Intravital imaging of the effects of 5-fluorouracil on the murine liver microenvironment using 2-photon laser scanning microscopy

Okigami,Masato; Tanaka,Koji; Inoue,Yasuhiro; Saigusa,Susumu; Okugawa,Yoshinaga; Toiyama,Yuji; Mohri,Yasuhiko; Kusunoki,Masato

doi:10.3892/ol.2016.4258

Intravital imaging of the effects of 5-fluorouracil on the murine liver microenvironment using 2-photon laser scanning microscopy

Authors:
- Masato Okigami
- Koji Tanaka
- Yasuhiro Inoue
- Susumu Saigusa
- Yoshinaga Okugawa
- Yuji Toiyama
- Yasuhiko Mohri
- Masato Kusunoki
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Affiliations: Department of Gastrointestinal and Pediatric Surgery, Division of Reparative Medicine, Institute of Life Sciences, Mie University Graduate School of Medicine, Tsu, Mie 514‑8507, Japan
Published online on: February 23, 2016 https://doi.org/10.3892/ol.2016.4258
Pages: 2433-2439

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Abstract

5-fluorouracil (5FU) is often used in the treatment of colorectal cancer. 5FU improves the median overall and disease‑free survival rates and reduces recurrence rates in patients who have undergone curative surgical resection. However, in the adjuvant setting, whether 5FU eradicates clinically undetectable micrometastases in target organs such as the liver, or whether 5‑FU inhibits the adhesion of circulating tumor cells has not yet been established. In the present study, 5FU was administered following the inoculation of red fluorescent protein-expressing HT29 cells into green fluorescent protein (GFP)-transgenic nude mice to examine its inhibitory effect. 2-photon laser scanning microscopy was performed at selected time points for time‑series imaging of liver metastasis of GFP‑transgenic mice. The cell number in vessels was quantified to evaluate the response of the tumor microenvironment to chemotherapy. HT29 cells were visualized in hepatic sinusoids at the single‑cell level. A total of 2 hours after the injection (early stage), time‑series imaging revealed that the number of caught tumor cells gradually reduced over time. In the 5FU treatment group, no significant difference was observed in the cell number in the early stage. One week after the injection (late stage), a difference in morphology was observed. The results of the present study indicated that 5FU eradicated clinically undetectable micrometastases in liver tissues by acting as a cytotoxic agent opposed to preventing adhesion. The present study indicated that time‑series intravital 2‑photon laser scanning microscopic imaging of metastatic tumor xenografts may facilitate the screening and evaluation of novel chemotherapeutic agents with less interindividual variability.

