Cell Density Research Articles

Comprehensive assessment of corneal microstructural changes following V4c implantable collamer lens surgery using in vivo confocal microscopy

BackgroundImplantable Collamer Lense (ICL) presents a viable alternative to conventional refractive surgeries, but their impact on corneal microstructure remains unclear. By employing in vivo confocal microscopy (IVCM), we examined changes in stromal and endothelial cells following the insertion of V4c ICLs, with the goal of enhancing post-surgical care and outcomes.MethodsIn this longitudinal investigation, we conducted detailed preoperative assessments on 103 eyes from 53 participants. Follow-up evaluations were carried out after surgery at set intervals: one day, one week, one month, three months, six months, and twelve months. We used IVCM to analyze changes in stromal and endothelial cells. To assess differences between pre- and post-surgery variables and to investigate correlations with age, axial length (AL), and spherical equivalent refraction (SER), we applied a repeated measures mixed-effects model, with statistical significance set at P < 0.05.ResultsNo vision-threatening complications were reported post-surgery. Significant reductions in stromal cell density (SCD) were observed postoperatively, with anterior and mid- SCD reaching their lowest values at 3 months and posterior SCD at 1 month, remaining below baseline at 12 months. endothelial cell density (ECD) and percentage of hexagonal cells (PHC) decreased initially, recovering by 12 months. Conversely, endothelial cellular area (ECA) and coefficient of variation of cell size (CoV) increased postoperatively, with the most significant change at 1 week. Endothelial deposits were detected in 49 of 101 eyes on postoperative day 1, half of them were absorbed within 3 months post-surgery. Changes in posterior SCD were negatively related to AL, while AL, SER, lens thickness showed associated with endothelium changes.ConclusionOur findings elucidate the corneal microstructural changes following V4c ICL implantation, particularly the significant early reductions in stromal and endothelial cell densities. We recommend careful management of viscoelastics during surgery to minimize endothelial deposits that may harm the endothelium. Enhanced early postoperative monitoring and these surgical adjustments can lead to improved surgical and post-surgical care, ultimately supporting better patient recovery.

&lt;i&gt;CDKN2B-AS1&lt;/i&gt; polymorphism rs1333049 is associated with advanced carotid artery atherosclerosis in a Slovenian population

Several studies have reported an association between the 9p21 region in the human genome and atherosclerosis. The rs1333049 polymorphism is a single nucleotide polymorphism (SNP) in CDKN2B-AS1, located in the 9p21 region. The aim of our study was to investigate the association between the rs1333049 polymorphism and advanced carotid atherosclerosis, as well as its effect on CDKN2B expression in endarterectomy sequesters. In our case-control study, we included 881 participants, divided into two groups. The case group comprised 308 participants with advanced atherosclerosis of the internal or common carotid artery (stenosis &gt; 75 %) who underwent a revascularization procedure. The control group included 573 participants without hemodynamically significant carotid atherosclerosis. We analyzed the rs1333049 polymorphism using the StepOne real-time polymerase chain reaction (PCR) and TaqMan SNP genotyping assay. We found a statistically significant association according to the co-dominant (P=0.014, OR=3.29, 95% CI: 1.32-8.91, and P=0.015, OR=2.50, 95% CI: 1.22-5.37) and dominant (P=0.006, OR=2.74, 95% CI: 1.36-5.71) models. We performed immunohistochemical analysis of CDKN2B expression on 26 endarterectomy sequesters. The C allele of rs1333049 was associated with a lower numerical area density of CDKN2B-positive cells in atherosclerotic plaques. In conclusion, the C allele of the rs1333049 SNP is associated with an increased risk of developing advanced carotid atherosclerosis and lower CDKN2B expression in the plaques.

A mathematical insight to control the disease psoriasis using mesenchymal stem cell transplantation with a biologic inhibitor

