Co-culture Model Research Articles

Extracellular vesicle-packaged PIAT from cancer-associated fibroblasts drives neural remodeling by mediating m5C modification in pancreatic cancer mouse models.

Perineural invasion (PNI) is a biological characteristic commonly observed in pancreatic cancer. Although PNI plays a key role in pancreatic cancer metastasis, recurrence, and poor postoperative survival, its mechanism is largely unclarified. Clinical sample analysis and endoscopic ultrasonographic elasticity scoring indicated that cancer-associated fibroblasts (CAFs) were closely related to the occurrence of PNI. Furthermore, CAF-derived extracellular vesicles (EVs) were involved in PNI in dorsal root ganglion coculture and mouse sciatic nerve models. Next, we demonstrated that CAFs promoted PNI through extracellular vesicle transmission of PNI-associated transcript (PIAT). Mechanistically, PIAT specifically bound to YBX1 and blocked the YBX1-Nedd4l interaction to inhibit YBX1 ubiquitination and degradation. Furthermore, PIAT enhanced the binding of YBX1 and PNI-associated mRNAs in a 5-methylcytosine (m5C)-dependent manner. Mutation of m5C recognition motifs in YBX1 or m5C sites in downstream target genes reversed PIAT-mediated PNI. Consistent with these findings, analyses using a KPC mouse model demonstrated that the PIAT/YBX1 axis enhanced PNI through m5C modification. Clinical data suggested that the PIAT expression in the serum EVs of patients with pancreatic cancer was associated with the degree of neural invasion and prognosis. Our study revealed the important role of the PIAT/YBX1 signaling axis in the tumor microenvironment (TME) in promoting tumor cell PNI and provided a new target for precise interference with CAFs and RNA methylation in the TME to suppress PNI in pancreatic cancer.

Drug resistant pancreatic cancer cells exhibit altered biophysical interactions with stromal fibroblasts in imaging studies of 3D co-culture models.

Interactions between tumor and stromal cells are well known to play a prominent roles in progression of pancreatic ductal adenocarcinoma (PDAC). As knowledge of stromal crosstalk in PDAC has evolved, it has become clear that cancer associated fibroblasts can play both tumor promoting and tumor suppressive roles through a combination of paracrine crosstalk and juxtacrine interactions involving direct physical contact. Another major contributor to dismal survival statistics for PDAC is development of resistance to chemotherapy drugs. Though less is known about how the acquisition of chemoresistance impacts upon tumor-stromal crosstalk. Here, we use 3D co-culture geometries to recapitulate juxtacrine interactions between epithelial and stromal cells. In particular, extracellular matrix (ECM) overlay cultures in which stromal cells (pancreatic stellate cells, or normal human fibroblasts) are placed adjacent to PDAC cells (PANC1), result in direct heterotypic cell adhesions accompanied by dramatic fibroblast contractility which leads to highly condensed macroscopic multicellular aggregates as detected using particle image velocimetry (PIV) analysis to quantify cell velocities over the course of time lapse movie sequences. To investigate how drug resistance impacts these juxtacrine interactions we contrast cultures in which PANC1 are substituted with a drug resistant subline (PANC1-OR) previously established in our lab. We find that heterotypic cell-cell interactions are highly suppressed in drug-resistant cells relative to the parental PANC1 cells. To investigate further we conduct RNA-seq and bioinformatics analysis to identify differential gene expression in PANC1 and PANC1-OR, which shows that negative regulation of cell adhesion molecules, consistent with increased epithelial mesenchymal transition (EMT), is also consistent with loss of hetrotypic cell-cell contact necessary for the contractile behavior observed in drug naïve cultures. Overall these findings elucidate the role of drug-resistance in inhibiting an avenue of stromal crosstalk which is associated with tumor suppression and also help to establish cell culture conditions useful for further mechanistic investigation.

