Changes In Cellular Morphology Research Articles

PET imaging for the early evaluation of ocular inflammation in diabetic rats by using [18F]-DPA-714

Ocular complications of diabetes mellitus (DM) are the leading cause of vision loss. Ocular inflammation often occurs in the early stage of DM; however, there are no proven quantitative methods to evaluate the inflammatory status of eyes in DM. The 18 kDa translocator protein (TSPO) is an evolutionarily conserved cholesterol binding protein localized in the outer mitochondrial membrane. It is a biomarker of activated microglia/macrophages; however, its role in ocular inflammation is unclear. In this study, fluorine-18-DPA-714 ([18F]-DPA-714) was evaluated as a specific TSPO probe by cell uptake, cell binding assays and micro positron emission tomography (microPET) imaging in both in vitro and in vivo models. Primary microglia/macrophages (PMs) extracted from the cornea, retina, choroid or sclera of neonatal rats with or without high glucose (50 mM) treatment were used as the in vitro model. Sprague-Dawley (SD) rats that received an intraperitoneal administration of streptozotocin (STZ, 60 mg/kg once) were used as the in vivo model. Increased cell uptake and high binding affinity of [18F]-DPA-714 were observed in primary PMs under hyperglycemic stress. These findings were consistent with cellular morphological changes, cell activation, and TSPO up-regulation. [18F]-DPA-714 PET imaging and biodistribution in the eyes of DM rats revealed that inflammation initiates in microglia/macrophages in the early stages (3 weeks and 6 weeks), corresponding with up-regulated TSPO levels. Thus, [18F]-DPA-714 microPET imaging may be an effective approach for the early evaluation of ocular inflammation in DM.

Biomechanism of abnormal stress on promoting osteoarthritis of temporomandibular joint through Piezo1 ion channel.

This study aimed to investigate whether flow fluid shear stress (FFSS)-mediated signal transduction affects the function of Piezo1 ion channel in chondrocyte and to further explore the role of mechanical overloading in development of temporomandibular joint osteoarthritis (TMJ OA). Immunohistochemical staining was used to determine the expression of Piezo1 in TMJ OA tissue collected from rat unilateral anterior crossbite (UAC) models. Chondrocytes harvested from normal adult SD rats were treated with FFSS (0, 4, 8, 12 dyn/cm2) invitro. Immunofluorescent staining, real-time polymerase chain reaction, western blotting, flow cytometry and phalloidin assay were performed to detect the changes of cellular morphology as well as the expression of Piezo1 and certain pro-inflammatory and degradative factors in chondrocyte. Immunohistochemical analysis revealed that significantly increased Piezo1 expression was associated with UAC stimulation (p < .05). As applied FFSS escalated (4, 8 and 12 dyn/cm2), the expression levels of Piezo1, ADAMTS-5, MMP-13 and Col-X gradually increased, compared with the non-FFSS group (p < .05). Administering Piezo1 ion channel inhibitor to chondrocytes beforehand, it was observed that expression of ADAMTS-5, MMP-13 and Col-X was substantially decreased following FFSS treatment (p < .05) and the effect of cytoskeletal thinning was counteracted. The activated Piezo1 ion channel enhanced intracellular Ca2+ excess in chondrocytes during abnormal mechanical stimulation and the increased intracellular Ca2+ thinned the cytoskeleton of F-actin. Mechanical overloading activates Piezo1 ion channel to promote pro-inflammation and degradation and to increase Ca2+ concentration in chondrocyte, which may eventually result in TMJ OA.

Metallothionein III Decreases the Proliferation of Astrocytes and Enhances Pathological Improvements in a Mouse Model of Alzheimer's Disease.

