Healthy Cells Research Articles

AI DRIVEN BLOOD CELL ANALYSIS FOR MALARIA AND BLOOD CANCER SCREENING

Malaria and blood Cancer is one of the deadliest diseases cross the globe. This is caused by the bite of female Anopheles mosquito that transmits the Plasmodium parasites. Some current malaria detection techniques include manual microscopic examination and RDT. These approaches are vulnerable to human mistakes. Early detection of malaria can help in reducing the death rates across the globe.Deep Learning can emerge as a highly beneficial solution in the diagnosis of disease. This model gives a faster and cheaper method for detecting plasmodium parasites. It Was Designed to Identify Malaria Parasites and Cancerous Cell Presence in Blood Using Images of Blood Samples which got Tested with Giemsa-stain. Convolutional neural networks (CNNs) are then trained on these extracted features to accurately classify blood cell images into disease positive or disease-negative categories. The CNN detects malaria parasites in microscopic images by classifying them into parasitized and healthy cells, enabling the detection of malaria parasites. The proposed model consists of three convolutional layers and fully connected layers each. The neural network presented is a cascade of several convolutional layers having multiple filters present in layers, which yields the exceptionally good accuracy as per the available resources. The experiment results demonstrate a significant improvement in malaria parasite and blood cancer recognition. Compared to traditional manual microscopy, the proposed system is more accurate and faster. Finally, this study demonstrates the need to provide robust and efficient solutions by leveraging state-of-the-art technologies to combat malaria and blood cancer. Keywords : Deep Learning, CNN, Image analysis , InceptionresnetV2

Biomedical Applications of Cold Atmospheric Plasma: Cancer Therapy and Virus Inactivation

Cold atmospheric plasma (CAP), known for its ability to generate Reactive Oxygen Species (ROS) and Reactive Nitrogen Species (RNS), has emerged as a promising tool in many biomedical applications, including cancer therapeutics and virus inactivation. In cancer therapy, ROS and RNS induced by plasma have been shown to selectively target cancer cells, triggering apoptosis and inhibiting tumor growth while sparing healthy cells. This selective cytotoxicity makes plasma an attractive option for localized cancer treatment with minimal side effects. Additionally, the antimicrobial properties of ROS and RNS make plasma an effective agent for virus inactivation. By connecting the dots between cancer therapy and virus disinfection, this review paper sheds light on the potential of plasma-generated ROS and RNS as versatile tools in medical applications, offering a dual benefit of targeted cancer treatment and effective virus control. Specifically, this review explores the effects of CAP treatment on B16 melanoma cells, assessing changes in cell viability, proliferation, apoptosis and oxidative stress response. Dichloroacetate (DCA), a mitochondrial modulator, has shown promise in inducing apoptosis in melanoma cells. However, its clinical use is limited by side effects related to its neurotoxicity and suspected carcinogenicity. The potential of CAP in enhancing the therapeutic efficacy of DCA on B16-F10 melanoma apoptosis is also explored. CAP therapeutics for other types of cancers are also explored in this review. Alongside its implications in oncology, CAP has gathered substantial attention for its extraordinary potential in neutralizing viruses. CAP’s exceptional precision in targeting viral envelopes, genetic material and structural components disrupts their integrity and vital functions. This orchestrated mechanism offers a swift and efficacious route to render viruses inactive, encompassing both enveloped and nonenveloped viral types. In parallel pursuit, this review examines CAP-generated reactive species’ proficiency in deactivating SARS-CoV-2 across various surfaces. The inquiry entails subjecting surfaces to CAP treatment, succeeded by quantifying viral load and scrutinizing viral integrity. The outcomes gleaned from these investigations provide pivotal insights into CAP’s potential as a robust surface disinfection strategy, poised to combat the propagation of viral transmission.

Extracellular Vesicles: A New Approach to Study the Brain's Neural System and Its Diseases.

In normal and pathophysiological conditions our cells secrete vesicular bodies known as extracellular particles. Extracellular vesicles are lipid-bound extracellular particles. A majority of these extracellular vesicles are linked to cell-to-cell communication. Brain consists of tightly packed neural cells. Neural cell releases extracellular vesicles in cerebrospinal fluid. Extracellular vesicle mediated crosstalk maintains neural homeostasis in the central nervous system via transferring cargos between neural cells. In neurodegenerative diseases, small extracellular vesicle transfer misfolded proteins to healthy cells in the neural microenvironment. They can also cross blood-brain barrier (BBB) and stimulate peripheral immune response inside central nervous system. In today's world different approaches employ extracellular vesicle in various therapeutics. This review gives a brief knowledge about the biological relevance of extracellular vesicles in the central nervous system and relevant advances in the translational application of EV in brain disorders.

