Search

During the initiation of invasive aspergillosis, inhaled Aspergillus fumigatus conidia are deposited on the epithelial cells lining the bronchi, terminal bronchioles, and alveoli. While the interactions of A. fumigatus with bronchial and type II alveolar cell lines have been investigated in vitro, little is known about the interactions of this fungus with terminal bronchiolar epithelial cells. Using the HSAEC1-KT human small airway epithelial (HSAE) cell line, we developed an in vitro model to study the interaction of two strains of A. fumigatus with these cells. We then compared the interactions of A. fumigatus with the A549 type II alveolar epithelial cell line and the HSAE cell line. We found that A. fumigatus conidia were poorly endocytosed by A549 cells, but avidly endocytosed by HSAE cells. A. fumigatus germlings invaded both cell types by induced endocytosis, but not by active penetration. A549 cell endocytosis of A. fumigatus was independent of fungal viability, more dependent on host microfilaments than microtubules, and induced by A. fumigatus CalA interacting with host cell integrin α5β1. By contrast, HSAE cell endocytosis required fungal viability, was more dependent on microtubules than microfilaments, and did not require CalA or integrin α5β1. HSAE cells were more susceptible than A549 cells to damage caused by direct contact with killed A. fumigatus germlings and by secreted fungal products. In response to A. fumigatus infection, A549 cells secreted a broader profile of cytokines and chemokines than HSAE cells. Taken together, these results demonstrate that studies of HSAE cells provide complementary data to A549 cells and thus represent a useful model for probing the interactions of A. fumigatus with bronchiolar epithelial cells in vitro. Importance During the initiation of invasive aspergillosis, Aspergillus fumigatus interacts with the epithelial cells that line the airways and alveoli. Previous studies of A. fumigatus-epithelial cell interactions in vitro used either large airway epithelial cell lines or the A549 type II alveolar epithelial cell line; the interactions of fungi with terminal bronchiolar epithelial cells were not investigated. Using the TERT-immortalized human small airway epithelial HSAEC1-KT (HSAE) cell line, we developed an in vitro model of the interactions of A. fumigatus with bronchiolar epithelial cells. We discovered that A. fumigatus invades and damages A549 and HSAE cell lines by distinct mechanisms. Also, the proinflammatory responses of the cell lines to A. fumigatus are different. These results provide insight into how A. fumigatus interacts with different types of epithelial cells during invasive aspergillosis and demonstrate that HSAE cells are useful in vitro model for investigating the interactions of this fungus with bronchiolar epithelial cells.

With most of the T cells residing in the tissue, not the blood, developing noninvasive methods for in vivo quantification of their biodistribution and kinetics is important for studying their role in immune response and memory. This study presents the first use of dynamic positron emission tomography (PET) and kinetic modeling for in vivo measurement of CD8+ T cell biodistribution in humans. A 89Zr-labeled CD8-targeted minibody (89Zr-Df-Crefmirlimab) was used with total-body PET in healthy individuals (N = 3) and coronavirus disease 2019 (COVID-19) convalescent patients (N = 5). Kinetic modeling results aligned with T cell-trafficking effects expected in lymphoid organs. Tissue-to-blood ratios from the first 7 hours of imaging were higher in bone marrow of COVID-19 convalescent patients compared to controls, with an increasing trend between 2 and 6 months after infection, consistent with modeled net influx rates and peripheral blood flow cytometry analysis. These results provide a promising platform for using dynamic PET to study the total-body immune response and memory.

Meaningful advances in targeted therapy for head and neck squamous cell carcinoma (HNSCC) have been hampered by limited availability of robust preclinical models for translational research. Using an impressive array of in vitro and in vivo preclinical HNSCC models, Smith and colleagues demonstrated the efficacy of alpelisib and tipifarnib combination therapy through sustained mTOR inhibition in PIK3CA/HRAS-dysregulated HNSCC, including preliminary evidence of robust antitumor activity in a patient enrolled in a precision medicine trial. This study in this issue of Cancer Research illustrates the value of preclinical avatars for informing biomarker-driven clinical trials to advance precision medicine in HNSCC and other cancers. See related article by Smith et al., p. 3252.

The mechanical properties of solid tumors influence tumor cell phenotype and the ability to invade surrounding tissues. Using bioengineered scaffolds to provide a matrix microenvironment for patient-derived glioblastoma (GBM) spheroids, this study demonstrates that a soft, brain-like matrix induces GBM cells to shift to a glycolysis-weighted metabolic state, which supports invasive behavior. We first show that orthotopic murine GBM tumors are stiffer than peritumoral brain tissues, but tumor stiffness is heterogeneous where tumor edges are softer than the tumor core. We then developed 3D scaffolds with μ-compressive moduli resembling either stiffer tumor core or softer peritumoral brain tissue. We demonstrate that the softer matrix microenvironment induces a shift in GBM cell metabolism toward glycolysis, which manifests in lower proliferation rate and increased migration activities. Finally, we show that these mechanical cues are transduced from the matrix via CD44 and integrin receptors to induce metabolic and phenotypic changes in cancer cells.

Bipolar disorder (BD) is a neuropsychiatric mood disorder manifested by recurrent episodes of mania and depression. More than half of BD patients are non-responsive to lithium, the first-line treatment drug, complicating BD clinical management. Given its unknown etiology, it is pertinent to understand the genetic signatures that lead to variability in lithium response. We discovered a set of differentially expressed genes (DEGs) from the lymphoblastoid cell lines (LCLs) of 10 controls and 19 BD patients belonging mainly to the immunoglobulin gene family that can be used as potential biomarkers to diagnose and treat BD. Importantly, we trained machine learning algorithms on our datasets that predicted the lithium response of BD subtypes with minimal errors, even when used on a different cohort of 24 BD patients acquired by a different laboratory. This proves the scalability of our methodology for predicting lithium response in BD and for a prompt and suitable decision on therapeutic interventions.

Dysregulation of the E3 ubiquitin ligase Parkin has been linked to various human cancers, indicating that Parkin is a tumor suppressor protein. However, the mechanisms of action of Parkin remain unclear to date. Thus, we aimed to elucidate the mechanisms of action of Parkin as a tumor suppressor in human lung and colorectal cancer cells. Results showed that Parkin overexpression reduced the viability of A549 human lung cancer cells by inducing G2/M cell cycle arrest. In addition, Parkin caused DNA damage and ATM (Ataxia telangiectasia mutated) activation, which subsequently led to p53 activation. It also induced the p53-mediated upregulation of p21 and downregulation of cyclin B1. Moreover, Parkin suppressed the proliferation of HCT-15 human colorectal cancer cells by a mechanism similar to that in A549 lung cancer cells. Taken together, our results suggest that the tumor-suppressive effects of Parkin on lung and colorectal cancer cells are mediated by DNA damage/p53 activation/cyclin B1 reduction/cell cycle arrest. [BMB Reports 2023; 56(10): 557-562].

Although tumor growth requires the mitochondrial electron transport chain (ETC), the relative contribution of complex I (CI) and complex II (CII), the gatekeepers for initiating electron flow, remains unclear. In this work, we report that the loss of CII, but not that of CI, reduces melanoma tumor growth by increasing antigen presentation and T cell-mediated killing. This is driven by succinate-mediated transcriptional and epigenetic activation of major histocompatibility complex-antigen processing and presentation (MHC-APP) genes independent of interferon signaling. Furthermore, knockout of methylation-controlled J protein (MCJ), to promote electron entry preferentially through CI, provides proof of concept of ETC rewiring to achieve antitumor responses without side effects associated with an overall reduction in mitochondrial respiration in noncancer cells. Our results may hold therapeutic potential for tumors that have reduced MHC-APP expression, a common mechanism of cancer immunoevasion.

