Indirect Co-culture Research Articles

LPA released from dying cancer cells after chemotherapy inactivates Hippo signaling and promotes pancreatic cancer cell repopulation.

The Hippo pathway in the tumorigenesis and progression of PDAC, with lysophosphatidic acid (LPA) regulating the Hippo pathway to facilitate cancer progression. However, the impact of the Hippo signaling pathway on tumor repopulation in PDAC remains unreported. Direct and indirect co-culture models to investigate gemcitabine-induced apoptotic cells can facilitate the repopulation of residual tumor cells. Mass spectrometry analysis was conducted to assess the impact of gemcitabine treatment on the lipid metabolism of pancreatic cancer cells. ELISA assays confirmed gemcitabine promotes the release of LPA from apoptotic pancreatic cancer cells. The expression of Yes-associated protein 1 (YAP1) elucidated the underlying mechanism by which dying cells induce tumor repopulation using qRT-PCR and Western blot. We studied the biological function of pancreatic cancer cells using CCK-8, colony formation, and transwell invasion assays in vitro. Co-culture models were used to validate the impact of Hippo pathway on tumor repopulation, while flow cytometry was employed to assess the sensitivity of pancreatic cancer cells to gemcitabine in the context of Hippo pathway. Gemcitabine-induced dying cells released LPA in a dose-dependent manner, which promoted the proliferation, clonal formation, and invasion of pancreatic cancer cells. Mechanistic studies showed that gemcitabine and LPA facilitated the translocation of YAP1 and induced the inactivation of the Hippo pathway. YAP1 overexpression significantly enhanced the activity of autotaxin, leading to stimulated pancreatic cancer cells to secrete LPA. This mechanism orchestrated a self-sustaining LPA-Hippo feedback loop, which drove the repopulation of residual tumor cells. Simultaneously, it was observed that suppressing LPA and YAP1 expression enhanced the sensitivity of pancreatic cancer cells to gemcitabine. Our investigation indicated that targeting the LPA-YAP1 signaling pathway could serve as a promising strategy to augment the overall therapeutic efficacy against PDAC.

Evaluation of the influence of radiation-induced cohort effect in cell populations receiving different doses

Purpose A non-targeted effect called radiation-induced cohort effect, which results in interactions among irradiated neighboring cells through cellular communication, has been reported. In high-precision radiotherapy, the dose is localized to the tumor, and rapid spatial changes occur in dose distribution. However, the effect of irradiating a population of cells with non-uniform doses remains unknown. In this study, we evaluated the influence of cohort effect by creating cell populations irradiated with different doses using human oral squamous cell carcinoma (SAS) and human lung (A549) cells. Materials and methods Cell populations irradiated with different doses were created in two ways: direct contact co-culture (DCC) using a cell tracer dye and indirect contact co-culture (ICC) using cell culture inserts to assess the effects of soluble factors. Target cells were irradiated with 4 Gy and co-cultured cells with 0, 0.8, 3.2, and 4 Gy. In DCC, cell proliferation assays were performed using a flow cytometer, and in ICC, modified high-density survival, clonogenic, and apoptosis assays were performed. Results In DCC, irradiation of co-cultured cells with X-rays increased the relative proliferation rate of the target cells. Similarly, irradiating co-cultured cells using ICC with X-rays increased the relative survival rate of target cells. Conclusions The results of this study showed that, even if there is a sharp decrease in dose near the tumor, the cytocidal effect on the tumor is not adversely affected. In addition, soluble factors were found to be involved in cohort effect.

SLAMF8 Disrupts Epithelial Barrier in Chronic Rhinosinusitis with Nasal Polyps via M1 Macrophage Polarization.

