The Critical Importance of Spatial and Temporal Scales in Designing and Interpreting Immune Cell Migration Assays

Intravital microscopy and other direct-imaging techniques have allowed for a characterisation of leukocyte migration that has revolutionised the field of immunology, resulting in an unprecedented understanding of the mechanisms of immune response and adaptive immunity. However, there is an assumption within the field that modern imaging techniques permit imaging parameters where the resulting cell track accurately captures a cell’s motion. This notion is almost entirely untested, and the relationship between what could be observed at a given scale and the underlying cell behaviour is undefined. Insufficient spatial and temporal resolutions within migration assays can result in misrepresentation of important physiologic processes or cause subtle changes in critical cell behaviour to be missed. In this review, we contextualise how scale can affect the perceived migratory behaviour of cells, summarise the limited approaches to mitigate this effect, and establish the need for a widely implemented framework to account for scale and correct observations of cell motion. We then extend the concept of scale to new approaches that seek to bridge the current “black box” between single-cell behaviour and systemic response.

Matricellular Protein Periostin Promotes Pericyte Migration in Fibrotic Airways

Introduction: Periostin is a matricellular protein that is currently used as a biomarker for asthma. However, its contribution to tissue remodeling in allergic asthma is currently unknown. We have previously demonstrated that tissue-resident mesenchymal stem cells known as pericytes are a key cell type involved in airway remodeling. This is thought to be caused the uncoupling of pericytes from the microvasculature supporting the large airways, facilitated by inflammatory growth factors and cytokines. It is hypothesized that periostin may be produced by profibrotic pericytes and contribute to the remodeling observed in allergic asthma.Methods: Lung sections from mice with allergic airway disease driven by exposure to house dust mite (HDM) were stained using an anti-periostin antibody to explore its involvement in fibrotic lung disease. Human pericytes were cultured in vitro and stained for periostin to assess periostin expression. Migration assays were performed using human pericytes that were pretreated with TGF-β or periostin. ELISAs were also carried out to assess periostin expression levels in bronchoalveolar lavage fluid as well as the induction of periostin production by IL-13.Results: Immunostaining indicated that pericytes robustly express periostin, with increased expression following treatment with TGF-β. Migration assays demonstrated that pericytes treated with periostin were more migratory. Periostin production was also increased in HDM exposed mice as well as in cultured pericytes treated with IL-13.Conclusion: Periostin is produced by pericytes in response to TGF-β or IL-13, and periostin plays a key role in inducing pericyte migration. The increase in periostin expression in TGF-β or IL-13 treated pericytes suggests that IL-13 may trigger periostin production in pericytes whilst TGF-β modulates periostin expression to promote pericyte migration in the context of tissue fibrosis.

NLRP3 inflammasome promoted the malignant progression of prostate cancer via the activation of caspase-1

It is widely accepted that inflammation is an important risk for the development of prostate cancer (PCa). The objective of this study was designed to investigate the potential molecular mechanism of NLR family, pyrin domain-containing protein 3 (NLRP3) inflammasome in the malignant progression of PCa. The expression level of NLRP3 was evaluated by quantitative real-time polymerase chain reaction (qRT-PCR) and fluorescence in situ hybridization. The effects of NLRP3 in the development of PCa by applying gain- and loss-of-function assays in LNCaP and PC3 cell lines were detected by CCK-8, TUNEL, and Transwell migration assays. The underlying mechanism of NLRP3 and caspase-1 in PCa was examined by the rescue experiments, western blotting, and qRT-PCR assays. In addition, the promoting effect of NLRP3 inflammasome was performed with an animal subcutaneous tumorigenesis experiment in vivo. The upregulation of NLRP3 was confirmed in PCa tissues and cell lines. Functionally, using CCK-8, TUNEL, and Transwell migration assays, these results showed that activation of NLRP3/caspase-1 inflammasome by LPS + ATP could enhance the ability of proliferation and migration; and decrease the apoptosis of LNCaP and PC3 cell lines. Western blotting assay showed that the activation of caspase-1 would increase after the stimulation of NLRP3 inflammasome by LPS + ATP. Moreover, the overexpression of NLRP3 promoted, while the knockdown of NLRP3 inhibited the malignant progression in PCa cell lines by positively regulating caspase-1. In addition, the rescue experiments revealed the association among NLRP3 and caspase-1, which showed that the overexpression vectors/inhibitors of caspase-1 could reverse the effect of knockdown/overexpression of NLRP3 in PCa cell lines in vitro. Finally, In in vivo experiment, the suppression of NLRP3 knockdown impaired tumor growth of PCa. Collectively, these results indicated that NLRP3 inflammasome played a vital role in promoting the malignant progression of PCa via the activation of caspase-1. Together, our findings provided insight into the mechanisms of NLRP3/caspase-1 inflammasome and revealed an alternative and potential target for the clinical diagnosis and treatment of PCa.