Pancreatic ductal adenocarcinoma (PDAC) is a lethal cancer with a low survival rate. Recently, new drugs that target KRASG12D, a common mutation in PDAC, have been developed. We studied one of these compounds, MRTX1133, and found it was specific and effective at low nanomolar concentrations in patient-derived organoid models and cell lines harboring KRASG12D mutations. Treatment with MRTX1133 upregulated the expression and phosphorylation of EGFR and HER2, indicating that inhibition of ERBB signaling may potentiate MRTX1133 antitumor activity. Indeed, the irreversible pan-ERBB inhibitor, afatinib, potently synergized with MRTX1133 in vitro, and cancer cells with acquired resistance to MRTX1133 in vitro remained sensitive to this combination therapy. Finally, the combination of MRTX1133 and afatinib led to tumor regression and longer survival in orthotopic PDAC mouse models. These results suggest that dual inhibition of ERBB and KRAS signaling may be synergistic and circumvent the rapid development of acquired resistance in patients with KRAS mutant pancreatic cancer.SignificanceKRAS-mutant pancreatic cancer models, including KRAS inhibitor-resistant models, show exquisite sensitivity to combined pan-ERBB and KRAS targeting, which provides the rationale for testing this drug combination in clinical trials.

Objective. To propose a mathematical model for applying ionization detail (ID), the detailed spatial distribution of ionization along a particle track, to proton and ion beam radiotherapy treatment planning (RTP).Approach. Our model provides for selection of preferred ID parameters (Ip) for RTP, that associate closest to biological effects. Cluster dose is proposed to bridge the large gap between nanoscopicIpand macroscopic RTP. Selection ofIpis demonstrated using published cell survival measurements for protons through argon, comparing results for nineteenIp:Nk,k= 2, 3, …, 10, the number of ionizations in clusters ofkor more per particle, andFk,k= 1, 2, …, 10, the number of clusters ofkor more per particle. We then describe application of the model to ID-based RTP and propose a path to clinical translation.Main results. The preferredIpwereN4andF5for aerobic cells,N5andF7for hypoxic cells. Significant differences were found in cell survival for beams having the same LET or the preferredNk. Conversely, there was no significant difference forF5for aerobic cells andF7for hypoxic cells, regardless of ion beam atomic number or energy. Further, cells irradiated with the same cluster dose for theseIphad the same cell survival. Based on these preliminary results and other compelling results in nanodosimetry, it is reasonable to assert thatIpexist that are more closely associated with biological effects than current LET-based approaches and microdosimetric RBE-based models used in particle RTP. However, more biological variables such as cell line and cycle phase, as well as ion beam pulse structure and rate still need investigation.Significance. Our model provides a practical means to select preferredIpfrom radiobiological data, and to convertIpto the macroscopic cluster dose for particle RTP.

As the era of omics continues to expand with increasing ubiquity and success in both academia and industry, omics-based experiments are becoming commonplace in industrial biotechnology, including efforts to develop novel solutions in bioprocess optimization and cell line development. Omic technologies provide particularly valuable observational insights for discovery science, especially in academic research and industrial R&D; however, biomanufacturing requires a different paradigm to unlock actionable insights from omics. Here, we argue the value of omic experiments in biotechnology can be maximized with deliberate selection of omic approaches and forethought about analysis techniques. We describe important considerations when designing and implementing omic-based experiments and discuss how systems biology analysis strategies can enhance efforts to obtain actionable insights in mammalian-based biologics production.

γδ T cells are potent anticancer effectors with the potential to target tumours broadly, independent of patient-specific neoantigens or human leukocyte antigen background1-5. γδ T cells can sense conserved cell stress signals prevalent in transformed cells2,3, although the mechanisms behind the targeting of stressed target cells remain poorly characterized. Vγ9Vδ2 T cells-the most abundant subset of human γδ T cells4-recognize a protein complex containing butyrophilin 2A1 (BTN2A1) and BTN3A1 (refs. 6-8), a widely expressed cell surface protein that is activated by phosphoantigens abundantly produced by tumour cells. Here we combined genome-wide CRISPR screens in target cancer cells to identify pathways that regulate γδ T cell killing and BTN3A cell surface expression. The screens showed previously unappreciated multilayered regulation of BTN3A abundance on the cell surface and triggering of γδ T cells through transcription, post-translational modifications and membrane trafficking. In addition, diverse genetic perturbations and inhibitors disrupting metabolic pathways in the cancer cells, particularly ATP-producing processes, were found to alter BTN3A levels. This induction of both BTN3A and BTN2A1 during metabolic crises is dependent on AMP-activated protein kinase (AMPK). Finally, small-molecule activation of AMPK in a cell line model and in patient-derived tumour organoids led to increased expression of the BTN2A1-BTN3A complex and increased Vγ9Vδ2 T cell receptor-mediated killing. This AMPK-dependent mechanism of metabolic stress-induced ligand upregulation deepens our understanding of γδ T cell stress surveillance and suggests new avenues available to enhance γδ T cell anticancer activity.

Drug-tolerant persister (DTP) cancer cells drive residual tumor and relapse. However, the mechanisms underlying DTP state development are largely unexplored. In a recent study, we determined that PINK1-mediated mitophagy favors DTP generation in the context of MAPK inhibition therapy. DTP cells that persist in the presence of a MAPK inhibitor exhibit mitochondriadependent metabolism. During DTP state development, MYC depletion alleviates the transcriptional repression of PINK1, resulting in PINK1 upregulation and mitophagy activation. PINK1-mediated mitophagy is essential for mitochondrial homeostasis in DTP cells. Either knockdown of PINK1 or inhibition of mitophagy eradicates DTP cells and achieves complete responses to MAPK inhibition therapy. This study reveals a novel role of mitophagy as a protective mechanism for DTP development.

PurposeTo test the hypothesis that antitumoral immunity can be induced after cryoablation (cryo) of hepatocellular carcinoma (HCC) through coadministration of the immunostimulant CpG and an immune checkpoint (programmed cell death 1 [PD-1]) inhibitor.Materials and methodsSixty-three immunocompetent C57BL/6J mice were generated with 2 orthotopic HCC tumor foci: 1 for treatment and 1 to observe for antitumoral immunity. Tumors were treated with incomplete cryo alone or intratumoral CpG and/or a PD-1 inhibitor. The primary endpoint was death or when the following criteria for sacrifice were met: tumor > 1 cm (determined using ultrasound) or moribund state. Antitumoral immunity was assessed using flow cytometry and histology (tumor and liver) as well as enzyme-linked immunosorbent assay (serum). Analysis of variance was used for statistical comparisons.ResultsAt 1 week, the nonablated satellite tumor growth was reduced by 1.9-fold (P = .047) in the cryo + CpG group and by 2.8-fold (P = .007) in the cryo + CpG + PD-1 group compared with that in the cryo group. Compared with cryo alone, the time to tumor progression to endpoints was also prolonged for cryo + CpG + PD-1 and cryo + CpG mice, with log-rank hazard ratios of 0.42 (P = .031) and 0.27 (P < .001), respectively. Flow cytometry and histology showed increased cytotoxic T-cell infiltration (P = .002) and serum levels of the proinflammatory cytokine interferon-γ (P = .015) in tumors and serum of cryo + CpG mice compared with those in tumors and serum of mice treated with cryo alone. High serum levels of the anti-inflammatory cytokine tumor growth factor-β and the proangiogenesis chemokine C-X-C motif chemokine ligand 1 were correlated with a shorter time to endpoints and faster tumor growth.ConclusionsCryo combined with the immunostimulant CpG promoted cytotoxic T-cell infiltration into tumors, slowed tumor growth, and prolonged the time to progression to endpoints in an aggressive murine HCC model.

