Ascites Cells Research Articles

Clinical significance of CD101+PD-1+CD8 T cells in malignant ascites among patients with advanced ovarian cancer

Clinical significance of CD101+PD-1+CD8 T cells in malignant ascites among patients with advanced ovarian cancer

Unleashing the antitumor power of cyclophosphamide by arabinogalactan and aptamer conjugation

Cyclophosphamide (CPA) (2-oxo-2-di(β-chloroethyl)amino tetrahydro-2,1,3-phosphoxazine) is an alkylating cytostatic compound with a broad spectrum of antitumor activity. Despite its efficacy, the clinical application of CPA is hindered by the significant occurrence of adverse side effects. To address these limitations, a promising approach involves the mechanochemical treatment of CPA with arabinogalactan (AG) to facilitate the dispersion of the drug within the AG matrix. AG stands out from other polymers due to its uniformity, low molecular weight, water solubility, and ability to form drug conjugates, thereby enhancing their therapeutic potency. Moreover, AG possesses immune-modulating properties that have the potential to counteract the immunosuppressive effects induced by CPA. By means of mechanical treatment, we successfully obtained CPA-AG complexes with a CPA:AG ratio of 1:10. These complexes were further modified with As42 aptamers that specifically target Erlich ascites cells. Aptamers, a novel class of oligonucleotide ligands obtained through SELEX technology, possess high affinity and specificity for binding to various receptors. An ascitic form of Ehrlich carcinoma was chosen as an in vitro and in vivo tumor model due to its notable drug resistance. In vitro and in vivo evaluations were conducted to compare the antitumor activity of both the CPA-AG and CPA-AG-As42 complexes with pure CPA. In vitro experiments revealed that the CPA-AG complex displayed superior antitumor activity compared to pure CPA, leading to complete tumor cell death primarily through necrosis. Notably, no toxic effects were observed with the CPA-AG and CPA-AG-As42 complexes, and they significantly prolonged the lifespan of tumor-bearing mice by more than 3.5 times. Histological studies further supported the antitumor efficacy of these complexes. These results underscore the potential of utilizing CPA-AG mechanocomposites, functionalized with aptamers, for the targeted delivery of CPA to tumors.

Increased peritoneal TGF-β1 is associated with ascites-induced NK-cell dysfunction and reduced survival in high-grade epithelial ovarian cancer.

Natural killer (NK) cell therapy represents an attractive immunotherapy approach against recurrent epithelial ovarian cancer (EOC), as EOC is sensitive to NK cell-mediated cytotoxicity. However, NK cell antitumor activity is dampened by suppressive factors in EOC patient ascites. Here, we integrated functional assays, soluble factor analysis, high-dimensional flow cytometry cellular component data and clinical parameters of advanced EOC patients to study the mechanisms of ascites-induced inhibition of NK cells. Using a suppression assay, we found that ascites from EOC patients strongly inhibits peripheral blood-derived NK cells and CD34+ progenitor-derived NK cells, albeit the latter were more resistant. Interestingly, we found that higher ascites-induced NK cell inhibition correlated with reduced progression-free and overall survival in EOC patients. Furthermore, we identified transforming growth factor (TGF)-β1 to correlate with ascites-induced NK cell dysfunction and reduced patient survival. In functional assays, we showed that proliferation and anti-tumor reactivity of CD34+ progenitor-derived NK cells are significantly affected by TGF-β1 exposure. Moreover, inhibition of TGF-β1 signaling with galunisertib partly restored NK cell functionality in some donors. For the cellular components, we showed that the secretome is associated with a different composition of CD45+ cells between ascites of EOC and benign reference samples with higher proportions of macrophages in the EOC patient samples. Furthermore, we revealed that higher TGF-β1 levels are associated with the presence of M2-like macrophages, B cell populations and T-regulatory cells in EOC patient ascites. These findings reveal that targeting TGF-β1 signaling could increase NK cell immune responses in high-grade EOC patients.

