Innovative treatment for hepatocellular carcinoma (HCC)

Introduction

Indocyanine green (ICG) is not new in the field of liver surgery. Early studies performed in the 1980s and 1990s revealed the value of the ICG clearance test in predicting post-hepatectomy morbidity and mortality (1,2). ICG clearance and retention tests are crucial for determining precise liver function before liver surgery and offer several benefits for safe surgery (3). Recent progress in infrared light technology over the last decade has highlighted another feature of ICG. In this article, we review three recently described features of ICG that may contribute to the development of innovative treatments for hepatocellular carcinoma (HCC).

Fluorescence-guided surgery for HCC

Anatomical resection

Liver resection is the first-line curative treatment for HCC (4). Anatomical liver resection was first reported by Makuuchi et al. in 1985 (5), who injected indigo-carmine into a branch of the portal vein. Several studies since then have demonstrated a survival benefit of anatomical liver resection compared to non-anatomical liver resection (6,7). Accurate Anatomical resection requires identification of the liver segments before parenchymal transection (8). Beside injection of indigo-carmine, segment boundaries can be identified by isolating and clamping the corresponding Glissonian sheaths (9,10). These techniques, however, do not always provide clear identification of the segment boundaries, especially in patients with cirrhosis and/or undergoing repeated hepatectomy (11,12).

To overcome the limitations of these methods, ICG injection into the corresponding branch of the portal vein was proposed (8,13). As initially reported by Aoki et al. (13,14), portal vein branches feeding the HCC-bearing hepatic segment are punctured under intraoperative ultrasonography and fluorescence images of the liver surfaces are obtained using an infrared camera and amplifier (Figure 1). Aoki et al. showed that the ICG staining technique identified stained subsegments and segments in 90% (73/81) of patients. They also reported that the detection rate of the liver segment boundaries did not differ significantly between non-cirrhotic liver and cirrhotic liver (9). Miyata et al. reported that identifying hepatic segments using ICG fluorescence imaging enhances the accuracy of anatomical segmentectomy compared with the conventional technique using indigo-carmine (8). In their study, the ICG staining technique was effective in all 30 patients (100%), whereas the indigo-carmine technique was effective in only 17 patients (57%). The ICG staining technique was especially effective in cases that underwent repeated liver resection for HCC recurrence (8). In cases in which multiple branches of the portal vein feed the HCC-bearing hepatic segment, multiple staining and counterstaining using ICG can also be performed, similar to conventional indigo-carmine staining (11,15). Fusion ICG fluorescence imaging, which provides both fluorescence images and macroscopic views on a single screen, may be more effective for obtaining a clear demarcation line to enhance the accuracy of parenchymal dissection (16). The feasibility and clinical utility of projection mapping with ICG fluorescence to identify anatomical landmarks for parenchymal dissection were recently reported (17).

Figure 1 Identification of liver segment boundaries by injecting indocyanine green into the inferior branch of the portal vein for Couinaud’s segment IV. (A) Without fluorescence imaging; (B) under fluorescence imaging.

Laparoscopic liver resection for HCC has developed rapidly and is considered a standard treatment strategy, especially for minor liver resection (18). Furthermore, in specialized centers for laparoscopic liver surgery, laparoscopic anatomical liver resection is also performed (19). Ishizawa et al. initially reported application of the ICG staining technique for laparoscopic anatomical resection (20). Recently, Ueno et al. reported a combination of an interventional radiology technique with laparoscopic liver segmentation (21). A catheter is inserted from the femoral artery into the targeted arterial branch feeding the segment in a hybrid operating room. After confirming the perfusion area by arteriography, an embolic solution containing ICG is injected and the demarcation line identified by ICG fluorescence imaging. Because the conventional puncture technique may sometimes be difficult in a laparoscopic setting due to limited access, this new procedure may be a good alternative in laparoscopic surgery for identifying the demarcation lines of liver segments.

