Tumor-Derived Exosomal PDLIM1 Promotes Angiogenesis and Tumor Progression in Papillary Thyroid Carcinoma: Insights From Integrated Single-Cell Transcriptomics and Exosomal Proteomics

BACKGROUNDThis study was done to see if there were correlations between anatomic and molecular parameters such as microvessel density (MVD), lymphatic vessel density (LVD), and vascular endothelial growth factor receptor (VEGFR)-3 expression and various clinical parameters for papillary thyroid carcinomas of size > 1.0 cm (PTCs) and size < or = 1.0 cm (papillary thyroid microcarcinomas, PTMCs). PTMCs were divided into two subgroups (0-5 mm and 6-10 mm).METHODSWe analyzed 197 thyroid carcinomas including 113 PTCs and 84 PTMCs. Tissue samples form 30 patients from each group matched for clinical characteristics were selected for immunostaining.RESULTSAlthough PTCs and PTMCs showed significant differences in clinical characteristics, they did not show significant difference in MVD, LVD, or VEGFR-3 expression. There was a significantly higher LVD in the PTMC subgroup with the larger tumors but no difference in clinical characteristics. LVD was higher in patients > 45 years old (more apparent in the PTC group) and LVD had suggestive correlations with multicentricity and extrathyroidal extension depending on analytic conditions.CONCLUSIONSSince LVD showed variable correlations with clinical variables for papillary carcinoma of the thyroid depending on analytic conditions, the individually planned treatments based on overall clinicopathological factors are advised.

Objective To study effect of metastasis of expressions on intratumour microvessel density (MVD), serine threonine kinase1 ,2( AKT2 ) 、VGEF-C in thyroid papillary carcinoma. Methods Immunohis to chemical method was used to detect the expressions of AKT2 and MVD in 85 thyroid papillary carcinoma. Results The positive expression rates of AKT2 and VGEF-C of papillary carcinomas of the thyroid were 75.7%(64/85)74% (63/85), and respectively. The positive expression of AKT2、VGEF-C were closely related to lymph nodemetastasis and intratumor MVD. Conclusion The over expression of AKT2 and VGEF-C are strongly related to the invasion and metastasis of papillary carcinomas of the thyroid, it shows that AKT2 、VGEF-C might have important significance in metastases of papillary carcinomas of thyroid. Key words: Thyroid neoplasms; AKT2; VGEF-C; Angiogenesis; Metastasis

Akt activation has been found in various human cancers, while experimental studies have suggested that Akt plays an important role in the development of tumor angiogenesis and lymphangiogenesis. Immunohistochemical analyses for VEGF-C and Akt and the lymphatic endothelial specific marker D2-40 were performed on a series of 242 invasive ductal carcinomas of the breast, in which VEGF-A expression and microvessel density (MVD) had been determined previously. Lymphatic vessel density (LVD) was estimated in three hot spots. A significant correlation was observed between the VEGF-C expression and LVD (p=0.0026) and between LVD and the lymph node status (p<0.0001). The VEGF-C expression, however, did not correlate significantly with the lymph nodes status, while a high VEGF-C expression was associated with a smaller tumor size (p=0.0188). There was a significant correlation between VEGF-C and VEGF-A expression (p=0.0079) and between LVD and MVD (p=0.0008). The VEGF-C expression correlated with MVD (p<0.0001), while the VEGF-A expression correlated with LVD (p=0.0155). The Akt expression correlated with VEGF-A (p=0.0173) and -C expression (p=0.0056) as well as MVD (p=0.0482) and LVD (p=0.0012), while the correlation of Akt expression to VEGF-C expression and LVD was stronger than that to VEGF-A expression and MVD. Although the patients with a high LVD have a poorer disease-free survival than those with a low LVD (p=0.0005), a multivariate analysis determined the lymph node status and MVD to be independently significant factors for the disease-free survival. In conclusion, the correlation of both VEGF-C and VEGF-A to LVD and MVD suggested the two growth factors to be involved in both angiogenesis and lymphangiogenesis in breast cancer. The correlation of the Akt expression to the VEGF-A and -C expression as well as MVD and LVD, thus, suggested Akt activation to contribute to both angiogenesis and lymphangiogenesis via VEGF-A and -C expression in breast cancer.

