Angiogenesis inhibition for the improvement of photodynamic therapy: The revival
of a promising idea
Andrea Weissa, Hubert van den Bergha, Arjan W. Griffioenb,⁎, Patrycja Nowak-Sliwinskab,c,⁎⁎
aMedical Photonics Group, Institute of Bioengineering, Swiss Federal Institute of Technology (EPFL), Lausanne, Switzerland
bAngiogenesis Laboratory, Department of Medical Oncology, VU University Medical Center, Amsterdam, The Netherlands
cUrology Department, University Hospital (CHUV), Lausanne, Switzerland
a b s t r a c t a r t i c l e i n f o
Received 18 December 2011
Received in revised form 13 March 2012
Accepted 14 March 2012
Available online 21 March 2012
Photodynamic therapy (PDT) is a minimally invasive form of treatment, which is clinically approved for the
treatment of angiogenic disorders, including certain forms of cancer and neovascular eye diseases. Although
the concept of PDT has existed for a long time now, it has never made a solid entrance into the clinical man-
agement of cancer. This is likely due to secondary tissue reactions, such as inflammation and neoangiogen-
esis. The recent development of clinically effective angiogenesis inhibitors has lead to the initiation of
research on the combination of PDT with such angiostatic targeted therapies. Preclinical studies in this re-
search field have shown promising results, causing a revival in the field of PDT. This review reports on the
current research efforts on PDT and vascular targeted combination therapies. Different combination strate-
gies with angiogenesis inhibition and vascular targeting approaches are discussed. In addition, the concept
of increasing PDT selectivity by targeted delivery of photosensitizers is presented. Furthermore, the current
insights on sequencing the therapy arms of such combinations will be discussed in light of vascular normal-
ization induced by angiogenesis inhibition.
© 2012 Published by Elsevier B.V.
Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
PDT and its vascular effects . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
PDT in combination with anti-angiogenesis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
3.1.Growth factor targeted agents . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
3.1.1. Vascular endothelial growth factor (VEGF) axis targeting agents . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
3.1.2.Other growth factor targeted agents . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
3.1.3.Tyrosine kinase inhibitors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
3.2. Non-growth factor targeted agents . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
3.2.1.Matrix metalloproteinases (MMPs). . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
3.2.2.Cyclooxygenase-2 (COX-2) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
3.2.3. Kinase inhibitor p21. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
PDT in combination with vascular normalization through
anti-VEGF therapy . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Biochimica et Biophysica Acta 1826 (2012) 53–70
Abbreviations: ALA, aminolevulinic acid; AMD, age-related macular degeneration; AP-1, activator protein-1; AlPcS4, tetrasulfonated aluminum phthalocyanine; APRPG, Ala-Pro-
Arg-Pro-Gly peptide; ATP, adenosine triphosphate; bFGF, basic fibroblast growth factor; CAM, chorioallantoic membrane; CCH, circumscribed choroidal haemangioma; COX-2, cy-
clooxygenase-2; DMXAA, 5,6-dimethylxanthenone-4-acetic acid; EGF, epidermal growth factor; GM-CSF, granulocyte-macrophage colony stimulating factor; HeLa, human cervix
carcinoma; HIF-1α, hypoxia-inducible factor 1-alpha; ICG, Indocyanine Green; IL-1β, interleukin 1-beta; MMP, matrix metalloproteinase; n.a., not applicable; NF-κB, nuclear factor
κB; NPC, nasopharyngeal carcinoma; NPC, nasopharyngeal carcinoma; NSCLC, non-small-cell lung cancer; PCI, photochemical internalization; PDGF(R), platelet derived growth fac-
tor (receptor); PDT, photodynamic therapy; PS, photosensitizer; PEG, polyethylene glycol; PGE2, prostaglandin E2; PMN, polymorphonuclear; PNET, Pancreatic Neuroectodermal
Tumor; RCH, retinal capillary haemangioma; RGD, Arg-Gly-Asp tri-peptide; RIF, radiation-induced fibrosarcoma; SCC, squamous-cell carcinoma; SIP, small immune protein; TNF-α,
tumor necrosis factor alpha; TTT, transpupillary thermotherapy; VDA, vascular disrupting agent; VEGF, vascular endothelial cell growth factor; vWF, von Willebrand factor
⁎ Correspondence to: A.W. Griffioen, Angiogenesis Laboratory, Department of Medical Oncology, VU University Medical Center Amsterdam, Amsterdam, The Netherlands.
Tel.: +31 20 4443374; fax: +31 20 4443844.
⁎⁎ Correspondence to: P. Nowak-Sliwinska, Urology Department, University Hospital (CHUV), Lausanne, CH-1011, Switzerland. Tel.: +41 21 6935169; fax: +41 21 6935110.
E-mail addresses: email@example.com (A.W. Griffioen), Patrycja.Nowak-Sliwinska@chuv.ch (P. Nowak-Sliwinska).
0304-419X/$ – see front matter © 2012 Published by Elsevier B.V.
Contents lists available at SciVerse ScienceDirect
Biochimica et Biophysica Acta
journal homepage: www.elsevier.com/locate/bbacan
PDT in combination with vascular disrupting agents (VDA) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 63
PDT with photosensitizers targeted specifically to the
tumor endothelium . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 63
6.1. ED-B domain of fibronectin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 63
αvβ3integrin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 64
Translation to the clinic . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 64
Back to the drawing board . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 66
Acknowledgements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 67
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 67
Photodynamic therapy (PDT) is a minimally invasive treatment
that utilizes the combination of a non-toxic dose of light-sensitive
molecules, known as a photosensitizer (PS), with the application of
light at a wavelength appropriate to excite the PS and tissue oxygen
in order to generate tissue damage. The earliest recorded use of a PS
and a light source for a medical effect can be found in records originat-
ingin ancientEgyptfrom asearly as3000 years ago. Information on the
probably containing psolarens (furanocumarins), can be found in the
so-called Ebers Papyrus. This document dates circa 1550 BC, but was
likely based on prior knowledge dating back as far as 3400 BC . The
use of a topically applied vegetable substance on the skin and subse-
quent irradiation by sunlight was used to produce photoreactions for
skin repigmentation. The first scientific evidence of the use of PSs was
reported over 100 years ago, when it was found that acridine orange
and the application of light could kill protozoan cells . Shortly after,
Jesionek experimented with eosin as a therapeutic compound for the
PDT treatment of cancer . In the 1970s, following some intermittent
PDT activity, Thomas Dougherty and co-workers clinically tested PDT
and were able to demonstrate total and partial eradicationof the growth
of variousmalignant tumors, includingmetastatic melanomas, recurrent
colon carcinomas, metastatic breast carcinomas on the chest wall, and
recurrent basal cell carcinomas . The latter publication, to a large ex-
tent, set off the current field of PDT research.
PDT is based on the generation of cytotoxic reactive oxygen spe-
cies that cause tissue damage in the treated area [5,6]. In this way,
PDT can be used to induce direct damage to tumor cells . In addi-
tion, PDT can exert its effect through damage to the endothelium.
This can lead to the disruption of the vasculature via endothelial cell
(EC) damage, vascular leakage, and induction of thrombocyte aggre-
gation and coagulation, eventually resulting in the occlusion of the
vessels  and having effects on the immune system . In current
medicine, PDT is approved and has been implemented extensively
in the treatment of various diseases of the eye, such as age-related
macular degeneration (AMD) . For therapy of oncological condi-
tions, however, the success of PDT has been limited with the excep-
tion of certain skin conditions . Although approved for some
cancers, such as squamous cell carcinoma [11–13] and centrally locat-
ed unresectable early stage lung cancer [14,15], major applications for
the treatment of solid tumors have not yet been developed. This may
be due to limitations of PDT for certain indications, such as the local na-
ture of the therapy and the limited depth of light penetration into tissue.
However, more important delimiters of the failure so far, may be the in-
evitable tissue responses to therapy, possibly enhancing and facilitating
regrowth or further outgrowth of tumors. PDT has been shownto induce
an inflammatory response in the tumor tissue. During and directly
donic acid metabolites, histamine discharged from mast cells, comple-
ment anaphylatoxins, cytokines, chemoattractants, leukocyte adhesion
sponse and to attract neutrophils and other inflammatory cells to the
treated site . All of these factors induce tumor vasculature alterations,
including increased permeability to allow the transport of blood proteins
and pro-adhesiveness to inflammatory cells. Interleukin (IL)-1β and IL-6
have been shown to play critical roles in this PDT-induced inflammatory
process [16,17]. Therefore, the modulation of different inflammatory fac-
tors can be used to enhance the effects of PDT, as elegantly reviewed by
Agostinis et al. .
The above mentioned plethora of produced cytokines can also lead to
direct tumor cell activation. Such cytokine storms, as well as attracted
macrophages, can also stimulate endothelial cells to increase angiogene-
sis. Additionally, initial damage to the tumor tissue causes hypoxia and
oxidative stress in the treated area , which induces hypoxia-
These processes are interpreted by the body as localized acute trauma
,which willthen leadtomoreunwantedangiogenesis,possiblylead-
was realized that the use of combination therapies which attempt to
counteract these tissue responses may improve the final outcome of
PDT. The main therapeutic modalities that are tested in this respect are
chemotherapy , pro-oxidant therapies (e.g. erythropoietin) [27,28],
anti-inflammation  and anti-angiogenesis therapies .
The combination of PDT with anti-angiogenic strategies is the
focus of the current review. The relationship between PDT-induced
damage, the activation of angiogenic pathways and subsequent vas-
cular proliferation were confirmed by the landmark study of Ferrario
et al. . In this study, Photofrin®-mediated PDT was performed
in vitro on breast carcinoma cells showing increased VEGF expres-
sion which may have been associated with treatment-induced hyp-
oxia and to a lesser extent with treatment-induced oxidative stress
. In addition, this study examined the effects of PDT followed by
the administration of anti-angiogenic drugs in vivo on tumor bear-
ing mice. This report showed that the combination therapy resulted
not only in a significant reduction in PDT-induced VEGF expression,
but also elicited a significant increase in the tumoricidal activity of PDT,
as measured by tumor cure rates. The results of this study have since
been confirmed and reproduced by an assortment of groups using var-
ious combinations of different PSs and anti-angiogenic or anti-vascular
will focus on what is known of this combination strategyand will give a
critical view of its future applications. The different approaches of an-
giogenesis inhibition and vascular targeting, as well as the timing and
sequencing of therapeutic strategies will be discussed. In addition, the
enhancement of the selectivity of PDT through the molecular targeting
of PSs will be reviewed and discussed. The importance of the subject of
combination of PDT with angiostasis may allow for a revival in the field
of PDT and holds promise for the application of PDT in cancer.
2. PDT and its vascular effects
Research over the past few decades investigating the mechanisms
and cellular effects of PDT has revealed that the vascular damage
caused by PDT is largely responsible for the therapeutic benefit of
the treatment, depending on the timing and the PS administered
A. Weiss et al. / Biochimica et Biophysica Acta 1826 (2012) 53–70
[30–32]. It has also been demonstrated that tumor cure rates are
strongly dependent on vessel damage in and around the treated
tumor . Tissue damage from PDT depends on the photosensitiz-
ing agent and the treatment regimen used. The vascular effects of
PDT, which eventually lead to vascular occlusion, will be described
in more detail. Early damage following the injection and illumina-
tion of a PS is mostly observed in the endothelial and sub-
endothelial cells . Endothelial cell damage, originating at the en-
dothelial luminal surface, begins with the influx of calcium into the
cells . An increase in the cytosolic calcium concentration leads
to conformational changes in adhesion molecules and alterations
in cytoskeletal components, including the depolarization of cyto-
plasmic microtubules. This induces changes in cell shape, such as
swelling, rounding up and contraction, as well as fragmentation,
resulting in complete eradication of the endothelial lining . Con-
traction of endothelial cells results in the loss of tight junctions be-
tween cells and the exposure of the vascular basement membrane
 to circulating blood. The interaction of blood platelets with
the denudated vascular wall induces the activation and aggregation
of platelets. Platelets have also been shown to accumulate PSs and
upon irradiation are damaged, losing serotonin and ATP proportion-
al to the PS dose administered [38,39]. Taken together, platelet
damage and the interaction of platelets with pro-coagulant extracel-
lular matrix components eventually leads to vessel constriction and
a thrombus formation [36,40,41].
The adhesion of granulocytes has also been observed following
endothelial cell damage, which can induce increased vascular perme-
ability and edema . Damage to platelets further stimulates the
release of thromboxane (a platelet pro-aggregation compound) and
leukotriene B4, both of which further contribute to enhanced vascular
permeability and the disruption of the endothelial cell lining of ex-
posed blood vessels [43,44]. These leukocytes have been shown to
bind to the vascular endothelium in PDT-treated normal tissue ,
but not to PDT-treated tumor microvasculature, indicating that
tumor vasculature damage following PDT is not related to leukocyte
adherence . It is likely that the anti-adhesive properties of
tumor endothelial cells, as have been previously described, explain
these observations [47,48]. Endothelial cells may also influence the
blood clotting balance through the release of clotting factors , in-
cluding von Willebrand factor (vWf) . All of these biochemical
changes disrupt the balance of platelet pro-aggregatory/constricting
compounds and anti-aggregatory/vasodilating agents (prostacyclins)
[50,51], resulting in smooth muscle constriction and further platelet
aggregation. In a parallel mechanism, PDT-induced damage to mem-
brane lipids elicits the release of arachidonic acid, which initiates a
series of reactions, also ending in the release of thromboxane. Vessel
constriction is further supported by the inhibition of nitric oxide pro-
duction in endothelial cells . Finally, a blood clot within the vessel
lumen could potentially cause obstruction to blood flow, leading to
the termination of vascular function and an increase in intratumoral
interstitial fluid pressure . A simplified scheme of the PDT-vascular
effects is shown in Fig. 1.
3. PDT in combination with anti-angiogenesis
Major problems associated with the application of PDT for cancer
are the secondary angiogenic and inflammatory responses to treat-
ment, which result in the revascularization of treated lesions and con-
tribute to tumor recurrence [25,32]. Tumor angiogenesis is a robust
physiological process, regulated by a variety of endogenous pro-
and anti-angiogenic factors. The process starts in many cases with
hypoxia in a malignantly transformed cell mass. This forces the angio-
genic switch in these cells, which will then express angiogenic
growth factors, such as vascular endothelial growth factor (VEGF).
Endothelial cells in preexisting blood vessels will then migrate into
the growth factor gradient, proliferate and form new vascular sprouts.
These initially immature blood vessels form a new basement mem-
brane and attract accessory cells to form mature blood vessels that
can transport blood. The angiogenesis cascade is schematically pre-
sented and summarized in Fig. 2.
3.1. Growth factor targeted agents
Over the last few decades, many angiogenesis mechanisms have
been delineated and treatment strategies against cancer, based on
angiogenesis inhibition, have been developed. The most well-
developed strategy of angiogenesis inhibition is the intervention
with angiogenic growth factor signaling. Both growth factor neutral-
izing antibodies or antibody-based constructs and inhibitors of
growth factor receptor signaling, which act through the inhibition
of tyrosine kinase activity, have been developed. Other drug targets
which are currently being clinically assessed as modulators of an-
giogenesis, include circulating endothelial progenitor cells, the cyto-
skeleton (see Section 5), cell adhesion molecules (intergrins, see
Section 6.2), hypoxia (Section 3.1), MMPs and cathepsins (Section
3.2). Procoagulant pathways are also upregulated in tumor vascula-
ture, a pathway which can be blocked with anti-thrombotic drugs
(fragmin). Platelets are often overstimulated by IL-6 in tumors and
provide a major source of VEGF. Additionally, the enzyme thymidine
phosphorylase is often upregulated in tumors and produces angio-
genic metabolites. The inhibition of its action may therefore poten-
tially lead to effective angiogenesis inhibition (Fig. 3).
