Rare Cell Subsets Research Articles

OMIP-109: 45-color full spectrum flow cytometry panel for deep immunophenotyping of the major lineages present in human peripheral blood mononuclear cells with emphasis on the T cell memory compartment.

The need for more in-depth exploration of the human immune system has moved the flow cytometry field forward with advances in instrumentation, reagent development and availability, and user-friendly implementation of data analysis methods. We developed a high-quality human 45-color panel, for comprehensive characterization of major cell lineages present in circulation including T cells, γδ T cells, NKT-like cells, B cells, NK cells, monocytes, basophils, dendritic cells, and ILCs. Assay optimization steps are described in detail to ensure that each marker in the panel was optimally resolved. In addition, we highlight the outstanding discernment of cell activation, exhaustion, memory, and differentiation states of CD4+ and CD8+ T cells using this 45-color panel. The panel enabled an in-depth description of very distinct phenotypes associated with the complexity of the T cell memory response. Furthermore, we present how this panel can be effectively used for cell sorting on instruments with a similar optical layout to achieve the same level of resolution. Functional evaluation of sorted specific rare cell subsets demonstrated significantly different patterns of immunological responses to stimulation, supporting functional and phenotypic differences within the T cell memory subsets. In summary, the combination of full spectrum profiling technology and careful assay design and optimization results in a high resolution multiparametric 45-color assay. This panel offers the opportunity to fully characterize immunological profiles present in peripheral blood in the context of infectious diseases, autoimmunity, neurodegeneration, immunotherapy, and biomarker discovery.

DEPICT-seq: Single-Cell Transcriptomic Analysis of Rare Cell Subsets Isolated via Nucleic Acid Cytometry.

The ability to dive deep into specific rare cell populations is critical for understanding tissue physiology and pathology across various biological domains. As single-cell RNA-seq flourishes, many newly discovered cell subtypes are defined by their transcriptomic markers. However, our ability to retrieve and analyze cells based on their nucleic acid markers remains underdeveloped. Here, we present Double Emulsion PCR-Initiated Cell sorting and Transcriptomic Sequencing (DEPICT-seq), a high-throughput droplet nucleic acid cytometry method that integrates batch cell fixation for cellular information preservation, double emulsion digital PCR-based cell sorting to target nucleic acid markers of interest, and in-depth full-length transcriptomic analyses at single-cell resolution. We utilize DEPICT-seq to isolate and characterize T cell receptor (TCR)-engineered T cells within a mixed population and also demonstrate a variation of the workflow by incorporating an RNase H-dependent PCR step to enrich full-length TCR sequences for paired single-cell TCR sequencing and transcriptomic profiling.

Emetine induces oxidative stress, cell differentiation and NF-κB inhibition, suppressing AML stem/progenitor cells

Acute myeloid leukemia (AML) is a fatal malignancy of the blood and bone marrow. Leukemic stem cells (LSCs) are a rare subset of leukemic cells that promote the development and progression of AML, and eradication of LSCs is critical for effective control of this disease. Emetine is an FDA-approved antiparasitic drug with antitumor properties; however, little is known about its potential against LSCs. Herein, we explored the antileukemic potential of emetine, focusing on its effects on AML stem/progenitor cells. Emetine exhibited potent cytotoxic activity both in hematologic and solid cancer cells and induced AML cell differentiation. Emetine also inhibited AML stem/progenitor cells, as evidenced by decreased expression of CD34, CD97, CD99, and CD123 in KG-1a cells, indicating anti-AML stem/progenitor cell activities. The administration of emetine at a dosage of 10 mg/kg for two weeks showed no significant toxicity and significantly reduced xenograft leukemic growth in vivo. NF-κB activation was reduced in emetine-treated KG-1a cells, as shown by reduced phospho-NF-κB p65 (S529) and nuclear NF-κB p65. DNA fragmentation, YO-PRO-1 staining, mitochondrial depolarization and increased levels of active caspase-3 and cleaved PARP (Asp214) were detected in emetine-treated KG-1a cells. Moreover, treatment with the pancaspase inhibitor Z-VAD(OMe)-FMK partially prevented the apoptotic cell death induced by emetine. Emetine treatment also increased cellular and mitochondrial reactive oxygen species, and emetine-induced apoptosis in KG-1a cells was partially prevented by the antioxidant N-acetylcysteine, indicating that emetine induces apoptosis, at least in part, by inducing oxidative stress. Overall, these studies indicate that emetine is a novel potential anti-AML agent with promising activity against stem/progenitor cells, encouraging the development of further studies aimed at its clinical application.

