Fig 1.
Cross sections of various types of SQ20B spheroid.
Spheroids from tumor-cells only were very small (upper left), those from mixed-cells (with MRC5) somewhat larger (lower left) and from tumor-fragments much larger still (right; all nuclei stained with Hoechst 33342). The larger TF spheroids developed central necrosis (absent or pyknotic nuclei) with a relatively thin rim of viable cells. The scale bars represent 100 μm for the two left panels and 250 μm for the right panel.
Fig 2.
Freshly-cut tumor fragments from SQ20B tumor (left panel) formed spheroids within 2–3 days (right panel). (representative of several experiments—scale bar is 1000 μm).
Table 1.
Characteristics of cells and spheroids used in this study.
Fig 3.
Stromal cells in TF spheroids.
6-day TF spheroids retained host tissue components, some of which migrate to characteristic positions in the spheroid. Macrophages (CD11B positive) migrated to the periphery (upper left), and, as in this example sometimes comprised a sizeable fraction of the total cells (indicated by Hoechst 33342 staining of nuclei) (upper right). CD31 positive endothelial cells remain grouped together (lower left) while vimentin positive cells tended to congregate within and around the central hypoxic/necrotic zone as well as the periphery (lower right. (representative of several TF spheroids from 2 preparations—scale bar is 250 μm).
Fig 4.
Cell cycling and EF5 binding in SQ20B TF spheroids.
Cycling Ki67 positive cells were peripherally localized (central panel), in contrast to the inverse location of EF5 binding cells (right panel). Hoechst 33342 staining of all cell nuclei shown in the left panel. For the larger, more necrotic TF spheroids (small portion seen in upper left of each panel), the EF5 positive cells lined the inside of the viable rim, since EF5 is only metabolized by viable cells (scale bar 250 μm). Ki67 positive cells were also found peripherally in U87 spheroids of all types, but no EF5 binding was observed (data not shown).
Fig 5.
CD11b cells in SQ20B subcutaneous tumors.
CD11b positive cells were minimally found in the tumor interior (left panel). Total cells illustrated in the right panel, showing Hoechst 33342 nuclear staining (scale bar 0.5 mm). The lower density of nuclei along the left edge of the section showed the transition from tumor to normal tissue.
Fig 6.
MRC5 fibroblasts form a surface capsule in MC spheroids.
In 10-day mixed cell spheroids, vimentin positive fibroblasts migrated to form a surface capsule (left U87+MRC5; right Panc1+MRC5—scale bar 0.25 mm). No surface vimentin staining was observed for spheroids consisting solely of either tumor cell type (data not shown).
Fig 7.
SQ20B tumors contained EF5 binding (upper panel) which was inversely correlated with the position of blood vessels (lower panel). (scale bar 500 μm).
Fig 8.
U87 tumors had a much higher density of vessels (upper panel) than did SQ20B tumors which explains their lack of EF5 binding. The lack of hypoxia was associated with a relatively uniform distributions of macrophages (CD11b positive; lower panel) but it is not known if this was causally associated (scale bar 500 μm).
Fig 9.
Respiration rates of U87 versus SQ20B cells.
The respiration rate of U87 cells (closed squares) was about one third that of SQ20B cells (open circles). pO2 was monitored as a function of time in a closed, stirred 10.0 ml vial containing medium at 37°. Cell numbers were similar for this experiment, but cells were added based on their pellet weight to avoid counting issues related to multiplicity/cell-clumping. Thus, each vial contained 10 mg of cell mass. These data are representative of 3 experiments, giving respiration rates for U87 and SQ20B cells (respectively) of 2.7±0.5 and 8.6±3.2 (x10^-17M/cell/sec).
Fig 10.
Microvesicles from tissue culture medium 24 hr following medium change to SQ20B cells.
Typical output from the flow cytometer showed two particle populations. Those with the polygon surrounding them had high phosphotidyl-serine content, as measured by Pacific Blue labeled Annexin V, while the rest did not stain for this surface marker: left panel, low speed supernatant, right panel medium speed pellet. There were eight-fold more MV in the pellet, which was expected based on the final volumes assuming collection of almost all MV. The fraction of Annexin V positive particles was about 0.09 for the left panel and 0.18 for the right panel.
Fig 11.
Quantification of MV production per 1000 SQ20B cells over a 24 hr period under four conditions of growth: LDC—low-density cells; HDC—high-density cells; SPH; tumor-cell spheroids; TFS—TF spheroids (error bars SD of 3 replicates).
Table 2.
Ordinal ranking of most abundant SQ20B cellular miRNA’s and corresponding ranks for SQ20B tumor and its derived TFS (6-day).