Ann Surg Oncol DOI 10.1245/s10434-015-4594-0
ORIGINAL ARTICLE – GASTROINTESTINAL ONCOLOGY
Theranostic Photosensitive Nanoparticles for Lymph Node Metastasis of Gastric Cancer Hironori Tsujimoto, MD, PhD1, Yuji Morimoto, MD, PhD2, Risa Takahata, MD, PhD1, Shinsuke Nomura, MD1, Kazumichi Yoshida, MD, PhD1, Shuichi Hiraki, MD, PhD1, Hiroyuki Horiguchi, MD1, Hiromi Miyazaki, PhD3, Satoshi Ono, MD, PhD3, Daizo Saito, MD, PhD3, Isao Hara, PhD4, Eiichi Ozeki, PhD4, Junji Yamamoto, MD, PhD1, and Kazuo Hase, MD, PhD1 Department of Surgery, National Defense Medical College, Tokorozawa, Saitama, Japan; 2Department of Integrative Physiology and Bio-Nano Medicine, National Defense Medical College, Tokorozawa, Saitama, Japan; 3Division of Traumatology, National Defense Medical College Research Institute, Tokorozawa, Saitama, Japan; 4Technology Research Laboratory, Shimadzu Corporation, Soraku-gun, Kyoto, Japan
1
ABSTRACT Background. Preoperative and intraoperative diagnoses of lymph node (LN) metastasis in patients with gastric cancer is essential to determine the extent of LN dissection in order to establish individualized treatment strategies. We investigated the theranostic value of a newly developed drug delivery system employing nanoparticles loaded with the indocyanine green (ICG) derivative ICG-loaded lactosome (ICGm) using a murine draining LN metastasis model of gastric cancer. Methods. In the experimental draining LN metastasis model of human gastric cancer, the right hind footpads of nude mice were injected with cancer cells. Three weeks later, either ICGm or ICG solution was injected through the tail vein. Forty-eight hours after the administration of a photosensitizer, in vivo and ex vivo imaging and photodynamic therapy (PDT) were performed, and size of the LNs was measured. Results. In vivo imaging revealed metastatic LNs in the ICGm-treated mice but not in the ICG-treated mice. PDT using ICGm induced apoptosis and significantly inhibited the growth of metastatic LNs.
Electronic supplementary material The online version of this article (doi:10.1245/s10434-015-4594-0) contains supplementary material, which is available to authorized users. Ó Society of Surgical Oncology 2015 First Received: 6 December 2014 H. Tsujimoto, MD, PhD e-mail: [email protected]
Conclusions. ICGm presents a novel theranostic nanodevice for LN metastasis of gastric cancer. Gastric cancer is the fourth most common malignancy and the second most frequent cause of cancer-related deaths, accounting for approximately 10 % of cancer-related deaths worldwide.1–3 Lymph node (LN) metastasis is an important prognostic factor for long-term survival.4 In patients with N0 gastric cancer, the 5-year survival rate is 89.0 %, whereas survival rates dramatically decrease to 58.3, 33.4, and 17.4 % for patients with N1, N2, and N3 staging, respectively.3 Patients with T1 tumors have a low risk of LN metastasis: 2.2 % in T1a (mucosal) and 17.9 % in T1b (submucosal) cancers.5 On the other hand, the risk of LN metastases increases to 44 and 64 % for patients with T2 and T3 tumors, respectively. Thus, the extent of optimal LN dissection remains debatable;6–8 therefore; it is necessary to establish individualized treatment strategies for surgery and lymphadenectomy on the basis of accurate preoperative and intraoperative staging.8 Nanotechnology has the potential to create intricate structures comparable in size to biomolecules with unprecedented chemical and physical functionality.9 In tumor tissues, submicron-sized defects exist on the vascular wall because of rapid angiogenesis, thereby enabling permeation of macromolecules. Furthermore, the lymph system around the tumor grows too slowly to remove foreign compounds from the tumor region, and nanocarriers in the range of 30–100 nm in size can passively accumulate in tumors, in a process known as enhanced permeability and retention (EPR).10,11 Hence nanoparticles, such as liposomes, polymeric micelles, dendrimers, superparamagnetic iron oxide
H. Tsujimoto et al.
crystals, and colloidal gold, have been employed in drug delivery systems (DDSs) for targeted cancer therapies.12 Recent advances in nano-DDS carriers have facilitated the integration of diagnostic and therapeutic functions of theranostic nanoparticles to enable simultaneous diagnosis, therapy, and monitoring of therapeutic responses.13 Recently, we established theranostic photosensitive nanoparticles composed of micelles assembled from poly (sarcosine)–poly (L-lactic acid) (PS–PLLA) block copolymers loaded with the indocyanine green (ICG) derivative ICG-loaded lactosome (ICGm).14,15 ICG is a well-established infrared fluorescence dye that also can be used as a photosensitizer, suggesting that the use of ICG-based nanoparticles is a promising theranostic system.16 Indeed, we previously elucidated the efficacy of ICGm for the diagnosis and treatment of experimental peritoneal dissemination of gastric cancer. In vivo imaging revealed that ICGm selectively accumulated in tumor tissues and photodynamic therapy (PDT) with ICGm reduced the total weight of the disseminated nodules and significantly improved weight loss and survival rate.15 We investigated the theranostic value of ICGm using a murine model of draining LN metastasis of gastric cancer. MATERIALS AND METHODS Cancer Cell Lines, Animals, and Ethics Statement Human gastric adenocarcinoma MKN45 cells were purchased from the Riken Bioresource Center (Cell Bank, Ibaraki, Japan) and then cultured in Roswell Park Memorial Institute 1640 medium (Sigma-Aldrich, St. Louis, MO) supplemented with 10 % heat-inactivated fetal bovine serum (Life Technologies, Carlsbad, CA), 100 U/mL of penicillin, 100 lg/mL of streptomycin, and 0.25 lg/mL of amphotericin B (Antibiotic-Antimycotic, Life Technologies) at 37 °C in 5 % CO2 with 95 % humidity. Male nude Balb/c nu/ nu mice at 8 weeks of age (CLEA Japan, Tokyo, Japan) were fed under specific pathogen-free conditions. All animal procedures followed the guidelines approved by the National Defense Medical College Animal Care and Use Committee. Experimental Draining LN Metastasis Model To develop the experimental draining LN metastasis model, mice were injected with 1.0 9 106 MKN45 cells suspended in 50 lL of phosphate-buffered saline into the right hind footpad under anesthesia as previously described.17 Three weeks after injection, in vivo and ex vivo images of the popliteal lymph nodes (PLNs) were obtained. For PDT, the PLNs of another group of mice were irradiated through the skin under anesthesia, and the size of the LNs was measured by ultrasonography.
