Combined Application of Indocyanine Green (ICG) and Laser Lead to Targeted Tumor Cell Destruction

Abstract

Purpose:

Precise excision of neuroblastoma is challenging, especially when tumors adhere to vital structures. Indocyanine green (ICG), an FDA-approved dye with absorption peaking at 800 nanometers, can absorb the near IR laser energy and release heat in the dyed tissue. We hypothesize that by injecting ICG at tumor sites followed by precise laser application, tumor cell death can be selectively targeted.

Methods:

Orthotopic neuroblastoma tumors were created in the adrenal gland of immunocompromised mice. Tumor, liver, kidney, and muscle tissues were chosen for ICG injection. Intervention variables included presence of tumor capsule, continuous vs. pulsed laser treatment and total energy delivered. Control groups included laser or ICG only. Tissues were stained with hematoxylin/eosin.

Results:

Continuous wave laser generated excessive heat, causing damage in all tissues. When using pulsed laser treatment, liver, kidney, muscle and intact tumor tissues showed no cell death when treated with laser alone or laser plus ICG. Tumor tissue with the capsule removed, however, showed cell death on histology.

Conclusions:

Pulsed laser treatment combined with ICG causes targeted tumor cell death in neuroblastoma tumor without capsule. No cell death was observed when tumor capsule was present, when only laser was used, or when applied over non-tumor tissues.

1. INTRODUCTION

Neuroblastoma is one of the most common pediatric tumors, affecting one in every 7,000 live births, or nearly 700 children annually in the United States.[1] Neuroblastoma is most commonly encountered in the adrenal medulla and is highly aggressive. Despite intensive treatment, high risk neuroblastomas have recurrence rates greater than 50%.[2] Management for high-risk neuroblastoma include induction chemotherapy, surgical resection, radiation, and possibly myeloablative therapy or immunotherapy. Not all tumors respond to induction chemotherapy however, and locally advanced tumors can be technically difficult to resect because of adhesions and proximity to critical vascular structures.[3, 4]

Radical surgical resection and debulking has become a common practice in the treatment of solid tumors, and has been shown to improve survival, even when residual disease remains.[5] La Quaglia and colleagues have demonstrated a benefit in survival and local progression with gross total resection in high risk neuroblastoma.[6] In this case series of 141 patients, more than 70% had successful gross total resection, defined as removal of all visible and palpable tumor. The remaining 27% failed to achieve a complete resection, underscoring the difficulty involved in the resection of these aggressive tumors.

We have previously shown that there is suppression of tumor growth by local administration of controlled released chemotherapy in tumor beds after an incomplete resection of orthotopic neuroblastoma.[7] Irreversible electroporation (IRE) is another technique that can improve tumor growth suppression in difficult-to-resect tumors by applying electric energy to tumors and leading them to undergo apoptosis. IRE has been studied in ex vivo metastatic osteosarcoma, metastatic colorectal carcinoma, hepatocellular carcinoma, and pancreatic cancer.[8] This method, however, can cause destruction to healthy tissue adjacent to tumors, and may not be suitable for locally advanced tumors.

Indocyanine green (ICG) is a water-soluble compound that is widely and safely used in medical diagnostics for its well-established fluorescence properties.[9] It has been used in fluorescence-guided surgery to identify critical structures, including intra-abdominal tumors. The aim of this study is to explore the use of ICG combined with the application of a near IR laser for the treatment of residual tumor. We hypothesize that utilizing ICG injections in combination with precise laser application can selectively cause tumor cell death in an orthotopic neuroblastoma tumor.

2. METHODS

2.1. Cell culture.

Human neuroblastoma KELLY cells (Sigma-Aldrich, St. Louis, MO, USA) were maintained in RPMI-1640+HEPES (HyClone) as previously described.[7] Cells were supplemented with 5% FBS, 50mg ml⁻¹ gentamicin, 1% OPI Media Supplement (Sigma-Aldrich), and 1% a,l-glutamine solution (Gibco). All cells were maintained in a 5% CO₂ atmosphere at 37°C and EDTA-passaged at 80% confluence.

