Progression to advanced stage metastatic disease, resistance to endocrine therapies, and failure of drug combinations remain major barriers in the breast cancer therapy. Tumor microenvironments play an important role in progression from non-invasive to invasive disease as well as in response to therapies. Development of physiologically relevant, three-dimensional (3D) controlled microenvironments that can reliably recapitulate tumor progression from the early noninvasive to advanced metastatic stage will contribute to our understanding of disease biology and serve as a tool for screening of drug regimens targeting different disease stages. We have recently engineered physicochemical microenvironments by precisely controlling the size of 3D microtumors of non-invasive T47D breast cancer cells. We hypothesized that the precise control over physiochemical microenvironments will generate unique molecular signatures in size-controlled microtumors (small 150 μm vs large 600 μm) leading to differential phenotypic features and drug responses. The results indicated that large (600 μm) T47D microtumors exhibited traits of clinically advanced tumors such as hypoxia, reactive oxygen species, mesenchymal marker upregulation and collective cell migration unlike non-hypoxic, non-migratory small microtumors (150 μm). Interestingly, large microtumors also lost estrogen receptor alpha (ER-α) protein, consequently showing resistance to 4-hydroxytamoxifen (4-OHT). On the other hand, large microtumors showed upregulation of pro-angiogenic marker, vascular endothelial growth factor (VEGF), and hence were more responsive than small microtumors to the growth inhibition by anti-VEGF antibody. Surprisingly, both small and large microtumors exhibited comparable levels of phosphorylated epidermal growth factor receptor (pEGFR) and downstream signaling molecules such as AKT. As a consequence, both small and large microtumors showed comparable growth inhibition in response to gefitinib (inhibitor preferentially targeting EGFR) independent of microtumor size. Thus, precise control over the microenvironmental factors successfully recapitulated molecular characteristics underlying early vs advanced stage disease using the same non-invasive T47D cells. Such unique molecular signatures further resulted in differential response of small and large microtumors to anti-estrogen, and anti-VEGF treatments with comparable response to the EGFR-targeted therapies, underlining the importance of such stage-specific disease progression models in cancer drug discovery. KEYWORDS: size-controlled microtumor model, three-dimensional in vitro models, breast cancer progression, in vitro drug screening, endocrine resistance, EGFR/VEGF targeted therapy

Background CRIPTO is a multi-functional signaling protein that promotes stemness and oncogenesis. We previously developed a CRIPTO antagonist, ALK4L75A-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 ALK4L75A-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 ALK4L75A-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 ALK4L75A-Fc, which represents a candidate therapeutic approach. Results ALK4L75A-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. ALK4L75A-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 ALK4L75A-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.