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Papers by Ludovic Bourre

Cancer Research

Introduction: Intrahepatic cholangiocarcinoma (iCCA) is a highly aggressive adenocarcinoma derive... more Introduction: Intrahepatic cholangiocarcinoma (iCCA) is a highly aggressive adenocarcinoma derived from bile ducts within the liver. This malignancy accounts for about 10% of cholangiocarcinoma cases and is the second most common primary liver cancer. Effective treatments for iCCA are limited, and they are primarily treated by tumor surgical resection. Regrettably, surgical resection is limited to early-stage disease and has an unfavorable prognosis. Development of novel therapeutic strategies for iCCA is needed to improve outcomes for non-resectable late stage iCCAs. Therefore, establishing suitable animal models for in vivo investigation of oncology and immuno-oncology treatment strategies for iCCA is critical. Methods: The Pten−/- GEMM was generated by CRISPR-Cas9 and crossed with Alb-Cre and LSL-KrasG12D/+ mice. Pathological examination of liver and blood biochemistry were investigated in thirteen GEMM mice. Liver fragments that were enriched with iCCA lesions were inoculated in...

Cancer Research

Introduction: Gliomas account for majority of primary brain tumors and are composed of different ... more Introduction: Gliomas account for majority of primary brain tumors and are composed of different grades, which present distinct molecular alterations, histological characteristics, and response to treatments. Patient-derived xenografts (PDXs) are the most “close-to-patient” models for oncology studies which largely maintain the histo- and molecular- pathology of the original tumors and are widely applied in preclinical evaluations. Subcutaneously inoculated glioma PDX models provide convenient evaluation of treatments’ efficacy but are difficult to mimic the in-situ microenvironment and diffuse invasive growth to peripheral tissue in the brain. Orthotopic implantation of bioluminescent labelled glioma models provides a more relevant preclinical model to evaluate and quantify treatment response. Methods: A glioma PDX model was established by subcutaneously engrafting tumor directly from patient following surgery into immunodeficient mice. Once established and expanded through passagi...

Cancer Research

T cell engagers show high efficacy in B cells malignances. High risk of immune-related adverse ev... more T cell engagers show high efficacy in B cells malignances. High risk of immune-related adverse events, including cytokine release syndrome (CRS), is reported in patients due to on-target off-site effects of T cell engagers. Thus, reliable and translational mouse models are required to predict potential safety issues and investigate their rescue. Here we describe two preclinical models mimicking to some extent CRS induction upon activation with anti-CD3 antibodies.BRGSF (Balb/C Rag2-/-, IL2Rγ-/-, SIRPαNOD and Flt3-/-) is a highly immunodeficient mouse featuring reduced murine myeloid cells. Development of human lymphoid (B and T cells) and myeloid (NK, cDC, pDC and monocytes) compartments upon CD34+ HSC-engraftment are observed in blood, spleen and bone marrow and are stable for over a year, without side effects. Anti-hCD3 mAb, OKT3, administration in BRGSF-HIS mice induced a rapid release of human cytokines (i.e., IL-6, TNF-α, IFN-γ, IL-2) in serum. Pretreatment with Flt3L, enhancin...

Photodynamic therapy (PDT), a treatment for small "early" carcinomas is based on the co... more Photodynamic therapy (PDT), a treatment for small "early" carcinomas is based on the combined action of a non toxic drug (photosensitizer) administered orally or systemically and visible light applied locally on tumor. Tumoral death is induced by light with release of toxic radicals. One of the most important parameter during PDT treatment is the pharmacokinetic of molecule, as pharmacokinetic must be perfectly known in order to irradiate the lesion when tissue concentration of photosensitizer is maximum. However the delay between injection and the maximum intratumoral concentration in photosensitizers must be as short as possible in order to avoid a costly hospitalisation. Moreover, photosensitizers must have a fast clearance to avoid too long cutaneous photosensitivity for the patient. The diphenylchlorin (SIM01), object of this study, has two phenyl rings around a central nucleus, which could allow a faster pharmacokinetic, with a maximum concentration reached more rapi...

Journal for ImmunoTherapy of Cancer, 2021

BackgroundThe breakthrough of immunotherapies has unleashed new hope and new success for cancer t... more BackgroundThe breakthrough of immunotherapies has unleashed new hope and new success for cancer therapy. However, the choice of a preclinical model is one of the main challenges as they are important for evaluation of translatability to help support testing in clinical studies, including potential efficacy and tolerability of immunotherapies during preclinical development.The development of mouse models featuring a human immune system (HIS) provides new paths for the investigation of the efficacy of immunotherapies in preclinical models engrafted with human tumors. Although these models provided a breakthrough in the assessment of immune targeting agents, they also come with a few significant caveats. These include: a lack of a mature human myeloid compartment in the mouse, and a short life span of the model when this compartment is promoted at non-physiological levels via the over-expression of human cytokines. Here, we report a novel mouse model (BRGSF-HIS), featuring functional h...

Journal of Clinical Oncology, 2012

e15161 Background: Docetaxel was the first cytotoxic treatment demonstrating a survival benefit i... more e15161 Background: Docetaxel was the first cytotoxic treatment demonstrating a survival benefit in metastatic castration-resistant prostate cancer (mCRPC) patients. Recently, cabazitaxel (a novel taxane) and abiraterone acetate (specific inhibitor of CYP17) have demonstrated a significant survival benefit in mCRPC patients progressing after receiving a docetaxel – containing regimen. Here we compare the antitumor efficacy of docetaxel, cabazitaxel and abiraterone acetate in the HID28 hormone-refractory human prostate carcinoma xenograft. Methods: HID28 tumor bearing nude mice received 20 mg/kg of docetaxel (IP, q3wk x 2), cabazitaxel at 15 and 20 mg/kg (IP, q3wk x 2) and abiraterone acetate at 50 mg/kg (PO, qdx21). Median tumor growth inhibition (T/C%) was evaluated as well as time course androgen receptor expression (4, 8 and 24h post dosing) by western blot analysis. Results: Docetaxel (20 mg/kg) inhibited tumor growth with a T/C%= 16.7% at D35, 2 partial (PR) and 1 complete (CR) ...