SuperCellCyto: enabling efficient analysis of large scale cytometry datasets

Advancements in cytometry technologies have enabled quantification of up to 50 proteins across millions of cells at single cell resolution. Analysis of cytometry data routinely involves tasks such as data integration, clustering, and dimensionality reduction. While numerous tools exist, many require extensive run times when processing large cytometry data containing millions of cells. Existing solutions, such as random subsampling, are inadequate as they risk excluding rare cell subsets. To address this, we propose SuperCellCyto, an R package that builds on the SuperCell tool which groups highly similar cells into supercells. SuperCellCyto is available on GitHub (https://github.com/phipsonlab/SuperCellCyto) and Zenodo (https://doi.org/10.5281/zenodo.10521294).

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 & 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.

Transplanted human photoreceptors transfer cytoplasmic material but not to the recipient mouse retina

BackgroundThe discovery of material transfer between transplanted and host mouse photoreceptors has expanded the possibilities for utilizing transplanted photoreceptors as potential vehicles for delivering therapeutic cargo. However, previous research has not directly explored the capacity for human photoreceptors to engage in material transfer, as human photoreceptor transplantation has primarily been investigated in rodent models of late-stage retinal disease, which lack host photoreceptors.MethodsIn this study, we transplanted human stem-cell derived photoreceptors purified from human retinal organoids at different ontological ages (weeks 10, 14, or 20) into mouse models with intact photoreceptors and assessed transfer of human proteins and organelles to mouse photoreceptors.ResultsUnexpectedly, regardless of donor age or mouse recipient background, human photoreceptors did not transfer material in the mouse retina, though a rare subset of donor cells (< 5%) integrated into the mouse photoreceptor cell layer. To investigate the possibility that a species barrier impeded transfer, we used a flow cytometric assay to examine material transfer in vitro. Interestingly, dissociated human photoreceptors transferred fluorescent protein with each other in vitro, yet no transfer was detected in co-cultures of human and mouse photoreceptors, suggesting that material transfer is species specific.ConclusionsWhile xenograft models are not a tractable system to study material transfer of human photoreceptors, these findings demonstrate that human retinal organoid-derived photoreceptors are competent donors for material transfer and thus may be useful to treat retinal degenerative disease.

Practical 10-Color T-Cell Panel for Phenotyping Diverse Populations Using Spectral Flow Cytometry: A Beginner's Guide.

Flow cytometry stands as the most employed high-throughput single-cell analysis technique, facilitating the profiling of remarkably diverse samples, such as blood, bone marrow and body fluids. In addition, it allows for the discrimination of diverse immune cell subsets, including infrequently encountered types like T regulatory cells and exhausted CD28Null T cells. However, analyzing rare immune cell subsets with conventional flow cytometry poses challenges stemming from factors like fluorophore overlap, compensation issues, and limited flexibility in fluorophore selection. Therefore, spectral flow cytometry offers advantages over traditional flow cytometry. It measures the full emission spectrum and then separates it to identify different fluorochromes. This enables the use of fluorochromes with significant overlap in a single test, allowing for the analysis of more protein markers. Following this, spectral technology employs precise calculations to separate individual fluorochromes, thereby enabling the detection and elimination of autofluorescent signals originating from cells within the entire emission spectrum. This capability is pivotal in achieving deep phenotyping of immune cells with the requisite sensitivity and resolution essential for monitoring the immune systems of patients with compromised immunity, such as cancer and autoimmune disorders. Additionally, it allows for the exploration of interactions between distinct immune subsets. In this context, we introduce an optimized protocol utilizing spectral flow cytometry for precise T-cell characterization and differentiation, encompassing the assessment of their activation states. Furthermore, this protocol extends its applicability to the identification of less common circulating T-cell populations, notably T-regulatory and CD28Null T cells, following autofluorescence correction within the spectrum. This protocol provides a set of steps and reagents for the surface and intracellular staining of human T cells using whole peripheral blood. The spectral-based design of this panel allows for its applicability to other spectral machines, providing a versatile and efficient tool for T-cell analysis. © 2024 Wiley Periodicals LLC. Basic Protocol 1: Achieving optimal staining through effective antibody titration Basic Protocol 2: Single-cell staining Basic Protocol 3: Comprehensive panel staining post-titration and spectral library integration.

