King's Health Partners' Prostate Cancer Biobank (KHP PCaBB).

The KHP PCaBB was established in 2013 and recruits donors from the Urology or Oncology Departments at Guy's Hospital in London (UK). Prostate cancer patients may be approached to give their consent for biobanking at any point in their treatment pathway, which allows residual material from their earlier diagnosis to be transferred and used by the Biobank. Currently, patients are specifically asked to donate samples of blood and surplus prostate tissue as well as permitting access to their clinical and pathological data that continues to be added throughout the course of their disease. Between 2013 and 2015, 549 prostate cancer patients gave their consent to the biobank and, the tissue repository collected 489 blood samples, 120 frozen prostate tissue samples and 1064 formalin fixed paraffin embedded diagnostic blocks.Prostate cancer has become a chronic disease in a large proportion of men, with many men receiving multiple subsequent treatments, and their treatment trajectory often spanning over decades. Therefore, this resource aims to provide an ideal research platform to explore potential variations in treatment response as well as disease markers in the different risk categories for prostate cancer.A recent audit of the KHP PCaBB revealed that between 2013 and 2015, 1796 patients were diagnosed with prostate cancer at King's Health Partners (KHP), out of which 549 (30.6%) gave their consent to KHP PCaBB. Comparisons between demographic and clinical characteristics of patients who had consented compared to the total patient population revealed that the KHP PCaBB is demographically representative of the total prostate cancer patient population seen in Guy's and St Thomas' NHS Foundation Trust (GSTT). We observed no differences in distribution of ethnicity (p = 0.507) and socioeconomic status (p = 0.097). Some differences were observed in clinical characteristics, specifically with treatment type - which differed significantly between the patients who had given consent and total patient population.The KHP PCaBB has thereby amassed a rich data and tissue repository that is largely reflective of both the demographic and clinical diversity within the total prostate cancer patient population seen at KHP, making it an ideal platform for prostate cancer research.

Lenalidomide enhances the anti-prostate cancer activity of docetaxel in vitro and in vivo.

BACKGROUND: In this study, we investigated the effects of combining lenalidomide and docetaxel on in vitro and in vivo models of prostate cancer as a potential strategy for treatment of castrate resistant prostate cancer (CRPC). METHODS: The effects of combining lenalidomide and docetaxel on proliferation, apoptosis, invasive potential, anchorage independent growth, and p53 activation in the PC3 and DU145 prostate cell lines were investigated. The effects of the lenalidomide and docetaxel combination on LNCaP prostate cancer cell growth and invasiveness in vitro was also studied. The combination of these two agents was finally tested on a xenograft model of PC3 tumor growth in nude mice. RESULTS: Lenalidomide decreased the IC(50) of docetaxel by up to 50% (P < 0.05) and also decreased invasion in PC3, LNCaP, and DU145 cells and anchorage independent growth in PC3 cells (P < 0.01). Apoptosis in lenalidomide/docetaxel-treated cells was increased by 2.2-fold over single agent docetaxel and a corresponding increase in p53, p38, and BAD activation was observed in Western blots (P < 0.001). When PC3 challenged mice were treated with lenalidomide and docetaxel, median survival increased from 48 to 59 days and the rate of tumor growth was significantly reduced (P < 0.05). CONCLUSIONS: Lenalidomide may be a promising candidate for combination with docetaxel in the treatment of CRPC.

T regulatory cells, the evolution of targeted immunotherapy.

T regulatory cells are able to suppress anti-tumour immunity in pre-clinical models and in patients. This review highlights the important discoveries in Treg immunology critical to the evolution of targeted immunotherapy. We also describe the therapeutic applications that are currently being assessed and their future potential.

The effect of colorectal cancer upon host peripheral immune cell function.

OBJECTIVE: Colorectal cancer is immunogenic. However, it is also associated with suppression of host immunity. Identifying the mechanisms involved in immune suppression is necessary to develop future immunotherapeutic strategies. The aim of this study was to assess immune cell function in colorectal cancer patients. METHOD: A total of 80 colorectal cancer patients (41 male) prior to treatment and 38 matched controls (21 male) were recruited. Venous blood samples were taken. White blood cell composition was determined using monoclonal antibodies. Levels of cytokines IFN-gamma, TNF-alpha, IL-2, IL-10, IL-4 and IL-6 were measured from the supernatants of activated peripheral blood mononuclear cells (PBMC) following thawing and re-suspension. Peripheral blood mononuclear proliferation was measured using 3H-Thymidine. RESULTS: Stage I-III cancer patients had elevated percentages of CD8 T cell (P = 0.004) whilst stage IV patients had low total lymphocyte percentages (P = 0.016). Monocyte and NKT cell percentage decreased with advanced tumour stages (P = 0.013 and P = 0.038). Patients had lower PBMC proliferation and production of the TH1 cytokines (IFN-gamma and TNF-alpha) (P < 0.001) than that of the controls. IL-6 and IL-4 production were not significantly different. IFN-gamma and TNF-alpha concentrations reduced with tumour vascular invasion (P = 0.011 and P = 0.019). CONCLUSION: Colorectal cancer induces an immunological response, shifting the cytokine balance. The most profound changes are seen once disease has spread systemically.