ObjectivesTo investigate the relationships between lymph node metastasis (LNM) and expression of CD31, D2-40 and vascular endothelial growth factors (VEGF)-A and -C in patients with papillary thyroid cancer (PTC).MethodsParaffin-embedded thyroid tissues of 72 patients were evaluated, which included 25 patients with thyroid nodular hyperplasia (TNH), 24 PTC patients without LNM, and 23 PTC patients with LNM. Three pathologists, who were blinded to the patient's clinical information, assessed the immunohistochemical staining results. The amount of expression was scored as high (>25% of cells stained) or low (0-25%).ResultsA higher level of VEGF-A expression was observed in the PTC groups regardless of LNM when compared to the group with TNH (91.3%, 79.2%, 4.0%, respectively). VEGF-C expression in the PTC with LNM group was significantly higher than the other two groups (P<0.05). No difference in microvessel density (MVD) scores was observed using CD31 among the three groups. The lymphatic vessel density (LVD) score using D2-40 was significantly higher in patients having PTC with LNM than the other groups (P<0.05).ConclusionVEGF-C and D2-40 were more highly expressed in patients having PTC with LNM than in patients having PTC without LNM or in those having TNH. Analysis of VEGF-C level and LVD using D2-40 may be helpful in the diagnosis of PTC and the evaluation of LNM potential in patients with PTC.

The aim of the present study was to evaluate total and membranous Na+/I- symporter (NIS) expressions in papillary thyroid carcinoma (PTC) tissue, correlation of NIS expression between primary and metastatic lymph node (LN) PTC tissues, and relationship of NIS expression with I131 whole body scan (WBS) uptake between primary and metastatic LN PTC tissues by analyzing 17 pairs of primary and metastatic LN PTC tissues. Staining positivity was calculated, and staining intensity was graded as negative (0), weak (1+), moderate (2+) and strong (3+). In primary PTC tissues, positivities and intensities of normal cells were higher than those of carcinoma cells but had no correlation with those in matched metastatic LN PTC tissues. In classic type, positivities, intensities and membranous intensities (mIS) were correlated between primary and matched metastatic LN PTC tissues. In patients aged younger than 45 yr, positivities and intensities in primary PTC tissues had correlation with those in matched metastatic LN PTC tissues. Positivities, intensities, mIS and pathological subtype of carcinoma cells in primary PTC tissues were not correlated with age, tumor size, TNM stage, MACIS score and thyroglobulin (Tg) levels at the time of I131 WBS. Sensitivity, specificity, as well as positive and negative predicted values of mIS in patients with I131 WBS uptake were 69.2, 75, 90 and 42.9% in primary PTC tissues, and 92.3, 100, 100 and 80% in metastatic LN PTC tissues. The results of mIS taken either as positive or negative were correlated with those of I131 WBS after controlling for age. Our results demonstrate that PTC tissues have altered total and membranous NIS expressions, suggesting that NIS expression in primary PTC tissues may predict NIS expression and I131 WBS uptake in matched metastatic LN PTC tissues.

