Serglycin Is Involved in TGF-ß Induced Epithelial-Mesenchymal Transition and Is Highly Expressed by Immune Cells in Breast Cancer Tissue.

Serglycin is a proteoglycan highly expressed by immune cells, in which its functions are linked to storage, secretion, transport, and protection of chemokines, proteases, histamine, growth factors, and other bioactive molecules. In recent years, it has been demonstrated that serglycin is also expressed by several other cell types, such as endothelial cells, muscle cells, and multiple types of cancer cells. Here, we show that serglycin expression is upregulated in transforming growth factor beta (TGF-ß) induced epithelial-mesenchymal transition (EMT). Functional studies provide evidence that serglycin plays an important role in the regulation of the transition between the epithelial and mesenchymal phenotypes, and it is a significant EMT marker gene. We further find that serglycin is more expressed by breast cancer cell lines with a mesenchymal phenotype as well as the basal-like subtype of breast cancers. By examining immune staining and single cell sequencing data of breast cancer tissue, we show that serglycin is highly expressed by infiltrating immune cells in breast tumor tissue.

Current Status of Circulating Tumor Cells, Circulating Tumor DNA, and Exosomes in Breast Cancer Liquid Biopsies.

Breast cancer is the most common cancer among women worldwide. Although the five-, ten- and fifteen-year survival rates are good for breast cancer patients diagnosed with early-stage disease, some cancers recur many years after completion of primary therapy. Tumor heterogeneity and clonal evolution may lead to distant metastasis and therapy resistance, which are the main causes of breast cancer-associated deaths. In the clinic today, imaging techniques like mammography and tissue biopsies are used to diagnose breast cancer. Even though these methods are important in primary diagnosis, they have limitations when it comes to longitudinal monitoring of residual disease after treatment, disease progression, therapy responses, and disease recurrence. Over the last few years, there has been an increasing interest in the diagnostic, prognostic, and predictive potential of circulating cancer-derived material acquired through liquid biopsies in breast cancer. Thanks to the development of sensitive devices and platforms, a variety of tumor-derived material, including circulating cancer cells (CTCs), circulating DNA (ctDNA), and biomolecules encapsulated in extracellular vesicles, can now be extracted and analyzed from body fluids. Here we will review the most recent studies on breast cancer, demonstrating the clinical potential and utility of CTCs and ctDNA. We will also review literature illustrating the potential of circulating exosomal RNA and proteins as future biomarkers in breast cancer. Finally, we will discuss some of the advantages and limitations of liquid biopsies and the future perspectives of this field in breast cancer management.

Circulating Tumor Cells as a Tool for Assessing Tumor Heterogeneity.

Tumor heterogeneity is the major cause of failure in cancer prognosis and prediction. Accurately detecting heterogeneity for the development of biomarkers and the detection of the clones resistant to therapy is one of the main goals of contemporary medicine. Metastases belong to the natural history of cancer. The present review gives an overview on the origin of tumor heterogeneity. Recent progress has made it possible to isolate and characterize circulating tumor cells (CTCs), which are the drivers of the disease between the primary sites and metastatic foci. The most recent methods for characterizing CTCs are summarized and we discuss the power of CTC profiling for analyzing tumor heterogeneity in early and advanced diseases.

The combined use of EFS, GPX2, and SPRR1A expression could distinguish favorable from poor clinical outcome among epithelial-like head and neck carcinoma subtypes.

BACKGROUND: We aimed at identifying molecular markers predictive of clinical outcome in patients with head and neck cancer based on the expression profile of cells showing epithelial-like (EL) or mesenchymal-like (ML) phenotypes. MATERIALS AND METHODS: We analyzed the association between EL and ML cells and migration, drug resistance, or tumor growth. The differential gene expression profile between cell types was used to build a model to stratify patients according to survival. RESULTS: EL cells were sensitive to cisplatin and cetuximab, showed low migration, and generated squamous differentiated tumors in mouse. A differential 93-gene expression signature between ML and EL cells was used to build a three-gene (EFS, GPX2, and SPRR1A) survival model by analyzing the RNA-seq data of the TCGA-HNSC project. Its prognostic value was confirmed in two independent cohorts. CONCLUSION: EFS, GPX2, and SPRR1A are prognostic markers able to distinguish clinical outcome among subtypes sharing an EL phenotype.