The Expression of Adipogenic Marker Is Significantly Increased in Estrogen-Treated Lipedema Adipocytes Differentiated from Adipose Stem Cells In Vitro

Lipedema is a chronic, idiopathic, and painful disease characterized by an excess of adipose tissue in the extremities. The goal of this study is to characterize the gene expression of estrogen receptors (ERα and ERβ), G protein-coupled estrogen receptor (GPER), and ER-metabolizing enzymes: hydroxysteroid 17-beta dehydrogenase (HSD17B1, 7, B12), cytochrome P450 (CYP19A1), hormone-sensitive lipase (LIPE), enzyme steroid sulfatase (STS), and estrogen sulfotransferase (SULT1E1), which are markers in Body Mass Index (BMI) and age-matched non-lipedema (healthy) and lipedema ASCs and spheroids. Flow cytometry and cellular proliferation assays, RT-PCR, and Western Blot techniques were used to determine the expression of ERs and estrogen-metabolizing enzymes. In 2D monolayer culture, estrogen increased the proliferation and the expression of the mesenchymal marker, CD73, in hormone-depleted (HD) healthy ASCs compared to lipedema ASCs. The expression of ERβ was significantly increased in HD lipedema ASCs and spheroids compared to corresponding healthy cells. In contrast, ERα and GPER gene expression was significantly decreased in estrogen-treated lipedema spheroids. CYP19A1 and LIPE gene expressions were significantly increased in estrogen-treated healthy ASCs and spheroids, respectively, while estrogen upregulated the expression of PPAR-ϒ2 and ERα in estrogen-treated lipedema-differentiated adipocytes and spheroids. These results indicate that estrogen may play a role in adipose tissue dysregulation in lipedema.

Development of a 3D in vitro human-sized model of cervical dysplasia to evaluate the delivery of ethyl cellulose-ethanol injection

Introduction: Cervical cancer, the second leading cause of cancer-related death for women worldwide, remains a preventable yet persistent disease that disproportionately affects women in low and middle-income countries (LMICs). While existing therapies for treating cervical dysplasia are effective, they are often inaccessible in LMICs. Ethanol ablation is an alternative low-cost, accessible therapy that we previously enhanced into ethyl cellulose (EC)-ethanol gel formulation to improve efficacy.Methods: To evaluate the efficacy of EC-ethanol, in this study, we developed a 3D in vitro model of cervical dysplasia featuring a central lesion of cervical cancer cells surrounded by fibroblasts and keratinocytes. Using a GelMA hydrogel formulation (8.7% w/v), we successfully built a 3D model that captured the architectural complexity of cervical dysplasia. We evaluated changes in cell coverage and cell viability. Then, we compared the viscoelastic properties of the GelMA hydrogels to human cervical tissue and using micro-CT imaging, we assessed EC-ethanol injection deposition in the hydrogel, revealing retention of virtually the entire injected volume near the injection site. Finally, we measured changes in cell viability and cell coverage after the EC-ethanol injection.Results: The developed 3D in vitro model successfully replicated the architectural complexity of cervical dysplasia, demonstrating high cell viability and capturing cell responses effectively. The GelMA hydrogel formulation (8.7% w/v) exhibited viscoelastic properties akin to human cervical tissue. Micro-CT imaging revealed efficient deposition of EC-ethanol within the hydrogel, with retention of the injected volume near the injection site. Furthermore, the EC-ethanol injection significantly reduced cervical cancer cell viability and cell coverage while preserving healthy cells within the model.Conclusion: Our findings indicate that our 3D in vitro model mirrored the architecture of cervical dysplasia and demonstrated the potential of EC-ethanol for localized treatment of cervical dysplasia.

Exploiting non-permissive CHO cells as a rapid and efficient method for recombinant HSV-1 isolation

