and correlating with different clinical features of patients. Additionally, we determined the potential value of these subsets as diagnostic biomarkers, thus positioning B cells as neglected key elements possibly participating in idiopathic inflammatory myopathies onset or development.

Development of a customizable mouse backbone spectral flow cytometry panel to delineate immune cell populations in normal and tumor tissues.

In vivo studies of cancer biology and assessment of therapeutic efficacy are critical to advancing cancer research and ultimately improving patient outcomes. Murine cancer models have proven to be an invaluable tool in pre-clinical studies. In this context, multi-parameter flow cytometry is a powerful method for elucidating the profile of immune cells within the tumor microenvironment and/or play a role in hematological diseases. However, designing an appropriate multi-parameter panel to comprehensively profile the increasing diversity of immune cells across different murine tissues can be extremely challenging. To address this issue, we designed a panel with 13 fixed markers that define the major immune populations -referred to as the backbone panel- that can be profiled in different tissues but with the option to incorporate up to seven additional fluorochromes, including any marker specific to the study in question. This backbone panel maintains its resolution across different spectral flow cytometers and organs, both hematopoietic and non-hematopoietic, as well as tumors with complex immune microenvironments. Having a robust backbone that can be easily customized with pre-validated drop-in fluorochromes saves time and resources and brings consistency and standardization, making it a versatile solution for immuno-oncology researchers. In addition, the approach presented here can serve as a guide to develop similar types of customizable backbone panels for different research questions requiring high-parameter flow cytometry panels.

Abstract 6533: Pivotal role of specific LYVE1+ tissue resident macrophages in PDAC progression &amp; immunotherapy resistance

Abstract Pancreatic ductal adenocarcinoma (PDAC) is characterized by dense fibrotic stroma and abundant myeloid cell infiltration, which render it resistant to any therapeutic interventions, including immunocheckpoint blockade (ICB) therapy. Understanding the composition and function of myeloid cells within the PDAC microenvironment may help identify promising targets for PDAC immunotherapy. Tissue resident macrophages (TRMs) contribute to organogenesis and tissue regeneration and metabolism. In this study, we discovered a distinct population of pancreas TRMs that highly express lymphatic vessel hyaluronan receptor 1 (LYVE-1). Single cell RNAseq data reveal that LYVE-1+ TRM cluster in the pancreas expresses high levels of complement activation genes and transcription factors such as c-Maf and Stab1. RNAseq data also confirm that LYVE-1+ TRMs express enriched genes related to mannose binding, endocytosis, and glutathione metabolic process, while LYVE-1 negative macrophages express enriched genes related to antigen processing and presentation, T cell activation, and phagocytosis. Multiparameter flow cytometric analysis demonstrates that LYVE-1+ TRMs express M2-like markers, including increased CD163, CD206 and decreased MHC Class II molecule. During PDAC development and progression, LYVE-1+ TRMs expand and localize around the tumor border, promoting fibrotic stroma formation. Imaging mass cytometry (IMC) also reveals abundant infiltration of LYVE-1+CD14+CD68+ macrophages in human PDAC tumors and colocalized with collagen deposition. This subset of macrophages also highly express c-Maf. Through the use of Lys-Cre-c-Maf f/f mice, we demonstrate that depletion of c-Maf from myeloid cells significantly reduces LYVE-1+ TRMs and polarizes them to resemble an M1-like phenotype. In conclusion, our findings suggest that LYVE-1+ macrophages are a unique subset of myeloid cells within the pancreas and likely play a critical role in PDAC progression and immunotherapeutic resistance. Future work will focus on targeting LYVE1+ TRMs using various approaches, such as conditional knockout mice and small molecule inhibitors, to determine their therapeutic potential for PDAC treatment. Presented by Dr. Hong Li Citation Format: Hong Li, Jun Yan. Pivotal role of specific LYVE1+ tissue resident macrophages in PDAC progression &amp; immunotherapy resistance [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 6533.

