Canine hemangiosarcoma: A tumor of

Cancer Therapy Vol 6, page 457
Cancer Therapy Vol 6, 457-462, 2008
Canine hemangiosarcoma: A tumor of
contemporary interest
Review Article
Stuart C. Helfand
Oncology, Oregon State University, Magruder Hall, Corvallis, Oregon, USA
__________________________________________________________________________________
*Correspondence: Stuart C. Helfand, D.V.M., Professor, Diplomate ACVIM (Oncology and Internal Medicine), Oregon State
University, Magruder Hall, Corvallis, OR 97331, USA; Tel: 541-737-4830; Fax: 541-737-4818; e-mail: Stuart.Helfand@oregonstate.edu
Key Words: Canine hemangiosarcoma, tumor, adjuvant chemotherapy, immunotherapy, receptor tyrosine kinase
Abbreviations: galectin-3, (Gal-3); hemangiosarcoma, (HSA); histone deacetylase, (HDAC); inducible protein-10, (IP-10); interleukin12, (IL-12); liposome-encapsulated muramyl tripeptide phosphotidylethanolamine, (L-MTP-PE); receptor tyrosine kinase, (RTK);
suberoylanilide hydroxamic acid, (SAHA)
Received: 9 June 2008; electronically published: August 2008
Presented in the Theilen Tribute Symposium at UC Davis 31st May- 1st June 2008.
Summary
Hemangiosarcoma is an endothelial cell-derived malignancy that continues to be a fatal cancer in dogs. Death is
almost always due to local and systemic metastases despite numerous attempts to alter the clinical course with
adjuvant chemotherapy. A benefit from doxorubicin-based chemotherapy, albeit modest, has been established
unambiguously and represents the current standard of adjuvant care following surgical removal of
hemangiosarcoma. Survival times, however, typically are not appreciably prolonged beyond six months. A small
number of studies employing immunotherapy approaches indicate the potential to develop this modality for better
tumor control. As more information detailing cellular and molecular aspects of hemangiosarcoma cell function is
revealed, new opportunities for development of novel treatments, with implications for antiangiogenic approaches
for cancer, are emerging. Targeting hemangiosarcoma cell surface adhesion molecules, differentiation molecules,
and growth factor receptor pathways may provide the opportunities needed to impact on hemangiosarcoma growth
and improve clinical outcome.
I. Introduction
survival time following removal of the primary (splenic)
HSA; 2) the benefit obtained by adjuvant chemotherapy is
short lived; 3) adjuvant chemotherapy protocols should
include doxorubicin. There is a consensus of opinion that
chemotherapy given during the micrometastasis phase of
HSA extends survival, but close inspection of various
reports fails to show much difference in outcome
regardless of the novel question studied.
Doxorubicin-containing protocols rarely extend life
much beyond six months compared to the two or three
month survival time of dogs not receiving chemotherapy
(Hammer et al, 1991; Sorenmo et al, 1993; Ogilvie et al,
1996). More recent efforts to improve survival employed a
variety of strategies including the destruction of intraabdominal metastasis by intraperitoneal administration of
liposome-doxorubicin (Sorenmo et al, 2007), continuous
low-dose oral chemotherapy (i.e., cyclophosphamide,
etoposide, piroxicam) (Lana et al, 2007) and
immunotherapy using a potentiated allogeneic HSA
antigen vaccine combined with doxorubicin (U'Ren et al,
2007), all failed to improve survival time compared to that
observed with systemically administered doxorubicin
containing protocols. Survival times of canine HSA
patients were similarly unaltered by modified adjuvant
doxorubicin protocols testing an accelerated (dose
intensified) protocol (Sorenmo et al, 2004) or the addition
of a putatively antiangiogenic antibiotic minocycline
(Sorenmo et al, 2000). Table 1 summarizes the median
Dogs develop hemangiosarcoma (HSA) more
frequently than any other species. Hemangiosarcoma
originates from transformed endothelial cells and while
endothelial malignancies such as angiosarcoma and
Kaposi sarcoma are seen in humans, their incidence is low
compared to canine HSA. At least 7% of all canine cancer
is due to HSA and its profoundly aggressive behavior
almost always portends an unfavorable outcome in
affected dogs. The veterinary profession has singularly
dealt with the challenge of this deadly cancer but despite
the plethora of novel treatment strategies, HSA has refused
to be tamed and remains a formidable clinical problem.
