Flow Cytometric Analysis Research Articles

Abstract 5816: An effective delivery platform for nucleic acid therapeutics: Enhanced cancer cell internalization and endosomal escape

Abstract Nucleic acid therapeutics (NATs), such as siRNA and mRNA, show great potential as cancer medicines. However, translation of these compounds has been held back by their limited bioavailability in target tissues. The aim of this study was to test the use of a novel cell-penetrating peptide (CPP)-based delivery platform to enhance uptake of NAT into cancer cells. This platform consists of a highly efficient CPP that combines the advantages of multimerization and cyclization of the archetypal CPP, TAT peptide1, plus a photochemical (PC) strategy to promote endosomal release of NAT into the cytoplasm2. Negatively charged NATs form complexes with cationic CPPs through charge:charge interaction. NATs (siRNA or mRNA) were incubated with CPP at different molar ratios and assessed in gel retardation assays. Efficiency of internalization of Cy3-NAT:CPP complexes was evaluated using live cell confocal imaging with quantification of intracellular fluorescence. The mechanism of cellular uptake of NAT:CPP was investigated using inhibitors of endocytosis (chlorpromazine, NaN3, amiloride and methyl-β-cyclodextrin). To trigger NAT release from intracellular vesicles, 420 nm/50 mA LED light irradiation was applied following pre-treatment of cells with a photosensitizer, fimaprofin. CPP-PC mediated internalization and endosomal release was used for delivery of glyceraldehyde 3-phosphate dehydrogenase (GAPDH) siRNA. GAPDH enzymatic activity assays (Sigma, USA) were conducted to confirm inhibition of GAPDH in HeLa, HEK293T and MDA-MB-231 cells. Transfection efficiency of mRNA encoding enhanced green fluorescence protein (eGFP) was evaluated by flow cytometry in HeLa cells. NAT:CPP complexes were found to be maximally efficient at a 10:1 molar ratio. Analysis of live cell confocal images indicated that GAPDH Cy3-siRNA:CPP complexes were more efficiently internalized compared to Cy3-siRNA + monomeric TAT or Cy3-siRNA + lipofectamine (****p<0.0001, one-way ANOVA). The combined CPP-PC platform delivers NAT via a clathrin-mediated endocytosis pathway. CPP-PC delivery of GAPDH siRNA caused marked GAPDH enzyme activity downregulation in HeLa (91%), MDA-MB-231 (51%) and HEK293T (44%) cells. These results were similar to the level of downregulation achieved with lipofectamine + GAPDH siRNA. Flow cytometric analysis revealed that 52.4% of HeLa cells expressed eGFP at 1 h following delivery of mRNA-eGFP by CPP-PC. Functional siRNA and mRNA were efficiently delivered into the cytoplasm of cancer cells through combined use of a multimeric/cyclized CPP plus photochemical-mediated endosomal release. This approach holds promise for clinical application, particularly in the treatment of intraluminal tumors. 1. Tietz, O. et al. Nat Chem. 14: 284-93, 2022. 2. Selbo, P. K. et al. J Control Release. 148, 2-12, 2010. Citation Format: Siqi Li, Ole Tietz, Afaf H. El-Sagheer, Tom Brown, Katherine A. Vallis. An effective delivery platform for nucleic acid therapeutics: Enhanced cancer cell internalization and endosomal escape [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 5816.

Abstract 105: Understanding the role of β2-adrenergic receptor signaling on type II innate lymphoid cells in cancer

Abstract Increased norepinephrine (NE) due to chronic stress can activate adrenergic receptors, specifically β2-adrenergic receptors (β2AR), to produce an immunosuppressive tumor microenvironment (TME). Previous work by our lab demonstrated that tumor burden can be reduced by blocking β2AR signaling. Immune cells express β2AR and we have shown that the increased β2AR signaling leads to increased T cell exhaustion and increased inhibitory activity of myeloid-derived suppressor cells (MDSCs). In general, the resulting immunosuppression leads to a modest but significant reduction in tumor growth. However, the lack of a dramatic reduction in tumor growth reveals the complex nature of adrenergic signaling dependent regulation of the cancer immune response. Recent literature revealed that innate lymphoid cells (ILCs), especially type II ILCs (ILC2s), express high levels of β2AR. Therefore, we evaluated whether β2AR regulates ILC2s within the TME and how that affects tumor prognosis. We generated a genetic conditional knockout murine model where β2AR expression in ILC2s was deleted by crossing IL5cre expressing mice to β2ARflox/flox mice (IL5creβ2ARf/f). AT3 breast cancer cell line was injected into the 4th mammary fat pad and tumor volume was measured every 2-3 days. Tumor infiltrating ILC2s (CD45+Lineage−CD90.2+CD127+ST2+) were analyzed by flow cytometry at D36 post-tumor implantation. Notably, IL5creβ2ARf/f mice had increased AT3 breast cancer tumor growth compared to control mice. Flow cytometric analysis of the tumor revealed an increased frequency of GATA3+ ILC2s within the TME. These GATA3+ ILC2s lacking β2AR expressed higher levels of the IL33 receptor, ST2, and a higher percentage of IL13+ cells compared to WT GATA3+ ILC2 cells. We also observe a trend toward increased MDSC population within the TME of mice lacking β2AR in ILC2s. These results reveal an anti-tumor function of β2AR signaling through the suppression of ILC2 activity within the TME that warrants further investigation. This work has been in part supported by NIH Grants F30 CA284763 (to J.C.), K99 HL155792 (to H.M.), and RO1 CA205246 (to E.R.) Citation Format: Jee Eun Choi, Cameron R. MacDonald, Saeed Daneshmandi, Jianmin Wang, Nathan T. Roberts, Caitlin M. James, Philip L. McCarthy, Hemn Mohammadpour, Elizabeth A. Repasky. Understanding the role of β2-adrenergic receptor signaling on type II innate lymphoid cells in cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 105.

