Cell Surface Protein Expression Research Articles

MM-253: Single Cell Multi-Omic Analysis And Immune Cell Type Profiling Of Multiple Myeloma With t(4;14)

Context Multiple myeloma (MM) is an incurable plasma cell (PC) malignancy and high risk (HR) MM remains an unmet clinical need. Translocation 4;14 occurs in 15% of MM and is associated with an adverse prognosis. A deeper understanding of the biology and immune micro-environment of t(4;14) MM is necessary for the development of effective targeted therapies. Objective Here we utilized Proteona’s ESCAPE™ single cell multi-omics platform to study a cohort of patients with t(4;14) MM with the goal of better understanding the biology underlying this high risk patient cohort. Design Diagnostic bone marrow (BM) samples from 13 patients with t(4;14) MM were analysed using the ESCAPE platform from Proteona which simultaneously measures gene and cell surface protein expression in single cells. Resulting data were analyzed with MapSuite tools from Proteona and Seurat. Results The patients had a median age of 63 years. All received novel agent based induction. Median progression free and overall survival (PFS and OS) were 22 and 34 months respectively. MMSET was overexpressed in all PCs while FGFR3 expression could be categorized into low expression ( 80% of cells expressing FGFR3). Variation in the immune microenvironment of the BM was seen across all patient samples with no correlation between cell types present in the BM and PFS or OS. Plasma cells from the combined samples formed clusters that could be differentiated by gene expression with each cluster having a unique combination of cells of varying PFS. Gene and protein expression differences were seen in PCs when samples were clustered by PFS suggesting a role for these molecules in disease progression. Finally, we identified gene expression changes in one patient between a diagnostic sample and relapse disease. Conclusions We present the first application of single cell multi-omics immune profiling in high-risk MM. Our results suggest that t(4;14) MM is a molecularly heterogeneous disease. That heterogeneity, coupled with our small sample size may explain the lack of correlation between gene or protein expression with clinical outcomes. Single cell analysis of larger cohorts is required to build on our findings.

Improved integration of single-cell transcriptome and surface protein expression by LinQ-View

SummaryMultimodal advances in single-cell sequencing have enabled the simultaneous quantification of cell surface protein expression alongside unbiased transcriptional profiling. Here, we present LinQ-View, a toolkit designed for multimodal single-cell data visualization and analysis. LinQ-View integrates transcriptional and cell surface protein expression profiling data to reveal more accurate cell heterogeneity and proposes a quantitative metric for cluster purity assessment. Through comparison with existing multimodal methods on multiple public CITE-seq datasets, we demonstrate that LinQ-View efficiently generates accurate cell clusters, especially in CITE-seq data with routine numbers of surface protein features, by preventing variations in a single surface protein feature from affecting results. Finally, we utilized this method to integrate single-cell transcriptional and protein expression data from SARS-CoV-2-infected patients, revealing antigen-specific B cell subsets after infection. Our results suggest LinQ-View could be helpful for multimodal analysis and purity assessment of CITE-seq datasets that target specific cell populations (e.g., B cells).

Abstract 129: An integrated multi-omic cellular atlas of human breast cancers

Abstract Breast cancers are complex cellular ecosystems where heterotypic interactions play central roles in disease progression and response to therapy. However, our knowledge of the cellular composition and organization of breast cancer remains limited. We present a comprehensive single cell and spatially resolved transcriptomic atlas of human breast cancers. The 10X Genomics Chromium platform was used to generate single cell transcriptomic data (scRNA-Seq) from more than 120,000 cells sampled from 26 breast cancers. CITE-Seq was employed to simultaneously generate protein measurements using a panel of 157 antibodies against immune, stromal and epithelial cell surface markers and analysed using Seurat. Using single cell signatures, we estimated the cellular composition of more than 2000 breast cancers in the Metabric cohort using deconvolution methods. Spatial transcriptomics was conducted on 12 frozen tissues (Luminal, Her2+ and triple negative breast cancer (TNBC)) using the 10X genomics Visium solution. We also used a novel Spatial Whole Transcriptome Panel, targeting 18,000+ genes on the Nanostring GeoMX platform, to profile T cells and malignant cells across multiple tissue niches from 16 TNBC FFPE cases. Integrative scRNA-Seq analysis identifies recurrent gene modules driving neoplastic cell heterogeneity, including interferon signaling, estrogen receptor function and mutually exclusive patterns of proliferation versus EMT. We also develop a single cell classifier of intrinsic subtype (scSubtype) to reveal frequent intra-tumoral heterogeneity for breast cancer intrinsic subtypes. CITE-Seq revealed immune profiles at high resolution, leading to the identification of novel macrophage populations with high expression of PD-L1 and PD-L2 immune checkpoint ligands and associations with clinical outcome. We also observe enrichment of exhausted and proliferative CD8 T cells in TNBC, with unique patterns of cell-surface checkpoint protein expression when compared to other subtypes. Targeted analysis using the GeoMX revealed spatial segregation of T cell phenotypes, with exhausted and proliferative CD8 T cells forming small clusters adjacent to tumor cells with high interferon pathway activity. Analysis of scRNA-Seq data revealed that stromal cells generate diverse functions and cell surface protein expression through differentiation within 3 major lineages: fibroblast, endothelial and perivascular-like. Subsets of stromal cells had features associated with immune regulation and Visium data revealed that stromal-immune niches were spatially organized in tumors, offering insights into anti-tumor immune suppression by stromal cells. Finally, deconvolution stratified >2000 breast cancer cases in Metabric into nine clusters, termed ‘ecotypes', with distinct cellular compositions and clinical outcomes. This study provides a comprehensive atlas of the cellular architecture of breast cancer. Citation Format: Sunny Z. Wu, Daniel Roden, Ghamdan Al Eryani, Simon Junankar, Elgene Lim, Aatish Thennavan, Alma Andersson, Stephen Williams, Jingjing Gong, Robin Fropf, Kit Fuhrman, Joakim Lundeberg, Chuck Perou, Alexander Swarbrick. An integrated multi-omic cellular atlas of human breast cancers [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 129.

