ORTHOTOPIC MODEL OF LUNG CANCER

Orthotopic models of cancers consist in the injection of tumor
cell suspension as well as in the implantation of fresh tumor
tissues directly into the appropriate organ of origin. Human
tumors and/or human cancer cells can be orthotopically implanted
in various organs, including stomach, colon, pancreas,
prostate, mammary gland, bladder, and lung. 25 The availability
of immunodeficient mouse strains such as the nude mice,
the Rag2 −/− mice, and the severe combined immunodeficient
(SCID) mice facilitated the establishment of human orthotopic
models of cancer because of the inefficiency of these
mice to reject human cells. The injection of tumor cells in the
organ of origin clearly allows a better understanding of the
role of the microenvironment in the development of primary
tumors. In addition, a major advantage of this model is that
it allows studying and recapitulating of the entire process of
tumor progression consisting of local tumor growth, vascular
and lymphatic invasion at the local site, flow in the vessels and
lymphatic, extravasation at the metastatic organs, and seeding
and growth at relevant metastatic sites. The availability of both
human NSCLC and small cell lung carcinoma (SCLC) cells
make the orthotopic model an attractive assay to study lung
cancer growth and development as, at present, there is only one
genetic mouse model of primary SCLC available (see discussion
later). In addition, based on the observation that the microenvironment
profoundly affects the phenotype and progression of
many tumor types, 26 the injection of lung cancer cells directly
into the organ of origin may recapitulate the events of lung
cancer growth and progression similar to those observed in humans.
Several orthotopic injection routes have been developed
for lung cancer, including intrabronchial, 27–29 intrathoracic, 30
intrapleural or intravenous, 31 and direct injection of tumor
cells 32,33 as well as implantation of fragments of subcutaneously
growing tumor tissues 34 into the lung parenchyma of recipient
mice. Of these models, the direct injection of tumor cells into
the lung parenchyma represents an exceptionally rapid procedure
with limited trauma to the mice and reduced intrapleural
leakage of tumor cells. In addition, this method had been successfully
used to produce a solitary tumor nodule in the lung
followed by metastasis to the mediastinal lymph nodes. It is
well recognized that the orthotopically growing tumors will
grow and metastasize to organs similarly to the human situation.
Thus, the presence of tumors in the contralateral noninjected
lung, as well as presence of metastases in lymph nodes,
liver, brain, and bones can be used to evaluate the metastatic
efficiency of different lung cancer cell lines. 33
One of the major issues related to cancer is the detection
of micrometastases (single cell or clusters of fewer than
10 cells). To improve the visualization of tumors in different
organs, tumor cells can be stably labeled with different fluorescent
or bioluminescent markers such as green fluorescent protein
(GFP) 35 and luciferase. 36 Single GFP-labeled tumor cells
can be detected in freshly isolated organs with a fluorescence
microscope, 37 and the number of tumors growing on the surface
and/or within the organs can be evaluated, and quantitative
measurements can be obtained using computer software
imaging programs. 33 Retroviral delivery of GFP has been successfully
used not only to label primary human tumors, but
also to analyze regional and distant metastases. 38 A significant
advantage of using GFP- or luciferase-expressing cells is that
tumors can be visualized and measured externally in live mice.
Hoffman 37 implanted highly metastatic human cell line expressing
GFP into the left lungs of nude mice and then followed
primary and metastatic growth in real time by analyzing
the mice under fluorescence light. Similarly, Rosol and colleagues
39 injected into the left ventricle luciferase- labeled metastatic
cells and followed in real time their localization in the
lungs by using in vivo bioluminescent imaging. More recently,
Acuff and colleagues 29 have used luciferase- labeled human
NSCLC cells to determine in real time the contribution of
host-derived matrix metalloproteinases to the survival and the
early establishment of tumors in the lung. Finally, noninvasive
in real-time imaging has been recently employed to determine
the effect of systemic delivery of capsid- modified adenoviruses
in an orthotopic model of advanced lung cancer. 40 Thus, the
use of labeled cells clearly facilitates in vivo imaging as well as
longitudinal studies as the same mouse can be monitored over
time (Fig. 13.2).
The different orthotopic models of lung cancers, in combination
with the intravital imaging of GFP-expressing cells,
clearly allow one to follow in real time the effects of chemopreventive
and/or antimetastatic drugs. In addition, they allow to
asses some critical parameters related to the use of these drugs
for cancer treatment and/or prevention, such as (a) the selection
of the tumor models that better resemble the phases of
tumor progression in humans, (b) the route of administration
of the drug, (c) the maximal dose tolerated, (d) the relationship
between the desired therapeutic benefits and the length
of the treatment, and (e) how long the beneficial effects of the
drug last upon withdrawal. It has been observed that orthotopically
transplanted human SCLC display a different chemosensitivity
pattern compared with the subcutaneously transplanted
model, 41 clearly suggesting a different pharmacodynamics between
the orthotopic lung and the ectopic subcutaneous sites.
Using an orthotopic human lung cancer model, Liu and colleagues
42 showed that KP-392, a potent selective inhibitor of
integrin-linked kinase, can be used in combination with cisplatin
to enhance tumor necrosis and decrease lung cancer progression.
In addition, in vivo inhibition of both vascular endothelial
growth factor receptor 2 (VEGFR2) and epidermal growth factor
receptor (EGFR) signaling pathways by ZD6474 resulted
in profound reduced angiogenesis as well as growth of human
lung adenocarcinoma cells orthotopically injected in mice, 43
suggesting that blocking both EGFR and VEGFR signaling
might be viewed as a valid tool for the management of locally
advanced lung cancer. Finally, aerosol nonviral gene delivery
system has been recently used to successfully deliver p53 gene
to mice intratracheally inoculated with H358 human NSCLC
cell line, 44 strongly indicating this therapeutic strategy might be
viewed as an option for patients with early lung cancer.
Thus, the orthotopic models of human cancer offer a tool
for the discovery of new chemopreventive and antimetastatic
agents, for the design of drug treatment regimes, and for investigating
their mechanism of actions in a model that better resembles
the phases of tumor progression observed in humans.