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Background The prostate cancer microenvironment is highly immunosuppressive; immune cells stimulated in the periphery by systemic immunotherapies will be rendered inactive once entering this environment. Immunotherapies for prostate cancer need to break this immune tolerance. We have previously identified interleukin-15 (IL-15) as the only cytokine tested that activates and expands immune cells in the presence of prostate cancer cells. In the current study, we aimed to identify a method of boosting the efficacy of IL-15 in prostate cancer. Methods We engineered, by conjugation to a myristoylated peptide, a membrane-localising form of IL-15 (cyto-IL-15) and the checkpoint inhibitor antibodies cytotoxic T lymphocyte antigen 4 (CTLA-4) and programmed death ligand 1 (PD-L1) (cyto-abs) to enable them to bind to cell surfaces by non-specific anchoring to the phospholipid bilayer. The efficacy of these agents was investigated by intratumoral administration either alone (cyto-IL-15 or cyto-abs) or in combination (cyto-combo) in subcutaneous TRAMP-C2 prostate tumors in C57BL/6J mice and compared with their non-modified equivalents in vivo. Following the survival endpoint, histological analyses and RNA sequencing were performed on the tumors. Results Intratumoral injection of cyto-IL-15 or cyto-combo delayed tumor growth by 50% and increased median survival to 28 and 25 days, respectively, compared with vehicle (17 days), whereas non-modified IL-15 or antibodies alone had no significant effects on tumor growth or survival. Histological analysis showed that cyto-IL-15 and cyto-combo increased necrosis and infiltration of natural killer (NK) cells and CD8 T cells in the tumors compared with vehicle and non-modified agents. Overall, the efficacy of cyto-combo was not superior to that of cyto-IL-15 alone. Conclusion We have demonstrated that intratumoral injection of cyto-IL-15 leads to prostate cancer growth delay, induces tumor necrosis and increases survival. Hence, cytotopic modification in combination with intratumoral injection appears to be a promising novel approach for prostate cancer immunotherapy.

Interleukin-15 (IL-15) is a cytokine previously suggested as a potential immunotherapy for cancer treatment. IL-15 can effectively reduce tumor growth in many preclinical tumor models including prostate cancer. This is due to its ability to expand and activate immune cells, such as CD8+ T cells and natural killer cells. To increase the potency of IL-15, we have engineered a protein variant that can be modified to localize and be retained in tissues where it is administered. However, the production of recombinant IL-15, the purity, and correct refolding of the final protein is not always ideal. In the current study, we aimed to optimize the methodology for production and purification of a modified recombinant human IL-15 and investigate the efficacy of the produced protein in the treatment of prostate tumors. Human IL-15 with its polypeptide sequence modified at the C-terminus to enable thiol conjugation with membrane localizing peptides, was produced in E. coli and purified using mild denaturing conditions (2M urea) from a washing step or from solubilization of inclusion bodies. The purified protein from the wash fraction was conjugated to a myristoylated peptide to form a membrane-localizing IL-15 (cyto-IL-15). The efficacy of cyto-IL-15 was investigated in subcutaneous TRAMP-C2 prostate tumors in mice and compared with cyto-IL-15 derived from protein purified from inclusion bodies (cyto-IL-15 Gen). When mild denaturing conditions were used for purification, the largest amount of IL-15 was collected from the wash fraction and a smaller amount from inclusion bodies. The protein from the wash fraction was mainly present as a monomer, whereas the one from inclusion bodies formed homodimers and higher complexes. After cytotopic modification, the purified IL-showed great efficacy in delaying prostate tumor growth (∼50%) and increased mice survival by ∼1.8-fold compared with vehicle. This study demonstrates an alternative, inexpensive and efficient method to produce and purify a modified version of IL-15 using mild denaturing conditions. This IL-15, when cytotopically modified, showed great efficacy as a monotherapy in prostate tumors in mice further highlighting the potential of IL-15 as a cancer immunotherapy.

Prostate cancer is the second most commonly diagnosed cancer in men with mortality rates, overtaking those for breast cancer in the last 2 years in the UK. Despite advances in prostate cancer treatments, over 25% of men do not survive over 5 years with advanced disease. Due to the success of immunotherapies in treating other cancers, this treatment modality has been investigated for Prostate cancer, however, the sole FDA approved immunotherapy so far (Provenge™) only extends life by a few months. Therefore, finding immunotherapeutic agents to treat prostate cancer is of major interest. Our group has previously shown that Interleukin-15 (IL-15), unlike other therapeutic cytokines such as IL-2 and IL-12, can stimulate expansion and activity of CD8 T cells and NK cells in vitro when they are exposed to prostate cancer cells, while studies in mice have shown a 50% reduction in tumor size with no apparent toxicity. In this study, we aim to examine potencies of IL-15 in combination with a cyclic dinucleotide (CDN) that activates the Stimulator of Interferon-Gene (STING) receptor. Selected CDNs (also known as STING agonists) have previously been shown to activate both T cells and dendritic cells through STING. We hypothesize that the combination of STING agonists and IL-15 can additively increase NK and T cell activity as they act to increase type I interferons (IFNs) through STING activation and IFN-γ through IL-15. In prostate cancer-lymphocyte co-cultures we now show that combination of IL-15 and the STING agonist ADU-S100 analog induces a marked killing of cancer cells above that seen with IL-15 or ADU-S100 alone. We show that this is related to a potent activation of NK cells resulting in increased perforin and CD69 expression, and up to a 13-fold increase in IFNγ secretion in the co-cultures. NK cells are responsible for killing of the cancer cells, as shown by a lack of cytotoxicity in NK depleted lymphocyte-tumor cell co-cultures, or in co-cultures of B and T cells with tumor cells. In summary, we propose that the combination of IL-15 and the sting agonist ADU-S100 analog may be potently effective in treatment of prostate cancer.

