3. Background:
Angiogenesis
Angiogenesis is essential for tumor growth and
metastasis
Pathological retinal angiogenesis occurs in several
diseases characterized by retinal ischemia
Angiogenesis may complicate the clinical
presentation by inducing glaucoma due to iris
neovascularization(NVI) with secondary peripheral
synechiae(PAS) formation
Inhibion of angiogenesis has been shown to kill
retinoblastoma cells (harbour ophtalmic research)
Avastin (anti VEGF antibody)
Tyrosine kinase inhibitors
VEGF inhibition may also help as a chemosensityzer
4. Background: VEGF
VEGF is a hypoxia inducible
cytokine required for retinal
vascularization
Produced by Rb tumor
There are several proangiogenic factors involved
in retinal angiogenesis
VEGF and its receptors are
expresed in nonoverlapping manner
(Prospero Ponce et al 2013)
8. Relevant background
Neovascularization in retinoblastoma is
associated with poorer prognosis and increased
chances of invasion
Neovascularization as well as angiogenesis is
driven by angiogenic factors.
VEGF is the most important and most studied
angiogenic factor
Several anti-VEGF therapies have demonstrated
good results in various types of cancer
No one has studied histopathological behavior of
VEGF in Rb
VEGF is a promising therapy in Retinoblastoma
10. Objective
To analyze the expression of angiogenic factors in
the eye (retina, iris, and tumor ) in
retinoblastoma with high-risk features (HRF) and
non-high risk features (Non-HRF). To correlate the
expression of angiogenic factors in tumors with
HRF and the expression of stem cell marker
Sox2. keep order same throughout. If
possible, use this order when discussing your results
as well.
17. Measurements: Image J
Color deconvolution
Stain separation using Ruifrok and Johnston's method1
Vimentin
VEGF
[1] Ruifrok AC, Johnston DA. Quantification of histochemical staining by color deconvolution. Anal
Quant Cytol Histol 23: 291-299, 2001
20. Figure 1.
*
100 μm
100 μm
A
B
C
VEGF
8000
VEGF+ Vimentin
Vimentin
p<0.05
8000
20000
HRF
NON-HRF
6000
HRF
Non-HRF
4000
p<0.05
10000
4000
E
Location
F
Iri
s
Tu
m
a
et
in
R
Tu
m
et
in
R
Tu
m
et
in
R
Iri
s
Location
Location
or
0
Iri
s
0
or
0
a
2000
or
5000
a
2000
D
HRF
Non-HRF
p<0.05
6000
Pixel Area
15000
Pixel Area
Pixel Area
100 μm
22. Figure 3.
*
*
*
T
*
*
100 μm
A
B
C
VEGFR2
CD105
3000
VEGFR2 + CD105
1500
HRF
Non-HRF
p<0.05
1000
HRF
Non-HRF
200
1000
Pixel Area
2000
250
HRF
Non-HRF
p<0.05
Pixel Area
Pixel Area
R
500
150
100
50
0
Location
D
E
F
Iri
s
Tu
m
or
R
et
in
a
Iri
s
Tu
m
et
in
R
Location
or
0
a
Iri
s
Tu
m
or
R
et
in
a
0
24. Summary
HRF tumor s express more VEGF and VEGFR2 than
non-HRF
Comparison with other stainings indicate that
VEGF secretion might be done by tumor cells
Neovascularization occures more in the iris
This expression is correlated with the expression of
stem cell marker SOX2
25. Conclusions
HRF tumors seem to be more “stem like”
They might regulate their invasiveness through a
VEGF feedback loop
Temporal expression of VEGF receptors should be
considered
Anti-VEGF therapy might be a promising therapy
for Rb and its side effects
Eyes with HRF show increased expression of VEGF and its receptor VEGFR-2 in both the tumor and the iris. CD105 expression(newly formed vessels) is elevated in tumors but is particularly higher in the iris of HRF eyes. Therefore in HRF eyes, treatments that target cells expressing angiogenic factors and/or the factors alone may be useful to prevent complications such as development of neovascular glaucoma.
VEGF expression in the retinawas lower in the non-HRF tumor compared to its expression in the retina of the non-HRF samples or to the HRF tumor. Its co-expression with the glial marker vimentin and the qualitative interpretation shows that a significant amount of VEGF is probably secreted by tumor cells. The VEGF Receptor 2 is highly expressed in the retina and the HRF tumor, unlike in the non-HRF tumor. The activated endothelial cell marker CD105 shows that the majority of angiogenesis takes place in the iris. Co-expression of VEGFR2 and CD105 seems to be predominant in the vessels of the tumor and the iris neovascular membranes. The neural stem cell marker SOX2 was more highly expressed in the perinecrotic areas of the tumor and the regions closest to the retina. It was also higher in HRFtumors compared to non-HRF tumors. Retina of what? THe HRF, non-HRF? The writing is very non-specific and thus hard to follow...
VEGF expression in the retinawas lower in the non-HRF tumor compared to its expression in the retina of the non-HRF samples or to the HRF tumor. Its co-expression with the glial marker vimentin and the qualitative interpretation shows that a significant amount of VEGF is probably secreted by tumor cells. The VEGF Receptor 2 is highly expressed in the retina and the HRF tumor, unlike in the non-HRF tumor. The activated endothelial cell marker CD105 shows that the majority of angiogenesis takes place in the iris. Co-expression of VEGFR2 and CD105 seems to be predominant in the vessels of the tumor and the iris neovascular membranes. The neural stem cell marker SOX2 was more highly expressed in the perinecrotic areas of the tumor and the regions closest to the retina. It was also higher in HRFtumors compared to non-HRF tumors. Retina of what? THe HRF, non-HRF? The writing is very non-specific and thus hard to follow...
VEGF expression in the retinawas lower in the non-HRF tumor compared to its expression in the retina of the non-HRF samples or to the HRF tumor. Its co-expression with the glial marker vimentin and the qualitative interpretation shows that a significant amount of VEGF is probably secreted by tumor cells. The VEGF Receptor 2 is highly expressed in the retina and the HRF tumor, unlike in the non-HRF tumor. The activated endothelial cell marker CD105 shows that the majority of angiogenesis takes place in the iris. Co-expression of VEGFR2 and CD105 seems to be predominant in the vessels of the tumor and the iris neovascular membranes. The neural stem cell marker SOX2 was more highly expressed in the perinecrotic areas of the tumor and the regions closest to the retina. It was also higher in HRFtumors compared to non-HRF tumors. Retina of what? THe HRF, non-HRF? The writing is very non-specific and thus hard to follow...tumors.
Conclusions and Relevance: Our results, for the first time, show that in HRF retinoblastoma eyes when compared with non-HRF eyes, there is overexpression of VEGFA and VEGFR2in the tumor cells, neovascularization of iris with increased amount of activated endothelial cells, and that overexpression of VEGFA is associated with an increase in SOX2-positive stem/progenitor cells in the tumor. The increasedexpression of angiogenic factors and concomitant increase in stem cells in the HRF tumors may partially explain the aggressiveness of these tumors. Further studies are needed to confirm this association. In non-HRF eyes the pattern of expression of VEGFA and VEGFR2 seems to be comparable to the recognized pattern of response of the non-neoplastic tissuesto injury . The complex interaction of pathways in these tumors, especially in HRF retinoblastoma, indicate that targeting vasculogenic tumor cells, genes, and signaling pathways may be necessary in anti-tumor vasculogenic and metastatic prevention drug development. ARE THERE CONTROLS?? I did not see them mentioned at any other point in the manuscript.