Using herpes simplex virus type 1 (HSV-1) as a therapeutic tool has recently emerged as a promising strategy for enhancing the treatment of various cancers, particularly those associated with the nervous system, which is the virus's natural site of infection. These viruses are specifically engineered to infect and eradicate tumor cells while leaving healthy cells unharmed. To introduce targeted mutations in specific viral genes, gene-modification techniques such as shuttle vector homologous recombination are commonly employed. Plaque purification is then utilized to select and purify the recombinant virus from the parental viruses. However, plaque purification becomes problematic when the insertion of the desired gene at the target site hampers progeny virus replication, resulting in a lower titer of cell-released virus than the parental virus. This necessitates a laborious initial screening process using approximately 10–15 tissue culture dishes (10 cm), making plaque purification time-consuming and demanding. Although the recently developed CRISPR-Cas9 system significantly enhances the efficiency of homologous integration and editing precision in viral genes, the purification of recombinant variants remains a tedious task. In this study, we propose a rapid and innovative method that employs non-permissive Chinese hamster ovary (CHO) cells, representing a remarkable improvement over the aforementioned arduous process. With this approach, only 1–2 rounds of plaque purification are required. Our proposed protocol demonstrates great potential as a viable alternative to current methods for isolating and purifying recombinant HSV-1 variants expressing fluorescent reporter genes using CHO cells and plaque assays.

Extremely Low-Frequency Electromagnetic Field (ELF-EMF) Induced Alterations in Gene Expression and Cytokine Secretion in Clear Cell Renal Carcinoma Cells.

The study aimed to investigate the influence of extremely low-frequency electromagnetic fields (ELF-EMF) on clear cell renal cell carcinoma (ccRCC) by assessing alterations in gene expression and the secretion of cytokines and chemokines. Three ccRCC cell lines (786-O, 769-P, and CAKI-1) and a healthy HEK293 cell line were subjected to ELF-EMF exposure (frequency 50 Hz, magnetic field strength 4.5 mT) for 30 min daily for 5 days. The study examined the expression of ADAM28, NCAM1, and VEGFC genes, along with the secretion of 30 cytokines and chemokines. Notably, primary tumor-derived cell lines, but not those from metastatic sites, exhibited ADAM28 gene expression, which increased following ELF-EMF exposure. A statistically significant reduction in VEGFC gene expression was observed in 769-P cells after ELF-EMF exposure. Additionally, NCAM1 gene expression was upregulated in HEK293, 769-P, and 786-O cells, representing normal embryonic kidney cells and primary tumor cells, but not in CAKI-1 cells, which model metastatic sites. After EMF exposure, there was a statistically significant decrease in transforming growth factor β1 (TGF-β1) concentration in the cell culture supernatants of HEK293 and CAKI-1 cell lines, with no other significant changes in the secretion of tested cytokines. Given the study's findings and available research, caution is warranted when drawing conclusions about the potential inhibitory effect of ELF-EMF on ccRCC progression. Standardization of experimental models is imperative when assessing the effects of EMF in a human context.

Heterogeneity and plasticity of tissue-resident memory T cells in skin diseases and homeostasis: a review

Skin tissue-resident memory T (Trm) cells are produced by antigenic stimulation and remain in the skin for a long time without entering the peripheral circulation. In the healthy state Trm cells can play a patrolling and surveillance role, but in the disease state Trm cells differentiate into various phenotypes associated with different diseases, exhibit different localizations, and consequently have local protective or pathogenic roles, such as disease recurrence in vitiligo and maintenance of immune homeostasis in melanoma. The most common surface marker of Trm cells is CD69/CD103. However, the plasticity of tissue-resident memory T cells after colonization remains somewhat uncertain. This ambiguity is largely due to the variation in the functionality and ultimate destination of Trm cells produced from memory cells differentiated from diverse precursors. Notably, the presence of Trm cells is not stationary across numerous non-lymphoid tissues, most notably in the skin. These cells may reenter the blood and distant tissue sites during the recall response, revealing the recycling and migration potential of the Trm cell progeny. This review focuses on the origin and function of skin Trm cells, and provides new insights into the role of skin Trm cells in the treatment of autoimmune skin diseases, infectious skin diseases, and tumors.

AN UPDATED REVIEW OF STEALTH LIPOSOMES AND ITS ABILITY TO EVADE THE IMMUNE SYSTEM: A NEW FRONTIER IN CANCER CHEMOTHERAPY

Liposomes have been the delivery of choice for the cancer targeting therapy for the last few decades. Since the 1990s, the development of sterically stabilized (stealth) liposomes has garnered interest for their long circulating half-life. PEGylated (Polyethylene Glycol) liposomes are most extensively studied for delivering cancer therapeutics in a sustained manner. Stealth liposomes are having a less intrinsic toxicity with higher efficacy in cancer treatment. There are numerous clinical trials on the liposomes in tackling cancer is evident for the better outcome of the delivery system. Stealth liposomes are extensively studied for their improved circulation time and better pharmacokinetic profile in cancer treatment. The steric hindrance of the stealth liposomes bypasses the reticuloendothelial system clearance. Further the ligands conjugation in the surface of the liposomes able to achieve better target to the cancer cells. The vascularization nature of the cancerous cells is readily making the liposomal delivery of the cancer drugs accumulate in the cancerous cells rather than healthy cells. There is an utmost need to understand the possible mechanism of stealth liposomes and the basic science behind the development of liposomal delivery system in advancing the cancer treatment with less toxicity. The present review addresses the various modalities of the liposomal development, liposome characterization, mechanism of PEGylated liposomes, the advancements and results of the liposomes in the treatment of various diseases, and the clinical trials and regulatory considerations of liposomal drug delivery system.