Psoriasis is a chronic, non-contagious, immune-mediated skin disorder. Inflammation of the skin’s surface is characterised by scaly white, red, or silvery spots that occur due to the hyper-proliferation of keratinocytes in the epidermal layer. Primarily, pharmaceutical drugs or immune therapy are used to treat psoriasis. We are all aware that, certain therapeutic strategies can have some adverse effects, and over time, that hidden inflammation may manifest. This article introduces a mathematical model for psoriasis, formulated by employing a set of nonlinear ordinary differential equations (ODEs) that describe the densities of T-cells, dendritic cells (DCs), keratinocytes, and mesenchymal stromal cells (MSCs) as basic cell populations. A tumor necrosis factor-α\\documentclass[12pt]{minimal} \\usepackage{amsmath} \\usepackage{wasysym} \\usepackage{amsfonts} \\usepackage{amssymb} \\usepackage{amsbsy} \\usepackage{mathrsfs} \\usepackage{upgreek} \\setlength{\\oddsidemargin}{-69pt} \\begin{document}$$\\alpha$$\\end{document} (TNF-α\\documentclass[12pt]{minimal} \\usepackage{amsmath} \\usepackage{wasysym} \\usepackage{amsfonts} \\usepackage{amssymb} \\usepackage{amsbsy} \\usepackage{mathrsfs} \\usepackage{upgreek} \\setlength{\\oddsidemargin}{-69pt} \\begin{document}$$TNF-\\alpha$$\\end{document}) inhibitor has been imposed from the initial stage of the treatment regime, using the optimal control theoretic approach, and the numerical results have been observed. After 80 days of monitoring using only biologic TNF-α\\documentclass[12pt]{minimal} \\usepackage{amsmath} \\usepackage{wasysym} \\usepackage{amsfonts} \\usepackage{amssymb} \\usepackage{amsbsy} \\usepackage{mathrsfs} \\usepackage{upgreek} \\setlength{\\oddsidemargin}{-69pt} \\begin{document}$$TNF-\\alpha$$\\end{document} inhibitors, if this approach did not provide the intended outcomes (when severity arises), stem cells are administered a few times in a pulsed manner as a cell replacement technique in addition to this anti TNF-α\\documentclass[12pt]{minimal} \\usepackage{amsmath} \\usepackage{wasysym} \\usepackage{amsfonts} \\usepackage{amssymb} \\usepackage{amsbsy} \\usepackage{mathrsfs} \\usepackage{upgreek} \\setlength{\\oddsidemargin}{-69pt} \\begin{document}$$TNF-\\alpha$$\\end{document} medicine. We have observed the combined therapeutic benefit of stem cell replacement with a TNF-α\\documentclass[12pt]{minimal} \\usepackage{amsmath} \\usepackage{wasysym} \\usepackage{amsfonts} \\usepackage{amssymb} \\usepackage{amsbsy} \\usepackage{mathrsfs} \\usepackage{upgreek} \\setlength{\\oddsidemargin}{-69pt} \\begin{document}$$TNF-\\alpha$$\\end{document} inhibitor from a mathematical point of view. The theoretical analysis and the numerical results revealed that stem cell transplantation, along with a TNF-α\\documentclass[12pt]{minimal} \\usepackage{amsmath} \\usepackage{wasysym} \\usepackage{amsfonts} \\usepackage{amssymb} \\usepackage{amsbsy} \\usepackage{mathrsfs} \\usepackage{upgreek} \\setlength{\\oddsidemargin}{-69pt} \\begin{document}$$TNF-\\alpha$$\\end{document} inhibitor, is a promising psoriasis treatment option moving forward.

A growth-based screening strategy for engineering the catalytic activity of an oxygen-sensitive formate dehydrogenase.

Enzyme engineering is a powerful tool for improving or altering the properties of biocatalysts for industrial, research, and therapeutic applications. Fast and accurate screening of variant libraries is often the bottleneck of enzyme engineering and may be overcome by growth-based screening strategies with simple processes to enable high throughput. The currently available growth-based screening strategies have been widely employed for enzymes but not yet for catalytically potent and oxygen-sensitive metalloenzymes. Here, we present a screening system that couples the activity of an oxygen-sensitive formate dehydrogenase to the growth of Escherichia coli. This system relies on the complementation of the E. coli formate hydrogenlyase (FHL) complex by Mo-dependent formate dehydrogenase H (EcFDH-H). Using an EcFDH-H-deficient strain, we demonstrate that growth inhibition by acidic glucose fermentation products can be alleviated by FHL complementation. This allows the identification of catalytically active EcFDH-H variants at a readily measurable cell density readout, reduced handling efforts, and a low risk of oxygen contamination. Furthermore, a good correlation between cell density and formate oxidation activity was established using EcFDH-H variants with variable catalytic activities. As proof of concept, the growth assay was employed to screen a library of 1,032 EcFDH-H variants and reduced the library size to 96 clones. During the subsequent colorimetric screening of these clones, the variant A12G exhibiting an 82.4% enhanced formate oxidation rate was identified. Since many metal-dependent formate dehydrogenases and hydrogenases form functional complexes resembling E. coli FHL, the demonstrated growth-based screening strategy may be adapted to components of such electron-transferring complexes.IMPORTANCEOxygen-sensitive metalloenzymes are highly potent catalysts that allow the reduction of chemically inert substrates such as CO2 and N2 at ambient pressure and temperature and have, therefore, been considered for the sustainable production of biofuels and commodity chemicals such as ammonia, formic acid, and glycine. A proven method to optimize natural enzymes for such applications is enzyme engineering using high-throughput variant library screening. However, most screening methods are incompatible with the oxygen sensitivity of these metalloenzymes and thereby limit their relevance for the development of biosynthetic production processes. A microtiter plate-based assay was developed for the screening of metal-dependent formate dehydrogenase that links the activity of the tested enzyme variant to the growth of the anaerobically grown host cell. The presented work extends the application range of growth-based screening to metalloenzymes and is thereby expected to advance their adoption to biosynthesis applications.

Physics-informed neural networks for biopharmaceutical cultivation processes: Consideration of varying process parameter settings.

We present a new modeling approach for the study and prediction of important process outcomes of biotechnological cultivation processes under the influence of process parameter variations. Our model is based on physics-informed neural networks (PINNs) in combination with kinetic growth equations. Using Taylor series, multivariate external process parameter variations for important variables such as temperature, seeding cell density and feeding rates can be integrated into the corresponding kinetic rates and the governing growth equations. In addition to previous approaches, PINNs also allow continuous and differentiable functions as predictions for the process outcomes. Accordingly, our results show that PINNs in combination with Taylor-series expansions for kinetic growth equations provide a very high prediction accuracy for important process variables such as cell densities and concentrations as well as a detailed study of individual and combined parameter influences. Furthermore, the proposed approach can also be used to evaluate the outcomes of new parameter variations and combinations, which enables a saving of experiments in combination with a model-driven optimization study of the design space.

