Cancer Res Treat.  2021 Jul;53(3):733-743. 10.4143/crt.2020.494.

The Establishment of a Fast and Safe Orthotopic Colon Cancer Model Using a Tissue Adhesive Technique

• Hu HT
• Wang Z
• Kim MJ
• Jiang LS
• Xu SJ
• Jung J
• Lee E
• Park JH
• Bakheet N
• Yoon SH
• Kim KY
• Song HY
• Chang S

• KMID: 2518397
• DOI: http://doi.org/10.4143/crt.2020.494

Abstract

Purpose
We aimed to develop a novel method for orthotopic colon cancer model, using tissue adhesive in place of conventional surgical method.
Materials and Methods
RFP HCT 116 cell line were used to establish the colon cancer model. Fresh tumor tissue harvested from a subcutaneous injection was grafted into twenty nude mice, divided into group A (suture method) and group B (tissue adhesive method). For the group A, we fixed the tissue on the serosa layer of proximal colon by 8-0 surgical suture. For the group B, tissue adhesive (10 μL) was used to fix the tumor. The mortality, tumor implantation success, tumor metastasis, primary tumor size, and operation time were compared between the two groups. Dissected tumor tissue was analyzed for the histology and immunohistochemistry. Also, we performed tumor marker analysis.
Results
We observed 30% increase in graft success and 20% decrease in mortality, by using tissue adhesive method, respectively. The median colon tumor size was significantly increased by 4 mm and operation time was shortened by 6.5 minutes. The H&E showed similar tumor structure between the two groups. The immunohistochemistry staining for cancer antigen 19-9, carcinoembryonic antigen, cytokeratin 20, and Ki-67 showed comparable intensities in both groups. Real-time quantitative reverse transcription analysis showed eight out of nine tumor markers are unchanged in the tissue adhesive group. Western blot indicated the tissue adhesive group expressed less p-JNK (apototic marker) and more p-MEK/p-p38 (proliferation marker) levels.
Conclusion
We concluded the tissue adhesive method is a quick and safe way to generate orthotopic, colon cancer model.

Keyword

Orthotopic; Colon neoplasms; Mouse model; Tissue adhesive technique

Figure

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  Fig. 1 The development of tissue adhesive method for orthotopic tumor implantation on mouse colon. (A) A representative pictures of the proximal colon with small incision to expose serosa. (B) Suturing prepared before the tumor tissue attachment. (C) One 1 mm3 tumor fragment was implanted on the damaged colon wall of the proximal colon. (D) Stitch the tissue on the colon wall with an 8-0 surgical suture. (E) The serosa of the proximal colon was exposed by small incision. (F) One 1 mm3 tumor fragment was attached on the damaged colon wall. (G) One small drop of tissue adhesive was applied to cover the tumor piece. The image was captured 10 seconds after application. (H) A picture of fixed tumor on colon after 60 seconds, showing the tissue adhesive was fully solidified (A–H, ×8). Arrows indicate the sites of incision and tumor placement, by suture or tissue adhesive glue.

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  Fig. 2 Tissue adhesive method maintains the gross histology of colon tumors, compared to conventional suture method. (A, B) H&E staining images of the tumor generated by suture method. (C, D) H&E staining images of the tumor generated by the tissue adhesive method. Representative images were taken at ×40 and ×200 magnification. (E, F) Liver metastasis (E) or mesenteric metastasis (F) of colon tumor from gluing method. (G–J) Representative pictures of organized tissue pattern and immune cell infiltration on suture (G, H) and tissue adhesive (I, J) methods. Scale bars=400 μm (A, C), 100 μm (B, D–J).

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  Fig. 3 Immunohistochemical staining for CA19-9, CEA, CK20, and Ki-67 reveals comparable expression pattern between tissue adhesive and suture method. (A) Representative pictures of immunohistochemistry from tumors of tissue adhesive or suturing method. CA-19-1, CEA, CK20, Ki-67, and TUNEL are examined. Tumors obtained from liver metastasis or abdominal metastasis were also included. For CA19-9, CEA, and CK20, the relative intensity is marked as +, ++, and +++. Scale bars=60 μM. (B) Graph showing the Ki-67 positive area of tissue adhesive and suture group (n=4). (C) Graph showing average TUNEL postive cells of tissue adhesive and suture group (n=4). CA19-9, cancer antigen 19-9; CEA, carcinoembryonic antigen; CK20, cytokeratin 20; TUNEL, terminal deoxynucleotidyl transferase dUTP nick end labeling. **p < 0.01, ***p < 0.001; n.s., not significant.

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  Fig. 4 Real-time PCR analysis from tumors obtained by tissue adhesive method and suture method show similar marker RNA expression pattern. RNA from three tumors of the two graft methods were analyzed by colon tumor markers (A, COX2; B, CEA; C, CD133), proliferation markers (D, JNK; E, ERK2; F, PKM2), cell cycle markers (G, CDK6; H, CCNA2) as well as migration markers (I, IGFBP2). Cell line (HCT-116) and Subcu (tumors from subcutaneous injection) tumor samples were compared together. *p < 0.05; n.s., not significant. CCNA2, cyclin A2; CDK6, cyclin-dependent kinase 6; CEA, carcinoembryonic antigen; COX2, cyclooxygenase-2; IGFBP2, insulin-like growth factor binding protein 2.

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  Fig. 5 Western blot analysis of marker expression revealed higher proliferation signal in tissue adhesive with retained tumor marker expression. Protein samples extracted from three tumors of the two graft methods were analyzed by western blot. Cell (HCT-116) and Subcu (tumors from subcutaneous injection) tumor samples were compared together. CA 19-9, cancer antigen 19-9; CEA, carcinoembryonic antigen; COX2, cyclooxygenase-2.

Reference

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