Recent studies show that M1 macrophages accumulate predominantly in non-eosinophilic chronic rhinosinusitis with nasal polyps (neCRSwNP). However, the precise mechanisms regulating M1 macrophages and their impact on the epithelial barrier remain unclear. We aim to investigate the expression and regulatory role of SLAMF8, a molecule exclusively expressed in myeloid cells, in M1 macrophage polarization and its potential contribution to neCRSwNP development. We evaluated SLAMF8 expression and its correlation with clinical variables using real-time quantitative polymerase chain reaction and Western blot in sinonasal mucosa samples from CRSwNP and control subjects. Immunofluorescence staining confirmed the co-expression of SLAMF8 with macrophages. After SLAMF8 knockdown, we explored the influence on macrophage M1 polarization and the effect on epithelial-mesenchymal transition (EMT) process and tight junction integrity in epithelial cells through an indirect co-culture system of M1 macrophages with human nasal epithelial cells. SLAMF8 was highly expressed on M1 macrophages in polyp tissues, notably in neCRSwNP, and correlated with disease severity indices only in neCRSwNP. SLAMF8 knockdown in THP-1 cells reduced M1 macrophage markers (CD86, iNOS, and NLRP3) and decreased secretion of inflammatory cytokines (IL-1β, IL-6, and TNF-α). Co-culture with M1 macrophage supernatant after SLAMF8 knockdown enhanced epithelial viability, reduced EMT and apoptosis, and upregulated tight junction markers Occludin and Claudin-4 in nasal epithelial cells. SLAMF8 elevation correlates with the EMT, epithelial tight junction and disease severity in neCRSwNP. The SLAMF8 upregulation promotes M1 macrophage polarization, by which facilitates EMT and impairs nasal epithelial barrier function. SLAMF8 may represent a novel therapeutic target for neCRSwNP.

Synergistic effects of mTOR inhibitors with VEGFR3 inhibitors on the interaction between TSC2-mutated cells and lymphatic endothelial cells.

Lymphangioleiomyomatosis (LAM) is a rare neoplastic disease affecting the lung, kidney, and lymphatic system with a molecular mechanism of tuberous sclerosis complex 2 (TSC2) mutations. Vascular endothelial growth factor D (VEGF-D), a ligand for vascular endothelial growth factor receptor 3 (VEGFR3), is a diagnostic biomarker of LAM and is associated with lymphatic circulation abnormalities. This study explored the interaction between LAM cells and lymphatic endothelial cells (LECs) and the effects of rapamycin on this interaction, which may help to identify new targets for LAM treatment. This study used direct and indirect cocultures of TSC2-null cells and LECs. The xenograft model was applied to explore the therapeutic feasibility. Single-cell sequencing revealed increased LECs in the lungs of LAM patients through activation of pathways involved in lymphangiogenesis. TSC2-null cells attracted LECs and promoted tube formation. VEGF-D/VEGFR3 was a key mediator of the above interaction. Rapamycin can directly inhibit recruitment but not the tube formation of LECs in vitro. The combination of rapamycin with VEGFR3 inhibitors not only enhanced the effect of rapamycin but also inhibited lymphatic related manifestations including the tube formation and the lymphatic metastasis. Our findings demonstrated that LAM cells recruit LECs and promote tube formation via VEGF-D/VEGFR3. Rapamycin only partially blocked this interaction. The synergistic effects of rapamycin and VEGFR3 inhibitors suggest a novel strategy for the treatment of LAM and other TSC2-mutated diseases.

Targeting Fibroblast-Derived Interleukin 6: A Strategy to Overcome Epithelial-Mesenchymal Transition and Radioresistance in Head and Neck Cancer.

Cancer-associated fibroblasts have been reported to play a central role in driving cancer progression, promoting metastasis, and conferring resistance to therapy in HNSCC. Indirect and direct co-culture models of HPV-positive and HPV-negative HNSCC cells with fibroblasts were developed to study the effect of fibroblasts on cancer cells. ELISA was used to measure IL-6 secretion in these models. To dissect the underlying signalling mechanisms, the effects of IL-6, an IL-6 receptor (IL-6R) inhibitor, a MAPK/ERK inhibitor, and a JAK/STAT inhibitor were evaluated. Epithelial-to-mesenchymal transition (EMT) was assessed by measuring EMT markers and conducting scratch assays and spheroid assays. Radioresistance was evaluated using clonogenic assays. Additionally, radioresistant (RR) cell lines were established from parental cells to examine the correlation between radioresistance and EMT. Fibroblasts were found to drive EMT-like changes and heightened radioresistance in HNSCC cells through IL-6 secretion. Remarkably, these Fb-driven effects were robustly reversed using IL-6R and MAPK/ERK inhibitors in both HPV-positive and HPV-negative cell lines, whereas JAK/STAT inhibitors proved effective only in HPV-negative cells. RR cell lines exhibit a more aggressive phenotype than their parental counterparts, marked by pronounced EMT features and heightened resistance to radiotherapy. Importantly, these aggressive characteristics were substantially attenuated by targeting IL-6R or MAPK/ERK pathways. This study highlights the critical role of fibroblast-secreted IL-6 in driving and maintaining EMT and radioresistance in HNSCC, resulting in a more aggressive tumour phenotype. Targeting the IL-6/IL-6R/ERK pathway emerges as a promising therapeutic approach for combating CAF-driven tumour progression and improving clinical outcomes in patients with aggressive, therapy-resistant HNSCC.