Highly susceptible fish cell lines are required to isolate and propagate fish nodavirus and the main purpose of the present study is to develop highly susceptible cell lines from Lates calcarifer which is highly vulnerable to viral nervous necrosis. Three permanent cell lines, brain (SBB), eye (SBE) and optic nerve (SBON) were developed from L. calcarifer by explant culture technique and sub-cultured for 97, 128 and 75 respectively, at 28 °C in Leibovitz's L-15 media supplemented with 10% fetal bovine serum (FBS). The plating efficiency of SBB, SBE and SBON cells was determined as 69.09, 63.19 and 48.22%, respectively, and the doubling time was 27, 29 and 38 h, respectively. All these cell lines were stored by cryopreservation at various passage levels, and a recovery rate of 75–95% was observed. Immunostaining of the SBB and SBON cell lines showed that they are of neuronal origin, whereas the SBE cell line is of retinal pigment epithelial origin. Amplification of mitochondrial 16S rRNA gene confirmed that the SBB, SBE and SBON cell lines were of L. calcarifer origin. Three established cell lines showed 15–18% of transfection efficiency. These cell lines were highly susceptible to striped jack nervous necrosis virus (SJNNV) and infection in these cells was confirmed by RT-PCR, immunocytochemistry, Western blot, ELISA and qRT-PCR. The cell lines established from L. calcarifer were continuous, stable and potentially valuable for virological and biomedical studies. [ABSTRACT FROM AUTHOR]

The matrix protein 2 (M2) is a preferred target for developing a universal vaccine against the influenza A virus (IAV). This study aimed to develop a method for assessing antibody-dependent cell-mediated cytotoxicity (ADCC) associated with M2-based immunization in mice. We first established a stable cell line derived from mouse lymphoma cells (YAC-1) expressing M2 of H3N2. This cell line, designated as YAC-1-M2, was generated using a second-generation lentiviral tricistronic plasmid system to transduce the M2 gene into YAC-1 cells. The ADCC effect induced by polyclonal antibodies targeting matrix protein 2 ectodomain (M2e) was demonstrated by YAC-1-M2 cell lysis by natural killer cells (NK) derived from mice, in the presence of anti-M2 antibodies obtained from mice immunized with an mRNA vaccine based on M2e. This ADCC effect was found to be stronger compared to the effect induced by monoclonal antibodies (14C2) against M2. Moreover, the ADCC effect was enhanced as the effector-to-target ratio of NK to YAC-1-M2 cells increased. In conclusion, we established a novel method to detect ADCC of M2 of IAV, which paves the way for the development of an M2-based universal vaccine against IAV and an in-depth analysis of its mechanism of broad-spectrum immune protection in mice.

Abstract Immunodeficient murine models are usually used as the preclinical models of osteosarcoma. Such models do not effectively simulate the process of tumorigenesis and metastasis. Establishing a suitable animal model for understanding the mechanism of osteosarcoma and the clinical translation is indispensable. The UMR-106 cell suspension was injected into the marrow cavity of Balb/C nude mice. Tumor masses were harvested from nude mice and sectioned. The tumor fragments were transplanted into the marrow cavities of SD rats immunosuppressed with cyclosporine A. Through muti-rounds selection in SD rats, we constructed orthotopic osteosarcoma animal models using rats with intact immune systems. The primary tumor cells were cultured in-vitro to obtain the immune-tolerant cell line. VX2 tumor fragments were transplanted into the distal femur and parosteal radius of New Zealand white rabbit to construct orthotopic osteosarcoma animal models in rabbits. The rate of tumor formation in SD rats (P1 generation) was 30%. After four rounds of selection and six rounds of acclimatization in SD rats with intact immune systems, we obtained immune-tolerant cell lines and established the orthotopic osteosarcoma model of the distal femur in SD rats. Micro-CT images confirmed tumor-driven osteolysis and the bone destruction process. Moreover, the orthotopic model was also established in New Zealand white rabbits by implanting VX2 tumor fragments into rabbit radii and femurs. We constructed orthotopic osteosarcoma animal models in rats with intact immune systems through muti-rounds in-vivo selection and the rabbit osteosarcoma model.

Colorectal cancer (CRC) is the third most common type of cancer worldwide. Its treatment options have had a limited impact on cancer remission prognosis. Therefore, there is an ongoing need to discover novel anti-cancer agents. Medicinal plants have gained recognition as a source of anti-cancer bioactive compounds. Recently, ethanolic extract of L. virginicum stems ameliorated dinitrobenzene sulfonic acid (DNBS)-induced colitis by modulating the intestinal immune response. However, no scientific study has demonstrated this potential cytotoxic impact on colon cancer cells. The objective of this study was to evaluate the cytotoxic effect of the methanolic extract of L. virginicum (ELv) on a human colorectal adenocarcinoma cell line (Caco-2) and to identify and quantify the phenolic compounds present in ELv extracts by liquid chromatography-mass spectrometry analysis. The cytotoxic activity was assessed using cell viability assays by reduction in the compound 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) and lactate dehydrogenase (LDH). MTT and LDH assays revealed that the ELv decreases cell viability in the Caco-2 cell line in a concentration-dependent manner. Cell death was a result of DNA fragmentation and p53-mediated apoptosis. Eight phenolic acids and five flavonoids were identified and quantified in the stems. In conclusion, our findings demonstrate that the extract of L. virginicum possesses cytotoxic properties on Caco-2 cell line, suggesting that it could be a potential source of new drugs against CRC. [ABSTRACT FROM AUTHOR]

In the present study, biodegradable poly (3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV) nanoparticles (NPs) containing insulin were loaded in sodium alginate/jeffamine (ALG/jeff) hydrogel for prolonged delivery of insulin. The main aim of this work was to fabricate an efficient insulin delivery system to improve patient adherence by decreasing the repetition of injections. Swelling and morphological properties and crosslinking efficiency of ALG/jeff hydrogel were assessed. The composite hydrogel was prepared by adding PHBV NPs to ALG/jeff hydrogel concurrently with crosslinking process. The morphology and loading capacity of composite hydrogel were analyzed. Circular dichroism measurement demonstrated that insulin remains stable following fabrication process. The release profile exhibited 54.6 % insulin release from composite hydrogel within 31 days with minor initial burst release equated to nanoparticles and hydrogels. MTT cell viability analysis was performed by applying L-929 cell line and no cytotoxic effect was observed. Favorable results clearly introduced fabricated composite hydrogel as an excellent candidate for drug delivery systems and also paves the route for prolonged delivery systems of other proteins. [Display omitted] • Synthesis of insulin-PHBV nanoparticles / alginate hydrogel composite system. • incorporation of insulin-PHBV nanoparticles into an ALG/jeff hydrogel. • Outstanding biocompatibility of insulin-PHBV nanoparticles / alginate hydrogel composite system. • fabricating injectable formulation for extended delivery of insulin. • provide a platform for the prolonged and controlled delivery of other proteins and peptides. [ABSTRACT FROM AUTHOR]

In this work, we performed anti-proliferative assays for the compound N-(2-hydroxyphenyl)-2-propylpentanamide (HO-AAVPA) on breast cancer (BC) cells (MCF-7, SKBR3, and triple-negative BC (TNBC) MDA-MB-231 cells) to explore its pharmacological mechanism regarding the type of cell death associated with G protein-coupled estrogen receptor (GPER) expression. The results show that HO-AAVPA induces cell apoptosis at 5 h or 48 h in either estrogen-dependent (MCF-7) or -independent BC cells (SKBR3 and MDA-MB-231). At 5 h, the apoptosis rate for MCF-7 cells was 68.4% and that for MDA-MB-231 cells was 56.1%; at 48 h, that for SKBR3 was 61.6%, that for MCF-7 cells was 54.9%, and that for MDA-MB-231 (TNBC) was 43.1%. HO-AAVPA increased the S phase in MCF-7 cells and reduced the G2/M phase in MCF-7 and MDA-MB-231 cells. GPER expression decreased more than VPA in the presence of HO-AAVPA. In conclusion, the effects of HO-AAVPA on cell apoptosis could be modulated by epigenetic effects through a decrease in GPER expression. [ABSTRACT FROM AUTHOR]

Phosphoribosylaminoimidazole carboxylase, phosphoribosylaminoimidazole succinocarboxamide synthetase (PAICS) is a de novo purine biosynthetic enzyme. It has been found to be overexpressed in various types of cancer and is related to cell proliferation, invasion, the epithelial–mesenchymal transition, and efficient tumor growth. In this study, we describe a rat monoclonal antibody (mAb) 6A10, which was generated as an antigen of human PAICS. This mAb was generated to interact with the N-terminal region of human PAICS and was found to recognize endogenous PAICS enzymes in several cancer cells. Our results also indicated that it can recognize monkey and dog PAICS, which possess the same amino acid sequence in the antigenic region as human PAICS, but it does not recognize rat and mouse PAICS. Furthermore, our data indicated that this mAb is suitable for immunoprecipitation and immunoblotting use for several cancer cell lines. We, therefore, anticipate that mAb 6A10 will be useful for functional analyses of human PAICS in several cancers and for diagnosis of malignant transformation. [ABSTRACT FROM AUTHOR]