Relative biomembrane fusogenicities of the tumor-selective liposomes of RGDK- and CGKRK-lipopeptides.

Cancer is the second leading cause of death globally after heart diseases. Currently used highly cytotoxic anti-cancer drugs not only kill cancer cells but also often kill non-cancerous healthy body cells, causing adverse side effects. Efforts are now being directed towards developing tumor-selective chemotherapy. Tumor/tumor endothelial cell selective peptide ligands are being covalently grafted onto the exo-surfaces of drug carriers such as liposomes, polymers, etc. A number of prior studies used conjugation of tumor/tumor endothelial cell-selective RGDK- or CGKRK-peptide ligands on the outer surfaces of liposomes, metal-based nanoparticles, single walled carbon nanotubes (SWNTs), etc. However, studies aimed at examining the relative cell membrane fusogenicities and the relative degrees of cellular uptake for the RGDK- and CGKRK-ligand-grafted nanometric drug carriers have not yet been undertaken. Herein, using the widely used liposomes of DOPC, DOPE, DOPS and cholesterol (45 : 25 : 20 : 15, w/w ratio) as the model biomembranes and the fluorescence resonance energy transfer (FRET) assay for measuring membrane fusogenicities, we show that the liposomes of the RGDK-lipopeptide are more biomembrane fusogenic than the liposomes of the CGKRK-lipopeptide. Notably, such FRET assay-derived relative biomembrane fusogenicities of the liposomes of RGDK- and CGKRK-lipopeptides were found to be consistent with their relative degrees of cellular uptake in cultured cancer cells. The present findings open the door for undertaking in-depth in vivo studies aimed at evaluating the relative therapeutic potential of different nanocarriers of drugs/genes/siRNA having tumor-targeting RGDK- and CGKRK-peptides on their exo-surfaces.

Epi-revolution in rheumatology: the potential of histone deacetylase inhibitors for targeted rheumatoid arthritis intervention.

Autoimmune diseases hold significant importance in the realm of medical research, prompting a thorough exploration of potential therapeutic interventions. One crucial aspect of this exploration involves understanding the intricate processes of histone acetylation and deacetylation. Histone acetylation, facilitated by histone acetyl transferases (HATs), is instrumental in rendering DNA transcriptionally active. Conversely, histone deacetylases (HDACs) are responsible for the removal of acetyl groups, influencing gene expression regulation. The upregulation of HDACs, observed in various cancers, has steered attention towards histone deacetylase inhibitors (HDACi) as promising anti-cancer agents. Beyond cancer, HDACi has demonstrated anti-inflammatory properties, prompting interest in their potential therapeutic applications for inflammatory diseases such as rheumatoid arthritis (RA). RA, characterized by the immune system erroneously attacking healthy cells, leads to joint inflammation. Recent studies suggest that HDACi could offer a viable therapeutic strategy for RA, with potential mechanisms including the inhibition of synovial tissue growth and suppression of pro-inflammatory cytokines. Furthermore, HDACi may exert protective effects on bone and cartilage, common targets in RA pathology. In-depth investigations through in vivo and histopathology studies contribute to the ongoing discourse on the therapeutic benefits of HDACis in the context of RA treatment.

Enhanced efficiency of photodynamic therapy-mediated the plant pigment hypericin through Folate-PEG functionalized chitosan nanoparticles