2.2. Animal procedures.

All animal work was performed at the University of Illinois at Chicago in accordance with recommendations for care and use of animals. Animal protocols were approved by the Institutional Animal Care and Use Committee. All procedures were performed on female NCr nude mice (Envigo, NJ, USA). Procedures and ultrasound were performed on mice anesthetized with isoflurane inhalation.

2.2.1. Mouse orthotopic neuroblastoma model.

As previously described, KELLY cells were injected into the left adrenal glands of immunocompromised, seven-week old female NCr nude mice (Envigo, NJ, USA) in order to create an orthotopic neuroblastoma tumor model.[7, 10] Briefly, mice were anesthetized with intraperitoneal injection of Ketamine 100 mg/kg and Xylazine 10 mg/kg and a transverse incision was made on the left flank. The adrenal gland was localized and 2μL of phosphate buffered saline (PBS) containing one million KELLY cells were injected into the adrenal gland. The fascia and skin were closed in separate layers. Tumor growth was followed post-injection with serial ultrasound until tumor size reached 1000mm³.

2.2.2. High frequency ultrasound.

A VisualSonics Vevo 2100 sonographic probe (Toronto, Ontario, Canada) was used to monitor and measure tumor growth as previously described.[7] Briefly, mice were anesthetized with inhaled isoflurane and placed in the prone position. The ultrasound probe was placed over the left flank to locate the left adrenal gland and the tumor. Serial cross-sectional images (0.076mm between images) were taken, and 3-D reconstruction software (Vevo Software v1.6.0, Toronto, Ontario, Canada) was used to calculate the tumor volume.

2.2.3. ICG injections and application of laser.

After tumor volume reached 1000mm³, mice were anesthetized with intraperitoneal injection of Ketamine 100 mg/kg and Xylazine 10 mg/kg and underwent laparotomy to expose the orthotopic neuroblastoma tumor, liver, and kidney; skin over the proximal leg was removed to expose the quadriceps (Figure 1). Exposed tumor and organs were treated at multiple locations with a pulse or continuous wave laser (805nm, Lingyun Photoelectronic Systems, Inc., Wuhan, Hubei, China), or laser application after an injection of 10μL of 0.857mg/mL ICG (Sigma-Aldrich, St. Louis, MO, USA). Laser exposure was limited to a 3mm diameter area for 20 – 120 seconds; total energy delivered per treatment area was recorded. Some of the tumors were treated with parts of their capsules intact and some parts had the capsules removed prior to treatment. Other organs were left with their capsule intact.

Figure 1.

Orthotopic neuroblastoma tumor seen (A) through a left flank incision and (B) at the time of laparotomy. Asterisk (*) indicates spleen just cephalad to tumor, which is being injected with ICG prior to laser treatment.

2.3. Morphologic and histologic examination.

Animals were euthanized immediately following treatment. Specimens from the treated tumor, liver, kidneys, and muscle were fixed in 10% buffered formalin, serially dehydrated and embedded in paraffin. Specimens were sectioned (5μm thickness) and affixed to glass slides for staining with hematoxylin and eosin (H/E).

3. RESULTS

3.1. Pulse vs continuous wave laser application.

Preliminary testing of the laser showed that a continuous wave laser application caused an excessive amount of heat and gross destruction of all tissues. Pulse laser application over a limited time was tested at various frequencies (10, 30, and 60Hz) and lengths of treatment (20 – 120 seconds); the energy was measured at 30 – 300 joules (J). The most effective laser treatment was found to be a 60Hz pulse laser applied for 45 – 60 seconds, delivering between 70 – 80 J to the tissue. These settings were used for all subsequent laser treatments.

3.2. Treatment with laser alone.

When applying the pulse laser (even up to 120 J) to the liver, kidney, or muscle, there was some evaporation of liquid from the tissues, but no evidence of tissue destruction on gross inspection (Figure 2) or histologic examination. Similarly, there was no gross destruction or cell death by histology when the laser was applied to the tumor with or without having the capsule surgically removed.

Figure 2.

Gross specimens after treatment: (A)neuroblastoma tumor treated with laser or (B) laser and ICG; (C) muscle treated with laser or (D) laser and ICG; (E) liver treated with laser or (F) laser and ICG; (G) kidney treated with laser or (H) laser and ICG.