Central to the growth factor targeting approach is the VEGF sig-
naling axis. VEGF is a rather specific mitogenic endothelial growth
factor, which is able to stimulate all steps in the angiogenesis cascade,
from the activation of the endothelium to produce proteases, to the
stimulation of migration and proliferation, and the maturation and
attraction of pericytes. In addition, its role in cancer is of pivotal im-
portance, as reflected by the overexpression of VEGF in practically
all types of cancer. The importance of the VEGF signaling axis is fur-
ther evidenced by the large interest within the field of PDT regard-
ing combination therapies that seek to inhibit this pathway. Ferrario
et al. performed the initial study that confirmed a link between
PDT-induced vascular damage, the activation of angiogenic path-
ways, and tumor revascularization. This study showed that this
combination treatment in a breast cancer model reduced VEGF ex-
pression and increased tumor cure rates from 39% with PDT alone
to 80–90% in the combination group . An overview of such tar-
geted combination studies will be given below, where a distinction
will be made between VEGF and other growth factor (−receptor)
targeted approaches and agents that directly target endothelial cell
function (Fig. 3B).
3.1.1. Vascular endothelial growth factor (VEGF) axis targeting agents
The first angiogenesis inhibitor approved for the treatment of can-
cer was the monoclonal antibody based compound bevacizumab
(Avastin®, Genentech). This drug, the activity of which is the neutral-
ization of VEGF, is currently approved as a first-line treatment, in
combination with chemotherapy, for patients suffering from ad-
vanced metastatic colorectal cancer . The initial success of clinical
trials based on the use of bevacizumab set a milestone in the field of
anti-angiogenic cancer therapy [55,56]. The combination of bevacizu-
mab with PDT is approved for the treatment of AMD , and has also
become of interest in the treatment of various forms of cancer .
Following their landmark study in 2000, the group of Ferrario
reported on the combination of Photofrin®-mediated PDT with beva-
cizumab, applied immediately after PDT, in human Kaposi's sarcoma
xenografts in nude mice . This study confirmed the angiogenic re-
sponse induced by PDT through the detection of increased expression
of HIF-1α, VEGF, prostaglandin E2 (PGE2), tumor necrosis factor
alpha (TNF-α) and interleukin 1-beta (IL-1β) following PDT treatment.
More interestingly, a significant increase in long-term tumor
A. Weiss et al. / Biochimica et Biophysica Acta 1826 (2012) 53–70
responses was noted after combination therapy, as compared to ei-
ther monotherapy (55% of mice saw long-term tumor response in
the combination treatment group, compared to 22% and 10% in the
PDT-only and bevacizumab-only treated groups). Additionally, there
was no significant increase in normal tissue toxicity associated with
the better tumor response. These results provided the first proof
that bevacizumab could be used to improve PDT and suggested that
VEGF inhibitors, in general, may ameliorate the clinical efficacy of
PDT in cancer .
Shortly after this study, a similar study examined tumor response
to hypericin-PDT in combination with bevacizumab on bladder carci-
noma xenografts in nude mice . Assessment of tumor volume
following treatment showed that combination therapy (with high or
low dose PDT) resulted in a significantly better tumor response
when compared to control (no treatment) or monotherapy treatment
groups. This study also showed that the treatment of bladder carci-
noma tumors with PDT and bevacizumab resulted in suppressed
expression of VEGF and downregulation in the expression of other
angiogenic molecules (angiogenin, bFGF, EGF, and interleukin-6 and
-8) as compared to PDT-only treated tumors. These findings strongly
support the hypothesis that PDT-induced angiogenesis can be coun-
teracted using subsequent anti-angiogenic therapy.
The same group implemented the use of confocal endomicroscopy to
visualize vasculature following the combination approach as described
above in order to evaluate the angiogenic responses of vasculature to
treatment . This study, which allowed in vivo surface and subsurface
the combination treatment group was significantly more efficient than
Fig. 1. Tumor endothelial responses after photodynamic therapy (PDT) leading to blood flow stasis. A simplified scheme of the different steps after injection of a photosensitizer and
exposure to light. (A) Tumor blood vessel before PDT. (B-E) Magnification of the junction of the tumor vessel. (B) Before PDT endothelial cells are tightly attached to the basement
membrane of the vessel wall, lining the blood vessel. Endothelial cells are connected through tight junctions. (C) After injection of photosensitizer and light exposure, cellular stress
inside the endothelial cells results in disruption of tight junctions, partial retraction and detachment from the vessel wall. (D) Blood gets in contact with the vessel wall collagen and
the clotting cascade is initiated, ultimately leading, through the interaction with fibrinogen, to the formation of a stabilized thrombus, leading to obstruction of the vessel. (E) Due to
the angiogenic switch, endothelial cell proliferation, migration and sprout formation is observed.
Fig. 2. The different steps of the angiogenic cascade.I. Outgrowth of a tumor will involve generation of hypoxia, leading to the onset of angiogenic genes, such as vascular endothelial
growth factor (VEGF) and fibroblast growth factor (FGF). II. The secretion of these factors activates endothelial cells of preexisting nearby capillaries to produce matrix metallopro-
teinases to breakdown the extracellular matrix. This will allow the endothelial cells to start migrating towards the stimulus. III. Endothelial cells proliferate and form vascular
sprouts that can transport blood but are initially very leaky. IV. Only after the formation of a new extracellular matrix and basement membrane, a new blood vessel is available
for oxygenation of the tissue and removal of waste products. New vessels in tumors can be leaky and may allow migration of tumor cells to distant sites.
A. Weiss et al. / Biochimica et Biophysica Acta 1826 (2012) 53–70
either monotherapy. Visualization of blood vessels also showed a de-
crease in tumor vessel density in the bevacizumab monotherapy group,
phologically normalized. On the other hand, blood vessels in the tumors
treated with the combination therapy were leaky, showed loss of func-
tion and revealed the greatest reduction in mean vessel area. Complete
cures were observed in the treatment group receiving PDT and contin-
ued bevacizumab therapy. Immunohistochemistry and immunofluores-
cence studies confirmed these observations, showing a significant
An entirely different approach to inhibiting the VEGF signaling
axis was undertaken in the study by Jiang et al. . Here, the use
of a mix of two antibodies directed to the VEGF-receptor-1 (MF1)
and -2 (DC101) were used as monotherapies or in combination with
Photofrin®-PDT to treat intracerebral U87 glioblastoma xenografts in
nude mice.Inthis study, theangiogenesisinhibitingdrugswereadmin-
istered through i.p. injection every other day from days 8 through 14
after tumor implantation, which corresponded to 24 h following PDT
performed on day 7 after implantation. Monotherapies of PDT or MF1
+DC101 significantly reduced the tumor volume and prolonged sur-
vival of tumor bearing mice. Anti-angiogenic therapy was observed to
decrease proliferation and increase apoptosis of tumor cells. Combina-
tion therapy also increased survival time, while decreasing tumor
volume, showing significantly better outcomes than either monother-
apy. The benefit of the combination, however, was reported to be addi-
tive and not synergistic. Again, a decrease in the expression of the
angiogenic growth factors VEGF and von Willebrand factor (vWF) was
seen in the combination therapy group as compared to the PDT-only
Anti-VEGF therapy combined with verteporfin-PDT has been suc-
cessfully used in the treatment of ocular tumors. Circumscribed cho-
roidal haemangioma (CCH) is an uncommon, benign vascular tumor
manifesting as a discrete smooth, round, orange-red mass located
posterior to the equator, normally in the macular and peripapillary
region. Sagong et al. discussed two patients with CCH who were
treated with intravitreal injection of Avastin® and subsequent
verteporfin-PDT . The result of this clinical application on a
Fig. 3. Main mechanisms involved in tumor angiogenesis on a vascular and cellular level. (A) Overview of tumor blood vessel including different components of its structure and
factors that can influence angiogenesis. (B) A simplified diagram of the angiogenesis targets and inhibitors discussed in this review. Green arrows represent induction and red in-
hibition of signaling. RGD, the tripeptide sequence ligand of (αvβ3-)integrin; EDB-FN, extra domain B containing splice variant of fibronectin associated to tumor blood vessels; L19
Ab, the anti-EDB-FN antibody; MMPs, matrix metalloproteinases; COX-2, cyclooxygenase-2.
A. Weiss et al. / Biochimica et Biophysica Acta 1826 (2012) 53–70
very limited number of patients turned out to be very successful, as
complete regression of lesions was observed for 6–9 months. Fur-
thermore, improved visual acuity was accompanied by complete re-
sorption of subretinal fluid due to partial vessel normalization. PDT
alone, however, has also been used in case studies to successfully treat
patients with CCH resulting in minimal side-effects . Randomized
controlled trials are therefore warranted to confirm the efficacy of
both verteporfin-PDT and combination therapy in circumscribed choroi-
dal haemangioma, and to further investigate the potential of combina-
tion therapies utilizing verteporfin-PDT with anti-VEFG or intravitreal
triamcinolone, for which there is little but encouraging data.
In another case study, verteporfin-PDT in combination with beva-
cizumab was used as an effective and well-tolerated option for juxta-
papillary retinal capillary haemangioma (RCH), another form of
vascular tumor located at the border of the optic nerve head and fre-
quently associated with von Hippel–Lindau syndrome . PDT is
considered the treatment of choice for juxtapapillary RCH, however
the need for multiple treatments can result in damage to surrounding
tissue and poor overall results for patients . The anti-exudative
effect of bevacizumab promises to provide better delineation of RCH
and, consequently, allows for more precise adjustment of PDT irradi-
ation to minimize collateral damage. In this study, a single combina-
tion therapy of bevacizumab and verteporfin-PDT showed long-
lasting effects and, thus, eliminated the necessity of further destruc-
tion by repeated PDT treatment. Long-term follow-up of other cases
is necessary to make a conclusive statement about the efficacy of
combination therapy in the treatment of RCH.
Taken together, these results suggest that the inhibition of VEGF
signaling, through bevacizumab or other molecules, can provide a
viable means to combat PDT-induced angiogenesis, and more im-
portantly, that these combination therapies can help to prevent
tumor recurrence, thereby increasing the overall efficacy of PDT
treatment. The study of bevacizumab in preclinical models, how-
ever, is not entirely straight forward, as controversy exists regarding
the binding affinity of bevacizumab to non-human VEGF. Bevacizu-
mab is a humanized (93% human) antibody, derived from the mu-
rine VEGF monoclonal antibody A4.6.1 [67,68]. A key difference
between human and mouse VEGF-A (mVEGF-A) is that the Gly-88
in human VEGF-A corresponds to Ser-87 in mVEGF-A, a difference
that is located at the core of the protein-antibody interface .
Crystal structure analysis of hVEGF-A when it is bound to bevacizu-
mab, shows a tightly packed interface between the protein and an-
tibody. In the mVEGF-A–antibody complex, where Ser-87 is present,
the addition of two non-hydrogen atoms at the interface prevents a
strong interaction. This indicates that bevacizumab would have a
weak affinity for mVEGF-A compared to hVEGF-A. The study by
Bock et al. , however, reported strong anti-angiogenic effects
of systemically administered bevacizumab on corneal angiogenesis in
ity of bevacizumab in murine models in a study published in 2008 .
They reported a very weak interaction between bevacizumab and
mVEGF-A in the western blot analysis, and no inhibitory effect on
mVEGF-stimulated endothelial cell proliferation or inhibitory action in
in vivo models. The authors of this paper concluded that it is “unlikely
that the in vivo findings described by Bock et al. resulted from a specific
immunoneutralization of mVEGF-A by bevacizumab” .
3.1.2. Other growth factor targeted agents
The epidermal growth factor (EGF) pathway also provides an in-
teresting target for anti-angiogenic and anti-cancer therapies, as it is
known to be involved in the regulation of normal cellular processes
and its overexpression is frequently correlated with the development
Fig. 4. Improvement of PDT by targeted delivery of photosensitizers. (A) Subcutaneous xenografts of human A431 epidermoid carcinoma in nude mice are ablated by PDT after
injection of a single dose of SIP(L19)–PS. Photographs are taken at several points in time after a single dose of irradiation with light. (B) Tumor responses in F9 teratocarci-
nomas after i.v. injections with SIP(L19)–PS (diamonds and circles), SIP(F16)–PS (triangles) or saline (squares) on days 0, 2, 4 and 6 of the treatment schedule. Irradiation
was given daily from days 1 to 8 (in diamonds-, triangles-, and squares graphs). *Pb0.01 vs not irradiated, **Pb0.01 vs saline. (C) Effect of PDT with SIP(L19)-PS on tumor
histology. Sections of F9 tumors excised 1 h after PDT, stained with haematoxylin/eosin and imaged at ×2.5 magnification. (Adapted with permission from Macmillan Pub-
lishers Ltd on behalf of Cancer Research UK: Palumbo et al., Br. J. Cancer, copyright 2011).
A. Weiss et al. / Biochimica et Biophysica Acta 1826 (2012) 53–70
of malignancy . EGF promotes cell cycle progression from G1 to S
phase, thus promoting an increase in the number of proliferating cells
. The overexpression of EGF has been shown to be associated with
a wide array of cancers, including head and neck-, breast-, colon-,
lung-, prostate-, kidney- and bladder carcinomas [72,74]. The use of
EGF inhibitors in combination with PDT has also become of particular
interest, as it has been shown that PDT can result in increased phos-
phorylation of the EGF receptor (EGFR) and increased activity of
downstream signals, such as ERK1 and ERK2, protecting cells from
PDT-induced damage. Recently, a review was published on studies
examining the activity of EGFR pathways in PDT induced cell death
. The studies indicated that the inhibition of EGFR plays an impor-
tant role in post-PDT effect and, that following PDT, both normal and
cancerous cells become less sensitive to EGF signaling. Additionally,
the activation of ERK 1/2 and EGFR-PI3K-Akt pathways following
PDT appeared to aid in cell survival. These results indicate that the
inhibition of EGFR in combination with PDT may provide potential
for clinical applications. The combination of PDT with EGF inhibi-
tors, particularly cetuximab (Erbitux®), an antibody which binds
competitively with the EGFR, has shown good potential to increase
the efficacy of therapy, not only through the inhibition of PDT-
induced angiogenesis, but also through the inhibition of other
PDT-activated pathways in tumor cells.
An early study examining the synergistic effects of EGFR inhibition
and verteporfin-PDT was performed by Del Carmen et al.  on an
ovarian carcinoma model in nude mice. Treatment of tumor bearing
mice with PDT followed by cetuximab resulted in synergistic inhibi-
tion of tumor growth and an overall reduction in tumor size (9.8%
of the control) as compared to verteporfin-PDT (38.2%) or cetuximab
alone (66.6%). The overall survival rate in this group was found to be
three times higher than non-treated mice, i.e. at day 180, 3/9 mice in
the combination group were alive compared to 0/12 in the drug only
group and 1/10 in the PDT-only group, while all non-treated mice
died by day 40. The in vivo effects of administering hypercin-PDT fol-
lowed by cetuximab in human bladder carcinoma-bearing nude mice
have also been reported . The results of this study showed strong
inhibition of tumor growth in the combination therapy group, with a
relative tumor inhibition of 93% compared to control mice, while
monotherapies of PDT and cetuximab showed only 57.8% and 74.8%
inhibition of tumor growth, respectively. Although an initial accelera-
tion in tumor growth was seen one week following treatment, this
was followed by a decrease in tumor size, eventually leading to com-
plete tumor regression. The combination therapy group showed an
increase in apoptosis and downregulation of EGFR expression, as
well as inhibition of phosphorylation at most of the EGFR phospho-
rylation sites and downregulation of EGFR target genes, such as cyclin
D1 and c-myc. These researchers also performed an additional study,
applying hypericin-PDT followed by the administration of the EGFR
inhibitor cetuximab, the VEGF inhibitor bevacizumab, or both drugs,
in vitro on bladder cancer and HUVEC cells and in vivo on a murine
bladder tumor model . Although both drugs showed efficient in-
hibition of cell migration when administered alone, the combination
of cetuximab and bevacizumab did not show a significant increase
in the inhibition of cell migration. Cell invasion and tube formation,
however, were suppressed by both drugs individually, while being
significantly more suppressed by the combination of both drugs,
even resulting in complete prevention of tube formation. Assess-
ment of tumor volume in treated mice revealed that mice receiving
combination therapies (PDT+bevacizumab, PDT+cetuximab, or
PDT+bevacizumab+cetuximab) showed significantly greater treat-
ment responses when compared to control and PDT-only treated
groups. Mice treated with bevacizumab and cetuximab only exhibited
tumor regression, but no complete cures, indicating that while anti-
angiogenic drug therapy is somewhat effective alone, its combination
with hypericin-PDT results in a much greater tumor response (see
Fig. 5). These results support the hypothesis that the combination of
hypericin-PDT with the angiogenesis inhibitors bevacizumab and/or
cetuximab can help to prevent the PDT-induced angiogenic process,
improving treatment efficiency and resulting in complete cures in
tumor bearing mice.
