Signal Regulatory Protein Alpha Research Articles

Mendelian Randomization Estimates the Effects of Plasma and Cerebrospinal Fluid Proteins on Intelligence, Fluid Intelligence Score, and Cognitive Performance.

Observational studies have revealed associations between levels of plasma and cerebrospinal fluid (CSF) proteins and cognition-related traits. However, these associations may be influenced by confounding factors inherent in observational research. This study aims to identify plasma and CSF proteins associated with intelligence, fluid intelligence score, and cognitive performance through the application of Mendelian randomization (MR). Proteomic quantitative trait locus (pQTL) data for plasma and CSF proteins were sourced from existing genome-wide association study (GWAS). Intelligence, fluid intelligence score, and cognitive performance GWAS summary statistics provided comprehensive data for two-sample MR analysis. Extensive sensitivity analyses, including Steiger testing, reverse MR analysis, and Bayesian co-localization, were conducted to validate associations and identify shared genetic variants. Phenotype scanning explored potential pleiotropic effects. MR analysis identified several proteins in plasma and CSF significantly associated with intelligence, fluid intelligence scores, and cognitive performance. For intelligence, negatively associated proteins in plasma include endoplasmic reticulum aminopeptidase 2 (ERAP2) and secretogranin III (SCG3), while positively associated proteins are myeloperoxidase (MPO), signal regulatory protein alpha (SIRPA), regulator of microtubule dynamics 1 (RMDN1), and endoplasmic reticulum lectin 1 (ERLEC1). In CSF, C1-esterase inhibitor and carboxypeptidase E (CBPE) both exhibited positive associations with intelligence. For fluid intelligence scores, negatively associated proteins in plasma are copine 1 (CPNE1) and SCG3, while positively associated proteins are nudix hydrolase 12 (NUDT12) and RMDN1.In CSF, Macrophage Stimulating Protein (MSP) demonstrated a significant negative impact. For cognitive performance, negatively associated proteins in plasma include ERAP2, tyrosine kinase with immunoglobulin-like and EGF-like domains 1 (TIE1), and SCG3, while positively associated proteins are NUDT12, RMDN1, ERLEC1, and ectonucleotide pyrophosphatase/phosphodiesterase family member 5 (ENPP5). In CSF, C1-esterase inhibitor was positively associated, while MSP and soluble tyrosine kinase with immunoglobulin-like and EGF-like domains 1(sTie-1) showed a negative association. Bayesian co-localization analysis revealed significant genetic overlaps between SIRPA, RMDN1, and ERLEC1 in plasma with intelligence; NUDT12 and SCG3 in plasma with fluid intelligence scores; and TIE1, NUDT12, RMDN1, ERLEC1, and ENPP5 in plasma with cognitive performance. Additionally, significant co-localization was identified between C1-esterase inhibitor and CBPE in CSF with intelligence, as well as between C1-esterase inhibitor and sTie-1 in CSF with cognitive performance. Reverse causality analysis confirmed the causal direction from proteins to cognitive traits. This study identifies specific plasma and CSF proteins that significantly impact intelligence, fluid intelligence scores, and cognitive performance. These proteins could serve as biomarkers and targets for future research and therapeutic interventions aimed at sustaining cognitive abilities and reducing impairment risks.

Facilitating cholangiocarcinoma inhibition by targeting CD47

Immune evasion is one of the mechanisms by which cancer cells acquire immunity during cancer development and progression. One of these is the increased expression of cluster of differentiation 47 (CD47), a transmembrane glycoprotein that protects cells from phagocytic elimination. The interaction between CD47 and signal regulatory protein alpha (SIRPα) on macrophages alleviates the phagocytic signal. The present group previously reported high CD47 expression in cholangiocarcinoma (CCA), a major health problem in Thailand and East Asia, and that blocking CD47 using anti-CD47 antibodies promoted the removal of CCA. However, the mechanism through which CD47 inhibition attenuates CCA growth remains unclear. This study explored the clinical significance of targeting CD47 in CCA. Expression levels of CD47 and the macrophage marker CD68 were determined in CCA tissues by immunohistochemistry and correlated with clinical parameters. The role of CD47 in CCA cells was established using CD47-deficient KKU-213A CCA clones in vitro and in vivo. The results showed that CD47 was highly expressed in CCA tissues and significantly correlated with lymph node metastasis (P = 0.038). Moderate-to-dense CD68-positive infiltrating cells in CCA tissues were significantly associated with shorter survival of patients (P = 0.019) and were an independent prognostic factor of CCA patients as determined by the Cox proportional hazard model (hazard ratio, 2.040; 95 % confidence interval, 1.109–3.752; P = 0.022). Three CD47-deficient KKU-213A clones (#19, #23, and #28) were generated. The elimination of CD47 did not affect cell proliferation but increased monocyte-derived macrophage-mediated phagocytosis in vitro. Decreased tumor weights and volumes were observed in mice injected with CD47-deficient CCA clones. This revealed a significant role for CD47 in CCA, with a focus on protecting cancer cells from macrophage phagocytosis.

The expression of signal regulatory protein alpha (SIRPα) in periodontal cells and tissue.

