Fluorescence-Guided Surgery and Fluorescence Laparoscopy for Gastrointestinal Cancers in Clinically-Relevant Mouse Models

There are many challenges that face surgeons when attempting curative resection for gastrointestinal cancers. The ability to properly delineate tumor margins for complete resection is of utmost importance in achieving cure and giving the patient the best chance of prolonged survival. Targeted tumor imaging techniques have gained significant interest in recent years to enable better identification of tumor lesions to improve diagnosis and treatment of cancer from preoperative staging modalities to optimizing the surgeon's ability to visualize tumor margins at the initial operation. Using unique characteristics of the tumor to fluorescently label the tissue can delineate tumor margins from normal surrounding tissue, allowing improved precision of surgical resection. In this paper, different methods of fluorescently labeling native tumor are discussed as well as the development of fluorescence laparoscopy and the potential role for fluorescence-guided surgery in the treatment of gastrointestinal cancers.


Introduction
The primary treatment modality for most patients with solid tumors is surgery. There are a multitude of factors that can significantly alter a patient's postoperative survival, such as tumor size, histological tumor grade, and vessel invasion [6,7]. However, it is lymph node status and a complete surgical resection (R0 resection) that provides the patient with the most valuable prognostic information with regard to postoperative survival [6,8].
Targeted tumor imaging techniques have gained significant interest in recent years to enable better identification of tumor lesions to improve diagnosis and treatment of cancer, from preoperative staging modalities [9][10][11][12] to optimizing the surgeon's ability to visualize tumor margins at the initial operation [1,2,[13][14][15][16][17][18][19]. Using unique characteristics of the tumor to fluorescently label the tissue can delineate a margin between tumor and adjacent tissue, allowing improved precision of surgical resection. One example is the use of activatable probes that rely on high tumor tissue enzymatic activity [17]. Other examples include using fluorophore-conjugated antibodies to unique surface markers expressed by individual tumor types [14,16], or the use of replication-competent viruses engineered to express the green fluorescent protein (GFP) in the presence of activated telomerase [10,15]. In this review, we will discuss the progression of fluorescence-guided surgery and laparoscopy as well as its future directions and its potential use in the clinical treatment of gastrointestinal cancers.

Development of Fluorescence Imaging Prototypes and Applications
Herpes simplex-1 virus, NV1066, a replication-competent virus was engineered to infect and lyse cancer cells selectively. In addition, the virus contained a transgene for green fluorescent protein (GFP) that would result in fluorescent cells upon infection. In vivo infection of NV1066 resulted in localized expression of GFP to the tumor, which could be visualized endoscopically with the use of a laparoscope and appropriate fluorescence filters. Furthermore, the NV1066 selectively infected and replicated within the esophageal cancer cells, killing the cells in vitro and in vivo [20]. NV1066 was used to infect mouse models with lymphatic metastasis of human mesothelioma cancer cells. NV1066 injected into primary tumors was able to locate and infect lymph node metastases, producing GFP-expressing metastases which were easily visualized under fluorescence imaging. The fluorescence thoracoscopy model used in this experiment [21] involved an excitation filter on the light source set at 470 ± 20 nm and an emission filter on a camera processor set at 510 nm.
Tumors were also selectively and accurately labeled with GFP using a telomerase-dependent adenovirus (OBP-401) containing the GFP gene [15,22,23] and subsequently resected under fluorescence guidance. Recurring cancer cells maintained GFP expression after fluorescence-guided surgery, enabling the detection of recurrence and future metastasis possible with OBP-401 GFP labeling [22]. Maintenance of label in recurrent tumors is not possible with nongenetic probes.

