Development of novel tracers for sentinel node identification in cervical cancer

1 INTRODUCTION

Sentinel lymph nodes (SLNs) are the first lymph nodes to receive lymphatic inflow from a primary tumor, and sentinel lymph node biopsy (SLNB) is used to determine whether lymph nodes associated with solid tumors are metastatic. Since SLNs were first reported in relation to malignant melanoma,¹ they have been clinically applied in gynecology, general surgery, urology, and otolaryngology for monitoring of several types of solid tumors.^2-6

In cervical cancer, pelvic lymph node dissection (PLND) is an important procedure for determining the stage of progression, and the presence or absence of lymph node metastasis is an important indicator of the need for adjuvant therapy.^{7, 8} However, since the rate of lymph node metastasis in early-stage cervical cancer is approximately 10%–20%,^9-11 many patients may be receiving unnecessary PLND. In addition to increasing operative time and blood loss, PLND can also cause postoperative complications such as lymphedema and lymphangitis that may decrease quality of life.^{12, 13} Such complications can be avoided by omitting PLND for cases without lymph node metastasis that are diagnosed intraoperatively by SLNB.¹³

Dye, radioisotope (RI), and fluorescent tracers are currently used to monitor SLN.¹⁴ Technetium-99m (99mTc)-labeled phytic acid used with RI methods has a high molecular weight and is retained for long periods in the first node of lymphatic flow from a tumor, thus making this method suitable for SLNB. In fact, we have reported a high detection rate for SLN metastases in cervical cancer patients using the 99mTc-based RI method alone.^{13, 15, 16} However, RI methods require specialized equipment and expertise, which limits their availability. To avoid these restrictions, clinical application of a simpler fluorescent method involving indocyanine green (ICG) alone is increasing not only for cervical cancer,^{14, 17, 18} but also for breast cancer,¹⁹ malignant melanoma,²⁰ and vulvar cancer.²¹ However, ICG aggregates in solution and its fluorescence intensity diminishes rapidly if injection does not occur within a few hours of preparation.²² In addition, ICG is a small molecule, and thus is quickly extravasated through collecting lymphatics or taken up by venous capillaries at the injection site or high endothelial veins in the lymph nodes.²³

Liposomes (LPs) having a wide range of compositions and surface properties as well as good safety can be generated. They can also be miniaturized or diluted without loss of stability.²⁴ Based on these characteristics, we hypothesized that LPs could be promising agents for imaging the lymphatic system as part of cancer therapy. Here, we describe the development of a new tracer, which is a complex between phytate and LPs carrying the fluorescent probe ICG that forms via chelation mediated by Ca²⁺ in bodily fluids. We investigated the efficacy and safety of this tracer in mice and pigs. The new tracer is expected to improve the stability of ICG in biological fluids while increasing its retention time in SLN.

2 MATERIALS AND METHODS

3 RESULTS

3.1 Stability of ICG in different media types

To select a suitable medium for preparation of LPs, we first examined the stability of ICG alone stored long term (>3 weeks) in several different types of aqueous media (Figure 1A). ICG was not stable in saline and phosphate-buffered saline due to the high ionic strength of these solutions. ICG was also not stable in pure water. The highest stability was seen for 10 mM PB. Next, we determined the optimum concentration of ICG in 10 mM PB (Figure 1B). Fluorescence intensity increased linearly up to 10 μM before reaching saturation at 20 μM. Above 20 μM, the fluorescence was quenched due to aggregation or concentration-dependent quenching. ICG at 20 μM was used for the LP preparation.

3.2 Preparation of ICG-LP coated with Ca²⁺/phytate complex

ICG-LP was prepared using a standard hydration method with hydration medium containing 10 mM PB and 20 μM ICG. The LP composition included a small amount of phosphatidic acid (DMPA), which has a phosphate group that interacts with the Ca²⁺/phytate complex (Figure 2A). The prepared LP was diluted with PB containing a physiological concentration of Ca²⁺ (1 mM) and/or phytate. The coating of ICG-LP with the Ca²⁺/phytate complex was confirmed based on DLS measurement of the size of ICG-LP (Figure 2B,C; Table 1). In the presence of Ca²⁺, the size of LPs without DMPA (DMPA[−] LP) was essentially unaffected, but LPs containing DMPA (DMPA[+] LP) were enlarged, indicating chelating activity of Ca²⁺ toward the DMPA phosphate group. In contrast, the presence of phytate caused an increase in the LP size whether or not DMPA was present, which may indicate an electrostatic interaction between the phytate and cationic ammonium group of HSPC. The presence of both phytate and Ca²⁺ resulted in particular enlargement of DMPA-containing LP, indicating the significant contribution of DMPA to the Ca²⁺/phytate complex. Together, these results suggest that DMPA-containing LP and spontaneous coating with the Ca²⁺/phytate complex could enhance retention of the LPs in lymph nodes.

TABLE 1. Characteristics of LPs in the presence of phytate and/or Ca²⁺.