Interleukin-15 (IL-15) is a cytokine that has been shown to expand CD8 T cell and natural killer (NK) cell populations, and therefore has potential for potentiating adoptive immune cell therapy for cancer. Previously, IL-15 has been shown to induce proliferation of CD8 memory T cells through activation of telomerase. Here, we investigated whether telomerase is also activated during the IL-15 mediated proliferation of NK and NKT-like (CD56+CD3+) cells. We also examined the extent that each of the three signaling pathways known to be stimulated by IL-2/IL-15 (JAK-STAT, PI3K-AKT Ras-RAF/MAPK) were activated and involved in the telomerase expression in the three cell types NK, NKT, or CD8 T cells. To assess cell proliferation and doubling, peripheral blood mononuclear cells (PBMCs) or isolated NK, NKT-like or CD8 T cells were incubated with varying concentrations of IL-15 or IL-2 for 7 days. CD8 T, NK, and NKT cell expansion was determined by fluorophore-conjugated antibody staining and flow cytometry. Cell doubling was investigated using carboxyfluorescein-succinimidyl-ester (CFSE). Telomerase expression was investigated by staining cells with anti-telomerase reverse transcriptase (anti-TERT). Telomerase activity in CD56+ and CD8 T cells was also measured via Telomerase Repeat Amplification Protocol (TRAP). Analysis of cellular expansion, proliferation and TERT expression concluded that IL-15 increased cellular growth of NK, NKT, and CD8 T cells more effectively than IL-2 using low or high doses. IL-15, increased TERT expression in NK and NKT cells by up to 2.5 fold, the same increase seen in CD8 T cells. IL-2 had effects on TERT expression only at high doses (100–1000 ng/ml). Proteome profiling identified that IL-15 activated selected signaling proteins in the three pathways (JAK-STAT, PI3K-AKT, Ras-MAPK) known to mediate IL-2/IL-15 signaling, more strongly than IL-2. Evaluation by signaling pathway inhibitors revealed that JAK/STAT and PI3K/AKT pathways are important in IL-15’s ability to upregulate TERT expression in NK and NKT cells, whereas all three pathways were involved in CD8 T cell TERT expression. In conclusion, this study shows that IL-15 potently stimulates TERT upregulation in NK and NKT cells in addition to CD8 T cells and is therefore a valuable tool for adoptive cell therapies.

Objectives To identify cytokines that can activate and expand NK cells in the presence of prostate cancer cells in order to determine whether these agents may be useful in future intra-tumoural administration in pre-clinical and clinical prostate cancer trials. Materials and methods Lymphocytes isolated from normal donor blood were set up in co-cultures with either cancer or non-cancerous prostate cell lines, together with each of the cytokines interleukin (IL)-2, IL-12, IL-15, interferon (IFN)-γ or IL-21 for a period of 7 days. Then, expansion of NK cells, NKT cells and CD8 T cells was measured by flow cytometry and compared with the expansion of the same cells in the absence of prostate cells. The cytotoxic activity of NK cells, as measured by perforin and tumour cell killing, was also assessed. NK cell receptors and their corresponding ligands on prostate tumour cells were analysed to determine whether any of these were modulated by co-culture. The role of the tumour-secreted heat shock proteins HSP90 and HSP70 in the expansion of NK cells in the co-cultures was also investigated because of their effects on NK and CD8 T-cell activation. Results We showed that, among a panel of cytokines known to cause NK cell activation and expansion, only IL-15 could actively induce expansion of NK, NKT and CD8 T cells in the presence of prostate cancer cell lines. Furthermore, the expansion of NK cells was far greater (up to 50% greater) in the presence of the cancer cells (LNCaP, PC3) than when lymphocytes were incubated alone. In contrast, non-cancerous cell lines (PNT2 and WPMY-1) did not exert any expansion of NK cells. The cytolytic activity of the NK cells, as measured by perforin, CD107a and killing of tumour cells, was also greatest in co-cultures with IL-15. Examination of NK cell receptors shows that NKG2D is upregulated to a greater degree in the presence of prostate cancer cells, compared with the upregulation with IL-15 in lymphocytes alone. However, blocking of NKG2D does not inhibit the enhanced expansion of NK cells in the presence of tumour cells. Conclusions Among a panel of NK cell-activating cytokines, IL-15 was the only cytokine that could stimulate expansion of NK cells in the presence of prostate cancer cells; therefore IL-15 may be a good candidate for novel future intra-tumoural therapy of the disease.

The immunosuppressive prostate cancer microenvironment renders most infiltrating effector immune cells anergic or regulatory. Immunotherapeutic agents localized to the prostate can break this immune tolerance, leading to greater efficacy and less toxicity than systemically administered drugs. We have previously shown that cytotopic modification of interleukin-15 (cyto-IL-15) can activate NK and CD8+ T-cells in the presence of prostate cancer cells. In the present study, we used this cyto-IL-15 and two immuno-checkpoint blocking antibodies modified in a similar approach (these antibodies have previously exhibited antitumor response in clinical trials) to investigate their efficacy when used alone or in combination in a syngeneic murine model of prostate cancer. IL-15 and antibodies were modified by conjugation to a myristoylated peptide that enables them to adhere to cell membranes (patents pending), and thus localize to any potential site of injection. The cytotopically modified IL-15 and antibodies were injected intratumorally either alone (cyto-IL-15 or cyto-abs) or in combination (cyto-combo) in subcutaneous TRAMP C2 prostate tumors in C57BL/6 mice and compared with their nonmodified equivalents and with a vehicle-treated control group. Histopathologic analysis and immunohistochemistry were performed on sections from excised frozen tumors in order to quantify necrosis (haematoxylin and eosin staining) and immune cell infiltration. Intratumoral injection of cyto-IL15, combo or cyto-combo significantly delayed tumor growth at day 14 post-treatment by 50%. However, only cyto-IL15 and cyto-combo significantly increased median survival after treatment to 28 (p < 0.05) and 24 days (p < 0.05) respectively, compared with 17 days in the vehicle group. Nonmodified IL15 or antibodies alone had no effect on tumor growth or survival. Mice showed no adverse effects or weight loss with either of the treatments. Moreover, histologic analysis showed that cyto-IL-15 increased necrosis and infiltration of NK cells and CD8+ T cells in the tumors compared with the vehicle- and nonmodified-IL-15-treated groups. We have demonstrated that cytotopically modified immunotherapeutic agents can lead to tumor growth delay in an in vivo murine model of prostate cancer, induce tumor cell death and increase mouse survival. Hence, cytotopic modification in combination with direct injection of the agents directly into the tumor site appears to be a promising novel approach for prostate cancer immunotherapy. Citation Format: Efthymia Papaevangelou, Dorota Smolarek, Oussama Elhage, Richard A. Smith, Prokar Dasgupta, Christine Galustian. Targeting prostate cancer using novel cytotopically modified immunotherapies intratumorally in a syngeneic murine model [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology and Immunotherapy; 2018 Nov 27-30; Miami Beach, FL. Philadelphia (PA): AACR; Cancer Immunol Res 2020;8(4 Suppl):Abstract nr A11.

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.

