Cancer Res Treat.  2016 Apr;48(2):727-737. 10.4143/crt.2014.350.

Altered Biological Potential and Radioresponse of Murine Tumors in Different Microenvironments

• Lee IJ
• Lee EJ
• Park H
• Kim W
• Ha SJ
• Shin YK
• Seong J

• KMID: 2454351
• DOI: http://doi.org/10.4143/crt.2014.350

Abstract

PURPOSE
This study was conducted to evaluate the biological features of murine hepatocarcinoma according to different tumor microenvironmental models and to determine the change in molecular and immunologic responses after radiation.
MATERIALS AND METHODS
Tumor models were established in the liver (orthotopic) and thigh (heterotopic) of male C3H/HeN mice. Tumor growth and lung metastasis were assessed in these models. To evaluate the radiation effect, the tumors were irradiated with 10 Gy. Factors associated with tumor microenvironment including vascular endothelial growth factor (VEGF), cyclooxygenase-2 (COX-2), transforming growth factor beta1 (TGF-β1), CD31, and serum interleukin-6 (IL-6) were evaluated. Tumor-infiltrating regulatory immune cells, regulatory T cells (Tregs), and myeloid-derived suppressor cells (MDSCs) were also analyzed.
RESULTS
A higher number of lung metastases were observed in the orthotopic tumor model than in the heterotopic tumor model. VEGF, CD31, COX-2, and TGF-β1 expression was more prominent in the orthotopic tumor model than in the heterotopic tumor model. Expression of the angiogenic factor VEGF and key regulatory molecules (TGF-β1 and COX-2) decreased following radiation in the orthotopic tumor model, while the serum IL-6 level increased after radiation. In the orthotopic tumor model, the number of both Tregs and MDSCs in the tumor burden decreased after radiation.
CONCLUSION
The orthotopic tumor model showed higher metastatic potential and more aggressive molecular features than the heterotopic tumor model. These findings suggest that the orthotopic tumor mouse model may be more reflective of the tumor microenvironment and suitable for use in the translational research of radiation treatment.

Keyword

Tumor microenvironment; Hepatocarcinoma; Radiation

MeSH Terms

Angiogenesis Inducing Agents
Animals
Cyclooxygenase 2
Humans
Interleukin-6
Liver
Lung
Male
Mice
Neoplasm Metastasis
Radiation Effects
T-Lymphocytes, Regulatory
Thigh
Transforming Growth Factor beta1
Translational Medical Research
Tumor Burden
Tumor Microenvironment
Vascular Endothelial Growth Factor A
Angiogenesis Inducing Agents
Cyclooxygenase 2
Interleukin-6
Transforming Growth Factor beta1
Vascular Endothelial Growth Factor A

Figure

• 

  Fig. 1. Tumor cells were implanted in the thigh muscle (arrow) in the heterotopic tumor model (A) and at a subcapsular site (arrow) in the liver in the orthotopic tumor model (B). The frequency of lung metastases (arrows) was determined after fixation with Bouin’s solution (C) under a light microscope. The number of metastatic lung nodules was higher in the orthotopic tumor model than in the heterotopic tumor model (D). The number of metastatic lung nodules was found to significantly increase in the orthotopic tumor model at 6 (p=0.03), 9 (p=0.02), 12 (p=0.01), and 15 days (p=0.01) after tumor implantation. Groups consisted of six mice each.

• 

  Fig. 2. (A) The orthotopic tumor model showed intense cytoplasmic immunoreactivity (×400). N, peritumor normal liver; T, tumor tissue. (B) The immunohistochemical stains of CD31 (arrows) for heterotopic and orthotopic tumor model (×400). (C) Vascular endothelial growth factor (VEGF) protein levels were elevated in the orthotopic tumor model (O) compared with the heterotopic tumor model (H). CD31 expression was also higher in the orthotopic model than in the heterotopic tumor model. GAPDH, glyceraldehyde 3-phosphate dehydrogenase. (D) Microvessel density using CD31 increased significantly in the orthotopic tumor model (**p < 0.01). Arrows indicate the microvessels of positive CD31 immunohistochemical staining. The number of mice was six per group.

• 

  Fig. 3. (A) Orthotopic and heterotopic tumor models demonstrated intense transforming growth factor beta1 (TGF-β1) immunoreactivity (×400). N, peritumor normal liver; T, tumor tissue. (B) The orthotopic tumor model showed predominant expression of cyclooxygenase-2 (COX-2) in the cytoplasm of peripheral tumor tissue (×400). (C) Western blot assays using antibodies against TGF-β1 and COX-2 in the orthotopic (O) and heterotopic (H) tumor models showed higher TGF-β1 and COX-2 expression in the orthotopic model than in the heterotopic tumor model. GAPDH, glyceraldehyde 3-phosphate dehydrogenase.

• 

  Fig. 4. Immunohistochemical staining with antibodies against vascular endothelial growth factor (VEGF), transforming growth factor beta1 (TGF-β1), and cyclooxygenase-2 (COX-2) in the heterotopic and orthotopic tumor models (A-C). After radiation, staining for VEGF, TGF-β1, and COX-2 decreased in the orthotopic tumor model (×400), while the tumor compartment in the heterotopic tumor model showed no significant responses after radiation (3 days after radiation). (D) These findings were confirmed in the orthotopic tumor model by western blot assay. Expression of VEGF, TGF-β1, and COX-2 in tumor tissues decreased after radiation, not in peritumoral normal tissue. (E) Serum interleukin-6 (IL-6) increased after irradiation in heterotopic and orthotopic tumor models (p=0.01 and p=0.01), and the orthotopic tumor model showed a significantly higher level of IL-6 than the heterotopic tumor model (p < 0.01). Increased serum VEGF levels were also measured in the orthotopic tumor model and this level was reduced after irradiation in the orthotopic tumor model, but the difference was not significant (F). The sampling number was six per group. N/L, normal liver of naive mouse; N, normal peritumor liver; T, tumor; RT, radiation treatment; GAPDH, glyceraldehyde 3-phosphate dehydrogenase.

• 

  Fig. 5. Immune cell expression after radiation (n=4 mice per each group). Irradiated orthotopic tumor samples were obtained on the third day after radiation. (A) The frequency of total CD4 and CD8 T cells in tumor-infiltrating lymphocytes are shown in the irradiated and non-irradiated orthotopic tumor model. In tumor bearing mice (TM-bearing) mice, the frequency of CD4 and CD8 T cells showed no significant difference after radiation. (B) The number of CD25+Foxp3 Treg cells in the tumor was significantly decreased after radiation (p < 0.05). (C) Myeloid-derived suppressor cell CD11b+Gr-1+ expression was also significantly decreased after radiation (p < 0.05). Treg, regulatory T cell; RT, radiation treatment; ns, not significant.

Reference

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