Andrew Gravett

The antimalarial agent artesunate possesses anticancer properties that can be enhanced by combination strategies

Artemisinins are a class of compounds that are first‐line treatment options for malaria. They also have potent antiproliferative activity, which makes them potential anticancer drugs. We have previously demonstrated anticancer activity of a number of these compounds in vitro; however, cytotoxic activities were compromised by drug‐induced cell cycle arrests. In this study, we have explored further the activity of the clinical lead artemisinin‐drug artesunate (ART), used either alone or in combination with established chemotherapy. Also, by using a cell line expressing polyploidy character, have also explored the impact of cell cycle arrest in determining overall drug activity. Results showed that ART caused dose‐dependent decreases in cell number, which were associated with either increased cytotoxicity or cytostasis. Cytostasis appeared to be a consequence of a simultaneous arrest at all phases of the cell cycle, a deduction that was supported by molecular profiling, which showed reductions in cell cycle transit proteins. ART appeared to maintain cells in this arrested state; however, reculturing these treated cells in drug‐free medium resulted in significant reductions in viabilities. We also showed that ART maintained activity in polyploidy cells, and that an impressive enhancement to its activity was achievable through a combination with the immunomodulatory drug lenalidomide. Taken together, these observations indicate ART and its related compounds may be effective for the treatment of tumours, and that activity is related to schedule. Therefore, it is important to carefully select the most appropriate schedule to maximise ART efficacy.

Supernatants derived from chemotherapy-treated cancer cell lines can modify angiogenesis

Background: There is evidence that tumours produce substances such as cytokines and microvesicular bodies bearing bioactive molecules, which support the carcinogenic process. Furthermore, chemotherapy has also been shown to modify these exudates and in doing so, neutralise their tumourigenic influence. Methods: In the current study, we have investigated the effect of chemotherapy agents on modifying the cytokine profile and microvesicular cargo of supernatants derived from cancer cell lines. In addition, we have explored the effect of these tumour-derived supernatants on angiogenesis, and how chemotherapy can alter the supernatants rendering them less pro-angiogenic. Results: Herein, we show that supernatants contain a rich cocktail of cytokines, a number of which are potent modulators of angiogenesis. They also contain microvesicular bodies containing RNA transcripts that code for proteins involved in transcription, immune modulation and angiogenesis. These supernatants altered intracellular signalling molecules in endothelial cells and significantly enhanced their tubulogenic character; however, this was severely compromised when supernatants from tumours treated with chemotherapy was used instead. Conclusion: This study suggests tumour exudates and bioactive material from tumours can influence cellular functions, and that treatment with some chemotherapy can serve to negate these pro-tumourigenic processes.

Supernatants from lymphocytes stimulated with Bacillus Calmette-Guerin can modify the antigenicity of tumours and stimulate allogeneic T-cell responses

Background:Reduced expression of class 1 human leucocyte antigens (HLA1) is often a mechanism by which tumours evade surveillance by the host immune system. This is often associated with an immune function that is unable to mount appropriate responses against disease, which can result in a state that favours carcinogenesis.Methods:In the current study, we have explored the effects of Bacillus Calmette-Guerin (BCG) on the cytokine output of leucocytes, which is a key determinant in generating antitumour action, and have also assessed the effect of these cytokine cocktails on HLA1 expression in solid tumour cell lines.Results:BCG potently activated a broad range of leucocytes, and also enhanced the production of cytokines that were Th1-predominant. Supernatants from BCG-treated leucocytes significantly increased the expression of HLA1 on the surface of cancer cell lines, which correlated with increased cytolytic T-cell activity. We also showed that the increased HLA1 expression was associated with activation of intracellular signalling pathways, which was triggered by the increases in the Th1-cytokines interferon-γ and tumour necrosis factor-α, as counteracting their effects negated the enhancement.Conclusion:These studies reaffirm the role of BCG as a putative immunotherapy through their cytokine-modifying effects on leucocytes and their capacity to enhance tumour visibility.

Gemcitabine alters the proteasome composition and immunopeptidome of tumour cells

ABSTRACT The antigenic makeup of tumour cells can have a profound effect on the progression of cancer and success of immunotherapies. Therefore, one strategy to improve the efficacy of cancer treatments is to augment the antigens displayed by tumours. The present study explores how the recognition of tumour cells may be altered by non-cytotoxic concentrations of gemcitabine (GEM). Testing a panel of chemotherapeutics in human cancer cell lines in vitro, it was found that GEM increased surface expression of HLA-A,B,C and that underlying this were specific increases in β-2-microglobulin and immunoproteasome subunit proteins. Furthermore, the peptide antigen repertoire displayed on HLA class I was altered, revealing a number of novel antigens, many of which that were derived from proteins involved in the DNA-damage response. Changes in the nature of the peptide antigens eluted from HLA-A,B,C after GEM treatment consisted of amino acid anchor-residue modifications and changes in peptide length which rendered peptides likely to favour alternative HLA-alleles and increased their predicted immunogenicity. Signalling through the MAPK/ERK and NFκB/RelB pathways was associated with these changes. These data may explain observations made in previous in vivo studies, advise as to which antigens should be used in future vaccination protocols and reinforce the idea that chemotherapy and immunotherapy could be used in combination.

Abstract B38: Non-cytotoxic effects of the chemotherapeutic agent gemcitabine can alter the immunophenotype of tumor cells in in vitro culture

In addition to their cytotoxic/cytostatic properties, some chemotherapeutics have shown a remarkable capacity to modulate immune response. One such chemotherapy is the antimetabolite, gemcitabine (GEM). GEM has been shown to influence the quantity and activation of certain immune cell subsets when used in vivo. It has also been demonstrated that administration of GEM in conjunction with immunotherapy, such as dendritic cell vaccination, is beneficial in terms of overall survival and progression-free survival. The purpose of this study is to investigate if GEM can influence the way that tumor cells surviving treatment are regarded by the immune system. Our group has found that short-term in vitro culture with GEM enhances expression of human leukocyte antigen (HLA) class I, CD95, NKG2D ligands and TRAIL receptors on tumor cell lines. These molecules are all cell surface proteins important for the efficient surveillance and effector function of the immune system. The HLA class I increase is beta-2-microglobulin dependent and changes are underpinned by ERK, JNK and RelB-NFkB signalling pathways. Furthermore, culture with GEM induces immunoproteasomal components LMP2 and MECL-1 in tumor cells and, subsequently, the peptide antigen repertoire displayed on HLA class I is altered. The changes in peptide characteristics include amino acid anchor residue modifications which render the peptides likely to favor binding alternative HLA-allotypes. The renovation of the peptidome displayed by HLA class I may reveal sub-dominant immunogens to T-cells. Taken together, these changes may render tumor cells more sensitive to immune cell-mediated lysis. These data help explain observations made in previous in vivo studies, advise as to which novel antigens could be utilised in future vaccination protocols and lend further credence to the idea that chemotherapy and immunotherapy could be used in combination. Citation Format: Andrew M. Gravett, John P. Copier, Wai M. Liu, Martin P. Cranage, Angus G. Dalgleish. Non-cytotoxic effects of the chemotherapeutic agent gemcitabine can alter the immunophenotype of tumor cells in in vitro culture. [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology and Immunotherapy; 2016 Oct 20-23; Boston, MA. Philadelphia (PA): AACR; Cancer Immunol Res 2017;5(3 Suppl):Abstract nr B38.