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Imaging Cleared Intact Biological Systems at a Cellular Level by 3DISCO

Tissue clearing and subsequent imaging of transparent organs is a powerful method to analyze fluorescently labeled cells and molecules in 3D, in intact ...

Genentech, Inc.

The efficacy of chemotherapy is usually viewed as the outcome of cancer-cell-autonomous processes while the contribution of stroma is being overseen. Here we show that p53 mutations in stromal fibroblasts, a genetic lesion that is detectable in primary breast, prostate and probably other cancers, while they accelerate tumorigenesis they also sensitize tumours against conventional chemotherapy by doxorubicin and cis-platinum. The mechanism by which p53 of stromal fibroblasts affects the response of a tumour against chemotherapy is likely to involve the induction of senescence in the fibroblasts which in turns results in the production of growth factors acting onto the cancer cells by paracrine mechanisms. Our findings identify stromal fibroblasts as important modulators of the efficacy of anticancer therapy.

P53 mutations in stromal fibroblasts sensitize tumors against chemotherapy.

GHRH, besides its neuroendocrine action in controlling the release of GH from the pituitary, stimulates the growth of various cancers in vivo and in vitro by direct mechanism(s). However, the molecular mechanism that mediates these proliferative effects of GHRH in extrapituitary tissues remains poorly characterized. In the present study, we investigated whether the tumor suppressor p21/waf1 is involved in the mediation of the proliferative effects of GHRH in A549 human lung cancer epithelial cells. Exposure of A549 cells to the GHRH antagonist JMR-132 caused a significant inhibition in the rate of cell proliferation. In A549 cells, GHRH suppressed while JMR-132 increased the levels of p21 expression in a dose-dependent manner. This suggests that GHRH could regulate p21 levels. We then evaluated whether p21 is required in A549 cells for the regulation of cell proliferation by GHRH. To this end, we knocked-down p21 expression in A549 cells by siRNA and assessed the effects of antagonist JMR-132 on cell proliferation. We found that the loss of p21 expression abolished the anti-proliferative effects of JMR-132. Suppression of p21 expression by siRNA in human HT29 colon cancer cells and non-transformed mouse osteoblasts KS483 also blocked the anti-proliferative effects of JMR-132 suggesting that the regulation of cell proliferation by GHRH is p21 dependent. These results shed light on the molecular mechanism of action of GHRH antagonists in tumor tissues and suggest that the antineoplastic activity of GHRH antagonists could be considered for the treatment of cancers expressing p21.

Essential role of p21/waf1 in the mediation of the anti-proliferative effects of GHRH antagonist JMR-132.

The synthesis of M(I)(CO)(3)(NNO) (M = Re, (99m)Tc) complexes conjugated to the antitumor agent 2-(4'-aminophenyl)benzothiazole and to its 6-methyl derivative, as well as their in vitro and in vivo biological evaluation as breast cancer radiopharmaceuticals, is reported. The Re complexes displayed under the fluorescence microscope clear uptake by the sensitive to the 2-(4'-aminophenyl)benzothiazole pharmacophore breast cancer cell lines MCF-7 and T47D, while uptake by less sensitive lines and by normal fibroblasts was much weaker. In accordance, uptake of the corresponding radioactive (99m)Tc complexes was clearly higher in the breast cancer cell lines MCF-7 and MDA-231 compared to normal fibroblasts. Biodistribution of the (99m)Tc complexes in SCID mice bearing MCF-7 xenografts showed appreciable tumor uptake. A tumor/muscle ratio of 2.2 was measured for the complex conjugated to 2-(4'-aminophenyl)benzothiazole that led to successful tumor imaging. The results render the 2-(4'-aminophenyl)benzothiazole complexes potential candidates for imaging ((99m)Tc) and targeted radiotherapy ((188)Re) of breast cancer.

Evaluation of Re and (99m)Tc complexes of 2-(4'-aminophenyl)benzothiazole as potential breast cancer radiopharmaceuticals.

In this study we evaluated the immunohistochemical expression of the receptors Notch 1 and Notch 2, the ligand Delta 1, and the transcription factors HES 1 and HES 5 in the epithelium of well-defined periapical cysts.

Immunohistochemical expression of Notch signaling in the lining epithelium of periapical cysts.

The conserved role of Notch signaling in controlling intestinal cell fate specification and homeostasis has been extensively studied. Nevertheless, the precise identity of the cells in which Notch signaling is active and the role of different Notch receptor paralogues in the intestine remain ambiguous, due to the lack of reliable tools to investigate Notch expression and function in vivo. We generated a new series of transgenic mice that allowed us, by lineage analysis, to formally prove that Notch1 and Notch2 are specifically expressed in crypt stem cells. In addition, a novel Notch reporter mouse, Hes1-EmGFP(SAT), demonstrated exclusive Notch activity in crypt stem cells and absorptive progenitors. This roster of knock-in and reporter mice represents a valuable resource to functionally explore the Notch pathway in vivo in virtually all tissues.

Notch lineages and activity in intestinal stem cells determined by a new set of knock-in mice.

Notch signalling is implicated in stem and progenitor cell fate control in numerous organs. Using conditional in vivo genetic labelling we traced the fate of cells expressing the Notch2 receptor paralogue and uncovered the existence of two previously unrecognized mammary epithelial cell lineages that we term S (Small) and L (Large). S cells appear in a bead-on-a-string formation and are embedded between the luminal and basal/myoepithelial layers in a unique reiterative pattern, whereas single or paired L cells appear among ductal and alveolar cells. Long-term lineage tracing and functional studies indicate that S and L cells regulate ipsi- and contralateral spatial placement of tertiary branches and formation of alveolar clusters. Our findings revise present models of mammary epithelial cell hierarchy, reveal a hitherto undescribed mechanism regulating branching morphogenesis and may have important implications for identification of the cell-of-origin of distinct breast cancer subtypes.

Notch2 genetic fate mapping reveals two previously unrecognized mammary epithelial lineages.

The identity of mammary stem and progenitor cells remains poorly understood, mainly as a result of the lack of robust markers. The Notch signaling pathway has been implicated in mammary gland development as well as in tumorigenesis in this tissue. Elevated expression of the Notch3 receptor has been correlated to the highly aggressive "triple negative" human breast cancer. However, the specific cells expressing this Notch paralogue in the mammary gland remain unknown. Using a conditionally inducible Notch3-CreERT2(SAT) transgenic mouse, we genetically marked Notch3-expressing cells throughout mammary gland development and followed their lineage in vivo. We demonstrate that Notch3 is expressed in a highly clonogenic and transiently quiescent luminal progenitor population that gives rise to a ductal lineage. These cells are capable of surviving multiple successive pregnancies, suggesting a capacity to self-renew. Our results also uncover a role for the Notch3 receptor in restricting the proliferation and consequent clonal expansion of these cells.

Daniel Lafkas

Department of Discovery Oncology

Genentech, Inc.

Imaging Cleared Intact Biological Systems at a Cellular Level by 3DISCO