Distribution-Agnostic Deep Learning Enables Accurate Single-Cell Data Recovery and Transcriptional Regulation Interpretation.

Single-cell RNA sequencing (scRNA-seq) is a robust method for studying gene expression at the single-cell level, but accurately quantifying genetic material is often hindered by limited mRNA capture, resulting in many missing expression values. Existing imputation methods rely on strict data assumptions, limiting their broader application, and lack reliable supervision, leading to biased signal recovery. To address these challenges, authors developed Bis, a distribution-agnostic deep learning model for accurately recovering missing sing-cell gene expression from multiple platforms. Bis is an optimal transport-based autoencoder model that can capture the intricate distribution of scRNA-seq data while addressing the characteristic sparsity by regularizing the cellular embedding space. Additionally, they propose a module using bulk RNA-seq data to guide reconstruction and ensure expression consistency. Experimental results show Bis outperforms other models across simulated and real datasets, showcasing superiority in various downstream analyses including batch effect removal, clustering, differential expression analysis, and trajectory inference. Moreover, Bis successfully restores gene expression levels in rare cell subsets in a tumor-matched peripheral blood dataset, revealing developmental characteristics of cytokine-induced natural killer cells within a head and neck squamous cell carcinoma microenvironment.

CyTOF Intracellular Cytokine Assays for Antigen-Specific T Cells.

T cells specific for a single antigen tend to be rare, even after expansion of memory cells. They are commonly detected by in vitro stimulation with peptides or protein, followed by staining for intracellular cytokines. In this protocol, CyTOF® mass cytometry is used to collect single-cell data on a large number of cytokines/chemokines, as well as cell-surface proteins that characterize T cells and other immune cells. A method for magnetic bead enrichment of antigen-stimulated T cells, based on their expression of CD154 and CD69, is also included. Coupling magnetic enrichment with highly multiparameter mass cytometry, this method enables the ability to dissect the frequency, phenotype, and function of antigen-specific T cells in greater detail than previously possible. Rare cell subsets can be examined, while minimizing run times on the CyTOF.

Platinum-Chimeric Carrier Cells for Ultratrace Cell Analysis in Mass Cytometry.

Mass cytometry by time-of-flight (CyTOF), a high-dimensional single-cell analysis platform, detects up to 50 biomarkers at single-cell resolution. However, CyTOF analysis of biological samples with a minimal number of available cells or rare cell subsets remains a major technical challenge due to the extensive loss of cells during cell recovery, staining, and acquisition. Here, we introduce a platinum-chimeric carrier cell strategy for mass cytometry profiling of ultratrace cell samples. Cisplatin can rapidly enter broken plasma membranes of dead cells and form a chimeric interaction with cellular proteins, peptides, and amino acids. Thus, 198Pt-cisplatin is adopted to tag carrier cells in the pretreatment stage. We investigated 8 cell lines that are commonly accessible in laboratories for their potential as carrier cells to preserve rare target cells for CyTOF analysis. We designed a panel of 35 protein biomarkers to evaluate the comprehensive single-cell subtype classification capability with or without the carrier cell strategy. We further demonstrated the detection and analysis of as few as 1 × 104 immune cells using our method. The proposed method thus allows CyTOF analysis on precious clinical samples with less abundant cells.

TLR4 sensitizes plasmacytoid dendritic cells for antiviral response against SARS-CoV-2 coronavirus.

