Multikinase inhibitor regorafenib inhibits the growth and metastasis of colon cancer with abundant stroma

Abstract

Interaction between tumor cells and stromal cells plays an important role in the growth and metastasis of colon cancer. We previously found that carcinoma-associated fibroblasts (CAFs) expressed platelet-derived growth factor receptor-β (PDGFR-β) and that PDGFR targeted therapy using imatinib or nilotinib inhibited stromal reaction. Bone marrow-derived mesenchymal stem cells (MSCs) migrate to tumor stroma and differentiate into CAFs. A novel oral multikinase inhibitor regorafenib inhibits receptor tyrosine kinases expressed on stromal cells (vascular endothelial growth factor receptor 1-3, TIE2, PDGFR-β, and fibroblast growth factors) and tumor cells (c-KIT, RET, and BRAF). These molecules are involved in tumor growth, angiogenesis, lymphangiogenesis, and stromal activation. Therefore, we examined whether regorafenib impaired the tumor-promoting effect of CAFs/MSCs. KM12SM human colon cancer cells alone or KM12SM cells with MSCs were transplanted into the cecal wall of nude mice. Co-implantation of KM12SM cells with MSCs into the cecal wall of nude mice produced tumors with abundant stromal component and promoted tumor growth and lymph node metastasis. Single treatment with regorafenib inhibited tumor growth and metastasis by inhibiting both tumor cells and stromal reaction. This tumor-inhibitory effect of regorafenib was more obvious in tumors developed by co-implanting KM12SM cells with MSCs. Our data suggested that targeting of the tumor microenvironment with regorafenib affected tumor cell-MSC interaction, which in turn inhibited the growth and metastasis of colon cancer.

Keywords: Colon cancer; mesenchymal stem cells; molecular target therapy; regorafenib; stroma.

Figure 1

In vitro cell proliferation and migration assay. Growth (a) and migration (b) of tumor cells in the presence or absence of regorafenib was measured using Incucyte Zoom, which monitors cell confluency in real time. Regorafenib inhibited tumor proliferation and migration irrespective of KRAS and BRAF mutation status. *P < 0.05 versus control.

Figure 2

(a) Effect of mesenchymal stem cells on tumor stromal reaction. Expression of α‐smooth muscle actin (α‐SMA) and collagen I was higher in mixed‐cell tumors than in KM12SM tumors. (b) Effect of regorafenib on tumor growth and lymph node metastasis in orthotopic colon tumor models. Regorafenib inhibited the growth of both KM12SM and mixed‐cell tumors. Average tumor volume of mice not treated with regorafenib was set as 100% for both the tumor types. The rate of tumor reduction after regorafenib treatment was significantly higher (78% vs 45%; P < 0.05) in mixed‐cell tumors than in KM12SM tumors. Data are expressed as mean ± SE; *P < 0.05.

Figure 3

Effect of regorafenib on KM12SM and mixed‐cell tumors. Expression of ERK (a) and pERK (b), analysis of cell proliferation (Ki‐67 labeling index) (c), apoptosis index (TUNEL assay) (d), H&E staining (e), expression of α‐smooth muscle actin (α‐SMA) (carcinoma‐associated fibroblasts) (f), angiogenesis (CD31 staining) (g), and lymphangiogenesis (LYVE‐1 staining) (h). Scale bar = 200 μm.

Figure 4

Western blot analysis showed that regorafenib inhibited the phosphorylation of ERK1/2.

Figure 5

Quantification of the effects of regorafenib on KM12SM and mixed‐cell tumors. Regorafenib decreased the Ki‐67 labeling index (a), α‐smooth muscle actin (α‐SMA)‐positive area (c), and lymphatic vessel area (e) in mixed‐cell tumors. Furthermore, regorafenib increased the apoptosis index (b) and decreased microvessel area (d) in KM12SM tumors. Data are expressed as mean ± SE; *P < 0.05. NS., not significant.

Figure 6

Effect of regorafenib on vascular and lymphatic endothelial cells in KM12SM and mixed‐cell tumors. Expression of CD31 and TUNEL in apoptotic vascular endothelial cells (a), LYVE‐1 and TUNEL in apoptotic lymphatic endothelial cells (b), CD31 and Ki‐67 in proliferating vascular endothelial cells (c), and LYVE‐1 and KI‐67 in proliferating lymphatic endothelial cells (d). Proliferating (Ki67‐positive) and apoptotic (TUNEL‐positive) cells are depicted in green. Vascular and lymphatic endothelial cells are depicted in red. Scale bar = 50 μm. (c) Average ratio of apoptotic vascular endothelial cells to the total number of vascular endothelial cells. (d) Average ratio of apoptotic lymphatic endothelial cells to the total number of lymphatic endothelial cells. (e) Average ratio of proliferating vascular endothelial cells to the total number of vascular endothelial cells. (f) Average ratio of proliferating lymphatic endothelial cells to the total number of lymphatic endothelial cells. Data are expressed as mean ± SE; *P < 0.05.