Determining the migratory and invasive capacity of tumor and stromal cells and clarifying the underlying mechanisms is most relevant for novel strategies in cancer diagnosis, prognosis, drug development and treatment. Here we shortly summarize the different modes of cell travelling and review in vitro methods, which can be used to evaluate migration and invasion. We provide a concise summary of established migration/invasion assays described in the literature, list advantages, limitations and drawbacks, give a tabular overview for convenience and depict the basic principles of the assays graphically. In many cases particular research problems and specific cell types do not leave a choice for a broad variety of usable assays. However, for most standard applications using adherent cells, based on our experience we suggest to use exclusion zone assays to evaluate migration/invasion. We substantiate our choice by demonstrating that the advantages outbalance the drawbacks e.g. the simple setup, the easy readout, the kinetic analysis, the evaluation of cell morphology and the feasibility to perform the assay with standard laboratory equipment. Finally, innovative 3D migration and invasion models including heterotypic cell interactions are discussed. These methods recapitulate the in vivo situation most closely. Results obtained with these assays have already shed new light on cancer cell spreading and potentially will uncover unknown mechanisms.

In vitro cell migration and invasion assays.

Since cancer stem cells (CSCs) were first identified in leukemia in 1994, they have been considered promising therapeutic targets for cancer therapy. These cells have self-renewal capacity and differentiation potential and contribute to multiple tumor malignancies, such as recurrence, metastasis, heterogeneity, multidrug resistance, and radiation resistance. The biological activities of CSCs are regulated by several pluripotent transcription factors, such as OCT4, Sox2, Nanog, KLF4, and MYC. In addition, many intracellular signaling pathways, such as Wnt, NF-κB (nuclear factor-κB), Notch, Hedgehog, JAK-STAT (Janus kinase/signal transducers and activators of transcription), PI3K/AKT/mTOR (phosphoinositide 3-kinase/AKT/mammalian target of rapamycin), TGF (transforming growth factor)/SMAD, and PPAR (peroxisome proliferator-activated receptor), as well as extracellular factors, such as vascular niches, hypoxia, tumor-associated macrophages, cancer-associated fibroblasts, cancer-associated mesenchymal stem cells, extracellular matrix, and exosomes, have been shown to be very important regulators of CSCs. Molecules, vaccines, antibodies, and CAR-T (chimeric antigen receptor T cell) cells have been developed to specifically target CSCs, and some of these factors are already undergoing clinical trials. This review summarizes the characterization and identification of CSCs, depicts major factors and pathways that regulate CSC development, and discusses potential targeted therapy for CSCs.

/pdf/targeting-cancer-stem-cell-pathways-for-cancer-therapy-26ugvhsqd5.pdf

Targeting cancer stem cell pathways for cancer therapy

Diffuse intrinsic pontine glioma (DIPG) is a fatal brain cancer that arises in the brainstem of children, with no effective treatment and near 100% fatality. The failure of most therapies can be attributed to the delicate location of these tumors and to the selection of therapies on the basis of assumptions that DIPGs are molecularly similar to adult disease. Recent studies have unraveled the unique genetic makeup of this brain cancer, with nearly 80% found to harbor a p.Lys27Met histone H3.3 or p.Lys27Met histone H3.1 alteration. However, DIPGs are still thought of as one disease, with limited understanding of the genetic drivers of these tumors. To understand what drives DIPGs, we integrated whole-genome sequencing with methylation, expression and copy number profiling, discovering that DIPGs comprise three molecularly distinct subgroups (H3-K27M, silent and MYCN) and uncovering a new recurrent activating mutation affecting the activin receptor gene ACVR1 in 20% of DIPGs. Mutations in ACVR1 were constitutively activating, leading to SMAD phosphorylation and increased expression of the downstream activin signaling targets ID1 and ID2. Our results highlight distinct molecular subgroups and novel therapeutic targets for this incurable pediatric cancer.

