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Determining the expression of genes in response to different classes of chemotherapeutic drugs may allow for a better understanding as to which may be used effectively in combination. In the present study, the human colorectal cancer cell line HCT116 was cultured with equi-active concentrations of a series of anti-cancer agents. Gene expression profiles were then measured by whole-genome microarray. Although each drug induced a unique signature of gene expression in tumour cells, there were marked similarities between certain drugs, even in those from different classes. For example, the antimalarial agent artesunate and the platinum-containing alkylating agent, oxaliplatin, produced a very similar mRNA expression pattern in HCT116 cells with ~14,000 genes being affected by the two drugs in the same way. Furthermore, the overall correlation of gene responses between two agents could predict whether their use in combination would lead to a greater or lesser effect on cell number, determined experimentally, than predicted by single agent experiments. The results indicated that even when working through different mechanisms, combining drugs that initiate a similar transcriptional response may constitute the best option for determining drug-combination strategies for the treatment of cancer.

BACKGROUND: Despite the majority of patients do not gain any benefit from dendritic cells (DC) vaccines, this approach has occasionally given rise to dramatic responses in melanoma. Biomarkers are crucial to identify which patients are more likely to respond. We looked for correlations between pre- or post- vaccination biomarkers and clinical outcomes to DC therapy in a cohort of patients with stage IV melanoma receiving a vaccine with autologous ex-vivo expanded DCs pulsed with allogeneic tumor cell lysate. METHODS: Serial serum samples were collected at baseline, week 4 and 12 and they were analyzed for a panel of different inflammatory markers using cytometric bead array technology and ELISA. RESULTS: Twenty-one patients were evaluable for response. Patients were separated into responders and non-responders based on clinical benefit. Responders were defined as patients who achieved a complete response, partial response or stable disease the latter lasting for at least 6 months. Responders (N = 9) showed a significantly longer Progression-free Survival (PFS; HR 0.23; 95% CI 0.08–062; P < .001) and Overall Survival (OS; HR 0.22; 95% CI 0.08–0.59; P < .001). The clinical non-responder phenotype correlated with an elevated pre-vaccination level of cytokines associated with inflammation compared to clinical responders (Apolipoprotein C111; IL-12 p40; MiP1α; Stem Cell Factor and TNFα). Apolipoprotein E (ApoE) was also significantly elevated in the pre-vaccine sera of the clinically non-responding group and in addition it was found to correlate with outcomes. Patients with increased levels of ApoE had a significantly shorter PFS (HR 3.02; 95% CI 1.09–8.35; P = .015) and OS (HR 2.40; 95% CI 0.9–6.3; P = .034). CONCLUSION: Our findings support the notion that treating the inflammatory background may have an impact on clinical outcome for patients receiving immunotherapy. A larger study is needed to confirm the significance of ApoE as a predictive biomarker for response to DC vaccines.

Much effort has been made to try to understand the relationship between chemotherapeutic treatment of cancer and the immune system. Whereas much of that focus has been on the direct effect of chemotherapy drugs on immune cells and the release of antigens and danger signals by malignant cells killed by chemotherapy, the effect of chemotherapy on cells surviving treatment has often been overlooked. In the present study, tumour cell lines: A549 (lung), HCT116 (colon) and MCF-7 (breast), were treated with various concentrations of the chemotherapeutic drugs cyclophosphamide, gemcitabine (GEM) and oxaliplatin (OXP) for 24 hours in vitro. In line with other reports, GEM and OXP upregulated expression of the death receptor CD95 (fas) on live cells even at sub-cytotoxic concentrations. Further investigation revealed that the increase in CD95 in response to GEM sensitised the cells to fas ligand treatment, was associated with increased phosphorylation of stress activated protein kinase/c-Jun N-terminal kinase and that other death receptors and activatory immune receptors were co-ordinately upregulated with CD95 in certain cell lines. The upregulation of death receptors and NKG2D ligands together on cells after chemotherapy suggest that although the cells have survived preliminary treatment with chemotherapy they may now be more susceptible to immune cell-mediated challenge. This re-enforces the idea that chemotherapy-immunotherapy combinations may be useful clinically and has implications for the make-up and scheduling of such treatments.

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.

We sought to establish if methylene homologues of artemisone are biologically more active and more stable than artemisone. The analogy is drawn with the conversion of natural O- and N-glycosides into more stable C-glycosides that may possess enhanced biological activities and stabilities. Dihydroartemisinin was converted into 10β-cyano-10-deoxyartemisinin that was hydrolyzed to the α-primary amide. Reduction of the β-cyanide and the α-amide provided the respective methylamine epimers that upon treatment with divinyl sulfone gave the β- and α-methylene homologues, respectively, of artemisone. Surprisingly, the compounds were less active in vitro than artemisone against P. falciparum and displayed no appreciable activity against A549, HCT116, and MCF7 tumor cell lines. This loss in activity may be rationalized in terms of one model for the mechanism of action of artemisinins, namely the cofactor model, wherein the presence of a leaving group at C10 assists in driving hydride transfer from reduced flavin cofactors to the peroxide during perturbation of intracellular redox homeostasis by artemisinins. It is noted that the carba analogue of artemether is less active in vitro than the O-glycoside parent toward P. falciparum, although extrapolation of such activity differences to other artemisinins at this stage is not possible. However, literature data coupled with the leaving group rationale suggest that artemisinins bearing an amino group attached directly to C10 are optimal compounds. A brief critique is made of proteomic studies purporting to demonstrate the alkylation of intraparasitic proteins by alkyne- and azide-tagged artemisinins and synthetic peroxides in relation to mechanism of action.

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.

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.

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.

Artemisinins are now established drugs for treatment of malaria. These agents have been shown to possess impressive anti-cancer properties. We have investigated the role of artemisone (ATM), a novel derivative of artemisinin (ART) in a cancer setting both alone and in combination with established chemotherapeutic agents. The anti-proliferative effects of ART and ATM were tested on a panel of human cancer cells in vitro using the methylthiazoletetrazolium assay, and the effect on cell cycling established by flow cytometry. Immunoblot analyses were performed to determine effects at the molecular level. Finally, ART and ATM were combined with the common anti-cancer agents oxaliplatin, gemcitabine and thalidomide. ART and ATM caused dose dependent decreases in cell number. ATM was consistently superior to ART, with IC50 s significantly lower in the former. Neither drug caused significant changes to the cell viability (%viable cells >95%), but arrested cell cycling. Blockade was either exclusively at the level of G1, or at all phases of the cell cycle, and associated with reductions in cyclin D1, CDK4 and pRb. Combination studies showed the anti-proliferative effect of ATM was often enhanced by addition of the other drugs, whilst ART exhibited antagonistic properties. ART and ATM are active in cancer cell lines, with ATM displaying the greater anti-proliferative effect when used alone. ATM also enhances the effects of the above drugs, with ART being less likely to improve activities. Taken together, ATM should be thought of as the ART-derived compound next in line for further study.

Meeting abstracts Chemotherapy and Immunotherapy were always considered incompatible in the treatment of cancer. However, analysis of sequential use has revealed that there may be significant additive potential if not synergistic, in enhancing the immune response to cancer. There are many reasons