Author Contributions
Conceptualization, M.R., A.Z. and G.F.; methodology, F.F. and M.L.D.A.; software, S.M. and V.D.; validation, E.P., X.L. and R.C.; formal analysis, M.R. and E.P.; investigation, M.R., E.P., S.M. and A.B.; resources, G.F.; data curation, M.R., G.F. and V.D.; writing—original draft preparation, M.R., S.M., F.F. and M.L.D.A.; writing—review and editing, M.R., A.Z. and G.F.; visualization, A.B.; supervision, A.Z. and G.F.; project administration, G.F. and M.R.; funding acquisition, G.F. and M.R., All authors have read and agreed to the published version of the manuscript.
Figure 1.
(A) Light microscopy H&E 100×. Histopathological examination shows the adenocarcinoma being poorly differentiated/high grade with cribriform architecture. (B) Light microscopy H&E 200×. the picture evidences the presence of juxtaposed gland lumens without stroma in between. (C) Light microscopy H&E 400×, at higher magnification loss of cell polarity is visible.
Figure 1.
(A) Light microscopy H&E 100×. Histopathological examination shows the adenocarcinoma being poorly differentiated/high grade with cribriform architecture. (B) Light microscopy H&E 200×. the picture evidences the presence of juxtaposed gland lumens without stroma in between. (C) Light microscopy H&E 400×, at higher magnification loss of cell polarity is visible.
Figure 2.
SEM micrographs of spheroids. (A) two spheroids are imagined at 700×, the right one has a smooth surface, the left one shows an entire cell emerging from the cell mass; c: bulging cell; (B) spheroid surface is irregular due to bulging cells, c, 1000×; (C) detailed image of spheroid surface (3000×) shows blebs, filled arrow and microvilli, empty arrow; (D) deep sulci delimit cells’ borders. Cells surface presents, several short processes, microvilli-like, empty arrow and pseudopodia-like, dotted arrow, together with single or multiple blebs, filled arrow.
Figure 2.
SEM micrographs of spheroids. (A) two spheroids are imagined at 700×, the right one has a smooth surface, the left one shows an entire cell emerging from the cell mass; c: bulging cell; (B) spheroid surface is irregular due to bulging cells, c, 1000×; (C) detailed image of spheroid surface (3000×) shows blebs, filled arrow and microvilli, empty arrow; (D) deep sulci delimit cells’ borders. Cells surface presents, several short processes, microvilli-like, empty arrow and pseudopodia-like, dotted arrow, together with single or multiple blebs, filled arrow.
Figure 3.
Light microscopy observations of spheroids’ semithin sections. Methylene blue staining. (A) Compact spheroid with several mitotic figures, arrows, cell nuclei appear dysmorphic, 400×. (B) In this spheroid cells are arranged circularly, forming an aberrant colonic gland, (asterisk in the lumen) 400×. (C) At high magnification, cells appear undifferentiated, with large nuclei, pale euchromatin and prominent nucleoli, 1000×. (D) Detail of an aberrant colonic gland, cells are arranged circularly around a central lumen, a brush border is visible, cells are differentiated as enterocyte-like cells, 1000×.
Figure 3.
Light microscopy observations of spheroids’ semithin sections. Methylene blue staining. (A) Compact spheroid with several mitotic figures, arrows, cell nuclei appear dysmorphic, 400×. (B) In this spheroid cells are arranged circularly, forming an aberrant colonic gland, (asterisk in the lumen) 400×. (C) At high magnification, cells appear undifferentiated, with large nuclei, pale euchromatin and prominent nucleoli, 1000×. (D) Detail of an aberrant colonic gland, cells are arranged circularly around a central lumen, a brush border is visible, cells are differentiated as enterocyte-like cells, 1000×.
Figure 4.
Light microscopy observations of xenograft semi-thin sections. Methylene blue staining. (A) In the upper part of the image cells form an aberrant gland (asterisk), in the center of the picture a nerve (n) and a blood vessel (v) are visible. Very scarce stroma is present, 400×. (B) Multiple aberrant glands (asterisks) are shown, they are separated by very scarce stroma, 400×. (C) At higher magnification, dysmorphic nuclei of cells delimiting an aberrant gland (asterisk) are evident, chromatin appears well stained and nucleoli are large. (D) Detail of an aberrant gland, enterocyte-like cells (on the right), less differentiated cells (on the left) and a mitotic figure (arrow) line the lumen.
Figure 4.
Light microscopy observations of xenograft semi-thin sections. Methylene blue staining. (A) In the upper part of the image cells form an aberrant gland (asterisk), in the center of the picture a nerve (n) and a blood vessel (v) are visible. Very scarce stroma is present, 400×. (B) Multiple aberrant glands (asterisks) are shown, they are separated by very scarce stroma, 400×. (C) At higher magnification, dysmorphic nuclei of cells delimiting an aberrant gland (asterisk) are evident, chromatin appears well stained and nucleoli are large. (D) Detail of an aberrant gland, enterocyte-like cells (on the right), less differentiated cells (on the left) and a mitotic figure (arrow) line the lumen.