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1	Desch CE, Benson AB III, Somerfield MR, Flynn PJ, Krause C, Loprinzi CL, Minsky BD, Pfister DG, Virgo KS and Petrelli NJ: American Society of Clinical Oncology: Colorectal cancer surveillance: 2005 update of an American society of clinical oncology practice guideline. J Clin Oncol. 23:8512–8519. 2005. View Article : Google Scholar : PubMed/NCBI
2	Manfredi S, Lepage C, Hatem C, Coatmeur O, Faivre J and Bouvier AM: Epidemiology and management of liver metastases from colorectal cancer. Ann Surg. 244:254–259. 2006. View Article : Google Scholar : PubMed/NCBI
3	No authors listed: Chemotherapy of metastatic colorectal cancer: Fluorouracil plus folinic acid and irinotecan or oxaliplatin. Prescrire Int. 14:230–233. 2005.PubMed/NCBI
4	Kong L, Wang X, Zhang K, Yuan W, Yang Q, Fan J, Wang P and Liu Q: Gypenosides synergistically enhances the anti-tumor effect of 5-fluorouracil on colorectal cancer in vitro and in vivo: A role for oxidative stress-mediated DNA damage and p53 activation. PLoS One. 10:e01378882015. View Article : Google Scholar : PubMed/NCBI
5	Moertel CG, Fleming TR, Macdonald JS, Haller DG, Laurie JA, Goodman PJ, Ungerleider JS, Emerson WA, Tormey DC and Glick JH: Levamisole and fluorouracil for adjuvant therapy of resected colon carcinoma. N Engl J Med. 322:352–358. 1990. View Article : Google Scholar : PubMed/NCBI
6	Wikman H, Vessella R and Pantel K: Cancer micrometastasis and tumour dormancy. APMIS. 116:754–770. 2008. View Article : Google Scholar : PubMed/NCBI
7	Denk W, Strickler JH and Webb WW: Two-photon laser scanning fluorescence microscopy. Science. 248:73–76. 1990. View Article : Google Scholar : PubMed/NCBI
8	Hickey MJ and Kubes P: Intravascular immunity: The host-pathogen encounter in blood vessels. Nat Rev Immunol. 9:364–375. 2009. View Article : Google Scholar : PubMed/NCBI
9	Grutzendler J, Yang G, Pan F, Parkhurst CN and Gan WB: Transcranial two-photon imaging of the living mouse brain. Cold Spring Harb Protoc. 2011:pdb.prot0654742011. View Article : Google Scholar : PubMed/NCBI
10	Sumen C, Mempel TR, Mazo IB and von Andrian UH: Intravital microscopy: Visualizing immunity in context. Immunity. 21:315–329. 2004. View Article : Google Scholar : PubMed/NCBI
11	Tanaka K, Okigami M, Toiyama Y, Morimoto Y, Matsushita K, Kawamura M, Hashimoto K, Saigusa S, Okugawa Y, Inoue Y, et al: In vivo real-time imaging of chemotherapy response on the liver metastatic tumor microenvironment using multiphoton microscopy. Oncol Rep. 28:1822–1830. 2012.PubMed/NCBI
12	Toiyama Y, Mizoguchi A, Okugawa Y, Koike Y, Morimoto Y, Araki T, Uchida K, Tanaka K, Nakashima H, Hibi M, et al: Intravital imaging of DSS-induced cecal mucosal damage in GFP-transgenic mice using two-photon microscopy. J Gastroenterol. 45:544–553. 2010. View Article : Google Scholar : PubMed/NCBI
13	Koike Y, Tanaka K, Okugawa Y, Morimoto Y, Toiyama Y, Uchida K, Miki C, Mizoguchi A and Kusunoki M: In vivo real-time two-photon microscopic imaging of platelet aggregation induced by selective laser irradiation to the endothelium created in the beta-actin-green fluorescent protein transgenic mice. J Thromb Thrombolysis. 32:138–145. 2011. View Article : Google Scholar : PubMed/NCBI
14	Morimoto Y, Tanaka K, Toiyama Y, Inoue Y, Araki T, Uchida K, Kimura K, Mizoguchi A and Kusunoki M: Intravital three-dimensional dynamic pathology of experi- mental colitis in living mice using two-photon laser scanning microscopy. J Gastrointest Surg. 15:1842–1850. 2011. View Article : Google Scholar : PubMed/NCBI
15	Tanaka K, Morimoto Y, Toiyama Y, Okugawa Y, Inoue Y, Uchida K, Kimura K, Mizoguchi A and Kusunoki M: Intravital dual-colored visualization of colorectal liver metastasis in living mice using two photon laser scanning microscopy. Microsc Res Tech. 75:307–315. 2012. View Article : Google Scholar : PubMed/NCBI