Despite the frustrations associated with numerous
unrewarding, yet novel treatment efforts for HSA, the
quest to improve survival time has provided a unique
opportunity to learn about endothelial cell biology
including subcellular pathways and the process of
angiogenesis, the formation of new blood vessels.
II. Lessons learned from adjuvant
chemotherapy
It has been more than 20 years since the first reports
of adjuvant chemotherapy for canine HSA were published.
Since then, numerous studies have appeared, all of which
confirm several apparent truths. These include: 1) adjuvant
chemotherapy extends the disease free interval and
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Helfand: Canine hemangiosarcoma: A tumor of contemporary interest
survival times reported in these studies. Death is always
due to widespread metastasis, often affecting the lungs
(Figure 1). Taken together, it is readily apparent that
major breakthroughs in controlling canine HSA will not
come from adjuvant chemotherapy alone.
dogs were treated with the macrophage activator
liposome-encapsulated
muramyl
tripeptide
phosphotidylethanolamine (L-MTP-PE) concurrently with
multiple chemotherapy cycles consisting of doxorubicin
and cyclophosphamide. The median survival time for all
18 dogs receiving chemoimmunotherapy was 277 days,
but of note were the eight dogs with stage I disease that
had a median survival time of 425 days, although not
significant in comparison to the placebo-treated dogs due
to the small numbers. The recent report by U’ren and
colleagues in 2007 provides evidence of the potential to
elicit humoral immune responses to unknown HSA
III. Potential of immunotherapy
Several lines of evidence indicate immunotherapy
may play a role in suppressing HSA micrometastases.
These include approaches that elicited both cell-mediated
and humoral responses. In fact, the most impressive
survival times reported for canine HSA patients were
those described by Vail and colleagues in 1995 in which
Table 1. Median survival times of dogs with hemangiosarcoma treated with splenectomy and chemotherapy.
Study
Year
Treatment
Wood and colleagues
Hammer and colleagues
1998
1991
Sorenmo and colleagues
Ogilvie and colleagues
Lana and colleagues*
Sorenmo and colleagues
Sorenmo and colleagues
Sorenmo and colleagues
Lana and colleagues*
1993
1996
2007
2000
2004
2007
2007
splenectomy, no chemotherapy
vincristine, doxorubicin,
cyclophosphamide
doxorubicin, cyclophosphamide
doxorubicin
doxorubicin
doxorubicin, minocycline
doxorubicin every 2 weeks
pegylated doxorubicin intraperitoneal
daily low dose etoposide,
cyclophosphamide, piroxicam
Median survival time
(days)
86
145
202
172
133
170
257(I)#, 210 (II), 107 (III)
131
178
*Same study comparing standard doxorubicin every three weeks vs. daily low dose etoposide, cyclophosphamide, and piroxicam.
#
Results reported by clinical stage in parenthesis.
Figure 1. Canine hemangiosarcoma pulmonary metastasis. Diffuse pulmonary metastatic hemangiosarcoma nodules are readily visible
(numerous small white densities) in the thoracic radiograph of a dog presented for a penile hemangiosarcoma (left). At postmortem
(right), the lungs are filled with large numbers of round maroon nodules comprised of metastatic hemangiosarcoma. A section of lung
has been excised (center) to establish a hemangiosarcoma cell line from metastatic lesions (see Figure 3).
antigenic determinants following vaccination with
allogeneic canine HSA cell lysates in combination with
liposome containing (non-coding) DNA complexes
(LDC). The inclusion of LDC with the tumor cell lysate
was intended as an adjuvant as LDC reportedly trigger
enhanced immune responses in a variety of vaccine
protocols (U'Ren et al, 2007). While the survival time of
the treated dogs was not much different than animals given
chemotherapy alone, the development of specific humoral
immune responses to the HSA vaccine is a promising step
in overcoming immune tolerance.
