Experimental Cell Research

Ewing sarcoma is a pediatric cancer that develops in skeletal elements. The majority of Ewing sarcoma patients carry the aberrant EWSR1-FLI1 fusion gene. Despite trisomy 8 being an additional common aberration associated with a poor prognosis for patients, its induction mechanism remains unknown. When the EWSR1-FLI1 gene is formed, the cell loses one wildtype EWSR1 allele. To elucidate the induction mechanism of trisomy 8, we generated a cell line that allows for the conditional induction of EWSR1-FLI1 expression and EWSR1 knockdown (derived from a single EWSR1 allele. Specifically, the conditional cell line was generated by integrating the Tet-on EWSR1-FLI1 construct into the AAVS locus and adding a miniAID tag at the 5 end of the EWSR1 locus using auxin-degron system. A combination of the EWSR1-FLI1 expression and degradation of one allele-derived EWSR1 induced a high incidence of trisomy 8 within eight days, enhancing colony formation. Mechanistically, trisomy 8 is induced by the haploinsufficiency of EWSR1, and the remaining EWSR1 proteins are likely inhibited by interaction with EWSR1-FLI1. Our data showed that the knockout of EWSR1 alone was sufficient to increase the incidence of trisomy 8. Expression of wild-type EWSR1 in EWSR1 knockout cells rescued the high incidence of trisomy 8. In contrast, the EWSR1:R565A mutant, which lacks the ability to interact with Aurora B kinase, failed to rescue this phenotype. We propose that the combination of EWSR1-FLI1 expression and loss of EWSR1 contributes to the induction of trisomy 8 through the compromised EWSR1-Aurora B pathway. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=135 SRC="FIGDIR/small/726567v1_ufig1.gif" ALT="Figure 1"> View larger version (28K): org.highwire.dtl.DTLVardef@999891org.highwire.dtl.DTLVardef@1ef6748org.highwire.dtl.DTLVardef@65e475org.highwire.dtl.DTLVardef@179da40_HPS_FORMAT_FIGEXP M_FIG C_FIG

Epigenetic regulation, particularly histone acetylation, plays a critical role in skeletal muscle differentiation by modulating gene expression programs without altering DNA sequence. Histone deacetylases (HDACs) tightly regulate myogenesis by controlling the timing of differentiation. Pharmacological inhibition of HDACs has shown context-dependent effects on muscle cells. We investigated the effects of 1 {micro}M SAHA (suberoylanilide hydroxamic acid) on C2C12 and L6 myoblasts during differentiation using morphological, immunofluorescence, transcriptomic, and proteomic analyses. SAHA delayed early differentiation, reducing myotube formation with partial recovery at later stages. Transcriptomic analysis revealed time-dependent changes in pathways related to cytoskeleton, cell cycle, and chromatin regulation. Proteomics showed increased mitochondrial metabolism and reduced cytoskeletal components in C2C12 cells, while L6 cells displayed alterations in muscle structural and extracellular matrix proteins. SAHA induces stage- and model-dependent reprogramming of myogenesis, highlighting the importance of timing and cellular context in HDAC-targeted therapies.

Ergosterol has multiple functions in filamentous fungi and yeasts, although only a part of the functions seems to be understood. An antifungal peptide, theonellamide A (TNM-A) induces drastic morphological changes in fission yeast cells by targeting plasma membrane ergosterol. TNM-A induces overproduction and ectopic accumulation of cell wall glucan at both growing tips and septum through a yet unknown mechanism. Here we show that TNM-A treatment causes accumulation of 1,3-{beta}-glucan at cell-polarity sites, not by increased activity of 1,3-{beta}-glucan synthase, but by an increased, persistent localization of the glucan synthase enzymes. Screening based on subcellular localization of proteins at periphery or polarity sites suggested the involvement of the Rho family GTPase Cdc42. In agreement, TNM-A induced both activation of Cdc42 and enhancement of membrane trafficking of glucan synthase enzymes. In conclusion, our chemical genetics analyses using TNM-A suggest that membrane ergosterol regulates the activity of Cdc42, which further regulates the localization of glucan synthases and cell wall biosynthesis. Highlights (four sentences)- Thenoellamide A (TNM-A) induces an ectopic overproduction of cell wall glucan. - TNM-A treatment causes increased, persistent localization of glucan synthases at the cell tips and septum. - TNM-A activates Cdc42 and upregulates membrane trafficking of glucan synthases. - Ergosterol is involved in proper activation/inactivation of Cdc42.