Yip et al. used photochemical internalization (PCI) in order to
achieve drug delivery and PDT . PCI is a drug delivery method
where endocytosed macromolecules can be directly delivered into
the cytosol of cells and PDT can be performed upon the activation of
PSs by light . In this study, cetuximab was linked to a type I
ribosome-inactivating protein called saporin (cetuximab-saporin),
thereby targeting the immunotoxin saporin to EGFR-expressing
cells. Results showed the selective binding and uptake of the drug in
EGFR-positive cells, as well as reduced levels of unspecific cetuximab
uptake. In addition, non-conjugated cetuximab therapy alone only
decreased cell viability by 10% at its highest concentration and
showed no enhancement of cytotoxicity with increased doses of
non-conjugated cetuximab when used in combination TPPS2a-PDT,
mimicking the PCI protocol. Combination of cetuximab and saporin
with TPPS2a-PDT resulted in a triplication of the cytotoxicity, due to
the action of cetuximab, PDT and PCI of saporin.
These studies have shown that the combination of PDT with EGF
inhibition has great potential in the treatment of certain types of can-
cer. This is of particular interest as the overexpression of EGFR in
tumor cells has been shown to be an indicator of aggressive and che-
motherapy resistant tumors . In addition, PDT has been shown to
be effective against epithelial ovarian carcinoma that is refractory to
Fig. 5. (A) Tumor growth inhibition by PDT, anti-angiogenesis or the combination. The combination therapy groups of PDT+Avastin®, PDT+Erbitux®, and PDT+Avastin®+Erbitux®
al., Lasers in Surgery and Medicine, 2011, after receiving permission from the publisher).
A. Weiss et al. / Biochimica et Biophysica Acta 1826 (2012) 53–70
chemotherapy and radiation therapy [79,80] and is known to over-
express EGFR. Overall, these studies indicate that synergistic effects
may be seen when combining individual monotherapies which target
and affect non-overlapping pathways , resulting in maximal
treatment benefits with minimal additional side effects.
3.1.3. Tyrosine kinase inhibitors
Complex signaling in malignant cells and cellular heterogeneity
within each tumor dictates the necessity of targeting multiple signal-
ing pathways . The above-mentioned strategies target only single
growth factor receptors. There is not only a theoretical advantage of
targeting multiple pathways, as for example, it has been demonstrat-
ed for non-small-cell lung cancer (NSCLC) that there is a strong rela-
tionship between the EGFR and VEGFR pathways . It is thus
reasonable to hypothesize that targeting both pathways with sepa-
rate drugs would have an additive or even a synergistic inhibitory
effect on tumor growth. With the development of small molecule
tyrosine kinase inhibitors, it is possible to target multiple growth
factor receptors by blocking their intracellular phosphorylation
sites and, thereby, their signaling capacity. Several of these drugs
have received FDA approval in the treatment of solid tumors, such as
sunitinib (Sutent®) for the treatment of renal cell carcinoma. Suniti-
nib targets and inhibits multiple kinase pathways, among which are
VEGFR2, platelet derived growth factor receptor (PDGFR), c-kit, FLT3
and RET. Sorafenib (Nexavar®) targets VEGFR2 and -3, PDGFR and
FLT-3 and is now registered for treatment of advanced renal cell car-
cinoma and hepatocellular carcinoma. Some of these compounds
have a more narrow spectrum of action, such as erlotinib (Tarceva®),
which more selectively inhibits epidermal growth factor receptor
(EGFR) and is approved for NSCLC and pancreatic cancer. These com-
pounds have been tested in combination with PDT to combat the sec-
ondary angiogenesis response in the expectation that broader
receptor targeting may result in more robust inhibition of angiogene-
sis, further enhancing the therapeutic benefits of PDT.
A recent study was performed by us aimed to compare the ability
of different anti-angiogenic compounds to prolong verteporfin-PDT-
induced vascular occlusion in the chicken chorioallantoic membrane
(CAM) model . In this study, the action of bevacizumab was com-
pared with various clinically approved TKIs with varying spectrums of
action, including sorafenib , erlotinib  and sunitinib . The
results of this study showed that all compounds tested were capable
of inhibiting both physiological angiogenesis in the CAM, as well as
verteporfin-PDT-induced angiogenesis, resulting in prolonged vascu-
lar occlusion in the treated areas. Image processing analysis of the
treated blood vessels revealed that sorafenib induced the strongest
anti-angiogenic effect, outperforming the other drugs at improving
verteporfin-PDT. Interestingly, the morphology of regrown blood ves-
sels in the PDT treated areas differed from untreated blood vessels
. In addition, there was also a clear difference in the morphology
of blood vessels growing after treatment with the different anti-
angiogenic drugs (see Fig. 6). The results of this study showed that
the inhibitor with the broadest spectrum of action resulted in the
strongest inhibition of PDT-induced angiogenesis in the CAM.
A study by Dimitroff et al.  verified the action of a broad
(PD166285, inhibiting c-src, FGFR-1, PDGFR-β and EGFR) and a nar-
row (PD173074, inhibiting selectively FGFR-1) spectrum experimen-
tal TKI in combination with Photofrin®-PDT, in vivo in the murine 16c
breast carcinoma model. Independent of their spectrums of action,
oral administration of either PD166285 (1–25 mg/kg) or PD173074
(25–100 mg/kg) generated dose-dependent inhibition of angiogene-
sis. Additionally, significantly prolonged tumor regression was
achieved with daily doses of PD166285 (5–10 mg/kg) or PD173074
(30–60 mg/kg) following Photofrin®-PDT, as compared to Photo-
The use of the TKIs SU5416 and SU6668 in combination with
hypericin-PDT was investigated by Zhou et al. in human nasopharyn-
geal carcinoma (NPC) bearing BALB/c athymic mice . Single
hypericin-PDT alone resulted in the increased expression of angio-
genic factors, including VEGF, HIF-1α, COX-2 and bFGF, when com-
pared to control mice. The combination of single hypericin-PDT
with subsequent administration of SU6668 resulted in the best thera-
peutic response. It increased the survival rate of treated mice to 100%,
Fig. 6. Sustained photodynamic vaso-occlusion by angiogenesis inhibitors. (A) Fluorescence angiograms taken before (A), 48 h after Visudyne®-PDT (0.20 mg/kg embryo weight,
λex=420 nm, λem>470 nm; light dose of 20 J/cm2and an irradiance of 50 mW/cm2, drug-light interval 1 min) alone (B), and combination therapy of PDT with topically admin-
istered angiogenesis inhibitors erlotinib (C) sunitinib (D), sorafenib (E), and bevacizumab (F). All agents were applied twice (immediately and 24 h post PDT). The vasculature
is visualized by FITC-dextran fluorescence angiography (25 mg/kg, 20 kDa, λex=470 nm, λem>520 nm). (G) Quantifications of 2 descriptors for two concentrations of the
drugs. Mean values are shown, error bars represent standard error of the mean.
A. Weiss et al. / Biochimica et Biophysica Acta 1826 (2012) 53–70
as compared to 0%, 33%, 75%, 33%, and 33% in the control, hypericin-
PDT alone, SU5416 alone, SU6668 alone and PDT followed by
SU5416 treatment groups, respectively. The better therapeutic re-
sponse of SU6668 with hypericin-PDT was, at least partially, attribut-
ed the broader spectrum of action of SU6668, blocking the signaling
of receptors for VEGF, PDGF and FGF. An interesting observation,
however, was that even though SU6668 was more effective at inhi-
biting tumor growth in combination with hypericin-PDT, following
the arrest of anti-angiogenic treatment, control of tumor volume
lasted much longer in the SU5416-treated mice than in SU6668-
treated mice. These findings are supported by an in vitro study
which showed that SU5416 resulted in long-lasting inhibition of
VEGFR phosphorylation and function, reducing proliferation of endo-
thelial cells , while similar studies did not show the same effect
for SU6668. This may also help to explain why SU6668 was less effec-
tive than SU5416 as a monotherapy to increase the overall survival
of treated mice.
Summarizing, angiogenesis is an intricately regulated biological
process, which may be more effectively inhibited through novel
drug strategies, such as with the use TKIs, which target multiple dif-
ferent pathways simultaneously. Even though many of these studies
seem to suggest that the inhibition of more than one angiogenesis
signal pathway could result in more efficacious inhibition of PDT-
induced angiogenesis, it is clear that more knowledge is needed to
substantiate this claim. This advantage, however, may in fact be a
double-edged sword, as kinase inhibitors frequently have off-target
interactions, which can result in drug related adverse side-effects
and toxicities. For this reason, the spectrum of the drug in question
must be carefully taken into account in order to maximize its thera-
peutic effects, while maintaining tolerable side effects.
3.2. Non-growth factor targeted agents
3.2.1. Matrix metalloproteinases (MMPs)
In addition to growth factor mediated signals stimulating angio-
genesis, there are many other, later steps in the angiogenic cascade
that are amenable for intervention to improve the effects of PDT.
First of all, the release of proteases, particularly matrix metalloprotei-
nases (MMPs), can be targeted. MMPs are produced by endothelial
cells in order to prepare the extra-cellular matrix for invasion by vas-
cular sprouts. In addition, specifically targeting the processes of endo-
thelial cell migration and proliferation provide attractive approaches
for angiogenesis inhibition, as well as interfering with vascular mat-
uration steps, such as the formation of the basal membrane and the
attraction of pericytes. MMPs are proteolytic enzymes, which are
known to aid in the degradation of components of the extracellular
matrix. They play a critical role in cancer cell invasion and metasta-
sis, as well as in angiogenesis [89,90].
A variety of studies have examined the effects of PDT on MMP
expression in various tumor cell lines and tumor models. In one
such study, Du et al.  performed hypericin-PDT on two nasopha-
ryngeal cancer (NPC) cell lines, well differentiated HK1 cells and
poorly differentiated CNE-2 cells, and in vivo on HK1-NPC tumors
in mice. It was found that PDT increased MMP-1 protein and
mRNA expression, both in cell lines and in vivo in tumor models.
A subsequent study by the same group  reported on the effects
of hypericin-PDT on MMP-9 expression in HK1 NPC in vitro and in
vivo, reporting a downregulation in the expression of MMP-9. This
study also reported that PDT inhibited the secretion of granulo-
cyte–macrophage colony stimulating factor (GM-CSF), resulting in
decreased transcriptional activity in two of its downstream proteins,
activator protein-1 (AP-1) and nuclear factor (NF)–κB. Additionally,
incubating cells in exogenous GM-CSF prior to PDT treatment
resulted in an additional decrease in MMP-9 production. This find-
ing indicate that PDT-induced downregulation of MMP-9 is mediat-
ed by the inhibition of GM-CSF and leads to the modulation of AP-1
and NF-kB. It is interesting to note here that decreased activity in
NF-κB has been associated with increased angiogenesis, as angio-
genesis inhibition involves NF-κB activity [93,94]. The decreased ex-
pression of MMP-9 following PDT has also been noted in two other
studies. One study by Au et al.  showed that the treatment of gli-
oma cells with ALA-, Photofrin®- and calphostin C-PDT resulted in a
reduction in cell migration, which was partially attributed to a reduc-
tion in MMP-9 production. The other study, performed by Sharwani
et al. , showed that PDT suppressed the production of MMP-9 in
keratinocyte cell lines from human oral squamous-cell carcinoma
(SCC). A study by Chu et al.  examined the effects of Hexyl-ALA
(ALA-H)-PDT and 5-ALA-PDT on MMP expression in the medulloblas-
toma cell line TE-671, showing a small, yet significant, decrease in
MMP-2 expression and an inhibition of cell migration 24 h post
PDT, which was believed to be, at least partially, attributed to MMP-
2 downregulation. Interestingly, this study did not report a change
in the expression of MMP-9 in PDT treated cells.
Although changes in MMP expression after PDT are not unidirec-
tional in the sense of upregulation, combinations with MMP inhibi-
tors have been performed. Ferrario et al.  studied the effects of
combining Photofrin®-PDT with Prinomastat®, an inhibitor of
MMP-2 and -9, on mouse mammary carcinoma, mouse brain endo-
thelial cells, mouse macrophages and human fibrosarcoma cells.
This study showed that PDT alone increased the expression and activ-
ity of MMP-9 (as well as MMP-1, -3, and -8) 24 h following therapy.
In this study, the expression and activity of MMP-2, however, was
not affected by PDT. In addition, PDT resulted in endothelial cell ex-
pression of MMP-9, as well as an influx of MMP-9 expressing inflam-
matory host cells. Administration of Prinomastat® significantly
improved PDT-mediated tumor response without affecting normal
skin photosensitization. While these results may seem contradictory
to the study by Du et al. in 2007, which showed a decrease in MMP-
9 expression following PDT, it should be stressed that the BA mam-
mary cells in the study of Ferrario et al. did not secrete any detect-
able levels of MMP-9, nor were they induced to do so by PDT. The
increase in MMP-9 seen in this study can be attributed to the induc-
tion of MMP-9 expression in endothelial cells and the influx of
MMP-9 expressing inflammatory host cells . Treatment with
the MMP inhibitor Prinomastat® alone reduced the rate of tumor
growth, but did not result in a significant decrease in tumor size
or long-term remissions. The combination of PDT with Prinoma-
stat® therapy, however, resulted in increased cure rates of 46%,
compared to only 20% in PDT-only treated mice, indicating the ther-
apeutic potential of combining PDT with MMP inhibiting drugs.
Sufficient evidence has been acquired to indicate a pivotal role of
MMPs, not only in the progression of many forms of cancer, but also
in the initiation of the angiogenic process. However, the role of
MMPs in the response to PDT is still not completely clarified. In fact,
the regulation of MMPs after PDT has been controversial in literature;
however several studies have reported a potential benefit for their
combination with PDT. Additionally, the fact that many MMP inhibi-
tors have failed for cancer treatment in translational development
during clinical studies is mainly due to unacceptable adverse side
effects. Therefore, MMP inhibitors should not be select as the first
choice of adjuvant therapy to PDT.
3.2.2. Cyclooxygenase-2 (COX-2)
Cyclooxygenase-2 (COX-2) is another known inducer of angio-
genesis, acting through the production of VEGF and resulting in in-
creased vascular sprouting, migration and tube formation [99,100].
COX-2 functions by performing the rate limiting reaction in the con-
version of arachidonic acid to prostaglandins (PGs). PGs are involved
in the regulation of biological processes ranging from immune func-
tion and kidney development, to modulating platelet aggregation,
inflammation , and the induction of VEGF expression. Through
these functions, it aids in the processes of tumor progression and
A. Weiss et al. / Biochimica et Biophysica Acta 1826 (2012) 53–70
metastasis . More importantly, COX-2 plays a role in tumor
growth and angiogenesis. It has been shown to be over-expressed
in many cancer types including colon , breast  and lung
 cancers, and studies have shown that overexpression of
COX-2 is sufficient to induce tumorigenesis in animal models
. The combination of PDT with COX-2 inhibitors has also
been examined in a variety of studies and has shown good thera-
peutic potential for increasing the efficacy of PDT treatment.
Ferrario et al.  examined the in vivo and in vitro effects of
Photofrin®- and chlorine(NPe6)-based PDT, alone and in combina-
tion with a selective COX-2 inhibitor, NS-398 (N-(2-cyclohexyloxy-
4-nitrophenyl)-methanesulfonamide), on radiation-induced fibro-
sarcoma (RIF) tumors. Results of in vitro assays showed that PDT
can effectively activate COX-2 expression in multiple cell lines. In
addition, PDT resulted in increased levels of PGs (particularly
PGE2), which were directly related to COX-2 activation, as selective
inhibition of COX-2 with NS-398 resulted in a reduction in the syn-
thesis of PGs. In vivo assays confirmed these findings showing in-
creased activity of COX-2 in PDT treated mice, as measured
through PGE2levels in tumor lysates. Inhibition of COX-2 by NS-
398 resulted in the reduction of the PDT-induced increase in PGE2
production in treated tumors. In addition, PDT induced an increase
in VEGF expression, which was inhibited in mice treated in combi-
nation with NS-398, indicating that COX-2 is indeed involved in
the PDT-induced expression of pro-angiogenic molecules. Finally,
PDT in combination with NS-398 resulted in a significant increase
in tumor cures when compared to PDT alone (NS-398 alone did
not affect tumor response), while having no significant effect on
normal tissue, indicating its potential for enhancing the efficacy of
PDT treatment with minimal added side-effects. Harvey et al. also
noted decreased tumor weights after treatment with NPe6-PDT in
single dose when combined with the COX-2 inhibitor NS-398 as
compared to PDT alone on colon-38 tumors in mice .