Annals of Oncology, 2012

Background: Levels of bone turnover markers (BTM) might be correlated with outcome in terms of sk... more Background: Levels of bone turnover markers (BTM) might be correlated with outcome in terms of skeletal related events (SRE), disease progression and death. The aim of this study was to determine the possible correlation between BTM, disease progression, SREs and death in patients with genitourinary cancer and bone metastases (BM) treated with zoledronic acid (ZA). Methods: Observational, prospective, multicenter study. Patients with genitourinary cancer (prostate, renal, bladder) and BM were included. BTM determined were: carboxiterminal telopeptide of type I collagen (β-CTX) and bone specific alkaline phosphatase (BALP) by ELISA (immunoenzymatic assay, IDS UK), and aminoterminal propeptide of type I collagen (P1NP) by automatised assays (Elecsys, Roche). All BTM were determined at baseline (V0) and every 3 mo of treatment until Month 18 (V6). All patients started treatment with ZA 4 mg IV every 3-4 weeks at the beginning of the study. Results: Data of 168 patients with genitourinary cancer were analyzed. In this work data of prostate cancer patients (n = 95) are presented. Population basal characteristics [35 new, without previous treatment (N) / 60 pre-treated, with anti-hormonal treatment (P)]: mean age (years): 72.1; median time from tumor diagnosis (months): 7.6 (N) / 11.6 (P); ECOG 0-1: 90% (N) / 87.5% (P). Patients with pathologic baseline levels were: 47.1% β-CTX, 64.7% BALP and 57.1% P1NP (N) and 48.3% β-CTX, 80.7% BALP and 60% P1NP (P). After 6 mo.: 28%, 48% and 32% (N) and 32%, 59.4% and 31.3% (P) presented pathologic values of β-CTX, BALP and P1NP respectively. Normalized levels remained steady throughout 18 mo follow-up. Reductions from baseline to month 3 in BALP levels correlated with lower risk of death (p = 0.0219) (Cox regression analysis). Conclusions: Elevation of baseline BTM levels is frequently present in advanced prostate cancer with bone metastasis. Addition of AZ to standard systemic therapy reduces BTM levels, even though in those highly hormone sensitive ones. A significant association was observed between elevated levels of BALP and death throughout follow-up. Disclosure: All authors have declared no conflicts of interest.

Oncotarget, 2019

Telomerase reverse transcriptase (TERT) is highly expressed in more than 90% of canine cancer cel... more Telomerase reverse transcriptase (TERT) is highly expressed in more than 90% of canine cancer cells and low to absent in normal cells. Given that immune tolerance to telomerase is easily broken both naturally and experimentally, telomerase is an attractive tumor associated antigen for cancer immunotherapy. Indeed, therapeutic trials using human telomerase peptides have been performed. We have developed an immunogenic yet catalytically inactive human telomerase DNA construct that is in clinical trials with patients presenting solid tumors. Paralleling this human construct, we have developed a canine telomerase DNA vaccine, called pDUV5. When administered intradermally to mice combined with electrogene transfer, pDUV5 induced canine TERT specific cytotoxic T-cells as measured by IFN-γ ELISpot assay. Intradermal vaccination of healthy dogs with 400 µg of pDUV5 generated strong, broad and long lasting TERT specific cellular immune responses. In vitro immunization with cTERT peptides revealed the maintenance of cTERT specific T-cells in PBMCs from tumor bearing dogs showing that this repertoire was not depleted. This study highlights the potential of pDUV5 as a cancer vaccine and supports its evaluation for the treatment of spontaneous canine tumors.

Journal of Clinical Oncology, 2017

3087 Background: INVAC-1 is an optimized plasmid encoding an inactive form of human telomerase re... more 3087 Background: INVAC-1 is an optimized plasmid encoding an inactive form of human telomerase reverse transcriptase (hTERT). hTERT is a prototype of shared tumor antigen expressed in more than 85% of human tumors. Telomerase activation is associated with maintenance of telomere length and accounts for the unlimited proliferative capacity of cancer cells. In preclinical models, INVAC-1 triggered Th1-polarized hTERT-specific CD8+ and CD4+T-cell immune responses and anti-tumor effects. Here, we report clinical and pharmacodynamics results of the first clinical study with INVAC-1 as a single agent in solid tumors. Methods: A 3+3 design phase 1 First in Human study evaluating INVAC-1 given monthly for 3 cycles using electroporation-based intra-dermal (ID) injection was conducted. Primary objectives included safety, tolerability and dose limiting toxicities to identify the maximum tolerated dose (MTD) and recommended phase 2 dose (RP2D). Secondary objectives included immune response and ...

Molecular Cancer Therapeutics, 2013

Background: The ability of tumor cells to spread and form metastases is one of the key parameters... more Background: The ability of tumor cells to spread and form metastases is one of the key parameters used to classify tumor aggressiveness. Metastasis detection at diagnosis or during followup post-surgery is associated with poor prognosis. The paucity of pertinent models to investigate the biology of metastasis renders problematic the set-up of anti-metastasis therapies. We took advantage of our human patient-derived xenograft (PDX) collection to develop models suitable to assess anti-metastatic therapy. To enable evaluation of metastasis response to treatment, we labeled tumors with lentivirus-mediated luciferase insertion, allowing longitudinal followup of metastasis formation by non-invasive imaging. A review of our metastatic bioluminescent PDX panel is described here with a particular focus on our most advanced bioluminescent breast model HBCx-14-Luc1. Methods: Tumor cells from freshly excised PDXs were dissociated and transduced with a defective lentivirus bearing the firefly lu...

Journal of Environmental Pathology, Toxicology and Oncology, 2006

Photodynamic therapy (PDT) is based on the selective light activation of an exogenously given dru... more Photodynamic therapy (PDT) is based on the selective light activation of an exogenously given drug to patients. PDT acts mainly on cell membranes either of neovascular endothelial cells or of cancer cells leading to cancer cell death. Six drugs are now marketed based on clinical assays in various indications, which showed a clear cost efficiency as compared to other classical procedures. PDT is easy to handle and can be performed in medical installations fitting the conditions of health care in developing countries. Its cost effectiveness could represent an appropriate solution to the increasing number of cancers of various origin. However despite all the clinical results now available, PDT development remains slow. The reasons for this situation include cost of development, intellectual property, and competition between pharmaceutical companies.

