Flow Cytometric Analysis Research Articles

7-O-tyrosyl Silybin Derivatives as a Novel Set of Anti-Prostate Cancer Compounds.

Silybin is a natural compound extensively studied for its hepatoprotective, neuroprotective and anticancer properties. Envisioning the enhancement of silybin potential by suitable modifications in its chemical structure, here, a series of new 7-O-alkyl silybins derivatives were synthesized by the Mitsunobu reaction starting from the silybins and tyrosol-based phenols, such as tyrosol (TYR, 3), 3-methoxytyrosol (MTYR, 4), and 3-hydroxytyrosol (HTYR, 5). This research sought to explore the antioxidant and anticancer properties of eighteen new derivatives and their mechanisms. In particular, the antioxidant properties of new derivatives outlined by the DPPH assay showed a very pronounced activity depending on the tyrosyl moiety (HTYR > MTYR >> TYR). A significant contribution of the HTYR moiety was observed for silybins and 2,3-dehydro-silybin-based derivatives. According to the very potent antioxidant activity, 2,3-dehydro-silybin derivatives 15ab, 15a, and 15b exerted the most potent anticancer activity in human prostate cancer PC-3 cells. Furthermore, flow cytometric analysis for cell cycle and apoptosis revealed that 15ab, 15a, and 15b induce strong G1 phase arrest and increase late apoptotic population in PC-3 cells. Additionally, Western blotting for apoptotic marker cleaved caspase-3 confirmed apoptosis induction by these silybin derivatives in PC-3 cells. These findings hold significant importance in the investigation of anticancer properties of silybin derivatives and strongly encourage swift investigation in pre-clinical models and clinical trials.

Dynamic Changes of the Gut Microbiota and Its Functional Metagenomic Potential during the Development of Non-Small Cell Lung Cancer.

The gut microbiota plays a significant role in tumor pathogenesis by regulating the host metabolism and immune response, and there are few studies focused on tracking changes in the gut microbiota from the onset of lung cancer. Therefore, the aim of our study is combining preclinical and clinical research to thoroughly analyze the signatures of fecal microbiota in lung cancer, which will be useful for early diagnosis and predicting the therapeutic efficacy of lung cancer. The first part of this study analyzed the fecal metagenomic differences between patients with non-small cell lung cancer and healthy subjects, and the second part of this work constructed a murine lung cancer model to monitor changes in mouse fecal metagenomics and T cell immunology during lung cancer progression. We found that the fecal microbiota was altered in both humans and mice with lung cancer, characterized by a significantly reduced microbial diversity and number of beneficial microbes, with increases in potential pathogens. The fecal level of Akkermansia muciniphila and the gut metabolic module of the secondary bile acid metabolism were diminished in both humans and mice with lung cancer compared with healthy subjects. Splenomegaly was observed in the lung cancer mice. Flow cytometer analysis of the splenocytes revealed substantial alterations in the proportions of T cell subsets in the lung cancer mice, characterized by significant increases in CD4+Foxp3+CD25+ T regulatory cells (p < 0.05) while significant decreases in CD3+ T cells (p < 0.001), CD4+ T cells (p < 0.001), and the CD4+/CD8+ ratio (p < 0.01). Vertical and longitudinal analyses of the fecal microbiota of the two mouse groups identified some lung cancer biomarkers (including Acutalibacter timonensis, Lachnospiraceae bacterium NSJ-38 sp014337195, etc.). The fecal microbiota of the lung cancer mice had a reduced metagenomic potential for neurotransmitters (melatonin, γ-aminobutyric acid, and histamine) compared with healthy mice. In summary, this study found that the diversity, structure, and composition of gut microbiota vary between cancer and healthy conditions, ultimately leading to changes in the potential for functional metagenomics.

The clinical association of programmed death-1/PD-L1 axis, myeloid derived suppressor cells subsets and regulatory T cells in peripheral blood of stable COPD patients.

Myeloid-derived suppressor cells (MDSCs) have crucial immunosuppressive role in T cell dysfunction in various disease processes. However, the role of MDSCs and their impact on Tregs in COPD have not been fully understood. The aim of the present study is to investigate the immunomodulatory role of MDSCs and their potential impact on the expansion and function of Tregs in COPD patients. Peripheral blood samples were collected to analyze circulating MDSCs, Tregs, PD-1/PD-L1 expression to assess the immunomodulatory role of MDSC and their potential impact on the expansion and function of Treg in COPD. A total of 54 COPD patients and 24 healthy individuals were enrolled in our study. Flow cytometric analyses were performed to identify granulocytic MDSCs (G-MDSCs), monocytic MDSCs (M-MDSCs), Tregs, and the expression of PD-1/PD-L1(L2) on MDSCs and Tregs in peripheral blood. Our results revealed a significantly higher percentage of G-MDSCs and M-MDSCs (p < 0.001) in COPD patients compared to the healthy controls. Additionally, a significantly higher proportion of peripheral blood Tregs was observed in COPD patients. Furthermore, an increased expression of cytotoxic T-lymphocyte-associated protein 4 (CTLA-4) on Tregs (p < 0.01) was detected in COPD patients. The expression of PD-1 on CD4+ Tcells and Tregs, but not CD8+Tcells, was found to be increased in patients with COPD compared to controls. Furthermore, an elevated expression of PD-L1 on M-MDSCs (p < 0.01) was also observed in COPD patients. A positive correlation was observed between the accumulation of M-MDSCs and Tregs in COPD patients. Additionally, the percentage of circulating M-MDSCs is positively associated with the level of PD-1 (r = 0.51, p < 0.0001) and CTLA-4 (r = 0.42, p = 0.0014) on Tregs in COPD. The recruitment of MDSCs, accumulation of Tregs, and up-regulation of CTLA-4 on Treg in COPD, accompanied by an increased level of PD-1/PD-L1, suggest PD-1/PD-L1 axis may be potentially involved in MDSCs-induced the expansion and activation of Treg at least partially in COPD.

