Fig 1.
General characteristics of tumor spheres formed from multiple cell lines.
A. Significant heterogeneity existed in the size of TS as shown by representative pictures of TS from H1299 and MCF7 cultured in TS medium for 8 days. Counting of smaller TS (solid arrow) was sometimes prone to subjectiveness. TS derived from certain cell lines (HCT116) might have strong tendency to fuse (hollow arrow). B. “size of spheres” for H1299 TS showed a skewed distribution with smaller spheres account for the majority of population. H1299 cells were plated in 24-well ULA plate (2000 per well in 2ml of TS medium) to allow TS formation for 4 days before microscopic assessment. The results were presented as Mean ± SEM from 6 replicates. C. Some tumor cell lines formed typical 3-dimensional spherical structures with sharp edges (A), while others were not, or require special supplement (hollow arrow).
Fig 2.
H1299 cells were cultured in TS forming condition for 8 days, before being harvested, fixed, paraffin embedded, sectioned, followed by H&E staining to show how single tumor cells were packed in TS.
Fig 3.
A general protocol for SPF calculation using H1299 TS.
Step 1: Spheres were harvested and seeded individually in each well. Step 2: The diameter of spheres was measured by computer-based imaging software, and documented. Note that the three labels in red are software generated measurement of distance between centers of two spheres (223.08 μm), radius of left sphere (165.45 μm) and radius of right sphere (248.03 μm), respectively (from left to right). Step 3: The volume of each sphere was calculated based on measured diameter obtained from step 2. Step 4: Spheres were disassociated enzymatically and mechanically to make single cell suspension, followed by precise cell counting by a cytometry. Meanwhile, the diameter of single floating cells from spheres was measured. Step 5: SPF was calculated by an indicated formula.
Fig 4.
Standardized Sphere Score (SSS) linearity validation.
A. H1299 cells (plating cell number was 1600 × 0.5i,i = 0,1,2…5 per well) were plated in TS medium in 24-well ULA plates for 8 days to allow for formation of TS. Number of spheres (n) and SSS were then measured and calculated. The results were presented with SSS×N and n as the Y-axis, and N as the X-axis. B. MCF7 cells (plating cell number was 3200 × 0.5i,i = 0,1,2…5, per well) were plated in TS medium supplemented with 1× B27 in 24-well ULA plates for 8 days to allow for formation of TS. Number of spheres (n) and SSS were then measured and calculated. The results were presented with SSS×N and n as the Y-axis, and N as the X-axis. C-F. mESC (plating cell number was 4000*0.8i,i = 0,1,2…11, per well) were plated in 24-well ULA plates in DMEM supplemented with LIF and, respectively, with 15% FBS (C), 3% FBS (D, E), and 1.5% FBS (F) for up to 8 days, to allow the formation of EB. Number of EBs (n) and SSS were similarly obtained. Since the numerical value of n is much smaller than that of SSS×N, the plots of n against N appeared to be very close to the X-axis. Each experiment was repeated at least 3 times.
Fig 5.
Dose dependent inhibition of sphere formation by anticancer drugs.
To compare two sphere scoring parameters of the SSS vs. “number of spheres”, H1299 (2000 cells per well) were plated in 24-well ULA plates in TS medium supplemented with indicated drugs or vehicle for 4 days. %SSSi and %ni were then calculated. Concentration of drugs were as the following: (A) CI-1033: + 2 μM; (B) Erlotinib: + 3 μM, ++ 6 μM, +++ 10 μM; (C) MK2206: + 0.25 μM, ++ 0.5 μM, +++ 1 μM; (D) Perifosine: + 1 μM, ++ 3 μM, +++ 5 μM; and (E) BEZ235: + 25 nM, ++ 125 nM, +++ 250 nM. The results were presented as Mean ± SEM.
Table 1.
Pros and cons of SSS in assessing sphere formation assay.