Inhibition of metastatic potential in colorectal carcinoma in vivo and in vitro using immunomodulatory drugs (IMiDs).

BACKGROUND: Thalidomide and lenalidomide are FDA approved for the treatment of multiple myeloma and, along with pomalidomide, are being investigated in various other cancers. Although these agents display immunomodulatory, anti-angiogenic and anti-apoptotic effects, little is known about their primary mode of therapeutic action in patients with cancer. METHODS: As part of a continuing research effort, we have investigated the effects of these agents on the metastatic capacity of murine colorectal cancer cell lines both in vivo and in vitro. Allied to these, we have studied their effects on the molecular pathways associated with metastasis. RESULTS: Results indicate that thalidomide, lenalidomide and pomalidomide significantly inhibit the metastatic capability of colorectal carcinoma cells. Anchorage-independent growth, used as a coarse indicator of transformation, was significantly reduced, as were migratory capacity and invasive competence. In addition, an in vivo experimental metastasis model also showed that treatment with the drugs resulted in a significantly lower number of metastatic pulmonary nodules relative to control mice. Allied to these cellular and phenotypic changes were alterations in molecular markers of metastasis and in intracellular signalling competency. CONCLUSIONS: These results provide evidence that in addition to their immunomodulatory effects, thalidomide, lenalidomide and pomalidomide can impair the metastatic capacity of tumours, and that this mechanism may involve alterations to cell signalling functionality.

T-regulatory cell modulation: the future of cancer immunotherapy?

T-regulatory cells suppress anti-tumour immunity in cancer patients and in murine tumour models. Furthermore, their activity is likely to have an effect on the effectiveness of immunotherapeutic treatments for cancer. Here we describe the current status of developing clinical strategies for modulating Treg activity in cancer patients.

Transcription factor NF kappa B expression and postsurgical organ dysfunction.

OBJECTIVE: To examine the role of neutrophil NF kappa B activation in organ dysfunction after major surgery. SUMMARY BACKGROUND DATA: NF kappa B is a transcription factor involved in the signal transduction of many stimuli that may participate in the pathogenesis of sepsis and resultant multiple organ dysfunction syndrome (MODS). It may therefore be a potential target for modulation in the reduction of postsurgical MODS. METHODS: Twenty-five patients undergoing major vascular surgery (thoracoabdominal aortic aneurysm repair) were studied. Perioperative levels of neutrophil NF kappa B, CD11b, and glutathione were measured. In vitro inhibition experiments using NF kappa B inhibitors were also performed. RESULTS: No differences in clinical parameters were apparent before surgery between the patients who subsequently developed MODS and those who did not. However, there was a significant difference in preoperative levels of NF kappa B between the patients who developed postoperative organ dysfunction and those who did not. There was also a significant preoperative difference between patients who survived surgery and those who did not. Glutathione levels were reduced both in patients who developed MODS and those who did not at the onset of surgery. NF kappa B inhibitors suppressed patient plasma-stimulated NF kappa B activation in healthy neutrophils. CONCLUSIONS: Preoperative neutrophil NF kappa B status may be a marker of postoperative outcome after major surgery, and therapy aimed at attenuating neutrophil NF kappa B activation may reduce postoperative sepsis and organ dysfunction.

Recombinant soluble human CD69 dimer produced in Escherichia coli: reevaluation of saccharide binding.

We reevaluate here an earlier report of monosaccharide binding by the C-type lectin-like, leukocyte surface protein CD69 in the form of a recombinant soluble dimer, and we examine polysaccharide binding by the protein. We have expressed in Escherichia coli a new construct of the extracellular part (Q(65)-K(199)) of human CD69. We describe the folding in vitro to produce, in good yield, the protein in a soluble, disulphide-linked, dimeric form, and the results of binding experiments with monosaccharides: glucose, galactose, mannose, fucose, N-acetylglucosamine, and N-acetylgalactosamine, linked to bovine serum albumin. Monosaccharide-binding signals are not detectable. Among the polysaccharides, heparin, chondroitin sulphates A, B, and C, fucoidan, and dextran sulphate, CD69 dimer gives a weak binding signal with fucoidan.