Alzheimer's disease (AD) affects the brain and causes difficulties with cognition and emotions. At present, there are no viable therapies to halt or slow down the advancement of AD. Metallothionein III (MT-III) exhibits antioxidant and anti-inflammatory characteristics, indicating possible therapeutic benefits. This study aimed to explore the influence of MT-III on AD pathological alterations and cognitive abilities. In this research, we employed the universally accepted AD mouse models (3xTg-AD) as test subjects and administrated vehicle or MT-III. The mice were subjected to the Morris water maze test to assess their spatial learning and memory capabilities. Moreover, to evaluate the consequent effects on neuronal groups in the hippocampus, the Nissl staining and neuronal nuclear antigen (NeuN) immunohistochemistry were used to identify the cellular morphology changes and density. Immunohistochemistry was also used to detect β-amyloid (Aβ) and glial fibrillary acidic protein (GFAP) to measure Aβ accumulation and astrocyte growth. Western blot was also used to measure Tau pathology-related PHD finger protein 1 (PHF-1), phosphorylated Tau (AT-8), and total Tau protein. The administration of MT-III notably enhanced spatial learning and memory function in 3xTg-AD mice, as evidenced by the Morris water maze test (p < 0.01). According to immunohistochemistry and the obtained findings, it was observed that brain tissues of mice treated with MT-III showed a notable increase of Nissl bodies and NeuN intensity (p < 0.01) while a remarkable decrease in Aβ accumulation and GFAP (p < 0.01). Additionally, MT-III largely decreased levels of Tau phosphorylation-related PHF-1 and AT-8 (p < 0.01) and slightly reduced the level of Tau 5 (p < 0.05). In summary, our research indicates that MT-III has the capacity to ameliorate pathological alterations in AD mouse models and safeguard their cognitive and emotional abilities. By decreasing β-amyloid accumulation and reducing the intensity of Tau pathology, MT-III protected hippocampal subfield neurons against pathological harm. Furthermore, MT-III reduced inflammation by inhibiting abnormal proliferation of astrocytes. Of utmost importance, MT-III greatly enhanced the cognitive abilities related to spatial learning and memory in mice, suggesting its promising therapeutic properties for AD.

Elimination of radiation-induced senescent cancer cells and stromal cells invitro by near-infrared photoimmunotherapy.

Therapy-induced senescent cancer and stromal cells secrete cytokines and growth factors to promote tumor progression. Therefore, senescent cells may be novel targets for tumor treatment. Near-infrared photoimmunotherapy (NIR-PIT) is a highly tumor-selective therapy that employs conjugates of a molecular-targeting antibody and photoabsorber. Thus, NIR-PIT has the potential to be applied as anovel senolytic therapy. This study aims to investigate the efficacy of NIR-PIT treatment on senescent cancer and stromal cells. Two cancer cell lines (human lung adenocarcinoma A549 cells and human pancreatic cancer MIA PaCa-2 cells) and two normal cell lines (mouse fibroblast transfected with human epidermal growth factor receptor 2 [HER2] cells and human fibroblast WI38 cells) were used. The cytotoxicity of NIR-PIT was evaluated using anti-epidermal growth factor receptor (EGFR) antibody panitumumab and anti-HER2 antibody transtuzumab. Cellular senescence was induced in A549 and MIA PaCa-2 cells by 10 Gy γ-irradiation. The up-regulation of cellular senescence markers and characteristic morphological changes in senescent cells, including enlargement, flattening, and multinucleation, were observed in cancer cells after 5 days of γ-irradiation. Then, NIR-PIT targeting EGFR was performed on these senescent cancer cells. The NIR-PIT induced morphological changes, including bleb formation, swelling, and the inflow of extracellular fluid, and induced a significant decrease in cellular viability. These results suggested that NIR-PIT may induce cytotoxicity using the same mechanism in senescent cancer cells. In addition, similar morphological changes were also induced in radiation-induced senescent 3T3-HER2 fibroblasts by NIR-PIT targeting human epidermal growth factor receptor 2. NIR-PIT eliminates both senescent cancer and stromal cells invitro suggesting it may be a novel strategy for tumor treatment.

DHM/SERS reveals cellular morphology and molecular changes during iPSCs-derived activation of astrocytes.

The activation of astrocytes derived from induced pluripotent stem cells (iPSCs) is of great significance in neuroscience research, and it is crucial to obtain both cellular morphology and biomolecular information non-destructively in situ, which is still complicated by the traditional optical microscopy and biochemical methods such as immunofluorescence and western blot. In this study, we combined digital holographic microscopy (DHM) and surface-enhanced Raman scattering (SERS) to investigate the activation characteristics of iPSCs-derived astrocytes. It was found that the projected area of activated astrocytes decreased by 67%, while the cell dry mass increased by 23%, and the cells changed from a flat polygonal shape to an elongated star-shaped morphology. SERS analysis further revealed an increase in the intensities of protein spectral peaks (phenylalanine 1001 cm-1, proline 1043 cm-1, etc.) and lipid-related peaks (phosphatidylserine 524 cm-1, triglycerides 1264 cm-1, etc.) decreased in intensity. Principal component analysis-linear discriminant analysis (PCA-LDA) modeling based on spectral data distinguished resting and reactive astrocytes with a high accuracy of 96.5%. The increase in dry mass correlated with the increase in protein content, while the decrease in projected area indicated the adjustment of lipid composition and cell membrane remodeling. Importantly, the results not only reveal the cellular morphology and molecular changes during iPSCs-derived astrocytes activation but also reflect their mapping relationship, thereby providing new insights into diagnosing and treating neurodegenerative diseases.

