Autophagy-deficient cancers don’t release ATP and fail to recruit immune cells to tumor site.

Autophagy of chemotherapy-treated dying cancer cells is necessary for triggering additional anticancer immune responses against remaining tumor cells, researchers claim.  In vitro and in vivo studies have shown that autophagy is dispensable for chemotherapy-induced cell death but is required to induce subsequent immunogenicity.

In effect, the French team led by scientists at INSERM Unit 848 and the Institute Gustave Roussy claim, suppressing autophagy inhibits the release of ATP from dying tumor cells. This lack of ATP release prevents the recruitment of anticancer immune cells to the tumor.

Subsequent experiments by Guido Kroemer, Ph.D., and Mikaël Michaud, Ph.D., et al., confirmed that boosting extracellular ATP levels in autophagy-deficient tumors effetively reestablished the recruitment of immune cells and restored chemotherapeutic immune responses in immunocompetent animals. The work is reported in Science in a paper titled “Autophagy-Dependent Anticancer Immune Responses Induced by Chemotherapeutic Agents in Mice.”

Studies have previously indicated that chemotherapy can induce a type of tumor cell stress and death that is immunogenic, which suggests that the patient’s dying cancer cells can act as a vaccine that stimulates an antitumor immune response against residual cancer cells, the authors explain. Dying cancer cells display calreticulin (CRT) on their cell surfaces, secrete ATP, and as a result of apoptosis release chromatin-binding protein high mobility group B1 (HMGB1). These factors interact with receptors on the surface of dendritic cells (DCs), triggering engulfment of the dying cells, and the presentation of tumor antigen and production of interleukin-1Β.

While previous research has found that CRT exposure results from a chemotherapy-induced endoplasmic stress response and HMGB1 release occurs following postapoptotic necrosis, the chemotherapy-related mechanism responsible for inducing ATP release has to date not been identified. The authors postulated whether autophagy might play a role in immunogenic signaling.

To investigate this more closely they first studied authophagy-deficient CT26 cells (Atg5KD or Atg7KD) by mass spectrometry and found that after treatment with mitoxantrone (MTX), the supernatants of these cells contained much less ATP than supernatants of MTX-treated autophagy-competent CT6 cells. Knocking down essential autophagy proteins such as Atg5 or Atg7 in previously autophagy-competent CT6 cells also led to the release of less ATP after MTX  or oxaliplatin therapy.  Importantly, however, both autophagy-deficient and autophagy-competent cancer cells demonstrated similar exposure of CRT and HMGB1 release in response to chemotherapy.

These results were replicated in mouse embryonic fibroblasts and in human osteosarcoma cells in which autophagy-essential genes were knocked down. In fact, in human osteosarcoma cells engineered to express a GFP-tagged LC3 (a marker of autophagosomes) fusion protein, the depletion of several Atg gene products significantly decreased the MTX-driven accumulation of autophagosomes as well as ATP release, whereas gene deletion didn’t reduce HMGB1 release or cell membrane CRT exposure. “Thus, autophagy is required for optimal ATP release but dispensable for the emission of other immunogenic signals,” the authors state.

Treating CT26 tumors with MTX led to the autophagic aggregation of a GFP-LCR reporter within 48 hours of chemotherapy, which indicated that anticancer treatment induces autophagy in vivo. Reporter tagging studies also indicated that the treated tumors released ATP release, whereas Atg5KD or Atg7KD knockout tumors didn’t release ATP.

The authors next injected mice with autophagy-competent CT26 cells [class I major histocompatibility complex (MHC) haplotype H-2d] that had been treated in vitro using MTX to induce apoptosis, without adjuvant. The cells effectively triggered a protective anticancer immune response and immunized syngenic BALB/c mice against subsequent inoculation with live tumor cells of the same type.

In contrast, while autophagy-incompetent cells treated with MTX in vitro also underwent apoptosis, when they were injected in mice they failed to induce antitumor immunity in recipient animals. These findings were confirmed using a second cancer cell type in a different strain of mouse.

Notably, this failure of MTX-treated autophagy-incompetent cells to generate a protective immune response in vivo could be reversed by co-injecting animals with either the apyrase inhibitor ARL67156 (ARL), which artificially increases extracellular ATP concentrations, or with recombinant IL-1β.

In vivo, MTX-treated autophagy-deficient tumors exhibited an apoptotic response to systemic MTX treatment. In vivo, they failed to attract DCs or elicit a local immune response characterized by CD4+ or CD8+ cells. This could be remedied by injecting the tumors directly with ARL or another apyrase inhibitor, NGXT191.

Indeed, the researchers state, “although ARL did not increase the cytotoxic potential of MTX, ARL injections restored the efficacy of MTX-based chemotherapy against autophagy-deficient CT26- or MCA205-derived cancers, hence significantly reducing tumor growth.” These findings were also replicated in mice carrying autophagy-deficient MCA205 fibrosarcomas treated using oxaliplatin: The cancers failed to respond to therapy unless the treatment was accompanied by ARL injections. 

“Together, these results, which have been obtained with immunogenic cancers, indicate that restoring extracellular ATP concentrations suffices to reestablish the chemotherapeutic response of autophagy-deficient tumors,” the authors conclude. “If our findings could be translated to humans, patients with autophagy-deficient cancers might profit from pharmacological strategies designed to compensate defective immunogenic signaling. Beyond the development of cancer-specific autophagy inducers, one such compensatory strategy might be to enhance local ATP concentrations through the application of apyrase inhibitors.”

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