To examine whether the self-renewing sphere-forming cells possess a hypothesized CSC chemoresistant property, the sensitivity of the PLC/PRF/5 parental cells versus the tertiary passage sphere-forming cells to cisplatin was assessed.
Afterwards, the eyes of some scholars were turned to solid tumors and intriguingly found that a small subset of cancer cells isolated from solid tumors were also able to generate new tumor in xenograft mice model and endowed with the capacity of serial passage, sphere-formation, and self-renewal in vitro as well [ 9, 13].
Briefly, RNA samples from both the PLC/PRF/5 parental cells (as control) and the tertiary passage spheres were prepared.
For each passage, spheres formed after 4 days in vitro (DIV) were enzymatically dissociated with Accutase (Stem Cell Technologies, Vancouver, BC, Canada) and reseeded in complete medium.
At the 5th and 6th passages, spheres were dissociated into a single-cell suspension using trypsin-EDTA.
For passaging spheres, the harvested spheres were trypsinized at 37°C for 5 min followed by quenching with FBS.
To further substantiate the notion that rhabdospheres are enriched for CSC, we quantified the expression levels of several known SC genes like oct4, nanog, c-myc, sox2 and pax3 with real-time PCR in different passages of sphere cultures (passages 3, 7, 10) compared to adherent cells.
For the first passage of sphere culture, 1 × 10 cancer cells were seeded into a T25 flask with 6 ml of sphere culture medium.
As we continue to passage this sphere (or population of cells) in this manner we would observe a 1-fold expansion, which is represented by a flat growth curve as seen in Figure 1a(i).
We were also able to passage the sphere generated from a single CD44+CD54+ cell several times in vitro, suggesting that rectospheres originated from a CD44+CD54+ single cell, rather than having developed by mere cell aggregation.