A study by Makowski et al.  examined the effects of Photo-
frin®-PDT on C-26 cells alone and in combination with COX-2 in-
hibitors NS-398, rofecoxib or nimesulide. Interestingly, treatment of
cells or C-26 tumor bearing mice with COX-2 inhibitors prior to PDT
treatment did not sensitize cells or tumors to PDT-induced damage.
In contrast, administration of COX-2 inhibitors following PDT in-
creased the antitumor effects of PDT significantly, resulting in com-
plete cures in 6 out of 8 mice. These results suggest that the
antitumor activities of COX-2 inhibitors in combination with PDT
are indirect, likely acting through the inhibition of angiogenesis. Fer-
rario et al.  also combined Photofrin®-PDT with the COX-2 in-
hibitors celecoxib or NS-398 in the mouse BA mammary carcinoma
model. In vitro experiments showed that combination therapy with
celecoxib or NS-398 increased the cyctotoxic effect of PDT and in-
creased apoptosis in the mouse mammary carcinoma cell line. Re-
duced expression of pro-inflammatory molecules interleukin-1β and
TNF-α and increased expression of the anti-inflammatory cytokine
interleukin-10, were also noted in the combination treatment group.
Yee et al. studied the in vivo effects of non-curative doses of
hypericin-PDT in combination with the COX-2 inhibitor celecoxib on
NPC in vivo . PDT monotherapy resulted in the hypoxia-
induced upregulation of COX-2 and HIF-1α genes and a reduction in
tumor size. Tumor inhibition, however, was followed by tumor
regrowth by 24–48 days after PDT treatment, which could likely be
attributed to COX-2 and HIF-1α upregulation. Tumors treated with
PDT and celecoxib showed a downregulation of COX-2, HIF-1α, and
VEGF-A isoforms 165 and 121 genes, compared to PDT-only treated
tumors. Interestingly, tumors first treated with celecoxib 6 h after
PDT showed the best control of tumor regrowth, while tumors first
treated with celecoxib 24 h after PDT showed no control of tumor
growth, indicating that the time of celecoxib administration is an im-
portant factor affecting tumor response. A similar and confirming
study was reported by Akita et al. .
The role of COX-2 suppression in PDT-induced angiogenesis, how-
ever, is still not completely understood and has been specifically in-
vestigated by many different groups. The group of Hendrickx
investigated the pathways involved in the PDT-induced expression
of COX-2, reporting that COX-2 regulation in tumor cells is achieved
through p38 MAPK and phospholipase A2 [113,114]. Additionally,
these studies indicated that the inhibition of p38α MAPK blocked
the release of VEGF and inhibited tumor-promoted endothelial cell
migration. The combination of PDT with PD169316, a selective P38
MAPK inhibitor, resulted in more effective inhibition of VEGF syn-
thesis than PDT in combination with NS398, a COX-2 inhibitor. More-
over, a genetic deficiency of p38α MAPK shifted the balance towards
cell death in a manner which could not be reproduced by COX-2 inhi-
bition, indicating that the targeting of p38α MAPK could surpass the
ability of COX-2 inhibition, as it most likely also targets additional an-
giogenic and cell survival signaling pathways.
The mechanisms of PDT-induced COX-2 expression, as well as
the effects of COX-2 expression on the activation of angiogenesis
and cell survival pathways, have been the topic of many studies
over the past decades. The combination of PDT with COX-2 inhibi-
tion appears to result in increased efficacy through the inhibition
of angiogenesis .
3.2.3. Kinase inhibitor p21
The studies presented above describe some of the endogenous
non-growth factor molecules, such as MMPs and COX-2, which are in-
volved in the activation of angiogenesis following PDT and can be
used as targets in the inhibition of PDT-induced angiogenesis. TNP-
470 is one of the first identified non-endogenous angiogenesis inhib-
itors. It is a synthetic analogue of fumagillin, which acts as a potent
angiogenesis inhibitor through strong inhibition of endothelial cell
proliferation and migration . TNP-470 has been shown to arrest
endothelial cell growth by activating P53 through a unique mecha-
nism in endothelial cells [116,117]. Yeh et al.  found that treat-
ment of endothelial cells with nanomolar concentrations of TNP-470
resulted in the accumulation of p21CIP/WAFproteins, correlating to
dose-specific inhibition of endothelial cell growth. It was shown
that TNP-470 engaged the p53 pathway to exert p21CIP/WAF-depen-
dent G1 checkpoint control in endothelial cells. These findings were
extended in vivo by showing that p21CIP/WAF−/−mice were unre-
sponsive to TNP-470 in the corneal micropocket angiogenesis assay.
Due to its potent anti-angiogenic activity, the use of TNP-470 in com-
bination with PDT has also been briefly investigated.
Solban et al. reported subcurative verteporfin-PDT-induced ex-
pression of VEGF in an orthotopic model of LNCaP prostate cancer
. It was shown that verteporfin-PDT followed by the administra-
tion of TNP-470 not only inhibited the increase in VEGF secretion,
which was seen in PDT-only treated animals, but also reduced local
tumor growth rate, tumor volume, and the fraction of animals with
lymph node metastases . Interestingly, administration of TNP-
470 prior to PDT resulted in less effective control of tumor growth.
It should be mentioned that certain anti-proliferative and cyto-
static agents, such as chemotherapeutics, have been shown to exhibit
intrinsic anti-angiogenic activity when administered over a long time
period at low dose . These so called metronomic dosing sched-
ules have been tested in clinical trials. Additionally, other experimen-
tal approaches have been tested in the context of PDT [121–124].
4. PDT in combination with vascular normalization through
There is a large body of research in the literature in regards to
anti-VEGF therapy in combination with conventional treatment regi-
mens, such as chemo- and radiation therapies. Tumor blood vessels
are known to be irregularly shaped, creating uneven blood flow to
different parts of the tumor. In addition, the capillary endothelial
A. Weiss et al. / Biochimica et Biophysica Acta 1826 (2012) 53–70
cells lining the inner surface of tumor capillaries are often discon-
tinuous, resulting in vessel leakiness. These structural characteristics
contribute to tumor vascular hyper-permeability, non-uniform
blood flow and distribution, and high interstitial pressure, which
together limit the effectiveness of anti-cancer chemo- and radio-
therapies by impairing the delivery of drugs and oxygen to the
tumor site. Interestingly, anti-angiogenic treatments have been
shown to have a temporary normalization effect on tumor vasculature
. This subsequently leads to normalization of the interstitial tissue
pressure and increased oxygenation. Therefore, it has been hypothe-
sized that the use of anti-angiogenic therapy prior to the application
of these conventional anti-cancer strategies may result in synergistic
anti-tumor activity. This may be a result of improved drug delivery
of chemotherapeutic agents, which can penetrate deeper into the
tumor tissue due to the lower tissue pressure. For radiotherapy,
which is known to be more efficacious in well-oxygenized tissues,
activity will increase due to normalized circulation in the tumor tis-
sue . PDT is also a strategy that is dependent on oxygenation of
the target tissue. Therefore, it has been hypothesized that PDT can be
more efficacious when applied after angiostatic therapy , as it is
the case for other treatment regimens [63,125,128]. Nevertheless, the
overall and eventual effect of anti-angiogenic therapy is to decrease
microvasculature, leading to a decrease in circulation and tissue oxy-
genation [126,127]. It is clear that a deeper understanding of the
mechanisms responsible for the formation of abnormal tumor vascu-
lature is required to further improve future therapeutic success .
5. PDT in combination with vascular disrupting agents (VDA)
Vascular disrupting agents (VDA) are a separate class of com-
pounds, which are comprised of agents that target and disrupt al-
ready matured and established tumor vasculature. These agents
cause an instantaneous vascular shutdown, leading to extensive
necrosis at the tumor core [129,130]. It is known that VDAs act well
in the tumor core, but leave cells at the rim of the tumor alive, prob-
ably as a result of the continuous diffusion of nutrients and oxygen
from the surrounding tissues . It has even been demonstrated
that such compounds may work better on larger tumors than on
smaller ones .
Siemann and Shi evaluated the antitumor efficacy of combining
bevacizumab with VDAs, combretastatin (CA4P) or OXi4503, in a
tumor model of human clear cell renal cell carcinoma, Caki-1.
This study demonstrated marked enhancement of tumor response
in mice treated with bevacizumab in combination with either CA4P
or OXi4503 in a preclinical setting, and provided an experimental
basis for the consideration of such a treatment strategy in the clinics.
VDAs have previously been combined with other treatment mo-
dalities, like chemotherapy, giving better results than the respective
single agents without increased host toxicity . Mechanistically,
vascular disruption would lead to decreased blood flow and pro-
longed entrapment of chemotherapeutic agents within the tumor
mass. Unfortunately, a recently published randomized phase III
placebo-controlled trial of carboplatin and paclitaxel with or without
the vadimezan (5,6-dimethylxanthenone-4-acetic acid; ASA404 or
DMXAA), although well tolerated, failed to improve frontline efficacy
in advanced NSCLC . However, the combination of vadimezan
with radiotherapy gave promising results in a preclinical model .
There are only a few combination studies reported on VDAs and
photodynamic therapy in preclinical models. Treatment with low
dose DMXAA two hours prior to short-duration/ high irradiance
photochlor-PDT in CT-26 colon carcinoma-bearing mice showed a
synergistic interaction, resulting in long-term cures in 60% of treated
animals . Moreover, the combination therapy resulted in signif-
icantly less peritumoral edema than the dose of PDT monotherapy re-
quired for an equivalent cure rate. These results indicate that this
combination has the potential to improve treatment efficacy and
He et al. tested the combination of verteporfin-PDT with combre-
tastatin in SVEC4-10 mouse endothelial cells and found it significantly
increased endothelial cell apoptosis as compared to the single therapy
. In a study on the PC-3 prostate tumor model, it was found that
combretastatin highly enhanced tumor response to verteporfin-PDT,
when both treatments were used in sub-lethal regimens. Pretreatment
with the VDA decreased the rate of blood flow in tumors, while making
them more sensitive to verteporfin-PDT. This observation is rather
counterintuitive and the mechanisms underlying this phenomenon re-
Promising effects were obtained for the combination of verteporfin-
cells and PC-3 prostate tumor cells . PI3K is an important enzyme
involved in extracellular signal transduction. The study showed an in-
creased extent of commitment to apoptotic cell death in SVEC, and
slower, but increased, commitment to autophagy in PC3 cells following
verteporfin-PDT with PI3 kinase inhibitors. This increase in endothelial
cell death would be of interest in the context of vascular-PDT aimed
at faster vessel ablation. A similar principle was used by Morrero et al.
who combined topically applied vadimezan with aminolevulinic acid
(ALA)-PDT  in Colon26 murine colon adenocarcinoma bearing
mice. The onset of blood flow reduction was rapid in tumors treated
with both ALA-PDT and vadimezan. CD31-immunostaining of tumor
sections confirmed vascular damage following the topical application
of vadimezan. Tumor weight measurements revealed enhanced tumor
growth inhibition with combination treatment, as compared with
ALA-based PDT or vadimezan treatment alone. In conclusion, vadime-
zan as a topical agent enhances treatment efficacy when combined
a promising therapeutic strategy, which is even more supported by the
finding that some of these compounds have intrinsic anti-angiogenic
6. PDT with photosensitizers targeted specifically to the
Tumor angiogenesis does not only provide a target for combi-
nation therapy, but also provides an altered vasculature with endo-
thelial cells that have adapted to the increased metabolic needs of
the tumor cells. This activated tumor endothelium has an altered mo-
lecular make-up [142,143] providing targets for selective delivery of
drugs, including PSs . Recently, the idea of selectively targeting
the tumor vasculature, through the conjugation of PSs to antibodies
or molecules which bind to markers of an angiogenic endothelium,
was challenged. Such strategies aim to increase the efficacy of PDT,
while reducing normal tissue toxicity .
6.1. ED-B domain of fibronectin
During certain stages of embryonic development, fibronectin can
be alternatively spliced to generate a variant that has an extra domain
B, which later in life is rarely expressed and confined to pathological
conditions, including cancer. This extra domain B (ED-B domain)
of fibronectin is also a marker of angiogenic vasculature in tumors
. Antibodies targeting ED-B-fibronectin, such as the scFv (L19)
antibody fragment, therefore can be used to selectively deliver thera-
peutic agents to angiogenic tumor endothelium. Such targeting has
been confirmed by the accumulation of radio-labeled L19 antibody
(L19(scFv)2) in glioblastoma and lung carcinomas . These find-
ings suggest the potential for increasing the selectivity and efficacy
of PDT through direct targeting of PSs to markers of angiogenic
This concept was tested by Birchler et al.  using PSs which
were chemically bound to L19 antibodies. These conjugates were
A. Weiss et al. / Biochimica et Biophysica Acta 1826 (2012) 53–70
shown to selectively target neovessels in a rabbit model of ocular an-
giogenesis. In this study, ED-B-fibronectin targeted PDT resulted in
complete and selective occlusion of neovessels, which corresponded
with endothelial cell apoptosis in targeted vessels, while blood ves-
sels of the conjunctiva and other ocular structures were left unaf-
fected . The same group also conjugated porphyrin-based PSs
to the L19 antibody in its small immune protein (SIP) format, SIP
(L19)-PS, which is believed to have better accumulation in tumor
tissue. These SIP(L19)-PS were used in the PDT treatment of aggres-
sive tumors, including an SCC model (A431) implanted in the skin
of nude mice. The results of this study showed that PDT using
these targeted PSs had a strong anti-cancer effect, resulting in com-
plete, long-term (100 days following treatment) tumor eradication
 (see also Fig. 4). The treatment induced extensive hemorrhage
and edema. Additionally, it was observed that natural killer cells
were required in order to achieve complete tumor ablation, as re-
moval of these cells resulted in a transient inhibition of tumor
growth followed by tumor progression.
Fabbrini et al. showed that intravenous injection and subsequent
irradiation of the porphyrin based PS SnChe6 conjugated to L19 anti-
bodies resulted in the arrest of tumor growth in mice with subcu-
taneous tumors (FE8 sarcoma, F9 teratocarcinoma and C51 colon
adenocarcinoma) . This study showed that the SIP form of the
L19 antibody was more effective at targeting tumor vasculature
than the scFv format, inducing a significant reduction in tumor mass
6 days after irradiation. Treating mice repetitively resulted in the sta-
sis of tumor growth until day 20. Additionally, the average tumor
weight in these mice was significantly lower than in mice treated
with PSs conjugated to an antibody lacking ED-B specificity (0.04 vs
1.10 g) . These studies indicate that the ED-B domain of fibro-
nectin can be used to selectively target the angiogenic endothelium
of tumor vasculature and, more importantly, that targeted PSs can in-
deed increase the anti-tumor activity of PDT.
Integrins are a group of adhesion molecules, which are involved in
a wide array of biological functions. Endothelial cells have integrins to
aid in their adherence to each other and to the extracellular matrix.
These integrins consist of an α and a β subunit, for which several dif-
ferent genes are available. Generally, integrins which contain a β1-
subunit are involved in matrix interactions. A specific integrin, the
αvβ3integrin, which binds to tri-peptide Arg-Gly-Asp (RGD) contain-
ing proteins, was found to be overexpressed in angiogenically stimu-
lated endothelial cells of tumors . The αvβ3integrin is believed
to play a critical role in angiogenesis by mediating the adhesion of
endothelial cells to fibronectin, fibrinogen, laminin, collagen, vWF
and osteopontin, through their RGD-moiety . Additionally, the
inhibition or blocking of αvβ3integrin, by RGD peptides or antibodies,
results in endothelial cell apoptosis, indicating that this integrin also
plays a role in endothelial cell survival [152,153]. Therefore, the tar-
geted delivery of PSs using RGD peptides has become a topic of inter-
est, as it may result in the increased selectivity of PDT, with the
possibility of additive anti-angiogenic effects due to the RGD peptide.