Aloe vera gel mediated green synthesis of ruthenium nanoparticles and their potential anticancer activity

Metal nanoparticles have a noteworthy future in cancer treatment research because of their smaller size and large active surface area. Though gold, silver, platinum, palladium, copper, zinc, iron and several other metal nanoparticles have been explored for their anticancer potential in different pathways, the main limitation of these particles is their toxicity which may be controlled through their size, surface modification and route of administration. Compared to other metal nanoparticles, ruthenium nanoparticles have high bio compatibility and they exhibit excellent photo-thermal effect. Though there are several reports in the literature on the anticancer potential of ruthenium complexes, ruthenium nanoparticles are not much investigated. In the present work, therefore, an attempt has been made to synthesize ruthenium nanoparticles in an easy and eco-friendly way using Aloe vera gel. Ruthenium chloride was used as a precursor and Aloe vera gel acted both as reducing and capping agent. The synthesized ruthenium nanoparticles were characterized using UV-Visible spectrophotometry, Fourier Transform Infrared Spectroscopy (FT-IR), High Resolution Transmission Electron Microscopy (HRTEM), Powder X-ray Diffraction (PXRD), Dynamic Light Scattering (DLS) and Field Emission Scanning Electron Microscopy (FESEM). The analyses confirmed the formation of nano globules of Aloe vera gel of diameter in the range 90–300 nm with ruthenium nanoparticles of average size 1.5 nm embedded in them. The synthesized Ru nanoparticles embedded in the nano globules of Aloe vera gel (ALV RuNPs) were explored for their anticancer potential in the Dalton's lymphoma ascites (DL) cell line using Trypan Blue assay. The results of the assay showed that the ALV RuNPs can induce concentration dependent cytotoxicity in DL cancer cells. Approximately 40 % cytotoxicity was obtained for concentration range 5–50 mg/mL of the sample while negligible cytotoxicity was observed for healthy PBMC cells. Theoretical study indicates significant interaction between the components present in Aloe vera and Ru-nanoparticles. The results showed that ruthenium nanoparticles can emerge as a promising bio-compatible candidate with the ability to selectively target cancer cells while sparing normal cells.

From Ficus recemosa Leaf Galls to Therapeutic Silver Nanoparticles: Antibacterial and Anticancer Applications.

The synthesis of silver nanoparticles (AgNPs) using environmentally friendly methods has become increasingly important due to its sustainability and cost-effectiveness. This study investigates the green synthesis of AgNPs using gall extracts from the plant Ficus recemosa, known for its high phytochemical content. The formation of AgNPs was verified through multiple analytical techniques, including UV-Vis spectroscopy, Fourier-transform infrared (FTIR) spectroscopy, transmission electron microscopy (TEM), zeta potential analysis, and dynamic light scattering (DLS). The UV-Vis spectroscopy results displayed a distinct surface plasmon resonance peak indicative of AgNP formation. FTIR analysis revealed specific interactions between silver ions and phytochemicals in the gall extract, while TEM images confirmed the nanoscale morphology and size of the synthesized particles. Zeta potential and DLS analyses provided insights into the stability and size distribution of the AgNPs, demonstrating good colloidal stability. Biological properties of the AgNPs were assessed through various assays. Antimicrobial activity was tested using the disc diffusion method against Escherichia coli and Staphylococcus aureus, showing significant inhibitory effects. The anticancer potential was evaluated using the trypan blue exclusion assay on Dalton's Lymphoma Ascites (DLA) cells, revealing considerable cytotoxicity. Additionally, antimitotic activity was studied in the dividing root cells of Allium cepa, where the AgNPs significantly inhibited cell division. This research highlights the effective use of F. recemosa gall extracts for the green synthesis of AgNPs, presenting an eco-friendly approach to producing nanoparticles with strong antimicrobial, anticancer, and antimitotic properties. The promising results suggest potential applications of these biogenic AgNPs in medical and agricultural sectors, paving the way for further exploration and utilization.

