Hayley Pye

Development of Photodynamic Antimicrobial Chemotherapy (PACT) for Clostridium difficile.

Background Clostridium difficile is the leading cause of antibiotic-associated diarrhoea and pseudo membranous colitis in the developed world. The aim of this study was to explore whether Photodynamic Antimicrobial Chemotherapy (PACT) could be used as a novel approach to treating C. difficile infections. Methods PACT utilises the ability of light-activated photosensitisers (PS) to produce reactive oxygen species (ROS) such as free radical species and singlet oxygen, which are lethal to cells. We screened thirteen PS against C. difficile planktonic cells, biofilm and germinating spores in vitro, and cytotoxicity of effective compounds was tested on the colorectal adenocarcinoma cell-line HT-29. Results Three PS were able to kill 99.9% of bacteria in both aerobic and anaerobic conditions, both in the planktonic state and in a biofilm, after exposure to red laser light (0.2 J/cm2) without harming model colon cells. The applicability of PACT to eradicate C. difficile germinative spores indirectly was also shown, by first inducing germination with the bile salt taurocholate, followed by PACT. Conclusion This innovative and simple approach offers the prospect of a new antimicrobial therapy using light to treat C. difficile infection of the colon.

Cathepsin B-degradable, NIR-responsive nanoparticulate platform for target-specific cancer therapy

Stimuli-responsive anticancer formulations can promote drug release and activation within the target tumour, facilitate cellular uptake, as well as improve the therapeutic efficacy of drugs and reduce off-target effects. In the present work, indocyanine green (ICG)-containing polyglutamate (PGA) nanoparticles were developed and characterized. Digestion of nanoparticles with cathepsin B, a matrix metalloproteinase overexpressed in the microenvironment of advanced tumours, decreased particle size and increased ICG cellular uptake. Incorporation of ICG in PGA nanoparticles provided the NIR-absorbing agent with time-dependent altered optical properties in the presence of cathepsin B. Having minimal dark toxicity, the formulation exhibited significant cytotoxicity upon NIR exposure. Combined use of the formulation with saporin, a ribosome-inactivating protein, resulted in synergistically enhanced cytotoxicity attributed to the photo-induced release of saporin from endo/lysosomes. The results suggest that this therapeutic approach can offer significant therapeutic benefit in the treatment of superficial malignancies, such as head and neck tumours.

Apparatus for Histological Validation of In Vivo and Ex Vivo Magnetic Resonance Imaging of the Human Prostate

This article describes apparatus to aid histological validation of magnetic resonance imaging studies of the human prostate. The apparatus includes a 3D-printed patient-specific mold that facilitates aligned in vivo and ex vivo imaging, in situ tissue fixation, and tissue sectioning with minimal organ deformation. The mold and a dedicated container include MRI-visible landmarks to enable consistent tissue positioning and minimize image registration complexity. The inclusion of high spatial resolution ex vivo imaging aids in registration of in vivo MRI and histopathology data.

Efficacy of photochemical internalisation using disulfonated chlorin and porphyrin photosensitisers: An in vitro study in 2D and 3D prostate cancer models

This study shows the therapeutic outcome of Photochemical Internalisation (PCI) in prostate cancer in vitro surpasses that of Photodynamic Therapy (PDT) and could improve prostate PDT in the clinic, whilst avoiding chemotherapeutics side effects. In addition, the study assesses the potential of PCI with two different photosensitisers (TPCS2a and TPPS2a) in prostate cancer cells (human PC3 and rat MatLyLu) using standard 2D monolayer culture and 3D biomimetic model. Photosensitisers were used alone for photodynamic therapy (PDT) or with the cytotoxin saporin (PCI). TPPS2a and TPCS2a were shown to be located in discrete cytoplasmic vesicles before light treatment and redistribute into the cytosol upon light excitation. PC3 cells exhibit a higher uptake than MatLyLu cells for both photosensitisers. In the 2D model, PCI resulted in greater cell death than PDT alone in both cell lines. In 3D model, morphological changes were also observed. Saporin-based toxicity was negligible in PC3 cells, but pronounced in MatLyLu cells (IC50 = 18 nM). In conclusion, the study showed that tumour features such as tumour cell growth rate or interaction with drugs determine therapeutic conditions for optimal photochemical treatment in metastatic prostate cancer.