/pdf/genomic-analysis-of-diffuse-intrinsic-pontine-gliomas-i9qtr1t1q0.pdf

Genomic analysis of diffuse intrinsic pontine gliomas identifies three molecular subgroups and recurrent activating ACVR1 mutations

Cancer is a complex multistep process involving genetic and epigenetic changes that eventually result in the activation of oncogenic pathways and/or inactivation of tumor suppressor signals. During cancer progression, cancer cells acquire a number of hallmarks that promote tumor growth and invasion. A crucial mechanism by which carcinoma cells enhance their invasive capacity is the dissolution of intercellular adhesions and the acquisition of a more motile mesenchymal phenotype as part of an epithelial-to-mesenchymal transition (EMT). Although many transcription factors can trigger it, the full molecular reprogramming occurring during an EMT is mainly orchestrated by three major groups of transcription factors: the ZEB, Snail and Twist families. Upregulated expression of these EMT-activating transcription factors (EMT-ATFs) promotes tumor invasiveness in cell lines and xenograft mice models and has been associated with poor clinical prognosis in human cancers. Evidence accumulated in the last few years indicates that EMT-ATFs also regulate an expanding set of cancer cell capabilities beyond tumor invasion. Thus, EMT-ATFs have been shown to cooperate in oncogenic transformation, regulate cancer cell stemness, override safeguard programs against cancer like apoptosis and senescence, determine resistance to chemotherapy and promote tumor angiogenesis. This article reviews the expanding portfolio of functions played by EMT-ATFs in cancer progression.

EMT-activating transcription factors in cancer: beyond EMT and tumor invasiveness.

Krüppel-like Factor 4 (KLF4): What We Currently Know

Background 
The aim of the present study was to analyze the expression of Zinc finger E-box Binding homeobox 2 (ZEB2) in glioma and to explore the molecular mechanisms of ZEB2 that regulate cell proliferation, migration, invasion, and apoptosis.


Methodology/Principal Findings 
Expression of ZEB2 in 90 clinicopathologically characterized glioma patients was analyzed by immunohistochemistry. Furthermore, siRNA targeting ZEB2 was transfected into U251 and U87 glioma cell lines in vitro and proliferation, migration, invasion, and apoptosis were examined separately by MTT assay, Transwell chamber assay, flow cytometry, and western blot.


Results 
The expression level of ZEB2 protein was significantly increased in glioma tissues compared to normal brain tissues (P<0.001). In addition, high levels of ZEB2 protein were positively correlated with pathology grade classification (P = 0.024) of glioma patients. Knockdown of ZEB2 by siRNA suppressed cell proliferation, migration and invasion, as well as induced cell apoptosis in glioma cells. Furthermore, ZEB2 downregulation was accompanied by decreased expression of CDK4/6, Cyclin D1, Cyclin E, E2F1, and c-myc, while p15 and p21 were upregulated. Lowered expression of ZEB2 enhanced E-cadherin levels but also inhibited β-Catenin, Vimentin, N-cadherin, and Snail expression. Several apoptosis-related regulators such as Caspase-3, Caspase-6, Caspase-9, and Cleaved-PARP were activated while PARP was inhibited after ZEB2 siRNA treatment.


Conclusion 
Overexpression of ZEB2 is an unfavorable factor that may facilitate glioma progression. Knockdown ZEB2 expression by siRNA suppressed cell proliferation, migration, invasion and promoted cell apoptosis in glioma cells.

/pdf/zeb2-mediates-multiple-pathways-regulating-cell-3j6he6hd1i.pdf

ZEB2 Mediates Multiple Pathways Regulating Cell Proliferation, Migration, Invasion, and Apoptosis in Glioma

The aim of the present study was to analyze the expression of matrix metalloproteinase 9 (MMP9) in nasopharyngeal carcinoma (NPC) and its correlation with clinicopathologic features, including the survival of patients with NPC. Using real-time PCR, we detected the mRNA expression of MMP9 in normal nasopharyngeal tissues and nasopharyngeal carcinoma (NPC) tissues. Using immunohistochemistry analysis, we analyzed MMP9 protein expression in clinicopathologically characterized 164 NPC cases (116 male and 48 female) with age ranging from 17 to 80 years (median = 48.4 years) and 32 normal nasopharyngeal tissues. Cases with greater than or equal to 6 and less than 6 of the score value of cytoplasmic MMP9 immunostaining were regarded as high expression and low expression, respectively. The relationship between the expression levels of MMP9 and clinical features was analyzed. The expression level of MMP9 mRNA was markedly greater in NPC tissues than that in the nasopharyngeal tissues. Immunohistochemical analysis revealed that the protein expression of MMP9 detected in NPC tissues was higher than that in the nasopharyngeal tissues (P = 0.004). In addition, high levels of MMP9 protein were positively correlated with the status of lymph node metastasis (N classification) (P = 0.002) and clinical stage (P < 0.001) of NPC patients. Patients with higher MMP9 expression had a significantly shorter overall survival time than did patients with low MMP9 expression. Multivariate analysis suggested that the level of MMP9 expression was an independent prognostic indicator (P = 0.008) for the survival of patients with NPC. High level of MMP9 expression is a potential unfavorable prognostic factor for patients with NPC.

