Strategies for Curative Fluorescence-Guided Surgery of Cancer

By Robert Hoffman

Strategies for Curative Fluorescence-Guided Surgery of Cancer is the first book to discuss how fluorescence-guided surgery can be successfully used during surgeries with several tumor types. FGS is one of the most exciting emerging modalities of surgery, especially cancer surgery, as it potentially allows the surgeon to visualize the actual margin of the tumor, thus greatly increasing the possibility of curative resection. The book discusses the applicability of FGS for several types of cancer, such as pancreatic cancer, liver metastasis, soft-tissue sarcoma, glioma, melanoma, and breast and lung cancer.

This book is a valuable resource for cancer surgeons, cancer researchers and members of several other areas in the biomedical field who are interested in understanding this powerful technique.

• Presents an overview of fluorescence-guided surgery
• Explains general strategies for curative fluorescence-guided surgery and their applicability for each major tumor type
• Discusses the current and future achievements of FGS as a precise technique for cancer surgeries

• Language: English
• Publisher: Academic Press
• Release date: May 27, 2020
• ISBN: 9780128126776

Book preview

States

Preface

Robert M. Hoffman and Michael Bouvet

Using fluorescent agents to guide surgery goes back at least to the late 1940s. However, the agents used were fluorescent dyes that were not tumor-specific. In the late 1990s the laboratory at AntiCancer Inc discovered that cancer cells expressing green fluorescent protein (GFP) could be imaged in live animals and under certain conditions, imaged non-invasively [1]. This breakthrough led to the idea that it was possible to have cancer cells specifically labeled with a fluorescent agent in order to improve cancer surgery. In 2007 a young doctor from Japan, Hiroyuki Kishimoto came to work at AntiCancer, and he brought with him a GFP-containing adenovirus, the replication of which depended on the host cells having high expression of telomerase, which in an adult would only be cancer cells. Dr. Kishimoto was able to show that he could infect tumors in orthotopic mouse models of cancer selectively with his GFP-containing virus [2]. The virus brightly lit up primary and metastatic tumors which were then resected under fluorescence guidance. This was the beginning of tumor-selective fluorescence-guided surgery (FGS). In 2012 a young doctor training to be a surgeon at UCSD came to work in the AntiCancer Laboratory, Cristina Metildi, who found that in orthotopic mouse of cancer expressing GFP, fluorescence-guided surgery inhibited cancer recurrence, resulting in extended survival [3]. Around that same time as Dr. Kishimoto’s experiment, collaboration began with oncologist George Luiken who was developing cancer-specific antibodies conjugated with fluorophores. The fluorescent antibodies were very effective to label cancer cells in orthotopic mouse models and such labeling enabled (FGS) that was subsequently shown to result in complete resection and extension of survival of the animals [4]. Later, infrared dyes conjugated to tumor-specific antibodies were found to be very effective for imaging of deep-seated tumors which could then be successfully resected [5]. Another young doctor from Japan, Shuya Yano came to AntiCancer and showed that color-coding of cancer and stromal cells also resulted in improved tumor eradication by FGS [6]. Another young Japanese doctor, Yukihiko Hiroshima showed that the combination of FGS and adjuvant chemotherapy could extend the survival time of tumor-bearing mice [7]. Laboratories all over the world now use these technologies to develop cancer-specific labeling for fluorescence-guided surgery. Fluorescent tumor-specific antibodies are now starting to enter the clinic for fluorescence-guided surgery. The present volume reviews pre-clinical development of tumor-specific labeling for FGS and also reviews the current state-of-the-art of FGS in the clinic. We hope that you find this book inspiring and enjoyable.

References

1. Hoffman RM. The multiple uses of fluorescent proteins to visualize cancer in vivo. Nat Rev Cancer. 2005;10(5):796–806.

2. Kishimoto H, et al. In vivo internal tumor illumination by telomerase-dependent adenoviral GFP for precise surgical navigation. PNAS. 2009;106(34):14514–14517.