Abstract 1053: The Influence of PD1 and TIM3 Expression on CD4+CD103+ and CD8+CD103+ Cells in Glioblastoma Progression

Abstract Glioblastoma (GB), IDH-wildtype, is the most common and aggressive high-grade primary malignant brain tumor with extremely poor outcome. Although the use of immune checkpoint inhibitors (ICIs) in GB has shown disappointing results, the identification of a small subgroup of responders underpins the role of the intratumoral immune contexture, in particular tumour-infiltrating lymphocytes (TILs), in determining disease outcome. This study aims to define the features of TIL subpopulations correlating with GB prognosis, and to establish in parallel the predictive roles of peripheral T cell subsets. A single cohort observational study, including patients undergoing GB resection and standard therapeutic and follow-up approaches in Neuro-Oncology, was conducted. Of the 45 patients, histologically confirmed WHO grade 4, 26 had MGMT promoter methylation (60.0%), 24 were male (53.3%) and the median age of young patients (≤63 years) vs elderly patients (&amp;gt;63 years) was 57 vs 71, respectively. Predictors of OS were KPS (≥70 vs &amp;lt;70: 14.7 vs 7.5 months, p=0.0007) and GTR (GTR vs non-GRT: 15 vs 9 months, p=0.004) whereas patients with methylated MGMT promoter had non-significant longer median OS compared to those with unmethylated promoter (14.5 vs 10.5 months). Patients were characterized for the expression of lineage, differentiation, memory, activation, and inhibition markers on intratumoral and peripheral CD4+ and CD8+ T cell subpopulations, and the correlation of an array of CD4+ and CD8+ T cell subsets with OS or PFS was evaluated by multiparametric flow cytometry and uni/multivariate analyses. Intratumoral immune infiltrate was positively associated with CD8+CD103+ tissue-resident memory T cells (Trm), and in particular with CD8+CD103+ Trm expressing PD1 and TIM3 rather than CD4+ Trm. Low CD8+CD103+PD1+ Trm resulted significantly associated with better OS and PFS (p=0.02 and 0.0031, respectively) and even higher significant was the correlation with better OS and PFS of low CD8+CD103+TIM3+ Trm (p=0.0002 and 0.0033, respectively) and CD8+CD103+PD1+TIM3+ Trm (p&amp;lt;0.0001, both). On multivariate analysis, low CD8+CD103+PD1+TIM3+ Trm and KPS ≥70 were confirmed to be the most predictive independent factors associated with longer OS (hazard ratios - HR [95%CI]: 0.14 [0.04 - 0.52] p˂0.001, 0.39 [0.16 - 0.96] p=0.04, respectively). Moreover, CD8+CD103+ Trm subpopulations resulted age-related predictors for GB survival. Consistent with this finding, in vitro PD1 and TIM3 blockade released the capability of intratumoral T cells to produce the effector cytokines IFN-γ and TNF-α, and the cytotoxic factor GrzB. Conversely, T cells expressing PD1 and TIM3 of peripheral blood did not show any prognostic significance. In conclusion, low frequency of Trm expressing PD1 or TIM3 or both markers defined TIL subsets as independent positive prognostic factors for patient survival. Citation Format: Lucia Gabriele, Giulia Romagnoli, Quintino Giorgio D'Alessandris, Imerio Capone, Andrea Tavilla, Irene Canini, Caterina Lapenta, Mariachiara Buccarelli, Martina Giordano, Valentina Tirelli, Massimo Sanchez, Alessandra Fragale, Stefano Giannetti, Rina Di Bonaventura, Liverana Lauretti, Mauro Biffoni, Lucia Ricci-Vitiani, Roberto Pallini. The Influence of PD1 and TIM3 Expression on CD4+CD103+ and CD8+CD103+ Cells in Glioblastoma Progression [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 1053.

Abstract 5278: Immunological control by PARP inhibitors for successful immunotherapy in metastatic ovarian carcinoma

