Distant Cell Research Articles

Radiation Therapy Improves Survival in Patients with Distant Metastatic Head and Neck Squamous Cell Carcinoma: A Retrospective Study.

The role of radiation therapy in patients with distant metastatic squamous cell carcinoma of the head and neck (mHNSCC) is unclear. In this study, we compare the differences in survival among mHNSCC patients treated with chemotherapy plus radiotherapy (RT) vs. chemotherapy alone. This study included patients with distant mHNSCC recruited from 2 cohorts: The Surveillance, Epidemiology, and End Results (SEER [N=885]) database and a Chinese single-institution registry in Sun Yat-sen University Cancer Center (SYSUCC [N=60]). The SEER cohort included 600 patients received RT plus chemotherapy and 285 patients received chemotherapy alone; in the SYSUCC cohort, 40 patients received RT plus chemotherapy and 20 patients received chemotherapy alone were recruited. The period of data collection for the SEER study was from January 2010 to December 2015, and that for SYSUCC was from January 2010 to December 2020. The study's primary outcome was overall survival (OS), with disease-specific survival (DSS) as a secondary outcome. Of the 885 patients in the SEER cohort, the addition of RT to chemotherapy increased one-year OS from 46.6% to 56.0% compared with chemotherapy alone (P =0.009) and from 10.4% to 25.1% for three-year OS (P <0.001). Patients who received RT in addition to chemotherapy were also more likely to have better three-year DSS than those who received chemotherapy alone (28.9% vs. 14.0%, P<0.001). Similarly, in the SYSUCC cohort, patients who received chemotherapy plus RT had better three-year OS than chemotherapy alone (62.8% vs. 21.0%, P=0.003). The addition of RT to chemotherapy increased median OS among patients with mHNSCC from 10 months to 13 months in the SEER cohort and from 14 months to 29 months in the SYSUCC cohort. Radiotherapy in addition to chemotherapy significantly improved OS and DSS in patients with mHNSCC.

DDEL-10. MICROBUBBLE-ENHANCED FOCUSED ULTRASOUND REGULATES THE ENRICHMENT OF INFLAMMATORY PATHWAYS IN THE BLOOD-BRAIN BARRIER AND IMPROVES CAR T CELL ACCUMULATION IN GLIOMAS

Abstract A major challenge to brain cancer immunotherapy is posed by the rate-limiting obstacles to the systemic delivery of cytotoxic T cells, such as chimeric antigen receptor (CAR) T cells, in brain tumors posed by the blood brain barrier (BBB). Although the BBB can be bypassed by direct infusion of CAR-T cells in brain tumors, these approaches offer only short-term benefits, since they cannot affect distant tumor cell infiltrations, as evidenced by tumor relapse. Circulating microbubbles (MB) upon ultrasound (US) exposure (sonication) can exert mechanical stress in brain vessels to trigger a range of responses, including local increase in the BBB permeability and activation of inflammatory signaling and phenotypes. We hypothesize that ultrasound frequency tuned at MB resonance inside brain capillaries can transiently modulate the BBB signaling and function to improve CAR-T cell trafficking in glioma tumors. RNAseq and immunostaining of protein expression reveal that at frequencies just above microbubble resonance inside vessels (1.5MHz for 2µm MB), where maximum vessel wall shear stress is observed, there is a marked enrichment of inflammatory markers in the endothelial cells of brain vessels, including P-selectin and ICAM-1. Consistent with these findings, we observed that under high frequency excitation (1.5MHz), there is a 5-fold increase in the accumulation of EGFRvIII CAR-T cells in orthotopic EGFRvIII murine glioma tumors as compared not sonicated controls. We also found that over 85% of the tumor-infiltrating CAR-T cells express PSGL-1 and LFA-1 receptors, which interact with P-selectin and ICAM-1 to facilitate T cell extravasation. Together, these findings demonstrate that tuning the US frequency can improve CAR-T cell trafficking, potentially through upregulation of cell adhesion molecules in the glioma tumor microenvironment, and further support the functional relevance and potential clinical significance of the observed immuno-mechano-biological changes.

Metabolic Profiling as an Approach to Differentiate T-Cell Acute Lymphoblastic Leukemia Cell Lines Belonging to the Same Genetic Subgroup.