Upregulation of mucin glycoprotein MUC1 in the progression to esophageal adenocarcinoma and therapeutic potential with a targeted photoactive antibody-drug conjugate

Background Mucin glycoprotein 1 (MUC1) is a glycosylated transmembrane protein on epithelial cells. We investigate MUC1 as a therapeutic target in Barrett’s epithelium (BE) and esophageal adenocarcinoma (EA) and provide proof of concept for a light based therapy targeting MUC1. RESULTS MUC1 was present in 21% and 30% of significantly enriched pathways comparing BE and EA to squamous epithelium respectively. MUC1 gene expression was x2.3 and x2.2 higher in BE (p=<0.001) and EA (p=0.03). MUC1 immunohistochemical expression increased during progression to EA and followed tumor invasion. HuHMFG1 based photosensitive antibody drug conjugates (ADC) showed cell internalization, MUC1 selective and light-dependent cytotoxicity (p=0.0006) and superior toxicity over photosensitizer alone (p=0.0022). Methods Gene set enrichment analysis (GSEA) evaluated pathways during BE and EA development and quantified MUC1 gene expression. Immunohistochemistry and flow cytometry evaluated the anti-MUC1 antibody HuHMFG1 in esophageal cells of varying pathological grade. Confocal microscopy examined HuHMFG1 internalization and HuHMFG1 ADCs were created to deliver a MUC1 targeted phototoxic payload. Conclusions MUC1 is a promising target in EA. Molecular and light based targeting of MUC1 with a photosensitive ADC is effective in vitro and after development may enable treatment of locoregional tumors endoscopically.

Immunohistochemical biomarker validation in highly selective needle biopsy microarrays derived from mpMRI-characterized prostates

Diagnosing prostate cancer routinely involves tissue biopsy and increasingly image guided biopsy using multiparametric MRI (mpMRI). Excess tissue after diagnosis can be used for research to improve the diagnostic pathway and the vertical assembly of prostate needle biopsy cores into tissue microarrays (TMAs) allows the parallel immunohistochemical (IHC) validation of cancer biomarkers in routine diagnostic specimens. However, tissue within a biopsy core is often heterogeneous and cancer is not uniformly present, resulting in needle biopsy TMAs that suffer from highly variable cancer detection rates that complicate parallel biomarker validation.

Using antibody directed phototherapy to target oesophageal adenocarcinoma with heterogeneous HER2 expression

Early oesophageal adenocarcinoma (OA) and pre-neoplastic dysplasia may be treated with endoscopic resection and ablative techniques such as photodynamic therapy (PDT). Though effective, discrete areas of disease may be missed leading to recurrence. PDT further suffers from the side effects of off-target photosensitivity. A tumour specific and light targeted therapeutic agent with optimised pharmacokinetics could be used to destroy residual cancerous cells left behind after resection. A small molecule antibody-photosensitizer conjugate was developed targeting human epidermal growth factor receptor 2 (HER2). This was tested in an in vivo mouse model of human OA using a xenograft flank model with clinically relevant low level HER2 expression and heterogeneity. In vitro we demonstrate selective binding of the conjugate to tumour versus normal tissue. Light dependent cytotoxicity of the phototherapy agent in vitro was observed. In an in vivo OA mouse xenograft model the phototherapy agent had desirable pharmacokinetic properties for tumour uptake and blood clearance time. PDT treatment caused tumour growth arrest in all the tumours despite the tumours having a clinically defined low/negative HER2 expression level. This new phototherapy agent shows therapeutic potential for treatment of both HER2 positive and borderline/negative OA.

Su1964 The Development of a Novel Endoscopic Orthotopic Tumour Model for Oesophageal Cancer

Introduction The incidence of oesophageal adenocarcinoma (OAC) continues to rise rapidly. A clinically relevant animal model of OAC is needed to help study the effectiveness of novel therapeutic strategies for OAC. Current animal models have many limitations. Models that involve the injection of cells into the flanks of rodents do not replicate the native environment of OAC. Surgical models producing permanent biliary reflux that can lead to cancer are limited by the need for highly skilled surgical techniques, difficult reproducibility and significant morbidity to the animals involved. We describe a novel orthotopic tumour model for OAC. Our model is based upon the injection of tumour cells into the rodent oesophagus under direct endoscopic visualisation. Method Two human tumour cell lines were used – OAC (OE19) and colon adenocarcinoma (HT29). Both lines were stably transfected to express luciferase and maintained in 5% CO2 at 370C. 6–8 week-old female nude athymic rats (RNU Rat) were used for all experiments. A high-resolution diagnostic endoscope was used to perform all endoscopic procedures. Animals were anaesthetised using Isoflurane with concomitant oxygen given. Under direct visualisation, OE19 or HT29 tumour cells were injected. Immediately after endoscopy, 1 ml of D-Luciferin (20 mg/ml) was injected i.p. to follow luciferase-expressing OE19/HT29 tumour implantation. After 15 min, rats were inserted into a whole body cooled charged-coupled device (CCD) camera Photon Imager system and images obtained. All animals had Bioluminescent imaging (BLI) and endoscopy performed regularly to look for evidence of tumour growth. Results Implanted tumour cells were detected immediately after injection using BLI. Weekly, non-invasive BLI and regular endoscopic observation detected successful orthotopic tumour growth (Figure 1). Histological tissue samples confirmed the presence of tumour ulcerating the overlying squamous mucosa and infiltrating into, and through, the muscularis propria. This mirrors closely the behaviour of a primary human OAC. Conclusion We have developed a novel, more clinically relevant, orthotopic tumour model for OAC. By utilising rodent gastroscopy, our model replicates the native environment in which OAC grows and provides an opportunity for us to better understand the natural history of OAC in a manner that confers considerably less morbidity to animals than current models allow. Disclosure of interest None Declared.