3. Metidi C, et al. Fluorescence-guided surgery allows for more complete resection of pancreatic cancer resulting in longer disease-free survival compared to standard surgery in orthotopic mouse models. J Am Coll Surg. 2012;215(1):126–135.

4. Hiroshima Y, et al. Successful fluorescence-guided surgery on human colon cancer patient-derived orthotopic xenograft mouse models using a fluorophore-conjugated anti-CEA antibody and a portable imaging system. J Laparoendosc Adv Surg Tech A. 2014;24(4):241–247.

5. Hiroshima Y, et al. Effective fluorescence-guided surgery of liver metastasis using a fluorescent anti-CEA antibody. J Sur Oncol. 2016;114(8):951–958.

6. Yano S, et al. Color-coding cancer and stromal cells with genetic reporters in a patient-derived orthotopic xenograft (PDOX) model of pancreatic cancer enhances fluorescence-guided surgery. Cancer Gene Ther. 2015;22(7):344–350.

7. Hiroshima Y, et al. Metastatic recurrence in a pancreatic cancer patient derived orthotopic xenograft (PDOX) nude mouse model is inhibited by neoadjuvant chemotherapy in combination with fluorescence-guided surgery with an anti-CA 19-9-conjugated fluorophore. PLoS One. 2014;9(12):e114310.

Part I

Preclinical Development of Fluorescence-Guided Surgery

Outline

Chapter 1 Fluorescence-guided surgery improved long-term survival in orthotopic nude mouse models of cancer

Chapter 2 Development of fluorescence-guided surgery for colorectal cancer in orthotopic mouse models using fluorescent tumor-specific antibodies to increase survival

Chapter 3 Comparison of fluorescence-labeling strategies of colon cancer for fluorescence-guided surgery of liver metastasis in orthotopic mouse models

Chapter 4 Efficacy of the combination of fluorescence-guided surgery and adjuvant therapy in orthotopic nude mouse models of cancer

Chapter 5 Fluorescence-guided surgery using patient-derived orthotopic xenograft models of cancer

Chapter 6 Current and new fluorescent probes for fluorescence-guided surgery

Chapter 7 Precise recurrence-free fluorescence-guided surgery with color-coded cancer and stromal cells in a patient-derived orthotopic xenograft model of pancreatic cancer

Chapter 8 Fluorescence-guided surgery for primary and metastatic bone tumors in orthotopic nude mouse models

Chapter 1

Fluorescence-guided surgery improved long-term survival in orthotopic nude mouse models of cancer

Robert M. Hoffman¹, ², Cristina A. Metildi¹, ², Takashi Murakami¹, ², Fuminari Uehara¹, ², Hiroto Nishino¹, ² and Michael Bouvet²,    ¹AntiCancer, Inc., San Diego, CA, United States,    ²Department of Surgery, University of California, San Diego, CA, United States

Abstract

Background: Negative surgical margins are vital to achieve cure and prolong survival in patients with cancer. We inquired if fluorescence-guided surgery (FGS) compared to bright-light surgery (BLS) could improve surgical outcomes and reduce recurrence rates in orthotopic mouse models of human cancer.

Methods: A randomized active-control pre-clinical trial comparing BLS to FGS was used. Orthotopic mouse models of human pancreatic cancer, colon cancer, including liver metastasis, and fibrosarcoma were established. The models were established with human cancer cell lines expressing fluorescent proteins. Pre- and postoperative images were obtained with the OV-100 Small Animal Imaging System to assess completeness of surgical resection in real time. Post-operatively, noninvasive whole-body imaging was done to assess recurrence and follow tumor progression.

Results: With the pancreatic cancer model, FGS resulted in significantly longer disease-free survival (DFS) than BLS (P=.02, hazard ratio=0.39, 95% CI 0.17, 0.88).

With the colon cancer model, FGS lengthened disease-free median survival from 9 to >36 weeks. The median overall survival (OS) increased from 16 weeks in the BLS group to 31 weeks in the FGS group. FGS resulted in a cure in 67% of mice compared with only 37% of mice that underwent BLS (P=.049).

