Flow Cytometry-based Immunophenotyping Research Articles

Abstract 6649: Tumor treating fields (TTFields) induce an anti-tumor immune response in a pancreatic cancer mouse model

Abstract Background: Pancreatic ductal adenocarcinoma (PDAC) is a deadly malignancy with few therapeutic options. Tumor Treating Fields (TTFields) are low-intensity electric fields that disrupt cellular processes critical for cancer cell division and tumor progression and are approved for the treatment of glioblastoma and pleural mesothelioma. A phase 3 clinical trial is in progress to test the efficacy and safety of TTFields concomitant with standard-of-care gemcitabine and nab-paclitaxel (GnP) as front-line treatment of locally advanced PDAC. TTFields have been shown to induce immunogenic cell death (ICD) in cancer cells and elicit a systemic anti-cancer immune response. Accordingly, the benefit of applying TTFields together with immune checkpoint inhibitors (ICI) was demonstrated in a phase 3 study in patients with non-small cell lung carcinoma. This preclinical study investigated TTFields-induced immune responses in PDAC. Methods: An orthotopic PDAC tumor model was established by inoculating 4 × 105 Panc02 pancreatic cancer cells with stable luciferase expression into the pancreas tail of immune-competent C57BL/6 mice. The mice were randomized into four groups following bioluminescence detection of tumor volume: control, TTFields (150 kHz, 10 days), GnP (50 mg/kg for each drug, i.p. injected once a week for a total of two administrations), and GnP + TTFields. At the end of treatment, tumor volume was measured using caliper, and blood was collected for spectral flow cytometry-based immunophenotyping and ELISA testing for the ICD marker HMGB-1. In vitro, Panc02 cells were treated for 72h with TTFields (150 kHz, 1.62 V/cm RMS) and examined by flow cytometry for apoptosis and ATP secretion (using quinacrine staining) and surface exposure of calreticulin. Results: TTFields markedly reduced the tumor size in Panc02 tumor-bearing mice compared to control mice. Analysis of peripheral blood effector (CD62L-CD44+) and central (CD62L+CD44+) memory T cells demonstrated the induction of circulating effector-memory cytotoxic CD8+ T cells. Additionally, serum HMGB-1 concentrations were significantly elevated in mice treated with TTFields compared to control mice, implying the involvement of ICD in the response to TTFields. In vitro, TTFields application induced apoptosis of Panc02 cells and increased ICD, as indicated by increased surface exposure of calreticulin and ATP depletion. When TTFields were used in Panc02 tumor bearing mice concomitantly with GnP, a substantial reduction in tumor size was detected, and a complete tumor regression was observed in a number of mice. Conclusions: Our results suggest that TTFields may enhance the effects of standard-of-care chemotherapy for the treatment of PDAC. Furthermore, TTFields were shown to induce ICD and activate an antitumor immune response in PDAC, supporting future studies evaluating the efficacy of TTFields together with ICI in PDAC. Citation Format: Tal Kan, Tharwat Haj Khalil, Yiftah Barsheshet, Tali Voloshin, Avital Vorontsov, Boris Brant, Lilach Koren, Roni Blatt, Shay Cahal, Efrat Zemer-Tov, Rom Paz, Anat Klein-Goldberg, Catherine Tempel Brami, Sara Jacobovitch, Alexandra Volodin, Bella Koltun, Cfir David, Adi Haber, Moshe Giladi, Uri Weinberg, Yoram Palti. Tumor treating fields (TTFields) induce an anti-tumor immune response in a pancreatic cancer mouse model [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 6649.

Hybrid Macromolecular Constructs as a Platform for Spectral Nanoprobes for Advanced Cellular Barcoding in Flow Cytometry.

Herein, an advanced bioconjugation technique to synthesize hybrid polymer-antibody nanoprobes tailored for fluorescent cell barcoding in flow cytometry-based immunophenotyping of leukocytes is applied. A novel approach of attachment combining two fluorescent dyes on the copolymer precursor and its conjugation to antibody is employed to synthesize barcoded nanoprobes of antibody polymer dyes allowing up to six nanoprobes to be resolved in two-dimensional cytometry analysis. The major advantage of these nanoprobes is the construct design in which the selected antibody is labeled with an advanced copolymer bearing two types of fluorophores in different molar ratios. The cells after antibody recognition and binding to the target antigen have a characteristic double fluorescence signal for each nanoprobe providing a unique position on the dot plot, thus allowing antibody-based barcoding of cellular samples in flow cytometry assays. This technique is valuable for cellular assays that require low intersample variability and is demonstrated by the live cell barcoding of clinical samples with B cell abnormalities. In total, the samples from six various donors were successfully barcoded using only two detection channels. This barcoding of clinical samples enables sample preparation and measurement in a single tube.

Multicolor flow cytometry-based immunophenotyping for preclinical characterization of nanotechnology-based formulations: an insight into structure activity relationship and nanoparticle biocompatibility profiles.