This study investigated the role of ultraviolet (UV) radiation and oxidation in high-density polyethylene microplastics (2–15 μm) and nanoplastics (0.2–9.9 μm) (NMPs) on particle chemistry, morphology, and reactivity with cadmium (Cd). Additionally, toxicity of NMPs alone and with Cd was evaluated using RTgutGC cells, a model of the rainbow trout (Oncorhynchus mykiss) intestine. The role on NMPs on Cd bioaccumulation in RTgutGC cells was also evaluated. Dynamic light scattering indicated that after UV radiation NPs agglomerated size increased from 0.8 to 28 µm, and to 8 µm when Cd was added. Oxidized MPs agglomerated size increased from 11 and 7 to 46 and 27 µm in non-UV- and UV-aged oxidized MPs when adding Cd, respectively. Cd-coated particles exhibited generally significantly higher zeta potential than non-Cd-coated particles, while attenuated total reflectance–Fourier transform infrared spectroscopy showed that the functional chemistry of the particles was oxidized and modified after being exposed to UV radiation. Presence of NMPs resulted in a significant decrease in Cd bioaccumulation in RTgutGC cells (100.5–87.9 ng Cd/mg protein) compared to Cd alone (138.1 ng Cd/mg protein), although this was not quite significant for co-exposures with UV-aged NPs (105.7 ng Cd/mg protein). No toxicity was observed in RTgutGC cells exposed to NMPs alone for 24 h. Moreover, co-exposures with Cd indicated that NMPs reduce the toxicity of Cd. Altogether these results show that UV aging enhances NMP surface reactivity, increasing Cd absorption in solution, which resulted in a reduction in Cd bioavailability and toxicity. [ABSTRACT FROM AUTHOR]

Core/shell Fe@Au nanostructures with their unique physicochemical properties have been widely studied in a wide variety of biomedical programs, such as imaging, biosensing, hyperthermia, drug delivery, and tissue engineering. Core/shell hematite (α-Fe2O3)@Au nanotruffles were synthesized using Rosmarinus officinalis leaf extract as natural reducing agent. First, hematite (α-Fe2O3) nanoparticles were fabricated using phenolic compounds in rosemary extract. Then, the Au nanoparticles were coated on the α-Fe2O3 core nanoparticles without utilizing any intermediate material. The physicochemical properties of the Fe2O3@Au nanotruffles were evaluated by XRD, FTIR, FESEM-EDAX, HR-TEM, and XPS. Due to the specific morphology of Fe2O3@Au nanotruffles, the Au shell thickness varied from 2.3 to 9 nm in different regions. The toxicity of the core/shell α-Fe2O3@Au nanotruffles with different concentrations was evaluated on human glioblastoma U87 cancer and PC12 pheochromocytoma cells based on MTT assay. The results established that these nanotruffles had the high toxicity effect on glioblastoma cells at a concentration of 500 μg/mL. Additionally, their antibacterial was evaluated on Escherichia coli and Streptococcus mutans by using the dilution method. [ABSTRACT FROM AUTHOR]

Purpose: Peritoneal carcinomatosis (PC) presents a major challenge in the treatment of late-stage, solid tumors, with traditional therapies limited by poor drug penetration. We evaluated a novel hyperthermic pressurized intraperitoneal aerosol chemotherapy (HPIPAC) system using a human abdominal cavity model for its efficacy against AGS gastric cancer cells. Materials and Methods: A model simulating the human abdominal cavity and AGS gastric cancer cell line cultured dishes were used to assess the efficacy of the HPIPAC system. Cell viability was measured to evaluate the impact of HPIPAC under 6 different conditions: heat alone, PIPAC with paclitaxel (PTX), PTX alone, normal saline (NS) alone, heat with NS, and HPIPAC with PTX. Results: Results showed a significant reduction in cell viability with HPIPAC combined with PTX, indicating enhanced cytotoxic effects. Immediately after treatment, the average cell viability was 66.6%, which decreased to 49.2% after 48 hours and to a further 19.6% after 120 hours of incubation, demonstrating the sustained efficacy of the treatment. In contrast, control groups exhibited a recovery in cell viability; heat alone showed cell viability increasing from 90.8% to 94.4%, PIPAC with PTX from 82.7% to 89.7%, PTX only from 73.3% to 74.8%, NS only from 90.9% to 98.3%, and heat with NS from 74.4% to 84.7%. Conclusions: The HPIPAC system with PTX exhibits a promising approach in the treatment of PC in gastric cancer, significantly reducing cell viability. Despite certain limitations, this study highlights the system’s potential to enhance treatment outcomes. Future efforts should focus on refining HPIPAC and validating its effectiveness in clinical settings. [ABSTRACT FROM AUTHOR]

Breast cancer (BC) cells characteristics have played a crucial role in clinical strategies decisionmaking and patient outcome prediction. Although there have been several studies examining this, there are still relatively few that analyze stem cell subpopulations or protein biomarkers. The conducted research sought to elucidate breast cancer subtype characteristics based on immunohistochemical analysis and cancer stem cell presence based on flow cytometry analysis in three breast cancer samples from Bandung, Indonesia. A flow cytometry analysis was conducted to determine how much CD44/CD24 was expressed as a marker of cancer stem cell in isolated BC cells using various cell references, such as cell lines of SKBR-3, MDA-MB-468, and Hs587T. Immunohistochemistry analysis was carried out to investigate the expression of p53, HER2, PGR, Ki67, and ER in sample tissue sections. According to histological analysis, 2 samples were solid mammary carcinoma, and 1 sample was mammary fibroadenoma. Immunohistochemistry and histological analysis showed that all BC tissues are classified as Luminal B. In addition, a large percentage of CD44+/CD24- subpopulation was found in isolated BC cells (> 90 % in patients 1 and 3; > 60 % in patient 2). All samples showed similarity to Hs587T cell line characteristic. This study has successfully characterized solid mammary tumors and mammary fibroadenoma with luminal B characteristics. All specimens contained a high proportion of cancer stem cells. [ABSTRACT FROM AUTHOR]

Introduction: Osteosarcoma (OS) is a rare and aggressive form of bone cancer that primarily affects the long bones of the body, such as the arms and legs. It is characterized by the uncontrolled growth of malignant cells in the bone tissue, leading to the formation of abnormal and painful bone masses. Steviol glycosides have been widely used as natural noncalorie sweeteners and are the collective name of the sweet substances found naturally in the plant Stevia rebaudiana, which is commonly called Stevia. Our study aimed to analyze the anticancer activity of Stevioside in OS. Materials and Methods: Stevioside was applied to OS cells, and the levels of Bcl xL, Bcl-2, and Bax were then estimated. The results of three separate studies, each carried out in triplicate, were expressed as the mean ± standard errors of the mean (SEM). One-way ANOVA was used for statistical analysis. Results: The findings showed that the effect of Stevioside on sarcoma osteogenic cells with mean ± SEM as 0.74 ± 0.05, 0.69 ± 0.09, 0.46 ± 0.09 for Bcl-xL gene, 0.98 ± 0.06, 0.58 ± 0.07, 0.5 ± 0.07 for Bcl-2 gene, and 1.2 ± 0.08, 1.45 ± 0.11, 1.67 ± 0.12 for Bax gene, respectively, when treated with untreated control cells. Conclusion: The study concludes its action against bone OS cells was significant with apoptotic induction. Stevia has a wide range of health benefits as well as being a plant-based diet it has less of side effects and promoting features even by intaking it daily along with other medicines. [ABSTRACT FROM AUTHOR]

Background: Exposure to particulate matter (PM) and house dust mite (HDM) can change the expression patterns of inflammation-, oxidative stress-, and cell death-related genes. We investigated the changes in gene expression patterns owing to PM exposure. Objective: This study examined the changes in gene expression patterns following PM exposure. Methods: We searched for differentially expressed genes (DEGs) following PM exposure using five cell line-based RNA-seq or microarray datasets and six human-derived datasets. The enrichment terms of the DEGs were assessed. Results: DEG analysis yielded two gene sets. Thus, enrichment analysis was performed for each gene set, and the enrichment terms related to respiratory diseases were presented. The intersection of six human-derived datasets and two gene sets was obtained, and the expression patterns following PM exposure were observed. Conclusions: Two gene sets were obtained for cells treated with PM and their expression patterns were presented following verification in human-derived cells. Our findings suggest that exposure to PM2.5 and HDM may reveal changes in genes that are associated with diseases, such as allergies, highlighting the importance of mitigating PM2.5 and HDM exposure for disease prevention. Highlights: Two gene sets with differences in expression were obtained after the treatment of cells with PM. Enrichment terms for each gene set were extracted. DEGs in patient-derived cells after PM treatment were compared with those in cell lines. When PBMC-derived cells were treated with PM, a relatively high overlap of DEGs was found. [ABSTRACT FROM AUTHOR]

