Parental Cells Research Articles

Exosomes and their distinct integrins transfer the characteristics of oxaliplatin- and 5-FU-resistant behaviors in colorectal cancer cells

BackgroundExosomes are communication carriers and suitable biomarker candidates due to their cargoes with specific dynamic profiles. Integrins, as valuable prognostic markers in cancer, have importance in exosome-cell interaction. However, the role of exosome integrins in chemoresistant colorectal cancer remained unclear.MethodsOxaliplatin- and 5-FU-resistant cells (OXR and FUR) were established from human HCT-116 cells of colorectal cancer. Exosomes were collected from untreated and treated cells with oxaliplatin or 5-FU. Exosomes were isolated via ultracentrifugation and characterized using DLS and electron microscopy to evaluate size and morphology. Western blot analysis was employed to identify exosomal markers. The effects of exosomes on parental cells were examined using various methods, including MTT assay for proliferation, wound healing assay for migration, flow cytometry for cell cycle and apoptosis analysis, Matrigel-coated transwell inserts for invasion, and western blot for integrin expression evaluation.ResultsExosome integrins determine resistance behaviors in cells. We observed that exosomes from OXR cells or OXR cells treated with oxaliplatin increased ITGβ3 expression and decreased ITGβ4 expression in parental cells, resulting in distinct resistance behaviors. Exosomes from FUR cells or FUR cells treated with 5-FU reduced ITGβ4 levels and elevated ITGαv levels in parental cells, leading to varying degrees of invasive resistance behaviors. These findings suggest that exosome integrins may affect these behaviors. High ITGβ3 exosomes induced oxaliplatin resistance behaviors in parental cells. Lowering ITGβ3 levels in these exosomes inhibited the resistance behaviors observed in these cells. FUR exosomes that overexpressed ITGαv or ITGβ4 resulted in invasive 5-FU resistance behaviors in parental cells. A reduction in these exosome integrin levels led to moderate invasive behaviors. The decrease of ITGβ4 in FUR cell exosomes inhibited resistant migration and proliferation in parental cells. A twofold reduction of ITGαv in FUR cell exosomes resulted in a threefold decrease in invasion and inhibited migration in parental cells compared to those treated with high ITGαv exosomes.ConclusionOur findings reveal that, despite discrepancies between cellular integrin patterns and cellular behaviors, the levels of exosomal ITGβ3, ITGαv, or ITGβ4 could serve as potential diagnostic and therapeutic markers for resistance to oxaliplatin and 5-FU in future cancer treatments.

Knocking out p38α+p38β+p38γ is required to abort the myogenic program in C2C12 myoblasts and to impose uncontrolled proliferation.

The p38 MAPKs' family includes four isoforms, of which only p38α has been considered essential for numerous important processes including mice embryogenesis. It is also considered essential for myoblast to myotube differentiation, as exposure of myoblasts to p38α/β inhibitors or to siRNA that targets p38α suppresses the process. The functions of p38β and p38γ in myoblast differentiation are not clear. We knocked out p38α in C2C12 myoblasts, assuming that the resulting C2p38α-/- cells would not differentiate. They did, however, form mature fibers. We found elevated levels and activation of the p38 activator MKK6 in the C2p38α-/- cells, leading to activation of p38β and p38γ, which are not active in differentiating parental C2C12 cells. Thus, p38α is an inhibitor of p38β+p38γ, that perhaps replace it in promoting differentiation. To test this notion, we generated C2p38α/γ-/- and C2p38α/β-/- cells and found that in both clones, the myogenic program was induced. C2p38β/γ-/- cells also formed myotubes. These observations could be interpreted in two ways: either each p38 isoform can promote, by itself, the myogenic program, or p38 activity is not required at all for the process. Generating C2p38α/β/γ-/- cells in which the myogenic program was shut-off altogether, showed that p38 activity is critical for differentiation. Notably, C2p38α/β/γ-/- cells proliferate uncontrollably and give rise to foci, reminiscence of oncogenically-transformed cells. In summary, our study shows that a crosstalk between p38 isoforms functions in C2C12 cells as a safeguard mechanism that ensures resilience of the p38 activity in promoting the myogenic program and enforcing cell cycle arrest.