T-cell acute lymphoblastic leukemia (T-ALL) is an aggressive tumor mainly affecting children and adolescents. It is driven by multiple genetic mutations that together define the leukemic phenotype. Interestingly, based on genetic alterations and/or deregulated expression, at least six genetic subgroups have been recognized. The TAL/LMO subgroup is one of the most represented genetic subgroups, characterizing 30-45% of pediatric T-ALL cases. The study of lipid and metabolic profiles is increasingly recognized as a valuable tool for comprehending the development and progression of tumors. In this study, metabolic and lipidomic analysis via LC/MS have been carried out on four T-ALL cell lines belonging to the TAL/LMO subgroup (Jurkat, Molt-4, Molt-16, and CCRF-CEM) to identify new potential metabolic biomarkers and to provide a subclassification of T-ALL cell lines belonging to the same subgroup. A total of 343 metabolites were annotated, including 126 polar metabolites and 217 lipid molecules. The statistical analysis, for both metabolic and lipid profiles, shows significant differences and similarities among the four cell lines. The Molt-4 cell line is the most distant cell line and CCRF-CEM shows a high activity in specific pathways when compared to the other cell lines, while Molt-16 and Jurkat show a similar metabolic profile. Additionally, this study highlighted the pathways that differ in each cell line and the possible enzymes involved using bioinformatic tools, capable of predicting the pathways involved by studying the differences in the metabolic profiles. This experiment offers an approach to differentiate T-ALL cell lines and could open the way to verify and confirm the obtained results directly in patients.

Surgery Adherence and Survival Outcomes in Patients With Locally Advanced (IIA-IVA) Cancer of the Lower Esophagus: A SEER Database-Based Study.

Esophageal cancer is a prevalent malignant tumor of the upper gastrointestinal tract. The aim of this study was to examine the impact of surgical adherence on the prognosis of patients with locally advanced lower esophageal cancer and to analyze the factors that affect surgical adherence. Patients diagnosed with locally advanced (IIA-IVA) lower esophageal cancer between 2004 and 2015 were screened from the Surveillance, Epidemiology, and End Results (SEER) database. We utilized multifactorial logistic regression to analyze the correlates affecting surgical adherence. Furthermore, we employed Kaplan-Meier curves and Cox regression to determine the impact of surgical adherence on cancer-specific survival (CSS) and overall survival (OS). Of the 4922 patients screened, 2372 individuals were advised to undergo surgery, out of which 2025 ultimately underwent the procedure while the remaining 347 refused. Lower surgical adherence was associated with older age, unmarried, SEER classification of "distant," and squamous cell carcinoma. Adherence to surgery proved to be an independent factor affecting OS and CSS. The Cox regression analysis showed that patients who refused surgery had lower OS (OR: 1.657; 95% CI: 1.429∼1.927; P < .001) and CSS (OR: 1.487; 95% CI: 1.309∼1.690; P < .001) than those who underwent surgery. Kaplan-Meier curves showed that patients who underwent surgical treatment had a better prognosis. Good surgical adherence can improve the prognosis of patients with locally advanced (IIA-IVA) lower esophageal cancer, while poor surgical adherence is associated with older age, unmarried, SEER classification of "distant," and squamous cell carcinoma.

Connexin-Containing Vesicles for Drug Delivery.

Connexin is a transmembrane protein present on the cell membrane of most cell types. Connexins assemble into a hexameric hemichannel known as connexon that pairs with another hemichannel present on a neighboring cell to form gap junction that acts as a channel or pore for the transport of ions and small molecules between the cytoplasm of the two cells. Extracellular vesicles released from connexin-expressing cells could carry connexin hemichannels on their surface and couple with another connexin hemichannel on a distant recipient cell to allow the transfer of the intravesicular content directly into the cytoplasm. Connexin-containing vesicles can be potentially utilized for intracellular drug delivery. In this review, we introduced cell-derived, connexin-containing extracellular vesicles and cell-free connexin-containing liposomes, methods of preparing them, procedures to load cargos in them, factors regulating the connexin hemichannel activity, (potential) applications of connexin-containing vesicles in drug delivery, and finally the challenges and future directions in realizing the promises of this platform delivery system for (intracellular) drug delivery.