Inflow and outflow evaluation

Inflow and outflow evaluation of the liver is also important for performing safe liver surgery, especially in living-donor liver transplantation (22,23). After reconstruction of the hepatic vessels, ICG administration allows for clear visualization of the reconstructed hepatic artery and portal vein on the fluorescence images (24,25). Kawaguchi et al. reported that veno-occlusive regions and non-veno-occlusive regions can be visualized after intravenous injection of ICG, with ICG concentrations significantly lower in the veno-occlusive regions than in the non-veno-occlusive regions (26). These findings indicate that intraoperative ICG-fluorescence imaging enables real-time visualization of non-veno-occlusive, veno-occlusive, and ischemic regions, and evaluation of the extent of the functional outflow decrease.

Cholangiography

Cholangiography is performed for intraoperative visualization of the biliary tract to avoid injury of the bile duct (27). Routine cholangiography is recommended during cholecystectomy and is an essential procedure during donor hepatectomy to divide the bile duct at the appropriate level (28,29). Conventional radiographic cholangiography has several drawbacks, however, including exposure of the medical staff to radiation, and the need for a large C-arm fluoroscopic machine and trans-cystic tube for injecting the contrast medium. Ishizawa et al. reported intraoperative fluorescence cholangiography by intravenous injection of ICG (30). In their study, the common hepatic duct was identified in all 10 patients after intravenous ICG injection, similar to the results of intrabiliary injection of ICG in 13 patients. Schols et al. reported that ICG was visible in the bile duct within 20 minutes after injection and remained there for up to approximately 2 h in 30 patients undergoing laparoscopic cholecystectomy (31,32). The common bile duct was identified in 83% of the cases and the cystic duct was identified in 97% of the cases using ICG cholangiography. The usefulness of ICG cholangiography has also been reported in donor hepatectomy and laparoscopic hepatectomy (33,34).

In addition to visualizing the biliary tracts, ICG is useful for real-time detection of bile leaks during hepatectomy (35,36). Kaibori et al. reported a randomized controlled trial enrolling 102 patients who underwent hepatic resection without biliary reconstruction. The patients were randomly divided in two groups with or without ICG fluorescence cholangiography and sites of bile leakage were closed by suture or ligation. Five patients developed postoperative bile leakage in the control group compared with no bile leakage in the ICG cholangiography group (10% vs. 0%, P=0.019). Thus, ICG cholangiography may be useful for preventing bile leakage after hepatic resection.

Tumor visualization

Identification of small HCCs is important for curative resection and improvement of patient outcomes. In addition, clear delineation between tumor and normal tissue is important to obtain safe surgical margins. In 2009, real-time identification of HCC using ICG fluorescence imaging was reported (37,38). This technique is based on the ICG accumulation characteristics of HCC. Gotoh et al. reported the ICG fluorescence technique in 10 patients with solitary HCC who underwent hepatectomy (37). ICG was injected intravenously several days before surgery. All the HCCs were detected by ICG fluorescence and completely removed with negative margins using the fluorescence images as a guide. In four cases, new HCC nodules were detected by ICG fluorescence. Ishizawa et al. reported the results of 20 HCC patients who underwent ICG fluorescence imaging of the liver surfaces before hepatic resection (38). ICG fluorescence imaging before resection identified 21 of 41 HCCs (51%). Tumors that were not identified by intraoperative ICG fluorescence imaging were smaller than the identifiable tumors and the tumor location was deeper. None of the tumors located deeper than 8 mm from the liver surface were detected. In the surgical specimens, ICG fluorescence imaging identified all HCCs. Well-differentiated HCCs appeared as uniformly fluorescing lesions with a higher lesion-to-liver contrast than poorly differentiated HCCs (39). Fluorescence microscopy revealed an accumulation of ICG in the cytoplasm of HCC cells (38). This ICG fluorescence imaging system can also be applied for intraoperative detection of extrahepatic metastasis in HCC patients (40).