Hypericin (Hy), an anthraquinone-derived pigment, has recently attracted attention as a safe and natural photosensitizer for photodynamic therapy (PDT). However, its clinical applications have been limited by its hydrophobicity and off-target photoactivity. This work aimed to enhance efficacy of Hy-based PDT using a high-efficiency and high-specificity vehicle for targeted delivery Hy to the cancer cells. Hy-loaded chitosan nanoparticles (Hy-CNs) were prepared using the ionic gelation technique, and further functionalized with folic acid (FA) and PEG. These generated nanoparticles (PEG-FA-Hy-CNs) were physicochemically characterized using, FT-IR spectroscopy, UV–Vis spectroscopy, dynamic light scattering, and SEM analysis and then compared with other Hy formulations in terms the singlet oxygen production, intra cellular uptake, and phototoxicity under suitable irradiation (590 nm, 10 Jcm−2). All the formed nanostructures presented a mean particle size about 200–300 nm and greater than 80 % entrapment efficiency. Drug release data of PEG-FA-Hy-CNs indicated a sustained release of Hy and a good correlation with Higuchi model. PEG-FA-Hy-CNs induced a much higher singlet oxygen formation, cellular uptake, photoactivity, and apoptosis compared to Hy-CN, Hy-FA, Hy-PEG, and free Hy. Additionally, these effects were much higher in FA receptors over-expressing breast cancer cells than healthy cell lines. Results suggest that PEG-FA-Hy-CNs can be used for controlled Hy release and active targeting of breast cancer cells, which are beneficial for Hy-based PDT.

The long non-coding RNAs (lncRNA) in the pathogenesis of gastric cancer cells: molecular mechanisms and involvement miRNAs.

A complex sequence of occurrences, including host genetic vulnerability, Helicobacter pylori infection, and other environmental variables, culminate in gastric cancer (GC). The development of several genetic and epigenetic changes in oncogenes and tumor suppressor genes causes dysregulation of several signaling pathways, which upsets the cell cycle and the equilibrium between cell division and apoptosis, leading to GC. Developments in computational biology and RNA-seq technology enable quick detection and characterization of long non-coding RNAs (lncRNAs). Recent studies have shown that long non-coding RNAs (lncRNAs) have multiple roles in the development of gastric cancer. These lncRNAs interact with molecules of protein, RNA, DNA, and/or combinations. This review article explores several gastric cancer-associated lncRNAs, such as ADAMTS9-AS2, UCA1, XBP-1, and LINC00152. These various lncRNAs could change GC cell apoptosis, migration, and invasion features in the tumor microenvironment. This review provides an overview of the most recent research on lncRNAs and GC cell apoptosis, migration, invasion, and drug resistance, focusing on studies conducted in cancer cells and healthy cells during differentiation.

Stain-free approach to determine and monitor cell heath using supervised and unsupervised image-based deep learning.

Cell-based medicinal products (CBMPs) are a growing class of therapeutics that promise new treatments for complex and rare diseases. Given the inherent complexity of the whole human cells comprising CBMPs, there is a need for robust and fast analytical methods for characterization, process monitoring, and quality control (QC) testing during their manufacture. Existing techniques to evaluate and monitor cell quality typically constitute labor-intensive, expensive, and highly specific staining assays. In this work, we combine image-based deep learning with flow imaging microscopy (FIM) to predict cell health metrics using cellular morphology "fingerprints" extracted from images of unstained Jurkat cells (immortalized human T-lymphocyte cells). A supervised (i.e., algorithm trained with human-generated labels for images) fingerprinting algorithm, trained on images of unstained healthy and dead cells, provides a robust stain-free, non-invasive, and non-destructive method for determining cell viability. Results from the stain-free method are in good agreement with traditional stain-based cytometric viability measurements. Additionally, when trained with images of healthy cells, dead cells and cells undergoing chemically induced apoptosis, the supervised fingerprinting algorithm is able to distinguish between the three cell states, and the results are independent of specific treatments or signaling pathways. We then show that an unsupervised variational autoencoder (VAE) algorithm trained on the same images, but without human-generated labels, is able to distinguish between samples of healthy, dead and apoptotic cells along with cellular debris based on learned morphological features and without human input. With this, we demonstrate that VAEs are a powerful exploratory technique that can be used as a process monitoring analytical tool.

Antileukemic potential of methylated indolequinone MAC681 through immunogenic necroptosis and PARP1 degradation

