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Per Borgström, Ph.D., Associate Professor
Dr. Borgstrom's Active Grants
Dr. Borgstrom's Publications
pborgstrom@skcc.org VASCULAR BIOLOGY AND ANGIOGENESIS Laboratory
Staff: Anders Angelborg, Raymond Winger, Randy Spencer, Linda Nyborg,
and Catherine Legras, PhD.
Research Interests Angiogenesis. Tumors can not grow beyond a certain size without angiogenesis.
Every successful increase in the tumor cell population must be preceded
by an increase in new capillaries that converge upon the tumor. Without
angiogenesis, potentially malignant tumors can stay in a "dormant" state
for a number of years without invading surrounding tissues. The critical
event converting such a dormant tumor into a rapidly growing malignancy,
is the switch to the angiogenic phenotype demarcating two stages in
the development of the tumor - the prevascular phase and the vascular
phase. Angiogenesis, a fundamental process by which new blood vessels are
formed, is rare in adult mammals under normal physiological conditions.
In adult physiological angiogenesis, i.e., wound healing, negative
inhibitory influences ultimately take over and the process of neovascularization
ceases at the completion of the wound healing. However, in pathophysiological
conditions like cancer and chronic inflammation, these inhibitory controls
fail and angiogenesis persists. The idea of anti-angiogenic drugs as a strategy for cancer therapy
was first proposed nearly 25 years ago. Since then, many compounds
have been shown to inhibit angiogenesis in various in vitro and in
vivo systems. To date, many angiostatic drugs are being tested in clinical
trials. The formation of new blood vessels during angiogenesis can be broken
down into a number of distinct yet overlapping processes. Angiogenesis
begins with the early inflammatory phase, characterized by dilated
and permeable vessels. This early inflammatory response is followed
by a proteolytic step where the basement membrane of the endothelium
is degraded through the action of a variety of proteases. All metastatic
tumor cells synthesize the required protease for remodeling endothelial
basement membranes. Several metalloproteinase inhibitors are currently
being tested in clinical trials. Proteolysis is followed by nonmitogenic
migration of capillary endothelial cells towards the angiogenic stimuli
such that endothelial cells migrate from the vascular wall, through
perivascular connective tissue and parenchyma. The next step in the
process of angiogenesis is the proliferation of endothelial cells behind
the leading front of migrating endothelial cells. A variety of soluble
polypeptides and other factors have been described that promote such
endothelial proliferation. One such factor is the vascular endothelial
growth factor (VEGF), an endothelial cell-specific mitogen and a growth
factor which is secreted by a variety of human tumors. Various antagonists
of the VEGF signaling pathway are currently in Phase II/III trials. To study the effects of anti-angiogenic substances, a variety of in
vitro assays, which model the different steps of angiogenesis have
been developed over the years. The traditional models include the cornea
pocket and the chorioallantoic membrane of the chick embryo (CAM).
A number of experimental models are also available for the study of
angiogenesis in normal and tumor tissues. However, no model allows
detailed description of early phases of tumor induced angiogenesis
with simultaneous visualization of tumor growth in vivo. To fill this
gap, we developed a system for non-invasive, in vivo and in situ study
of tumor angiogenesis in conscious mice, using video-microscopy of
tumor spheroids implanted in dorsal skin fold chambers in nude mice. 
Cartoon of the dorsal skinfold chamber adapted to nude
mice. The dorsal skinfold chamber technique was developed already in
the 1943 by Algire. (Algire, G.H. (1943). An adaptation of the transparent
chamber technique to the mouse. J. Natl. Cancer Inst. 4, 1-11.). 
Click to view an MPEG illustrating the usefulness of histone H2B-GFP
labeling of tumor cells.
Comparative Evaluation of Systemic Inhibitors. Currently we are using
this system to study prostate cancer angiogenesis. Our in vivo model
permits the evaluation of a molecule’s angiostatic potential
as well as its consequences on cell cycle and apoptosis. Several metastatic
variants of prostate carcinoma are being evaluated in this micrometastasis
model environment using intravital microscopy in the dorsal skinfold
chamber of athymic mice. Three variants of PC3 and LNCAP, which vary
in their metastatic potential, have been transduced with a retroviral
vector expressing Histone-H2B as a fusion protein with enhanced green
fluorescent protein. This fusion protein, implemented in our laboratory,
through collaboration with Dr. Geoff Wahl’s group at the Salk
Institute in La Jolla, permits the evaluation of mitotic and apoptotic
indices by way of its association with chromatin in a non-invasive
manner. Valuable mechanistic information regarding cell cycle and apoptosis
may therefore be obtained from sample fields of the prostate cancer
micrometastasis model, growing in the dorsal skinfold chamber of a
nude mouse.
This model permits the evaluation of angiostatic agents within a 14
- 21 day period. In other orthotopic or experimental metastasis models,
this process requires several months per experiment, is laborious to
perform, and may lead to ambiguous results given the inherent latency
of prostate carcinogenesis. Moreover, this model is being developed
with the goal of comparative analysis of different angiostatic agents
currently in preclinical development. 
Prostate Angiogenesis Model Variants of different metastatic potential from LnCAP
and PC3 are transduced with VSV pseudotyped histone H2B-GFP retrovirus.
Tumor spheroids prepared using the liquid overlay technique ensures
reproducibility of tumor spheroid size. Spheroids are implanted in
dorsal skinfold chambers in nude mice, and animals are observed at
certain intervals during a two week observation period, and evaluated
for tumor area, vascular density as well as mitotic and apoptotic indices.