Chen et al.  investigated RGD targeted Photofrin® encapsu-
lated in a surfactant-like tetra-tail amphiphilic nanoparticle. The ef-
fects of the Photofrin®-loaded micelles were tested in vitro on two
different cell lines, human cervix carcinoma (HeLa) and human em-
bryonic kidney transformed 239 (293 T), the first of which is known
to over-express αvβ3integrin. The porphyrin-loaded micelles showed
no apparent dark toxicity in HeLa cells, but showed significant photo-
toxicity upon irradiation with light. The amount of Photofrin® inter-
nalized by the HeLa cells was shown to be much greater than that
internalized by the 293 T cells, suggesting the success of the RGD tar-
geting ligand. Furthermore, it was shown that cell death was due to
the presence of reactive oxygen species, and that Photofrin® had
accumulated in the nucleus of these cells, indicating successful tar-
geted drug delivery.
Integrins can also be targeted using adenovirus type 2 structural
proteins, such as hexon and penton bases and fiber antigens, as
these are known to contain the RGD peptide sequence . Allen
et al.  covalently coupled such adenoviral proteins to tetrasulfo-
nated aluminum phthalocyanine (AlPcS4) PSs and tested their effects
in vitro and in vivo. All of the AlPcS4- protein complexes induced
greater cytotoxicity than the unconjugated AlPcS4PSs. Additionally,
in vivo experiments in nude mice with EMT-6 tumors showed en-
hanced anti-tumor activity with the targeted PSs.
Angiogenic blood vessels can also be targeted using the 5-mer
peptide Ala-Pro-Arg-Pro-Gly (APRPG), which has been shown to spe-
cifically bind to angiogenic tumor blood vessels . Ichikawa et al.
 reported the use of polyethylene glycol (PEG)-modified lipo-
somes to encapsulate verteporfin, which were then functionalized
using the APRPG pentapeptide (APRPG-PEG-lip verteporfin). The
APRPG-PEG-lip verteporfin was shown to selectively accumulate
in vivo in the tumors of Meth-A sarcoma bearing mice to a degree
which was approximately 4-fold higher than verteporfin delivered
in non-modified liposomes. In addition, PDT using the APRPG-PEG-
lip verteporfin resulted in strong suppression of tumor growth and
prolonged life in treated mice compared to the untargeted PEG-lip
verteporfin, which resulted in a small amount of tumor growth
suppression but no increase in the survival time of treated mice.
More recently, Oku et al. performed a confirmatory study using
these targeted nanoparticles. The results of this study demonstrated
that enhanced efficacy can be attained using liposomes which are
targeted to angiogenic endothelial cells for the selective delivery
of PSs .
7. Translation to the clinic
Most of the studies discussed above describe new experimental
approaches to combination therapies tested in vitro on cell cultures
or in vivo in different animal models. The ultimate goal in this field
of research, however, is to identify viable new treatments that can
be translated and applied in clinical settings. As past experience in
the field of angiogenesis research has shown, promising pre-clinical
results in mice do not always lead to similar effects in clinical trials
. This leaves open the question of how one should go about
translating pre-clinical results into clinical applications. Recent ad-
vances have allowed for the development of genetically engineered
mouse models, which can be made to mimic both the pathophysio-
logical and molecular characteristics of human tumors . These
models, which more accurately reflect human disease, will hopefully
provide results that more precisely predict success in clinical
Recently, two different inhibitors of angiogenesis, sunitinib and
everolimus, have shown potential for the treatment of human pan-
creatic neuroectodermal tumors (PNET) in phase II clinical trials
[162,163]. The potential of both of these drugs for this indication
was initially predicted by promising results of preclinical studies
 performed in a genetically engineered mouse model, the RIP-
TAG2 model, which has similar features as human PNET tumors
[162,163,165]. In this case, the preclinical model helped to identify
not only a potential new therapy, but also the possible limitations of
this therapy. The phase II clinical trial revealed a significant increase
in the progression free survival of treated patients, however, a lack
of increase in overall survival (seen thus far in the study as it is still
ongoing), a limitation which was predicted by tumor shrinkage and
long-term survival in the mouse model [163,166]. Additionally, pre-
clinical models have also predicted the eventual failure of sunitinib
therapy alone , due the development of drug resistance, which
is now also being seen in clinical trials [168,169].
A. Weiss et al. / Biochimica et Biophysica Acta 1826 (2012) 53–70
Brief description of selected studies examining the effects of PDT in combination with various angiogenesis and vascular targeted agents.
Target Photosensitizer Angiostatic agent SequenceModel ObservationsReference
Hypericin Avastin®PDT+AI Human bladder
Leaky vasculature with functional alteration
in combination PDT and Avastin® and greater
reduction in vessel area than PDT-only;
decreased vessel area and intact vasculature
in Avastin® only group.
Significant increase in long-term tumor
response in mice treated with combination
therapy without increase in normal
Significant improvement of tumor response
in combination group. Relationship to
downregulated expression of angiogenic
molecules (VEGF, angiogen, bFGF, EGF
and IL-6 and −8).
Complete regression of the lesions for
Photofrin®Avastin®PDT+AI Human Kaposi's
sarcoma in nude mice
HypericinAvastin® PDT+AIBladder carcinoma
in nude mice
The single combination therapy of Avastin®
and PDT had a long-lasting effect and thus
eliminated the necessity of further destruction
by repeated PDT.
Significant inhibition of tumor growth and
increased long-term survival in combination
group associated with decreased expression
of angiogenic factors (VEGF and VWF).
Synergistic effect of combination PDT+
Increased tumor response in combination
group and increased apoptosis and
down-regulation of EGFR expression in
Significant difference between control
(no therapy), PDT-only and PDT combinations
(PDT+Avastin®, PDT+Erbitux®, PDT+
Combination therapy inhibited PDT-induced
increase of VEGF and significantly reduced
tumor weight and volume.
Increased therapeutic response in
combination therapy treated mice
(SU6668+PDT had better response than
Improved overall survival outcome and
inhibition of PDT-induced angiogenesis
with both inhibitors.
HypericinErbitux®PDT+AIOvarian carcinoma in
carcinoma in nude
Hypericin Erbitux® PDT+AI
PDT+AI Bladder carcinoma in
HypericinSU5416 SU6668PDT+AI Nasopharyngeal
carcinoma In BALB/c
Murine mammary 16c
Combination with COX-2 inhibitors
following PDT, but not prior to PDT,
increased antitumor activity.
Combination of PDT with COX-2 inhibitors
significantly reduced tumor growth in
cell line over-expressing COX-2.
Combination of PDT and COX-2 inhibitors
resulted in increased tumor growth inhibition
due to down-regulation of COX-2, HIF-1
Significant increase in long-term cures in
Improved efficacy and selectivity, and
decreased phototoxicity over PDT alone.
Increased apoptotic endothelial cell death
rate in combination group.
ALA NimesulidePDT+AI Oral squamous cell
HypericinPrinomastat PDT+AIMouse mammary
carcinoma (BA) in mice
CT colon carcinoma in
cells, PC-3 prostate
Colon26 murine colon
FE8 sarcoma bearing
CD-1 nude mice
Visudyne® Pl3/mTOR kinase
ALAVadimezan n.a. Increased tumor growth inhibition.
SnChe6Human antibody L19 n.a.Average tumor weight in treated mice
was significantly lower than in mice treated
with PS conjugated to an antibody lacking
The amount of Photofrin® internalized by
the HeLa cells was shown to be much greater
than that internalized by the 293 T cells,
indicating the success of the RGD
Photofrin® Surfactant-like tetra-
tail amphiphilic with
an RGD ligand
n.a. HeLa human
transformed (293 T)
(continued on next page)
A. Weiss et al. / Biochimica et Biophysica Acta 1826 (2012) 53–70
8. Back to the drawing board
The concept of PDT, i.e. the treatment of disease through the ad-
ministration of a photosensitive drug and the application of light, is
an old idea, but nowadays is successfully implemented in a limited
number of ocular applications. This includes the treatment of exuda-
tive age-related macular degeneration (AMD) or polypoidal choroidal
vasculopathy (PCV), and for the treatment of certain cancerous and
precancerous skin conditions, including basal cell carcinoma and ac-
tinic keratosis, as well as rheumatoid arthritis. The limited number
of successful applications for the PDT treatment of cancer may, in
part, be due to the local nature of the therapy making it an inappro-
priate form of treatment for disease in advanced stages, which has al-
ready progressed to multiple locations in the body. Additionally,
treatment of early stage localized disease is limited, as cancer is fre-
quently not detected until its later stages, due to the asymptomatic
nature of many early forms of cancer. Adding to complications associ-
ated with the local nature of PDT is the difficult, but necessary re-
quirement of achieving optimal light exposure to all parts of the
tumor, including deeper tissue areas. This problem tends to limit
the application of PDT in oncology to superficially growing tumors
in the skin, or in hollow organs. It is now being realized, however,
that there may be opportunities to overcome some of these limita-
tions that have precluded the development of efficient PDT-based
Although PDT is largely regarded as a local treatment, several
more recent studies have recognized that localized PDT can result in
the activation of a systemic anti-tumor immune response, which
will attack distant tumor growth [170–172], as well as inhibit new
growth [173,174]. One study showed that the PDT treatment of BCC
in humans resulted in the activation of a systemic anti-tumor immu-
nity to a BCC associated tumor antigen, Hip1, which was much greater
than in patients whose lesions had been surgically removed . A
case study also showed the activation of anti-tumor immunity in
the treatment of a patient suffering from a multifocal angiosarcoma
of the head and neck, which was associated with the regression of
distant untreated tumors . Similarly promising results have
also been obtained from the PDT treatment of gliomas in mouse
models . Gaining a better understanding of the mechanisms
and pathways involved in PDT-induced anti-tumor immunity may
allow for the development of new treatment regiments which en-
hance this effect and may lead to more diverse oncological applica-
tions for PDT.
Additionally, the recent development of new light distributing fi-
bers and the availability of improved PSs with long-wavelength ab-
sorption have allowed for improved light exposure, particularly in
deeper tissues . Furthermore, for some applications, like pros-
tate cancer, the device industry has made great strides to develop
methods to achieve localized light delivery deep in the tissue, for ex-
ample with the use of multifiber technology .
As mentioned above, the local nature of light delivery during the
administration of PDT is frequently incompatible with systemic dis-
ease. However, increasing the selectivity of the photosensitive drug
for neoplastic tissues is another viable method to help overcome
this limitation. In this sense, some of the advancements in the field
of PDT can be attributed to increasing knowledge in the field of angio-
genesis research, which is providing the main opportunities for tar-
geted delivery of PSs, i.e. new markers of tumor angiogenesis have
been identified, to which the PS can be targeted. Thus, specific target-
ing of PSs to the tumor endothelium is an attractive approach to en-
hance the selectivity and specificity of PDT. New targets are
continuously being identified, which can be used to selectively deliv-
er PS to the tumor endothelium. This strategy also holds promise for
the future, as the field of anti-angiogenesis research has become
even more active in recent years. We and others have identified the
gene expression profile of tumor endothelial cells. Only a limited
number of genes are overexpressed in tumor vessels, as compared
to normal vessels and vessels in angiogenically stimulated normal tis-
sue, such as placenta tissue [142,143]. These genes are currently being
tested for targeted delivery. We have recently shown that antibody
mediated targeting of HMGB1 is an effective strategy to prolong the
PDT-induced anti-vascular effect . Future studies will investigate
improved selectivity of PDT, when PSs are targeted to HMGB1. Re-
cently obtained knowledge has indicated that dual targeting of parti-
cles to more than one marker of the tumor endothelium may result in
significantly enhanced targeting capacity. For instance, it was shown
by Kluza et al.  that nanoparticles directed to galectin-1 by the
angiostatic peptide anginex [181,182] and, simultaneously, to αvβ3
integrin by a cyclic RGD-peptide showed increased targeting capacity.
It is consequently suggested that targeting PSs simultaneously to
more than one endothelial cell marker can increase the anti-tumor
selectivity and activity of PDT, while simultaneously reducing the
dose of PS needed compared to non-targeted PDT . Specific de-
livery of PSs will lead to better options for selective PDT and salvation
of adjacent normal tissues. Still, systemic treatment will be difficult,
but it should be realized that many anti-cancer therapies are suffi-
cient when applied locally, for instance to the surface of a given hol-
Finally, and probably most importantly, the secondary induction
of angiogenesis by PDT is likely responsible for a significant reduction
in the success rates of treatment. It is now realized that this limitation
can be overcome by simultaneous treatment of the cancer with
Table 1 (continued)
Target PhotosensitizerAngiostatic agent SequenceModel Observations Reference
n.a. n.a. Nude mice with EMT-6
Enhanced PS localization and accumulation in
the tumor tissue as a result of being targeted
to the αvβ3integrin.
PDT with the APRPG-PEG-lip verteporfin
resulted in strong suppression of tumor
growth resulting in prolonged life of treated
mice and in vascular damage.
The APRPG-PEG-modified verteporfin
liposomes were shown to selectively
accumulate in tumor tissue and strongly
inhibit tumor growth upon irradiation.
Selective in vivo localization around tumor
blood vessels. The conjugate with a
photosensitizer allows selective disruption
of tumor vasculature upon
irradiation, leading to complete and long-lasting
n.a.n.a. Meth-A sarcoma
APRPG-verteporfin n.a. n.a. n.a.
AI, angiogenesis inhibitor; RGD, Arg-Gly-Asp tri-peptide; VDA, vascular disrupting agent; n.a., not applicable
A. Weiss et al. / Biochimica et Biophysica Acta 1826 (2012) 53–70
angiogenesis inhibitors, many of which are currently becoming clini-
cally available. We favor the view that such a combination strategy
may allow for a revival of the field of PDT, leading to the development
of effective new therapies for a number of cancers. In this spirit, we
reviewed a series of efforts that were undertaken to give the com-
bination of anti-angiogenesis with PDT a go (see also Table 1).
Many of these studies were undertaken using compounds, which
are now known to not be the best inhibitors of angiogenesis. This
holds an intrinsic promise for further testing of combination thera-
pies using newer and more successful angiostatic compounds. In
this respect, it is especially interesting that several tyrosine kinase in-
hibitors (TKIs) have shown preclinical promise in combination with
PDT. Several new TKIs were recently developed, that represent a
second generation of angiostatic compounds with better activity
and fewer side effects. Examples of such compounds are axitinib
and tivozanib, and results obtained with these agents in combina-
tion with PDT are eagerly expected.
It should be realized, however, that new combination treat-
ments will only be efficient when designed in an optimal way.
In order to do this, the sequencing of both therapies is an impor-
tant issue. Although it is logical to start anti-angiogenic therapy
after the PDT treatment, because of the angiogenesis induction fol-
lowing PDT, it is remarkable that little effort has been undertaken
to test the efficacy of scheduling anti-angiogenic therapy before
PDT. Here one may note that it has been demonstrated that anti-
angiogenic therapy can normalize the vasculature in a tumor,
resulting in less permeable vessels, reduction of hypoxia and im-
proved blood flow . This was found to explain the synergism
of angiostasis with conventional therapies, such as chemo- and ra-
diotherapies. Similar reasoning is valid for PDT, which depends on
both efficient delivery of the PS, as well as the oxygenation of the
target tissue . We conclude that it is consequently also of ex-
treme importance to do a systematic search for optimal sequencing
of angiogenesis inhibition for improvement of the PDT approach.
The authors are grateful for financial support from Dr. J. Jacobi and
the Dutch Science Foundation (NWO grant # 040.11.195).
 P. Ghaliounghui, The Ebers Papyrus: A New English Translation, Commentaries
and Glossaries, Egyptian Academy of Scientific Research and Technology,
 O. Raab, Ueber die Wirkung Fluorescierenden Stoffe auf Infusorien, Z. Biol. 39
 H. Jesionek, H. von Tappeiner, Zur Behandlung der Hautcarcinome mit fluores-
zierenden Stoffen, Dtsch. Arch. Klin. Med 82 (1905) 223–226.
 T.J. Dougherty, J.E. Kaufman, A. Goldfarb, K.R. Weishaupt, D. Boyle, A. Mittleman,
Photoradiation therapy for the treatment of malignant tumors, Cancer Res. 38
 H.B. Van den Bergh, J.-P., Photodynamic therapy: basic principles and mecha-
nisms, Lasers Opthalmol. (2003) 183–195.
 T.J. Dougherty, C.J. Gomer, B.W. Henderson, G. Jori, D. Kessel, M. Korbelik, J.
