Fluorescent Multiplex Immunohistochemistry Coupled With Other State-Of-The-Art Techniques to Systematically Characterize the Tumor Immune Microenvironment.

Our expanding knowledge of the interactions between tumor cells and their microenvironment has helped to revolutionize cancer treatments, including the more recent development of immunotherapies. Immune cells are an important component of the tumor microenvironment that influence progression and treatment responses, particularly to the new immunotherapies. Technological advances that help to decipher the complexity and diversity of the tumor immune microenvironment (TIME) are increasingly used in translational research and biomarker studies. Current techniques that facilitate TIME evaluation include flow cytometry, multiplex bead-based immunoassays, chromogenic immunohistochemistry (IHC), fluorescent multiplex IHC, immunofluorescence, and spatial transcriptomics. This article offers an overview of our representative data, discusses the application of each approach to studies of the TIME, including their advantages and challenges, and reviews the potential clinical applications. Flow cytometry and chromogenic and fluorescent multiplex IHC were used to immune profile a HER2+ breast cancer, illustrating some points. Spatial transcriptomic analysis of a luminal B breast tumor demonstrated that important additional insight can be gained from this new technique. Finally, the development of a multiplex panel to identify proliferating B cells, Tfh, and Tfr cells on the same tissue section demonstrates their co-localization in tertiary lymphoid structures.

MiR-7-5p inhibits thyroid cell proliferation by targeting the EGFR/MAPK and IRS2/PI3K signaling pathways.

The aberrant expression of miRNAs is often correlated to tumor development. MiR-7-5p is a recently discovered downregulated miRNA in thyroid papillary carcinoma (PTC). The goal of this project was to characterize its functional role in thyroid tumorigenesis and to identify the targeted modulated pathways. MiR-7-5p overexpression following transfection in TPC1 and HT-ori3 cells decreased proliferation of the two thyroid cell lines. Analysis of global transcriptome modifications showed that miR-7-5p inhibits thyroid cell proliferation by modulating the MAPK and PI3K signaling pathways which are both necessary for normal thyroid proliferation and play central roles in PTC tumorigenesis. Several effectors of these pathways are indeed targets of miR-7-5p, among which EGFR and IRS2, two upstream activators. We confirmed the upregulation of IRS2 and EGFR in human PTC and showed the existence of a negative correlation between the decreased expression of miR-7-5p and the increased expression of IRS2 or EGFR. Our results thus support a tumor-suppressor activity of miR-7-5p. The decreased expression of miR-7-5p during PTC tumorigenesis might give the cells a proliferative advantage and delivery of miR-7-5p may represent an innovative approach for therapy.

miRNA expression and function in thyroid carcinomas: a comparative and critical analysis and a model for other cancers.

As in many cancer types, miRNA expression profiles and functions have become an important field of research on non-medullary thyroid carcinomas, the most common endocrine cancers. This could lead to the establishment of new diagnostic tests and new cancer therapies. However, different studies showed important variations in their research strategies and results. In addition, the action of miRNAs is poorly considered as a whole because of the use of underlying dogmatic truncated concepts. These lead to discrepancies and limits rarely considered. Recently, this field has been enlarged by new miRNA functional and expression studies. Moreover, studies using next generation sequencing give a new view of general miRNA differential expression profiles of papillary thyroid carcinoma. We analyzed in detail this literature from both physiological and differential expression points of view. Based on explicit examples, we reviewed the progresses but also the discrepancies and limits trying to provide a critical approach of where this literature may lead. We also provide recommendations for future studies. The conclusions of this systematic analysis could be extended to other cancer types.

New global analysis of the microRNA transcriptome of primary tumors and lymph node metastases of papillary thyroid cancer.