British journal of cancer, Jan 10, 2009

Intracellular generation of the photosensitiser, protoporphyrin IX, from a series of dipeptide de... more Intracellular generation of the photosensitiser, protoporphyrin IX, from a series of dipeptide derivatives of the haem precursor, 5-aminolaevulinic acid (ALA), was investigated in transformed PAM212 murine keratinocytes, together with studies of their intracellular metabolism. Porphyrin production was substantially increased compared with equimolar ALA using N-acetyl terminated phenylalanyl, leucinyl and methionyl ALA methyl ester derivatives in the following order: Ac-L-phenylalanyl-ALA-Me, Ac-L-methionyl-ALA-Me and Ac-L-leucinyl-ALA-Me. The enhanced porphyrin production was in good correlation with improved photocytotoxicity, with no intrinsic dark toxicity apparent. However, phenylalanyl derivatives without the acetyl/acyl group at the N terminus induced significantly less porphyrin, and the replacement of the acetyl group by a benzyloxycarbonyl group resulted in no porphyrin production. Porphyrin production was reduced in the presence of class-specific protease inhibitors, namel...

Research in Experimental Medicine, 1999

Photodynamic therapy (PDT) induces cell-membrane damage and alterations in cancer-cell adhesivene... more Photodynamic therapy (PDT) induces cell-membrane damage and alterations in cancer-cell adhesiveness, an important parameter in cancer metastasis. These alterations result from cell sensitivity to photosensitizers and the distribution of photosensitizers in cells. The efficacy of photosensitizers depends on their close proximity to targets and thus on their pharmacokinetics at the cellular level. We studied the cellular distribution of photosensitizers with a confocal microspectrofluorimeter by analysing the fluorescence emitted by benzoporphyrin derivative-monoacid ring A (BPD-MA) and Photofrin relative to their cell sensitivity. Two cancer cell lines of colonic origin, but with different metastatic properties, were used: PROb (progressive) and REGb (regressive). For BPD-MA (1.75 µg/ml), maximal fluorescence intensity (8,300 cts) was reached after 2 h for PROb and after 1 h (4,900 cts) for REGb. For Photofrin (10 µg/ml), maximal fluorescence intensity (467 cts) was reached after 5 h for PROb and after 3 h (404 cts) for REGb. Intracellular studies revealed stronger cytoplasmic than nuclear fluorescence for both BPD and Photofrin. Both of the sensitizers induced a dose-dependent phototoxicity; LD 50 with BPD-MA was 93.3 ng/ml for PROb and 71.1 ng/ml for REGb, under an irradiation of 10 J/cm 2. With Photofrin, LD 50 was 1,270 ng/ml for PROb and 1,200 ng/ml for REGb under an irradiation of 25 J/cm 2. The photosensitizer effect within PROb and REGb cancer cells was assessed by incorporation kinetics and toxicity-phototoxicity tests. The intracellular concentration of the photosensitive agent was one important factor in the effectiveness of PDT, but not the only one contributing to the photodynamic effect. In conclusion, this study showed that there was a clear difference between sensitizer uptake and phototoxicity, even in cancer cells of the same origin. This could induce cell-killing heterogeneity in clinics.

Surgical Neurology, 2007

Background: Glioblastomas are the third most common cause of cancer death in patients between 15 ... more Background: Glioblastomas are the third most common cause of cancer death in patients between 15 and 35 years old. Literature suggests that PDT could represent a promising treatment, providing that sensitizers could accumulate within the cancer tissues despite the blood-brain barrier. Methods: Distribution and PDT effect of SIM01, a promising photosensitizer, have been evaluated on orthotopic C6 tumor model in rats by comparison with HPD and m-THPC. Pharmacokinetics had been analyzed with fluorescence and ROS. Photodynamic treatment was done using a 630-nm light with an energy density of 100 J cm À2 for HPD and a 652-nm light with an energy density of 20 J cm À2 for m-THPC and SIM01. Results: The correlation between fluorescence and ROS dosimetry was found to be excellent. An optimal concentration was found after 12 hours for SIM01 (4 mg/kg), 24 hours for HPD (10 mg/kg), and 48 hours for m-THPC (4 mg/kg). The best normal tissue/cancer ratio of concentration had been found after 12 hours for SIM01 and 48 hours for HPD and m-THPC. Pathological examinations after PDT showed that the criteria for histology of glioblastic origin were absent in SIM01-treated rats 12 hours after injection but were present in 50% of rats treated 24 hours after injection and in all after a 48-hour delay. Mean survival of rats treated 12 or 24 hours after SIM01 injection was significantly improved compared with controls, HPD-, or m-THPC-treated groups. Survival of rats treated 12 or 24 hours after SIM01 injection reached 20 days but decreased for longer delays. On the contrary, survival reached 18 days at the maximum for rats treated 48 hours after m-THPC or HPD injection. Conclusions: Our results confirm that PDT is a promising treatment for glioblastomas. SIM01 efficacy is as efficient as m-THPC but with much more favorable pharmacokinetics.

Photodiagnosis and Photodynamic Therapy, 2005

PDT had been proposed in gastroenterology for various indications and the esophageal cancer treat... more PDT had been proposed in gastroenterology for various indications and the esophageal cancer treatment had been among the very first having been approved. However, PDT failed to be a real breakthrough. One reason for it was that although it had been approved for the palliative treatment of advanced tumors, PDT only has by nature a limited in-depth efficacy fitting better to the treatment and often the cure of ''early cancers''. For this reason PDT has also been proposed for the treatment of Barrett's esophagus (BE) with high-grade dysplasias. Barrett's mucosa (BM) is a field of a specialized metaplastic columnar epithelium replacing the normal stratified squamous epithelium or mucosa lining the distal esophagus. In this case, PDT has to destroy an area of thin tissues spread eventually over a wide area instead of a mass of tissues. Something important is that existing treatments allow the treatment of foci of dysplastic tissues but not the regression of the whole BM. BE is thus an unsolved medical problem having medical as well as economic consequences as BM being likely to transform into a cancer has to be carefully surveyed. The esophageal cancer, an adenocarcinoma, has to be surgically removed when it is possible something pretty heavy with a high morbidity. Economic burnt is also important with high survey costs independently to the additional surgical costs in case of diagnosed cancer. Treatments proposed for non or mild dysplastic BM regression have in common to have an inhomogenous impact on the target. Treatments for high-grade dysplasia (HGD, the ultimate pathological step before cancer) are based on mucosectomy and are limited to small areas of tissues. Recently circumferential mucosectomy had been proposed but at a higher risk making it suitable only to highly experienced hands in infrequent indications.

