Metastasis and immune evasion from extracellular cGAMP hydrolysis

Cytosolic DNA is characteristic of metastatic and chromosomally unstable cancer cells and it results in constitutive activation of the cGAS-STING innate immune pathway. How cancer cells co-opt inflammatory signaling while evading immune surveillance remains unknown. Here we show that the ectonucleotidase ENPP1 promotes metastasis by selectively de-grading extracellular cGAMP, an immune stimulatory metabolite whose breakdown products include the immune sup-pressor, adenosine. ENPP1 depletion restores tumor immune infiltration, suppresses metastasis, and potentiates tumor response to immune checkpoint blockade (ICB) therapy in tumor cell cGAS and host STING-dependent manners. Con-versely, ENPP1 overexpression renders otherwise sensitive tumors completely resistant to ICB. In human cancers, ENPP1 expression correlates with reduced immune cell infiltration, increased metastasis, and resistance to anti-PD1/PD-L1 therapy. Thus, cGAMP hydrolysis by ENPP1 enables metastatic cancer cells to transmute an immune stimulatory pathway into an immune suppressive mechanism and it represents a therapeutic vulnerability in tumors with pervasive chromosomal instability. reported (39). Immunohistochemistry for ENPP1 in breast cancer cohort 1 was performed on the automated Discovery XT processor (Ventana Medical Systems) by the Molecular Cytology Core Facility at MSKCC. Briefly, after deparaffinized and tumor tissue conditioning, the antigen was retrieved using standard CC1 (Ventana Medical Systems). Following blockage with Background Buster (Inno-vex), the slides were incubated with 2.5 µ g/ml anti-ENPP1 antibody for 5 hr, and then incubated with the biotinylated secondary antibody for 60 minutes. The Streptavidin-HRP D (DABMap kit, Ventana Medical Systems) and the DAB detection kit (Ventana Medical Systems) were used to detect the signal according to the manufacturer instructions. Then the slides were counterstained with hematoxylin and were mounted with Permount mounting medium. Slides of immunofluorescence and immunohistochemistry were scanned with Pannoramic Flash 250 (3DHistech, Budapest, Hungary) with 20x/0.8 NA air objective by the Molecular Cytology Core Facility at MSKCC. ENPP1 protein expression levels were performed by a board-certified breast pathologist who was blinded to other clinicopathological characteristics and outcome. ENPP1 protein expression levels were assessed manually using scores of 0 (absent), 1 (weak), 2 (moderate) and 3 (strong) for both stromal and tumor compartments. Given this analysis was performed on small core material, ENPP1 expression was considered when >1% of cells showed a given staining pattern. Distant metastasis-free survival data were collected by reviewing medical records available at MSKCC. TILs were scored according to the recommendations of the international TILs working group (40) based on the original hematoxylin and eosin-stained sections corresponding to each of the tumors present in the TMA. Tumors were stratified as having low (negative or weak) or high (moderate or strong) ENPP1 expression. Independent validation studies were performed on a tissue microarray of n = 91 estrogen receptor (ER) negative (Cohort 2) and n= 115 ER positive (Cohort 3) Dako dilution retrieval, for EPR22262-72, ab24538, Abcam at 1:1000 dilution. were scanned on an Aperio AT2 Digital scanner at 40x. CD8+ T cell infiltration was reported as CD8+ cell density per mm 2 based on the total number of cells in each core and determined using the open-source digital pathological analysis software QuPath v0.1. 2 (43,44). Cores with < 100 tumor cells were removed from analysis and multiple core data were averaged. Rigorous quality control steps were taken to remove necrosis or keratin, tissue folds and entrapped normal structures; this was confirmed by a second reviewer with frequent consultation following an established method. ENPP1 protein expression levels were assessed manually using scores of 0 (absent), 1 (weak), 2 (mod-erate) and 3 (strong) for both stromal and tumor compartments as described above. Both analyses were performed blinded to other clinicopathological characteristics and outcome. Survival analysis was restricted to tumors with low nodal disease burden (N0-1). For OS analysis, ER- tumors were stratified as either positive (n= 59) or negative (n = 15) for ENPP1 staining.