Assessment of cell death in the liver of Labeo rohita on exposure to an emerging contaminant aspirin: an immunofluorescent, flow cytometric and biochemical investigation.

Aspirin is one of the most frequently detected non-steroidal anti-inflammatory drugs in aquatic environments. Despite its prevalence, toxicity possessed by aspirin to non-target organisms like fish is poorly explored. In the present study, cell death induced by different concentrations of aspirin (1, 10, and 100µg/L) has been investigated in the liver of fish, Labeo rohita exposed for 28days. A significant increase (p < 0.05) in the density of caspase-3 positive cells in a dose and duration-dependent manner assessed through immunofluorescent staining indicates caspase-dependent pathway of cell death which may be either through intrinsic or extrinsic pathway. The flow cytometric analysis, in addition, revealed a significant (p < 0.05) decline in the live cells and an increase in apoptotic cells in the liver of fish exposed to aspirin. Cell death due to apoptosis is further indicated by a significant increase (p < 0.05) in the Kupffer cells and tumor necrosis factor-α. The decrease in the activity of cyclooxygenase (COX) enzyme significantly (p < 0.05) in all three exposure concentrations of aspirin suggests COX-dependent pathway of cell death. The present study provides in-depth insights into aspirin-induced cell death in the liver of fish at environmentally realistic concentrations.

Gut mucosa alterations after kidney transplantation: a cross sectional study.

Kidney transplant recipients (KTRs) rely on immunosuppressants like mycophenolate to prevent organ rejection. However, mycophenolate often causes intestinal symptoms and inflammation in various organs, including the skin and the colon. While KTRs have an increased risk for skin cancer, the risk of colorectal cancer is not increased. Elucidating the histological alterations in the colon of KTRs and comparing these changes with known skin alterations would help understand how immunosuppressants influence cancer development and progression. Whole slide images from gut biopsies (Non-transplanted subjects n = 35, KTRs n = 49) were analyzed using the ImageJ and R programming environment. A total of 22,035 epithelial cells, 38,870 interstitial cells, 3465 epithelial cell mitoses, and 7477 endothelial cells, each characterized by multiple microscopy parameters, from a total of 1788 glands were analyzed. The large database was subsequently analyzed to verify the changes of inflammatory milieu in KTRs and in cancer. KTRs without colon-cancer showed a significantly higher density of interstitial cells in the colon compared to non-transplanted patients. Moreover, the increase in interstitial cell number was accompanied by subtle modifications in the architecture of the colon glands, without altering the epithelial cell density. We could not identify significant structural modifications in cancer samples between KTRs and non-transplanted patients. Our findings demonstrate an increased number of resident interstitial cells in the colon of KTRs, as in other patients treated with mycophenolate. These changes are associated with subtle alterations in the architecture of colon glands.

SnF2-Catalyzed Lithiophilic-Lithiophobic Gradient Interface for High-Rate PEO-based All-Solid-State Batteries.

Polyethylene oxide (PEO)-based all solid-state lithium metal batteries (ASSLMBs) are strongly hindered by the fast dendrite growth at the Li metal/electrolyte interface, especially under large rates. The above issue stems from the suboptimal interfacial chemistry and poor Li+ transport kinetics during cycling. Herein, a SnF2-catalyzed lithiophilic-lithiophobic gradient solid electrolyte interphase (SCG-SEI) of LixSny/LiF-Li2O is in-situ formed. The superior ionic LiF-Li2O rich upper layer (17.1 nm) possesses high interfacial energy and fast Li+ diffusion channels, wherein lithiophilic LixSny alloy layer (8.4 nm) could highly reduce the nucleation overpotential with lower diffusion barrier and promote rapid electron transportation for reversible Li+ plating/stripping. Simultaneously, the insoluble SnF2-coordinated PEO promotes the rapid Li+ ion transport in the bulk phase. As a result, an over 46.7 and 3.5 times improvements for lifespan and critical current density of symmetrical cells are achieved, respectively. Furthermore, LiFePO4-based ASSLMBs deliver a recorded cycling performance at 5 C (over 1000 cycles with a capacity retention of 80.0%). More importantly, impressive electrochemical performances and safety tests with LiNi0.8Mn0.1Co0.1O2 and pouch cell with LiFePO4, even under extreme conditions (i.e., 100 ℃), are also demonstrated, reconfirmed the importance of lithiophilic-lithiophobic gradient interfacial chemistry in the design of high-rate ASSLMBs for safety applications.