Abstract Epithelial ovarian carcinoma (EOC) is among the top five causes of cancer-related death in women. Most women with EOC achieve indeed complete remission after primary or interval cytoreductive surgery combined with chemotherapy based on a platinum-taxane doublet. Homologous recombination (HR) defects imposed by germline or somatic BRCA1 DNA repair-associated (BRCA1) or BRCA2 mutations are not only key determinants of platinum sensitivity in EOC patients but also provide a strong rationale for maintenance therapy based on poly(ADP-ribose) polymerase (PARP) inhibitors, which is generally associated with improved progression-free survival (PFS). Besides direct cytotoxic and cytostatic properties, PARPi have been shown to mediate multipronged immunostimulatory effects, largely reflecting their ability to inhibit DNA repair in malignant cells and indicating the possibility to synergy with immune-check point inhibitors (ICIs). Here, using multiparametric flow cytometry, multiplex immunostaining, single cells transcriptomics and functional studies in experimental syngeneic BR5Brca1-/- mouse model and EOC tumor samples, we discuss PARPi immunomodulatory activity, with a specific focus on molecular and cellular pathways that can be harnessed to develop superior combinatorial regimens for clinical management. Thus, PARPi might enhance the mutational load of EOCs as consequence of unrepaired DNA damage and DAMPs exposure, favoring T cell infiltration, but also appear to drive robust type I IFN secretion downstream of cyclic GMP-AMP synthase and stimulator of IFN response cGAMP interactor 1 activation. In addition, the combination of PARPi with ICIs positively regulate the balance between adaptive anti-tumor immunity and innate myeloid components and significantly improve the cytotoxic T cell profile as shown in both experimental mice model and HGSOC tumor samples. We surmise that rationally designed combinations of PARPi and immunotherapeutic agents might be critical to unlock immunosuppression in the EOC microenvironment in support of clinical efficacy. Citation Format: Peter Holicek, Sarka Vosahlikova, Irena Moserova, Sandra Orsulic, Romana Mikyskova, Michal Hensler, Lenka Kasikova, Tereza Lanickova, Ondrej Novotny, Milan Reinis, Jana Drozenova, Ales Ryska, Jan Laco, Lukas Rob, Michael Halaska, David Cibula, Radek Spisek, Jitka Fucikova. Immunological control by PARP inhibitors for successful immunotherapy in metastatic ovarian carcinoma [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 5278.

Abstract 4987: TREM2 is a radiation-induced target for immunomodulation of tumor-associated macrophages in the prostate cancer tumor microenvironment

Abstract Background: The prostate cancer (PCa) tumor microenvironment (TME) is characterized by a predominance of myeloid cells that may have a key role in immunosuppression and disease progression. Stereotactic Body Radiation Therapy (SBRT) is utilized for treatment of localized and metastatic PCa and promotes inflammatory remodeling of the TME. We analyzed human PCa specimens at acute post-SBRT timepoints to identify radiation-induced targets in the PCa TME and used in vitro and in vivo models to test and validate our findings. Methods: We performed 10X single-cell RNA sequencing (scRNA-seq) on prostatectomy specimens from men with high-risk localized PCa; 12,988 cells from 4 patients that underwent pre-operative SBRT and 47,314 cells from 6 patients that underwent prostatectomy alone. THP-1 monocytes polarized into macrophage (Mɸ) states [M0, M1, M2 and tumor-associated macrophages (TAM), were used to characterize TREM2 expression and immune responses to radiation or conditioned media (CM) derived from irradiated PC3 cells. In vivo studies with C57BL/6 mice inoculated with MC38 flank tumors or PTEN-/- SPOPmut CHD1del mouse prostate organoid line were used to analyze intratumoral myeloid populations in response to SBRT (37.5Gy in 5 fractions). Results: scRNA-seq analysis (Cell Ranger 7.1.0 pipelines; Seurat v5 R package) revealed enrichment of a cluster of myeloid cells defined by a damage-associated Mɸ gene signature (TREM2, APOE, SPP1, LGALS3, CD9) in response to SBRT compared with non-irradiated specimens (35.6% vs 5.7%). TREM2 expression was significantly upregulated in THP-1 cells polarized in M2 (p&amp;lt;0.001) and TAM (p&amp;lt;0.001) conditions, compared to M1 conditions. Irradiation (20Gy x1) of THP-1 cells significantly increased TREM2 expression in M0 (p&amp;lt;0.01), M1 (p&amp;lt;0.001), and TAM (p&amp;lt;0.01) conditions. Proliferation of PC3 cells exposed to CM from irradiated TAMs was significantly increased (p&amp;lt;0.0001) compared to CM from non-irradiated TAM. Alternatively, CM derived from irradiated PC3 cells significantly increased (p&amp;lt;0.05) TREM2 expression in M0-polarized Mɸ. In vivo studies, in MC38 and organoid models, noted a significant reduction (p&amp;lt;0.0001) in tumor volume at day 7 post-SBRT. Multi-parametric flow cytometry demonstrated robust myeloid infiltration in response to SBRT dominated by CD11b+F4/80+ TAMs in irradiated tumors. Among irradiated tumors, TREM2 expression was markedly elevated in TAMs (organoid and MC38, p&amp;lt;0.0001). Conclusion: Preliminary data in human irradiated PCa and validation studies in preclinical models suggest TREM2 expression on myeloid cells is a radiation-induced target. Further studies are underway to understand the functional and immunological implications of SBRT-induced TREM2 expression on TAMs and whether this can be therapeutically targeted to enhance anti-tumor immunity in PCa. Citation Format: Janny A. Villa-Pulgarin, Un In Chan, Francesca Khani, Fabio Socciarelli, Jiansheng Wu, Ryan Carelli, Jeffrey Kraynak, Silvia C. Formenti, Luigi Marchionni, Massimo Loda, Himanshu Nagar, Ariel E. Marciscano, Christopher E. Barbieri. TREM2 is a radiation-induced target for immunomodulation of tumor-associated macrophages in the prostate cancer tumor 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 4987.