Introduction & Objectives: Prostate cancer (PC) is the second most commonly diagnosed cancer in men and the eighth leading cause of cancer-related mortality in the world. The only successful immunotherapy for the disease (ProvengeTM) only extends life by a few months. Therefore the finding of immunotherapeutic agents for the treatment of PC is of major interest. It has been previously shown by our group that Interleukin-15 (IL-15), unlike other therapeutic cytokines such as IL-2 and IL-12, can stimulate expansion and activity of CD8 and NK cells in vitro when they are exposed to prostate cancer cells, while studies in mice have shown a reduction in tumor size by 50% with no apparent toxicity. In this study, we aim to examine potencies of IL-15 in combination with a cyclic dinucleotide (CDN) that activates the Stimulator of Interferon-Gene (STING) receptor. CDNs have previously been shown to activate both T cells and Dendritic cells through STING. We hypothesize that combination of CDNs and IL-15 can additively increase NK and T cell activity as they act to increase type 1 interferons through STING activation and IFN gamma through IL-15. Material & Methods: Peripheral blood mononuclear cells (PBMCs) were isolated and co-cultured with cancer cells (LNCaP, ATCC® CRL-1740™) in the presence of either IL-15, the CDN 2’3’-c-di-AM(PS)2, or a mixture of IL-15 with CDN. Control experiments were also prepared in which IL-15 was replaced by PBS and the CDN by a linear dinucleotide. Co-cultures were incubated for 48 hours at 37°C and 5% CO2. After this period, all cells from each well were collected and stained with fluorophore conjugated anti-CD3, -CD8 and -CD56 to identify CD8 T cells and NK cells, and anti-NKG2D and -perforin to analyze activation of NK and T lymphocytes. Tumor cell death was also evaluated and quantified using Live/Dead staining. All samples were acquired on FACS Canto. Results We observed that 1µg/ml of 2’3’-c-di-AM(PS)2 in combination with 2.5 ng/ml of IL-15 caused 81% of LNCaP killing in prostate cancer-lymphocyte co-cultures, while IL-15 by itself led to approximately 45% of cancer cell death. Little or no cancer cell killing was seen without IL-15 or CDN. Interestingly, the mixture of IL-15 with the CDN did not expand NK cells more than IL-15 alone. This suggests that the activity of NK cells is increased despite lack of their expansion. Preliminary data suggests that the level of perforin is increased by up to 35% in NK cells cultured with the mixture of both therapeutic drugs when compared to IL-15 alone in the cocultures. Of note, cell culture of either LNCaP or PMBCs with 1µg/ml of CDN did not affect cell growth. Conclusion: The combination of two immunotherapeutic drugs, IL-15 and 2’3’-c-di-AM(PS)2, lead to an increase in the activity of NK cells, with an apparent increase in the levels of perforin and doubling of cell death mediated by immune effector cells compared with IL-15 alone. Citation Format: Ana M. Esteves, Efthymia Papaevangelou, Prokar Dasgupta, Richard A.G. Smith, Christine Galustian. Combination of Interleukin-15 with a STING (Stimulator of Interferon Gene) agonist: A potential immunotherapy for prostate cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 4070.

In the last few years there have been concerted attempts at using the power of the immune system as an effective treatment option for cancer. This has become possible since our understanding of the workings of the immune system improved. Tumours form because of failure of the organism to destroy a rogue, mutated cell in an appropriate way.This article is protected by copyright. All rights reserved.

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 IC50 of docetaxel by up to 50% (P

Prostate adenocarcinoma is present in over 80% of men over the age of 80 and is by far the most common cancer of men. Although radical prostatectomy is curative in early disease, the risks of incontinence and impotence can affect the quality of life of patients. Early intervention with localized immunotherapy represents a potential solution as lymphocyte infiltration does occur in prostate cancer lesions, and immunotherapy with dendritic cell vaccines can significantly increase survival in late stage disease. However, lymphocytic infiltrates in the cancerous prostates have an anergic character arising from the suppressive effects of the microenvironment resulting from a conversion of effector cells into regulatory T-cells. Although TGFβ (transforming growth factor β) and IL-10 (interleukin-10) are known to be strong suppressor molecules associated with prostate cancer, they are among many possible suppressive factors. We discuss the possible role of alternative suppressor molecules, including the WAP (whey acidic protein) homologue ps20 that is expressed on prostate stroma and other WAP domain-containing proteins in the immunosuppressive prostate cancer milieu and discuss novel immunotherapeutic strategies to combat this disease.

Lenalidomide (REVLIMID®) CC-5013 (Celgene, NJ, USA) is approved, in both the USA and Europe, in combination with dexamethasone for the treatment of multiple myeloma patients who have received at least one prior therapy, and is rapidly being accepted worldwide for this condition. Lenalidomide is also approved in the USA and Canada for use in transfusion-dependent anemia in patients with low- and intermediate-1-risk myelodysplastic syndromes associated with del (5q) abnormality with or without additional abnormalities. Lenalidomide is an IMiD® immunomodulatory compound, incorporating structural modification of the drug thalidomide, which is active against a wide variety of autoimmune Th-2-dependent disorders, including erythema nodosum of leprosy, leishmaniasis, as well as severe ulcerative disorders such as Behcet’s syndrome. Unfortunately, long-term use of thalidomide is limited, particularly by neurotoxicity. To date, results suggest that lenalidomide is more active than thalidomide and does not cause the neurotoxicity seen with thalidomide. Lenalidomide has multiple properties, including anti-inflammatory, antiangiogenic and costimulatory effects, as well as being able to inhibit T-regulatory cells, all of which are properties deemed desirable for anticancer activity. This article covers the evidence that lenalidomide may have a major role in the treatment and control of many cancer types other than del (5q) myelodysplastic syndrome and multiple myeloma.

Recent years have seen improvements in strategies to treat pancreatic cancer. Pancreatic cancer is a leading cause of cancer-related death in the world, and is characterised by rapid disease progression, highly invasive tumour phenotype and resistance to chemotherapy. Patient prognosis is extremely grim, with a one-year survival rate of just 10% and only a 5% chance of surviving beyond five years. There has been little change in the treatment regimen, with 5-FU-based therapies being the usual route. Gemcitabine has also offered some relief over the past two decades, with modest improvements in median survival. This lack of choice has increased the call for new treatments, and indeed, novel drugs are now being investigated for their use in pancreatic cancer. These include those agents classically applied to other indications such as doxycycline and doxorubicin, as well as dietary components such as curcumin and genistein. Each of these drugs possesses different levels of activity both as single agents and in combinatorial approaches with gemcitabine. This review will discuss the difficulties in treating pancreatic cancer, and will summarise the progress and latest development in drugs used within this field of cancer.

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.

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.

Over the past 5 years, lenalidomide (Revlimid, Celgene Co., Summit, NJ, USA), a member of a class of drugs termed immunomodulatory drugs, has emerged as a significant weapon in the arsenal of cancer-therapeutics. It is a lead therapeutic in multiple myeloma and del-5q myelodysplastic syndromes and has also been trialed for acute leukaemia and chronic lymphocytic leukaemia, relapsed or refractory Hodgkin's lymphoma, T-cell non-Hodgkin's lymphoma, prostate cancer, non-small cell lung cancer, malignant melanoma, renal cancer, advanced ovarian and peritoneal carcinoma. The most significant development for lenalidomide has been its FDA approval (US and Europe) for previously treated multiple myeloma in combination with dexamethasone. The following review describes key clinical and mechanistic breakthroughs that have made lenalidomide a leading cancer therapeutic.