A Comparison of Oral and Intravenous Treatments in Managing Spontaneous Bacterial Peritonitis

Abstract Background: In cirrhotics, spontaneous bacterial peritonitis (SBP) is a potentially fatal illness that requires immediate antibiotic administration. This meta-analysis aimed to examine the effectiveness of IV and oral antibiotics in patients with SBP. Objectives: We compared the efficacy, safety, and cost-effectiveness of oral vs intravenous antibiotic therapy for the treatment of SBP using data from a retrospective cohort analysis. Study design: Retrospective cohort study Place and duration of study: A tertiary care Hospital in Peshawar from Jan 2022 to Jan 20243 Methods:At Tertiary Care Hospital Peshawar, a Retrospective Cohort Study was conducted including all patients diagnosed with SBP between January 2022 and January 2023. The patients were split into two groups based on the kind of antibiotics administered: Group B: patients in need of ampicillin, cephalosporines, or IV IV therapy; Group A: patients on oral antibiotics and ciprofloxacin/cotrimoxazole with norfloxacin (patient 44). Patients who were eighteen years of age or older were eligible for inclusion. Based on an ascitic PMN cell count of ≥250 cells/mm³ and positive bacterial cultures, they were diagnosed with hospital-acquired SBP. Secondary peritonitis and serious co-morbidities requiring non-study antibiotic therapy were excluded criteria. Results:Group B, which received metronidazole orally, had an 85% resolution rate and an in-hospital mortality rate.The highest mortality rate and length of hospital stay were determined to be 10% and 7 ±3 days, respectively. With a 90% resolution rate and a roughly 8% overall mortality rate, Group B only needed to stay in the hospital for 10 (±4) days. 15% recurrences in Group A Group B: 12 percent recurrences Conclusion:Oral antibiotics are statistically no different from IV in terms of mortality, length of stay, or other outcomes when used to treat SBP. The proven outcomes back up the introduction of oral therapy for individuals who qualify in order to improve adherence and save costs. Keywords: Oral antibiotics, Systematic & individual, IV therapy

Integrated miRNA Profiling of Extracellular Vesicles from Uterine Aspirates, Malignant Ascites and Primary-Cultured Ascites Cells for Ovarian Cancer Screening.

Extracellular vesicles (EVs) are of growing interest in the context of screening for highly informative cancer markers. We have previously shown that uterine aspirate EVs (UA EVs) are a promising source of ovarian cancer (OC) diagnostic markers. In this study, we first conducted an integrative analysis of EV-miRNA profiles from UA, malignant ascitic fluid (AF), and a conditioned medium of cultured ascites cells (ACs). Using three software packages, we identified 79 differentially expressed miRNAs (DE-miRNAs) in UA EVs from OC patients and healthy individuals. To narrow down this panel and select miRNAs most involved in OC pathogenesis, we aligned these molecules with the DE-miRNA sets obtained by comparing the EV-miRNA profiles from OC-related biofluids with the same control. We found that 76% of the DE-miRNAs from the identified panel are similarly altered (differentially co-expressed) in AF EVs, as are 58% in AC EVs. Interestingly, the set of miRNAs differentially co-expressed in AF and AC EVs strongly overlaps (40 out of 44 miRNAs). Finally, the application of more rigorous criteria for DE assessment, combined with the selection of miRNAs that are differentially co-expressed in all biofluids, resulted in the identification of a panel of 29 miRNAs for ovarian cancer screening.

ATR, CHK1 and WEE1 inhibitors cause homologous recombination repair deficiency to induce synthetic lethality with PARP inhibitors

PurposePARP inhibitors (PARPi) are effective in homologous recombination repair (HRR) defective (HRD) cancers. To (re)sensitise HRR proficient (HRP) tumours to PARPi combinations with other drugs are being explored. Our aim was to determine the mechanism underpinning the sensitisation to PARPi by inhibitors of cell cycle checkpoint kinases ATR, CHK1 and WEE1.Experimental designA panel of HRD and HRP cells (including matched BRCA1 or 2 mutant and corrected pairs) and ovarian cancer ascites cells were used. Rucaparib (PARPi) induced replication stress (RS) and HRR (immunofluorescence microscopy for γH2AX and RAD51 foci, respectively), cell cycle changes (flow cytometry), activation of ATR, CHK1 and WEE1 (Western Blot for pCHK1S345, pCHK1S296 and pCDK1Y15, respectively) and cytotoxicity (colony formation assay) was determined, followed by investigations of the impact on all of these parameters by inhibitors of ATR (VE-821, 1 µM), CHK1 (PF-477736, 50 nM) and WEE1 (MK-1775, 100 nM).ResultsRucaparib induced RS (3 to10-fold), S-phase accumulation (2-fold) and ATR, CHK1 and WEE1 activation (up to 3-fold), and VE-821, PF-477736 and MK-1775 inhibited their targets and abrogated these rucaparib-induced cell cycle changes in HRP and HRD cells. Rucaparib activated HRR in HRP cells only and was (60-1,000x) more cytotoxic to HRD cells. VE-821, PF-477736 and MK-1775 blocked HRR and sensitised HRP but not HRD cells and primary ovarian ascites to rucaparib.ConclusionsOur data indicate that, rather than acting via abrogation of cell cycle checkpoints, ATR, CHK1 and WEE1 inhibitors cause an HRD phenotype and hence “induced synthetic lethality” with PARPi.

