Summary Tumor endothelial cells (ECs) promote cancer progression in ways beyond their role as conduits supporting metabolism. However, it is not understood how vascular niche-derived paracrine factors, known as angiocrine factors, provoke tumor aggressiveness. Here, we show that FGF4 produced by B-Cell lymphoma cells (LCs) through activating FGFR1 upregulates the Notch-ligand Jagged1 (Jag1) on neighboring tumor ECs. In turn, upregulation of Jag1 on ECs reciprocally induces Notch2-Hey1 in LCs. This crosstalk enforces aggressive CD44+IGF1R+CSF1R+ LC phenotypes, including extra-nodal invasion and chemoresistance. Inducible EC-selective deletion of Fgfr1 or Jag1 in the Eμ-Myc lymphoma model or impairing Notch2 signaling in mouse and human LCs diminished lymphoma aggressiveness and prolonged mouse survival. Thus, targeting the angiocrine FGF4-FGFR1/Jag1-Notch2 loop could inhibit LC aggressiveness and enhance chemosensitivity.

Purpose: Bone marrow-derived progenitor cells, including VEGFR2þ endothelial progenitor cells (EPCs) and copper-dependent pathways, model the tumor microenvironment. We hypothesized that copper depletion using tetrathiomolybdate would reduce EPCs in high risk for patients with breast cancer who have relapsed. We investigated the effect of tetrathiomolybdate on the tumor microenvironment in preclinical models. Experimental Design: Patients with stage II triple-negative breast cancer (TNBC), stage III and stage IV without any evidence of disease (NED), received oral tetrathiomolybdate to maintain ceruloplasmin (Cp) between 8 and 17 mg/dL for 2 years or until relapse. Endpoints were effect on EPCs and other biomarkers, safety, event-free (EFS), and overall survival (OS). For laboratory studies, MDA-LM2-luciferase cells were implanted into CB17-SCID mice and treated with tetrathiomolybdate or water. Tumor progression was quantified by bioluminescence imaging (BLI), copper depletion status by Cp oxidase levels, lysyl oxidase (LOX) activity by ELISA, and collagen deposition.Results: Seventy-five patients enrolled; 51 patients completed 2 years (1,396 cycles). Most common grade 3/4 toxicity was neutropenia (3.7%). Lower Cp levels correlated with reduced EPCs (P ¼ 0.002) and LOXL-2 (P < 0.001). Two-year EFS for patients with stage II-III and stage IV NED was 91% and 67%, respectively. For patients with TNBC, EFS was 90% (adjuvant patients) and 50% (stage IV NED patients) at a median follow-up of 6.3 years, respectively. In preclinical models, tetrathiomolybdate decreased metastases to lungs (P ¼ 0.04), LOX activity (P ¼ 0.03), and collagen crosslinking (P ¼ 0.012).Conclusions: Tetrathiomolybdate is safe, well tolerated, and affects copper-dependent components of the tumor microenvironment. Biomarker-driven clinical trials in high risk for patients with recurrent breast cancer are warranted.

Emerging studies have begun to demonstrate that reprogrammed stromal cells play pivotal roles in tumor growth, metastasis, and resistance to therapy. However, the contribution of stromal cells to non-small-cell lung cancer (NSCLC) has remained underexplored. We used an orthotopic model of Kras-driven NSCLC to systematically dissect the contribution of specific hematopoietic stromal cells in lung cancer. RNA deep-sequencing analysis of individually sorted myeloid lineage and tumor epithelial cells revealed cell-type-specific differentially regulated genes, indicative of activated stroma. We developed a computational model for crosstalk signaling discovery based on ligand-receptor interactions and downstream signaling networks and identified known and novel tumor-stroma paracrine and tumor autocrine crosstalk-signaling pathways in NSCLC. We provide cellular and molecular insights into components of the lung cancer microenvironment that contribute to carcinogenesis. This study has the potential for development of therapeutic strategies that target tumor-stroma interactions and may complement conventional anti-cancer treatments.

Metastatic tumors have been shown to establish permissive microenvironments for metastases via recruitment of bone marrow (BM)- derived cells. Here, we show that metastasis-incompetent tumors are also capable of generating such microenvironments. However, in these situations the otherwise pro-metastatic Gr1+ myeloid cells create a metastasis-refractory microenvironment via the induction of thrombospondin-1 (Tsp-1) by tumor-secreted prosaposin. (BM)-specific genetic deletion of Tsp-1 abolished the inhibition of metastasis, which was restored by BM transplant from Tsp-1+ donors. We also developed a 5-amino acid peptide from prosaposin as a pharmacological inducer of Tsp-1 in Gr1+ BM cells, which dramatically suppresses metastasis. These results provide mechanistic insights into why certain tumors are deficient in metastatic potential and implicate recruited Gr1+ myeloid cells as the main source of Tsp-1. The results underscore the plasticity of Gr1+ cells, which, depending on the context, promote or inhibit metastasis, and suggest that the peptide could be a potential therapeutic agent against metastatic cancer.