Pasteurized form of a potential probiotic lactobacillus brevis IBRC-M10790 exerts anti-inflammatory effects on inflammatory bowel disease in vitro

BackgroundInflammatory bowel disease (IBD) is a chronic, relapsing inflammatory disorder of the gastrointestinal system. So far, no treatment has been identified that can completely cure IBD. Lactobacillus brevis is hypothesized to be beneficial in preventing inflammation. This study aimed to evaluate the potential probiotic effects of live and pasteurized L. brevis IBRC-M10790 on the in vitro cell co-culture model of IBD.MethodsAn in vitro intestinal model was established using a transwell co-culture system of Caco-2 intestinal epithelial cells and RAW264.7 macrophages. Inflammatory conditions were induced in RAW264.7 cells using lipopolysaccharide. The effects of live and pasteurized L. brevis IBRC-M10790 on inflammatory mediators and epithelial barrier markers were investigated.ResultsL. brevis IBRC-M10790 was able to significantly decrease the proinflammatory cytokines (IL-6, IL-1β, and TNF-α) and increase the anti-inflammatory cytokine (IL-10) in the in vitro co-culture system. In addition, L. brevis increased adherens and tight junction (TJ) markers (ZO-1, E-cadherin, and Occludin) in Caco-2 intestinal epithelial cells. Based on the results, pasteurized L. brevis showed a higher protective effect than live L. brevis.ConclusionsOur findings suggest that live and pasteurized forms of L. brevis possess probiotic properties and can mitigate inflammatory conditions in IBD.

Simulated human digestion and fermentation of a high-molecular weight polysaccharide from Lentinula edodes mushroom and protective effects on intestinal barrier

Lentinula edodes (Shiitake) is an important edible mushroom and polysaccharides are its major constituents with proven health benefits. The study was to investigate the gut bacterial fermentation and subsequent effects on gut barrier function of a glucan-rich polysaccharide, LePS40 precipitated from the mushroom water extract with 40 % (v/v) ethanol. LePS40 consisted of a β-(1→3)-glucan main chain with substitution in the C-6 position with side chains mainly composed of (1 → 6)-linked β-Glcp residues, (1 → 6)-linked α-Galp residues and terminal residues of β-Glcp. LePS40 was found highly resistant to digestive enzymes and gastric acid in simulated human gastrointestinal tract, but highly fermentable during in vitro human fecal fermentation. The fecal fermentation degradation of LePS40 appeared to selectively break the glucoside linkage in view of the dramatic decrease in the glucose molar ratio (12.68 to 1.07). Compared with the prebiotic reference FOS, LePS40 led to much higher levels of butyric, and propionic acid and a lower level of acetic acid. Moreover, LePS40 enhanced the abundance of some beneficial bacterial populations, but decreased the bacteria possibly linked with fatty-liver disease and colorectal cancer. Furthermore, the fecal fermentation products of LePS40 showed a potential protective effect on intestinal barrier function against inflammatory damage in Caco-2/Raw264.7 co-culture model. These findings suggest the potential of LePS40 for improvement of gut health through modulation of gut microbiota.

A novel CAF-cancer cell crosstalk-related gene prognostic index based on machine learning: prognostic significance and prediction of therapeutic response in head and neck squamous cell carcinoma