Characterization of circulating tumor cells as a reflection of the tumor heterogeneity: myth or reality?

The current main goal of diagnostic medicine is to detect crucial events in 'infinitely' small samples. The key question now is how to determine whether the rare cell events isolated and characterized from these samples reliably reflect the disease and heterogeneity of the tumor. In this review, we provide a short overview of the most recent methods developed for the isolation and characterization of rare cell events in clinical practice, with a specific focus on circulating tumor cells. We discuss the biological value to studying these cells at the single cell level and how these rare cell events can reflect tumor heterogeneity. The potential biomedical applications are also critically discussed in light of precision medicine.

Exosomal lncRNAs: the newest promising liquid biopsy.

LncRNAs are defined as RNA transcripts greater than 200 nucleotides in length that have no or limited protein-coding potential. Basal expression of lncRNAs appeared important for various homeostatic processes, like gene imprinting cell differentiation and organogenesis. Moreover, it has been demonstrated that lncRNAs play an important role in tumorigenesis and metastasis. Some lncRNAs were stably detected in exosomes, which are widely found in body fluids. Several studies validated the use of exosomal lncRNAs as minimally invasive diagnostic and prognostic markers in several types of cancers. In addition, exosomal lncRNAs have been associated with drug resistance of tumor cells, suggesting a clinical application in cancer-targeted therapy. Despite the recent increase of studies on exosomal lncRNAs, their clinical significance in cancer diagnosis, prognosis and treatment needs to be fully explored. The methodologies for their detection with high purity and accuracy must be also improved in order to implement their use in clinical routine. This review aims to summarize the main recent technologies available for the isolation of exosomal lncRNAs, their status as a liquid biopsy as well as their future perspectives.

Circulating Tumor Cell-Derived Pre-Clinical Models for Personalized Medicine.

The main cause of death from cancer is associated with the development of metastases, resulting from the inability of current therapies to cure patients at metastatic stages. Generating preclinical models to better characterize the evolution of the disease is thus of utmost importance, in order to implement effective new cancer biomarkers and therapies. Circulating Tumor Cells (CTCs) are good candidates for generating preclinical models, making it possible to follow up the spatial and temporal heterogeneity of tumor tissues. This method is a non-invasive liquid biopsy that can be obtained at any stage of the disease. It partially summarizes the molecular heterogeneity of the corresponding tumors at a given time. Here, we discuss the CTC-derived models that have been generated so far, from simplified 2D cultures to the most complex CTC-derived explants (CDX models). We highlight the challenges and strengths of these preclinical tools, as well as some of the recent studies published using these models.

Isolation of circulating tumor cells in a preclinical model of osteosarcoma: Effect of chemotherapy.

Osteosarcoma is a rare primary bone tumor, which mainly affects children and adolescents and has a poor prognosis, especially for patients with metastatic disease. A poor therapeutic response to the conventional chemotherapy is observed with the development of lung metastases, highlighting the need for improving the current regimens and the identification of early markers of the recurrent and metastatic disease. Circulating Tumour Cells (CTCs) play a key role in the metastatic process and could be powerful biomarkers of the progressive disease. The present study aimed to isolate CTCs by using a pre-clinical model of human osteosarcoma and to monitor their kinetic of release and their modulation by ifosfamide. CTCs were detectable into the bloodstream before any palpable primary tumors. Ifosfamide increased CTCs count and in contrast decreased the number of lung tumor nodules. On established tumors, ifosfamide slowed down the tumour growth and did not modulate CTC count that could be explained by a release of cancer cells from the primary tumour with reduced properties for inducing lung metastases. This report highlights the biological interest of CTCs in osteosarcoma.

Dendritic cells combined with tumor cells and α-galactosylceramide induce a potent, therapeutic and NK-cell dependent antitumor immunity in B cell lymphoma.