Abstract 3863: Flow cytometry using stabilized whole blood to assess innate and adaptive immune cells (Monocytes, NK, T, and B cells) in immunotherapies

Abstract Flow cytometry assays using fresh whole blood which are interrogating either an extremely rare cell subset or a depleting immunotherapy, may require samples to be processed from global clinical sites over a wide geographical area, which can pose a major challenge for clinical operations. Precision for Medicine (PfM) has addressed these operational challenges using commercial stabilizers which are added to the blood allowing samples to be frozen at -80°C and subsequently batch processed for sample testing. We have performed a fit for purpose assay qualification of a multiparameter flow cytometry panel allowing the immunophenotypic analysis of monocyte, T cell, B cell and NK immune populations using Cytodelics™ fixed/stabilized healthy donor whole blood. Additionally, the assay was verified in whole blood samples from multiple solid tumor disease states including melanoma, colorectal cancer, head &amp; neck, non-small cell lung cancer to demonstrate clinical application. In these oncology samples, we were able to assess both immune cell frequencies and their activation status in both fresh whole blood as well as fixed whole blood over a short-term sample stability study, where the samples had been stored at -80°C for several months. This data provides a representative example of the use of commercial stabilizing fixatives for clinical studies to extend the stability window of whole blood for subsequent assessment by flow cytometry. Fixed whole blood can be stored at -80°C, shipped on dry ice allowing multiple timepoints in a cohort to be assessed together, thus minimizing assay variability as compared to fresh whole blood analysis, and mitigating the impact of shipping delays and temperature excursions on the samples. With sufficient time for assay development there are several commercial stabilizers that can be tested to determine if the markers in the flow assay are compatible with these reagents, providing a valuable option to assess the impact of immunotherapies on the immune cells of interest and reducing operational challenges in global studies. Citation Format: Jackie Benko, Samantha Splitt, Angelina Bisconte, Laura Lozza, Rachel Owen, Deborah Phippard. Flow cytometry using stabilized whole blood to assess innate and adaptive immune cells (Monocytes, NK, T, and B cells) in immunotherapies [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 3863.

Abstract 2647: Topical corticosteroids stimulate T cell-dependent melanoma growth control