Metformin Suppresses Both PD-L1 Expression in Cancer Cells and Cancer-Induced PD-1 Expression in Immune Cells to Promote Antitumor Immunity.

Metformin, a drug prescribed for patients with type 2 diabetes, has potential efficacy in enhancing antitumor immunity; however, the detailed underlying mechanisms remain to be elucidated. Therefore, we aimed to identify the inhibitory molecular mechanisms of metformin on programmed death ligand 1 (PD-L1) expression in cancer cells and programmed death 1 (PD-1) expression in immune cells. We employed a luciferase reporter assay, quantitative real-time PCR, immunoblotting analysis, immunoprecipitation and ubiquitylation assays, and a natural killer (NK) cell-mediated tumor cell cytotoxicity assay. A mouse xenograft tumor model was used to evaluate the effect of metformin on tumor growth, followed by flow-cytometric analysis using tumor-derived single-cell suspensions. Metformin decreased AKT-mediated β-catenin S552 phosphorylation and subsequent β-catenin transactivation in an adenosine monophosphate-activated protein kinase (AMPK) activation-dependent manner, resulting in reduced CD274 (encoding PD-L1) transcription in cancer cells. Tumor-derived soluble factors enhanced PD-1 protein stability in NK and T cells via dissociation of PD-1 from ubiquitin E3 ligases and reducing PD-1 polyubiquitylation. Metformin inhibited the tumor-derived soluble factor-reduced binding of PD-1 to E3 ligases and PD-1 polyubiquitylation, resulting in PD-1 protein downregulation in an AMPK activation-dependent manner. These inhibitory effects of metformin on both PD-L1 and PD-1 expression ameliorated cancer-reduced cytotoxic activity of immune cells in vitro and decreased tumor immune evasion and growth in vivo. Metformin blocks both PD-L1 and PD-1 within the tumor microenvironment. This study provided a mechanistic insight into the efficacy of metformin in improving immunotherapy in human cancer.

Flow Cytometric Assessment of CD26-Positive Leukemic Stem Cells: A Rapid and Valuable Tool in the Diagnosis and Follow-Up of Chronic Myeloid Leukemia.

Context Chronic myeloid leukemia (CML) is a clonal myeloproliferative neoplasm. Recent studies have suggested that CD26-positive leukemic stem cells (LSCs) circulating in peripheral blood are specific for CML. Objective This study was undertaken to determine the proportion of CD26-positive LSCsat diagnosis and its change during tyrosine kinase inhibitor therapy. Design This prospective study was conducted on 43 cases of CML at diagnosis. For flow cytometry, peripheral blood cells were stained with CD45, CD34, CD38, CD3, and CD26. A sequential gating strategy with CD45/SSC (side scatter), CD34/SSC, and CD34/CD38 was applied to identify CD45+/34+/38- populations, from which CD26-positive stem cells were identified and compared with controls. Data analysis was done with Kaluza software. Results All patients diagnosed with CML were detected with CD26-positive LSCs. The median percentage of CD26-positive CML LSCs was 0.02 with a range of 0.001 to 1.77. None of the control samples showed CD26 positivity. The percentage and absolute count of CD26-positive CML LSCs were reduced after six months of tyrosine kinase therapy in patients with complete hematological remission. Conclusion Flow cytometric analysis of circulating CD26-positive CML LSCs is a non-invasive, rapid, and useful tool in the diagnosis and follow-up of CML.

Spectral Flow Cytometry Methods and Pipelines for Comprehensive Immunoprofiling of Human Peripheral Blood and Bone Marrow.

This study introduces optimized methods and analysis algorithms that enhance capabilities in comprehensive immunophenotyping of human blood and bone marrow using spectral flow cytometry. This approach facilitates detection of rare cell types, enables measurement of cell variations across donors, and provides proof-of-concept in identifying known hematologic malignancies. By unlocking complexities of hematopoietic and immune landscapes at the single-cell level, this advancement holds potential for understanding disease states and therapeutic responses.

Abstract 3851: Beta-lapachone promotes tumor associated neutrophils (TANs) polarized towards anti-tumor (N1) phenotype

Abstract NAD(P)H:quinone oxidoreductase1 (NQO1) is overexpressed in most solid cancers, emerging as a promising target for tumor selective killing. β-lapachone (β-lap), an NQO1bioactivatable drug, exhibits significant antitumor effects on NQO1+ human solid cancer cell lines. It also induces immunogenic cell death in the tumor microenvironment (TME)and synergizes with anti-PD-L1 immunotherapy. However, the interaction between β-lap-mediated anti-tumor immune response and neutrophils, a novel antigen presenting cells(APCs), remains unknown. This study found β-lap treatment increased intracellular ROS formation and DNA damage to kill NQO1+ murine tumor cell lines and dicoumarol spared the lethality. Furthermore, flow cytometric analysis of immune cells in TME of subcutaneous tumor models indicated that there is a significant infiltration of neutrophils(CD11b+ Gr1high cells) or Tumor Associated Neutrophils (TANs) following β-lap treatment. Depletion of neutrophils by co-addition of anti-Ly6G antibody abolished β-lap-induced anti-tumor efficacy in immune competent mice. Neutrophil mediated tumor cell killing determined ex vivo using bone marrow derived neutrophils and it was observed that in the presence of β-lap induced antigen, neutrophils were killing murine tumor cells. Moreover, exposure of these neutrophils to β-lap induced antigen medium induce antitumor N1-like properties, include increase in certain surface markers such as CD95(Fas) and CD54 (ICAM-1) as well as increase in intracellular cytokine IFN-β and decrease in TGF-β revealed by flow cytometric analysis. Corroborating to this, there is a significant increase of CD95 and IFN-β and a decrease in TGF-β were observed with TANs infiltrated into β-lap treated TME compared to vehicle treated tumors. Additionally, TLR/MyD88signaling deficiency decreased β-lap induced neutrophil tumor infiltration. BlockingHMGB1, depleting neutrophils, or TLR4/MyD88 deficiency lessened the antigen-specific T cell response. This study highlights that β-lap kills NQO1+ murine tumor cells through ROS production and DNA damage, and neutrophils play a pivotal role in its antitumor effects. Citation Format: Xiumei Huang, Soumya Tumbath, Liangxiang Jiang, Yang-Xin Fu. Beta-lapachone promotes tumor associated neutrophils (TANs) polarized towards anti-tumor (N1) phenotype [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 3851.