BackgroundCancer-associated fibroblast (CAF)-cancer cell crosstalk (CCCT) plays an important role in tumor microenvironment shaping and immunotherapy response. Current prognostic indexes are insufficient to accurately assess immunotherapy response in patients with head and neck squamous cell carcinoma (HNSCC). This study aimed to develop a CCCT-related gene prognostic index (CCRGPI) for assessing the prognosis and response to immune checkpoint inhibitor (ICI) therapy of HNSCC patients.MethodsTwo cellular models, the fibroblast-cancer cell indirect coculture (FCICC) model, and the fibroblast-cancer cell organoid (FC-organoid) model, were constructed to visualize the crosstalk between fibroblasts and cancer cells. Based on a HNSCC scRNA-seq dataset, the R package CellChat was used to perform cell communication analysis to identify gene pairs involved in CCCT. Least absolute shrinkage and selection operator (LASSO) regression was then applied to further refine the selection of these gene pairs. The selected gene pairs were subsequently subjected to stepwise regression to develop CCRGPI. We further performed a comprehensive analysis to determine the molecular and immune characteristics, and prognosis associated with ICI therapy in different CCRGPI subgroups. Finally, the connectivity map (CMap) analysis and molecular docking were used to screen potential therapeutic drugs.ResultsFCICC and FC-organoid models showed that cancer cells promoted the activation of fibroblasts into CAFs, that CAFs enhanced the invasion of cancer cells, and that CCCT was somewhat heterogeneous. The CCRGPI was developed based on 4 gene pairs: IGF1-IGF1R, LGALS9-CD44, SEMA5A-PLXNA1, and TNXB-SDC1. Furthermore, a high CCRGPI score was identified as an adverse prognostic factor for overall survival (OS). Additionally, a high CCRGPI was positively correlated with the activation of the P53 pathway, a high TP53 mutation rate, and decreased benefit from ICI therapy but was inversely associated with the abundance of various immune cells, such as CD4+ T cells, CD8+ T cells, and B cells. Moreover, Ganetespib was identified as a potential drug for HNSCC combination therapy.ConclusionsThe CCRGPI is reliable for predicting the prognosis and immunotherapy response of HSNCC patients and may be useful for guiding the individualized treatment of HNSCC patients.

Glutamine metabolic competition drives immunosuppressive reprogramming of intratumour GPR109A+ myeloid cells to promote liver cancer progression

ObjectiveThe metabolic characteristics of liver cancer drive considerable hurdles to immune cells function and cancer immunotherapy. However, how metabolic reprograming in the tumour microenvironment impairs the antitumour immune response remains...

PBMC-mediated modulation of macrophage polarization in RAW264.7 cells through STAT1/STAT6 signaling cascades

Peripheral blood mononuclear cells (PBMC), sourced autologously, offer numerous advantages when procured: easier acquisition process, no in vitro amplification needed, decreased intervention and overall increased acceptability make PBMC an attractive candidate for cell therapy treatment. However, the exact mechanism by which PBMC treat diseases remains poorly understood. Immune imbalance is the pathological basis of many diseases, with macrophages playing a crucial role in this process. However, research on the role and mechanisms of PBMC in regulating macrophages remains scarce. This study employed an in vitro co-culture model of PBMC and RAW264.7 macrophages to explore the role and mechanisms of PBMC in regulating macrophages. The results showed that the co-culturing led to decreased expression of inflammatory cytokines and increased expression of anti-inflammatory cytokines in RAW264.7 or in the culture supernatant. Additionally, the pro-inflammatory, tissue matrix-degrading M1 macrophages decreased, while the anti-inflammatory, matrix-synthesizing, regenerative M2 macrophages increased in both RAW264.7 and monocytes within PBMC. Moreover, co-cultured macrophages exhibited a significantly decreased p-STAT1/STAT1 ratio, while the p-STAT6/STAT6 ratio significantly increased. This suggests that PBMC may inhibit M1 macrophage polarization by blocking STAT1 signaling cascades and may promote M2 macrophage polarization through the activation of STAT6 signaling cascades. Overall, this study sheds light on the role and mechanism of PBMC in regulating macrophages. Moreover, it was found that monocytes within co-cultured PBMC differentiated into M2 macrophages in the presence of macrophages. This finding provides experimental evidence for the use of PBMC in treating inflammatory diseases, especially macrophage-depleting inflammatory diseases such as osteoarthritis.

A Fibroblast-Derived Secretome Stimulates the Growth and Invasiveness of 3D Plexiform Neurofibroma Spheroids.

