Journal of Surgical Research 136, 288 –293 (2006) doi:10.1016/j.jss.2006.05.008
Establishment of a Syngeneic Model of Hepatic Colorectal Oligometastases James J. Mezhir, M.D.,* Kerrington D. Smith, M.D.,* Eric T. Kimchi, M.D.,* James O. Park, M.D.,* Carlos A. Lopez, M.D.,* Helena J. Mauceri, Ph.D.,† Micheal A. Beckett, M.S.,† Samual Hellman, M.D.,† Ralph R. Weichselbaum, M.D.,† and Mitchell C. Posner, M.D.*,1 *Department of Surgery and †Department of Radiation and Cellular Oncology, The University of Chicago, Chicago, Illinois Submitted for publication April 3, 2006
Background. Regional and systemic therapies aimed at improving the outcome for patients with colorectal hepatic metastases have met with modest yet tangible success. Currently, liver resection remains the only curative treatment, but only a minority of patients are candidates for surgery. Animal models are an ideal way to study new treatments for patients with metastatic colorectal cancer. We propose a syngeneic animal model of hepatic colorectal metastases that simulates oligometastases, which is a clinical state considered amenable to regional therapeutic strategies. Materials and methods. BDIX (BD-9) rats underwent intrasplenic injection of DHD/K12/TRb (Prob/K12) cells to create hepatic metastases via the portal system. After injection of 5 ⴛ 106 cells, rats underwent laparotomy to determine metastatic burden. Histological analysis confirmed the presence of metastases from resected tumors. Results. Fifty-three animals were prospectively treated and observed for the development of oligometastases defined as between 1 and 10 hepatic lesions. Thirty-six (68%) of the animals developed detectable metastases while 32 (60%) developed oligometastases (average ⴝ 4.40 ⴞ 2.67). Four animals had overwhelming metastatic liver and peritoneal disease. All animals underwent peritoneal examination and thoracotomy to ensure localized disease. Histological analysis of five hepatectomy specimens confirmed the presence of metastatic cancer. Animals with oligometastases were healthy as evidenced by normal feeding and grooming behavior. Conclusions. An animal model of oligometastatic colorectal cancer to the liver can reproducibly 1
To whom correspondence and reprint requests should be addressed at Section of General Surgery, The University of Chicago Hospitals, 5841 South Maryland Avenue, Chicago, IL 60637. E-mail:
[email protected].
0022-4804/06 $32.00 © 2006 Elsevier Inc. All rights reserved.
mimic the stage IV state in humans conducive to regional therapy and can be used reliably to test novel treatments and mechanisms of metastatic colorectal cancer. © 2006 Elsevier Inc. All rights reserved. Key Words: oligometastases; metastasis; animal model; syngeneic model; hepatic metastases; colorectal cancer; colorectal hepatic metastases; BDIX rats; DHD/K12/TRb (PROb) cells. INTRODUCTION
Approximately 70% of the patients diagnosed with colorectal cancer present either at the time of initial diagnosis or some time thereafter with hepatic metastases. Resection has proven to be the most effective therapy to produce a long-term disease-free state in patients with limited, liver isolated disease [1– 6]. Despite its proven efficacy, the vast majority of patients are not amenable to resection because of either excessive tumor burden or inaccessible location. Other regional forms of therapy such as hepatic artery infusion chemotherapy and radiofrequency ablation have been used in patients with unresectable disease with variable success [3, 7–11]. Systemic therapy for colorectal hepatic metastasis has improved substantially over the past 5 years with enhanced disease free survival and prolonged time to progression demonstrated with combined regimens using oxaliplatin, irinotecan, and/or targeted agents such as bevacizumab or cetuximab [12, 13]. Another advantage of systemic therapy is potential down staging of patients with unresectable disease [14, 15]. In the setting of numerous, ill-located or large metastatic lesions, a response to combination chemotherapy may render patients resectable by downsizing tumors and reducing tumor burden [14, 16 –18].
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Oligometastases is a clinical state characterized by metastatic tumors limited in size, number, and location [19]. These patients reflect a clinical entity that exists within a spectrum of disease, which includes at one end those with overwhelming metastatic burden and at the other end, patients with disease limited in scope, usually to single organ site. The latter group is targeted with systemic and regional therapy that can potentially result in a prolonged, disease free state while the intent of treatment for the former group is palliative in nature. Novel treatments that are more effective are needed for patients with metastatic colorectal cancer and therefore pre-clinical models that simulate the clinical situation would prove invaluable to test these experimental therapies [20, 21]. Ideally, animal models should imitate the human condition being studied in terms of biological behavior and morphology [20]. Syngeneic animal models in particular can mimic the immune system response to disease, similar to that observed in human cancer [21]. Models to date typically have overwhelming liver metastases that preclude applying regional and systemic therapy with similar intent to that delivered to patients with limited, liver only disease [22, 23]. The objective of this study was to develop a reliable and feasible syngeneic model of hepatic colorectal cancer oligometastases in the rat. MATERIALS AND METHODS Laboratory Animals and Cell Culture Inbred male BDIX (BD-9) rats (Charles River Laboratories, Wilmington, MA), 8 weeks old and weighing 220 to 250 g, were held for 7 days, housed in a pathogen-free animal facility, and kept in accordance with University of Chicago Animal Care and Use Committee. DHD/K12/TRb (PROb/K12), a rat colon adenocarcinoma established in syngeneic BD-IX rats by 1,2-dimethylhydrazine induction (obtained from Francois Martin, University of Dijon, France) [24] were maintained in DMEM (Invitrogen Corporation, Carlsbad, CA) supplemented with FBS (10% vol/vol) (Intergen Corporation, Purchase, NY), penicillin (100 IU/mL), and streptomycin (100 g/mL) (Invitrogen) at 37°C with 7.5% CO 2. Before injection, cells were harvested using versene (0.02% EDTA in HBSS) and trypsin-EDTA (0.25% trypsin, 1 mM EDTA ● 4Na) (Invitrogen).