How cells and their organelles are positioned in three-dimensional, organ level, anatomical context, is rarely investigated. Here we focus on cells, centrioles and primary cilia in the growing limb. Through the ciliums mechanobiological role in skeletal development, we explored the mechanobiology of morphogenesis. A transgenic mouse model (Centrin 2-GFP.ARL13B-mCherry), combined with an image analysis pipeline, can map cellular size, positions and orientations, centriole position and ciliary axoneme orientation, all with respect to the anatomy of the epiphysis or growth plate. The line was crossed with an ift88fl/flCreERT2 line to enable ciliary ift88 deletion. We used limb immobilization, to test for a role of mechanical forces associated with ambulatory loading, in the organization of these elements and transcriptomics to understand the role of forces in regulating growth plate morphogenic programs. The pipeline can accurately quantify expected patterns of cell orientation and size through zones of the growth plate. Analysis across thousands of cells, through regions and zones of multiple murine growth plates, reveals cilia prevalence is increased in the periphery, highest in the resting zone in the outer limb, harboring stem cells. Cilia length is greatest in the hypertrophic cells about to die or transdifferentiate, as part of the formation of bone from cartilage by endochondral ossification. The inducible and cartilage-specific, deletion of ciliary gene ift88, alters cell orientation and sizes and reduces ciliation in the areas where endochondral ossification is most disrupted, the periphery and expanded hypertrophic zones, linking changes in structure to function. Most strikingly, centriole position, including that of the basal body, from which the ciliary axoneme is extended, is not preferentially organised. In contrast, cilia axoneme orientation is preferentially organised. Axonemes are directed posterior or anterior, 45 degrees to the axis of the limb, irrespective of their position, which is defined by basal body position. Immobilization of the limb for 2 weeks markedly alters the transcriptomic profile of the growth plate, with changes to size and orientation of cells and alterations in matrix and cytoskeletal profiles. Within altered genes, primary cilia genes themselves are regulated, including those indicative of altered cilia signaling such as hedgehog signaling. However, despite the role of cilia in mechanobiology of the growing limb, and ciliary signature within changes to loading of the limb, cilia orientation is unaltered by the removal of ambulatory associated forces. Patterns of ciliation in control and IFT88cKO mice help reconcile the previously observed anisotropic effects of cilia perturbation, focusing study on stem cell-resting chondrocytes and hypertrophy, when considering the mechanobiological role of cilia in limb development. Endochondral ossification is apparently highly sensitive to ambulatory loading at transcriptomic level, including effects on ciliary genes and signaling. A highly organized orientation of these putative antennae is governed by centriole position-independent mechanisms and is independent to changes to ambulatory loading, indicating a cell intrinsic mechanism. The resilient position of axonemes in the limb, points to mechano-regulatory mechanisms for how cilia integrate biophysical signals. We propose that predominant ventral or dorsal orientation at 45 degrees to horizonal plane but never parallel to cranial-chordal or medial-lateral axis, ensures multiple signal integration and avoids single signal blindness.

