Differentiation and cancer 221 A DCIS B 21% C 1% Figure 1. A: Ductal carcinoma in situ of the breast, the comedo form, with several cell layers of epithelial cells surrounding a central necrotic area. The inner cell layers, adjacent to the necrosis, show low differentiation with unorganized structures and increased nucleus-to-cytoplasm ratio (scale bar: 500 mm). B: Non-malignant mammary epithelial cells cultured in a three-dimensional differentiation-inducing assay. After 21 days of culture at normoxia (21% O2), the mammary epithelial cells (MCF-10A) differentiate into growth-arrested, organized acini structures with polarized cells surrounding a hollow lumen, resembling the in vivo mammary gland appearance. The differentiated mammary cells have a polarized expression pattern of proteins, here laminin V (green), and small compact nuclei (blue, actin in red) in a palisade structure. C: At hypoxia (1% O2) the MCF-10A mammary epithelial cells fail to arrange into organized structures and appear as cell aggregates without lumen or polarized protein localization. The hypoxic cells have larger nuclei, remain proliferative, and express markers of undifferentiated cell stage—characteristics often seen in breast carcinoma (scale bar: 20 mm). Cell layers close to the central necrosis are hypoxic and have increasing protein levels of e.g. HIF-1. We have reported that the breast cancer cells close to the necrotic core have a less differentiated phenotype as assessed by histopathological criteria (23). The hyp- oxic cells have increased nuclear-to-cytoplasmic ratio and decreased expression of markers of differentia- tion, including estrogen receptor (ER) in ER-positive lesions. Furthermore, small duct-like structures fre- quently form within DCIS lesions and represent transformed epithelial cells striving to organize them- selves into polarized epithelial structures, structures that are rarely encountered in the hypoxic peri- necrotic regions of the DCIS lesions. Thus, intra- lesional hypoxia has direct bearing on two of the elements of the Nottingham grading system, nuclear phenotype and tubule formation. Our hypothesis is that the hypoxic conditions lead to a less differen- tiated tumor cell with lost ability to form organized and polarized structures. In support of this hypo- thesis, cells of established human breast cancer cell lines cultured at hypoxic conditions showed down- regulated expression of ER and up-regulation of CK19, indicating that, both in vitro and in vivo, hypoxic conditions promote a less differentiated breast cancer phenotype (23). DCIS is an instance of a typical carcinoma in situ lesion where the basal membrane has not been breached, meaning that the cancer is non-invasive. Initially regarded as a fairly homogeneous disease divided into non-comedo versus comedo type, DCIS is now seen as a multi-faceted entity where the degree of differentiation within the DCIS lesion has a large impact on the outcome. Cases displaying low differentiation most often progress to invasive carcinoma, whereas cases of high differentiation are less likely to do so. This underscores the importance of assessing differentiation also in pre-invasive can- cers. As a whole, DCIS may be seen as an interme- diate step in the maligniﬁcation process of tumors, and the typical hallmark trait of low differentiation in this form of neoplasia is the presence of comedo- type necrosis. This means that the cancer tissue has a necrotic and hypoxic center. This implies that hypoxia might contribute to the conversion of DCIS cells into invading tumor cells. One question we have addressed is whether the observed hypoxic impairment of differentiation in DCIS lesions is restricted to cancer cells. A related question is whether hypoxia can arrest normal breast epithelial cells at an immature differentiation stage and by doing so contribute to onset of tumorigenesis. We have studied the non-malignant immortalized mam- mary epithelial MCF-10A cells in 3D cultures where these cells form mammary acini with polarized rim cells and evacuated lumen. Hypoxia impaired both polarization and lumen formation as demonstrated in MCF-10A cells (Figure 1B and C) (Vaapil et al., unpublished paper, 25). Furthermore, a fraction of hypoxic MCF-10A cells remained in cell cycle, while the vast majority of the normoxic cells entered a differentiated post-mitotic state. Still the hypoxic structures were smaller, which we could attribute to increased apoptosis. We conclude that hypoxia confers a cancer-like phenotype to the mammary epithelial cells. It is nowadays well established that intra-tumor hypoxia correlates to a worse prognosis in many