Abstract 7260: An HSV-1-based vector for local delivery of IL-12 and IL-2 reshapes the immune landscape leading to tumor clearance and systemic immune surveillance

Abstract Clinical use of recombinant interleukin (IL)-12 and -2 therapy has been hindered due to unfavorable kinetics and toxicity associated with systemic exposure. Durable targeted delivery to the tumor microenvironment (TME) is a promising therapeutic strategy to overcome these clinical limitations, prompting the development of KB707, a replication-defective herpes simplex virus type 1 (HSV-1)-based vector encoding human IL-12 and -2, for the treatment of solid tumors. Prior studies in mice demonstrated that local vector-encoded cytokine administration enhanced murine IL-12/IL-2 expression while limiting systemic exposure compared to conventional protein therapy. In a syngeneic, B16-F10 melanoma mouse model, intratumoral murine KB707-equivalent treatment led to improved survival in a unilateral tumor model and an abscopal effect in a bilateral tumor model. Additionally, the majority of previously treated animals survived rechallenge with B16-F10 in the absence of further therapy, indicative of systemic anti-tumor immune memory. Together, these results suggested that vector-mediated delivery of IL-12 and -2 could elicit both local and systemic anti-tumor immune responses. To elucidate the underlying changes to the immune landscape, B16-F10 tumor-bearing animals were treated intratumorally with vector or vehicle control on day 0 and were sacrificed on day 7; a second cohort of tumor-bearing animals received two vector doses (days 0 and 7) and were sacrificed on days 8, 10, 12, or 14. Immune cell profiling of samples taken from the tumor, tumor draining lymph node (dLN), spleen, and whole blood were analyzed by flow cytometry, and serum cytokine analysis was conducted by multiplex ELISA. Flow cytometric analysis of tumors revealed that vector treatment resulted in increased frequencies of proliferating and activated effector CD8+ and CD4+ T cells, peaking 24 hours post-second dose. These results correlated with high levels of the proinflammatory cytokines IFNγ and TNFα in the serum of vector recipient animals. The blood, spleens, and dLNs also showed increased frequencies of activated effector CD8+ and CD4+ T cells, peaking 24-72 hours post-second dose. Interestingly, although IL-2 alone has been shown to promote immune suppression and tumor outgrowth by mediating regulatory T cell (Treg) expansion and infiltration in certain contexts, the frequency of Tregs in treated vs. control tumors was substantially reduced after first and repeat dosing. Together, these results support a mechanism by which vector-driven local expression of IL-12 and IL-2 can expand and activate effector T cells both in the TME and systemically while simultaneously repressing Tregs, consistent with the previously described inhibition of primary tumor outgrowth, abscopal effects on secondary tumors, and prevention of recurrence observed with this therapeutic approach. Citation Format: Dana M. Previte, Trevor J. Parry, Suma M. Krishnan. An HSV-1-based vector for local delivery of IL-12 and IL-2 reshapes the immune landscape leading to tumor clearance and systemic immune surveillance [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 7260.

Encapsulating Epigallocatechin-3-Gallate (EGCG) in Chitosan Nanoparticles Influenced Multiple Genes Controlling Oncostatic Signaling Pathways in HepG2 Cells

AbstractThe primary polyphenol in green tea, epigallocatechin-3-gallate (EGCG), is the primary active polyphenol in green tea that showed a remarkable anticancer effect in a variety of cancer types including liver cancer. However, its anticancer impact on gene expressions related to liver cancer proliferation and apoptosis is limited. The current study investigated the oncostatic influence of chitosan encapsulating EGCG (Chit-nanoEGCG) on human hepatocellular carcinoma HepG2 cells compared with its native form and the conventional anticancer drug cisplatin in vitro. The Chit-nanoEGCG caused a dose-dependent decrease in cell viability and significantly induced apoptosis compared with control. Flow cytometric analysis confirmed the results of RT-qPCR, displaying a significant increase in the expression of transcriptomes (P53, Bax, Caspase-3, Caspase-9, and PARP) with a significant decrease in the antiapoptotic Bcl-2 gene. Also, there was a significant decrease in transcription genes (OCT4 and SOX2) and receptor genes (CD133, NOTCH1, c-MET, and Ezrin) but an increase in CD95 expression. Furthermore, there was a highly significant decrease in oncogenes (mTOR, PI3K, RALA, and BMI) in HepG2 cells after Chit-nanoEGCG treatment when compared with the control group. The current findings indicate that Chit-nanoEGCG had a significant impact on a number of genes involved in controlling signaling pathways to inhibit the proliferation and development of HepG2 cells. In addition, encapsulating EGCG in chitosan nanoparticles increases its antitumor effectiveness compared to its native form, which encourages the use of various polyphenols in nanochemotherapy for cancer treatment. The finding that the nanoparticles (NPs) influenced the expression of genes involved in apoptosis and proliferation enables the development of medical chemotherapeutic agent for the treatment of hepatocellular carcinoma. Graphical Abstract

Abstract 6330: A novel monoclonal antibody for the detection, enrichment and activation of cells expressing scFv-based chimeric antigen receptors