L-selectin interactions with novel mono- and multisulfated Lewisx sequences in comparison with the potent ligand 3'-sulfated Lewisa.

The cell adhesion molecule L-selectin binds to 3'-sialyl-Lewis (Le)x and -Lea and to 3'-sulfo-Lex and -Lea sequences. The binding to 3'-sialyl-Lex is strongly affected by the presence of 6-O-sulfate as found on oligosaccharides of the counter receptor, GlyCAM-1; 6-O-sulfate on the N-acetylglucosamine (6-sulfation) enhances, whereas 6-O-sulfate on the galactose (6'-sulfation) virtually abolishes binding. To extend knowledge on the specificity of L-selectin, we have investigated interactions with novel sulfo-oligosaccharides based on the Lex pentasaccharide sequence. We observe that, also with 3'-sulfo-Lex, the 6-sulfation enhances and 6'-sulfation suppresses L-selectin binding. The 6'-sulfation without 3'-sialyl or 3'-sulfate gives no binding signal with L-selectin. Where the 6-sulfo,3'-sialyl-Lex is on an extended di-N-acetyllactosamine backbone, additional 6-O-sulfates on the inner galactose and inner N-acetylglucosamine do not influence the binding. Although binding to the 6,3'-sulfo-Lex and 6-sulfo, 3'-sialyl-Lex sequences is comparable, the former is a more effective inhibitor of L-selectin binding. This difference is most apparent when L-selectin is in paucivalent form (predominantly di- and tetramer) rather than multivalent. Indeed, as inhibitors of the paucivalent L-selectin, the 3'-sulfo-Lex series are more potent than the corresponding 3'-sialyl-Lex series. Thus, for synthetic strategies to design therapeutic oligosaccharide analogs as antagonists of L-selectin binding, those based on the simpler 3'-sulfo-Lex (and also the 3'-sulfo-Lea) would seem most appropriate.

The First Total Synthesis of 6-Sulfo-de-N-acetylsialyl Lewis(x) Ganglioside: A Superior Ligand for Human L-Selectin.

Originally discovered as a minor by-product of 6-sulfo-N-acetylsialyl Lewis(x) , the de-N-acetylated form 1 is a superior L-selectin ligand to the N-acetyl form. To substantiate the extraordinary reactivity of 1, it was synthesized for the first time and its binding to L-selectin investigated. Compound 1 and related structures may be high-affinity endogenous ligands for L-selectin that are involved in the interaction of leukocytes with the vascular endothelium.

Sialyl-Lewis(x) sequence 6-O-sulfated at N-acetylglucosamine rather than at galactose is the preferred ligand for L-selectin and de-N-acetylation of the sialic acid enhances the binding strength.

Oligosaccharide sequences based on sialyl-Lewis(x) with 6-O-sulfation at galactose (6'-sulfo) or at N-acetylglucosamine (6-sulfo) and expressed on high endothelial venules are considered likely endogenous ligands for the leukocyte adhesion molecule, L-selectin. In the course of high performance TLC of three hexaglycosylceramides 6'-sulfo sialyl Lewis(x), 6-sulfo sialyl Lewis(x), and 6',6-bis-sulfo sialyl Lewis(x), synthesized chemically for selectin recognition studies, two minor byproducts were detected and isolated from each parent compound. By liquid secondary ion mass spectrometry these were identified as isomers containing a de-N-acetylated sialic acid or having a modified carboxyl group. Binding experiments with the parent compounds and the non-sulfated sialyl Lewis(x) glycolipid show that 6-sulfation potentiates, whereas 6'-sulfation virtually abolishes L-selectin binding. Thus the hierarchy of binding strengths were 6-sulfo sialyl > sialyl = 6',6-bis-sulfo sialyl >> 6'-sulfo sialyl Lewis(x). Whereas modification of the sialic acid carboxyl group markedly impaired L-selectin binding, de-N-acetylation resulted in enhanced binding. The natural occurrence on high endothelial venules of this 'super-active' de-N-acetylated form of 6-sulfo sialyl Lewis(x), and related structures, now deserves investigation.

Valency dependent patterns of binding of human L-selectin toward sialyl and sulfated oligosaccharides of Le(a) and Le(x) types: relevance to anti-adhesion therapeutics.