Abstract Melanoma is the deadliest form of skin cancer. Treatment with immune checkpoint blockade (ICB) has transformed outcomes. However, half of melanoma patients do not derive long-term benefit. Side-effects of ICB are frequent, and typically managed by systemic or topical treatment with corticosteroids, widely known for their immunosuppressive effects. However, clinical data is contradictory as to the effect on prognosis of patients receiving ICB that also receive corticosteroids, with some studies suggesting a negative effect of steroid use, and others suggesting no effect or even positive outcomes with steroid use. Additionally, our lab recently showed that combining corticosteroids with ICB improves treatment responses in certain preclinical cancer models. Thus, understanding the role of corticosteroids in cancer progression, and their effect on the tumor immune microenvironment, is critical. In an intradermal murine melanoma model unresponsive to ICB, we found that topical corticosteroid treatment significantly impairs tumor growth. After just two doses, glucocorticoid-treated tumors shrank, whereas control-treated tumors doubled in volume. Intriguingly, this effect was lost in Rag1−/− mice (deficient in B and T cells) or in mice depleted of CD8+ T cells, uncovering a key role for T cells in corticosteroid-induced tumor growth control. Genetic ablation of the glucocorticoid receptor in tumor cells abrogated this effect, suggesting corticosteroids act directly on tumor cells to stimulate anti-tumor immunity. Analysis of TCGA RNA sequencing data showed melanoma patients with high glucocorticoid receptor expression have better overall survival than those with low expression, a finding repeated in a large, independent cohort of primary melanoma specimens of the Leeds Melanoma Cohort (n=703). Through cellular and molecular profiling using multiparametric flow cytometry and whole exome RNA sequencing, we have identified promising candidate pathways that might underlie the immune-dependent tumor control induced by steroids. Steroids induce tumor control in other, but not all, melanoma and non-melanoma tumor models studied, suggesting this may be a conserved immune-evasive pathway in a subset of tumors. Ongoing work aims to characterize the molecular and cellular effects of topical corticosteroid treatment, and to investigate their relevance in human melanoma. Given the widespread use of corticosteroids in patients receiving ICB, these unexpected findings may have significant clinical impact. Citation Format: Charles H. Earnshaw, Shih-Chieh Chiang, Agrin Moeini, Maria Koufaki, Eduardo Bonavita, Laetitia Nebot-Bral, Massimo Russo, Charlotte Bell, Theophile Bigirumurame, Jérémie Nsengimana, Julia Newton-Bishop, Christopher E. Griffiths, Santiago Zelenay. Topical corticosteroids stimulate T cell-dependent melanoma growth control [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 2647.

Abstract 2902: CA-4948 (emavusertib) improves treatment response of preclinical metastatic brain melanoma to anti-PD-1 immune checkpoint blockade

Abstract Background: Although recent advances in immunotherapy for metastatic melanoma have transformed the treatment paradigm for a disease that once carried a grim prognosis, nearly 50% of patients fail to reach a complete response to immune checkpoint blockade (ICB) due to multiple mechanisms of tumor-induced immune resistance. These challenges are amplified in the CNS tumor microenvironment (TME) where metastatic brain melanoma (MBM) demonstrates diminished clinical benefit to ICB treatment. A deeper understanding of these mechanisms are required to improve therapeutic response to ICB for these patients. Our group has previously identified that aberrant activation of the myddosomal cascade through downstream interleukin (IL)-1 receptor-associated kinase (IRAK-4) signaling results in chronic stimulation of MAPK and NF-κB in MBM. Further investigation reveals myddosome signaling emanates in large part from the TME, resulting in reflexive immune suppression. CA-4948 (emavusertib), a small molecule inhibitor of IRAK-4, is capable of reaching therapeutic dose levels in the brain where it demonstrates on-target inhibition of this pathway. These data highlight a unique therapeutic opportunity where selective inhibition of immune-suppressive signaling via targeted IRAK-4 blockade may sensitize MBM to anti-PD-1 ICB. Methods Multispectral imaging of patient MBM samples were analyzed for myddosome expression in the TME. Melanoma preclinical models, including both primary site (cutaneous) and metastatic brain disease, were evaluated for tumor burden and survival in response to CA-4948 treatment alone and in combination with anti-PD-1 ICB. Immunological responses of tumor to treatment were evaluated in two transgenic reporter models, CCR2RFPCX3CR1GFP and IFNγIRES-EYFP, to determine both the distribution and activation of infiltrating immune cells. Tumor-associated myeloid cells were assessed for phenotypic markers by multiparameter flow cytometry. Results Myddosome signaling was found to be elevated in tumor-associated immune cells in patient MBM specimens. Treatment of preclinical melanoma with CA-4948 combined with anti-PD-1 ICB resulted in significantly reduced tumor growth and improved survival in three independent models. CA-4948 treatment was associated with reduced tumor recruitment of peripherally derived tumor-associated macrophages, as well as increased lymphocyte infiltration and improved pro-inflammatory signaling measured by increased IFNγ expression in the combinatorial setting. Conclusion Targeted inhibition of IRAK-4 with CA-4948 improves immune surveillance and activation in preclinical models when combined with anti-PD-1 ICB, resulting in reduced tumor growth and increased survival. Thus, CA-4948 may be an effective adjuvant treatment strategy alongside anti-PD-1 ICB for the treatment of metastatic melanoma. Citation Format: Christina A. von Roemeling, Bently P. Doonan, Amanda M. Acevedo, Jeet A. Patel, Savannah L. Carpenter, Alisha Bhatia, Reinhard W. von Roemeling, Jamison Hoffman, Maureen Lane, Duane A. Mitchell. CA-4948 (emavusertib) improves treatment response of preclinical metastatic brain melanoma to anti-PD-1 immune checkpoint blockade [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 2902.