Abstract Introduction: Chimeric Antigen Receptor (CAR)-T cell therapy is a highly innovative form of immunotherapy that has proven to be successful in the treatment of B cell malignancies and multiple myeloma. As this treatment modality continues to evolve toward the targeting of novel tumor antigens and engineering of CAR constructs that enhance cell persistence, there is a need for reagents that can be leveraged to selectively interrogate multiple aspects of CAR-T cell biology. Here, we report on a novel and highly versatile recombinant rabbit monoclonal antibody raised against the Gly4Ser peptide linker, which is commonly integrated into single-chain variable fragment (scFv)-based CARs. This antibody can be leveraged for flow cytometry-based CAR detection, bead-based CAR-T cell enrichment, and selective activation of CAR transduced cells. Methods: The recombinant monoclonal antibody, E7O2V, was generated by immunizing rabbits with a synthetic peptide containing multiple repeats of the Gly4Ser core pentapeptide. E7O2V was directly conjugated to a panel of fluorophores and validated for specificity in a live cell flow cytometry assay using non-transduced and CAR-transduced primary human T cells. To assess its utility in cell enrichment using bead-based sorting, a biotinylated conjugate of E7O2V was used in combination with streptavidin releasable magnetic meads to immunoaffinity purify CAR transduced cells from a mixed population. Finally, a plate-bound antibody stimulation assay was leveraged to assess the ability of E7O2V to selectively activate CAR transduced cells. Results: Live cell flow cytometric analysis of CAR-transduced primary human T cells revealed that E7O2V could detect surface expressed CARs, independent of scFv specificity. No specific staining was observed on non-transduced cells. Furthermore, flow cytometric analysis of bead-free, purified cells revealed that biotinylated E7O2V could enrich CAR transduced cells to a high degree of purity and viability. E7O2V was found to selectively activate CAR transduced cells, but not non-transduced cells, as demonstrated by upregulation of cell surface activation markers, including CD69 and CD25. Conclusions: Exploiting the commonly used Gly4Ser linker of scFv-based CARs for antibody discovery, we identified a novel and highly versatile monoclonal antibody, E7O2V. This antibody binds to CARs of varying antigen specificity and can be leveraged in multiple assay formats to interrogate various attributes of CAR transduced cells, including CAR expression, CAR signaling, and transcriptome profiling enabled by bead-based enrichment. Citation Format: Amrik Singh, Sarah L'Heureux, Jeremy Fisher. A novel monoclonal antibody for the detection, enrichment and activation of cells expressing scFv-based chimeric antigen receptors [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 6330.

Abstract 125: Development of humanized VEGFA models to assess preclinical efficacy of novel therapeutics in vivo

Abstract VEGFA is an essential growth factor required for angiogenesis in the context of normal wound healing as well as malignant tissue growth, ultimately facilitating the delivery of oxygen and nutrients to fuel cellular survival, expansion, and repair. VEGFA signaling has also been reported to exert effects on cancer cells and immune cells directly, with evidence suggesting that the pathway can both support tumor growth and inhibit immune surveillance. Thus, targeting VEGFA has been intensely explored as a therapeutic strategy for several cancer indications. Here, we generated a novel humanized VEGFA model (B-hVEGFA mice) to explore the ability of novel VEGFA-targeting therapeutics to curb cancer cell growth and angiogenesis in vivo. This was achieved by replacing the murine Vegfa gene with the corresponding human VEGFA coding region. We confirmed exclusive expression of human VEGF in lung homogenates of the humanized model at levels comparable to murine VEGF quantified in wild-type C57BL/6 mice. Immunohistochemical analysis at 8 weeks of age demonstrates no significant difference in the CD31+ area in several organs of the humanized mice when compared to wild-type C57BL/6 mice, including kidney, heart, liver, and eye. To assess the functional utility of this model in evaluating VEGFA modulators, tumor studies were conducted: murine colon cancer MC38 cells that were also engineered to express VEGFA in a similar manner (B-hVEGFA MC38 cells) were subcutaneously implanted into homozygous B-hVEGFA mice. Treatment with bevacizumab analog essentially abolished tumor growth in the humanized mice, while control tumors continued to expand over the 3 week study. To further evaluate the role of novel combination therapies or bispecific antibodies targeting VEGFA and PD-1, we also generated a triple humanized model expressing human VEGF, PD-1, and PD-L1 (B-hPD-1/hPD-L1/hVEGFA mice) by crossing B-hVEGFA mice with our B-hPD-1/hPD-L1 mice model, which expresses the extracellular domains of human PD-1 and PD-L1. Flow cytometric analysis of this model is currently underway to validate the model for further efficacy testing. Together, these humanized VEGF models provide robust in vivo platforms to evaluate novel VEGFA therapeutic candidates. Citation Format: Shujin Zhang, Chonghui Liu, Jin Guo, Zhiyuan Shen, James Jin, Bahetiyaer Huwatibieke, Yang Shen. Development of humanized VEGFA models to assess preclinical efficacy of novel therapeutics in vivo [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 125.

Abstract 100: Blockade of CCL5 and CXCL10 signaling as a novel immunotherapy for epithelial ovarian cancer