Immunophenotyping, which is the identification of immune cell subsets based on antigen expression, is an integral technique used to determine changes of cell composition and activation in various disease states or as a response to different stimuli. As nanoparticles are increasingly utilized for diagnostic and therapeutic applications, it is important to develop methodology that allows for the evaluation of their immunological impact. Therefore, the development of techniques such as immunophenotyping are desirable. Currently, the most common technique used to perform immunophenotyping is multicolor flow cytometry. We developed two distinct multicolor flow cytometry immunophenotyping panels which allow for the evaluation of the effects of nanoparticles on the composition and activation status of treated human peripheral blood mononuclear cells. These two panels assess the presence of various lymphoid and myeloid-derived cell populations as well as aspects of their activation statuses-including proliferation, adhesion, co-stimulation/presentation, and early activation-after treatment with controls or nanoparticles. To conduct assay performance qualification and determine the applicability of this method to preclinical characterization of nanoparticles, we used clinical-grade nanoformulations (AmBisome, Doxil and Feraheme) and research-grade PAMAM dendrimers of different sizes (G3, G4 and G5) and surface functionalities (amine-, carboxy- and hydroxy-). We found that formulations possessing intrinsic fluorescent properties (e.g., Doxil and AmBisome) interfere with accurate immunophenotyping; such interference may be partially overcome by dilution. In the absence of interference (e.g., in the case of dendrimers), nanoparticle size and surface functionalities determine their effects on the cells with large amine-terminated dendrimers being the most reactive.

Abstract 1872: A DLL3/CD3/CD137 trispecific T cell engager shows potent antitumor activity in small cell lung cancer models

Abstract Background: Despite the approval of immune checkpoint inhibitors (ICIs), prognosis of small cell lung cancer (SCLC) remains poor. DLL3 is upregulated in SCLC whereas expression in normal tissues is minimal, representing the favorable profile as a therapeutic target. T cell engager (TCE) is a potent immunotherapy that redirects T cells to tumors expressing a specific antigen. Unlike ICIs, TCEs do not require the recognition of tumor antigens by T cells and thus could be an alternative approach to target tumors where the benefit of ICIs is limited such as SCLC. CD137 (4-1BB) is a costimulatory molecule that promotes T cell activation, proliferation, and survival. Since CD137 agonists synergize with CD3-mediated T cell activation, inducing concurrent CD137 costimulation is a promising strategy to unleash the potential of TCEs. We therefore developed a DLL3/CD3/CD137 trispecific T cell engager composed of two CD3/CD137 dual specific Fabs and one extra DLL3 Fab (DLL3 trispecific, RG6524). Results: We initially investigated the CD3 and CD137 signal transduction in Jurkat cells harboring NFAT or NF-κB reporter cocultured with DLL3 positive cells. DLL3 trispecific showed dose-dependent increase in NFAT and NF-κB activity, indicating that target engagement successfully activated CD3 and CD137 signaling. In the in vitro assay using human PBMC, DLL3 trispecific induced cytotoxicity against SCLC cell lines with EC50s in the two-digit pM range. In vivo efficacy was evaluated in SCLC xenograft models established by engrafting SCLC cell lines into immune humanized NOG mice. DLL3 trispecific showed greater tumor growth inhibition compared to a traditional bispecific T cell engager. Furthermore, flow cytometry-based immunophenotyping revealed that DLL3 trispecific increased T cell number in tumors and improved IFN-γ production from CD8 T cells. We also explored the combination with platinum-based drugs, which are widely used for SCLC, and showed that DLL3 trispecific enhanced the efficacy of platinum-based drugs. These data demonstrated that DLL3 trispecific has the potent in vivo efficacy and the potential for clinical use. We next evaluated whether cytokine release syndrome (CRS) mitigating approaches affect antitumor efficacy. Although prophylactic use of a steroid significantly reduced cytokine production, tumor growth inhibition by DLL3 trispecific remained unchanged. Likewise, tocilizumab treatment did not reduce the efficacy, suggesting that CRS mitigation did not abrogate the therapeutic benefit. We finally assessed the tolerability in non-human primates. DLL3 trispecific did not reach the maximum tolerated dose and was well tolerated up to 16 mg/kg Q2D, the highest dose tested. Conclusion: Our data showed that DLL3 trispecific has potent activity and is well suited for clinical application in SCLC. These findings provide a rationale for the clinical testing of DLL3 trispecific. Citation Format: Hirofumi Mikami, Shu Feng, Sotaro Naoi, Yumiko Azuma, Yoko Kayukawa, Toshiaki Tsunenari, Kentaro Asanuma, Ryutaro Iwabuchi, Hiroaki Nagano, Junko Shinozuka, Masaki Yamazaki, Haruka Kuroi, Siok Wan Gan, Priyanka Chichili, Shun Shimizu, Yutaka Matsuda, Shinya Ishii, Shogo Kamikawaji, Yasuko Kinoshita, Yuichiro Shimizu, Akihisa Sakamoto, Masaru Muraoka, Noriyuki Takahashi, Tatsuya Kawa, Hirotake Shiraiwa, Kenji Kashima, Futa Mimoto, Mika Kamata-Sakurai, Takehisa Kitazawa, Tomoyuki Igawa. A DLL3/CD3/CD137 trispecific T cell engager shows potent antitumor activity in small cell lung cancer models [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 1872.

Abstract 9504: Endothelial Shear Stress Metrics Correlate With Inflammatory Markers at the Culprit Site of Erosion in Patients With an Acute Coronary Syndrome: An OPTICO-ACS Substudy

