The Immune Interplay between Thyroid Papillary Carcinoma and Hepatic Fibrosis

Abstract

Background: A high prevalence of thyroid papillary cancer was reported in hepatitis-C-virus (HCV) positive patients. However, the mechanistic role of hepatic-fibrosis in thyroid malignancy progressions is still unclear.

Aim: We aimed to study the immune-modulatory interactions between thyroid papillary carcinoma and hepatic-fibrosis.

Methods: Hepatic-fibrosis was induced in nude-nu-male mice by intra-peritoneal administration of carbon-tetrachloride. To induce thyroid-tumor, a thyroid papillary carcinoma cell line (NPA) was injected subcutaneously in the backs. Fibrotic profile was estimated by α-smooth-muscle-actin (αSMA) expression in liver tissue extracts using western-blots and RT-PCR. Intra-hepatic NK cells were isolated and stained for NK activity (CD107a) by flow cytometry. Liver histopathology (H&E staining), thyroid tumor mass and serum alanine aminotransferase (ALT), serum vascular endothelial growth factor (VEGF) and free-T4 levels were also assessed.

Results: Ex-vivo: NPA cells were co-cultured with intra-hepatic NK cells isolated from fibrotic mice with/without the tumor were analyzed for CFSE-proliferations. Both tumor groups (with/without hepatic-fibrosis) excreted higher serum free T4 levels. Hepatic-fibrosis increased tumor weight and size and serum free-T4 levels. In addition, tumor induction increased liver injury (both hepatic-fibrosis, necro-inflammation and serum ALT levels). In addition, tumor-bearing animals with hepatic-fibrosis had increased NK activity. NPA tumor-bearing animals increased fibrosis in spite of increased NK activity; probably due to a direct effect through increased serum free-T4 excretions. Serum VEGF levels were significantly increased in the fibrotic- bearing tumor groups compared to the non-fibrotic groups. In-vitro, NK cells from fibrotic tumor-bearing animals reduced proliferation of NPA cells. This decrease is attributed to increase NK cells activity in the fibrotic animals with the NPA tumors.

Conclusions: Our results propose that NK cells although were stimulated in advanced fibrosis with tumor, they lost their anti-tumor and anti-fibrotic activity probably due to secretions of T4 and VEFG and may explain increased risk of thyroid tumors in chronic HCV patients.

Fig 1. Hepatic fibrosis increase NPA tumor weight and size.

In vivo S.C injection of NPA cells model was performed as described in M&M. In this model; S.C NPA-cells tumor was induced in fibrotic and naïve (no fibrosis) animals. S.C tumors were explanted at the end of 6 weeks post S.C injection, and evaluated for tumor weight and volume. (A) and (B) show the external appearance of the tumor in the animal’s back. Tumor weight (C) and volume (D) was increased significantly from (0.13±0.06gr) and (0.28±0.18ml) in the fibrotic group to (0.05±0.025 gr) and (0.09±0.01ml) in the non-fibrotic group; p-value = 0.02 and 0.04, respectively.

Fig 2. NPA Tumor inductions increase the severity of CCl4 related hepatic injury.

CCl4-hepatic injury was evaluated by hematoxylin and eosin (H&E) staining of necro-inflammatory liver lesions and ALT serum levels. Immunohistochemical staining with H&E (5X magnification) for the four major animal groups showed necro-inflammatory lesions and cell infiltrations that were increased in the fibrotic mice receiving the NPA-tumor cells (D) as compared to fibrotic alone (C). Arrows indicate the area with lymphocyte infiltrations. No inflammatory infiltrates were seen in H&E staining of (A) naïve WT and (B) naïve mice receiving the NPA-tumor cells. (E) Serum ALT levels were in line with histological findings and showed increase from (60±25/L) in fibrotic animals without tumor to (85.5 ± 20.5 U/L) in animals with tumor and hepatic fibrosis; p-value = 0.021.

Fig 3. Tumor increase severity of hepatic fibrosis.

Fibrotic profile was estimated by (A) Western blot quantitations and (B) RT-PCR expressions of α smooth muscle actin (αSMA). (A) (Lower panel) displays a representative membrane with examples of αSMA expression as a marker for the HSCs activation (upper bands) and GAPDH (lower bands) expression by western blotting in the harvested liver protein extracts. (A) (Upper panel) shows the calculated ratio of αSMA/β-actin based on the densitometry readings of the bands. (B) Real-time PCR data reflect changes in gene expression of αSMA mRNA expressed as fold change compared with naïve mice. Expression of αSMA mRNA corresponded to the western blot results. Experiments were repeated 4 times, in each time 4 mice were included in each group. Averages of the 4 experiments were included in the quantitations of western blots and RT PCR.

Fig 4. NK cell activity and T4 excretions increased in the fibrotic group with NPA tumor induction.

(A) Intra-hepatic NK cells as well as (B) NK from spleen from four groups were isolated and stained for NK activity (CD107a) using flow-cytometry. The data showed a significant activation of the intrahepatic and splenic NK from the fibrotic group with NPA tumor induction compared to the non-tumor injected animals. (C) Shows serum T4 levels significantly elevated in animal models of fibrosis following NPA tumor injections.

Fig 5. In vitro co-culture of lymphocytes with NPA cell line.

Adhered-NPA cells post co-culture with NK cells from different animal groups were analyzed for proliferation by CFSE using flow cytometry. (A) Direct co-culture of NPA cells with spleen NKs from fibrotic mice with tumor significantly decreased NPA tumor cell proliferation compared to the fibrotic mice without tumor, indicating highly stimulated NK cells effects; p-value = 0.001. (B) A representative histogram of the NPA cells following incubations with NK cells of fibrosis and tumor mice. The histogram shows CSFE-proliferations changes in day 3 and day 5 as compared to day 0 of CSFE staining. Proliferations fold changes were calculated by divided day 0 to day 5.

Fig 6. VEGF serum levels.

Quantikine Mouse VEGF immunoassay serum levels was significantly increased in the fibrotic group bearing tumor as compared to tumor alone (p = 0.01) or fibrosis alone groups (p = 0.04). No statistical significant differences were found in VEGF serum levels between the fibrotic and non-fibrotic group with tumor induction.