Abstract C056: Characterization of prostate cancer secretomes for therapeutic intervention

Abstract Introduction: Prostate cancer (PC) is a common malignancy among men, often driven by dysregulated androgen receptor (AR) signaling. Earlier stages of PC can be favorably treated with androgen deprivation therapies or AR signaling inhibitors. Unfortunately, metastatic PC (mPC) is currently incurable. Neuroendocrine prostate cancer (NEPC) is a subgroup of mPC that may be derived as result of transdifferentiation of adenocarcinomas, after AR-directed treatment. Previous studies using preclinical models have suggested a potentially beneficial relationship between NEPC and AR-active prostate cancer (ARPC) cells. We hypothesize that secreted factors derived from NEPC cells influence local, and distant ARPC cell types to promote treatment resistance. Methods: Five NEPC in vitro cell models: NCI-H660, MSKCC-EF1, LTL331R, LuCaP-49 and LuCaP-173.1 were maintained in serum free media for 24 hours. The conditioned media (CM) was then collected and admixed with growth medium for ARPC cells. The ARPC growth medium was either supplemented with Fetal Bovine Serum (FBS) or Charcoal Stripped fetal bovine Serum (CSS). GFP-labelled ARPC cells: LnCaP and C42B were seeded in the respective media admixed with the CM. The viability of cells was measured using a Cytation5 microplate reader. We implemented a transcriptomic (RNAseq) and mass-spectrometry based proteomic analysis to identify differentially expressing secretory proteins that could have an impact on the treatment resistance of ARPC cell types. Results: All 5 NEPC-CM showed qualitative growth promoting effects towards ARPC C42B cells grown in CSS supplemented media. Only CM from EF1 and H660 promoted the growth of ARPC C42B in FBS supplemented media. NEPC-CM did not affect growth of LnCaP ARPC cells. Mass-spectrometry for secreted proteins was performed on 3 NEPC cell models. Analysis of the proteins against the PANTHER gene ontology database shows: (i) cell communication, and (ii) cellular response to stimulus, as two important biological processes active in NEPC. Genes involved in the two pathways were further compared against a comprehensive transcriptomic dataset spanning distinct mPC phenotypes and genotypes. Neurotrophic growth factors: Brain-derived growth factor (BDNF), Nerve-growth factor (NGF), VGF and neurotrophin-3 (NT-3) emerged as important targets that were differentially expressed in NEPC versus ARPC cells. Conclusions: NEPC cells have the potential to influence ARPC phenotypes that include enhanced proliferation, AR targeting, and potentially other drug resistance programs. As mPC exhibits substantial intra-individual and intra-tumor heterogeneity, understanding a targeting heterotypic cell interactions could lead to new druggable targets and address treatment resistance resulting from complex inter-cellular interactions. (Funding: PC220408) Citation Format: Tarana Arman, Lisa A. Jones, Ilsa M. Coleman, Philip R. Gafken, Peter S. Nelson. Characterization of prostate cancer secretomes for therapeutic intervention [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2023 Oct 11-15; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2023;22(12 Suppl):Abstract nr C056.

Non-Surgical Therapeutic Strategies for Non-Melanoma Skin Cancers.

Non-melanoma skin cancer (NMSC) is a globally prevalent skin disease, with basal cell carcinoma and squamous cell carcinoma accounting for 99% of NMSC cases. While surgical excision is the most common approach, numerous non-surgical therapies have rapidly advanced in recent years. In cases of low-risk NMSC, alongside surgical excision, priority should be given to physical therapy and photodynamic therapy. Physical therapy modalities, exemplified by electrodessication and curettage, emerge as safe and efficacious alternatives. In juxtaposition, photodynamic therapy, albeit relatively more costly, assumes preference for patients exhibiting heightened cosmetic concerns owing to the scarring risks inherent to physical therapy and surgical excision. Notably, the combination of curettage and photodynamic therapy has exhibited remarkable efficacy in the treatment of nodular basal cell carcinoma. Additionally, for elderly patients who may be intolerant to stimulation, modified photodynamic therapy offers an almost painless option. When surgery is unavoidable, photodynamic therapy can be a valuable adjunct, allowing for a more conservative surgical approach, either before or after the procedure. Radiotherapy holds a prominent role in comprehensive treatment strategies, especially for patients ineligible for surgical intervention or those with lesions precluding further surgical measures. In cases of NMSC exhibiting perineural invasion or lymphovascular involvement, adjunctive radiotherapy is advised; however, potential adverse effects necessitate careful consideration. For advanced NMSC cases where surgery and physical therapy fall short, immunotherapy provide viable solutions. Systemic therapy employing Hedgehog pathway inhibitors can be considered for patients with distant metastatic basal cell carcinoma, despite its low incidence, or individuals with locally advanced lesions who are not surgical candidates, or those encountering recurrences after resection and radiotherapy. However, close monitoring of disease progression and adverse reactions is crucial. In this evolving landscape of NMSC treatment, personalized and multidisciplinary approaches are key, ensuring optimal outcomes while prioritizing patient safety and satisfaction.

