Live Tissue Research Articles

Deep-Red and Near-Infrared Compact Cyanine Dyes for Sensitive NAD(P)H Sensing in Live Cells and Kidney Disease Tissues.

Cyanine dyes constructed for NAD(P)H near-infrared sensing utilize extended π-conjugation but often exhibit delayed fluorescence responses to NAD(P)H due to reduced positive charge density in 3-quinolinium acceptors. This study introduces deep-red and near-infrared compact cyanine dyes represented by probes A and B for mitochondrial NAD(P)H detection in live cells. Probes A and B feature a unique structural design with a double bond connection linking 3-quinolinium to strategically positioned 1-methylquinolinium acceptor units at 2- and 4-positions, correspondingly. Probe A absorbs at 359 and 531 nm, while probe B absorbs at 324 and 370 nm, emitting subtle fluorescence at 587 and 628 nm, respectively, with no NADH present. Upon NADH exposure, probes A and B exhibit significant emission enhancements at 612 and 656 nm, correspondingly, attributed to the efficient reduction of 3-quinolinium units to electron-donative 1-methyl-1,4-dihydroquinoline units. Probe B, chosen for its near-infrared emission and fast response to NAD(P)H, effectively monitored dynamic intracellular NAD(P)H levels throughout diverse experimental conditions. In HeLa cells, minimal basal fluorescence increased upon NADH stimulation. It also identified increased NAD(P)H levels following chemical treatments with acesulfame potassium, cisplatin, carboplatin, and temozolomide, CoCl2-induced hypoxia, and TLR4 activation in macrophages and in disease models of kidney pathology, where diseased tissues exhibited higher fluorescence than normal tissues. In fruit fly larvae under starvation conditions, probe B tracked NAD(P)H increases triggered by exogenous NADH, demonstrating its in vivo applicability for metabolic studies. These findings highlight probe B's utility in elucidating dynamic NAD(P)H fluctuations in diverse biological contexts, offering insights into mitochondrial function and cellular metabolism.

Alginate/Chitosan Complex Fibers Reinforcement and Their Mechanical Transition Continuum With Water Uptake Increasing.

Living tissues span a remarkable spectrum of modulus ranging from the level of Pa to GPa in a water-rich environment. Constructing soft and hard materials that match the mechanics of tissues and researching mechanical transition in water, are beneficial for their biological applications. Here, using polyelectrolyte complex fiber as a model system and reinforcing the fiber by stepwisely introducing additional coordination and covalent bonds, this investigated that the water effect on mechanical transition behaviors. Alginate/chitosan fiber (AC fiber) has a single electrostatic bond and shows continuous mechanical transition containing a glassy state, rubbery state, and terminal relaxation (initial modulus lower than 10MPa) in aqueous solution. Alginate/chitosan/calcium fiber (ACC fiber) has both electrostatic and coordination bonds, which shows the behavior of hard rubber (initial modulus 100MPa) when water reaches equilibrium. Alginate/chitosan/calcium/polydopamine fiber (ACCP fiber) with triple bonds, including electrostatic, coordination, and covalent bonds, exhibits the behavior like ductile plastics in aqueous solution (initial modulus 1000MPa). This work not only provides important insight into the toughening mechanism of polyelectrolyte complexes in water but also contributes to the preparation of tissue adaptive implantations.