BACKGROUND: Invariant natural killer T (iNKT) cells are a small population of lymphocytes with unique specificity for glycolipid antigens presented by non-polymorphic CD1d receptor on dendritic cells (DCs). iNKT cells play a central role in tumor immunology since they are implicated in the coordination of innate and adaptive immune responses. These cells can be activated with the prototypic lipid α-galactosylceramide (α-GalCer), stimulating interferon gamma (IFN-γ) production and cytokine secretion, which contribute to the enhancement of T cell activation. METHODS: We evaluated the antitumor effect of a combination of dendritic cells (DCs) and tumor cells with the iNKT cell agonist α-GalCer in a therapeutic model of B cell lymphoma. iNKT, NK and T cell phenotype was determined by flow cytometry. Serum cytokines were analyzed by Luminex technology. Significant differences between survival curves were assessed by the log-rank test. For all other data, Mann-Whitney test was used to analyze the differences between groups. RESULTS: This vaccine induced a potent (100% survival), long-lasting and tumor-specific antitumor immune response, that was associated with an increase of both Th1 cytokines and IFN-γ secreting iNKT cells (4.59 ± 0.41% vs. 0.92 ± 0.12% in control group; p = 0.01) and T cells (CD4 IFN-γ+: 3.75 ± 0.59% vs. 0.66 ± 0.18% p = 0.02; CD8 IFN-γ+: 10.61 ± 0.84% vs. 0.47 ± 0.03% p = 0.002). Importantly, natural killer (NK) cells played a critical role in the antitumor effect observed after vaccination. CONCLUSIONS: This study provides clinically relevant data for the development of iNKT-cell based immunotherapy treatments for patients with B cell malignancies.

Analysis of gap junctional intercellular communications using a dielectrophoresis-based microchip.

Gap junctions are transmembrane structures that directly connect the cytoplasm of adjacent cells, making intercellular communications possible. It has been shown that the behaviour of several tumours - such as bone tumours - is related to gap junction intercellular communications (GJIC). Several methodologies are available for studying GJIC, based on measuring different parameters that are useful for multiple applications, such as the study of carcinogenesis for example. These methods nevertheless have several limitations. The present manuscript describes the setting up of a dielectrophoresis (DEP)-based lab-on-a-chip platform for the real-time study of Gap Junctional Intercellular Communication between osteosarcoma cells and the main cells accessible to their microenvironment. We conclude that using the DEParray technology for the GJIC assessment has several advantages comparing to current techniques. This methodology is less harmful for cells integrity; cells can be recovered after interaction to make further molecular analysis; it is possible to study GJIC in real time; we can promote cell interactions using up to five different populations. The setting up of this new methodology overcomes several difficulties to perform experiments for solving questions about GJIC process that we are not able to do with current technics.

Cancer stem cells in osteosarcoma.

Osteosarcoma is the most common primary bone tumour in children and adolescents and advanced osteosarcoma patients with evidence of metastasis share a poor prognosis. Osteosarcoma frequently gains resistance to standard therapies highlighting the need for improved treatment regimens and identification of novel therapeutic targets. Cancer stem cells (CSC) represent a sub-type of tumour cells attributed to critical steps in cancer including tumour propagation, therapy resistance, recurrence and in some cases metastasis. Recent published work demonstrates evidence of cancer stem cell phenotypes in osteosarcoma with links to drug resistance and tumorigenesis. In this review we will discuss the commonly used isolation techniques for cancer stem cells in osteosarcoma as well as the identified biochemical and molecular markers.

Tumour Heterogeneity: The Key Advantages of Single-Cell Analysis.

Tumour heterogeneity refers to the fact that different tumour cells can show distinct morphological and phenotypic profiles, including cellular morphology, gene expression, metabolism, motility, proliferation and metastatic potential. This phenomenon occurs both between tumours (inter-tumour heterogeneity) and within tumours (intra-tumour heterogeneity), and it is caused by genetic and non-genetic factors. The heterogeneity of cancer cells introduces significant challenges in using molecular prognostic markers as well as for classifying patients that might benefit from specific therapies. Thus, research efforts for characterizing heterogeneity would be useful for a better understanding of the causes and progression of disease. It has been suggested that the study of heterogeneity within Circulating Tumour Cells (CTCs) could also reflect the full spectrum of mutations of the disease more accurately than a single biopsy of a primary or metastatic tumour. In previous years, many high throughput methodologies have raised for the study of heterogeneity at different levels (i.e., RNA, DNA, protein and epigenetic events). The aim of the current review is to stress clinical implications of tumour heterogeneity, as well as current available methodologies for their study, paying specific attention to those able to assess heterogeneity at the single cell level.

