I am working with spheroid models. Cells of Head and Neck squamous carcinoma are seeded in special U bottom plates where they form spheroids.
I treat my spheroids with Cetuximab and/or Patritumab.
In monolayer, cetuximab has a very good anti proliferative effect on the cells, and the same level of effect when treated with combination.
However, in spheroid model, the spheroid looks intact and alive. When I provided Calcein AM dye (live cell stain), only a small dot appeared which was difficult to explain considering the integrity of the spheroid.
So if anyone has experience with spheroids, does spheroids always fall apart when the cells are dead or Does Calcein Am cannot be cleaved to flouresce even in some live cells?
Here are pictures describing what I meant.
I checked the intregity of the spheroid by agitating it with pipetting and it is quite stable. It did not shatter or became smaller.
HI, structural integrity doesn't (always) mean viability with spheroids. Indeed, the treated one is no more spherical according to me. But yes, Calcein stains living cells, so it's likely that your treated spheroids is mainly composed of dead cells. During cell death, spheroids can either disintegrate, shrink, change shapes, display 'escaping cells', it really depends on cell type and treatment. For antiproliferative agents, keep in minf that cellular proliferation is often lower in 3D compared to 2D so it' s likely that IC50 would be different in 2D and 3D for a given cell type and spheroid 'age '(time in culture) could also modulate this: the older, the less proliferative. You should try a double staining with calcein and IP (stains dead cells) or you can assess the metabolic acitivity by measuring ATP pool after spheroid lysis.
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