Comparative Assessment of the Anti-Helicobacter pylori Activity and Gastroprotective Effects of Three Herbal Formulas for Functional Dyspepsia In Vitro.

Helicobacter pylori has been implicated in various gastrointestinal disorders, including functional dyspepsia. This study aimed to compare the anti-H. pylori activity and gastroprotective effects of three typical herbal formulas used for gastrointestinal disorders in Korea: Shihosogan-tang (ST), Yijung-tang (YT), and Pyeongwi-san (PS). Firstly, we assessed the total phenolic and flavonoid contents, as well as the antioxidative capacity. Additionally, we evaluated the antibacterial effect on H. pylori using an ammonia assay, minimum inhibitory concentration (MIC) test, and the disk agar diffusion method. Furthermore, we examined alterations in the gene expression of tight junction proteins, pro-inflammatory cytokines, and cellular vacuolation using an AGS cell model infected with H. pylori. While ST exhibited a higher total phenolic content, superior free radical scavenging, and inhibition of H. pylori compared to YT and PS, YT more evidently inhibited gastric cellular morphological changes such as vacuolation. All formulations significantly ameliorated changes in inflammatory and gastric inflammation-related genes and cellular morphological alterations induced by H. pylori infection. Overall, the present in vitro study suggests that all three herbal formulas possess potential for ameliorating gastrointestinal disorders, with ST relatively excelling in inhibiting H. pylori infection and inflammation, while YT potentially shows greater efficacy in directly protecting the gastric mucosa.

Stable Isotope Probing-nanoFTIR for Quantitation of Cellular Metabolism and Observation of Growth-Dependent Spectral Features.

This study utilizes nanoscale Fourier transform infrared spectroscopy (nanoFTIR) to perform stable isotope probing (SIP) on individual bacteria cells cultured in the presence of 13C-labelled glucose. SIP-nanoFTIR simultaneously quantifies single-cell metabolism through infrared spectroscopy and acquires cellular morphological information via atomic force microscopy. The redshift of the amide I peak corresponds to the isotopic enrichment of newly synthesized proteins. These observations of single-cell translational activity are comparable to those of conventional methods, examining bulk cell numbers. Observing cells cultured under conditions of limited carbon, SIP- nanoFTIR is used to identify environmentally-induced changes in metabolic heterogeneity and cellular morphology. Individuals outcompeting their neighboring cells will likely play a disproportionately large role in shaping population dynamics during adverse conditions or environmental fluctuations. Additionally, SIP-nanoFTIR enables the spectroscopic differentiation of specific cellular growth phases. During cellular replication, subcellular isotope distribution becomes more homogenous, which is reflected in the spectroscopic features dependent on the extent of 13C-13C mode coupling or to specific isotopic symmetries within protein secondary structures. As SIP-nanoFTIR captures single-cell metabolism, environmentally-induced cellular processes, and subcellular isotope localization, this technique offers widespread applications across a variety of disciplines including microbial ecology, biophysics, biopharmaceuticals, medicinal science, and cancer research.

Glucose-Induced Cellular Morphological Changes Across Mammalian Species

All organisms encounter environmental fluctuations and must contend with these changes to maintain homeostasis. Some mammals tolerate a wide range of intracellular conditions while others do not. Altered glucose levels are an example of a physiological challenge that can be encountered by cells. We studied responses of dermal fibroblasts obtained from skin biopsies to altered glucose treatments in culture using a comparative approach. Specifically, we investigated responses of mammals known to tolerate a range of blood glucose levels (fruit-eating geladas and Egyptian rousettes) as well as a hibernator (ground squirrel), which exhibits a marked winter decrease in glucose metabolism. As a comparison, we investigated species that maintain fairly stable blood glucose (human and rat). Fibroblasts were cultured at baseline (8mM glucose), then subjected to hypoglycemic (2.5mM) and hyperglycemic (30mM) treatment over 24h. We assayed glycolytic rate in human and rat fibroblasts using a Seahorse XFp analyzer, and found no significance between oxygen consumption or glycolysis across glucose treatments (all p&gt;0.05). To investigate underlying cell phenotypes, we used Cell Painting, employing fluorescent stains to mark organelles. Hoechst 34580 stained DNA, to quantify number and size of nuclei, offering insights into proliferation. MitoTracker Deep Red visualized mitochondria, providing information about energy generation. Alexa Fluor 488 Concanavalin A conjugate marked endoplasmic reticulum. F-actin was stained with Alexa Fluor 555 Phalloidin conjugate, detecting cytoskeletal organization and overall cell structure. Images were analyzed with CellProfiler software, which extracts ~1400 morphological cell features, including area, compactness, fluorescence intensity, and cell connectivity to quantify subtle changes in phenotype. Each extracted feature will be compared across treatments and within a comparative context. We predict that fibroblasts exposed to hyperglycemia exhibit mitochondrial fragmentation and redistribution (MitoTracker) and increased production of reactive oxygen species (assessed with MitoSOX), which is linked to the opening of mitochondrial permeability transition pores. Cell Profiler analysis will identify structural changes that could indicate negative outcomes, such as mitophagy. Hyperglycemia may also induce endoplasmic stress, increasing lipid droplets and altering shape and size of the endoplasmic reticulum. In contrast, hypoglycemia will cause mitochondrial degradation. In both hyper- and hypoglycemia, proliferation rates are predicted to decrease, resulting in fewer nuclei and reduced cell connectivity. Phenotypic differences across species, evaluated in the context of their organismal phenotype, will help explain the mechanisms of cell function in mammals under environmental stress. Funding by NSF #2022046. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Melatonin prevents pulmonary fibrosis caused by PM2.5 exposure by targeting epithelial-mesenchymal transition