Signal regulatory protein alpha (SIRPα) is mainly expressed by cells of myeloid origin. This membrane glycoprotein is shown to be involved in regulation of different inflammatory conditions, such as colitis and arthritis. However, SIRPα has not been investigated in relationship to periodontitis, an inflammatory condition affecting the tooth supporting tissues. We aim to investigate if resident cells in the periodontium express SIRPα and whether a possible expression is affected by inflammatory conditions. Primary human keratinocytes, fibroblasts, periodontal ligament cells, and osteoblasts were cultured with or without the pro-inflammatory cytokines tumor necrosis factor alpha (TNF-α) or interleukin-1-beta (IL-1β). All different periodontal cell types showed a basal mRNA expression of SIRPα. Pro-inflammatory cytokines induced a 2-3-fold significant increase in SIRPα expression in both cultured human gingival fibroblasts and osteoblasts but neither in keratinocytes nor in periodontal ligament cells. Tissue sections from human gingival tissue biopsies were histochemically stained for SIRPα. Epithelial keratinocytes and gingival fibroblasts stained positive in sections from periodontally healthy as well as in sections from periodontitis. In periodontitis sections, infiltrating leukocytes stained positive for SIRPα. We highlight our finding that oral keratinocytes, gingival fibroblasts, and periodontal ligament cells do express SIRPα, as this has not been presented before. The fact that inflammatory stimulation of gingival fibroblasts increased the expression of SIRPα, while an increased expression by gingival fibroblasts in periodontitis tissue in situ could not be detected, is indeed contradictory.

Engineered Nanoparticles for Enhanced Antitumoral Synergy Between Macrophages and T Cells in the Tumor Microenvironment.

T cells and macrophages have the potential to collaborate to eliminate tumor cells efficiently. Macrophages can eliminate tumor cells through phagocytosis and subsequently activate T cells by presenting tumor antigens. The activated T cells, in turn, can kill tumor cells and redirect tumor-associated macrophages toward an antitumoral M1 phenotype. However, checkpoint molecules expressed on tumor cells impede the collaborative action of these immune cells. Meanwhile, monotherapy with a single immune checkpoint inhibitor (ICI) for either macrophages or T cells yields suboptimal efficacy in cancer patients. To address this challenge, here a nanoparticle capable of efficiently delivering dual ICIs to tumors for both macrophages and T cells is developed. These programmed cell death protein 1 (PD-1)-transfected macrophage membrane-derived nanoparticles (PMMNPs) can target tumors and provide signal-regulatory protein alpha and PD-1 to block CD47 and programmed cell death-ligand 1 (PD-L1), respectively, on tumor cells. PMMNPs enhance macrophage-mediated cancer cell phagocytosis and antigen presentation, promote T cell activation, and induce the reprogramming of macrophages toward an antitumoral phenotype. In syngeneic tumor-bearing mice, PMMNPs demonstrate superior therapeutic efficacy compared to nanoparticles delivering single ICIs and non-targeted delivery of anti-CD47 and anti-PD-L1 antibodies. PMMNPs capable of augmenting the antitumoral interplay between macrophages and T cells may offer a promising avenue for cancer immunotherapy.

Abstract P222: O-GlcNAcylation induces aortic stiffness in aging female rats via CD47-SIRPα -1 signaling

A reduction in aortic compliance serves as an early indicator of incipient vascular disease. While extracellular matrix modifications, such as elastin fragmentation, are the major mediators of aortic stiffness, the mechanisms that underlie these phenotypes remain to be elucidated. O-GlcNAc, the attachment of β-N-acetylglucosamine to serine and threonine residues of proteins, is a highly dynamic and widespread post-translational modification. This modification plays a pivotal role in modulating the function, activity, localization, and stability of target proteins across nuclear, cytoplasmic, and mitochondrial compartments. Primarily involved in intracellular signaling and transcriptional regulation, O-GlcNAc responds to nutrient availability and cell stress. However, chronic O-GlcNAc is consistently linked to vascular impairments. Indeed, our preliminary data has demonstrated that O-GlcNAc is elevated in aorta of Fisher-334 female at 16 months of age (16MO) than 3MO and pharmacological induction of O-GlcNAc (TMG 100 uM 24h) in 3MO rats can cause aortic stiffness via increased elastase activity and elastin fragmentation. However, how this inherently intracellular cellular signaling could impact aorta remodeling is unknown. CD47 (Cluster of Differentiation 47), also known as integrin-associated protein, is a transmembrane protein that acts as a “do not eat me” signal to immune cells. RNAseq data from our lab suggested that TMG (1 uM, 24 h) increased CD47 expression in vascular smooth muscle cells (n =3, p=0.08). Therefore, we hypothesized that CD47 could be an important downstream mediator of O-GlcNAc-induced aortic stiffness. Our findings revealed that CD47 inhibitors, [(RRx 10 uM) and SEN 177 (100 nM)] effectively mitigated the aortic stiffness and elastase activity (Ctrl 2302±38; TMG 4058±328; RRx 1776±121; Sen177 3032±50 A.U.) induced by TMG, suggesting that CD47 is a novel mechanism of O-GlcNAc-induced aortic stiffness. As aging is a major risk factor for aortic stiffness, we also revealed that CD47 was increased in the aorta of 16MO rats compared to 3MO controls (0.1±0.03 vs. 0.3±0.06). Potential ligands for CD47 include thrombospondin-1 (TSP-1) and signal-regulatory protein alpha (SIRPα). We observed that in 16MO rats, circulating SIRPα is increased, but not TSP1, suggesting a receptor-ligand interaction that is applicable in aging. Overall, these data reveal a previously unidentified and significant role for CD47 in O-GlcNAc-induced aortic stiffness.