A short ncRNA modulates gene expression and affects stress response and parasite differentiation in Leishmania braziliensis

The protozoan parasite Leishmania spp. is a causative agent of leishmaniasis, a disease that affects millions of people in more than 80 countries worldwide. Apart from its medical relevance, this organism has a genetic organization that is unique among eukaryotes. Studies of the mechanisms regulating gene expression in Leishmania led us to investigate noncoding RNAs (ncRNAs) as regulatory elements. We previously identified differentially expressed (DE) ncRNAs in Leishmania braziliensis with potential roles in the parasite biology and development. Herein, we present a functional analysis of one such DE ncRNA, the 147-nucleotide-long transcript ncRNA97, which is preferentially expressed in amastigotes, the replicative form within mammalian phagocytes. By RT-qPCR the ncRNA97 was detected in greater quantities in the nucleus under physiological conditions and in the cytoplasm under nutritional stress. Interestingly, the transcript is protected at the 5’ end but is not processed by the canonical trypanosomatid trans-splicing mechanism, according to the RNA circularization assay. ncRNA97 knockout (KO) and addback (AB) transfectants were generated and subjected to phenotypic analysis, which revealed that the lack of ncRNA97 impairs the starvation response and differentiation to the infective form. Comparative transcriptomics of ncRNA97KO and parental cells revealed that transcripts encoding amastigote-specific proteins were affected. This pioneering work demonstrates that ncRNAs contribute to the developmental regulatory mechanisms of Leishmania.

Cancer stem cell populations are resistant to 5-aminolevulinic acid-photodynamic therapy (5-ALA-PDT)

Photodynamic therapy (PDT) is a minimally invasive treatment approved for many types of cancers. PDT involves the administration of photoactive substances called photosensitizers (PS) that selectively accumulate in cancer cells and are subsequently excited/activated by irradiation with light at wavelengths of optimal absorbance. Activated PS leads to the generation of singlet oxygen and other reactive oxygen species (ROS), promoting cancer cell death. 5-aminolevulinic acid (5-ALA) is a naturally occurring PS precursor, which is metabolically converted to the PS, protoporphyrin IX (PPIX). Although 5-ALA-PDT is effective at killing cancer cells, in prior studies conducted by our group we normally observed in in vitro experiments that approximately 5–10% of cells survive 5-ALA-PDT, which served as an impetus for further investigation. Identifying the mechanisms of resistance to 5-ALA-PDT-mediated cell death is important to prevent tumor recurrence following 5-ALA-PDT. Previously, we reported that oncogenic activation of Ras/MEK promotes PPIX efflux and reduces cellular sensitivity to 5-ALA-PDT through increased expression of ABCB1 transporter. As cancer stem cells (CSCs) are known to drive resistance to other cancer treatments and have high efflux of chemotherapeutic agents via ABC-family transporters, we hypothesize that CSCs underlie 5-ALA-PDT resistance. In this study, we determined (1) if CSCs are resistant to 5-ALA-PDT and (2) if CSCs play roles in establishing resistant populations of 5-ALA-PDT. When we compared CSC populations before and after 5-ALA-PDT, we found that CSCs were less susceptible to 5-ALA-PDT. Moreover, we found that the CSC population was enriched in 5-ALA-PDT-resistant cell lines compared to the parental cell line. Our results indicate that CSCs are not sensitive to 5-ALA-PDT, which may contribute to establishment of 5-ALA-PDT resistance.

CD19.CAR-T cell-derived extracellular vesicles express CAR and kill leukemic cells, contributing to antineoplastic therapy.

Chimeric Antigen Receptor (CAR) T cell-derived Extracellular Vesicles (EVs) might represent a new therapeutic tool for boosting CAR-T cell antileukemic effects. Here, a cohort of 22 patients infused with CD19.CAR-T cells was monitored for the presence of circulating CD19.CAR+-T cell-derived EVs (CD19.CAR+EVs), which were then separated and functionally characterized for their killing abilities. A GMP-compliant separation method was also developed. Results demonstrated that CD19.CAR+EVs were detectable in peripheral blood up to 2 years after infusion indicating long-lasting persistence of their parental cells. Notably, early decreases of circulating CD19.CAR+EVs concentrations correlated with failure of CAR-T therapy. Circulating CD19.CAR+EVs displayed a median size (SD) of 133.1±65.5 nm and carried a pro-apoptotic protein cargo. These EVs expressed higher CAR levels than their parental cells. Furthermore, CD19.CAR+EVs did not activate heterologous T cells and produced significant, specific and dose-dependent cytotoxic effects on CD19+ cell lines and on primary cells. The new GMP-compliant EV isolation method allowed a recovery of 63±5.7 % of CD19.CAR+EVs. A deeper analysis of the different protein cargoes carried by EVs derived from different CAR-T cell subpopulations identified a pro-apoptotic functional pathway linked to CD8+LAG-3+ EVs. Overall, our data indicate that CD19.CAR+EVs may be proposed as promising dynamic new biomarkers of CAR-T cell activity and, contributing to the direct killing of the leukemic target, represent a new product with a strong therapeutic potential that could be infused independently from CAR-T cells.

A novel acquired resistance mechanism to 5-aminolevulinic acid-mediated photodynamic therapy with ABCG2 inhibition.

