Introduction: Fluorescence-guided surgery could potentially reduce local recurrence after pancreatic cancer resection. However, the ideal contrast agent for this purpose is not yet determined. The monoclonal antibody cetuximab targets the EGFR receptor, which is overexpressed in 64% of pancreatic cancers. We investigated the efficacy of near-infrared emitting silver sulphide Quantum Dot (QD)-cetuximab nanoconjugates for targeting EGFR-positive pancreatic cancer. Methods: 2-Mercaptopropionic acid-coated QDs were prepared from AgNO3 and Na2S. Pancreatic cancer cell lines PANC-1 and CFPAC-1 were confirmed EGFR-positive using a commercial AlexaFluor488-cetuximab probe. Nonconjugated QD and cetuximab-conjugated QD (QD-cetuximab) toxicity was assessed after 24 and 48 hours using MTT assay. Fluorescence microscopy was performed following a) formaldehyde-fixed immunofluorescence and b) live staining with QD-cetuximab for four hours at concentrations corresponding to 0, 10, 50, 100, 200, 400 and 600μg ml-1 of silver. Results: Untargeted QDs were non-toxic in both cell lines after 48 hours at all investigated concentrations, whereas QDcetuximab was toxic at 100µg ml-1 after 24 hours in PANC-1 and at 10µg ml-1 in CFPAC-1. Fixed immunofluorescence demonstrated EGFR targeting by QD-cetuximab at concentrations of 50μg ml-1 upwards in both cell lines. Live staining demonstrated similar efficacy of EGFR targeting up to 50μg ml-1 , although a reduction of fluorescence at higher concentrations was observed when compared to fixed immunofluorescence. Conclusion: Silver sulphide QD-cetuximab nanoconjugates have the potential to target live EGFR-positive pancreatic cancer cells at doses of up to 50 μg ml-1 . The reduction in QD fluorescence observed at higher concentrations is likely to be secondary to cetuximab toxicity.
Introduction: 5-aminolevulinic acid (ALA) generates protoporphyrin IX (PpIX)-induced fluorescence by acting as a substrate for the haem biosynthesis pathway. Despite suggestions that ALA could be used for pancreatic cancer photodiagnostics, the pancreatic cancer cell line PANC-1 only shows weak fluorescence following ALA administration. A possible explanation was that the haem biosynthesis pathway varies between cancers. Methods: We compared the mRNA expression of the haem biosynthesis pathway of PANC-1 (weak fluorescence) with the pancreatic cancer cell line CFPAC-1 (strong fluorescence) and the pancreatic ductal cell line H6c7 (control) with or without 24 hours ALA incubation. Cells were seeded on day one, fresh media with or without ALA (0.5mM) added on day two, and RNA extracted on day three. Quantitative real-time polymerase chain reaction was performed to assess the relative mRNA expression of four membrane transporters and eight enzymes responsible for haem biosynthesis. Results: Post-ALA incubation, CFPAC-1 demonstrated significant downregulation of cell membrane ALA influx transporter PEPT1, downregulation of ALA synthase and upregulation of the mitochondrial membrane transporter ABCB6. PANC-1, whilst showing similar changes to ALA synthase and ABCB6, showed significant upregulation of the PpIX efflux transporter ABCG2. PANC-1 also had minimal PEPT1 expression pre- and post-ALA. H6c7 demonstrated significant up- or downregulation of three transporters and five enzymes. Conclusion: Poor PpIX-induced fluorescence in PANC-1 is likely to be secondary to decreased ALA influx from low PEPT1 expression and increased ABCG2 expression. The use of nanocarriers to deliver ALA and/or ABCG2 inhibitors may improve ALA-induced fluorescence in PANC-1 and other ALA-resistant cancers.
Introduction: Pancreatic cancer often recurs following surgery suggesting new operative approaches are required. Fluorescence-guided surgery aims to assist surgeons in identifying tumour intraoperatively to facilitate complete resection. However, the ideal contrast agent for this purpose is not yet determined. The Rose criterion states that accurate imageguided surgery requires a Tumour-to-Background Ratio of contrast agent greater than 5. We investigated the potential of 5-aminolevulinic acid (ALA) for this purpose. Methods: Pancreatic cancer cell lines CFPAC-1 and PANC-1 were compared with the control pancreatic ductal cell line H6c7. Cells were seeded on day 1 and fluorescence measured on day 4 following 4, 8, 24 or 48 hours incubation with 0.25, 0.50, 0.75 or 1.00mM ALA. Fluorescence was measured using a plate reader and microscopy. Results: The maximum ALA-induced fluorescence for CFPAC-1 and PANC-1 was achieved after 48 hours incubation with 0.50mM ALA. Compared to cells incubated without ALA, a relative fluorescence increase of 39.4-fold in CFPAC-1 and 2.7-fold in PANC-1 was seen. ALA concentrations above 0.50mM did not result in higher fluorescence. In contrast, the control cell line H6c7 showed progressively increasing fluorescence with increasing ALA concentrations. The highest cancer/control cell fluorescence ratios for ALA were after 48 hours incubation with 0.25mM ALA; 122.9 in CFPAC-1 and 9.7 in PANC-1. Conclusion: ALA-induced fluorescence in CFPAC-1 is significantly higher than the control cell line H6c7. PANC-1 achieved only mildly increased fluorescence compared to H6c7. ALA has the potential to provide an adequate level of fluorescence for image-guided pancreatic surgery in ALA-susceptible cancers.