Figure 5.
Transmission electron microscopy images of spheroids cell types. (A) Type-A cell, large nucleus, oval shape very finely dispersed chromatin, small cytoplasm amount, poor of organelles, 16,000×. (B) Cluster of type B cells, the cytoplasm is more abundant than in type A, presence of RER, 10,500×. (C) Group of type C cells, intercellular spaces are visible, in which cells protrusions develop. Numerous mitochondria characterize the cytoplasm. 14,000×. (D) A type D cell, enterocyte-like is observed. It has microvilli on the apical pole and a large nucleus (with two prominent nucleoli) in the basal part. Intercellular spaces separate this cell from its surroundings. Altered junctional complexes in the lateral domain and the absence of basal binding complexes are observed 16,000×. (E) Type E cell is represented. It has a large nucleus, a nucleolus, cytoplasm with RER and mitochondria, its peculiar characteristic is the presence of lipid droplets in the cytoplasm, 14,000×.
Figure 5.
Transmission electron microscopy images of spheroids cell types. (A) Type-A cell, large nucleus, oval shape very finely dispersed chromatin, small cytoplasm amount, poor of organelles, 16,000×. (B) Cluster of type B cells, the cytoplasm is more abundant than in type A, presence of RER, 10,500×. (C) Group of type C cells, intercellular spaces are visible, in which cells protrusions develop. Numerous mitochondria characterize the cytoplasm. 14,000×. (D) A type D cell, enterocyte-like is observed. It has microvilli on the apical pole and a large nucleus (with two prominent nucleoli) in the basal part. Intercellular spaces separate this cell from its surroundings. Altered junctional complexes in the lateral domain and the absence of basal binding complexes are observed 16,000×. (E) Type E cell is represented. It has a large nucleus, a nucleolus, cytoplasm with RER and mitochondria, its peculiar characteristic is the presence of lipid droplets in the cytoplasm, 14,000×.
Figure 6.
Transmission electron microscopy images of xenograft cell types. (A) Type-A cell (asterisk), among Type-D cells, 16,000×. (B) Cluster of type B cells, the cytoplasm is more abundant than in type A, presence of RER, 10,500×. (C) A type C cells, loosely attached to its surroundings, the cell has an irregularly columnar shape. Numerous mitochondria and abundant RER characterize the cytoplasm. 14,000×. (D) Type-D cells, enterocyte-like, are observed. A longitudinally sectioned cell is visible; it shows microvilli on the apical pole and numerous mitochondria in the apical cytoplasm. It has a large nucleus (with chromatin aggregates along the nuclear membrane) in the basal part. It is strictly adherent to its surroundings, but it does not lie on a basal membrane. 16,000×. (E) Cluster of type-E cells are represented. They have a large nucleus, a nucleolus, cytoplasm with RER and a variable amount of mitochondria, abundant and lipid-filled cytoplasmic droplets characterize this cell type, 14,000×.
Figure 6.
Transmission electron microscopy images of xenograft cell types. (A) Type-A cell (asterisk), among Type-D cells, 16,000×. (B) Cluster of type B cells, the cytoplasm is more abundant than in type A, presence of RER, 10,500×. (C) A type C cells, loosely attached to its surroundings, the cell has an irregularly columnar shape. Numerous mitochondria and abundant RER characterize the cytoplasm. 14,000×. (D) Type-D cells, enterocyte-like, are observed. A longitudinally sectioned cell is visible; it shows microvilli on the apical pole and numerous mitochondria in the apical cytoplasm. It has a large nucleus (with chromatin aggregates along the nuclear membrane) in the basal part. It is strictly adherent to its surroundings, but it does not lie on a basal membrane. 16,000×. (E) Cluster of type-E cells are represented. They have a large nucleus, a nucleolus, cytoplasm with RER and a variable amount of mitochondria, abundant and lipid-filled cytoplasmic droplets characterize this cell type, 14,000×.
Figure 7.
Both spheroids and xenograft cell types series were normally distributed. SA: Spheroid cell type A; SB: Spheroid cell type B; SC: Spheroid cell type C; SD: Spheroid cell type D; SE: Spheroid cell type E. XA: Xenograft cell type A; XB: Xenograft cell type B; XC: Xenograft cell type C; XD: Xenograft cell type D; XE: Xenograft cell type D.
Figure 7.
Both spheroids and xenograft cell types series were normally distributed. SA: Spheroid cell type A; SB: Spheroid cell type B; SC: Spheroid cell type C; SD: Spheroid cell type D; SE: Spheroid cell type E. XA: Xenograft cell type A; XB: Xenograft cell type B; XC: Xenograft cell type C; XD: Xenograft cell type D; XE: Xenograft cell type D.
Figure 8.
