Summary: Photodynamic therapy (PDT) is becoming an evermore useful tool in oncology but is frequently limited by side‐effects caused by a lack of targeting of the photosensitizer. This problem can often be circumvented by the conjugation of photosensitizers to tumour‐specific monoclonal antibodies. An alternative is the use of single chain (sc) Fv fragments which, whilst retaining the same binding specificity, are more efficient at penetrating tumour masses because of their smaller size; and are more effectively cleared from the circulation because of the lack of an Fc domain. Here we describe the conjugation of two isothiocyanato porphyrins to colorectal tumour‐specific scFv, derived from an antibody phage display library. The conjugation procedure was successfully optimized and the resulting immunoconjugates showed no loss of cell binding. In vitro assays against colorectal cell lines showed these conjugates had a selective photocytotoxic effect on cells. Annexin V and propidium iodide staining of treated cells confirmed cell death was mediated principally via an apoptotic pathway. This work suggests that scFv : porphyrin conjugates prepared using isothiocyanato porphyrins show promise for use as targeted PDT agents.

Head and neck squamous cell carcinoma (HNSCC) is an aggressive epithelial malignancy. It is the most common neoplasm arising in the upper aerodigestive tract.Interleukin (IL) 12 and IL-18 are cytokines which have a major anti-tumour activity via stimulation of a T-helper type 1 (Th1) immune response. Interleukin 10, a potent antagonist of IL-12, is a cytokine which possesses immunosuppressive activity mainly produced via T-helper type 2 (Th2) cells. Studies of other types of cancer have shown that the level of IL-12 in serum or tissues is suppressed and/or the IL-10 level is increased, suggesting that there is an impaired cell-mediated anti-tumour response.The aim of this study was to measure pre-operative serum cytokine concentrations in HNSCC patients in order to detect any changes in IL-10, IL-12 and IL-18, compared with non-tumour controls. The relationship between cytokine levels and standard clinicopathological features, including tumour site, tumour stage and presence of nodal metastasis, was also examined.Fifty-seven patients with primary HNSCC were prospectively recruited, together with 40 non-tumour control patients with a similar age and sex distribution. Serum cytokine levels were measured using commercial quantitative enzyme-linked immunosorbent assay.The HNSCC patients had significantly lower IL-12 levels (median; interquartile range) than controls (42.8 pg/ml, 26.2–61.6 vs 52.3 pg/ml, 37.5–113.7; p=0.018). Also, patients were more likely to have detectable IL-10 levels than were controls, as IL-10 was positive in 27/55 patients but in only 9/39 controls (p=0.011). Furthermore, IL-10 detectability varied according to primary site, being more commonly observed in hypopharyngeal and laryngeal tumours, and IL-10 was more likely to be detected with advanced tumour stage (T₃ and T₄). No differences in IL-18 levels were observed between patients and controls (p=0.169).These results suggest (in agreement with studies on other solid malignancies) that HNSCC causes a significant change in the serum levels of specific Th1 and Th2 cytokines, producing an in vivo environment that is unlikely to promote an effective cell-mediated anti-tumour response.

A microfluidic based experimental methodology has been developed that offers a biomimetic microenvironment in which pseudo in vivo tissue studies can be carried out under in vitro conditions. Using this innovative technique, which utilizes the inherent advantages of microfluidic technology, liver tissue has been kept in a viable and functional state for over 70 h during which time on-chip cell lysis has been repeatedly performed. Tissue samples were also disaggregated in situ on-chip into individual primary cells, using a collagenase digestion procedure, enabling further cell analysis to be carried out off-line. It is anticipated that this methodology will have a wide impact on biological and clinical research in fields such as cancer prognosis and treatment, drug development and toxicity, as well as enabling better fundamental research into tissue/cell processes.

A total of 45 primary head and neck squamous cell carcinomas were analysed by comparative genomic hybridisation to identify regions of chromosomal deletion and gain. Multiple regions of copy number aberration were identified including gains affecting chromosomes 3q, 8q, 5p, 7q, 12p and 11q and deletion of material from chromosomes 3p, 11q, 4p, 5q, 8p, 10q, 13q and 21. Kaplan–Meier survival analysis revealed significant correlations between gain of 3q25–27 and deletion of 22q with reduced disease-specific survival. In addition, gain of 17q and 20q, deletion of 19p and 22q and amplification of 11q13 were significantly associated with reduced disease-free survival. A Cox proportional hazards regression model identified deletion of 22q as an independent prognostic marker. The data presented here provide further evidence that the creation of a genetically based tumour classification system will soon be possible, complementing current histopathological characterisation.

A microfluidic device has been developed to maintain viable heart tissue samples in a biomimetic microenvironment. This device allows rat or human heart tissue to be studied under pseudo in vivo conditions. Effluent levels of lactate dehydrogenase and hydrogen peroxide were used as markers of damaged tissue in combination with in situ electrochemical measurement of the release of reactive oxygen species (ROS). The parameters for perfusion were optimized to maintain biopsies of rat right ventricular or human right atrial tissue viable for up to 5 and 3.5 hours, respectively. Electrochemical assessment of the oxidation current of total ROS, employing cyclic voltammetry, gave results in real-time that were in good agreement to biochemical assessment using conventional, off-chip, commercial assays. This proof-of-principle, integrated microfluidic device, may be exploited in providing a platform technology for future cardiac research, offering an alternative approach for investigating heart pathophysiology and facilitating the development of new therapeutic strategies.

Development of personalised cancer models to predict response to radiation would benefit patient care; particularly in malignancies where treatment resistance is prevalent. Herein, a robust, easy to use, tumour-on-a-chip platform which maintains precision cut head and neck cancer for the purpose of ex vivo irradiation is described. The device utilises sintered discs to separate the biopsy and medium, mimicking in vivo microvascular flow and diffusion, maintaining tissue viability for 68 h. Integrity of tissues is demonstrated by the low levels of lactate dehydrogenase release and retained histology, accompanied by assessment of cell viability by trypan blue exclusion and flow cytometry; fluid dynamic modelling validates culture conditions. An irradiation jig is described for reproducible delivery of clinically-relevant doses (5 × 2 Gy) to newly-presenting primary tumours (n = 12); the addition of concurrent cisplatin is also investigated (n = 8) with response analysed by immunohistochemistry. Fractionated irradiation reduced proliferation (BrdU, p = 0.0064), increased DNA damage (ƴH2AX, p = 0.0043) and caspase-dependent apoptosis (caspase-cleaved cytokeratin-18) compared to control; caspase-dependent apoptosis was further increased by concurrent cisplatin compared to control (p = 0.0063). This is a proof of principle study showing the response of cancer tissue to irradiation ex vivo in a bespoke system. The novel platform described has the potential to personalise treatment for patients in a cost-effective manner with applicability to any solid tumour.