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Chapter 8 / Tumor Site Implantation 8 151 Tumor Site Implantation and Animal Model Selection in Oncology Anibal A. Arjona, PhD, and Enrique Alvarez, DVM, MA CONTENTS INTRODUCTION ORTHOTOPIC MODELS BACKGROUND AND CURRENT CHALLENGES IMPLANT SITE SELECTION AND TUMOR MODEL KINETICS CONCLUSIONS REFERENCES SUMMARY The goal of this chapter is to present several lines of evidence as to the importance of tumor site selection in oncology drug development. Tumor–host interactions differ according to the anatomical location of the tumor and can alter the pharmacodynamic effects of a drug candidate. In some instances, failure of a promising new drug to exhibit efficacy is attributed to drug resistance when instead, the lack of efficacy is a consequence of poor model characterization and selection. Orthotopic models are now presenting us with more-complex models to evaluate the activity of novel drug candidates. We present examples that demonstrate how implant site influences tumor growth kinetics and behavior; as a consequence of these influences, our interpretation of result with early stage drug candidates must be carefully considered. In this chapter, we review a number of studies that support the notion that tumor implantation site represents a critical determinant for the successful and meaningful efficacy evaluation of chemotherapeutic agents. Key Words: Tumor site; subcutaneous implantation; intradermal tumors; angiogenesis; hypoxia. 1. INTRODUCTION The vast majority of in vivo drug development programs in oncology relies on transplantable tumor models. Recently, the broad screening of agents using syngeneic rodent tumors has been mostly replaced by the use of human tumor xenografts. Within the spectrum of models currently in use, orthotopic tumor models are adding to our underFrom: Cancer Drug Discovery and Development: Cancer Drug Resistance Edited by: B. Teicher © Humana Press Inc., Totowa, NJ 151 152 Arjona and Alvarez standing of tumor–host interactions and drug response. Although relatively specialized and costly, orthotopic tumor models represent an important tool in drug development. A number of issues particular to cancer drug development have the potential to create challenges to the successful testing of a drug candidate. Some of these early scientific decisions include the selection of tumor lines as well as validated in vivo models. The former defines drug activity in vivo. Tumor–host interactions differ according to the anatomical location of the tumor and can alter the pharmacodynamic effects of a drug candidate. In some instances, failure of a promising new drug to exhibit efficacy is attributed to drug resistance when instead, the lack of efficacy is a consequence of poor model characterization and selection. The application of novel technologies has led to the development and characterization of animal models to a fine resolution. This increases resolution can be used to guide the selection of tumor models that best reflect the drug target under evaluation. This particular concept applies well to the current development of targeted drug therapies in cancer. The goal of this chapter is to present several lines of evidence as to the importance of tumor site selection in oncology drug development. Moreover, it presents a brief discussion on the background and supporting evidence found in the field of orthotopic models, a tool increasingly used to characterize new targeted therapies in oncology. 2. ORTHOTOPIC MODELS BACKGROUND AND CURRENT CHALLENGES Currently, the cost of bringing drugs to market reaches in to the hundreds of millions of dollars (1,2). The primary goal of drug development programs is to advance compounds that have the greatest potential to ameliorate or to cure human disease. Therefore, there is a great need to establish screening programs that select efficacious from nonefficacious compounds early in the development process. To date, a number of chemotherapeutic agents shown to be highly effective in preclinical animal models either lack or display reduced efficacy in clinical trials. These results can be attributed to the inherent limitations of today’s pharmaceutical screening pathways (3,4). Tumor modeling has a long history in cancer research. It is a rapidly evolving field where many areas of research and efforts to “synthesize” the applicability of models continue. Killion and coworkers (5) outlined the characteristics of a successful preclinical animal tumor model. They stated that the model must reproduce the biology of human cancer; it should allow the study of relevant cellular and molecular events associated with growth and metastasis of tumors. Moreover, it must adequately reproduce the problems associated with a specific type