We report the occurrence of acquired tumor cell resistance to 5-aminolevulinic acid (ALA)-mediated photodynamic therapy (PDT) in combination with ABCG2 inhibition. ALA-PDT in combination with either an ABCG2 tool inhibitor Ko143 or a repurposed clinically-relevant ABCG2 inhibitor lapatinib was highly effective in eradicating the H4 human glioma cells, resulting in minimal cell survival after treatment. However, after seven rounds of repeated treatments with light dose escalation, the resultant tumor cells became resistant to the combination therapy. The resistant sublines and the parental cell line showed similar ABCG2 activities and protein levels, indicating that it was not ABCG2 that caused the resistance. They also exhibited similar responses to PpIX-PDT and mTOR inhibitor AZD2014, suggesting that alterations in PDT sensitivity and mTOR pathway had little contribution to the development of resistance phenotype. By determining the intracellular and extracellular PpIX levels, the activities and protein levels of heme biosynthesis enzymes, we found that porphobilinogen deaminase (PBGD) activity and protein level were significantly reduced in the resistant sublines, causing resistance to PDT by substantially reducing PpIX biosynthesis. A novel acquired resistance mechanism to ALA-PDT with ABCG2 inhibition has been uncovered.

Bcl‑xL‑specific BH3 mimetic A‑1331852 suppresses proliferation of fluorouracil‑resistant colorectal cancer cells by inducing apoptosis.

BH3 mimetics are small‑molecule inhibitors of the antiapoptotic Bcl‑2 family and have therapeutic efficacy against hematological malignancies. BH3 mimetic A‑1331852 suppresses colorectal cancer cell proliferation. Progressive resistance to the widely used anticancer agent fluorouracil (5‑FU) is a key reason for colorectal cancer recurrence; therefore, the present study tested if A‑1331852 can suppress the proliferation of 5‑FU‑resistant colorectal cancer cells. A 5‑FU‑resistant colorectal cancer cell line was derived from HCT116 cells and compared with the parental line. Expression levels of the antiapoptotic Bcl‑2 proteins Bcl‑xL and myeloid cell leukemia 1 (Mcl‑1) were determined via western blotting, proliferation in the presence of 5‑FU and following small interfering (si)RNA‑mediated Bcl‑xL or Mcl‑1 knockdown was assessed by WST‑1 assay and sensitivity to A‑1331852‑induced apoptosis was assessed via western blotting and DNA fragmentation assay. In addition, a xenograft mouse model of 5‑FU‑resistant colorectal cancer was established via subcutaneous inoculation of 5‑FU‑resistant HCT116 cells to examine the in vivo antitumor efficacy of A‑1331852. Compared with the parental line, 5‑FU‑resistant cells overexpressed Bcl‑xL. Knockdown of Bcl‑xL by siRNA and treatment with A‑1331852 suppressed proliferation and induced the apoptosis of both 5‑FU‑resistant and parental HCT116 cells, but the potency of both effects was stronger in 5‑FU‑resistant than parental HCT116 cells. Furthermore, A‑1331852 suppressed the growth of xenograft tumors derived from 5‑FU‑resistant cells by inducing apoptosis. Overall, the present findings suggested that Bcl‑xL upregulation contributes to 5‑FU resistance of colorectal cancer and targeted inhibition by A‑1331852 may be an effective treatment strategy.

Cabergoline-induced NDFIP1 upregulation in pituitary neuroendocrine tumor cells activates mTOR signaling and contributes to cabergoline resistance.

To investigate the molecular mechanisms underlying resistance to dopamine agonists (DA). LC-MS/MS analysis was performed on rat pituitary neuroendocrine tumors (PitNET) cell line GH3 to identify differentially expressed proteins induced by cabergoline (CAB) treatment. A total of 180 human PITNET samples were subjected to transcriptome analysis. Immunohistochemistry (IHC) was conducted on 29 tumor samples to validate NDFIP1 alteration. A xenograft mouse model was established by subcutaneously injecting GH3 cells, with or without NDFIP1 overexpression, into nude mice to investigate tumor growth. PitNET cell lines were treated with CAB. Cell proliferation was assessed using the CCK-8, and protein expression levels were examined through Western blot analysis. CAB treatment upregulated FDFT1 and NDFIP1 protein expression in GH3 cells, with NDFIP1 showing a significant positive correlation with tumor size, as confirmed by IHC results. MMQ and GH3 cells overexpressing NDFIP1 exhibited enhanced viability and reduced sensitivity to CAB. In vivo experiments demonstrated that subcutaneous injection of NDFIP1-overexpressing GH3 cells led to enhanced tumor growth compared to parental GH3 cells. Although the total levels of PTEN remained unaltered, NDFIP1 overexpression induced PTEN nuclear translocation, potentially activating the mTOR pathway. This was supported by increased phosphorylation of key mTOR pathway components, including p-AKT and p-4EBP1, in cells overexpressing NDFIP1. CAB treatment induces the upregulation of NDFIP1 in PitNET cells, which correlates with tumor size and contributes to reduced CAB sensitivity, potentially through activation of the mTOR pathway. NDFIP1 as a potential therapeutic target for overcoming DA resistance in PitNET patients.