Investigations of the distant metastatic non-small cell lung cancer without local lymph node involvement: Real world data from a large database.

This study aimed to investigate the presentations and survival outcomes of the distant metastatic non-small cell lung cancer (NSCLC) without lymph node involvement to obtain a clearer picture of this special subgroup of metastatic NSCLC. A least absolute shrinkage and selection operator (LASSO) penalized Cox regression analysis was used to select the prognostic variables. A nomogram and corresponding risk-classifying systems were constructed. The C-index and calibration curves were used to evaluate the performance of the model. Overall survival (OS) curves were plotted using the Kaplan-Meier method, and the log-rank test was used to compare OS differences between groups. Propensity score matching (PSM) was performed to reduce bias. A total of 12 610 NSCLC patients with M1 category (N0 group: 3045 cases; N1-3 group: 9565 cases) were included. Regarding the N0 group, multivariate analysis demonstrated that age, sex, race, surgery, grade, tumor size, and M category were independent prognostic factors. A nomogram and corresponding risk-classifying systems were formulated. Favorable validation results were obtained from the C-index, calibration curves, and survival comparisons. Survival curves demonstrated that N0 NSCLC patients had better survival than N1-3 NSCLC patients both before and after PSM. Furthermore, the survival of resected N0M1 patients was superior to that of those without surgery. In this study, a prognostic nomogram and risk-classifying systems designed for the T1-4N0M1 NSCLC patients showed acceptable performance. Primary lung tumor resection might be a feasible treatment for this population subset. Additionally, we proposed that lymph node stage might have a place in the forthcoming tumor-node-metastasis (TNM)staging proposal for NSCLC patients with M1 category.

Preprint Highlight: Constriction forces imposed by basement membranes regulate developmental cell migration

Extracellular matrix (ECM) stiffness affects many processes of ECM-associated cells, such as migration and differentiation. Whether ECM mechanical properties also affect distant cell populations remains largely unexplored. This preprint studies migration of border cells within Drosophila ovarian follicles as a model system. Border cells migrate toward the center of the follicle by moving between nurse cells, which are surrounded by follicle cells that are themselves encased by the basement membrane ECM. Decreasing stiffness of the follicle ECM reduced cortical tension of follicle cells, and of nurse cells indirectly, accelerating border cell migration. Increasing follicle cell cortical tension slowed border cell migration. These results may inform how ECM stiffness affects cell migration within other complex tubular tissues, such as blood vessels. This preprint has been assigned the following badges: New Hypothesis, New Materials, Cross-Validation Read the preprint on bioRxiv ( Molina López et al., 2022 ): https://doi.org/10.1101/2022.12.19.520983 .

PD04-12 RADIOTHERAPEUTIC TREATMENT OF PROSTATE CANCER CELLS PROMOTES THE RELEASE OF POTENTIALLY DELETERIOUS AND IMMUNOLOGICALLY ACTIVE EXTRACELLULAR VESICLES