Our laboratory has investigated the potential to
develop interleukin-12 (IL-12) as an adjuvant therapy for
canine HSA. This line of research evolved from our
interest in exploiting the immunotherapeutic potential of
IL-12 coupled with the discovery that IL-12 also mediates
antiangiogenic activity. Interleukin-12 is a potent inducer
of cell-mediated immune responses making it attractive as
part of immunotherapeutic strategies for cancer. It is a
potent inducer of interferon-γ, which in turn stimulates
production of interferon inducible protein-10 (IP-10) and
the chemokine Mig (monokine induced by interferon-γ),
both of which mediate angiostatic activity. Since HSA
cells likely share functional properties in common with
neoangiogenic endothelium within tumors, i.e., mitotically
active endothelial cells that form new blood vessels within
the tumor microenvironment, agents that inhibit
angiogenesis may also be of value in suppressing HSA
growth.
Using a combination of in vitro and in vivo methods,
we demonstrated that IL-12 could be targeted to adhesion
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We have just begun to scratch the surface in
understanding the characteristics of the transformed cells
that give rise to canine HSA. Learning more about them
on a cellular and subcellular level will ultimately reveal
clues as to where they may be vulnerable which in turn,
will facilitate design of strategies that can lead to
improved therapeutic outcomes. Information is gradually
accruing relating to HSA cell dependency on external
growth factors, expression of surface receptors that
interact with the extracellular environment, signaling
pathways, stem cells, and genetic abnormalities. A variety
of contemporary research tools are helping with these
discoveries, including cellular, molecular, protein, and
genomic interrogation techniques.
Table 2 shows a partial list of surface molecules
reportedly expressed by canine HSA cells. Assuredly,
more features of HSA cells will come to light as research
progresses. Several of these have been the subjects of
studies exploring unique therapeutic strategies.
molecules (i.e., αvβ3 integrin) expressed by dividing
endothelial cells comprising the neovasculature induced
by canine HSA (and other tumor) cells. This in turn,
resulted in marked suppression of vascular ingrowth
normally triggered by the malignant cells (Akhtar et al,
2004; Dickerson et al, 2004). Figure 2 shows an example
of canine HSA-induced neovascularization in a corneal
angiogenesis assay. Furthermore, using a canine HSA
xenograft model in which HSA was transplanted into
immunoincompetent
mice,
we
demonstrated
unambiguously the potential for IL-12 to suppress growth
of canine HSA (Akhtar et al, 2004).
Although there are, as yet, not many studies
exploring a role for immune modulation in controlling
HSA, it appears that such strategies may offer promise,
especially when combined with other modalities.
IV. Form provides clues to function
Figure 2. Canine hemangiosarcoma cells are inducers of angiogenesis. In this corneal angiogenesis assay, a polyvinyl sponge containing
canine hemangiosarcoma cells surgically placed into a corneal pocket of a BALB/c mouse elicited ingrowth of new blood vessels arising
from the normal limbic vessel (bottom). After binding to receptors expressed on mature endothelial cells comprising the limbic vessel,
proangiogenic proteins released by the hemangiosarcoma cells induce angiogenesis with arborized neovessels clearly homing towards
the hemangiosarcoma cells, the source of the angiogenic stimulus. Details of this assay can be found in Dickerson et al (Dickerson et al,
2004).
Table 2. Surface molecules of potential interest in canine hemangiosarcoma.
Molecule
αvβ3 integrin (adhesion molecule)
(Akhtar et al, 2004; Fosmire et al, 2004)
ICAM-1
(Thamm et al, 2006)
c-kit (CD117)
(Akhtar et al, 2004; Fosmire et al, 2004)
VEGFRs
(Akhtar et al, 2004)
PDGFR
(Helfand, unpublished)
Galectin-3
(Johnson et al, 2007)
CD34, CD133, c-kit
(Lamerato-Kozicki et al, 2006)
PTEN (cytoplasmic)
(Dickerson et al, 2005)
Importance
RGD targeting (cytokines,
chemotherapy)
Adhesion molecule
Tyrosine kinase inhibition
Tyrosine kinase inhibition, mAb target
Tyrosine kinase inhibition
Gal-3 inhibition (modified citrus pectin,
lactulosyl-L-leucine)
HSA stem cells
Tumor suppressor protein
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Helfand: Canine hemangiosarcoma: A tumor of contemporary interest
As mentioned earlier, the author’s laboratory investigated
a novel approach targeting IL-12 to the adhesion molecule
αvβ3 integrin by molecular engineering of a fusion protein
consisting of the peptide ligand for αvβ3 integrin (RGD4C) combined with IL-12 (Dickerson et al, 2004). This
strategy resulted in a vastly superior anti-antiangiogenic
effect against neovascularization induced by canine HSA
(and other tumor histologies) compared to that observed
with untargeted IL-12. More importantly, IL-12
suppressed growth of canine HSA in a xenograft model,
but the αvβ3 integrin-targeted IL-12 was vastly more
potent than untargeted IL-12 in suppressing the growth of
tumors in a murine neuroblastoma model (Dickerson et al,
2004). Taken together, it would seem that there is merit in
pursuing novel therapeutic constructs that target αvβ3
integrin. An informative review of this concept was
recently published describing benefits of targeting a
variety of molecules capable of arresting tumor cell
growth to αvβ3 integrin including radioisotopes, TNF-α,
doxorubicin, IL-12, and others (Temming et al, 2005). We
are continuing to investigate this approach in our
laboratory for canine HSA.
