The mammary gland consists of cells with gene expression patterns reflecting their cellular origins, function, and spatiotemporal context. However, knowledge of developmental kinetics and mechanisms of lineage specification is lacking. We address this significant knowledge gap by generating a single-cell transcriptome atlas encompassing embryonic, postnatal, and adult mouse mammary development. From these data, we map the chronology of transcriptionally and epigenetically distinct cell states and distinguish fetal mammary stem cells (fMaSCs) from their precursors and progeny. fMaSCs show balanced co-expression of factors associated with discrete adult lineages and a metabolic gene signature that subsides during maturation but reemerges in some human breast cancers and metastases. These data provide a useful resource for illuminating mammary cell heterogeneity, the kinetics of differentiation, and developmental correlates of tumorigenesis.

CRIPTO (or CR-1 or TDGF1) is a protein that plays an active role in tumor initiation and progression. We have confirmed that increased expression of CRIPTO is associated with clinical and prostate-specific antigen (PSA) progression in human prostate tissues. Our approach involved gaining insight into the role of CRIPTO signaling in castration-resistant Nkx3.1-expressing cells (CARNs), targets for oncogenic transformation in prostate cancer (PCa), by integrating the existing Cripto ^flox/flox into CARNs model. The most aggressive stage was modeled using an inducible Cre under control of the Nkx3.1 promoter conferring Nkx3.1 inactivation and driving Pten inactivation, oncogenic Kras activation, and lineage tracing with yellow fluorescence protein (EYFP) upon induction. Our findings provide evidence that selective depletion of Cripto in epithelial cells in vivo reduced the invasive phenotype, particularly in more advanced tumor stages. Moreover, in vitro experiments with Cripto overexpression demonstrated alterations in the physical features of organoids, which correlated with increased tumorigenic activity. Transcriptomic analyses revealed a unique CRIPTO/MYC co-activation signature associated with PSA progression in a human PCa cohort. Taken together, our data highlights a role for CRIPTO in tumor invasiveness and progression, which carries implications for biomarkers and targeted therapies.

Background: CRIPTO is a multi-functional signaling protein that promotes stemness and oncogenesis. We previously developed a CRIPTO antagonist, ALK4^L75A-Fc, and showed that it causes loss of the stem cell phenotype in normal mammary epithelia suggesting it may similarly inhibit CRIPTO-dependent plasticity in breast cancer cells. Methods: We focused on two triple negative breast cancer cell lines (MDA-MB-231 and MDA-MB-468) to measure the effects of ALK4^L75A-Fc on cancer cell behavior under nutrient deprivation and endoplasmic reticulum stress. We characterized the proliferation and migration of these cells in vitro using time-lapse microscopy and characterized stress-dependent changes in the levels and distribution of CRIPTO signaling mediators and cancer stem cell markers. We also assessed the effects of ALK4^L75A-Fc on proliferation, EMT, and stem cell markers in vivo as well as on tumor growth and metastasis using inducible lentiviral delivery or systemic administration of purified ALK4^L75A-Fc, which represents a candidate therapeutic approach. Results: ALK4^L75A-Fc inhibited adaptive responses of breast cancer cells under conditions of nutrient and ER stress and reduced their proliferation, migration, clonogenicity, and expression of EMT and cancer stem cell markers. ALK4^L75A-Fc also inhibited proliferation of human breast cancer cells in stressed tumor microenvironments in xenografts and reduced both primary tumor size and metastatic burden. Conclusions: Cancer cell adaptation to stresses such as nutrient deprivation, hypoxia, and chemotherapy can critically contribute to dormancy, metastasis, therapy resistance, and recurrence. Identifying mechanisms that govern cellular adaptation, plasticity, and the emergence of stem-like cancer cells may be key to effective anticancer therapies. Results presented here indicate that targeting CRIPTO with ALK4^L75A-Fc may have potential as such a therapy since it inhibits breast cancer cell adaptation to microenvironmental challenges and associated stem-like and EMT phenotypes.