Low-temperature Co-precipitation Synthesis of ZnAl2O4 Nanophosphors Probing its Luminescent, Antibacterial and Anticancer Potentials.

Metal nanoparticles and their binary oxides are well-known for their interactions with biomolecules and their applications in the biomedical field. However, the potential of ternary oxide nanophosphors remains underexplored in these fields due to challenges associated with high-temperature synthesis procedures and the use of toxic chemicals. ZnAl2O4, a ternary oxide matrix, being recognized for its adjustable wide bandgap, impressive surface properties, mechanical strength, thermal stability, and high quantum yield, is chosen for the present work. This study aims to comprehensively investigate the structural, morphological, optical, and cytotoxic properties of zinc aluminate nano phosphors synthesized through a co-precipitation method followed by low-temperature calcination. Analysis using X-ray diffraction spectroscopy (XRD) and Fourier-transform infrared spectroscopy (FTIR) revealed that the formation of the ZnAl2O4 spinel phase initiates at 300°C and completes at 750°C.SEM-EDAX measurements provided further confirmation of the compositional integrity of the synthesized sample. The average crystallite size, determined to be 11.47nm through a W-H plot, along with a higher bandgap value of 4.49eV compared to bulk ZnAl2O4 from the diffuse reflectance spectra (DRS), attests to the success of the nanophosphor synthesis. The self-activated blue luminescent centers of ZnAl2O4 can be fine-tuned to emit light in the green and red regions of the electromagnetic spectrum through appropriate rare earth (RE) doping, utilizing Tb3+ and Eu3+ respectively. Furthermore, the particles underwent short-term in-vitro cytotoxicity testing using Dalton's Lymphoma Ascites cells (DLA) and normal cells, demonstrating high activity against DLA cells while maintaining compatibility with normal cells.

Expression of CD39 is associated with T cell exhaustion in ovarian cancer and its blockade reverts T cell dysfunction

ABSTRACT Immune exhaustion is a hallmark of ovarian cancer. Using multiparametric flow cytometry, the study aimed to analyze protein expression of novel immunological targets on CD3+ T cells isolated from the peripheral blood (n = 20), malignant ascites (n = 16), and tumor tissue (n = 6) of patients with ovarian cancer (OVCA). The study revealed an increased proportion of effector memory CD8+ T cells in OVCA tissue and malignant ascites. An OVCA-characteristic PD-1high CD8+ T cell population was detected, which differed from PD-1lowCD8+ T cells by increased co-expression of TIGIT, CD39, and HLA-DR. In addition, these OVCA-characteristic CD8+ T cells showed reduced expression of the transcription factor TCF-1, which may also indicate reduced effector function and memory formation. On the contrary, the transcription factor TOX, which significantly regulates terminal T cell-exhaustion, was found more frequently in these cells. Further protein and gene analysis showed that CD39 and CD73 were also expressed on OVCA tumor cells isolated from solid tumors (n = 14) and malignant ascites (n = 9). In the latter compartment, CD39 and CD73 were also associated with the expression of the “don’t eat me” molecule CD24 on tumor cells. Additionally, ascites-derived CD24+EpCAM+ tumor cells showed a higher frequency of CD39+ or CD73+ cells. Furthermore, CD39 expression was associated with unfavorable clinical parameters. Expression of CD39 on T cells was upregulated through CD3/CD28 stimulation and its blockade by a newly developed nanobody construct resulted in increased proliferation (eFluor), activation (CD25 and CD134), and production of cytotoxic cytokines (IFN-γ, TNF-α, and granzyme-B) of CD8+ T cells.