You have accessJournal of UrologyCME1 Apr 2023PD04-12 RADIOTHERAPEUTIC TREATMENT OF PROSTATE CANCER CELLS PROMOTES THE RELEASE OF POTENTIALLY DELETERIOUS AND IMMUNOLOGICALLY ACTIVE EXTRACELLULAR VESICLES Ryan Molony, Sarah Kerns, Brian Marples, Emmanuel Oshodi, YuhChyau Chen, and Yi-Fen Lee Ryan MolonyRyan Molony More articles by this author , Sarah KernsSarah Kerns More articles by this author , Brian MarplesBrian Marples More articles by this author , Emmanuel OshodiEmmanuel Oshodi More articles by this author , YuhChyau ChenYuhChyau Chen More articles by this author , and Yi-Fen LeeYi-Fen Lee More articles by this author View All Author Informationhttps://doi.org/10.1097/JU.0000000000003228.12AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookLinked InTwitterEmail Abstract INTRODUCTION AND OBJECTIVE: One in four prostate cancer (PCa) patients will develop late hematuria and other delayed bladder toxicities following radiotherapy (RT) through poorly understood mechanisms. Membrane-enclosed extracellular vesicles (EVs) are released from tumor and non-tumor cells at baseline, and their composition can change markedly in response to stimuli. The impact of RT-induced EV (RT-EV) release from PCa cells or other cell populations in the tumor microenvironment, including immune cells, have yet to be characterized. Here, we examined the dynamics and composition of RT-EVs released by PCa cells and immune cells in response to irradiation, and their association with bladder tissue toxicity. METHODS: Urinary EV cargo proteins in pre- and post-RT PCa patient samples were analyzed and compared by LC-MS/MS. The impact of RT on EV release kinetics was analyzed by nanoparticle tracking analysis. RT-EVs and naïve EVs were collected from PCa patients and PCa cells lines (C4-2 and LNCaP). Their mechanistic effects on normal SV-HUC urothelial cells were assessed, including cellular viability, reactive oxygen species (ROS) production, and cytokine release. The effects of RT-EVs and RT on the activation of immune cell populations were monitored by treating Jurkat T cells or HL-60-derived neutrophils with RT-EVs or irradiating these cells and evaluating activation, cytokine release, and ROS production. RESULTS: The post-RT urinary EVs of a PCa patient who later developed hematuria were enriched for potentially caustic neutrophil-associated proteins relative to EVs from a hematuria-free patient. RT significantly enhanced the release of EVs from PCa cell lines and neutrophils, and these RT-EVs or direct irradiation promoted increased ROS production by HL-60 neutrophils, Jurkat T cells, and SV-HUC urothelial cells while also increasing interleukin (IL)-1β release from SV-HUC cells consistent with the induction of a damage-associated innate immune response. CONCLUSIONS: These results suggest RT can provoke the release of immunologically active and potentially deleterious EVs from PCa cells and local immune cell populations that can contribute to oxidative stress and inflammatory response induction in local and distant cell populations including neutrophils, T cells, and urothelial cells, potentially driving a feedback loop exacerbating RT-associated tissue damage. Source of Funding: University of Rochester Medical Center Departmental Funding © 2023 by American Urological Association Education and Research, Inc.FiguresReferencesRelatedDetails Volume 209Issue Supplement 4April 2023Page: e146 Advertisement Copyright & Permissions© 2023 by American Urological Association Education and Research, Inc.MetricsAuthor Information Ryan Molony More articles by this author Sarah Kerns More articles by this author Brian Marples More articles by this author Emmanuel Oshodi More articles by this author YuhChyau Chen More articles by this author Yi-Fen Lee More articles by this author Expand All Advertisement PDF downloadLoading ...

Direct reprogramming of human fibroblasts into insulin-producing cells using transcription factors

Direct lineage reprogramming of one somatic cell into another without transitioning through a progenitor stage has emerged as a strategy to generate clinically relevant cell types. One cell type of interest is the pancreatic insulin-producing β cell whose loss and/or dysfunction leads to diabetes. To date it has been possible to create β-like cells from related endodermal cell types by forcing the expression of developmental transcription factors, but not from more distant cell lineages like fibroblasts. In light of the therapeutic benefits of choosing an accessible cell type as the cell of origin, in this study we set out to analyze the feasibility of transforming human skin fibroblasts into β-like cells. We describe how the timed-introduction of five developmental transcription factors (Neurog3, Pdx1, MafA, Pax4, and Nkx2-2) promotes conversion of fibroblasts toward a β-cell fate. Reprogrammed cells exhibit β-cell features including β-cell gene expression and glucose-responsive intracellular calcium mobilization. Moreover, reprogrammed cells display glucose-induced insulin secretion in vitro and in vivo. This work provides proof-of-concept of the capacity to make insulin-producing cells from human fibroblasts via transcription factor-mediated direct reprogramming.

Size-optimized nuclear-targeting phototherapy enhances the type I interferon response for "cold" tumor immunotherapy.

