In work with Andy Feinberg and Rafael Irizarry, published in Nature Genetics,“Increased methylation variation in epigenetic domains across cancer types” (PMID: 21706001), we demonstrated a universal dysregulation of the epigenetic state in cancer compared to normal tissue. We found that when we examine the differentially methylated regions previously discovered, by the Feinberg Lab, in colorectal cancer in other types of cancer, there seems to be a difference in other types of cancer too. Specifically, there seems to be a loss of regulation and an increase in variation (see left) of the epigenetic signature in cancer compared to normal samples (more detail here).
In work with Oliver McDonald and Andy Feinberg, we tested the effect of signaling on epigenetics using the TGF-β induced epithelial to mesenchymal transition model, published in Nature Structural and Molecular Biology “Genome-scale epigenetic reprogramming during epithelial-to-mesenchymal transition” (PMID: 21725293). In this work we found substantial changes in the chromatin state after triggering EMT – specifically a shift from heterochromatin marks to euchromatin marks, with LOCKs (Large Organized Chromatin K9) modifications being altered as measured by ChIP data. Pictured is immunofluorescent microscopy of some of these cells stained for heterochromatin marks.
Working with Andre Levchenko and Andy Feinberg we investigated the effects of epigenetics on signaling, specifically how loss of imprinting of insulin-like growth factor 2 (LOI of IGF2) affects the signaling response. LOI is thought to significantly (5x) increase the risk of colon cancer, and we have shown that it has a strong effect on colon neoplastic development in a mouse model in PNAS: “Enhanced sensitivity to IGF-II signaling links loss of imprinting of IGF2 to increased cell proliferation and tumor risk.” (PMID: 18087038). The issues were explored a bit further in our review article in Cell Cycle “A new link between epigenetic progenitor lesions in cancer and the dynamics of signal transduction” (PMID: 19177016) (more detail here).
Cell Positioning: Heterotypic Cell Signaling: Using 3D patterning technology described below, we used a simple test case to observe cellular signaling. Following Weiss et al. we used a genetically engineered system extracted from V. Fischeri to watch preciously positioned individual bacteriasignal each other through the use of small soluble factors. By using a simple microfluidic device, we were able to separately deliver the different bacteria to an assembly area, knowing which bacteria were senders and which receivers by which microfluidic channel they originated from. A side benefit to the microfluidics was the ability to controllably apply external ligands to induce the transmission of signal, and detect any emitted chemicals by the effluent of the microfluidic device. We were also able to control the transport characteristics in and around the bacteria by altering the flow rate, demonstrating that this system in V. Fischeri may be used as an environmental detection mechanism as well as a measure of bacterial density (see more).
Cell Positioning: Photopolymerizable Hydrogel and Optical Trapping: Using the combination of two techniques – photopolymerizable hydrogel and optical trapping, we demonstrated that it was possible to position living cells with submicron precision and trap them in a extracellular matrix analog. The advantage of this method compared to more large scale methods as developed by Bhatia et al. is the precision – specific cells may be selected and arranged with submicron precision. In collaboration with the G. Timp lab, I was able to trap and position multiple cells in 3D in hydrogel (see more)
Cell Positioning: Microfluidics and Surface Patterning: We have also tried using microfluidics and surface patterning to create patterns and culture cells. These methods may be useful for future work.
3D Cell Imaging: Another issue was imaging in 2D vs. 3D. Work in the literature has demonstrated that cells move and behave drastically differently on a 2D substrate, such as a glass coverslip, than in a 3D gel. In is believed that the 3D environment is a closer approximation to the in vivo microenvironment of the cells. This is especially important for cancer related work, where it has been conclusively shown that the microenvironment of the cells is very important to their behavior To that end, we have investigated several different gels – from natural products to the more useful photopolymerizable PEGDA derivatives – to identify the different advantages and disadvantages of each. Though photopolymerizable gels offer great utility for control, natural ECM analogs are usually more straightforward, and sometimes more physiologically relevant; there are pros and cons to both types of gel.
Cell Motility: Cell motility is a useful metric of cellular behavior, specifically interesting in wound healing and metastasis. We are specifically interested in investigating angiogenic and metastatic cell motion. The first issue with this work is how to properly imaging cell motion since fluorescent stains have a phototoxic effect on many cell types. I solved this problem by using cell lines stably transduced with a fluorescent protein (e.g., GFP).
Wet SEM with Quantum Dots: Correlative electron and fluorescent microscopy has high utility – the advantages of both can be used to investigate the same cells. We used Wet SEM – using Quantomix capsules to image cells in liquid – to investigate cytoskeletal structure. Specifically, quantum dots (as an electron dense and fluorescent material) were used to stain actin, allowing the sample to be imaged with both light and electron microscopy at the same time. This work was published in “Wet Electron Microscopy with Quantum Dots” in Biotechniques (PMID: 16989089).