BACKGROUND: Papillary Thyroid Cancer (PTC) is the most prevalent type of endocrine cancer. Its incidence has rapidly increased in recent decades but little is known regarding its complete microRNA transcriptome (miRNome). In addition, there is a need for molecular biomarkers allowing improved PTC diagnosis. METHODS: We performed small RNA deep-sequencing of 3 PTC, their matching normal tissues and lymph node metastases (LNM). We designed a new bioinformatics framework to handle each aspect of the miRNome: whole expression profiles, isomiRs distribution, non-templated additions distributions, RNA-editing or mutation. Results were validated experimentally by qRT-PCR on normal samples, tumors and LNM from 14 independent patients and in silico using the dataset from The Cancer Genome Atlas (small RNA deepsequencing of 59 normal samples, 495 PTC, and 8 LNM). RESULTS: We performed small RNA deep-sequencing of 3 PTC, their matching normal tissues and lymph node metastases (LNM). We designed a new bioinformatics framework to handle each aspect of the miRNome: whole expression profiles, isomiRs distribution, non-templated additions distributions, RNA-editing or mutation. Results were validated experimentally by qRT-PCR on normal samples, tumors and LNM from 14 independent patients and in silico using the dataset from The Cancer Genome Atlas (small RNA deep-sequencing of 59 normal samples, 495 PTC, and 8 LNM). We confirmed already described up-regulations of microRNAs in PTC, such as miR-146b-5p or miR-222-3p, but we also identified down-regulated microRNAs, such as miR-7-5p or miR-30c-2-3p. We showed that these down-regulations are linked to the tumorigenesis process of thyrocytes. We selected the 14 most down-regulated microRNAs in PTC and we showed that they are potential biomarkers of PTC samples. Nevertheless, they can distinguish histological classical variants and follicular variants of PTC in the TCGA dataset. In addition, 12 of the 14 down-regulated microRNAs are significantly less expressed in aggressive PTC compared to non-aggressive PTC. We showed that the associated aggressive expression profile is mainly due to the presence of the BRAF V600E mutation. In general, primary tumors and LNM presented similar microRNA expression profiles but specific variations like the down-regulation of miR-7-2-3p and miR-30c-2-3p in LNM were observed. Investigations of the 5p-to-3p arm expression ratios, non-templated additions or isomiRs distributions revealed no major implication in PTC tumorigenesis process or LNM appearance. CONCLUSIONS: Our results showed that down-regulated microRNAs can be used as new potential common biomarkers of PTC and to distinguish main subtypes of PTC. MicroRNA expressions can be linked to the development of LNM of PTC. The bioinformatics framework that we have developed can be used as a starting point for the global analysis of any microRNA deep-sequencing data in an unbiased way.

2-Iodohexadecanal inhibits thyroid cell growth in part through the induction of let-7f microRNA.

UNLABELLED: It is well known that pituitary TSH exerts the major task in the regulation of thyroid function. However, this gland is capable of certain degree of autonomy, independently of TSH control. Iodine plays an important role in thyroid physiology and biochemistry. The thyroid is capable of producing different iodolipids such as 2-iodohexadecanal (2-IHDA). It was shown that this iodolipid mimic some of the inhibitory effects of excess iodide on several thyroid parameters. OBJECTIVES: To identify the miRNAs regulated by 2-IHDA in rat thyroid cells and likely characterize their role in thyroid cell proliferation and function. RESULTS: FRTL-5 cells were grown in the presence of TSH and treated with 2-IHDA. Among the miRNAs up-regulated by 2-IHDA we focused on miR-let-7f and miR-138. When we transfected the miRNAs, miR-let-7f but not miR-138 overexpression inhibited proliferation of FRTL 5 cells, while miR-let-7f inhibition restored cell growth in 2-IHDA treated cultures. Analysis of cell cycle by flow cytometric DNA analysis revealed that miR-let-7f inhibition reduced the percentage of 2-IHDA treated cells in G1 phase and an increased of the percentage of cells in S phase was observed upon anti-let-7f transfection. The expresion of Cyclin D1 and Cyclin D3 were reduced after the transfection of miR-let-7f and miR-138, respectively. In in vivo studies we observed that miR-let-7f and miR-138 were up regulated by 2-IHDA during goiter involution. CONCLUSION: These results suggest that the inhibitory effects of 2-IHDA on FRTL-5 thyroid cell proliferation are mediated in part through the induction of let-7f microRNA.

MiRNA expression may account for chronic but not for acute regulation of mRNA expression in human thyroid tumor models.