The accuracy of intraoperative diagnosis in laparoscopic liver resection might be inferior to that in open surgery because the ability to visualize and palpate the liver surface during laparoscopy is relatively limited. Kudo et al. reported the results of ICG-fluorescence imaging in 10 HCC patients who underwent laparoscopic hepatectomy (41). Similar to the open technique, ICG was administered intravenously within the 2 weeks prior to surgery (38). ICG fluorescence images of the liver surfaces were obtained intraoperatively during mobilization of the liver using a laparoscopic fluorescence imaging system. In 16 resected HCCs, laparoscopic ICG fluorescence imaging identified 12 HCCs (75%) on the liver surfaces distributed over all Couinaud’s segments, including tumors that had not been identified in normal color images. Similar to the results of open ICG fluorescence imaging, tumors that were identified by fluorescence imaging were located closer to the liver surface than tumors that were not identified by fluorescence imaging (38,41). Like palpation during open hepatectomy, laparoscopic ICG fluorescence imaging enables real-time identification of subcapsular liver cancers (Figure 2). Terasawa et al. reported fusion-fluorescence imaging using ICG in laparoscopic hepatectomy (42). This technique provides real-time identification of the tumor and may facilitate estimation of the required extent of hepatic mobilization and determination of the appropriate liver transection line.

Figure 2 Identification of hepatocellular carcinoma using indocyanine green fluorescence imaging (PINPOINT, Stryker Japan K.K., Tokyo, Japan).

New fluorescence agents for liver surgery

Since the approval of ICG by the US Food and Drug Administration, it has been widely used for liver function analysis in HCC patients with well-established safety, and fluorescence imaging techniques for HCC mainly use ICG (43). Several other agents have been proposed for fluorescence imaging in liver surgery, but their clinical use and evidence are limited. The porphyrin precursor 5-aminolevulinic acid, approved as a fluorescence imaging agent in the clinical setting, is useful for intraoperative detection of liver tumors and bile leakage (44,45). Kaibori et al. reported that fluorescence imaging using 5-aminolevulinic acid may provide better specificity for detecting surface-invisible malignant liver tumors than ICG fluorescence imaging alone (44). In a porcine model, Matsui et al. reported the usefulness of methylene blue for visualizing extrahepatic bile ducts during cholecystectomy (46). In a rat model, sodium fluorescein, which can be visualized without a specific fluorescence-detecting camera when exposed to blue light, is useful for visualizing hepatobiliary structures (47).

In addition to the already approved fluorescence agents, new agents have also been reported in a preclinical setting. Zeng et al. synthesized arginine-glycine-aspartic acid-conjugated highly loaded ICG mesoporous silica nanoparticles as a fluorescent probe and explored its application to intraoperative imaging of liver tumors (48). In a mouse model, the probe accurately delineated tumor margins and detected microtumors (<2 mm) that could not be detected by ICG fluorescence imaging. Andreou et al. synthesized silica-encapsulated surface-enhanced Raman scattering nanoparticles that act as a molecular imaging agent for liver tumors (49). This imaging system delineated tumors more accurately and was less susceptible to photobleaching than fluorescence imaging using ICG in a mouse model. Although the findings require clinical confirmation, these new materials could be promising fluorescence agents to improve the accuracy of fluorescence imaging in liver surgery.

Mechanism of ICG accumulation in HCC cells

Hepatocyte metabolism is complex. Various transporter proteins are involved in hepatic clearance of compounds. The accumulation of ICG, an anionic compound, is thought to be induced by altering those transporters as well as the morphologic changes to HCC tissues due to cancer progression. The biologic mechanism of uptake, intracellular transportation, and biliary excretion of ICG in hepatocytes are suggested as shown in Figure 3, but the exact pathway of hepatic metabolism of ICG remains to be elucidated.

Figure 3 The suggested mechanism of hepatic metabolism of ICG. Hepatic uptake of ICG is facilitated by OATP1B3 and NTCP. During the intracellular transportation of ICG, it binds to the GST isozyme, forming phospholipid vesicles and bile salt/lipid hybrid micelles. Biliary excretion is facilitated by MDR3 and MRP2. ICG, indocyanine green; NTCP, Na⁺-taurocholate co-transporting polypeptide; GST, glutathione S-transferase.

