Background and PurposeIn this study, we investigate the plasticity of tumor-associated macrophages, which originate from circulating monocytes and are associated with poor cancer prognosis. The differentiation of monocytes into macrophages is a dynamic and spatiotemporal process, as is the resulting macrophages’ polarization. However, traditional methods for measuring polarization, such as qPCR and flow cytometry, provide only static information about polarization. To supplement these methods, we present a novel bioluminescent method that allows for time-resolved measurement of NFκB activation in macrophages while in co-culture with cancer cells. By using a monocyte cell line whose NFκB responsive element is labeled with firefly luciferase, we obtain a quantitative and temporal characterization of macrophage polarization in response to tumor-derived signals.Materials and MethodsTo quantify the effect of tumor cell signaling THP-1 monocytes encoded with a firefly luciferase NFκB response element were co-cultured with cancer cells. We investigated the impact of the following factors on NFκB signaling cancer cell type (HCT116 or MDA-MB-231), ratio of the number of cancer cells to macrophages in co-culture, and the THP-1 cell differentiation state (monocyte or monocyte-derived macrophage). Bioluminescence was measured over three days. Descriptive features of the bioluminescence response curves were then extracted to compare effects between cancer types.ResultsWe observed that the MDA-MB-231 cancer cells induced lower but more persistent NFκB activation in THP-1 monocyte-derived macrophages than was observed in HCT116 co-culture. Higher number of cancer cells (lower macrophage ratio) elicited higher AUC values in HCT116 co-culture compared to low cancer cell conditions. There was no difference between high and low macrophage ratios within the MDA-MB-231 co-culture condition. Moreover, the addition of macrophage differentiation stimuli modulated the NFκB profile in the co-culture. PMA-differentiated macrophages expressed higher and faster peaks of NFκB activation.ConclusionCancer cells can modulate monocyte/macrophage NFκB transcriptional activity, impacting the overall tumor microenvironment. Using NFκB reporter cells, we found that HCT116 colorectal cancer cells induced fast and strong NFκB activation profiles. In contrast, MDA-MB-231 cancer cells elicited lower but more persistent NFκB activation profiles. This study highlights how bioluminescence reporter assays can be used to extract meaningful metrics about monocyte/macrophage behavior during tumor progression. This approach could also be used to understand the crosstalk between cancer cells and monocytes/macrophages that could be useful in a therapeutic of diagnostic context.Graphical Supplementary InformationThe online version contains supplementary material available at 10.1007/s12195-025-00870-1.

The annulus fibrosus (AF), as an important component of the intervertebral disc (IVD), contributes to the structural integrity and functional normality of IVD. Degenerative disc diseases (DDD), due to AF impairment, are common problems that could lead to low back pain or neck pain, resulting in considerable disability and financial costs globally. The exact causes and underlying mechanisms of AF impairment, however, remain complex and unclear. A literature search was conducted to identify relevant articles published between 1952 and 2024. We summarize the current literature on the potential etiologies of AF damage, while also providing a brief overview of the basic characteristics of the AF and current therapeutic strategies for AF impairment. The findings suggest that several factors could induce or exacerbate AF impairment. We categorize them into distinct groups as physical and chemical stimuli, nutritional or metabolic disorders, immune and inflammatory responses, and genetic abnormalities. Various factors could lead to AF impairment, such as particular physical and chemical stimuli, nutritional or metabolic disorders, immune and inflammatory responses, and genetic abnormalities. Meanwhile, enhancing our understanding and management of AF impairment could help discover potential preventive or therapeutic interventions for DDD.

PurposeWe previously demonstrated that hyperosmotic stress, which acts as mechanical stress, induces autophagy of tubular epithelial cells. This study aims to elucidate the molecular mechanisms of hyperosmolarity-induced autophagy. The research question addresses how hyperosmotic stress activates autophagy through transcription factor EB (TFEB) and Ca2+ signaling pathways, contributing to understanding cellular responses to mechanical stress.MethodsNRK-52E normal rat kidney cells were subjected to hyperosmotic stress using mannitol-containing medium. Fluorescence microscopy was utilized to observe TFEB nuclear translocation, a crucial event in autophagy regulation. An intracellular Ca2+ chelator, BAPTA-AM, and a calcineurin inhibitor were used to dissect the Ca2+ signaling pathway involved in TFEB translocation. The phosphorylation of p70S6K, a substrate of the mammalian target of rapamycin complex 1 kinase, was analyzed to explore its role in TFEB localization. Additionally, the function of transient receptor potential mucolipin 1 (TRPML1), an intracellular Ca2+ channel, was assessed using pharmacological inhibition to determine its impact on TFEB translocation and autophagy marker LC3-II levels.ResultsMannitol-induced hyperosmotic stress promoted the nuclear translocation of TFEB, which was completely abolished by treatment with BAPTA-AM. Inhibition of calcineurin suppressed TFEB nuclear translocation under hyperosmolarity, indicating that a signaling pathway governed by intracellular Ca2+ is involved in TFEB’s nuclear translocation. In contrast, hyperosmotic stress did not significantly alter p70S6K phosphorylation. Pharmacological inhibition of TRPML1 attenuated both TFEB nuclear translocation and LC3-II upregulation in response to hyperosmotic stress.ConclusionsHyperosmotic stress promotes TFEB nuclear localization, and TRPML1-induced activation of calcineurin is involved in the mechanism of hyperosmolarity-induced autophagy.