TMOD-16. ZUCLOPENTHIXOL REDUCES ACTIVITY-DEPENDENT GLIOBLASTOMA PROLIFERATION

Abstract A major challenge in modeling glioblastoma in the laboratory is the complex microenvironment in which tumors evolve in humans. The varied architecture and immune privilege of the brain limits the external validity of many experiments performed in modern cell biology laboratories. Organotypic human cortical slices present an opportunity to model glioblastoma and test the effect of drugs against glioma in ex-vivo human tissue. Cortical slice cultures maintain the cellular organization of the human brain, more closely approximating glioblastoma proliferation in patients. In this study, we test the effect zuclopenthixol, a repurposed antipsychotic inhibitor of glioblastoma-induced neuronal hyperexcitability, on the proliferation of glioblastoma cells in an organotypic human cortical slice culture. Primary patient-derived glioblastoma cultures were established, and human cortical tissues were emersed in oxygenated ACSF agarose gel before preparing 250 µm thick slices. Slices were either exogenously seeded with glioblastoma cells or stained for endogenous tumor infiltration. Slices with either endogenous or exogenous seeding were then treated with zuclopenthixol or regular media change and cultured for 10 days. Proliferation was measured with Ki67 staining and quantified via automated pixel summation using the Pillow library in Python. 0.1 µM zuclopenthixol treated slices had significantly lower proliferation index in both the endogenous tumor condition (0.04 vs 0.30, P < 0.05) and the exogenous seeding condition (0.0013 vs 0.028, P = 0.033). Reduced tissue integrity was observed beginning day 8 of culture, as tissue surrounding tumor seeding sites eroded. Here, we model glioblastoma proliferation in live human tissue and quantify the anti-proliferative effect of zuclopenthixol. We show that zuclopenthixol treatment significantly reduced the proliferation index. Our experimental model allowed the optimization of organotypic human cortical slice cultures for immunofluorescence studies and live-cell imaging.

Oral Mucosa Capillaroscopy: A Narrative Review.

The oral cavity may suffer from diseases and lesions of different natures that can result in changes to the underlying microvasculature. These changes are typically observed during the examination of biopsy samples, but there is still a need to investigate methods for characterizing them in live tissues. Capillaroscopy, a medical imaging technique using polarized light and magnification, has shown promise in providing enhanced imaging of the oral mucosa microvasculature in preliminary studies. The present work proposed to review the literature on what capillaroscopy is, its applications in the imaging of oral mucosa microvasculature, and its diagnostic and prognostic significance in various diseases and conditions. While there is the limited literature available, further research in oral capillaroscopy, particularly in the field of oral oncology, is needed to determine its potential benefits in diagnosing and predicting outcomes for potentially malignant oral disorders and cancers.

Culturally responsive strategies and practical considerations for live tissue studies in Māori participant cohorts.

Indigenous communities globally are inequitably affected by non-communicable diseases such as cancer and coronary artery disease. Increased focus on personalized medicine approaches for the treatment of these diseases offers opportunities to improve the health of Indigenous people. Conversely, poorly implemented approaches pose increased risk of further exacerbating current inequities in health outcomes for Indigenous peoples. The advancement of modern biology techniques, such as three-dimensional (3D) in vitro models and next generation sequencing (NGS) technologies, have enhanced our understanding of disease mechanisms and individualized treatment responses. However, current representation of Indigenous peoples in these datasets is lacking. It is crucial that there is appropriate and ethical representation of Indigenous peoples in generated datasets to ensure these technologies can be used to maximize the benefit of personalized medicine for Indigenous peoples. This project discusses the use of 3D tumor organoids and single cell/nucleus RNA sequencing to study cancer treatment responses and explore immune cell roles in coronary artery disease. Using key pillars from currently available Indigenous bioethics frameworks, strategies were developed for the use of Māori participant samples for live tissue and sequencing studies. These were based on extensive collaborations with local Māori community, scientific leaders, clinical experts, and international collaborators from the Broad Institute of MIT and Harvard. Issues surrounding the use of live tissue, genomic data, sending samples overseas and Indigenous data sovereignty were discussed. This paper illustrates a real-world example of how collaboration with community and the incorporation of Indigenous worldviews can be applied to molecular biology studies in a practical and culturally responsive manner, ensuring fair and equitable representation of Indigenous peoples in modern scientific data.

The genomic landscape of papillary thyroid carcinoma on next-generation sequencing in patients undergoing total thyroidectomy.