Abstract Background: Cancer immune evasion remains a major barrier in developing enduring therapies for epithelial ovarian cancer (EOC). Expression of programmed death-ligand 1 (PD-L1) is known for its immunosuppressive role in cancer, thus interferences to PD-L1 have shown marked efficacy against many advanced cancers. However, the mechanisms by which PD-L1-negative cancers evade immune response remain unelucidated. We have found chemokine (C-C motif) ligand 5 (CCL5) and (C-X-C motif) ligand 10 (CXCL10) to up-regulate in PD-L1-negative EOC. Therefore, the present study aims to investigate blockade of CCL5/CXCL10 signaling to overcome immune evasion of EOC. Methods: Isogenic BRCA2-mutated PEO1 and BRCA2-wild type PEO4 EOC cells were treated with IFNγ, olaparib, or both in combination for western blot and flow cytometric analyses of PD-L1 expression. The conditioned medium of PEO1 and PEO4 cells was analyzed for immune array analysis to determine and compare the levels of 36 cytokines. PEO1 and PEO4 cells were co-cultured with CD3/CD28 antibodies-activated human peripheral blood mononuclear cells (PBMCs). Then, PBMCs were stained with anti-CD8 or CD4 and CD25 antibodies for flow cytometric analyses of cytotoxic or helper T cells, respectively. PEO1 and PEO4 cells were co-cultured with activated PBMCs in the presence and absence of TAK-779 or AMG-487. Thereafter, PBMCs were stained with anti-CD4, CD25, and FOXP3 antibodies for flow cytometric analyses of regulatory T cells (Tregs). PEO1 and PEO4 cells were transfected with CCL5- or CXCL10-siRNA and subsequently co-cultured with activated SUPT1 cells or PBMCs. The conditioned medium was collected and analyzed for the levels of IFNγ by ELISA. Results: PEO1 cells exhibited an up-regulation of basal and IFNγ-induced PD-L1 compared with PEO4 cells. PEO4 cells displayed a pronounced increase in the CCL5 and IFNγ-induced CXCL10 compared with PEO1 cells. Co-culture of PBMCs with PEO1 and PEO4 cells led to a significant decrease in activated CD8+ effector T and CD4+ helper T cells. However, co-culture of PBMCs with PEO4 cells caused a significant increase in activated CD4+ helper T cells. Co-culture of PBMCs with PEO4 cells caused a significant increase in FOXP3+CD4+CD25+ Tregs. This increase in Tregs was significantly reduced by the treatment with the CCL5/CCR5 antagonist TAK-779 or the CXCL10/CXCR3 antagonist AMG-487. Silencing of CCL5 or CXCL10 in PEO4 cells by siRNA significantly increased IFNγ production by SUPT1 cells and PBMCs. Conclusions: PEO4 cells secrete CCL5 and IFNγ-induced CXCL10 to activate Tregs, enabling tumor cells to evade host immune response by dysregulating their microenvironment. PEO4 cells-induced Tregs are reduced by CCL5/CXCL10 signaling blockade with TAG-779 or AMG-487. Furthermore, silencing of CCL5 or CXCL10 in PEO4 cells with siRNA abolishes Treg-mediated immunosuppression and restores the antitumor activity of effector T cells. Citation Format: Annabelle C. Lin, Jake Moscarelli, Z. Ping Lin, Elena S. Ratner. Blockade of CCL5 and CXCL10 signaling as a novel immunotherapy for epithelial ovarian cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 100.

Abstract 6596: BRMS1 downregulation alters the tumor microenvironment and induces immunotherapy resistance in lung adenocarcinoma

Abstract Background: Lung cancer is the leading cause of cancer-related deaths globally. During cancer progression, tumor cells interact with tumor-infiltrating immune cells (TIICs) creating an immunosuppressive microenvironment. Therefore, it is essential to delineate the communication dynamics between cancer cells and TIICs to identify novel pathways for therapeutic targeting. BRMS1 is a metastasis suppressor gene that is frequently downregulated in lung adenocarcinoma (LUAD). We, and others, have shown that loss of BRMS1 results in increased distant metastatic disease. Given the strong correlation between immune suppression, metastasis, and immunotherapy resistance we sought to elucidate if BRMS1 contributes to these processes. Specifically, we hypothesized that loss of BRMS1 in LUAD cells influences the immune cell composition, shaping an immunosuppressive microenvironment and decreasing tumor response to immunotherapy. Methods: To experimentally address our hypothesis, we generated KrasG12D P53fl/fl Brms1−/− mice inducing spontaneous tumors with Ad-Cre intratracheal inoculation. RNA-seq scRNA-seq on Brms1+/+ and Brms1−/− tumors, coupled with cell type annotations using mMCP-counter and DCATS were used to identify diverse TIIC subsets. To validate our observation, we performed immunofluorescence and flow cytometry to assess the number of TIICs. To explore the impact of Brms1 loss on the efficacy of anti-PD1 immunotherapy, a syngeneic CMT167 mouse model with BRMS1KD cells implanted in the flank was treated with an anti-PD1 antibody. Results: Differential gene expression analysis of RNA-seq data revealed a reduction in CCL9, CCL24, CLEC1B, PYCARD and other proinflammatory molecules in Brms1−/− tumors. Gene ontology and GSEA highlighted a diminished immune response signature in Brms1−/- tumors. In contrast, hallmark gene sets like IFNγ response, IL6 signaling, and inflammation are highly enriched in Brms1+/+ tumors. mMCP-counter suggested a decrease in CD8+ T cells in Brms1−/- tumors. Likewise, scRNA-seq data confirmed a significant decrease in cytotoxic CD8+ T cells in the Brms1−/− mice. Immunofluorescence and flow cytometric analysis showed a reduction in cytotoxic CD8+ T cells with low proliferative potential (Ki67+) in the Brms1−/− mice. Postmortem on our syngeneic mouse tumors after a 30-day anti-PD1 antibody treatment revealed a significantly larger mean tumor size in the BRMS1KD group compared to the WT group (942.4 mm³ vs. 184 mm³; p<0.023). Immunophenotyping on these tumors using flow cytometry also demonstrated reduced cytotoxic CD8+ T cells in the KD group post-treatment. Conclusion: Our data suggest that reduced BRMS1 expression in LUAD alters CD8+ T cell composition and primary tumor response to immunotherapy. Implications of our findings underscore the potential of BRMS1 as a therapeutic target in the context of immunotherapy-resistant LUAD. Citation Format: Manendra B. Lankadasari, Yuan Liu, Brooke Mastrogiacomo, Yingjie Zhu, Elizabeth G. Dunne, Cameron N. Fick, Choudhary Harsha, Jorge S. Reis-Filho, David R. Jones. BRMS1 downregulation alters the tumor microenvironment and induces immunotherapy resistance in lung adenocarcinoma [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 6596.