Abstract 1609: Peripheral NK-cell immunophenotypes predict progression in nasopharyngeal carcinoma patients

Abstract Objective: The tumorigenesis is closely associated with the alteration in the expression of cell-surface proteins. As NK cells play a vital role in cancer immunotherapy, we thus conducted this study to characterize the immunophenotypes of circulating NK cells and to explore the association between NK phenotypes and disease prognosis. Methods: 249 nasopharyngeal carcinoma (NPC) patients and 11 healthy individuals were recruited in the study, whose peripheral blood samples were collected before treatment. Multi-color flow cytometry analysis was performed. The expression of four inhibitory receptors, PD-1, TIGIT, LAG-3, TIM-3, and four stimulatory receptors, NKp46, NKp30, CD226, NKG2D, were measured in three NK subsets, identified by the co-expression of CD56 and CD16 when gated on CD3-. The LASSO (least absolute shrinkage and selection operator) logistic regression model was used to screen out potential prognostic NK subtypes and calculate the NK-function index. Results: NPC patients exhibited distinct peripheral NK-cell distribution and altered expression of key receptors, as characterized by increased expression of PD-1 and TIGIT on CD56+CD16- cells and decreased expression of NKp46 on CD56+CD16- and CD56-CD16- cells. Four markers were selected from the LASSO model to calculate the NK-function index: PD1 and NKp46 on CD56-CD16+ NK cells, NKp46 on CD56+CD16- cells and NKG2D on CD56+CD16+ cells. Patients with low NK index had significantly shorter progression-free survival (PFS) and distant metastasis-free survival (DMFS), and the NK-function index was an independent prognostic factor for NPC patients. Conclusions: The immunophenotype of peripheral NK cells and its association with survival outcomes of NPC patients revealed its potential as a strong prognosticator for PFS and DMFS, and highlighted the important role of circulating immune environment, especially for the application of immunotherapy. Citation Format: Xiao-Yun Li, Zi-Jian Lu, Hai-Qiang Mai. Peripheral NK-cell immunophenotypes predict progression in nasopharyngeal carcinoma patients [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1609.

Personalized Therapy Design for Early T-Cell Precursor Acute Lymphoblastic Leukemia (ETP-ALL) Using Computational Biology Modeling with in Vitro Validation

ETP-ALL is a high-risk subtype of T-ALL with poor prognosis and high risk of relapse in patients treated with standard of care (SOC). ETP-ALL patients have more complex cytogenetic profiles and a unique immunophenotype compared to other T-ALL cases. ETP-ALL is characterized by the expression of myeloid and hematopoietic stem cell (HSC) surface proteins, as well as gene expression profiles similar to myeloid progenitors and HSCs, including frequent mutations in myeloid oncogenes. Targeted therapies for ETP-ALL are limited because identified gene mutations are not actionable by currently available drugs. Recently we developed a predictive simulation modeling method that projects cancer patient-specific protein network maps based on the patient's unique cancer genomics (PMID 27855285). Here, we present a computational biology approach to analyze the genomic profile of ETP-ALL and identify novel treatment strategies using available FDA approved drugs. In this study, we used the LOUCY cell line as a model of ETP-ALL due to its high expression of characteristic ETP-ALL markers HHEX, LYL1, MEF2C and ZEB2.Aim: To identify and validate novel drug or drug combinations for ETP-ALL using an integrated genomics and computational biology modeling (CBM) approach.Methods: The LOUCY cell line was used as a model of ETP-ALL. Publicly available genomic profiling data (cBioPortal, Sanger) were collected. All available genomic data for the LOUCY cell line were entered into a CBM program (Cellworks Group) that generates a disease-specific protein network map using PubMed and other online resources. Disease-specific biomarkers unique to the LOUCY cell line were identified within the protein network maps. A digital drug library of FDA approved therapies and drugs in clinical development was developed and digitally simulated in the computational model at varying doses. Drug impact was quantitatively assessed by a cell growth score, which is a composite of cell proliferation, viability, apoptosis, along with the impact on disease-specific biomarkers. This CBM's predictions for drug response were compared with publicly available in vitro experimental data. Novel drug options and combinations to treat ETP-ALL were also identified by CBM. The novel drugs and drug combinations identified by CBM were then tested on the LOUCY cells at varying concentrations to calculate IC50 values. Drug activity was measured by XTT assay, using the Jurkat cell line as a non-ETP-ALL control and the KG-1 AML cell line as a non-ALL control.Results: CBM predicted extensive dysregulation of multiple protein signaling pathways in the LOUCY cell line: Cell cycle upregulation was predicted due to copy number loss of CDKN1B, CDKN2B and CDKN2A genes. BCL2 expression was predicted to be increased due to the presence of a NUP214 - SET fusion protein, activating the BCL2 transcription factor HOXA9. Knockdown of PTEN and TSC2 was predicted to activate the AKT-mTOR pathway, while knockdown of ETV6 and SOCS1 was predicted to activate the JAK-STAT pathway (Fig 1). The response predictions of 31 drugs were compared with data from in vitro experiments. LOUCY cells were predicted to respond to 9 of the drugs, and in vitro testing showed sensitivity to 8 out of 9 drugs (88.8%). LOUCY cells were predicted to show no response to 22 of the drugs, and in vitro testing showed resistance to all 22 drugs (100%) (Table 1). Additionally, CBM predicted everolimus, nelfinavir, and palbociclib as novel therapies for the ETP-ALL representative LOUCY cell line, and were validated (100%) by additional in vitro XTT assays (Fig 2). Key statistical correlations for drug efficacy were observed (Table 2).Conclusions: Predictive simulation modeling of the unique genomic abnormalities found in the ETP-ALL LOUCY cell line led to the creation of an ETP-ALL protein network map that shows multiple interacting and cooperative protein network disturbances including cell cycle, anti-apoptosis, AKT-mTOR and JAK-STAT pathways. Digital drug screening using this distinctive protein network map led to the discovery of new drugs with predicted efficacy. In vitro studies validated the computational method with high accuracy. Results from this work inform future clinical trials for patients with ETP-ALL. This CBM technology may be a useful tool for clinicians to personalize treatment for patients with relapsed/refractory ETP-ALL. [Display omitted] DisclosuresKumar:Cellworks: Employment. K:Cellworks: Employment. Kamath:cellworks: Employment. Nair:cellworks: Employment. Raju:cellworks: Employment. Abbasi:Cellworks Group Inc.: Employment. Vali:Cellworks Group Inc.: Employment. Cogle:Celgene: Other: Membership on Steering Committee for Connect MDS/AML Registry.

