Metastasis is the leading cause of cancer-related deaths; metastatic lesions develop from disseminated cancer cells (DCCs) that can remain dormant1. Metastasis-initiating cells are thought to originate from a subpopulation present in progressed, invasive tumours2. However, DCCs detected in patients before the manifestation of breast-cancer metastasis contain fewer genetic abnormalities than primary tumours or than DCCs from patients with metastases3–5. These findings, and those in pancreatic cancer6 and melanoma7 models, indicate that dissemination might occur during the early stages of tumour evolution3,8,9. However, the mechanisms that might allow early disseminated cancer cells (eDCCs) to complete all steps of metastasis are unknown8. Here we show that, in early lesions in mice and before any apparent primary tumour masses are detected, there is a sub-population of Her2+p-p38lop-Atf2loTwist1hiE-cadlo early cancer cells that is invasive and can spread to target organs. Intra-vital imaging and organoid studies of early lesions showed that Her2+ eDCC precursors invaded locally, intravasated and lodged in target organs. Her2+ eDCCs activated a Wnt-dependent epithelial–mesenchymal transition (EMT)-like dissemination program but without complete loss of the epithelial phenotype, which was reversed by Her2 or Wnt inhibition. Notably, although the majority of eDCCs were Twist1hiE-cadlo and dormant, they eventually initiated metastasis. Our work identifies a mechanism for early dissemination in which Her2 aberrantly activates a program similar to mammary ductal branching that generates eDCCs that are capable of forming metastasis after a dormancy phase.

Hypoxia is a poor-prognosis microenvironmental hallmark of solid tumours, but it is unclear how it influences the fate of disseminated tumour cells (DTCs) in target organs. Here we report that hypoxic HNSCC and breast primary tumour microenvironments displayed upregulation of key dormancy (NR2F1, DEC2, p27) and hypoxia (GLUT1, HIF1α) genes. Analysis of solitary DTCs in PDX and transgenic mice revealed that post-hypoxic DTCs were frequently NR2F1hi/DEC2hi/p27hi/TGFβ2hi and dormant. NR2F1 and HIF1α were required for p27 induction in post-hypoxic dormant DTCs, but these DTCs did not display GLUT1hi expression. Post-hypoxic DTCs evaded chemotherapy and, unlike ER− breast cancer cells, post-hypoxic ER+ breast cancer cells were more prone to enter NR2F1-dependent dormancy. We propose that primary tumour hypoxic microenvironments give rise to a subpopulation of dormant DTCs that evade therapy. These post-hypoxic dormant DTCs may be the source of disease relapse and poor prognosis associated with hypoxia.

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Stem-like" TCF1 + CD8 + T cells (T SL ) are necessary for long-term maintenance of T cell responses and the efficacy of immunotherapy but, as tumors contain signals that should drive T-cell terminal-differentiation, how these cells are maintained in tumors remains unclear. In this study, we found that a small number of TCF1 + tumor-specific CD8 + T cells were present in lung tumors throughout their development. Yet, most intratumoral T cells differentiated as tumors progressed, corresponding with an immunologic shift in the tumor microenvironment (TME) from "hot" (T cell-inflamed) to "cold" (non-T cell-inflamed). By contrast, most tumor-specific CD8 + T cells in tumor-draining lymph nodes (dLNs) had functions and gene expression signatures similar to T SL from chronic LCMV infection, and this population was stable over time, despite the changes in the TME. dLN T cells were the developmental precursors of, and were clonally related to, their more differentiated intratumoral counterparts. Our data support the hypothesis that dLN T cells are the developmental precursors of the TCF1 + T cells in tumors which are maintained by continuous migration. Finally, CD8 + T cells similar to T SL were also present in LNs from lung adenocarcinoma patients, suggesting a similar model may be relevant in human disease. Thus, we propose that the dLN T SL reservoir has a critical function in sustaining antitumor T cells during tumor development and protecting them from the terminal differentiation that occurs in the TME.

In the bone marrow (BM) microenvironment, NG2 + /Nestin + mesenchymal stem cells (MSCs) promote hematopoietic stem cell (HSC) quiescence 1,2 . Importantly, the BM can also harbour disseminated tumour cells (DTCs) from multiple cancers, which, like HSCs, can remain dormant 3 . The BM signals are so growth-restrictive that dormant BM DTCs can persist for years to decades only to awaken and fuel lethal metastasis 3-10 . The mechanisms and niche components regulating DTC dormancy remain largely unknown. Here, we reveal that periarteriolar BM-resident NG2 + /Nestin + MSCs can instruct breast cancer (BC) DTCs to enter dormancy. NG2 + /Nestin + MSCs produce TGFβ2 and BMP7 and activate a quiescence pathway dependent on TGFBRIII and BMPRII, which via p38-kinase result in p27-CDK inhibitor induction. Importantly, genetic depletion of the NG2 + /Nestin + MSCs or conditional knock-out of TGFβ2 in the NG2 + /Nestin + MSCs led to awakening and bone metastatic expansion of otherwise dormant p27 + /Ki67 -DTCs. Our results provide a direct proof that HSC dormancy niches control BC DTC dormancy. Given that aged NG2 + /Nestin + MSCs can lose homeostatic control of HSC dormancy, our results suggest that aging or extrinsic factors that affect the NG2 + /Nestin + MSC niche may result in a break from dormancy and BC bone relapse.Metastases, which are derived from disseminated tumour cells (DTCs), are the major source of cancer-related deaths from solid cancers 11 . Ample evidence supports that post-extravasation DTCs can remain in a dormant state from prolonged periods dictating the timing of metastasis initiation 3 .Since years to decades can lapse before dormant DTCs emerge as overt lesions, we postulate that targeting their biology is the shortest path to change patient outcomes by curtailing DTC conversion into metastasis. However, to achieve this goal we must understand the cancer cell intrinsic and microenvironmental mechanisms that control DTC dormancy and reactivation.The bone marrow (BM) is a common site where dormant DTCs are found and where metastasis can develop in various cancers after prolonged periods of clinical "remission" [3][4][5][6][7][8][9][10] . In trying to understand how the BM microenvironment might control DTC dormancy, we 9,12 and others 3,5,13,14 found that in both humans and mice, this microenvironment is a highly restrictive site for metastasis initiation. This is due to the presence of several cues, such as TGFβ2 12 , BMP7 15,16 , and LIF 14,20 , which induce DTC dormancy in different cancer types. Studies in mostly 2D or 3D in vitro models, proposed that mesenchymal stem cells (MSCs) 21 , vascular endothelial cells 13,22 and/or osteoblasts 18,23,24 may be the source of dormancy cues for different cancers. However, the function of such niche cells in vivo has not been formally tested.There is a long-standing hypothesis that the niches that control hematopoietic stem cell (HSC) dormancy may instruct DTCs to become dormant 25 . A prior study in prostate cancer has drawn a