Introduction: The pathobiological mechanisms of coronary plaque erosion are unclear. Low endothelial shear stress (ESS) is a proinflammatory/proatherogenic stimulus associated with coronary plaque progression/destabilization. Intravascular imaging studies suggest that high ESS gradient (low ESS areas adjacent to high ESS areas), and steepness of plaque upslope/downslope correlate with plaque erosion. We investigated the relationship of local fluid hemodynamics to the inflammatory microenvironment at the culprit site of erosion in patients with an acute coronary syndrome. Hypothesis: ESS metrics associate with proinflammatory/proatherogenic cells and cytokines, and contribute to plaque erosion. Methods: We studied 30 patients with erosion from the OPTIcal-COherence Tomography in Acute Coronary Syndrome study (OPTICO-ACS). OCT images were segmented, co-registered with the angiogram to create a 3D-reconstruction of the coronary artery. ESS metrics were calculated by Computational Fluid Dynamics. Systemic and local blood samples and thrombectomy specimens were collected at the culprit lesion and analyzed by flow cytometry-based immunophenotyping and plasma cytokine and chemokine profiling, and statistically tested for correlations of continuous variables using Spearman rank correlation (r). Results: Proinflammatory cytokines (IL6, MIP-1, IL1β, IL2) and local concentration of T-cells, including subsets of T-cells (CD4+, CD8+, and NKT-cells), were significantly higher at the culprit site of erosion and correlate with local adverse ESS metrics (Min ESS, Max ESS, Plaque Topographical Slope) (Table). Conclusion: Biomechanical features likely trigger activation of the adaptive immune system, including T-lymphocytes and their cytotoxic effector molecules. These results provide novel insights into the links between fluid hemodynamics, inflammatory activation, and mechanisms involved in the pathogenesis of coronary plaque erosion.

Comparative flow cytometry-based immunophenotyping analysis of peripheral blood leukocytes before and after fixation with paraformaldehyde

Flow cytometry based immunophenotyping provides prime insight into cellular population composition and characteristics, and is widely used in basic and clinical research. Challenges in processing peripheral blood samples in a timely manner necessitate protocol adaptations and utilization of fixatives. Fixation, however, may introduce artifacts to the flow cytometry readout. We performed a comparative flow cytometry immunophenotyping analysis of 13 immune cell populations in the whole blood using a staining protocol with and without fixation step. Freshly procured human peripheral blood samples were stained with a panel of 33 fluorochrome-conjugated antibodies. Samples were processed using a protocol with or without a paraformaldehyde-based fixation step, and matching sample pairs were analyzed by flow cytometry. Our results show that paraformaldehyde-based fixation, in comparison to matched unfixed samples, did not significantly affect population distribution and frequency for: B cells, Plasmablasts, Dendritic cells, NK cells, Granulocytes, Neutrophils, Eosinophils, or Hematopoietic Stem/Progenitor Cells. However, fixation led to significant marker shifts in the subpopulation distribution in CD4, T regulatory, CD8, Monocytes, and Basophils. These results indicate the importance of pre-experimental assessment of fixation-introduced artifacts in the flow cytometry output when considering the feasibility of fresh processing. This is especially important for samples analyzed using comprehensive exploratory immunoprofiling panels.

Traumatic brain injury alters dendritic cell differentiation and distribution in lymphoid and non-lymphoid organs

BackgroundPathophysiological consequences of traumatic brain injury (TBI) mediated secondary injury remain incompletely understood. In particular, the impact of TBI on the differentiation and maintenance of dendritic cells (DCs), which are regarded as the most professional antigen presenting cells of the immune system, remains completely unknown. Here, we report that DC-differentiation, maintenance and functions are altered on day 3 and day 7 after TBI.MethodsLong bones, spleen, peripheral lymph nodes (pLNs), mesenteric lymph nodes (mLNs), liver, lungs, skin and blood were collected from mice with either moderate-level cortical impact (CCI) or sham on day 1, day 3 or day 7 after TBI. Bone marrow cells were isolated from the tibias and femurs of hind limb through flushing. Tissues were digested with Collagenase-D and DNase I. Skin biopsies were digested in the presence of liberase + DNase I. Single cell suspensions were made, red blood cells were lysed with Ammonium chloride (Stem Cell Technology) and subsequently filtered using a 70 μM nylon mesh. DC subsets of the tissues and DC progenitors of the BM were identified through 10-color flow cytometry-based immunophenotyping studies. Intracellular reactive oxygen species (ROS) were identified through H2DCFDA staining.ResultsOur studies identify that; (1) frequencies and absolute numbers of DCs in the spleen and BM are altered on day 3 and day 7 after TBI; (2) surface expression of key molecules involved in antigen presentation of DCs were affected on day 3 and day 7 after TBI; (3) distribution and functions of tissue-specific DC subsets of both circulatory and lymphatic systems were imbalanced following TBI; (4) early differentiation program of DCs, especially the commitment of hematopoietic stem cells to common DC progenitors (CDPs), were deregulated after TBI; and (5) intracellular ROS levels were reduced in DC progenitors and differentiated DCs on day 3 and day 7 after TBI.ConclusionsOur data demonstrate, for the first time, that TBI affects the distribution pattern of DCs and induces an imbalance among DC subsets in both lymphoid and non-lymphoid organs. In addition, the current study demonstrates that TBI results in reduced levels of ROS in DCs on day 3 and day 7 after TBI, which may explain altered DC differentiation paradigm following TBI. A deeper understanding on the molecular mechanisms that contribute to DC defects following TBI would be essential and beneficial in treating infections in patients with acute central nervous system (CNS) injuries, such as TBI, stroke and spinal cord injury.

Flow cytometry-based immunophenotyping of myeloid-derived suppressor cells in human breast cancer patient blood samples

Multi-color flow cytometry is the standard approach for immunophenotyping clinical samples. With the recent advances in cancer immunotherapy, myeloid-derived suppressor cells (MDSC), immature myeloid-lineage cells in cancer patient blood and the tumor microenvironment, are highlighted as an important immune cell population that correlates with prognosis and therapeutic efficacy. In contrast to their clear functions and existence, immunophenotyping of MDSC is not consistent among investigators due to surface antigens overlapping with many normal hematopoietic lineage cell populations. We performed a clinical study and analyzed more than 1000 breast cancer patients blood samples to quantitate MDSC during breast cancer progression. In this methodology manuscript, we described detailed procedures for study design, sample logistics and handling, staining and flow cytometric analysis. This protocol used a 7-color fluorochrome-conjugated antibody panel to analyze polymorphonuclear (PMN)- and monocytic (M)-MDSC subsets simultaneously. The interim analysis results of this study showed that both PMN and M-MDSC populations are increased in patients with bone metastasis compared with patients with visceral organ metastasis. In conclusion, this work provides a versatile, comprehensive, and practical protocol to measure MDSC in patient blood samples.