Development of ICGm We successfully synthesized a ‘‘lactosome,’’ which is a micelle assembled from block copolymers, PS–PLLA, synthesized as previously reported.14,15 Because PS–PLLA is composed of a biodegradable polypeptide, its toxicity is negligible, thus suggesting that it is safe for human use. The ICGm used in this study contained 22 % of ICG– PLLA and 78 % of PS–PLLA. Regarding ICG–PLLA synthesis, the terminal end of PLLA was chemically modified using ICG as follows: 1.0 mg ICG–OSu was added to the dimethylformamide (DMF) solution of the free amino group bearing PLLA (2.46 mg), which contains an amino group designed as an indicator of sarcosine N-carboxyanhydride (NCA) polymerization during the synthesis of amphiphilic PS–PLLA-block copolymers. The reaction mixture was stirred at room temperature overnight under light-shielding conditions. The reaction mixture was purified by size-exclusion chromatography using a Sephadex LH–20 column (GE Healthcare Japan Corporation, Tokyo, Japan) with DMF as the eluent. Chloroform solutions of PS–PLLA and ICG–PLAA were mixed at a ratio of 0.78:0.22. The solvent was removed under reduced pressure and the thin film formed was dissolved in 10-mM Tris–HCl buffer (pH 7.4). The resulting aqueous solution was purified by Sephacryl S-100 size-exclusion chromatography by elution with 10-mM Tris–HCl buffer (pH 7.4) to obtain ICGm. Dynamic lightscattering analysis revealed that the hydrodynamic diameter of ICGm was 40–50 nm, and the zeta potential of ICGm was -0.51 mV. ICGm solutions (91 mg/mL H2O) were administered intravenously or intraperitoneally to 7-week-old BALB/c mice. Both intravenous and intraperitoneal administrations of ICGm to male and female mice (2000 mg/kg) caused no weight loss and the mice showed no obvious abnormal findings by macroscopic observation at autopsy (sFig. 1). In Vivo and Ex Vivo Imaging To visualize PLNs in mice, either 100 lL of ICGm, in which the ICG concentration was 281 lM, or 100 lL of ICG solution (281 lM) was injected through the tail vein. Then, macroscopic in vivo fluorescence images were obtained using an in vivo imaging system (IVIS; PerkinElmer, Inc., Waltham, MA) as described previously.18 For fluorescence imaging, the right hind foot and dissected PLNs of the mice were illuminated with an excitation light at the wavelength of 780 nm and the fluorescence was captured using an 845-nm filter. The exposure time was 1000 ms. A photographic image of the animal was taken in the chamber under dim illumination,
Nanoparticle for Lymph Node Metastasis
followed by acquisition and overlay of pseudocolor images representing the spatial distribution of radiant efficacy within the animal. In vivo and ex vivo imaging were performed 48 h after ICG or ICGm injection, because our preliminary data revealed that 48–72 h after the ICG injection offered the best contrast of tumor sites to normal tissue (sFig. 2). PDT Three weeks after the injection of the cancer cells, either 100 lL of ICGm or 100 lL of ICG solution (both containing 281 lM of ICG) was injected intravenously through the tail vein. Forty-eight hours after the administration of the photosensitizer, photoirradiation was performed using a fiber-coupled laser system with a laser diode at 808 nm (model FC-W-808, maximum output: 10 W; Changchun New Industries Optoelectronics Technology Co., Ltd., Jilin, China). The fiber probe was placed just above the knee so that the irradiated area on the PLN was 0.79 cm2 (diameter, 1.0 cm). The fluence rate and irradiation period were set to 500 mW/cm2 and 1000 s, respectively, corresponding to a fluence of 500 J/cm2 (sFig. 3).
followed by the application of antidigoxigenin–peroxidase solution. The color was developed with diaminobenzidine, after which the sections were lightly counterstained with hematoxylin. To confirm staining specificity of the TUNEL method, a positive control section was prepared. The substitution of equilibration buffer for TdT was used as a negative control. The apoptotic index was expressed as the ratio of positively stained cells to the total number of tumor cells for each case. To determine the apoptotic index, 10 representative areas comprising at least 1000 cells without inflammation or necrosis were counted for each sample using a light microscope (9400 magnification). Statistical Analysis Data are represented as the mean ± SD. Statistical analyses were performed using the Mann–Whitney U test or Chi square test with the Fisher’s exact test, where appropriate. Calculations were performed using StatView statistical software (version 5.0; SAS Institute, Inc., Cary, NC). A p value of \0.05 was considered statistically significant. RESULTS
Measurement of LN Size Development of Draining LN Metastasis of Human Gastric Cancer Cells
PLNs were measured using an ultrasound imaging system (Vevo 770; VisualSonics, Inc., Toronto, Canada) before and 7 days after PDT. A 40-MHz ultrasonic probe (RMV704, VisualSonics) with a focal depth of 6 mm and a spatial resolution of 50 lm was used (sFig. 4). The longitudinal and transverse dimensions of LN were measured, and LN volume was obtained by the longitudinal and transverse size of LN, i.e., volume = (longitudinal size) 9 (transverse size) 9 (transverse size) 9 1/2.