SnF2‐Catalyzed Lithiophilic–Lithiophobic Gradient Interface for High‐Rate PEO‐Based All‐Solid‐State Batteries

AbstractPolyethylene oxide (PEO)‐based all‐solid‐state lithium metal batteries (ASSLMBs) are strongly hindered by the fast dendrite growth at the Li metal/electrolyte interface, especially under large rates. The above issue stems from the suboptimal interfacial chemistry and poor Li+ transport kinetics during cycling. Herein, a SnF2‐catalyzed lithiophilic‐lithiophobic gradient solid electrolyte interphase (SCG‐SEI) of LixSny/LiF‐Li2O is in situ formed. The superior ionic LiF‐Li2O rich upper layer (17.1 nm) possesses high interfacial energy and fast Li+ diffusion channels, wherein lithiophilic LixSny alloy layer (8.4 nm) could highly reduce the nucleation overpotential with lower diffusion barrier and promote rapid electron transportation for reversible Li+ plating/stripping. Simultaneously, the insoluble SnF2‐coordinated PEO promotes the rapid Li+ ion transport in the bulk phase. As a result, an over 46.7 and 3.5 times improvements for lifespan and critical current density of symmetrical cells are achieved, respectively. Furthermore, LiFePO4‐based ASSLMBs deliver a recorded cycling performance at 5 C (over 1000 cycles with a capacity retention of 80.0 %). More importantly, impressive electrochemical performances and safety tests with LiNi0.8Mn0.1Co0.1O2 and pouch cell with LiFePO4, even under extreme conditions (i.e., 100 °C), are also demonstrated, reconfirmed the importance of lithiophilic‐lithiophobic gradient interfacial chemistry in the design of high‐rate ASSLMBs for safety applications.

Fed-batch strategies for intensified rVSV vector production in high cell density cultures of suspension HEK293 cells.

Vesicular stomatitis virus (VSV) has been increasingly demonstrated as a promising viral vector platform. As the interest over this modality for vaccine and gene therapy applications increases, the need for intensified processes to produce these vectors emerge. In this study, we develop fed-batch-based operations to intensify the production of a recombinant VSV-based vaccine candidate (rVSV-SARS-CoV-2) in suspension cultures of HEK293 cells. A feeding strategy, in which a commercial concentrated medium was added to cultures based on cell growth through a fixed cell specific feeding rate (CSFR), was applied for the development of two different processes using Ambr250 modular bioreactors. Cultures operated in hybrid fed-batch/perfusion (FB/P) or fed-batch (FB) were able to sustain infections performed at 8.0 × 106 cells/mL, respectively resulting in 3.9 and 5.0-fold increase in total yield (YT) and 1.7 and 5.6-fold increase in volumetric productivity (VP) when compared with a batch reference. A maximum viral titer of 4.5 × 1010 TCID50/mL was reached, which is comparable or higher than other processes for VSV production in different cell lines. Overall, our study reports efficient fed-batch options to intensify the production of a rVSV-based vaccine candidate in suspension HEK293 cells.

Predictive and dynamic signature for anti-angiogenics in combination with PD-1 inhibitor in soft-tissue sarcoma: correlative studies linked to the IMMUNOSARC trial.

IMMUNOSARC trial combined an anti-angiogenic agent (sunitinib) with a PD-1 inhibitor (nivolumab) in advanced sarcomas. Here we present the first correlative studies of the STS cohort enrolled in this trial. Formalin-fixed paraffin-embedded (FFPE) and peripheral blood samples were collected at baseline and week 13. FFPE were used for transcriptomics and multiplex immunofluorescence, while peripheral blood samples were used for multiplexed immunoassays. Flow cytometry and Luminex assays were performed to validate translational findings in tumor-isolated cells and peripheral blood mononuclear cells derived from patients. The density of intratumoral CD8+ T cells, measured by multiplexed immuno-phenotyping, was significantly increased after treatment. This augment was accompanied by the dynamic significant increase in gene expression of CD86, CHI3L1, CXCL10, CXCL9, LAG3, and VCAM1, and the decrease in the expression levels of NR4A1. In peripheral blood, 12 proteins were significantly modulated by treatment at W13. A score integrating the dynamic expression of the 7 genes and the 12 soluble factors separated two groups with distinct progression-free survival (PFS): 4.1 months (95% CI 3.5-NR) vs 17 months (95% CI 12.0 - NR), p=0.014. This molecular score was predictive of PFS when applied to the normalized data determined in the baseline samples. Treatment with sunitinib and nivolumab inflamed the sarcoma microenvironment, increasing CD8+ T cell density and the expression of several genes/ proteins with relevance in the response to PD-1 inhibitors. A molecular signature identified two groups of patients with distinct PFS for the combination of anti-angiogenics plus PD-1 inhibitor.

Environmental pH and compound structure affect the activity of short-chain carboxylic acids against planktonic growth, biofilm formation, and eradication of the food pathogen Salmonella enterica.