and location of primary and metastatic cancer; it must also possess objective and quantitative end points of therapeutic responses. Finally, it must be reliable, reproducible, available, and affordable. A perceived limitation of current models of transplantable tumors is that they do not possess a high degree of predictive value in identifying clinically active compounds (6). One potential reason for this relative lack of predictive ability is the tendency to use concentrations of chemotherapeutic agents that represent maximum tolerated doses for mice rather than humans. As it has been suggested, the predictive value of some of the current preclinical tumor models increases and is reflective of the clinical response once the doses used are equivalent to the “clinically equivalent dose” (7). Another major limitation of current preclinical tumor models is that they often do not accurately replicate the stage of tumor development during which the chemotherapeutic Chapter 8 / Tumor Site Implantation 153 agent is administered in clinical situations. In humans, most administration of a chemotherapeutic agent occurs in situations where there is advanced, high-volume metastatic disease, whereas in mice a compound is generally given to animals exhibiting primary tumors with minimal metastatic disease. In cancer research today, the subcutaneous xenograft tumor models represent the workhorse for testing the efficacy of new chemotherapeutic agents. This animal model possesses a number of advantages, namely it is rapid and reproducible (when compared to other models), it requires relatively minimal labor, and it is relatively inexpensive. In addition, tumor kinetics can be easily quantified (tumor size), and it is relatively easy to alter schedule conditions relative to tumor burden. Although useful, this model also generates an abundance of false-positive responses to drugs, probably a reflection of the dosages (maximum tolerated dose) and schedules that may not reflect the conditions use in the clinic. Another major disadvantage of subcutaneous xenograft tumor models is the general lack of metastasis. Studies have shown that subcutaneous implantation of cultured tumor cells or tumor fragments rarely leads to metastatic disease, a response that is in contrast to the natural course of human neoplastic disease (8,9). Since Paget postulated the “seed and soil” hypothesis (10), cancer researchers have strived to generate animal models that resemble the course of human disease. Orthotopic implantation of tumors can generate tumors that growth and metastasize as their human counterparts. A response attributed to the effect that the environment exerts on the tumor cell’s ability to express a particular set of genes. Keyes and coworkers (11) showed that, depending on the site of implantation (subcutaneous vs intraperitoneal), tumors produced substantially higher levels of angiogenic cytokines (vascular endothelial growth factor [VEGF] and basic fibroblast growth factor [bFGF]) when implanted intraperitoneally than subcutaneously. In addition, numerous studies using orthotopic models show the site-specific dependence of therapy. For example, Onn and coworkers (12) reported significant differences in the response of various human lung cell lines to chemotherapeutic agents when implanted orthotopically vs subcutaneously. They showed that in lung cancer cell lines implanted subcutaneously, paclitaxel induced tumor regression, whereas only a limited therapeutic response to paclitaxel occurred in tumors implanted orthotopically in the lung. These differences are probably the result of tissue–tumor interactions inducing the expression of specific genes. Farre and coworkers (13) assessed the influence of implantation site (orthotopic vs subcutaneous) on cell cycle and apoptotic gene regulation. In addition, they compared the effect of implantation site on influencing the metastatic process by comparing the behavior of tumor aliquots of two human pancreatic xenografts (NP18 and NP-9) implanted orthotopically, at the site of metastasis (liver) or in a nonmetastatic site (subcutaneous). They observed that implantation site changes tumor growth by altering apoptotic or cell cycle regulation in a tumor-specific manner. Whereas the NP18 tumor exhibited changes in Bcl2-antagonist of cell death /Bcl-XL/caspase3 pathway, the NP9 tumor exhibited changes in proteins that regulate the cell cycle (extracellular signal-related kinase, proliferating cell nuclear antigen, and cyclin B1). Furthermore, the site of tumor implantation influenced the location of the resulting metastasis. These advantages of orthotopic-tumor xenograft models make them highly useful in preclinical development programs, as they are generally reflective of the clinical situations. Orthotopic implantation of cells or tumor fragments is effective in inducing primary tumor growth as well as metastasis. It has been suggested that surgical orthotopic implantation of tumor fragments may result in greater success rate regarding tumor take and metastasis than implantation of cell suspensions (14,15). 