There exists a set of factors termed oncofetal proteins that play key roles in ontogeny before they decline or disappear as the organism’s tissues achieve homeostasis, only to then re-emerge in cancer. Although the unique therapeutic potential presented by such factors has been recognized for more than a century, their clinical utility has yet to be fully realized1. This review highlights the small signaling protein CRIPTO encoded by the tumor derived growth factor 1 (TDGF1/Tdgf1) gene, an oft cited oncofetal protein whose presence in the cancer literature as a tumor promoter, diagnostic marker and viable therapeutic target continues to grow. We touch lightly on features well established and well-reviewed since its discovery more than 30 years ago, including CRIPTO’s early developmental roles and modulation of SMAD2/3 activation by a selected set of transforming growth factor β (TGF-β) family ligands. We predominantly focus instead on more recent and less well understood additions to the CRIPTO signaling repertoire, on its potential upstream regulators and on new conceptual ground for understanding its mode of action in the multicellular and often stressful contexts of neoplastic transformation and progression. We ask whence it re-emerges in cancer and where it ‘hides’ between the time of its fetal activity and its oncogenic reemergence. In this regard, we examine CRIPTO’s restriction to rare cells in the adult, its potential for paracrine crosstalk, and its emerging role in inflammation and tissue regeneration—roles it may reprise in tumorigenesis, acting on subsets of tumor cells to foster cancer initiation and progression. We also consider critical gaps in knowledge and resources that stand between the recent, exciting momentum in the CRIPTO field and highly actionable CRIPTO manipulation for cancer therapy and beyond.

Stem-like T cells selectively contribute to autoimmunity, but the activities that promote their pathogenicity are incompletely understood. Here, we identify the transcription coregulator OCA-B as a driver of the pathogenic maturation of stem-like CD4 ⁺ T cells to promote autoimmune demyelination. Using 2 human multiple sclerosis (MS) datasets, we show that POU2AF1 , the gene encoding OCA-B, is elevated in CD4 ⁺ T cells from patients with MS. We show that T cell–intrinsic OCA-B loss protects mice from experimental autoimmune encephalomyelitis (EAE) while preserving responses to viral CNS infection. In EAE models driven by antigen re-encounter, OCA-B deletion nearly eliminates CNS infiltration, proinflammatory cytokine production, and clinical disease. OCA-B–expressing CD4 ⁺ T cells of mice primed with autoantigen express an encephalitogenic gene program and preferentially confer disease. In a relapsing-remitting EAE model, OCA-B loss protects mice specifically at relapse. During remission, OCA-B promotes the expression of Tcf7 , Slamf6 , and Sell in proliferating CNS T cell populations. At relapse time points, OCA-B loss results in both the accumulation of an immunomodulatory CD4 ⁺ T cell population expressing Ccr9 and Bach2 , and loss of proinflammatory gene expression from Th17 cells. These results identify OCA-B as a driver of pathogenic CD4 ⁺ T cells.

Cell state control is crucial for normal tissue development and cancer cell mimicry of stem/progenitor states, contributing to tumor heterogeneity, therapy resistance, and progression. Here, we demonstrate that the cell surface glycoprotein Mcam maintains the tumorigenic luminal progenitor (LP)-like epithelial cell state, leading to Basal-like mammary cancers. In the Py230 mouse mammary carcinoma model, Mcam knockdown (KD) destabilized the LP state by deregulating the Ck2/Stat3 axis, causing a switch to alveolar and basal states, loss of an estrogen-sensing subpopulation, and resistance to tamoxifen—an effect reversed by Ck2 and Stat3 inhibitors. In vivo, Mcam KD blocked generation of Basal-like tumors and Sox10+Krt14+ cells. In human tumors, MCAM loss was largely exclusive of the Basal-like subtype, linked instead to proliferative Luminal subtypes, including often endocrine-resistant Luminal B cancers. This study has implications for developing therapies targeting MCAM, CK2, and STAT3 and their likely effective contexts.