Abstract 1403: The role of Dclk1-expression cells in normal pancreas and in pancreatic tumor progression

Abstract Background: Tuft cells, marked by Doublecortin-like kinase-1 (Dclk1), are rare in the healthy pancreas but increase markedly during pancreatic tumorigenesis. While Group 2 innate lymphoid cells (ILC2s) are known to modulate inflammation and immunity, their specific roles in cancer-related immunity and immunotherapy interactions with pancreatic tuft cells remain unknown. Methods: To investigate tuft cells during early pancreatic neoplasia, we used a Dclk1 reporter mouse model and single-cell RNA-seq to study Dclk1-expressing cells in normal pancreas and neoplasia using Mist1/Kras mouse. We treated mice with IL-13 peptides and anti-IL-13 antibodies to explore the IL-13-ILC2 axis in tuft cell regulation. Results: Within the normal pancreas, Dclk1 expression marked several specialized epithelial cell types, including tuft-like cells, neuroendocrine cells, and a small number of acinar progenitor cells. A notable increase in Dclk1-expressing cells was observed at the onset of early PanIN lesions, with a subsequent decline in advanced PanIN stages and PDAC. Using flow cytometric analysis, we observed an increase in ILC2 cells in the Mist1/Kras pancreas in response to IL33 produced by pancreatic cancer-associated fibroblasts (CAFs). Blocking ILC2 activation with anti-IL33 antibodies reduced Dclk1 cell hyperplasia during PanIN progression. Given the known association between tuft cells, ILC2 cells, and the IL13/IL4 signaling pathways in the intestine, we discovered that Dclk1-expressing cells highly expressed the IL4 receptor (IL4ra). IL-13 levels increased during PanIN progression, and we found that ILC2s comprised the primary source of IL-13 within pancreatic tumors. In vitro, studies showed that the presence of IL-13 maintained the expansion of Dclk1-expressing tuft-like cells in pancreatic organoids derived from Dclk1-DTR-ZsGreen mice. Correspondingly, the in vivo administration of anti-IL-13 resulted in reduced Dclk1 expression. Conclusion: Previous studies have shown that pancreatic tuft cells inhibit pancreatic cancer progression. Our study reveals a novel regulatory mechanism wherein IL-13 secreted by ILC2s modulates Dclk1 tuft cell expression during pancreatic tumorigenesis. The IL13-ILC2 axis appears to be a potential therapeutic target to regulate Dclk1 cell activity, which could influence the course of pancreatic tumorigenesis. Future investigations will further explore the interaction between the IL13/ILC2 axis and Dclk1 in pancreatic cancer development. Citation Format: Giovanni Valenti, Pasquale Laise, Feijing Wu, Hiroki Kobayashi, Quin T. Waterbury, Ryota Takahashi, Tuo Ruan, Zhengyu Jiang, Yosuke Ochiai, Leah B. Zamechek, Timothy C. Wang. The role of Dclk1-expression cells in normal pancreas and in pancreatic tumor progression [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 1403.

Abstract 6744: Activating anti-MDS immunity with polarized dendritic cell vaccines

Abstract Less than 50% of patients with higher-risk myelodysplastic syndromes (MDS) show clinical response to standard-of-care hypomethylating (HMA) therapy. Significant enthusiasm existed for immunotherapy/HMA combinations for these patients, but studies have failed to deliver on the initial promise. We have demonstrated that patients with MDS have a deficiency in antigen-presenting cells, called CD141Hi dendritic cells (DC). DC-deficient MDS patients have reduced response to HMAs, worse overall survival, and show less response to cancer vaccination. We hypothesized that restoration of this population using a novel DC vaccination approach - alpha-type-1 polarized dendritic cells (αDC1) - might compensate for this deficiency and restore immunity. To generate αDC1s, we collected monocytes from MDS patients and healthy donors (HD) and cultured them for 6 days in vitro in the presence of recombinant human GM-CSF and IL-4 followed by an additional 18-24 hours in the presence of αDC1 maturation factors (poly-I:C/IFNα/IFNγ/TNFα/IL-1β). Overall, we attempted to generate αDC1s from 15 MDS samples; 13/15 exhibited intermediate to very high-risk disease and 10/15 are currently receiving HMA therapy. We successfully cultured cells from 8/15 samples (53%). Of the 8 MDS samples, 5 were collected from patients receiving HMA therapy (62.5%). We were unable to successfully culture αDC1s from the other 5 patients receiving HMA therapy, and thus the effect of HMA therapy on aDC1 generation remains unclear. To test the function of MDS αDC1s, we focused on two signals necessary to activate immune responses: 1) the expression of co-stimulatory molecules and 2) the production of IL-12p70. Flow cytometric analysis on αDC1s and immature DCs from MDS samples revealed intra-patient variation in the fold-change surface expression of CD40/80/83/86 and HLA-DR proteins. Additionally, there was a trend towards decreased up-regulation of CD80 and CD86 in MDS specimens compared to HD. To assess production of IL-12p70, we stimulated immature DCs and αDC1s with CD40L and performed ELISA analysis on the media supernatant. We found that 3 out of 4 samples could produce IL-12p70 (range 27 - 5,480 pg/ml). To date, our results suggest that generation of MDS patient-derived αDC1 cells is feasible and additional work is being performed to fully characterize their ability to activate anti-MDS immune responses. Since αDC1s have an established safety profile in patients with solid tumors, we anticipate that we will be able to rapidly translate our results to the clinic. Citation Format: Daniel Alvarado, Jennifer Peresie, Adam Brinkman, Cheryl Allen, Weijian Jiang, Pawel Kalinski, Tiffany Tanaka, Elizabeth Griffiths, Michael Nemeth. Activating anti-MDS immunity with polarized dendritic cell vaccines [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 6744.