Bioorthogonal click chemistry and aptamer-targeting enables highly selective fluorescence labeling of exosomal glycosylated EpCAM for super resolved imaging.

Exosomes, which are known to transport diverse proteins from parent cells to recipient cells, consequently influence the biological activities of the recipient cells. Among those proteins, the epithelial cell adhesion molecule (EpCAM), plays a crucial role as it is implicated in cell adhesion and signaling processes. As exosomal EpCAM potentially affects the migration of recipient cells, direct visualization with high spatial resolution is essential to better understand this impact and the role of exosomal EpCAM in recipient cells. Such understanding may provide valuable insights into the mechanisms underlying various diseases and potential treatment strategies. (94) RESULTS: This work focus on the selective labeling and fluorescent imaging of glycosylated EpCAM on tumor-derived exosomes using bioorthogonal click chemistry and aptamer-targeting strategies. To commence, exosomes with EpCAM overexpression, EpCAM N-glycosylation mutation, EpCAM silencing, or wildtype, were obtained by genetic manipulation. Subsequently, the glycosylation of exosomal EpCAM was directly visualized by capitalizing on the intramolecular fluorescence resonance energy transfer (FRET) that takes place between fluorescent EpCAM aptamers and fluorescent tags bound to glycans. As a result, this approach demonstrated its efficacy in investigating both the existence and the glycosylation state of exosomal EpCAM. Importantly, we proceeded to observe the uptake of tumor-derived exosomes by their recipient cells. It was then remarkably found that the expression and glycosylation levels of EpCAM in the co-cultured exosomes have a significant and substantial impact on the migratory ability of the recipient immune cells. (139) SIGNIFICANCE: We set up a novel labeling strategy for exosomal glycosylated EpCAM. This approach enabled us to realize the direct observation of exosomal EpCAM and its glycosylation with high spatial resolution. Based on this method, we find a significant role that the expression and the glycosylation of exosomal EpCAM in recipient cell adhesion. (52).

CRISPR editing of candidate host factors that impact influenza A virus infection.

Influenza A virus (IAV) is a respiratory pathogen with a segmented negative-sense RNA genome that can cause epidemics and pandemics. The host factors required for the complete IAV infectious cycle have not been fully identified. Here, we examined three host factors for their contributions to IAV infectivity. We performed CRISPR-mediated knockout of cytidine monophosphate N-acetylneuraminic acid synthetase (CMAS) as well as CRISPR-mediated overexpression of beta-1,4 N-acetylgalactosaminyltransferase 2 (B4GALNT2) and adenosine deaminase acting on RNA 1 (ADAR1) in the human bronchial epithelial A549 cell line and evaluated the impact on IAV and other RNA viruses. We confirmed that knockout of CMAS or overexpression of B4GALNT2 restricts IAV infection by diminishing binding to the cell surface but has no effect on vesicular stomatitis virus infection. Although ADAR1 overexpression does not significantly inhibit IAV replication, it has a pro-viral effect with coxsackie B virus (CVB) infection. This pro-viral effect is not likely secondary to reduced type I interferon (IFN) production, as the induction of the IFN-stimulated genes ISG15 and CXCL10 is negligible in both parent and ADAR1-overexpressing A549 cells following CVB challenge. In contrast, ISG15 and CXCL10 production is robust and equal for parent and ADAR1-overexpressing A549 cells challenged with IAV. Taken together, these data provide insights into how host factors can be further explored to understand the dynamics of pro- and anti-viral factors.IMPORTANCEInfluenza A virus (IAV) remains a global threat due to its ability to cause pandemics, making the identification of host factors essential for developing new antiviral strategies. In this study, we utilized CRISPR-based techniques to investigate host factors that impact IAV infectivity. Knockout of CMAS, a key enzyme in sialic acid biosynthesis, significantly reduced IAV binding and infection by disrupting sialic acid production on the cell surface. Overexpression of B4GALNT2 had similar effects, conferring resistance to IAV infection through diminished cell-surface binding. Overexpression of ADAR1, known for its role in RNA editing and immune regulation, impacted IAV replication minimally but enhanced coxsackie B virus replication. Such findings reveal the diverse roles of host factors in viral infection, offering insights for targeted therapeutic development against IAV and other pathogens.

Application of a CYP1B1-Targeted NIR Probe for Breast Cancer Diagnosis, Surgical Navigation, and CYP1B1-Associated Chemotherapy Resistance Monitoring.