Hypoxia and anoxia are known to suppress cell proliferation due to an increase in replication stress and activation of DNA damage checkpoints. Embryos of the annual killifish Austrofundulus limnaeus show a strong tolerance to extended anoxic exposure, indicating an improved genomic stability under oxygen starvation. Here we investigate the cell cycle regulation of the anoxia tolerant killifish embryonic cell line PSU-AL-WS40NE during anoxic exposure. Live cell imaging confirms continued cell proliferation of WS40NE cells for the first 24 hours of anoxic exposure with minimal cell death. Fluorescent imaging shows that cells begin to accumulate in G1 after the first day in anoxia with a pronounced and rapid entry into the S phase upon reoxygenation. Pharmacological inhibition tests show that this response appears to be reliant more on ATR signaling then ATM, suggesting that increased {gamma}H2AX levels are driven by increased replication stress instead of DNA damage. This conclusion is further supported by an apparent lack of induction of a G2 checkpoint in these cells suggesting that DNA damage during anoxic replication is minimal. Maintaining cellular proliferation during initial exposure to anoxia and accumulating cells in the G1 phase for extended anoxic exposure is likely one way that embryos of the annual killifish are able to survive prolonged anoxia and provides insight into mechanisms that enable cells to proliferate under metabolic stress.

Emerin (EMD), an inner nuclear membrane protein essential for nuclear architecture integrity, gene expression, cellular signaling, and chromatin stability, interacts with the LINC complex and participates in cytoskeleton-nucleoskeleton communication by binding to nuclear actin filaments. EMD is implicated in migration, invasion, and metastasis in some tumors, but its role in glioblastoma (GBM) remains unclear. This study evaluated the effects of EMD knockdown and overexpression in GBM cell lines following genotoxic treatment with cisplatin. In both wild-type p53 (U87-MG) and mutant p53 (U138-MG) GBM cells, EMD expression is high, and cisplatin treatment did not affect these protein levels. EMD knockdown in U87-MG cells significantly increased cisplatin IC50, viability, and proliferation. Conversely, stable overexpression of EMD in U87-MG cells led to reduced cisplatin IC50, viability, proliferation, and migration. EMD knockdown or overexpression did not affect any U138-MG phenotypes, with or without cisplatin treatment. Modulation of EMD levels causes morphological changes in stress fiber cytoskeleton, whereas overexpression of EMD in U87-MG cells promotes an increase and a decrease in nuclear and cytoplasmic actin levels, respectively. These biological responses of U87-MG cells overexpressing EMD were coincidentally associated with alterations in the levels of pH2AX(Ser139), p-p53(Ser15), p53, and p21Kip1 proteins after cisplatin exposure. In sum, modulation of EMD levels affects the viability, migration, and proliferation of wild-type p53 GBM cells treated with cisplatin, suggesting unknown roles in the DNA damage response and repair. This work highlights EMD as a potential regulator of GBM chemoresistance and a target for therapeutic intervention.

PurposeGlasdegib is a Sonic Hedgehog (SHH) pathway inhibitor used for treating newly diagnosed acute myeloid leukemia in elders or patients unfit for intensive chemotherapy. This study sought to demonstrate growth inhibition and increased apoptosis of B-cell acute lymphoblastic leukemia (B-ALL) in vitro under glasdegib, alone and combined with inotuzumab, using a novel co-culture system and validated chemosensitivity testing model to determine whether glasdegib with and without inotuzumab may represent a promising treatment strategy in B-ALL. MethodsSeven blood and marrow samples from B-ALL patients were co-cultured with HS-5 stromal cells in a co-culturing system designed to mimic the tumor microenvironment to maintain B-ALL cell viability for chemosensitivity testing under glasdegib and inotuzumab. ResultsCo-culturing improved B-ALL viability from four to nine days. Dosage-dependent responses to glasdegib were consistent among B-ALL samples on day four based on culture viability, and varied based on expressions of SSH genes GLI1, GLI3, SMO, and PTCH1. Combination with inotuzumab had varied effects on treatment response. ConclusionCo-culturing B-ALL cells with HS-5 stromal cells improves B-ALL growth and viability. Glasdegib with and without inotuzumab treatments impact the viability of co-cultured B-ALL cells by day four. SHH gene expressions suggest different B-ALL patients may be sensitive or resistant to glasdegib and inotuzumab.