A human ovarian clear cell carcinoma cell line was established from a 46-year-old Japanese woman. That line, designated MTC-22, has proliferated continuously for over 6 months in conventional RPMI 1640 medium supplemented with 10% foetal bovine serum and has been passaged over 50 times. MTC-22 doubling-time is ~ 18 h, which is much shorter than most ovarian clear cell carcinoma lines reported to date. Morphologically, MTC-22 cells exhibit polygonal shapes and proliferate to form a monolayer in a jigsaw puzzle-like arrangement without contact inhibition. Ultrastructurally, cells exhibit numerous intracytoplasmic glycogen granules and well-developed mitochondria. G-band karyotype analysis indicated that cells have a complex karyotype close to tetraploid. We observed that the expression pattern of a series of ovarian carcinoma-related molecules in MTC-22 cells was identical to that seen in the patient's tumour tissue. Notably, MTC-22 cells, and the patient's carcinoma tissue, expressed low-sulphated keratan sulphate recognised by R-10G and 294-1B1 monoclonal antibodies, a hallmark of non-mucinous ovarian carcinoma, and particularly of clear cell ovarian carcinoma. Moreover, characteristic point mutations—one in ARID1A, which encodes the AT-rich interaction domain containing protein 1A, and the other in PIK3CB, which encodes the catalytic subunit of phosphoinositide 3-kinase—were seen in the patient's tumour tissue and retained in MTC-22 cells. Collectively, these findings indicate that MTC-22 cells could serve as a valuable tool for investigating the pathophysiology of ovarian clear cell carcinoma, particularly that harbouring PIK3CB mutations, and for developing and validating new diagnostic and therapeutic approaches to this life-threatening malignancy. [ABSTRACT FROM AUTHOR]

Immunodeficient murine models are usually used as the preclinical models of osteosarcoma. Such models do not effectively simulate the process of tumorigenesis and metastasis. Establishing a suitable animal model for understanding the mechanism of osteosarcoma and the clinical translation is indispensable. The UMR-106 cell suspension was injected into the marrow cavity of Balb/C nude mice. Tumor masses were harvested from nude mice and sectioned. The tumor fragments were transplanted into the marrow cavities of SD rats immunosuppressed with cyclosporine A. Through muti-rounds selection in SD rats, we constructed orthotopic osteosarcoma animal models using rats with intact immune systems. The primary tumor cells were cultured in-vitro to obtain the immune-tolerant cell line. VX2 tumor fragments were transplanted into the distal femur and parosteal radius of New Zealand white rabbit to construct orthotopic osteosarcoma animal models in rabbits. The rate of tumor formation in SD rats (P1 generation) was 30%. After four rounds of selection and six rounds of acclimatization in SD rats with intact immune systems, we obtained immune-tolerant cell lines and established the orthotopic osteosarcoma model of the distal femur in SD rats. Micro-CT images confirmed tumor-driven osteolysis and the bone destruction process. Moreover, the orthotopic model was also established in New Zealand white rabbits by implanting VX2 tumor fragments into rabbit radii and femurs. We constructed orthotopic osteosarcoma animal models in rats with intact immune systems through muti-rounds in-vivo selection and the rabbit osteosarcoma model. [ABSTRACT FROM AUTHOR]

Simple Summary: Simple Summary: In this study, a new cell line derived from red hybrid tilapia brain tissue, RHTiB, was established. This fibroblast-like cell line was maintained for over 50 passages with optimal growth at 25 °C in Leibovitz-15 medium with 10% fetal bovine serum at pH 7.4. Genetic and chromosomal analyses confirmed its origin from red hybrid tilapia. Additionally, immunofluorescence and RT-qPCR tests revealed successful TiLV propagation in RHTiB cell lines. The development of this novel cell line offers valuable prospects in enhancing diagnostic techniques in red hybrid tilapia. Tilapia lake virus (TiLV) presents a substantial threat to global tilapia production. Despite the development of numerous cell lines for TiLV isolation and propagation, none have been specifically derived from red hybrid tilapia (Oreochromis spp.). In this study, we successfully established a new cell line, RHTiB, from the red hybrid tilapia brain. RHTiB cells were cultured for 1.5 years through over 50 passages and demonstrated optimal growth at 25 °C in Leibovitz-15 medium supplemented with 10% fetal bovine serum at pH 7.4. Morphologically, RHTiB cells displayed a fibroblast-like appearance, and cytochrome oxidase I gene sequencing confirmed their origin from Oreochromis spp. Mycoplasma contamination testing yielded negative results. The revival rate of the cells post-cryopreservation was observed to be between 75 and 80% after 30 days. Chromosomal analysis at the 25th passage revealed a diploid count of 22 pairs (2n = 44). While no visible cytopathic effects were observed, both immunofluorescence microscopy and RT-qPCR analysis demonstrated successful TiLV propagation in the RHTiB cell line, with a maximum TiLV concentration of 107.82 ± 0.22 viral copies/400 ng cDNA after 9 days of incubation. The establishment of this species-specific cell line represents a valuable advancement in the diagnostic and isolation tools for viral diseases potentially impacting red hybrid tilapia. [ABSTRACT FROM AUTHOR]

Capmatinib and savolitinib, selective MET inhibitors, are widely used to treat various MET-positive cancers. In this study, we aimed to determine the effects of these inhibitors on MET-amplified gastric cancer (GC) cells. Methods: After screening 37 GC cell lines, the following cell lines were found to be MET-positive with copy number variation >10: SNU-620, ESO51, MKN-45, SNU-5, and OE33 cell lines. Next, we assessed the cytotoxic response of these cell lines to capmatinib or savolitinib alone using cell counting kit-8 and clonogenic cell survival assays. Western blotting was performed to assess the effects of capmatinib and savolitinib on the MET signaling pathway. Xenograft studies were performed to evaluate the in vivo therapeutic efficacy of savolitinib in MKN-45 cells. Savolitinib and capmatinib exerted anti-proliferative effects on MET-amplified GC cell lines in a dose-dependent manner. Savolitinib inhibited the phosphorylation of MET and downstream signaling pathways, such as the protein kinase B (AKT) and extracellular signal-regulated kinase (ERK) pathways, in MET-amplified GC cells. Additionally, savolitinib significantly decreased the number of colonies formed on the soft agar and exerted dose-dependent anti-tumor effects in an MKN-45 GC cell xenograft model. Furthermore, a combination of trastuzumab and capmatinib exhibited enhanced inhibition of AKT and ERK activation in human epidermal growth factor receptor-2 (HER2)- and MET-positive OE33 cells. Targeting MET with savolitinib and capmatinib efficiently suppressed the growth of MET-amplified GC cells. Moreover, these MET inhibitors exerted synergistic effects with trastuzumab on HER2- and MET-amplified GC cells. [ABSTRACT FROM AUTHOR]

Interference in cell cycle progression has been noted as one of the important properties of anticancer drugs. In this study, we developed the cell cycle prediction model using high‐content imaging data of recipient cells after drug exposure and DNA‐staining with a low‐toxic DNA dye, SiR‐DNA. For this purpose, we exploited HeLa and MCF7 cells introduced with a fluorescent ubiquitination‐based cell cycle indicator (Fucci). Fucci‐expressing cancer cells were subjected to high‐content imaging analysis using OperettaCLS after 36‐h exposure to anticancer drugs; the nuclei were segmented, and the morphological and intensity properties of each nucleus characterized by SiR‐DNA staining were calculated using imaging analysis software, Harmony. For the use of training, we classified cells into each phase of the cell cycle using the Fucci system. Training data (n = 7500) and validation data (n = 2500) were randomly sampled and the binary classification prediction models for G1, early S, and S/G2/M phases of the cell cycle were developed using four supervised machine learning algorithms. We selected random forest as the model with the best performance through 10‐fold cross‐validation; the accuracy rate was approximately 75%–87%. Regarding feature importance, variables expected to be biologically related to the cell cycle, for example, signal intensity and nuclear size, were highly ranked, suggesting the validity of the model. These results showed that the cell cycle can be predicted in cancer cells by simply exploiting the current prediction model using fluorescent images of DNA‐staining dye, and the model could be applied for the use of future ex vivo drug sensitivity diagnosis. [ABSTRACT FROM AUTHOR]