401. CLINICAL SIGNIFICANCE OF SIGNAL REGULATORY PROTEIN ALPHA AND CLUSTER OF DIFFERENTIATION 47 EXPRESSION IN ESOPHAGEAL SQUAMOUS CELL CARCINOMA

Abstract Background This study focuses on assessing the expression of Signal Regulatory Protein Alpha (SIRPα) and Cluster of Differentiation 47 (CD47) in Esophageal Squamous Cell Carcinoma (ESCC) and their impact on prognosis. Given the prominence of the CD47-SIRPα axis in cancer for inhibiting macrophage-mediated phagocytosis, this research aims to explore their role and significance in the development and treatment of ESCC. Methods In this study, 100 ESCC patients who underwent esophagectomy were included. Resected specimens were stained immunohistochemical staining for SIRPα and CD47. SIRPα expression was classified as high or low with cut-off value of 3%. CD47 expression was evaluated by Combined Positive Score (CPS) and Tumor Proportion Score (TPS) methods with setting a 10% threshold for high and low expression. The five-year survival rates were compared across groups over a median observation period of 61 months. This study reveals the correlation between these biomarkers and postoperative prognosis. Results Total 79 males and 21 females were included in this study and mean age was 68.7-year-old. The high SIRPα expression group (48 patients) revealed significantly worse 5-year overall survival rate than the low expression group (52 patients) (56.4 vs 82.8%, P=0.0098). For CD47, both evaluated by using CPS and TPS, the high expression group also showed significantly worse 5-year overall survival rate than its low group (48.8 vs 89.5%, P<0.0001, and 48.4 vs 87.5%, P<0.0001 respectively). Relapse free survival also showed significantly worse survival rate in high expression group of SIRPα and CD47. Conclusion The expression levels of SIRPα and CD47 serve as important biomarkers for predicting the prognosis of ESCC. They may influence prognosis by inhibiting macrophage-mediated tumor cell phagocytosis and modulating anti-tumor immune responses, offering new targets for ESCC treatment.

SIRPα engagement regulates ILC2 effector function and alleviates airway hyperreactivity via modulating energy metabolism

Group-2 innate lymphoid cells (ILC2) are part of a growing family of innate lymphocytes known for their crucial role in both the development and exacerbation of allergic asthma. The activation and function of ILC2s are regulated by various activating and inhibitory molecules, with their balance determining the severity of allergic responses. In this study, we aim to elucidate the critical role of the suppressor molecule signal regulatory protein alpha (SIRPα), which interacts with CD47, in controlling ILC2-mediated airway hyperreactivity (AHR). Our data indicate that activated ILC2s upregulate the expression of SIRPα, and the interaction between SIRPα and CD47 effectively suppresses both ILC2 proliferation and effector function. To evaluate the function of SIRPα in ILC2-mediated AHR, we combined multiple approaches including genetically modified mouse models and adoptive transfer experiments in murine models of allergen-induced AHR. Our findings suggest that the absence of SIRPα leads to the overactivation of ILC2s. Conversely, engagement of SIRPα with CD47 reduces ILC2 cytokine production and effectively regulates ILC2-dependent AHR. Furthermore, the SIRPα-CD47 axis modulates mitochondrial metabolism through the JAK/STAT and ERK/MAPK signaling pathways, thereby regulating NF-κB activity and the production of type 2 cytokines. Additionally, our studies have revealed that SIRPα is inducible and expressed on human ILC2s, and administration of human CD47-Fc effectively suppresses the effector function and cytokine production. Moreover, administering human CD47-Fc to humanized ILC2 mice effectively alleviates AHR and lung inflammation. These findings highlight the promising therapeutic potential of targeting the SIRPα-CD47 axis in the treatment of ILC2-dependent allergic asthma.

Clinical Significance of SIRPα Expression on Tumor-Associated Macrophages in Patients with Lung Squamous Cell Carcinoma.

Signal-regulatory protein alpha (SIRPα) is an immune checkpoint molecule expressed on macrophages that functions to inhibit phagocytosis by binding to CD47 expressed on tumor cells. SIRPα has attracted increasing attention as a novel target for cancer immunotherapy; however, the expression and immune function of SIRPα in lung squamous cell carcinoma (LUSC) remain unclear. Therefore, this study aimed to identify the clinical importance of SIRPα expression in LUSC and to explore the factors that elevate SIRPα expression. Primary LUSC specimens surgically resected from 172 patients underwent immunohistochemical evaluation of the association of SIRPα expression on tumor-associated macrophages with clinicopathological features and clinical outcomes. Furthermore, we analyzed the association of SIRPα expression with tumor-infiltrating lymphocytes and the expression of programmed cell death ligand 1 (PD-L1). In vitro, monocytes were treated with cytokines, and SIRPα protein expression was assessed by flow cytometry. There were no differences in SIRPα expression and clinicopathological factors. High SIRPα expression was significantly associated with PD-L1-positive expression, and high CD8, PD-1, and CD163 expression. The high SIRPα expression group showed significantly shorter recurrence-free survival (RFS) and overall survival (OS). On multivariate analysis, high SIRPα expression was an independent poor prognostic factor for RFS and OS. The expression of SIRPα protein in monocytes was upregulated by treatment with IFNγ. Our analysis revealed that high SIRPα expression significantly predicts poor prognosis in patients with surgically resected LUSC.

