The Use of Tissue-on-Chip Technology to Focus the Search for Extracellular Vesicle miRNA Biomarkers in Thyroid Disease.

Small extracellular vesicles (sEVs) contain microRNAs (miRNAs) which have potential to act as disease-specific biomarkers. The current study uses an established method to maintain human thyroid tissue ex vivo on a tissue-on-chip device, allowing the collection, isolation and interrogation of the sEVs released directly from thyroid tissue. sEVs were analysed for differences in miRNA levels released from benign thyroid tissue, Graves' disease tissue and papillary thyroid cancer (PTC), using miRNA sequencing and quantitative reverse transcriptase polymerase chain reaction (qRT-PCR) to identify potential biomarkers of disease. Thyroid biopsies from patients with benign tissue (n = 5), Graves' disease (n = 5) and PTC (n = 5) were perfused with medium containing sEV-depleted serum for 6 days on the tissue-on-chip device. During incubation, the effluents were collected and ultracentrifuged to isolate sEVs; miRNA was extracted and sequenced (miRNASeq). Out of the 15 samples, 14 passed the quality control and miRNASeq analysis detected significantly higher expression of miR-375-3p, miR-7-5p, miR-382-5p and miR-127-3p in the sEVs isolated from Graves' tissue compared to those from benign tissue (false discovery rate; FDR p < 0.05). Similarly, miR-375-3p and miR-7-5p were also detected at a higher level in the Graves' tissue sEVs compared to the PTC tissue sEVs (FDR p < 0.05). No significant differences were observed between miRNA in sEVs from PTC vs. those from benign tissue. These results were supported by Quantitative Reverse Transcriptase Polymerase Chain Reaction (qRT-PCR). The novel findings demonstrate that the tissue-on-chip technology is a robust method for isolating sEVs directly from the tissue of interest, which has permitted the identification of four miRNAs, with which further investigation could be used as biomarkers or therapeutic targets within thyroid disease.

A novel microfluidic device capable of maintaining functional thyroid carcinoma specimens ex vivo provides a new drug screening platform.

BACKGROUND: Though the management of malignancies has improved vastly in recent years, many treatment options lack the desired efficacy and fail to adequately augment patient morbidity and mortality. It is increasingly clear that patient response to therapy is unique to each individual, necessitating personalised, or 'precision' medical care. This demand extends to thyroid cancer; ~ 10% patients fail to respond to radioiodine treatment due to loss of phenotypic differentiation, exposing the patient to unnecessary ionising radiation, as well as delaying treatment with alternative therapies. METHODS: Human thyroid tissue (n = 23, malignant and benign) was live-sliced (5 mm diameter × 350-500 µm thickness) then analysed or incorporated into a microfluidic culture device for 96 h (37 °C). Successful maintenance of tissue was verified by histological (H&E), flow cytometric propidium iodide or trypan blue uptake, immunohistochemical (Ki67 detection/ BrdU incorporation) and functional analysis (thyroxine [T4] output) in addition to analysis of culture effluent for the cell death markers lactate dehydrogenase (LDH) and dead-cell protease (DCP). Apoptosis was investigated by Terminal deoxynucleotidyl transferase dUTP nick end labelling (TUNEL). Differentiation was assessed by evaluation of thyroid transcription factor (TTF1) and sodium iodide symporter (NIS) expression (western blotting). RESULTS: Maintenance of gross tissue architecture was observed. Analysis of dissociated primary thyroid cells using flow cytometry both prior to and post culture demonstrated no significant change in the proportion of viable cells. LDH and DCP release from on-chip thyroid tissue indicated that after an initial raised level of release, signifying cellular damage, detectable levels dropped markedly. A significant increase in apoptosis (p < 0.01) was observed after tissue was perfused with etoposide and JNK inhibitor, but not in control tissue incubated for the same time period. No significant difference in Ki-67 positivity or TTF1/NIS expression was detected between fresh and post-culture thyroid tissue samples, moreover BrdU positive nuclei indicated on-chip cellular proliferation. Cultured thyroid explants were functionally viable as determined by production of T4 throughout the culture period. CONCLUSIONS: The described microfluidic platform can maintain the viability of thyroid tissue slices ex vivo for a minimum of four days, providing a platform for the assessment of thyroid tissue radioiodine sensitivity/adjuvant therapies in real time.

Somatostatin receptor expression in thyroid disease.