Johnson and colleagues chose to explore targeting
galectin-3 (Gal-3) they believed to be expressed on the
surface of canine HSA cells (Johnson et al, 2007). Gal-3 is
a member of a group of surface carbohydrate binding
proteins, overexpressed in numerous human malignancies,
and participates in a variety of cellular processes important
to cancer including cell differentiation, cell-cell and
extracellular matrix adhesions, metastasis, and apoptosis
(Johnson et al, 2007). It also participates in angiogenesis
providing a rationale to examine it as a target in canine
HSA. The authors identified Gal-3 expression on naturally
occurring canine HSA biopsies and showed that
proliferation of a Gal-3 positive murine HSA cell line
could be inhibited in vitro by Gal-3 inhibitors. Clearly,
more needs to be done to determine if Gal-3 blockade can
be developed as a meaningful therapy for canine HSA, but
the novelty of this approach bears obvious consideration.
Another recent report by Lamerato-Kozicki and
colleagues in 2006 was the first to clarify that a small
subset (i.e., <1%) of canine HSA cells coexpress surface
markers of hematopoietic stem cells (CD133, CD34, c-kit)
with commitment to endothelial differentiation. This study
lends credence to the idea that HSA is really a bone
marrow derived malignancy with progenitor cells arising
from lineage committed stem cells that circulate and
eventually give rise to HSA in specific organs. The
significance of this finding is not trivial because it implies
that complete eradication of HSA in affected dogs will
require elimination of these apparent HSA stem cells.
Cancer stem cells have proven to be the most resilient and
resistant to therapy and are capable of regenerating a
tumor even from a residual microscopic tumor population.
This may help to explain, in part, the poor results obtained
with chemotherapy of HSA in the microscopic disease
setting that is routine after splenectomy for a primary
splenic HSA.
Information regarding the HSA genome is gradually
emerging as well, which may facilitate identification of
numerous genes in tumor cells that are either over- or
under-expressed compared to non-cancerous endothelial
cells. The advent of genomics, sequencing of the entire
canine genome, and commercially available canine gene
chips that allow vast numbers of genes to be examined in
microarrays, is beginning to reveal the complexity of
canine HSA at the gene level. To this end, splenic HSAs
from Golden retrievers, a breed at above average risk for
developing this cancer, have recently been found to have a
unique pattern of gene under expression (Dr. Jaime
Modiano, personal communication 2008). It is tempting
to wonder if some of these genes may actually code for
tumor suppressor proteins that may have been silenced
either as a consequence or as a cause of malignant
transformation. The single case report of a dog with
splenic HSA that was treated with the histone deacetylase
(HDAC) inhibitor suberoylanilide hydroxamic acid
(SAHA), that lived >1000 days post splenectomy, more
than four times longer than most dogs with this
malignancy, may possibly be explained on the basis of
upregulation of silenced tumor suppressor genes by the
SAHA treatment (Cohen et al, 2004). HDAC inhibitors are
a class of new anticancer agents capable of inhibiting
promiscuous histone deacetylation in cancer cells that
results in gene silencing. There are alternative
explanations for the favorable results in this case report,
such as the potential for a good outcome regardless of
treatment as this dog’s tumor was reported to be low grade
(Thamm, 2005). There is continuing interest in developing
HDAC inhibitors for canine HSA and the example of
genomic investigation of canine HSA in Golden retrievers
will be instructional in exploring the effects of HDAC
inhibitors on HSA from a mechanistic perspective.