Three weeks after the injection of the cancer cells, an increase in the thickness of the right hind footpad was evident because of tumor growth. The ipsilateral (metastatic) PLNs were enlarged compared with the contralateral (nonmetastatic) PLNs (Fig. 1a). Microscopic examination of the ipsilateral PLNs confirmed metastatic adenocarcinoma (Fig. 1b).
Detection of Apoptotic Cells
Imaging of Metastatic LN
Apoptotic cells were detected by the terminal deoxynucleotidyl transferase (TdT)-mediated deoxyuridine triphosphate biotin nick-end labeling (TUNEL) method. The TACS2 TdT Blue Label apoptosis detection kit (Trevigen, Gaithersburg, MD) also was used in this study. Briefly, paraffin sections were deparaffinized in xylene and rehydrated in graded ethanol. After washing in Tris-buffered saline (TBS; pH 7.4), the sections were placed in 3% H2O2 for 10 min and then digested with proteinase K (20 mg/mL in TBS) at 37 °C for 10 min to enhance nuclear staining of the apoptotic cells. After digestion was stopped, the sections were washed in TBS and then treated with terminal transferase enzyme and digoxigenin-labeled nucleotides,
The right hind footpad tumor and PLNs of the ICGmtreated mice were detectable through the skin using the IVIS. After removing the skin from the hind leg, enlargement of the PLNs due to metastasis was confirmed by both macroscopic observation and using an IVIS (Fig. 2a, left lower panel). However, no fluorescence signal was observed in the ICG-treated mice (Fig. 2a, right lower panel). Bilateral PLNs were harvested and ex vivo images of the ICG- and ICGm-treated mice were obtained (Fig. 2b). Metastatic PLNs were clearly identified in the ICGmtreated mice but not the ICG-treated mice. Contralateral (nonmetastatic) LNs in both groups were undetectable by ex vivo imaging.
H. Tsujimoto et al.
FIG. 1 Development of a draining lymph node (LN) metastasis model using human gastric cancer cells. a Comparison of ipsilateral popliteal lymph nodes (PLNs) (left) and contralateral PLNs (right) 3 weeks after the injection of 1.0 9 106 MKN45 cells in the right hind footpad. Each experiment was independently performed four times with similar results. Representative data are depicted. b Microscopic view of the ipsilateral PLNs (left panel: 940, right panel: 9200 magnification). Each experiment was independently performed four times with similar results. Representative data are depicted
Effect of PDT on Metastatic LNs The size of the metastatic PLNs in the ICG-treated mice increased 7 days after PDT; however, in the ICGm-treated mice, no such enlargement of the ipsilateral PLNs was observed. There was a significant difference in the longitudinal and transverse sizes of PLN between the two groups 7 days after PDT (Fig. 3, p \ 0.05). Calculated PLN volumes after PDT in the ICGm-treated mice were significantly smaller than those in ICG-treated mice (3.1 ± 0.3 versus 6.8 ± 2.4 mm3, p \ 0.05). The apoptotic cells were remarkably visible in the metastatic LNs of the ICGm-treated mice (Fig. 4a). The apoptotic index was significantly higher in the ICGmtreated mice (11.0 ± 2.2 %) than in the ICG-treated mice (3.9 ± 3.4 %; p \ 0.05; Fig. 4b). DISCUSSION The results of this study demonstrated that in vivo and ex vivo imaging using ICGm clearly visualized metastatic PLNs. Moreover, PDT using ICGm induced apoptosis and significantly suppressed enlargement of the PLNs. The Japanese Gastric Cancer Association guidelines recommend partial or total gastrectomy with reduced lymphadenectomy, depending on the clinical and surgical
FIG. 2 In vivo and ex vivo imaging of the metastatic lymph nodes (LNs). a In vivo bright-field and fluorescent imaging of the right hind footpad tumor and the popliteal lymph nodes (PLNs) after removal of the skin (bottom panels). Enlargement of the PLNs due to metastasis was confirmed by both macroscopic observation and IVIS in the indocyanine green (ICG) derivative ICG-loaded lactosome (ICGm)treated mice (left panels). However, no fluorescence was observed in the ICG-treated mice (right panels). Arrowheads indicate popliteal lymph nodes. Each experiment was independently performed four times with similar results. Representative data are depicted. b Ex vivo fluorescent imaging of the bilateral PLNs in ICGm-treated mice (left panels) and ICG-treated mice (right panels). Ipsilateral (metastatic) PLNs of the ICGm-treated mice were detectable using the IVIS, but not the ICG-treated mice. Contralateral (nonmetastatic) LNs in both groups were undetectable by ex vivo imaging. Each experiment was independently performed four times with similar results. Representative data are depicted
findings, for the treatment of gastric cancer.19 However, because of the limitations in the sensitivity of preoperative diagnostic imaging methods to detect pathologic metastasis in regional LNs, systemic (D2) gastrectomy has become a standard procedure to achieve a curative effect, even for clinically node-negative patients.20 Thus, accurate preoperative and intraoperative diagnoses of LN metastasis helps to determine the LN site to be dissected. In this regard, we demonstrated that ICGm administered intravenously accumulates in metastatic LNs but not in nonmetastatic LNs, indicating the diagnostic value of ICGm.