Spartalizumab or placebo in combination with dabrafenib and trametinib in patients with BRAF V600-mutant melanoma: exploratory biomarker analyses from a randomized phase 3 trial (COMBI-i)

BackgroundThe randomized phase 3 COMBI-i trial did not meet its primary endpoint of improved progression-free survival (PFS) with spartalizumab plus dabrafenib and trametinib (sparta-DabTram) vs placebo plus dabrafenib and trametinib...

Protective Effect of Saffron in Mouse Colitis Models Through Immune Modulation.

People with inflammatory bowel disease (IBD) including ulcerative colitis are at risk for colorectal cancer. Despite available effective drugs used to treat IBD, many patients fail or lose response over time with some displaying drug-induced adverse events. Saffron (Crocus sativus) has been reported to have anti-inflammatory properties. Its protective role in IBD has not been explored extensively. To establish whether saffron treatment alleviates inflammation in experimental colitis. Colitis was induced in C57BL/6 mice with 3% DSS and treated with either saffron doses (7.5, 15, 20, 25mg/kg body weight) or vehicle through daily gavage. On day 11, mice were euthanized and analyzed for gross and microscopic inflammation. Distal colon segments were collected for mRNA and protein expression of HO-1 protein and GPX2, (the downstream targets of NRF-2). Nrf-2 translocation from cytosol to nucleus was confirmed by immunofluorescence, and further Nrf-2 protein expression in nuclear and cytosolic fraction of colon was analyzed by immunoblot. Immune cells were isolated from the lamina propria of mouse colon for flow cytometry-based immunophenotyping. Colitis was also induced in C57BL/6 Ahr knockout and wild type mice to explore the involvement of Ahr-dependent pathways in saffron's protective effect(s). The therapeutic effect of saffron was further validated in another TNBS model of colitis. Saffron 20mg/kg body weightshowed improved colon gross and histology features and led to better body weight, colon length, histology score, and reduced disease activity index (DAI). Saffron significantly decreased pro-inflammatory macrophages (M1), while increasing anti-inflammatory macrophages (M2) and IL10 + dendritic cells. Saffron treatment also enhanced CD3 + T and CD3 + CD8 + T cells followed by increase in different CD3 + CD4 + T cells subsets like CD25 + T cells, FoxP3 + CD25 + regulatory T cells, and CD4 + FOXP3 + CD25-regulatory T cells. Immunoblot analysis showed a significant increase in HO-1/GPX2 protein expression. With saffron treatment, Nrf-2 translocation into nucleus from cytosol also supports the involvement of Nrf-2 and its downstream targets in the protective effect of saffron. Further, we demonstrated that saffron in part exert anti-inflammatory effect through activation of aryl hydrocarbon receptor (AhR)-nuclear factor erythroid 2-related factor 2 (Nrf2)-dependent pathways. These data demonstrate saffron's therapeutic potential and its protective role in part via Ahr/Nrf-2 pathways and regulatory innate and adaptive immune cells.

Differential transcriptional and functional properties of regulatory T cells in HIV-infected individuals on antiretroviral therapy and long-term non-progressors.

ObjectivesRegulatory T cells (Tregs) are widely recognised as a subset of CD4+CD25+FOXP3+ T cells that have a key role in maintaining immune homeostasis. The impact of HIV‐1 infection on immunological properties and effector functions of Tregs has remained the topic of debate and controversy. In the present study, we investigated transcriptional profile and functional properties of Tregs in HIV‐1‐infected individuals either receiving antiretroviral therapy (ART, n = 50) or long‐term non‐progressors (LTNPs, n = 24) compared to healthy controls (HCs, n = 38).MethodsRNA sequencing (RNAseq), flow cytometry‐based immunophenotyping and functional assays were performed to study Tregs in different HIV cohorts.ResultsOur RNAseq analysis revealed that Tregs exhibit different transcriptional profiles in HIV‐infected individuals. While Tregs from patients on ART upregulate pathways associated with a more suppressive (activated) phenotype, Tregs in LTNPs exhibit upregulation of pathways associated with impaired suppressive properties. These observations may explain a higher propensity for autoimmune diseases in LTNPs. Also, we found substantial upregulation of HLA‐F mRNA and HLA‐F protein in Tregs from HIV‐infected subjects compared to healthy individuals. These observations highlight a potential role for this non‐classical HLA in Tregs in the context of HIV infection, which should be investigated further in other chronic viral infections and cancer.ConclusionOur study has provided a novel insight into Tregs at the transcriptional and functional levels in different HIV‐infected groups.