The Challenges of Detecting Circulating Tumor Cells in Sarcoma.

Sarcomas are a heterogeneous group of malignant neoplasms of mesenchymal origin, many of which have a propensity to develop distant metastases. Cancer cells that have escaped from the primary tumor are able to invade into surrounding tissues, to intravasate into the bloodstream to become circulating tumor cells (CTCs), and are responsible for the generation of distant metastases. Due to the rarity of these tumors and the absence of specific markers expressed by sarcoma tumor cells, the characterization of sarcoma CTCs has to date been relatively limited. Current techniques for isolating sarcoma CTCs are based on size criteria, the identification of circulating cells that express either common mesenchymal markers, sarcoma-specific markers, such as CD99, CD81, or PAX3, and chromosomal translocations found in certain sarcoma subtypes, such as EWS-FLI1 in Ewing's sarcoma, detection of osteoblast-related genes, or measurement of the activity of specific metabolic enzymes. Further studies are needed to improve the isolation and characterization of sarcoma CTCs, to demonstrate their clinical significance as predictive and/or prognostic biomarkers, and to utilize CTCs as a tool for investigating the metastatic process in sarcoma and to identify novel therapeutic targets. The present review provides a short overview of the most recent literature on CTCs in sarcoma.

ΔNp63α Silences a miRNA Program to Aberrantly Initiate a Wound-Healing Program That Promotes TGFß-Induced Metastasis.

Primary cancer cell dissemination is a key event during the metastatic cascade, but context-specific determinants of this process remain largely undefined. Multiple reports have suggested that the p53 (TP53) family member p63 (TP63) plays an antimetastatic role through its minor epithelial isoform containing the N-terminal transactivation domain (TAp63). However, the role and contribution of the major p63 isoform lacking this domain, ΔNp63α, remain largely undefined. Here, we report a distinct and TAp63-independent mechanism by which ΔNp63α-expressing cells within a TGFß-rich microenvironment become positively selected for metastatic dissemination. Orthotopic transplantation of ΔNp63α-expressing human osteosarcoma cells into athymic mice resulted in larger and more frequent lung metastases than transplantation of control cells. Mechanistic investigations revealed that ΔNp63α repressed miR-527 and miR-665, leading to the upregulation of two TGFß effectors, SMAD4 and TßRII (TGFBR2). Furthermore, we provide evidence that this mechanism reflects a fundamental role for ΔNp63α in the normal wound-healing response. We show that ΔNp63α-mediated repression of miR-527/665 controls a TGFß-dependent signaling node that switches off antimigratory miR-198 by suppressing the expression of the regulatory factor, KSRP (KHSRP). Collectively, these findings reveal that a novel miRNA network involved in the regulation of physiologic wound-healing responses is hijacked and suppressed by tumor cells to promote metastatic dissemination. Cancer Res; 76(11); 3236-51. ©2016 AACR.

CKMT1 and NCOA1 expression as a predictor of clinical outcome in patients with advanced-stage head and neck squamous cell carcinoma.

BACKGROUND: We studied the association between the expression of a subset of previously identified genes and clinical outcome in patients with head and neck cancer. METHODS: We analyzed by reverse transcriptase-polymerase chain reaction (RT-PCR) the expression of 89 genes in tumor biopsies from stage III to IVa/b chemotherapy treated patients (n = 46). Two additional cohorts analyzed by RNAseq (The Cancer Genome Atlas [TCGA] project; n = 371) or immunohistochemistry (IHC; n = 73) were used to validate results. RESULTS: Thirty genes were associated with local-recurrence or progression-free survival. The best multi-gene decision-tree model to predict local recurrence included nuclear receptor coactivator 1 (NCOA1) and serum-amyloid A2 (SAA2) expression, whereas the best model to predict disease recurrence included creatine kinase mitochondrial 1 (CKMT1) and metal-regulatory transcription factor 1 (MTF1). Both models were associated with cancer-specific survival. Results were confirmed analyzing the RNAseq data included in the TCGA project. CKMT1 and NCOA1 were identified as independent risk factors for survival in an independent cohort analyzed by immunohistochemistry. CONCLUSION: CKMT1 and NCOA1 expression has prognostic significance in advanced-stage head and neck carcinoma. © 2015 Wiley Periodicals, Inc. Head Neck 38: E1392-E1403, 2016.