Nanoparticle for Lymph Node Metastasis mm
Longitudinal dimension
mm
Transverse dimension
6.0 2.0
5.0
* *
4.0 3.0
1.5 1.0
2.0 0.5
1.0
0
0
Pre PDT
7 days after PDT
FIG. 3 Effect of photodynamic therapy (PDT) on metastatic lymph nodes (LNs) The longitudinal and transverse sizes of the metastatic popliteal lymph nodes (PLNs) of the indocyanine green (ICG)-treated mice (blue bars) and the ipsilateral PLNs of ICG derivative ICG-
A %
Apoptotic index
15.0
*
Pre PDT
7 days after PDT
loaded lactosome (ICGm)-treated mice (orange bars) pre-PDT and 7 days after PDT. *p \ 0.05. Each experiment was independently performed four times
vasculature, and sentinel node mapping for gastric cancer. 21,22 The increasing interest in ICG as a photosensitizer in PDT is because of its strong absorption band (between 700 and 800 nm), which allows deeper tissue penetration.16 In addition, ICGm used in this study was 40–50 nm in diameter and selectively accumulated in the tumor tissues through the EPR effect.10,11 Thus, ICGm may be a safe and ideal photosensitizer of PDT for the treatment of metastatic LNs. PDT elicits both necrosis and apoptosis depending on the light/drug dose, cell type, genetic and metabolic potential, nature of the photosensitizer used, and subcellular localization.23,24 We demonstrated that enhanced apoptosis in metastatic LNs in the ICGm-treated mice compared with that in the ICG-treated mice resulted in the growth suppression of the metastatic LNs.15
10.0
CONCLUSION 5.0
0 ICG
ICGm
B FIG. 4 Apoptosis in metastatic lymph nodes (LNs) after photodynamic therapy (PDT). a Apoptotic cells in the metastatic LNs of indocyanine green (ICG) derivative ICG-loaded lactosome (ICGm)treated mice were visualized (9 400 magnification). Each experiment was independently performed 4 times with similar results. Representative data are depicted. b Apoptotic index in the ICGm-treated mice (orange bar) and the ICG-treated mice (blue bar). *p \ 0.05
Here, as a photosensitizer, we employed ICG, which is a tricarbocyanine dye approved by the United States Food and Drug Administration for medical diagnostic studies and is widely used to evaluate cardiac output and liver function, visualization of the retinal and choroidal
ICGm selectively accumulated in metastatic LN because of the EPR effect, which enabled ICG fluorescence-based visualization of the metastatic LNs and PDT using ICGminduced apoptosis and significantly inhibited the growth of the metastatic LNs. These findings suggest that ICGm is a useful and novel theranostic tool for LN metastasis of gastric cancer. PDT is advantageous, because it can be used repeatedly without inducing resistance, unlike chemotherapy and radiotherapy. PDT using ICGm presents a promising theranostic approach for LN metastases in the clinical setting. ACKNOWLEDGMENTS This work was supported by JSPS KAKENHI (grant number 24591892). The authors thank Enago (www.enago.jp) for the English language review. DISCLOSURE All authors certify that they have no commercial associations that might pose a conflict of interest in connection with the submitted article.
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REFERENCES 1. Tsujimoto H, Ono S, Ichikura T et al. Roles of inflammatory cytokines in the progression of gastric cancer: friends or foes? Gastric Cancer. 2010;13:212–21. 2. Jemal A, Siegel R, Ward E et al. Cancer statistics, 2008. CA Cancer J Clin. 2008;58:71–96. 3. Isobe Y, Nashimoto A, Akazawa K et al. Gastric cancer treatment in Japan: 2008 annual report of the JGCA nationwide registry. Gastric Cancer. 2011;14:301–16. 4. Siewert JR, Bottcher K, Stein HJ, Roder JD. Relevant prognostic factors in gastric cancer: ten-year results of the German Gastric Cancer Study. Ann Surg. 1998;228:449–61. 5. Gotoda T, Yanagisawa A, Sasako M et al. Incidence of lymph node metastasis from early gastric cancer: estimation with a large number of cases at two large centers. Gastric Cancer. 2000;3:219–25. 6. Hohenberger P, Gretschel S. Gastric cancer. Lancet. 2003; 362: 305–15. 7. Ichikura T, Chochi K, Sugasawa H et al. Individualized surgery for early gastric cancer guided by sentinel node biopsy. Surgery. 2006; 139: 501–07. 8. Tsujimoto H, Sugasawa H, Ono S et al. Has the accuracy of preoperative diagnosis improved in cases of early-stage gastric cancer? World J Surg. 2010; 34: 1840–46. 9. Wong IY, Bhatia SN, Toner M. Nanotechnology: emerging tools for biology and medicine. Genes Dev. 2013; 27: 2397–08. 10. Lukyanov AN, Hartner WC, Torchilin VP. Increased accumulation of PEG-PE micelles in the area of experimental myocardial infarction in rabbits. J Control Release. 2004; 94: 187–93. 11. Torchilin VP. Micellar nanocarriers: pharmaceutical perspectives. Pharm Res. 2007; 24: 1–16. 12. Bechet D, Couleaud P, Frochot C et al. Nanoparticles as vehicles for delivery of photodynamic therapy agents. Trends Biotechnol. 2008; 26: 612–21. 13. Ryu JH, Koo H, Sun IC et al. Tumor-targeting multi-functional nanoparticles for theragnosis: new paradigm for cancer therapy. Adv Drug Deliv Rev. 2012; 64: 1447–58.
