220 A. Jögi et al. breast cancer as described below. Our observation provides one mechanism behind phenotypic hetero- geneity in solid tumors and, importantly, highlights how hypoxia can contribute to the aggressive behavior of tumors with an overall low grade of oxygenation. HIF-2a-deﬁned pseudohypoxic phenotype and tumor aggressiveness in neuroblastoma According to well-established models, both HIF-1a and HIF-2a subunits become degraded via the proteasomes’ conditions of proper oxygenation (16). However, in clinical samples we found that HIF-2a but not HIF-1a protein was highly expressed in a small subset of neuroblastoma cells close to blood vessels (17-19). In cell lines we could establish that HIF-2 was active at a subphysiological level of oxygenation (5% oxygen) and transcribing known hypoxia-driven genes such as VEGF. Importantly, presence of tumor cells with strong immunohisto- chemical staining for HIF-2a correlates to high clinical neuroblastoma stage and unfavorable out- come (17). A closer examination of the HIF-2a- positive, peri-vascular cells revealed that they are immature and have a neural crest-like phenotype (18). They further express VEGF, and we specu- late that this subset of tumor cells actively attracts vascular endothelial cells. Glioma tumor stem/tumor- initiating cells as deﬁned functionally are immature, have neural stem cell properties, and express high HIF-2a protein levels (20). Like the strongly HIF-2a and VEGF-positive neuroblastoma cells, glioma stem cells are located in a peri-vascular niche and express VEGF (20). Thus, these two neutrally derived tumors contain subsets of cells that are immature, and their presence is associated with an aggressive, unfavorable disease. HIF-2 appears to be involved in keeping the stemness of both neuroblastoma and glioma cells, and, as discussed below, HIF-2a expression in breast cancer is also associated to unfavorable disease, sug- gesting HIF-2a as a potential treatment target in these tumor forms. As shown in neuroblastoma, by down- regulating HIF-2a in stem cell-like cells, sympathetic neuronal differentiation can be induced (21), and we speculate that HIF-2 inhibition could be a strategy to push immature stem cell-like cells into a more differ- entiated, bulk-like cell population that can be treated by established treatment protocols. Breast cancer and tumor cell differentiation Breast cancer is an additional tumor type where impaired development is an important component of the malignant process and where the tumor cell differentiation stage is used in histopathological tumor grading. The Nottingham grading system, widely employed for diagnosis and prognosis of breast tumors, is based on three parameters: extent of tubule formation, mitosis frequency, and nuclear pleomor- phism, where at least the ﬁrst and last elements are related to differentiation stage. Tubule formation requires presence of differentiated polarized epithelial cells and that these cells orientate in relation to adjacent cells. Thus, the capacity to form tubules is indicative of the differentiation stage of a group of breast cancer cells. In breast cancers cells are unable to distinguish between apical and basal surface and organize themselves into tubule-like organoid structures, have a low stage of differentiation, and receive a high score in the Nottingham grading system. Increased nuclear-to-cytoplasmic ratio is also a marker of low differentiation, and when it is associated with variation in nuclear size between cancer cells the tumor gets a high score also in this element of the Nottingham grading system. A high Nottingham score (8–9, or grade III) is associated with poor prognosis and progressing disease. The relation between differentiation stage and prognosis in breast cancer implies that the understanding of processes of cancer initiation and progression should be paralleled by insight into mechanisms of normal breast development and function. The tissue origin in breast cancer, the mammary gland, is continuously in a developmental phase in fertile women. Puberty and pregnancy are phases of intense epithelial cell growth, migration, and differ- entiation. The life cycle of the mammary gland in addition holds phases of epithelial regression and tissue remodeling, i.e. the dramatic involution after lactation and tissue regression after menopause. The involution process involves extensive cell death in the epithelial compartment, intense remodeling of the stromal compartment, and inﬁltration of inﬂamma- tory cells. In this apparently chaotic environment the mammary epithelial tissue stem cells must be pre- served to ensure rebuilding of the functional gland in future pregnancies. All these processes of the mam- mary gland functional cycle, proliferation, differenti- ation, and regression also occur in each monthly estrous cycle but to a lesser extent. There is a strong correlation between the total number of estrous cycles during lifetime (depending on early menarche, late menopause, and number of child-births) and the risk of breast cancer, implying that stem cell activation and differentiation processes are linked to breast tumorigenesis (22). In breast ductal carcinoma in situ (DCIS) with comedo lesions, layers of transformed epithelial cells ﬁll up the ductal space and due to intra-lesional hypoxia a central necrotic core arises (Figure 1A).