Influence of Seven Ion Species on Osteogenic Differentiation of Mesenchymal Stem Cells Stimulated by Macrophages in Indirect and Direct Coculture Systems.

Implanted biomaterials release inorganic ions that trigger inflammatory responses, which recruit immune cells whose biochemical signals affect bone tissue regeneration. In this study, we evaluated how mouse macrophages (RAW264, RAW) and mesenchymal stem cells (KUSA-A1, MSCs) respond to seven types of ions (silicon, calcium, magnesium, zinc, strontium, copper, and cobalt) that reportedly stimulate cells related to bone formation. The collagen synthesis, alkaline phosphatase activity, and osteocalcin production of the MSCs varied by ion dose and type after culture in the secretome of RAW cells. However, DNA production was relatively unaffected. The MSC secretome may also stimulate RAW cells in coculture and, therefore, affect osteogenic differentiation of MSCs. Overall, the ions often exerted different effects on each cell type. This study guides future work that explores the mechanisms behind ion-dependent osteogenic differentiation and cell functions.

Chemokine (C-C Motif) Ligand 2/CCR2/Extracellular Signal-Regulated Kinase Signal Induced through Cancer Cell-Macrophage Interaction Contributes to Hepatocellular Carcinoma Progression.

Tumor-infiltrating macrophages, known as tumor-associated macrophages, play a crucial role in the tumor microenvironment. Immunohistochemistry revealed that intratumoral CD68-positive macrophages are associated with poor prognosis and clinicopathologic factors in patients with hepatocellular carcinoma (HCC). Subsequently, an indirect co-culture system involving HCC cells and peripheral blood-derived macrophages was developed. cDNA microarray analysis revealed that chemokine (C-C motif) ligand 2 (CCL2) was highly expressed in HCC cells co-cultured with macrophages. CCL2 neutralization suppressed proliferation, migration, and phosphorylation of extracellular signal-regulated kinase (Erk) in HCC cells and macrophages enhanced through co-culture. In contrast, recombinant human CCL2 (rhCCL2) addition facilitated these malignant phenotypes and increased Erk phosphorylation levels in HCC cells and macrophages. The primary CCL2 receptor, CCR2, was expressed in HCC cells and macrophages and was up-regulated in co-cultured HCC cells. CCR2 inhibition suppressed malignant phenotypes and reduced phosphorylated levels of Erk enhanced by rhCCL2. Additionally, the inhibition of Erk signal suppressed rhCCL2-enhanced malignant phenotypes. Moreover, serum CCL2 levels were higher in patients with HCC than those in healthy donors. On the basis of immunohistochemistry, CCL2-positive cases with high CCR2 expression and phosphorylated Erk-positive cases exhibited poor survival outcomes. Therefore, CCL2 up-regulation through interactions between HCC cells and macrophages contributed to HCC progression, making the CCL2/CCR2/Erk signal a potential target for HCC treatment.

Neuronal TRPV1-CGRP axis regulates peripheral nerve regeneration through ERK/HIF-1 signaling pathway.