Receptor tyrosine kinases: Characterisation, mechanism of action and therapeutic interests for bone cancers.

Bone cancers are characterised by the development of tumour cells in bone sites, associated with a dysregulation of their environment. In the last two decades, numerous therapeutic strategies have been developed to target the cancer cells or tumour niche. As the crosstalk between these two entities is tightly controlled by the release of polypeptide mediators activating signalling pathways through several receptor tyrosine kinases (RTKs), RTK inhibitors have been designed. These inhibitors have shown exciting clinical impacts, such as imatinib mesylate, which has become a reference treatment for chronic myeloid leukaemia and gastrointestinal tumours. The present review gives an overview of the main molecular and functional characteristics of RTKs, and focuses on the clinical applications that are envisaged and already assessed for the treatment of bone sarcomas and bone metastases.

Enhanced cell migration and apoptosis resistance may underlie the association between high SERPINE1 expression and poor outcome in head and neck carcinoma patients.

High SERPINE1 expression is a common event in head and neck squamous cell carcinoma (HNSCC); however, whether it plays a role in determining clinical outcome remains still unknown. We studied SERPINE1 as a prognostic marker in two HNSCC patient cohorts. In a retrospective study (n = 80), high expression of SERPINE1 was associated with poor progression-free (p = 0.022) and cancer-specific (p = 0.040) survival. In a prospective study (n = 190), high SERPINE1 expression was associated with poor local recurrence-free (p = 0.022), progression-free (p = 0.002) and cancer-specific (p = 0.006) survival. SERPINE1 expression was identified as an independent risk factor for progression-free survival in patients treated with chemo-radiotherapy or radiotherapy (p = 0.043). In both patient cohorts, high SERPINE1 expression increased the risk of metastasis spread (p = 0.045; p = 0.029). The association between SERPINE1 expression and survival was confirmed using the HNSCC cohort included in The Cancer Genome Atlas project (n = 507). Once again, patients showing high expression had a poorer survival (p < 0.001). SERPINE1 over-expression in HNSCC cells reduced cell proliferation and enhanced migration. It also protected cells from cisplatin-induced apoptosis, which was accompanied by PI3K/AKT pathway activation. Downregulation of SERPINE1 expression had the opposite effect. We propose SERPINE1 expression as a prognostic marker that could be used to stratify HNSCC patients according to their risk of recurrence.

High RAB25 expression is associated with good clinical outcome in patients with locally advanced head and neck squamous cell carcinoma.

Currently there are no molecular markers able to predict clinical outcome in locally advanced head and neck squamous cell carcinoma (HNSCC). In a previous microarray study, RAB25 was identified as a potential prognostic marker. The aim of this study was to analyze the association between RAB25 expression and clinical outcome in patients with locally advanced HNSCC treated with standard therapy. In a retrospective immunohistochemical study (n = 97), we observed that RAB25-negative tumors had lower survival (log-rank, P = 0.01) than patients bearing positive tumors. In an independent prospective mRNA study (n = 117), low RAB25 mRNA expression was associated with poor prognosis. Using classification and regression tree analysis (CART) we established two groups of patients according to their RAB25 mRNA level and their risk of death. Low mRNA level was associated with poor local recurrence-free (log-rank, P = 0.005), progression-free (log-rank, P = 0.002) and cancer-specific (log-rank, P < 0.001) survival. Multivariate Cox model analysis showed that low expression of RAB25 was an independent poor prognostic factor for survival (hazard ratio: 3.84, 95% confidence interval: 1.93-7.62, P < 0.001). Patients whose tumors showed high RAB25 expression had a low probability of death after treatment. We also found lower RAB25 expression in tumors than in normal tissue (Mann-Whitney U, P < 0.001). Moreover, overexpression of RAB25 in the UM-SCC-74B HNSCC cell line increased cisplatin sensitivity, and reduced cell migration and invasion. Our findings support a tumor suppressor role for RAB25 in HNSCC and its potential use to identify locally advanced patients with a high probability of survival after genotoxic treatment.