Naringenin Inhibits Acid Sphingomyelinase-Mediated Membrane Raft Clustering to Reduce NADPH Oxidase Activation and Vascular Inflammation.

Macrophage inflammation and oxidative stress promote atherosclerosis progression. Naringenin is a naturally occurring flavonoid with antiatherosclerotic properties. Here, we elucidated the effects of naringenin on monocyte/macrophage endothelial infiltration and vascular inflammation. We found naringenin inhibited oxidized low-density lipoprotein (oxLDL)-induced pro-inflammatory cytokines such as IL-1β, IL-6, and TNF-α toward an M2 macrophage phenotype and inhibited oxLDL-induced TLR4 (Toll-like receptor 4) membrane translocation and downstream NF-κB transcriptional activity. Results from flow cytometric analysis showed that naringenin reduced monocyte/macrophage infiltration in the aorta of high-fat-diet-treated ApoE-deficient mice. The aortic cytokine levels were also inhibited in naringenin-treated mice. Further, we found that naringenin reduced lipid raft clustering and acid sphingomyelinase (ASMase) membrane gathering and inhibited the TLR4 and NADPH oxidase subunit p47phox membrane recruitment, which reduced the inflammatory response. Recombinant ASMase treatment or overexpression of ASMase abolished the naringenin function and activated macrophage and vascular inflammation. We conclude that naringenin inhibits ASMase-mediated lipid raft redox signaling to attenuate macrophage activation and vascular inflammation.

Abstract 4099: AMP-peptide vaccination against multiple p53 mutant epitopes promotes lymph node delivery to generate potent, functional T cell immunity

Abstract Background p53 is the most commonly mutated gene in human cancer, prevalent in nearly all tumor types. Despite its significant medical relevance and the knowledge that human T cells recognize these mutations[1,2], immunotherapies designed to promote responses against mutated p53 (mp53) have not had the hoped-for clinical impact[3]. The Amphiphile (AMP) platform improves the potency of vaccine immunotherapy by programming the delivery of vaccine components to the lymph nodes where efficient uptake by immune cells initiates tumor-targeted immune responses. AMP-modification of vaccine components (peptide antigens, molecular adjuvants) results in covalent conjugation to albumin-binding lipids. Upon injection, AMP-vaccines associate with tissue-resident albumin which efficiently distributes to draining lymph nodes. This approach was shown to promote activation of polyfunctional, cytotoxic T cells with promising safety and improved clinical outcomes in a Phase 1 trial of ELI-002, an mKRAS AMP therapeutic vaccine (AMPLIFY-201 NCT05726864). Application of this strategy to p53 mutations with ELI-008 offers the potential for improved immunotherapeutic activity in a setting of significant unmet need. Methods Following immunization of C57BL/6J mice with AMP-modified or soluble comparator vaccines consisting of mp53 peptides and CpG adjuvant, analyses were performed 7 days after the third bi-weekly dose. To assess antigen-specific T cell responses, ELISpot (IFNγ, GzmB), multiplexed proteomic, and flowcytometric analysis of effector cytokines (IFNγ, TNFα, IL-2) were performed following antigenic stimulation. Responses were determined in secondary lymphoid tissues and lung. Cytolytic capabilities of antigen-specific T cells were evaluated by monitoring specific killing of IV-transferred antigen-pulsed target cells. Results AMP-immunization generated robust immune responses yielding strong T cell activation against common p53 hot spot mutations (R248W, R248Q, R175H, G245S, R273H, Y220C, C135Y, R158H, H214R). Responses to AMP-vaccination were characterized by the generation of increased frequency of polyfunctional T cells (IFNγ, TNFα, IL2) specific to mp53 epitopes. Induced T cells demonstrated significant levels of cytolytic activity including GzmB production and elimination of target cells in vivo. Non-lymph targeted vaccines using soluble comparators were inactive. Conclusions AMP-conjugation permits efficient delivery directly to the lymph nodes and thus improves the immunogenicity of peptide vaccination. These substantially improved immune responses induced by ELI-008 represent a promising therapeutic opportunity for targeting cancer in a large fraction of human tumors. The AMP-platform technology is simple, rapid and scalable, promising broad clinical, off-the-shelf application for treating p53 mutated tumors. Citation Format: Martin P. Steinbuck, Xavier Cabana-Puig, Erica Palmer, Mimi M. Jung, Thomas Williams, Kristen Osaer, Jeff Zhang, Christopher M. Haqq, Peter C. DeMuth. AMP-peptide vaccination against multiple p53 mutant epitopes promotes lymph node delivery to generate potent, functional T cell 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 4099.

Abstract 656: A novel dual-targeting peptide HS1002 enhances anti-tumor activity via GnRH/human telomerase reverse transcriptase (hTERT) interaction in human prostate cancer cells