The system allows evaluation of systemic as well as local treatment
regimens. By comparing the efficacy of
a candidate therapeutic to other treatment regimens within the same
model, a given therapeutic may be evaluated on the competitive basis
of 1) the extent of angiostasis 2) the effects of such angiostasis
on cell cycle progression within the core and periphery of a micrometastasis
and 3) the relative cell death as a result of angiostasis. The ability
to weigh all three parameters gives a more balanced perspective to
the potential of a candidate therapeutic in this relatively new field.
The use of the LNCAP and PC3 variant models will hopefully permit
the evaluation of the potential synergy of angiostatic therapy combined
with androgen ablation and/or chemotherapy regimens. In earlier experiments
we implanted tumor spheroids from the human prostate carcinoma cell
line DU 145. Results from these experiments demonstrated that anti-VEGF
treatment results in complete inhibition of tumor angiogenesis and
no growth beyond an initial prevascular phase. 
DU-145 Photos Photomicrographs illustrating
the complete inhibition of tumor angiogenesis after anti VEGF treatment
of nude mice implanted with the human prostate cell carcinoma cell
line DU 145. In tumors of control animals, vascular networks with
high vdensity were induced, wheras in tumors treated with an anti-VEGF
moAb (A4.6.1, 200 ug twice weekly IP, Genentech Inc.,) angiogenic
activity was reduced to at most budding. Tetracycline Regulated Expression Vectors: With the
rapid advancement of novel gene therapy based strategies for angiostasis,
and given the
obvious potential for potent "bystander effects", we have
constructed a new expression system to generate tetracycline regulated
cDNA constructs of angiostatic agents. This vector eliminates much
of the labor intensive screening of multiple clones in the presence
and absence of tetracycline. pTRE contains a minimal CMV promoter which
is fused to an upstream tetracycline response element, TRE. For the
rapid generation TET-regulated expression of candidate genes in TET-OFF
cell lines, we have included an internal ribosomal entry site (IRES)
with green fluorescent protein (TRE-IRES-eGFP). This vector allows
the generation of TET regulated cell lines which express essentially
any gene of interest. As the candidate gene is located between the
promoter and the eGFP, transcription of the candidate gene is required
to allow the transcription of the downstream reporter gene eGFP using
the IRES. Other vectors that use reporter genes under separate promoters
frequently result in varied levels of expression of the reporter and
the gene of interest. By performing first positive and then negative
FACs sort on the drug resistant population of cells, in the absence
and presence of tetracycline respectively, a GFP inducible population
of cells can be isolated that is regulated by the presence of tetracycline
or doxycycline. Moreover, as the eGFP and the gene of interest are
transcribed under the same mRNA, levels of GFP florescence correlate
with levels of expression of the candidate gene of interest, allowing
a sort of cells with a specific level of maximal gene induction. RT-PCR
/ Western Blot analysis of the gene of interest from the sorted population
can then be used to assure the inducible expression of the gene of
interest. Initial generation of Tet-Off cell lines is achieved using
the TET-OFF vector (Clontech, Palo Alto, CA). We have used this TETOFF
IRES2GFP model to evaluate the angiostatic potential of several candidate
angiostatic genes in the highly angiogenic HT1080 model. 
Candidate Angiostatic Genes Illustration of the TET-OFF
system using the highly angiogenic cell line HT1080. When implanted in
dorsal skinfold chambers in nude mice, these cells induce a very
high density vascular network (Panel A). Panel B demonstrates that
the gene is effectively turned OFF, i.e., no GFP fluorescence. Panel
C illustrates the angiostatic activity when the gene is turned on
as illustrated by the GFP fluorescence (Panel D). In addition to this plasmid-based model, we have now successfully
generated and tested a 2-vector tetracycline regulated retroviral system
using the TRE-IRES2GFP model and a modified TET-ON system. In this
system, the TETON retroviral vector contains the VP16 TET transactivator
followed by an IRES sequence and the TTS sequence which encodes a novel
repressor protein that prevents gene expression until the levels of
doxycycline reach 10ng/ml, after which the TETON transactivator drives
gene expression. This model results in a more tightly regulated expression
system than the older "leaky" TETON vectors. The retroviral
system is packaged in an HEK-293 system pseudotyped with the VSV envelope,
allowing concentration of virions to very high titers (>109). Evaluation of the angiogenic potential of tumor tissues derived from
human prostate cancer thin needle biopsies: In ongoing investigations,
we are also using our in vivo system to evaluate the angiogenic activity
of tumor tissues derived from human prostate cancer thin needle biopsies
and are comparing these with the established prostate cancer cell lines
mentioned above. We also examine the capacity of a neutralizing anti-VEGF
moAb, and a soluble VEGF receptor fusion protein (fIt-IgG) to inhibit
the angiogenesis and growth of the thin needle biopsies. While these
experiments are designed to examine the dependence of heterogeneous
prostate tumor xenograff biopsies on VEGF mediated angiogenesis, we
also intend to examine whether neutralization of VEGF is synergistic
or antagonistic to some of the chemotherapy regimens used for prostate
cancer. We will combine anti-VEGF treatment with the following two
agents:
a) Estramustine phosphate (EMP), a unique antitumour agent, which is
selectively taken up by prostate cells and exerts antineoplastic effects
by interfering with microtubule dynamics and by reducing plasma levels
of testosterone. It has been demonstrated that the antitumour response
obtained with oral EMP as a single agent is comparable with that obtained
with the best intravenous chemoregimens but EMP induces no myelotoxicity,
and other adverse effects of treatment are usually mild.
b) Taxol (paclitaxel) is a chemotherapeutic agent derived from the
yew tree that show important activity in metastatic prostate cancer.
Paclitaxel, the first of a new class of compounds, antimicrotubule
agents called taxanes, has a unique mechanism of action in blocking
cells at multiple phases of the cell cycle, inhibiting cell division
and rendering cells non-functional. Interestingly, combining paclitaxel
with EMP, results in enhancement of the effect of paclitaxel.
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