Thyroidectomy is increasingly performed for suspected malignancy. This cohort study aimed to identify genetic markers associated with malignancy and determine the molecular landscape of papillary thyroid carcinoma (PTC) through next-generation sequencing (NGS) in patients undergoing total thyroidectomy. Among 116 surgical candidates, 103 patients (age=42.9±13.7years; Male: Female=14:89) with benign or malignant thyroid nodules were eligible. Live thyroid tissue samples harvested intraoperatively with adequate DNA and RNA yield were subjected to NGS on the Illumina NovaSeq 6000 platform for genomic and transcriptomic analysis, respectively. Histopathology comprised 20 malignant (19.4%) and 83 benign (80.6%) cases, including 16 PTC (15.5%) cases. On NGS, single nucleotidepolymorphisms (SNPs) in NTRK1 at NC_000001.11:156879016 on chromosome 1 and ALK at NC_000002.12:29717663 on chromosome 2 were frequent in malignant lesions (p<0.05). A SNP in ALK at NC_000002.12:29193706 was consistently a homozygous alternate alleleacross the cohort. DNA-sequencing of PTC lesions identified recurrentsomatic mutations in BRAF (100%), ALK (56.3%), RET (18.8%), PIK3CA (12.5%), NTRK1 (12.5%), NTRK2 (87.5%), NTRK3(12.5%), NRAS(6.3%), and PTCH1 (31.3%) genes. RNA sequencing revealednovelfusiongenes, including MKRN1-BRAF, RN7SL1-CDH1, IRF2BPL-MED12, MED12-IRF2BPL, CPM-MDM2, and AC005895.3-PDGFRB. Inreceiveroperative characteristics analysis, the AUCs ofALKmutationpredictingrecurrence and metastases were 0.818 and 0.783. This Indian study identified novel somatic mutations and fusion genes in PTC, revealing a distinct genomic landscape with implications in precision diagnostics and personalized therapies. NGS with intraoperative live sampling shows promise in prognostication and therapeutic optimization of advanced/metastatic PTC cases. CTRI/2020/09/027607dt 04/09/2020; REF/2020/08/036119.

Piezoelectricity and flexoelectricity in biological cells: the role of cell structure and organelles.

Living tissues experience various external forces on cells, influencing their behaviour, physiology, shape, gene expression, and destiny through interactions with their environment. Despite much research done in this area, challenges remain in our better understanding of the behaviour of the cell in response to external stimuli, including the arrangement, quantity, and shape of organelles within the cell. This study explores the electromechanical behaviour of biological cells, including organelles like microtubules, mitochondria, nuclei, and cell membranes. A two-dimensional bio-electromechanical model for two distinct cell structures has been developed to analyze the behavior of the biological cell to the external electrical and mechanical responses. The piezoelectric and flexoelectric effects have been included via multiphysics coupling for the biological cell. All the governing equations have been discretized and solved by the finite element method. It is found that the longitudinal stress is absent and only the transverse stress plays a crucial role when the mechanical load is imposed on the top side of the cell through compressive displacement. The impact of flexoelectricity is elucidated by introducing a new parameter called the maximum electric potential ratio ( ). It has been found that depends upon the orientation angle and shape of the microtubules. The magnitude of exhibit huge change when we change the shape and orientation of the organelles, which in some cases (boundary condition (BC)-3) can reach to three times of regular shape organelles. Further, the study reveals that the number of microtubules significantly impacts effective elastic and piezoelectric coefficients, affecting cell behavior based on structure, microtubule orientation, and mechanical stress direction. The insight obtained from the current study can assist in advancements in medical therapies such as tissue engineering and regenerative medicine.