There is growing interest in the effect of innate immune silencing in "cold" tumors, which always fail in the immune checkpoint blockade monotherapy using PD-L1 monoclonal antibodies (aPD-L1). Combination of aPD-L1 with photodynamic therapy, i.e., photoimmunotherapy, is a promising strategy to improve the mono immunotherapy. Nuclear-targeting nanoparticles could elicit a type I interferon (IFN)-mediated innate immune response and reverse the immunosuppressive microenvironment for long-term immunotherapy of "cold" tumors. Photosensitizers such as zinc phthalocyanine (ZnPc) have limited ability to target the nucleus and activate innate sensing pathways to minimize tumor recurrence. Additionally, the relationship between nanoparticle size and nuclear entry capacity remains unclear. Herein, graphene quantum dots (GQDs) were employed as aPD-L1 and ZnPc carriers. Three particle sizes (200 nm, 32 nm and 5 nm) of aPD-L1/ZnPc/GQD-PEG (PZGE) were synthesized and tested. The 5 nm nanoparticles achieved the best nuclear enrichment capacity contributing to their ultrasmall size. Notably, 5 nm PZGE-based photodynamic therapy enabled an amplification of the type I IFN-mediated innate immune response and could convert "immune-cold" tumors into "immune-hot" ones. Utilizing their size advantage to target the nucleus, 5 nm nanoparticles induced DNA damage and activated the type I IFN-mediated innate immune response, subsequently promoting cytotoxic T-lymphocyte infiltration and reversing negative PD-L1 expression. Furthermore, the nanoplatform we designed is promising for the effective suppression of distant oral squamous cell carcinoma. Thus, for the first time, this study presents a size design strategy for nuclear-targeted photo-controlled immune adjuvants and the nuclear-targeted phototherapy-mediated immunomodulatory functions of type I IFN innate immune signalling for "immune-cold" tumors. STATEMENT OF SIGNIFICANCE: The potential of commonly used photosensitizers to activate innate sensing pathways for producing type I IFNs is limited due to the lack of nuclear targeting. Facilitating the nuclear-targeting of photosensitizers to enhance innate immune response and execute long-term tumor killing effect would be a promising strategy for "cold" tumor photoimmunotherapy. Herein, we report an optimal size of PZGE nanoparticles that enable the nuclear-targeting of ZnPc, which reinforces the type I IFN-mediated innate immune response, synergistically reversing "cold tumors" to "hot tumors" for effective primary and distant tumor photoimmunotherapy. This work highlights the marked efficacy of ultrasmall nuclear-located nanocarriers and offers new insight into "immune-cold tumors" via prominent innate immune activation mediated by nuclear-targeting photoimmunotherapy.

Collective directional migration drives the formation of heteroclonal cancer cell clusters.

Metastasisation occurs through the acquisition of invasive and survival capabilities that allow tumour cells to colonise distant sites. While the role of multicellular aggregates in cancer dissemination is acknowledged, the mechanisms that drive the formation of multiclonal cell aggregates are not fully elucidated. Here, we show that cancer cells of different tissue of origins can perform collective directional migration and can actively form heteroclonal aggregates in 3D, through a proliferation-independent mechanism. Coalescence of distant cell clusters is mediated by subcellular actin-rich protrusions and multicellular outgrowths that extend towards neighbouring aggregates. Coherently, perturbation of cytoskeletal dynamics impairs collective migration while myosin II activation is necessary for multicellular movements. We put forward the hypothesis that cluster attraction is mediated by secreted soluble factors. Such a hypothesis is consistent with the abrogation of aggregation by inhibition of PI3K/AKT/mTOR and MEK/ERK, the chemoattracting activity of conditioned culture media and with a wide screening of secreted proteins. Our results present a novel collective migration model and shed light on the mechanisms of formation of heteroclonal aggregates in cancer.

Spatiotemporal tracking of small extracellular vesicle nanotopology in response to physicochemical stresses revealed by HS-AFM.

Small extracellular vesicles (sEVs) play a crucial role in local and distant cell communication. The intrinsic properties of sEVs make them compatible biomaterials for drug delivery, vaccines, and theranostic nanoparticles. Although sEV proteomics have been robustly studied, a direct instantaneous assessment of sEV structure dynamics remains difficult. Here, we use the high-speed atomic force microscopy (HS-AFM) to evaluate nanotopological changes of sEVs with respect to different physicochemical stresses including thermal stress, pH, and osmotic stress. The sEV structure is severely altered at high-temperature, high-pH, or hypertonic conditions. Surprisingly, the spherical shape of the sEVs is maintained in acidic or hypotonic environments. Real-time observation by HS-AFM imaging reveals an irreversible structural change in the sEVs during transition of pH or osmolarity. HS-AFM imaging provides both qualitative and quantitative data at high spatiotemporal resolution (nanoscopic and millisecond levels). In summary, our study demonstrates the feasibility of HS-AFM for structural characterization and assessment of nanoparticles.

High-resolution atomic force microscopy as a tool for topographical mapping of surface budding.

