Malcolm D. Mason

Analysis of antigen presenting cell derived exosomes, based on immuno-magnetic isolation and flow cytometry.

We present a simple yet powerful method for the isolation and analysis of exosomes released by antigen-presenting cells (APC). Exosomes are small vesicles (40-90 nm) released by APC, and may have an immuno-regulatory function in vivo. Such exosomes originate from MHC class II peptide loading compartments and, as such, express high levels of MHC Class II. We have utilised magnetic beads, coated with monoclonal antibodies specific for HLA DP, DQ, DR for the specific isolation of exosomes from cell-free supernatants. Beads coated with exosomes are subsequently stained with conjugated antibodies, and analysed by flow cytometry. Characterisation of exosomes by this method demonstrated that exosomes derived from B-lymphocytes express abundant MHC Class I and II molecules. Other immunologically important molecules detected included the co-stimulatory molecules B7.1 (CD80) and B7.2 (CD86). The adhesion molecule ICAM-1 (CD54) was also detected. These exosomes also expressed the B cell marker CD20, and the complement inhibitory protein CD59. The expression of CD63, a lysosomal marker, was variable, and there was no detectable expression of transferrin receptor (CD71). Monocyte derived dendritic cells (cultured for 7 days in GM-CSF/IL-4), demonstrated an immature phenotype, and secreted exosomes with a similar phenotype, with abundant MHC molecules. The expression of CD63 was consistently strong, and the MHC Class I-like molecule CD1a was also present, suggesting a possible function in the presentation of lipid antigens. Again CD59 was expressed suggesting a possible role for APC exosomes in complement regulation. There was no detectable CD71, CD40, CD14, CD20 or CD83. Modification of the extraction protocol allowed a comparative analysis of exosome secretion under various conditions. Treatment of cells with calcium ionophore, or phorbol ester resulted in apparent increases in exosome release, while the phosphatidyl inositol 3-kinase inhibitor, wortmannin, reduced exosome secretion. The immuno-magnetic isolation and analysis of exosomes is a versatile and rapid tool for the analysis of APC exosomes, and may prove a valuable tool for the study of exosome biology.

Antigen‐presenting cell exosomes are protected from complement‐mediated lysis by expression of CD55 and CD59

Exosomes are secreted nanometer‐sized vesicles derived from antigen‐presenting cells, which have attracted recent interest as they likely play important roles in immune regulation, and their use as cell‐free tools for immunotherapy has been proposed. Liposomes used clinically as transport vehicles can activate the complement system, leading to their rapid degradation and significant inflammatory toxicity. The use of isolated exosomes in therapy, therefore, may also elicit complement activation, reducing their potential efficacy. We have examined the expression and functional roles of the membrane regulators of complement (CD46, CD55 and CD59) on antigen‐presenting cell‐derived exosomes. Exosomes express the glycosylphosphatidylinositol (GPI)‐anchored regulators CD55 and CD59,but not the transmembrane protein CD46. Antibody blocking of CD55 in the presence of sensitizing antibody (w6/32) and human serum resulted in increased C3b deposition and significantly increased exosome lysis. Blockade of CD59 also resulted in significant lysis, while blocking both CD55 and CD59 increased lysis still further. We conclude that exosomes express GPI‐anchored complement regulators in order to permit their survival in the extracellular environment.