Pulmonary fibrosis is a lung disorder characterized by the accumulation of abnormal extracellular matrix, scar tissue formation, and tissue stiffness. Type II alveolar epithelial cells (AEII) play a critical role in repairing lung tissue after injury, and repeated injury to these cells is a key factor in the development of pulmonary fibrosis. Chronic exposure to PM2.5, a type of air pollution, has been shown to increase the incidence and severity of pulmonary fibrosis by enhancing the activation of EMT in lung epithelial cells. Melatonin, a hormone with antioxidant properties, has been shown to prevent EMT and reduce fibrosis in previous studies. However, the mechanism through which melatonin targets EMT to prevent pulmonary fibrosis caused by PM2.5 exposure has not been extensively discussed before. In this current study, we found that melatonin effectively prevented pulmonary fibrosis caused by prolonged exposure to PM2.5 by targeting EMT. The study demonstrated changes in cellular morphology and expression of EMT markers. Furthermore, the cell migratory potential induced by prolonged exposure to PM2.5 was greatly reduced by melatonin treatment. Finally, in vivo animal studies showed reduced EMT markers and improved pulmonary function. These findings suggest that melatonin has potential clinical use for the prevention of pulmonary fibrosis.

Neuronal microstructural changes in the human brain are associated with neurocognitive aging.

Gray matter (GM) alterations play a role in aging-related disorders like Alzheimer's disease and related dementias, yet MRI studies mainly focus on macroscopic changes. Although reliable indicators of atrophy, morphological metrics like cortical thickness lack the sensitivity to detect early changes preceding visible atrophy. Our study aimed at exploring the potential of diffusion MRI in unveiling sensitive markers of cortical and subcortical age-related microstructural changes and assessing their associations with cognitive and behavioral deficits. We leveraged the Human Connectome Project-Aging cohort that included 707 participants (394 female; median age = 58, range = 36-90 years) and applied the powerful mean apparent diffusion propagator model to measure microstructural parameters, along with comprehensive behavioral and cognitive test scores. Both macro- and microstructural GM characteristics were strongly associated with age, with widespread significant microstructural correlations reflective of cellular morphological changes, reduced cellular density, increased extracellular volume, and increased membrane permeability. Importantly, when correlating MRI and cognitive test scores, our findings revealed no link between macrostructural volumetric changes and neurobehavioral performance. However, we found that cellular and extracellular alterations in cortical and subcortical GM regions were associated with neurobehavioral performance. Based on these findings, it is hypothesized that increased microstructural heterogeneity and decreased neurite orientation dispersion precede macrostructural changes, and that they play an important role in subsequent cognitive decline. These alterations are suggested to be early markers of neurocognitive performance that may distinctly aid in identifying the mechanisms underlying phenotypic aging and subsequent age-related functional decline.