Early detection and precise treatment for breast cancer are crucial, given its high global incidence rate. Hence, the development of novel imaging targets is essential for diagnosing and monitoring resistance to chemotherapy, which is pivotal for achieving precise and personalized treatment for breast cancer patients. In our previous work, we successfully developed a near-infrared (NIR) probe 1 for CYP1B1-targeted imaging. In this study, we aimed to investigate the utility of the probe as a NIR fluorescence and photoacoustic dual-mode imaging probe for the detection and surveillance of breast cancer. Western blotting of cancer cell lines has confirmed that CYP1B1 is widely expressed in breast cancer and gynecological cancer. In vitro NIR fluorescence imaging capability of the probe for tracking CYP1B1-positive tumor cells was validated by using confocal microscopy. Further studies, including in vivo fluorescence and photoacoustic dual-model imaging and ex vivo biological distribution analysis on a triple-negative breast cancer xenograft mouse model, demonstrated that the probe selectively accumulated in tumor tissue within as early as 0.5 h postinjection. The results of the surgical resection experiment revealed that the tumor could be entirely removed under the guidance of NIR imaging, thereby indicating the probe's efficacy in surgical navigation. CYP1B1 expression was found to be upregulated in adriamycin (ADR)-resistant breast cancer cells, MCF-7/ADR. Consequently, the sensitivity of CYP1B1 overexpressed cells, MCF-7/1B1, to ADR was significantly reduced, with an IC50 value of 0.586 ± 0.0934 μM, compared to the parental MCF-7 cells with an IC50 value of 0.183 ± 0.0444 μM. In vivo and ex vivo imaging assays conducted on MCF-7/ADR tumor-bearing mice demonstrated that the probe was specifically enriched in tumor sites, suggesting its potential for monitoring chemotherapy resistance in breast cancer. This study expands the scope of application for NIR probe 1, establishing its utility in breast cancer diagnosis through fluorescence-photoacoustic dual-model imaging, monitoring of chemotherapy resistance, and guidance for surgical resection. This strategy paves the way for novel approaches to precise and personalized treatment for breast cancer patients.

Epigenetic memory as crucial contributing factor in directing the differentiation of human iPSC into pancreatic β-cells in vitro.

Impaired insulin secretion contributes to the pathogenesis of type 1 diabetes mellitus through autoimmune destruction of pancreatic β-cells and the pathogenesis of severe forms of type 2 diabetes mellitus through β-cell dedifferentiation and other mechanisms. Replenishment of malfunctioning β-cells via islet transplantation has the potential to induce long-term glycemic control in the body. However, this treatment option cannot widely be implemented in clinical due to healthy islet donor shortage. Emerging β-cell replacement with human-induced pluripotent stem cell (iPSC) provides high remedial therapy hopes. Thus, tremendous progress has been made in developing β-cell differentiation protocols in vitro; however, most of the differentiated iPSC-derived β-cells showed immature phenotypes associated with low efficiency depending on the iPSC lines used, creating a crucial barrier for their clinical implementation. Multiple mechanisms including differences in genetic, cell cycle patterns, and mitochondrial dysfunction underlie the defective differentiation propensity of iPSC into insulin-producing β-cells. Accumulating evidence recently indicated that, following the reprogramming, epigenetic memory inherited from parental cells substantially affects the differentiation capacity of many iPSC lines. Therefore, differences in epigenetic signature are likely to be essential contributing factors influencing the propensity of iPSC differentiation. In this review, we will document the impact of the epigenome on the reprogramming efficacy and differentiation potential of iPSCs and how targeting the epigenetic residual memory could be an additional strategy to improve the differentiation efficiency of existing protocols to generate fully functional hPSC-derived pancreatic β-cells for diabetes therapy and drug screening.

Investigation into drug resistance to cisplatin in cancer stem cell-enriched population in non-small cell lung cancer

Abstract Objectives understanding drug resistance in cancer is of importance in treatment. Cancer stem cells are main factor for drug resistance. Therefore, the possible gene/gene interactions/proteins were explored in our study using a cancer stem cell-enriched population (H1299/S) derived from a parental non-small cell lung cancer cell line (H1299/P). Methods response to cisplatin, which is the main drug for the treatment of lung cancer, was evaluated with the Adenosine triphosphate (ATP) viability test. As a result of the gene expression analysis, while 14 genes were not evaluated, expression profiles were obtained for 37 genes out of 51 genes. By the drug-protein interaction analyses, Topoisomerase I (TOPI), Topoisomerase 2 alpha (TOP2A), Topoisomerase 2 beta (TOP2B), Cyclin-dependent kinases 4 (CDK4), Cyclin-dependent kinases 6 (CDK6), ATP binding cassette subfamily B member 1 (ABCB1), ATP binding cassette subfamily C member 1 (ABCC1), ATP binding cassette subfamily C member 3 (ABCC3), B-cell leukemia/lymphoma 2 (BCL2), Poly (ADP-ribose) polymerase 1 (PARP1), Breast cancer gene 1 (BRCA1) and Cyclin dependent kinase inhibitor 1A (CDKN1A) genes and protein products were statistically significantly found to be in association with drug resistance. Results and discussion in bioinformatics analyses, it was observed that 13 pathways were affected due to expression changes and 12 genes related to these pathways were determined to activate multidrug resistance mechanisms. Conclusions platinum-based drugs, as well as a broad range of other agents including topoisomerase and PARP1 inhibitors, and anthracyclines, have been shown to potentially possess multiple drug resistance.