Plasmacytoid dendritic cells are a rare subset of dendritic cells that exhibit antiviral functions in response to toll-like receptor 7/8 stimulations. Alternative toll-like receptors such as TLR4 have been known to be active in plasmacytoid dendritic cells for immune regulatory functions. However, it is unclear whether these toll-like receptors differentially activate plasmacytoid dendritic cells as compared with canonical toll-like receptor 7/8 stimulation. Here, we assessed alternative plasmacytoid dendritic cell activation states mediated by toll-like receptors other than endosomal toll-like receptors via the RNA sequencing approach. We found that toll-like receptor 4 stimulation induced a high degree of similarity in gene expression pattern to toll-like receptor 7/8 stimulation in plasmacytoid dendritic cells. Despite high resemblance to toll-like receptor 7/8, we discovered unique genes that were activated under toll-like receptor 4 activation only, as well as genes that were induced at a higher magnitude in comparison to toll-like receptor 7/8 activation. In comparison between toll-like receptor 4-activated plasmacytoid dendritic cells and conventional dendritic cells, we revealed that plasmacytoid dendritic cells and conventional dendritic cells expressed distinct gene sets, whereby conventional dendritic cells mostly favored antigen presentation functions for adaptive immune response regulation while plasmacytoid dendritic cells leaned toward immune response against infectious diseases. Last, we determined that toll-like receptor 4 activation sensitized plasmacytoid dendritic cells against SARS-CoV-2 (COVID-19) single-stranded RNA by enhancing antiviral-related responses and type I interferon production. These findings provided greater insights into the toll-like receptor 4 activation state in plasmacytoid dendritic cells, which can be beneficial for alternative therapeutic interventions involving plasmacytoid dendritic cells for various diseases.

MiR-30e-5p regulates leukemia stem cell self-renewal through the Cyb561/ROS signaling pathway.

Leukemia stem cells (LSC) represent a crucial and rare subset of cells present in acute myeloid leukemia (AML); they play a pivotal role in the initiation, maintenance, and relapse of this disease. Targeting LSC holds great promise for preventing AML relapse and improving long-term outcomes. However the precise molecular mechanisms governing LSC self-renewal are still poorly understood. Here, we present compelling evidence that the expression of miR-30e-5p, a potential tumor-suppressive microRNA, is significantly lower in AML samples than in healthy bone marrow samples. Forced expression of miR- 30e effectively inhibits leukemogenesis, impairs LSC self-renewal, and delays leukemia progression. Mechanistically, Cyb561 acts as a direct target of miR-30e-5p in LSC, and its deficiency restricts the self-renewal of LSC by activating reactive oxygen series signaling and markedly prolongs recipients' survival. Moreover, genetic or pharmacological overexpression of miR-30e-5p or knockdown of Cyb561 suppresses the growth of human AML cells. In conclusion, our findings establish the crucial role of the miR-30e-5p/Cyb561/ROS axis in finely regulating LSC self-renewal, highlighting Cyb561 as a potential therapeutic target for LSC-directed therapies.

Role of POU1F1 promoting the properties of stemness of gastric carcinoma through ENO1-mediated glycolysis reprogramming.

Cancer stem cells (CSCs), a rare subset of tumor cells, have been recognized as promotive role on tumor initiation and propagation. Among, aerobic glycolysis, widely clarified in multiple tumor cells, is the key for maintaining cancer stemness. Regrettably, it is largely unknown about the connection of cellular metabolic reprogramming and stemness in gastric carcinoma (GC). Two GC parental cells lines PAMC-82 and SNU-16 and their spheroids were obtained to determine the expression status of POU1F1 using quantitative real-time PCR (qRT-PCR) and western blotting analysis, respectively. Gain or loss-of-function assay was employed to assess its biological effects. Sphere formation and transwell assays were performed to evaluate the stem cell-like traits, including self-renewal capacity, migration and invasion. Chromatin immunoprecipitation (ChIP) and luciferase reporter assays were conducted for determining the binding relationship of POU1F1 on ENO1 promoter region. Herein, aberrantly upregulated POU1F1 was observed in spheroids, compared with the parental PAMC-82 and SNU-16 cells, which promoted stem cell-like traits, as representing increasing sphere formation, enhanced cell migration and invasion. Additionally, POU1F1 expression was positively with glycolytic signaling, as displaying increasing glucose consumption, lactic acid production, and extracellular acid ratio (ECAR). Furthermore, POU1F1 was identified to be a transcriptional activator of ENO1, of which overexpression remarkably abolished POU1F1 knockdown-mediated blocking effects. Taken together, we draw a conclusion that POU1F1 facilitated the stem cell-like properties of GC cells through transcriptionally augmenting ENO1 to enhance glycolysis.