Short-chain carboxylic acids (SCCAs) that are naturally produced by microbial fermentation play an essential role in delaying microbial spoilage. SCCAs are structurally diverse, but only a few of them are routinely used in food biopreservation. This study investigated the effects of environmental pH and intrinsic properties of 21 structurally different SCCAs on the antimicrobial and antibiofilm activity against Salmonella enterica. Inhibition of SCCA toward planktonic and biofilm growth of S. enterica was higher in an acidic environment (pH 4.5) that is common in fermented products, and for SCCA that possessed both a high acid dissociation strength (pKa) (>4.0) and a positive hydrophobicity [octanol/water partition coefficient (log Kow)]. Crotonic and caproic acids were identified as SCCAs with potential as biopreservatives even at near-neutral pH. SCCA with hydrophilic groups such as lactic acid did not inhibit S. enterica at concentrations up to 50 mM, while SCCA with benzene or methyl groups or a double bond prevented S. enterica growth and biofilm formation. Stimulation of biofilm formation was observed for formic, acetic, and propionic acid close to the minimum inhibitory concentration to reduce 50% of cell density (MIC50) of planktonic cells, and for citric and isocitric acid with an MIC50 of ≥50 mM. The presence of low concentrations of formic and propionic acids during biofilm formation conferred protection during eradication possibly due to a pre-adaptation effect, yet two consecutive acid treatments were successful in eradicating biofilms if the first acid treatment was two- to threefold of the MIC50.IMPORTANCEThis study provides a systematic comparison on the antimicrobial and antibiofilm activity of more than 20 structurally different SCCAs against a common food pathogen. We tested the antimicrobial activity at controlled pH and identified the structure-dependent antimicrobial effects of SCCA without the confounding influence of acidification. The combined effect of pKa and log Kow was identified as an important feature that should be considered when deciding for a specific SCCA in the application as antimicrobial. Our results imply that additional phenomena such as the use of SCCA as substrate and cellular pre-adaption effects have to be taken into consideration. We finally present a two-step treatment as an efficient approach to eradicate biofilms, which can be applied for the disinfection of contact surfaces and manufacturing equipment. Results obtained here can serve as guidelines for application of SCCA to avoid the growth of food pathogens and/or to develop biopreserved food systems.

Carbon nanotubes perpendicularly grown on graphene oxide nanosheets derived from metal-organic frameworks: Synergistic reinforcement of poly(l-lactic acid) scaffold

The construction of the nanohybrid composed of two carbonaceous nanomaterials is a promising strategy to inhibit their aggregation, and effectively exert the advantages of their excellent mechanical properties as reinforcement for polymers. Here, carbon nanotubes (CNTs) were in situ perpendicularly growing on the surface of graphene oxide (GO) through metal-organic frameworks (MOF)-derived strategy by chemical vapor deposition (CVD), aiming to facilitate their dispersion on poly(l-lactic acid) (PLLA) bone scaffold fabricated by selective laser sintering (SLS). Specifically, GO provided nucleation sites for the growth of MOF, and worked as the templates when CNTs grew, in which MOF provided catalysts and carbon sources simultaneously. The precursor was heated to 550 °C and kept for 2 h, then heated to 900 °C and kept for 2 h before cooling. The obtained nanohybrid (GO@CNT) exhibited a superior dispersion state than the pure GO in the scaffold at the same loading, especially when the loading was 0.75 wt%. Adding 0.75 wt% GO@CNT endowed the scaffold with a remarkable increment in tensile strength and compressive strength of 13.36 MPa and 24.72 MPa with enhancement of 55.35% and 18.85%, respectively, compared to the scaffold containing 0.75 wt% GO. A crack extension model combined with experiment results was proposed to better understand reinforcement mechanisms. Additionally, the scaffold containing GO@CNT exhibited benign cytocompatibility with a cell-spreading area of 86.31% and cell density of 564 cells/mm2 after culturing for 5 d according to the results of cell adhesion and immunofluorescence tests, making it a promising candidate for bone defect repair.

Abstract B081: In situ multi-modal characterization of pancreatic ductal adenocarcinoma reveals tumor cell identity as a defining factor of the surrounding microenvironment