Abstract PO-046: Dissecting the role of tumor-intrinsic Cxcl1 in mediating immune exclusion in Ras-p53 cooperative pancreatic cancer

Abstract INTRODUCTION: Ras-p53 cooperative mutations, which define >50% of human pancreatic ductal adenocarcinoma (PDAC), promote an immune-excluded tumor microenvironment. In Ras-p53 murine models that phenocopy human PDAC, overexpression of the myeloid chemo-attractant Cxcl1 is associated with T-cell exclusion. We sought to dissect the role of tumor-intrinsic Cxcl1 in mediating immune exclusion in Ras-p53 PDAC. METHODS: Single-cell immune deconvolution and Cxcl1 expression were queried in TCGA PDAC samples (n=150). Single-cell RNA sequencing (scRNAseq) data in KrasG12D/+;Trp53fl/+;Pdx1Cre (KPC WT) genetically engineered mice (GEM) were interrogated. CRISPR/Cas9-based genome editing was used to silence Cxcl1 in KPC tumor cells (KPC-Cxcl1KO). Multiparameter flow cytometry-based immunophenotyping of tumor-infiltrating and circulating myeloid/T-cell populations was performed in in-vivo orthotopic models utilizing KPCWT / KPC-Cxcl1KO cells. RESULTS: In human TCGA samples, Cxcl1 overexpression was strongly associated with expression of its cognate receptor CXCR2, reduced CD4+/CD8+ T-cells, and increased CD33+ myeloid-derived suppressor cells (MDSCs). In KPC GEMs, scRNAseq revealed that CXCR2 expression was exclusively present on the polymorphonuclear MDSC subset (PMN-MDSC). Exogenous Cxcl1 conditioning in murine bone marrow-derived cells induced PMN-MDSC polarization and upregulated CXCR2. Genetic ablation of Cxcl1 in KPC tumor cells disrupted transwell migration of splenocyte-enriched MDSC in-vitro; this effect was dependent on CXCR2 expression on MDSCs. There was a dramatic reduction in tumor weights and metastatic outgrowth (p<0.0001), as well as circulating PMN-MDSC and CXCR2+-MDSC populations in KPC-Cxcl1KO compared with KPCWT mice (p=0.0006). Significantly fewer PMN-MDSC and M2-like macrophages infiltrated KPC-Cxcl1KO tumors, which was accompanied by dramatically higher CD4+/CD8+ T-cell infiltration (p<0.0001). While cytolytic effector CD8+ T-cell populations (CD44+62L-CD107a+) remained unchanged, non-cytolytic “exhausted” PD-1+CD8+ T-cells (PD-1+CD107a-) increased in KPC-Cxcl1KO vs. KPCWT tumors, providing rationale for addition of checkpoint blockade to reverse T-cell exhaustion. CONCLUSION: Tumor-intrinsic Cxcl1 appears a major contributor to immune exclusion in Ras-p53 cooperative PDAC. Targeting Cxcl1 represents a promising strategy to overcome MDSC-mediated immunosuppression and improve susceptibility to checkpoint blockade in PDAC. Citation Format: Anna Bianchi, Siddharth Mehra, Daisy Dai, Iago de Castro Silva, Prateek Sharma, Antonio Colaprico, Austin R. Dosch, Samara Singh, Nagaraj S. Nagathihalli, Nipun B. Merchant, Jashodeep Datta. Dissecting the role of tumor-intrinsic Cxcl1 in mediating immune exclusion in Ras-p53 cooperative pancreatic cancer [abstract]. In: Proceedings of the AACR Virtual Special Conference on Pancreatic Cancer; 2020 Sep 29-30. Philadelphia (PA): AACR; Cancer Res 2020;80(22 Suppl):Abstract nr PO-046.

Abstract 3144: Using artificial intelligence and flow cytometry to confirm the need for a prostate biopsy in at-risk men

Abstract The goal of this study was to create a blood based, liquid biopsy test for confirming the need for a prostate biopsy by objectively analyzing flow cytometry-based immunophenotyping data using machine learning (ML) by evaluating whether a patient is at-risk for having intermediate-/high-grade prostate cancer PCa [Gleason score (GS) ≥ 7]. Currently, there is a lack of blood-based biomarkers that can accurately predict high-risk PCa in at-risk men making it difficult for clinicians to assess disease status. Furthermore, a large percentage of men undergo unnecessary prostate biopsy procedures each year since the majority of biopsies return either benign pathologies, such as benign prostatic hyperplasia (BPH), or low-grade PCa [Gleason score (GS) = 6). Flow cytometry-based immunophenotyping of peripheral blood is an accessible and non-invasive technology, but as more biomarkers are included, new methods must be developed for the efficient analysis and utilization of these large datasets for clinical applications. Here, we used ML to create pattern recognition neural networks (PRNNs) to identify patients with intermediate-/high-grade PCa from those that have either low-grade PCa or BPH based upon the immunophenotyping of 27 different myeloid and lymphoid cell populations. A key population of interest are myeloid-derived suppressor cells (MDSCs) since they are known to be key contributors in supporting tumor progression and escape through their ability to suppress anti-tumor responses mediated through T cell and natural killer (NK) cell activity. We used standard multiparametric flow cytometry to measure MDSCs and other immune cell populations found in the peripheral blood of 114 biopsy-confirmed PCa and 58 biopsy-confirmed BPH patients that were collected in Streck Cyto-Chex® BCT. PRNNs were trained with raw flow cytometry from two data sets: 37 patients with PCa GS ≥ 7 and 40 patients with BPH/PCa GS6. Predictions were evaluated using the performance of the trained PRNNs on known PCa GS ≥ 7 and BPH/PCa GS6 not used in the PRNN training set (holdout samples). In the holdout samples, we identified 21 out of 23 patients who were identified as PCa GS ≥ 7 (Sens = 91.3%, 95%CI = 72.0% to 98.9%; PPV = 33.3%, 95%CI = 28.4% to 38.7%) while 30 out of 72 patients were identified as BPH/PCa GS6 (Spec = 41.7%, 95%CI = 30.2% to 53.9%; NPV = 93.8%, 95%CI = 79.5% to 98.3%) for a combined accuracy of 53.7% (95%CI = 43.2% to 64.0%). The PRNN classified 17 out of 33 BPH, 13 out of 26 GS6 PCa, 13 out of 14 GS7 PCa, and 8 out of 9 > GS7 PCa samples correctly. For this study, out of the 112 patients who were ultimately diagnosed with BPH or low-grade PCa, approximately 47 procedures could have been avoided or delayed. In a clinical setting, we believe that this technology, in use with other known clinical factors, would allow for clinicians to have a more informed decision when recommending their patients for a prostate biopsy procedure while reducing the number of unnecessary biopsy procedures performed each year. Citation Format: George A. Dominguez, John Roop, Anthony J. Campisi, Thomas Schlumpberger, Amit Kumar. Using artificial intelligence and flow cytometry to confirm the need for a prostate biopsy in at-risk men [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 3144.