BackgroundKATNIP (Katanin-interacting protein), also known as KIAA0556, is one of the human genes with pathogenic variants linked to Joubert syndrome, an archetypal neurodevelopmental ciliopathy. KATNIP is a scaffolding protein with a critical role in ciliogenesis. In this study, we characterized the ciliopathy phenotypes due to KATNIP gene deletion. ResultsWe produced a Katnip null mouse model using CRISPR-Cas12a (Cpf1). The null heterozygotes appeared normal while the homozygotes died around postnatal day 9, showing severe hydrocephalus and deficiency in neuroprogenitor cell proliferation. Katnip-deficient cells in the brain have a higher rate of cilia formation and longer cilia than wild type cells. ConclusionKATNIP loss of function gives rise to hydrocephalus found in Joubert syndrome. The results indicate that KATNIP restricts ciliogenesis and cilia extension and supports proliferation of neuroprogenitor cells in the brain.

Osteocytes and adipocytes represent cells with disparate functions. Osteocytes regulate bone metabolism (remodeling) and bone homeostasis, while adipocytes regulate energy metabolism and energy storage. Here, we demonstrate that osteocyte phenotype consists of adipocytic features which are under control of peroxisome proliferator-activated receptor gamma (PPARG), a master regulator of adipocyte differentiation and function. Using a mouse model with osteocyte-specific deletion of PPARG (OT{gamma}KO) and osteocyte cellular model of MLO-Y4 cells edited with CRISPR/Cas9 for PPARG deficiency, we are demonstrating that under PPARG control osteocytes produce and secrete adiponectin (ADIPOQ), and they are equipped in adipocyte-specific mechanisms for lipid-storage and their metabolism. Under PPARG, osteocytes accumulate lipid droplets which correlate with their capability to cover up to 20% of energy requirements from fatty acids metabolism. Although osteocytes like osteoblasts mainly express perilipin 2 (Plin2), however similarly to adipocytes, lipid droplets accumulation is associated with expression of perilipin 1 (Plin1) under PPARG control. Similarly, lipids accumulation and metabolism involve adipocyte-specific activities including fatty acids binding protein 4 (Fabp4), hormone-specific lipase (Hsl) and adipocyte-specific triglyceride lipase (Atgl), which expression are under PPARG control. These studies provide a new understanding of osteocyte biology which include adipocyte-like endocrine and lipid metabolism features probably reflecting an adaptation to their unique localization and a need for a maintenance of functional fitness in these conditions. They deepen our comprehension of the crossroads of osteocyte and adipocyte function and underscore the therapeutic potential of targeting common molecular pathways in both cell types for managing metabolic disorders and skeletal diseases.

Lung adenocarcinomas frequently harbour actionable oncogenic mutations that are vulnerable to treatment with targeted therapies. While responses to targeted therapies are often initially dramatic, relapse is almost inevitable and prevents durable responses in advanced-stage patients. Relapse is, in part, caused by drug tolerant persister cells (DTPs) which are able to survive treatment by entering a reversible, dormant state. Although long non-coding RNAs (lncRNAs) regulate processes thought to allow DTPs to survive and become stably resistant, the potential roles of lncRNAs in DTPs are largely unknown. In this study, we sought to investigate the expression of lncRNAs in in vitro DTP models of lung adenocarcinoma. We found that the lncRNAs Metastasis-Associated Lung Adenocarcinoma Transcript 1 (MALAT1) and Nuclear Paraspeckle Assembly Transcript 1 (NEAT1) were enriched in DTPs and that knocking down MALAT1 enhanced the effect of targeted therapies in both EGFR- and KRAS-mutant DTP models. To better understand pathways that MALAT1 might regulate in DTPs, bulk RNA-sequencing was performed and several pathways that may contribute to the actions of MALAT1 in DTPs were identified. Overall, our work describes a role for the lncRNA MALAT1 in DTPs in NSCLC and suggests that MALAT1 may be a novel target for the prevention of drug tolerance and subsequent resistance to targeted therapy in NSCLC.