Abstract Pancreatic Ductal Adenocarcinoma (PDAC) is heterogeneous with low tumor purity, prominent microenvironment and complex architecture, which preclude identification of shared tumor intrinsic biology within and across patients. We overcame these challenges by applying complementary spatial omics approaches – providing necessary resolution and context – to primary untreated PDAC tumors from 39 patients capturing 341,949 low-bulk and 531,718 single-cell spatial transcriptomes. Of these, 59,403 low-bulk profiles and 205,665 single-cells represented tumor cells. We leveraged this data to build on prior reports of tumor cell subtypes in PDAC. We identify 5 distinct malignant subtypes, excluding ductal-like and intraepithelial neoplasia cells, that span the classical-to-basal spectrum. One tumor subtype is highly proliferative and enriched for the DNA synthesis-condensation-mitosis transcriptional network suggesting this subset disproportionately contribute to tumor growth. Strikingly, the spectrum of tumor cell subtypes was present within all patients, suggesting that subtype-based treatment stratification may need refinement. We find that each subtype has its own distinct regulators and histology. The classical subtype has extensive cell intrinsic regulation, higher cell density and lower extracellular matrix content. In contrast, the basal subtype phenotype results from a combination of cell intrinsic and tumor microenvironment (TME) interactions, wherein the basal subtype has a lower cell density, is surrounded by activated stroma and dense collagen matrix, and is poised for hypoxic adaptation. We derive the composition of cellular neighborhoods providing an explanation for the complex architecture seen in PDAC. Tumor cell neighborhoods stratified based on which side of the classical-to-basal spectrum they fell. For example, cells towards the classical-side of the spectrum had more homogenous tumor neighborhoods that generally lacked intermediate and basal tumor cells. In contrast, cells towards the basal-side of the spectrum were likely to contain classical neighbors. More broadly, neighborhoods along this continuum also contained different proportions of immune and stromal cells, cell densities, collagen deposition, and TME adaptation. These observations support a model where tumor subtypes exist within disparate niches. Lastly, our atlas unifies the catalog of previously reported stromal cells (fibroblasts and stellate cells versus myofibroblastic and inflammatory cancer associated fibroblasts (CAF)) and describes a novel interferon stimulated gene (ISG) resistance signature (ISG.RS) fibroblast. We map the location of these various stromal cells relative to tumor subtypes. For example, myofibroblast CAFs and ISG.RS fibroblasts are enriched near the basal tumor subtype whereas inflammatory CAF abundance increases with distance away from tumor cells. In sum, our atlas provides a lens for stratifying tumor complexity in a cancer cell centric manner and provides a path for testing therapeutic hypothesis in the right cell subtype and context. Citation Format: Brian Rabe, Anna Lyubetskaya, Andrew Kavran, Yulong Bai, Andrew Fisher, Hannah Pliner, Alba Font-Tello, Anne Lewin, Ruifeng Hu, Alexandre P Alloy, Yelena Cheng, Chao Dai, Yunfan Fan, Constance Brett, Todd Brett, Lauren Giampapa, Soeren Stahlschmidt, Fayaz Seifuddin, Steven Vasquez Grinnell, Daniel Carrera, Carlos Rios, Tom Lila, Pradeep Kar, Abhishek Shukla, Rachael Bashford Rogers, Lara Heij, Mike Mason, Ryan Golhar, Isaac Neuhaus, Enas Abu Shah, Shivan Sivakumar, Jimena Trillo Tinoco, Benjamin J Chen, Konstantinos J Mavrakis, Eugene Drokhlyansky. In situ multi-modal characterization of pancreatic ductal adenocarcinoma reveals tumor cell identity as a defining factor of the surrounding microenvironment [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Advances in Pancreatic Cancer Research; 2024 Sep 15-18; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2024;84(17 Suppl_2):Abstract nr B081.

SMAC mimetic drives microglia phenotype and glioblastoma immune microenvironment

Tumor-associated macrophages/microglia (TAMs) are highly plastic and heterogeneous immune cells that can be immune-supportive or tumor-supportive depending of the microenvironment. TAMs are the most abundant immune cells in glioblastoma (GB), and play a key role in immunosuppression. Therefore, TAMs reprogramming toward immune-supportive cells is a promising strategy to overcome immunosuppression. By leveraging scRNAseq human GB databases, we identified that Inhibitor of Apoptosis Proteins (IAP) were expressed by TAMs. To investigate their role in TAMs-related immunosuppression, we antagonized IAP using the central nervous system permeant SMAC mimetic GDC-0152 (SMg). On explants and cultured immune cells isolated from human GB samples, SMg modified TAMs activity. We showed that SMg treatment promoted microglia pro-apoptotic and anti-tumoral function via caspase-3 pro-inflammatory cleavage and the inhibition of tumoroids growth. Then we designed a relevant immunogenic mouse GB model to decipher the spatio-temporal densities, distribution, phenotypes and function of TAMs with or without SMg treatment. We used 3D imaging techniques, a transgenic mouse with fluorescent TAM subsets and mass cytometry. We confirmed that SMg promoted microglia activation, antigen-presenting function and tumor infiltration. In addition, we observed a remodeling of blood vessels, a decrease in anti-inflammatory macrophages and an increased level of monocytes and their mo-DC progeny. This remodeling of the TAM landscape is associated with an increase in CD8 T cell density and activation. Altogether, these results demonstrated that SMg drives the immunosuppressive basal microglia toward an active phenotype with pro-apoptotic and anti-tumoral function and modifies the GB immune landscape. This identifies IAP as targets of choice for a potential mechanism-based therapeutic strategy and SMg as a promising molecule for this application.

Abstract A040: Multiplex fluorescent immunohistochemistry reveals immunosuppressive tumor microenvironment in COMPASS-like complex gene mutated pancreatic cancer