Abstract 298: Contribution of cancer associated fibroblasts to treatment response and resistance in high-risk multifocal prostate cancer

Abstract Patients with multifocal, locally advanced prostate cancer are at the highest risk of recurrence and death. Neoadjuvant therapies have limited response rates in this patient cohort and there remains a critical need to develop curative treatments. Prostate cancer (PC) shows marked heterogeneity, which is not limited to tumor cells but extends across immune and stromal compartments and has been linked to metastatic disease and therapeutic resistance. Cancer associated fibroblasts (CAFs) have been proposed to play a critical role in PC progression and invasion, but it is still unclear how CAFs interact with tumor cells. We evaluated CAFs from prostatectomy specimens as drivers of resistance to neoadjuvant androgen receptor signaling inhibitor (ARSi) and chemotherapy and assessed treatment response in co-culture systems in the context of CAF presence. We have utilized a novel radio-pathology tool that integrates PSMA PET/MRI scans to identify regions of interest that associate with heterogenous treatment response. MRI scans were utilized to print patient-specific 3D molds to micro dissect live tissue for subsequent cell sorting and molecular analyses. We have established a multi-parameter flow cytometry panel to characterize primary prostate fibroblasts from tumor foci and adjacent normal prostate tissue. In addition, co-culture assays were performed including immortalized CAF (hPrCSC-44) and MSC-derived fibroblast. To examine the tumor-promoting role of CAFs, fibroblasts were co-cultured with androgen-sensitive 3D tumor PC spheroids (LNCaP, C42B and LAPC4) in the integrated microfluidic STACKs platform that allows for the assessment of spatio-temporal interaction in a variety of culture microenvironments to model complex interactions. Cytotoxicity in 3D PC spheroids was assessed after Apalutamide, Darolutamide, or Docetaxel treatment by confocal microscopy. Integrated DNA, RNA, and radiology analysis of tumor biopsies have identified gene signatures associated with early biochemical recurrence. Most biopsies had low tumor purity, evidence of the cytotoxic impact of chemohormonal therapy while high tumor purity predicted high tumor expression. Poor clinical outcome was significantly associated with elevated stromal scores while epithelial and stromal enrichment were inversely correlated (rho= -0.80, p&amp;lt; 5 × 10−16). The presence of CAFs significantly decreased Docetaxel (C42B only vs C42B+CAF, 86.07% vs. 44.57%, respectively, p&amp;lt;0.001; LAPC4 only vs LAPC4+CAF, 70.98% vs 26.64%, respectively, p&amp;lt;0.001) and Darolutamide-induced (C42B only vs C42B+CAF, 52.59% vs 37.87%, respectively, p=0.0407). In conclusion, recurrent PC is linked to increased stromal content and the presence of CAFs supported tumor survival in response to ARSi and Docetaxel treatment. The molecular drivers of this phenomenon are currently being investigated. Citation Format: Nikolett Lupsa, Erika Heninger, Adeline Ding, Shannon R. Reese, Xavier T. Hazelberg, Aaron M. LeBeau, Brian P. Johnson, Peter P. Geiger, David J. Beebe, David A. Quigley, Joshua M. Lang. Contribution of cancer associated fibroblasts to treatment response and resistance in high-risk multifocal 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 298.