Generation of Half-Life Extended Anti-BCMA Bite® Antibody Construct Compatible with Once-Weekly Dosing for Treatment of Multiple Myeloma (MM)

Multiple myeloma (MM) is a hematologic malignancy characterized by accumulation of abnormal plasma cells in the bone marrow. Although several treatment options are available, patients frequently develop relapsed/refractory disease leading to significant morbidity and mortality. More effective induction and elimination of residual disease are required for curative treatments. One potential strategy for improving patient outcomes is the use of T cell engaging agents such as BiTE® antibody constructs and CAR-T cells.The bispecific T cell engager (BiTE®) antibody construct blinatumomab, which targets CD19-positive cells, has demonstrated efficacy in treating certain CD19-positive hematological malignancies. Blinatumomab comprises a single chain variable fragment (scFv) that binds CD19 on B cells and a scFv that targets the T cell CD3 protein. The short serum half-life of blinatumomab necessitates administration by continuous IV infusion to maintain therapeutic serum concentrations. To enable once-weekly dosing, next generation BiTE® antibody constructs have been modified by fusion to an Fc domain to extend half-life.BCMA, a member of the TNF receptor super family, supports plasma cell survival, and is widely expressed on MM and normal plasma cells. A half-life extended (HLE) anti-BCMA BiTE® was generated and evaluated in vitro, in mouse xenograft models and in non-human primates (NHP). In vitro assays evaluated 1) binding to both human and NHP BCMA and CD3 proteins, and 2) cytotoxicity using human and NHP target and effector cells. The anti-BCMA BiTE® demonstrated picomolar binding affinity and efficacy in cytotoxicity assays. In a mouse xenograft model in which BCMA-positive NCI-H929 cells and human PBMC were co-administered SC, the anti-BCMA HLE BiTE® completely inhibited tumor formation in contrast to vehicle control-treated groups with or without PBMC. BCMA cell surface protein expression was assessed by flow cytometry and observed on NHP bone marrow plasma cells but not on blood or bone marrow B cells. The serum half-life in NHP was 112 hours following a single 15 µg/kg dose. The PK/PD relationship in NHP was subsequently evaluated in a repeat-dose study. To assess elimination of BCMA-positive plasma cells in blood and bone marrow, a sensitive and specific ddPCR-based method was developed. Robust depletion of BCMA mRNA was observed in NHP following anti-BCMA BiTE® treatment, indicating effective elimination of BCMA-positive plasma cells from blood and bone marrow. These results demonstrate that HLE anti-BCMA BiTE® is effective in vitro and in vivo, and may be suitable for once-weekly dosing in MM patients. DisclosuresGoyos:Amgen: Employment. Li:Amgen, Inc.: Employment. Deegen:Amgen: Employment. Bogner:Amgen: Employment. Thomas:Amgen Research (Munich): Employment. Matthias:Amgen: Employment. Wahl:Amgen Research (Munich): Employment. Goldstein:Amgen: Employment. Coxon:Amgen, Inc.: Employment. Balazs:Amgen Inc.: Employment, Equity Ownership. Chapman-Arvedson:Amgen, Inc.: Employment, Equity Ownership.