Exploring the anticancer potential of double-active pharmaceutical ingredient, metforminium ibuprofenate: Integrating in vivo and in silico investigations

Rationale: A novel double active pharmaceutical ingredient (d-API), metforminium ibuprofenate (MtHIb) was synthesised by combining metformin hydrochloride (MtHCl) and sodium ibuprofen (NaIb). This formulation exhibits anti-diabetic and anti-inflammatory properties similar to its parent compounds, along with a remarkable antiproliferative effect on diverse cancer cells. AimThe present study is aimed to analyse the anticancer potential of MtHIb by mouse tumour model and molecular docking. MethodsSolid tumour was induced by Dalton's Lymphoma Ascites (DLA) cells in Swiss albino mice, and d-API was administered orally. The tumour volume was measured using Vernier calliper and the activity of γ-glutamyl transferase (GGT) was assessed. The potential interactions of MtHIb with target proteins mTOR, HER2, EGFR, CDK4, CDK6, ERα/β, progesterone, COX2, BUB1, and KEAP2 were analyzed in silico. ResultsA significant reduction in tumour size was observed in mice treated with MtHIb. On the 30th day, the tumour volume, which was 8.12 cm3 in untreated animals, decreased to 1.311 cm3 with the treatment of 5 mg/kg b.wt of MtHIb, representing an 83.8 % reduction. The activity of GGT, a marker enzyme of proliferative activity, was considerably reduced from 394.87 to 51.26 U/mg protein/L. Molecular docking studies indicated that MtHIb exhibited strong binding affinity towards all the screened target proteins, specifically HER2, EGFR, CDK4, and BUB1. DiscussionThe findings suggest that MtHIb has promising anti-tumour effect, as evidenced by its significant reduction in tumour size and GGT activity in mice. The strong binding affinity of MtHIb towards the key cancer related proteins further supports its potential as a therapeutic agent against cancer, particularly in targeting pathways involved in cell proliferation and survival. Further preclinical and clinical studies are required to validate these findings and to explore the full therapeutic potential of MtHIb in cancer treatment.

Cordyceps militaris: A Comprehensive Study on Laboratory Cultivation and Anticancer Potential in Dalton's Ascites Lymphoma Tumor Model.

Cancer, a predominant cause of mortality, poses a formidable challenge in our pursuit of elevating life expectancy. Throughout history, individuals have sought natural remedies with minimal side effects as an appealing substitute for chemotherapeutic drugs. One such remedy is Cordyceps militaris, a renowned medicinal mushroom deeply entrenched in Asian ethnomedicine. Revered for its rejuvenating and curative attributes, it relied upon for ages. The mushroom's soaring demand outpaced natural availability, necessitating controlled laboratory cultivation as the core focus and exploring the potential of methanolic extracts from harvested Cordyceps militaris fruiting bodies against Dalton's Lymphoma Ascites (DLA) cells in vitro, with a specific emphasis on its anticancer traits. For cultivation, we employed a diverse range of rice substrates, among which bora rice showed promising growth of C. militaris fruiting bodies. To assess DLA cell cytotoxicity, several assays, including trypan blue exclusion assay, MTT assay, and LDH assay, were employed at different time points (24-96 h), which provided valuable insights on DLA cell viability and proliferation, shedding light on its therapeutic potential against cancer. Our studies unveiled that methanolic extract prompts apoptosis in DLA cells via AO/EB dual staining, manifesting consistent apoptosis indicators such as membrane blebbing, chromatin condensation, nuclei fragmentation, and cellular shrinkage at 48-96 h of treatment. Furthermore, these striking repercussions of apoptosis were comprehended by an in silico approach having molecular docking simulation against antiapoptotic proteins like BCL-2, BCL-XL, MCL-1, BFL-1 & HSP100. Methanolic C. militaris extracts exhibited cytotoxicity and apoptotic alterations in DLA cells.

Strongyloides stercoralis infection masquerading as malignant ascites in a case of angiosarcoma.