P-1974. Surface Protein Profiling of Extracellular Vesicles: A Novel Biomarker Approach for Long COVID Diagnosis?

Abstract Background Extracellular vehicles (EVs) are lipid bilayer vesicles secreted by all cell types into the circulation. EVs carry an array of biomolecules indicative of their origin cells and act as cargo vesicles capable of delivering proteins, mitochondria, and viral particles. However, whether long COVID affects the profile of plasma EVs remains unknown. Methods Plasma samples were selected from the Stanford repositories of Long COVID participants—seventy-six plasma samples from patients with documented SARS-CoV-2 infection. Twenty-five participants had full recovery (10 months post-infection, Stanford Lambda Trial), 24 patients with Long COVID with CFS phenotype and 27 with ME/CFS from the Stanford Long COVID clinic. Plasma EVs were isolated using size-exclusion columns (qEV, Izon Science). The positivity for surface markers and mitochondrial membrane potentials of the plasma EVs were evaluated by flow cytometry. Samples were stained with pre-titrated volumes of the following fluorochrome-conjugated monoclonal antibodies: CD45 leukocytes, CD3/T cells, CD20/B cells, CD14/Monocytes, CD16/NK cells, CD144/endothelial cells, CD41a/Platelets, CD56/neural cell adhesion molecule (NCAM), and Mitotracker deep red/mitochondrial membrane potential. Results The total EV counts and the proportion of mitochondria-positive were decreased in Long COVID group compared to the COVID recovery group (5.02 x 106 vs. 1.92 x 107 /mL, and 42.7 vs. 56.7%, respectively). Compared with the recovery group, Monocyte and NK cell-derived EVs were significantly increased, whereas Endothelial cell-derived EVs were decreased in the Long COVID group. The EVs from the Long COVID group consistently exhibited lower positivity of mitochondrial membrane potential regardless of the parental cells. No difference was observed in plasma EVs derived from NCAM positive cells. NK cell proportion is the only difference between LC/CSF and CSF (P= 0.046). Conclusion Patients with Long COVID showed alterations in plasma EV profiles. These findings may offer insights into the pathogenesis of long COVID. Disclosures All Authors: No reported disclosures

Extracellular vesicles as drug and gene delivery vehicles in central nervous system diseases.

Extracellular vesicles (EVs) are secreted by almost all cell types and contain DNA, RNA, proteins, lipids and other metabolites. EVs were initially believed to be cellular waste but now recognized for their role in cell-to-cell communication. Later, EVs from immune cells were discovered to function similarly to their parent cells, paving the way for their use as gene and drug carriers. EVs from different cell types or biological fluids carry distinct cargo depending on their origin, and they perform diverse functions. For instance, EVs derived from stem cells possess pluripotent properties, reflecting the cargo from their parent cells. Over the past two decades, substantial preclinical and clinical research has explored EVs-mediated drug and gene delivery to various organs, including the brain. Natural or intrinsic EVs may be effective for certain applications, but as drug or gene carriers, they demonstrate broader and more efficient potential across various diseases. Here, we review research on using EVs to treat central nervous system (CNS) diseases, such as Alzheimer's Disease, Parkinson diseases, depression, anxiety, dementia, and acute ischemic strokes. We first reviewed the naïve EVs, especially mesenchymal stem cell (MSC) derived EVs in CNS diseases and summarized the clinical trials of EVs in treating CNS diseases and highlighted the reports of two complete trials. Then, we overviewed the preclinical research of EVs as drug and gene delivery vehicles in CNS disease models, including the most recent two years' progress and discussed the mechanisms and new methods of engineered EVs for targeting CNS. Finally, we discussed challenges and future directions and of EVs as personalized medicine for CNS diseases.