Melanoma, one of the most metastatic and multidrug resistant cancer, is the first leading cause of death from skin cancer. This complex disease requires identification of additional cooperating events that contribute to progression, invasion and metastasis to reinforce therapeutics. RhoGTPases play key roles in cancer development and metastasis. Rhophilin-2 (RHPN2), a Rho effector, is amplified in various human cancers and its role in melanoma remains unexplored. Here, we combined knock-down experiments in human melanoma cells, with knock-out and overexpression experiments in zebrafish to uncover the roles of RHPN2 in melanoma development. We show that in human melanoma cells RHPN2 contributes to growth, and to clonogenic, migratory and invasive properties of the cells. Using NRASQ61L and BRAFV600E zebrafish models, we provide the first in vivo evidence that Rhpn2 promotes melanoma onset and development. Histological analysis of the Rhpn2 deficient tumors showed decreased cellular density and absence of primary cilia structures at the invasive tumor/stroma borders. Transcriptomic profiling of the Rhpn2-KO melanoma revealed increased expression of the IFN1-responsive genes and modulation of genes involved in lipid metabolism and cilia function. Together these findings position RHPN2 as a modulator of melanoma, offering new perspectives in considering it as a target to impair the development of the tumor.

Adults with Down syndrome (DS) are two times more likely to be diagnosed with chronic heart failure post-anthracycline chemotherapy compared to age and sex-matched adults without DS. They have an elevated lifetime risk of cardiovascular diseases, increasing their likelihood of anthracycline-induced chronic cardiovascular toxicity. We investigated the chronic effects of daunorubicin on the cardiovascular system of the adult Ts65Dn mouse model of DS compared to wild type euploid mice (WT). WT and Ts65Dn mice received two doses of 2mg/kg or 4mg/kg of daunorubicin or saline and were monitored for up to 117 days. Cardiac and vascular function were evaluated using left ventricular catheterization, histology, pulse wave velocity, and cardiac troponin tests. Survival significantly decreased in the Ts65Dn 4mg/kg group compared to saline controls (p<0.001). Further experiments were carried out with the saline and 2mg/kg groups, which exhibited lower mortality, more consistent with chronic toxicity. Body weight (p=0.001), end-diastolic pressure (p=0.016), and left ventricular mass (p=0.021) decreased in treated mice. The effect of treatment differed significantly between strains for ejection fraction (p=0.029). Pulse wave velocity increased over time (p<0.001). A significant interaction between treatment and strain was observed for collagen in both the left ventricles and thoracic aorta (p=0.002 and p<0.001, respectively). There was a strain difference for cardiac troponin I, indicating an increase in Ts65Dn mice (p=0.020). Daunorubicin treatment results in a distinct cardiovascular remodeling phenotype in Ts65Dn mice. More mechanistic studies are warranted to outline the pathophysiology of anthracycline cardiovascular toxicity in DS.

Zebrafish (Danio rerio) possess remarkable regenerative capacity, making them an ideal model for studying the molecular mechanisms underlying tissue regeneration. In this article we report the identification of blastema linked exosome like extracellular vesicles (EVs) in zebrafish, that to the vesicles were plausibly being translocated in the proximo-distal axis through filipodia. We further thoroughly examined the exosome like EVs isolated from regenerating tissues of zebrafish caudal fins to characterize their nucleic acid cargo and evaluate their potential regulatory functions in regeneration. Caudal fins were amputated and allowed to regenerate and exosome like EVs isolated from blastema tissues displayed increased abundance compared to non-amputated controls. RNA sequencing identified a dynamic cluster of EV linked microRNAs (miRs). These differentially expressed miRs, including dre-miR-21, dre-miR-200b, dre-miR-218a and dre-let-7e were upregulated and associated with promoting proliferation, migration, differentiation, and tumour suppression pathways. Moreover, dre-miR-100, dre-miR-146a and dre-miR-200c regulated osteogenic differentiation, inflammatory signalling, epithelial-mesenchymal transition, and cell adhesion. Regeneration is generally believed to be coordinated only by local morphogen diffusion. Through this study it is indicative that filipodia bound EVs might have a pivotal role in long-range communication between blastema and the proximal tissues during the regeneration process. A detailed analyses of the miR targets and their validation could potentially lead to novel advancement and solutions in the field of regeneration and regenerative medicine in the near future.

