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Metastasis is the leading cause of cancer-related deaths. It is unclear how intratumor heterogeneity (ITH) contributes to metastasis and how metastatic cells adapt to distant tissue environments. The study of these adaptations is challenged by the limited access to patient material and a lack of experimental models that appropriately recapitulate ITH. To investigate metastatic cell adaptations and the contribution of ITH to metastasis, we analyzed single-cell transcriptomes of matched primary tumors and metastases from patient-derived xenograft models of breast cancer. We found profound transcriptional differences between the primary tumor and metastatic cells. Primary tumors upregulated several metabolic genes, whereas motility pathway genes were upregulated in micrometastases, and stress response signaling was upregulated during progression. Additionally, we identified primary tumor gene signatures that were associated with increased metastatic potential and correlated with patient outcomes. Immune-regulatory control pathways were enriched in poorly metastatic primary tumors, whereas genes involved in epithelial-mesenchymal transition were upregulated in highly metastatic tumors. We found that ITH was dominated by epithelial-mesenchymal plasticity (EMP), which presented as a dynamic continuum with intermediate EMP cell states characterized by specific genes such as CRYAB and S100A2. Elevated expression of an intermediate EMP signature correlated with worse patient outcomes. Our findings identified inhibition of the intermediate EMP cell state as a potential therapeutic target to block metastasis.

TNF inhibitors are widely used to treat inflammatory diseases; however, 30%-50% of treated patients develop new autoantibodies, and 0.5%-1% develop secondary autoimmune diseases, including lupus. TNF is required for formation of germinal centers (GCs), the site where high-affinity autoantibodies are often made. We found that TNF deficiency in Sle1 mice induced TH17 T cells and enhanced the production of germline encoded, T-dependent IgG anti-cardiolipin antibodies but did not induce GC formation or precipitate clinical disease. We then asked whether a second hit could restore GC formation or induce pathogenic autoimmunity in TNF-deficient mice. By using a range of immune stimuli, we found that somatically mutated autoantibodies and clinical disease can arise in the setting of TNF deficiency via extrafollicular pathways or via atypical GC-like pathways. This breach of tolerance may be due to defects in regulatory signals that modulate the negative selection of pathogenic autoreactive B cells.