DMPA (%)	Phytate (0.88 mM)	Ca2+ (1 mM)	Size (nm)	PdI	Z-potential (mV)
0	−	−	89.7	0.13	−3.8
0	−	+	89.4	0.076	-
0	+	−	113	0.28	-
0	+	+	106	0.19	−6.7
5	−	−	98.5	0.036	−10
5	−	+	109	0.16	-
5	+	−	138	0.31	-
5	+	+	146	0.27	−13

• Note: Hyphen (-) means not measured.
• Abbreviations: DMPA, dimyristoyl-sn-glycero-3-phosphatidic acid; LP, liposome; PdI, polydispersity index.

3.3 Lymph node retention of tracers in mice

ICG, ICG-LP, and ICG-phytate-LP (20 μL of a 20 μM solution) were administered subcutaneously in the right inguinal region of mice. At the indicated time after administration, the right inguinal lymph node as a SLN and the right axillary lymph node as a secondary lymph node (SN) were resected to follow the migration of each tracer from SLN to SN (Figure S1). In the SLN of mice injected with ICG alone, the fluorescence of ICG reached maximum intensity 1 h after administration and thereafter rapidly disappeared. In the SN, ICG fluorescence intensity was observed beginning 1 h after administration and peaked between 3 and 6 h, before rapidly disappearing. These results showed the rapid migration of ICG from the injected region to the SLN and then to the SN. Meanwhile, in mice treated with ICG-LP or ICG-phytate-LP, the fluorescence intensity increased more slowly than that seen for ICG alone, and the fluorescence was retained for a longer period in the SLN. ICG-phytate-LP showed a clear peak 6 h after administration, whereas a constant weak fluorescence signal was seen for ICG-LP during the 24 h after administration. The ICG-phytate-LP group showed significantly enhanced fluorescence intensity relative to the ICG-LP group (Figure 3). Both tracers had a similar migration rate to the SN, and this rate was much slower than that for ICG alone. Based on the clear maximum in fluorescence intensity and long retention in the SLN, ICG-phytate-LP appears to be the most useful tracer among the three tested.

3.4 Imaging performance and safety of ICG tracers in pigs

We next confirmed the safety of ICG and ICG-phytate-LP in a larger animal model involving pigs. A total of three pigs were used for each tracer. Trocars were implanted under general anesthesia and the abdominal cavity was observed. The cysto-uterine fossa peritoneum was incised and expanded, and 2 mL of tracer (ICG concentration was set at 20 μM) was administered directly from the peritoneal cavity to the uterine cervix. After tracer administration, the retroperitoneal cavity was expanded to check lymphatic flow (Figure 4A). The uterine cervix and retroperitoneum with lymphatic flow were checked with a NIR laparoscopic system immediately after drug administration, and 1, 2, and 3 h later. In the region of the cervix, no differences were observed grossly between the fluorescence of ICG and that of ICG-phytate-LP (Figure 4B). In the lymphatic flow after retroperitoneal delivery to the left PLN region, fluorescence of ICG was observed immediately after administration and 1 h later. At 2 and 3 h, fluorescent intensity has disappeared in the proximal lymph node. On the other hand, in the group treated with ICG-phytate-LP, fluorescence intensity persisted even 2 and 3 h after administration in proximal lymph node. (Figure 4C; Video S1).

The blood test results and body weight on days 1, 8, 15, and 29 after surgery are shown in Figure 5. No weight loss or death was observed on the day after surgery, or 8, 15, and 29 days after surgery, for either tracer (Figure 5). Although results of blood tests did have some variation, differences between the ICG and ICG-phytate-LP groups were not significant and the overall safety was good.

4 DISCUSSION

Here, we designed a new tracer agent termed ICG-phytate-LP, and investigated its efficacy and safety relative to ICG. NIR fluorescence imaging measurements using wavelengths between 700 and 900 nm have been used to observe ICG, and NIR has also been used for optical imaging of the lymphatic system.²⁵ However, ICG is unstable in solution and can rapidly enter venous capillary lymph vessels after local injection, making use of ICG alone unsuitable for some applications like quantitative lymph node imaging.²⁴ Moreover, ICG aggregates in solution and rapidly loses fluorescence if not injected within a few hours of preparation.²² The low molecular weight of ICG allows it to quickly drain out of vessels through the collecting lymph vessels or to be taken up by venous capillaries at the injection site or high endothelial veins in the lymph nodes.²³ Therefore, soon after injection into the uterine cervix, ICG may spread via lymphatic flow to the secondary and third nodes too rapidly to permit confirmation of vascular and lymphatic routes of transport or identification of more than one node. The rapid transport of ICG may also allow inadequate time to perform appropriate SLNB.