BackgroundDespite advancements in chronic myeloid leukemia (CML) therapy with tyrosine kinase inhibitors (TKIs), resistance and intolerance remain significant challenges. Leukemia stem cells (LSCs) and TKI-resistant cells rely on altered mitochondrial metabolism and oxidative phosphorylation. Targeting rewired energy metabolism and inducing non-apoptotic cell death, along with the release of damage-associated molecular patterns (DAMPs), can enhance therapeutic strategies and immunogenic therapies against CML and prevent the emergence of TKI-resistant cells and LSC persistence.MethodsTranscriptomic analysis was conducted using datasets of CML patients' stem cells and healthy cells. DNA damage was evaluated by fluorescent microscopy and flow cytometry. Cell death was assessed by trypan blue exclusion test, fluorescent microscopy, flow cytometry, colony formation assay, and in vivo Zebrafish xenografts. Energy metabolism was determined by measuring NAD+ and NADH levels, ATP production rate by Seahorse analyzer, and intracellular ATP content. Mitochondrial fitness was estimated by measurements of mitochondrial membrane potential, ROS, and calcium accumulation by flow cytometry, and morphology was visualized by TEM. Bioinformatic analysis, real-time qPCR, western blotting, chemical reaction prediction, and molecular docking were utilized to identify the drug target. The immunogenic potential was assessed by high mobility group box (HMGB)1 ELISA assay, luciferase-based extracellular ATP assay, ectopic calreticulin expression by flow cytometry, and validated by phagocytosis assay, and in vivo vaccination assay using syngeneic C57BL/6 mice.ResultsTranscriptomic analysis identified metabolic alterations and DNA repair deficiency signatures in CML patients. CML patients exhibited enrichment in immune system, DNA repair, and metabolic pathways. The gene signature associated with BRCA mutated tumors was enriched in CML datasets, suggesting a deficiency in double-strand break repair pathways. Additionally, poly(ADP-ribose) polymerase (PARP)1 was significantly upregulated in CML patients’ stem cells compared to healthy counterparts. Consistent with the CML patient DNA repair signature, treatment with the methylated indolequinone MAC681 induced DNA damage, mitochondrial dysfunction, calcium homeostasis disruption, metabolic catastrophe, and necroptotic-like cell death. In parallel, MAC681 led to PARP1 degradation that was prevented by 3-aminobenzamide. MAC681-treated myeloid leukemia cells released DAMPs and demonstrated the potential to generate an immunogenic vaccine in C57BL/6 mice. MAC681 and asciminib exhibited synergistic effects in killing both imatinib-sensitive and -resistant CML, opening new therapeutic opportunities.ConclusionsOverall, increasing the tumor mutational burden by PARP1 degradation and mitochondrial deregulation makes CML suitable for immunotherapy.

The role of singlet oxygen and hydroxyl radical in the photobleaching of meso-substituted cationic pyridyl porphyrins in the presence of folic acid

Using a photosensitizer (PS), light, and oxygen, photodynamic therapy creates cytotoxic reactive oxygen species, such as singlet oxygen (1O2), that kill cancer cells. Many cancer cell lines have up to 300 times more folic acid receptors than healthy cells. Therefore, folic acid is often used to improve selectivity of PSs. Photobleaching poses a disadvantage for PSs. In this paper, we have studied the photoinduced changes of meso-substituted cationic pyridyl porphyrins in the presence of folic acid using fluorescence and absorption spectroscopy. In this work, it was demonstrated that L-histidine, which is a 1O2 quencher, and D-mannitol, which is a hydroxyl radical quencher, can reduce photobleaching of cationic porphyrins and their interaction products with FA. This implies both singlet oxygen and hydroxyl radicals are involved in photobleaching. Additionally, our study revealed certain important features of the photobleaching of cationic porphyrins in the presence of folic acid.

Generation of a healthy heavy smoker patient-derived induced pluripotent stem cell line UHOMi007-A from peripheral blood mononuclear cells

Human pluripotent stem cells (hiPSC) represent a unique opportunity to model lung development and chronic bronchial diseases. We generated a hiPSC line from a highly characterized healthy heavy smoker male donor free from emphysema or tobacco related disease. Peripheral blood mononuclear cells (PBMCs) were reprogrammed using integration-free Sendai virus. The cell line had normal karyotype, expressed pluripotency hallmarks, and differentiated into the three primary germ layers.The reported UHOMi007-A iPSC line may be used as a control to model lung development, study human chronic bronchial diseases and drug testing.

Synthesis, Characterization, and Cytotoxicity Evaluation of Novel Water-Soluble Cationic Platinum(II) Organometallic Complexes with Phenanthroline and Imidazolic Ligands.