Extracellular vesicles (EVs) are membranous nanoparticles secreted by almost all cell types. Reflecting the physiopathological state of the parental cell, EVs circulate in all body fluids, reaching distant cell targets and delivering different bioactive cargoes. As biological carriers, EVs influence their microenvironment altering cellular responses, being considered promising biomarkers for both physiological and pathological conditions. EVs are heterogeneous in terms of size and composition, depending on cell type and exposure to stimuli, and different methods have been developed to characterize their morphological, biophysical, and biochemical features. Among them, electron microscopy (EM) is the main technique used, however, the lack of standardized protocols makes it difficult to characterize EVs with a good reproducibility, thus using multiple approaches may represent a way to obtain more precise information. Furthermore, the relationship between architecture and function, not only in a molecular, but also in a cellular level, is gaining growing emphasis, characterizing morphometric parameters may represent a distinct, but effective approach to study the physiopathological state of the cell. Atomic force microscopy (AFM), may represent a promising method to study in detail EVs dynamics throughout the cell surface and its variations related to the physiological state, overcoming the limits of EM, and providing more reliable information. In this study, human neuroblastoma SH-SY5Y cell line, a cellular model to investigate neurodegeneration and oxidative stress, has been used to perform a comparative morphological and quantitative analysis of membrane budding and isolated large vesicles-enriched (microvesicles-like vesicles; MVs) fraction from control or oxidative stressed cells. Our main goal was to build up a methodology to characterize EVs morphology and spatial distribution over the cell surface in different physiological conditions, and to evaluate the efficacy of AFM against conventional EM. Interestingly, both microscopy techniques were effective for this analysis, but AFM allowed to reveal a differential profiling of plasma membrane budding between the physiological and the stress condition, indicating a potential relationship between mechanical characteristics and functional role. The results obtained may provide interesting perspectives for the use of AFM to study EVs, validating a morphometric approach to understand the pathophysiological state of the cell related to EVs trafficking.

Exosome‐based cancer stem cell communication: Implication for detecting and eliminating cancer stem cells

AbstractExosomes are bilayered nano‐dimensional bio‐vesicles shed by all types of cells including transformed malignant cells. They are primarily involved in cell‐to‐cell communication and resemble the contents of their parental cells. They transport specific contents, like DNA, proteins, and lipids, which is selectively absorbed by proximate and distant cell from the site of their release, conditioning the recipient cells. Cancer stem cells (CSCs) are the root cause behind tumor development, immune evasion, and relapse also produce and secrete large quantities of exosomes that aid and abet CSCs. Interaction of CSCs and neighboring cells occurs through the exosomal exchange of biological materials. Exosomes promote immune evasion, metastasis, and self‐renewal within the tumor microenvironment. These properties of exosomes are exploited to detect the presence of malignant cells via noninvasive techniques. These exosomes can be modified to target CSCs. In this review, we highlight the unique role of exosomes in disease progression and how they can be exploited to detect CSCs or be used as a vehicle for medicinal drugs and genetic components to destroy CSCs. Exosome research is still in its inception, in‐depth analysis of the components and mechanistic details involved in exosome synthesis and precise bio‐engineering for cell‐targeting will help us discern it as a next‐generation cancer therapy option.

A Rapid and Efficient Method for Isolation and Transformation of Cotton Callus Protoplast.

Protoplasts, which lack cell walls, are ideal research materials for genetic engineering. They are commonly employed in fusion (they can be used for more distant somatic cell fusion to obtain somatic hybrids), genetic transformation, plant regeneration, and other applications. Cotton is grown throughout the world and is the most economically important crop globally. It is therefore critical to study successful extraction and transformation efficiency of cotton protoplasts. In the present study, a cotton callus protoplast extraction method was tested to optimize the ratio of enzymes (cellulase, pectinase, macerozyme R-10, and hemicellulase) used in the procedure. The optimized ratio significantly increased the quantity and activity of protoplasts extracted. We showed that when enzyme concentrations of 1.5% cellulase and 1.5% pectinase, and either 1.5% or 0.5% macerozyme and 0.5% hemicellulase were used, one can obtain increasingly stable protoplasts. We successfully obtained fluorescent protoplasts by transiently expressing fluorescent proteins in the isolated protoplasts. The protoplasts were determined to be suitable for use in further experimental studies. We also studied the influence of plasmid concentration and transformation time on protoplast transformation efficiency. When the plasmid concentration reaches 16 µg and the transformation time is controlled within 12–16 h, the best transformation efficiency can be obtained. In summary, this study presents efficient extraction and transformation techniques for cotton protoplasts.

Energy Sources for Exosome Communication in a Cancer Microenvironment.