A microphysiological system for studying barrier health of live tissues in real time

Epithelial cells create barriers that protect many different components in the body from their external environment. Increased gut barrier permeability (leaky gut) has been linked to several chronic inflammatory diseases. Understanding the cause of leaky gut and effective interventions are elusive due to the lack of tools that maintain tissue’s physiological environment while elucidating cellular functions under various stimuli ex vivo. Here we present a microphysiological system that records real-time barrier permeability of mouse colon in a physiological environment over extended durations. The system includes a microfluidic chamber; media composition that preserves microbiome and creates necessary oxygen gradients across the barrier; and integrated sensor electrodes for acquiring transepithelial electrical resistance (TEER). Our results demonstrate that the system can maintain tissue viability for up to 72 h. The TEER sensors can distinguish levels of barrier permeability when treated with collagenase and low pH media and detect different thickness in the tissue explant.

Applications of bioactive glass: A review

The scope of research on dental materials has significantly increased throughout the last century. Bioactive glass has become an attractive option in biomaterials because of its unique qualities that promote advantageous biological responses. Compared to traditional dental glasses, bioactive glasses are innovative dental materials. As bioactive glass can form bonds with live tissues, mainly composed of calcium, phosphorus, and silicon, it is an excellent choice for many biomedical uses. At the vanguard of biomaterials, bioactive glass provides a unique blend of bioactivity, flexibility, and versatility. The bioactivity of bioactive glass is one of its primary characteristics. Bioactive glass generates a hydroxyapatite coating on its surface when it comes into contact with physiological fluids. The potential for introducing growth factors and bioactive substances to enhance the material's capacity to communicate with biological tissues has been studied. Various uses of bioactive glass have been discovered, including wound healing, bone grafts, dentin hypersensitivity, regeneration, implant devices, etc. Cement was used for dental applications, and the attachment of joint prostheses to bone was also made using bioactive glass. Bioactive glasses are present in the composition of several dental and oral hygiene products. Because of their many benefits—such as their capacity to maintain biological tissues' structural integrity, their usefulness as scaffolds, and their capacity to inhibit bacterial growth—bioactive glasses are becoming increasingly valuable in several dental specialties.

Autoinduction-Based Quantification of In Situ TGF-β Activity in Native and Engineered Cartilage.

Transforming growth factor beta (TGF-β) is a potent growth factor that regulates the homeostasis of native cartilage and is administered as an anabolic supplement for engineered cartilage growth. The quantification of TGF-β activity in live tissues in situ remains a significant challenge, as conventional activity assessments (e.g., Western blotting of intracellular signaling molecules or reporter cell assays) are unable to measure absolute levels of TGF-β activity in three-dimensional tissues. In this study, we develop a quantification platform established on TGF-β's autoinduction response, whereby active TGF-β (aTGF-β) signaling in cells induces their biosynthesis and secretion of new TGF-β in its latent form (LTGF-β). As such, cell-secreted LTGF-β can serve as a robust, non-destructive, label-free biomarker for quantifying in situ activity of TGF-β in live cartilage tissues. Here, we detect LTGF-β1 secretion levels for bovine native tissue explants and engineered tissue constructs treated with varying doses of media-supplemented aTGF-β3 using an isoform-specific ELISA. We demonstrate that: 1) LTGF-β secretion levels increase proportionally to aTGF-β exposure, reaching 7.4- and 6.6-fold increases in native and engineered cartilage, respectively; 2) synthesized LTGF-β exhibits low retention in both native and engineered cartilage tissue; and 3) secreted LTGF-β is stable in conditioned media for 2 weeks, thus enabling a reliable biological standard curve between LTGF-β secretion and exposed TGF-β activity. Accordingly, we perform quantifications of TGF-β activity in bovine native cartilage, demonstrating up to 0.59 ng/mL in response to physiological dynamic loading. We further quantify the in situ TGF-β activity in aTGF-β-conjugated scaffolds for engineered tissue, which exhibits 1.81 ng/mL of TGF-β activity as a result of a nominal 3 μg/mL loading dose. Overall, cell-secreted LTGF-β can serve as a robust biomarker to quantify in situ activity of TGF-β in live cartilage tissue and can be potentially applied for a wide range of applications, including multiple tissue types and tissue engineering platforms with different cell populations and scaffolds.