Abstract 3904: Multiplexed flow cytometry based immunophenotyping paired with functional ex vivo (MFLEX) drug profiling informs potential efficacy of therapeutic agents in acute myeloid leukemia

Abstract Introduction: Acute Myeloid Leukemia (AML) is a heterogenous hematological malignancy with a complex clonal architecture that requires in-depth immunophenotyping for disease characterization. Conventional AML ex vivo models for preclinical drug screening lack functional readouts that provide single cell level data to identify drug activity in defined AML subsets with unique immune signatures. Here, we present a novel platform that links Multiparameter Flow cytometry with high-throughput EX vivo drug screening (MFLEX) in AML patient-derived cell models to simultaneously detect phenotypic changes and predict cell-subset specific drug responses in AML. Method: We developed a 21-color high-dimensional immune profiling panel that captures AML heterogeneity by delineating primitive and mature AML blasts along with normal hematopoietic lineages. The panel also includes markers for functional measurement of apoptosis, proliferation, differentiation, and expression of BCL2 family proteins in lymphoid and myeloid cell subsets. Ex vivo culture conditions were optimized using physiologically relevant stroma-free cytokine-rich media that supports primary AML cells with minimal phenotypic shift, allowing for high throughput functional drug sensitivity testing. Timepoints and dosing conditions for AML standard of care (SoC) agents were optimized to support combination screens. Multi-parameter datasets were analysed for functional differences and visualized using dimension reduction methods. Results: We tested MFLEX on clinically annotated AML samples to understand sensitivity profiles to venetoclax and combination partners: 5-azacytidine, cytarabine, fludarabine and gilteritinib. We identified distinct drug sensitivity profiles for venetoclax in AML patients with varying leukemic differentiation states where M0/M1 subtypes were more sensitive to therapy than M4/M5. We also observed cell lineage specific sensitivities, with myeloid blast being more sensitive to venetoclax compared to autologous CD4+ and CD8+ T-cells and this observed difference was correlated with higher expression of BCL2. Our platform demonstrated that patients who previously failed venetoclax therapy could benefit from a combination with 5-azacytidine or fludarabine and this was patient-specific. We further evaluated MFLEX’s predictive value for patient clinical response by systematically comparing venetoclax sensitivity in patient-derived xenograft models in vivo vs ex vivo and found a high correlation between drug responses. Conclusion: We have established a unique, immune drug screening platform, MFLEX, that has high translational value and can be implemented in clinical trials to identify drug response patterns, novel combinations, and potential biomarkers of response for patient stratification in AML. Citation Format: Reecha Shah, Heba Wander, Michelle Hsueh, Muskan Floren, Wei Liu, Patricia Cheung, Courtney Andersen, Michael White, Nathan Kingston, Pablo Morentin Gutierrez, Lisa Drew, Giulia Fabbri, Elena Bibikova, Daniel Auclair. Multiplexed flow cytometry based immunophenotyping paired with functional ex vivo (MFLEX) drug profiling informs potential efficacy of therapeutic agents in acute myeloid leukemia [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 3904.

Abstract 6409: A multiparametric flow cytometry assay to monitor regeneration of B-cells following anti B-cell therapy in cancer and autoimmune patients

Abstract Purpose Functional dysregulation of B-cells leads to a variety of autoimmune disorders and blood cancers. Multiple B-cell depleting therapies (e.g., BTK inhibitors, antibodies or CAR-Ts) are currently in active clinical trial investigation. Following therapeutic elimination of pathogenic B-cells, it is critical to monitor the productive regeneration of fully functional B-cells as they mediate innate and adaptive immune responses. To this end, we developed a multiparametric flow cytometry assay for exquisite monitoring of early stages of B cell development, maturation, and antibody production to correlate it with treatment outcomes and determine if continuous depletion of B-cells is necessary to maintain clinical benefits for patients. Study Design We designed a high throughput 12-parameter flow cytometry panel incorporating primary differentiation markers namely, CD45, CD3, CD38, CD10, CD27, CD19, CD20, IgD, Lambda &amp; Kappa Light Chains required for identification of different developmental stages of B-cells in peripheral blood using an in vitro diagnostic grade flow cytometry system (BD FACSLyric). The optimized method underwent robust fit-for-purpose analytical validation per CLSI H62 guidelines with emphasis on establishing assay sensitivity, specificity, and reproducibility across experimental days, operators, and instruments. Results We were able to reliably identify different stages of B-cell development, namely, Naïve (CD19+CD20+IgD+CD27-), Unswitched/Marginal (CD19+CD20+IgD+CD27+), Memory (CD19+CD20+IgD-CD27+), Transitional (CD19+CD20+IgD+CD27-CD10+), Antibody Secreting Cells (CD19+CD20-IgD-CD27+CD38hi) as well as clonality (CD19+CD20+Lambda+ or Kappa+). The lower limit of quantitation was determined to be around 60 events, based upon analysis of 90 data points with good reproducibility (CV range: 0-11.7%) across experimental days, operators and instruments demonstrating sensitive detection and robust performance. Conclusion In summary, we have developed and validated a 12-parameter flow cytometry assay focusing on monitoring the regeneration of different subsets of B-cells in various B-cell malignancies (Non-Hodgkin’s Lymphoma, B CLL) and autoimmune disorders (Multiple Sclerosis, Rheumatoid Arthritis, Systemic Lupus Erythematosus). The high reproducibility/robustness of the assay demonstrates its suitability for use in clinical trials as a valuable tool for safety monitoring and disease evaluation enabling development of novel therapeutic agents. Citation Format: Amit Kumar Mehta, Ruby Tandon, Anil Pahuja, Jane Gao, Kevin Nguyen, Naveen Dakappagari, Zeni Alfonso. A multiparametric flow cytometry assay to monitor regeneration of B-cells following anti B-cell therapy in cancer and autoimmune patients [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 6409.