Lenalidomide (Revlimid; CC-5013) and pomalidomide (CC-4047) are IMiDs proprietary drugs having immunomodulatory properties that have both shown activity in cancer clinical trials; lenalidomide is approved in the United States for a subset of MDS patients and for treatment of patients with multiple myeloma when used in combination with dexamethasone. These drugs exhibit a range of interesting clinical properties, including anti-angiogenic, anti-proliferative, and pro-erythropoietic activities although exact cellular target(s) remain unclear. Also, anti-inflammatory effects on LPS-stimulated monocytes (TNF-alpha is decreased) and costimulatory effects on anti-CD3 stimulated T cells, (enhanced T cell proliferation and proinflammatory cytokine production) are observed. These drugs also cause augmentation of NK-cell cytotoxic activity against tumour-cell targets. Having shown that pomalidomide confers T cell-dependent adjuvant-like protection in a preclinical whole tumour-cell vaccine-model, we now show that lenalidomide and pomalidomide strongly inhibit T-regulatory cell proliferation and suppressor-function. Both drugs inhibit IL-2-mediated generation of FOXP3 positive CTLA-4 positive CD25high CD4+ T regulatory cells from PBMCs by upto 50%. Furthermore, suppressor function of pre-treated T regulatory cells against autologous responder-cells is abolished or markedly inhibited without drug related cytotoxicity. Also, Balb/C mice exhibit 25% reduction of lymph-node T regulatory cells after pomalidomide treatment. Inhibition of T regulatory cell function was not due to changes in TGF-beta or IL-10 production but was associated with decreased T regulatory cell FOXP3 expression. In conclusion, our data provide one explanation for adjuvant properties of lenalidomide and pomalidomide and suggest that they may help overcome an important barrier to tumour-specific immunity in cancer patients.

Hairy and enhancer of split homolog-1 (HES1) is a part of an extensive family of basic helix-loop-helix (bHLH) proteins and plays a crucial role in the control and regulation of cell cycle, proliferation, cell differentiation, survival and apoptosis in neuronal, endocrine, T-lymphocyte progenitors as well as various cancers. HES1 is a transcription factor which is regulated by the NOTCH, Hedgehog and Wnt signalling pathways. Aberrant expression of these pathways is a common feature of cancerous cells. There appears to be a fine and complicated crosstalk at the molecular level between the various signalling pathways and HES1, which contributes to its effects on the immune response and cancers such as leukaemia. Several mechanisms have been proposed, including an enhanced invasiveness and metastasis by inducing epithelial mesenchymal transition (EMT), in addition to its strict requirement for tumour cell survival. In this review, we summarize the current biology and molecular mechanisms as well as its use as a clinical target in cancer therapeutics.

Cells used in adoptive cell-transfer immunotherapies against cancer include dendritic cells (DCs), natural-killer cells, and CD8+ T-cells. These cells may have limited efficacy due to their lifespan, activity, and immunosuppressive effects of tumor cells. Therefore, increasing longevity and activity of these cells may boost their efficacy. Four cytokines that can extend immune effector-cell longevity are IL-2, IL-7, IL-21, and IL-15. This review will discuss current knowledge on effector-cell lifespans and the mechanisms by which IL-2, IL-7, IL-15, and IL-21 can extend effector-cell longevity. We will also discuss how lifespan and efficacy of these cells can be regulated to allow optimal clinical benefits.

Multiple organ dysfunction syndrome (MODS) is a term used to describe the sequential failure of single organ systems leading ultimately to a general failure of multiple organs. 1 MODS is responsible for 50% to 80% of all deaths in the surgical intensive care unit and has remained so for the past 20 years. 2–4 It develops secondary to severe systemic inflammation (sepsis), in which the activated neutrophil plays a central role. 4–6 The neutrophil is therefore subject to intensive investigation as a target for modulation in the reduction of MODS. 7–9 Various strategies have been used to reduce neutrophil activation in both experimental and clinical MODS. Because neutrophil activation usually involves ligation of surface receptors, 10 most strategies have tried to target various receptors or ligands. Such strategies have met with limited success clinically, 11–14 which probably reflects the complex nature of neutrophil activation in clinical MODS, where the neutrophil is stimulated by a variety of simultaneous extracellular signals, including various cytokines and endotoxin. A common event occurring as a result of many ligand–receptor interactions is the activation of the transcription factor NFκB (Fig. 1). NFκB is a dimer of two DNA binding subunits (RelA and p50). Inactive NFκB exists in the cytoplasm bound to IκB, which prevents NFκB from translocating to the nucleus and binding DNA. NFκB activation is the process by which NFκB is dissociated from ΙκB and translocated to the nucleus, where it binds DNA and ultimately initiates transcription. This very complex process occurs rapidly (within minutes of extracellular stimulation) and involves phosphorylation, proteolysis, and redox changes. Figure 1. Hypothetical pathway for NFκB activation. Any one of a number of extracellular inducers results in the activation of a number of protein kinases that phosphorylate IκB/p105 (shown in red; RelA, dark blue; p50, light blue). Phosphorylated ... NFκB is rapidly activated in response to many pathologic signals that may be relevant during surgical trauma, including cytokines, 15,16 adhesion molecules, 17,18 endotoxin, 19 hypoxia, 20,21 and shear stress. 22 These stimuli activate several different protein kinases (protein kinase C, MAP kinase, ceramide kinase, IκB kinase) 23 that initiate NFκB activation. It has been postulated that the common mechanism by which these signals result in the activation of NFκB is by a change in the redox status of the cells. 24,25 Increased levels of hydrogen peroxide have been implicated in IκB degradation and NFκB DNA binding. The increase in intracellular reactive oxygen species (hydrogen peroxide) is also accompanied by a depletion of antioxidants such as glutathione. 26 It has been reported that replenishing glutathione levels by treatment with the antioxidant N-acetyl-L-cysteine (NAC) reduces activation of NFκB. 27 Other antioxidants, including pyrrolidine dithiocarbamate (PDTC) and diethyldithiocarbamate (DDTC), have also been shown to abrogate activation of NFκB. 28 Activation of NFκB results in the transcription of genes that can participate in the inflammatory reaction by inducing the production of cytokines, immunoreceptors, and cell adhesion molecules. 29 This transcription factor is therefore a potentially attractive target for immunomodulation. Information on the role of NFκB in human sepsis and MODS is limited but shows that the activity of this transcription factor is increased during inflammation and sepsis. 30,31 There is, however, no published information about neutrophil NFκB status and the development of organ dysfunction. In this study we investigated the role of neutrophil NFκB activation in the development of postsurgical MODS and potential modulation of neutrophil NFκB activation in the prevention of MODS-related neutrophil activation.