/pdf/increased-expression-of-mmp9-is-correlated-with-poor-3cche6aiwb.pdf

Increased expression of MMP9 is correlated with poor prognosis of nasopharyngeal carcinoma

Expression of vimentin and the epithelial to mesenchymal transition (EMT) markers E-cadherin, β-catenin is essential for the progression of various human cancers. Our study aimed to investigate the aberrant localization E-cadherin, β-catenin and vimentin, and their prognostic significance in 122 nasopharyngeal carcinoma (NPC) patients by immunohistochemistry and immunofluorescence. Our results showed that both membranous and cytoplasmic localization of E-cadherin staining were associated with lymph node metastasis (p = 0.000 and 0.005, respectively) and clinical stage (p = 0.000 and 0.007, respectively). High cytoplasmic β-catenin correlated significantly with larger tumor size (p = 0.020), lymph node metastasis (p = 0.000) and advanced clinical stage (p = 0.036). However, no significant difference was observed between membranous β-catenin and clinicopathologic features (p ≥ 0.05). High nuclear vimentin expression correlated significantly with positive lymph node metastasis (p = 0.000) and advanced clinical stage (p = 0.000). Multivariate analysis showed that nuclear vimentin and cytoplasmic E-cadherin were independent prognostic factors (p = 0.016 and 0.001, respectively), as well as M classification (p = 0.001). More importantly, patients with high coexpression of nuclear vimentin and cytoplasmic E-cadherin had shorter survival time (p = 0.000). Furthermore, high coexpression of these two proteins was closely associated with lymph node metastasis (p = 0.000) and advanced clinical stage (p = 0.000). Our studies provide convincing evidence that EMT may play an important role in the biological progression of NPC, and nuclear vimentin and cytoplasmic E-cadherin might have independent prognostic value in NPC patients and serve as novel targets for prognostic therapeutics.

https://onlinelibrary.wiley.com/doi/pdf/10.1002/ijc.27467

Aberrant expression of nuclear vimentin and related epithelial–mesenchymal transition markers in nasopharyngeal carcinoma

The aim of the present study was to analyze the expression of Cyclin-dependent kinase 4 (CDK4) in lung cancer and its correlation with clinicopathologic features. Furthermore, the involvement of CDK4-mediated cell cycle progression and its molecular basis were investigated in the pathogenesis of lung cancer. Using immunohistochemistry analysis, we analyzed CDK4 protein expression in 89 clinicopathologically characterized lung cancer patients (59 males and 30 females) with ages ranging from 36 to 78 years and compared them to 23 normal lung tissues. Cases with cytoplasmic and nuclear CDK4 immunostaining score values greater than or equal to 7 were regarded as high expression while scores less than 7 were considered low expression. The correlation between the expression level of CDK4 and clinical features was analyzed. Furthermore, we used lentiviral-mediated shRNA to suppress the expression of CDK4 and investigate its function and molecular mechanism for mediating cell cycle progression. The expression level of CDK4 protein was significantly increased in lung cancer tissues compared to normal tissues (P < 0.001). In addition, high levels of CDK4 protein were positively correlated with the status of pathology classification (P = 0.047), lymph node metastasis (P = 0.007), and clinical stage (P = 0.004) of lung cancer patients. Patients with higher CDK4 expression had a markedly shorter overall survival time than patients with low CDK4 expression. Multivariate analysis suggested the level of CDK4 expression was an independent prognostic indicator (P < 0.001) for the survival of patients with lung cancer. Use of lentiviral-mediated shRNA to inhibit the expression of CDK4 in lung cancer cell line A549 not only inhibited cell cycle progression, but also dramatically suppressed cell proliferation, colony formation, and migration. Furthermore, suppressing CDK4 expression also significantly elevated the expression of cell cycle regulator p21 Overexpressed CDK4 is a potential unfavorable prognostic factor and mediates cell cycle progression by regulating the expression of p21 in lung cancer

/pdf/elevated-expression-of-cdk4-in-lung-cancer-3lhqyhgp57.pdf

Weiren Luo

Papers

ZEB2 Mediates Multiple Pathways Regulating Cell Proliferation, Migration, Invasion, and Apoptosis in Glioma

Increased expression of MMP9 is correlated with poor prognosis of nasopharyngeal carcinoma

Aberrant expression of nuclear vimentin and related epithelial–mesenchymal transition markers in nasopharyngeal carcinoma

Elevated expression of CDK4 in lung cancer

Aldehyde dehydrogenase 1, a functional marker for identifying cancer stem cells in human nasopharyngeal carcinoma