Clinical studies of human papilloma vaccines in pre-invasive and invasive cancer

Cervical cancer is the second most common cause of cancer death in women worldwide. It is almost invariably associated with infection with human papilloma virus (HPV) particularly types 16 and 18. The ubiquitous expression of E6 and E7 oncogene products has been recognised as an attractive target for CTL-mediated immunotherapy. In-vivo expansion of an HPV oncogene product specific MHC class 1 restricted response has been demonstrated using intradermally administered live vaccinia virus HPV 16 and 18 E6/E7 gene construct (TA-HPV, Cantab Pharmaceuticals). Responses have been seen in 1/3 evaluable patients with advanced cervical cancer, and 3/12 CIN3 volunteers, and in 4/29 patients with early invasive cervical cancer (Rankin et al. Proceedings of 91st AACR Meeting, San Francisco, April 2000). In addition, the adoptive transfer of ex vivo HPV 16 or 18 positive autologous tumour lysate pulsed dendritic cells is currently being tested as an alternative means of expanding HPV specific CTL in advanced cervical cancer patients. So far an HLA-A*O201 restricted CD8 T cell response has been recorded in the single HLA-A*O201 patient whose tumour was shown to be HPV16 positive. It appears therefore feasible to induce HPV specific CTL responses in patients with cervical cancer using several vaccine strategies. However, further clinical trials are needed to determine the full anti-tumour potential of this vaccine based immunotherapy.

Dendritic cell (DC) based therapy for cervical cancer: use of DC pulsed with tumour lysate and matured with a novel synthetic clinically non-toxic double stranded RNA analogue poly [I] :poly [C12U] (Ampligen®)

Human papilloma virus (HPV) found in 99.7% of cervical cancers represents an attractive immunotherapeutic target for novel adjuvant dendritic cell (DC) immunotherapy. DC primed with HPV antigens have been shown to be capable of inducing CTL responses powerful enough to eradicate established murine tumours expressing HPV16 antigen. The use of tumour lysate has been found to be an effective means of priming DC with tumour associated antigens in animal models and in clinical trials leading to significant anti-tumour responses. Autologous DC primed with sonicated HPV expressing tumour lysate have been shown to be capable of inducing HPV specific classes I and II T-cell immunity in a pilot clinical study.Synthetic double stranded polyribonucleotides are effective in vitro activation/maturation agents capable of inducing a stable mature DC phenotype producing high levels of IL12. However, the prototype polymer poly [I]:poly [G] has proved to be clinically toxic. Preliminary in vitro data have demonstrated that a novel clinically non-toxic analogue polymer poly [I]:poly [C(12)U] (Ampligen R) can effectively induce in vitro maturation of human monocyte derived DC with sustained bioactive IL12 production. Human monocyte derived DC primed with tumour lysate and matured with synthetic dsRNA may therefore offer an effective way of optimising Th1 specific anti-cancer T-cell responses in cancer patients. This strategy is currently being tested in a clinical trial in patients with cervical cancer.

CELL-CELL ADHESION MOLECULES AND SIGNALING INTERMEDIATES AND THEIR ROLE IN THE INVASIVE POTENTIAL OF PROSTATE CANCER CELLS

PURPOSEnThe highly variable natural history of prostate carcinoma may be reflected in heterogeneity of invasive potential between tumors.nnnMATERIALS AND METHODSnWe have examined two prostate cancer cell lines of low invasive potential (CAHPV10 and PZHPV7) and three cell lines of high invasive potential (DU-145, PC-3, LNCapFGC), to determine whether specific adhesion molecule profiles correlated with their invasive behavior.nnnRESULTSnUsing an in vitro invasion assay, we demonstrated that DU-145, LNCapFGC and PC-3 cells were highly invasive compared with CA-HPV-10 and PZ-HPV-7 cells. LNCapFGC cells expressed high levels of E-cadherin, alpha-, beta- and gamma-catenin, desmoglein, desmoplakin and GSK3beta using immunoblotting. This was, in general, comparable to immunohistochemical staining. PC-3 cells had no E-cadherin or alpha-catenin, but expressed a high level of the HGF/SF receptor c-Met. In contrast, DU-145 cells were found to express E-cadherin and low levels for all other protein molecules, except c-Met. The DU-145 cell line also lacked alpha-catenin expression. In CA-HPV-10 and PZ-HPV-7 cells, there was no detection of APC, PECAM-1, P-cadherin or Wnt-1. DU-145, LNCapFGC and PC-3 cells formed cell-cell aggregates, which were reduced by inclusion of anti-E-cadherin antibody and the motogen HGF/SF.nnnCONCLUSIONnThese results show that prostate cancer cells exhibit a diverse expression of cell-cell adhesion molecules and their signaling intermediates. The expression of these adhesion molecules bears an important relationship with the invasive phenotype of these cells.