Severe trauma frequently leads to nerve damage. Peripheral nerves possess a degree of regenerative ability, and actively promoting their recovery can help restore the sensory and functional capacities of tissues. The neuropeptide calcitonin gene-related peptide (CGRP) is believed to regulate the repair of injured peripheral nerves, with neuronal transient receptor potential vanilloid type 1 (TRPV1) potentially serving as a crucial upstream factor. In this study, we established a mouse model of sciatic nerve (SN) crush injury and found that intrathecal injection of capsaicin (Cap) activated the neuronal TRPV1-CGRP axis, thereby promoting SN repair. Conversely, the application of capsazepine (Cpz), which inhibits the neuronal TRPV1-CGRP axis, delayed SN repair. Local restoration of CGRP expression at the injury site enhanced the repair process. Invitro experiments, we employed the rat Schwann cell (SC) line RSC96 to establish an indirect co-culture model of neurons and SCs. We observed that the proliferation, migration, expression of myelination-associated proteins, and neurotrophic secretion functions of RSC96 cells are positively correlated with the degree of activation of neuronal TRPV1. Inhibition of neuronal TRPV1, followed by the restoration of CGRP levels, improved these functions in RSC96 cells. Furthermore, activation of the neuronal TRPV1-CGRP axis resulted in an upregulation of extracellular signal-regulated kinases 1/2 (ERK1/2) phosphorylation levels and an increase in hypoxia-inducible factor 1α (HIF-1α) accumulation in RSC96 cells, thereby promoting their proliferation and migration. In summary, this study demonstrates that neuronal TRPV1-CGRP axis can regulate biological behavior of SCs and axon regeneration by activating the ERK/HIF-1 signaling pathway following peripheral nerve injury. This finding clarifies the role of CGRP in neuroregulatory networks and provides a novel reference point for the development of drugs and biomaterials for treating nerve damage.

Cucurbita ficifolia regulates the secretomes of adipocytes and macrophages in co-culture, breaking meta-inflammation

AbstractCucurbita ficifolia’s fruit aqueous extract has been attributed with antidiabetic and anti-inflammatory properties, offering a promising alternative in addressing meta-inflammation. Meta-inflammation plays a pivotal role in the development of metabolic dysfunction, contributes to conditions such as obesity, diabetes mellitus, and metabolic syndrome. The interactions between adipocytes and macrophages within adipose tissue cause the onset of meta-inflammation, leading to disruptions in the secretomes and transcriptomes of inflammatory cytokines among other factors. We evaluated the effects of the aqueous extract from the fruit of Cucurbita ficifolia on the secretomes and transcriptomes of 3T3-L1 adipocytes and RAW-264.7 macrophages, by exchange of their conditioned mediums and in co-culture conditions. Adipocytes incubated with conditionate medium from macrophages treated with the extract exhibited an increase in GLUT-4 expression, and the protein release of TNFα, accompanied by a reduction in IL-1β. In contrast, macrophages incubated with conditionate medium from adipocytes treated with the extract exhibited inhibition the release of TNFα, IL-6, and IL-10, with TLR-4 expression reduction. Interestingly, under the conditions of indirect simultaneous co-culture, the release of TNFα, IL-6, and IL-1β was disrupted, increasing PPARγ expression in 3T3-L1 adipocytes. The salicin, compound reported in Cucurbita ficifolia extract, had a similar effect in this last model. The aqueous extract of Cucurbita ficifolia demonstrates the ability to suppress meta-inflammation by correcting the secretion patterns of TNFα and IL-6 in co-culture conditions. It effectively modulates the interactions between 3T3-L1 adipocytes and RAW-264.7 macrophages, leading to an improvement in the inflammatory cytokines profile.

Differential vasoproliferative traits of Bartonella henselae strains associated with autotransporter BafA variants.