BACKGROUND: For thyroid tumorigenesis, two main human in vitro models are available: primary cultures of human thyrocytes treated with TSH or EGF/serum as models for autonomous adenomas (AA) or papillary thyroid carcinomas (PTC) respectively, and human thyroid tumor derived cell lines. Previous works of our group have assessed properties of those models, with a special emphasis on mRNA regulations. It is often assumed that miRNA may be one of the primary events inducing these mRNA regulations. METHODS: The purpose of this study was to investigate the representativity of those models to study microRNA regulations and their relation with mRNA expression. To achieve this aim, the miRNA expressions profiles of primary cultures treated with TSH or EGF/serum and of 6 thyroid cancer cell lines were compared to the expression profiles of 35 tumor tissues obtained by microarrays. RESULTS: Our data on primary cultures have shown that the TSH or EGF/serum treatment did not greatly modify the microRNA expression profiles, which is contrary to what is observed for mRNA expression profiles, although they still evolved differently according to the treatment. The analysis of miRNA and mRNA expressions profiles in the cell lines has shown that they have evolved into a common, dedifferentiated phenotype, closer to ATC than to the tumors they are derived from. CONCLUSIONS: Long-terms TSH or EGF/serum treatments do not mimic AA or PTC respectively in terms of miRNA expression as they do for mRNA, suggesting that the regulations of mRNA expression induced by these physiological agents occur independently of miRNA. The general patterns of miRNA expression in the cell lines suggest that they represent a useful model for undifferentiated thyroid cancer. Mirna probably do not mediate the rapid changes in gene expression in rapid cell biology regulation.

miRNA expression in anaplastic thyroid carcinomas.

Anaplastic thyroid carcinoma (ATC) is the most lethal form of thyroid neoplasia and represents an end stage of thyroid tumor progression. No effective treatment exists so far. In this study, we analyzed the miRNA expression profiles of 11 ATC by microarrays and their relationship with the mRNA expression profiles of the same 11 ATC samples. ATC show distinct miRNA expression profiles compared to other less aggressive thyroid tumor types. ATC show 18 commonly deregulated miRNA compared to normal thyroid tissue (17 downregulated and 1 upregulated miRNA). First, the analysis of a combined approach of the mRNA gene expression and of the bioinformatically predicted mRNA targets of the deregulated miRNA suggested a role for these regulations in the epithelial to mesenchymal transition (EMT) process in ATC. Second, the direct interaction between one of the upregulated mRNA target, the LOX gene which is an EMT key player, and a downregulated miRNA, the miR-29a, was experimentally validated by a luciferase assay in HEK cell. Third, we confirmed that the ATC tissue is composed of about 50% of tumor associated macrophages (TAM) and suggested, by taking into account our data and published data, their most likely direct or paracrine intercommunication between them and the thyroid tumor cells, amplifying the tumor aggressiveness. Finally, we demonstrated by in situ hybridization a specific thyrocyte localization of 3 of the deregulated miRNA: let-7g, miR-29a and miR-30e and we pointed out the importance of identifying the cell type localization before drawing any conclusion on the physiopathological role of a given gene.

Thyroid cancer cell lines: an overview.

Human thyroid cancer cell lines are the most used models for thyroid cancer studies. They must be used with detailed knowledge of their characteristics. These in vitro cell lines originate from differentiated and dedifferentiated in vivo human thyroid tumors. However, it has been shown that mRNA expression profiles of these cell lines were closer to dedifferentiated in vivo thyroid tumors (anaplastic thyroid carcinoma, ATC) than to differentiated ones. Here an overview of the knowledge of these models was made. The mutational status of six human thyroid cancer cell lines (WRO, FTC133, BCPAP, TPC1, K1, and 8505C) was in line with previously reported findings for 10 genes frequently mutated in thyroid cancer. However, the presence of a BRAF mutation (T1799A: V600E) in WRO questions the use of this cell line as a model for follicular thyroid carcinoma (FTC). Next, to investigate the biological meaning of the modulated mRNAs in these cells, a pathway analysis on previously obtained mRNA profiles was performed on five cell lines. In five cell lines, the MHC class II pathway was down-regulated and in four of them, ribosome biosynthesis and translation pathways were up-regulated. mRNA expression profiles of the cell lines were also compared to those of the different types of thyroid cancers. Three datasets originating from different microarray platforms and derived from distinct laboratories were used. This meta-analysis showed a significant higher correlation between the profiles of the thyroid cancer cell lines and ATC, than to differentiated thyroid tumors (i.e., PTC or FTC) specifically for DNA replication. This already observed higher correlation was obtained here with an increased number of in vivo tumors and using different platforms. In summary, this would suggest that some papillary thyroid carcinoma or follicular thyroid carcinoma (PTC or FTC) cell lines (i.e., TPC-1) might have partially lost their original DNA synthesis/replication regulation mechanisms during their in vitro cell adaptation/evolution.