Plexiform neurofibromas (PNs) occur in about a half of neurofibromatosis type 1 (NF1) patients and have garnered significant research attention due to their capacity for growth and potential for malignant transformation. NF1 plexiform neurofibroma (pNF1) is a complex tumor composed of Schwann cell-derived tumor cells (Nf1-/-) and the tumor microenvironment (TME). Although it has been widely demonstrated that the TME is involved in the formation of neurofibromas, little is known about the effects of the TME on the subsequent progression of human pNF1. Elucidating the molecular interactions between tumor cells and the TME may provide new therapeutic targets to reduce the progression of pNF1. In the present study, we focused on the contributions of fibroblasts, the most abundant cell types in the TME, to the growth of pNF1. To simulate the TME, we used a three-dimensional (3D) coculture model of immortalized pNF1 tumor cells (Nf1-/-) and primary fibroblasts (Nf1+/-) derived from pNF1 patients. We performed live-cell imaging of 3D/4D (3D in real-time) cultures through confocal microscopy followed by 3D quantitative analyses using advanced imaging software. The growth of pNF1 spheroids in 3D cocultures with fibroblasts was significantly greater than that of pNF1 spheroids in 3D monocultures. An increase in the growth of pNF1 spheroids also occurred when they were cultured with conditioned media (CM) from fibroblasts. Moreover, fibroblast-derived CM increased the invasive outgrowth and further local invasion of pNF1 spheroids. Interestingly, when small extracellular vesicles (sEVs) were depleted from the fibroblast-derived CM, the stimulation of the growth of pNF1 spheroids was lost. Our results suggest that fibroblast-derived sEVs are a therapeutic target for reducing the growth of pNF1.

M2-type tumor-associated macrophages upregulated PD-L1 expression in cervical cancer via the PI3K/AKT pathway

Background and purposePD-1/PD-L1 inhibitors have become a promising therapy. However, the response rate is lower than 30% in patients with cervical cancer (CC), which is related to immunosuppressive components in tumor microenvironment (TME). Tumor-associated macrophages (TAMs), as one of the most important immune cells, are involved in the formation of tumor suppressive microenvironment. Therefore, it will provide a theoretical basis for curative effect improvement about the regulatory mechanism of TAMs on PD-L1 expression.MethodsThe clinical data and pathological tissues of CC patients were collected, and the expressions of PD-L1, CD68 and CD163 were detected by immunohistochemistry. Bioinformatics was used to analyze the macrophage subtypes involved in PD-L1 regulation. A co-culture model was established to observe the effects of TAMs on the morphology, migration and invasion function of CC cells, and the regulatory mechanism of TAMs on PD-L1.ResultsPD-L1 expression on tumor cells could predict the poor prognosis of patients. And there was a strong correlation between PD-L1 expression with CD163+TAMs infiltration. Similarly, PD-L1 expression was associated with M1/M2-type TAMs infiltration in bioinformatics analysis. The results of cell co-culture showed that M1/M2-type TAMs could upregulate PD-L1 expression, especially M2-type TAMs may elevate the PD-L1 expression via PI3K/AKT pathway. Meanwhile, M1/M2-type TAMs can affect the morphological changes, and enhance migration and invasion abilities of CC cells.ConclusionsPD-L1 expression in tumor cells can be used as a prognostic factor and is closely related to CD163+TAMs infiltration. In addition, M2-type TAMs can upregulate PD-L1 expression in CC cells through PI3K/AKT pathway, enhance the migration and invasion capabilities, and affect the tumor progression.

Development of Imiquimod-induced HaCaT-THP-1 co-culture for modeling of psoriasis

Psoriasis is one of the most prevalent and chronic inflammatory disease of the skin, associated with disrupted barrier function. Currently, a widely accepted, generally usable cell culture model has not been developed yet. In the present work, we aimed to establish a co-culture model with human keratinocyte (HaCaT) and human monocyte cells (THP-1) induced by Imiquimod (IMQ), which acts on the TLR7 receptor. The role of TLR7 expressed on THP-1 cells was confirmed by immunofluorescence staining of NF-κB activation. Chloroquine (CH) was used as a receptor inhibitor, in the presence or absence of which the NF-κB pathway was activated. We determined the most effective proliferation-stimulating IMQ concentration by RTCA method and the hyperproliferative effect was investigated by wound-healing test. The effect of IMQ was compared with the effects of the anthocyanin (AC) components from the anti-inflammatory sour cherry extract that we have already studied. We found that IMQ significantly increased the migration rate however, the combined treatment resulted in a decreased migration rate compared to the IMQ treatment alone. Inflammatory cytokines were measured from the supernatant of co-culture by ELISA. During the development of the co-culture intended to model psoriasis, we confirmed the induction effect of IMQ and in the case of AC treatment, we supported the stabilizing effect of the barrier.