MicroRNA-124 Alleviates Retinal Vasoregression via Regulating Microglial Polarization

Microglial activation is implicated in retinal vasoregression of the neurodegenerative ciliopathy-associated disease rat model (i.e., the polycystic kidney disease (PKD) model). microRNA can regulate microglial activation and vascular function, but the effect of microRNA-124 (miR-124) on retinal vasoregression remains unclear. Transgenic PKD and wild-type Sprague Dawley (SD) rats received miR-124 at 8 and 10 weeks of age intravitreally. Retinal glia activation was assessed by immunofluorescent staining and in situ hybridization. Vasoregression and neuroretinal function were evaluated by quantitative retinal morphometry and electroretinography (ERG), respectively. Microglial polarization was determined by immunocytochemistry and qRT-PCR. Microglial motility was examined via transwell migration assays, wound healing assays, and single-cell tracking. Our data showed that miR-124 inhibited glial activation and improved vasoregession, as evidenced by the reduced pericyte loss and decreased acellular capillary formation. In addition, miR-124 improved neuroretinal function. miR-124 shifted microglial polarization in the PKD retina from the pro-inflammatory M1 phenotype to the anti-inflammatory M2 phenotype by suppressing TNF-α, IL-1β, CCL2, CCL3, MHC-II, and IFN-γ and upregulating Arg1 and IL-10. miR-124 also decreased microglial motility in the migration assays. The transcriptional factor of C/EBP-α-PU.1 signaling, suppressed by miR-124 both in vivo (PKD retina) and in vitro (microglial cells), could serve as a key regulator in microglial activation and polarization. Our data illustrate that miR-124 regulates microglial activation and polarization. miR-124 inhibits pericyte loss and thereby alleviates vasoregression and ameliorates neurovascular function.

Tumor cell-derived SPON2 promotes M2-polarized tumor-associated macrophage infiltration and cancer progression by activating PYK2 in CRC

Background Tumor-associated macrophages (TAMs) are key regulators of the complex interplay between cancer and the immune microenvironment. Tumor cell-derived spondin 2 (SPON2) is an extracellular matrix glycoprotein that has complicated roles in recruitment of macrophages and neutrophils during inflammation. Overexpression of SPON2 has been shown to promote tumor cell migration in colorectal cancer (CRC). However, the mechanism by which SPON2 regulates the accumulation of TAMs in the tumor microenvironment (TME) of CRC is unknown. Methods Immunohistochemistry was used to examine SPON2 expression in clinical CRC tissues. In vitro migration assays, transendothelial migration assays (iTEM), and cell adhesion assays were used to investigate the effects of SPON2 on monocyte/macrophage migration. Subcutaneous tumor formation and orthotopic implantation assays were performed in C57 BL/6 mice to confirm the effects of SPON2 on TAM infiltration in tumors. Results SPON2 expression is positively correlated with M2-TAM infiltration in clinical CRC tumors and poor prognosis of CRC patients. In addition, SPON2 promotes cytoskeletal remodeling and transendothelial migration of monocytes by activating integrin β1/PYK2 axis. SPON2 may indirectly induce M2-polarization through upregulating cytokines including IL10, CCL2 and CSF1 expression in tumor cells. Blocking M2 polarization and Macrophage depletion inhibited the SPON2-induced tumors growth and invasion. Furthermore, blocking the SPON2/integrin β1/PYK2 axis impairs the transendothelial migration of monocytes and cancer-promoting functions of TAMs in vivo. Conclusions Our findings demonstrate that SPON2-driven M2-TAM infiltration plays an important role during CRC tumor growth and metastasis. SPON2 may be a valuable biomarker guiding the use of macrophage-targeting strategies and a potential therapeutic target in advanced CRC.