14. Makino A, Yamahara R, Ozeki E, Kimura S. Preparation of novel polymer assemblies, ‘‘lactosome’’, composed of poly(L-lactic acid) and poly(sarcosine). Chem Lett. 2007; 36: 1220–21. 15. Tsujimoto H, Morimoto Y, Takahata R et al. Photodynamic therapy using nanoparticle loaded with indocyanine green for experimental peritoneal dissemination of gastric cancer. Cancer Sci. 2014; 105: 1626–30. 16. Skrivanova K, Skorpikova J, Svihalek J et al. Photochemical properties of a potential photosensitiser indocyanine green in vitro. J Photochem Photobiol B 2006; 85: 150–54. 17. Hayashi K, Jiang P, Yamauchi K et al. Real-time imaging of tumor-cell shedding and trafficking in lymphatic channels. Cancer Res. 2007; 67: 8223–28. 18. Vooijs M, Jonkers J, Lyons S, Berns A. Noninvasive imaging of spontaneous retinoblastoma pathway-dependent tumors in mice. Cancer Res. 2002; 62: 1862–67. 19. Nakajima T. Gastric cancer treatment guidelines in Japan. Gastric Cancer. 2002; 5: 1–5. 20. Maruyama K, Gunven P, Okabayashi K et al. Lymph node metastases of gastric cancer. General pattern in 1931 patients. Ann Surg. 1989; 210: 596–02. 21. Kitagawa Y, Takeuchi H, Takagi Y et al. Sentinel node mapping for gastric cancer: a prospective multicenter trial in Japan. J Clin Oncol. 2013; 31: 3704–10. 22. Ichikura T, Sugasawa H, Sakamoto N et al. Limited gastrectomy with dissection of sentinel node stations for early gastric cancer with negative sentinel node biopsy. Ann Surg. 2009; 249: 942–47. 23. Granville DJ, McManus BM, Hunt DW. Photodynamic therapy: shedding light on the biochemical pathways regulating porphyrinmediated cell death. Histol Histopathol. 2001; 16: 309–17. 24. Zhao B, Yin JJ, Bilski PJ et al. Enhanced photodynamic efficacy towards melanoma cells by encapsulation of Pc4 in silica nanoparticles. Toxicol Appl Pharmacol. 2009; 241: 163–72.
ORIGINAL ARTICLE – GASTROINTESTINAL ONCOLOGY
Theranostic Photosensitive Nanoparticles for Lymph Node Metastasis of Gastric Cancer Hironori Tsujimoto, MD, PhD1, Yuji Morimoto, MD, PhD2, Risa Takahata, MD, PhD1, Shinsuke Nomura, MD1, Kazumichi Yoshida, MD, PhD1, Shuichi Hiraki, MD, PhD1, Hiroyuki Horiguchi, MD1, Hiromi Miyazaki, PhD3, Satoshi Ono, MD, PhD3, Daizo Saito, MD, PhD3, Isao Hara, PhD4, Eiichi Ozeki, PhD4, Junji Yamamoto, MD, PhD1, and Kazuo Hase, MD, PhD1 Department of Surgery, National Defense Medical College, Tokorozawa, Saitama, Japan; 2Department of Integrative Physiology and Bio-Nano Medicine, National Defense Medical College, Tokorozawa, Saitama, Japan; 3Division of Traumatology, National Defense Medical College Research Institute, Tokorozawa, Saitama, Japan; 4Technology Research Laboratory, Shimadzu Corporation, Soraku-gun, Kyoto, Japan
1
ABSTRACT Background. Preoperative and intraoperative diagnoses of lymph node (LN) metastasis in patients with gastric cancer is essential to determine the extent of LN dissection in order to establish individualized treatment strategies. We investigated the theranostic value of a newly developed drug delivery system employing nanoparticles loaded with the indocyanine green (ICG) derivative ICG-loaded lactosome (ICGm) using a murine draining LN metastasis model of gastric cancer. Methods. In the experimental draining LN metastasis model of human gastric cancer, the right hind footpads of nude mice were injected with cancer cells. Three weeks later, either ICGm or ICG solution was injected through the tail vein. Forty-eight hours after the administration of a photosensitizer, in vivo and ex vivo imaging and photodynamic therapy (PDT) were performed, and size of the LNs was measured. Results. In vivo imaging revealed metastatic LNs in the ICGm-treated mice but not in the ICG-treated mice. PDT using ICGm induced apoptosis and significantly inhibited the growth of metastatic LNs.
Electronic supplementary material The online version of this article (doi:10.1245/s10434-015-4594-0) contains supplementary material, which is available to authorized users. Ó Society of Surgical Oncology 2015 First Received: 6 December 2014 H. Tsujimoto, MD, PhD e-mail: [email protected]
Conclusions. ICGm presents a novel theranostic nanodevice for LN metastasis of gastric cancer. Gastric cancer is the fourth most common malignancy and the second most frequent cause of cancer-related deaths, accounting for approximately 10 % of cancer-related deaths worldwide.1–3 Lymph node (LN) metastasis is an important prognostic factor for long-term survival.4 In patients with N0 gastric cancer, the 5-year survival rate is 89.0 %, whereas survival rates dramatically decrease to 58.3, 33.4, and 17.4 % for patients with N1, N2, and N3 staging, respectively.3 Patients with T1 tumors have a low risk of LN metastasis: 2.2 % in T1a (mucosal) and 17.9 % in T1b (submucosal) cancers.5 On the other hand, the risk of LN metastases increases to 44 and 64 % for patients with T2 and T3 tumors, respectively. Thus, the extent of optimal LN dissection remains debatable;6–8 therefore; it is necessary to establish individualized treatment strategies for surgery and lymphadenectomy on the basis of accurate preoperative and intraoperative staging.8 Nanotechnology has the potential to create intricate structures comparable in size to biomolecules with unprecedented chemical and physical functionality.9 In tumor tissues, submicron-sized defects exist on the vascular wall because of rapid angiogenesis, thereby enabling permeation of macromolecules. Furthermore, the lymph system around the tumor grows too slowly to remove foreign compounds from the tumor region, and nanocarriers in the range of 30–100 nm in size can passively accumulate in tumors, in a process known as enhanced permeability and retention (EPR).10,11 Hence nanoparticles, such as liposomes, polymeric micelles, dendrimers, superparamagnetic iron oxide