Cells in the polyaneuploid cancer cell (PACC) state have increased metastatic potential

Although metastasis is the leading cause of cancer deaths, it is quite rare at the cellular level. Only a rare subset of cancer cells (~ 1 in 1.5 billion) can complete the entire metastatic cascade: invasion, intravasation, survival in the circulation, extravasation, and colonization (i.e. are metastasis competent). We propose that cells engaging a Polyaneuploid Cancer Cell (PACC) phenotype are metastasis competent. Cells in the PACC state are enlarged, endocycling (i.e. non-dividing) cells with increased genomic content that form in response to stress. Single-cell tracking using time lapse microscopy reveals that PACC state cells have increased motility. Additionally, cells in the PACC state exhibit increased capacity for environment-sensing and directional migration in chemotactic environments, predicting successful invasion. Magnetic Twisting Cytometry and Atomic Force Microscopy reveal that cells in the PACC state display hyper-elastic properties like increased peripheral deformability and maintained peri-nuclear cortical integrity that predict successful intravasation and extravasation. Furthermore, four orthogonal methods reveal that cells in the PACC state have increased expression of vimentin, a hyper-elastic biomolecule known to modulate biomechanical properties and induce mesenchymal-like motility. Taken together, these data indicate that cells in the PACC state have increased metastatic potential and are worthy of further in vivo analysis.

A Rare Subset of Primary Tumor Cells with Concomitant Hyperactivation of Extracellular Matrix Remodeling and dsRNA-IFN1 Signaling Metastasizes in Breast Cancer.

Transcriptional lineage tracing coupled with single-cell transcriptomics defined the transcriptional programs underlying metastatic progression in breast cancer, identifying prognostic signatures and prevention strategies.

Identifiation of novel effector regulatory T cells through simulaneous single cell epigenetic and transcriptomic profiling

Abstract Regulatory T cells (Tregs) harness a central role in peripheral immune tolerance. Since its discovery, there have been numerous efforts to dissect the role of Tregs in the context of disease condition. Recently, Treg fields are moving from the academia to industry since many evidences have been accumulated for the potential of treatment of inflammatory disease by administering genetically engineered Tregs. However, in the pre-clinical or clinical trials, this rare cell subset needs to be expanded in ex-vivo condition to achieve a sufficient number for the controlling of the disease. Our group previously established ex-vivo Treg expansion condition with 25 mer oligonucleotide to maintain the stability and function of Tregs. However, non-biased investigations on the perturbation of transcriptomic and epigenetic changes during the culture condition has not been explored. Since cutting edge multiomics tools which can simultaneously profile epigenetic and transcript signatures in a single cell resolution has been commercialized, we adopt 10x multiome technique to investigate our frozen ex-vivo expanded Tregs(eTreg) compared to freshly isolated frozen human Treg(fTreg) from the same donor. Compared to fTreg cells, we have identified a new subset that exists only in the eTreg cells and named it eeTreg. We found that the eeTreg resembles the phenotype of both effector Tregs and tumor-infiltrated Tregs. eeTreg is characterized by a significantly increased expression and chromatin accessibility score of FOXP3, STAT5B, ETS1 and RUNX1, which are known to be the essential transcription factors of regulatory T cell function. In summary, we suggest a novel human treg subset which might be manufactured by manipulating ex-vivo culture condition.