Hepatic uptake of anionic compounds is accomplished by organic anion transporting polypeptides (OATP), organic anion transporters (OAT), and Na⁺-taurocholate co-transporting polypeptide (NTCP) (50-52). Biochemical studies revealed that ICG inhibits the activity of OATP1B1, OATP1B3, OATP2B1, and NTCP in a dose-dependent manner (53,54). Therefore, those transporter proteins are suggested to interact with ICG. Genetic analysis was performed to elucidate the transporters that take-up ICG into the hepatocytes. Cell biology studies using Chinese hamster ovary cells showed that ICG uptake is enhanced in OATP1B3-transfected cells and NTCP-transfected cells compared with wild-type cells (54). These findings suggested that OATP1B3 and NTCP mainly contribute to ICG uptake in hepatocytes. Subclinical studies also demonstrated a relationship between the expression of those transporters and ICG clearance. A case report of an HCC patient with an ICG excretory defect showed that OATP1B3 was downregulated in both the cancerous and non-cancerous tissues (55). In this case, however, the expression of other transporters, including NTCP, was not downregulated. Patients with Rotor syndrome presenting with conjugating hyperbilirubinemia are also negative for OATP1B3 expression (56). The cause of the OATP1B3 downregulation in these patients is suggested to be a homozygous insertion of long-interspersed element-1 in intron 5 of the Slco1b3 gene as well as a homozygous mutation of the Slco1b1 gene (56). As described above, OATP1B1 is not a main contributor to ICG uptake in hepatocytes. Therefore, genetic alteration of the Slco1b3 gene is suggested to induce the ICG excretory defect via OATP1B3 downregulation. Another genetic study using tissues of patients with liver diseases also revealed that all patients were positive for long-interspersed element-1 insertion in intron 5 of the Slco1B3 gene and negative for OATP1B3 protein expression (57). These results indicated that alterations of OATP1B3 expression significantly affect the hepatic clearance of ICG in patients with liver disease, suggesting that the accumulation of ICG in HCC tissues is induced by OATP1B3 overexpression. Gene set enrichment analysis revealed upregulation of Slco1B3 and NTCP in cancerous tissue compared with non-cancerous tissue in HCC patients with ICG accumulation (58). The protein expression of OATP1B3 and NTCP was higher in the HCC tissues of patients showing cancerous-type ICG fluorescence compared with those showing rim-type ICG fluorescence (58). On the basis of these biologic and subclinical studies, the accumulation of ICG in HCC tissues is suggested to be induced by enhanced ICG uptake via the overexpression of OATP1B3 and NTCP.

On the other hand, alterations of the ICG excretion system have also been examined. ICG is a hydrophobic organic anion excreted in bile (59), and it is associated with phospholipid vesicles and bile salt/lipid hybrid micelles (60). Microscopic analysis shows higher ICG fluorescence in the perinuclear granule/vesicles of wild-type Chinese hamster ovary cells (54). Series of transporters including the multidrug resistance (Mdr) genes and multidrug resistance-associated protein (Mrp) genes mediate biliary excretion of various substances. Huang et al. studied the role of Mdr2 in ICG excretion using their established Mdr2^-/- mice and clarified that the biliary excretion of ICG in Mdr2^-/- mice is significantly decreased by 90% compared with the wild-type (61). Immunoblot analysis of the expression of MDR3, the human homologous gene of Mdr2, in HCC tissues indicated that MDR3 expression is significantly higher in HCC tissues with ICG accumulation than in those without ICG accumulation (62). In immunohistochemical analysis, however, the expression of MDR3 was not significantly related with ICG accumulation in HCC tissues (62). According to the results of these studies, alterations in MDR3 expression do not always induce ICG accumulation. The gene set enrichment analysis performed by Ishizawa et al. also indicated MRP2 upregulation in cancerous tissue compared with non-cancerous tissue in HCC patients with ICG accumulation (58). MRP2 is a canalicular multispecific OAT and functions to excrete various substrates into the bile (63). Thus, MRP2 was considered to contribute to the bile excretion of ICG. In a study using Mrp2-deficient rats, however, bile excretion of ICG decreased only 25% to 75% in those rats compared with the wild-type, and the cause of this partial decrease was suggested to be alterations of intracellular transportation properties of ICG and not decreased transportation across the hepatocyte canalicular membrane (64-66). ICG is suggested to bind some glutathione S-transferase (GST) isozymes, which contributes to the intracellular transportation of organic anions (66). A biochemical study using mutant rats with hyperbilirubinemia indicated that the decreased binding of ICG to cytosolic proteins, e.g., GSTs, may impair the biliary excretion of ICG (66). The gene set enrichment analysis performed by Ishizawa et al., however, indicated the upregulation of GST theta-1 (GSTT1) in cancerous tissue compared with non-cancerous tissue in HCC patients with ICG accumulation (58). On the basis of this result, expression of the target cytosolic protein that interacts with ICG is not considered to decrease in HCC cells. Further studies are necessary to clarify the mechanism of intracellular transportation of ICG, which has an important relation to the biliary excretion of that compound. Although multiple pathways for the transport of organic anions across the canalicular membrane were clarified, the mechanism of biliary excretion of those anions with high hydrophobicity such as ICG remains controversial.