With the colon cancer liver-metastasis model, the FGS-treated mice had significantly prolonged disease-free survival (DFS) (P=.001) and OS (P=.027) compared to BLS-treated mice.

With the fibrosarcoma model the combination of BLS+FGS significantly decreased recurrence compared to BLS-only treated mice (P<.001). Mice treated with BLS+FGS had a significantly higher DFS rate than mice treated with BLS-only.

Conclusion: These results demonstrate the feasibility and efficacy of FGS of primary and metastatic cancer compared to BLS.

Keywords

BLS; bright-light surgery; FGS; fluorescence-guided surgery; colorectal cancer; CRC; disease-free survival; overall survival

1.1 Introduction

Currently, surgical resection of pancreatic cancer remains the only curative option for this disease [1]. Surgical resection has the greatest potential to offer prolonged disease-free survival (DFS) and thus an overall survival (OS) benefit for all stages [1–7]. Surgical margins appear to be a highly significant factor in predicting survival [7].

The single most important prognostic factor for OS in patients with colorectal cancer (CRC) is not only the microscopic status of the resected margin but also the complete resection of tumor at all sites (R0) [8,9]. Achieving an R0 resection can significantly improve 5-year survival rates of patients with CRC [10].

Liver metastases develop in approximately 60% of patients with CRC [11]. Liver resection of colon cancer metastasis, in combination with chemotherapy, improves survival which ranges from 27% to 45% for 5 years [12]. Current indications for resectability include the potential to obtain a complete resection with negative margins, the ability to functionally preserve two adjacent liver segments, and the ability to functionally preserve the liver remnant.

Most tumors in the retroperitoneum are malignant, and about one-third of these are soft tissue sarcomas [13,14]. Retroperitoneal tumors present several therapeutic challenges because of their relatively late presentation and anatomical location [15]. Complete tumor resection can potentially be a curative treatment modality for retroperitoneal soft tissue sarcoma patients [16], but local recurrence occurs in a large proportion of patients and is responsible for as many as 75% of sarcoma-related deaths [17].

Local recurrence often occurs following attempted curative resection of the primary tumor because all cancer cells are not removed by the surgeon due to the inability to see them.

1.2 Materials and methods

1.2.1 Animal care

Female athymic nu/nu nude mice were maintained in a barrier facility on high-efficiency particulate air-filtered racks. The animals were fed with autoclaved laboratory rodent diet (Teklad LM-485; Western Research Products, Orange, California). All surgical procedures were performed under anesthesia with an intramuscular injection of 100 μL of a mixture of 100 mg/kg ketamine and 10 mg/kg xylazine. For each procedure, 20 μL of 1 mg/kg buprenorphine was administered for pain control. Euthanasia was achieved by 100% carbon dioxide inhalation, followed by cervical dislocation. All animal studies were conducted in accordance with the principles and procedures outlined in the National Institutes of Health (NIH) Guide for the Care and Use of Animals under assurance number A3873-01 [18–21].

1.2.2 Noninvasive imaging of tumor recurrence and progression

To assess for recurrence and to follow tumor progression postoperatively, weekly whole-body imaging of the mice was obtained with the Olympus OV-100 Small Animal Imaging System (Olympus Corp, Tokyo, Japan), containing an MT-20 light source (Olympus Biosystems) and DP70 CCD camera (Olympus Corp.). Seven weeks after resection, the mice were sacrificed, and intravital images were taken to evaluate primary and metastatic tumor burden. All images were analyzed with Image J v1.440 (NIH) [18].

1.2.3 Tissue histology

For histologic examination of surgical margins, tumor samples were surgically removed en bloc with surrounding tissue at the time of resection in an additional mouse that was not included in the randomization of the 40 mice. OV-100 imaging of the surgical area was performed prior to resection, and the mouse was then returned to the surgeon. Following resection, both the surgical field and the resected specimens were again imaged on the OV-100 using bright field and fluorescence light settings. The mouse was then sacrificed, the surgical margins inked, and the tissues processed for permanent sectioning. Fresh tissue samples were fixed in Bouin’s solution and regions of interest embedded in paraffin prior to sectioning and staining with H&E for standard light microscopy. H&E-stained permanent sections were examined using an Olympus BX41 microscope equipped with a Micropublisher 3.3 RTV camera (QImaging, Surrey, BC, Canada). All images were acquired using QCapture software (QImaging) without postacquisition processing. The investigators who evaluated the histology and the images were blinded to treatment assignment [18].