Abstract B50: Using pattern recognition neural networks to detect prostate cancer: A new method to analyze flow cytometry-based immunophenotyping using machine learning

Abstract Prostate cancer (PCa) screening and detection relies heavily upon prostate-specific antigen (PSA) testing, but PSA testing has a high rate of false positives, leading to increased risks for overdiagnosis and overtreatment; thus, additional blood-based biomarkers for PCa detection are needed. Flow cytometry-based immunophenotyping of peripheral blood is an accessible and noninvasive technology, but as more parameters are included, new computational methods must be developed for the efficient analysis and utilization of these large datasets for clinical applications. Machine learning algorithms, specifically pattern recognition neural networks (PRNNs), have the potential to assist in these types of analyses, but the flow cytometry data need to be transformed into a usable input format. The goal of this study was to use our newly developed “hypervoxelation of cytometry events” computational technique, or HyperVOX, to transform flow cytometry data into a useable format for input into a series of PRNNs to detect PCa of all Gleason scores (GS) from circulating immune cells. We used standard multiparametric flow cytometry techniques to measure 16 different myeloid and lymphoid cell populations found in the peripheral blood of 156 biopsy-confirmed PCa (GS6 n = 59, GS7 n = 68, GS8 n = 12, GS9 n = 16, and GS10 n = 1; median age = 68 ± 8.7 years) along with 99 male healthy donors (HD) (median age = 53 ± 8.5 years). Flow cytometry data were then transformed using HyperVOX in order to create hypervoxels that can be used as the common feature across all samples. Briefly, each channel was used as an axis in a multidimensional space and divided into four segments, with each event being defined by its location within each segment of each axis. The resulting count of events that fall within each hypervoxel for each sample is then used as the input for the PRNN. With this, a screening-type assay was developed to detect PCa compared against HD. PRNNs were trained using raw flow cytometry data processed using HyperVOX from 97 PCa patients and 67 HD controls. Predictions were evaluated using the performance of the trained PRNNs on 59 PCa patients and 32 HD that were not used for PRNN training (holdout test set). The PRNN classified 28 out of 32 HD and 57 out of 59 PCa samples correctly, resulting in a sensitivity of 96.6% (95% CI, 88.3–99.6), specificity of 87.5% (95% CI, 71.0–96.5), positive predictive value (PPV) of 93.4% (95% CI, 85.1–98.2), negative predictive value (NPV) of 93.3% (95% CI, 78.1–98.2), and an AUC of 0.9656 (95% CI, 0.9202–1). Upon Gleason score stratification, the NN classified 27 out of 28 GS6, 18 out of 19 GS7, and 11 out of 11 >GS7 samples correctly. In a clinical setting, this technology would improve PCa detection and allow clinicians to have a more informed decision when recommending their patients for a prostate biopsy procedure and subsequent medical interventions to help reduce overdiagnosis and overtreatment. Citation Format: George A. Dominguez, John Roop, Alexander Polo, Anthony Campisi, Dmitry I. Gabrilovich, Amit Kumar. Using pattern recognition neural networks to detect prostate cancer: A new method to analyze flow cytometry-based immunophenotyping using machine learning [abstract]. In: Proceedings of the AACR Special Conference on Advances in Liquid Biopsies; Jan 13-16, 2020; Miami, FL. Philadelphia (PA): AACR; Clin Cancer Res 2020;26(11_Suppl):Abstract nr B50.

A novel liquid biopsy aid for identifying clinically significant prostate cancer in at-risk men: Combining artificial intelligence with flow cytometry-based immunophenotyping.

e17508 Background: Current screening methods for prostate cancer (PCa) result in a large number of false positives making it difficult for clinicians to assess disease status thus warranting advancements in screening and early detection methods. The goal of this study was to design a liquid biopsy assay that uses flow cytometry-based immunophenotyping and artificial intelligence (AI) analysis to detect clinically significant PCa in at-risk men. Methods: Standard multiparametric flow cytometry techniques were used to immunophenotype 27 different MDSC, myeloid, and lymphocyte cell populations found in the peripheral blood of 114 biopsy-confirmed PCa (GS6 = 54; ≥ GS7 = 60) and 58 biopsy-confirmed benign prostatic hyperplasia (BPH) patients. Pattern recognition neural networks (PRNNs), a type of AI, were trained with flow cytometry data from two data sets: patients with confirmed clinically significant PCa (≥ GS7) versus patients with BPH or clinically insignificant PCa (GS6). Predictions were evaluated using the performance of the trained PRNNs on 37 known ≥ GS7 PCa patients and 40 known BPH/ < GS7 PCa patients not used in the PRNN training set (holdout samples). Results: In the holdout data set, 21 out of the 23 patients with clinically significant PCa were identified correctly (Sens = 91.3%, 95%CI = 72.0%-98.9%; PPV = 33.3%, 95%CI = 28.4%-38.7%) while 30 out of the 72 patients at low-risk or no-risk were identified correctly (Spec = 41.7%, 95%CI = 30.2%-53.9%; NPV = 93.8%, 95%CI = 79.5%-98.3%) for an accuracy of 53.7% (95%CI = 43.2%-64.0%). For all patients in this study, our data suggests that approximately 47 out of the 112 patients with BPH or GS6 PCa could have avoided an unnecessary prostate biopsy procedure. Conclusions: Pairing our PRNN with surface biomarker blood-based analyses of various leukocyte populations has enabled the ability to objectively identify patterns indicative for the existence of clinically significant PCa (≥ GS7). In a clinical setting, we believe that this technology, in use with other known clinical factors, would allow for clinicians to have a more informed decision when recommending their patients for a prostate biopsy procedure.