Supramolecular Guest Exchange in Cucurbit[7]uril for Bioorthogonal Fluorogenic Imaging across the Visible Spectrum.

Fluorogenic probes that unmask fluorescence signals in response to bioorthogonal reactions are a powerful new addition to biological imaging. They can significantly reduce background fluorescence and minimize nonspecific signals, potentially enabling real-time, high-contrast imaging without the need to wash out excess fluorophores. While diverse classes of highly refined synthetic fluorophores are now readily available, integrating them into a bioorthogonal fluorogenic scheme still requires extensive design efforts and customized structural alterations to optimize quenching mechanisms for each specific fluorophore scaffold. Herein, we present a highly generalizable strategy that can produce an efficient bioorthogonal fluorogenic response from essentially any readily available fluorophore without further structural alterations. We designed this strategy based on the macrocyclic cucurbit[7]uril (CB7) host, where a fluorogenic response is achieved by programming a guest exchange reaction within the macrocyclic cavity. We employed this strategy to rapidly create fluorogenic probes across the visible spectrum from diverse fluorophore scaffolds, which enabled no-wash imaging in live cells and tissues with minimal background signal. Finally, we demonstrated that this strategy can be combined with metabolic labeling for fluorogenic detection of metabolically tagged mycobacteria under no-wash conditions and paired with covalently clickable probes for high-contrast super-resolution and multiplexed imaging in cells and tissues.

NADPH-cytochrome P450 reductase involved in the lambda-cyhalothrin susceptibility on the green mirid bug Apolygus lucorum.

NADPH-cytochrome P450 reductase (CPR) is crucial for the detoxification process catalysed by cytochrome P450, which targets various exogenous xenobiotics, as well as pesticides. In our research, we successfully obtained the complete cDNA sequence of Apolygus lucorum's CPR (AlCPR) using reverse transcription PCR along with rapid amplification of cDNA ends technology. Bioinformatics analysis exhibited that the inferred amino acid sequence of AlCPR is characteristic of standard CPRs, featuring an N-terminal membrane anchor and three conserved FMN, FAD and NADP binding sites. Phylogenetic result revealed that AlCPR was positioned within the Hemiptera cluster, showing a close evolutionary relationship with the CPR of Cimex lectularius. The real-time quantitative PCR results demonstrated widespread expression of AlCPR across various life stages and tissues of A. lucorum, with the most prominent expression in adults and the abdominal region. Injecting double-stranded RNA of AlCPR only significantly increased the lambda-cyhalothrin susceptibility in lambda-cyhalothrin-resistant strain rather than the susceptible strain. These findings suggest a potential link between AlCPR and the P450-dependent defence mechanism against lambda-cyhalothrin in A. lucorum.

Electric Pulp Test Threshold Responses in Healthy Mature Permanent Teeth

Electric pulp testers assess only the neural component of pulp sensibility in dental practice, while vascular status is evaluated with laser Doppler flowmeters and pulp oximeters, which are more reliable for determining vitality in traumatic teeth. Objective: To determine the accuracy of the electric pulp test in healthy mature permanent teeth. Methods: This cross-sectional study was conducted at the Liaquat University of Medical and Health Sciences Jamshoro by consecutive sampling on 220 participants aged 18-35, who had healthy, mature permanent central incisors, canines, first premolars, and first molars, with no history of cardiac pacemakers and metallic restorations. Teeth were isolated, dried, and tested with a COXO C-Pulse electric pulp tester using toothpaste as a conducting medium. Threshold responses were recorded at increasing currents, with each tooth tested twice to determine the mean value. Accuracy was assessed using additional metrics post-therapy. Chi-square tests were employed to compare accuracy across gender and age groups, with significance set at p&lt;0.05. Results: The mean age of the patients was 25.55 ± 5.41 years. 132 (60%) subjects were female, 88 (40%) subjects were male in this study. Accuracy of the electric pulp test in healthy mature permanent teeth was detected in 181 (82.3%) subjects in this study. Conclusion: It was concluded that the electric pulp tester method seems to be a reliable way to evaluate how sensitive the live nerve tissue inside a tooth is for healthy permanent teeth that have fully formed.