Phenotypic analysis by flow cytometry of spheroids and xenograft cells. (A) Flow cytometry analysis of CD133 expression of spheroids (upper panel) and CD133/EpCAM expression of xenograft (lower panel). (B) Flow cytometry analysis of CD44v6 expression of spheroids (upper) panel and CD44v6/EpCAM expression of xenograft (lower panel). (C) Flow cytometry analysis of TOP-GFP expression of spheroids (upper) panel and xenograft (lower panel).
Figure 8.
Phenotypic analysis by flow cytometry of spheroids and xenograft cells. (A) Flow cytometry analysis of CD133 expression of spheroids (upper panel) and CD133/EpCAM expression of xenograft (lower panel). (B) Flow cytometry analysis of CD44v6 expression of spheroids (upper) panel and CD44v6/EpCAM expression of xenograft (lower panel). (C) Flow cytometry analysis of TOP-GFP expression of spheroids (upper) panel and xenograft (lower panel).
Table 1.
Morphological parameters that are considered in cell type evaluation.
Table 1.
Morphological parameters that are considered in cell type evaluation.
Cell Compartment | Morphological Parameters |
---|
Membrane | Microvilli, apical binding complex; basal and lateral domains |
Cytoplasm | Types and morphology of organelles, filaments and inclusions. |
Nucleus | Shape, number of nucleoli; chromatin aspect |
Table 2.
This table shows summary statistics values for spheroids shape descriptors.
Table 2.
This table shows summary statistics values for spheroids shape descriptors.
Descriptors | Mean | Std. Error | 95% CI |
---|
Circularity | 0.94 | 0.006 | 0.92 to 0.95 |
Roundness | 0.87 | 0.008 | 0.86 to 0.89 |
Aspect Ratio | 1.17 | 0.02 | 1.12 to 1.22 |
Solidity | 0.99 | 0.001 | 0.98 to 0.99 |
Table 3.
This table shows summary statistics values for spheroids size parameters.
Table 3.
This table shows summary statistics values for spheroids size parameters.
Parameter | Mean | Std. Error | 95% CI |
---|
Area | 967.75 µm2 | 59.95 µm | 844.26 to 1091.24 µm |
Perimeter | 112.21 µm | 3.46 µm | 105.07 to 119.35 µm |
Feret diameter | 39.21 µm | 1.51 µm | 36.10 to 42.32 µm |
Min Feret diameter | 32.84 µm | 1.11 µm | 30.55 to 35.14 µm |
Table 4.
This table shows summary statistics values for spheroids and xenograft cell types.
Table 4.
This table shows summary statistics values for spheroids and xenograft cell types.
Factor | Mean | Std. Error | 95% CI |
---|
Spheroid cell type A | 7.1250 | 0.7425 | 5.3692 to 8.8808 |
Spheroid cell type B | 11.1250 | 1.7159 | 7.0676 to 15.1824 |
Spheroid cell type C | 10.3750 | 1.5691 | 6.6647 to 14.0853 |
Spheroid cell type D | 8.6250 | 1.4134 | 5.2828 to 11.9672 |
Spheroid cell type E | 1.2500 | 0.3660 | 0.3846 to 2.1154 |
Xenograft cell type A | 1.6250 | 0.3750 | 0.7383 to 2.5117 |
Xenograft cell type B | 9.6250 | 1.3879 | 6.3431 to 12.9069 |
Xenograft cell type C | 10.1250 | 1.5748 | 6.4013 to 13.8487 |
Xenograft cell type D | 10.1250 | 1.3016 | 7.0472 to 13.2028 |
Xenograft cell type E | 5.7500 | 0.4119 | 4.7761 to 6.7239 |
Table 5.
Repeated measures ANOVA-Test of Within-Subjects Effects.
Table 5.
Repeated measures ANOVA-Test of Within-Subjects Effects.
Source of Variation | Sum of Squares | DF | Mean Square | F | P |
---|
Sphericity assumed | 941,550 | 9 | 104,671 | 9.69 | <0.001 |
Greenhouse Geisser | 941,550 | 3.711 | 253,701 | 9.69 | <0.001 |
Huynh-Feldt | 941,550 | 8.420 | 111,827 | 9.69 | <0.001 |
Table 6.
Repeated measures ANOVA-Pairwise comparisons.
Table 6.
Repeated measures ANOVA-Pairwise comparisons.
Factor | Mean Difference | Std. Error | p | 95% CI |
---|
Spheroid cell type A vs. | Xenograft cell type A | 5.500 | 0.732 | 0.0061 | 1.613 to 9.387 |
Spheroid cell type B vs. | Xenograft cell type B | 1.500 | 2.619 | 1.0000 | −12.405 to 15.405 |
Spheroid cell type C vs. | Xenograft cell type C | 0.250 | 1.461 | 1.0000 | −7.507 to 8.007 |
Spheroid cell type D vs. | Xenograft cell type D | −1.500 | 1.195 | 1.0000 | −7.847 to 4.847 |
Spheroid cell type E vs. | Xenograft cell type E | −4.500 | 0.732 | 0.0211 | −8.387 to −0.613 |