Moan, Q. Peng, Photodynamic therapy, J. Natl. Cancer Inst. 90 (1998) 889–905.
 P. Nowak-Sliwinska, A. Karocki, M. Elas, A. Pawlak, G. Stochel, K. Urbanska, Ver-
teporfin, photofrin II, and merocyanine 540 as PDT photosensitizers against mel-
anoma cells, Biochem. Biophys. Res. Commun. 349 (2006) 549–555.
 B. Chen, B.W. Pogue, J.M. Luna, R.L. Hardman, P.J. Hoopes, T. Hasan, Tumor vas-
cular permeabilization by vascular-targeting photosensitization: effects, mecha-
nism, and therapeutic implications, Clin. Cancer Res. 12 (2006) 917–923.
 M. Korbelik, PDT-associated host response and its role in the therapy outcome,
Lasers Surg. Med. 38 (2006) 500–508.
 F. Fantini, A. Greco, C. Del Giovane, A.M. Cesinaro, M. Venturini, C. Zane, T.
Surrenti, K. Peris, P.G. Calzavara-Pinton, Photodynamic therapy for basal cell car-
cinoma: clinical and pathological determinants of response, J. Eur. Acad. Derma-
tol. Venereol. 25 (2011) 896–901.
 M.A. Biel, Photodynamic therapy treatment of early oral and laryngeal cancers,
Photochem. Photobiol. 83 (2007) 1063–1068.
 N.R. Rigual, K. Thankappan, M. Cooper, M.A. Sullivan, T. Dougherty, S.R. Popat,
T.R. Loree, M.A. Biel, B. Henderson, Photodynamic therapy for head and neck
dysplasia and cancer, Arch. Otolaryngol. Head Neck Surg. 135 (2009) 784–788.
 A. Klein, P. Babilas, S. Karrer, M. Landthaler, R.M. Szeimies, Photodynamic ther-
apy in dermatology—an update 2008, J. Dtsch. Dermatol. Ges. 6 (2008)
 K. Moghissi, K. Dixon, J.A. Thorpe, M. Stringer, C. Oxtoby, Photodynamic therapy
(PDT) in early central lung cancer: a treatment option for patients ineligible for
surgical resection, Thorax 62 (2007) 391–395.
 J. Usuda, H. Kato, T. Okunaka, K. Furukawa, H. Tsutsui, K. Yamada, Y. Suga, H.
Honda, Y. Nagatsuka, T. Ohira, M. Tsuboi, T. Hirano, Photodynamic therapy
(PDT) for lung cancers, J. Thorac. Oncol. 1 (2006) 489–493.
 J. Sun, I. Cecic, C.S. Parkins, M. Korbelik, Neutrophils as inflammatory and im-
mune effectors in photodynamic therapy-treated mouse SCCVII tumours, Photo-
chem. Photobiol. Sci. 1 (2002) 690–695.
 S.O. Gollnick, S.S. Evans, H. Baumann, B. Owczarczak, P. Maier, L. Vaughan, W.C.
Wang, E. Unger, B.W. Henderson, Role of cytokines in photodynamic therapy-
induced local and systemic inflammation, Br. J. Cancer 88 (2003) 1772–1779.
 P. Agostinis, K. Berg, K.A. Cengel, T.H. Foster, A.W. Girotti, S.O. Gollnick, S.M.
Hahn, M.R. Hamblin, A. Juzeniene, D. Kessel, M. Korbelik, J. Moan, P. Mroz, D.
Nowis, J. Piette, B.C. Wilson, J. Golab, Photodynamic therapy of cancer: an up-
date, CA Cancer J. Clin. 61 (2011) 250–281.
 I.P. van Geel, H. Oppelaar, P.F. Rijken, H.J. Bernsen, N.E. Hagemeier, A.J. van der
Kogel, R.J. Hodgkiss, F.A. Stewart, Vascular perfusion and hypoxic areas in RIF-
1 tumours after photodynamic therapy, Br. J. Cancer 73 (1996) 288–293.
 B.W. Henderson, T.J. Dougherty, How does photodynamic therapy work? Photo-
chem. Photobiol. 55 (1992) 145–157.
 C. Hopper, Photodynamic therapy: a clinical reality in the treatment of cancer,
Lancet Oncol. 1 (2000) 212–219.
 P. Nowak-Sliwinska, G. Wagnieres, H. van den Bergh, A.W. Griffioen, Angiosta-
sis-induced vascular normalization can improve photodynamic therapy, Cell.
Mol. Life Sci. 67 (2010) 1559–1560.
 P. Nowak-Sliwinska, A. Weiss, J.R. Beijnum, T.J. Wong, J.P. Ballini, B. Lovisa, H.V.
Bergh, A.W. Griffioen, Angiostatic kinase inhibitors to sustain photodynamic
angio-occlusion, J. Cell. Mol. Med., in press, PMID: 21880113.
 A.C. Moor, Signaling pathways in cell death and survival after photodynamic
therapy, J. Photochem. Photobiol. B 57 (2000) 1–13.
 A. Ferrario, K.F. von Tiehl, N. Rucker, M.A. Schwarz, P.S. Gill, C.J. Gomer, Antian-
giogenic treatment enhances photodynamic therapy responsiveness in a mouse
mammary carcinoma, Cancer Res. 60 (2000) 4066–4069.
 M.Y. Nahabedian, R.A. Cohen, M.F. Contino, T.M. Terem, W.H. Wright, M.W.
Berns, A.G. Wile, Combination cytotoxic chemotherapy with cisplatin or doxoru-
bicin and photodynamic therapy in murine tumors, J. Natl. Cancer Inst. 80
 J. Golab, D. Olszewska, P. Mroz, K. Kozar, R. Kaminski, A. Jalili, M. Jakobisiak,
Erythropoietin restores the antitumor effectiveness of photodynamic therapy
in mice with chemotherapy-induced anemia, Clin. Cancer Res. 8 (2002)
 Z. Luksiene, A. Kalvelyte, R. Supino, On the combination of photodynamic ther-
apy with ionizing radiation, J. Photochem. Photobiol. B 52 (1999) 35–42.
 R. Bhuvaneswari, Y.Y. Gan, K.C. Soo, M. Olivo, The effect of photodynamic thera-
py on tumor angiogenesis, Cell. Mol. Life Sci. 66 (2009) 2275–2283.
 V.H. Fingar, P.K. Kik, P.S. Haydon, P.B. Cerrito, M. Tseng, E. Abang, T.J. Wieman,
Analysis of acute vascular damage after photodynamic therapy using benzopor-
phyrin derivative (BPD), Br. J. Cancer 79 (1999) 1702–1708.
 B.W. Henderson, S.M. Waldow, T.S. Mang, W.R. Potter, P.B. Malone, T.J. Dougherty,
Tumor destruction and kinetics of tumor cell death in two experimental mouse
tumors following photodynamic therapy, Cancer Res. 45 (1985) 572–576.
 P. Nowak-Sliwinska, J.R. van Beijnum, M. van Berkel, H. van den Bergh, A.W.
Griffioen, Vascular regrowth following photodynamic therapy in the chicken
embryo chorioallantoic membrane, Angiogenesis 13 (2010) 281–292.
 W.M. Star, H.P. Marijnissen, A.E. van den Berg-Blok, J.A. Versteeg, K.A. Franken,
H.S. Reinhold, Destruction of rat mammary tumor and normal tissue microcircu-
lation by hematoporphyrin derivative photoradiation observed in vivo in sand-
wich observation chambers, Cancer Res. 46 (1986) 2532–2540.
 H. van den Bergh, J.P. Ballini, Photodynamic therapy: basic principle, in: S.
F.F.a.K. (Ed.), Lasers in Ophthalmology—Basic, diagnostic and Surgical Aspects,
Kugler Publications, The Hague, 2003, pp. 183–195.
 T.H. Foster, M.C. Primavera, V.J. Marder, R. Hilf, L.A. Sporn, Photosensitized re-
lease of von Willebrand factor from cultured human endothelial cells, Cancer
Res. 51 (1991) 3261–3266.
 J.S. Nelson, L.H. Liaw, M.W. Berns, Tumor destruction in photodynamic therapy,
Photochem. Photobiol. 46 (1987) 829–835.
 U. Schmidt-Erfurth, T. Hasan, E. Gragoudas, N. Michaud, T.J. Flotte, R. Birngruber,
Vascular targeting in photodynamic occlusion of subretinal vessels, Ophthal-
mology 101 (1994) 1953–1961.
 H.M. Solomon, P.D. Zieve, J.R. Krevans, The effect of hematoporphyrin and light
on human platelets. II. Uptake of hematoporphyrin, J. Cell. Physiol. 67 (1966)
 P.D. Zieve, H.M. Solomon, J.R. Krevans, The effect of hematoporphyrin and light
on human platelets. I. Morphologic, functional, and biochemical changes, J. Cell.
Physiol. 67 (1966) 271–279.
 B.W. Henderson, B. Owczarczak, J. Sweeney, T. Gessner, Effects of photodynamic
treatment of platelets or endothelial cells in vitro on platelet aggregation, Photo-
chem. Photobiol. 56 (1992) 513–521.
 M.W. Reed, F.N. Miller, T.J. Wieman, M.T. Tseng, C.G. Pietsch, The effect of photo-
dynamic therapy on the microcirculation, J. Surg. Res. 45 (1988) 452–459.
 V.H. Fingar, Vascular effects of photodynamic therapy, J. Clin. Laser Med. Surg. 14
A. Weiss et al. / Biochimica et Biophysica Acta 1826 (2012) 53–70
 J.M. Klausner, I.S. Paterson, G. Goldman, L. Kobzik, C. Rodzen, R. Lawrence, C.R.
Valeri, D. Shepro, H.B. Hechtman, Postischemic renal injury is mediated by neu-
trophils and leukotrienes, Am. J. Physiol. 256 (1989) F794–F802.
 J. Doukas, H.B. Hechtman, D. Shepro, Vasoactive amines and eicosanoids interac-
tively regulate both polymorphonuclear leukocyte diapedesis and albumin per-
meability in vitro, Microvasc. Res. 37 (1989) 125–137.
 V.H. Fingar, T.J. Wieman, S.A. Wiehle, P.B. Cerrito, The role of microvascular
damage in photodynamic therapy: the effect of treatment on vessel constriction,
permeability, and leukocyte adhesion, Cancer Res. 52 (1992) 4914–4921.
 M. Dellian, C. Abels, G.E. Kuhnle, A.E. Goetz, Effects of photodynamic therapy on
leucocyte–endothelium interaction: differences between normal and tumour
tissue, Br. J. Cancer 72 (1995) 1125–1130.
 A.W. Griffioen, C.A. Damen, S. Martinotti, G.H. Blijham, G. Groenewegen, En-
dothelial intercellular adhesion molecule-1 expression is suppressed in
human malignancies: the role of angiogenic factors, Cancer Res. 56 (1996)
 A.E. Dirkx, M.G. Oude Egbrink, M.J. Kuijpers, S.T. van der Niet, V.V. Heijnen, J.C.
Bouma-ter Steege, J. Wagstaff, A.W. Griffioen, Tumor angiogenesis modulates
leukocyte-vessel wall interactions in vivo by reducing endothelial adhesion mol-
ecule expression, Cancer Res. 63 (2003) 2322–2329.
 E. Ben-Hur, E. Heldman, S.W. Crane, I. Rosenthal, Release of clotting factors from
photosensitized endothelial cells: a possible trigger for blood vessel occlusion by
photodynamic therapy, FEBS Lett. 236 (1988) 105–108.
 W.H. Chang, J.J. Petry, Platelets, prostaglandins, and patency in microvascular
surgery, J. Microsurg. 2 (1980) 27–35.
 M.L. Ogletree, Overview of physiological and pathophysiological effects of
thromboxane A2, Fed. Proc. 46 (1987) 133–138.
 M.J. Gilissen, L.E. van de Merbel-de Wit, W.M. Star, J.F. Koster, W. Sluiter, Effect
of photodynamic therapy on the endothelium-dependent relaxation of isolated
rat aortas, Cancer Res. 53 (1993) 2548–2552.
 V.H. Fingar, T.J. Wieman, K.W. Doak, Changes in tumor interstitial pressure in-
duced by photodynamic therapy, Photochem. Photobiol. 53 (1991) 763–768.
 R.K. Jain, Normalization of tumor vasculature: an emerging concept in antian-
giogenic therapy, Science (New York, N.Y.) 307 (2005) 58–62.
 D. Cao, M. Hou, Y.S. Guan, M. Jiang, Y. Yang, H.F. Gou, Expression of HIF-1alpha
and VEGF in colorectal cancer: association with clinical outcomes and prognos-
tic implications, BMC Cancer 9 (2009) 432.
 N.H. Fernando, H.I. Hurwitz, Targeted therapy of colorectal cancer: clinical expe-
rience with bevacizumab, Oncologist 9 (Suppl. 1) (2004) 11–18.
 S.M. Couch, S.J. Bakri, Review of combination therapies for neovascular age-
related macular degeneration, Semin. Ophthalmol. 26 (2011) 114–120.
 G.Y. R.Y. Bhuvaneswari, S.K. Chee, M. Olivo, Antiangiogenesis agents Avastin and
Erbitux enhance the efficacy of photodynamic therapy in a murine bladder
tumor model, Lasers Surg. Med. 43 (2011) 651–662.
 A. Ferrario, C.J. Gomer, Avastin enhances photodynamic therapy treatment of
Kaposi's sarcoma in a mouse tumor model, J. Environ. Pathol. Toxicol. Oncol.
25 (2006) 251–259.
 R. Bhuvaneswari, G.Y. Yuen, S.K. Chee, M. Olivo, Hypericin-mediated photody-
namic therapy in combination with Avastin (bevacizumab) improves tumor re-
sponse by downregulating angiogenic proteins, Photochem. Photobiol. Sci. 6
 R. Bhuvaneswari, P.S. Thong, Y.Y. Gan, K.C. Soo, M. Olivo, Evaluation of
hypericin-mediated photodynamic therapy in combination with angiogenesis
inhibitor bevacizumab using in vivo fluorescence confocal endomicroscopy, J.
Biomed. Opt. 15 (2010) 011114.
 F. Jiang, X. Zhang, S.N. Kalkanis, Z. Zhang, H. Yang, M. Katakowski, X. Hong, X.
Zheng, Z. Zhu, M. Chopp, Combination therapy with antiangiogenic treatment
and photodynamic therapy for the nude mouse bearing U87 glioblastoma,
Photochem. Photobiol. 84 (2008) 128–137.
 M. Sagong, J. Lee, W. Chang, Application of intravitreal bevacizumab for circum-
scribed choroidal hemangioma, Korean J. Ophthalmol. 23 (2009) 127–131.
 F.D. Verbraak, R.O. Schlingemann, M.D. de Smet, J.E. Keunen, Single spot PDT in
patients with circumscribed choroidal haemangioma and near normal visual
acuity, Graefes Arch. Clin. Exp. Ophthalmol. 244 (2006) 1178–1182.
 T. Baba, M. Kitahashi, M. Kubota-Taniai, T. Oshitari, S. Yamamoto, Subretinal
hemorrhage after photodynamic therapy for juxtapapillary retinal capillary
hemangioma, Case Rep. Ophthalmol. 2 (2011) 134–139.
 F. Ziemssen, M. Voelker, W. Inhoffen, K.U. Bartz-Schmidt, F. Gelisken, Com-
bined treatment of a juxtapapillary retinal capillary haemangioma with intra-
vitreal bevacizumab and photodynamic therapy, Eye (Lond.) 21 (2007)
 K.J. Kim, B. Li, K. Houck, J. Winer, N. Ferrara, The vascular endothelial growth fac-
tor proteins: identification of biologically relevant regions by neutralizing
monoclonal antibodies, Growth Factors 7 (1992) 53–64.
 N. Ferrara, K.J. Hillan, H.P. Gerber, W. Novotny, Discovery and development of
bevacizumab, an anti-VEGF antibody for treating cancer, Nat. Rev. Drug Discov.
3 (2004) 391–400.
 H.P. Gerber, X. Wu, L. Yu, C. Wiesmann, X.H. Liang, C.V. Lee, G. Fuh, C. Olsson, L.
Damico, D. Xie, Y.G. Meng, J. Gutierrez, R. Corpuz, B. Li, L. Hall, L. Rangell, R.