Simple SummaryExosomal communication in the tumor microenvironment plays a crucial role in cancer development, progression, and metastasis, and is achieved by either short-distance communication with neighboring cells or long-distance communication with distant organs. Nevertheless, how exosomes gain energy to establish such communication and the different sources of energy are unclear. Recently, a handful of studies have demonstrated the presence of mitochondria, adenosine triphosphate, and glycolytic enzymes, which may serve as potential energy sources for exosomes. This review clarifies how exosomes maintain their structural integrity and stability during their intracellular communication, and reviews evidence of their energy source. Exosomes are crucial extracellular vesicles (EVs) with a diameter of approximately 30–200 nm. They are released by most cell types in their extracellular milieu and carry various biomolecules, including proteins and nucleic acids. Exosomes are increasingly studied in various diseases, including cancer, due to their role in local and distant cell–cell communication in which they can promote tumor growth, cancer progression, and metastasis. Interestingly, a tremendous number of exosomes is released by malignant cancer cells, and these are then taken up by autologous and heterologous recipient stromal cells such as immune cells, cancer stem cells, and endothelial cells. All these events demand an enormous amount of energy and require that exosomes remain stable while having the capacity to reach distant sites and cross physical barriers. Nevertheless, there is a dearth of research pertaining to the energy sources of exosomes, and questions remain about how they maintain their motility in the tumor microenvironment (TME) and beyond. Moreover, exosomes can produce adenosine triphosphate (ATP), an important energy molecule required by all cells, and mitochondria have been identified as one of the exosomal cargoes. These findings strengthen the prospect of exosomal communication via transfer of mitochondria and the bioenergetics of target recipient cells. In the TME, the accumulation of ATP and lactate may facilitate the entry of exosomes into cancer cells to promote metastasis, as well as help to target cancer cells at the tumor site. This review highlights how exosomes obtain sufficient energy to thrive in the TME and communicate with distant physiological destinations.

Cerebral Metastasis Secondary to a Distant Malignant Cutaneous Squamous Cell Carcinoma in an Immunosuppressed Patient

Squamous cell carcinoma of cutaneous origin is an insidiously developing cancer, and seldom metastasises. Patient and lesion characteristics that predispose to more aggressive biological behaviours include immune incompetence, recurrence following excision and those demonstrating invasion of local lymphovascular and neural structures.

A Pseudorabies Virus Serine/Threonine Kinase, US3, Promotes Retrograde Transport in Axons via Akt/mToRC1.

ABSTRACTInfection of peripheral axons by alpha herpesviruses (AHVs) is a critical stage in establishing a lifelong infection in the host. Upon entering the cytoplasm of axons, AHV nucleocapsids and associated inner-tegument proteins must engage the cellular retrograde transport machinery to promote the long-distance movement of virion components to the nucleus. The current model outlining this process is incomplete, and further investigation is required to discover all viral and cellular determinants involved as well as the temporality of the events. Using a modified trichamber system, we have discovered a novel role of the pseudorabies virus (PRV) serine/threonine kinase US3 in promoting efficient retrograde transport of nucleocapsids. We discovered that transporting nucleocapsids move at similar velocities in both the presence and absence of a functional US3 kinase; however, fewer nucleocapsids are moving when US3 is absent, and they move for shorter periods of time before stopping, suggesting that US3 is required for efficient nucleocapsid engagement with the retrograde transport machinery. This led to fewer nucleocapsids reaching the cell bodies to produce a productive infection 12 h later. Furthermore, US3 was responsible for the induction of local translation in axons as early as 1 h postinfection (hpi) through the stimulation of a phosphatidylinositol 3-kinase (PI3K)/Akt-mToRC1 pathway. These data describe a novel role for US3 in the induction of local translation in axons during AHV infection, a critical step in transport of nucleocapsids to the cell body.IMPORTANCE Neurons are highly polarized cells with axons that can reach centimeters in length. Communication between axons at the periphery and the distant cell body is a relatively slow process involving the active transport of chemical messengers. There is a need for axons to respond rapidly to extracellular stimuli. Translation of repressed mRNAs present within the axon occurs to enable rapid, localized responses independently of the cell body. AHVs have evolved a way to hijack local translation in the axons to promote their transport to the nucleus. We have determined the cellular mechanism and viral components involved in the induction of axonal translation. The US3 serine/threonine kinase of PRV activates Akt-mToRC1 signaling pathways early during infection to promote axonal translation. When US3 is not present, the number of moving nucleocapsids and their processivity are reduced, suggesting that US3 activity is required for efficient engagement of nucleocapsids with the retrograde transport machinery.