Abstract 4550: Assessing efficacy and immune-stimulatory effects of tumor-derived dendritic cell reprogramming using immuno-competent 3D tumor spheroid model

Abstract Immunotherapy has brought hope for cancer treatment, but its clinical success remains limited. Recently, overexpression of the transcription factors PU.1, IRF8 and BATF3 (PIB) was shown to induce direct reprogramming of tumor cells into antigen-presenting type 1 conventional dendritic cells (cDC1s), a rare subset of immune cells with pivotal role in anti-cancer immunity. This strategy might open avenues to enhance cancer cell recognition and elimination by the immune system. However, currently existing in-vitro and in-vivo testing platforms do not qualify to reproduce all complex cell interactions essential for the approbation of this hypothesis. Here, we report the development of the InSphero 3D InSight™ Oncology Platform for in-vitro assessment of efficacy and immune-stimulatory effects of this novel cancer immunotherapy approach. The feasibility of 3D spheroid formation for several GFP-expressing tumor cell lines was evaluated by varying seeding conditions in AKURA 96 well plate. We have measured the growth (ATP content) and GFP signal overtime and analyzed the morphology of the spheroids by IHC. With this, we have established spheroid models of T98G (glioblastoma), PK59 (pancreatic cancer), and A375 (melanoma) cell lines that are growing and viable for at least 10 days. In parallel, using 2D cultures, we have identified the optimal multiplicity of infection of a lentiviral vector encoding for PIB and mCherry to enable high transduction (mCherry+ cells), reprogramming efficiency (mCherry+CD45+HLA-DR+ cells), and cell viability, quantified by flow cytometry and IHC. Then, we have demonstrated that cDC1 reprogramming progresses in the context of 3D cancer spheroids and tumor cells acquire expression of CD45+ and HLA-DR+ cells using IHC and confocal microscopy analysis. We developed an algorithm enabling automated analysis of confocal images and quantification of cDC1 reprogramming efficiency from individual image stacks calculated as a ratio of mCherry+, CD45+ and HLA-DR+ cells versus the number of DAPI+ nuclei. Using the new algorithm we have evaluated the reprogramming efficacy of the different virus dosages in all three types of 3D tumor spheroids. Lastly, we have cocultured tumor spheroids transduced with PIB with naïve or activated HLA-matched PBMCs and evaluated cytokine secretion as a readout of immune cell activation. We observed that reprogramming induces activation of T cells and correlated it to the number of reprogrammed cells in the tumor spheroid, evaluated by the HC imaging. In summary, we developed the InSphero 3D InSight™ Oncology Platform that allowed us to demonstrate the effects of direct reprogramming of tumor cells into immunogenic dendritic cells. Combined with high-content imaging analysis, this platform offers a powerful solution for preclinical translational research. Citation Format: Irina Agarkova, Nadezhda Rotankova, Michal Rudnik, Silvan Strebel, Olga Zimmermannova, Andre Rosa, Fabio F. Rosa, Wolfgang Moritz, Carlos-Filipe Pereira, Christiana F. Pires. Assessing efficacy and immune-stimulatory effects of tumor-derived dendritic cell reprogramming using immuno-competent 3D tumor spheroid model. [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 4550.

Abstract 4420: Carbohydrate-CD11b engagement enhances differentiation of tumor-associated myeloid cells in immunotherapy of solid cancers