ALX148 Is a High Affinity Sirpα Fusion Protein That Blocks CD47, Enhances the Activity of Anti-Cancer Antibodies and Checkpoint Inhibitors, and Has a Favorable Safety Profile in Preclinical Models

CD47 is a widely expressed cell surface protein thought to function as an immune checkpoint in cancer. CD47 binds SIRPα on myeloid cells, suppressing tumor cell phagocytosis and other innate immune functions. CD47-SIRPα signaling has also been implicated in the suppression of adaptive antitumor responses, but the relevant cellular functions have yet to be elucidated. Abundant CD47 expression has been observed in tumors and may aid tumor cells in the evasion of immune surveillance.Therapeutic blockade of the CD47 pathway may stimulate antitumor immunity and improve cancer therapy. To this end, the novel CD47-blocking molecule ALX148 was generated by fusing a modified SIRPα N-terminal D1 domain to an inactive Fc. Surface plasmon resonance analysis demonstrated that ALX148 binds human and murine CD47 with high affinity, with respective KDs of 0.14 nM and 12 nM. ALX148 also bound multiple CD47-expressing cell populations in flow cytometry assays. ALX148 blocks the interaction between CD47 and wild type SIRPα, enhancing antibody-dependent phagocytosis of hematologic tumor cell lines by daratumumab, rituximab, and obinutuzumab.ALX148 increased the efficacy of antibodies targeting hematologic tumors, such as rituximab and obinutuzumab, in xenograft models of hematologic malignancies. Because ALX148 also binds murine CD47 with high affinity, syngeneic tumor models were used to examine the activity of ALX148 in the context of an intact immune system. ALX148 enhanced the antitumor activities of checkpoint inhibitors. Specifically, the immune response induced by ALX148 bridged innate and adaptive immune responses. ALX148 stimulated antitumor properties of innate immune cells, promoting dendritic cell activation and a shift of tumor associated macrophages toward a proinflammatory phenotype. ALX148 in combination with checkpoint inhibitors caused increased cytotoxic T cell infiltrates, with greater CD8 to Treg ratios, and greater amounts of interferon gamma production by T cells in both tumors and the spleen. These findings reveal multiple mechanisms by which adaptive immunity is stimulated by CD47 blockade.Notably, ALX148 had no adverse effect on CD47-expressing red blood cells and platelets. In vitro, ALX148 did not induce erythrocyte hemagglutination. In non-human primate toxicology studies, ALX148 multi-dose administration was well tolerated and did not affect levels of circulating red blood cells, white blood cells, or platelets. This is in contrast to other published CD47-blocking molecules, which have caused anemia and thrombocytopenia in preclinical and clinical studies. Thus, ALX148 may have minimal hematologic toxicity in humans.ALX148 enhances adaptive and innate immune responses, demonstrates enhanced preclinical anti-tumor activity in combination with multiple standard of care cancer therapies, and is well tolerated. It is currently being investigated in a first-in-patient, phase 1 dose escalation/expansion, multi-center study in patients with advanced solid tumors and lymphoma. (NCT03013218). DisclosuresKauder:Alexo Therapeutics: Employment. Kuo:Alexo Therapeutics: Employment. Chen:Alexo Therapeutics: Employment. Harrabi:Alexo Therapeutics: Employment. Rocha:Alexo Therapeutics: Employment. Doyle:Alexo Therapeutics: Employment. Bollini:Alexo Therapeutics: Employment. Han:Alexo Therapeutics: Employment. Sangalang:Novartis: Employment; Alexo Therapeutics: Employment. Sim:Alexo Therapeutics: Employment. Randolph:VenBio: Consultancy; Carrick Therapeutics: Membership on an entity's Board of Directors or advisory committees; Alexo Therapeutics: Employment. Pons:Alexo Therapeutics: Employment, Equity Ownership. Wan:Alexo Therapeutics: Employment; Pfizer: Equity Ownership.

Coupling of Cell Surface Biotinylation and SILAC-Based Quantitative Proteomics Identified Myoferlin as a Potential Therapeutic Target for Nasopharyngeal Carcinoma Metastasis.

Distant metastasis is a major cause of treatment failure in nasopharyngeal carcinoma (NPC) patients. Cell surface proteins represent attractive targets for cancer diagnosis or therapy. However, the cell surface proteins associated with NPC metastasis are poorly understood. To identify potential therapeutic targets for NPC metastasis, we isolated cell surface proteins from two isogenic NPC cell lines, 6-10B (low metastatic) and 5-8F (highly metastatic), through cell surface biotinylation. Stable isotope labeling by amino acids in cell culture (SILAC) based proteomics was applied to comprehensively characterize the cell surface proteins related with the metastatic phenotype. We identified 294 differentially expressed cell surface proteins, including the most upregulated protein myoferlin (MYOF), two receptor tyrosine kinases(RTKs) epidermal growth factor receptor (EGFR) and ephrin type-A receptor 2 (EPHA2) and several integrin family molecules. These differentially expressed proteins are enriched in multiple biological pathways such as the FAK-PI3K-mTOR pathway, focal adhesions, and integrin-mediated cell adhesion. The knockdown of MYOF effectively suppresses the proliferation, migration and invasion of NPC cells. Immunohistochemistry analysis also showed that MYOF is associated with NPC metastasis. We experimentally confirmed, for the first time, that MYOF can interact with EGFR and EPHA2. Moreover, MYOF knockdown could influence not only EGFR activity and its downstream epithelial–mesenchymal transition (EMT), but also EPHA2 ligand-independent activity. These findings suggest that MYOF might be an attractive potential therapeutic target that has double effects of simultaneously influencing EGFR and EPHA2 signaling pathway. In conclusion, this is the first study to profile the cell surface proteins associated with NPC metastasis and provide valuable resource for future researches.