SRGN promotes macrophage recruitment through CCL3 in osteoarthritis

ABSTRACT Background Osteoarthritis (OA) is a degenerative disease that affects synovial joints and leads to significant pain and disability, particularly in older adults. Infiltration of macrophages plays a key role in the progression of OA. However, the mechanisms underlying macrophage recruitment in OA are not fully understood. Methods The Serglycin (SRGN) expression pattern was analyzed, along with its association with macrophage infiltration in OA, using bioinformatic methods. SRGN expression in chondrocytes was altered by small interfering RNA (siRNA) and plasmids. Conditioned media (CM) was obtained from transfected chondrocytes to establish a co-culture model of chondrocytes and THP-1 derived macrophages. The impact of SRGN on macrophage recruitment was evaluated using a transwell assay. Furthermore, the regulatory effect of SRGN on CCL3 was validated through qPCR, WB, and ELISA experiments. Results In OA patients, the upregulation of SRGN positively correlated with K-L grade and macrophage infiltration. It was found that SRGN expression and secretion were up-regulated in OA and that it can promote macrophage migration in vitro. Further investigation showed that SRGN affects macrophage migration by regulating the expression of CCL3. Conclusion SRGN in chondrocytes plays a role in promoting the recruitment of THP-1 derived macrophages in vitro by regulating production of CCL3.

63 Developing a 3D in Vitro Schwann Cell Model of Myelination for Peripheral Nerve Injury

Abstract Aim This study investigated the effectiveness of fabricating engineered neural tissue (EngNT) to develop a 3D in vitro model of myelination for peripheral nerve injury. Additionally, the effect of metformin and sulindac sulfide in nerve regeneration was evaluated by assessing neurite growth in both monolayer and 3D co-culture systems. Method Neuronal cells (NG108-15 and SH-SY5Y cell lines) were treated with metformin for 72 hrs in an Incucyte® live-cell analysis system. EngNT cellular collagen gels were fabricated using Schwann cells (SCL4.1/F7), SCL4.1/F7:NG108-15 (1:1) cells or harvested rat dorsal root ganglion cells. NG108–15 cells were co-cultured with SCL4.1/F7 cellular gels. 3D EngNT co-cultures were treated with metformin and sulindac sulfide for 72 hrs. EngNT cellular gels were analysed with transmission electron microscopy (TEM). Results TEM evaluation of EngNT co-cultures demonstrated the enfoldment of neurones by Schwann cells resulting in cell-cell contacts and myelination was identified. NG108-15 and SH-SY5Y cells exhibited significant neurite growth with 100 μM metformin being the optimal dose (151.4 ± 3 μM and 108.0 ± 12 μM respectively). Increased neurite growth of NG108-15 neurones was demonstrated with 100 μM (179.3 ± 2 μM) metformin treatment in 3D EngNT. Additionally, significant neurite growth was demonstrated following treatment with 100 μM (203.9 ± 32 μM) sulindac sulfide. Conclusions A 3D EngNT in vitro co-culture model of Schwann cell myelination was developed illustrating the cell-cell interaction between Schwann cells and neurones and the formation of myelin in the peripheral nervous system. Metformin elicits significant positive results on neurite growth in vitro highlighting its therapeutic potential in PNI.