Lung cancer cell-intrinsic IL-15 promotes cell migration and sensitizes murine lung tumors to anti-PD-L1 therapy

BackgroundIL-15 plays a vital role in enhancing NK cell- and T-cell-mediated antitumor immune responses; however, the direct effect of IL-15 on tumor cells has not been fully elucidated. Herein, we investigated the effect of IL-15 on lung adenocarcinoma cells.MethodsSilencing and overexpression techniques were used to modify endogenous IL-15 expression in tumor cells. Transwell assays were used to assess tumor cell migration and invasion; a live-cell analysis system was used to evaluate cell motility; cellular morphological changes were quantified by confocal fluorescence microscopy; the molecular mechanisms underlying the effect of IL-15 on tumor cells were analyzed by western blotting; and RhoA and Cdc42 activities were evaluated by a pulldown assay. NCG and C57BL/6 mouse models were used to evaluate the functions of IL-15 in vivo.ResultsCancer cell-intrinsic IL-15 promoted cell motility and migration in vitro and metastasis in vivo via activation of the AKT-mTORC1 pathway; however, exogenous IL-15 inhibited cell motility and migration via suppression of the RhoA-MLC2 axis. Mechanistic analysis revealed that both the intracellular and extracellular IL-15-mediated effects required the expression of IL-15Rα by tumor cells. Detailed analyses revealed that the IL-2/IL-15Rβ and IL-2Rγ chains were undetected in the complex formed by intracellular IL-15 and IL-15Rα. However, when exogenous IL-15 engaged tumor cells, a complex containing the IL-15Rα, IL-2/IL-15Rβ, and IL-2Rγ chains was formed, indicating that the differential actions of intracellular and extracellular IL-15 on tumor cells might be caused by their distinctive modes of IL-15 receptor engagement. Using a Lewis lung carcinoma (LLC) metastasis model, we showed that although IL-15 overexpression facilitated the lung metastasis of LLC cells, IL-15-overexpressing LLC tumors were more sensitive to anti-PD-L1 therapy than were IL-15-wild-type LLC tumors via an enhanced antitumor immune response, as evidenced by their increased CD8+ T-cell infiltration compared to that of their counterparts.ConclusionsCancer cell-intrinsic IL-15 and exogenous IL-15 differentially regulate cell motility and migration. Thus, cancer cell-intrinsic IL-15 acts as a double-edged sword in tumor progression. Additionally, high levels of IL-15 expressed by tumor cells might improve the responsiveness of tumors to immunotherapies.

Abstract P70: HIGH-THROUGHPUT IMAGING-BASED PHENOTYPIC PROFILING OF IMMUNE CELLS FROM BRCA2 VARIANT CARRIERS AND NON-CARRIERS

Abstract Mutated germline alleles in the DNA damage repair (DDR) genes BRCA1/2 are major risk factors for breast and ovarian cancers. Recent studies have found that BRCA1-mutant tumors tend to have higher abundance of immune cells within the tumor microenvironment, but much better response was observed from the BRCA2-mutant tumors when given immune-checkpoint inhibitors, suggesting possible functional and phenotypic effects of germline BRCA2 mutations on the immune cells. However, most existing studies focus on investigating the genomic instability and phenotypes of hereditary mutations in breast and ovarian cancer cells. Thus, the impact of these mutations on immune cells’ phenotypes and functions remains largely unknown. Here, we report a study of immune cells with and without BRCA2 mutations using High-throughput Imaging-based Phenotypic Profiling (HIPP), which can identify changes in cellular morphology and protein subcellular localization patterns that are not obvious from other traditional functional assays. We performed HIPP on immune cells from nine BRCA2 pathogenic variant carriers and eight non-carriers. Automatic quantification of ~200 phenotypic features from each single cell revealed that CD8+ T cells from BRCA2 variant carriers have distinct and quantifiable changes in their morphological features (such as cytoskeleton structures) and immunological features (such as Granzyme B expressions). Moreover, we found that BRCA2-mutant CD8+ T cells proliferate faster than normal CD8+ T cells. Our results suggest that BRCA2 mutations affect the functions and phenotypes of immune cells. Some of these phenotypic features and markers may be used to classify BRCA2 variants and predict the pathogenicity of variants of uncertain significance (VUS). Our study provides new insight into the immune phenotyping of cancer patients at individual level and may help to stratify patients, leading to the development of more effective therapeutic strategies. This method can potentially extend to other germline variants and overcome the challenge of VUS interpretation in clinical management. Citation Format: Guorui Zhong, Oscar Fu, Claresta Yeo, Siao Ting Chong, Sock Hoai Chan, Joanne Ngeow, Lit-Hsin Loo. HIGH-THROUGHPUT IMAGING-BASED PHENOTYPIC PROFILING OF IMMUNE CELLS FROM BRCA2 VARIANT CARRIERS AND NON-CARRIERS [abstract]. In: Proceedings of Frontiers in Cancer Science; 2023 Nov 6-8; Singapore. Philadelphia (PA): AACR; Cancer Res 2024;84(8_Suppl):Abstract nr P70.

Harnessing Microbial Effectors for Macrophage-Mediated Drug Delivery.