The concept that cancer can be eliminated by the immune system has been put forward over 100 years ago [1]. At this time, it was already thought that immune effector cells can recognize cancer cells as non-self and can eliminate them in the same way as viral or microbial pathogens. Both the innate immune system and the adaptive immune system have a major role in the control of tumor cell growth. The innate immune system consists of nonantigen-specific cells including macrophages, dendritic cells, neutrophils, natural killer cells, gamma delta T cells, and complement. The adaptive immune system consists of cells such as antigen-specific cytotoxic and helper T cells and antibody-producing B cells which can obtain a memory phenotype against specific antigenic challenge. The result is the ability of the different immune cell types to recognize cancer cells as foreign [2]. Antigens produced by tumor cells are known to be recognized by T cells and B cells, and both tumor antigen-specific T cells and antibodies against tumor antigens can be detected in patients with cancers such as melanoma, ovarian cancer, colorectal carcinoma, and hepatocellular cell carcinoma [3, 4]. Tumor-related antigens fall into a number of types including unique patient or shared tumor-specific antigens, antigens which are in both tumors and normal tissues, and antigens derived from tumor-associated viruses. In prostate cancer, a number of antigens are expressed which can be used for prostate cancer diagnosis or monitoring (Table 5.1).

The IMiDs(®) immunomodulatory compounds lenalidomide and pomalidomide are agents with anti-inflammatory, immunomodulatory and anti-cancer activity. An excellent success rate has been shown for multiple myeloma in phase I/II clinical trials leading to Food and Drug Administration approval of lenalidomide. One mechanism by which these drugs could enhance anti-tumour immunity may be through enhanced dendritic cell (DC) function. Thalidomide, a compound structurally related to lenalidomide and pomalidomide, is known to enhance DC function, and we have investigated whether its analogues, pomalidomide and lenalidomide, also have functional effects on DCs. We used mouse bone marrow-derived DCs treated with 5 or 10 μm pomalidomide, or lenalidomide from day 1 of culture. Treatment with IMiD(®) immunomodulatory compounds increased expression of Class I (H2-Kb), CD86, and pomalidomide also increased Class II (I-Ab) expression in bone marrow-derived DCs, as measured by flow cytometry. Fluorescent bead uptake was increased by up to 45% when DCs were treated with 5 or 10 μm pomalidomide or lenalidomide compared with non-treated DCs. Antigen presentation assays using DCs primed with ovalbumin, and syngeneic T cells from transgenic OTI and OTII mice (containing MHC restricted, ovalbumin-specific, T cells) showed that both pomalidomide and lenalidomide effectively increased CD8(+) T-cell cross-priming (by up to 47%) and that pomalidomide alone was effective in increasing CD4(+) T-cell priming (by 30%). Our observations suggest that pomalidomide and lenalidomide enhance tumour antigen uptake by DCs with an increased efficacy of antigen presentation, indicating a possible use of these drugs in DC vaccine therapies.

INTRODUCTION AND OBJECTIVES: Infiltration of cytotoxic T effector and NK cells in the tumour microenvironment has been a major goal for effective immunotherapy. The prostate cancer microenviroment is a challenge considering its poor immunogenicity. Although T and NK cells infiltrate these tumours, they are frequently anergic or suppressive. Secretion of TGF beta and IL-10 by prostate cancer cells is thought to influence the suppressive microenvironment. Cytokines that enhance effector T and NK cell infiltration without regulatory T cell expansion are therefore desirable. The aim of this study was to identify cytokines that can expand effector cells in prostate cancer-effector cell co-cultures. METHODS: Non adherent PBMCs were isolated and cultured for 7 days with irradiated PC3 cancer cells in a 8:1 ratio with cytokines IL-2, IFN gamma, IL-12, IL-15 and IL-21 used at ED50 doses. Expansion of effector cells was measured using anti-CD3, CD56, CD4, CD8, CD25 and FOXP3. Results were analyzed on a FACSCalibur flow cytometer. RESULTS: In the absence of tumour cells, IL-2, IL-15, IL-21 and IFN gamma had similar effects on expanding effector T and NK cells resulting in a population of 10 –11% NK cells, 20 –21% CD8 effector T cells, and 35– 40% CD4 CD25T cells ( % of gated lymphocytes). The cytokine IL-12 expanded NK cells (13%), CD8 T cells (24%) and CD4 CD25effector cells (43%) to a greater extent in the absence of tumour cells compared to other cytokines (p 0.05 by 1 way anova and Newman Keuls). Similar numbers of CD4 CD25 FOXP3 Tregs arose with all cytokines (25–30% of the CD4 CD25 cells). However, with tumour cells present, there were less NK cells (2–7%), CD8 T cells (6–9%) and CD4 CD25T cells (13–19%) after 7 days: IL21 or IFN gamma did not affect NK cell numbers compared to PBS control, (3% of the gated lymphocytes); IL-2 and IL-12 gave a significant increase (3–4%) but the greatest effect was with IL-15 (7%).CD8 cells were also increased significantly by IL-15 (9% compared to 6% with the other cytokines) and CD4 25cells were increased by IL-12 and IL-15 (19% and 18% respectively compared to 13% with the other cytokines). Treg expansion occurred with IL-2 (44% compared to 30% of CD4 CD25 FOXP3 T cells in the PBS control) but Tregs were decreased by IL-12(14%)and IFN gamma (17%). CONCLUSIONS: IL-15 and IL-12 are most effective in restoring effector cell numbers in the immunosuppressive prostate tumour environment, with reduced Tregs in the presence of IL-12 and increased CD8 T cells in the presence of IL-15. Combinations of these cytokines may therefore be the basis for new immunotherapeutic modalities in prostate cancer.

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.

Background Surgical trauma suppresses host immune function, potentially creating an environment vulnerable to tumor cell growth. This study compared immune function after laparoscopy, minilaparotomy, and conventional colorectal tumor resections. Methods Seventy-one patients underwent surgery (20 laparoscopy, 21 minilaparotomy, and 30 conventional). Blood samples were taken before surgery and at 3 hours, 24 hours, and 5 days after surgery. White blood cell constitution was determined using monoclonal antibodies. Levels of TH1 cytokines interferon-γ, tumor necrosis factor-α, and interleukin (IL)-2 and TH2 cytokines IL-10, -4, and -6 were measured in plasma and from supernatants of activated peripheral blood mononuclear cells. Results At 5 days after surgery, lymphocyte counts remained low in the conventional and minilaparotomy groups (P = .001 and P = .008) but had resolved in laparoscopic patients. Three-hour postoperative serum IL-6 concentrations were lower in laparoscopic than in conventional patients (P = .028). Production of TH1 cytokines 3 hours after surgery were significantly increased in laparoscopic patients (interferon-γ P = .018, tumor necrosis factor-α P = .011, and IL-2 P = .037). Conclusions TH1 lymphocyte function is improved transiently and immune homeostasis restored earlier in patients undergoing laparoscopic colorectal cancer resection, which may influence disease recurrence.