Pancreatic ductal adenocarcinoma remains one of the worst types of cancers mainly due to its late diagnosis, lack of effective therapies for advance disease and high chemoresistance. Novel therapeutic options that could improve patient quality of life and overall survival are therefore imperative. In this study, we describe the use of an original strategy based on photochemical internalisation (PCI) technology for pancreatic cancer treatment. Subcellular localisation of the photosensitiser meso-tetraphenylporphine-disulfonate (TPPS2a) was performed in PANC-1 cells, showing its preferential accumulation in lysosomes. Treatments with increasing concentrations of the ribosome-inactivating protein saporin or TPPS2a alone were compared with PCI-saporin. Metabolic activity and cell viability of PANC-1 cells were determined 96h post-illumination by MTT and trypan blue assays, respectively. Our results show that PCI using the photosensitiser TPPS2a, synergistically enhances the cytotoxic effects of saporin in PANC- 1 cells and could offer more effective treatment options for pancreatic cancer.
Pancreatic tumors are characterized by large interstitial hypertension from enhanced deposition of extracellular matrix components, resulting in widespread vascular collapse and reduced molecular uptake of systemically delivered therapies. Although the origins of hypoperfusion is debated amongst researchers, spatial distribution of collagen density and hyaluronic acid content have shown to be a key metric in understanding the lack of efficacy for both acute and chronic therapies in these tumors. In this study, the AsPC-1 tumor model was used both subcutaneously and orthotopically to study the measurable factors which are related to this. A conventional piezoelectric pressure catheter was used to measure total tissue pressure (TTP), defined as a combination of solid stress (SS) and interstitial fluid pressure (IFP), TTP = SS + IFP, in multiple locations within the tumor interstitium. Matrix components such as collagen and hyaluronic acid were scored using masson’s trichrome stain and hyaluronic acid binding protein (HABP), respectively, and co-registered with values of TTP. The results show that these key measurements are related to the spatial distribution of verteporfin in the same tumors. Photodynamic treatment with verteporfin is known to ablate large regions of tumor tissue and also allow better permeability for chemotherapies. The study of spatial distribution of verteporfin in relation to stromal content and TTP will help us better control these types of combination therapies.
Bryan Spring, R. Bryan Sears, Lei Zheng, Zhiming Mai, Reika Watanabe, Margaret Sherwood, David Schoenfeld, Brian Pogue, Stephen Pereira, Elizabeth Villa, Tayyaba Hasan
We introduce photoactivatable multi-inhibitor nanoliposomes (PMILs) for photodynamic tumor cell and microvessel damage in synchrony with photo-initiation of tumor-confined, multikinase inhibitor release. The PMIL is a biodegradable delivery system comprised of a nanoliposome carrying a photoactivable chromophore (benzoporphyrin derivative monoacid A, BPD) in its bilayer. A multikinase inhibitor-loaded PEG-PLGA nanoparticle is encapsulated within the liposome, which acts a barrier to nanoparticle erosion and drug release. Following intravenous PMIL administration, near infrared irradiation of tumors triggers photodynamic therapy and initiates tumor-confined drug release from the nanoparticle. This talk presents promising preclinical data in mouse models of pancreatic cancer utilizing this concept to suppress the VEGF and MET signaling pathways—both critical to cancer progression, metastasis and treatment escape. A single PMIL treatment using low doses of a multikanse inhibitor (cabozantinib, XL184) achieves sustained tumor reduction and suppresses metastatic escape, whereas combination therapy by co-administration of the individual agents has significantly reduced efficacy. The PMIL concept is amenable to a number of molecular inhibitors and offers new prospects for spatiotemporal synchronization of combination therapies whilst reducing systemic drug exposure and associated toxicities.