Clinicopathological analysis of immunohistochemical CD47 and signal-regulatory protein-α expression in Extranodal Natural killer/T-cell lymphoma.

The interaction between CD47 and signal-regulatory protein-alpha (SIRPα) inhibits phagocytosis, and their clinicopathological characteristics have been evaluated in various diseases. However, the significance of CD47 and SIRPα expression, as well as the combined effect, in Extranodal Natural killer/T-cell Lymphoma (ENKTL) remains uncertain. In total, 76 newly diagnosed ENKTL patients (mean age 49.9 years, 73.7% male) were included in this study. CD47 and SIRPα expression were examined by immunohistochemistry. Survival analyses were conducted through Kaplan-Meier curves and the Cox regression model. Seventy-one (93.4%) cases were categorized as the CD47 positive group and 59 (77.6%) cases were categorized as the SIRPα positive group. CD47-negative cases had more advanced-stage illness (P = 0.001), while SIRPα-positive cases showed significantly lower levels of high-density lipoprotein (P < 0.001). In univariable analysis, CD47, SIRPα expression, and their combination were significantly associated with prognosis (P < 0.05). In multivariable analysis, only positive SIRPα expression remained significantly associated with superior overall survival (Hazard ratio [HR] 0.446; 95% confidence interval [CI] 0.207-0.963; P = 0.004). Furthermore, SIRPα expression could re-stratify the survival of patients in ECOG (< 2), advanced CA stage, PINK (HR), CD38-positive, PD1-positive, and CD30-positive groups. SIRPα status was a potential independent prognostic factor for ENKTL. The prognostic significance of CD47 expression and the interaction between CD47 and SIRPα in ENKTL need further investigation.

A phase I/II study of maplirpacept in combination with pegylated liposomal doxorubicin (PLD) in patients with platinum-resistant ovarian cancer (OC): Phase 1 results.

e17546 Background: CD47, an overexpressed factor correlated with poor clinical characteristics and prognosis in OC, is an innate immune checkpoint that binds signal regulatory protein alpha (SIRPα) and delivers a signal to suppress macrophage phagocytosis. Maplirpacept (PF-07901801) is a fusion protein consisting of the CD47-binding domain of human SIRPα linked to the Fc region of human IgG4, enhancing phagocytosis by blocking CD47. Blockade of CD47, both alone and combined with doxorubicin, has shown anti-tumor activity in vitro and in vivo in xenograft animal models. PLD 40mg/m2 conveyed poor efficacy (PFS 4 months; ORR 8%), as did bevacizumab + PLD (PFS 5 months; ORR 14%) (AURELIA), highlighting the need for new treatments for platinum-resistant OC. C4971002 [NCT05261490] is an open label, multicenter dose-escalation and expansion study evaluating the safety, preliminary anti-tumor activity, pharmacokinetics and -dynamics of maplirpacept in combination with PLD in patients with platinum-resistant recurrent OC. Methods: The study included adults with platinum-resistant recurrent epithelial OC (PD ≤6 months after platinum-based therapy or unable/unwilling to receive platinum-based therapy). PLD while platinum-resistant or directly prior to study was not permitted. Patients received maplirpacept (12mg/kg Dose Level [DL] 1; 24mg/kg DL2; 48mg/kg DL3) weekly in Cycle 1 and every 2 weeks in Cycle 2+, and PLD (40mg/m2) on Day 1 of each 4-week cycle. Assessments of adverse events (AEs) were based on CTCAE v5.0. Response assessments (RECIST v1.1) were performed every 8 weeks. Results of the Phase I portion, aiming to evaluate the DLTs, the MTD and the RP2D, are presented as of 18 December 2023, after all patients completed the 28-day DLT assessment period. Results: 10 patients were treated (DL1 n=3; DL2 n=3; DL3 n=4) with a median (min, max) age of 69 (53, 77) years, 80% ECOG 1, 60% serous adenocarcinoma. Median (min, max) treatment duration was 14 (8, 32) weeks. Most common adverse events are shown in the table. No DLTs occurred in the assessment period. 1 serious AE (gait disturbance &amp; muscle weakness) related to maplirpacept was reported (DL3). 2 deaths occurred &gt;120 days after 1st dose, both due to disease. 5 (50%) patients had a best response of stable disease, 2 of which were treated into at least Cycle 6. Conclusions: Despite immature anti-tumor activity, maplirpacept + PLD was tolerable at all dose levels, not reaching the MTD. The encouragingly tolerable dose of 48mg/kg thus represents the RP2D which could support further development for this population. Clinical trial information: NCT05261490 . [Table: see text]