H. Tsujimoto et al.
crystals, and colloidal gold, have been employed in drug delivery systems (DDSs) for targeted cancer therapies.12 Recent advances in nano-DDS carriers have facilitated the integration of diagnostic and therapeutic functions of theranostic nanoparticles to enable simultaneous diagnosis, therapy, and monitoring of therapeutic responses.13 Recently, we established theranostic photosensitive nanoparticles composed of micelles assembled from poly (sarcosine)–poly (L-lactic acid) (PS–PLLA) block copolymers loaded with the indocyanine green (ICG) derivative ICG-loaded lactosome (ICGm).14,15 ICG is a well-established infrared fluorescence dye that also can be used as a photosensitizer, suggesting that the use of ICG-based nanoparticles is a promising theranostic system.16 Indeed, we previously elucidated the efficacy of ICGm for the diagnosis and treatment of experimental peritoneal dissemination of gastric cancer. In vivo imaging revealed that ICGm selectively accumulated in tumor tissues and photodynamic therapy (PDT) with ICGm reduced the total weight of the disseminated nodules and significantly improved weight loss and survival rate.15 We investigated the theranostic value of ICGm using a murine model of draining LN metastasis of gastric cancer. MATERIALS AND METHODS Cancer Cell Lines, Animals, and Ethics Statement Human gastric adenocarcinoma MKN45 cells were purchased from the Riken Bioresource Center (Cell Bank, Ibaraki, Japan) and then cultured in Roswell Park Memorial Institute 1640 medium (Sigma-Aldrich, St. Louis, MO) supplemented with 10 % heat-inactivated fetal bovine serum (Life Technologies, Carlsbad, CA), 100 U/mL of penicillin, 100 lg/mL of streptomycin, and 0.25 lg/mL of amphotericin B (Antibiotic-Antimycotic, Life Technologies) at 37 °C in 5 % CO2 with 95 % humidity. Male nude Balb/c nu/ nu mice at 8 weeks of age (CLEA Japan, Tokyo, Japan) were fed under specific pathogen-free conditions. All animal procedures followed the guidelines approved by the National Defense Medical College Animal Care and Use Committee. Experimental Draining LN Metastasis Model To develop the experimental draining LN metastasis model, mice were injected with 1.0 9 106 MKN45 cells suspended in 50 lL of phosphate-buffered saline into the right hind footpad under anesthesia as previously described.17 Three weeks after injection, in vivo and ex vivo images of the popliteal lymph nodes (PLNs) were obtained. For PDT, the PLNs of another group of mice were irradiated through the skin under anesthesia, and the size of the LNs was measured by ultrasonography.
Development of ICGm We successfully synthesized a ‘‘lactosome,’’ which is a micelle assembled from block copolymers, PS–PLLA, synthesized as previously reported.14,15 Because PS–PLLA is composed of a biodegradable polypeptide, its toxicity is negligible, thus suggesting that it is safe for human use. The ICGm used in this study contained 22 % of ICG– PLLA and 78 % of PS–PLLA. Regarding ICG–PLLA synthesis, the terminal end of PLLA was chemically modified using ICG as follows: 1.0 mg ICG–OSu was added to the dimethylformamide (DMF) solution of the free amino group bearing PLLA (2.46 mg), which contains an amino group designed as an indicator of sarcosine N-carboxyanhydride (NCA) polymerization during the synthesis of amphiphilic PS–PLLA-block copolymers. The reaction mixture was stirred at room temperature overnight under light-shielding conditions. The reaction mixture was purified by size-exclusion chromatography using a Sephadex LH–20 column (GE Healthcare Japan Corporation, Tokyo, Japan) with DMF as the eluent. Chloroform solutions of PS–PLLA and ICG–PLAA were mixed at a ratio of 0.78:0.22. The solvent was removed under reduced pressure and the thin film formed was dissolved in 10-mM Tris–HCl buffer (pH 7.4). The resulting aqueous solution was purified by Sephacryl S-100 size-exclusion chromatography by elution with 10-mM Tris–HCl buffer (pH 7.4) to obtain ICGm. Dynamic lightscattering analysis revealed that the hydrodynamic diameter of ICGm was 40–50 nm, and the zeta potential of ICGm was -0.51 mV. ICGm solutions (91 mg/mL H2O) were administered intravenously or intraperitoneally to 7-week-old BALB/c mice. Both intravenous and intraperitoneal administrations of ICGm to male and female mice (2000 mg/kg) caused no weight loss and the mice showed no obvious abnormal findings by macroscopic observation at autopsy (sFig. 1). In Vivo and Ex Vivo Imaging To visualize PLNs in mice, either 100 lL of ICGm, in which the ICG concentration was 281 lM, or 100 lL of ICG solution (281 lM) was injected through the tail vein. Then, macroscopic in vivo fluorescence images were obtained using an in vivo imaging system (IVIS; PerkinElmer, Inc., Waltham, MA) as described previously.18 For fluorescence imaging, the right hind foot and dissected PLNs of the mice were illuminated with an excitation light at the wavelength of 780 nm and the fluorescence was captured using an 845-nm filter. The exposure time was 1000 ms. A photographic image of the animal was taken in the chamber under dim illumination,
Nanoparticle for Lymph Node Metastasis
followed by acquisition and overlay of pseudocolor images representing the spatial distribution of radiant efficacy within the animal. In vivo and ex vivo imaging were performed 48 h after ICG or ICGm injection, because our preliminary data revealed that 48–72 h after the ICG injection offered the best contrast of tumor sites to normal tissue (sFig. 2). PDT Three weeks after the injection of the cancer cells, either 100 lL of ICGm or 100 lL of ICG solution (both containing 281 lM of ICG) was injected intravenously through the tail vein. Forty-eight hours after the administration of the photosensitizer, photoirradiation was performed using a fiber-coupled laser system with a laser diode at 808 nm (model FC-W-808, maximum output: 10 W; Changchun New Industries Optoelectronics Technology Co., Ltd., Jilin, China). The fiber probe was placed just above the knee so that the irradiated area on the PLN was 0.79 cm2 (diameter, 1.0 cm). The fluence rate and irradiation period were set to 500 mW/cm2 and 1000 s, respectively, corresponding to a fluence of 500 J/cm2 (sFig. 3).