Abstract 6528: Integrin ɑvβ6 upregulation as a mechanism of T-cell evasion in head and neck squamous cell carcinoma

Abstract Head and neck squamous cell carcinoma (HNSCC) encompasses malignancies of the mucosal epithelium in the oral cavity, pharynx, and larynx. The lack of effective screening strategies and the typically late stage of detection are contributors to a considerable annual death toll of HNSCC. Additionally, the anatomical proximity of the malignancy to delicate structures of the head and neck leads to tremendous treatment-related morbidity. Therefore, there is great interest in developing life-saving therapies and strategies for treatment de-escalation. The immune checkpoint inhibitor pembrolizumab (αPD-1) has been approved for recurrent or metastatic HNSCC and as a frontline therapy for unresectable disease. However, only about a quarter of patients respond to αPD-1 therapy. The immunosuppressive effect of TGFβ has been widely described as relevant to the HNSCC tumor microenvironment. However, attempts to blockade TGFβ have failed mainly due to the widespread side effects of disrupting a highly promiscuous cytokine. Integrin αvβ6 is a major activator of the inert latent TGFβ into the active, immunosuppressive form. With its high relative specificity to HNSCC tissue, inhibition of αvβ6 could offer a safer approach for disrupting TGFβ to overcome resistance to immune checkpoint blockade. After screening ɑvβ6 expression levels of HNSCC cell lines via flow cytometry, cell lines FaDu and CAL27 were selected for further investigation due to high endogenous ITGB6 expression (FaDu 35.57% cells ITGB6+, CAL27 95.69% cells ITGB6+). The cell lines were transduced with a doxycycline-inducible short hairpin RNA (shRNA) knockdown of αvβ6 using a lentiviral system. Both knockdown and control shRNA cell lines were pretreated with 1 μg/mL of doxycycline for five days to induce the shRNA and subsequently co-cultured with TALL-104 T-cells. Quantitative fluorescence microscopy of the co-culture revealed that the knockdown of αvβ6 substantially increased T-cell killing over 24hrs (FaDu CTRL shRNA 6.72%±2.49 dead vs. FaDu ITGB6 shRNA 12.80%±3.16 dead, p&amp;lt;0.001). However, treatment of FaDu and CAL27 cell lines with active-TGFβ showed no change in PD-L1 expression via flow cytometric analysis. To determine whether the T cell evasive effect of αvβ6 is driven by other immune checkpoints on the cancer cells or by the direct effect that TGFβ has on immune cells, we show the generation of relevant mouse αvβ6 shRNA knockdown cells. These syngeneic in vivo studies will facilitate cytokine profiling to uncover the dynamics of tumor-immune cell interactions in response to αvβ6 inhibition. Citation Format: William J. MacDonald, Praveen R. Srinivasan, Maximilian Pinho-Schwermann, Shengliang Zhang, Vida Tajiknia, Connor Purcell, Jillian Strandberg, Nolan Stubbs, Wafik S. El-Deiry. Integrin ɑvβ6 upregulation as a mechanism of T-cell evasion in head and neck squamous cell carcinoma [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 6528.

Abstract 4559: SMAD3 mediates oxaliplatin resistance in esophageal adenocarcinoma

Abstract Background: Resistance to conventional treatments is a significant barrier to esophageal adenocarcinoma (EAC) therapy. TGF-β signaling appears to be important for EAC tumorigenesis; however, its role in EAC chemoresistance remains understudied. This study investigates the role of SMAD3 in EAC resistance to the frontline chemotherapeutic drug oxaliplatin. Methods: Publicly available datasets were analyzed to determine SMAD3 expression in EAC versus normal samples. Western blot, quantitative real-time polymerase chain reaction, immunofluorescence staining, and flow cytometric analyses were utilized to validate bioinformatic results. Human EAC cell lines (OE33 and OE19) were treated with pharmacologic inhibitors or transfected with small interfering RNA and assessed for DNA damage and apoptotic cell death. Patient-derived organoids and patient-derived xenograft models of EAC were used to confirm our findings in vivo. Results: SMAD3 was significantly upregulated in EAC versus normal tissue samples. Total and phosphorylated SMAD3 protein levels were upregulated in EAC tissues accompanied by increased expression of several genes regulated by SMAD3. Notably, oxaliplatin-resistant EAC cells exhibited overexpression of SMAD3. Chemotherapy non-responding patients showed enrichment of SMAD3 gene expression when compared to responders in the clinical dataset GSE165252. Knockdown or inhibition of SMAD3 hindered DNA repair and sensitized EAC cells to oxaliplatin treatment in vitro and in vivo. Mechanistically, SMAD3 promoted ATM phosphorylation by interacting with protein phosphatase 2A (PP2A) and sequentially inhibiting the binding of PP2A to ATM. Conclusion: Our results reveal a pivotal role for SMAD3 in mediating oxaliplatin resistance in EAC and thus hold a significant potential in guiding future drug development and combination strategies. Citation Format: Farah Ballout, Heng Lu, Nadeem Bhat, Lei Chen, Dunfa Peng, Zheng Chen, Alexander Zaika, Oliver McDonald, Wael El-Rifai. SMAD3 mediates oxaliplatin resistance in esophageal 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 4559.