Resolvin E1 improves efferocytosis and rescues severe aplastic anemia in mice

Severe aplastic anemia (SAA) is a rare, fatal disease characterized by severe cytopenias and loss of hematopoietic stem cells (HSCs). Immune-mediated destruction and inflammation are known drivers of SAA, however, the underlying mechanisms driving persistent inflammation are unknown. Current treatments for SAA rely on immunosuppressive therapies or HSC transplantation, however, these treatments are not always effective. Using an established mouse model of SAA, we observed a significant increase in apoptotic cells within the bone marrow (BM) and impaired efferocytosis in SAA mice, relative to radiation controls. Single-cell transcriptomic analysis revealed heterogeneity among BM monocytes and unique populations emerged during SAA characterized by increased inflammatory signatures and significantly increased expression of Sirpa and Cd47. CD47, a “don’t eat me” signal, was increased on both live and apoptotic BM cells, concurrent with markedly increased expression of signal regulatory protein alpha (SIRPα) on monocytes. Functionally, SIRPα blockade improved cell clearance and reduced accumulation of CD47-positive apoptotic cells. Lipidomic analysis revealed a reduction in the precursors of specialized pro-resolving lipid mediators (SPMs) and increased prostaglandins in the BM during SAA, indicative of impaired inflammation resolution. Specifically, 18-HEPE, a precursor of E-series resolvins, was significantly reduced in SAA-induced mice relative to radiation controls. Treatment of SAA mice with Resolvin E1 (RvE1) improved efferocytic function, BM cellularity, platelet output, and survival. Our data suggest that impaired efferocytosis and inflammation resolution contributes to SAA progression and demonstrate that SPMs, such as RvE1, offer new and/or complementary treatments for SAA that do not rely on immune suppression.

Interrogating the CD47-SIRP axis in chronic T cell stimulation for the translation of Graft-vs.-Host disease therapeutics.

Abstract Graft-vs.-Host disease (GvHD) is the major complication associated with hematopoietic stem cell transplants (HSCTs), characterized by severe alloreactive immunotoxicity of the graft towards the immunocompromised patient. GvHD affects 40-50% of HSCT patients with an average 3-year survival of just 43%, underlining the need for novel therapies. CD47 the 'don't-eat-me' signal, interacts with Signal Regulatory Protein alpha (SIRPa), an inhibitory transmembrane receptor on macrophages, preventing the phagocytosis of the target cell. In cancer therapy, CD47-blockers are investigated as novel immunotherapies promoting phagocytosis of cancer cells. Serendipitously, we found that either CD47 blockade or pan-SIRP blockade effectively alleviates GvHD onset in a murine xeno-GvHD model. Early phagocytosis in vivo was found not to be the driver of GvHD alleviation, leading us to investigate SIRP proteins as potential regulators of chronic T cell stimulation. Particularly SIRP gamma (SIRPg) is exclusively expressed on T cells, and is known to be the only other SIRP to ligate CD47, however it is regarded understudied and its function, signaling, its regulators and downstream effectors are not known. In this work we could show that SIRPg is upregulated in a setting of chronic T cell stimulation, that its expression levels correlate with exhaustion marker expression in clinical scRNA seq data sets, and that primary human SIRPg KO T cells show a delayed onset of GvHD similar to SIRP blockade.

Enhancing IgA-mediated neutrophil cytotoxicity against neuroblastoma by CD47 blockade

BackgroundApproximately half of the neuroblastoma patients develop high-risk neuroblastoma. Current treatment involves a multimodal strategy, including immunotherapy with dinutuximab (IgG ch14.18) targeting GD2. Despite achieving promising results, the recurrence rate...

Proteomic study of vitreous in proliferative diabetic retinopathy patients after treatment with aflibercept: a quantitative analysis based on 4D label-free technique.

To identify different metabolites, proteins and related pathways to elucidate the causes of proliferative diabetic retinopathy (PDR) and resistance to anti-vascular endothelial growth factor (VEGF) drugs, and to provide biomarkers for the diagnosis and treatment of PDR. Vitreous specimens from patients with diabetic retinopathy were collected and analyzed by Liquid Chromatography-Mass Spectrometry (LC-MS/MS) analyses based on 4D label-free technology. Statistically differentially expressed proteins (DEPs), Gene Ontology (GO), Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway representation and protein interactions were analyzed. A total of 12 samples were analyzed. The proteomics results showed that a total of 58 proteins were identified as DEPs, of which 47 proteins were up-regulated and 11 proteins were down-regulated. We found that C1q and tumor necrosis factor related protein 5 (C1QTNF5), Clusterin (CLU), tissue inhibitor of metal protease 1 (TIMP1) and signal regulatory protein alpha (SIRPα) can all be specifically regulated after aflibercept treatment. GO functional analysis showed that some DEPs are related to changes in inflammatory regulatory pathways caused by PDR. In addition, protein-protein interaction (PPI) network evaluation revealed that TIMP1 plays a central role in neural regulation. In addition, CD47/SIRPα may become a key target to resolve anti-VEGF drug resistance in PDR. Proteomic analysis is an approach of choice to explore the molecular mechanisms of PDR. Our data show that multiple proteins are differentially changed in PDR patients after intravitreal injection of aflibercept, among which C1QTNF5, CLU, TIMP1 and SIRPα may become targets for future treatment of PDR and resolution of anti-VEGF resistance.