Peritumoral Lymphatic Microvessel Density Associated with Tumor Progression and Poor Prognosis in Gastric Carcinoma

Tyrosine kinase inhibitors (TKIs) are evaluated for treatment of radioiodine refractory thyroid cancer. Their effects in this setting are based on blockade of proangiogenic signaling mediated by receptors for vascular endothelial growth factors (VEGFs) and platelet-derived growth factors (PDGF). Most TKIs also block other cancer-relevant kinases, such as B-type Raf kinase (BRAF), which are constitutively activated in approximately half of papillary thyroid carcinomas (PTCs), but the impact of these effects is not clear. The aim of our study was to investigate the impact of BRAF(V600E) on proangiogenic gene expression and microvascular features of PTCs. mRNA levels for VEGFA, VEGF receptors, and coreceptors (VEGFRs 1, 2, and 3, neuropilin-1), and PDGF receptor β (PDGFRβ or PDGFRB) were measured with real-time PCR in BRAF(V600E) (n=55) and wild-type BRAF (BRAF-wt; n=35) PTCs. VEGF and VEGFR protein expression and microvessel densities (MVD) and lymphatic vessel densities (LVDs) were assessed by immunohistochemistry in 22 of the 90 PTCs (including 11 BRAF(V600E) cases). Angiogenic gene expression was also studied in vitro after induction/silencing of the BRAF(V600E) mutation in thyrocyte lines. Transcript levels of proangiogenic factors were significantly lower in BRAF(V600E) PTCs versus BRAF-wt PTCs (P<0.0001), but MVD and LVDs were not significantly different. VEGFA mRNA levels in thyroid cell lines decreased when BRAF(V600E) mutation was induced (P=0.01) and increased when it was silenced (P=0.01). Compared with BRAF-wt PTCs, those harboring BRAF(V600E) exhibit downregulated VEGFA, VEGFR, and PDGFRβ expression, suggesting that the presence of BRAF mutation does not imply a stronger prediction of response to drugs targeting VEGF and PDGFB signaling pathways.

Lymphatic and blood vessel density in the follicular patterned lesions of thyroid

Molecular Rearrangements and Morphology in Thyroid Cancer

Background/AimPapillary thyroid carcinoma (PTC) is the most common type of thyroid cancer accounting for 75-85% of cases. Despite its favorable prognosis, 30-50% of patients develop regional lymph node metastases. Lymphatic vessel density (LVD) is a potential predictor of tumor progression, metastasis, and patient survival in PTC. This systematic review and meta-analysis evaluate the prognostic significance of LVD in PTC, focusing on intratumoral and peritumoral LVD and their association with nodal metastasis.Materials and MethodsA systematic review and meta-analysis were conducted following PRISMA guidelines and the Cochrane Handbook. Eligible studies included patients with PTC who underwent tumor resection and had LVD assessed via immunohistochemistry (D2-40, LYVE-1). Literature search was performed in MEDLINE, Cochrane Library, and PubMed. Two independent reviewers screened studies and extracted data, including survival outcomes and LVD measurements. Hazard ratios (HRs) and mean differences were calculated using fixed-effects or random-effects models, with heterogeneity assessed via I2 statistics.ResultsA total of 21 studies were identified, with nine meeting eligibility criteria. Meta-analysis demonstrated a significant association between high overall LVD and increased nodal metastasis (summary mean difference: 2.64; 95%CI=1.45, 3.82; p<0.001, I2=30.6%). No statistically significant association was observed for intratumoral (summary HR=1.33; 95%CI=0.88-2.02; p=0.176, I2=74.3%) or peritumoral LVD (summary HR=1.58; 95%CI=0.51-4.89; p=0.429, I2=74.7%). Heterogeneity across studies suggested potential variability in LVD measurement techniques and patient populations.ConclusionThis meta-analysis highlights the prognostic role of overall LVD in predicting nodal metastasis in PTC. However, intratumoral and peritumoral LVD did not show a significant correlation, indicating the need for further research. Standardization of LVD assessment and integration with molecular markers could enhance risk stratification and personalized treatment approaches in PTC management.