Reduced B7-H3 expression by PAX3-FOXO1 knockdown inhibits cellular motility and promotes myogenic differentiation in alveolar rhabdomyosarcoma

B7-H3 (also known as CD276) is associated with aggressive characteristics in various cancers. Meanwhile, in alveolar rhabdomyosarcoma (ARMS), PAX3-FOXO1 fusion protein is associated with increased aggressiveness and poor prognosis. In the present study, we explored the relationship between PAX3-FOXO1 and B7-H3 and the biological roles of B7-H3 in ARMS. Quantitative real time PCR and flow cytometry revealed that PAX3-FOXO1 knockdown downregulated B7-H3 expression in all the selected cell lines (Rh-30, Rh-41, and Rh-28), suggesting that PAX3-FOXO1 positively regulates B7-H3 expression. Gene expression analysis revealed that various genes and pathways involved in chemotaxis, INF-γ production, and myogenic differentiation were commonly affected by the knockdown of PAX3-FOXO1 and B7-H3. Wound healing and transwell migration assays revealed that both PAX3-FOXO1 and B7-H3 were associated with cell migration. Furthermore, knockdown of PAX3-FOXO1 or B7-H3 induced myogenin expression in all cell lines, although myosin heavy chain induction varied depending on the cellular context. Our results indicate that PAX3-FOXO1 regulates B7-H3 expression and that PAX3-FOXO1 and B7-H3 are commonly associated with multiple pathways related to an aggressive phenotype in ARMS, such as cell migration and myogenic differentiation block.

LncRNA HOXA11-AS Aggravates The Keloid Formation By Targeting miR-148b-3p/IGFBP5 Axis

Background: LncRNA homeobox (HOX) A11 antisense (HOXA11-AS) mediates cell-biological phenotypes of keloid fibroblasts and influence the keloid progression, yet the underlying mechanism is not fully understood.Methods: HOXA11-AS, miR-148b-3p and IGFBP5 expression were detected by RT-qPCR or western blots. CCK8 and colony formation assays were applied to examine the cell proliferation. The cell migration was determined via Transwell migration assays. The cell apoptosis was determined by western blots with anti-Bax antibodies and anti-Cleaved Caspase-3 antibodies. The interplay between miR-148b-3p HOXA11-AS and IGFBP5 was confirmed by luciferase reporter assays or RNA immunoprecipitation.Results: The amplification of HOXA11-AS and IGFBP5 was detected in keloid and keloid fibroblasts, while miR-148b-3p expression was reduced. Moreover, downregulation of HOXA11-AS in keloid fibroblasts inhibited cell proliferation, migration and triggered apoptosis. Mechanically, HOXA11-AS was proved to sponge miR-148b-3p and abrogate the inhibition on miR-148b-3p target, IGFBP5 mRNA, thus promoting keloid fibroblasts proliferation, migration and inhibiting apoptosis.Conclusion: These results find that HOXA11-AS promotes keloid progression by miR-148b-3p/IGFBP5 axis, suggesting the potential of targeting HOXA11-AS/miR-148b-3p/IGFBP5 axis to combat keloid.

P2Y2 promotes fibroblasts activation and skeletal muscle fibrosis through AKT, ERK, and PKC