Pluripotent stem cells derived from livestock species represent valuable systems for studying early mammalian development and for establishing renewable, well-defined cell sources; however, direct comparative characterization of distinct pluripotent stem cell platforms in sheep remains limited. In this study, we established and evaluated two ovine pluripotent stem cell types: reprogrammed induced pluripotent stem cells (siPSCs) and embryonic disc-derived stem cells (sEDSCs). Both siPSCs and sEDSCs exhibited core features of pluripotency, including compact colony morphology, alkaline phosphatase activity, expression of key pluripotency-associated markers, and maintenance of a normal ovine karyotype. Flow cytometry and quantitative RT-PCR analyses revealed broadly overlapping yet distinguishable pluripotency marker expression profiles between the two cell types. Functional pluripotency was confirmed by embryoid body formation and in vitro differentiation into derivatives of all three germ layers. To further assess lineage-specific differentiation competence and compare functional outputs relevant to mesodermal differentiation, both pluripotent stem cell types were directed towards the adipogenic lineage. While siPSCs and sEDSCs were each capable of adipogenic differentiation, differences in differentiation efficiency and marker expression were observed. Together, these findings demonstrate that ovine siPSCs and sEDSCs share core pluripotency characteristics while retaining distinct molecular and functional properties, providing a robust comparative framework for studies of ovine pluripotency, lineage specification, and stem cell biology.

Although calmodulin is best known as an intracellular calcium sensor, it also possesses calcium-independent functions in unicellular organisms. This is exemplified by the budding yeast S. cerevisiae calmodulin, which binds its essential targets, the pericentrin-like protein Spc110 and type I and V myosins, without needing calcium. Whether such calcium-independent cellular functions are conserved in other yeasts and vertebrates nevertheless remains an open question. Here, we examined the calcium-independent functions of the fission yeast S. pombe calmodulin Cam1 by measuring its intracellular distribution. Using quantitative fluorescence microscopy, we assessed the intracellular localization of two cam1 mutants, where binding of Ca2+ had been compromised by mutations in their EF hands, compared to the wild type protein. Both Cam1-2V and -3V reduced their localization by 90% to the yeast microtubule-organizing center spindle pole bodies (SPB). In contrast, these two mutants did not affect the myosin-dependent localization to the equatorial division plane and to the cell tips. Replacing the endogenous cam1 with cam1-2V decreased the SPB localization of pericentrin Pcp1 by 69%, without changing the localization of either type V or I myosins. Over-expression of Pcp1 rescued the mitotic defects of cam1-2V cells at the restrictive temperature. Surprisingly, the cytokinesis of this cam1 mutant was largely normal. We concluded that fission yeast calmodulin Cam1 depends on Ca2+to be a component of SPBs, suggesting that calcium plays a critical role in the assembly of SPBs.