followed by the application of antidigoxigenin–peroxidase solution. The color was developed with diaminobenzidine, after which the sections were lightly counterstained with hematoxylin. To confirm staining specificity of the TUNEL method, a positive control section was prepared. The substitution of equilibration buffer for TdT was used as a negative control. The apoptotic index was expressed as the ratio of positively stained cells to the total number of tumor cells for each case. To determine the apoptotic index, 10 representative areas comprising at least 1000 cells without inflammation or necrosis were counted for each sample using a light microscope (9400 magnification). Statistical Analysis Data are represented as the mean ± SD. Statistical analyses were performed using the Mann–Whitney U test or Chi square test with the Fisher’s exact test, where appropriate. Calculations were performed using StatView statistical software (version 5.0; SAS Institute, Inc., Cary, NC). A p value of \0.05 was considered statistically significant. RESULTS
Measurement of LN Size Development of Draining LN Metastasis of Human Gastric Cancer Cells
PLNs were measured using an ultrasound imaging system (Vevo 770; VisualSonics, Inc., Toronto, Canada) before and 7 days after PDT. A 40-MHz ultrasonic probe (RMV704, VisualSonics) with a focal depth of 6 mm and a spatial resolution of 50 lm was used (sFig. 4). The longitudinal and transverse dimensions of LN were measured, and LN volume was obtained by the longitudinal and transverse size of LN, i.e., volume = (longitudinal size) 9 (transverse size) 9 (transverse size) 9 1/2.
Three weeks after the injection of the cancer cells, an increase in the thickness of the right hind footpad was evident because of tumor growth. The ipsilateral (metastatic) PLNs were enlarged compared with the contralateral (nonmetastatic) PLNs (Fig. 1a). Microscopic examination of the ipsilateral PLNs confirmed metastatic adenocarcinoma (Fig. 1b).
Detection of Apoptotic Cells
Imaging of Metastatic LN
Apoptotic cells were detected by the terminal deoxynucleotidyl transferase (TdT)-mediated deoxyuridine triphosphate biotin nick-end labeling (TUNEL) method. The TACS2 TdT Blue Label apoptosis detection kit (Trevigen, Gaithersburg, MD) also was used in this study. Briefly, paraffin sections were deparaffinized in xylene and rehydrated in graded ethanol. After washing in Tris-buffered saline (TBS; pH 7.4), the sections were placed in 3% H2O2 for 10 min and then digested with proteinase K (20 mg/mL in TBS) at 37 °C for 10 min to enhance nuclear staining of the apoptotic cells. After digestion was stopped, the sections were washed in TBS and then treated with terminal transferase enzyme and digoxigenin-labeled nucleotides,
The right hind footpad tumor and PLNs of the ICGmtreated mice were detectable through the skin using the IVIS. After removing the skin from the hind leg, enlargement of the PLNs due to metastasis was confirmed by both macroscopic observation and using an IVIS (Fig. 2a, left lower panel). However, no fluorescence signal was observed in the ICG-treated mice (Fig. 2a, right lower panel). Bilateral PLNs were harvested and ex vivo images of the ICG- and ICGm-treated mice were obtained (Fig. 2b). Metastatic PLNs were clearly identified in the ICGmtreated mice but not the ICG-treated mice. Contralateral (nonmetastatic) LNs in both groups were undetectable by ex vivo imaging.