154 Arjona and Alvarez One of the major drawbacks for the large-scale use of orthotopic models in preclinical screening programs remains the high level of technical skill required for successful implantation (15). Another disadvantage of orthotopic-tumor xenograft models results from their ability to replicate the course of human disease, as this makes monitoring the kinetics of tumor growth and chemotherapeutic activity more complex. However, a number of groups continue to develop novel methodologies aimed at monitoring tumor kinetics in response to the chemotherapeutic agents’ action. Katz and coworkers (16) demonstrated the feasibility of using a red fluorescent protein orthotopic pancreatic cancer cell model for the preclinical evaluation of chemotherapeutics. These authors use the MIA-PaCa-2 human pancreatic cancer cell line transduced with red fluorescent protein and grown subcutaneously. Tissue fragments from the subcutaneous implants were then implanted into the pancreas of nude mice. The authors then compare the effects of gemcitabine (intraperitoneally) and irinotecan (intravenously) on tumor growth with that of control mice by imaging the tumors sequentially. In this tumor mouse model, control animals exhibited a mean survival time of 21 d, whereas gemcitabine- and irinotecantreated animals had mean survival times of 32.5 and 72 d, respectively. The authors concluded that this tumor is a highly metastatic model that reliably simulated the aggressive course of human pancreatic cancer. Another approach used to monitor tumor kinetics is to measure tumor-specific markers or to engineer tumors to secrete a number of cytokines, which are then use to assess tumor growth and drug efficacy. Pesce and coworkers (17) suggested lactic dehydrogenase (LDH) isoenzymes as a useful indicator for detecting the presence and assessing the growth of human tumors in athymic mice. Circulating LDH cleared rapidly following an intravenous or intraperitoneal administration, decreasing to about 10% of the initial value by 12 hr. Solid tumors of HEp-2, T24, and SW733 cells implanted subcutaneously continuously released amounts of LDH that correlated with tumor mass. More recently, Shih and coworkers (18) engineered tumors to express β-human chorionic gonadotropin hormone. Expression of this protein by the tumor and its secretion in the mouse urine served as a surrogate marker for tumor-growth kinetics and chemotherapeutic agent efficacy. Engineered cells were injected subcutaneously, intraperitoneally, intravenously and intrasplenic. β-Human chorionic gonadotropin levels were detected in the mice urine following 2, 1, 7 and 4 d after subcutaneous, intraperitoneal, intravenous, and intrasplenic injections, respectively. Furthermore, the levels continued to increase until the mice became moribund. Although useful in enabling researchers to monitor the progression and effect of chemotherapeutic agents, this tumor model does not provided the ability to assess the extent and location of tumor cells. 3. IMPLANT SITE SELECTION AND TUMOR MODEL KINETICS Since Paget’s postulated seed and soil hypothesis (10), a number of studies have shown that tumor kinetics and responses to therapeutic agents differ according to their site of implantation. Cancer researchers continue to streamline screening pathways and to develop preclinical animal models aim at enhancing the model’s ability to predict a compounds efficacy in the clinic. This volume clearly outlines our current knowledge and views regarding mechanisms of drug resistance. These mechanisms are various; however, it is possible to group them into several general categories such as pharmacodynamic, cellular, and molecular mechanisms. Whereas a tumor model might be intrinsically resistant—from a cellular or molecular perspective—to an experimental agent, the com- Chapter 8 / Tumor Site Implantation 155 bined effect of the tumor–host interaction and anatomical implant site also affect the overall drug response. Improperly conducted studies or studies performed using a poorly characterize model could lead to the conclusion that the experimental agent is less active than otherwise predicted. A difference in response can result from changes in the pharmacodynamic profile of the experimental agent as the result of the tumor–host response. For example, Teicher and coworkers (19) reported a pharmacokinetic alteration of alkylating agents by a tumor–host response. In this study, in vivo selective