Ferrando, H. Lowman, F. Peale, N. Ferrara, Mice expressing a humanized form
of VEGF-A may provide insights into the safety and efficacy of anti-VEGF anti-
bodies, Proc. Natl. Acad. Sci. U. S. A. 104 (2007) 3478–3483.
 F. Bock, J. Onderka, T. Dietrich, B. Bachmann, F.E. Kruse, M. Paschke, G. Zahn, C.
Cursiefen, Bevacizumab as a potent inhibitor of inflammatory corneal angiogen-
esis and lymphangiogenesis, Investig. Ophthalmol. Vis. Sci. 48 (2007)
 L. Yu, X. Wu, Z. Cheng, C.V. Lee, J. LeCouter, C. Campa, G. Fuh, H. Lowman, N.
Ferrara, Interaction between bevacizumab and murine VEGF-A: a reassessment,
Investig. Ophthalmol. Vis. Sci. 49 (2008) 522–527.
 R.S. Herbst, D.M. Shin, Monoclonal antibodies to target epidermal growth factor
receptor-positive tumors: a new paradigm for cancer therapy, Cancer 94 (2002)
 M.G. del Carmen, I. Rizvi, Y. Chang, A.C. Moor, E. Oliva, M. Sherwood, B. Pogue, T.
Hasan, Synergism of epidermal growth factor receptor-targeted immunothera-
py with photodynamic treatment of ovarian cancer in vivo, J. Natl. Cancer Inst.
97 (2005) 1516–1524.
 E.S. Kim, F.R. Khuri, R.S. Herbst, Epidermal growth factor receptor biology (IMC-
C225), Curr. Opin. Oncol. 13 (2001) 506–513.
 P.A. Martinez-Carpio, M.A. Trelles, The role of epidermal growth factor receptor
in photodynamic therapy: a review of the literature and proposal for future in-
vestigation, Lasers Med. Sci. 25 (2010) 767–771.
 R. Bhuvaneswari, Y.Y. Gan, K.C. Soo, M. Olivo, Targeting EGFR with photodynam-
ic therapy in combination with Erbitux enhances in vivo bladder tumor re-
sponse, Mol. Cancer 8 (2009) 94.
 W.L. Yip, A. Weyergang, K. Berg, H.H. Tonnesen, P.K. Selbo, Targeted delivery and
enhanced cytotoxicity of cetuximab-saporin by photochemical internalization
in EGFR-positive cancer cells, Mol. Pharm. 4 (2007) 241–251.
 K. Berg, P.K. Selbo, L. Prasmickaite, T.E. Tjelle, K. Sandvig, J. Moan, G. Gaudernack,
O. Fodstad, S. Kjolsrud, H. Anholt, G.H. Rodal, S.K. Rodal, A. Hogset, Photochem-
ical internalization: a novel technology for delivery of macromolecules into cy-
tosol, Cancer Res. 59 (1999) 1180–1183.
 R.A. Lustig, T.J. Vogl, D. Fromm, R. Cuenca, R. Alex Hsi, A.K. D'Cruz, Z. Krajina, M.
Turic, A. Singhal, J.C. Chen, A multicenter Phase I safety study of intratumoral
photoactivation of talaporfin sodium in patients with refractory solid tumors,
Cancer 98 (2003) 1767–1771.
 M.J. Manyak, K. Ogan, Photodynamic therapy for refractory superficial bladder
cancer: long-term clinical outcomes of single treatment using intravesical diffu-
sion medium, J. Endourol. 17 (2003) 633–639.
 Y. Cao, Opinion: emerging mechanisms of tumour lymphangiogenesis and lym-
phatic metastasis, Nat. Rev. Cancer 5 (2005) 735–743.
 R.S. Herbst, D.H. Johnson, E. Mininberg, D.P. Carbone, T. Henderson, E.S. Kim, G.
Blumenschein Jr., J.J. Lee, D.D. Liu, M.T. Truong, W.K. Hong, H. Tran, A. Tsao, D.
Xie, D.A. Ramies, R. Mass, S. Seshagiri, D.A. Eberhard, S.K. Kelley, A. Sandler,
Phase I/II trial evaluating the anti-vascular endothelial growth factor monoclo-
nal antibody bevacizumab in combination with the HER-1/epidermal growth
factor receptor tyrosine kinase inhibitor erlotinib for patients with recurrent
non-small-cell lung cancer, J. Clin. Oncol. 23 (2005) 2544–2555.
 R. Iyer, G. Fetterly, A. Lugade, Y. Thanavala, Sorafenib: a clinical and pharmaco-
logic review, Expert. Opin. Pharmacother. 11 (2010) 1943–1955.
 M. Van den Eynde, J.F. Baurain, F. Mazzeo, J.P. Machiels, Epidermal growth factor
receptor targeted therapies for solid tumours, Acta Clin. Belg. 66 (2011) 10–17.
 T. Powles, S. Chowdhury, R. Jones, M. Mantle, P. Nathan, A. Bex, L. Lim, T. Hutson,
Sunitinib and other targeted therapies for renal cell carcinoma, Br. J. Cancer 104
 C.J. Dimitroff, W. Klohs, A. Sharma, P. Pera, D. Driscoll, J. Veith, R. Steinkampf, M.
Schroeder, S. Klutchko, A. Sumlin, B. Henderson, T.J. Dougherty, R.J. Bernacki,
Anti-angiogenic activity of selected receptor tyrosine kinase inhibitors,
PD166285 and PD173074: implications for combination treatment with photo-
dynamic therapy, Invest. New Drugs 17 (1999) 121–135.
 Q. Zhou, M. Olivo, K.Y. Lye, S. Moore, A. Sharma, B. Chowbay, Enhancing the ther-
apeutic responsiveness of photodynamic therapy with the antiangiogenic
agents SU5416 and SU6668 in murine nasopharyngeal carcinoma models, Can-
cer Chemother. Pharmacol. 56 (2005) 569–577.
 D.B. Mendel, R.E. Schreck, D.C. West, G. Li, L.M. Strawn, S.S. Tanciongco, S. Vasile,
L.K. Shawver, J.M. Cherrington, The angiogenesis inhibitor SU5416 has long-
lasting effects on vascular endothelial growth factor receptor phosphorylation
and function, Clin. Cancer Res. 6 (2000) 4848–4858.
 S. Curran, G.I. Murray, Matrix metalloproteinases in tumour invasion and metas-
tasis, J. Pathol. 189 (1999) 300–308.
 W.G. Stetler-Stevenson, Matrix metalloproteinases in angiogenesis: a moving
target for therapeutic intervention, J. Clin. Invest. 103 (1999) 1237–1241.
 H.Du, M. Olivo, R.Mahendran,B.H.Bay,Modulationof Matrix metalloproteinase-1
in nasopharyngeal cancer cells by photoactivation of hypericin, Int. J. Oncol. 24
 H.Y. Du, M. Olivo, R. Mahendran, Q. Huang, H.M. Shen, C.N. Ong, B.H. Bay, Hyper-
icin photoactivation triggers down-regulation of matrix metalloproteinase-9 ex-
pression in well-differentiated human nasopharyngeal cancer cells, Cell. Mol.
Life Sci. 64 (2007) 979–988.
 S.P. Tabruyn, S. Memet, P. Ave, C. Verhaeghe, K.H. Mayo, I. Struman, J.A. Martial,
A.W. Griffioen, NF-kappaB activation in endothelial cells is critical for the activ-
ity of angiostatic agents, Mol. Cancer Ther. 8 (2009) 2645–2654.
 S.P. Tabruyn, A.W. Griffioen, NF-kappa B: a new player in angiostatic therapy,
Angiogenesis 11 (2008) 101–106.
 C.M. Au, S.K. Luk, C.J. Jackson, H.K. Ng, C.M. Yow, S.S. To, Differential effects of
photofrin, 5-aminolevulinic acid and calphostin C on glioma cells, J. Photochem.
Photobiol. B 85 (2006) 92–101.
 A. Sharwani, W. Jerjes, C. Hopper, M.P. Lewis, M. El-Maaytah, H.S. Khalil, A.J.
Macrobert, T. Upile, V. Salih, Photodynamic therapy down-regulates the invasion
promoting factors in human oral cancer, Arch. Oral Biol. 51 (2006) 1104–1111.
 E.S. Chu, T.K. Wong, C.M. Yow, Photodynamic effect in medulloblastoma: down-
regulation of matrix metalloproteinases and human telomerase reverse tran-
scriptase expressions, Photochem. Photobiol. Sci. 7 (2008) 76–83.
A. Weiss et al. / Biochimica et Biophysica Acta 1826 (2012) 53–70
 A. Ferrario, C.F. Chantrain, K. von Tiehl, S. Buckley, N. Rucker, D.R. Shalinsky, H.
Shimada, Y.A. DeClerck, C.J. Gomer, The matrix metalloproteinase inhibitor pri-
nomastat enhances photodynamic therapy responsiveness in a mouse tumor
model, Cancer Res. 64 (2004) 2328–2332.
 S. Gately, The contributions of cyclooxygenase-2 to tumor angiogenesis, Cancer
Metastasis Rev. 19 (2000) 19–27.
 S. Gately, W.W. Li, Multiple roles of COX-2 in tumor angiogenesis: a target for
antiangiogenic therapy, Semin. Oncol. 31 (2004) 2–11.
 M.T. Wang, K.V. Honn, D. Nie, Cyclooxygenases, prostanoids, and tumor progres-
sion, Cancer Metastasis Rev. 26 (2007) 525–534.
 M. Romano, J. Claria, Cyclooxygenase-2 and 5-lipoxygenase converging func-
tions on cell proliferation and tumor angiogenesis: implications for cancer ther-
apy, FASEB J. 17 (2003) 1986–1995.
 T. Kinoshita, Y. Takahashi, T. Sakashita, H. Inoue, T. Tanabe, T. Yoshimoto,
Growth stimulation and induction of epidermal growth factor receptor by over-
expression of cyclooxygenases 1 and 2 in human colon carcinoma cells, Biochim.
Biophys. Acta 1438 (1999) 120–130.
 B. Singh, J.A. Berry, A. Shoher, V. Ramakrishnan, A. Lucci, COX-2 overexpression in-
 D.K. Petkova, C. Clelland, J. Ronan, L. Pang, J.M. Coulson, S. Lewis, A.J. Knox, Over-
expression of cyclooxygenase-2 in non-small cell lung cancer, Respir. Med. 98
 Y. Cao, S.M. Prescott, Many actions of cyclooxygenase-2 in cellular dynamics and
in cancer, J. Cell. Physiol. 190 (2002) 279–286.
 A. Ferrario, K. Von Tiehl, S. Wong, M. Luna, C.J. Gomer, Cyclooxygenase-2 inhib-
itor treatment enhances photodynamic therapy-mediated tumor response, Can-
cer Res. 62 (2002) 3956–3961.
 E.H. Harvey, J. Webber, D. Kessel, D. Fromm, Killing tumor cells: the effect of
photodynamic therapy using mono-L-aspartyl chlorine and NS-398, Am. J.
Surg. 189 (2005) 302–305.
 M. Makowski, T. Grzela, J. Niderla, M. L.A., P. Mroz, M. Kopee, M. Legat, K.
Strusinska, K. Koziak, D. Nowis, P. Mrowka, M. Wasik, M. Jakobisiak, J. Golab, In-
hibition of cyclooxygenase-2 indirectly potentiates antitumor effects of photo-
dynamic therapy in mice, Clin. Cancer Res. 9 (2003) 5417–5422.
 A. Ferrario, A.M. Fisher, N. Rucker, C.J. Gomer, Celecoxib and NS-398 enhance
photodynamic therapy by increasing in vitro apoptosis and decreasing in vivo in-
flammatory and angiogenic factors, Cancer Res. 65 (2005) 9473–9478.
 K.K. Yee, K.C. Soo, M. Olivo, Anti-angiogenic effects of Hypericin-photodynamic
therapy in combination with Celebrex in the treatment of human nasopharyn-
geal carcinoma, Int. J. Mol. Med. 16 (2005) 993–1002.
 Y. Akita, K. Kozaki, A. Nakagawa, T. Saito, S. Ito, Y. Tamada, S. Fujiwara, N.
Nishikawa, K. Uchida, K. Yoshikawa, T. Noguchi, O. Miyaishi, K. Shimozato, S.
Saga, Y. Matsumoto, Cyclooxygenase-2 is a possible target of treatment ap-
proach in conjunction with photodynamic therapy for various disorders in
skin and oral cavity, Br. J. Dermatol. 151 (2004) 472–480.
 N. Hendrickx, M. Dewaele, E. Buytaert, G. Marsboom, S. Janssens, M. Van Boven,
J.R. Vandenheede, P. de Witte, P. Agostinis, Targeted inhibition of p38alpha
MAPK suppresses tumor-associated endothelial cell migration in response to
hypericin-based photodynamic therapy, Biochem. Biophys. Res. Commun. 337
 N. Hendrickx, C. Volanti, U. Moens, O.M. Seternes, P. de Witte, J.R. Vandenheede,
J. Piette, P. Agostinis, Up-regulation of cyclooxygenase-2 and apoptosis resis-
tance by p38 MAPK in hypericin-mediated photodynamic therapy of human
cancer cells, J. Biol. Chem. 278 (2003) 52231–52239.
 D. Ingber, T. Fujita, S. Kishimoto, K. Sudo, T. Kanamaru, H. Brem, J. Folkman, Syn-
thetic analogues of fumagillin that inhibit angiogenesis and suppress tumour
growth, Nature 348 (1990) 555–557.
 Y. Hama, T. Shimizu, S. Hosaka, A. Sugenoya, N. Usuda, Therapeutic efficacy of
the angiogenesis inhibitor O-(chloroacetyl-carbamoyl) fumagillol (TNP-470;
AGM-1470) for human anaplastic thyroid carcinoma in nude mice, Exp. Toxicol.
Pathol. 49 (1997) 239–247.
 J.R. Yeh, R. Mohan, C.M. Crews, The antiangiogenic agent TNP-470 requires p53
and p21CIP/WAF for endothelial cell growth arrest, Proc. Natl. Acad. Sci. U. S. A.
97 (2000) 12782–12787.
 N. Solban, P.K. Selbo, A.K. Sinha, S.K. Chang, T. Hasan, Mechanistic investigation
and implications of photodynamic therapy induction of vascular endothelial
growth factor in prostate cancer, Cancer Res. 66 (2006) 5633–5640.
 B. Kosharskyy, N. Solban, S.K. Chang, I. Rizvi, Y. Chang, T. Hasan, A mechanism-
based combination therapy reduces local tumor growth and metastasis in an
orthotopic model of prostate cancer, Cancer Res. 66 (2006) 10953–10958.
 M.W. Kieran, C.D. Turner, J.B. Rubin, S.N. Chi, M.A. Zimmerman, C. Chordas, G.
Klement, A. Laforme, A. Gordon, A. Thomas, D. Neuberg, T. Browder, J.
Folkman, A feasibility trial of antiangiogenic (metronomic) chemotherapy in pe-
diatric patients with recurrent or progressive cancer, J. Pediatr. Hematol. Oncol.
27 (2005) 573–581.
 S.H. Lim, P. Nowak-Sliwinska, F.A. Kamarulzaman, H. van den Bergh, G.
Wagnieres, H.B. Lee, The neovessel occlusion efficacy of 15-hydroxypurpurin-
7-lactone dimethyl ester induced with photodynamic therapy, Photochem.
Photobiol. 86 (2010) 397–402.
 S.H. Lim, C. Thivierge, P. Nowak-Sliwinska, J. Han, H. van den Bergh, G.
Wagnieres, K. Burgess, H.B. Lee, In vitro and in vivo photocytotoxicity of boron
dipyrromethene derivatives for photodynamic therapy, J. Med. Chem. 53
 P. Nowak-Sliwinska, J.R. van Beijnum, A. Casini, A.A. Nazarov, G. Wagnieres, H.
van den Bergh, P.J. Dyson, A.W. Griffioen, Organometallic ruthenium(II) arene
compounds with antiangiogenic activity, J. Med. Chem. 54 (2011) 3895–3902.
 M.F. Zuluaga, N. Lange, Combination of photodynamic therapy with anti-cancer
agents, Curr. Med. Chem. 15 (2008) 1655–1673.