Development of Fluorescence Laparoscopy
With new techniques emerging to fluorescently label tumors, fluorescence laparoscopy is becoming an exciting field of investigation. An optimal fluorescence laparoscopy model should maximize the fluorescence signal of the tumor for easy and rapid imaging and also provide adequate background illumination to visualize surrounding tissues to allow for spatial orientation without compromising the tumor-tobackground contrast.
Our group developed a fluorescence laparoscopy model with the use of a Xenon light source that permitted facile, real-time imaging and localization of tumors labeled with fluorescent proteins within the abdomen of a mouse [18]. A standard laparoscopic system was easily modified by placing a 480 nm short-pass excitation filter between the light cable and the laparoscope. A 2 mm-thick emission filter was placed between the laparoscope and camera. The use of proper filters enabled simultaneous visualization of fluorescent tumor and non-fluorescent normal tissue and greatly enhanced the diagnostic capabilities of staging laparoscopy ( Figure 1) [1].
Fluorophore-conjugated antibodies directed at unique tumor antigens were also used to fluorescently label tumor [2]. Kaushal et al. [14,16] used antibodies directed against common tumor antigens to deliver fluorophores for enhanced detection of tumors during laparotomy in orthotopic mouse models of pancreatic and colon cancer. Fluorescence laparoscopy significantly enhanced the sensitivity and positive predictive value of diagnostic laparoscopy. Tumor detection was quicker and more accurate with very few false positives ( Figure 2).
However, due to the lack of intensity from the filtered Xenon light, adjustments to exposure time and gain were necessary. However, increasing the exposure time and gain resulted in significant dynamic delay that impaired surgical navigation. Replacing the Xenon light source with an LED   Figure 3: Stryker laparoscopic setup. A standard laparoscopic tower was slightly modified to achieve a fluorescence light mode that permitted detection of fluorescence signals while still allowing visualization of the background. The LED light source (Stryker L9000 LED lamp) was filtered through a glass emission filter (Schott GG495) that was placed between the laparoscope and the 1288 HD camera. With alterations to red, blue, and green components of the light source, we were able to visualize tumors of different fluorescent wavelengths. A Stryker X8000 Xenon light source was used for bright field laparoscopy [3].
lamp virtually eliminated the need for an excitation filter between the light cable and laparoscope ( Figure 3) [3]. With only the use of an 495 nm emission filter along with adjustments to the red, blue, and green components of the LED, no adjustments to exposure time or gain was necessary, and rapid detection of fluorescent tumor was greatly improved while also allowing visualization of surrounding tissue which can enable surgical navigation (Figure 4). This new model of fluorescence laparoscopy, with maximal blue light and adjustments to red and green light, produced a spectrum of light transmission that resulted in proper color balance and adequate background illumination. This enhanced the fluorescence signal-to-background ratio, enabling real-time simultaneous detection of tumors with different fluorescent colors ( Figure 5). The ability to visualize differently fluorescent tumors simultaneously resulted in the identification of an optimal fluorophore for fluorescence laparoscopy [3]. The combination of dually labeling nonfluorescent tumor with Alexa 488 and Alexa 555 greatly enhanced the fluorescence signal allowing for better detection of sub-millimeter deposits throughout the abdomen ( Figure 6). The combination of red and green fluorophores optimized the fluorescence signal of tumor allowing accurate distinction of tumor margins without compromising background illumination. This permitted laparoscopic resection of tumors in mouse models of pancreatic cancer. The improved visualization of surrounding structures for surgical navigation without compromising tumor detection further demonstrates the potential therapeutic uses of fluorescence laparoscopy.

Fluorescently Labeling Native Tumors
In addition to using fluorophore-conjugated antibodies and GFP-containing viruses, there have been a variety of methods described to fluorescently label native tumor.
Activatable cell penetrating peptides (ACPPs) have been used as targeting agents for cancer cells. Polycationic cell penetrating peptides (CPPs) are connected via a cleavable linker to a neutralizing polyanion whose adsorption and uptake into cells are inhibited until the linker is proteolyzed. With the upregulation of MMP-2 and MMP-9 in most solid tumors, exposure to these proteases results in cleavage and dissociation of the inhibitory peptide, allowing the CPP to bind to and enter cancer cells. Conjugating CPPs to a fluorophore then enables improved visualization of the tumor. Further conjugating dendrimers to ACPPs (ACPPDs) results in a higher absolute tumor fluorescence and tumor-tobackground fluorescence contrast than free ACPPs [17].

Future Directions of Fluorescence-Guided Surgery and Laparoscopy
Our recent work with fluorophore-conjugated antibodies (FCAs) directed against the tumor antigen CEA has shown to be a method of labeling, detecting and subsequently resecting tumor to improve surgical outcomes in mouse models of pancreatic and colon cancer [3,14]. The significant improvement in resection of primary tumor lesions achieved under fluorescence-guided surgery significantly reduces the postoperative tumor burden in mouse models of human cancer ( Figure 7). Furthermore, the greater incidence of achieving an R0 resection in these mouse models results in longer disease-free survival and overall survival. The goal is to improve methods of fluorescently labeling native tumor to permit better preoperative detection of metastatic tumor and to further enhance the surgeon's ability to delineate tumor margins and allow more objective means of identifying and resecting all tumor at the initial operation.  [4,5].