pressures placed on a tumor by in vivo exposure to alkylating agents, and development of drug resistance, directly affected the pharmacokinetic profile of alkylating agents. They showed that tumor resistance in vivo resulted from broad pharmacodynamic alterations. They hypothesized that resistance arose from cytokine release from the resistant tumor and a concurrent host response. It is doubtful that these pronounced pharmacokinetic changes could have been anticipated a priori. The pharmacokinetic alterations observed in this study raise questions whether other model factors in addition to direct cellular resistance in vivo can alter the response to a drug in more subtle ways, thus altering the overall profile of a drug. Before one considers how to select a particular tumor model and its implant site to develop our understanding on drug resistance, one must reflect on the model’s fundamental role in research. As outlined by Harrison (20), one finds a context with which to frame the use of the models in oncology research: “Models have provided a means to study not only the therapy of cancer but the biology of cancer as well.” According to Harrison, tumor models allow for the description of four fundamental research areas: discovery, biology, mechanism, and development. Briefly, discovery refers to general drug screening, biology to cellular characteristics, mechanism to pharmacodynamics and finally development to prediction of clinical drug activity. We can frame the consideration of tumor implantation site and drug resistance within these four areas and evaluate how tumor site selection can affect each particular area. Even though not a widely used in vivo model today, the VX2 rabbit carcinoma line represents an excellent example of how tumor implant site is an important factor to consider when studying drug effects. The VX2 rabbit model was initially described by Kidd and Rous in 1940 (21) and has been extensively used as a model of hypercalcemia of malignancy (22,23). A paraneoplastic syndrome associated with alterations of calcium homeostasis. Clinical management of hypercalcemia is an important consideration as it adversely affects clinical outcome in cancer patients. Hubbard and coworkers (24) described how the implantation site for the VX2 rabbit tumor directly affected the development of hypercalcemia in vivo. They evaluated endocrine changes and calcium levels associated with intramuscular vs intra-abdominal tumor implantation in rabbits. In this animal model, clinical hypercalcemia results from tumor implantation intramuscularly but not intra-abdominally. To characterize fully the model, the authors performed a direct comparison between animals implanted intra-abdominally and intramuscularly. The animal’s serum calcium levels measured to establish the presence and degree of hypercalcemia. In addition, serum levels of 15-keto-13, 14-dihydroprostaglandin E2 levels were determined using gas chromatography/mass spectrometry. The studies took place over a 5-wk period but did not directly measure tumor burden in the rabbits. The results showed that only animals with intramuscular tumor implants were significantly hypercalcemic when compared to animals with intra-abdominal tumor implants. Furthermore, calcium levels in naive animals did not differ significantly from intra-abdominally tumor implanted rabbits. Interestingly, plasma levels of 15-keto-13, 156 Arjona and Alvarez 14-dihydro-prostaglandin E2 were equivalent for both tumor-implanted groups (but 10to 20-fold higher than naive rabbits). The authors hypothesized that the observed hypercalcemia in rabbits resulted from the general metabolism of prostaglandin E2 in the lungs. They suggested that intramuscular tumor implantation could promote venous drainage directing the tumor outflow to the lungs, whereas venous drainage from the intra-abdominal tumor implantation could allow for the metabolism of effluent through the liver before reaching the lungs, thereby explaining the overall differences in calcium homeostasis in this model. Whereas the authors did not attempt to alter the levels of hypercalcemia in the intramuscular or the abdominally implanted tumors, one can assume that the mechanisms involved in the resulting hypercalcemia would be specific to the implant site, and that they can exert a significant effect on potential therapeutics modalities. Another example of the importance of tumor implantation site to the outcome of host response is the study of Malave and coworkers (25), who evaluated the Lewis lung carcinoma model. They assessed the immune response of B6 male mice to the tumor as a function of the tumors’ implantation site (the flank or the footpad [fp]). The authors stated: The incidence of 3LL carcinoma was lower in B6 mice inoculated