Abstract 75: Luciferase reporter cell lines allow simultaneous incorporation of tumor cells, innate immune cells, and adaptive immune cells for in-depth immune checkpoint studies

Abstract Despite the considerable success of immune checkpoint therapies targeting T cells, a sizable proportion of patients experience resistance or relapse due to the immunosuppressive nature of the tumor microenvironment. Myeloid cells, a major component that suppresses effector lymphocytes, have emerged as an alternative and promising therapeutic target. However, there is a deep lack of widely accessible immunological models capable of representing the intricate three-way interaction between tumor cells, T cells, and myeloid cells. To address this need, we conducted a comprehensive protein profiling of human tumor and immune cell lines available at ATCC for various established and novel immune checkpoint molecules. Cell lines with high endogenous expression of the immune checkpoint proteins, such as programmed death-ligand 1 and 2 (PD-L1 and PD-L2), cluster of differentiation 155 (CD155), B7 homolog 3 (B7-H3), sialic acid-binding Ig-like lectin 10 (Siglec-10), or signal-regulatory protein alpha (SIRPα), were selected and constructed into luciferase reporter cell lines. For tumor reporter cell lines, a gamma interferon activation site (GAS) response element was placed upstream of the luciferase gene in the lentiviral vector, enabling the activation of the JAK-STAT signaling pathway within tumor cells to induce luciferase expression. In myeloid reporter cell lines, a nuclear factor kappa B (NF-κB) response element replaced GAS to monitor the activation of the NF-κB signaling pathway. In the presence of corresponding immune checkpoint inhibitors that enhance T cell-mediated anti-tumor activity, these reporter cell lines produce a bioluminescent signal based on luciferase expression. This signal can be easily detected and quantified to assess the efficacy of the inhibitor. Our data revealed that bioluminescence intensity in the tumor and myeloid reporter cell lines increased by &amp;gt;100-fold in a dose-dependent manner in response to interferon gamma (IFN-γ) and tumor necrosis factor alpha (TNF-α) stimulation, respectively, and by &amp;gt;50-fold in response to the conditioned media collected from activated primary T cells. Furthermore, in co-culture assays involving various combinations of immune cell and tumor cell types with corresponding immune checkpoint inhibitors, these reporter cell lines demonstrated a significant increase in bioluminescence intensity. In conclusion, these newly established luciferase reporter cell lines offer an excellent ex vivo model for cancer immunotherapy. These cell lines naturally express immune checkpoint proteins, enabling the sensitive and reproducible monitoring of combinatorial responses from various immune cell types. Citation Format: Hyeyoun Chang, Alicia C. Walker, John G. Foulke, Luping Chen, Fang Tian, Zhizhan Gu. Luciferase reporter cell lines allow simultaneous incorporation of tumor cells, innate immune cells, and adaptive immune cells for in-depth immune checkpoint studies [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 75.

Abstract 7472: Melanoma-intrinsic expression of signal-regulatory protein alpha (SIRPα) confers tumor cells resistance to anti-tumor adaptive immunity

Abstract Introduction: Melanoma patient survival rates have significantly improved recently owing to the development of both targeted therapies and immunotherapies. However, there are still around 50% of patients relapse. The limited response to immunotherapies and global rise in melanoma incidence, with around 290,000 cases newly diagnosed each year, highlight the need for identifying new biomarkers, modulatory pathways, and risk factors for melanoma development. Earlier studies have characterized that aberrant expression of CD47 (a.k.a. “don’t eat me” signal) is one of the mechanisms that tumors exploit to avoid innate immune surveillance. CD47 blockade has been demonstrated to restore myeloid effector function by reinvigorating phagocytosis of tumor cells, which further enhances anti-tumor T cell responses. However, melanoma tumors are resistant to CD47 blockade. Interestingly, we and others have discovered that human and mouse melanoma cells express high levels of SIRPα, the receptor for CD47, which is typically expressed in myeloid compartments. Experimental Procedure: To investigate the role of tumor SIRPα, we designed gRNAs to CRISPR out the SIRPα N-terminal region, where it interacts with CD47, in B16 melanoma and generated SIRPα+/+ (WT) and SIRPα-/- (KO) B16 tumors. We conducted multiple in vitro and in vivo experiments to investigate whether tumor SIRPα played a role in tumor growth and modulated anti-tumor immunity. The preclinical tumor model we used is B16-F10 melanoma and the murine strains we used are the immune-competent (C57BL/6j; B6) and the immune-deficient (RAGnull) mice. For phenotypic and functional assessments, we performed analysis using flow cytometry, immunohistochemistry (IHC) and single cell RNA-sequencing (scRNA-seq) derived from live-sorted CD45+ tumor infiltrating lymphocytes (TILs) of SIRPα-WT and SIRPα-KO B16 tumors. Summary of Unpublished Data Our preliminary data in a preclinical B16-F10 melanoma model revealed that surface SIRPα expression is necessary for optimal B16-F10 melanoma growth in immune-competent mice, but not in immune-deficient RAGnull mice, suggesting a potential crosstalk between tumor SIRPα and the host adaptive immune system. Intriguingly, we found that selective SIRPα deletion in melanoma cells results in a significant increase of immune infiltrates within melanoma tumors that further suppresses tumor growth. Concluding Remarks: SIRPα is required for optimal melanoma development by suppressing anti-tumor adaptive immunity. Ethics Approval: All mice were maintained in microisolator cages and treated in accordance with the NIH and American Association of Laboratory Animal Care regulations. All mouse procedures and experiments for this study were approved by the Weill Cornell Medicine Institutional Animal Care and Use Committee. Citation Format: Chien-Huan Weng, Chana Schochet, Linda Hamadene, Shabnam Eghbali, Sébastien Monette, Levi Mangarin, Cailian Liu, Fadi Samaan, Jedd D. Wolchok, Taha Merghoub. Melanoma-intrinsic expression of signal-regulatory protein alpha (SIRPα) confers tumor cells resistance to anti-tumor adaptive immunity [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 7472.