Background & AimsColorectal cancer liver metastasis is the leading cause of mortality in affected patients, with liver sinusoidal endothelial cells playing a pivotal role in metastatic niche formation. Proprotein convertase subtilisin/kexin type 9 has emerged as a regulator of tumor biology, but its function in the hepatic microenvironment remains poorly defined. This study aimed to characterize the role and subcellular localization of PCSK9 in liver sinusoidal endothelial cells and to evaluate the therapeutic potential of its endothelial-specific inhibition in colorectal liver metastasis. MethodsIn vitro studies were performed using human and murine liver sinusoidal endothelial cells stimulated with conditioned media from metastatic colorectal cancer cells and cancer stem cells. Subcellular localization was assessed by immunofluorescence, immunogold electron microscopy, and biochemical fractionation. Protein interactions were investigated using co-immunoprecipitation and proteomic analyses. For in vivo validation, a murine model of colorectal liver metastasis was generated by intrasplenic injection of tumor cells, followed by systemic administration of chondroitin sulfate-targeted nanoparticles delivering PCSK9 siRNA every 5 days for 18 days. ResultsPCSK9 was consistently expressed in liver sinusoidal endothelial cells and displayed a predominant nuclear localization, which increased upon tumor-induced activation. Proteomic integration identified multiple candidate interacting proteins involved in metabolic and tumor-related pathways. Targeted nanoparticle-mediated delivery achieved efficient PCSK9 silencing in vitro. In vivo, endothelial-specific PCSK9 inhibition significantly reduced liver metastatic tumor burden compared with control groups, whereas free siRNA showed no significant effect. ConclusionsPCSK9 exhibits a novel nuclear localization in liver sinusoidal endothelial cells and potentially interacts with proteins implicated in tumor mediated pathways. Selective inhibition of endothelial PCSK9 using targeted nanoparticles significantly reduces colorectal liver metastasis, highlighting a novel therapeutic strategy focused on the hepatic microenvironment. Impact and ImplicationsThis study provides mechanistic insight into how PCSK9 contributes to colorectal liver metastasis by identifying its novel nuclear localization and potential function in liver sinusoidal endothelial cells. These findings are important for researchers and clinicians seeking to understand microenvironment-driven metastasis and resistance to current therapies. The demonstration that endothelial-specific targeting of PCSK9 reduces metastatic burden suggests a new avenue for therapeutic development beyond systemic inhibition. Such strategies could be translated into precision nanomedicine approaches to improve outcomes in patients with metastatic colorectal cancer while minimizing off-target effects.

The surface layer of articular cartilage provides for low-friction joint movement and protects the tissue from mechanical wear. The superficial zone chondrocytes (SZCs) of the surface layer produce proteoglycan-4 (PRG4), which is a lubricant that is necessary to reduce friction. Articular cartilage has limited capacity for self-repair and cell-based therapies, such as autologous chondrocyte implantation (ACI), is used to stimulate repair. However, in ACI, cells are expanded on tissue culture polystyrene where SZC poorly attach, proliferate slowly and dedifferentiate. Consequently, expanded SZC produce fibrocartilage tissue with insufficient PRG4. We previously demonstrated that culturing SZC on chondrocyte-derived decellularized extracellular matrix (CM) enhances SZC attachment and preserves phenotype. Since fibronectin (FN) was identified as the most abundant matrix protein within CM, here we tested the hypothesis that FN-coated culture surfaces would partially reproduce the beneficial effects of CM. We found that, similar to CM, SZC on FN-coated polystyrene increased SZC attachment and proliferation. However, unlike CM, SZCs expanded on FN-coated polystyrene remained more dedifferentiated as indicated by spread cells, elevated fibroblastic and contractile mRNA levels, and increased formation of SMA positive stress fibers. Consistent with the dedifferentiated phenotype, SZC on FN-coated polystyrene displayed extensive stress fibers, and higher nuclear myocardin-related-transcription-factor-a (MRTF-A). In contrast, CM reduced stress fiber formation and diminished nuclear MRTF-A in SZC. CM provides matrix cues beyond FN that suppress dedifferentiation and preserve the SZC phenotype. Identifying the matrix cues necessary to improve SZC expansion could lead to the generation of a superior surface in ACI repair tissue.

