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

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<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>

<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>

In order to delineate the underlying molecular mechanisms responsible for intra tumoral enrichment of BCSCs in aggressive breast tumors, firstly we evaluated the frequency and characteristics of breast cancer stem cells (BCSCs) within the tumor mass as well as in pathologically normal adjacent tissues in primary breast carcinomas of various clinical and histological grades. Then, we evaluated the expression profiles of various genes in non-cancer stem cells from these tumors to delineate the role played by cellular niche in de novo origin and/or expansion of intra-tumoral cancer stem cells.The study included primary tumor and adjacent normal breast tissue specimens from chemotherapy-naïve breast carcinoma patients. The BCSCs, identified as Lin-CD44+CD24- and aldehyde dehydrogenase 1 A1 positive were enumerated. The frequency of intra-tumoral BCSCs was correlated with various clinicopathological parameters of breast cancer. The flow-cytometrically sorted stromal cells and cancer cells from treatment naïve primary breast tumors were processed for gene expression profiling using a custom designed PCR array of genes known to facilitate cancer cell proliferation and disease progression.The frequency of BCSCs within the tumor mass as well as in the adjacent normal tissue correlated significantly with histopathological and molecular grades of tumors indicating a direct relationship of BCSC with aggressive behavior of breast cancer. A significantly higher number of BCSCs was also detected in metastatic LN group as compared to non-metastatic LN. Further, a significantly increased expression of the genes associated with growth factors, cytokines & matricellular proteins in tumors with high BCSCs content (> 5%; Hi-BCSCs tumors) as compared to Lo-BCSC tumors (with <5% intratumoral BCSC content) suggested the possible contribution of stromal cells and cancer cells in intra-tumoral expansion of CSCs. Similarly, a significant up-regulation of genes associated with hypoxia and angiogenesis in Hi-BCSCs tumors further supported the role of hypoxic environment. The expression levels of genes associated with epithelial to mesenchymal transition also followed a similar pattern. On the other hand, downregulated SNAI1 gene (generally upregulated in onset of EMT) in stromal cells of Hi-BCSCs tumors suggests a post EMT environment in Hi-BCSCs tumors.The findings suggest that the molecular crosstalk between the non-BCSC niche cells and the cancer stem cells within the breast cancer microenvironment directly contribute to formation of biologically conducive conditions for expansion of cancer stem cells.

Purpose In-vivo CXCR4 receptor quantification in different lung cancer (LC) sub-types using 68Ga-Pentixafor PET/CT and correlation with quantitative CXCR4-receptors’ tissue density by immunochemistry analyses. Methods 68Ga-Pentixafor PET/CT imaging was performed prospectively in 94 (77M: 17F, mean age 60.15 ± 10.12 yrs) LC patients. CXCR4 receptors’ expression was estimated in all the patients on lung tissue by immunohistochemistry (IHC) and FACS analyses. SUVmax on PET, Intensity score on IHC and Mean fluorescence Index (MFI) on FACS analyses were measured. Results 75/94 (79.8%) cases had NSCLC, 14 (14.9%) had SCLC and 5 (5.3%) had lung NETs. All LC types showed increased CXCR4 expression on PET (SUVmax) and FACS (MFI). However, both these parameters (mean SUVmax = 10.30 ± 5.0; mean MFI = 349.0 ± 99.0) were significantly (p = 0.005) higher in SCLC as compared to NSCLC and lung NETs. PET SUVmax in adenocarcinoma (n = 16) were 8.00 ± 1.9 which was significantly (p = 0.003) higher than in squamous cell carcinoma (n = 54; 6.2 ± 2.15) and NOS (n = 5; 5.8 ± 1.5) subtypes of NSCLC. A significant correlation (r = 0.697; p = 001) was seen between SUVmax and MFI values in squamous cell NSCLC as well as in NSCLC-adenocarcinoma (r = 0.538, p = 0.031) which supports the specific uptake of 68Ga-Pentixafor by CXCR4 receptors. However, this correlation was not significant in SCLC (r = 0.435, p = 0.121) and NET (r = 0.747,p = 0.147) which may be due to the small sample size. 68Ga-Pentixafor PET/CT provided good sensitivity (85.7%) and specificity (78.1%) for differentiating SCLC from NSCLC (ROC cut-off SUVmax= 7.24). Almost similar sensitivity (87.5%) and specificity (71.4%) were observed (ROC cut-off SUVmax= 6.67) for differentiating adenocarcinoma and squamous cell variants of NSCLC. Conclusion Higher CXCR4 expression was seen in SCLC as compared to NSCLC and NETs on 68Ga-Pentixafor PET imaging. The findings may potentially supplement the existing data for inclusion and expanding CXCR4 -based radioligand therapies in LC beyond haematological malignancies.