The murine cell line MH-22A was originally established at least 50 years ago from a non-metastasizing tumor induced by 3-methylcholanthrene in C3HA mice and further adapted for in vitro growth. MH-22A cells grow predominantly epithelioid and they lead to the formation of transplantable tumors when injected subcutaneously into mice. In silico translation of genome data of this nearly triploid cell line to the human genome has recently characterized MH-22A as a suitable model for hepatoblastoma or early hepatocellular carcinoma. Here we established a multi-loci short tandem repeat signature for this cell line that allows quick and reliable cell line authentication. [ABSTRACT FROM AUTHOR]

Simple Summary: Enzootic nasal adenocarcinoma is an infectious, chronic tumor disease of goats and sheep. Tumor cell lines are very important tools in tumor research. This manuscript describes the isolation, culture, and characterization of a new enzootic nasal adenocarcinoma cell line from goats. This is the first report about an enzootic nasal adenocarcinoma cell line. ENA-1 cells are stable in passaging, have strong proliferative potential and tumorigenicity in nude mice, express primitive tumor phenotypes, and carry enzootic nasal tumor virus. Enzootic nasal adenocarcinoma (ENA) is a contagious tumor disease of goats and sheep, which is caused by enzootic nasal tumor virus (ENTV). To better understand the pathogenesis of ENA, this study aimed to establish a goat ENA cell line (ENA-1). The cells have been characterized with regard to morphology, growth rate, ultrastructural features, chromosome number, expression of CK7 and CK18, tumorigenicity, species, and mycoplasma contamination. ENA-1 had an epithelioid cell morphology with an unstable chromosome number under a light microscope. Under an electron microscope, the cell nuclear heterogeneity was not obvious, and there were more intermediate filaments and a small number of immature retrovirus-like particles in the cytoplasm. ENA-1 had strong proliferative potential, and the cell multiplication time was about 36 h, which could make BALB/c nude mice develop tumors. CK7 and CK18 were expressed in the cytoplasm of primary goat tumors, in transplanted tumors from nude mice, and un ENA-1 cells with the same intensity. PCR revealed that ENA-1 continuously carried ENTV-2 up to the 17th generation with no germline contamination or mycoplasma contamination. In conclusion, using a serum-containing culture system, ENA-1 cells were successfully isolated, cultured, and purified from goat tumor tissues. The isolated ENA-1 cells retained robust proliferation potential and maintained their phenotype, indicating the potential application of the ENA-1 cell line as an in vitro model of ENA. [ABSTRACT FROM AUTHOR]

CRISPR-Cas9 genome editing has promising therapeutic potential for genetic diseases and cancers, but safety could be a concern. Here we use whole genomic analysis by 10x linked-read sequencing and optical genome mapping to interrogate the genome integrity after editing and in comparison to four parental cell lines. In addition to the previously reported large structural variants at on-target sites, we identify heretofore unexpected large chromosomal deletions (91.2 and 136 Kb) at atypical non-homologous off-target sites without sequence similarity to the sgRNA in two edited lines. The observed large structural variants induced by CRISPR-Cas9 editing in dividing cells may result in pathogenic consequences and thus limit the usefulness of the CRISPR-Cas9 editing system for disease modeling and gene therapy. In this work, our whole genomic analysis may provide a valuable strategy to ensure genome integrity after genomic editing to minimize the risk of unintended effects in research and clinical applications.

Perineural invasion (PNI) is the phenomenon whereby cancer cells invade the space surrounding nerves. PNI occurs frequently in epithelial malignancies, but is especially characteristic of pancreatic ductal adenocarcinoma (PDAC). The presence of PNI portends an increased incidence of local recurrence, metastasis and poorer overall survival. While interactions between tumor cells and nerves have been investigated, the etiology and initiating cues for PNI development is not well understood. Here, we used digital spatial profiling to reveal changes in the transcriptome and to allow for a functional analysis of neural-supportive cell types present within the tumor-nerve microenvironment of PDAC during PNI. We found that hypertrophic tumor-associated nerves within PDAC express transcriptomic signals of nerve damage including programmed cell death, Schwann cell proliferation signaling pathways, as well as macrophage clearance of apoptotic cell debris by phagocytosis. Moreover, we identified that neural hypertrophic regions have increased local neuroglial cell proliferation which was tracked using EdU tumor labeling in KPC mice, as well as frequent TUNEL positivity, suggestive of a high turnover rate. Functional calcium imaging studies using human PDAC organotypic slices confirmed nerve bundles had neuronal activity, as well as contained NGFR+ cells with high sustained calcium levels, which are indicative of apoptosis. This study reveals a common gene expression pattern that characterizes solid tumor-induced damage to local nerves. These data provide new insights into the pathobiology of the tumor-nerve microenvironment during PDAC as well as other gastrointestinal cancers.

The therapeutic efficacy of metformin in prostate cancer (PCa) appears uncertain based on various clinical trials. Metformin treatment failure may be attributed to the high frequency of transcriptional dysregulation, which leads to drug resistance. However, the underlying mechanism is still unclear. In this study, we found evidences that metformin resistance in PCa cells may be linked to cell cycle reactivation. Super-enhancers (SEs), crucial regulatory elements, have been shown to be associated with drug resistance in various cancers. Our analysis of SEs in metformin-resistant (MetR) PCa cells revealed a correlation with Prostaglandin Reductase 1 (PTGR1) expression, which was identified as significantly increased in a cluster of cells with metformin resistance through single-cell transcriptome sequencing. Our functional experiments showed that PTGR1 overexpression accelerated cell cycle progression by promoting progression from the G0/G1 to the S and G2/M phases, resulting in reduced sensitivity to metformin. Additionally, we identified key transcription factors that significantly increase PTGR1 expression, such as SRF and RUNX3, providing potential new targets to address metformin resistance in PCa. In conclusion, our study sheds new light on the cellular mechanism underlying metformin resistance and the regulation of the SE-TFs-PTGR1 axis, offering potential avenues to enhance metformins therapeutic efficacy in PCa.

Pancreatic ductal adenocarcinoma (PDAC) is an insidious disease with a low 5-year survival rate. PDAC is characterized by infiltration of abundant tumor-associated macrophages (TAM), which promote immune tolerance and immunotherapeutic resistance. Here we report that macrophage spleen tyrosine kinase (Syk) promotes PDAC growth and metastasis. In orthotopic PDAC mouse models, genetic deletion of myeloid Syk reprogrammed macrophages into immunostimulatory phenotype, increased the infiltration, proliferation, and cytotoxicity of CD8+ T cells, and repressed PDAC growth and metastasis. Furthermore, gemcitabine (Gem) treatment induced an immunosuppressive microenvironment in PDAC by promoting protumorigenic polarization of macrophages. In contrast, treatment with the FDA-approved Syk inhibitor R788 (fostamatinib) remodeled the tumor immune microenvironment, "re-educated" protumorigenic macrophages towards an immunostimulatory phenotype and boosted CD8+ T-cell responses in Gem-treated PDAC in orthotopic mouse models and an ex vivo human pancreatic slice culture model. These findings illustrate the potential of Syk inhibition for enhancing the antitumor immune responses in PDAC and support the clinical evaluation of R788 either alone or together with Gem as a potential treatment strategy for PDAC.SignificanceSyk blockade induces macrophage polarization to an immunostimulatory phenotype, which enhances CD8+ T-cell responses and improves gemcitabine efficacy in pancreatic ductal adenocarcinoma, a clinically challenging malignancy.

TGFβ signaling is associated with non-response to immune checkpoint blockade in patients with advanced cancers, particularly in the immune-excluded phenotype. While previous work demonstrates that converting tumors from excluded to inflamed phenotypes requires attenuation of PD-L1 and TGFβ signaling, the underlying cellular mechanisms remain unclear. Here, we show that TGFβ and PD-L1 restrain intratumoral stem cell-like CD8 T cell (TSCL) expansion and replacement of progenitor-exhausted and dysfunctional CD8 T cells with non-exhausted T effector cells in the EMT6 tumor model in female mice. Upon combined TGFβ/PD-L1 blockade IFNγhi CD8 T effector cells show enhanced motility and accumulate in the tumor. Ensuing IFNγ signaling transforms myeloid, stromal, and tumor niches to yield an immune-supportive ecosystem. Blocking IFNγ abolishes the anti-PD-L1/anti-TGFβ therapy efficacy. Our data suggest that TGFβ works with PD-L1 to prevent TSCL expansion and replacement of exhausted CD8 T cells, thereby maintaining the T cell compartment in a dysfunctional state.