Photodiagnosis and Photodynamic Therapy, 2005

Photodynamic therapy (PDT) is a new approach to cancer treatment for a variety of malignant tumor... more Photodynamic therapy (PDT) is a new approach to cancer treatment for a variety of malignant tumors. A new photosensitizer, 2,3-dihydro-5,15-di(3,5dihydroxyphenyl)porphyrin (SIM01), had been evaluated for its genotoxic effects on glioma cells (C6). Comet assay had been used to evaluate the potential genotoxic effect induced by SIM01 on the C6 cells. When SIM01 had been shown to be a powerful sensitizer no DNA strand break was detected in the absence of light. SIM01 localized in cytoplasm but not in the nucleus of the tumors cells, which supported the finding of undetectable DNA damage under darkness and low photodynamic dose. Cell exposure to 20 J cm −2 after an incubation time of 2 h with 0, 0.25, 0.5, 2 or 4 g mL −1 induced less than 25% of cell death but significant Tail Moment changes. If DNA damage intensity increased according to SIM01 doses under light exposure, importance of repair seemed to increase proportionally to PDT-induced damage. Positive controls consisted of doxorubicin-treated C6 cells this mutagen being known to induce genetic damage. Whatever the conditions used SIM01 appeared to be less deleterious than doxorubicin. As the comet assay can not give us the certitude that no mutation, photoadducts or oxidative damage had been developed under light exposure this point will have to be verified with another mutagenicity assay. SIM01 appears to be safe from a mutagenic point of view something of importance as tumors of small volume in patients with a long lifespan are at first indicated for PDT.

Photodiagnosis and Photodynamic Therapy, 2006

Delta amino levulinic acid photodynamic therapy (ALA-PDT) represents one of the most prominent ad... more Delta amino levulinic acid photodynamic therapy (ALA-PDT) represents one of the most prominent advances in PDT. ALA itself or its derivatives are marketed for a variety of clinical indications. Despite the development of clinical applications, experimental ALA results are very heterogeneous and experimentally used parameters are still not standardized. This suggests that some problems remain unsolved that are likely to impair experiments to be performed but also that clinical results obtained could be greatly improved. Frequently unmentioned or imprecise data concern solvents, pH of ALA solutions, storage time, ALA degradation or ALA efficacy. In addition, diversity of experimental model is huge while capabilities of ALA transformation into PpIX are known to vary from one cell to the other. Thus, the aim of the present paper was to quantify the level of ALA degradation or changes in ALA efficacy using one single cell line without presuming of the mechanisms and determine the conditions of storage inducing the best transformation into PpIX and/or cell phototoxicity. We added ALA diluted in water, PBS or RPMI to C6 cells, a murine brain tumour cell line that can be used in vivo as an orthotopic graft. We measured in cells used as tools for final bio efficacy estimation, both the induced fluorescence and phototoxicity in various conditions of storage before use chosen to be as close as possible to the real lab conditions. Water had been found to better preserve ALA than, respectively, PBS and RPMI and this for any temperature or storage durations. The lowest temperature and the shortest duration for storage used had also been shown to better preserve ALA-induced fluorescence and phototoxicity. The fact that these properties were found to be better preserved in 7.4 buffered solvent could be in relationship with a fast ALA condensation occurring at neutral or lightly acidic pH modifying its availability for an optimal transformation into PpIX.

Photochemistry and Photobiology, 2007

Synthesis of ␦-aminolevulinic acid (ALA) derivatives is a promising way to improve the therapeuti... more Synthesis of ␦-aminolevulinic acid (ALA) derivatives is a promising way to improve the therapeutic properties of ALA, particularly cell uptake or homogeneity of protoporphyrin IX (PpIX) synthesis. The fluorescence emission kinetics and phototoxic properties of ALA-n-pentyl ester (E1) and R,S-ALA-2-(hydroxymethyl) tetrahydrofuranyl ester (E2) were compared with those of ALA and assessed on C6 glioma cells. ALA (100 g/mL), E1 and E2 (10 g/mL) induced similar PpIX-fluorescence kinetics (maximum between 5 and 7 h incubation), fluorescence being limited to the cytoplasm. The 50% lethal dose occurred after 6 h with 45, 4 and 8 g/mL of ALA, E1 and E2, respectively. ALA, E1 and E2 induced no dark toxicity when drugs were removed after 5 min of incubation. However, light (25 J/cm 2) applied 6 h after 5 min incubation with 168 g/mL of each compound induced 85% survival with ALA, 27% with E1 and 41% with E2. Increasing the incubation time with ALA, E1 and E2 before washing increased the phototoxicity, but E1 and E2 remained more efficient than ALA, regardless of incubation time. ALA-esters were more efficient than ALA in inducing phototoxicity after short incubation times, probably through an increase of the amount of PpIX synthesized by C6 cells.

Photochemical & Photobiological Sciences, 2010

Given that cell-penetrating peptides (CPP) are cationic and often amphipathic, similar to membran... more Given that cell-penetrating peptides (CPP) are cationic and often amphipathic, similar to membrane-active antimicrobial peptides, it may be possible to use CPP conjugation to improve the delivery of photosensitisers for antimicrobial photodynamic therapy (antimicrobial PDT). We investigated the possibility of using a Tat peptide to deliver the photosensitiser, tetrakis(phenyl)porphyrin (TPP) and kill bacteria. The Tat peptide is a positively-charged mammalian cell-penetrating peptide with potent antimicrobial activity but no haemolytic activity. Fluorescence spectroscopy revealed that the bioconjugate can bind to and/or be incorporated into all bacterial species tested. All species were susceptible to the Tat-porphyrin, with the bactericidal effect being dependent on both the concentration and the light dose. Using the highest light dose, treatment with the Tat-porphyrin achieved reductions of 6.6 log 10 and 6.37 log 10 in the viable counts of Staphylococcus aureus and Streptococcus pyogenes, and reductions of 5.74 log 10 and 6.6 log 10 in the viable counts of Pseudomonas aeruginosa and Escherichia coli. Moreover, the Tat moiety appears to confer antimicrobial properties to the conjugate, particularly for the Gram positive strains, based on the observation of dark toxicity using 1 mM of Tat-porphyrin. Finally, the conjugate induced membrane destabilization by synergistic action of the peptide and PDT, resulting in carboxyfluorescein leakage from bacterial membrane-mimicking liposomes. These findings demonstrate that the use of CPP to deliver a photosensitiser is an effective way of improving the uptake and the treatment efficacy of antimicrobial PDT.