H. Tsujimoto et al.
FIG. 1 Development of a draining lymph node (LN) metastasis model using human gastric cancer cells. a Comparison of ipsilateral popliteal lymph nodes (PLNs) (left) and contralateral PLNs (right) 3 weeks after the injection of 1.0 9 106 MKN45 cells in the right hind footpad. Each experiment was independently performed four times with similar results. Representative data are depicted. b Microscopic view of the ipsilateral PLNs (left panel: 940, right panel: 9200 magnification). Each experiment was independently performed four times with similar results. Representative data are depicted
Effect of PDT on Metastatic LNs The size of the metastatic PLNs in the ICG-treated mice increased 7 days after PDT; however, in the ICGm-treated mice, no such enlargement of the ipsilateral PLNs was observed. There was a significant difference in the longitudinal and transverse sizes of PLN between the two groups 7 days after PDT (Fig. 3, p \ 0.05). Calculated PLN volumes after PDT in the ICGm-treated mice were significantly smaller than those in ICG-treated mice (3.1 ± 0.3 versus 6.8 ± 2.4 mm3, p \ 0.05). The apoptotic cells were remarkably visible in the metastatic LNs of the ICGm-treated mice (Fig. 4a). The apoptotic index was significantly higher in the ICGmtreated mice (11.0 ± 2.2 %) than in the ICG-treated mice (3.9 ± 3.4 %; p \ 0.05; Fig. 4b). DISCUSSION The results of this study demonstrated that in vivo and ex vivo imaging using ICGm clearly visualized metastatic PLNs. Moreover, PDT using ICGm induced apoptosis and significantly suppressed enlargement of the PLNs. The Japanese Gastric Cancer Association guidelines recommend partial or total gastrectomy with reduced lymphadenectomy, depending on the clinical and surgical
FIG. 2 In vivo and ex vivo imaging of the metastatic lymph nodes (LNs). a In vivo bright-field and fluorescent imaging of the right hind footpad tumor and the popliteal lymph nodes (PLNs) after removal of the skin (bottom panels). Enlargement of the PLNs due to metastasis was confirmed by both macroscopic observation and IVIS in the indocyanine green (ICG) derivative ICG-loaded lactosome (ICGm)treated mice (left panels). However, no fluorescence was observed in the ICG-treated mice (right panels). Arrowheads indicate popliteal lymph nodes. Each experiment was independently performed four times with similar results. Representative data are depicted. b Ex vivo fluorescent imaging of the bilateral PLNs in ICGm-treated mice (left panels) and ICG-treated mice (right panels). Ipsilateral (metastatic) PLNs of the ICGm-treated mice were detectable using the IVIS, but not the ICG-treated mice. Contralateral (nonmetastatic) LNs in both groups were undetectable by ex vivo imaging. Each experiment was independently performed four times with similar results. Representative data are depicted
findings, for the treatment of gastric cancer.19 However, because of the limitations in the sensitivity of preoperative diagnostic imaging methods to detect pathologic metastasis in regional LNs, systemic (D2) gastrectomy has become a standard procedure to achieve a curative effect, even for clinically node-negative patients.20 Thus, accurate preoperative and intraoperative diagnoses of LN metastasis helps to determine the LN site to be dissected. In this regard, we demonstrated that ICGm administered intravenously accumulates in metastatic LNs but not in nonmetastatic LNs, indicating the diagnostic value of ICGm.
Nanoparticle for Lymph Node Metastasis mm
Longitudinal dimension
mm
Transverse dimension
6.0 2.0
5.0
* *
4.0 3.0
1.5 1.0
2.0 0.5
1.0
0
0
Pre PDT
7 days after PDT
FIG. 3 Effect of photodynamic therapy (PDT) on metastatic lymph nodes (LNs) The longitudinal and transverse sizes of the metastatic popliteal lymph nodes (PLNs) of the indocyanine green (ICG)-treated mice (blue bars) and the ipsilateral PLNs of ICG derivative ICG-
A %
Apoptotic index
15.0
*
Pre PDT
7 days after PDT
loaded lactosome (ICGm)-treated mice (orange bars) pre-PDT and 7 days after PDT. *p \ 0.05. Each experiment was independently performed four times
vasculature, and sentinel node mapping for gastric cancer. 21,22 The increasing interest in ICG as a photosensitizer in PDT is because of its strong absorption band (between 700 and 800 nm), which allows deeper tissue penetration.16 In addition, ICGm used in this study was 40–50 nm in diameter and selectively accumulated in the tumor tissues through the EPR effect.10,11 Thus, ICGm may be a safe and ideal photosensitizer of PDT for the treatment of metastatic LNs. PDT elicits both necrosis and apoptosis depending on the light/drug dose, cell type, genetic and metabolic potential, nature of the photosensitizer used, and subcellular localization.23,24 We demonstrated that enhanced apoptosis in metastatic LNs in the ICGm-treated mice compared with that in the ICG-treated mice resulted in the growth suppression of the metastatic LNs.15
10.0
CONCLUSION 5.0
0 ICG
ICGm
B FIG. 4 Apoptosis in metastatic lymph nodes (LNs) after photodynamic therapy (PDT). a Apoptotic cells in the metastatic LNs of indocyanine green (ICG) derivative ICG-loaded lactosome (ICGm)treated mice were visualized (9 400 magnification). Each experiment was independently performed 4 times with similar results. Representative data are depicted. b Apoptotic index in the ICGm-treated mice (orange bar) and the ICG-treated mice (blue bar). *p \ 0.05
Here, as a photosensitizer, we employed ICG, which is a tricarbocyanine dye approved by the United States Food and Drug Administration for medical diagnostic studies and is widely used to evaluate cardiac output and liver function, visualization of the retinal and choroidal
ICGm selectively accumulated in metastatic LN because of the EPR effect, which enabled ICG fluorescence-based visualization of the metastatic LNs and PDT using ICGminduced apoptosis and significantly inhibited the growth of the metastatic LNs. These findings suggest that ICGm is a useful and novel theranostic tool for LN metastasis of gastric cancer. PDT is advantageous, because it can be used repeatedly without inducing resistance, unlike chemotherapy and radiotherapy. PDT using ICGm presents a promising theranostic approach for LN metastases in the clinical setting. ACKNOWLEDGMENTS This work was supported by JSPS KAKENHI (grant number 24591892). The authors thank Enago (www.enago.jp) for the English language review. DISCLOSURE All authors certify that they have no commercial associations that might pose a conflict of interest in connection with the submitted article.
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