Strongyloides stercoralis is a parasitic nematode that infects millions of people worldwide. It primarily infects humans but can also be found in domestic animals and primates. The severity of infection varies from asymptomatic to life-threatening complications. We present a case of a 56-year-old male with a known case of angiosarcoma liver with massive ascites and low-grade fever. He was clinically diagnosed as having malignant ascites and was planned for chemotherapy. During therapeutic cum diagnostic ascitic tap, cell block sections revealed the presence of cross sections of nematode S. stercoralis gravid uterus with eggs. Later, stool for ova and cysts also revealed multiple larval forms of the nematode. S. stercoralis hyperinfection is often accompanied by sepsis or meningitis with enteric organisms. Patient was started on tab. ivermectin 12 mg once a day for two days (in the standard dosage of 200 mcg/kg) was then repeated in the same dosage for another two days after two weeks, and gradually the ascites settled. Post-treatment ascitic tap cell block preparations did not show any parasites. Patient has been on follow-up for 6 months, and he remains asymptomatic.

CXCR6+CD69+ CD8+ T cells in ascites are associated with disease severity in patients with cirrhosis

CXCR6+CD69+ CD8+ T cells in ascites are associated with disease severity in patients with cirrhosis

Abstract 1273: Tumor progression and tumor microenvironment of pancreas cancer ascites revealed by scRNA-seq and spatial transcriptomics

Abstract Peritoneal metastasis of pancreas cancer is one of the ultimate modes of spread in the cancer. Little is known about how cancer cells spread into abdominal cavity and what is different from hematogenous metastasis. It is also not well characterized how tumor microenvironment prevent cancer cells from spreading in abdominal cavity. We performed single-cell RNA-seq analysis and special transcriptomic analysis to clarify the progression and environment of the cancer. We collected and processed ascites samples of 20 PDAC samples from 19 patients. Sixteen ascites samples were obtained from abdominal paracentesis and two cases in the postmortem period. For one patient ascites was collected both pre and postmortem. All but one patient received chemotherapy prior to ascites collection. Routine clinical cytology evaluation of ascites samples from 17 patients indicated it was malignant in 11 patients and nonmalignant in six patients. All ascites samples were purified by gradient centrifuge using Ficoll-Paque Plus to remove erythrocytes and necrotic debris. For the platform, we adopted the 10x Genomics Chromium Single Cell Gene Expression platform for single-cell RNA-seq library preparation. Spatial transcriptomics was performed for eight FFPE samples from the two autopsy patients with paired cancer ascites using 10x Visium. The CytAssist was used to transfer transcripts of pre-sectioned and pre-stained tissues with 11 × 11 mm Visium Capture Area slides. The sample locations were metastatic tumors to bowel wall, rib bone, mesentery, iliac nerve, diaphragm, peritoneum, pelvis, and primary pancreas tumor. A total of 248,263 cells were analyzed in single-cell RNA-seq. Cell population in pancreas cancer ascites consisted of macrophages, lymphocytes, dendritic cells, plasma cells, plasmacytoid dendritic cells, mesothelial cells, and cancer cells. Cancer cells were detected in twelve samples whereas no cancer cells were detected in eight samples. Gene ontology and gene set enrichment analyses showed macrophages in ascites with cancer cells involved neutrophil activation while those in ascites without cancer cells retained antigen presentation capacity and inducing interferon gamma pathway. Mesothelial cells in ascites with cancer cells showed more apoptosis and lymphocyte proliferation while those in ascites without cancers cells triggered fibrosis and mitosis. These data suggest that macrophage and mesothelial cells could be categorized into anti-tumor cells and tumor associated cells. Ongoing investigations are exploring ligand-receptor relationships. We are going to compare the expression profile of tumor cells in ascites fluid and in tissue from various organs by integrating single cell RNA-seq data and special transcriptomics data. Citation Format: Shigeaki Umeda, Elias-Ramzey Karnoub, Fiyinfolu Balogun, Alejandro Jiménez-Sánchez, Christine A. Iacobuzio-Donahue. Tumor progression and tumor microenvironment of pancreas cancer ascites revealed by scRNA-seq and spatial transcriptomics [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 1273.