16	Tanaka K, Morimoto Y, Toiyama Y, Matsushita K, Kawamura M, Koike Y, Okugawa Y, Inoue Y, Uchida K, Araki T, et al: In vivo time-course imaging of tumor angiogenesis in colorectal liver metastases in the same living mice using two-photon laser scanning microscopy. J Oncol. 2012:2654872012. View Article : Google Scholar : PubMed/NCBI
17	Tanaka K, Toiyama Y, Inoue Y, Uchida K, Araki T, Mohri Y, Mizoguchi A and Kusunoki M: Intravital imaging of gastrointestinal diseases in preclinical models using two-photon laser scanning microscopy. Surg Today. 43:123–129. 2013. View Article : Google Scholar : PubMed/NCBI
18	De Gramont A, Krulik M, Cady J, Lagadec B, Maisani JE, Loiseau JP, Grange JD, Gonzalez-Canali G, Demuynck B and Louvet C: High-dose folinic acid and 5-fluorouracil bolus and continuous infusion in advanced colorectal cancer. Eur J Cancer Clin Oncol. 24:1499–1503. 1988. View Article : Google Scholar : PubMed/NCBI
19	Torimura T, Ueno T, Taniguchi E, Masuda H, Iwamoto H, Nakamura T, Inoue K, Hashimoto O, Abe M, Koga H, et al: Interaction of endothelial progenitor cells expressing cytosine deaminase in tumor tissues and 5-fluorocytosine administration suppresses growth of 5-fluorouracil-sensitive liver cancer in mice. Cancer Sci. 103:542–548. 2012. View Article : Google Scholar : PubMed/NCBI
20	Hoffmann D, Bayer W and Wildner O: In situ tumor vaccination with adenovirus vectors encoding measles virus fusogenic membrane proteins and cytokines. World J Gastroenterol. 13:3063–3070. 2007.PubMed/NCBI
21	Wilmanns C, Fan D, Obrian C, Radinsky R, Bucana C, Tsan R and Fidler I: Modulation of doxorubicin sensitivity and level of P-glycoprotein expression in human colon-carcinoma cells by ectopic and orthotopic environments in nude-mice. Int J Oncol. 3:413–422. 1933.
22	Wilmanns C, Fan D, O'Brian CA, Bucana CD and Fidler IJ: Orthotopic and ectopic organ environments differentially influence the sensitivity of murine colon carcinoma cells to doxorubicin and 5-fluorouracil. Int J Cancer. 52:98–104. 1992. View Article : Google Scholar : PubMed/NCBI
23	Martin MD, Kremers GJ, Short KW, Rocheleau JV, Xu L, Piston DW, Matrisian LM and Gorden DL: Rapid extravasation and establishment of breast cancer micrometastases in the liver microenvironment. Mol Cancer Res. 8:1319–1327. 2010. View Article : Google Scholar : PubMed/NCBI
24	Warusavitarne J, Ramanathan P, Kaufman A, Robinson BG and Schnitzler M: 5-fluorouracil (5FU) treatment does not influence invasion and metastasis in microsatellite unstable (M SI-H) colorectal cancer. Int J Colorectal Dis. 21:625–631. 2006. View Article : Google Scholar : PubMed/NCBI
25	Kerbel RS and Kamen BA: The anti-angiogenic basis of metronomic chemotherapy. Nat Rev Cancer. 4:423–436. 2004. View Article : Google Scholar : PubMed/NCBI
26	Colleoni M, Orlando L, Sanna G, Rocca A, Maisonneuve P, Peruzzotti G, Ghisini R, Sandri MT, Zorzino L, Nolè F, et al: Metronomic low-dose oral cyclophosphamide and methotrexate plus or minus thalidomide in metastatic breast cancer: Antitumour activity and biological effects. Ann Oncol. 17:232–238. 2006. View Article : Google Scholar : PubMed/NCBI
27	Ishii M, Egen JG, Klauschen F, Meier-Schellersheim M, Saeki Y, Vacher J, Proia RL and Germain RN: Sphingosine-1-phosphate mobilizes osteoclast precursors and regulates bone homeostasis. Nature. 458:524–528. 2009. View Article : Google Scholar : PubMed/NCBI
28	Hickey MJ and Kubes P: Intravascular immunity: The host-pathogen encounter in blood vessels. Nat Rev Immunol. 9:364–375. 2009. View Article : Google Scholar : PubMed/NCBI
29	Bousso P, Bhakta NR, Lewis RS and Robey E: Dynamics of thymocyte-stromal cell interactions visualized by two-photon microscopy. Science. 296:1876–1880. 2002. View Article : Google Scholar : PubMed/NCBI
30	Yang M, Baranov E, Jiang P, Sun FX, Li XM, Li L, Hasegawa S, Bouvet M, Al-Tuwaijri M, Chishima T, et al: Whole-body optical imaging of green fluorescent protein-expressing tumors and metastases. Proc Natl Acad Sci USA. 97:1206–1211. 2000. View Article : Google Scholar : PubMed/NCBI