Cytosolic DNA is characteristic of chromosomally unstable metastatic cancer cells, resulting in constitutive activation of the cGAS–STING innate immune pathway. How tumors co-opt inflammatory signaling while evading immune surveillance remains unknown. Here, we show that the ectonucleotidase ENPP1 promotes metastasis by selectively degrading extracellular cGAMP, an immune-stimulatory metabolite whose breakdown products include the immune suppressor adenosine. ENPP1 loss suppresses metastasis, restores tumor immune infiltration, and potentiates response to immune checkpoint blockade in a manner dependent on tumor cGAS and host STING. Conversely, overexpression of wild-type ENPP1, but not an enzymatically weakened mutant, promotes migration and metastasis, in part through the generation of extracellular adenosine, and renders otherwise sensitive tumors completely resistant to immunotherapy. In human cancers, ENPP1 expression correlates with reduced immune cell infiltration, increased metastasis, and resistance to anti–PD-1/PD-L1 treatment. Thus, cGAMP hydrolysis by ENPP1 enables chromosomally unstable tumors to transmute cGAS activation into an immune-suppressive pathway. Significance: Chromosomal instability promotes metastasis by generating chronic tumor inflammation. ENPP1 facilitates metastasis and enables tumor cells to tolerate inflammation by hydrolyzing the immunotransmitter cGAMP, preventing its transfer from cancer cells to immune cells. This article is highlighted in the In This Issue feature, p. 995

Murine tissues harbor signature γδ T cell compartments with profound yet differential impacts on carcinogenesis. Conversely, human tissue-resident γδ cells are less well defined. In the present study, we show that human lung tissues harbor a resident Vδ1 γδ T cell population. Moreover, we demonstrate that Vδ1 T cells with resident memory and effector memory phenotypes were enriched in lung tumors compared with nontumor lung tissues. Intratumoral Vδ1 T cells possessed stem-like features and were skewed toward cytolysis and helper T cell type 1 function, akin to intratumoral natural killer and CD8+ T cells considered beneficial to the patient. Indeed, ongoing remission post-surgery was significantly associated with the numbers of CD45RA−CD27− effector memory Vδ1 T cells in tumors and, most strikingly, with the numbers of CD103+ tissue-resident Vδ1 T cells in nonmalignant lung tissues. Our findings offer basic insights into human body surface immunology that collectively support integrating Vδ1 T cell biology into immunotherapeutic strategies for nonsmall cell lung cancer.

Murine tissues harbour signature, resident γδ T cell compartments with profound yet differential impacts on carcinogenesis. γδ T cell knockout mice have heightened susceptibility to carcinogenesis. However, it is now clear in the murine setting that IFNγ-producing γδ T cells reject tumours whilst IL-17-producing γδ T cells promote them. Nonetheless, many human γδ T cell compartments are distinct from that in mice and vice versa. The extent to which human tissues and tumours harbour resident γδ T cells, their effector function and their role in cancer is less clear. Although a large scale in silico study of 5000+ patients with cancer found that intratumoural γδ T cells were the most important correlate of survival, smaller studies have found that these cells to be associated with either survival or progression. Many historical studies have however been limited by the availability of technologies to rigorously identify, isolate, and examine tissue-resident γδ T cells. To address these issues, we present data from stage I-III non-small cell lung cancers and paired non-tumour (NT) tissue obtained at primary surgery from 25 patients enrolled in the TRACERx Study. Using flow cytometry and quantitative T cell receptor sequencing, we demonstrate that NT lung tissues harbour a resident population of Vδ1 γδ T cells, entirely distinct to blood. Compared with NT lung tissues, resident-memory and effector-memory Vδ1 T cells are enriched in tumours. RNA sequencing revealed that intratumoural Vδ1 T cells are skewed towards cytolysis and T-helper-1 functions, akin to intratumoural NK and CD8+ T cells. Importantly, we found no evidence of T-helper-17 skew that has been implicated in tumour promotion in murine models. Ongoing remission after surgery was significantly associated with the presence of CD103+ tissue-resident Vδ1 T cells in non-malignant lung tissues and the presence of CD45RA-/CD27+ effector-memory Vδ1 T cells in tumours. Moreover, patients with a greater proportion of intratumoural Vδ1 T cell clones shared with paired NT tissues were more likely to remain in remission, consistent with the cells’ proposed immunosurveillance function in steady state epithelial tissues. Whilst immunotherapies modulating αβ T cells have been successful for some patients, including those with non-small cell lung cancer, clinical trials of γδ T cells have so far demonstrated poor efficacy in solid cancers. These trials have hitherto exclusively utilised Vδ2 T cells, a subset which is found predominantly in peripheral blood and more commonly associated, albeit still rarely, with IL-17 production. The first-in-human clinical trial of Vδ1 T cell immunotherapy has just opened for patients with acute myeloid leukaemia. Thus, our findings have immediate translational relevance and support the utilisation of these as-yet-untapped Vδ1 T cells in solid cancer immunotherapy. Citation Format: Yin Wu, Dhruva Biswas, Ieva Usaite, Angelova Mihaela, Stefan Boeing, Takahiro Karasaki, Selvaraju Veeriah, Justyna Czyzewska-Khan, James Reading, Andrew Georgiou, Maise Al-Bakir, Nicholas McGranahan, Mariam Jamal-Hanjani, Allan Hackshaw, TRACERx Consortium, Sergio Quezada, Adrian Hayday, Charles Swanton. V-delta-1 T cells are resident in the human lung and associate with survival in patients with non-small cell lung cancer in the TRACERx Study [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 5636.