Cancer Research

Introduction: Intrahepatic cholangiocarcinoma (iCCA) is a highly aggressive adenocarcinoma derive... more Introduction: Intrahepatic cholangiocarcinoma (iCCA) is a highly aggressive adenocarcinoma derived from bile ducts within the liver. This malignancy accounts for about 10% of cholangiocarcinoma cases and is the second most common primary liver cancer. Effective treatments for iCCA are limited, and they are primarily treated by tumor surgical resection. Regrettably, surgical resection is limited to early-stage disease and has an unfavorable prognosis. Development of novel therapeutic strategies for iCCA is needed to improve outcomes for non-resectable late stage iCCAs. Therefore, establishing suitable animal models for in vivo investigation of oncology and immuno-oncology treatment strategies for iCCA is critical. Methods: The Pten−/- GEMM was generated by CRISPR-Cas9 and crossed with Alb-Cre and LSL-KrasG12D/+ mice. Pathological examination of liver and blood biochemistry were investigated in thirteen GEMM mice. Liver fragments that were enriched with iCCA lesions were inoculated in...

Cancer Research

Introduction: Gliomas account for majority of primary brain tumors and are composed of different ... more Introduction: Gliomas account for majority of primary brain tumors and are composed of different grades, which present distinct molecular alterations, histological characteristics, and response to treatments. Patient-derived xenografts (PDXs) are the most “close-to-patient” models for oncology studies which largely maintain the histo- and molecular- pathology of the original tumors and are widely applied in preclinical evaluations. Subcutaneously inoculated glioma PDX models provide convenient evaluation of treatments’ efficacy but are difficult to mimic the in-situ microenvironment and diffuse invasive growth to peripheral tissue in the brain. Orthotopic implantation of bioluminescent labelled glioma models provides a more relevant preclinical model to evaluate and quantify treatment response. Methods: A glioma PDX model was established by subcutaneously engrafting tumor directly from patient following surgery into immunodeficient mice. Once established and expanded through passagi...

Cancer Research

T cell engagers show high efficacy in B cells malignances. High risk of immune-related adverse ev... more T cell engagers show high efficacy in B cells malignances. High risk of immune-related adverse events, including cytokine release syndrome (CRS), is reported in patients due to on-target off-site effects of T cell engagers. Thus, reliable and translational mouse models are required to predict potential safety issues and investigate their rescue. Here we describe two preclinical models mimicking to some extent CRS induction upon activation with anti-CD3 antibodies.BRGSF (Balb/C Rag2-/-, IL2Rγ-/-, SIRPαNOD and Flt3-/-) is a highly immunodeficient mouse featuring reduced murine myeloid cells. Development of human lymphoid (B and T cells) and myeloid (NK, cDC, pDC and monocytes) compartments upon CD34+ HSC-engraftment are observed in blood, spleen and bone marrow and are stable for over a year, without side effects. Anti-hCD3 mAb, OKT3, administration in BRGSF-HIS mice induced a rapid release of human cytokines (i.e., IL-6, TNF-α, IFN-γ, IL-2) in serum. Pretreatment with Flt3L, enhancin...

Photodynamic therapy (PDT), a treatment for small "early" carcinomas is based on the co... more Photodynamic therapy (PDT), a treatment for small "early" carcinomas is based on the combined action of a non toxic drug (photosensitizer) administered orally or systemically and visible light applied locally on tumor. Tumoral death is induced by light with release of toxic radicals. One of the most important parameter during PDT treatment is the pharmacokinetic of molecule, as pharmacokinetic must be perfectly known in order to irradiate the lesion when tissue concentration of photosensitizer is maximum. However the delay between injection and the maximum intratumoral concentration in photosensitizers must be as short as possible in order to avoid a costly hospitalisation. Moreover, photosensitizers must have a fast clearance to avoid too long cutaneous photosensitivity for the patient. The diphenylchlorin (SIM01), object of this study, has two phenyl rings around a central nucleus, which could allow a faster pharmacokinetic, with a maximum concentration reached more rapi...

Journal for ImmunoTherapy of Cancer, 2021

BackgroundThe breakthrough of immunotherapies has unleashed new hope and new success for cancer t... more BackgroundThe breakthrough of immunotherapies has unleashed new hope and new success for cancer therapy. However, the choice of a preclinical model is one of the main challenges as they are important for evaluation of translatability to help support testing in clinical studies, including potential efficacy and tolerability of immunotherapies during preclinical development.The development of mouse models featuring a human immune system (HIS) provides new paths for the investigation of the efficacy of immunotherapies in preclinical models engrafted with human tumors. Although these models provided a breakthrough in the assessment of immune targeting agents, they also come with a few significant caveats. These include: a lack of a mature human myeloid compartment in the mouse, and a short life span of the model when this compartment is promoted at non-physiological levels via the over-expression of human cytokines. Here, we report a novel mouse model (BRGSF-HIS), featuring functional h...

Journal of Clinical Oncology, 2012

e15161 Background: Docetaxel was the first cytotoxic treatment demonstrating a survival benefit i... more e15161 Background: Docetaxel was the first cytotoxic treatment demonstrating a survival benefit in metastatic castration-resistant prostate cancer (mCRPC) patients. Recently, cabazitaxel (a novel taxane) and abiraterone acetate (specific inhibitor of CYP17) have demonstrated a significant survival benefit in mCRPC patients progressing after receiving a docetaxel – containing regimen. Here we compare the antitumor efficacy of docetaxel, cabazitaxel and abiraterone acetate in the HID28 hormone-refractory human prostate carcinoma xenograft. Methods: HID28 tumor bearing nude mice received 20 mg/kg of docetaxel (IP, q3wk x 2), cabazitaxel at 15 and 20 mg/kg (IP, q3wk x 2) and abiraterone acetate at 50 mg/kg (PO, qdx21). Median tumor growth inhibition (T/C%) was evaluated as well as time course androgen receptor expression (4, 8 and 24h post dosing) by western blot analysis. Results: Docetaxel (20 mg/kg) inhibited tumor growth with a T/C%= 16.7% at D35, 2 partial (PR) and 1 complete (CR) ...

