A ß-galactosidase probe for the detection of cellular senescence by mass cytometry.

Mass cytometry (MC) is a powerful method that combines the cellular resolution of flow cytometry with the isotopic resolution of inductively coupled plasma mass spectrometry (ICP-MS). This combination theoretically allows for the simultaneous quantification of >80 different parameters at the single cell level, in turn allowing for the deep profiling of heterogeneous cell populations. The majority of available reagents for MC are antibodies labeled with heavy metal isotopes, allowing for the quantification of static biomarkers. To complement these reagents, we aim to develop small molecule reporters of cellular metabolism that are compatible with MC. Here we report a probe of ß-galactosidase activity capable of detecting cellular senescence. The galactoside probe contains a tellurophene reporter group and, when hydrolyzed, generates a quinone alkide. This reactive alkylating agent forms covalent tellurophene bearing conjugates with local nucleophiles, allowing for the quantification of ß-galactosidase activity in individual cells. Difluoromethyl and monofluoroethyl quinone alkide generating warheads were examined for their activities and compared in vitro and in vivo. We showed that the difluoromethyl derivative gave higher tellurium labelling in vitro and that the quinone methide was more reactive towards thiols than amines. In vivo the difluoromethyl derivative successfully labeled senescent cells with comparable selectivity to the commonly used fluorescent senescence probe C12FDG.

Integration of hypoxic HIF-α signaling in blood cancers.

Hypoxia (low O2) is a fundamental microenvironmental determinant of bone marrow (BM) pathophysiology. Recent data from molecular and clinical studies indicate that hematopoiesis and leukemogenesis are dependent upon hypoxia-inducible factors (HIFs), a family of essential transcriptional activators mediating the metazoan hypoxic response. In blood cancers, the synergism between HIF overexpression and stabilization within the hypoxic BM microenvironment promotes disease progression, therapy resistance and relapse. In this review, we will summarize current advances in the understanding of HIF signaling in blood cancers and its translational implications for hypoxic-targeted therapy.

HIF-1-dependent expression of angiopoietin-like 4 and L1CAM mediates vascular metastasis of hypoxic breast cancer cells to the lungs.

Most cases of breast cancer (BrCa) mortality are due to vascular metastasis. BrCa cells must intravasate through endothelial cells (ECs) to enter a blood vessel in the primary tumor and then adhere to ECs and extravasate at the metastatic site. In this study we demonstrate that inhibition of hypoxia-inducible factor (HIF) activity in BrCa cells by RNA interference or digoxin treatment inhibits primary tumor growth and also inhibits the metastasis of BrCa cells to the lungs by blocking the expression of angiopoietin-like 4 (ANGPTL4) and L1 cell adhesion molecule (L1CAM). ANGPTL4 is a secreted factor that inhibits EC-EC interaction, whereas L1CAM increases the adherence of BrCa cells to ECs. Interference with HIF, ANGPTL4 or L1CAM expression inhibits vascular metastasis of BrCa cells to the lungs.