Abstract Human telomerase reverse transcriptase (hTERT) is overexpressed in most human cancers. hTERT is considered as an important target for cancer therapy. In the present study, a new peptide HS1002 were synthesized with sequence of pGlu-His-Trp-Ser-Tyr-Arg-Leu-Arg-Phe-Ile-Pro-NHEt based on the GnRH agonist and hTERT. The aim of this study was to evaluate HS1002 for its anticancer activity by dual-targeting to GnRH/hTERT in prostate cancer cells. Upon comparison with different prostate cancer cell lines, HS1002 exhibited highest cytotoxicity against LNCaP cells. The hTERT expression correlated with telomerase activity in LNCaP cells, was significantly inhibited by HS1002, resulting in reduction of proliferation, metastasis and increase in apoptosis. Additionally, HS1002 suppressed c-Myc and AKT/ERK pathway in LNCaP cells. In the tumor-bearing nude mice model, HS1002 significantly inhibited tumor growth and downregulated the expression of hTERT and Ki-67 in tumor tissue as evidenced by western blot and immunohistochemistry analysis, respectively. Moreover, repeated subcutaneous injection of HS1002 resulted in the reduction of both serum testosterone levels and seminal vesicle weight. HS1002 also increased cytosolic calcium influx and CRE-luciferase activity in GnRHR-overexpressing HEK293 cells compared to mock-transfected transfected cells. Furthermore, a potent cytotoxicity in LNCaP cells was exhibited by HS1002-activated peripheral blood mononuclear cell (PBMC) with interleukin (IL)-2 using LDH release assay. In addition, a flow-cytometric analysis confirmed an elevation in the production of granzyme B and IFN-γ in CD8+ T cells in the MC38 syngeneic mouse model by HS1002. These data indicate that the inhibition of hTERT expression by HS1002 suppresses prostate cancer cell growth and induced anti-cancer immunity. Therefore, HS1002 might be used as a novel therapeutic drug for prostate cancer. Citation Format: Jae Hyeon Park, Ju Ri Kim, Hwa Young Cha, Hyung Sik Kim. A novel dual-targeting peptide HS1002 enhances anti-tumor activity via GnRH/human telomerase reverse transcriptase (hTERT) interaction in human prostate cancer cells [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 656.

Cx3Mab-4: A Novel Anti-Mouse CXCR3 Monoclonal Antibody for Flow Cytometry.

C-X-C motif chemokine receptor 3 (CXCR3, CD183) is a G-protein-coupled receptor for CXCL9, CXCL10, and CXCL11. CXCR3 induces chemotaxis of immune cells and promotes inflammation. Various mouse models have been developed to mimic the pathogenesis of diseases and used in the evaluation of therapeutics for these diseases. Although CXCR3 is an attractive target to suppress inflammation, anti-CXCR3 therapeutic agents have not been approved. In this study, we established a novel anti-mouse CXCR3 (mCXCR3) monoclonal antibody, Cx3Mab-4 (rat IgG1, kappa), using the Cell-Based Immunization and Screening method. Flow cytometric analysis demonstrated that Cx3Mab-4 bound to mCXCR3-overexpressed Chinese hamster ovary-K1 (CHO/mCXCR3) cells, but did not react to parental CHO-K1 cells. The dissociation constant of Cx3Mab-4 was determined as 1.3 × 10-9 M, indicating that Cx3Mab-4 possesses a high affinity to mCXCR3-expressing cells. Cx3Mab-4 could be useful for targeting CXCR3-expressing cells in preclinical mouse models.

TMEM120B strengthens breast cancer cell stemness and accelerates chemotherapy resistance via β1-integrin/FAK-TAZ-mTOR signaling axis by binding to MYH9

BackgroundBreast cancer stem cell (CSC) expansion results in tumor progression and chemoresistance; however, the modulation of CSC pluripotency remains unexplored. Transmembrane protein 120B (TMEM120B) is a newly discovered protein expressed in human tissues, especially in malignant tissues; however, its role in CSC expansion has not been studied. This study aimed to determine the role of TMEM120B in transcriptional coactivator with PDZ-binding motif (TAZ)-mediated CSC expansion and chemotherapy resistance.MethodsBoth bioinformatics analysis and immunohistochemistry assays were performed to examine expression patterns of TMEM120B in lung, breast, gastric, colon, and ovarian cancers. Clinicopathological factors and overall survival were also evaluated. Next, colony formation assay, MTT assay, EdU assay, transwell assay, wound healing assay, flow cytometric analysis, sphere formation assay, western blotting analysis, mouse xenograft model analysis, RNA-sequencing assay, immunofluorescence assay, and reverse transcriptase-polymerase chain reaction were performed to investigate the effect of TMEM120B interaction on proliferation, invasion, stemness, chemotherapy sensitivity, and integrin/FAK/TAZ/mTOR activation. Further, liquid chromatography–tandem mass spectrometry analysis, GST pull-down assay, and immunoprecipitation assays were performed to evaluate the interactions between TMEM120B, myosin heavy chain 9 (MYH9), and CUL9.ResultsTMEM120B expression was elevated in lung, breast, gastric, colon, and ovarian cancers. TMEM120B expression positively correlated with advanced TNM stage, lymph node metastasis, and poor prognosis. Overexpression of TMEM120B promoted breast cancer cell proliferation, invasion, and stemness by activating TAZ-mTOR signaling. TMEM120B directly bound to the coil-coil domain of MYH9, which accelerated the assembly of focal adhesions (FAs) and facilitated the translocation of TAZ. Furthermore, TMEM120B stabilized MYH9 by preventing its degradation by CUL9 in a ubiquitin-dependent manner. Overexpression of TMEM120B enhanced resistance to docetaxel and doxorubicin. Conversely, overexpression of TMEM120B-∆CCD delayed the formation of FAs, suppressed TAZ-mTOR signaling, and abrogated chemotherapy resistance. TMEM120B expression was elevated in breast cancer patients with poor treatment outcomes (Miller/Payne grades 1–2) than in those with better outcomes (Miller/Payne grades 3–5).ConclusionsOur study reveals that TMEM120B bound to and stabilized MYH9 by preventing its degradation. This interaction activated the β1-integrin/FAK-TAZ-mTOR signaling axis, maintaining stemness and accelerating chemotherapy resistance.