Introduction & Objectives: Prostate cancer progression can arise by tolerance to, and evasion of tumour cells from immune effector cells such as CD8 T cells and NK cells due to the immunosuppressive tumour microenvironment. Immunotherapeutic drugs can eliminate tumours by breaking this tolerance, and such drugs that can be localized to the prostate will have greater efficacy and less systemic toxicity than drugs administered systemically. It has also been shown that a localized stimulation of the immune system can boost immunity all over the body to clear distant metastases. However, in the highly immunosuppressive prostate cancer microenvironment, most activated effector immune cells are rendered anergic or even regulatory. We have shown that IL-15 in a localizable form retains its ability to activate NK and CD8 T cells in the presence and absence of prostate cancer cells. We have now used a similar approach to modify two antibodies (patent pending) which have been shown previously to be potent in inhibiting checkpoint blockade of the immune response in clinical trials. Material & Methods: The antibodies were modified to enable their conjugation to a cytotopic “tail” molecule. The “tail” consists of three regions - a thiol reactive region which interacts with free cysteines on the protein of interest, a cationic region, which binds to the negatively charged cell membrane surface, and a hydrophobic region that enters the cell membrane. The cytotopic antibodies can then adhere to cell membranes and can thus localize to any lesion where it is potentially injected. The tailed agents were then compared to non-tailed ones in in-vitro assays of T cell inhibition to measure their neutralizing ability and an in-vivo prostate tumour challenge model in C57/BL6 mice. Results: We have shown that the modified antibodies can attach to cell membranes through their cytotopic tails. The two cytotopically modified antibodies were both active in reconstituting T cell activation as shown by IL-2 secretion assays in Jurkats and EL4 by upto 50% (p Furthermore we have also demonstrated in vivo in a TRAMP-C2 tumour challenge model using C57/BL6 wild type mice model that a cocktail of tailed antibodies and cytotopically modified IL-15 can completely clear animals of tumour by day 73 whereas tumour size is only reduced by 50% with the non-tailed cocktail. Conclusion: We have identified that a cocktail of a modified cytokine, IL-15, together with two antibodies directed to immune checkpoint proteins can break tolerance to immunity in the prostate cancer microenvironment and cause disease regression. A tailed form of these molecules, has equivalent or greater activity in in vivo model and makes it therefore a promising new agent for immunotherapy of prostate cancer. Citation Format: Dorota Smolarek, Christina A. Sakellariou, Prokar Dasgupta, Richard A. G. Smith, Christine Galustian. Cytotopically modified antibodies to checkpoint proteins can actively reconstitute immune checkpoint blockade and inhibit tumor growth in a prostate cancer mouse model. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 268. doi:10.1158/1538-7445.AM2015-268

INTRODUCTION The immunosuppressive Prostate Cancer (PCa) microenvironment has a major influence on the progression of the disease and is a key challenge for immunotherapy considering that immune effector cells activated by such modalities, will be rendered anergic or regulatory once they enter this microenvironment. We have previously shown that IL-15 can activate NK and NKT cells to mediate cytotoxicity against PCa cells in lymphocyte-PCa cocultures and also that IL-15 can modulate NK receptors to shift the balance between activation and inhibition of NK cells. From initial data in PCa-lymphocyte cocultures, we also observed that IL-15 downregulated the inhibitory NK ligand HLA-BW4 on PCa cells, whereas the ligands MICA/MICB were not significantly affected. Due to the critical role of MICA and other activatory NK receptor ligands (aNKRL) such as Nectin-2 and ULBP1 on NK cell function we have now studied effects of IL-15 on the expression of aNKRLs MICA, Nectin-2 and ULBP1 both on the cell surface of PCa cells and in the supernatants of PCa-lymphocyte co-cultures. METHODS PC-3 and LNCaP PCa cell-lines were incubated with non-adherent PBMCs at effector:target ratios of 8:1 and cytokines IL-2 or IL-15. After 1 week, tumour cells were stained with antibodies to Nectin-2, MICA, and ULBP1 ligands. Flow cytometric analysis followed staining. MICA, Nectin-2 and ULBP1 ELISAs were performed with supernatants from these co-cultures of PCa cells and lymphocytes (and from PCa cells alone, treated with or without cytokines) to determine whether there was any effect of IL-15 on shedding of these ligands from the PCa cells. RESULTS We now show that MICA, Nectin-2 and ULBP1 ligands expressed on PC-3 cells were not significantly changed with IL-15 treatment in PCa-lymphocyte cocultures, however, on LNCaP cells, IL-15, (but not IL-2) downregulated MICA, Nectin-2 and ULBP1 ligands by upto 90%* (* = p In ELISAs measuring concentrations of MICA and Nectin-2 in the PCa-lymphocyte co-culture sups, IL-15, but not IL-2, upregulated concentrations of MICA by upto 145%* (* = p CONCLUSIONS In contrast to previous findings with IL-15 where aNKRLs (e.g. MICA, ULBP1) on certain cell types are upregulated, we see a downregulation of MICA, ULBP1 and Nectin-2 ligand expression on LNCaP cells with a concomitant increase in shed MICA. Although the current paradigm has been that shed MICA can inhibit NK cell function, other groups are now showing that the shed ligand may actually activate NKG2D on NK cells and our findings that IL-15 dramatically increases NK cell functions against PCa cells may confirm this alternative paradigm. Citation Format: Christina Alexandra Sakellariou, Oussama Elhage, Richard A. Smith, Prokar Dasgupta, Christine Galustian. IL-15 increases NK functions in the PCa-lymphocyte microenvironment by a profound increase in shedding of MICA from PCa cells - a novel paradigm. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 2340. doi:10.1158/1538-7445.AM2015-2340

Thalidomide, a drug originally used to treat morning sickness, was removed from the market place in the early 1960s after it was found to cause serious congenital birth defects. However, thalidomide has recently been investigated in a new light following its activity in a number of chronic diseases. Moreover, like thalidomide itself, its second-generation immunomodulatory drug (IMiD) analogues have been shown to act as powerful anticancer agents and are clearly active in the treatment of patients with relapsed multiple myeloma. These new drugs, in particular the second-generation IMiDs, lenalidomide (CC-5013, REVLIMID; Celgene Corp., NJ, USA) and CC-4047 (ACTIMID; Celgene Corp.), offer improvements over thalidomide (a first-generation IMiD) in terms of efficacy and safety in human studies. The key to the therapeutic potential of IMiDs lies in the fact that the drugs have multiple mechanisms of action, which may produce both anti-inflammatory and antitumour effects. These effects are probably contextual, depending both on the cell type and the stimulus involved. Mechanisms associated with IMiD activity include TNF-alpha-inhibitory, T cell costimulatory and antiangiogenic activities. Studies of the mechanisms of action of these drugs are ongoing and will facilitate the continued development of this class of compound in a number of diseases.