Abstract 6527: Fucosyltransferase 5 promotes immune evasion in ovarian cancer by suppressing double negative T cells

Abstract Emerging evidence suggests that aberrant glycosylation in cancer impact on various stages in the disease progression including immunomodulation, but the underlying mechanisms remain poorly understood. Our previous data demonstrated that fucosyltransferase 5 (FUT5), a glycosyltransferase responsible for the terminal step in synthesizing Lewis antigens, may contribute to peritoneal metastasis of ovarian cancer. In this study, using clinical samples of high grade serous ovarian cancer (HGSOC), the most common subtype, we found that high expression of FUT5 is associated with a worse outcome. By employing multiplex immunohistochemistry (IHC) to examine T cell subsets, we found a significant decrease in CD3+CD4−CD8− (double negative, DN) T cells in FUT5high (n=10) compared to FUT5low samples (n=10), while there is no difference in the abundance of CD8+ cytotoxic T cells, CD4+ helper T cells or CD4+Foxp3+ regulatory T cells. Tumor-infiltrating DN T cells has been shown to mediate potent anti-tumor response. In addition to their cytotoxic capabilities, DN T cells largely exhibit modulatory functions on other T cell subsets. To further investigate, we performed additional IHC staining and observed a significant increase in proliferating (Ki67+) CD8+ tissue resident memory cells, which are known to be important for cancer immunity. In addition, we observed a decrease in metastases in the FUT5 knockdown group, accompanied by an increase in DN T cells in ascites and omental metastatic tumors in humanised mice, as revealed by flow cytometry, confirming a direct immunomodulatory role of FUT5. Together, these findings reveal a novel role of FUT5 in promoting immune evasion by suppressing anti-tumor DN T cells, which could provide novel insights for immunotherapy in HGSOC, which has a low response rate to immune checkpoint inhibitors (This research is supported by RGC SRFS2223-7S05, HMRF 08192286 and LSCCB under the Health@InnoHK Program launched by ITC, HKSAR). Citation Format: S. W. Fung, Sally K. Y. To, A. A. Hassan, Phillip P.C. Ip, Alice S. T. Wong. Fucosyltransferase 5 promotes immune evasion in ovarian cancer by suppressing double negative T 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 6527.

Abstract 3725: Exosomal Galactin-3 promotes pre-metastatic niche formation that accelerates peritoneal metastases of gastric adenocarcinoma

Abstract Peritoneal carcinomatosis (PC) in gastric adenocarcinoma (GAC) lead to a short median survival (≤6 months). Pre-metastatic niche (PMN) formation, in a distant organ to prepare for tumor seeding and colonization, has long been recognized. However, the precise molecular and mechanistic contributions of exosomes in PMN formation of PC remain unclear. We identify that exosomes promote PMN formation in the peritoneal cavity by patient-derived orthotopic mouse model. Using an in-depth proteomic profiling of paired ascites cells and ascites-derived exosomes from PC patients, we discover that Galectin-3 as the exosomal payload is responsible for PC by promoting PMN formation in both patient-derived orthotopic model and syngeneic mouse model. Inhibition of exosomal Galectin-3 reverses PC. Furthermore, exosomal Galectin-3 activates cancer-associated fibroblasts (CAFs), which further activate the CXCL12/CXCR4 axis to orchestrate the immunosuppressive PMN. Simultaneous inhibition of exosomal Galectin-3, CXCR4, and PD-1 leads to tumor regression and durable anti-tumor responses. Thus, this study provides a rationale for the novel clinical strategy for GAC patients with PC. Citation Format: Yibo Fan, Shumei Song, Melissa Pool Pizzi, Katsuhiro Yoshimura, Jody V. Vykoukal, Samir Hanash, Jaffer Ajani. Exosomal Galactin-3 promotes pre-metastatic niche formation that accelerates peritoneal metastases of gastric 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 3725.

Impacts of cryopreservation on phenotype and functionality of mononuclear cells in peripheral blood and ascites.