with small number of tumor cells in the flank than in those receiving a similar number of tumor cells in the fp. Lung metastases appeared earlier, and the number of metastatic nodules was significantly higher in mice bearing tumors in the flank than those in the fp. These observations could be the result of tumor implant efficacy, as the histological properties of both sites are different. The authors also compared and evaluated the host lymphatic organ weight of both implant sites. Again the authors described their findings: . . . the flank 3LL carcinoma implant was followed by early and marked enlargement of the spleen, whereas the increase in spleen weight was delayed after fp 3LL implant. Thymus weight decreased gradually in either group, though thymus involution was faster in mice bearing flank tumors. The study does not contain a detailed explanation for the differences in tumor growth. These differences may result from immunogenicity, circulatory effects, and/or paracrine factor release. From a drug development standpoint, it is noteworthy how an a priori assumption regarding tumor implant site can adversely affect the outcome of a study. The VX2 rabbit and the Lewis lung carcinoma models lead us to conclude that a therapeutic agent applied to either model without a full understanding of the differential drug responses resulting from tumor site implantation can result in drawing erroneous conclusions regarding a novel agent’s efficacy on a tumor. It is clear from both examples that implant site influences tumor growth kinetics and behavior. These two studies represent broad scientific efforts to described tumor–host interactions. A specific example of how tumor implant site results in a differential response to antitumor interventions can found in the work of Hill and Denekamp (26). The authors used the sarcoma F syngeneic tumor of the CBA mouse to evaluate the response of the tumor to hyperthermia, misonidazole and radiation therapy when implanted at various anatomical areas (ventral wall of thorax, distal tail, dorsal foot, and intramuscularly). As part of the initial characterization effort, the investigators measured latent time, tumor-doubling time, and tumor temperatures of the tumor at all implant sites. The results of this study indicate that the tumor implant site alters tumor growth kinetics. For tumors Chapter 8 / Tumor Site Implantation 157 implanted in the chest, tail, foot and leg the doubling times were 1.2, 1.7, 1.6, and 0.6 d respectively. Basal tumor temperature was also influence by site of implantation. Again, for tumors implanted in the chest, tail, foot, and leg the recorded tumor temperatures were 34.9, 22.1, 27.5, and 35.8°C respectively. The authors noted that: Tumors on the tail were consistently different from all other sites; they appeared later, grew slower, had the poorest blood flow, the lowest natural temperature, low drug concentration, and highest thermal enhancement ratio. Some of these features, but not all, were shared by tumors on the foot, which was also thought to be a constricted site. The growth rate and normal tumor temperature of the foot and tail tumors were similar, but in most other respects, the foot tumors matched the chest and leg tumors more closely. These data serve as a warning that the choice of an implant site for experimental hyperthermia studies should not be made lightly. That choice will carry with it many changes in the biological characteristics of the tumor; these should be considered alongside the obviously greater ease of experimentation and the reduced risk of whole body warming if tumors in the extremities are used. This study also demonstrated that the degree blood perfusion to the tumor resulting from the selection of implant site alters the tumor’s temperature, and its response to radiation therapy with or without a radiosensitizer. Although these differences are not intuitively difficult to establish, the work of characterizing these differences is essential for the description of the model and its future application in the area of hyperthermia and radiation therapy research. As discussed earlier, the application of modern technologies to the overall characterization of established models has increased our understanding of the available animal models. Preclinical model selection and knowledge of its limitations are crucial determinants for the establishment of a successful drug development program. The recent work of Keyes and coworkers (27) best exemplifies model characterization. They monitor the angiogenic cytokine profile of several well-established cell lines grown subcutaneously in vivo using Luminex technology. This system enabled them to simultaneously quantified circulating levels of basic fibroblast growth factor, vascular endothelial