Macrophage-based drug delivery systems are promising, but their development is still in its infancy, with many limitations remaining to be addressed. Our aim was to design a system harnessing microbial effectors to facilitate controlled drug cargo expulsion from macrophages to enable the use of more toxic drugs without adding to the risk of off-target detrimental effects. The pore forming and actin polymerizing Listeria monocytogenes effectors listeriolysin-O (LLO) and actin assembly-inducing protein (ActA) were synthesized using a novel green fluorescent protein (GFP)-linked heterologous expression system. These effectors were coated onto polystyrene beads to generate "synthetic cargo" before loading into primary M1 macrophages. Bead uptake and release from macrophages were evaluated by using high-throughput quantitative imaging flow cytometry and confocal microscopy. In vitro results confirmed appropriate activity of synthesized effectors. Coating of these effector proteins onto polystyrene beads (simulated drug cargo) resulted in changes in cellular morphology, bead content, and intracellular bead localization, which may support an interpretation of the induced release of these beads from the cells. This forms the basis for further investigation to fully elucidate any potential release mechanisms. Bacterial effectors ActA and LLO successfully effectuated actin polarization and protrusions from cell membranes similar to those seen in cells infected with Listeria spp., illustrating the potential of using these effectors and production methods for the development of an endogenous drug delivery system capable of low-risk, targeted release of high potency drugs.

BRD4L cooperates with MYC to block local tumor invasion via suppression of S100A10

Targeted therapy based on BRD4 and MYC shows promise due to their well-researched oncogenic functions in cancer, but their tumor-suppressive roles are less understood. In this study, we employ a systematic approach to delete exons that encode the low-complexity domain (LCD) of BRD4L in cells by using CRISPR–Cas9. In particular, the deletion of exon 14 (BRD4–E14) results in cellular morphological changes towards spindle-shaped and loosely packed. BRD4–E14 deficient cells show increased cell migration and reduced cell adhesion. The expression of S100A10 was significantly increased in cells lacking E14. BRD4L binds with MYC via the E14-encoded region of the LCD to inhibit the expression of S100A10. In cancer tissues, there is a positive correlation between BRD4 and MYC, while both of these proteins are negatively associated with S100A10 expression. Finally, knocking out the BRD4–E14 region or MYC promotes tumor growth in vivo. Together, these data support a tumor-suppressive role of BRD4L and MYC in some contexts. This discovery emphasizes the significance of a discreetly design and precise patient recruitment in clinical trials that testing cancer therapy based BRD4 and MYC.

Phenotypic comparison and the potential antitumor function of immortalized bone marrow-derived macrophages (iBMDMs).

Macrophages are an important component of innate immunity and involved in the immune regulation of multiple diseases. The functional diversity and plasticity make macrophages to exhibit different polarization phenotypes after different stimuli. During tumor progression, the M2-like polarized tumor-associated macrophages (TAMs) promote tumor progression by assisting immune escape, facilitating tumor cell metastasis, and switching tumor angiogenesis. Our previous studies demonstrated that functional remodeling of TAMs through engineered-modifying or gene-editing provides the potential immunotherapy for tumor. However, lack of proliferation capacity and maintained immune memory of infused macrophages restricts the application of macrophage-based therapeutic strategies in the repressive tumor immune microenvironment (TIME). Although J2 retrovirus infection enabled immortalization of bone marrow-derived macrophages (iBMDMs) and facilitated the mechanisms exploration and application, little is known about the phenotypic and functional differences among multi kinds of macrophages. HE staining was used to detect the biosafety of iBMDMs, and real-time quantitative PCR, immunofluorescence staining, and ELISA were used to detect the polarization response and expression of chemokines in iBMDMs. Flow cytometry, scratch assay, real-time quantitative PCR, and crystal violet staining were used to analyze its phagocytic function, as well as its impact on tumor cell migration, proliferation, and apoptosis. Not only that, the inhibitory effect of iBMDMs on tumor growth was detected through subcutaneous tumor loading, while the tumor tissue was paraffin sectioned and flow cytometry was used to detect its impact on the tumor microenvironment. In this study, we demonstrated iBMDMs exhibited the features of rapid proliferation and long-term survival. We also compared iBMDMs with RAW264.7 cell line and mouse primary BMDMs with in vitro and in vivo experiments, indicating that the iBMDMs could undergo the same polarization response as normal macrophages with no obvious cellular morphology changes after polarization. What's more, iBMDMs owned stronger phagocytosis and pro-apoptosis functions on tumor cells. In addition, M1-polarized iBMDMs could maintain the anti-tumor phenotypes and domesticated the recruited macrophages of receptor mice, which further improved the TIME and repressed tumor growth. iBMDMs can serve as a good object for the function and mechanism study of macrophages and the optional source of macrophage immunotherapy.

