Fig 1: Enpp1 and NET in human TNBC. A,In silico analysis of the prognostic value of gene-expression levels using the Kaplan–Meier plotter (www.kmplot.com) of ENPP1 (Affy ID: 229088_at) and HP (Affy ID: 206697_s_at) in TNBC. Differences between groups were evaluated using the log-rank test. B, Left, quantification of ENPP1 immunostaining in a TMA of human breast cancer primary and relapsed breast tumors. Nonparametric Wilcoxon paired sample test was used for comparison. Right, representative images. C, Representative images of NET evaluation by multispectral imaging with an automated quantitative pathology imaging system (Vectra Polaris) in primary nontreated breast cancer tumors and their paired relapsed TNBC tumors from formalin-fixed, paraffin-embedded tissue sections of a TMA. Representative composite images immunostained with CD15 (clone Carb-3, blue), MPO (clone C8/144B, red), anticitrullinated histone (H3Cit; green, citrulline R2 + R8 + R17), cytokeratin (wide spectrum screening, cyan), and DAPI (gray). NET are costained with CD15, MPO, and H3Cit. NET are visualized as white structures in the full composite color images. Square is magnified in the bottom plots. Top: scale bar, 100 µm. Bottom: scale bar, 50 µm. D, Segmented fluorescence images for CD15, MPO, and H3Cit for magnified squares shown in C, showing coimmune localization of three markers, indicating bona fide NET. Scale bar, 50 µm. E, Quantification of the NET area of the multispectral images obtained. Nonparametric Wilcoxon paired sample test was used for comparison. F, Schematic of the mechanism of LRF at the cellular and molecular levels. Top, primary breast tumors release CTC. After surgery, the RTB constitutes a fertile soil for CTC. Enpp1hi CTC are more competent to engraft and efficiently colonize the RTB. Enpp1hi cells promote the intrusion of PMN-MDSC and strong immunosuppressed microenvironment in the recurrent tumor. PMN-MDSC create NET, which also abolish the immune attack. Bottom, at the molecular level, ENPP1 hydrolyzes ATP into AMP and pyrophosphate (PPi). Similarly, CD39 in the microenvironment hydrolyzes ATP to AMP. AMP is hydrolyzed to adenosine (Ado) by CD73 in the adenosinergic pathway (CD39–CD73). The accumulation of AMP in the TME induces the secretion of HP through P1 purinergic receptors. Hp and other components act as chemoattractant factors for PMN-MDSC that infiltrate and contribute to the incipient tumor colonization by an immunosuppressive TME from early stages of tumor formation. This also leads to immune landscape remodeling by impairing NK and CD8+ T-cell infiltration. Secretion of HP also induces NET formation, which further promotes an immunosuppressive microenvironment. Radiation obliterates the early engraftment of CTC. ENPP1 inhibition decreases the extracellular AMP and Ado levels and decreases HP with an enhancement of an immunocompetent microenvironment. ENPP1 blockade radiosensitizes the tumor, triggers the infiltration of CD8 and NK, and decreases the infiltration of Tregs and myeloid suppressor cells. In this scenario, tumor cells are less competent to colonize the RTB. Subsequent fractionated irradiation contributes to eradicate engrafted tumor cells, preventing locoregional failure, but does not prevent the subsequent homing of CTC post-IR.