Unlike the conventional mature neutrophils, immature neutrophils have been investigated for their regenerative properties; however, their limited availability necessitates alternative generation strategies. Here, we used a combination of dimethylsulfoxide (DMSO) and 1,25-dihydroxyvitamin D3 (D3) to differentiate myeloid leukemia (HL-60) cells into immature neutrophil-like cells. Differentiated cells exhibited reduced cell size, loss of uniformity, decreased nuclear-to-cytoplasmic ratio, band-shaped nuclei, increased proportion of CD11b+CD14+ cells (indicative of immature neutrophils), decreased proportion of CD11b+CD16+ cells (indicative of mature neutrophils), higher levels of arginase 1, TGF{beta}1 (markers of immature neutrophils), and no expression of CD16, MRC1 (markers of mature neutrophils and M2 macrophages, respectively). Proteomic analysis revealed enrichment of proteins associated with immature neutrophils and wound healing. Functionally, these cells supported limbal stem cell growth and wound closure in vitro, indicating relevance for corneal regeneration. Administration of these cells to ex-vivo and in-vivo alkali-injured corneas, resulted in significant effect on promotion of wound healing, with epithelial regeneration and decreased fibrotic markers, proving that such cells hold promise for clinical translation as a therapeutic tool for tissue repair.

Up to 50% of adults with colorectal cancer (CRC) are at risk of progressive involuntary loss of skeletal muscle mass and function known as cachexia. Available options to prevent and treat cachexia in cancer survivors are currently limited. Long-chain polyunsaturated fatty acids (LC-PUFAs) and their bioactive metabolites, termed specialized pro-resolving lipid mediators (SPMs), promote the resolution of inflammation and support muscle growth and repair. However, prior studies of cachexia have mainly focused on fish oil supplements, and it is not fully understood how different individual omega-3 (n-3) and omega-6 (n-6) LC-PUFAs mediate CRC-induced muscle wasting. In addition, the crosstalk between cancer cells, the host immune system, and skeletal muscle cells in response to LC-PUFA treatments remains unclear. This study aimed to examine the effects of n-3 and n-6 LC- PUFAs on CRC-induced muscle wasting and the underlying cellular and molecular mechanisms involved. Using murine C2C12 skeletal muscle cells and CT26 colorectal carcinoma cells, we investigated the impacts of LC- PUFAs including arachidonic acid (ARA, 20:4n-6), eicosapentaenoic acid (EPA, 20:5n-3), docosapentaenoic acid (DPA, 22:5n-3), and docosahexaenoic acid (DHA, 22:6n-3) on CT26-induced muscle cell wasting in the presence or absence of lipoxygenase (LOX) inhibitors such as NDGA or BLX-3887. We also examined the lipidomic profile of C2C12-CT26 co-cultures in response to individual LC-PUFA treatments. Our results suggest that LC- PUFAs including ARA, EPA, DHA, and DPA each individually protect against CRC-induced muscle cell wasting in vitro, and these protective effects are dependent on 15-LOX activity. Furthermore, we found that C2C12-CT26 co-culture produced mature SPMs in response to individual PUFA treatments. Taken together, this study suggests that individual n-3 and n-6 LC-PUFAs can mitigate CRC-associated cachexia primarily by producing 15-LOX- derived bioactive lipid mediators.

Chemoresistance and recurrence of osteosarcoma (OS) can be attributed to a subpopulation of OS cells known as OS Cancer Stem-like Cells (OS-CSCs). We hypothesized that certain mechano-structurally distinct niches within the bone and tumor might be more conducive to OS-CSC formation. Using biomaterial-based 3D scaffolds of distinct structural and mechanical properties (anisotropic soft [~]10 kPa vs isotropic stiff [~]35 kPa), we discovered that OS cells growing in softer, anisotropic scaffolds were rounder, softer, had higher stemness gene expression, showed increased chemoresistance to doxorubicin and cisplatin, and were more tumorigenic in vivo. Mechanistically, biological reprogramming of OS cells on these scaffolds occurred through niche-driven transcriptional changes and differences in chemoresistance-associated epigenetic pathways. Our study showed that a softer, anisotropic niche is more conducive for maintaining OS-CSCs and these findings could be translated to designing therapeutic strategies targeting heterogeneous tumor populations or modifying the microarchitecture to reduce CSC-favoring niches.