 R.P. Dings, M. Loren, H. Heun, E. McNiel, A.W. Griffioen, K.H. Mayo, R.J. Griffin,
Scheduling of radiation with angiogenesis inhibitors anginex and Avastin im-
proves therapeutic outcome via vessel normalization, Clin. Cancer Res. 13
 R. Murata, Y. Nishimura, M. Hiraoka, An antiangiogenic agent (TNP-470) inhib-
ited reoxygenation during fractionated radiotherapy of murine mammary carci-
noma, Int. J. Radiat. Oncol. Biol. Phys. 37 (1997) 1107–1113.
 J. Ma, H.Q. Mai, M.H. Hong, H.Q. Min, Z.D. Mao, N.J. Cui, T.X. Lu, H.Y. Mo, Results
of a prospective randomized trial comparing neoadjuvant chemotherapy plus
radiotherapy with radiotherapy alone in patients with locoregionally advanced
nasopharyngeal carcinoma, J. Clin. Oncol. 19 (2001) 1350–1357.
 Y. Kakeji, B.A. Teicher, Preclinical studies of the combination of angiogenic inhib-
itors with cytotoxic agents, Invest. New Drugs 15 (1997) 39–48.
 B.C. Baguley, Antivascular therapy of cancer: DMXAA, Lancet Oncol. 4 (2003)
 S.M. Galbraith, R.J. Maxwell, M.A. Lodge, G.M. Tozer, J. Wilson, N.J. Taylor, J.J.
Stirling, L. Sena, A.R. Padhani, G.J. Rustin, Combretastatin A4 phosphate has
tumor antivascular activity in rat and man as demonstrated by dynamic mag-
netic resonance imaging, J. Clin. Oncol. 21 (2003) 2831–2842.
 M.J. McKeage, B.C. Baguley, Disrupting established tumor blood vessels: an
emerging therapeutic strategy for cancer, Cancer 116 (2010) 1859–1871.
 P. Lambin, W. Landuyt, Vascular targeting: a potential additional anti-cancer
treatment, Verh. K. Acad. Geneeskd. Belg. 65 (2003) 29–46.
 D.W. Siemann, W. Shi, Dual targeting of tumor vasculature: combining Avastin
and vascular disrupting agents (CA4P or OXi4503), Anticancer. Res. 28 (2008)
 B.G. Siim, A.E. Lee, S. Shalal-Zwain, F.B. Pruijn, M.J. McKeage, W.R. Wilson,
Marked potentiation of the antitumour activity of chemotherapeutic drugs by
the antivascular agent 5,6-dimethylxanthenone-4-acetic acid (DMXAA), Cancer
Chemother. Pharmacol. 51 (2003) 43–52.
 P.N. Lara Jr., J.Y. Douillard, K. Nakagawa, J. von Pawel, M.J. McKeage, I. Albert, G.
Losonczy, M. Reck, D.S. Heo, X. Fan, A. Fandi, G. Scagliotti, Randomized phase III
placebo-controlled trial of carboplatin and paclitaxel with or without the vas-
cular disrupting agent vadimezan (ASA404) in advanced non-small-cell lung
cancer, J. Clin. Oncol. 29 (2011) 2965–2971.
 R. Murata, D.W. Siemann, J. Overgaard, M.R. Horsman, Improved tumor response
by combining radiation and the vascular-damaging drug 5,6-dimethylxanthe-
none-4-acetic acid, Radiat. Res. 156 (2001) 503–509.
 M. Seshadri, D.A. Bellnier, The vascular disrupting agent 5,6-dimethylxanthe-
none-4-acetic acid improves the antitumor efficacy and shortens treatment
time associated with Photochlor-sensitized photodynamic therapy in vivo,
Photochem. Photobiol. 85 (2009) 50–56.
 C. He, B. Fateya, B. Chen, Combination of vascular targeting PDT with combretas-
tatin A4 phosphate, Proc. SPIE 7380 (2009) 7380321–7380326.
 B. Fateye, B. Chen, combination of PI3K/Akt/mTOR inhibitors and PDT in endo-
thelial and tumor cells, Proc. SPIE 7886 (2009) 78860B78861-78816.
 A. Marrero, T. Becker, U. Sunar, J. Morgan, D. Bellnier, Aminolevulinic acid-
photodynamic therapy combined with topically applied vascular disrupting
agent vadimezan leads to enhanced antitumor responses, Photochem. Photo-
biol. 87 (2011) 910–919.
 B. Ahmed, L.I. Van Eijk, J.C. Bouma-Ter Steege, D.W. Van Der Schaft, A.M. Van
Esch, S.R. Joosten-Achjanie, P. Lambin, W. Landuyt, A.W. Griffioen, Vascular tar-
geting effect of combretastatin A-4 phosphate dominates the inherent angio-
genesis inhibitory activity, Int. J. Cancer 105 (2003) 20–25.
 J.R. van Beijnum, R.P. Dings, E. van der Linden, B.M. Zwaans, F.C. Ramaekers,
K.H. Mayo, A.W. Griffioen, Gene expression of tumor angiogenesis dissected:
specific targeting of colon cancer angiogenic vasculature, Blood 108 (2006)
 B. St Croix, C. Rago, V. Velculescu, G. Traverso, K.E. Romans, E. Montgomery,
A. Lal, G.J. Riggins, C. Lengauer, B. Vogelstein, K.W. Kinzler, Genes expressed
in human tumor endothelium, Science (New York, N.Y.) 289 (2000)
 S. Folli, P. Westermann, D. Braichotte, A. Pelegrin, G. Wagnieres, H. van den
Bergh, J.P. Mach, Antibody-indocyanin conjugates for immunophotodetection
of human squamous cell carcinoma in nude mice, Cancer Res. 54 (1994)
 M.L. Yarmush, W.P. Thorpe, L. Strong, S.L. Rakestraw, M. Toner, R.G. Tompkins,
Antibody Targeted Photolysis, Crit. Rev. Ther. Drug Carrier Syst. 10 (1993)
 M. Santimaria, G. Moscatelli, G.L. Viale, L. Giovannoni, G. Neri, F. Viti, A. Leprini, L.
Borsi, P. Castellani, L. Zardi, D. Neri, P. Riva, Immunoscintigraphic detection of
the ED-B domain of fibronectin, a marker of angiogenesis, in patients with can-
cer, Clin. Cancer Res. 9 (2003) 571–579.
 M. Birchler, F. Viti, L. Zardi, B. Spiess, D. Neri, Selective targeting and photocoag-
ulation of ocular angiogenesis mediated by a phage-derived human antibody
fragment, Nat. Biotechnol. 17 (1999) 984–988.
 A. Palumbo, F. Hauler, P. Dziunycz, K. Schwager, A. Soltermann, F. Pretto, C.
Alonso, G.F. Hofbauer, R.W. Boyle, D. Neri, A chemically modified antibody me-
diates complete eradication of tumours by selective disruption of tumour
blood vessels, Br. J. Cancer 104 (2011) 1106–1115.
 M. Fabbrini, E. Trachsel, P. Soldani, S. Bindi, P. Alessi, L. Bracci, H. Kosmehl, L.
Zardi, D. Neri, P. Neri, Selective occlusion of tumor blood vessels by targeted de-
livery of an antibody-photosensitizer conjugate, Int. J. Cancer 118 (2006)
A. Weiss et al. / Biochimica et Biophysica Acta 1826 (2012) 53–70
 A.W. Griffioen, G. Molema, Angiogenesis: potentials for pharmacologic interven- Download full-text
tion in the treatment of cancer, cardiovascular diseases, and chronic inflamma-
tion, Pharmacol. Rev. 52 (2000) 237–268.
 D.A. Cheresh, Structure, function and biological properties of integrin alpha v
beta 3 on human melanoma cells, Cancer Metastasis Rev. 10 (1991) 3–10.
 P.C. Brooks, R.A. Clark, D.A. Cheresh, Requirement of vascular integrin alpha v
beta 3 for angiogenesis, Science (New York, N.Y.) 264 (1994) 569–571.
 E. Garanger, D. Boturyn, P. Dumy, Tumor targeting with RGD peptide ligands-
design of new molecular conjugates for imaging and therapy of cancers, Anti-
cancer Agents Med. Chem. 7 (2007) 552–558.
 J.X. Chen, H.Y. Wang, C. Li, K. Han, X.Z. Zhang, R.X. Zhuo, Construction of
surfactant-like tetra-tail amphiphilic peptide with RGD ligand for encapsula-
tion of porphyrin for photodynamic therapy, Biomaterials 32 (2011)
 M.J. Goldman, J.M. Wilson, Expression of alpha v beta 5 integrin is necessary for
efficient adenovirus-mediated gene transfer in the human airway, J. Virol. 69
 C.M. Allen, W.M. Sharman, C. La Madeleine, J.M. Weber, R. Langlois, R. Ouellet,
J.E. van Lier, Photodynamic therapy: tumor targeting with adenoviral proteins,
Photochem. Photobiol. 70 (1999) 512–523.
 T. Asai, K. Shimizu, M. Kondo, K. Kuromi, K. Watanabe, K. Ogino, T. Taki, S. Shuto,
A. Matsuda, N. Oku, Anti-neovascular therapy by liposomal DPP-CNDAC tar-
geted to angiogenic vessels, FEBS Lett. 520 (2002) 167–170.
 K. Ichikawa, T. Hikita, N. Maeda, S. Yonezawa, Y. Takeuchi, T. Asai, Y. Namba, N.
Oku, Antiangiogenic photodynamic therapy (PDT) by using long-circulating
liposomes modified with peptide specific to angiogenic vessels, Biochim. Bio-
phys. Acta 1669 (2005) 69–74.
 N. Oku, T. Ishii, Antiangiogenic photodynamic therapy with targeted liposomes,
Methods Enzymol. 465 (2009) 313–330.
 R. Kerbel, J. Folkman, Clinical translation of angiogenesis inhibitors, Nat. Rev.
Cancer 2 (2002) 727–739.
 K.K. Frese, D.A. Tuveson, Maximizing mouse cancer models, Nat. Rev. Cancer 7
 E. Raymond, L. Dahan, J.L. Raoul, Y.J. Bang, I. Borbath, C. Lombard-Bohas, J. Valle,
P. Metrakos, D. Smith, A. Vinik, J.S. Chen, D. Horsch, P. Hammel, B. Wiedenmann,
E. Van Cutsem, S. Patyna, D.R. Lu, C. Blanckmeister, R. Chao, P. Ruszniewski, Suni-
tinib malate for the treatment of pancreatic neuroendocrine tumors, N. Engl. J.
Med. 364 (2011) 501–513.
 J.C. Yao, M.H. Shah, T. Ito, C.L. Bohas, E.M. Wolin, E. Van Cutsem, T.J. Hobday, T.
Okusaka, J. Capdevila, E.G. de Vries, P. Tomassetti, M.E. Pavel, S. Hoosen, T.
Haas, J. Lincy, D. Lebwohl, K. Oberg, Everolimus for advanced pancreatic neuro-
endocrine tumors, N. Engl. J. Med. 364 (2011) 514–523.
 M. Paez-Ribes, E. Allen, J. Hudock, T. Takeda, H. Okuyama, F. Vinals, M. Inoue, G.
Bergers, D. Hanahan, O. Casanovas, Antiangiogenic therapy elicits malignant
progression of tumors to increased local invasion and distant metastasis, Cancer
Cell 15 (2009) 220–231.
 D. Hanahan, Heritable formation of pancreatic beta-cell tumours in transgenic
mice expressing recombinant insulin/simian virus 40 oncogenes, Nature 315
 D. Tuveson, D. Hanahan, Translational medicine: cancer lessons from mice to
humans, Nature 471 (2011) 316–317.
 J.M. Ebos, C.R. Lee, W. Cruz-Munoz, G.A. Bjarnason, J.G. Christensen, R.S. Kerbel,
Accelerated metastasis after short-term treatment with a potent inhibitor of
tumor angiogenesis, Cancer Cell 15 (2009) 232–239.
 K.J. Gotink, H.J. Broxterman, M. Labots, R.R. de Haas, H. Dekker, R.J. Honeywell,
M.A. Rudek, L.V. Beerepoot, R.J. Musters, G. Jansen, A.W. Griffioen, Y.G. Assaraf,
R. Pili, G.J. Peters, H.M. Verheul, Lysosomal sequestration of sunitinib: a novel
mechanism of drug resistance, Clin. Cancer Res. 17 (2011) 7337–7346.
 A.W. Griffioen, L. Mans, A.M. de Graaf, P. Nowak-Sliwinska, S. Bosch, C. de Hoog,
T. Dellemijn, F. Vyth-Dreese, A. Bex, E. Jonasch, Preoperative sunitinib treatment
of RCC patients inhibits angiogenesis in the primary tumor; rapid angiogenesis
onset after discontinuation of treatment, Clin. Cancer Res., in press.
 E. Kabingu, L. Vaughan, B. Owczarczak, K.D. Ramsey, S.O. Gollnick, CD8+ T cell-
mediated control of distant tumours following local photodynamic therapy is
independent of CD4+ T cells and dependent on natural killer cells, Br. J. Cancer
96 (2007) 1839–1848.
 P.S. Thong, K.W. Ong, N.S. Goh, K.W. Kho, V. Manivasager, R. Bhuvaneswari, M.
untreated tumours in recurrent angiosarcoma, Lancet Oncol. 8 (2007) 950–952.
 P.S. Thong, M. Olivo, K.W. Kho, R. Bhuvaneswari, W.W. Chin, K.W. Ong, K.C. Soo,
Immune response against angiosarcoma following lower fluence rate clinical
photodynamic therapy, J. Environ. Pathol. Toxicol. Oncol. 27 (2008) 35–42.
 S.O. Gollnick, C.M. Brackett, Enhancement of anti-tumor immunity by photody-
namic therapy, Immunol. Res. 46 (2010) 216–226.
 A.P. Castano, P. Mroz, M.R. Hamblin, Photodynamic therapy and anti-tumour
immunity, Nat. Rev. Cancer 6 (2006) 535–545.
 E. Kabingu, A.R. Oseroff, G.E. Wilding, S.O. Gollnick, Enhanced systemic immune
reactivity to a Basal cell carcinoma associated antigen following photodynamic
therapy, Clin. Cancer Res. 15 (2009) 4460–4466.
 F. Li, Y. Cheng, J. Lu, R. Hu, Q. Wan, H. Feng, Photodynamic therapy boosts anti-
glioma immunity in mice: a dependence on the activities of T cells and comple-
ment C3, J. Cell. Biochem. 112 (2011) 3035–3043.
new photosensitizer Tookad (WST09) for photodynamic vessel occlusion of the
choroidal tissue in rabbits, Invest. Ophthalmol. Vis. Sci. 47 (2006) 5437–5446.
 C.M. Moore, C.A. Mosse, C. Allen, H. Payne, M. Emberton, S.G. Bown, Light pene-
tration in the human prostate: a whole prostate clinical study at 763 nm, J.
Biomed. Opt. 16 (2011) 015003.
 J.R. van Beijnum, P. Nowak-Sliwinska, E. van den Boezem, P. Hautvast, W. Buur-
man, A. Griffioen, Tumor angiogenesis is enforced by autocrine regulation of
high-mobility group box 1, Oncogene, in press, PMID:22391561.
 E. Kluza, D.W. van der Schaft, P.A. Hautvast, W.J. Mulder, K.H. Mayo, A.W.
Griffioen, G.J. Strijkers, K. Nicolay, Synergistic targeting of alphavbeta3 integrin
and galectin-1 with heteromultivalent paramagnetic liposomes for combined
MR imaging and treatment of angiogenesis, Nano Lett. 10 (2010) 52–58.
 V.L. Thijssen, R. Postel, R.J. Brandwijk, R.P. Dings, I. Nesmelova, S. Satijn, N.
Verhofstad, Y. Nakabeppu, L.G. Baum, J. Bakkers, K.H. Mayo, F. Poirier, A.W.
Griffioen, Galectin-1 is essential in tumor angiogenesis and is a target for antian-
giogenesis therapy, Proc. Natl. Acad. Sci. U. S. A. 103 (2006) 15975–15980.
 A.W. Griffioen, D.W. van der Schaft, A.F. Barendsz-Janson, A. Cox, H.A. Struijker
Boudier, H.F. Hillen, K.H. Mayo, Anginex, a designed peptide that inhibits angio-
genesis, Biochem. J. 354 (2001) 233–242.
A. Weiss et al. / Biochimica et Biophysica Acta 1826 (2012) 53–70