Bartonella henselae, a Gram-negative facultative intracellular bacterium, is the etiological agent of cat-scratch disease and also causes bacillary angiomatosis in immunocompromised individuals. Although the ability to promote vascular endothelial cell proliferation differs among Bartonella species, variations among strains within B. henselae remain unclear. Bartonella angiogenic factor A (BafA) and Bartonella adhesin A (BadA) have been identified as autotransporters of B. henselae that are involved in endothelial cell proliferation. Although strain-specific differences in the expression of BadA and the VirB/D4 type IV secretion system have been reported, BafA expression among B. henselae strains has yet to be examined. Therefore, the present study investigated the proliferation-promoting ability of 13 B. henselae strains from several sources in human umbilical vein endothelial cells (HUVECs). We identified BafA variants 1 and 2 based on the deduced amino acid sequences of its passenger domain. The recombinant proteins of both variants exhibited similar proliferation activity against HUVECs. However, BafA variant 2 strains showed cytotoxicity at a high bacterial inoculum in a direct coculture with HUVECs, which was attenuated in an indirect coculture. These strains, in contrast to BafA variant 1 strains, highly expressed BadA and exhibited bacterial aggregation. Based on a core genome SNP analysis of 50 B. henselae strains, the BafA variant types corresponded to clades 1-4. These results indicate that vasoproliferative traits differ among B. henselae clades based on the variant types. Therefore, this study provides a new conceptual framework in which the clades of B. henselae may predict their pathogenicity in humans.IMPORTANCEBartonella species including Bartonella henselae, Bartonella quintana, and Bartonella bacilliformis cause vasoproliferative lesions. Their proliferation-promoting ability in vascular endothelial cells differs among Bartonella species; however, it is unclear whether these differences exist among B. henselae strains. We herein showed that B. henselae strains exhibited variable proliferation-promoting ability and cytotoxicity in vascular endothelial cells, which corresponded to the bafA gene variants possessed by the strains. The expression levels of Bartonella angiogenic factor A (BafA) and Bartonella adhesin A, as well as the degree of proliferation-promoting ability and cytotoxicity in endothelial cells, varied among the strains. A core genome SNP analysis of strains using whole genome sequencing data divided B. henselae strains into four clades, with each clade corresponding to BafA variants 1-4. These results suggest the differential vasoproliferative potency of B. henselae, with potential implications in clinical management, including risk stratification and predictions of the clinical course.

Associations between glycan signature alterations and the cellular antigenic properties of passaged chondrocytes.

Cartilage repair is a significant clinical challenge because of the limited intrinsic healing capacity. Current therapeutic strategies, such as cell transplantation therapy, aim to overcome this challenge by replacing damaged tissue with healthy cells. However, the long-term survival and functionality of transplanted cells remain major hurdles. This study investigated the impact of chondrocyte passaging on glycan profiles and their antigenic properties. We hypothesized that alterations in glycan composition due to passaging may contribute to the enhanced ability to activate macrophages, thereby affecting the outcome of cell transplantation therapy. Peritoneal macrophages and primary articular chondrocytes were isolated from C57BL/6 mice to establish direct and indirect coculture models. Macrophage activation was assessed by measuring the concentrations of IL-6 and nitric oxide in the culture supernatants or their gene expression. Glycome analysis of various glycoconjugates was performed by glycoblotting methods combined with the SALSA procedure for N-glycans and GSLs and the BEP method for O-glycans. Our results revealed that direct coculture of macrophages with passaged chondrocytes increased the production of proinflammatory cytokines, including IL-6 and NO, as the number of passages increased. With increasing passage number, the expression of GD3 substantially decreased, and the expression of GM3, especially GD1a, significantly increased. Coculturing passaged GM3S knockout chondrocytes with macrophages significantly suppressed IL-6 expression, implying reduced macrophage activation. The observed activation of macrophages due to alterations in the glycan profile of chondrocytes provides a possible explanation for the antigenicity and immune rejection of transplanted cells.

Deciphering influence of donor age on adipose-derived stem cells: in vitro paracrine function and angiogenic potential

Background: As fat grafting is commonly used as a filler, Adipose-derived stem/stromal cells (ASC) have been reported to be key player in retention rate. Paracrine and differentiation potential of those cells confer them strong pro-angiogenic capacities. However, a full characterization of the influence of aging on ASC has not been reported yet. Here we’ve investigated the effect of age on paracrine function, stemness and angiogenic potential of ASC. Methods: ASC were extracted from young and old adult donors. We assessed stromal vascular fraction cell populations repartition, ASC stemness potential, capability to differentiate into mesenchymal lineages as well as their secretome. Angiogenic potential was assessed using a sprouting assay, an indirect co-culture of ASC and dermal microvascular endothelial cells (EC). Total vascular sprout length was measured, and co-culture soluble factors were quantified. Pro-angiogenic factors alone or in combination as well as ASC-conditioned medium (CM) were added to EC to assess sprouting induction. Results: Decrease of endothelial cells yield and percentage is observed in cells extracted from adipose tissue of older patients, whereas ASC percentage increased with age. Clonogenic potential of ASC is stable with age. ASC can differentiate into adipocytes, chondrocytes and osteoblasts, and aging does not alter this potential. Among the 25 analytes quantified, high levels of pro-angiogenic factors were found, but none is significantly modulated with age. ASC induce a significantly longer vascular sprouts compared to fibroblasts, and no difference was found between young and old ASC donors on that parameter. Higher concentrations of FGF-2, G-CSF, HGF and IL-8, and lower concentrations of VEGF-C were quantified in EC/ASC co-cultures compared to EC/fibroblasts co-cultures. EC/ASC from young donors secrete higher levels of VEGF-A compared to old ones. Neither soluble factor nor CM without cells are able to induce organized sprouts, highlighting the requirement of cell communication for sprouting. CM produced by ASC supporting development of long vascular sprouts promote sprouting in co-cultures that establish shorter sprouts. Conclusion: Our results show cells from young and old donors exhibit no difference in all assessed parameters, suggesting all patients could be included in clinical applications. We emphasized the leading role of ASC in angiogenesis, without impairment with age, where secretome is a key but not sufficient actor.