Matrix stiffening is ubiquitous in solid tumors and can direct epithelial-mesenchymal transition (EMT) and cancer cell migration. Stiffened niche can even cause poorly invasive oral squamous cell carcinoma (OSCC) cell lines to acquire a less adherent, more migratory phenotype, but mechanisms and durability of this acquired "mechanical memory" are unclear. Here, we observed that contractility and its downstream signals could underlie memory acquisition; invasive SSC25 cells overexpress myosin II (vs. noninvasive Cal27 cells) consistent with OSCC. However, prolonged exposure of Cal27 cells to a stiff niche or contractile agonists up-regulated myosin and EMT markers and enabled them to migrate as fast as SCC25 cells, which persisted even when the niche softened and indicated "memory" of their prior niche. Stiffness-mediated mesenchymal phenotype acquisition required AKT signaling and was also observed in patient samples, whereas phenotype recall on soft substrates required focal adhesion kinase (FAK) activity. Phenotype durability was further observed in transcriptomic differences between preconditioned Cal27 cells cultured without or with FAK or AKT antagonists, and such transcriptional differences corresponded to discrepant patient outcomes. These data suggest that mechanical memory, mediated by contractility via distinct kinase signaling, may be necessary for OSCC to disseminate.

Prostate cancer is initially regulated by the androgen receptor (AR), a ligand-activated, transcription factor, and is in a hormone-dependent state (hormone-sensitive prostate cancer (HSPC)), but eventually becomes androgen-refractory (castration-resistant prostate cancer (CRPC)) because of mechanisms that bypass the AR, including by activation of ErbB3, a member of the epidermal growth factor receptor family. ErbB3 is synthesized in the cytoplasm and transported to the plasma membrane for ligand binding and dimerization, where it regulates downstream signaling, but nuclear forms are reported. Here, we demonstrate in prostatectomy samples that ErbB3 nuclear localization is observed in malignant, but not benign prostate, and that cytoplasmic (but not nuclear) ErbB3 correlated positively with AR expression but negatively with AR transcriptional activity. In support of the latter, androgen depletion upregulated cytoplasmic, but not nuclear ErbB3, while in vivo studies showed that castration suppressed ErbB3 nuclear localization in HSPC, but not CRPC tumors. In vitro treatment with the ErbB3 ligand heregulin-1β (HRG) induced ErbB3 nuclear localization, which was androgen-regulated in HSPC but not in CRPC. In turn, HRG upregulated AR transcriptional activity in CRPC but not in HSPC cells. Positive correlation between ErbB3 and AR expression was demonstrated in AR-null PC-3 cells where stable transfection of AR restored HRG-induced ErbB3 nuclear transport, while AR knockdown in LNCaP reduced cytoplasmic ErbB3. Mutations of ErbB3's kinase domain did not affect its localization but was responsible for cell viability in CRPC cells. Taken together, we conclude that AR expression regulated ErbB3 expression, its transcriptional activity suppressed ErbB3 nuclear translocation, and HRG binding to ErbB3 promoted it.

Many anticancer drugs exhibit high systemic off-target toxicities causing severe side effects. Peptide-drug conjugates (PDCs) that target tumor-specific receptors such as integrin αvβ6 are emerging as powerful tools to overcome these challenges. The development of an integrin αvβ6-selective PDC was achieved by combining the therapeutic efficacy of the cytotoxic drug monomethyl auristatin E with the selectivity of the αvβ6-binding peptide (αvβ6-BP) and with the ability of positron emission tomography (PET) imaging by copper-64. The [64Cu]PDC-1 was produced efficiently and in high purity. The PDC exhibited high human serum stability, integrin αvβ6-selective internalization, cell binding, and cytotoxicity. Integrin αvβ6-selective tumor accumulation of the [64Cu]PDC-1 was visualized with PET-imaging and corroborated by biodistribution, and [64Cu]PDC-1 showed promising in vivo pharmacokinetics. The [natCu]PDC-1 treatment resulted in prolonged survival of mice bearing αvβ6 (+) tumors (median survival: 77 days, vs αvβ6 (-) tumor group 49 days, and all other control groups 37 days).

MYC transcription factors have critical roles in facilitating a variety of cellular functions that have been highly conserved among species during evolution. However, despite circumstantial evidence for an involvement of MYC in animal osmoregulation, mechanistic links between MYC function and osmoregulation are missing. Mozambique tilapia (Oreochromis mossambicus) represents an excellent model system to study these links because it is highly euryhaline and highly tolerant to osmotic (salinity) stress at both the whole organism and cellular levels of biological organization. Here, we utilize an O. mossambicus brain cell line and an optimized vector-based CRISPR/Cas9 system to functionally disrupt MYC in the tilapia genome and to establish causal links between MYC and cell functions, including cellular osmoregulation. A cell isolation and dilution strategy yielded polyclonal myca (a gene encoding MYC) knockout (ko) cell pools with low genetic variability and high gene editing efficiencies (as high as 98.2%). Subsequent isolation and dilution of cells from these pools produced a myca ko cell line harboring a 1-bp deletion that caused a frameshift mutation. This frameshift functionally inactivated the transcriptional regulatory and DNA-binding domains predicted by bioinformatics and structural analyses. Both the polyclonal and monoclonal myca ko cell lines were viable, propagated well in standard medium, and differed from wild-type cells in morphology. As such, they represent a new tool for causally linking myca to cellular osmoregulation and other cell functions.

Targeting common oncogenic drivers of glioblastoma multiforme (GBM) in patients has remained largely ineffective, raising the possibility that alternative pathways may contribute to tumor aggressiveness. Here we demonstrate that Vangl1 and Fzd7, components of the non-canonical Wnt planar cell polarity (Wnt/PCP) signaling pathway, promote GBM malignancy by driving cellular proliferation, migration, and invasiveness, and engage Rho GTPases to promote cytoskeletal rearrangements and actin dynamics in migrating GBM cells. Mechanistically, we uncover the existence of a novel Vangl1/Fzd7 complex at the leading edge of migrating GBM cells and propose that this complex is critical for the recruitment of downstream effectors to promote tumor progression. Moreover, we observe that depletion of FZD7 results in a striking suppression of tumor growth and latency and extends overall survival in an intracranial mouse xenograft model. Our observations support a novel mechanism by which Wnt/PCP components Vangl1 and Fzd7 form a complex at the leading edge of migratory GBM cells to engage downstream effectors that promote actin cytoskeletal rearrangements dynamics. Our findings suggest that interference with Wnt/PCP pathway function may offer a novel therapeutic strategy for patients diagnosed with GBM.

In continuation of our previous efforts for the development of potent small molecules against brain cancer, herein we synthesized seventeen new compounds and tested their anti-gliomapotential against established glioblastoma cell lines, namely, D54MG, U251, and LN-229 as well as patient derived cell lines (DB70 and DB93). Among them, the carboxamide derivatives, BT-851 and BT-892 were found to be the most active leads in comparison to our established hit compound BT#9.The SAR studies of our hit BT#9 compound resulted in the development of two new lead compounds by hit to lead strategy. The detailed biological studies are currently underway. The active compounds could possibly act as template for the future development of newer anti-glioma agents.