Annals of Oncology, 2012

Background: Levels of bone turnover markers (BTM) might be correlated with outcome in terms of sk... more Background: Levels of bone turnover markers (BTM) might be correlated with outcome in terms of skeletal related events (SRE), disease progression and death. The aim of this study was to determine the possible correlation between BTM, disease progression, SREs and death in patients with genitourinary cancer and bone metastases (BM) treated with zoledronic acid (ZA). Methods: Observational, prospective, multicenter study. Patients with genitourinary cancer (prostate, renal, bladder) and BM were included. BTM determined were: carboxiterminal telopeptide of type I collagen (β-CTX) and bone specific alkaline phosphatase (BALP) by ELISA (immunoenzymatic assay, IDS UK), and aminoterminal propeptide of type I collagen (P1NP) by automatised assays (Elecsys, Roche). All BTM were determined at baseline (V0) and every 3 mo of treatment until Month 18 (V6). All patients started treatment with ZA 4 mg IV every 3-4 weeks at the beginning of the study. Results: Data of 168 patients with genitourinary cancer were analyzed. In this work data of prostate cancer patients (n = 95) are presented. Population basal characteristics [35 new, without previous treatment (N) / 60 pre-treated, with anti-hormonal treatment (P)]: mean age (years): 72.1; median time from tumor diagnosis (months): 7.6 (N) / 11.6 (P); ECOG 0-1: 90% (N) / 87.5% (P). Patients with pathologic baseline levels were: 47.1% β-CTX, 64.7% BALP and 57.1% P1NP (N) and 48.3% β-CTX, 80.7% BALP and 60% P1NP (P). After 6 mo.: 28%, 48% and 32% (N) and 32%, 59.4% and 31.3% (P) presented pathologic values of β-CTX, BALP and P1NP respectively. Normalized levels remained steady throughout 18 mo follow-up. Reductions from baseline to month 3 in BALP levels correlated with lower risk of death (p = 0.0219) (Cox regression analysis). Conclusions: Elevation of baseline BTM levels is frequently present in advanced prostate cancer with bone metastasis. Addition of AZ to standard systemic therapy reduces BTM levels, even though in those highly hormone sensitive ones. A significant association was observed between elevated levels of BALP and death throughout follow-up. Disclosure: All authors have declared no conflicts of interest.

Oncotarget, 2019

Telomerase reverse transcriptase (TERT) is highly expressed in more than 90% of canine cancer cel... more Telomerase reverse transcriptase (TERT) is highly expressed in more than 90% of canine cancer cells and low to absent in normal cells. Given that immune tolerance to telomerase is easily broken both naturally and experimentally, telomerase is an attractive tumor associated antigen for cancer immunotherapy. Indeed, therapeutic trials using human telomerase peptides have been performed. We have developed an immunogenic yet catalytically inactive human telomerase DNA construct that is in clinical trials with patients presenting solid tumors. Paralleling this human construct, we have developed a canine telomerase DNA vaccine, called pDUV5. When administered intradermally to mice combined with electrogene transfer, pDUV5 induced canine TERT specific cytotoxic T-cells as measured by IFN-γ ELISpot assay. Intradermal vaccination of healthy dogs with 400 µg of pDUV5 generated strong, broad and long lasting TERT specific cellular immune responses. In vitro immunization with cTERT peptides revealed the maintenance of cTERT specific T-cells in PBMCs from tumor bearing dogs showing that this repertoire was not depleted. This study highlights the potential of pDUV5 as a cancer vaccine and supports its evaluation for the treatment of spontaneous canine tumors.

Journal of Clinical Oncology, 2017

3087 Background: INVAC-1 is an optimized plasmid encoding an inactive form of human telomerase re... more 3087 Background: INVAC-1 is an optimized plasmid encoding an inactive form of human telomerase reverse transcriptase (hTERT). hTERT is a prototype of shared tumor antigen expressed in more than 85% of human tumors. Telomerase activation is associated with maintenance of telomere length and accounts for the unlimited proliferative capacity of cancer cells. In preclinical models, INVAC-1 triggered Th1-polarized hTERT-specific CD8+ and CD4+T-cell immune responses and anti-tumor effects. Here, we report clinical and pharmacodynamics results of the first clinical study with INVAC-1 as a single agent in solid tumors. Methods: A 3+3 design phase 1 First in Human study evaluating INVAC-1 given monthly for 3 cycles using electroporation-based intra-dermal (ID) injection was conducted. Primary objectives included safety, tolerability and dose limiting toxicities to identify the maximum tolerated dose (MTD) and recommended phase 2 dose (RP2D). Secondary objectives included immune response and ...

Molecular Cancer Therapeutics, 2013

Background: The ability of tumor cells to spread and form metastases is one of the key parameters... more Background: The ability of tumor cells to spread and form metastases is one of the key parameters used to classify tumor aggressiveness. Metastasis detection at diagnosis or during followup post-surgery is associated with poor prognosis. The paucity of pertinent models to investigate the biology of metastasis renders problematic the set-up of anti-metastasis therapies. We took advantage of our human patient-derived xenograft (PDX) collection to develop models suitable to assess anti-metastatic therapy. To enable evaluation of metastasis response to treatment, we labeled tumors with lentivirus-mediated luciferase insertion, allowing longitudinal followup of metastasis formation by non-invasive imaging. A review of our metastatic bioluminescent PDX panel is described here with a particular focus on our most advanced bioluminescent breast model HBCx-14-Luc1. Methods: Tumor cells from freshly excised PDXs were dissociated and transduced with a defective lentivirus bearing the firefly lu...

Journal of Environmental Pathology, Toxicology and Oncology, 2006

Photodynamic therapy (PDT) is based on the selective light activation of an exogenously given dru... more Photodynamic therapy (PDT) is based on the selective light activation of an exogenously given drug to patients. PDT acts mainly on cell membranes either of neovascular endothelial cells or of cancer cells leading to cancer cell death. Six drugs are now marketed based on clinical assays in various indications, which showed a clear cost efficiency as compared to other classical procedures. PDT is easy to handle and can be performed in medical installations fitting the conditions of health care in developing countries. Its cost effectiveness could represent an appropriate solution to the increasing number of cancers of various origin. However despite all the clinical results now available, PDT development remains slow. The reasons for this situation include cost of development, intellectual property, and competition between pharmaceutical companies.