growth factor and transforming growth factor beta in nude mice bearing several human tumors. This effort generated an “angiogenic agent in vivo profile” for said models. In addition, the authors attempted to evaluate the correlation between tumor volume and cytokine levels. Several of the tumors tested show a positive correlation between VEGF levels and tumor volume (e.g., Calu-6 NSCLC, SW2 SCLC, HCT116 colorectal carcinoma, Caki1 renal cell carcinoma, and HS746T gastric carcinoma). Interestingly, there was little evidence of VEGF production in animals bearing tumors <800 mm3. Additionally, the levels of tumor growth factor-β also correlated with tumor volume in animals bearing GC3 colorectal carcinoma, HS746T gastric carcinoma, and the MX-1 breast carcinoma line. Whereas the work helps define the in vivo cytokine profile for several cell lines in vivo, of importance is the understanding of the cytokine levels in relation to a potential antiangiogenic agent. A well-characterized model enables researchers to select objectively a model to suit the molecular pathway targeted by a drug candidate. Subcutaneously implanted Caki-1 renal cell carcinoma tumors can serve as an example of the complexity of model selection and its impact on establishing agent efficacy. Keyes and coworkers (25) showed that maximal VEGF plasma level for this tumor type reach approximately 200 pg/mL. This result presents us with some important questions regard- 158 Arjona and Alvarez ing the development of targeted therapies. For example, how would the knowledge of a circulating angiogenic factor profile in the model, guide the selection of a specific antiangiogenic factor inhibitor? Would it be more (or less) reasonable to test a specific VEGF neutralizing agent against a tumor model of high or low VEGF expression? How does the presence/absence of such an angiogenic profile influences one’s interpretation of the overall agent efficacy? A logical progression of the above referenced study was to evaluate the effect of implant site on the angiogenic cytokine profile of a number of tumors cell lines. Keyes and coworkers (28) measured VEGF, bFGF, and tumor necrosis factor-α circulating levels as well as tumor volumes of mice implanted with various human ovarian (A2780, OVCAR-3, and SKOV-3) or human pancreatic (BxPC-3, Panc-1, and AsPC3-3) carcinomas using Luminex technology. The tumors were implanted either subcutaneously or intraperitoneally. The data show that intraperitoneal implantation resulted in significantly elevated VEGF levels when compared to subcutaneously implanted tumors. For example, subcutaneously implanted A2780 and the SKOV-3 lines produced VEGF plasma levels of 350 pg/mL and 1500 pg/mL, respectively, whereas intraperitoneal implantation of these cell lines resulted in plasma levels of 1500 pg/mL and 3000 pg/mL, respectively. In contrast, one of the pancreatic tumor lines, BxPC-3, produced low levels of VEGF when implanted at either site. Another pancreatic tumor line, AsPC-1, responded similarly to the ovarian cell lines as subcutaneously implanted tumors produced plasma VEGF levels of 500 pg/mL, whereas intraperitoneal implantation resulted in a threefold increase in the overall VEGF levels. Of particular interest was the observation that when ascites production was evident, VEGF, and bFGF levels this fluid was manyfold higher than plasma levels. The researchers concluded that: Different sites of tumor implantation will result in differences in levels of angiogenic cytokines secreted into the plasma of tumor bearing animals. These findings may be valuable for determining the model of choice for the in vivo evaluation of antiangiogenic agents. 4. CONCLUSIONS One of the major goals of an animal model in all indications is that it should display a similar course and involvement as that seen in humans. This has been, in most cases, very difficult to achieve. Historically, gross pathology has solely described differences in the natural course of a tumor model. The elucidation of the various cellular and biochemical differences associated with the various sites of tumor implantation presents a more challenging yet attainable goal. In this chapter, we reviewed a number of studies that support the notion that tumor implantation site represents a critical determinant for the successful and meaningful efficacy evaluation of chemotherapeutic agents. Some key points include that it is of the utmost importance to recognize that the therapy of cancer is the therapy of metastatic disease and that it is essential to use or develop animal models to address specific questions with a clear understanding of an animal model’s limitation. As new technologies become available, rational tumor model selection should become the norm in drug developing/screening programs. Cancer researchers will continue to streamline screening pathways and to develop preclinical animal models capable of enhancing the model’s ability to predict a compounds efficacy in the clinic. Chapter 8 / Tumor Site Implantation 159 REFERENCES 1. Dimasi JA, Hansen RW, Grabowski HG. The price of innovation: new estimates of drug development costs. J Health Econ 2003; 835:1–35. 