AEBP1 is a negative regulator of skeletal muscle cell differentiation in oral squamous cell carcinoma

The tumor microenvironment plays a pivotal role in cancer development. We recently reported that in oral squamous cell carcinoma (OSCC), adipocyte enhancer-binding protein 1 (AEBP1) is abundantly expressed in cancer-associated fibroblasts (CAFs), leading to CAF activation and inhibition of CD8 + T cell infiltration. In the present study, we investigated whether AEBP1 contributes to the destruction and atrophy of muscle tissues in OSCC. By analyzing human skeletal muscle myoblasts (HSMMs), we found that AEBP1 is downregulated during muscle cell differentiation. Transcriptome analysis revealed that AEBP1 knockdown significantly upregulates myogenesis-related genes in HSMMs, and qRT-PCR and western blot analyses confirmed the induction of muscle-related genes, including MYOG, in HSMMs after AEBP1 knockdown. Conversely, ectopic expression of AEBP1 strongly suppressed myogenesis-related genes in HSMMs. Notably, indirect co-culture of HSMMs with OSCC cells led to AEBP1 upregulation and robust suppression of muscle-related genes in HSMMs. Treatment with TGF-β1 also upregulated AEBP1 and suppressed expression of muscle-related genes in HSMMs. Our findings suggest that AEBP1 is a negative regulator of skeletal muscle cell differentiation and that OSCC cells inhibit muscle cell differentiation, at least in part, by inducing AEBP1.

Zinc and gallium doped borate bioactive glasses influence in-vitro angiogenesis: New evidence in cell co-culture studies

Borate bioactive glasses doped with 1 mol% ZnO or Ga2O3 were produced by melt-quenching. The cytocompatibility of fibroblast and endothelial cells in response to glass extracts was assessed. The angiogenic potential was examined through fibroblast-derived vascular endothelial growth factor secretion and tubular formation analysis in an indirect co-culture of fibroblast and endothelial cells. The results confirm that Zn and Ga ion-releasing bioactive glasses are attractive biomaterials with angiogenic properties.

Glucotoxicity suppresses function of pancreatic beta and duct cells via miR-335-targeted Runx2 and insulin-mediated mechanism.

Pancreatic cell dynamics have important contributions to the development of type 2 diabetes and related diseases such as nonalcoholic fatty pancreas disease. The aim of this study was to investigate the effects of prolonged excessive glucose exposure on the functions of pancreatic beta cells and duct cells in single and co-culture conditions. In this study, we focused on the effects of glucotoxicity on insulin secretion which is the main function of beta cells and on progenitor functions of duct cells. Rat primary INS1 beta cells and ARIP duct cells were exposed to glucose (25mM) for 72h under single or indirect co-culture conditions. Glucotoxicity stimuli increased insulin secretion and decreased insulin expression in single beta cells while stimulating beta-cell differentiation and adipogenesis in single duct cells. On the other hand, glucotoxicity caused functional loss and increased proliferation and apoptosis in beta cells while increasing proliferation but suppressed beta-cell differentiation and adipogenesis in duct cells under co-culture conditions. The expression level of miR-335, a microRNA known to be upregulated by leptin and target Runx2, was measured. As a result, unlike single-cell culture, glucotoxicity upregulated miR-335, downregulated Runx2, and decreased insulin signaling in beta cells while downregulating miR-335 and upregulating Runx2, and decreased insulin signaling in duct cells under co-culture conditions. When the results of single and co-culture experiments are compared, insulin and miR-335 may be seen as important mediators for setting up the relation between beta and duct cells. Our findings are important for preventing the development of type 2 diabetes and nonalcoholic fatty pancreas disease, even developing new diagnosis and treatment strategies.