225Ac-targeted α-radiotherapy is a promising approach to treating malignancies, including prostate cancer. However, α-emitting isotopes are difficult to image because of low administered activities and a low fraction of suitable γ-emissions. The in vivo generator 134Ce/134La has been proposed as a potential PET imaging surrogate for the therapeutic nuclides 225Ac and 227Th. In this report, we detail efficient radiolabeling methods using the 225Ac-chelators DOTA and MACROPA. These methods were applied to radiolabeling of prostate cancer imaging agents, including PSMA-617 and MACROPA-PEG4-YS5, for evaluation of their in vivo pharmacokinetic characteristics and comparison to the corresponding 225Ac analogs. Methods: Radiolabeling was performed by mixing DOTA/MACROPA chelates with 134Ce/134La in NH4OAc, pH 8.0, at room temperature, and radiochemical yields were monitored by radio-thin-layer chromatography. In vivo biodistributions of 134Ce-DOTA/MACROPA.NH2 complexes were assayed through dynamic small-animal PET/CT imaging and ex vivo biodistribution studies over 1 h in healthy C57BL/6 mice, compared with free 134CeCl3 In vivo, preclinical imaging of 134Ce-PSMA-617 and 134Ce-MACROPA-PEG4-YS5 was performed on 22Rv1 tumor-bearing male nu/nu-mice. Ex vivo biodistribution was performed for 134Ce/225Ac-MACROPA-PEG4-YS5 conjugates. Results: 134Ce-MACROPA.NH2 demonstrated near-quantitative labeling with 1:1 ligand-to-metal ratios at room temperature, whereas a 10:1 ligand-to-metal ratio and elevated temperatures were required for DOTA. Rapid urinary excretion and low liver and bone uptake were seen for 134Ce/225Ac-DOTA/MACROPA. NH2 conjugates in comparison to free 134CeCl3 confirmed high in vivo stability. An interesting observation during the radiolabeling of tumor-targeting vectors PSMA-617 and MACROPA-PEG4-YS5-that the daughter 134La was expelled from the chelate after the decay of parent 134Ce-was confirmed through radio-thin-layer chromatography and reverse-phase high-performance liquid chromatography. Both conjugates, 134Ce-PSMA-617 and 134Ce-MACROPA-PEG4-YS5, displayed tumor uptake in 22Rv1 tumor-bearing mice. The ex vivo biodistribution of 134Ce-MACROPA.NH2, 134Ce-DOTA and 134Ce-MACROPA-PEG4-YS5 corroborated well with the respective 225Ac-conjugates. Conclusion: These results demonstrate the PET imaging potential for 134Ce/134La-labeled small-molecule and antibody agents. The similar 225Ac and 134Ce/134La-chemical and pharmacokinetic characteristics suggest that the 134Ce/134La pair may act as a PET imaging surrogate for 225Ac-based radioligand therapies.

Although induction of differentiation represents an effective strategy for neuroblastoma treatment, the mechanisms underlying neuroblastoma differentiation are poorly understood. We generated a computational model of neuroblastoma differentiation consisting of interconnected gene clusters identified based on symmetric and asymmetric gene expression relationships. We identified a differentiation signature consisting of series of gene clusters comprised of 1251 independent genes that predicted neuroblastoma differentiation in independent datasets and in neuroblastoma cell lines treated with agents known to induce differentiation. This differentiation signature was associated with patient outcomes in multiple independent patient cohorts and validated the role of MYCN expression as a marker of neuroblastoma differentiation. Our results further identified novel genes associated with MYCN via asymmetric Boolean implication relationships that would not have been identified using symmetric computational approaches and that were associated with both neuroblastoma differentiation and patient outcomes. Our differentiation signature included a cluster of genes involved in intracellular signaling and growth factor receptor trafficking pathways that is strongly associated with neuroblastoma differentiation, and we validated the associations of UBE4B, a gene within this cluster, with neuroblastoma cell and tumor differentiation. Our findings demonstrate that Boolean network analyses of symmetric and asymmetric gene expression relationships can identify novel genes and pathways relevant for neuroblastoma tumor differentiation that could represent potential therapeutic targets.

MicroRNA-22 (miR-22) can be induced by beneficial metabolites that have metabolic and immune effects, including retinoic acids, bile acids, vitamin D3, and short-chain fatty acids. The tumor suppressor effects of miR-22 have been suggested, but whether miR-22 treats orthotopic hepatocellular carcinoma (HCC) is not established. The role of miR-22 in regulating tumor immunity is also poorly understood. Our data showed that miR-22 delivered by adeno-associated virus serotype 8 effectively treated HCC. Compared with FDA-approved lenvatinib, miR-22 produced better survival outcomes without noticeable toxicity. miR-22 silenced hypoxia-inducible factor 1 (HIF1α) and enhanced retinoic acid signaling in both hepatocytes and T cells. Moreover, miR-22 treatment improved metabolism and reduced inflammation. In the liver, miR-22 reduced the abundance of IL17-producing T cells and inhibited IL17 signaling by reducing the occupancy of HIF1α in the Rorc and Il17a genes. Conversely, increasing IL17 signaling ameliorated the anti-HCC effect of miR-22. Additionally, miR-22 expanded cytotoxic T cells and reduced regulatory T cells (Treg). Moreover, depleting cytotoxic T cells also abolished the anti-HCC effects of miR-22. In patients, miR-22 high HCC had upregulated metabolic pathways and reduced IL17 pro-inflammatory signaling compared with miR-22 low HCC. Together, miR-22 gene therapy can be a novel option for HCC treatment.

PurposeSmall molecule inhibitors that target oncogenic driver kinases are an important class of therapies for non-small cell lung cancer (NSCLC) and other malignancies. However, these therapies are not without their challenges. Each inhibitor works on only a subset of patients, the pharmacokinetics of these inhibitors is variable, and these inhibitors are associated with significant side effects. Many of these inhibitors lack non-invasive biomarkers to confirm pharmacodynamic efficacy, and our understanding of how these inhibitors block cancer cell growth remains incomplete. Limited clinical studies suggest that early (

Sprouty-related EVH-1 domain-containing (SPRED) proteins are a family of proteins that negatively regulate the RAS-Mitogen-Activated Protein Kinase (MAPK) pathway, which is involved in the regulation of the mitogenic response and cell proliferation. However, the mechanism by which these proteins affect RAS-MAPK signaling has not been elucidated. Patients with mutations in SPRED give rise to unique disease phenotypes; thus, we hypothesized that distinct interactions across SPRED proteins may account for alternative nodes of regulation. To characterize the SPRED interactome and evaluate how members of the SPRED family function through unique binding partners, we performed affinity purification mass spectrometry. We identified 90-kDa ribosomal S6 kinase 2 (RSK2) as a specific interactor of SPRED2 but not SPRED1 or SPRED3. We identified that the N-terminal kinase domain of RSK2 mediates the interaction between amino acids 123 to 201 of SPRED2. Using X-ray crystallography, we determined the structure of the SPRED2-RSK2 complex and identified the SPRED2 motif, F145A, as critical for interaction. We found that the formation of this interaction is regulated by MAPK signaling events. We also find that this interaction between SPRED2 and RSK2 has functional consequences, whereby the knockdown of SPRED2 resulted in increased phosphorylation of RSK substrates, YB1 and CREB. Furthermore, SPRED2 knockdown hindered phospho-RSK membrane and nuclear subcellular localization. We report that disruption of the SPRED2-RSK complex has effects on RAS-MAPK signaling dynamics. Our analysis reveals that members of the SPRED family have unique protein binding partners and describes the molecular and functional determinants of SPRED2-RSK2 complex dynamics.

Pancreatic ductal adenocarcinoma (PDA) is a lethal disease notoriously resistant to therapy1,2. This is mediated in part by a complex tumour microenvironment3, low vascularity4, and metabolic aberrations5,6. Although altered metabolism drives tumour progression, the spectrum of metabolites used as nutrients by PDA remains largely unknown. Here we identified uridine as a fuel for PDA in glucose-deprived conditions by assessing how more than 175 metabolites impacted metabolic activity in 21 pancreatic cell lines under nutrient restriction. Uridine utilization strongly correlated with the expression of uridine phosphorylase 1 (UPP1), which we demonstrate liberates uridine-derived ribose to fuel central carbon metabolism and thereby support redox balance, survival and proliferation in glucose-restricted PDA cells. In PDA, UPP1 is regulated by KRAS-MAPK signalling and is augmented by nutrient restriction. Consistently, tumours expressed high UPP1 compared with non-tumoural tissues, and UPP1 expression correlated with poor survival in cohorts of patients with PDA. Uridine is available in the tumour microenvironment, and we demonstrated that uridine-derived ribose is actively catabolized in tumours. Finally, UPP1 deletion restricted the ability of PDA cells to use uridine and blunted tumour growth in immunocompetent mouse models. Our data identify uridine utilization as an important compensatory metabolic process in nutrient-deprived PDA cells, suggesting a novel metabolic axis for PDA therapy.