Photodynamic therapy (PDT) for HCC

PDT is a noninvasive treatment that combines a photosensitizer and an activating light source. Although it is effective for only a limited area, PDT is a well-established clinical treatment for various cancers developing in limited and superficial tissues, such as skin cancer (67), Barrett’s esophagus and/or esophageal cancer (68), bladder cancer (69), and early-stage lung cancer (70). There are, however, two important obstacles. One is that the photosensitizer has weak tumor selectivity, and the other is the shallow light penetration ability. Consequently, PDT is not optimal for treating solid tumors or cancers.

Although the prognosis of patients with HCC has remarkably improved over the last 50 years, new treatment methods using next generation medicine are eagerly anticipated. ICG fluorescence in HCC was discovered in 2009 (38), and researchers have fervently evaluated this phenomenon. Because ICG has high HCC specificity, it is a photosensitizer and absorbs near-infrared (NIR) light. Although these features lead to the hypothesis that PDT may become a reality for HCC, reports supporting this hypothesis are still scarce.

NIR light has some advantages. The penetration depth of NIR light ranges from 7 to 10 mm (71,72), up to several centimeters, which is deeper than any other light sauce. In a clinical setting, dermatologists use PDT in combination with ICG and NIR light to treat port-wine stains (73). There are no reports, however, of the use ICG and NIR light to treat cancer in a clinical setting.

A recent report suggests that the HuH-7 human HCC cell line preferentially takes up ICG as a new mouse experimental model (74). The authors report that PDT using ICG-NIR is effective for the human HCC cell line. They evaluated three groups: ICG administration only (ICG+NIR− group), ICG administration and NIR laser exposure (ICG+NIR+ group), and NIR laser exposure without ICG administration (ICG−NIR+ group). ICG (5 mg/kg Diagnogreen; Daiichi Sankyo Co. Ltd., Tokyo, Japan) was administered intravenously via a tail vein in mice with HuH-7 cell xenografts. The HuH-7 xenograft mice were irradiated with 823 nm NIR laser 24 h after ICG administration (prototype NIR Diode Laser System, Hamamatsu Photonics, Hamamatsu, Japan). The power density was 160 mW/cm² for 3 min. HuH-7 tumor size was measured every 3 days. Unlike in the two control groups [ICG alone (ICG+NIR−) and neither ICG nor NIR (ICG−NIR−)], the tumor volume of the ICG+NIR+ group did not increase in the first 3 days followed by ICG-NIR PDT. Mean tumor volume did not differ significantly between the ICG−NIR+ and ICG+NIR− groups (P=0.9), but was significantly different between the ICG+NIR+ group and both the ICG−NIR+ and ICG+NIR− groups (P<0.01, both).

In their reports, developing HuH-7 tumors were observed in the center of the abdomen of the xenograft mice with no fluorescence under the fluorescence imaging system in a darkroom (Figure 4A). Twenty-four hours after ICG administration, the HuH-7 xenograft tumor emitted strong fluorescence under the fluorescence imaging system (Figure 4B). Under fluorescence microscopy, normal tissue surrounding the HuH-7 tumors produced no fluorescence, and fluorescence was observed in the cytoplasm of HuH-7 cells, but not in the nucleus (Figure 5).