Abstract Background: Pancreatic ductal adenocarcinoma (PDAC) is highly resistant to chemoradiation and immune therapies. There is limited biomarker to identify subsets of PDAC that responds to immunotherapy or targeted therapy. We have identified a subset of PDACs (approximately 25%) carrying COMPASS-like complex (CLC) gene mutations. Analysis of the clinicopathologic and molecular features of this PDAC subset revealed that these tumors are an aggressive group of PDACs with poor or squamous histologic differentiation, increased rates of TP53 mutations, and poor patient survival compared to control PDACs. Our preliminary data showed that PDACs carrying mutations in two members of the CLC genes (KDM6A and KMT2D) have immunosuppressive tumor microenvironment (TME), suggesting that CLC genes are candidate biomarkers for immunotherapy. However, a comprehensive analysis of the immune composition, immune checkpoint expression, and the relationship between tumor cells and immune cells in CLC gene mutated PDACs compared to controls has not been performed. In this study, we characterized the immune composition in CLC gene mutated PDACs compared to paired control PDACs using tyramide signal amplification multiplex fluorescent immunohistochemistry (mfIHC). Methods: Human PDAC tissue slides were stained with 2 mfIHC panels and cell types were defined by different markers: panel 1 (PanCK, CD163, PD-L1, CD3, CD8, FoxP3) and panel 2 (PanCK, CD163, CD3, CD8, TIGIT, TIM3). Random image sections were selected in each sample (5∼8 sections per sample) and divided into wild-type (WT) group (n=57) and mutant group (n=41) and analyzed using InForm Cell Analysis software. Tissue segmentation training was performed to separate stromal and epithelial elements. Cell percentage, cell density, and nearest neighbor distance of each cell type were calculated using R program. Results: The CLC gene mutation is associated with elevated epithelial cells (ECs) and PDL1+ ECs and increased infiltration of exhausted cytotoxic T cells (TIGIT+/TIM3+ CD8 T cells). CLC gene mutated PDACs are characterized by spatial proximity between exhausted T cells (TIGIT+) and epithelial cells/antigen presenting cells (APCs). Higher number of epithelial cells is associated with reduced infiltration of helper T cells in the mutant PDAC tissue. We also observed that high numbers of APC in proximity to CD4 T cells were positively correlated with the proximity of EC and CD8 cytotoxic T cells as well as the proximity of CD4 T cells and cytotoxic T cells in the WT group but not in the CLC gene mutant group. Conclusion: Our results provide insight into the effects of CLC gene mutations on shaping the immune microenvironment of PDAC and suggest that CLC gene mutated PDACs have an immunosuppressive immune microenvironment than other PDACs and may respond differently to immunotherapy. Citation Format: Shungang Zhang, Elaina Daniels, Jake McGue, Hong Sun Kim, Dafydd Thomas, Timothy Frankel, Jiaqi Shi. Multiplex fluorescent immunohistochemistry reveals immunosuppressive tumor microenvironment in COMPASS-like complex gene mutated pancreatic cancer [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Advances in Pancreatic Cancer Research; 2024 Sep 15-18; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2024;84(17 Suppl_2):Abstract nr A040.

Protective role of transforming growth factor-Β3 (TGF-Β3) in the formation of radiation-induced capsular contracture around a breast implant: In vivo experimental study

Postmastectomy radiotherapy causes capsular contracture due to fibroproliferation of the capsular tissue around the implant. In fibrosis, unlike normal wound healing, structural and functional disorders are observed in the tissues caused by excessive/irregular accumulation of extracellular matrix proteins. It has been reported that transforming growth factor-β3 (TGF-β3) prevents and reverses fibrosis in various tissues or provides scarless healing with its antifibrotic effect. Additionally, TGF-β3 has been shown to reduce fibrosis in radiotherapy-induced fibrosis syndrome. However, no study in the literature investigates the effects of exogenously applied TGF-β3 on capsular contracture in aesthetic or reconstructive breast implant application. TGF-β3, which has a very short half-life, has low bioavailability with parenteral administration. Within the scope of this study, free TGF-β3 was loaded into the nanoparticles to increase its low bioavailability and extend its duration of action by providing controlled release. The aim of this study is to investigate the preventive/improving effects of radiation induced capsular contracture using chitosan film formulations containing TGF-β3 loaded poly(lactic-co-glycolic acid)-b-poly(ethylene glycol) (PLGA-b-PEG) nanoparticles in implant-based breast reconstruction. In the characterization studies of nanoparticles, the particle size and zeta potential of the TGF-β3-loaded PLGA-b-PEG nanoparticle formulation selected to be used in the treatment group were found to be 123.60 ± 2.09 nm and −34.87 ± 1.42 mV, respectively. The encapsulation efficiency of the formulation was calculated as 99.91 %. A controlled release profile was obtained in in vitro release studies. Chitosan film formulations containing free TGF-β3 or TGF-β3-loaded PLGA-b-PEG nanoparticles were used in in vivo studies. In animal studies, rats were randomly distributed into 6 groups (n = 8) as sham, implant, implant + radiotherapy, implant + radiotherapy + chitosan film containing unloaded nanoparticles, implant + radiotherapy + chitosan film containing free TGF-β3, implant + radiotherapy + chitosan film containing TGF-β3 loaded nanoparticle. In all study groups, a 2 cm incision was made along the posterior axillary line at the thoracic vertebral level in rats to reach the lateral edge of the latissimus dorsi. The fascial attachment to the chest wall was then bluntly dissected to create a pocket for the implants. In the treatment groups, the wound was closed after films were placed on the outer surface of the implants. After administering prophylactic antibiotics, rats were subjected to irradiation with 10 Gy photon beams targeted to each implant site. Each implant and the surrounding excised tissue were subjected to the necessary procedures for histological (capsule thickness, cell density), immunohistochemical, and biochemical (α-SMA, vimentin, collagen type I and type III, TGF-β1 and TGF-β3: expression level/protein level) examinations. It was determined that the levels of TGF-β1 and TGF-β3 collagen type III, which decreased as a result of radiotherapy, were brought to the control level with free TGF-β3 film and TGF-β3 nanoparticle film formulations. Histological analyses, consistent with biochemical analyses, showed that thick collagen and fibrosis, which increased with radiotherapy, were brought to the control level with free TGF-β3 film and TGF-β3 nanoparticle film treatments.In biochemical analyses, the decrease in thick collagen was compatible with the decrease in the collagen type I/type III ratio in the free TGF-β3 film and TGF-β3 nanoparticle film groups. Changes in protein expression show that TGF-β3 loaded nanoparticles are more successful than free TGF-β3 in wound healing. In line with these results and the literature, it is thought that the balance of TGF-β1 and TGF-β3 should be maintained to ensure scarless wound healing with no capsule contracture.