Photodynamic therapy can be a highly complex treatment with more than one parameter to control, or in some cases it is easily implemented with little control other than prescribed drug and light values. The role of measured dosimetry as related to clinical adoption has not been as successful as it could have been, and part of this may be from the conflicting goals of advocating for as many measurements as possible for accurate control, versus companies and clinical adopters advocating for as few measurements as possible, to keep it simple. An organized approach to dosimetry selection is required, which shifts from mechanistic measurements in pre-clinical and early phase I trials, towards just those essential dose limiting measurements and a focus on possible surrogate measures in phase II/III trials. This essential and surrogate approach to dosimetry should help successful adoption of clinical PDT if successful. The examples of essential dosimetry points and surrogate dosimetry tools which might be implemented in phase II and higher trials are discussed for solid tissue PDT with verteporfin and skin lesion treatment with aminolevulinc acid.
The use of in vivo contrast-enhanced magnetic resonance (MR) imaging as a surrogate for photosensitizer (verteporfin) dosimetry in photodynamic therapy of pancreas cancer is demonstrated by correlating MR contrast uptake to ex vivo fluorescence images on excised tissue. An orthotopic pancreatic xenograft mouse model was used for the study. A strong correlation (r=0.57 ) was found for bulk intensity measurements of T1-weighted gadolinium enhancement and verteporfin fluorescence in the tumor region of interest. The use of contrast-enhanced MR imaging shows promise as a method for treatment planning and photosensitizer dosimetry in human photodynamic therapy (PDT) of pancreas cancer.
The goal of this work was to develop and validate a pancreas tumor animal model to investigate the relationship between photodynamic therapy (PDT) effectiveness and photosensitizer drug delivery. More specifically, this work lays the foundation for investigating the utility of dynamic contrast enhanced blood perfusion imaging to be used to inform subsequent PDT. A VX2 carcinoma rabbit cell line was grown in the tail of the pancreas of three New Zealand White rabbits and approximately 3-4 weeks after implantation the rabbits were imaged on a CT scanner using a contrast enhanced perfusion protocol, providing parametric maps of blood flow, blood volume, mean transit time, and vascular permeability surface area product.
We undertook a phase I dose-escalation study of verteporfin photodynamic therapy (PDT) in 15 patients with locally advanced pancreatic cancer. Needle placement and laser delivery were technically successful in all patients. Thirteen patients were treated with a single laser fibre. Three treatments were carried out each at 5, 10 and 20 J/cm2; and 5 treatments (4 patients) at 40 J/cm2. A further 2 patients were treated with 2 or 3 laser fibres at 40 J/cm2. Tumour necrosis was measured on CT (computed tomography) by two radiologists 5 days after treatment. There was a clear dosedependent increase in necrosis with a median area of 20 x 16 mm (range 18 x 16 to 35 x 30 mm) at 40 J/cm2. In the 2 patients treated with multiple fibres, necrosis was 40 x 36 mm and 30 x 28 mm, respectively. There were no early complications in patients treated with a single fibre. Both patients treated with multiple fibres had evidence on CT of inflammatory change occurring anterior to the pancreas but without clinical deterioration. These results suggest that single fibre verteporfin PDT is safe in a clinical setting up to 40J/cm2 and produces a dose-dependent area of pancreatic necrosis.
The goal of this work was to determine the light dose required to induce necrosis in verteporfin-based photodynamic therapy, in the VERTPAC-1 trial. Patient CT scans were obtained of the abdomen, including the entire treatment zone of pancreas and surrounding tissues, before and after treatment, as well as fast scans during needle placement. These scans were used to estimate arterial and venous blood content, and provide structural information of the pancreas and nearby blood vessels. Using NIRFAST, a finite-element based package for modeling diffuse near-infrared light transport in tissue, simulations were run to create maps of light fluence within the pancreas. These maps provided visualizations of light dose overlaid on the original CT scans, and were used to estimate light dose at the boundary of the zone of necrosis, as observed in follow up treatment outcome CT scans. The aim of these simulation studies was to assist pre-treatment planning by informing the light treatment parameters. This paper presents a case study of the process used on a single patient.