Targeting HDAC6 improves anti-CD47 immunotherapy

BackgroundCancer cells can overexpress CD47, an innate immune checkpoint that prevents phagocytosis upon interaction with signal regulatory protein alpha (SIRPα) expressed in macrophages and other myeloid cells. Several clinical trials have reported that CD47 blockade reduces tumor growth in hematological malignancies. However, CD47 blockade has shown modest results in solid tumors, including melanoma. Our group has demonstrated that histone deacetylase 6 inhibitors (HDAC6is) have immunomodulatory properties, such as controlling macrophage phenotype and inflammatory properties. However, the molecular and cellular mechanisms controlling these processes are not fully understood. In this study, we evaluated the role of HDAC6 in regulating the CD47/SIRPα axis and phagocytosis in macrophages.MethodsWe tested the role of HDAC6is, especially Nexturastat A, in regulating macrophage phenotype and phagocytic function using bone marrow-derived macrophages and macrophage cell lines. The modulation of the CD47/SIRPα axis and phagocytosis by HDAC6is was investigated using murine and human melanoma cell lines and macrophages. Phagocytosis was evaluated via coculture assays of macrophages and melanoma cells by flow cytometry and immunofluorescence. Lastly, to evaluate the antitumor activity of Nexturastat A in combination with anti-CD47 or anti-SIRPα antibodies, we performed in vivo studies using the SM1 and/or B16F10 melanoma mouse models.ResultsWe observed that HDAC6is enhanced the phenotype of antitumoral M1 macrophages while decreasing the protumoral M2 phenotype. In addition, HDAC6 inhibition diminished the expression of SIRPα, increased the expression of other pro-phagocytic signals in macrophages, and downregulated CD47 expression in mouse and human melanoma cells. This regulatory role on the CD47/SIRPα axis translated into enhanced antitumoral phagocytic capacity of macrophages treated with Nexturastat A and anti-CD47. We also observed that the systemic administration of HDAC6i enhanced the in vivo antitumor activity of anti-CD47 blockade in melanoma by modulating macrophage and natural killer cells in the tumor microenvironment. However, Nexturastat A did not enhance the antitumor activity of anti-SIRPα despite its modulation of macrophage populations in the SM1 tumor microenvironment.ConclusionsOur results demonstrate the critical regulatory role of HDAC6 in phagocytosis and innate immunity for the first time, further underscoring the use of these inhibitors to potentiate CD47 immune checkpoint blockade therapeutic strategies.

Blockade of SIRPα-CD47 axis by anti-SIRPα antibody enhances anti-tumor activity of DXd antibody-drug conjugates.

Signal regulatory protein alpha (SIRPα) is an immune inhibitory receptor on myeloid cells including macrophages and dendritic cells, which binds to CD47, a ubiquitous self-associated molecule. SIRPα-CD47 interaction is exploited by cancer cells to suppress anti-tumor activity of myeloid cells, therefore emerging as a novel immune checkpoint for cancer immunotherapy. In blood cancer, several SIRPα-CD47 blockers have shown encouraging monotherapy activity. However, the anti-tumor activity of SIRPα-CD47 blockers in solid tumors seems limited, suggesting the need for combination therapies to fully exploit the myeloid immune checkpoint in solid tumors. Here we tested whether combination of SIRPα-CD47 blocker with antibody-drug conjugate bearing a topoisomerase I inhibitor DXd (DXd-ADC) would enhance anti-tumor activity in solid tumors. To this end, DS-1103a, a newly developed anti-human SIRPα antibody (Ab), was assessed for the potential combination benefit with datopotamab deruxtecan (Dato-DXd) and trastuzumab deruxtecan (T-DXd), DXd-ADCs targeting human trophoblast cell-surface antigen 2 and human epidermal growth factor receptor 2, respectively. DS-1103a inhibited SIRPα-CD47 interaction and enhanced antibody-dependent cellular phagocytosis of Dato-DXd and T-DXd against human cancer cells. In a whole cancer cell vaccination model, vaccination with DXd-treated cancer cells led to activation of tumor-specific T cells when combined with an anti-mouse SIRPα (anti-mSIRPα) Ab, implying the benefit of combining DXd-ADCs with anti-SIRPα Ab on anti-tumor immunity. Furthermore, in syngeneic mouse models, both Dato-DXd and T-DXd combination with anti-mSIRPα Ab showed stronger anti-tumor activity over the monotherapies. Taken together, this study provides a preclinical rationale of novel therapies for solid tumors combining SIRPα-CD47 blockers with DXd-ADCs.