Scarring from traumatic injury, burns, and other complications remains a significant problem that diminishes quality of life for millions of people worldwide. A common target for the development of new therapies to promote healing and reduce scarring are myofibroblasts because of their central role in pathological scarring. Recent work indicates that adipocyte lineage cells also contribute to the wound healing process,including clinical reports that indicate that the placement of autologous adipose micrografts at the surgical site improves the appearance and pliability of existing scars. To better understand how adipocyte lineage cells interact with fibroblasts to promote healing, we firstutilized anin vitromodel of wound healing to visualize fibroblast spheroid collagen deposition via time-lapse imaging. We then introduced pre-adipocyte and adipocyte spheroids to visualize pair-wise spheroid interactions and collagen deposition among all three cell types. Finally, we quantified differences in the extracellular matrix (ECM) proteins produced using liquid chromatography with tandem mass spectrometry (LC-MS/MS). We found that all three cell-types contribute to ECM deposition and that the composition of the ECM proteins, or matrisome, was significantly different depending on which cells were co-cultured together. By better understanding the interactions among these cell types, novel adipose-tissue-based therapeutic approaches can be developed to improve wound healing and reduce scar tissue. The online version contains supplementary material available at 10.1007/s12195-024-00829-8.

Kidney cancer is a significant global health problem that affects nearly 1 in 25 of cancer patients. Prevalence, morbidity and mortality data associated with kidney cancer continue to increase every year, revealing a heavy economic and social burden. Organoid culture is a new research tool with great potential for many applications, particularly in cancer research. The integration of organoids with other emerging technologies has simultaneously expanded their potential applications. However, there is no thorough assessment of organoids in the field of renal cancer research. This paper presents a comprehensive review of the current development of renal cancer organoids and discusses the corresponding solutions and future directions of renal cancer organoids. In this study, we have compared the operating procedures of different organoid culture protocols and proposed a summary of constituents in culture media. Extensive discussions of renal cancer organoids, including generation and maintenance approaches, application scenarios, current challenges and prospects, have also been made. The information required for this study is extracted from literature databases such as PubMed, SCOPUS and Web of Science. In this article, we systematically review thirteen successful methods for generating organoids to kidney cancer and provide practical guidelines for their construction as a reference. In addition, we also elucidate the clinical application of organoids, address the existing challenges and limitations, and highlight promising prospects. Ultimately, we firmly believe that as kidney tumour organoids continue to develop and improve, they will become a crucial tool for treating kidney cancer.

Fibroblasts, an abundant cell type in the breast tumor microenvironment, interact with cancer cells and orchestrate tumor progression and drug resistance. However, the mechanisms by which fibroblast-derived factors impact drug sensitivity remain poorly understood. Here, we develop rational combination therapies that are informed by proteomic profiling to overcome fibroblast-mediated therapeutic resistance in HER2+ breast cancer cells. Drug sensitivity to the HER2 kinase inhibitor lapatinib was characterized under conditions of monoculture and exposure to breast fibroblast-conditioned medium. Protein expression was measured using reverse phase protein arrays. Candidate targets for combination therapy were identified using differential expression and multivariate regression modeling. Follow-up experiments were performed to evaluate the effects of HER2 kinase combination therapies in fibroblast-protected cancer cell lines and fibroblasts. Compared to monoculture, fibroblast-conditioned medium increased the expression of plasminogen activator inhibitor-1 (PAI1) and cell cycle regulator polo like kinase 1 (PLK1) in lapatinib-treated breast cancer cells. Combination therapy of lapatinib with inhibitors targeting either PAI1 or PLK1, eliminated fibroblast-protected cancer cells, under both conditions of direct coculture with fibroblasts and protection by fibroblast-conditioned medium. Analysis of publicly available, clinical transcriptomic datasets revealed that HER2-targeted therapy fails to suppress PLK1 expression in stroma-rich HER2+ breast tumors and that high PAI1 gene expression associates with high stroma density. Furthermore, we showed that an epigenetics-directed approach using a bromodomain and extraterminal inhibitor to globally target fibroblast-induced proteomic adaptions in cancer cells, also restored lapatinib sensitivity. Our data-driven framework of proteomic profiling in breast cancer cells identified the proteolytic degradation regulator PAI1 and the cell cycle regulator PLK1 as predictors of fibroblast-mediated treatment resistance. Combination therapies targeting HER2 kinase and these fibroblast-induced signaling adaptations eliminates fibroblast-protected HER2+ breast cancer cells. The online version contains supplementary material available at 10.1007/s12195-024-00823-0.