The fluorochrome-to-protein ratio is crucial for the flow cytometric detection of tissue factor on extracellular vesicles

Extracellular vesicles (EVs) have crucial roles in hemostasis and coagulation. They sustain coagulation by exposing phosphatidylserine and initiate clotting by surface expression of tissue factor (TF) under inflammatory conditions. As their relevance as biomarkers of coagulopathy is increasingly recognized, there is a need for the sensitive and reliable detection of TF+ EVs, but their flow cytometric analysis is challenging and has yielded controversial findings for TF expression on EVs in the vascular system. We investigated the effect of different fluorochrome-to-protein (F/P) ratios of anti-TF-fluorochrome conjugates on the flow cytometric detection of TF+ EVs from activated monocytes, mesenchymal stem cells (MSCs), and in COVID-19 plasma. Using a FITC-labeled anti-TF antibody (clone VD8), we show that the percentage of TF+ EVs declined with decreasing F/P ratios. TF was detected on 7.6%, 5.4%, and 1.1% of all EVs derived from activated monocytes at F/P ratios of 7.7:1, 6.6:1, and 5.2:1. A similar decline was observed for EVs from MSCs and for EVs in plasma, whereas the detection of TF on cells remained unaffected by different F/P ratios. We provide clear evidence that next to the antibody clone, the F/P ratio affects the flow cytometric detection of TF+ EVs and should be carefully controlled.

Antiproliferative and immunomodulative potential of Citrullus colocynthis and its bioactive compounds in human lymphocytes and lung cells

Ethnopharmacological relevanceCitrullus colocynthis (L.) Schrad is a member of the Cucurbitaceae plant family which has been used in traditional medicine for the treatment of lung diseases such as asthma and bronchitis. Aim of the studyThe study was conducted to investigate antiproliferative and immunomodulating effects of C. colocynthis and isolated cucurbitacins on human T lymphocytes and lung epithelial cells in order to evaluate their potential in the treatment of airway diseases. Materials and methodsDifferent concentrations of an ethanolic extract of C. colocynthis fruits and cucurbitacins B (CuB), E (CuE) and E-glucopyranoside (CuE-Glu) were analysed for their cytotoxicity and immunomodulatory potential on Peripheral Blood Mononuclear Cells (PBMCs) of healthy donors and on the epithelial lung cancer cell line A549. Viability and proliferation were tested using WST1 and CFSE assays. Flow cytometric analysis of AnnexinV/PI staining was used to investigate cell death through apoptosis/necrosis. Effects on regulatory mechanisms of T lymphocytes, such as CD69 and CD25 marker activation, cytokine production of the cytokines interleukin 2 (IL2), tumor necrosis factor α (TNFα) and interferon γ (IFNy) were also analysed via flow cytometry. Influences on the activator protein 1 (AP1), nuclear factor of activated T-cells (NFAT) or nuclear factor ‘kappa-light-chain-enhancer’ of activated B-cells (NFκB) pathways were analysed in the Jurkat reporter cell line. Cytokine secretion in A549 cells stimulated with virus-like particles was analysed using the bead-based Legendplex™ assay. ResultsNon-toxic concentrations of C. colocynthis and CuE-Glu showed dose-dependent effects on viability and proliferation in both T lymphocytes and A549 cells. The extracts inhibited lymphocyte activation and suppressed T cell effector functions, which was also shown by lower production of cytokines IL2, TNFα and IFNy. A dose dependent inhibition of the pathways NFκB, NFAT and AP1 in Jurkat cells could be observed. In A549 cells, especially CuE and CuE-Glu showed inhibitory effects on cytokine production following a simulated viral infection. Unglycosylated cucurbitacins were more effective in suppressing the immune function in lymphocytes than glycosylated cucurbitacins, however this activity is limited to cytotoxic concentrations. ConclusionIn our study we could confirm the immunmodulating effect of C. colocynthis and cucurbitacins B, E and E-glucopyranoside in vitro by suppression of different pathways of inflammation and T cell proliferation. Activity in a lung cell model using a virus-like stimulation shows promise for further research regarding cucurbitacins in airway diseases.

Chromosomal and genome size variations in Opium poppy (Papaver somniferum L.) from Afghanistan

The genus Papaver classified in the Papaveraceae family, is a valuable, non-alternative medicinal plant which has illustrated a massive variety of pharmacologically important alkaloids. Chromosomal and monoploid genome size diversity of seven populations collected from different districts of Balkh Province in northern parts of Afghanistan were studied. All populations were diploid, six of which (P1-P6) had 22 chromosomes, while P7 had 20 larger chromosomes. The mean chromosome length (CL) of P1-P6 populations was 1.32 μm (0.91-1.74 μm), but that of P7 population was 2.24 μm. The results of flow cytometric analysis showed that the mean monoploid 2Cx DNA of P1-P6 populations was 5.701 pg (5.574-5.901 pg), whereas that of P7 population was 5.795 pg, confirming intraspecific variation. This study is being reported for the first time from the northern part of Afghanistan’s opium cultivation area, and P7 population is also being reported for the first time in terms of chromosome number. Valuable information on Cytogenetics can be used in some research fields, including polygenetic analysis, taxonomic relationships, evolutionary characteristics, and plant breeding.

MiR-200a-3p inhibits the PDGF-BB-induced proliferation of VSMCs by affecting their phenotype-associated proteins.