JAK2 as a surface marker for enrichment of human pluripotent stem cells-derived ventricular cardiomyocytes

BackgroundHuman pluripotent stem cell (hPSC)-derived cardiomyocytes (CMs) hold great promise for cardiac disease modelling, drug discovery and regenerative medicine. Despite the advancement in various differentiation protocols, the heterogeneity of the generated population composed of diverse cardiac subtypes poses a significant challenge to their practical applications. Mixed populations of cardiac subtypes can compromise disease modelling and drug discovery, while transplanting them may lead to undesired arrhythmias as they may not integrate and synchronize with the host tissue's contractility. It is therefore crucial to identify cell surface markers that could enable high purity of ventricular CMs for subsequent applications.MethodsBy exploiting the fact that immature CMs expressing myosin light chain 2A (MLC2A) will gradually express myosin light chain 2 V (MLC2V) protein as they mature towards ventricular fate, we isolated signal regulatory protein alpha (SIRPA)-positive CMs expressing intracellular MLC2A or MLC2V using MARIS (method for analysing RNA following intracellular sorting). Subsequently, RNA sequencing analysis was performed to examine the gene expression profile of MLC2A + and MLC2V + sorted CMs. We identified genes that were significantly up-regulated in MLC2V + samples to be potential surface marker candidates for ventricular specification. To validate these surface markers, we performed immunostaining and western blot analysis to measure MLC2A and MLC2V protein expressions in SIRPA + CMs that were either positive or negative for the putative surface markers, JAK2 (Janus kinase 2) or CD200. We then characterized the electrophysiological properties of surface marker-sorted CMs, using fluo-4 AM, a green-fluorescent calcium indicator, to measure the cellular calcium transient at the single cell level. For functional validation, we investigated the response of the surface marker-sorted CMs to vernakalant, an atrial-selective anti-arrhythmic agent.ResultsIn this study, while JAK2 and CD200 were identified as potential surface markers for the purification of ventricular-like CMs, the SIRPA+/JAK2+ population showed a higher percentage of MLC2V-expressing cells (~ 90%) compared to SIRPA+/CD200+ population (~ 75%). SIRPA+/JAK2+ sorted CMs exhibited ventricular-like electrophysiological properties, including slower beating rate, slower calcium depolarization and longer calcium repolarization duration. Importantly, vernakalant had limited to no significant effect on the calcium repolarization duration of SIRPA+/JAK2+ population, indicating their enrichment for ventricular-like CMs.ConclusionOur study lays the groundwork for the identification of cardiac subtype surface markers that allow purification of cardiomyocyte sub-populations. Our findings suggest that JAK2 can be employed as a cell surface marker for enrichment of hPSC-derived ventricular-like CMs.

Induced pluripotent stem cell-derived cardiac myocytes to assess anthracycline cardiotoxicity

Abstract Background Current oncological treatments improved survival but also increased awareness of cardiovascular side-effects following cancer therapy. Anthracyclines are a class of highly effective chemotherapeutics, yet their use is regularly complicated by anthracycline-induced cardiotoxicity (AIC). The advent of induced pluripotent stem cell-derived cardiac myocytes (iPSC-CMs) offers the potential for disease modeling and prediction of genetic cardiomyopathies. Purpose We aim to use iPSC-CMs to assess susceptibility to cardiotoxicity induced by anti-cancer treatments on an individual patient level. Furthermore, the assay could be exploited to unravel cellular processes and pathways involved in AIC. Methods Five heart transplant patients following AIC and five anthracycline treated patients without AIC were included in our study. We reprogrammed patient-derived lymphocytes from both groups toward iPSCs using Sendai Virus; afterwards, patient derived iPSCs were differentiated into CM-like cells. Cytotoxicity following chemotherapy exposure was assessed using flow cytometry (FC), in which iPSC-CM-specific cells were first selected based on the expression of signal-regulatory protein alpha (SIRPA) surface marker; thereafter the percentage of apoptotic versus viable cells following doxorubicin exposure was quantitatively assessed. Ultrastructural changes were evaluated using electron microscopy (EM) images, comparing doxorubicin treated and untreated iPSC-CMs. Reactive Oxygen Species (ROS) generation following doxorubicin exposure was assessed using a luciferin precursor-based plate reader assay (ROS-GLO) and FC. Furthermore, whole genome sequencing (WGS) was performed and variants were filtered based on allele frequency, pathogenicity using HumVar trained variant effect predictors and presence in patients versus controls. Results The cytotoxic effect of increasing concentrations of doxorubicin was quantifiable using FC on SIRPA positive iPSC-CMs (Figure 1 panel A). On the ultrastructural level, doxorubicin treated iPSC-CMs manifested darker and more condensed mitochondria as seen with EM (Figure 1 panel B). Increased ROS production was observed following doxorubicin exposure as assessed using ROS-GLO plate reader assay and FC (Figure 1 panel C). We used iPSC-CMs of transplant patients and matched controls to assess inter-individual susceptibility to anthracycline-induced cardiotoxicity. Included patient profiles can be found in Figure 2 panel A. There was a higher rate of cell death at any concentration of doxorubicin in patients with proven AIC as opposed to controls (Figure 2 panel B). WGS analysis showed 2 pathogenic variants in the PRAMEF6 gene present in cases and not in controls (Figure 3 panel C). Conclusion iPSC-CMs represent a robust disease model to study cancer therapy-induced cardiotoxicity. Furthermore, iPSC-CMs allow to study inter-individual differences in susceptibility to cancer therapy-induced cardiotoxicity.Mechanism of AICSusceptibility to AIC