Introduction & Objectives: Prostate cancer progression can arise by tolerance to, and evasion of tumor cells from immune effector cells such as CD8 T cells and NK cells due to the immunosuppressive tumor microenvironment. Immunotherapeutic drugs can eliminate tumors by breaking this tolerance, and such drugs that can be localized to the prostate will have greater efficacy and less systemic toxicity than drugs administered systemically. It has also been shown that a localized stimulation of the immune system can boost immunity all over the body to clear distant metastases. However, in the highly immunosuppressive prostate cancer microenvironment, most activated effector immune cells are rendered anergic or even regulatory. We have previously shown that IL-15, unlike other therapeutic cytokines such as IL-2 and IL-12, can stimulate expansion and activity of CD8 and NK cells when they are exposed to prostate cancer cells. Therefore, we decided to create a localizable form of this cytokine to study its activity on NK and CD8 T cells in the presence and absence of prostate cancer cells. Material & Methods: The cytokine IL-15 was produced in a modified form to enable its conjugation to a cytotopic “tail” molecule. The “tail” consists of three regions - a thiol reactive region which interacts with free cysteines on the protein of interest, a cationic region, which binds to the negatively charged cell membrane surface, and a hydrophobic region that enters the cell membrane. The cytotopic IL-15 can therefore adhere to cell membranes and can thus localize to any lesion where it is potentially injected. The tailed IL-15 was then compared to non-tailed IL-15 and other cytokines in assays to measure T cell proliferation, and NK and CD8 T cell expansion in the presence and absence of prostate cancer cells. Results: Compared to non-tailed forms of IL-15, the tailed IL-15 induced greater proliferation of a murine T cell line, CTLL-2 and PBMCs . Proliferation levels were up to 20 times greater with the tailed IL-15. In initial experiments, activity of the tailed IL-15 was also greater or equivalent to non tailed IL-15 in its ability to expand NK cells in the presence of a prostate cancer cell line (TRAMP-C1). Conclusion: We have identified a cytokine, IL-15, that can break tolerance to immunity in the prostate cancer microenvironment. A tailed form of this molecule, has equivalent or greater activity in expanding immune effector cells in the presence or absence of prostate cancer cells and its potential for regioselective action makes it therefore a promising new agent for immunotherapy of prostate cancer. Citation Format: Dorota A. Smolarek, Christina A. Sakellariou, Oussama Elhage, Osamu Ukimura, Inderbir Gill, Angus G. Dalgleish, Prokar Dasgupta, Richard A. G. Smith, Christine Galustian. Cytotopic IL-15 as a novel therapeutic for prostate cancer. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 2582. doi:10.1158/1538-7445.AM2014-2582

Background The prostate cancer microenvironment profoundly inhibits effector immune-cell activity. We have shown that interleukin (IL) 15, unlike other cytokines proposed for anti-tumour immunotherapy such as IL12 and IL21, can increase natural killer (NK) cell and CD8 T-cell activity within the prostate cancer microenvironment. To investigate mechanisms of this activation, we studied the effects of IL15 on inhibitory and activatory receptors on NK cells and their corresponding ligands on prostate cancer cells. NK receptors known as killer-Ig-like receptors (KIRs) have a crucial role in NK-mediated killing of tumour cells. NK-receptor ligands on tumour cells can attenuate NK activity by binding their corresponding inhibitory receptors. Conversely, increased HLA-related ligand expression can increase the recognition and killing of tumour cells by T cells. Methods The prostate cancer cell-lines PC3 and LNCaP were incubated with non-adherent peripheral blood mononuclear cells (PBMCs) at effector-to-target ratios of 8:1 and cytokines IL2 or IL15. After 1 week, NK cells in the co-cultures were stained with antibodies to inhibitory KIRs (KIR2DL1, KIR2DL2, KIR3DL1) and activatory KIRs (NKp44, NKG2D, NKp46, DNAM1). Tumour cells in the co-cultures were stained with antibodies to inhibitory NK-receptor ligands HLA-class1-Bw4 and HLA-G, and activatory receptor ligands Nectin-2, and MICA/B. Antibody to HLA-ABC was also used. Staining was followed by flow cytometric analysis. Groups were compared with one-way ANOVA and post-hoc Newman-Keuls (five repeated experiments). Findings IL15, but not IL2, inhibited expression of KIR2DL1 and KIR3DL1 by up to 50% (p Interpretation One mechanism by which IL15 increases prostate cancer killing by NK cells in PBMC-prosate cancer co-cultures is through upregulating activatory NKG2D and decreasing inhibitory KIR2DL1 and KIR3DL1 receptors. In addition, inhibitory ligands on prostate cancer cells are downregulated, suggesting strong shifts towards NK activation versus inhibition in the co-cultures. Increased HLA-ABC also favours stronger cytotoxic-T-cell recognition. Funding Heathside Trust, UK Medical Research Council, National Institute for Health Research, Prostate cancer UK. The research was funded and supported by the National Institute for Health Research (NIHR) Biomedical Research Centre based at Guy's and St Thomas' NHS Foundation Trust and King's College London.