Figure 4 HuH-7 xenograft tumors were observed in the center of abdomen of the transplanted mice (A). Twenty-four hours after ICG administration, HuH-7 xenograft tumors in mice exhibited uniform fluorescence under the fluorescence imaging system (B). ICG, indocyanine green.

Figure 5 Under fluorescence microscopy, normal tissue surrounding HuH-7 xenograft tumors produced no fluorescence. All HuH-7 cells, however, were fluorescent. Fluorescence was observed in the cytoplasm of HuH-7 cells, but not in the nucleus.

In the same group, Shirata et al. focused on the mechanism of PDT and effectiveness of repeated laser irradiation (75). They found that the temperature of HuH-7 tumors increased to 48.5 °C in mice administered ICG (Figure 6) and 43.4 °C in mice without ICG. Terminal deoxynucleotidyl transferase dUTP nick end labeling analysis detected apoptosis in tumor tissues after ICG-NIR irradiation, while apoptosis was not detected in the ICG alone group. They thought that the photothermal effects contributed to the anti-tumor effect of the ICG-NIR model. Furthermore, reactive oxygen species (ROS) production was strongly detected in ICG-NIR irradiated cells compared to cells treated with ICG alone or NIR alone, or control cells in vitro. To analyze the production of ROS by ICG-NIR irradiation in vivo, they evaluated the expression of 8-hydroxy-2’-deoxyguanosine (8-OHdG) in the xenograft tumor. Positive 8-OHdG staining was detected in tumor sections after ICG-NIR irradiation, but not in tumor sections after ICG administration alone. ROS production of ICG-NIR was inhibited under the cooling condition in vitro. In their ICG-NIR model, the anti-tumor effect of ICG-NIR therapy against human HCC is due to apoptosis through photothermal effects and oxidative stress, and suppresses HCC cell growth by repeated NIR irradiation (Figure 7A,B).

Figure 6 Temperature calibration analysis (lower panel, center of crossshaped sign) using thermography during ICG-NIR PDT. The temperature was 41.1 °C within 60 s after starting ICG-NIR PDT. ICG, indocyanine green; NIR, near-infrared; PDT, photodynamic therapy.

Figure 7 Large HuH-7 xenograft tumor observed in the mouse abdomen (A, triangular arrow). Later, a thin and obscure tumor was observed in the abdomen of the transplanted mice after ICG-NIR PDT (B, triangular arrow). ICG, indocyanine green; NIR, near-infrared; PDT, photodynamic therapy.

In another recent experiment, Tsuda et al. suggested the therapeutic efficacy of ICG-loaded lactosomes for PDT for HCC in a similar mouse model (76). Lactosomes are a core-shell-type nanocarrier made of biocompatible and biodegradable material. The ICG-lactosomes accumulated in HuH-7 cell xenograft tumors and were fluorescent under NIR imaging 24 h after injection. During ICG-NIR PDT, the temperature in the xenograft tumor increased from 34.0 to 47.7 °C and from 34.5 to 51.7 °C within 0±200 s in the ICG and ICG-lactosome groups, respectively. Based on observations of tumor volume, PDT had antineoplastic effects on HuH-7 cells treated with ICG-lactosomes. Thus, several recent experiments using ICG-NIR PDT for human HCC cell line tumors in a mouse model portend a new innovative treatment for HCC, but further studies are needed to advance the clinical application.

Conclusions

In conclusion, ICG fluorescence-guided surgery may change the next generation of liver surgery. Several aspects of the mechanisms of ICG accumulation in HCC cells have been revealed by important basic research studies. In the near future, ICG-NIR PDT is expected to become a new treatment method for HCC. Although ICG is well-known and has long history in medicine, new imaging technologies and mechanistic findings keep ICG in the spotlight.

Acknowledgements

We thank Dr. Chikara Shirata and Sho Kiritani for important experiments to prove the mechanism of Indocyanine green with near-infrared light photodynamic therapy.

Funding: This work was supported by grants 17H04279 from the Ministry of Education, Culture, Sports, Science, and Technology of Japan.

Footnote

Conflicts of Interest: The authors have no conflicts of interest to declare.

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