Hyper-energy metabolism of oxidative phosphorylation and enhanced glycolysis contributes to radioresistance in glioma cells

The concept of dual-state hyper-energy metabolism characterized by elevated glycolysis and OxPhos has gained considerable attention during tumor growth and metastasis in different malignancies. However, it is largely unknown how such metabolic phenotypes influence the radiation response in aggressive cancers. Therefore, the present study aimed to investigate the impact of hyper-energy metabolism (increased glycolysis and OxPhos) on the radiation response of a human glioma cell line. Modulation of the mitochondrial electron transport chain was carried out using a 2,4-dinitrophenol (DNP). Metabolic characterization was carried out by assessing glucose uptake, lactate production, mitochondrial mass, membrane potential, and ATP production. The radiation response was examined by cell growth, clonogenic survival, and cell death assays. Macromolecular oxidation was assessed by DNA damage, lipid peroxidation, and protein carbonylation assay. Hypermetabolic OPM-BMG cells exhibited a significant increase in glycolysis and OxPhos following irradiation as compared to the parental BMG-1 cells. Enhanced radioresistance of OPM-BMG cells was evidenced by the increase in α/β ratio (9.58) and D1 dose (4.18 Gy) as compared to 4.36 and 2.19 Gy in BMG-1 cells respectively. Moreover, OPM-BMG cells were found to exhibit increased resistance against radiation-induced cell death, and macromolecular oxidation as compared to BMG-1 cells. Inhibition of glycolysis and mitochondrial complex-II significantly enhanced the radiosensitivity of OPM-BMG cells compared to BMG-1 cells. Our results demonstrate that the hyper-energy metabolism of increased glycolysis and OxPhos confer radioresistance. Consequently targeting glycolysis and OxPhos in combination with radiation may overcome therapeutic resistance in aggressive cancers like glioma.

Polo-like Kinase 1 Inhibitors Demonstrate In Vitro and In Vivo Efficacy in Preclinical Models of Small Cell Lung Cancer

Objective: To investigate the preclinical efficacy and identify predictive biomarkers of polo-like kinase 1 (PLK1) inhibitors in small cell lung cancer (SCLC) models. Methods: We tested the cytotoxicity of selective PLK1 inhibitors (rigosertib, volasertib, and onvansertib) in a panel of SCLC cell lines. We confirmed the therapeutic efficacy of subcutaneous xenografts of representative cell lines and in four patient-derived xenograft models generated from patients with platinum-sensitive and platinum-resistant SCLC. We employed an integrated analysis of genomic and transcriptomic sequencing data to identify potential biomarkers of the activity and mechanisms of resistance in laboratory-derived resistance models. Results: Volasertib, rigosertib, and onvansertib showed strong in vitro cytotoxicity at nanomolar concentrations in human SCLC cell lines. Rigosertib, volasertib, and onvansertib showed equivalent efficacy to that of standard care agents (irinotecan and cisplatin) in vivo with significant growth inhibition superior to cisplatin in PDX models of platinum-sensitive and platinum-resistant SCLC. There was an association between YAP1 expression and disruptive or inactivation TP53 gene mutations, with greater efficacy of PLK1 inhibitors. Comparison of lab-derived onvansertib-resistant H526 cells to parental cells revealed differential gene expression with upregulation of NAP1L3, CYP7B1, AKAP7, and FOXG1 and downregulation of RPS4Y1, KDM5D, USP9Y, and EIF1AY highlighting the potential mechanisms of resistance in the clinical setting. Conclusions: We established the efficacy of PLK1 inhibitors in vitro and in vivo using PDX models of platinum-sensitive and resistant relapsed SCLC. An ongoing phase II trial is currently testing the efficacy of onvansertib in patients with SCLC (NCT05450965).

Size-Dependent Separation of Extracellular Vesicle Subtypes with Exodisc Enabling Proteomic Analysis in Prostate Cancer.

Extracellular vesicles (EVs) are emerging as crucial biomarkers in cancer diagnostics and therapeutics with their heterogeneity presenting both challenges and opportunities in prostate cancer research. However, existing methods for isolating and characterizing EV subtypes have been limited by inefficient separation and inadequate proteomic analysis. Here we show an optimized centrifugal microfluidic device, Exodisc, that efficiently isolates large quantities of EV subtypes from particle-enriched medium, enabling comprehensive proteomic analysis of small (EV-S, 20-200 nm) and large (EV-L, >200 nm) EVs. Using this device, we successfully separated EV-S and EV-L from prostate cancer cell lines LNCaP and PC3. Mass spectrometry-based proteomics revealed that EV proteins reflect parental cell characteristics more than EV size, with EV-L demonstrating increased expression of PSMA-correlated proteins. Our optimized protocol addresses challenges in EV isolation and characterization, providing a more effective method for studying cellular and molecular mechanisms of specific EV subtypes. This study extends the potential use of EVs as a liquid biopsy for cancer theranostics, paving the way for more precise isolation of EV subtypes and potentially leading to improved biomarker discovery and the development of personalized treatments.