COVID-19 vaccination in cancer patients: Immune responses one year after the third dose

Cancer patients (CPs), being immunosuppressed due to the treatment received or to the disease itself, are more susceptible to infections and their potential complications, showing therefore an increased risk of developing severe COVID-19 compared to the general population.We evaluated the immune responses to anti-SARS-CoV-2 vaccination in patients with solid tumors one year after the administration of the third dose and the effect of cancer treatment on vaccine immunogenicity was assessed. Healthy donors (HDs) were enrolled. Binding and neutralizing antibody (Ab) titers were evaluated using chemiluminescence immunoassay (CLIA) and Plaque Reduction Neutralization Test (PRNT) respectively. T-cell response was analyzed using multiparametric flow cytometry.CPs who were administered three vaccine doses showed lower Ab titers than CPs with four doses and HDs. Overall, a lower cell-mediated response was found in CPs, with a predominance of monofunctional T-cells producing TNFα. Lower Ab titers and a weaker T-cell response were observed in CPs without prior SARS-CoV-2 infection when compared to those with a previous infection. While no differences in the humoral response were found comparing immunotherapy and non-immunotherapy patients, a stronger T-cell response in CPs treated with immunotherapy was observed.Our results emphasize the need of booster doses in cancer patients to achieve a level of protection similar to that observed in healthy donors and underlines the importance of considering the treatment received to reach a proper immune response.

Immunophenotypic characterization of leukemic stem cells in acute myeloid leukemia using single tube 10-colour panel by multiparametric flow cytometry: Deciphering the spectrum, complexity and immunophenotypic heterogeneity.

Despite extensive research, comprehensive characterization of leukaemic stem cells (LSC) and information on their immunophenotypic differences from normal haematopoietic stem cells (HSC) is lacking. Herein, we attempted to unravel the immunophenotypic (IPT) characteristics and heterogeneity of LSC using multiparametric flow cytometry (MFC) and single-cell sequencing. Bone marrow aspirate samples from patients with acute myeloid leukaemia (AML) were evaluated using MFC at diagnostic and post induction time points using a single tube-10-colour-panel containing LSC-associated antibodies CD123, CD45RA, CD44, CD33 and COMPOSITE (CLL-1, TIM-3, CD25, CD11b, CD22, CD7, CD56) with backbone markers that is, CD45, CD34, CD38, CD117, sCD3. Single-cell sequencing of the whole transcriptome was also done in a bone marrow sample. LSCs and HSCs were identified in 225/255 (88.2%) and 183/255 (71.6%) samples, respectively. Significantly higher expression was noted for COMPOSITE, CD45RA, CD123, CD33, and CD44 in LSCs than HSCs (p < 0.0001). On comparing the LSC specific antigen expressions between CD34+ (n = 184) and CD34- LSCs (n = 41), no difference was observed between the groups. More than one sub-population of LSC was demonstrated in 4.4% of cases, which further revealed high concordance between MFC and single cell transcriptomic analysis in one of the cases displaying three LSC subpopulations by both methods. A single tube-10-colour MFC panel is proposed as an easy and reproducible tool to identify and discriminate LSCs from HSCs. LSCs display both inter- and intra-sample heterogeneity in terms of antigen expressions, which opens the facets for single cell molecular analysis to elucidate the role of subpopulations of LSCs in AML progression.