For this reason, we sought to develop an ICG-containing material that had a larger size which was comparable to that of 99mTc used in the RI method. A larger probe with a longer retention time would allow broader application of SLNB even at facilities without RI capabilities. Here, we developed a tracer that combined ICG with LPs, which are expected to be retained in the SLN for long periods. LPs are colloidal particles that can interact with or incorporate ICG and improve its spectral properties. For example, large phospholipids in LPs can decrease self-quenching of dyes and slightly increase their quantum yield.^{24, 26} The safety of LPs as well as their relatively small size, their stability after dilution, and range of composition and surface properties suggest that they would be promising agents for use as novel tracers for lymphatic system imaging.

Phytate is known to form colloidal aggregates with Ca²⁺ in biofluids in vivo.²⁷ Coadministration of RIs such as Tc with phytate results in their incorporation into colloidal aggregates of Ca²⁺/phytate in situ via electrostatic interaction.²⁸ Such aggregates promote enhanced retention of these isotopes in SLN. Ca²⁺/phytate complexes have previously been successfully used to prolong retention of ICG in tissues.²⁹ Here, coadministration of ICG with phytate resulted in increased retention of ICG fluorescence in lymph nodes.

Nanoparticles containing PEGylated LPs have also been used to improve retention of ICG in lymph nodes.³⁰ ICG aggregates in physiological saline conditions due to its hydrophobicity, and this aggregation results in quenching of its fluorescence. However, incorporation of ICG in the inner phase of PEGylated LPs avoids such aggregation by protecting ICG from the saline.^{24, 31, 32} Recently, the Akita group reported that the negatively charged lipid bilayers of PEGylated LPs are associated with enhanced retention in SLN.^{33, 34}

Here we tested retention and safety of ICG-encapsulating LPs coated with a Ca²⁺/phytate complex, which is anchored to phosphatidic acid in the LP membrane. Thus, coadministration of these LPs with phytate will result in interactions with Ca²⁺ present in body fluids and spontaneous coating with Ca²⁺/phytate complexes. The negative charge of the complex may also promote its uptake by macrophages in the lymph nodes and in turn enhance retention in SLN. The animal studies also indicated that the tracer has good overall safety that may in part be due to its composition of only compounds that are approved for in vivo injection.

As mentioned above, ICG has been successfully used in gynecology for reduction surgery that involves SLN.^{14, 17, 18} Several reports described use of ICG in robot-assisted surgery not only for gynecological cancers but also for complete resection of deep endometriosis lesions while preserving pelvic nerves.³⁵ In gynecologic cancers SLNs are located deep in the retroperitoneum, and, as has also been reported for head and neck cancers,³⁶ can require identification of deep regional lymph nodes. The detection of SLNs can also be challenging due to their location in deep areas where tissue attenuation can limit signal transmission of fluorescent probes. Thus, ICG could flow to secondary or tertiary lymph nodes before ICG fluorescence is confirmed. In the present study, we showed that combining ICG and LPs with phytate resulted in a longer residence time in SLNs compared with ICG alone, and detectable fluorescence intensity was maintained for at least 6 h after administration. Taken together, this new tracer for SLNB for gynecological cancers, in contrast to those for breast cancer and malignant melanoma, which are close to the skin, could allow more time for identification of SLNs, particularly in the presence of retroperitoneal expansion.

This study does have several limitations. Injection of the tracer in mice and pigs could demonstrate its overall safety, but confirming the flow of ICG fluorescence and lymphatic flow over time in pigs was challenging. Moreover, at our facilities we do not have the capability to carry out RI studies in large animals. Thus, in a future study the performance of our new tracer should be compared with that of the conventional RI method.^{15, 16} In addition, further experiments involving more animals are needed to confirm the SLN model.

In conclusion, our results indicate that ICG-phytate-LP may be a useful tracer for gynecological cancers that require time for lymph node identification due to a retroperitoneal approach. We think that our novel tracer may contribute to the clinical application and dissemination of SLN in the future.

FUNDING INFORMATION

Our study was supported in part by a Grant-in-Aid for Scientific Research from Kyushu University School of Medicine Obstetrics and Gynecology Research Fund (grant number: FAKF401232) and the Japan Society for the Promotion of Science (number: JP19K09804).

CONFLICT OF INTEREST STATEMENT

The corresponding author (Kiyoko Kato) is an Editorial Board Member of Cancer Science. The corresponding author has received funding from Astra Zeneca K.K. and Fuji Pharma Co., Ltd. Other authors have no conflict of interest.

ETHICS STATEMENT

The study protocol was approved by the Institutional Review Board of Kyushu University.

Animal Studies: All procedures involving mice were conducted under guidelines approved by the Animal Care and Use Committee of Kyushu University (approval reference A19-265-0, A21-127-0), and all experiments involving pigs were approved by the Intervention Technical Center Animal Welfare Committee, Kobe Japan (IVT20-142, IVT-21-15, IVT21-144). The study was conducted according to the Animal Research Reporting In Vivo Experiments (ARRIVE) guidelines.

Informed Consent: N/A.

Registry and the Registration No. of the study/trial: N/A.