Antineoplastic chemotherapies are particularly efficient when they elicit immunogenic cell death, thus provoking an anticancer immune response. Here we demonstrate that autophagy, which is often disabled in cancer, is dispensable for chemotherapy-induced cell death but required for its immunogenicity. In response to chemotherapy, autophagy-competent, but not autophagy-deficient, cancers attracted dendritic cells and T lymphocytes into the tumor bed. Suppression of autophagy inhibited the release of adenosine triphosphate (ATP) from dying tumor cells. Conversely, inhibition of extracellular ATP-degrading enzymes increased pericellular ATP in autophagy-deficient tumors, reestablished the recruitment of immune cells, and restored chemotherapeutic responses but only in immunocompetent hosts. Thus, autophagy is essential for the immunogenic release of ATP from dying cells, and increased extracellular ATP concentrations improve the efficacy of antineoplastic chemotherapies when autophagy is disabled.

BackgroundThere is growing awareness that tumour cells build up a “self-advantageous” microenvironment that reduces effectiveness of anti-tumour immune response. While many different immunosuppressive mechanisms are likely to come into play, recent evidence suggests that extracellular adenosine acting at A2A receptors may have a major role in down-modulating the immune response as cancerous tissues contain elevated levels of adenosine and adenosine break-down products. While there is no doubt that all cells possess plasma membrane adenosine transporters that mediate adenosine uptake and may also allow its release, it is now clear that most of extracellularly-generated adenosine originates from the catabolism of extracellular ATP.Methodology/Principal FindingsMeasurement of extracellular ATP is generally performed in cell supernatants by HPLC or soluble luciferin-luciferase assay, thus it generally turns out to be laborious and inaccurate. We have engineered a chimeric plasma membrane-targeted luciferase that allows in vivo real-time imaging of extracellular ATP. With this novel probe we have measured the ATP concentration within the tumour microenvironment of several experimentally-induced tumours.Conclusions/SignificanceOur results show that ATP in the tumour interstitium is in the hundrends micromolar range, while it is basically undetectable in healthy tissues. Here we show that a chimeric plasma membrane-targeted luciferase allows in vivo detection of high extracellular ATP concentration at tumour sites. On the contrary, tumour-free tissues show undetectable extracellular ATP levels. Extracellular ATP may be crucial for the tumour not only as a stimulus for growth but also as a source of an immunosuppressive agent such as adenosine. Our approach offers a new tool for the investigation of the biochemical composition of tumour milieu and for development of novel therapies based on the modulation of extracellular purine-based signalling.

Danger signals released upon cell damage can cause excessive immune-mediated tissue destruction such as that found in acute graft-versus-host disease (GVHD), allograft rejection and systemic inflammatory response syndrome. Given that ATP is found in small concentrations in the extracellular space under physiological conditions, and its receptor P2X(7)R is expressed on several immune cell types, ATP could function as a danger signal when released from dying cells. We observed increased ATP concentrations in the peritoneal fluid after total body irradiation, and during the development of GVHD in mice and in humans. Stimulation of antigen-presenting cells (APCs) with ATP led to increased expression of CD80 and CD86 in vitro and in vivo and actuated a cascade of proinflammatory events, including signal transducer and activator of transcription-1 (STAT1) phosphorylation, interferon-γ (IFN-γ) production and donor T cell expansion, whereas regulatory T cell numbers were reduced. P2X(7)R expression increased when GVHD evolved, rendering APCs more responsive to the detrimental effects of ATP, thereby providing positive feedback signals. ATP neutralization, early P2X(7)R blockade or genetic deficiency of P2X(7)R during GVHD development improved survival without immune paralysis. These data have major implications for transplantation medicine, as pharmacological interference with danger signals that act via P2X(7)R could lead to the development of tolerance without the need for intensive immunosuppression.

P2X7 is the largest member of the P2X subfamily of purinergic receptors. A typical feature is the carboxyl tail, which allows formation of a large pore. Recently a naturally occurring truncated P2X7 splice variant, isoform B (P2X7B), has been identified. Here we show that P2X7B expression in HEK293 cells, a cell type lacking endogenous P2X receptors, mediated ATP-stimulated channel activity but not plasma membrane permeabilization, raised endoplasmic reticulum Ca(2+) content, activated the transcription factor NFATc1, increased the cellular ATP content, and stimulated growth. In addition, P2X7B-transfected HEK293 cells (HEK293-P2X7B), like most tumor cells, showed strong soft agar-infiltrating ability. When coexpressed with full-length P2X7 (P2X7A), P2X7B coassembled with P2X7A into a heterotrimer and potentiated all known responses mediated by this latter receptor. P2X7B mRNA was found to be widely distributed in human tissues, especially in the immune and nervous systems, and to a much higher level than P2X7A. Finally, P2X7B expression was increased on mitogenic stimulation of peripheral blood lymphocyte. Altogether, these data show that P2X7B is widely expressed in several human tissues, modulates P2X7A functions, participates in the control of cell growth, and may help understand the role of the P2X7 receptor in the control of normal and cancer cell proliferation.

ATP is emerging as an ubiquitous extracellular messenger. However, measurement of ATP concentrations in the pericellular space is problematic. To this aim, we have engineered a firefly luciferase-folate receptor chimeric protein that retains the N-terminal leader sequence and the C-terminal GPI anchor of the folate receptor. This chimeric protein, named plasma membrane luciferase (pmeLUC), is targeted and localized to the outer aspect of the plasma membrane. PmeLUC is sensitive to ATP in the low micromolar to millimolar level and is insensitive to all other nucleotides. To identify pathways for nonlytic ATP release, we transfected pmeLUC into cells expressing the recombinant or native P2X7 receptor (P2X7R). Both cell types release large amounts of ATP (100-200 microM) in response to P2X7R activation. This novel approach unveils a hitherto unsuspected nonlytic pathway for the release of large amounts of ATP that might contribute to spreading activation and recruitment of immune cells at inflammatory sites.