Macrophage-derived amphiregulin promoted the osteogenic differentiation of chondrocytes through EGFR/Yap axis and TGF-β activation

Endochondral ossification represents a crucial biological process in skeletal development and bone defect repair. Macrophages, recognized as key players in the immune system, are now acknowledged for their substantial role in promoting endochondral ossification within cartilage. Concurrently, the epidermal growth factor receptor (EGFR) ligand amphiregulin (Areg) has been documented for its contributory role in restoring bone tissue homeostasis post-injury. However, the mechanism by which macrophage-secreted Areg facilitates bone repair remains elusive. In this study, the induction of macrophage depletion through in vivo administration of clodronate liposomes was employed in a standard open tibial fracture mouse model to assess bone healing using micro-computed tomography (micro-CT) analysis, histomorphology, and ELISA serum evaluations. The investigation revealed sustained expression of Areg during the fracture healing period in wild-type mice. Macrophage depletion significantly reduced the number of macrophages on the local bone surface and vital organs. This reduction led to diminished Areg secretion, decreased collagen production, and delayed fracture healing. However, histological and micro-CT assessments at 7 and 21 days post-local Areg treatment exhibited a marked improvement of bone healing compared to the vehicle control. In vitro studies demonstrated an increase of Areg secretion by the Raw264.7 cells upon ATP stimulation. Indirect co-culture of Raw264.7 and ATDC5 cells indicated that Areg overexpression enhanced the osteogenic potential of chondrocytes, and vice versa. This osteogenic promotion was attributed to Areg's activation of the membrane receptor EGFR in the ATDC5 cell line, the enhanced phosphorylation of transcription factor Yap, and the facilitation of the expression of bioactive TGF-β by chondrocytes. Collectively, this research elucidates the direct mechanistic effects of macrophage-secreted Areg in promoting bone homeostasis following bone injury.

YKL40/Integrin β4 Axis Induced by the Interaction between Cancer Cells and Tumor-Associated Macrophages Is Involved in the Progression of High-Grade Serous Ovarian Carcinoma.

Macrophages in the tumor microenvironment, termed tumor-associated macrophages (TAMs), promote the progression of various cancer types. However, many mechanisms related to tumor-stromal interactions in epithelial ovarian cancer (EOC) progression remain unclear. High-grade serous ovarian carcinoma (HGSOC) is the most malignant EOC subtype. Herein, immunohistochemistry was performed on 65 HGSOC tissue samples, revealing that patients with a higher infiltration of CD68+, CD163+, and CD204+ macrophages had a poorer prognosis. We subsequently established an indirect co-culture system between macrophages and EOC cells, including HGSOC cells. The co-cultured macrophages showed increased expression of the TAM markers CD163 and CD204, and the co-cultured EOC cells exhibited enhanced proliferation, migration, and invasion. Cytokine array analysis revealed higher YKL40 secretion in the indirect co-culture system. The addition of YKL40 increased proliferation, migration, and invasion via extracellular signal-regulated kinase (Erk) signaling in EOC cells. The knockdown of integrin β4, one of the YKL40 receptors, suppressed YKL40-induced proliferation, migration, and invasion, as well as Erk phosphorylation in some EOC cells. Database analysis showed that high-level expression of YKL40 and integrin β4 correlated with a poor prognosis in patients with serous ovarian carcinoma. Therefore, the YKL40/integrin β4 axis may play a role in ovarian cancer progression.