RFC3 drives the proliferation, migration, invasion and angiogenesis of colorectal cancer cells by binding KIF14.

Colorectal cancer (CRC) is a deadly and aggressive type of cancer that has a high fatality rate. The expression levels of replication factor C subunit 3 (RFC3) and kinesin family member 14 (KIF14) have been reported to be increased in CRC. The current study aimed to explore the effects of RFC3 on the malignant behaviors of CRC cells and its possible underlying mechanism involving KIF14. RFC3 and KIF14 expression levels in CRC tissues were analyzed using TNMplot database and Gene Expression Profiling Interactive Analysis database bioinformatics tools. RFC3 and KIF14 levels in CRC cells were examined using reverse transcription-quantitative PCR and western blotting. Cell Counting Kit-8 and 5-ethynyl-2'-deoxyuridine assays were performed to assess cell proliferation. Cell apoptosis was determined using flow cytometric analysis. Wound healing and Transwell assays were adopted for the evaluation of cell migration and invasion. Tube formation assay in human umbilical vein endothelial cells was used to measure angiogenesis. Western blotting analysis was performed to determine the expression of apoptosis-, migration- and angiogenesis-associated proteins. Additionally, bioinformatics tools predicted the co-expression and interaction of RFC3 and KIF14, which was verified by a co-immunoprecipitation assay. RFC3 displayed elevated expression in CRC tissues and cells, and depletion of RFC3 halted the proliferation, migration, invasion and angiogenesis, while increasing the apoptosis of CRC cells; this was accompanied by changes in the expression levels of related proteins. In addition, RFC3 bound to KIF14 and interference with RFC3 reduced KIF14 expression. Moreover, KIF14 upregulation reversed the effects of RFC3 depletion on the aggressive cellular behaviors in CRC. In conclusion, RFC3 might interact with KIF14 to function as a contributor to the malignant development of CRC.

Abstract 7144: BR0L075 (SCB01A), a microtubule inhibitor targeting the colchicine binding site, induces immunogenic cell death

Abstract BPR0L075 (also known as SCB01A in phase II clinical trial) is a novel anti-microtubule drug with potent anti-tumor and anti-angiogenic activity in vitro and in vivo. BPR0L075 inhibits tubulin polymerization (microtubule de-stabilizer) by binding to the colchicine-binding site of tubulin. We previously reported that BPR0L075 overcomes paclitaxel (microtubule stabilizer) resistance and induces vascular-disruption in vitro and in vivo. We observed that breast and ovarian cancer cells treated with BPR0L075 in vitro induced key hallmarks of immunogenic cell death (ICD). In both human and murine, ovarian (OVCAR3, ID8) and breast cancer (MDA-MB-231, 4T1) cell lines, treatment with low nanomolar concentration of BPR0L075 significantly decreased the viability of cells evidenced by SRB assay. Using a known ICD inducer mitoxantrone as a positive control, both flow cytometric analysis and immunofluorescence staining assays demonstrated that BPR0L075 treatment led to a time- and dose-dependent translocation of calreticulin, an endoplasmic reticulum chaperone protein, on the plasma membrane of dying cells which sends an “eat me” signal. The BPR0L75 treatment also led to secretion of High Mobility Group Box 1 (HMGB1), a non-histone chromatin-binding nuclear protein, as well as adenosine triphosphate (ATP) from the cell into the extracellular space using ELISA assay. These damage-associated molecular patterns (DAMPs) are known to lead to antigen-presenting cell recruitment, phagocytosis, maturation of dendritic cells, and chemotaxis. The appearance of the ICD biomarkers (calreticulin exposure, HMGB1 and ATP release) supports BPR0L075 as an ICD-inducing antimitotic agent. Further in vivo studies are warranted to delineate the effect of BPR0L075 on immune activation and recruitment of immune cells to the tumor microenvironment. Citation Format: Ayesha Bano, Xinli Liu. BR0L075 (SCB01A), a microtubule inhibitor targeting the colchicine binding site, induces immunogenic cell death [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 7144.

Abstract 4147: Chitosan luminescent rare-earth doped nanoparticles as cancer cell tracking for imaging tumors and their immune response

Abstract Background: Cancer is a group of diseases characterized by the unregulated growth of cells, which can be hard to diagnose due to the high false negatives or positives derived from technical and human mistakes.The detection of solid tumors during resection surgery is also a common issue, so contrast agents are used for both cancer diagnosis and tumor visualization during surgery, facilitating the tumor resection procedure. Nevertheless, they cause long-term problems because they accumulate in specific tissues, low penetration, and rapid clearance from the circulation. It is necessary to study new contrast agents that improve tumor detection to address these issues.Here, we synthesized two different kinds of luminescent nanoparticles (LNPs) conjugated with chitosan (Ch) and functionalized with folic acid (FA) for breast cancer detection. We compared in vitro its physicochemical characteristics and effects on the proliferation of cancer cells. Evaluated its impact on the immune system in vivo and implemented a differential imaging process to resemble a refined 3D visualization of cluster cells using Light Sheet Microscopy. Results: Luminescent nanoparticles (LNPs) synthesized by sol-gel method and functionalized with FA using EDC-NHS coupling revealed high-intensity yellow emission at 540 nm wavelength with a quantum yield of ~ 36%. In vitro viability assay at different times with 293T epithelial cells, MDA-MB-231, and T-47D cancer cells demonstrated biocompatibility and high improvement of cellular uptake by LNP functionalized with FA for cancer cells versus normal cells. Light-sheet microscopy analysis of cell-LNP interactions to visualize tagged MDA-MB-231 clusters with LNP/Ch-FA showed positive interactions due to higher stability and adherence. Biodistribution and effects on the immune system by LNP/Ch-FA nanoparticles inoculated in mice were evaluated by flow cytometer analysis. LNP/Ch-FA were well tolerated in vivo. No adverse effects were observed macroscopically. Peripheral blood analysis from animals injected with LNP/Ch-FA after 2 or 6 days of the treatment showed that a single injection of the different NPs did not have any effects on the amount of circulating neutrophils or monocytes in peripheral blood when assessed 2 or 6 days after the injection. With T cells, the core NP induced a significant decrease in circulating T cells (-31%, p=0.045) that seemed specific to the CD4+ T cells (-33%, p=0.0479). However, conjugation of the NP to chitosan prevented this effect. Conclusions: Our work presented significant evidence that could allow a better understanding of LNPs conjugated with Ch and functionalized with FA for possible upcoming clinical applications that could work singly or in synergy with the present detection techniques to boost accuracy and precision for cancer detection without compromise the inmunosystem. Citation Format: Rodrigo Osorio-Arciniega, Eduardo Lucero-Meza, Bonifacio Can-Uc, Israel Rocha-Mendoza, Pierrick G.J. Fournier, Gustavo A Hirata, Patricia Juarez. Chitosan luminescent rare-earth doped nanoparticles as cancer cell tracking for imaging tumors and their immune response [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 4147.