<div>Abstract<p>Cytosolic DNA is characteristic of chromosomally unstable metastatic cancer cells, resulting in constitutive activation of the cGAS–STING innate immune pathway. How tumors co-opt inflammatory signaling while evading immune surveillance remains unknown. Here, we show that the ectonucleotidase ENPP1 promotes metastasis by selectively degrading extracellular cGAMP, an immune-stimulatory metabolite whose breakdown products include the immune suppressor adenosine. ENPP1 loss suppresses metastasis, restores tumor immune infiltration, and potentiates response to immune checkpoint blockade in a manner dependent on tumor cGAS and host STING. Conversely, overexpression of wild-type ENPP1, but not an enzymatically weakened mutant, promotes migration and metastasis, in part through the generation of extracellular adenosine, and renders otherwise sensitive tumors completely resistant to immunotherapy. In human cancers, ENPP1 expression correlates with reduced immune cell infiltration, increased metastasis, and resistance to anti–PD-1/PD-L1 treatment. Thus, cGAMP hydrolysis by ENPP1 enables chromosomally unstable tumors to transmute cGAS activation into an immune-suppressive pathway.</p>Significance:<p>Chromosomal instability promotes metastasis by generating chronic tumor inflammation. ENPP1 facilitates metastasis and enables tumor cells to tolerate inflammation by hydrolyzing the immunotransmitter cGAMP, preventing its transfer from cancer cells to immune cells.</p><p><i>This article is highlighted in the In This Issue feature, p. 995</i></p></div>

<p>Supplementary Figures and Methods in PDF format</p>

<p>Supplementary Figures and Methods in PDF format</p>

<div>Abstract<p>Cytosolic DNA is characteristic of chromosomally unstable metastatic cancer cells, resulting in constitutive activation of the cGAS–STING innate immune pathway. How tumors co-opt inflammatory signaling while evading immune surveillance remains unknown. Here, we show that the ectonucleotidase ENPP1 promotes metastasis by selectively degrading extracellular cGAMP, an immune-stimulatory metabolite whose breakdown products include the immune suppressor adenosine. ENPP1 loss suppresses metastasis, restores tumor immune infiltration, and potentiates response to immune checkpoint blockade in a manner dependent on tumor cGAS and host STING. Conversely, overexpression of wild-type ENPP1, but not an enzymatically weakened mutant, promotes migration and metastasis, in part through the generation of extracellular adenosine, and renders otherwise sensitive tumors completely resistant to immunotherapy. In human cancers, ENPP1 expression correlates with reduced immune cell infiltration, increased metastasis, and resistance to anti–PD-1/PD-L1 treatment. Thus, cGAMP hydrolysis by ENPP1 enables chromosomally unstable tumors to transmute cGAS activation into an immune-suppressive pathway.</p>Significance:<p>Chromosomal instability promotes metastasis by generating chronic tumor inflammation. ENPP1 facilitates metastasis and enables tumor cells to tolerate inflammation by hydrolyzing the immunotransmitter cGAMP, preventing its transfer from cancer cells to immune cells.</p><p><i>This article is highlighted in the In This Issue feature, p. 995</i></p></div>