29 research outputs found
Modelling temperature variability in batch retorts and its impact on lethality distribution
Single-cell transcriptomics identifies a WNT7A-FZD5 signaling axis that maintains fallopian tube stem cells in patient-derived organoids
Summary: The study of fallopian tube (FT) function in health and disease has been hampered by limited knowledge of FT stem cells and lack of in vitro models of stem cell renewal and differentiation. Using optimized organoid culture conditions to address these limitations, we find that FT stem cell renewal is highly dependent on WNT/β-catenin signaling and engineer endogenous WNT/β-catenin signaling reporter organoids to biomark, isolate, and characterize these cells. Using functional approaches, as well as bulk and single-cell transcriptomics analyses, we show that an endogenous hormonally regulated WNT7A-FZD5 signaling axis is critical for stem cell renewal and that WNT/β-catenin pathway-activated cells form a distinct transcriptomic cluster of FT cells enriched in extracellular matrix (ECM) remodeling and integrin signaling pathways. Overall, we provide a deep characterization of FT stem cells and their molecular requirements for self-renewal, paving the way for mechanistic work investigating the role of stem cells in FT health and disease
Intracellular Routing in Breast Cancer Cells of Streptavidin-Conjugated Trastuzumab Fab Fragments Linked to Biotinylated Doxorubicin-Functionalized Metal Chelating Polymers
We
describe the synthesis of a heterotelechelic metal-chelating
polymer (Bi-MCP-Dox), a polyacrylamide with a number average degree
of polymerization DP<sub>n</sub> = 50 (PDI = 1.2), with biotin (Bi)
and doxorubicin (Dox) as functional chain ends and diethylenetriaminepentaacetic
acid (DTPA) pendant groups as the binding sites for metal ions. We
compared its behavior in cell-uptake experiments with a similar polymer
(Bi-MCP) without Dox. These MCPs were complexed with trastuzumab Fab
(tmFab) fragments covalently linked to streptavidin (SAv) to form
tmFab-SAv-Bi-MCP-Dox and tmFab-SAv-Bi-MCP via the strong affinity
between Bi and SAv. tmFab targets human epidermal growth factor receptor-2
(HER2), which is overexpressed on certain human breast cancer cells.
Surface plasmon resonance (SPR) experiments with the extracellular
domain (ECD) of HER2 showed that incorporation of the MCPs in these
complexes had no significant effect on the association or dissociation
rate with the HER2 ECD and the dissociation constants. The tmFab-complexed
MCPs were subsequently labeled with <sup>111</sup>In (an Auger electron
emitting radionuclide). Auger electrons can cause lethal DNA double
strand breaks (DSBs) but only if they are emitted intracellularly
and especially, in close proximity to the nucleus. To evaluate the
cellular and nuclear uptake of tmFab-SAv-Bi-MCP-Dox, we incubated
HER2+ SK-BR-3 human breast cancer cells with the complexes saturated
with stable In<sup>3+</sup> and visualized their distribution by confocal
fluorescence microscopy, monitoring the fluorescence of Dox. In parallel,
we carried out cell fractionation studies on tmFab-SAv-Bi-MCP-Dox
and on tmFab-SAv-Bi-MCP labeled with <sup>111</sup>In. Both radiolabeled
complexes showed cell internalization and nuclear localization. We
conclude that metal-chelating polymers with this composition appear
to encourage internalization, nuclear uptake, and chromatin (DNA)
binding of trastuzumab fragments modified with streptavidin in human
breast cancer cells expressing HER2. Further study is needed to understand
the impact of polymer charge on cellular uptake and distribution to
intracellular compartments