A phase-1 study of sequential mitomycin C and 5-aminolaevulinic acid-mediated photodynamic therapy in recurrent superficial bladder carcinoma.

To report a phase‐1 study of patients with recurrent superficial bladder cancer treated with photodynamic therapy (PDT) using sequential mitomycin C and 5‐aminolaevulinic acid (ALA).

Cell adhesion molecules and adhesion abnormalities in prostate cancer

Prostate cancer, the leading male cancer in Western countries, has accelerated in its incidence in the past decade. Patients with prostate cancer frequently have a poor prognosis as a result of local or distant spread of cancer. This review summarises some of the recent progress made in understanding the biology of cancer metastasis with a special emphasis on the role of cell adhesion molecules and adhesion abnormalities. The molecular and cellular function of cell adhesion molecules, their role in cancer and cancer progression, the clinical impact of these molecules, and therapeutic considerations are also discussed.

NAPHTHYL PHOSPHORAMIDATE DERIVATIVES OF BVdU AS POTENTIAL ANTICANCER AGENTS: DESIGN, SYNTHESIS AND BIOLOGICAL EVALUATION

The phosphoramidate technology we have developed has been recently applied to BVdU, leading to NB1011 (NewBiotics Inc., California), a novel potential anticancer compound recently entered into phase 2 of the clinical trials for colon cancer. We report in this work a new series of derivatives containing naphthol as aryl masking group on the phosphate moiety, which has shown a significant increase in anticancer activity in preliminary biological evaluations.

Phospholipase-C gamma-1 (PLCγ-1) is critical in hepatocyte growth factor induced in vitro invasion and migration without affecting the growth of prostate cancer cells

BACKGROUNDnPhospholipase C gamma-1 (PLCgamma-1) is an intracellular signalling molecule regulating a number of biological processes including transporter mechanisms, transcription factors, and scaffolding proteins mediating cytoskeleton and membrane trafficking. Hepatocyte growth factor (HGF) is a pleiotrophic factor and a mediator of metastatic spread. This study sought to determine the effect of HGF on the invasive and migratory potential of prostate cancer cells targeted by a ribozyme transgene to PLCgamma-1.nnnMETHODSnA ribozyme transgene consisting of hammerhead ribozyme and antisense specific to PLCgamma-1 was cloned into a PEF6 expression vector and transfected into PC-3 cells. RT-PCR and Western blotting confirmed knock down of PLCgamma-1. In vitro invasion and a cytodex-2 bead motility assays with in vitro and in vivo growth models were used to assess the impact of PLCgamma-1 manipulation.nnnRESULTSnPC-3 cells stably transfected with PLCgamma-1 ribozyme transgene (PC-3(DeltaPLC)gamma) manifested a reduction of PLCgamma-1 expression at mRNA/protein levels. HGF/SF increased invasiveness (P < 0.01) and motility (P < 0.0001) of PC-3(WT) and PC-3(PEF6) cells. In contrast, PLCgamma-1 knock down PC-3(DeltaPLC)gamma cells had reduced invasiveness (P < 0.05) and motility (P < 0.01) compared with PC-3(WT) and PC-3(PEF6) cells. Although there was a marginal change of cell growth in vitro, there was no difference in the rate of growth between PC-3(DeltaPLC)gamma, PC-3(WT), and PC-3(PEF6) cells.nnnCONCLUSIONnTargeting PLCgamma-1 by way of a hammerhead ribozyme to PLCgamma-1 is an effective method in reducing invasive phenotype of prostate cancer. PLCgamma-1 is a signalling intermediate that has prime influence on HGF induced cellular invasion and migration without affecting the growth of the cells.

Clinical studies of human papilloma vaccines in cervical cancer

Cervical cancer is the second most common cause of cancer death world wide’ and is a particular problem in developing countries. Even with optimum treatment approximately 40% continue to die from this disease.2There is therefore a need for new strategies to reduce global mortality, including immunotherapeutic intervention.