Utilizing human cerebral organoids to model breast cancer brain metastasis in culture

BackgroundMetastasis, the spread, and growth of malignant cells at secondary sites within a patient’s body, accounts for over 90% of cancer-related mortality. Breast cancer is the most common tumor type diagnosed and the leading cause of cancer lethality in women in the United States. It is estimated that 10–16% breast cancer patients will have brain metastasis. Current therapies to treat patients with breast cancer brain metastasis (BCBM) remain palliative. This is largely due to our limited understanding of the fundamental molecular and cellular mechanisms through which BCBM progresses, which represents a critical barrier for the development of efficient therapies for affected breast cancer patients.MethodsPrevious research in BCBM relied on co-culture assays of tumor cells with rodent neural cells or rodent brain slice ex vivo. Given the need to overcome the obstacle for human-relevant host to study cell-cell communication in BCBM, we generated human embryonic stem cell-derived cerebral organoids to co-culture with human breast cancer cell lines. We used MDA-MB-231 and its brain metastatic derivate MDA-MB-231 Br-EGFP, other cell lines of MCF-7, HCC-1806, and SUM159PT. We leveraged this novel 3D co-culture platform to investigate the crosstalk of human breast cancer cells with neural cells in cerebral organoid.ResultsWe found that MDA-MB-231 and SUM159PT breast cancer cells formed tumor colonies in human cerebral organoids. Moreover, MDA-MB-231 Br-EGFP cells showed increased capacity to invade and expand in human cerebral organoids.ConclusionsOur co-culture model has demonstrated a remarkable capacity to discern the brain metastatic ability of human breast cancer cells in cerebral organoids. The generation of BCBM-like structures in organoid will facilitate the study of human tumor microenvironment in culture.

Immobilized NRG1 Accelerates Neural Crest like Cell Differentiation Toward Functional Schwann Cells Through Sustained Erk1/2 Activation and YAP/TAZ Nuclear Translocation.

Neural Crest cells (NC) are a multipotent cell population that give rise to a multitude of cell types including Schwann cells (SC) in the peripheral nervous system (PNS). Immature SC interact with neuronal axons via the neuregulin 1 (NRG1) ligand present on the neuronal surface and ultimately form the myelin sheath. Multiple attempts to derive functional SC from pluripotent stem cells have met challenges with respect to expression of mature markers and axonal sorting. Here, they hypothesized that sustained signaling from immobilized NRG1 (iNRG1) might enhance the differentiation of NC derived from glabrous neonatal epidermis towards a SC phenotype. Using this strategy, NC derived SC expressed mature markers to similar levels as compared to explanted rat sciatic SC. Signaling studies revealed that sustained NRG1 signaling led to yes-associated protein 1 (YAP) activation and nuclear translocation. Furthermore, NC derived SC on iNRG1 exhibited mature SC function as they aligned with rat dorsal root ganglia (DRG) neurons in an in vitro coculture model; and most notably, aligned on neuronal axons upon implantation in a chick embryo model in vivo. Taken together their work demonstrated the importance of signaling dynamics in SC differentiation, aiming towards development of drug testing platforms for de-myelinating disorders.

Parsing the effect of co-culture with brain organoids on Diffuse Intrinsic Pontine Glioma (DIPG) using quantitative proteomics

Diffuse Intrinsic Pontine Gliomas (DIPGs) are deadly brain cancers in children for which there is no effective treatment. This can partly be attributed to preclinical models that lack essential elements of the in vivo tissue environment, resulting in treatments that appear promising preclinically, but fail to result in effective cures. Recently developed co-culture models combining stem cell-derived brain organoids with brain cancer cells provide tissue dimensionality and a human-relevant tissue-like microenvironment. As these models are technically challenging, we aimed to establish whether interaction with the organoid influences DIPG biology and thus warrants their use. To address this question DIPG24 cells were cultured with pluripotent stem cell-derived cortical organoids. We created “mosaic” co-cultures enriched for tumour cell-neuronal cell interactions versus “assembloid” co-cultures enriched for tumour cell-tumour cell interactions. Sequential window acquisition of all theoretical mass spectra (SWATH-MS) was used to analyse the proteomes of DIPG fractions isolated by flow-assisted cell sorting. Control proteomes from DIPG spheroids were compared with DIPG cells isolated from mosaic and assembloid co-cultures. This suggested changes in cell interaction with the external environment reflected by decreased gene ontology terms associated with adhesion and extracellular matrix, and increased DNA synthesis and replication, in DIPG24 cells under either co-culture condition. By contrast, the mosaic co-culture was associated with neuron-specific brahma-associated factor (nBAF) complex signalling, a process associated with neuronal maturation. We propose that co-culture with brain organoids is a valuable tool to parse the contribution of the brain microenvironment to DIPG tumour biology.