Lymph vessel density (LVD) and microvessel density (MVD) correlate with the malignant potential of tumors and patient survival. Vascular endothelial growth factors (VEGF)-A, VEGF-C, and VEGF-D could modulate LVD and MVD. We investigated the clinical and prognostic significance of LVD and MVD on lymphangiogenic and angiogenic function of VEGF-A, VEGF-C, and VEGF-D in human bladder cancer. We reviewed tissue samples from patients with nonmetastatic bladder cancer who had undergone transurethral resections (n = 126). The densities of D2-40-positive vessels (LVD) and CD34-positive vessels (MVD) were measured by a computer-aided image analysis system. Expression of VEGF-A, VEGF-C, and VEGF-D was examined by immunohistochemistry; survival analyses and their independent roles were investigated using multivariate analysis models. LVD was associated with tumor grade but not with pT stage. LVD was associated with metastasis-free survival (log rank P = 0.039), but was not an independent prognostic factor. Although MVD affected survival, the combination of high LVD and high MVD in tumors was an independent predictor of metastasis-free survival. Although VEGF-C expression was positively associated with both LVD and MVD, VEGF-D was associated only with LVD. VEGF-A expression was associated with MVD in univariate analysis, however, it was not an independent factor. Lymphangiogenesis and angiogenesis influence metastasis-free survival, and are regulated by VEGF-C and/or VEGF-D. Our results suggest that LVD and MVD are useful tools for the selection of postoperative management and treatment strategies in patients with bladder cancer.

BackgroundMicroRNA (MiRNA) is a small non-coding RNA which is implicated in a cohort of biological function in cancer, including proliferation, metastasis, apoptosis and invasion. MiR-96 has been reported to be involved in many cancers, including papillary thyroid cancer. However, the role of miR-96-3p in papillary thyroid cancer metastasis is still unclear.MethodsqRT-PCR is used to detect the level of miR-96-3p and mRNA of SDHB in PTC tissues and cell lines. Western blot assays are used to verify the protein expression of SDHB. The transwell assays are performed to identify the migration ability of PTC cell lines. Moreover, dual-luciferase 3′-UTR reporter assays are chosen to illuminate the direct target of miR-96-3p.ResultsThe relative miR-96-3p upregulate in PTC tissues and three PTC cell lines (B-CPAP, K-1 and TPC-1 cells) while the relative SDHB is opposite. Our results revealed that the miR-96-3p promotes metastasis and invasion in PTC cell lines (K-1 and TPC-1 cells) by direct targeting SDHB and influence the downstream protein AKT.ConclusionsTaken together, the miR-96-3p is involved in PTC metastasis and invasion by direct targeting SDHB and the downstream molecule AKT and mTOR.

Simple SummaryThis study was conducted to investigate the clinical significance and prognostic value of KLF5 in a large cohort of Middle Eastern PTC patients and explore its functional role and mechanism in PTC cell lines in vitro and in vivo. We found KLF5 over-expression in PTC patient cases and this was significantly associated with aggressive clinico-pathological parameters and worse outcome. We also found a significant association between KLF5 and HIF-1α in PTC patients and cell lines. Functionally, KLF5 promoted cell growth, stemness, invasion, migration, and angiogenesis, while its inhibition reverses its action in PTC cell lines. Finally, the depletion of KLF5 regressed PTC tumor growth in nude mice. These data suggest that KLF5 may potentially be a suitable therapeutic target in PTC, and pharmacological inhibition of KLF5 might be a viable therapeutic option for the treatment of patients with an aggressive subtype of PTC.The Krüppel-like factor 5 (KLF5), a zinc-finger transcriptional factor, is highly expressed in several solid tumors, but its role in PTC remains unclear. We investigated the expression of KLF5 protein in a large cohort of PTC patient samples and explored its functional role and mechanism in PTC cell lines in vitro and in vivo. KLF5 overexpression was observed in 65.1% of all PTC cases and it was significantly associated with aggressive clinico-pathological parameters and poor outcome. Given the significant association between KLF5 and HIF-1α overexpression in PTC patients, we investigated the functional correlation between KLF5 and HIF-1α in PTC cells. Indeed, the analysis revealed the co-immunoprecipitation of KLF5 with HIF-1α in PTC cells. We also identified KLF5-binding sites in the HIF-1α promoter that specifically bound to KLF5 protein. Mechanistically, KLF5 promoted PTC cell growth, invasion, migration, and angiogenesis, while KLF5 downregulation via specific inhibitor or siRNA reverses its action in vitro. Importantly, the silencing of KLF5 decreases the self-renewal ability of spheroids generated from PTC cells. In addition, the depletion of KLF5 reduces PTC xenograft growth in vivo. These findings suggest KLF5 can be a possible new molecular therapeutic target for a subset of PTC.