Platinum-based chemotherapeutic agents are widely used in the treatment of cancer. However, their effectiveness is limited by severe adverse reactions, drug resistance, and poor water solubility. This study focuses on the synthesis and characterization of new water-soluble cationic monofunctional platinum(II) complexes starting from the [PtCl(η1-C2H4OEt)(phen)] (1, phen = 1,10-phenanthroline) precursor, specifically [Pt(NH3)(η1-C2H4OEt)(phen)]Cl (2), [Pt(1-hexyl-1H-imidazole)(η1-C2H4OEt)(phen)]Cl (3), and [Pt(1-hexyl-1H-benzo[d]imidazole)(η1-C2H4OEt)(phen)]Cl (4), which deviate from traditional requirements for antitumor activity. These complexes were evaluated for their cytotoxic effects in comparison to cisplatin, using immortalized cervical adenocarcinoma cells (HeLa), human renal carcinoma cells (Caki-1), and normal human renal cells (HK-2). While complex 2 showed minimal effects on the cell lines, complexes 3 and 4 demonstrated higher cytotoxicity than cisplatin. Notably, complex 4 displayed the highest cytotoxicity in both cancer and normal cell lines. However, complex 3 exhibited the highest selectivity for renal tumor cells (Caki-1) among the tested complexes, compared to healthy cells (HK-2). This resulted in a significantly higher selectivity than that of cisplatin and complex 4. Therefore, complex 3 shows potential as a leading candidate for the development of a new generation of platinum-based anticancer drugs, utilizing biocompatible imidazole ligands while demonstrating promising anticancer properties.

Abstract PO4-11-03: Impact of Short Term Fasting (STF) on Fatigue developing in primary breast cancer (BC) patients receiving the Henderson or Sparano Scheme in the prospective multicentre FIT2- cohort

Abstract Background: Experimentally, it has been shown that short-term fasting (STF) induces a “protective mode” in healthy cells against chemotherapeutic toxicity while it enhances the susceptibility of tumor cells to chemotherapy. In healthy cells, fasting triggers distinct protective metabolic and gene expression changes, such as upregulation of DNA repair mechanisms, autophagy, and downshift of metabolic pathways including IGF-1-dependent signaling. This study was conducted to assess feasibility, quality of life (QoL) including chemotherapy-induced fatigue (CTF) comparing STF around each chemotherapy (4xAC or 4xEC) to a plant-based low-sugar diet (PBD). Methods: Newly diagnosed BC-patients treated by the Henderson or Sparano scheme were included in this randomised, controlled, multicenter study. CTF was assessed by the FACIT-F questionnaire before (day 0) and after chemotherapy (day 7) as well as at months 4 (V1) and 6 (V2). Results: Of the included n=106 patients, 52 were randomized to STF and 54 to PBD. n=90 (84.9%) patients obtained AC/EC+Pac and 16 (15.1%) AC/EC+Doc. FACIT-F showed no significant difference at baseline (STF 42.8; PBD 41.3; p=0.708). The differences between the corresponding FACIT-F Trial Outcome Index resp. Total FACIT-F at cycle 4 day 7 were 13.1 (SE 4.7) resp. 15.8 (SE 5.6), both in favor of STF (statistically significant (p < 0.007) and clinically relevant (minimal important difference (MID) > 5). This difference was visible for all breast cancer subtypes, i.e. luminal A & B, as well as HER2 overexpressed and triple negative tumors, with the STF group showing no clinically relevant fatigue after 4 cycles of chemotherapy (FACIT-F > 34), across all subtypes. The opposite was true for the PBD group (FACIT-F < 34) across all subtypes. FACIT-F (22.7) for PBD in HER2 overexpressed and triple negative tumors was significantly worse than those in luminal A and B patients (FACIT-F= 29.1; difference 6.4 > MID=3). STF was well tolerated and there were no serious adverse effects. Conclusion: STF during chemotherapy is well tolerated and appears to improve QoL including fatigue during chemotherapy compared to a plant-based low-sugar diet, independent of intrinsic subtypes. Citation Format: Daniela Koppold, Christian Kessler, Manfred Wischnewsky, Ixquic Alvarado, Nico Steckhan, Cornelia Kempter, Marion Paul, Barbara Brückner, Dorothea Fischer, Aysen Wunschel, Rainer Stange, Etienne Hanslian, Michael Jeitler, Andreas Michalsen. Impact of Short Term Fasting (STF) on Fatigue developing in primary breast cancer (BC) patients receiving the Henderson or Sparano Scheme in the prospective multicentre FIT2- cohort [abstract]. In: Proceedings of the 2023 San Antonio Breast Cancer Symposium; 2023 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2024;84(9 Suppl):Abstract nr PO4-11-03.