Tu1895 Antibody Directed Phototherapy (ADP) With HuHMFG1:PS1 Is a Novel and Potentially Curative Therapeutic Modality for MUC1 Expressing Oesophageal Adenocarcinomas

Aim: The aim of the present study was to evaluate whether chronomodulated administration of capecitabine would reduce toxicity of the drug in patients with metastatic colorectal cancer. Patients & Methods: 27 patients with advanced colorectal cancer were randomized to receive capecitabine (2500mg/KOF daily) either in standard administration with 50% of the dose in themorning and 50% in the evening separated by 12 hours or as chronomodulated dose-regime with 25% morning-dose and 75% late evening-dose. Treatment was continued until thirteen treatment-cycles were finished or progress of cancer or limitation of therapy due to side effects occurred. Results: Overall, response rates to chemotherapy were similar in both treatment groups (p= 0,296). However, within the group of patients with chronomodulated application of capecitabine, reduction of drug-doses due to side effects was less frequently necessary (14.8 vs. 19.3%, p=0,026) and more patients were able to finish all thirteen chemotherapy-cycles (41.7 vs. 7.1%, p=0,007). While there was no statistically significant difference in the frequency of hand-foot-syndrome in both treatment-arms (51.7 vs. 41.5%, p=0,119) other side effects as nightly nausea, tiredness and sleeplessness were significantly reduced in patients receiving chronomodulated therapy (p=0,035, p=0,001 and 0,009, respectively). Additionally, there was a trend to lower frequency of nausea, diarrhea and stomatitis within this group compared to standard treatment (9.0 vs. 19.5%, 19.1 vs. 25.6%, and 4.5 vs. 8.5%) Conclusion: The chronomodulated capecitabine schedule achieved similar response rate and better tolerability regarding various side effects compared with standard application of the drug in patients with colorectal cancer. This schedule could enable patients to sustain on capecitabine therapy for a longer time-period.

Su1883 HER-2 Expression in the Barrett's Metaplasia-Dysplasia-Carcinoma Sequence and Potential Targeting In-Vitro With the Single Chain Antibody Fragment C6.5

Introduction There is increasing attention on the integration of targeted agents for oesophageal adenocarcinoma (OA) therapy. The most notable example of success of oesophagogastric targeted therapy was the addition of a HER2 targeting agent in the Phase III ToGA study. However, there is limited data on the expression of HER2 in the progression from Barrett9s (BE) to OA. This study aimed to clarify expression in this sequence, and to show binding of C6.5, a single chain HER2 targeting human antibody Fv fragment (scFv), to known HER2 expressing OA cell lines in vitro. Efficacy of antibody based therapies can be enhanced by scFv9s which penetrate tumours more quickly and demonstrate better tumour: normal tissue specificity. Methods 33 paraffin embedded oesophageal tissue specimens were selected from patients with squamous (n=4), non-dysplastic BE (NDBE; n=4), low grade dysplasia (LGD; n=6), high grade dysplasia (HGD; n=8) and OA (n=12). Sections were immunostained with the automated Oracle Bond system (Leica, UK) for consistency. Staining was then scored by 2 expert pathologists according to the proportion of cells in the tissue staining positively as negative ( 10%). In phase 2, binding of C6.5 scFv to the cancer cell lines SKOV-3 (ovarian), OE-19, OE-33 (oesophageal) and HT-29 (colon) was identified with flow cytometry using a mouse secondary followed by tertiary anti-mouse FITC. Data were then analysed with FlowJo software. Results Significant expression of HER2 (>10% cells positive according to National Guidelines) was only seen in HGD (25%) and cancer (25%) specimens. Borderline staining (5%–10%) was seen in LGD (17%), HGD (13%) and cancer (8%). All NDBE and the remaining LGD, HGD and cancer samples were negative. Flow cytometry demonstrated C6.5 binding to SKOV-3, OE19 and OE-33 cells but not HT-29 cells. Conclusion This study demonstrated that significant HER2 expression is only seen in roughly a quarter of patients with HGD and OA and not in NDBE or LGD. We also found that the HER2 targeting scFv C6.5 binds to breast cancer and OA cell lines but not colon cancer, the negative control. HER2 targeting therapies could therefore be postulated for patients with BE with HGD and OA, and these may be enhanced with scFv9s such as C6.5. Competing interests None declared.