Isolation of Circulating Tumour Cells in Patients With Glioblastoma Using Spiral Microfluidic Technology - A Pilot Study.

Glioblastoma (GBM) is the most common and aggressive type of tumour arising from the central nervous system. GBM remains an incurable disease despite advancement in therapies, with overall survival of approximately 15 months. Recent literature has highlighted that GBM releases tumoural content which crosses the blood-brain barrier (BBB) and is detected in patients’ blood, such as circulating tumour cells (CTCs). CTCs carry tumour information and have shown promise as prognostic and predictive biomarkers in different cancer types. Currently, there is limited data for the clinical utility of CTCs in GBM. Here, we report the use of spiral microfluidic technology to isolate CTCs from whole blood of newly diagnosed GBM patients before and after surgery, followed by characterization for GFAP, cell-surface vimentin protein expression and EGFR amplification. CTCs were found in 13 out of 20 patients (9/20 before surgery and 11/19 after surgery). Patients with CTC counts equal to 0 after surgery had a significantly longer recurrence-free survival (p=0.0370). This is the first investigation using the spiral microfluidics technology for the enrichment of CTCs from GBM patients and these results support the use of this technology to better understand the clinical value of CTCs in the management of GBM in future studies.

IMMU-16. TARGETING GLYPICAN 2 (GPC2) ON PEDIATRIC MALIGNANT BRAIN TUMORS WITH MRNA CAR T CELLS

Glypican 2 (GPC2) is a cell-surface oncoprotein initially identified in neuroblastoma, retinoblastoma, and medulloblastoma as an ideal target for immunotherapy (Cancer Cell, 2017). Here we evaluated GPC2 expression across the spectrum of pediatric brain tumors using RNA sequencing from specimens in the Children’s Brain Tumor Network (CBTN). High GPC2 expression, defined as >10 FPKM, was found in 100% of embryonal tumors with multilayered rosettes (ETMRs) (n=6), 95% of medulloblastomas (n=122), 86% of other embryonal tumors (n=21), 50% of choroid plexus carcinomas (n=4), 42% of high grade gliomas (HGG) (n=117), and 37% of diffuse midline gliomas (DMG) (n=65). Within medulloblastoma subtypes, group 4 tumors had the highest expression, and within the HGG tumor cohort H3.3 G34 mutated gliomas had the highest GPC2 expression. High GPC2 protein expression was validated with medulloblastoma and HGG/DMG primary tumors and cell lines using IHC, Western blot, and flow cytometry. We next developed two potent CAR T cell constructs using the D3 specific scFv directed against GPC2 for testing in brain tumor models. GPC2-directed CAR T cells were tested in vitro against medulloblastoma and HGG cells lines, and in vivo using two patient-derived medulloblastoma xenograft models: Rcmb28 (group 3) and 7316-4509 (group 4). GPC2-directed mRNA CAR T cells induced significant GPC2-specific cell death in medulloblastoma and HGG cellular models with concomitant T cell degranulation compared to CD19-directed mRNA CAR T cells. In vivo, GPC2-directed mRNA CAR T cells delivered locoregionally induced significant tumor regression measured by bioluminescence after 4–6 intratumoral infusions of 4 x 106 CAR T cells (p<0.0001 for Rcmb28, p<0.05 for 7316-4509). No GPC2-directed CAR T cell related toxicity was observed. GPC2 is a highly differentially expressed cell surface protein on multiple malignant pediatric brain tumors that can be targeted safely with local delivery of mRNA CAR T cells.

Single cell multiomic analysis and immune cell type profiling of multiple myeloma with t(4;14).

e20013 Background: Multiple Myeloma (MM) is an incurable plasma cell (PC) malignancy and high risk (HR) MM remains an unmet clinical need. Translocation 4;14 occurs in 15% of MM and is associated with an adverse prognosis. A deeper understanding of the biology and immune micro-environment of t(4;14) MM is necessary for the development of effective targeted therapies. Single Cell multi-omics provides a new tool for phenotypic characterization of MM. Here we used Proteona’s ESCAPE™ single cell multi-omics platform to study a cohort of patients with t(4;14) MM. Methods: Diagnostic bone marrow (BM) samples from 14 patients with t(4;14) MM were analysed using the ESCAPE platform from Proteona which simultaneously measures gene and cell surface protein expression in single cells. Cryopreserved BM samples were stained with 65 DNA barcoded antibodies and subsequently sorted on CD138 expression. The CD138 positive and negative fractions were recombined at a known ratio for analysis using the 10x Genomics 3’ RNAseq kit. Resulting data were analyzed with Seurat and MapCell. Results: The patients had a median age of 63 years. All received novel agent based induction. Median progression free and overall survival (PFS and OS) were 22 and 34 months respectively. MMSET was overexpressed in all PCs while FGFR3 expression could be categorized into zero cells expressing FGFR3, low expression (&lt; 10% of cells expressing FGFR3) or high expression (&gt; 80% of cells expressing FGFR3). We also found heterogeneity in the expression of cancer testis antigens (CTA) such as FA133A and CTAG2 between PC clusters across samples. Variation in the immune microenvironment of the BM was seen across all patient samples with no correlation between cell types and PFS or OS. However, an analysis of BM samples at diagnosis and relapse in one patient showed a shift in the ratio of T cells to CD14 monocytes with a ratio of 5.7 at diagnosis compared to 0.6 at relapse. Further analysis of PCs in this patient found 8 PC populations, each containing variable numbers of cells from both the diagnostic and relapse samples. This suggests that all populations present at relapse were also present at diagnosis, although at variable proportions. Increased expression of RCAN3 (associated with cereblon depletion) was detected at relapse. Conclusions: We present the first application of single cell multi-omics immune profiling in high risk MM. The heterogeneity in expression of CTA has implications for the application of immunotherapies, while the upregulation of RCAN3 may explain failure of immunomodulatory therapy. Our small sample size may explain the lack of correlation between gene or protein expression with clinical outcomes. We propose that t(4;14) MM is a genomically and immunologically heterogeneous disease. Single cell analysis of larger cohorts is required to build on our findings.