Molecular Detection of Human parvovirus B19 in Patients with Thalassemia in Wasit Province, Iraq

Background: Human B19 (PVB19) isssmall, non-envelope single strand DNA viruses from family of "Parvoviridae" it cause infections in human. In thalassemia are a heterogeneous grouping of genetic disorders that result from a decreased synthesis of alpha or beta chains of hemoglobin (Hb). Numerous factors contribute to functional abnormality found in βeta thalassemia patient like decrease red cells life span, rapidsiron turnover, and tissue deposition of excess iron B19 targets the erythroid progenitors in the bone marrow by binding to the glycosphingolipid. Materials and Methods: This case cross sectional study and the patients aged between (5-40) years , study has been carried outsin Al-Kut teaching hospitals in Al-Iraq-Wasit in a period between September 2023 and March 2024. This study followed the cross-sectional design. The populations of our study included 120 patients’ samples with thalassemia infected by Human Parvovirus B19 in blood transfusion patients 64 male and 56 female. Nested-PCR used to detect of B19V by using primers. PCR reaction was done by using special sets of primers mentioned previously and special components and specific program and procedure according to components of PCR reaction. Result: Human parvovirus 19 Positive case percentage using polymerase chain reaction indicated that the positive cases was reported according to polymerase chain reaction (PCR) as a percentage of case related to the main characteristic of patients, current study data has been indicated as following: in total of 120 patient sample with thalassemia has been included in this study, PCR positive case was 17 (14.2%), 6 (5%), 2 (1.7%) and 0 (0%) in the age groups (5-15), (16-25), (26-35) and (36-45) respectively. Male was more prevalent 17 (14.2%), than female 8 (6.7%), So as to records of major type in comparision to intermediate 19 (15.8%) and 6 (5%). All positive cases were 25 cases. Conclusion: Current data showed prevalence of Human parvovirus 19 in Iraqi patients. This prevalence should be alarming public health.

NPCoronaPredict: A Computational Pipeline for the Prediction of the Nanoparticle-Biomolecule Corona.

The corona of a nanoparticle immersed in a biological fluid is of key importance to its eventual fate and bioactivity in the environment or inside live tissues. It is critical to have insight into both the underlying bionano interactions and the corona composition to ensure biocompatibility of novel engineered nanomaterials. A prediction of these properties in silico requires the successful spanning of multiple orders of magnitude of both time and physical dimensions to produce results in a reasonable amount of time, necessitating the development of a multiscale modeling approach. Here, we present the NPCoronaPredict open-source software package: a suite of software tools to enable this prediction for complex multicomponent nanomaterials in essentially arbitrary biological fluids, or more generally any medium containing organic molecules. The package integrates several recent physics-based computational models and a library of both physics-based and data-driven parametrizations for nanomaterials and organic molecules. We describe the underlying theoretical background and the package functionality from the design of multicomponent NPs through to the evaluation of the corona.

Dioxythiophene/Nafion Polymer Composite Membranes for Tunable Size-Based Selectivity in the Voltammetric Detection of Small Neuropeptides.