MiR-NPs-RVG promote spinal cord injury repair: implications from spinal cord-derived microvascular endothelial cells

BackgroundSpinal cord injury (SCI) often leads to a loss of motor and sensory function. Axon regeneration and outgrowth are key events for functional recovery after spinal cord injury. Endogenous growth of axons is associated with a variety of factors. Inspired by the relationship between developing nerves and blood vessels, we believe spinal cord-derived microvascular endothelial cells (SCMECs) play an important role in axon growth.ResultsWe found SCMECs could promote axon growth when co-cultured with neurons in direct and indirect co-culture systems via downregulating the miR-323-5p expression of neurons. In rats with spinal cord injury, neuron-targeting nanoparticles were employed to regulate miR-323-5p expression in residual neurons and promote function recovery.ConclusionsOur study suggests that SCMEC can promote axon outgrowth by downregulating miR-323-5p expression within neurons, and miR-323-5p could be selected as a potential target for spinal cord injury repair.Graphical

Optimization of an autaptic culture system for studying cholinergic synapses in sympathetic ganglia.

An autaptic synapse (or 'autapse') is a functional connection between a neuron and itself, commonly used in studying the molecular mechanisms underlying synaptic transmission and plasticity in central neurons. Most previous studies on autonomic synaptic functions have relied on spontaneous connections among neurons in mass cultures. However, growing evidence supports the utility of microcultures cultivating autaptic neurons for examining cholinergic transmission within sympathetic ganglia. Despite these advancements, standardized protocols for culturing autaptic sympathetic neurons have yet to be established. Drawing on historical literature, this study delineates optimal experimental conditions to efficiently and reliably produce cholinergic synapses in sympathetic neurons within a short time frame. Our research emphasizes five key factors: (i) the generation of uniformly sized microislands of growth permissive substrates; (ii) the addition of nerve growth factor, ciliary neurotrophic factor (CNTF), and serum to the culture medium; (iii) independence from specific serum and neuronal medium types; (iv) the reciprocal roles of CNTF and glial cells; and (v) the promotion of cholinergic synaptogenesis in SCG neurons through indirect glia co-cultures, rather than direct glial feeder layer cultures. In conclusion, glia-free monocultures of SCG neurons are relatively simple to prepare and yield robust and reliable synaptic currents. This makes them an effective model system for straightforwardly addressing fundamental questions about neurogenic mechanisms involved in cholinergic synaptic transmission in autonomic ganglia. Furthermore, autaptic culture experiments could eventually be implemented to investigate the roles of functional neuron-satellite glia units in regulating cholinergic functions under physiological and pathological conditions.

The Role of Breastmilk in Macrophage-Tumour Cell Interactions in Postpartum Breast Cancer

Background: Lactation is associated with long-term reduced risk of breast cancer. However, there is a transient increased risk of breast cancer in the 5 to 10 years postpartum and this is associated with a high incidence of metastasis and mortality. Breastmilk is a physiological fluid secreted by the mammary glands intimately connected with breast cells and the microenvironment that may affect postpartum breast cancer development and progression. This study aims to investigate the effect of breastmilk on interactions between breast cancer cells and macrophages in vitro. Methods: Human breastmilk from healthy donors (n = 7) was pooled and incubated with breast cancer (MCF-7 and MDA-MB-231) and macrophage (RAW264.7) cell lines to assess cell proliferation, viability, migration, and expression of key genes associated with epithelial-mesenchymal transition (EMT) and macrophage phenotype. Indirect co-culture studies assessed the effect of breastmilk on interactions between breast cancer cells and macrophages. Results: Breastmilk increased the proliferation and viability of breast cancer cells, reduced EMT markers, and reduced cell migration in MDA-MB-231 cells. Breastmilk decreased mRNA expression of interleukin 1B (IL1B) and interleukin 10 (IL10) in macrophages. Reduced EMT marker expression was observed in breast cancer cells co-cultured with macrophages pre-treated with breastmilk. Macrophages co-cultured with breast cancer cells pre-treated with breastmilk exhibited increased expression of a pro-inflammatory cytokine tumor necrosis factor A (TNFA) and pro-inflammatory nitric oxide synthase 2 (NOS2), and reduced expression of cytokines IL10 and transforming growth factor B1 (TGFB1) which are associated with the alternatively-activated macrophage phenotype. Conclusions: Breastmilk has the potential to promote breast cancer proliferation, however, it can also reduce breast cancer progression through inhibition of breast cancer cell migration and regulation of macrophage polarisation. These findings suggest that breastmilk has potential to shape the tumour microenvironment in postpartum breast cancer.