Mesothelin is a novel cell surface disease marker and potential therapeutic target in acute myeloid leukemia

AbstractIn an effort to identify acute myeloid leukemia (AML)-restricted targets for therapeutic development in AML, we analyzed the transcriptomes of 2051 children and young adults with AML and compared the expression profile with normal marrow specimens. This analysis identified a large cohort of AML-restricted genes with high expression in AML, but low to no expression in normal hematopoiesis. Mesothelin (MSLN), a known therapeutic target in solid tumors, was shown to be highly overexpressed in 36% of the AML cohort (range, 5-1077.6 transcripts per million [TPM]) and virtually absent in normal marrow (range, 0.1-10.7 TPM). We verified MSLN transcript expression by quantitative reverse transcription polymerase chain reaction, confirmed cell surface protein expression on leukemic blasts by multidimensional flow cytometry, and demonstrated that MSLN expression was associated with promoter hypomethylation. MSLN was highly expressed in patients with KMT2A rearrangements (P < .001), core-binding factor fusions [inv(16)/t(16;16), P < .001; t(8;21), P < .001], and extramedullary disease (P = .001). We also demonstrated the presence of soluble MSLN in diagnostic serum specimens using an MSLN-directed enzyme-linked immunosorbent assay. In vitro and in vivo preclinical efficacy of the MSLN-directed antibody-drug conjugates (ADCs) anetumab ravtansine and anti-MSLN–DGN462 were evaluated in MSLN+ leukemia cell lines in vitro and in vivo, as well as in patient-derived xenografts. Treatment with ADCs resulted in potent target-dependent cytotoxicity in MSLN+ AML. In this study, we demonstrate that MSLN is expressed in a significant proportion of patients with AML and holds significant promise as a diagnostic and therapeutic target in AML, and that MSLN-directed therapeutic strategies, including ADCs, warrant further clinical investigation.

Changes in Density and Localization of GLUT1 and MCT1 Transporters of Cancer Cells Induced by Ketones

Ketone supplementation has been investigated as a potential therapy and/or as adjuvant with traditional cancer treatments. Previous studies have indicated that ketone supplementation decreases viability and proliferation of cultured tumor cells, as well as prolongs survival of mice with metastatic cancer. The primary fuel for cancer cells is glucose that enters the cells through glucose transporters (GLUT), while lactate, another key energy source for cancer cells that has been shown to enhance tumor angiogenesis, is exported through monocarboxylate transmembrane transporters (MCT). The inhibition of these transporters could lead to promising anti-cancer therapies, therefore, we investigated the changes in the density and localization of GLUT1 and MCT1 transporters in various cancer cell lines in response to R,S-sodium-3-hydroxybutyrate (βHB) in the culture media, in order to identify underlying mechanisms of the previously observed anti-cancer effects. We studied mice metastatic cancer (VM-M3), human brain cancer (U87MG) and human lung cancer (A549) cells using indirect immunofluorescence assay with microplate photometer and confocal microscopy. Cells were cultured in 12 or 96-well plates, and after 2 days they were treated with graded concentrations of βHB for 24 hrs, followed by immunofluorescent reaction. In VM-M3 cells only 1 nM βHB treatment decreased GLUT1 density, but not higher concentrations. A statistically significant decrease in GLUT1 density was detected with increasing βHB concentration starting at 2 nM in U87 cells and at 5nM in A549 cells. The density of MCT1 transporter decreased in all cell types with βHB treatment, and less protein was localized in the cell nucleus. These results suggest a possible mechanism for ketone bodies to decrease cancer cell viability via decreased ability to take up glucose and export lactate. However, the lowest effective concentration was different in each cell type and it is possible that higher concentrations might induce compensatory mechanisms. Further exploration of the effects of ketones on the expression of cell surface proteins and transporters relevant to cancer cell metabolism may improve our understanding of the clinical relevance of ketone supplementation in the treatment of cancer.

Technological advancements in multiomic single cell immune profiling and analysis

Abstract Continued advances in single cell gene detection sensitivity and the ability to analyze data quickly with biological context are critical for discoveries in therapeutic research. The second version of the Chromium Single Cell Immune Profiling Solution by 10x Genomics enables highly sensitive detection of gene expression, full-length paired T-cell alpha- and beta-chain and immunoglobulin sequences, T-cell antigen specificity, and cell surface protein expression from the same single cells, allowing a comprehensive view of the immune response at the cellular level. The new workflow provides a 45% increase in the number of genes detected per cell and up to a 25% increase in the cells detected with paired full-length V(D)J receptor sequences in melanoma tumor derived cells. Using a new version of Cell Ranger (v5.0), clonotypes were grouped from &amp;gt;30,000 cells, increasing the power to detect small clonotype expansions with fewer total cells. In addition, the new software enabled comparison of pre- and post-influenza vaccination B-cell receptor sequences from a single donor, identifying post-vaccination specific clonotypes. These technological and informatics advancements enhance a researcher’s ability to perform a broad characterization of immune cell populations at unprecedented throughput and resolution.