Cancer cell migration depends on adjacent ASC and adipose spheroids in a 3D bioprinted breast cancer model

Breast cancer develops in close proximity to mammary adipose tissue and interactions with the local adipose environment have been shown to drive tumor progression. The specific role, however, of this complex tumor microenvironment in cancer cell migration still needs to be elucidated. Therefore, in this study, a 3D bioprinted breast cancer model was developed that allows for a comprehensive analysis of individual tumor cell migration parameters in dependence of adjacent adipose stroma. In this co-culture model, a breast cancer compartment with MDA-MB-231 breast cancer cells embedded in collagen is surrounded by an adipose tissue compartment consisting of adipose-derived stromal cell (ASC) or adipose spheroids in a printable bioink based on thiolated hyaluronic acid. Printing parameters were optimized for adipose spheroids to ensure viability and integrity of the fragile lipid-laden cells. Preservation of the adipogenic phenotype after printing was demonstrated by quantification of lipid content, expression of adipogenic marker genes, the presence of a coherent adipo-specific extracellular matrix, and cytokine secretion. The migration of tumor cells as a function of paracrine signaling of the surrounding adipose compartment was then analyzed using live-cell imaging. The presence of ASC or adipose spheroids substantially increased key migration parameters of MDA-MB-231 cells, namely motile fraction, persistence, invasion distance, and speed. These findings shed new light on the role of adipose tissue in cancer cell migration. They highlight the potential of our 3D printed breast cancer-stroma model to elucidate mechanisms of stroma-induced cancer cell migration and to serve as a screening platform for novel anti-cancer drugs targeting cancer cell dissemination.

9-cis-retinoic acid signaling in Sertoli cells regulates their immunomodulatory function to control lymphocyte physiology and Treg differentiation

BackgroundTestis is an immune privileged organ, which prevents the immune response against sperm antigens and inflammation. Testicular cells responsible for immune tolerance are mainly Sertoli cells, which form the blood-testis barrier and produce immunosuppressive factors. Sertoli cells prevent inflammation in the testis and maintain immune tolerance by inhibiting proliferation and inducing lymphocyte apoptosis. It has been shown that 9-cis-retinoic acid (9cRA) blocks ex vivo apoptosis of peripheral blood lymphocytes and promotes the differentiation of Treg cells in the gut. However, the role of retinoid signaling in regulating the immune privilege of the testes remains unknown.ObjectiveThe aim of this study was to determine whether 9cRA, acting via the retinoic acid receptors (RAR) and the retinoic X receptors (RXR), controls the immunomodulatory functions of Sertoli cells by influencing the secretion of anti-inflammatory/pro-inflammatory factors, lymphocyte physiology and Treg cell differentiation.MethodsExperiments were performed using in vitro model of co-cultures of murine Sertoli cells and T lymphocytes. Agonists and antagonists of retinoic acid receptors were used to inhibit/stimulate retinoid signaling in Sertoli cells.ResultsOur results have demonstrated that 9cRA inhibits the expression of immunosuppressive genes and enhances the expression of pro-inflammatory factors in Sertoli cells and lymphocytes, increases lymphocyte viability and decreases apoptosis rate. Moreover, we have found that 9cRA blocks lymphocyte apoptosis acting through both RAR and RXR and inhibiting FasL/Fas/Caspase 8 and Bax/Bcl-2/Caspase 9 pathways. Finally, we have shown that 9cRA signaling in Sertoli cells inhibits Treg differentiation.ConclusionCollectively, our results indicate that retinoid signaling negatively regulates immunologically privileged functions of Sertoli cells, crucial for ensuring male fertility. 9cRA inhibits lymphocyte apoptosis, which can be related to the development of autoimmunity, inflammation, and, in consequence, infertility.