British journal of cancer, Jan 10, 2009

Intracellular generation of the photosensitiser, protoporphyrin IX, from a series of dipeptide de... more Intracellular generation of the photosensitiser, protoporphyrin IX, from a series of dipeptide derivatives of the haem precursor, 5-aminolaevulinic acid (ALA), was investigated in transformed PAM212 murine keratinocytes, together with studies of their intracellular metabolism. Porphyrin production was substantially increased compared with equimolar ALA using N-acetyl terminated phenylalanyl, leucinyl and methionyl ALA methyl ester derivatives in the following order: Ac-L-phenylalanyl-ALA-Me, Ac-L-methionyl-ALA-Me and Ac-L-leucinyl-ALA-Me. The enhanced porphyrin production was in good correlation with improved photocytotoxicity, with no intrinsic dark toxicity apparent. However, phenylalanyl derivatives without the acetyl/acyl group at the N terminus induced significantly less porphyrin, and the replacement of the acetyl group by a benzyloxycarbonyl group resulted in no porphyrin production. Porphyrin production was reduced in the presence of class-specific protease inhibitors, namel...

Research in Experimental Medicine, 1999

Photodynamic therapy (PDT) induces cell-membrane damage and alterations in cancer-cell adhesivene... more Photodynamic therapy (PDT) induces cell-membrane damage and alterations in cancer-cell adhesiveness, an important parameter in cancer metastasis. These alterations result from cell sensitivity to photosensitizers and the distribution of photosensitizers in cells. The efficacy of photosensitizers depends on their close proximity to targets and thus on their pharmacokinetics at the cellular level. We studied the cellular distribution of photosensitizers with a confocal microspectrofluorimeter by analysing the fluorescence emitted by benzoporphyrin derivative-monoacid ring A (BPD-MA) and Photofrin relative to their cell sensitivity. Two cancer cell lines of colonic origin, but with different metastatic properties, were used: PROb (progressive) and REGb (regressive). For BPD-MA (1.75 µg/ml), maximal fluorescence intensity (8,300 cts) was reached after 2 h for PROb and after 1 h (4,900 cts) for REGb. For Photofrin (10 µg/ml), maximal fluorescence intensity (467 cts) was reached after 5 h for PROb and after 3 h (404 cts) for REGb. Intracellular studies revealed stronger cytoplasmic than nuclear fluorescence for both BPD and Photofrin. Both of the sensitizers induced a dose-dependent phototoxicity; LD 50 with BPD-MA was 93.3 ng/ml for PROb and 71.1 ng/ml for REGb, under an irradiation of 10 J/cm 2. With Photofrin, LD 50 was 1,270 ng/ml for PROb and 1,200 ng/ml for REGb under an irradiation of 25 J/cm 2. The photosensitizer effect within PROb and REGb cancer cells was assessed by incorporation kinetics and toxicity-phototoxicity tests. The intracellular concentration of the photosensitive agent was one important factor in the effectiveness of PDT, but not the only one contributing to the photodynamic effect. In conclusion, this study showed that there was a clear difference between sensitizer uptake and phototoxicity, even in cancer cells of the same origin. This could induce cell-killing heterogeneity in clinics.

Surgical Neurology, 2007

Background: Glioblastomas are the third most common cause of cancer death in patients between 15 ... more Background: Glioblastomas are the third most common cause of cancer death in patients between 15 and 35 years old. Literature suggests that PDT could represent a promising treatment, providing that sensitizers could accumulate within the cancer tissues despite the blood-brain barrier. Methods: Distribution and PDT effect of SIM01, a promising photosensitizer, have been evaluated on orthotopic C6 tumor model in rats by comparison with HPD and m-THPC. Pharmacokinetics had been analyzed with fluorescence and ROS. Photodynamic treatment was done using a 630-nm light with an energy density of 100 J cm À2 for HPD and a 652-nm light with an energy density of 20 J cm À2 for m-THPC and SIM01. Results: The correlation between fluorescence and ROS dosimetry was found to be excellent. An optimal concentration was found after 12 hours for SIM01 (4 mg/kg), 24 hours for HPD (10 mg/kg), and 48 hours for m-THPC (4 mg/kg). The best normal tissue/cancer ratio of concentration had been found after 12 hours for SIM01 and 48 hours for HPD and m-THPC. Pathological examinations after PDT showed that the criteria for histology of glioblastic origin were absent in SIM01-treated rats 12 hours after injection but were present in 50% of rats treated 24 hours after injection and in all after a 48-hour delay. Mean survival of rats treated 12 or 24 hours after SIM01 injection was significantly improved compared with controls, HPD-, or m-THPC-treated groups. Survival of rats treated 12 or 24 hours after SIM01 injection reached 20 days but decreased for longer delays. On the contrary, survival reached 18 days at the maximum for rats treated 48 hours after m-THPC or HPD injection. Conclusions: Our results confirm that PDT is a promising treatment for glioblastomas. SIM01 efficacy is as efficient as m-THPC but with much more favorable pharmacokinetics.

Photodiagnosis and Photodynamic Therapy, 2005

PDT had been proposed in gastroenterology for various indications and the esophageal cancer treat... more PDT had been proposed in gastroenterology for various indications and the esophageal cancer treatment had been among the very first having been approved. However, PDT failed to be a real breakthrough. One reason for it was that although it had been approved for the palliative treatment of advanced tumors, PDT only has by nature a limited in-depth efficacy fitting better to the treatment and often the cure of ''early cancers''. For this reason PDT has also been proposed for the treatment of Barrett's esophagus (BE) with high-grade dysplasias. Barrett's mucosa (BM) is a field of a specialized metaplastic columnar epithelium replacing the normal stratified squamous epithelium or mucosa lining the distal esophagus. In this case, PDT has to destroy an area of thin tissues spread eventually over a wide area instead of a mass of tissues. Something important is that existing treatments allow the treatment of foci of dysplastic tissues but not the regression of the whole BM. BE is thus an unsolved medical problem having medical as well as economic consequences as BM being likely to transform into a cancer has to be carefully surveyed. The esophageal cancer, an adenocarcinoma, has to be surgically removed when it is possible something pretty heavy with a high morbidity. Economic burnt is also important with high survey costs independently to the additional surgical costs in case of diagnosed cancer. Treatments proposed for non or mild dysplastic BM regression have in common to have an inhomogenous impact on the target. Treatments for high-grade dysplasia (HGD, the ultimate pathological step before cancer) are based on mucosectomy and are limited to small areas of tissues. Recently circumferential mucosectomy had been proposed but at a higher risk making it suitable only to highly experienced hands in infrequent indications.