Abstract 956: NCX 4040 and napabucasin synergistically inhibits stemness in human prostate cancer (PCa) cellular models and potently targets transgenic murine PCa cells via inducing oxidative stress

Abstract Prostate cancer (PCa) continues to have a substantial impact on men’s wellbeing and quality of life regardless of recent progress in diagnosis and treatments and it continues to have the second highest occurrence rate following lung cancer. The strategy of combination drug therapy has gradually become a valuable choice for treating various cancer types, addressing the heterogeneity within tumors, and surpassing the constraints of existing treatment methods. In our previous work, when we combined two drugs NCX 4040 (an oxidative stress inducer) and napabucasin (a STAT3 signaling inhibitor), we discovered that the combination treatment synergistically works to inhibit the malignant characteristics of human PCa cells. Also, the combination upregulated the cGAS-STING immunogenic pathway in PCa cells which could potentially activate the cancer-targeting cytotoxic T-cells. In the current study, we aimed at investigating the anti-stemness properties of the drug duo which can not only inhibit cancer cell differentiation but also can be effective in preventing cancer relapse. We used two cell lines i.e., BPH-Cd which served as a model of cadmium induced PCa and DU145 as a model of advanced PCa to examine the anti-stemness properties of the drug combination. Based on western-blot analysis, we found the downregulation of transcription factors such as Oct4, Sox2, Nanog, β-catenin and cell surface markers like CD44, CD133 and ALDHA1, which are responsible for stem-cell like properties. In addition, we have tested the combinatory anticancer effects in transgenic mouse derived PCa cell line (Myc-Cap) as a prelude to conduct future studies in FVB mouse syngeneic model to evaluate the tumor suppressive and the antitumor immunogenic responses of combination treatment as it activated cGAS-STING pathway. By performing the cell viability assay, we found that the IC50 value of NCX 4040 is ~10µM and that of napabucasin is ~1μM, however when used in combination, ~90% inhibition is seen at 10µM of NCX and 250nM of napabucasin dual treatment. The drug combination also showed synergy in inhibiting tumorigenicity in clonogenic assay and migratory potential in wound-healing assay. By investigating the ROS production, cell death and cell cycle mechanism using flow cytometric analysis, it was observed that the combination therapy synergistically induces cellular oxidative stress which causes cancer cells to undergo late apoptosis and halts the growth of Myc-Cap cells in G2M phase of cell cycle. In conclusion, our data suggests that combination therapy of NCX 4040 and napabucasin can be potential treatment strategy for PCa warranting further evaluation in in vivo models. Citation Format: Shrushti Shah, Gnanasekar Munirathinam. NCX 4040 and napabucasin synergistically inhibits stemness in human prostate cancer (PCa) cellular models and potently targets transgenic murine PCa cells via inducing oxidative stress [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 956.

Abstract 2840: Development, characterization, and validation of a systemic human mantle cell lymphoma model utilizing luciferase expressing Jeko-1 tumor cells

Abstract Mantle cell lymphoma (MCL) is an aggressive subtype of B-cell non-Hodgkin lymphoma that is clinically and biologically heterogenous. MCL murine models are scarce and reliable in vivo models are necessary for pre-clinical testing and identification of improved novel therapeutics. To address this need, we established and characterized a human MCL in vivo model by systemic implantation of the luciferase-expressing Jeko-1 cell line (Jeko-1-luc) into immunodeficient NCG mice. Tumor growth kinetics was evaluated by testing multiple cell inoculums, and disease progression was monitored by in vivo serial bioimaging, three-dimensional optical imaging (3D), and ex-vivo endpoint analyses. Jeko-1-luc tumor growth profile revealed moderate latency with a median survival of 40 days post implant. Subsequently, we utilized 3D optical imaging and ex-vivo imaging that confirmed the localization of tumor growth predominantly in lungs, spleen, and skeletal regions. Additionally, we determined the surface expression of selected B-cell lineage markers including CD19, CD20, and CD22. Next, we evaluated the efficacy and effect on survival in response to various standard of care agents including Vincristine, Rituximab, Palbociclib, Temsirolimus, and Cyclophosphamide. The quantification of tumor burden by in vivo and ex-vivo bioluminescent imaging demonstrated that the highest therapeutic activity and overall survival was achieved by Palbociclib and Vincristine. Flow cytometric analyses further demonstrated that tumor cells obtained from blood and selected tissues including spleen and bone marrow showed reduced tumor burden in treatment groups relative to the control group. Taken together, these results illustrate the translational value of the Jeko-1-luc model and provides a platform for evaluating novel treatment modalities for mantle cell lymphoma. Citation Format: David Harris, Andrew Wong, Elizabeth Rainbolt, Nicole Westrick, Justin Avery, Joseph Kolb, Aswani Vunnava, Zachary Ward, Chassidy Hall, Bincy John. Development, characterization, and validation of a systemic human mantle cell lymphoma model utilizing luciferase expressing Jeko-1 tumor 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 2840.

