Week 2: Pieces of the lymphangiogenesis puzzle

In my second week here at Memorial Sloan Kettering Cancer Center, part of the larger conglomerate Weill Cornell Medicine, I continued my investigations into the intracellular mechanisms of lymphangiogenesis in the generation of lymphedema. Early in the week, I bore witness to the students of the Mehrara lab performing in vivo lymphatic surgeries on mice depleted for inducible nitric oxide synthase (iNOS) in lymphatic endothelial cells (LECs), a far more complex procedure than the non-living PDMS constructs I create back in Ithaca for modeling the lymphatics. It is hypothesized that reactive oxygen species proliferated by iNOS in the LEC microenvironment lead to immune cell inflammation and fibrosis in lymphedema. Thus, the research students formed a small lymphatic injury in the tails of mice to mimic the lymphatic damage that occurs in lymphedema, which will over the course of six weeks lead to lymphatic fluid buildup and swelling in the mice tails. 

Continuing the experiments from last week, a graduate mentor and I developed the Western blots from LECs cultured in vitro, stepping into a nearly pitch-black room devoid of outside light to visualize the bands of PTEN, AKT, and ERK proteins, otherwise invisible to the human eye, on autoradiographic instruments. However, this method is only capable of measuring the protein expression within the intracellular pathways we are interested in, necessitating further analysis of the downstream RNA expression of the associated genes. Therefore, the majority of this week was spent performing PCR on RNA isolated from our cultured LECs exposed to differing concentrations of lymphangiogenic factor VEGFC. Treating the cells with Trizol to dually lyse them and protect the genetic material, I scraped off the cells from their culture wells with an instrument similar to the a miniature cell culture rake and added chloroform to the solution to separate the RNA from impurities. This was also my first time quantifying the RNA concentration from the cells using a nanodrop device, a procedure akin to spectrophotometry that is based on the principle of single stranded RNA absorbing select wavelengths. For the final portion of the experiment, complementary DNA was created from the RNA template and amplified in a daunting 384 well plate to find the relative expression levels of RNA for each relevant protein. 

For the coming weeks, I hope to observe ICG imaging of lymphatics and learn how to obtain clinical samples.

-Samantha Kraus

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