Background: Blood and lymph vessel invasion are well-recognized markers of tumor aggressiveness, as these are the routes that lead to metastases. Thyroid tumors, depending on the histological variant, tend to have distinctive biological behaviors and use different vascular routes to metastasize, yet the mechanisms underlying the metastatic process are still poorly understood. Objectives: The aim of this study was to assess how the lymph vessel density (LVD) in different histological types of thyroid tumors, and in their surrounding tissue, correlate with the presence of lymph node metastases (LNM) and tumor pathological features. Methods: Lymph vessels of papillary thyroid carcinomas (PTC), of the classical (CVPTC, n = 50) and follicular variants (FVPTC, n = 18), and medullary thyroid carcinomas (MTC, n = 34) were immunohistochemically stained against antigen D2-40. The stained area was quantified using a computerized morphometric analysis tool and correlated with the tumor pathological characteristics. Results: LVD within all analyzed thyroid tumor subtypes was significantly lower than in the surrounding thyroid tissues (p < 0.001). Despite intratumoral LVD being significantly higher in CVPTC than in FVPTC, and peritumoral LVD being significantly higher in MTC than in PTC (p < 0.05), no correlations were found between LVD (either intratumoral or peritumoral) and the presence of lymph node metastasis. Conclusions: As no LVD differences were found amongst thyroid tumors with or without LNM, dissemination is more likely to depend on the tumor ability to invade the abundant lymph vessel network of the surrounding thyroid tissue than on the ability of the tumor to promote de novo lymphangiogenesis.

Objective To investigate the correlation between elasticity score, strain ratio(SR)of real-time ultrasound elastrography(RTE)and microvessel density(MVD)in papillary thyroid carcinoma(PTC). Methods A retrospective study was performed on 54 cases(58 nodules)of patients with PTC who were admitted from January 2016 to December 2017.All patients underwent standard RTE examination preoperatively, CD 34 immunohistochemical staining and MVD count were performed postoperatively.The nodules were divided into ≤10 mm, 10~20 mm and >20 mm groups.The difference of elasticity score, SR and MVD in the three groups was compared, and the correlation between elasticity score, SR and MVD in PTC nodules was analyzed. Results Among the 58 PTC nodules, 54 had elastic scores greater than or equal to 3 points(93.1%), and the average SR value was(3.6±1.3). There was no statistically significant difference between the maximum diameter ≤10 mm group, 10 ~ 20 mm group and >20 mm group(P>0.05). Among the 58 PTC nodules, the expression rate of CD 34 was 100%.Nodules of MVD in the 3 groups were(47.7±8.8)/HP, (51.1±8.5)/HP and(56.1±5.8)/HP, respectively, with statistically significant differences(P 0.05). PTC elastic score was negatively correlated with MVD, and the difference was statistically significant(r=-0.567, P=0.000); SR and MVD of PTC were also negatively correlated, with statistically significant differences(r=-0.570, P=0.000). Conclusion Both elasticity score and SR have significantly negative correlation with MVD in PTC nodules.RTE may have potential clinical value in predicting aggressiveness and prognosis of PTC preoperatively. Key words: Papillary thyroid carcinoma; Real-time ultrasound elastography; Elasticity score; Strain ratio; Microvessel density