1.2.4 Data processing and statistical analysis

PASW Statistics 18.0 (SPSS, Inc.) or R v. 2.11.0 was used for statistical analyses. Tumor burden is expressed as either median (range) or mean±SEM. A Welch’s t-test was used to compare continuous variables between two groups; ANOVA models were used to compare multiple groups. Comparisons between categorical variables were analyzed using Fisher’s exact test. Survival curves were constructed using the Kaplan–Meier method and survival outcomes were compared using log-rank tests with Brookmeyer and Crowley [22] A P-value of ≤.05 was considered statistically significant for all comparisons [18].

1.3 Pancreatic cancer model

1.3.1 Cell line and cell culture

The BxPC-3 pancreatic cancer cell line was obtained from the American Type Culture Collection (Manassas, Virginia). Cells were maintained in DMEM media supplemented with 10% heat-inactivated fetal bovine serum (FBS), and 1% penicillin and streptomycin (Life Technologies, Inc., Grand Island, New York). Cells were cultured at 37°C in a 5% CO2 incubator [18].

1.3.2 Orthotopic tumor implantation

Human BxPC-3-RFP (red fluorescent protein) cancer cells (AntiCancer, Inc., San Diego, CA) were harvested by trypsinization and washed twice with serum-free medium. Viability was verified to be greater than 95% using the Vi-Cell XR automated cell viability analyzer (Beckman Coulter, Brea, California). The cells were resuspended at 10⁶ cells per 10 μL of serum-free medium. Orthotopic human pancreatic cancer xenografts were established in nude mice by direct injection of fluorescent BxPC-3-RFP pancreatic cancer cells into the pancreas. A small 6- to 10-mm transverse incision was made on the left flank of the mouse through the skin and peritoneum. The tail of the pancreas was exposed through this incision, and 1×10⁶ cells, mixed 1:1 with matrigel (BD Biosciences, Bradford, Massachusetts) in a 10-μL final volume, were injected into the pancreatic tail using a Hamilton syringe (Hamilton Co, Reno, Nevada). Upon completion the pancreas was returned to the abdomen and the incision was closed in two layers using 6.0 Ethibond nonabsorbable sutures (Ethicon Inc., Somerville, New Jersey) [18].

1.3.3 Fluorescence-guided surgery of pancreatic cancer

A total of 41 mice were used in the experiments; 20 of them underwent fluorescence-guided surgery (FGS) and the other 20 mice underwent bright-light surgery (BLS). One mouse was sacrificed immediately after resection in order to use to assess surgical margins by histology. Two weeks following orthotopic implantation of human pancreatic cancer cells, mice bearing BxPC-3 tumors were randomly assigned to the BLS group or to the FGS group. Prior to resection of the pancreatic tumor, mice were anesthetized as described, and their abdomens were sterilized. The tail of the pancreas was delivered through a midline incision, and the exposed pancreatic tumor was imaged preoperatively with the OV-100 under both standard bright field and fluorescence illumination. The investigators who evaluated the histology and the images were blinded to treatment assignments. Resection of the primary pancreatic tumor was performed using the MVX-10 fluorescence-dissecting microscope (Olympus) under bright-light illumination for the BLS group and under fluorescence illumination through an RFP filter (excitation HQ 545/30×; emission 620/60 m) for the FGS group. Postoperatively, the surgical resection bed was imaged with the OV-100 Small Animal Imaging System under both standard bright field and fluorescence illumination to assess the completeness of surgical resection [18].