Carbon-fiber microelectrodes are proven and powerful sensors for electroanalytical measurements in a variety of environments, including complex systems such as the brain. They are used to detect and quantify a range of biological molecules, including neuropeptides, which are of broad interest for understanding physiological function. The enkephalins (met- and leu-) are endogenous opioid peptides that are involved in both pain and motivated behavior. Each is comprised of only five amino acids including tyrosine, an electroactive species. Electroanalytical measurements targeting tyrosine can reveal the dynamics of endogenous enkephalin transients in live tissue. However, when using electrochemistry in a biological system, selectivity is always a concern. Many larger neuropeptides also contain tyrosine. As such, they could generate a redox signature similar to that of the enkephalins, potentially confounding the measurement. In this work, three distinctly sized dioxythiophene monomers were mixed with Nafion and electrodeposited onto cylindrical carbon-fiber microelectrodes to form composite polymer films that allow for the tunable, size-based exclusion of larger molecules. The dioxythiophene monomers 3,4-ethylenedioxythiophene (EDOT), 3,4-propylenedioxythiophene (ProDOT), and 3,4-(2',2'-diethylpropylene) dioxythiophene (ProDOT-Et2) were used to create nanostructured pores of increasing size. The dioxythiophene/Nafion modified electrodes were characterized in the voltammetric detection of dopamine, a classic small molecule neurotransmitter, and a series of tyrosine containing neuropeptides of increasing size: met-enkephalin (M-ENK; 5 residues), oxytocin (OXY; 9 residues), neurotensin (NT; 13 residues), and neuropeptide Y (NPY; 36 residues). The modified electrodes exhibited enhanced selectivity for smaller peptide species over larger peptides in a manner consistent with the size of the dioxythiophene monomer incorporated into the polymeric film, allowing for tunability in terms of size-based selective detection.

Family-Wide Photoproximity Profiling of Integrin Protein Social Networks in Cancer.

Integrin family transmembrane receptors mediate dynamic interactions between cells and their extracellular microenvironment. The heterogeneous interaction partners of integrins directly regulate cell adhesion, motility, proliferation, and intracellular signaling. Despite the recognized importance of protein-protein interactions and the formation of signaling hubs around integrins, the ability to detect and quantify these dynamic binding partners with high spatial and temporal resolution remains challenging. Here, we developed an integrin-family-directed quantitative photoproximity protein interaction (PhotoPPI) profiling method to detect and quantify native integrin-centered protein social networks on live cells and tissues without the need for genetic manipulation, antibodies, or non-physiologic cell culture conditions. We drafted quantitative maps of integrin-centered protein social networks, highlighting conserved and unique binding partners between different cell types and cellular microenvironments. Comparison of integrin social networks in cancer cell lines of diverse tissue of origin and disease state identified specific AND-gate binding partners involved cell migration, microenvironmental interactions and proliferation that serve as markers of tumor cell metastatic state. Finally, we identified unique combinations - or barcodes - of integrin-proximal proteins on the surface of pre- and post-metastatic triple negative breast cancer (TNBC) cells whose expression strongly correlate with both positive and negative disease progression and outcomes in TNBC patients. Taken together, these data provide the first family-wide high-resolution maps of native protein interactors on live cells and identify dynamic integrin-centered social networks as potential AND-gate markers of cell identity, microenvironmental context and disease state.

Abstract A057: Investigating Fibroblast-Epithelial crosstalk in pre-malignant and cancerous microenvironment