PSLBII-27 Investigating the emergence of antimicrobial resistance in Mycoplasma bovis from feedlot cattle

Abstract Bovine respiratory disease (BRD) is the most significant disease affecting feedlot cattle in North America. Mycoplasma bovis is among the main BRD pathogenic bacteria. Infection control of M. bovis is hampered by a lack of effective vaccines and increasing antimicrobial resistance (AMR). Integrative and conjugative elements (ICEs) have been documented as a mechanism of horizontal gene transfer (HGT) in mycoplasmas. Mycoplasma ICEs (MICE) do not carry AMR genes but have been associated with the HGT of genes that confer AMR through point mutations. Hence, there is a need to gain a better understanding of the role of MICE in M. bovis AMR. Randomly selected M. bovis isolates (n = 54) collected from Western Canadian feedlot cattle (AB and SK) underwent whole-genome sequencing by short-reads (Illumina), with a subset (n = 6) selected based on AMR and ICE profiles for Oxford Nanopore long-read sequencing. Sequences were screened for the presence of MICE. MICE circularization (cMICE) is the first step of conjugation, and an in-house collection of 472 M. bovis field isolates (feedlot cattle just from AB) was screened by PCR for the presence of cMICE as a proxy for conjugation capabilities. Environmental conditions that could impact cMICE quantity, including growth phase, temperature, cell density, pH, starvation, UV light, CO2 concentration, and sub-inhibitory concentrations of mitomycin C were investigated by qPCR, followed by growth of M. bovis isolates under the most favorable conditions identified for cMICE-detection and screening by a high throughput qPCR method that eliminated the need for DNA extraction. Nineteen different mating pairs were carried out and 4 different environmental factors were tested for their possible impact on conjugation occurrence (incubation time, incubation volume, conjugation parent proportions, atmosphere). All isolates carried MICEs with 23/54 having a structure/sequence comparable to previously described functional MICE in mycoplasma, 43/54 possessing non-functional ICE, and 23 having &amp;gt;1 ICE. The presence of more than one MICE/genome suggests a lack of MICE (entry) exclusion systems in M. bovis, a trait that may promote the flow of genetic information among M. bovis isolates including AMR. Even though some factors including growth phase (early stationary phase) and pH (9.0) generated greater levels (≥1.5 fold) of cMICE, none of the examined parameters were statistically significant. However, as early stationary growth phase generated the greatest cMICE quantity (2.3 fold), M. bovis isolates were cMICE-screened at this growth stage for the expeditious identification of M. bovis conjugation candidates. A total of 25.7% M. bovis were cMICE-positive. Of 19 mating pairs tested, transconjugants were recovered from only 2 different mating pairs, possibly reflective of the need for further optimization of conjugation conditions.

The effect of aging on mast cell density in human skin: a comparative analysis of photoexposed and photoprotected regions.

Mast cells are mononuclear cells originating from bone marrow. They produce various biologically active substances, which allow them to actively participate in immune and inflammatory processes associated with intrinsic and extrinsic skin aging. This research focused on distribution and density of mast cells in healthy skin in different stages of skin aging. This project included samples of photoexposed and photoprotected skin, obtained from 90 cadavers aged 0-82 years. The samples were classified into five age groups: newborns, young age, middle age, senior age and the oldest age. In order to visualize the mast cells, we have employed several histochemical staining protocols. The number of mast cells of the photoexposed skin significantly correlated to the individual's age. The number of mast cells of the photoprotected skin was in general statistically significantly lower in younger compared to older groups; however, the correlation of the mast cell density in photoprotected skin and the age did not reach statistical significance. In middle age, senior age and the oldest age groups, a significantly higher number of mast cells was recorded in the skin of the photoexposed compared to photoprotected region. The increase in mast cell density correlated with age only in photoexposed skin. Age-related higher accumulation of dermal mast cells in photoexposed skin can be an important factor in the photoaging process, as well as the contributing factor in the occurrence of skin cancer.

Research on the biophysical properties of circulating tumor cells

This article reviews the biophysical properties of circulating tumor cells (CTCs) and their significance in tumor metastasis and liquid biopsy. CTCs are tumor cells that have shed from solid tumors or metastatic foci and entered the bloodstream, possessing various biological markers. They may be cleared by the immune system, enter a dormant state, or form clusters to facilitate metastasis. The physical properties of CTCs, including cell size, density, rigidity, and dielectric properties, differ from normal blood cells, providing a theoretical basis for separation and detection. The biological characteristics of CTCs include epithelial cell properties, expression of specific surface marker proteins, and the characteristics of epithelial-mesenchymal transition, which are crucial for the aggressiveness and metastatic potential of tumors. The development of CTC detection technologies, such as the Cell Search system, has been clinically applied, but the heterogeneity of tumor cells remains a major challenge in research. The development of composite separation technologies that combine various properties of CTCs is expected to improve the sensitivity, specificity, and efficiency of CTC detection, thereby playing an important role in the personalized treatment and prognostic assessment of tumors.