1.4 Colon cancer model

1.4.1 Cell culture

Human colon cancer cell line HT-29-DsRed, expressing RFP (AntiCancer, Inc., San Diego, CA), were maintained in DMEM (Gibco-BRL, Grand Island, New York) supplemented with 10% FBS (Hyclone, Logan, Utah). The cell culture medium was supplemented with penicillin or streptomycin (Gibco-BRL), sodium pyruvate (Gibco-BRL), sodium bicarbonate (Cell-gro, Manassas, Virginia), L-glutamine (Gibco-BRL), and minimal essential medium nonessential amino acids (Gibco-BRL). Cells were incubated at 37°C with 5% carbon dioxide [19].

1.4.2 Orthotopic tumor implantation

For cellular orthotopic implantation, HT-29-DsRed human colon cancer cells were harvested by trypsinization and washed twice with serum-free medium. Viability was verified to be >95% using the Vi-Cell XR automated cell viability analyzer (Beckman Coulter, Brea, California). The HT-29-DsRed cells were resuspended at concentrations of 2×10⁹ cells per 10 mL of serum-free medium. A midline abdominal incision was made, and the cecum was delivered through the incision. Orthotopic human colon cancer models were established in nude mice by direct injection of fluorescent HT-29-DsRed cancer cells into the wall of the cecum using a Hamilton syringe (Hamilton Co, Reno, Nevada) [19].

1.4.3 Fluorescence-guided surgery of colon cancer

A total of 51 mice were used in the experiments: 21 of them underwent FGS, and the other 24 mice underwent BLS. Once tumors reached 2–4 mm in diameter, tumor-bearing mice were randomly assigned to the BLS or FGS group. Before resection of the colon tumor, mice were anesthetized as described previously, and their abdomens were sterilized. The cecum was delivered through a midline incision, and the exposed colon tumor was imaged preoperatively with the OV-100 under both standard bright field and fluorescence illumination. Resection of the primary colon tumor was performed using the MVX-10 fluorescence-dissecting microscope under bright-light illumination for the BLS group and under fluorescence illumination through an RFP filter (excitation HQ 545/30× and emission 620/60 nm) or GFP filter (excitation HQ 470/10 nm and emission 525/50 nm) for the FGS group. For primary tumor resection the cecal stump was sutured closed in a running fashion with 8-0 nylon surgical sutures. In order to develop a carcinomatosis model a total of six mice harboring orthotopic human HCT-116-GFP colon cancer were allowed to progress to advanced metastatic disease. These mice were then randomly assigned to the FGS or BLS groups. Before surgical resection the mice were terminated, and a large midline incision was made to expose the entire abdomen, and the preoperative images were taken as described previously. Resection of primary and metastatic lesions was performed under bright-light illumination for the BLS group and under fluorescence illumination through a GFP filter for the FGS group. Post-operatively, the surgical resection bed in all mice was imaged with the OV-100 under both standard bright field and fluorescence illumination to assess the completeness of surgical resection [19].

1.5 Colon cancer liver metastasis model

1.5.1 Cell culture

The human colon cancer cell line HT-29, expressing GFP, was maintained in DMEM (Irvine Scientific, Irvine, California) supplemented with heat-inactivated 10% FBS (Gemini Biologic Products, Calabasas, California), 2 mM glutamine, 100 U/mL penicillin, 100 µg/mL streptomycin, and 0.25 µg/mL amphotericin B (Life Technologies, Inc., Grand Island, New York). The cells were incubated at 37°C in 5% CO2 [20].

1.5.2 Establishment of liver metastasis

HT-29 cancer cells expressing GFP were harvested by trypsinization and washed twice with serum-free medium. Cells (5×10⁵ in 50 µL serum-free medium with 50% Matrigel) were injected into the spleen in mice. Subsequent experimental liver metastases were resected and cut into fragments. In order to develop an orthotopic liver metastasis model a small 6–8 mm midline incision was made in other 14 nude mice to access the liver. The left lobe of the liver was exposed through this incision, and a single tumor fragment was orthotopically implanted to the left lobe of the liver. Three weeks after splenic injection of HT-29 cells expressing GFP, liver metastases were established. The liver metastases had strong GFP expression