Preparation of Cells for Injection The optimal timing for obtaining cells that when introduced into the portal circulation yield hepatic metastases is to harvest cells from culture when they are subconfluent. Cells were maintained in 150-cm2 flasks and split 2 to 3 days before injection. Cells are trypsinized as described above and then transferred to warm PBS. Cells are centrifuged for 5 min at 1,000 rpm and resuspended again in warm PBS and a cell count is performed using a hemocytometer. Cells are resuspended in PBS at a concentration of 1 ⫻ 107 cells per milliliter for a 500-L injection of 5 ⫻ 106 cells.
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FIG. 1. Creation of hepatic oligometastases. (A) Through a left subcostal incision, the spleen is mobilized and the left gastric artery ligated. (B) Injection of 5 ⫻ 10 6 cells in 500 L of warm PBS are injected directly into the inferior pole of the spleen over 60 s. Tumor cells are carefully visualized during injection to ensure passage through the portal system. (C) The spleen is removed to prevent local tumor growth. (Color version of figure is available online.)
they remain in PBS before being injected. Animals were shaven and the skin was cleansed with 95% ethanol and draped with sterile towels. A 1 cm subcostal incision was made and the spleen was exposed (Fig. 1A). The spleen is placed onto the surface of the abdomen and draped in sterile gauze soaked in sterile PBS. The placement of the sterile gauze helps contain tumor spillage preventing wound and peritoneal contamination. The short gastric artery was isolated and ligated using 4-0 silk sutures. Using a 1-mL syringe and 28-gauge needle (Fisher Scientific, Chicago, IL) 500-L of admixture (5 ⫻ 106 total cells) was injected directly into the inferior pole of the spleen over a 60-s interval (Fig. 1B). During injection of cells, attention is on the splenic hilum to ensure visualization of tumor cells moving through the splenic arcades into the portal circulation, confirming that cells are targeting the liver and not the splenic parenchyma. The syringe and needle are left in place for at least 2 min while the splenic vessels are carefully ligated with 4-0 silk sutures. The spleen is then removed to prevent tumor growth within the splenic parenchyma (Fig. 1C). The wound was copiously irrigated with sterile PBS and then closed in two layers using 4-0 vicryl sutures.
Tumor Assessment Three to 8 weeks after inoculation with K12/PRob cells, animals were anesthetized, shaven, and prepped for surgery in the same fashion as described above. However, a transverse laparotomy incision was made to allow for exposure of abdominal contents to facilitate inspection for liver metastases.
Establishment of Hepatic Metastases
Histological Assessment of Tumor Tissue
Animals were anesthetized by intraperitoneal injection of 50 mg/kg of pentobarbital. Animals are inoculated with tumor in groups of four to keep cells fresh and to minimize the amount of time that
Tumor and hepatic tissues from five animals with hepatic metastases were fixed in 10% formalin immediately after removal. Then, 48 h later, the tissues were embedded in paraffin, sectioned, and
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stained with hematoxylin and eosin (H&E) for light-microscopic study. Cancer cells were identified based on their characteristic appearance and morphology.
RESULTS
Of the 53 rats treated with intrasplenic injection of Prob/K12 cells, 32 of 53 (60%) had between 1 and 10 metastatic lesions (mean ⫾ S.D. ⫽ 4.40 lesions ⫾ 2.67 lesions). The range of time delay between injection and exploration for visualization of the hepatic metastasis was 21 to 56 days (mean ⫾ S.D. ⫽ 31.2 days ⫾ 11.45 days. Seventeen (32%) of the animals developed no visible metastases. Four animals (8%) were explored and were found to have overwhelming peritoneal seeding and large, bulky liver disease too innumerable to count. Animals were observed before exploration and exhibited normal grooming and feeding behavior. All animals underwent laparotomy as well as thoracotomy to assure that there was no metastatic disease elsewhere. Animals were explored at different time points from the initial inoculation with cells. There was no correlation between the number and size of metastatic lesions and the amount of time to exploration. It appears that those animals who do develop metastases will begin to show growth of their lesions at about 3 weeks, and that the lesions will continue to grow slowly. Additionally, the distribution of the metastatic lesions revealed a random pattern, all of which is similar to that seen in human colorectal metastatic disease (Fig. 2A and 2B) [5]. To confirm the presence of cancer in the nodules counted, tumors were removed and fixation performed to be stained and examined by light microscopy (data not shown). DISCUSSION
Oligometastases has been described as a clinical state that exists between two extremes of cancer progression [19]. At one end of that spectrum is the patient with cancer limited to the primary site, while at the other end is a patient with an aggressive, unrelenting metastatic phenotype that is typically only amenable to palliative therapy. The oligometastatic state is defined as disseminated cancer that is limited in size, location, and number of metastatic nodules [19]. Patients with oligometastases have the potential to undergo hepatic resection for curative intent with five year survival upwards of 30% [5, 6]. In addition to surgery, patients with oligometastases have the potential to be treated with regional forms of therapy, such as radiofrequency ablation and ionizing radiation [1, 7, 10, 25]. One of the goals of treating patients who are deemed unresectable is to reduce tumor burden in order render them amenable to these local therapies [14]. A measurable response to these therapies is a key factor leading to resectability [17, 26].