Abstract 3272: PJA1 inhibits docetaxel-induced pyroptosis and results in chemoresistance in nasopharyngeal carcinoma

Abstract Background: Nasopharyngeal carcinoma (NPC) is highly prevalent in South China. More than 70% of patients are diagnosed with locoregionally advanced NPC (LA-NPC) at initial presentation, and the addition of induction chemotherapy (IC) to concurrent chemoradiotherapy is recommended for these patients. However, chemoresistance is a main reason for treatment failure in NPC patients, and the exact regulatory mechanism underlying chemoresistance in NPC remains to be elucidated. Methods: A gene expression dataset and STRING database were used to identify core module and gene that are related to the efficacy of docetaxel-cisplatin-5-fluorouracil (TPF) IC in NPC patients. In vitro and in vivo functional experiments were used to test the effects of PJA1 on docetaxel chemoresistance of NPC cells. The flow cytometric analysis, western blot, and lactate dehydrogenase (LDH) release assays were performed to determine the effects of PJA1 on GSDME-mediated cell pyroptosis. Co-immunoprecipitation, mass spectrometry, immunofluorescence staining, and ubiquitination assays were performed to explore the regulatory manners between PJA1 and PGAM5-DRP1 axis. Finally, multiplex immunofluorescence and immunohistochemical staining were used to evaluate the clinical significance of PJA1 -PGAM5-DRP1 axis in patients with NPC. Results: PJA1 was identified as a key E3 ligase of the top-ranked module ubiquitin-proteasome system dysregulation that involved in NPC chemoresistance, which was highly expressed in NPC patients with nonresponse to the TPF IC. PJA1 facilitated the docetaxel resistance of NPC cells through inhibiting the GSDME-mediated pyroptosis. Mechanistically, PJA1 promoted the degradation of the mitochondrial protein PGAM5 by increasing its K48-linked ubiquitination at K88, further increasing the phosphorylation of DRP1 at S637 and reducing the mitochondrial reactive oxygen species (ROS) production, in turn resulting in a suppressive of GSDME-mediated pyroptosis. PGAM5 knockdown fully restored the docetaxel sensitization effect of PJA1 knockdown. Moreover, RTA402, a pharmacological inhibitor of PJA1, enhanced the docetaxel sensitivity of NPC cells. Clinically, high PJA1 expression indicated resistance to TPF IC an inferior survival in NPC patients. Conclusions: Our study emphasizes the essential role of E3 ligase PJA1 in regulating chemoresistance in NPC and provides new therapeutic strategies for NPC patients based on targeting the ubiquitin-proteasome system. Citation Format: Na Liu, Ying-Qing Li, Sheng-Yan Huang. PJA1 inhibits docetaxel-induced pyroptosis and results in chemoresistance in nasopharyngeal carcinoma [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 3272.

Abstract 551: Investigation of the antipoliferative effect of the Xylopia Aethiopicaextract in triple-negative breast cancer cells

Abstract Triple-negative breast cancer (TNBC) is the most aggressive subtype of breast cancer and has very limited treatment options. Xylopia Aethiopica (XA) is a common spice that is widely used in the regions of central and western Africa. It is known to have various pharmacological properties, one of which is anti-inflammatory activity. The anti-inflammatory effect may portend antiproliferative activity. We hypothesized that the extract of XA will exert antiproliferative activity against TNBC cells. The study aimed to establish the molecular mechanism underlying the antiproliferative activity of XA extract (XAE) against TNBC. The dried fruits of XA were crushed in a mixer and macerated using diethyl ether for 72 h. The macerate was subjected to vacuum and rotary evaporation to remove the organic solvent, and a stock solution was prepared in dimethyl sulfoxide (DMSO). Assessment of the efficacy of XAE was performed using serial dilutions (100 µg/ml, 50 µg/ml, 10 µg/ml, 5 µg/ml, and 1µg/ml) of the XAE stock solution, followed by in vitro testing against two TNBC cell lines (MDA-MB-231, and MDA-MB-468 cells). The cytotoxic effect of the XAE was then measured using the resazurin dye assay. XAE resulted in a concentration-dependent cell death in both cell lines. At the highest concentration (100 µg/ml), XAE induced cytotoxic cell death of 89% and 91% in MDAMB231 and MDAMB468 cells, respectively. This was consistent with IC50 values of 32.95 ± 1.32 µg/ml (MDAMB231) and 20.83 ± 1.05 µg/ml (MDAMB468). At the molecular level, western blot analysis of mitogenic markers associated with MAPK oncogenic signaling pathway demonstrated the inhibition of EGFR and AKT protein levels after a 48-h treatment. Acridine orange-ethidium bromide (AO/EB) staining showed a concentration-dependent induction of apoptosis in both cell lines. Flow cytometric analysis was performed to quantify the apoptotic cells. A significant increase of apoptotic cells was observed in treatment groups as compared to control after 72 h. Apoptotic induction was associated with increased p53 protein levels after 48 h treatment compared to control. The study results underscore the anticancer effects of XAE in TNBC cells mediated through induction of apoptosis. Citation Format: Shailvi Soni, Shail Rakesh Modi, Terrick Andey. Investigation of the antipoliferative effect of the Xylopia Aethiopicaextract in triple-negative breast 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 551.

