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Isolation and characterization of squamous cell carcinoma-derived stem-like cells: role in tumor formation.

Dallaglio K, Petrachi T, Marconi A, Truzzi F, Lotti R, Saltari A, Morandi P, Puviani M, Maiorana A, Roop DR, Pincelli C - Int J Mol Sci (2013)

Bottom Line: RAD cells expressed higher levels of survivin, a KSC marker, as compared to non-rapidly adhering (NRAD) cells.Moreover, RAD cells proliferated to a greater extent and were more efficient in forming colonies than NRAD cells.RAD cells also migrated significantly better than NRAD cells.

View Article: PubMed Central - PubMed

Affiliation: Department of Dermatology, University of Modena and Reggio Emilia, Via del Pozzo 71, 41124 Modena, Italy. carlo.pincelli@unimore.it.

ABSTRACT
In human epidermis, keratinocyte stem cells (KSC) are characterized by high levels of β1-integrin, resulting in the rapid adhesion to type IV collagen. Since epithelial tumors originate from KSC, we evaluated the features of rapidly adhering (RAD) keratinocytes derived from primary human squamous cell carcinoma of the skin (cSCC). RAD cells expressed higher levels of survivin, a KSC marker, as compared to non-rapidly adhering (NRAD) cells. Moreover, RAD cells proliferated to a greater extent and were more efficient in forming colonies than NRAD cells. RAD cells also migrated significantly better than NRAD cells. When seeded in a silicone chamber and grafted onto the back skin of NOD SCID mice, RAD cells formed tumors 2-4 fold bigger than those derived from NRAD cells. In tumors derived from RAD cells, the mitotic index was significantly higher than in those derived from NRAD cells, while Ki-67 and survivin expression were more pronounced in RAD tumors. This study suggests that SCC RAD stem cells play a critical role in the formation and development of epithelial tumors.

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Related in: MedlinePlus

Analysis of stem-cell features in RAD and NRAD cells in vitro. (A) Clonal growth assessment of cSCC subpopulations by CFE. CFE was performed in triplicate in three independent experiments and quantification is shown in the upper panel. At the bottom, representative pictures of CFE obtained by growing cells at clonal density and stained with CV are shown; (B) mRNA expression of Nanog, Oct4 and Sox-2 by Real Time PCR. The different levels of gene expression in RAD vs. NRAD cells are shown. *p < 0.05; **p < 0.01.
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f4-ijms-14-19540: Analysis of stem-cell features in RAD and NRAD cells in vitro. (A) Clonal growth assessment of cSCC subpopulations by CFE. CFE was performed in triplicate in three independent experiments and quantification is shown in the upper panel. At the bottom, representative pictures of CFE obtained by growing cells at clonal density and stained with CV are shown; (B) mRNA expression of Nanog, Oct4 and Sox-2 by Real Time PCR. The different levels of gene expression in RAD vs. NRAD cells are shown. *p < 0.05; **p < 0.01.

Mentions: Colony forming efficiency (CFE) assay assesses the capability of cells to generate progeny. It has been employed to evaluate clonogenic ability of cancer cell subtypes and as a surrogate to analyze putative enrichments of stem cell-like cells [16,31]. CFE analysis of cells re-plated at clonal density immediately after separation showed that RAD cells have a significantly higher CFE as compared to NRAD and total cells (Figure 4A). This is in line with the highest total cell output and proliferation observed in RAD cells, as shown in Figure 2. In addition, the stem cell markers Nanog, Oct4 and Sox-2 were significantly more expressed in RAD cells, further confirming the stem cell nature of these cSCC cells (Figure 4B). Interestingly, the transcription factor Sox-2 directly increases survivin levels in neural stem cells [26], suggesting that it may contribute to sustain RAD stemness also by upregulating survivin in cSCC cells.


Isolation and characterization of squamous cell carcinoma-derived stem-like cells: role in tumor formation.

Dallaglio K, Petrachi T, Marconi A, Truzzi F, Lotti R, Saltari A, Morandi P, Puviani M, Maiorana A, Roop DR, Pincelli C - Int J Mol Sci (2013)

Analysis of stem-cell features in RAD and NRAD cells in vitro. (A) Clonal growth assessment of cSCC subpopulations by CFE. CFE was performed in triplicate in three independent experiments and quantification is shown in the upper panel. At the bottom, representative pictures of CFE obtained by growing cells at clonal density and stained with CV are shown; (B) mRNA expression of Nanog, Oct4 and Sox-2 by Real Time PCR. The different levels of gene expression in RAD vs. NRAD cells are shown. *p < 0.05; **p < 0.01.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3821572&req=5

f4-ijms-14-19540: Analysis of stem-cell features in RAD and NRAD cells in vitro. (A) Clonal growth assessment of cSCC subpopulations by CFE. CFE was performed in triplicate in three independent experiments and quantification is shown in the upper panel. At the bottom, representative pictures of CFE obtained by growing cells at clonal density and stained with CV are shown; (B) mRNA expression of Nanog, Oct4 and Sox-2 by Real Time PCR. The different levels of gene expression in RAD vs. NRAD cells are shown. *p < 0.05; **p < 0.01.
Mentions: Colony forming efficiency (CFE) assay assesses the capability of cells to generate progeny. It has been employed to evaluate clonogenic ability of cancer cell subtypes and as a surrogate to analyze putative enrichments of stem cell-like cells [16,31]. CFE analysis of cells re-plated at clonal density immediately after separation showed that RAD cells have a significantly higher CFE as compared to NRAD and total cells (Figure 4A). This is in line with the highest total cell output and proliferation observed in RAD cells, as shown in Figure 2. In addition, the stem cell markers Nanog, Oct4 and Sox-2 were significantly more expressed in RAD cells, further confirming the stem cell nature of these cSCC cells (Figure 4B). Interestingly, the transcription factor Sox-2 directly increases survivin levels in neural stem cells [26], suggesting that it may contribute to sustain RAD stemness also by upregulating survivin in cSCC cells.

Bottom Line: RAD cells expressed higher levels of survivin, a KSC marker, as compared to non-rapidly adhering (NRAD) cells.Moreover, RAD cells proliferated to a greater extent and were more efficient in forming colonies than NRAD cells.RAD cells also migrated significantly better than NRAD cells.

View Article: PubMed Central - PubMed

Affiliation: Department of Dermatology, University of Modena and Reggio Emilia, Via del Pozzo 71, 41124 Modena, Italy. carlo.pincelli@unimore.it.

ABSTRACT
In human epidermis, keratinocyte stem cells (KSC) are characterized by high levels of β1-integrin, resulting in the rapid adhesion to type IV collagen. Since epithelial tumors originate from KSC, we evaluated the features of rapidly adhering (RAD) keratinocytes derived from primary human squamous cell carcinoma of the skin (cSCC). RAD cells expressed higher levels of survivin, a KSC marker, as compared to non-rapidly adhering (NRAD) cells. Moreover, RAD cells proliferated to a greater extent and were more efficient in forming colonies than NRAD cells. RAD cells also migrated significantly better than NRAD cells. When seeded in a silicone chamber and grafted onto the back skin of NOD SCID mice, RAD cells formed tumors 2-4 fold bigger than those derived from NRAD cells. In tumors derived from RAD cells, the mitotic index was significantly higher than in those derived from NRAD cells, while Ki-67 and survivin expression were more pronounced in RAD tumors. This study suggests that SCC RAD stem cells play a critical role in the formation and development of epithelial tumors.

Show MeSH
Related in: MedlinePlus