Background Gemcitabine resistance is a major challenge in pancreatic cancer where the drug is considered a “gold standard” treatment. Although many studies have attempted to address mechanisms of gemcitabine resistance, these are not yet clear. The complete elimination of these resistant cells will be a major breakthrough in treatment. In many cancers, curcumin is of interest due to its inherently low toxicity to patients even at high doses: The drug can inhibit pancreatic cancer cell growth, and also acts in synergy with gemcitabine reducing viability of several established cancer cell lines. Identifying curcumins efficacy after transition from gemcitabine sensitive to resistant disease is therefore a key question for progress toward treatment. We have created a novel cell line from a parental MIA-PaCa-2 pancreatic adenocarcinoma which is 286 times more resistant to gemcitabine exposure than the parent line. Here we investigated the cell cycling properties of this cell line and its ability to be killed by curcumin. Methods The gemcitabine resistant MIA-PaCa-2 cell line was created by continuous gemcitabine dosing which increased its EC50 for the drug to 286-fold greater than that of the parent line. The extent of resistance was confirmed by MTT for viability and BrdU ELISA for proliferation. The sensitive and resistant Mia-PaCa-2 cells were then incubated in the absence, or presence of varying doses of curcumin (0.01-150μM). Cells were analysed with respect to their cell cycling profiles by staining initially with EdU over a time course, followed by 7-AAD DNA staining of permeabilised cells with subsequent flow cytometric analysis. Early apoptotic, late apoptotic and necrotic fractions of these cells were determined by staining with annexin-V and propidium iodide. Results: Curcumin inhibited growth of both gemcitabine sensitive and resistant cell lines to a similar extent (IC50s =13.7 μM and 16.6 μM for sensitive and resistant lines respectively). Whereas gemcitabine was incapable of causing apoptosis in the resistant strain, curcumin induced similar levels of apoptosis in both lines at doses between 1 and 56μM. With respect to the cell cycle, gemcitabine caused complete S-phase arrest in the sensitive strain and had no effect on the resistant strain, whereas curcumin caused G2/M phase arrest (no increase in G1 and 100% loss of second cycle S-phase) in both cell lines. Conclusions Curcumin is effective as a chemotherapeutic drug against gemcitabine resistant cells due to its ability to inhibit the cell cycle at the G2/M boundary after gemcitabine fails to activate the restriction point. The ability of curcumin to avoid cross resistance after gemcitabine is rendered ineffective should be studied as this could provide mechanisms for the creation of new drugs or highlight the possibilities of curcumin analogues as a second-line therapeutic for the placation of pancreatic adenocarcinoma. Citation Format: Alan Levett, Rachel Allen, Christine Galustian, Angus George Dalgleish. Curcumin is effective in killing gemcitabine resistant pancreatic cancer cells: Studies of the cell cycle, and apoptosis. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 3426. doi:10.1158/1538-7445.AM2013-3426

Introduction The protein ps20 encoded by the WFDC1 gene, is a member of a protein family containing conserved whey-4-disulphide-core (WFDC) domains. Ps20 is upregulated in prostate cancer epithelium and expression is thought to correlate with disease progression. The aim of this study was to determine whether ps20 expressed by prostate cancer cells also increases docetaxel resistance. Methods The cell-lines LNCaP and PC3 express low endogenous ps20, but were transfected with empty vector (EV) or WFDC1 gene vectors that increase WFDC1 RNA and ps20 protein expression by over 100% as measured by qPCR and ELISA. Docetaxel was added from 0.001nM –1µM with or without ps20 antibody (1G7) and cell-killing was assessed by MTT after 48 hours. Results EV-transfected PC3 and LNCaP cells have docetaxel IC50s of 0.223+/-0.05 and 0.16+/- 0.09nM respectively. In WFDC1-transfected PC3 and LNCAP, IC50s are increased to 0.677+/-0.11 and 0.326+/-0.05nM respectively (p Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 4233. doi:1538-7445.AM2012-4233

Introduction The protein ps20 encoded by the WFDC1 gene contains a highly conserved whey 4-disulphide core (WFDC) domain and is a member of a protein family that regulates inflammation. Ps20/WFDC1 is expressed by prostate cancer stromal cells but its expression by cancerous prostate epithelium is associated with tumour progression in patients. The aim of this study was to determine how WFDC1expression by prostate cancer cells alters their tumourigenic properties. Methods The cell-lines LNCAP and PC3 were transfected with WFDC1 vector or empty vector (EV) and their growth, adhesive and migratory characteristics were investigated by MTT proliferation, transwell migration and ICAM-1 adhesion assays. Results PC3 and LNCAP cells transfected with WFDC1 increased in numbers by 3148 +/−151% and 523+/−311% after 48 hours respectively compared to EV transfected cell lines (p Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 2833. doi:1538-7445.AM2012-2833

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.

The tissue factor-thrombin pathway is known to mediate proliferation, adhesion, invasion and metastasis of many types of cancer cells. These effects are additional to the thromboembolic processes commonly occurring in cancer, particularly in patients on hormonal therapies. In prostate cancer, there is an overexpression of the thrombin receptor PAR1 correlating with tumor progression. Anticoagulants such as heparin and hirudin are known to inhibit metastasis of tumors and improve survival rates in prostate cancer patients with localized disease. However, anticoagulants given systemically have an associated risk of bleeding which is higher in cancer patients and can be compounded by an increased risk of thrombocytopenia. Anticoagulants which can be localized to a tumor lesion are therefore highly favorable. The aim of the following study was to determine the efficacy of membrane-tethered anticoagulants on thrombin mediated migration and proliferation of prostate cancer cells in vitro. The hirudin cytotopic analogs PTL004 and PTL006, previously designed for use with anti-complement therapeutics in transplantation, were assessed as inhibitors of proliferation and migration of prostate cancer cells, in comparison to non-tailed hirudin-derived peptide, a PAR-1 antagonist and hirudin itself. Methods: For proliferation assays, 2000 PC3 prostate cancer cells were seeded in 96 well plates in serum free culture medium. Cells were preincubated for 30 mins with anticoagulant inhibitors at concentrations from 1 µM to 0.001 µM, and then 0.5 nM thrombin was added. Proliferation was measured after 48 hrs using MTT. To measure effects on cell migration, 10,000 PC3 cells were seeded onto porous membrane inserts in 24 well plates in serum free medium and then incubated with the inhibitors at 1 µM for 30 mins followed by 0.5 nM thrombin. Migration of cells through the membrane was measured after a 24 hr period. Results: Thrombin increased proliferation of PC3 cells in serum-free medium with a 1.5 fold increase in cell number after 48h. The tailed derivative PTL004 inhibited proliferation with an IC50 of 1 µM, equivalent to hirudin and the PAR1 antagonist. Liposomes prepared with PTL004 on the surface were more active than the tailed preparation in free solution with an IC50 10-50 fold lower. However, the non-tailed hirudin-derived peptide, and the derivative PTL006, which is known to have a more rapid dissociation rate when used with other cell types, were not active under these assay conditions. In the migration assays, PTL004 was also most active among the derivatives we tested. Conclusions: The membrane localizing anti-coagulant PTL004 has comparable anti-migratory and anti-proliferative properties to hirudin, and liposomes bearing this compound are even more effective at inhibition of proliferation of PC3 cells. The results demonstrate the potential of a cytotopic approach using tailed anticoagulants in localized prostate cancer therapy. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 673. doi:10.1158/1538-7445.AM2011-673

e13630 Background: Gemcitabine is the leading therapeutic for the treatment of pancreatic cancer, despite emergence of resistance. Understanding the mechanisms of gemcitabine resistance and discove...

e13155 Background: Docetaxel in combination with prednisone is approved for castrate-resistant metastatic prostate cancer (CRPC). However, docetaxel-based drug combinations may exhibit significant ...