The human L-selectin is known to bind to immobilized 3'-sialyl-Le(x) and -Le(a) oligosaccharides both under static and physiological flow conditions. Here the reactivities toward 3'-sulfated and 3'-sialyl-Le(a) and -Le(x) pentasaccharides are compared by in-vitro binding and inhibition assays using preparations of human L-selectin-IgG-Fc chimera in which the selectin is predominantly in di- and tetrameric form (paucivalent) or in the form of a complex with anti-IgG (multivalent). Affinity for the sulfated ligands is marginally greater than for the sialyl ligands, as judged by concentrations required to give 50% inhibition of the multivalent selectin binding to the immobilized sulfated and sialyl ligands. There is a striking difference, however, in the avidities of binding of the two L-selectin forms toward the sulfated and sialyl ligands when these are immobilized in the clustered state: the paucivalent selectin gives detectable binding only to the sulfated ligands when these are immobilized as neoglycolipids on plastic microwells (up to 100 pmol immobilized per well) whereas the multivalent L-selectin binds well to both classes of ligand. Moreover, binding of the paucivalent selectin form is effectively inhibited only by the sulfated ligand, although binding of the multivalent selectin is inhibitable by both the sulfated and sialyl ligands. Such striking valency-dependent differences in ligand binding avidity and inhibitability may be manifest in vivo with the membrane-bound L-selectin, as marked variations occur in its density of expression on leukocytes. Thus, for the purpose of selecting inhibitors for development of therapeutic anti-inflammatory compounds, experimental designs based on the paucivalent L-selectin would more clearly single out compounds with broad spectrum anti-adhesive activities toward the both the high- and low-avidity interactions of the cell adhesion protein.

Actin cytoskeletal isoforms in human endothelial cells in vitro: alteration with cell passage.

The microfilamentous actin component of the cytoskeleton is crucial to endothelial angiogenesis and vascular permeability. Differences in actin cytoskeletal profiles in cultured human endothelial cells were explored: when first isolated, both primary human umbilical vein endothelial cells (HUVEC) and primary human placental microvascular endothelial cells (HPMEC) expressed F-actin, but not beta-actin or alpha-smooth muscle actin. A similar endothelial actin profile was observed in cryo-sections of freshly delivered term umbilical cord and placenta. In subsequent cell culture, although the actin cytoskeleton of HUVEC remained unchanged, the actin profiles of HPMEC altered after the second passage with the induction of alpha-smooth muscle actin expression, which was intercellularly heterogeneous and increased to 20% at P4. This behaviour occurred in HPMEC monolayers cultured on a variety of extracellular matrices. Comparisons with a spontaneously immortalized human microvascular cell-line, HGTEN 21, revealed that in prolonged passage, both alpha-smooth muscle actin and beta-actin were expressed, whereas HPMEC at P4 showed a lower level of beta-actin expression. Therefore, in comparison with large vessels, microvascular cells are more likely to dedifferentiate. This may reflect the ability of microvascular cells to remodel according to changing requirements for new vessel formation. In conclusion, passage of human microvascular endothelial cells, but not of larger vessel endothelial cells, alters the expression of actin isoforms. This may be important in relation to comparisons of in vitro and in vivo vascular permeability; higher passage microvascular endothelial cells should thus be used with caution in such studies.

Swainsonine, a glycosylation inhibitor, enhances both lymphocyte efficacy and tumour susceptibility in LAK and NK cytotoxicity.

Swainsonine (SW) inhibits the formation of N-linked complex oligosaccharides and has previously been shown to inhibit experimental metastasis in nude mice models. The present studies with human effector cells have shown that SW enhanced both lymphokine activated killer cell (LAK) and natural killer (NK) cytotoxicity in standard 51Cr-release assays. SW also increased the susceptibility of human K562 and Colo 320 target cells to NK and LAK cytotoxicity. The peak response of both LAK effectors and targets to SW occurred at 1-2 micrograms/ml SW. A novel finding was that SW enhanced the interleukin 2 (IL-2) beta chain receptor subunit expression on both LAK and NK cells to a greater extent than its enhancement of the IL-2R alpha (CD25 or TAC) receptor expression on LAK effectors. In addition, increases in both these receptors occurred at the doses of SW which augmented LAK cytotoxicity. We conclude that the anti-metastatic effects of SW have an immunological component which is maximal at 1-2 micrograms/ml SW. This suggests that dosage may be an important consideration to obtain optimal potential of SW in any future human cancer therapy.