A physics-informed neural network framework to investigate nonlinear and heterogenous shrinkage of drying plant cells

This paper introduces a novel Physics-Informed Neural Network-based (PINN-based) multi-domain computational framework to analyse nonlinear and heterogeneous morphological variations of plant cells during drying. Here, two distinct models are involved: PINN-MT to simulate mass transfer; and PINNNS to simulate nonlinear shrinkage. The models are coupled to examine cellular morphological changes resulting from moisture loss during drying. Firstly, the coupled framework, in tandem with homogeneous conditions, operates in parallel, allowing the mutual parameters to update between models. This approach demonstrates ability to approximate homogeneous cellular shrinkage within a tissue, factoring in the influence of surrounding plant cells. Secondly, non-uniform cell wall properties and heterogeneous boundary conditions are incorporated into this computational framework through domain decomposition. Inherent capabilities of neural networks allow for seamless integration of multiple domains, with additional loss terms introduced at interfaces. The framework shows capacity to account for drastic and non-uniform morphological variations of plant cells even under extreme drying conditions, which is the key novelty and has been a challenging task for existing traditional computational methods. Hence, the proposed computational approach offers an innovative avenue for understanding nonlinear and heterogeneous morphological variations not only for plant cells, but also for soft matter in general.

Effectiveness of Pheophorbide-a mediated Photodynamic Therapy against Doxorubicin Resistant Breast Cancer

SignificanceEmploying mechanistically distinct therapies like Photodynamic Therapy (PDT) can be a promising strategy to overcome the multidrug- resistance in breast cancer resulting in the killing of chemotherapeutic drug-resistant cancer cells. ApproachIn this study, Pheophorbide a (Pheo-a), is being used as a potential photosensitizer to induce cell death in Doxorubicin (DOX) resistant breast cancer MCF-7 cells. Efficacy of Pheo-a mediated PDT against MCF-7/DOX cancer cells was evaluated by MTT proliferation assay and analyzing cellular morphological changes, live dead cell death assay. ResultsAll experiments were performed thrice in triplicate, and the results obtained were analysed for statistical significance. Results showed that the Pheo-a can induce photodynamic cell death in resistant cancer cells treated with a 2.5 μM concentration of Pheo-a followed by irradiation with a 660 nm red laser at a light dose of 10 J/cm2. ConclusionsThus, the results from this study suggest the utilization of chlorophyll-based PSs as a rational therapeutic intervention for the eradication of chemo drug-resistance in cancers.

Effectiveness of Pheophorbide-a mediated Photodynamic Therapy against Doxorubicin Resistant Breast Cancer

SignificanceEmploying mechanistically distinct therapies like Photodynamic Therapy (PDT) can be a promising strategy to overcome the multidrug- resistance in breast cancer resulting in the killing of chemotherapeutic drug-resistant cancer cells. ApproachIn this study, Pheophorbide a (Pheo-a), is being used as a potential photosensitizer to induce cell death in Doxorubicin (DOX) resistant breast cancer MCF-7 cells. Efficacy of Pheo-a mediated PDT against MCF-7/DOX cancer cells was evaluated by MTT proliferation assay and analyzing cellular morphological changes, live dead cell death assay. ResultsAll experiments were performed thrice in triplicate, and the results obtained were analysed for statistical significance. Results showed that the Pheo-a can induce photodynamic cell death in resistant cancer cells treated with a 2.5 μM concentration of Pheo-a followed by irradiation with a 660 nm red laser at a light dose of 10 J/cm2. ConclusionsThus, the results from this study suggest the utilization of chlorophyll-based PSs as a rational therapeutic intervention for the eradication of chemo drug-resistance in cancers.