The diagnosis and treatment of pancreaticobiliary malignancy is of major interest to our group. Building on prior work,
we undertook a phase I study of verteporfin photodynamic therapy in patients with locally advanced, unresectable,
pancreatic cancer. We also initiated an optical diagnostic study using elastic scattering spectroscopy (ESS) of the
normal-appearing periampullary duodenal mucosa in vivo to investigate the hypothesis of a field effect in
pancreaticobiliary malignancy. In a phase I dose escalation study, patients were treated with interstitial verteporfin PDT
via a single fibre, to determine its general safety profile and the optimum treatment parameters needed to achieve
effective and safe necrosis of tumour, With increasing light doses, there was a linear increase in the extent of tumour
necrosis around the fibre, without serious adverse events. Follow-on studies using multiple fibres are planned. In 30
patients with benign or malignant pancreaticobiliary disease undergoing clinically-indicated endoscopy, ESS spectra
were collected from the normal-appearing duodenum and antrum and a diagnostic algorithm generated by principle
component and linear discriminant analysis. Pooled data from duodenal sites distal to the ampulla gave a sensitivity of
86% and a specificity of 72% (82% AUC) for the detection of malignancy, whereas those from the periampullary region
had a sensitivity of 77% and a specificity of 61% (72% AUC); antral measurements were not able to discriminate with
such accuracy. These early results suggest that ESS of the duodenal mucosa could represent a novel minimally invasive
diagnostic test for pancreaticobiliary malignancy.
Pancreatic adenocarcinoma ranks as the fourth most common cause of cancer death in the USA. Patients usually present late with advanced disease, limiting attempted curative surgery to 10% of
cases. Overall prognosis is poor with one-year survival rates of less than 10% with palliative chemotherapy and/or radiotherapy. Given these dismal results, a minimally invasive treatment capable
of local destruction of tumor tissue with low morbidity may have a place in the treatment of this disease. In this paper we review the preclinical photodynamic therapy (PDT) studies which have shown
that it is possible to achieve a zone of necrosis in normal pancreas and implanted tumour tissue. Side effects of treatment and evidence of a potential survival advantage are discussed. We describe the only
published clinical study of pancreatic interstitial PDT, which was carried out by our group (Bown et al Gut 2002), in 16 patients with unresectable locally advanced pancreatic adenocarcinoma. All patients
had evidence of tumor necrosis on follow-up imaging, with a median survival from diagnosis of 12.5 months. Finally, we outline a phase I dose-escalation study of verteporfin single fibre PDT followed by
standard gemcitabine chemotherapy which our group is currently undertaking in patients with locally advanced pancreatic cancer. Randomized controlled studies are also planned.
Photodynamic therapy (PDT) mediated with Verteporfin is being investigated as a pancreatic cancer treatment in the
cases for non-surgical candidates. Tissue response to PDT is based on a number of parameters including photosensitizer
(PS) dose, light dose and time interval between light application and PS injection.
In this study, PS uptake and distribution in animal leg muscle, oral cavity tissues, pancreas and tumor was measured
in vivo using light-induced fluorescence spectroscopy (LIFS) via an Aurora Optics Inc. PDT fluorescence dosimeter. An
orthotopic pancreatic cancer model (AsPC-1) was implanted in SCID mice and treated with the PS. Probe measurements
were made using a surface probe and an interstitial needle probe before and up to one hour after intravenous tail vein
injection of the PS.
The study demonstrated that it is possible to correlate in-vivo LIFS measurements of the PS uptake in the pancreas
with measurements taken from the oral cavity indicating that light dosimetry of PDT of the pancreas can be ascertained
from the LIFS measurements in the oral cavity. These results emphasize the importance of light dosimetry in improving
the therapeutic outcome of PDT through light dose adaptation to the relative in situ tissue PS concentration.
KEYWORDS: Photodynamic therapy, Receptors, Cell death, Proteins, Simulation of CCA and DLA aggregates, Cancer, Modulation, In vitro testing, Resistance, Confocal microscopy
EGFR, a member of the ERBB family, plays a pivotal role in carcinogenesis. EGFR overexpression is
implicated in DNA repair and synergistic interactions between EGFR-targeting drugs and conventional
chemo/radiotherapy have been reported in preclinical studies for different cancers but not
cholangiocarcinoma (CCA). To date there are no in vitro data available on the cellular response and
effect of either photodynamic therapy (PDT) or EGFR-targeting drugs on CCA. Therefore, we aimed
to study the: (i) response to Verteporfin PDT and to EGFR-targeting drugs, as single agents; (ii) effect
of PDT on ERBBs expression, phosporylation status and activation of its signaling pathways; (iii)
response to combination of PDT and EGFR-targeting agents.