Abstract 2706: Identification and characterization of cancer-associated polymorphonuclear cell subsets in mice

Abstract Background: Neutrophils are the main type of polymorphonuclear cells (PMN) and comprise most immune cells in the body. PMN cells are upregulated in cancer and accumulate in the tumors. Tumor-associated PMNs comprise a heterogeneous population consisting of immunologically active mature neutrophils and immature polymorphonuclear myeloid-derived suppressor cells (PMN-MDSCs). The phenotype of these cells evolves with disease progression and is implicated in modulating immune response. However, there is yet to be a consensus to define PMN populations in tumors and blood. Methods: Here, we used single-cell RNA-seq, bulk RNA sequencing, flow cytometry, and immunohistochemistry (IHC) analysis to characterize cancer-associated PMN-MDSCs in tumor-bearing mice. Results: Single-cell RNA-seq data from lung tumors and healthy individuals revealed five metaclusters (MC): MCA, MCB, MCC, MCD, and MCE. MCA and MCB made up 34.4 and 65.1% of PMNs in healthy tissues, respectively. MCA, MCB, MCC, MCD, and MCE comprised 3.8%, 23.5% 3.3% 14.6%, and 54.7% of PMNs in tumors, respectively. Genes enriched in MCA and MCE were associated with MDSCs and myeloid progenitor cells. MCB and MCC were associated with inflammatory responses mediated by TNF-alpha signaling and neutrophil degranulation. MCD was associated with a CD33 myeloid cell phenotype and had high expression levels of CD274, Pdcd1lg2, Havcr2, and Tigit genes. Cells from MCE were associated with protein translation signatures and had elevated levels of Nt5e. Gene expression signatures from single-cell RNA-seq data were compared with bulk transcriptomic data from healthy mouse prostates and Pten-null prostate tumors characterized by high PMN cell infiltration. Gene set enrichment analysis showed enrichments after androgen deprivation therapy and Cluster MCE was associated with castration-resistant tumors. Flow cytometric and IHC analysis confirmed the enrichment of PMN cells expressing elevated levels of proteins identified from gene expression analysis including CD73, PD-L1, and PD-L2. Conclusion: This study offers further insights into the distinct subsets of PMN-MDSCs present in tumors. Citation Format: Marco A. De Velasco, Yurie Kura, Kazuko Sakai, Takashi Kikuchi, Yasunori Mori, Takafumi Minami, Kazuhiro Yoshimura, Kazutoshi Fujita, Kazuto Nishio, Hirotsugu Uemura. Identification and characterization of cancer-associated polymorphonuclear cell subsets in mice [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 2706.

Abstract 2138: Understanding the link between breastfeeding and the risk of breast cancer through comparative analysis of the murine-based model of mammary gland involution

Abstract Introduction: A meta-analysis of 47 global epidemiological studies highlights a higher breast cancer risk in women who did not breastfeed or breastfed for a short time. Further studies showed this is especially true for triple-negative breast cancer (TNBC) patients. Premenopausal AA women (AAW) have a lower prevalence of breastfeeding and a higher incidence of TNBC and mortality. Our previous study compares short-term breastfeeding, abrupt involution (AI) with prolonged breastfeeding called gradual involution (GI), revealing that AI alone induces ductal hyperplasia four months postpartum. Our current investigation delves into early events during AI versus GI, employing a comprehensive approach encompassing histology, gene expression, and myeloid cell involvement. Methods: Utilizing FVB female mice, we conducted a comparative analysis of AI and GI. AI involved early pup removal, while GI was achieved through staggered weaning. The evaluation included analysis of histomorphology, gene/protein expression, and myeloid cell infiltration. Sequential mammary gland (MG) changes were monitored through H&amp;E staining, TUNEL assay, and DNA damage analysis. 3D-organoid cultures of luminal progenitors (LPs) were employed to assess the impact of AI versus GI. qRT-PCR, IHC, Western blot, and flow cytometry/multiplex imaging were employed for the differential expression analysis of molecular and cellular factors associated with AI/GI. Results: Our research showed that AI had early adipocyte repopulation, rapid cell death, DNA repair, and myeloid cell infiltration, resulting in a chronically inflamed microenvironment. In contrast, the GI triggers a controlled immune response and prolonged cell death, facilitating comprehensive remodeling of the MG. Our flow cytometric or multiplexing imaging analyses revealed that AI-affected glands exhibit an enrichment of CCL9-producing CD206+ M2-like macrophages and CD11b+Gr1+ myeloid-derived suppressor cells. Moreover, exogenous CCL9 treatment on LPs in 3D-organoid culture results in disorganized acinar-type organoids, mirroring morphological differences observed in LPs from AI mammary glands on day56 PPM. Further analysis of CCL9 treated organoids revealed the expansion of Esr1- LPs population in ex-vivo organoid culture which might indicate an increase in the putative cells of origin of TNBC. Conclusion: Our studies comparing AI and GI demonstrate that AI is producing a pro-tumorigenic environment in the breast. It is important to note that prolonged breastfeeding protects the breast, although this cannot be a singular risk factor for TNBC. Therefore, understanding the mechanism will lead to prevention strategies to improve outcomes for all women but, has the potential to have a significant benefit in AAW. Citation Format: Sanjay Mishra, Neelam Shinde, Maria Cuitino, Morgan Bauer, Dinesh Ahirwar, Vijaya Bharti, Kate Ormiston, Resham Mawalkar, Sara Alsammerai, Gautam Sarathy, Xiaoli Zhang, Anna Vilgelm, Ramesh Ganju, Sarmila Majumder, Bhuvaneswari Ramaswamy. Understanding the link between breastfeeding and the risk of breast cancer through comparative analysis of the murine-based model of mammary gland involution [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 2138.