Purple W/F Research Proposal
1. Begin to define your research proposal by making a wiki page to collect your ideas and resources (you can do this on one page with your partner or split the effort and each turn in an individual page). Keep in mind that your presentation to the class will ultimately need:
-a brief project overview -> We are looking into implementing a Mod2 type screening, but instead of cell viability measured through flow cytometry, we would screen mechanistic properties. In this way we can see how cells change with time over a full dosing schedule. The other main idea we were looking into is using this as a screening technique for cancer (see below for more details). -sufficient background information for everyone to understand your proposal -> Alejandro has most of this. We have not yet discussed what we will focus on, one idea is cancer screening. Could this technique be sensitive enough to pre-screen for metastases?
http://www.rsc.org/images/loc/2012/pdf/4.A1-2.pdf; Example of Screening by mechanistic properties http://www.sciencedirect.com/science/article/pii/S0006349506724808; Specifically relevant to Leukemia screenings
High-throughput mechanophenotyping for holistic systems approaches:
Introduction: Alterations to the mechanical properties of cells serve as key events in the etiologies of a variety of disorders from sickle cell disease, to malaria, to cancer metastasis. Therefore, being able to characterizing these properties in biological systems is an important step in diagnosing and understanding these diseases. A variety of devices aid in this burgeoning field of research such as atomic force microscopy, optical trapping, microfluidic stretching, micropipette aspiration, etc. Due to the nature of these analyses, single-cell mechanophenotyping has not allowed for high throughput experimentation, particularly useful for both clinical diagnoses of these disorders (cancers mostly, but malaria as well, perhaps SCD too) and systems approaches in studying these disorders.
Conceptual Basis: Recently, methods have been shown for cytological mechanophenotyping at rates comparable to traditional fluorescent activated cell sorting cytometry (FACS) of ~1000 cells/s. Some depend on measurements based on viscoelastic behavior (ie. the time-dependent elastic behavior of cells), whereby measurements of periodic signal-response phase lag allows for far more rapid mechanophenotyping of cell stiffness than conventional measurements of cell deformability, which requires force equilibration (on the order of seconds).
Cell deformability cytometry is an attractive alternative to traditional FACS stemming from the fact that biophysical markers are label-free and inherent to the cells. As such, mechanophenotyping forgoes the need to introduce non-native species in samples, which both reduces the time and cost of sample preparation, particularly attractive for clinical applications. Furthermore, since it relies on high-frequency periodic loading, cells “feel” little perturbation due to their viscoelastic creep. This is particularly important for post-sorting analyses.
Experimental Setup/Design: Since cancer cells have been shown to exhibit less stiffness than normal cells (speculated to function in their proclivity to migrate), they serve as an obvious target for high-throughput mechanophenotyping combined with a systems biology approach (very similar to Mod2).
Recall one of the motivating questions underlying our analysis of the efficacy of combinatorial drug therapy was cancer cells acquiring drug resistance over time. Although, we were able to show how dual pathway inhibition decreases cell viability overall, we did little to understand how it affects acquisition of drug resistance over time. Thus we could perform a similar experiment, but mechanophenotyping the cells over the course of their life (utilizing the non-destructible and non-invasive aspects of this form of cytometry) to find the time until drug resistance acquisition of many cancer cells treated with different drug regimens.
Mechanophenotyping for diagnosis: As stated before various disorders exhibit aberrant elastic properties, making them assayable through high-throughput mechanophenotyping. In particular, predicting cancer cell migration is a very attractive target for this form of cytometry as it is quite difficult to do so using traditional FACS. Thus, we could employ the same methodology as described above based on systems biology approaches to test the efficacy of combinatorial drug therapies on cancer metastasis.
Links to referenced material: - http://www.sciencedirect.com/science/article/pii/S0006349513011247?np=y# -> Viscoelasticity not elasticity as the biomarker for cell viability -http://stm.sciencemag.org/content/5/212/212ra163.full -> Groundwork for cell deformability cytometry for cancer migration analysis -http://stm.sciencemag.org/content/4/123/123ra26.full.pdf - example of how sickle cell disease can be mechanophenotyped (not yet at high throughput rates -http://www.sciencedirect.com/science/article/pii/S0006349513011363 -> Example of mechanism by which cells stiffen (through dynamic fibrin regulation)