Waterborne pathogens pose a significant threat to public health, emphasizing the continuous necessity for advancing robust detection techniques, particularly in preventing outbreaks associated with these pathogens. This study focuses on cholera, an infectious disease caused by Vibrio cholerae, serogroups O1 and O139, often transmitted through contaminated water and food, raising significant public health concerns in areas with poor sanitation and limited access to clean water. We developed a colorimetric biosensor using aptamer-functionalized gold nanoparticles to identify Vibrio cholerae O139 and address this issue. The detection mechanism relies on the color change of gold nanoparticles (AuNPs) from red to blue-purple induced by NaCl after the pathogen incubation and aptamer-target binding. Initial steps involved synthesizing and characterizing AuNPs, then exploring the impact of aptamer and NaCl concentrations on AuNP agglomeration. Optimization procedures for aptamer concentration and salt addition identified the optimal conditions for detection as 120 pM aptamers and 1 M NaCl. The aptasensor demonstrated a robust linear relationship, detecting V. cholerae concentrations from 103 to 108 CFU/mL, with a limit of detection (LOD) of 587 CFU/mL. Specificity tests and accurate sample analyses confirmed the efficiency of the AuNPs aptasensor, showcasing its reliability and speed compared to traditional culture examination methods. Moreover, we extended the aptasensor to a paper-based sensing platform with similar detection principles. The change in color upon target binding was captured with a smartphone and analyzed using image processing software. The paper-based device detected the target in less than 2 min, demonstrating its convenience for on-field applications.

Glioblastoma (GBM) is an aggressive malignant brain tumor with 2year survival rates of 6.7% (Stupp et al. in J Clin Oncol Off J Am Soc Clin Oncol 25:4127-4136, 2007; Mohammed et al. in Rep Pract Oncol Radiother 27:1026-1036, 2002). One key characteristic of the disease is the ability of glioblastoma cells to migrate rapidly and spread throughout healthy brain tissue (Lefranc et al. in J Clin Oncol Off J Am Soc Clin Oncol 23:2411-2422, 2005; Hoelzinger et al. in J Natl Cancer Inst 21:1583-1593, 2007). To develop treatments that effectively target cell migration, it is important to understand the fundamental mechanism driving cell migration in brain tissue. Several models of cell migration have been proposed, including the motor-clutch, bleb-based motility, and osmotic engine models. Here we utilized confocal imaging to measure traction dynamics and migration speeds of glioblastoma cells in mouse organotypic brain slices to identify the mode of cell migration. We found that nearly all cell-vasculature interactions reflected pulling, rather than pushing, on vasculature at the cell leading edge, a finding consistent with a motor-clutch mode of migration, and inconsistent with an osmotic engine model or confined bleb-based migration. Reducing myosin motor activity, a key component in the motor-clutch model, was found to decrease migration speed at high doses for all cell types including U251 and 6 low-passage patient-derived xenograft lines (3 proneural and 3 mesenchymal subtypes). Variable responses were found at low doses, consistent with a motor-clutch mode of migration which predicts a biphasic relationship between migration speed and motor-to-clutch ratio. Targeting of molecular clutches including integrins and CD44 slowed migration of U251 cells. Overall we find that glioblastoma cell migration is most consistent with a motor-clutch mechanism to migrate through brain tissue ex vivo, and that both integrins and CD44, as well as myosin motors, play an important role in constituting the adhesive clutch. The online version contains supplementary material available at 10.1007/s12195-024-00799-x.

We are pleased to present this year's twelve Young Innovators of Cellular and Molecular Bioengineering, whose original research is featured in this August issue. It is now the tenth year of the Young Innovators competition, and we continue to receive a large number of compelling nominations. All potential authors who hold the rank of Assistant Professor (or equivalent) at the time of nomination are eligible for selection, and while many of the authors are active members of the Biomedical Engineering Society (BMES), membership in BMES is not a requirement for inclusion. As in previous years, awardees will present the papers of this issue in a pair of invited plenary sessions at the 2023 BMES Annual Meeting, held on Friday, October 13, 2023. The articles in this issue represent some of the most innovative and impactful bioengineering studies carried out by junior faculty in our field. This year's Young Innovators are all clearly recognized as rising stars in their field. With a pioneering spirit, and consistent with the enthusiasm for large language models expressed in the pages of this journal, we invited ChatGPT to organize the 12 papers in this issue into two sections and generate text to introduce these papers to readers. This is perhaps a first in the history of scientific publishing and professional conferences, to turn over the reins of a journal table of contents and BMES session organization to a large language model. The output from ChatGPT is presented below in unedited form, appearing in italics to set it apart from the human-generated text elsewhere.