Abstract 582: Oncogenic PIK3CA fortifies immune determinants in vascular cancers

Abstract Angiosarcomas are a group of vascular cancers that form malignant blood vessels. These malignancies are seemingly inflamed primarily due to their pathognomonic nature, which consists of irregular endothelium and tortuous blood channels. The malignant vascular spaces host a variety of blood and immune components, where a controlled scavenging system is necessary for tumor maintenance. Oncogenes may convey molecular signals to foster the tumor immune niche, and PIK3CA mutations that induce continuous activation of the PI3K pathway occur in a subset of angiosarcomas. Despite the undoubtedly oncogenic properties of PIK3CA mutations, the mechanistic role of these mutations - particularly the potential for controlling the hemato-endothelial system in angiosarcomas - remains undetermined. In this study, we demonstrate the functional consequences of oncogenic PIK3CA and its molecular properties in angiosarcoma cells. We created isogenic cell clones from two angiosarcoma cell lines engineered to harbor hotspot mutations in PIK3CA using CRISPR/Cas9: two homozygous PIK3CA H1047R cell clones (C8 and C35) and one wild-type (WT) clone were generated from DHSA-1426 angiosarcoma cells; and one homozygous mutant clone was created from COSB cells. We found that PIK3CA mutant cells constitutively activated the PI3K/AKT/mTOR pathway at a greater level than that of WT cells. Bulk- and single cell(sc)-RNA-seq data revealed that mutant cells enriched gene sets associated with PI3K signaling and other oncogenic pathways including KRAS, MAPK, and TGF-beta. Also, mutant cells significantly increased cytokines regulating immune cell migration and chemotaxis such as IL-8 and MCP-1. In addition to the common features, we identified functional and molecular divergence between mutant clones. Noticeable changes in cellular morphology, growth kinetics, and tube-forming capacity were observed between two mutant clones. C35 mutant cells were more sensitive to both pan-PI3K and PI3K-alpha specific inhibitor, and DNA damage was greater than in the C8 clone. C35 cells enhanced activation of IL-6/STAT3 signaling compared to C8, despite the increment of the aforementioned immune cytokines in both mutants. Moreover, our comprehensive analysis of bulk- and sc-RNA-seq, ATAC-seq, and metabolomics data identified molecular signatures associated with metabolic vulnerabilities of C35 cells. In summary, we demonstrate that oncogenic PIK3CA perpetuates PI3K signaling activation and reinforces a supply for immune signaling. Furthermore, our data suggest that PIK3CA mutations contribute to establishing cellular heterogeneity that reveals a spectrum of vascular, immunogenic, and metabolic activities. Our ongoing work involves developing an experimental model of angiosarcoma using induced pluripotent stem cells to examine if oncogenic PIK3CA controls immune determinants during malignant endothelial transformation. Citation Format: Donghee Lee, Emma Kozurek, Md Abdullah, Rong Li, Erin B. Dickerson, Jong Hyuk Kim. Oncogenic PIK3CA fortifies immune determinants in vascular cancers [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 582.

Abstract 1430: Development of transparent transgenic zebrafish model to study anti-cancer drug induced in vivo cardiotoxicity

Abstract Cardiotoxicity is the second leading cause of death among cancer survivors and can manifest as arrhythmia, acute coronary syndrome, acute heart failure, or pericarditis/myocarditis. Chronic myocyte damage and apoptosis can transition from an asymptomatic phase to adaptive myocardial hypertrophy, followed by decreased left ventricular ejection fraction, and eventual heart failure. Cardio-oncology is an emerging field that studies cancer therapy-associated cardiotoxicities. The development of reliable and effective preclinical models for cardiotoxicity prediction and prevention is essential to improving the long-term cardiac health of cancer patients. Zebrafish are a promising animal model for the field of cardio-oncology and have emerged as an invaluable tool for investigating genetic and molecular aspects of cardiovascular research. Zebrafish heart cells are like human heart cells at the molecular level, making them an ideal system for studying the genetic underpinning of cardiac function. Zebrafish myl7 gene, a myosin light chain-7 gene, is a regulatory gene of heart orthologs to human MYL7. In-vivo imaging is limited due to the opacification of skin and subdermal structures. To conduct in-vivo whole organism imaging studies, skin transparency is a primary requirement, necessitating the blocking of pigmentation. The Zebrafish Casper mutant maintains skin transparency throughout its life, offering the ideal combination of sensitivity and resolution for in-vivo analyses and imaging. In this study, we have developed a novel transparent transgenic zebrafish model and established time-lapse in-vivo confocal microscopy to study cellular phenotype/pathologies of cardiomyocytes. The newly developed homozygous transparent transgenic Casper/myl7:RFP; annexin-5:YFP/NF-kB: GFP/mpeg1:mcherryFP, allows us to study cell death process (Annexin5) and inflammatory activity (NF-kB) in microglia and cardiomyocytes. We have successfully established in vivo fluorescence and confocal microscopy in this newly developed strain, enabling the study of cellular morphology and function changes under various conditions. This will allow us to elucidate cell death patterns, and cardio-inflammatory and cardiotoxicity pathways. Citation Format: Surendra K. Rajpurohit, Taitum Gossman, Sai Nasanally, Keshu Bhatt, Aiden Van Derhei, Simran Hotwani, Devan-Anmol Manhiani, Rohan Pasi, Jiaqi Mi, Vishal Arora, Balakrishna Lokeshwar. Development of transparent transgenic zebrafish model to study anti-cancer drug induced in vivo cardiotoxicity [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 1430.