Abstract Pancreatic ductal adenocarcinoma (PDAC) originates from precursor lesions, the most common of which are known as pancreatic intraepithelial neoplasia (PanIN). PanIN formation and progression is accompanied by accumulation of a precursor microenvironment (PME) composed of desmoplastic stroma that plays an important role in promoting tumor growth and immune evasion. The crosstalk between tumor cells and stromal cells is an area of active investigation. While PanIN and the PME have been extensively studied in mouse models, their nature in humans have remained elusive as there is no clinical indication to sample healthy pancreas. Through a unique collaboration with the Gift of Life Organ and Tissue donation program, we performed transcriptomic analysis on normal pancreata declined for transplant. We noted that epithelial cells in the PanIN and cancer microenvironment were transcriptomically similar; however, their surrounding stromal fibroblasts were markedly distinct. Given that most precursor lesions remain quiescent while some progress to tumor, we sought out to elucidate how the transcriptional programs in epithelial cells influence fibroblast cell fate. To do so, we developed a 3D ex-vivo organoid-fibroblast coculture system using primary organoid and fibroblast cultures successfully derived from Gift of Life pancreata or PDAC patients undergoing surgical resection. Our coculture system retains histological features of heterogeneity observed in vivo. Using single cell RNA-sequencing analysis of matched fibroblast-organoid cocultures we identified factors that were highly expressed in fibroblasts in tumor cocultures, but not in normal cocultures. We are currently interrogating candidate ligand/receptor interactions in our cocultures to determine the mediators of this epithelial-mediated fibroblast polarization. This model will allow us to gain an understanding about how components of the microenvironment restrain or promote malignant transformation and serve as a great tool to study therapies targeting tumor and stromal cells in a patient specific manner. Citation Format: Padma Kadiyala, Ahmed Elhossiny, Jianhua Liu, Alexander Bray, Katelyn Donahue, Arvind Rao, Jiaqi Shi, Yaqing Zhang, Filip Bednar, Timothy Frankel, Eileen Carpenter, Marina Pasca Di Magliano. Investigating Fibroblast-Epithelial crosstalk in pre-malignant and cancerous 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 A057.

Monte Carlo investigation of the nucleus size effect and cell’s oxygen content on the damage efficiency of protons

Living tissues could suffer different types of DNA damage as a result of being exposed to ionizing radiations. Monte Carlo simulations of the underlying interactions have been instrumental in predicting the damage types and the processes involved. In this work, we employed Geant4-DNA and MCDS for extracting the initial DNA damage and investigating the dependence of damage efficiency on the cell’s oxygen content. The frequency-mean lineal ( y¯F ) and specific ( z¯F ) energies were derived for a spherical volume of water of various diameters between 2 and 11.1 μm. This sphere would serve as the nucleus of a cell of 100 μm diameter, engulfed by a homogeneous beam of protons. These microdosimetric quantities were calculated assuming spherical samples of 1 μm diameter in MCDS. The simulation results showed that for 230 MeV protons, an increase in the oxygen content from 0 by 10% raised the frequency of single- and double-strand breaks and lowered the base damage frequency. The resulting damage frequencies appeared to be independent of nucleus diameter. For proton energies between 2 and 230 MeV, y¯F showed no dependence on the cell diameter and an increase of the cell size resulted in a decrease in z¯F. An increase in the proton energy slowed down the decreasing rate of z¯F as a function of nucleus diameter. However, the ratio of y¯F values corresponding to two proton energies of choice showed no dependence on the nucleus size and were equal to the ratio of the corresponding z¯F values. Furthermore, the oxygen content of the cell did not affect these microdosimetric quantities. Contrary to damage frequencies, these quantities appeared to depend only on direct interactions due to deposited energies. Our calculations showed the near independence of DNA damages on the nucleus size of the human cells. The probabilities of different types of single and double-strand breaks increase with the oxygen content.

Photobleaching analysis of fluorescent proteins in two-photon microscopy at high repetition rates

Severe photobleaching in two-photon excitation fluorescence microscopy (TPM) has limited its potential for long-term and quantitative imaging of live cells and tissues. One solution is to excite fluorophores with a high-repetition rate which reduces the peak intensity of irradiance. However, there is a lack of knowledge about the general utility of this strategy for fluorescent proteins. Here, using simple photobleaching assay, we studied the photobleaching of EGFP and tdTomato at 80 MHz and 640 MHz repetition rates. Our analysis shows that the impact of high repetition rate excitation on reducing photobleaching in TPM is highly dependent on the excitation wavelength and fluorophores. Our results are useful for selecting optimal parameters to minimize photobleaching and photodamage in TPM.