Abstract A71: Resistance mechanisms limiting the immunostimulatory and antitumor activity of anti-CSF-1 receptor-mediated macrophage depletion

Abstract Tumor-associated macrophages (TAMs) are present within the stroma of most solid tumors where they contribute to disease progression through production of tumor and angiogenic growth factors, extracellular matrix remodeling, and suppression of immune responses. As such, depletion of tumor-associated macrophages, for instance through the use of blocking antibodies against the CSF-1 receptor (CSF1R), has been widely explored as a therapeutic approach to both inhibit tumor growth/metastasis and stimulate antitumor immune responses. In the context of immunotherapy, multiple preclinical murine studies have demonstrated that anti-CSF1R meditated macrophage depletion can promote antitumor T-cell responses, synergizing with immune checkpoint inhibitor therapy, such as PD1 blockade, and leading to tumor regression. However, some tumor models appear resistant to these beneficial effects of CSF1R blockade. In order to define potential mechanisms of resistance to CSF1R inhibitors, we evaluated the effects of anti-CSF1R treatment in mice bearing subcutaneous Renca renal cell adenocarcinoma tumors. Despite robust macrophage depletion, this model shows only a modest decrease in tumor volume and minimal effector T-cell recruitment or activation in response to anti-CSF1R therapy. Comprehensive flow cytometry-based immunophenotyping and single-cell RNA sequencing on immune cells isolated from anti-CSF1R- and isotype control-treated Renca tumors revealed shifts in gene expression patterns related to vascular remodeling, hypoxic responses, and enhanced regulatory T-cell activity. These studies indicate macrophage depletion may, under some conditions, have negative effects on antitumor immune responses that could limit the clinical benefit of this therapeutic approach. Citation Format: Katarzyna M. Skrzypczynska, Sarah A. O'Brien, Brian Belmontes, Hong Tan, Jessica Orf, Daniel Lu, Ian Driver, Jackson G. Egen. Resistance mechanisms limiting the immunostimulatory and antitumor activity of anti-CSF-1 receptor-mediated macrophage depletion [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology and Immunotherapy; 2018 Nov 27-30; Miami Beach, FL. Philadelphia (PA): AACR; Cancer Immunol Res 2020;8(4 Suppl):Abstract nr A71.

Abstract P5-01-16: Combining HyperVOX with pattern recognition neural networks: A new method for analyzing flow cytometry-based immunophenotyping data for increased early detection of stage I/II breast cancer (BCa)

Abstract Breast cancer screening, detection, and diagnosis relies heavily upon mammography, but due to breast pathology and the intrinsic limits of detection, mammogram-based procedures result in many false positives causing an increased risk for overdiagnosis and overtreatment. Therefore, blood-based biomarkers that can accurately predict early stage breast cancer (BCa) in at-risk women are needed. Flow cytometry-based immunophenotyping of peripheral blood is an accessible and non-invasive technology, but as more parameters are included in these panels, new methods must be developed for the efficient analysis and utilization of large sets of flow cytometry data for clinical applications. The goal of this study was to use our newly developed ‘hypervoxelation of cytometry events’ computational technique, or HyperVOX, to transform flow cytometry data into a useable format for input into a series of pattern recognition neural networks (PRNNs) to detect early stage breast cancer (I/II). We used standard multiparametric flow cytometry techniques to immunophenotype myeloid-derived suppressor cell (MDSC), myeloid, and lymphocyte cell populations (14 markers total) found in the peripheral blood of 99 biopsy-confirmed BCa patients (stage I = 70; stage II = 29) with varying hormone receptor expression and 88 healthy donor female (HDF) controls. BCa patients recommended for biopsy by their physician were included in this study and excluded if they had a previous history of cancer or have received any type of treatment for breast cancer in the past 6 months. PRNNs were trained using raw flow cytometry data processed using HyperVOX from 64 BCa patients (stage I = 44; stage II = 20) and 69 HDF controls. Predictions were evaluated using the performance of the trained PRNNs on 35 early stage BCa patients (stage I = 26; stage II = 9) and 19 HDF that were not used for PRNN training (holdout test set). Myeloid-derived suppressor cells (MDSCs) are known to be key contributors in supporting tumor progression and have also been shown to increase in peripheral blood as tumor progression occurs. For this study, manually gated percentages of MDSCs were used for comparison. In the holdout test set, we identified 32 out of 35 subjects with either stage I or II BCa (Sens. = 91.4%, 95%CI = 76.9% to 98.2%; PPV = 97.0%, 95%CI = 82.6% to 99.5%) while 18 out of 19 HDF were identified (Spec. = 94.7%, 95%CI = 74.0% to 99.9%; NPV = 85.7%, 95%CI = 66.9% to 94.7%) for a combined accuracy of 92.6% (95%CI = 82.1% to 97.9%); the resulting AUC = 0.9098 (95%CI = 0.8031 to 1.000). For stage I BCa alone, 24 out of 26 were identified correctly with an AUC = 0.9069 (95%CI = 0.7928 to 1.000). For stage II BCa alone, 8 out of 9 were identified correctly with an AUC = 0.9181 (95%CI = 0.8028 to 1.000). Additionally, we tested 26 samples collected from patients with biopsy confirmed ductal carcinoma in situ (DCIS). Even though they are clinically deemed as pre-cancerous (stage 0), 18 out of 26 (AUC = 0.8421; 95%CI = 0.7163 to 0.9679) were classified as BCa suggesting utility for detecting the existence of even a non-invasive cancerous lesion. Although further study is needed, we believe that this technology, in conjunction with other known clinical risk factors, would allow for clinicians to make a more informed diagnosis and treatment recommendation when screening and for recommending subsequent interventions for early stage breast cancer. Citation Format: George A Dominguez, John Roop, Alexander Polo, Anthony Campisi, Dmitry I Gabrilovich, Amit Kumar. Combining HyperVOX with pattern recognition neural networks: A new method for analyzing flow cytometry-based immunophenotyping data for increased early detection of stage I/II breast cancer (BCa) [abstract]. In: Proceedings of the 2019 San Antonio Breast Cancer Symposium; 2019 Dec 10-14; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2020;80(4 Suppl):Abstract nr P5-01-16.