Somatostatin analogues are commercially available and used for the management of acromegaly and neuroendocrine tumours, but the expression of the receptors as a target in thyroid disease has not been explored. To assess somatostatin (SST) and somatostatin receptor (SSTR1-5) expression in both normal and thyroid disorders, as a potential target for somatostatin analogue therapy, 67 thyroid tissue specimens were reviewed: 12 differentiated thyroid carcinomas, 14 follicular adenomas, 17 multinodular goitres, 14 Graves disease, 10 Hashimotos thyroiditis specimens and five normal thyroids. Tissue was immunostained for SST and SSTR1-5. Positivity and the degree of positivity were recorded by double-blinded observers. Somatostatin receptor expression was highly expressed in normal tissue for SSTR1, 3, 4 and 5 (5 of 5, 4 of 5, 4 of 5 and 5 of 5 respectively) whilst SST and SSTR 2a and b were not expressed at all. The commonest receptor expressed for all pathological subtypes grouped together was SSTR2b (63 specimens). The commonest receptors expressed in differentiated thyroid cancer were SSTR5 (11 of 12 specimens) and SSTR2b (10 of 12 specimens). The commonest receptor expressed in benign disease was SSTR2b (53 of 55 specimens). SSTR5 was significantly under-expressed in Graves disease (P < 0.05). This study illustrates that SSTR 1, 3, 4 and 5 are highly expressed in normal, benign and malignant thyroid tissue. SSTR 2a and 2b appear absent in normal tissue and present in benign and malignant thyroid tissue (P < 0.02). This suggests that focussed SSTR2 treatment may be a potential therapeutic target.

Nodular fasciitis of the submandibular gland.

A woman in her 20s presented with a non-tender swelling of the right submandibular gland. Ultrasound was suggestive of pleomorphic adenoma. The histology result of the excised tumour later confirmed a diagnosis of nodular fasciitis which is extremely rare in the submandibular gland. Postoperatively, she has made good recovery. Due to the similarity of the radiological image of pleomorphic adenoma and nodular fasciitis, it poses difficulty in diagnosing nodular fasciitis without cytology or histology of the salivary gland. Although being extremely rare, it would be worth to consider nodular fasciitis as one of the differential diagnosis in future cases of benign submandibular lesions.

Maintenance of head and neck tumor on-chip: gateway to personalized treatment?

AIM: Head and neck squamous cell carcinomas (HNSCC) are solid tumors with low overall survival (40-60%). In a move toward personalized medicine, maintenance of tumor biopsies in microfluidic tissue culture devices is being developed. METHODOLOGY/RESULTS: HNSCC (n = 15) was dissected (5-10 mg) and either analyzed immediately or cultured in a microfluidic device (37°C) for 48 h. No difference was observed in morphology between pre- and postculture specimens. Dissociated samples were analyzed using trypan blue exclusion (viability), propidium iodide flow cytometry (death) and MTS assay (proliferation) with no significant difference observed highlighting tissue maintenance. Computational fluid dynamics showed laminar flow within the system. CONCLUSION: The microfluidic culture system successfully maintained HNSCC for 48 h, the culture system will allow testing of different treatment modalities with response monitoring.

Biphasic synovial sarcoma of the posterior pharyngeal wall: a case report.

Synovial sarcoma is not common in the head and neck region. Because its histopathologic features are many and varied, it is often misdiagnosed. We report a case of biphasic synovial sarcoma of the posterior pharyngeal wall, and we discuss the clinical and pathologic features of this case.

Vascular endothelial growth factor (VEGF), VEGF receptors expression and microvascular density in benign and malignant thyroid diseases.

Angiogenesis is critical for the growth and metastatic spread of tumours. Vascular endothelial growth factor (VEGF) is the most potent inducer of neovasculature, and its increased expression has been related to a worse clinical outcome in many diseases. The purpose of this study was to evaluate the relation between VEGF, its receptors (VEGFR-1 and VEGFR-2) and microvessel density (MVD) in thyroid diseases. Immunostaining for VEGF and VEGF receptors was performed in 66 specimens of thyroid tissue, comprising 17 multinodular goitre (MNG), 14 Graves' disease, 10 follicular adenoma, 8 Hashimoto's thyroiditis, 7 papillary carcinoma and 10 normal thyroid specimens. Thyrocyte positivity for VEGF and VEGF receptors was scored 0-3. Immunohistochemistry for CD31, and CD34 on the same sections was performed to evaluate MVD. Immunohistochemical staining of VEGF in thyrocytes was positive in 92% of all the thyroid tissues studied. Using an immunostaining intensity cut off of 2, increased thyrocyte staining was seen in follicular adenoma specimens, MNG and normal thyroids compared with Hashimoto's thyroiditis and Graves' disease (P < 0.05). Similarly, VEGF thyrocyte expression in Graves' disease was less than other pathologies (P < 0.05). VEGFR-1 expression and the average MVD score did not differ between the different thyroid pathologies. VEGF expression was lower in autoimmune pathologies compared to autonomous growth processes. Conversely, both VEGFR-1 and VEGFR-2 were widely expressed in benign and neoplastic thyroid disease, suggesting that the up-regulation of VEGF and not its receptors occurs as tissue becomes autonomous. There was no clear relationship between MVD measurement and thyroid pathology.