IFNγ, and to a Lesser Extent TNFα, Provokes a Sustained Endothelial Costimulatory Phenotype

BackgroundVascular endothelial cells (EC) are critical for regulation of local immune responses, through coordination of leukocyte recruitment from the blood and egress into the tissue. Growing evidence supports an additional role for endothelium in activation and costimulation of adaptive immune cells. However, this function remains somewhat controversial, and the full repertoire and durability of an enhanced endothelial costimulatory phenotype has not been wholly defined.MethodsHuman endothelium was stimulated with continuous TNFα or IFNγ for 1-48hr; or primed with TNFα or IFNγ for only 3hr, before withdrawal of stimulus for up to 45hr. Gene expression of cytokines, costimulatory molecules and antigen presentation molecules was measured by Nanostring, and publicly available datasets of EC stimulation with TNFα or IFNγ were leveraged to further corroborate the results. Cell surface protein expression was detected by flow cytometry, and secretion of cytokines was assessed by Luminex and ELISA. Key findings were confirmed in primary human endothelial cells from 4-6 different vascular beds.ResultsTNFα triggered mostly positive immune checkpoint molecule expression on endothelium, including CD40, 4-1BB, and ICOSLG but in the context of only HLA class I and immunoproteasome subunits. IFNγ promoted a more tolerogenic phenotype of high PD-L1 and PD-L2 expression with both HLA class I and class II molecules and antigen processing genes. Both cytokines elicited secretion of IL-15 and BAFF/BLyS, with TNFα stimulated EC additionally producing IL-6, TL1A and IL-1β. Moreover, endothelium primed for a short period (3hr) with TNFα mostly failed to alter the costimulatory phenotype 24-48hr later, with only somewhat augmented expression of HLA class I. In contrast, brief exposure to IFNγ was sufficient to cause late expression of antigen presentation, cytokines and costimulatory molecules. In particular HLA class I, PD-1 ligand and cytokine expression was markedly high on endothelium two days after IFNγ was last present.ConclusionsEndothelia from multiple vascular beds possess a wide range of other immune checkpoint molecules and cytokines that can shape the adaptive immune response. Our results further demonstrate that IFNγ elicits prolonged signaling that persists days after initiation and is sufficient to trigger substantial gene expression changes and immune phenotype in vascular endothelium.

Absence of Batf3 reveals a new dimension of cell state heterogeneity within conventional dendritic cells

SummaryConventional dendritic cells (cDCs) are traditionally subdivided into cDC1 and cDC2 lineages. Batf3 is a cDC1-required transcription factor, and we observed that Batf3−/− mice harbor a population of cDC1-like cells co-expressing cDC2-associated surface molecules. Using single-cell RNA sequencing with integrated cell surface protein expression (CITE-seq), we found that Batf3−/− mitotic immature cDC1-like cells showed reduced expression of cDC1 features and increased levels of cDC2 features. In wild type, we also observed a proportion of mature cDC1 cells expressing surface features characteristic to cDC2 and found that overall cDC cell state heterogeneity was mainly driven by developmental stage, proliferation, and maturity. We detected population diversity within Sirpa+ cDC2 cells, including a Cd33+ cell state expressing high levels of Sox4 and lineage-mixed features characteristic to cDC1, cDC2, pDCs, and monocytes. In conclusion, these data suggest that multiple cDC cell states can co-express lineage-overlapping features, revealing a level of previously unappreciated cDC plasticity.

Integration of Transcriptome and Cell Surface Protein Expression by LinQ-View Identifies Unique Immune Subpopulations

Single cell RNA sequencing is a powerful tool for characterizing the molecular and cellular heterogeneity of immune cells during their development and activation. Multimodal advances in single cell sequencing have enabled the simultaneous quantification of cell surface protein expression alongside unbiased transcriptional profiling. Here we present LinQ-View, a toolkit designed for multimodal single cell data visualization and analysis that links transcriptional and cell surface protein expression profiling data. Further, we propose a quantitative metric for cluster purity of CITE-seq data, enabling effective determination of clustering algorithms and their parameters, and finally demonstrate the utility of our toolkit through seamless integration with standard single cell analysis workflows on several public datasets. Through comparison to existing multimodal methods, we demonstrate that LinQ-View generates more accurate cell clusters and is highly efficient, even with massive datasets. LinQ-View is specialized in handling CITE-seq data with routine numbers of surface protein features (e.g. less than 50), by preventing variations in a single surface protein feature from affecting results. Finally, we utilized this method to integrate single cell transcriptional and protein expression data from COVID-19-infected patients and influenza-immunized subjects, revealing antigen-specific B cell subsets and previously unknown T cell subsets post-infection and vaccination.