We showed that two cholangiocarcinoma cell lines (HuCCT1 and TFK1 cells, intra- and extrahepatic,
respectively) differentially respond to verteporfin-PDT treatment and are resistant to EGFR-targeting
agents. A constitutive activation of EGFR in both cell lines was also observed, which could partly
account for the observed resistance to EGFR-targeting drugs. In addition, verteporfin-PDT induced
further phosphorylation of both EGFR and other Receptor Tyrosine Kinases. Mitochondria-independent
apoptosis was induced by PDT in both CCA cell lines; in particular, PDT modulated the
expression of members of the Inhibitor of Apoptosis (IAP) family of proteins. Interestingly, there was a
PDT-induced EGFR nuclear translocation in both cell lines; co-treatment with either an EGFR-inhibitor
(Cetuximab) or a nuclear import blocking agent (Wheat Germ Agglutinin) had an additive
effect on PDT cell killing, thus implying a role of EGFR in repairing the potential PDT-induced DNA
damage.
There is a need for better management strategies to improve survival and quality of life in patients with biliary tract
cancer (BTC). We compared treatment outcomes in 321 patients (median age 65 years, range 29-102; F:M; 1:1) with a
final diagnosis of BTC (cholangiocarcinoma n=237, gallbladder cancer n=84) seen in a tertiary referral cancer centre
between 1998-2007. Of 89 (28%) patients who underwent surgical intervention with curative intent, 38% had R0
resections and had the most favourable outcome, with a 3 year survival of 57%. Even though PDT patients had more
advanced clinical T-stages, their survival was similar to those treated with attempted curative surgery which resulted in
R1/2 resections (median survival 12 vs. 13 months, ns). In a subgroup of 36 patients with locally advanced BTC treated
with PDT as part of a prospective phase II study, the median survival was 12 (range 2-51) months, compared with 5
months in matched historical controls treated with stenting alone (p < 0.0001). In this large UK series, long-term survival
with BTC was only achieved in surgical patients with R0 resection margins. Palliative PDT resulted in similar survival to
those with curatively intended R1/R2 resections.
KEYWORDS: Simulation of CCA and DLA aggregates, Photodynamic therapy, Receptors, Cancer, Tumors, In vitro testing, Proteins, Solids, Therapeutic agents, Oncology
Accumulating preclinical and clinical evidence supports a pro-oncogenic function for Notch signaling in several solid
tumors. Therefore, Notch inhibitory agents, such as gamma-secretase inhibitors (GSI), are being investigated as cancer
therapeutic agents and a potential adjuvant to conventional chemo/radiotherapy. To date, no in vitro data are available on
the cellular response and effect of either photodynamic therapy (PDT) or GSI on human cholangiocarcinoma (CCA).
Consequently, we aimed to study the: (i) constitutive expression of Notch signaling pathway in CCA cell lines; (ii)
response to Verteporfin-PDT and to GSI, as single agents on CCA cell lines; (iii) effect of Verteporfin-PDT on Notch
signaling pathway expression.
Expression of Notch signaling components was studied in two cholangiocarcinoma cell lines, HuCCT1 and
TFK-1 (intra- and extrahepatic, respectively). No difference in basal expression of Notch1, 2 and Jagged1 was observed
in either cell line. In contrast, Notch3 was found to be weakly and highly expressed in HuCCT1 and TFK-1 cells,
respectively - supporting our recent microarray data which showed Notch3 overexpression in biliary brushings from
patients with extrahepatic CCA. HuCCT1 and TFK-1 differentially responded to Verteporfin-PDT treatment; preliminary
data showed no clear effect of GSI on proliferation/apoptosis in either cell line following short exposure (6 and 24h).
Following Verteporfin-PDT, Notch1, 2 and Jagged-1 expression was down-regulated in both cell lines, while Notch3
expression was unaffected in HuCCT1 cells and down-regulated in TFK-1 cells. The Notch signaling pathway could
represent a potential target for combination therapy in CCA treatment.
Patients with non-resectable pancreatic and biliary tract cancer (cholangiocarcinoma and gallbladder cancer) have a
dismal outlook with conventional palliative therapies, with a median survival of 3-9 months and a 5 year survival of less
than 3%. Surgery is the only curative treatment but is appropriate in less than 20% of cases, and even then is associated
with a 5-year survival of less than 30%. Although most applications of photodynamic therapy (PDT) in gastroenterology
have been on lesions of the luminal gut, there is increasing experimental and clinical evidence for its efficacy in cancers
of the pancreas and biliary tract. Our group has carried out the only clinical study of PDT in pancreatic carcinoma
reported to date, and showed that PDT is feasible for local debulking of pancreatic cancer. PDT has also been used with
palliative intent in patients with unresectable cholangiocarcinoma, with patients treated with stenting plus PDT reporting
improvements in cholestasis, quality of life and survival compared with historical or randomized controls treated with
stenting alone. Further controlled studies are needed to establish the influence of PDT and chemotherapy on the survival
and quality of life of patients with pancreatic and biliary tract carcinoma.
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