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Chapter 8 / Tumor Site Implantation
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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
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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
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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).
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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-
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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,
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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
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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-
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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.
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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.