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IntroductionNormal stem cells are proliferative, undifferentiated cellsthat have the ability to differentiate into different types ofcells in the body.

Previous studies on cancer cells suggest that there is asmall tumor-initiating cell population which can give rise tothe entire tumor mass; these cells are known as cancerstem cells (1). They are thought to initiate tumorigenesisand are the putative cause for disease recurrence andmetastasis. Therapeutic targeting of these cells istherefore important for cancer treatment.

Cancer cells, like normal stem cells, are highlyproliferative. Previous work suggests that both cancerstem cells and normal stem cells may share a commonmetabolism, one in which the natural pathway for glucosebreakdown, glycolysis, is shunted away from themitochondria, and pushed toward lactate production. Thisphenomenon is known as the Warburg effect (2).

Less differentiated cells (such as stem cells) areproliferative and produce lactate, while differentiated cellsare non-proliferative and depend less on fermentation. Ourhypothesis is that normal and cancer stem cells may beinduced to differentiate by modulating their sharedmetabolic dependency.

Our goal has been to understand how normal stem cellsand cancer stem cells can be induced to differentiate bymodulating the glycolytic pathway. Our studies hope toelucidate a novel mechanism for differentiation therapy ofcancer stem cells and may have further implications in ourunderstanding of normal stem cell biology.

My Specific Objective: To characterize the effects ofchemical modulation of glycolysis on differentiation byusing chemical inhibitors of glycolysis and measuring theeffects on C2C12 mouse myoblast (a precursor cell line)differentiation.

Exploring the role of glycolysis in mouse stem cell differentiationJulia, Abigail Bracha, Arvind Ramanathan, and Stuart Schreiber

Broad Institute of MIT and Harvard, Chemical Biology Platform, Cambridge, MA, USA

Materials and MethodsFor our experiments we used two different cell lines:

• C2C12 mouse myoblast cells: Myoblast cells are adultprecursor stem cells. These cells fuse to form mature musclefibers when differentiated. We added compounds (chemicalinhibitors of glycolysis) to the myoblast cells, and measuredeffects on differentiation. This is a robust model system forstudying differentiation, since muscle cell differentiation can beinduced and scored with high reproducibility.• Mouse embryonic stem cells (ES cells): Mouse ES cells areundifferentiated pluripotent cells that can differentiate into anytype of cell in the body. We used the mouse CCE stem cell linefor our assays.

Mouse ES cells were passaged every other day and mouseC2C12 cells were passaged every 3-4 days upon reachingconfluency.Compounds used to inhibit glycolysis were:

• Specific enzyme inhibitors of lactate dehydrogenase (theenzyme which is responsible for the conversion of pyruvate tolactate):

Oxamic Acid Silver Nitrate

• Compounds (found from a screen performed in the Schreiberlab) that inhibit lactate production:

Crinamine Lycorine Tomatine 5HT DOI 6ANThe mechanism by which these compounds work is unknown.

Assay Development: Mouse Myoblast CellsWe found that on day 3 of culture C2C12 cells differentiated withoutsignificant background noise, i.e. above the level of spontaneousdifferentiation which occurs despite non-differentiating conditions.

Control: Cells in Non-differentiated Medium DAY 3

Cells in Differentiation Medium DAY 3

ConclusionsCompounds: From our preliminary studies on metabolicinhibitors, we see that at least two of the compounds(crinamine and lycorine) primarily affect cell viability; this isapparent in reduced cell number as compared to controlcells. Three of the eight compounds (5HT, DOI, and 6AN)have modest effects on differentiation. We are currentlyrepeating these experiments to verify preliminary results,and we will quantify differentiation by immunofluorescencestaining against myosin heavy chain.

Lactate assay: From our studies on lactate production indifferentiating C2C12 cells, it appears that there is an initialincrease in lactate production (day 3) which tapers as thecells differentiate (day 5). This is likely to correlate with cellproliferation which we also believe initially increases beforecells terminally differentiate and exit the cell cycle.

Literature cited1. Reya, Tannishtha, Sean K. Morrison, Michael F.

Clarke, Irving L. Weissman. "Stem cells, cancer, andcancer stem cells." Nature 414(2001): 105-111.

2. Warburg, Otto. "On the Origin of Cancer Cells."Science 123(1956): 309-314.

AcknowledgmentsI would like to thank my mentors Abby Bracha andArvind Ramanathan for giving me this project to work onand assisting me throughout it. I would like to thankMegan Rokop, Julie Boehm, and Kate MacSwain forallowing me this opportunity through the EducationalOutreach Program. I would also like to thank ConnieWang for the use of her glycolytic inhibitors.

Results: Mouse Myoblast CellsWe tested 8 inhibitors of lactate production on our mousemyoblast cells to see if they induced differentiation.Differentiation was measured by myotube formation asdetermined by microscopy. The positive control was grown

Measurement of Lactate Production: Media was changed 24hours prior to assaying for lactate production. Supernatantwas collected the following day. Lactate production wasmeasured by fluorescence, with three differentconditions:Undifferentiated cells, differentiated cells day 3 ofculture and differentiated cells day 5 of culture.

Future DirectionsIn the future we hope to apply this system toembryonic stem cells, which we have begun toexplore. Using tissue culture methods and removal ofLIF, we tried to induce differentiation in mouseembryonic stem cells (CCE cell line). We tested fordifferentiation using alkaline-phosphatase and oct4antibody staining. We are still working on assaydevelopment with this system. We have not been ableto differentiate these cells, as evidenced by continuedexpression of alkaline phosphatase and oct4.

Cells in Differentiation Medium DAY 1

Cells in Differentiation Medium DAY 3

In the future, we intend to establish a genetic approachfor modulating glycolysis. RNA interference tools(RNAi) will be used to screen for target metabolicgenes which may induce differentiation.

Figure from (1)

+O2WARBURGEFFECT(LDH)

Pos. control Neg. control

Crinamine Lycorine

5HT DOI 6AN

Of the 8 compounds tested, the five below appeared tohave effects on either cell viability or differentiation.

in 2% horse serum, andthe negative control wasgrown in 10% horseserum.

Phase Contrast(cell outlines)

Hoechst(nuclear staining)

Phalloidin(cytoskeleton staining)

Alkaline-phosphatase(stem cell marker)

Hoechst(nuclear staining)

OCT4(stem cell marker)