2. Rawlins, M D. Cutting the cost of drug development. Nat Rev Drug Disc 2004; 3:360–364. 3. Schuh JCL. Trials, tribulations, and trends in tumor modeling in mice. Toxicol Pathol 2004; 32:53–66. 4. Bibby MC. Orthotopic models of cancer for preclinical evaluation: advantages and disadvantages. Eur J Cancer 2004; 40:852–857. 5. Killion JJ, Radinsky R, Fiddler IJ. Orthotopic models are necessary to predict therapy of transplantable tumors in mice. Cancer Metastasis Rev 1999; 17:279–284. 6. Kelland LR. “Of mice and men”: values and liabilities of the athymic nude mouse model in anticancer drug development. Eur J Cancer 2004; 40:827–836. 7. Kerbel RS. What is the optimal rodent model for anti-tumor drug testing. Cancer Metastasis Rev 1999; 17:301–304. 8. Naito S, von Eschenbach AC, Fidler IJ. Different growth pattern and biologic behavior of human renal cell carcinoma implanted into different organs in nude mice. J Natl Cancer Inst 1987; 78:377–385. 9. Kozlowski JM, Fidler IJ, Campbell D, Xu ZL, Kaighn ME, Hart IR. Metastatic behavior of human tumor cell lines grown in the nude mouse. Cancer Res 1984; 44:3522–3529. 10. Paget S. The distribution of secondary growths in cancer of the breast. Lancet 1889; 1:571–573. 11. Keyes KA, Mann L, Teicher B, Alvarez E. Site-dependent angiogenic cytokine production in human tumor xenografts. Cytokine 2003; 21:98–104. 12. Onn A. Isobe T, Itasaka S, et al. Development of an orthotopic model to study the biology and therapy of primary human lung cancer in mice. Clin Cancer Res 2003; 9:5532–5539. 13. Farre L, Casanova I, Guerrero S, Trias M, Capella G, Mangues R. Heterotopic implantation alters the regulation of apoptosis and the cell cycle and generates a new metastatic site in a human pancreatic tumor xenograft model. FASEB J 2002; 16:975–982. 14. Fu X., Guadagni F, Hoffman RM. A metastatic nude-mouse model of human pancreatic cancer constructed orthotopically from histologically intact patient specimens. Proc Natl Acad Sci U S A 1992; 89:5645–5649. 15. Hoffman RM. Orthotopic metastatic models for anticancer drug discovery and evaluation: a bridge to the clinic. Invest New Drugs 1999; 17:343–359. 16. Katz MH, Takimoto S, Spivack D, Moossa AR, Hoffman RM, Bouvet M. A novel red fluorescent protein orthotopic pancreatic model for the preclinical evaluation of chemotherapeutics. J Surg Res 2003; 113:151–160. 17. Pesce A, Blubel HC, DiPersio L, Michael JG. Human lactic dehydrogenase as a marker for human tumor cells grown in athymic mice. Cancer Res 1977; 37:1998–2003. 18. Shih I-M, Torrance C, Sokoll LJ, Chan DW, Kinzler KW. Vogelstein B. Assessing tumors in living animals through measurement of urinary b-human chorionic gonadotropin. Nat Med 2000; 6:711–714. 19. Teicher BA, Herman TS, Holden SA, et al. Tumor resistance to alkylating agents conferred by mechanisms operative only in vivo. Science 1990; 247(4949 Pt 1):1457–1461. 20. Harrison S. Perspective on the history of tumor models. In: Teicher BA, ed. Tumor models in cancer research. Totowa: Humana Press, 2002:3–22. 21. Kidd JW, Rous P. A transplantable rabbit carcinoma originating in a virus-induced papilloma and containing the virus is masked or altered form. J Exp Med 1940; 71:813–837. 22. Doppelt SH, Slovik DM, Neer RM, Nolan J, Zusman RM, Potts JT. Gut-mediated hypercalcemia in rabbits bearing VX2 carcinoma: new mechanism for tumor-induced hypercalcemia. Proc Natl Acad Sci U S A 1982; 79:640–644. 23. Shilling T. In vivo models of hypercalcemia of malignancy. Recent Results Cancer Res 1994; 137:4475. 24. Hubbard WC, Hough AJ, Johnson RM, Oates JA. The site of VX2 tumor transplantation affects the development of hypercalcemia in rabbits. Prostaglandins 1980; 19:881–889. 25. Malave I, Blanca I, Fuji H. Influence of inoculation site on development of the Lewis lung carcinoma and suppressor cell activity in syngeneic mice. J Natl Cancer Inst 1979; 62:83–88. 26. Hill SA, Denekamp J. Site dependent response of tumours to combined heat and radiation. British J Radiol 1982; 55:905–912. 27. Keyes KA, Mann L, Cox K, Treadway P, Iversen P, Chen Y, Teicher BA. Circulating angiogenic growth factor levels in mice bearing human tumors using Luminex multiplex technology. Cancer Chemother Pharmacol 2003; 51:321–327. 28. Keyes KA, MannL, Teicher BA, Alvarez E. Site-dependent angiogenic cytokine production in human tumor xenografts. Cytokine 2003; 21: 98–104.