Background Skeletal muscle atrophy and fibrosis are pathological conditions that contribute to morbidity in numerous conditions including aging, cachexia, and denervation. Muscle atrophy is characterized as reduction of muscle fiber size and loss of muscle mass while muscle fibrosis is due to fibroblasts activation and excessive production of extracellular matrix. Purinergic receptor P2Y2 has been implicated in fibrosis. This study aims to elucidate the roles of P2Y2 in sleketal muscle atrophy and fibrosis. Methods Primary muscle fibroblasts were isolated from wild type and P2Y2 knockout (KO) mice and their proliferating and migrating abilities were assessed by CCK-8 and Transwell migration assays respectively. Fibroblasts were activated with TGF-β1 and assessed by western blot of myofibroblast markers including α-SMA, CTGF, and collagen I. Muscle atrophy and fibrosis were induced by transection of distal sciatic nerve and assessed using Masson staining. Results P2Y2 KO fibroblasts proliferated and migrated significantly slower than WT fibroblasts with or without TGF-β1.The proliferation and ECM production were enhanced by P2Y2 agonist PSB-1114 and inhibited by antagonist AR-C118925. TGF-β1 induced fibrotic activation was abolished by P2Y2 ablation and inhibited by AKT, ERK, and PKC inhibitors. Ablation of P2Y2 reduced denervation induced muscle atrophy and fibrosis. Conclusions P2Y2 is a promoter of skeletal muscle atrophy and activation of fibroblasts after muscle injury, which signaling through AKT, ERK and PKC. P2Y2 could be a potential intervention target after muscle injury.

Exosomal let-7f-5p Derived from Mineralized Osteoblasts Promotes the Angiogenesis of Endothelial Cells via DUSP1 / Erk1/2 Signaling Pathway

Background: Angiogenesis is essential for the tissue engineering bone formation, and osteoblasts (OBs) has been proved to play an important role in angiogenesis via various pro-angiogenic factors. However, whether the mineralized osteoblast derived exosomes (MOB-Exos) and containing let-7f-5p can promote the angiogenesis of endothelial cells (ECs) is still unknown.Methods: MOB-Exos, let-7f-5p mimicked MOB-Exos (miR mimic group) and let-7f-5p inhibited MOB-Exos (miR inhibitor group) were respectively harvested from mineralized osteoblasts (MOBs) and then co-cultured with bEnd.3. Besides, the Erk1/2 signaling pathway in ECs in miR mimic group was inhibited. Subsequently, CCK-8 assays, wound healing assays, transwell migration assays and tube formation assays were performed to detect the angiogenic capability of ECs. Dual luciferase reporter assays were conducted to verify the target genes of exosomal let-7f-5p. Results: The results showed that MOB-Exos could significantly promote the angiogenesis of ECs, which could be enhanced by mimicking exosomal let-7f-5p, and attenuated by inhibiting exosomal let-7f-5p. And the angiogenic capability of ECs was partly impaired after inhibiting the Erk1/2 signaling pathway despite co-cultured with let-7f-5p mimicked MOB-Exos. Moreover, let-7f-5p suppressed the luciferase activity of wide-type DUSP1, while mutation of DUSP1 abrogated the repressive ability of let-7f-5p. Conclusion: Based the results, our study concluded that exosomal let-7f-5p derived from MOBs could promote the angiogenesis of ECs via activating DUSP1/Erk1/2 signaling pathway, which might be a promising target for tissue engineering bone formation.

The Origin of Stroma Influences the Biological Characteristics of Oral Squamous Cell Carcinoma

Normal stromal cells surrounding the tumor parenchyma, such as the extracellular matrix (ECM), normal fibroblasts, mesenchymal stromal cells, and osteoblasts, play a significant role in the progression of cancers. However, the role of gingival and periodontal ligament tissue-derived stromal cells in OSCC progression is unclear. In this study, the effect of G-SCs and P-SCs on the differentiation, proliferation, invasion, and migration of OSCC cells in vitro was examined by Giemsa staining, Immunofluorescence (IF), (3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium) (MTS), invasion, and migration assays. Furthermore, the effect of G-SCs and P-SCs on the differentiation, proliferation, and bone invasion by OSCC cells in vivo was examined by hematoxylin-eosin (HE) staining, immunohistochemistry (IHC), and tartrate-resistant acid phosphatase (TRAP) staining, respectively. Finally, microarray data and bioinformatics analyses identified potential genes that caused the different effects of G-SCs and P-SCs on OSCC progression. The results showed that both G-SCs and P-SCs inhibited the differentiation and promoted the proliferation, invasion, and migration of OSCC in vitro and in vivo. In addition, genes, including CDK1, BUB1B, TOP2A, DLGAP5, BUB1, and CCNB2, are probably involved in causing the different effects of G-SCs and P-SCs on OSCC progression. Therefore, as a potential regulatory mechanism, both G-SCs and P-SCs can promote OSCC progression.