The Human Neonatal Skin Fibroblast, an Available Cell Source for Tissue Production and Transplantation, Exhibits Low Risk of Immunogenicity In Vitro.

The immunogenicity of allogeneic skin fibroblasts in transplantation has been controversial. Whether this controversy comes from a natural heterogeneity among fibroblast subsets or species-specific differences between human and mouse remains to be addressed. In this study, we sought to investigate whether fibroblasts derived from either adult or neonatal human skin tissues could induce different immune responses toward phagocytosis and T cell activation using in vitro co-culture models. Our results indicate that both phagocytosis and T cell proliferation are reduced in the presence of neonatal skin fibroblasts compared to adult skin fibroblasts. We also show that neonatal skin fibroblasts secrete paracrine factors that are responsible for reduced T cell proliferation. In addition, we show that neonatal skin fibroblasts express less class II human leukocyte antigen (HLA) molecules than adult skin fibroblasts after interferon gamma priming, which might also contribute to reduced T cell proliferation. In conclusion, this study supports the use of allogeneic neonatal skin fibroblasts as a readily available cell source for tissue production and transplantation to treat patients with severe injuries.

Targeting MDSC-HTR2B to Improve Immune Checkpoint Inhibitors in Breast to Brain Metastasis.

Myeloid Derived Suppressor Cells (MDSCs) support breast cancer growth via immune suppression and non-immunological mechanisms. Although 15% of patients with breast cancer will develop brain metastasis, there is scant understanding of MDSCs' contribution within the breast-to-brain metastatic microenvironment. Utilizing co-culture models mimicking a tumor-neuron-immune microenvironment and patient tissue arrays, we identified serotonergic receptor, HTR2B, on MDSCs to upregulate pNF-κB and suppress T cell proliferation, resulting in enhanced tumor growth. In vivo murine models of metastatic and intracranial breast tumors treated with FDA-approved, anti-psychotic HTR2B antagonist, clozapine, combined with immunotherapy anti-PD-1 demonstrated a significant increase in survival and increased T cell infiltration. Collectively, these findings reveal a previously unknown role of MDSC-HTR2B in breast-to-brain metastasis, suggesting a novel and immediate therapeutic approach using neurological drugs to treat patients with metastatic breast cancer.

Pathogen-Mimicking Nanoparticles Based on Rigid Nanomaterials as an Efficient Subunit Vaccine Delivery System for Intranasal Immunization.

Despite the safety profile of subunit vaccines, the inferior immunogenicity hinders their application in the nasal cavity. This study introduces a novel antigen delivery and adjuvant system utilizing mucoadhesive chitosan-catechol (Chic) on silica spiky nanoparticles (Ssp) to enhance immunity through multiple mechanisms. The Chic functionalizes the Ssp surface and incorporates with SARS-CoV-2 spike protein receptor-binding domain (RBD) and toll-like receptor (TLR)9 agonist unmethylated cytosine-guanine (CpG) motif, forming uniform virus-like nanoparticles (Ssp-Chic-RBD-CpG) via electrostatic and covalent interactions. Ssp-Chic-RBD-CpG, mimicking the morphology and function of inactive virions, effectively prolongs the retention time of RBD in the nasal mucosa by 3.92-fold compared to RBD alone, enhances the maturation of dendritic cells (DCs), and facilitates the antigen trafficking to the draining lymph nodes, which subsequently induces a stronger mucosal immunity. Mechanistically, the enhanced chemokine chemokine (C-C motif) ligand 20 (CCL20)-driven DCs recruitment and maturation by Ssp-Chic-RBD-CpG are evidenced by a cell co-culture model. In addition, the overexpression of TLR4/9 and activation of MYD88/NF-κB signaling pathway in activation of DCs are observed. Proof of principle is obtained for RBD, but similar delivery mechanisms can be applied in other protein-based subunit vaccines as well when intranasal administration is needed.