Accumulating evidence shows that the abnormal proliferation and migration of vascular smooth muscle cells (VSMCs) can significantly affect the long-term prognosis of coronary artery bypass grafting. This study aimed to explore the factors affecting the proliferation, migration, and phenotypic transformation of VSMCs. First, we stimulated VSMCs with different platelet-derived growth factor-BB (PDGF-BB) concentrations, analyzed the expression of phenotype-associated proteins by Western blotting, and examined cell proliferation by scratch wound healing and the 5-ethynyl-2-deoxyuridine (EdU) assay. VSMC proliferation was induced most by PDGF-BB treatment at 20 ng/mL. miR-200a-3p decreased significantly in A7r5 cells stimulated with PDGF-BB. The overexpression of miR-200a-3p reversed the downregulation of α-SMA (p < 0.001) and the upregulation of vimentin (p < 0.001) caused by PDGF-BB. CCK8 and EdU analyses showed that miR-200a-3p overexpression could inhibit PDGF-BB-induced cell proliferation (p < 0.001). However, flow cytometric analysis showed that it did not significantly increase cell apoptosis. Collectively, the overexpression of miR-200a-3p inhibited the proliferation and migration of VSMCs induced by PDGF-BB, partly by affecting phenotypic transformation-related proteins, providing a new strategy for relieving the restenosis of vein grafts.

Curcumin Analog L48H37 Induces Apoptosis in Human Oral Cancer Cells by Activating Caspase Cascades and Downregulating the Inhibitor of Apoptosis Proteins through JNK/p38 Signaling.

L48H37 is a synthetic curcumin analog that has anticancer potentials. Here, we further explored the anticancer effect of L48H37 on oral cancer cells and its mechanistic acts. Cell cycle distribution was assessed using flow cytometric analysis. Apoptosis was elucidated by staining with PI/Annexin V and activation of the caspase cascade. Cellular signaling was explored using apoptotic protein profiling, Western blotting, and specific inhibitors. Our findings showed that L48H37 significantly reduced the cell viability of SCC-9 and HSC-3 cells, resulting in sub-G1 phase accumulation and increased apoptotic cells. Apoptotic protein profiling revealed that L48H37 increased cleaved caspase-3, and downregulated cellular inhibitor of apoptosis protein 1 (cIAP1) and X-linked inhibitor of apoptosis protein (XIAP) in SCC-9 cells, and the downregulated cIAP1 and XIAP in both oral cancer cells were also demonstrated by Western blotting. Meanwhile, L48H37 triggered the activation of caspases and mitogen-activated protein kinases (MAPKs). The involvement of c-Jun N-terminal kinase (JNK) and p38 MAPK (p38) in the L48H37-triggered apoptotic cascade in oral cancer cells was also elucidated by specific inhibitors. Collectively, these findings indicate that L48H37 has potent anticancer activity against oral cancer cells, which may be attributed to JNK/p38-mediated caspase activation and the resulting apoptosis. This suggests a potential benefit for L48H37 for the treatment of oral cancer.

Generation and characterization of mAb 61H9 against junctional adhesion molecule-a with potent antitumor activity.

Junctional adhesion molecule-A (JAM-A) is an adhesion molecule that exists on the surface of certain types of cells, including white blood cells, endothelial cells, and dendritic cells. In this study, the cDNA sequences of JAM-A-Fc were chemically synthesized with optimization for mammalian expression. Afterward, we analyzed JAM-A protein expression through transient transfection in HEK293 cell lines. Mice were immunized with JAM-A-Fc protein, and hybridoma was prepared by fusing myeloma cells and mouse spleen cells. Antibodies were purified from the hybridoma supernatant and four monoclonal strains were obtained and numbered 61H9, 70E5, 71A8, and 74H3 via enzyme-linked immunosorbent assay screening. Immunofluorescence staining assay showed 61H9 was the most suitable cell line for mAb production due to its fluorescence signal being the strongest. Flow cytometric analysis proved that 61H9 possessed high affinity. Moreover, antagonism of JAM-A mAb could attenuate the proliferative, migrative, and invasive abilities of ESCC cells and significantly inhibit tumor growth in mice. By examining hematoxylin-eosin staining mice tumor tissues, we found inflammatory cells infiltrated lightly in the anti-JAM-A group. The expression of BCL-2 and IκBα in the anti-JAM-A group were decreased in mice tumor tissues compared to the control group. Ultimately, a method for preparing high-yield JAM-A-Fc protein was created and a high affinity mAb against JAM-A with an antitumor effect was prepared.

IFNγ insufficiency during mouse intra-vaginal Chlamydia trachomatis infection exacerbates alternative activation in macrophages with compromised CD40 functions

Chlamydia trachomatis (C.tr), an obligate intracellular pathogen, causes asymptomatic genital infections in women and is a leading cause of preventable blindness. We have developed in vivo mouse models of acute and chronic C. trachomatis genital infection to explore the significance of macrophage-directed response in mediating immune activation/suppression. Our findings reveal that during chronic and repeated C. trachomatis infections, Th1 response is abated while Treg response is enhanced. Additionally, an increase in exhaustion (PD1, CTLA4) and anergic (Klrg3, Tim3) T cell markers is observed during chronic infection. We have also observed that M2 macrophages with low CD40 expression promote Th2 and Treg differentiation leading to sustained C. trachomatis genital infection. Macrophages infected with C. trachomatis or treated with supernatant of infected epithelial cells drive them to an M2 phenotype. C. trachomatis infection prevents the increase in CD40 expression as observed in western blots and flow cytometric analysis. Insufficient IFNγ, as observed during chronic infection, leads to incomplete clearance of bacteria and poor immune activation. C. trachomatis decapacitates IFNγ responsiveness in macrophages via hampering IFNγRI and IFNγRII expression which can be correlated with poor expression of MHC-II, CD40, iNOS and NO release even following IFNγ supplementation. M2 macrophages during C. trachomatis infection express low CD40 rendering immunosuppressive, Th2 and Treg differentiation which could not be reverted even by IFNγ supplementation. The alternative macrophages also harbour high bacterial load and are poor responders to IFNγ, thus promoting immunosuppression. In summary, C. trachomatis modulates the innate immune cells, attenuating the anti-chlamydial functions of T cells in a manner that involves decreased CD40 expression on macrophages.