Abstract P006: Partial tumor volume irradiation by 177Lu-PNT6555 induces diverse immune responses in a syngeneic murine tumor model

Abstract Purpose/Objective: Radiopharmaceutical therapy (RPT) enables the systemic delivery of an inherently heterogeneous dose of radiation. The RPT dose heterogeneity is created by a tumor’s non-uniform perfusion and the variation in RPT target expression. Radiation therapy is known to immunomodulate the tumor microenvironment (TME) in a dose-dependent manner. The hypothesis is that RPT with a heterogeneous distribution, delivering a low to high dose gradient in a single TME, will activate multiple dose dependent immune effects that are more effective in promoting priming of adaptive immunity compared to a more homogenous distribution of RPT. Methods: RPT was administered using PNT6555, a fibroblast activation protein alpha targeting agent, and uptake was confirmed with B16 murine melanoma. The B16 cell line is engineered to overexpress mouse fibroblast activation protein alpha (mFAP). The engineered B16 was implanted either alone to form a tumor with 100% mFAP expression (B16-mFAP+) or in a mixture with the parental cell line to create a heterogeneous expression of mFAP (mixed B16-mFAP+/B16-mFAP-). Utilizing the SPECT/CT data and ex vivo biodistribution, image-based dosimetry for 177Lu-PNT6555 was performed using the Monte-Carlo-based RAPID platform. C57BL/6 mice bearing B16-mFAP+ or mixed B16-mFAP+/mFAP- tumors were treated with non-radiolabeled PNT6555, 5, 10, 20 or 40 Gy on day 0. Tumors were harvested on day 7 post-treatment. RT-qPCR was performed to evaluate the type I interferon response and tumor immune susceptibility markers. Mice bearing B16-mFAP+ or mixed B16-mFAP+/mFAP- received a mean tumor dose of 5 Gy 177Lu-PNT6555. Mice were monitored for tumor growth and overall survival. In a separate cohort, tumors and tumor draining lymph nodes (TDLN) were harvested at days 4, 7, and 12 post-injection. Flow cytometry was performed on the tumor to evaluate immune infiltration and TDLN to evaluate dendritic cell and B cell activation. Statistical significance was determined by a one-way ANOVA (flow) or Kaplan-Meier with log-rank testing (therapy). Results: Optimal upregulation of immune-related genes by 177Lu-PNT6555 occurred at 2.5 Gy for Icam, 10 Gy for Ifnβ1, and 20 Gy for H2-K1 (murine gene coding MHCI). A heterogeneous delivery of 5 Gy to mice bearing mixed B16-mFAP+/mFAP- resulted in upregulation of all three genes in a single TME. For the therapy study, a heterogeneous distribution of 5 Gy 177Lu-PNT6555 significantly increased overall survival compared to a homogenous distribution. Flow cytometry revealed a significant increase in dendritic cell activation by CD80 and MHCII expression in the 5 Gy 177Lu-PNT6555 with heterogeneous distribution. Additionally, there was a significant increase in infiltration of CD4+ cells, CD8+ cells, NK cells and B cells as well as preservation of CD8+ cell in the 5 Gy 177Lu-PNT6555 with heterogeneous distribution. Conclusions: These preclinical studies demonstrate the capacity of a heterogeneous dose of RPT to enhance the immunomodulatory effect of RPT and improve overall survival in a monotherapy setting. Citation Format: Maya E. Takashima, Ohyun Kwon, Shin Hye Ahn, Anthony Belanger, Won Jong Jin, Charlotte Sawicki, Paul A. Clark, Tessa Chen, Ria Kumari, Bryan Bednarz, Zachary S. Morris. Partial tumor volume irradiation by 177Lu-PNT6555 induces diverse immune responses in a syngeneic murine tumor model.[abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Translating Targeted Therapies in Combination with Radiotherapy; 2025 Jan 26-29; San Diego, CA. Philadelphia (PA): AACR; Clin Cancer Res 2025;31(2_Suppl):Abstract nr P006

Exosome: an overview on enhanced biogenesis by small molecules.

Exosomes are extracellular vesicles that received attention for their potential use in the treatment of various injuries. They communicate intercellularly by transferring genetic and bioactive molecules from parent cells. Although exosomes hold immense promise for treating neurodegenerative and oncological diseases, their actual clinical use is very limited because of their biogenesis and secretion. Recent studies have shown that small molecules can significantly enhance exosome biogenesis, thereby remarkably improving yield, functionality, and therapeutic effects. These molecules modulate critical pathways toward optimum exosome production in a mode that is either ESCRT dependent or ESCRT independent. Improved exosome biogenesis may provide new avenues for targeted cancer therapy, neuroprotection in neurodegenerative diseases, and regenerative medicine in wound healing. This review explores the role of small molecules in enhancing exosome biogenesis and secretion, highlights their underlying mechanisms, and discusses emerging clinical applications. By addressing current challenges and focusing on translational opportunities, this study provides a foundation for advancing cell-free therapies in regenerative medicine and beyond.