Abstract PO1-07-02: Sensitive tumor detection, accurate quantification, and ER subtyping using low-pass methylome of liquid biopsy samples from patients (pts) with metastatic breast cancer (mBC)

Abstract Background The presence and amount of blood circulating tumor DNA (Tumor Content, TC) is emerging as a clinically relevant factor in many cancers, including BC. In the metastatic setting, a commonly used tumor tissue agnostic approach involves analyzing Copy Number Alterations (CNA) from low-pass whole-genome sequencing (lpWGS) by ichorCNA. However, with a lower limit of detection of 3%, it is unsuitable for low TC applications, such as monitoring disease in response to treatment. Here we report on the use of low-pass whole genome bisulfite sequencing (lpWGBS) coupled with a novel tool, METER (METhylation analyzER), to overcome current limitations of lpWGS, enabling sensitive TC detection, accurate quantification, and ER subtyping from mBC liquid biopsies. Methods METER is a computational tool to analyse TC exploiting BC-specific Differentially Methylated Sites (DMS) and Regions (DMR) in lpWGBS data. To define BC DMSs and DMRs, Rocker-meth was applied to the WGBS profiles of 30 BC tissue (BASIS dataset) and 23 healthy donor cell free DNA (cfDNA) (Fox-Fisher 2021). METER consists of three modules: 1) METER-quant, a DMS-based quantifier of TC; 2) METER-detect, a DMR-based z-score method to classify samples as TC+ (METER+) or TC- (METER-); 3) METER-subtype, to infer ER status via Robust Partial Correlation applied to ER+ vs. ER- BC DMR. We generated lpWGBS data (coverage 0.5-1X) of 135 cfDNA samples from 58 pts (45 ER+, 13 ER-, 55 (95%) up to 3 previous lines of treatment; before starting treatment (T0, n=58), after the first cycle (T1, n=25), and at progression (T2, n=22)) and 30 from healthy individuals as controls. ichorCNA using sensitive parameters (allowing normal fraction up to 0.99) was used to estimate reference TC. A leave-one-out strategy applied to the control samples was used to estimate the false discovery rate (FDR) of METER-detect classification. METER+/- pts were tested for association with OS and PFS using the log-rank test and compared with ichorCNA detection (TC by ichorCNA above or below 3%, ichorCNA+/-). Results TC of 105 cfDNA BC samples by METER-quant was concordant with estimates from the state-of-the-art tool ichorCNA (R >0.90, p< 1e-10). In 22 pts with complete longitudinal data, METER-quant median TC of 0.05 at T0 (IQR 0.02-0.12), 0.02 at T1 (0.01-0.07), and 0.06 at T2 (0.03-0.11) were obtained, with significant difference observed for T0 vs T1 and T2 vs T1 (paired Wilcoxon p< 0.05). Across all time points, METER-detect classified 43% of the n=42 ichorCNA- samples as METER+. The reliability and prognostic performance of METER and ichorCNA detection classification were then compared using clinical outcome data. Based on T0 data, METER+ pts had significantly worse OS than METER- (HR=4.2 CI=2.0-8.9, p< 0.001). This effect was stronger than using ichorCNA (HR=2.3 CI=1.3-4.0, p=0.006). Worse PFS at T0 was observed for METER+ compared with METER- pts (HR=3.7 CI=1.8-7.9, p< 0.001), while no significant association was observed for ichorCNA+ pts (HR=1.4 CI=0.8-2.5, p=0.2). Of note, ichorCNA-/METER+ pts (N=11) at T0 had worse PFS compared with ichorCNA-/METER- (N=11) (HR=3.7 CI 1.4-9.7, p=0.005). Considering the 73 METER+ and 53 samples with METER-quant >5%, METER-subtype showed an accuracy of 0.84 (CI=0.73-0.92) and 0.94 (CI=0.83-0.99) respectively in classifying ER status based on IHC. Discussion lpWGBS enables tumor tissue agnostic analysis and concurrent investigation of complementary molecular features, including CNA and DNA methylation patterns. In a small and heterogeneous cohort, METER showed comparable performance to state-of-the-art tools in terms of TC quantification (ichorCNA) and ER subtyping (IHC), while offering enhanced sensitivity and ensuring a low FDR. METER showed promising prognostic stratification capabilities, particularly for patients with low TC. Validation in an extended prospective cohort is currently ongoing. Citation Format: Marta Paoli, Agostina Nardone, Francesca Galardi, Chiara Biagioni, Dario Romagnoli, Francesca De Luca, Gian Marco Franceschini, Ilenia Migliaccio, Marta Pestrin, Giuseppina Sanna, Emanuela Risi, Luca Livraghi, Erica Moretti, Laura Biganzoli, Luca Malorni, Francesca Demichelis, Matteo Benelli. Sensitive tumor detection, accurate quantification, and ER subtyping using low-pass methylome of liquid biopsy samples from patients (pts) with metastatic breast cancer (mBC) [abstract]. In: Proceedings of the 2023 San Antonio Breast Cancer Symposium; 2023 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2024;84(9 Suppl):Abstract nr PO1-07-02.