Animal models play a critical role in the pre-clinical testing of novel therapies and approaches to the treatment of cancer [20, 21]. Translational research depends upon the utilization of models of disease that are compatible with the human pathology under investigation. Not only do laboratory animals serve as a testing ground for novel therapies, but also as a model for studying cancer development and progression [20]. Herein we describe a reproducible model of oligometastatic colon cancer. This model was developed primary by using a tumor cell injection that is far less in number compared with others used in the literature without direct orthotopic implantation [20, 27]. In our study, animals developed a mean number of four tumor nodules over a period of 3 to 8 weeks. Before the initiation of this study, several animals were injected with higher numbers of tumor cells (data not shown). Whenever greater than 5 ⫻ 106 tumor cells were used, animals invariably developed an uncountable number of nodules, peritoneal seeding, and ascites. These animals were visibly sicker than the animals with oligometastases. There were four animals in the study resembling this pattern, which is likely because of a technical error in cell count. Several animals in our study developed no metastatic lesions (32%). This number is too significant to attribute solely to an error in cell count; however, the percentage of animals that did not develop any detectable metastases decreased as our technique of cell preparation improved over time. The development of oligometastasis in our model raises several interesting questions. First, what is the threshold of tumor cells injected that result in oligometastases? We did not perform dose-response studies, so we are unable to answer this question definitively. However, one may speculate that there is a balance between the tumor cell burden and the host’s ability to fight seeding in the liver, possibly a function of immunological clearance (i.e., hepatic Kupfer cells) [28, 29]. Second, why do animals only develop metastases in the liver? All animals underwent thoracotomy when sacrificed and their lungs carefully inspected. Being that the lung is the most common site of extra-abdominal metastases [30, 31], it is surprising that this is not observed in this model. However, one explanation may be that the tumor cells were delivered by direct portal injection, and the first pass effect and removal of tumor cells from portal circulation occurred [32, 33]. This observation also indicates that no metastases are developing from the primary liver lesions, simulating the patient with isolated hepatic metastases and no disease in other distant sites. The utility of this model spans the spectrum as an investigative tool for the treatment and development of isolated colorectal hepatic metastases. It can be used to study new therapies or novel combinations of current approaches for the treatment of hepatic metastasis by
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FIG. 2. Hepatic oligometastases. Six weeks after intrasplenic injection of 5 ⫻ 10 6 Prob/K12 cells, exploratory laparotomy was performed to determine tumor burden. The presence of isolated metastases within the liver is noted. These animals exhibited normal grooming and eating behavior before laparotomy. (A) Several metastatic nodules are visualized across the liver surface. The liver was removed and tumor nodules were excised to verify the presence of cancer cells by histological examination. (B) A solitary metastatic hepatic nodule. (Color version of figure is available online.)
simulating human patients with hepatic metastases without disease involvement of other sites. This includes currently available, investigational treatments such as HAI chemotherapy, RFA, monoclonal antibodies, or novel combinations of these treatments with and
without resection [7, 10, 12]. Because of the animal’s relatively large size, they can be used to test different methods of agent delivery, such as portal vein and intraperitoneal injection. Experimental agents such as oncolytic viruses and transcriptional gene therapy tech-
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niques can be tested in this model to improve targeting strategies for isolated metastatic lesions [34, 35]. In addition to the study of novel therapeutic strategies, this is an invaluable model to study the biology and evolution of metastasis. For instance, stable transfection of the Prob/K12 cell line with fluorescent proteins would function as a non-invasive method for studying the development of oligometastasis in these animals [36, 37]. Animal models have been used in translational, preclinical research for metastatic colorectal cancer, and include xenograft models in immune-incompetent animals, spontaneous models, and chemically induced syngeneic models [20]. Our model represents a syngeneic representation of colorectal hepatic oligometastases using the Prob/K12 cells developed in BD-IX rats. Further study of hepatic metastases with the intent of improving curative therapy requires an appropriate lab model that corresponds with the behavior, morphology, and biology of a human cancer.
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ACKNOWLEDGMENTS The authors would like to thank Lydia Johns for medical illustration and Marija Pejovic for assistance with cell culture. Funding in part was provided by Varian, Incorporated.
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