Photodiagnosis and Photodynamic Therapy, 2005

Photodynamic therapy (PDT) is a new approach to cancer treatment for a variety of malignant tumor... more Photodynamic therapy (PDT) is a new approach to cancer treatment for a variety of malignant tumors. A new photosensitizer, 2,3-dihydro-5,15-di(3,5dihydroxyphenyl)porphyrin (SIM01), had been evaluated for its genotoxic effects on glioma cells (C6). Comet assay had been used to evaluate the potential genotoxic effect induced by SIM01 on the C6 cells. When SIM01 had been shown to be a powerful sensitizer no DNA strand break was detected in the absence of light. SIM01 localized in cytoplasm but not in the nucleus of the tumors cells, which supported the finding of undetectable DNA damage under darkness and low photodynamic dose. Cell exposure to 20 J cm −2 after an incubation time of 2 h with 0, 0.25, 0.5, 2 or 4 g mL −1 induced less than 25% of cell death but significant Tail Moment changes. If DNA damage intensity increased according to SIM01 doses under light exposure, importance of repair seemed to increase proportionally to PDT-induced damage. Positive controls consisted of doxorubicin-treated C6 cells this mutagen being known to induce genetic damage. Whatever the conditions used SIM01 appeared to be less deleterious than doxorubicin. As the comet assay can not give us the certitude that no mutation, photoadducts or oxidative damage had been developed under light exposure this point will have to be verified with another mutagenicity assay. SIM01 appears to be safe from a mutagenic point of view something of importance as tumors of small volume in patients with a long lifespan are at first indicated for PDT.

Photodiagnosis and Photodynamic Therapy, 2006

Delta amino levulinic acid photodynamic therapy (ALA-PDT) represents one of the most prominent ad... more Delta amino levulinic acid photodynamic therapy (ALA-PDT) represents one of the most prominent advances in PDT. ALA itself or its derivatives are marketed for a variety of clinical indications. Despite the development of clinical applications, experimental ALA results are very heterogeneous and experimentally used parameters are still not standardized. This suggests that some problems remain unsolved that are likely to impair experiments to be performed but also that clinical results obtained could be greatly improved. Frequently unmentioned or imprecise data concern solvents, pH of ALA solutions, storage time, ALA degradation or ALA efficacy. In addition, diversity of experimental model is huge while capabilities of ALA transformation into PpIX are known to vary from one cell to the other. Thus, the aim of the present paper was to quantify the level of ALA degradation or changes in ALA efficacy using one single cell line without presuming of the mechanisms and determine the conditions of storage inducing the best transformation into PpIX and/or cell phototoxicity. We added ALA diluted in water, PBS or RPMI to C6 cells, a murine brain tumour cell line that can be used in vivo as an orthotopic graft. We measured in cells used as tools for final bio efficacy estimation, both the induced fluorescence and phototoxicity in various conditions of storage before use chosen to be as close as possible to the real lab conditions. Water had been found to better preserve ALA than, respectively, PBS and RPMI and this for any temperature or storage durations. The lowest temperature and the shortest duration for storage used had also been shown to better preserve ALA-induced fluorescence and phototoxicity. The fact that these properties were found to be better preserved in 7.4 buffered solvent could be in relationship with a fast ALA condensation occurring at neutral or lightly acidic pH modifying its availability for an optimal transformation into PpIX.

Photochemistry and Photobiology, 2007

Synthesis of ␦-aminolevulinic acid (ALA) derivatives is a promising way to improve the therapeuti... more Synthesis of ␦-aminolevulinic acid (ALA) derivatives is a promising way to improve the therapeutic properties of ALA, particularly cell uptake or homogeneity of protoporphyrin IX (PpIX) synthesis. The fluorescence emission kinetics and phototoxic properties of ALA-n-pentyl ester (E1) and R,S-ALA-2-(hydroxymethyl) tetrahydrofuranyl ester (E2) were compared with those of ALA and assessed on C6 glioma cells. ALA (100 g/mL), E1 and E2 (10 g/mL) induced similar PpIX-fluorescence kinetics (maximum between 5 and 7 h incubation), fluorescence being limited to the cytoplasm. The 50% lethal dose occurred after 6 h with 45, 4 and 8 g/mL of ALA, E1 and E2, respectively. ALA, E1 and E2 induced no dark toxicity when drugs were removed after 5 min of incubation. However, light (25 J/cm 2) applied 6 h after 5 min incubation with 168 g/mL of each compound induced 85% survival with ALA, 27% with E1 and 41% with E2. Increasing the incubation time with ALA, E1 and E2 before washing increased the phototoxicity, but E1 and E2 remained more efficient than ALA, regardless of incubation time. ALA-esters were more efficient than ALA in inducing phototoxicity after short incubation times, probably through an increase of the amount of PpIX synthesized by C6 cells.

Photochemical & Photobiological Sciences, 2010

Given that cell-penetrating peptides (CPP) are cationic and often amphipathic, similar to membran... more Given that cell-penetrating peptides (CPP) are cationic and often amphipathic, similar to membrane-active antimicrobial peptides, it may be possible to use CPP conjugation to improve the delivery of photosensitisers for antimicrobial photodynamic therapy (antimicrobial PDT). We investigated the possibility of using a Tat peptide to deliver the photosensitiser, tetrakis(phenyl)porphyrin (TPP) and kill bacteria. The Tat peptide is a positively-charged mammalian cell-penetrating peptide with potent antimicrobial activity but no haemolytic activity. Fluorescence spectroscopy revealed that the bioconjugate can bind to and/or be incorporated into all bacterial species tested. All species were susceptible to the Tat-porphyrin, with the bactericidal effect being dependent on both the concentration and the light dose. Using the highest light dose, treatment with the Tat-porphyrin achieved reductions of 6.6 log 10 and 6.37 log 10 in the viable counts of Staphylococcus aureus and Streptococcus pyogenes, and reductions of 5.74 log 10 and 6.6 log 10 in the viable counts of Pseudomonas aeruginosa and Escherichia coli. Moreover, the Tat moiety appears to confer antimicrobial properties to the conjugate, particularly for the Gram positive strains, based on the observation of dark toxicity using 1 mM of Tat-porphyrin. Finally, the conjugate induced membrane destabilization by synergistic action of the peptide and PDT, resulting in carboxyfluorescein leakage from bacterial membrane-mimicking liposomes. These findings demonstrate that the use of CPP to deliver a photosensitiser is an effective way of improving the uptake and the treatment efficacy of antimicrobial PDT.