Since we discovered transcripts in canine HSA cells
that encode c-kit and VEGFR-1 and -2 as well as their
respective ligands, stem cell factor and VEGF (Akhtar et
al, 2004; Fosmire et al, 2004), we have been interested in
examining the possibility that these cell surface receptors,
members of the receptor tyrosine kinase (RTK) family,
may be important players in this malignancy. Much has
been written about RTKs and their role in various
malignancies. Briefly, RTKs are at the interface of the cell
and its extracellular environment and respond to external
stimuli by triggering intracellular growth promoting
pathways (London, 2004). In health, they are
protooncogenes in which their activity is tightly regulated,
but numerous transforming mutations have been reported
in a variety of cancers in which they function as
oncogenes. Indeed, many malignancies appear to be
addicted to mutated tyrosine kinases as a means to
promote their immortality. Activated RTKs (as well as
cytoplasmic tyrosine kinases), rely on phosphorylation of
tyrosine residues for their activity (London, 2004).
We are continuing to explore the role of certain
RTKs in canine HSA and have found that PDGFR-β, an
RTK important in endothelial differentiation and vascular
development (Zhu, 2006), is expressed by canine HSA
cells. Development of PDGFR-β antagonists is an active
area of cancer research because of the potential to arrest
neoangiogenesis in a broad range of cancers. In regards to
HSA, this concept is entirely compatible with the idea that
canine HSA is a malignancy of primitive endothelial cells
(Fosmire et al, 2004). The ligand for PDGFR-β is PDGFBB and when stimulated by PDGF-BB, PDGFR-β is
activated through phosphorylation of various tyrosine
residues. Figure 3 shows a western blot illustrating
activation (i.e., phosphorylation) of PDGFR-β in response
to stimulation by exogenous PDGF-BB in a canine HSA
cell line developed in the author’s laboratory. We are
continuing to examine the importance of this pathway in
canine HSA.
It would seem that we are now finally able to begin
to develop potentially meaningful therapeutic approaches
based on specific information about the composition of
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HSA cells. The stage is set through the processes of
discovery and innovation, for advancements that are likely
to improve the prognosis for dogs with HSA. As more is
revealed about the dysregulated processes driving canine
HSA, it is likely that previously unimaginable treatment
strategies will continue to come forth. Most likely,
progress will be the result of multimodality interventions
as canine HSA has proven itself to be a stubborn foe.
Biotechnology, Santa Cruz, CA) that recognizes total PDGFR-β
regardless of phosphorylation status (bottom).
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Acknowledgments
The author would like to thank numerous
collaborators and colleagues for their contributions to
various aspects of the hemangiosarcoma research
summarized here including Drs. Erin Dickerson, Jaime
Modiano (and his laboratory team), Nasim Akhtar, Robert
Auerbach, Matthew Breen, John Wojcieszyn, Valerie
MacDonald, and Michelle Turek. The author would also
like to thank Marcia Padilla, Wade Edris, and Kevin
Marley for their excellent contributions to some of the
studies mentioned in this review. Support for studies from
the Helfand laboratory came from the National Institutes
of Health (R01 CA86264), Morris Animal Foundation
(D03CA-71), AKC Canine Health Foundation (2025),
Midwest Athletes Against Childhood Cancer, and the
University of Wisconsin School of Veterinary Medicine
Companion Animal Fund.
Figure 3. Response of PDGFR-β to stimulation by PDGF-BB in
a canine hemangiosarcoma cell line (Rio-HSA). Cell lysates
from hemangiosarcoma cells (Rio-HSA) derived from a
pulmonary hemangiosarcoma metastatic lesion in a dog (Figure
1) were examined by western blot analysis following overnight
culture in serum starved medium without (0 min) or with PDGFBB (30 ng/ml) after 10 (middle lane) and 60 minutes (right lane)
and probed with an antibody specific for canine PDGFR-β
phosphotyrosine position 857 located in the kinase domain. The
cellular response is normal in this line in that the cells were not
autophosphorylated (time 0), but responded rapidly to PDGF-BB
stimulation by phosphorylating tyrosine after 10 minutes with
down regulation of the phosphotyrosine response by 60 minutes
despite further stimulation. As a loading control, the blot was
stripped and reprobed with an antibody (Santa Cruz
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Stuart C. Helfand
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