Detecting Prostate Cancer Using Pattern Recognition Neural Networks With Flow Cytometry-Based Immunophenotyping in At-Risk Men.

Current screening methods for prostate cancer (PCa) result in a large number of false positives making it difficult for clinicians to assess disease status, thus warranting advancements in screening and early detection methods. The goal of this study was to design a liquid biopsy test that uses flow cytometry–based immunophenotyping and artificial neural network (ANN) analysis to detect PCa. Numerous myeloid and lymphoid cell populations, including myeloid-derived suppressor cells, were measured from 156 patients with PCa, 123 with benign prostatic hyperplasia (BPH), and 99 male healthy donor (HD) controls. Using pattern recognition neural network (PRNN) analysis, a type of ANN, PCa detection compared against HD resulted in 96.6% sensitivity, 87.5% specificity, and an area under the curve (AUC) value of 0.97. Detecting patients with higher risk disease (⩾Gleason 7) against lower risk disease (BPH/Gleason 6) resulted in 92.0% sensitivity, 42.7% specificity, and an AUC of 0.72. This study suggests that analyzing flow cytometry immunophenotyping data with PRNNs may prove to be a useful tool to improve PCa detection and reduce the number of unnecessary prostate biopsies performed each year.

Identification and Immunophenotypic Characterization of Normal and Pathological Mast Cells.

Mast cells (MCs) are secretory cells that are central players in human allergic disease and immune responses. With the exception of a few pathological situations, MCs are usually present at relatively low frequencies in most tissues. Since their first description, MCs in tissues were identified mostly using their morphological characteristics and their typical coloration when stained with aniline dyes. However, increasing availability of highly specific antibodies now permits the use of fluorescence-based flow cytometry as the method of choice for the quantification, characterization, and purification of cells in suspension. This technique allows for a rapid analysis of thousands of events and for the identification of cells present at frequencies as low as one event in 106 unwanted cells. This method also permits for simultaneous characterization of multiple antigens at a single cell level, which is ideal in order to study rare populations of cells like MCs. Here we describe the basis of flow cytometry-based immunophenotyping applied to the study of MC. The protocol focuses on the study of human MCs present in body fluids (mainly bone marrow) but can easily be adapted to studying MCs from other tissues and species.

The Immune Response to Chronic Pseudomonas aeruginosa Wound Infection in Immunocompetent Mice.

Objective: Our goal was to develop a chronic wound model in mice that avoids implantation of foreign material or impaired immunity and to use this to characterize the local and systemic immune response associated with Pseudomonas aeruginosa infection. Approach: We generated bilateral full-thickness dermal wounds in healthy 10-12-week-old C57Bl6 mice. We waited 24 h to inoculate the developing wound eschar at these sites. We performed careful titration experiments with luminescent strains of P. aeruginosa to identify bacterial inoculation concentrations that consistently established stable infections in these animals. We performed flow cytometry-based immunophenotyping of immune cell infiltrates at the wound site, spleen, and draining lymph nodes over time. Finally, we compared inflammatory responses seen in wound inoculation with planktonic bacteria, preformed biofilm, and heat-killed (HK) P. aeruginosa. Results: Using this delayed inoculation model and 7.5 ± 2.5 × 102 CFU/mL of PAO1 we consistently established stable infections that lasted at 10 days in duration. During early infection, we detected a strong upregulation of inflammatory cytokines and neutrophil infiltration at the wound site, while natural killer (NK) cells and dendritic cells (DCs) were reduced. At the systemic level, only plasmacytoid DCs were increased early in infection. During later stages, there was systemic upregulation of B cells, T cells, and macrophages, whereas NK cells and interferon killer DCs were reduced. Infections with P. aeruginosa biofilms were not more virulent than infections with planktonic P. aeruginosa, whereas treatment with HK P. aeruginosa only induces a short-term inflammatory state. Innovation: We describe a versatile wound model of chronic P. aeruginosa infection that lasts 10 days without causing sepsis or other excessive morbidity. Conclusion: This model may facilitate the study of chronic wound infections in immunocompetent mice. Our findings also highlight the induction of early innate immune cell populations during P. aeruginosa infection.