3.3. Treatment with laser and ICG.

When tumor with intact capsule was treated with an injection of 10μL of ICG-saline solution followed by the application of a 60Hz pulse laser for 45 – 60 seconds, there was no evidence of gross tissue destruction or cell death on histology. After the tumor capsule had been surgically removed, the application of the laser following the injection of ICG caused tumor destruction and cell death (Figure 3G–J). Applying the same energy from the pulse laser after ICG injection did not cause any destruction or cell death to the treated muscle, kidney, or liver (Figure 3A–F). Histologic examination results are summarized in Table 1.

Figure 3.

Hematoxylin and eosin (H/E) stains of (A, B) liver, (C, D) kidney, (E, F) muscle, or (G-J) orthotopic neuroblastoma following treatment with laser or laser and ICG. Tumor treated with laser and ICG after capsule has been removed (J) shows cellular destruction.

Table 1.

Summary of tissue cell death by status of capsule and treatment group

Tissue	No. samples	Capsule	Laser alone	ICG + Laser
Tumor	13	Intact	−−−	−−−
	8	Removed	−−−	+++
Liver	5	Intact	−−−	−−−
Kidney	9	Intact	−−−	−−−
Muscle	2	NA	−−−	−−−

−−−, no histologic evidence of cell death

+++, cell death, tissue destruction by histology

4. DISCUSSION

We treated orthotopic neuroblastoma tumors in mice by injecting ICG solution and applying a pulse laser, which caused localized tissue destruction and cellular death. This effect was specific for a 60Hz pulse laser, wavelength of 805nm, with a total energy application of 70–80J. We observed an important differential response based on the presence or absence of a capsule and the tissue being treated. Cell death and tissue destruction was only observed in tumor samples that were treated with the combination of ICG and laser, and only after the tumor capsule had been removed.

Treatment options for solid tumors are being investigated on many fronts, so that interventions can take place at the time of diagnosis, during surgical resection with or without local drug delivery, and when metastatic disease is encountered.[5] Currently, surgical resection is of significant importance to the survival and local control in the treatment of advanced staged neuroblastoma.[6] The proximity to vital structures often limits the success of gross total resection in these patients; however, our method could offer a highly precise way to safely extend the resection. ICG infiltration combined with exact laser application could improve surgical resection in neuroblastoma without sacrificing healthy tissue or risking injury to adjacent structures.

Lasers of various intensities and heat dissipation profiles are currently used in numerous surgeries and endoscopic procedures. The Nd:YAG laser has been used in pediatrics for endoscopically controlling upper gastrointestinal bleeds and for tumor ablation.[11] ICG has been used as a nonradioactive tracer in medical diagnostics for more than 50 years. More recently it has been used in neurosurgery, breast cancer sentinel lymph navigation surgery, plastic surgery, cardiovascular surgery (SPY® System), and gastrointestinal surgery.[9] Given its binding affinity and biliary excretion pattern, ICG is often used to assess for adequate tissue perfusion or hepatobiliary anatomy. Fluorescence-guided surgery is an emerging technology that has led to the development of tumor-selective fluorescence labeling, which has improved tumor detection and surgical navigation.[12] This study is the first published data that combines ICG infiltration with the application of a laser for the treatment of a solid tumor, specifically neuroblastoma.

This novel method of injecting ICG and applying a pulse laser to cause tumor tissue destruction has numerous potential advantages for the treatment of advanced-staged neuroblastoma. The cell death appears to be selective for the tumor cells in our model, while sparing various non-tumor tissues. The focus of the laser combined with careful injections of ICG enable the operator to have excellent spatial precision, which could improve our ability to achieve negative margins. This method could potentially be used in other solid tumors, though further testing is needed.

There are limitations to this study, which was designed as a proof of concept model within an animal xenograft. While our observations support the idea that surgical margins can be safely extended by the application of our method, it is unknown what effect this would ultimately have on patient survival, local control, or disease progression. The long-term effects from the ICG and laser treatment on the tumor bed are also unknown, as the animals were sacrificed immediately following treatment. Future studies can examine the interplay between the ICG and tumor cells that cause them to be more susceptible to the energy from the laser.