Abstract Background: Methods to perturb tumor-associated myeloid cell function are being actively pursued to overcome these challenges in immunotherapy and search for a cure. CD11b is a potential therapeutic target to modulate suppressive myeloid derived cells and induce tumor-reactive T-cell responses. However, CD11b can bind to multiple carbohydrate ligands that trigger a variety of myeloid cell functions like adhesion, migration, and even phagocytosis and proliferation. This has created a major challenge to understand the molecular basis by which CD11b converts differences in receptor-ligand binding into subsequent immune signaling responses, and to use this knowledge for therapeutic development. Methods: This study investigated a previously reported carbohydrate ligand, BG34-200, that showed anti-tumor effect through modulating CD11b+ cells. Applying modern approaches of peptide microarray, multi-parameter FACS analysis, cellular/molecular immunological technology, advanced microscopic imaging, and transgenic mouse models of solid cancers, we studied the BG34-200-CD11b carbohydrate-protein engagement and immunological changes it triggered in the context of solid cancers including osteosarcoma, melanoma and pancreatic cancer. Results: Our results show that BG34-200 can mediate direct, specific, multi-site and multivalent binding to integrin CD11b to trigger cellular differentiation in a suppressive tumor-associated myeloid cell subset. In melanoma, osteosarcoma, and pancreatic cancer, we detected this rare myeloid cell subset in blood, which displayed an inflammatory monocyte phenotype and exhibited immunosuppressive behavior. BG34-200-CD11b engagement could induce differentiation of these tumor-associated inflammatory monocytes into dendritic cells by inducing phagocytosis that effectively trigger F-actin cytoskeletal rearrangement and intrinsic ICAM-1 clustering important for antigen presentation and effector T cell stimulation. Conclusions: Our findings improve understanding of molecular basis by which CD11b converts difference in carbohydrate ligands into subsequent immune signaling responses, may lead to development of safe and novel therapies modulating myeloid derived cell function for immunotherapy of solid cancers. Citation Format: Mei Zhang. Carbohydrate-CD11b engagement enhances differentiation of tumor-associated myeloid cells in immunotherapy of solid cancers. [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 4420.

High-throughput spatiotemporal monitoring of single-cell secretions via plasmonic microwell arrays

Methods for the analysis of cell secretions at the single-cell level only provide semiquantitative endpoint readouts. Here we describe a microwell array for the real-time spatiotemporal monitoring of extracellular secretions from hundreds of single cells in parallel. The microwell array incorporates a gold substrate with arrays of nanometric holes functionalized with receptors for a specific analyte, and is illuminated with light spectrally overlapping with the device’s spectrum of extraordinary optical transmission. Spectral shifts in surface plasmon resonance resulting from analyte–receptor bindings around a secreting cell are recorded by a camera as variations in the intensity of the transmitted light while machine-learning-assisted cell tracking eliminates the influence of cell movements. We used the microwell array to characterize the antibody-secretion profiles of hybridoma cells and of a rare subset of antibody-secreting cells sorted from human donor peripheral blood mononuclear cells. High-throughput measurements of spatiotemporal secretory profiles at the single-cell level will aid the study of the physiological mechanisms governing protein secretion.

A single-cell atlas identifies pretreatment features of primary imatinib resistance in chronic myeloid leukemia

AbstractPrimary resistance to tyrosine kinase inhibitors (TKIs) is a significant barrier to optimal outcomes in chronic myeloid leukemia (CML), but factors contributing to response heterogeneity remain unclear. Using single-cell RNA (scRNA) sequencing, we identified 8 statistically significant features in pretreatment bone marrow, which correlated with either sensitivity (major molecular response or MMR) or extreme resistance to imatinib (eventual blast crisis [BC] transformation). Employing machine-learning, we identified leukemic stem cell (LSC) and natural killer (NK) cell gene expression profiles predicting imatinib response with >80% accuracy, including no false positives for predicting BC. A canonical erythroid-specifying (TAL1/KLF1/GATA1) regulon was a hallmark of LSCs from patients with MMR and was associated with erythroid progenitor [ERP] expansion in vivo (P < .05), and a 2- to 10-fold (6.3-fold in group A vs 1.09-fold in group C) erythroid over myeloid bias in vitro. Notably, ERPs demonstrated exquisite TKI sensitivity compared with myeloid progenitors (P < .001). These LSC features were lost with progressive resistance, and MYC- and IRF1-driven inflammatory regulons were evident in patients who progressed to transformation. Patients with MMR also exhibited a 56-fold expansion (P < .01) of a normally rare subset of hyperfunctional adaptive-like NK cells, which diminished with progressive resistance, whereas patients destined for BC accumulated inhibitory NKG2A+ NK cells favoring NK cell tolerance. Finally, we developed antibody panels to validate our scRNA-seq findings. These panels may be useful for prospective studies of primary resistance, and in assessing the contribution of predetermined vs acquired factors in TKI response heterogeneity.