Copper serves as a co-factor for a host of metalloenzymes that contribute to malignant progression. The orally bioavailable copper chelating agent tetrathiomolybdate (TM) has been associated with a significant survival benefit in high-risk triple negative breast cancer (TNBC) patients. Despite these promising data, the mechanisms by which copper depletion impacts metastasis are poorly understood and this remains a major barrier to advancing TM to a randomized phase II trial. Here, using two independent TNBC models, we report a discrete subpopulation of highly metastatic SOX2/OCT4+ cells within primary tumors that exhibit elevated intracellular copper levels and a marked sensitivity to TM. Global proteomic and metabolomic profiling identifies TM-mediated inactivation of Complex IV as the primary metabolic defect in the SOX2/OCT4+ cell population. We also identify AMPK/mTORC1 energy sensor as an important downstream pathway and show that AMPK inhibition rescues TM-mediated loss of invasion. Furthermore, loss of the mitochondria-specific copper chaperone, COX17, restricts copper deficiency to mitochondria and phenocopies TM-mediated alterations. These findings identify a copper-metabolism-metastasis axis with potential to enrich patient populations in next-generation therapeutic trials.

Highlights d Blockade of EZH2 catalytic activity reduces dissemination and metastasis d EZH2 high tumor subpopulation exhibits enhanced metastatic potential d EZH2-mediated GATA3 suppression maintains EZH2 high metastatic cells d TNBC subtypes exhibit differential sensitivity to EZH2 therapy

Success of immune checkpoint inhibitors in advanced non-small-cell lung cancer (NSCLC) has invigorated their use in the neoadjuvant setting for early-stage disease. However, the cellular and molecular mechanisms of the early immune responses to therapy remain poorly understood. Through an integrated analysis of early-stage NSCLC patients and a Kras mutant mouse model, we show a prevalent programmed cell death 1/programmed cell death 1 ligand 1 (PD-1/PD-L1) axis exemplified by increased intratumoral PD-1+ T cells and PD-L1 expression. Notably, tumor progression was associated with spatiotemporal modulation of the immune microenvironment with dominant immunosuppressive phenotypes at later phases of tumor growth. Importantly, PD-1 inhibition controlled tumor growth, improved overall survival, and reprogrammed tumor-associated lymphoid and myeloid cells. Depletion of T lymphocyte subsets demonstrated synergistic effects of those populations on PD-1 inhibition of tumor growth. Transcriptome analyses revealed T cell subset-specific alterations corresponding to degree of response to the treatment. These results provide insights into temporal evolution of the phenotypic effects of PD-1/PD-L1 activation and inhibition and motivate targeting of this axis early in lung cancer progression.

◥ Metastases are responsible for the majority of breast cancerassociated deaths. The contribution of epithelial-to-mesenchymal transition (EMT) in the establishment of metastases is still controversial. To obtain in vivo evidence of EMT in metastasis, we established an EMT lineage tracing (Tri-PyMT) model, in which tumor cells undergoing EMT would irreversibly switch their fluorescent marker from RFP þ to GFP þ due to mesenchymal-specific Cre expression. Surprisingly, we found that lung metastases were predominantly derived from the epithelial compartment of breast tumors. However, concerns were raised on the fidelity and sensitivity of RFP-to-GFP switch of this model in reporting EMT of metastatic tumor cells. Here, we evaluated Tri-PyMT cells at the single-cell level using single-cell RNAsequencing and found that the Tri-PyMT cells exhibited a spectrum of EMT phenotypes, with EMT-related genes concomitantly expressed with the activation of GFP. The fluorescent color switch in these cells precisely marked an unequivocal change in EMT status, defining the pre-EMT and post-EMT compartments within the tumor. Consistently, the pre-EMT cells played dominant roles in metastasis, while the post-EMT cells were supportive in promoting tumor invasion and angiogenesis. Importantly, the post-EMT (GFP þ) cells in the Tri-PyMT model were not permanently committed to the mesenchymal phenotype; they were still capable of reverting to the epithelial phenotype and giving rise to secondary tumors, suggesting their persistent EMT plasticity. Our study addressed major concerns with the Tri-PyMT EMT lineage tracing model, which provides us with a powerful tool to investigate the dynamic EMT process in tumor biology. Significance: These findings confirm the fidelity and sensitivity of the EMT lineage tracing (Tri-PyMT) model and highlight the differential contributions of pre-and post-EMT tumor cells in breast cancer metastasis. See related commentary by Bunz, p.