Single-cell analysis of erythropoiesis in Rpl11 haploinsufficient mice reveals insight into the pathogenesis of Diamond–Blackfan anemia

Rpl11 haploinsufficient mice develop a macrocytic anemia similar to patients with DBA. Here, we fully characterize this model from clinical and pathophysiological perspectives. Early erythroid precursors have increased heme content and high cytoplasmic reactive oxygen species, impairing erythroid differentiation at the colony-forming unit-erythroid (CFU-E)/proerythroblast stage and subsequently. Using single-cell analyses that link a cell's surface protein expression to its total transcriptome and unbiased analyses, we found GATA1, GATA1 target gene, and mitotic spindle pathway gene transcription were the pathways that decreased the most. Expression of ribosome protein and globin genes was amplified. These changes, as well as the other transcriptional changes that were identified, closely resemble findings in mice that lack the heme export protein FLVCR and, thus, suggest that heme excess and toxicity are the primary drivers of the macrocytic anemia. Consistent with this, treating Rpl11 haploinsufficient mice with corticosteroids increased the numbers of earliest erythroblasts but failed to overcome heme toxicity and improve the anemia. Rpl11 haploinsufficient mice uniquely upregulated mitochondrial genes, p53 and CDKN1A pathway genes, and DNA damage checkpoint genes, which should contribute further to erythroid marrow failure. Together our data establish Rpl11 haploinsufficient mice as an excellent model of DBA that can be used to study DBA pathogenesis and test novel therapies.

A multicenter retrospective analysis examining the effect of dipeptidyl protease 4 (DPP4) inhibitors on progression-free survival in patients with prostate cancer.

109 Background: Dipeptidyl peptidase IV (DPP4), also known as Cluster of differentiation 26 (CD26) is a commonly expressed cell surface protein found in many cell types that can function as a tumor suppressor or activator, depending on the cancer type its interactions with the tumor microenvironment. DPP4 has been studied as a tumor biomarker due to its expression in various primary tumors and metastases. Recent literature established benefits of DPP4 inhibitors on progression free survival (PFS) of diabetic patients with advanced colorectal and airway cancers. In this study, we examine the effect of DPP4 inhibitors on PFS in diabetic patients (pts) with advanced-stage prostate cancer. Methods: We performed a multi-center retrospective analysis at Moffitt Cancer Center and the University of Florida of diabetic pts with advanced stage (III or IV) prostate cancer. All pts were on anti-hyperglycemic therapy and received either surgery, radiation therapy, or both for their prostate cancer. The control group included pts on metformin and a sulfonylurea, and the study group included pts on a DPP4 inhibitor and metformin. PFS was calculated for both groups, and Cox proportional hazards regression was used to determine statistical significance. Kaplan-Meier survival analysis was done to predict the effect of DPP4 inhibition and radiation therapy on PFS. Results: Our study population consisted of 161 pts, with 120 pts in the control group and 41 in the study group. No significant difference in progression of prostate cancer was found between those on DPP4 inhibitor vs. Metformin. Specifically, 24 of 41 (59%) of patients on DPP4 inhibitors progressed, while 73 of 120 (61%) of patients on Metformin had progression of prostate cancer (HR 1.01, 95% CI 0.64 to 1.61, p= 0.955). Median time to progression was 3.3 years in the DPP4 group versus 3.5 years in the Metformin group. However, radiation therapy was associated with improved PFS (HR 0.56, 95% CI 0.37 to 0.84, p= 0.0006). Conclusions: Contrary to what has been shown in pts with diabetes and advanced colorectal or airway cancers, exposure to DPP4 inhibitors did not lead to statistically significant improvement in PFS in pts with advanced stage prostate cancer. Recent data suggests that DPP4 may act as a tumor suppressor gene to the androgen receptor (AR) pathway, and that DPP4 inhibition can result in emergence of resistance to androgen deprivation therapy (ADT). Further analysis with larger sample sizes is necessary to elucidate if this is applicable to prostate cancer as a whole, or if this effect is specific to castrate-sensitive prostate cancer (CSPC) vs. castrate-resistant prostate cancer (CRPC).

Hemozoin Promotes Lung Inflammation via Host Epithelial Activation.

Respiratory distress in severe malaria is associated with high mortality, but its pathogenesis remains unclear. The malaria pigment hemozoin (HZ) is abundant in target organs of severe malaria, including the lungs, and is known to be a potent innate immune activator of phagocytes. We hypothesized that HZ might also stimulate lung epithelial activation and thereby potentiate lung inflammation. We show here that airway epithelium stimulated with HZ undergoes global transcriptional reprogramming and changes in cell surface protein expression that comprise an epithelial activation phenotype. Proinflammatory signaling is induced, and key cytoadherence molecules are upregulated, including several associated with severe malaria, such as CD36 and ICAM1. Epithelial and extracellular matrix remodeling pathways are transformed, including induction of key metalloproteases and modulation of epithelial junctions. The overall program induced by HZ serves to promote inflammation and neutrophil transmigration, and is recapitulated in a murine model of HZ-induced acute pneumonitis. Together, our data demonstrate a direct role for hemozoin in stimulating epithelial activation that could potentiate lung inflammation in malaria.IMPORTANCE Respiratory distress (RD) is a complication of severe malaria associated with a particularly high risk for death in African children infected with the parasite Plasmodium falciparum The pathophysiology underlying RD remains poorly understood, and the condition is managed supportively. The parasite-derived factor HZ accumulates in target organs of severe malaria, including the lungs, and is a potent stimulator of immune cells. Our findings demonstrate that HZ causes global activation of lung epithelial cells, a response that directly promotes lung inflammation. HZ stimulates expression of key proinflammatory and cell surface molecules, alters signaling pathways involved in epithelial-matrix remodeling, and promotes neutrophil transmigration and airway inflammation. The lung epithelial activation induced by HZ mimics patterns seen in malarial lung injury and provides new insights into the molecular pathogenesis of RD.