Mononuclear cells in peripheral blood and ascites are important clinical resources commonly used in translational and basic research. However, the impact of different cryopreservation durations and extra freeze-thaw cycles on the number and function of mononuclear cells is unknown. Peripheral blood samples (n = 21) and ascites samples (n = 8) were collected from healthy volunteers and ovarian cancer patients. Mononuclear cells were isolated, frozen, and thawed at 6 and 12 months. The impact of cryopreservation on cell viability, the phenotype, and the activation and proliferation of T cells were analyzed by flow cytometry. Single-cell sequencing was applied to investigate the underlying mechanism. The cell number and viability of mononuclear cells in peripheral blood and ascites were significantly decreased after cryopreservation. The T lymphocytes, especially CD4+ T cells, were affected the most significantly. By contrast, monocytes, natural killer (NK) cells, natural killer T (NKT) cells, and B cells were more tolerant. Meanwhile, T cell proliferation and IL-2 secretion are significantly affected after long-term cryopreservation. Mechanistically, the cell death induced by elevated reactive oxygen species (ROS) was involved in the reduction of CD4+ T cells after cryopreservation. Our data indicates that different subtypes of mononuclear cells exhibit different tolerance capacities upon cryopreservation. Thus, our research can provide evidence and support for individuals who are conducting experiments using frozen clinical patient-derived mononuclear cells, for basic research or clinical trials. In addition, extra caution is worthwhile when researchers compare immune cell functionality from peripheral blood or ascites across datasets obtained in different cryopreservation conditions.

Specific lineage transition of tumor-associated macrophages elicits immune evasion of ascitic tumor cells in gastric cancer with peritoneal metastasis.

Gastric cancer with peritoneal metastasis (PM-GC), recognized as one of the deadliest cancers. However, whether and how the tumor cell-extrinsic tumor microenvironment (TME) is involved in the therapeutic failure remains unknown. Thus, this study systematically assessed the immunosuppressive tumor microenvironment in ascites from patients with PM-GC, and its contribution to dissemination and immune evasion of ascites-disseminated tumor cells (aDTCs). Sixty-three ascites and 43 peripheral blood (PB) samples from 51 patients with PM-GC were included in this study. aDTCs in ascites and circulating tumor cells (CTCs) in paired PB were immunophenotypically profiled. Using single-cell RNA transcriptional sequencing (scRNA-seq), crosstalk between aDTCs and the TME features of ascites was inspected. Further studies on the mechanism underlying aDTCs-immune cells crosstalk were performed on in vitro cultured aDTCs. Immune cells in ascites interact with aDTCs, prompting their immune evasion. Specifically, we found that the tumor-associated macrophages (TAMs) in ascites underwent a continuum lineage transition from cathepsinhigh (CTShigh) to complement 1qhigh (C1Qhigh) TAM. CTShigh TAM initially attracted the metastatic tumor cells to ascites, thereafter, transitioning terminally to C1Qhigh TAM to trigger overproliferation and immune escape of aDTCs. Mechanistically, we demonstrated that C1Qhigh TAMs significantly enhanced the expression of PD-L1 and NECTIN2 on aDTCs, which was driven by the activation of the C1q-mediated complement pathway. For the first time, we identified an immunosuppressive macrophage transition from CTShigh to C1Qhigh TAM in ascites from patients with PM-GC. This may contribute to developing potential TAM-targeted immunotherapies for PM-GC.

Artificial-Intelligence-Assisted Detection of Metastatic Colorectal Cancer Cells in Ascitic Fluid.

Ascites cytology is a cost-effective test for metastatic colorectal cancer (CRC) in the abdominal cavity. However, metastatic carcinoma of the peritoneum is difficult to diagnose based on biopsy findings, and ascitic aspiration cytology has a low sensitivity and specificity and a high inter-observer variability. The aim of the present study was to apply artificial intelligence (AI) to classify benign and malignant cells in ascites cytology patch images of metastatic CRC using a deep convolutional neural network. Datasets were collected from The OPEN AI Dataset Project, a nationwide cytology dataset for AI research. The numbers of patch images used for training, validation, and testing were 56,560, 7068, and 6534, respectively. We evaluated 1041 patch images of benign and metastatic CRC in the ascitic fluid to compare the performance of pathologists and an AI algorithm, and to examine whether the diagnostic accuracy of pathologists improved with the assistance of AI. This AI method showed an accuracy, a sensitivity, and a specificity of 93.74%, 87.76%, and 99.75%, respectively, for the differential diagnosis of malignant and benign ascites. The diagnostic accuracy and sensitivity of the pathologist with the assistance of the proposed AI method increased from 86.8% to 90.5% and from 73.3% to 79.3%, respectively. The proposed deep learning method may assist pathologists with different levels of experience in diagnosing metastatic CRC cells of ascites.