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Ultrasonic Surgical Aspirate is a Reliable Source For Culturing Glioblastoma Stem Cells

View Article: PubMed Central - PubMed

ABSTRACT

Glioma stem cells (GSCs) are thought to be the source of tumor growth and therapy resistance. To understand the biology of GSCs, and target these tumors therapeutically, we need robust strategies for in vitro expansion of primary GSCs. To date, tumor core biopsies have been the main established source of GSCs. Since these samples are used for diagnostic purposes, the available tissue for cell culture and therapeutic targeting can be limited. In addition, a core biopsy is usually taken from one part of the tumor, thus would be unlikely to represent intra-tumor heterogeneity. To overcome these problems, tissue fragments from all over the tumor can be collected using an ultrasonic aspirator during surgery, thus assembling a “global tumor biopsy”. Usually, this ultrasonic aspirate (UA) sample is considered as biological waste after operations. Here, we show that UA samples offer a large and reliable source of live cells. Similar to core biopsies, UA samples enriched for GSCs that differentiated into neural lineages, showed inter-individual variation of GSC markers, and induced tumors. Molecular profiling showed that UA samples cover tumor heterogeneity better than core biopsies. These results suggest that UA samples can be used to establish large scale cultures for therapeutic applications.

No MeSH data available.


Related in: MedlinePlus

Gene expression profiling of the UA and Co samples.(A) Global profiling of gene expression. To visualize similarities or dissimilarities between Core (Co) and Ultrasonic aspirate (UA) samples, we used principal component analysis (PCA) of gene expression. In this 3D PCA plot each individual sample is represented with a ball of a certain colour. Principal components PC1, PC2 and PC3 were represented as axes 1, 2 and 3. While the PC1 separated spheres (right) from the fresh tissues (left) the second PC separated fresh tissue UA samples (dark blue) from the Fresh tissue Co samples (light blue) and the sphere (Sp) cultures expanded from UAs (darker shades of green) from the Sp cultures expanded from the core tissues (lighter shades of green). This was the case both for so-called “adherent” spheres and the spheres grown as “floating spheres”22. (B) Global PCA analysis according to the patient annotations. Individual sets of UA and Co samples were matched as they originated from the same patient. This was the case both for spheres (Sp) and Fresh tissues (Fr). The samples originating from core samples (Co) were clearly separated (pointing towards “north-east” from the samples originating from the ultrasound aspirates (UAs) (pointing towards “south-west”). This was the case both for fresh samples (right quadrants) and spheres (left quadrants). Each ball represents one sample. Each colour represents one patient. (C) Hierarchical clustering (HCL) plot showing subtyping of the sphere and fresh cultures according to the 12 gene signature22. Included in this analysis are: a) the new samples from this study (GSE84707) and b) several UA samples from the previous analysis22. The dendrogram colours red, blue and green specify mesenchymal, neural/proneural and classical subtypes, respectively as previously determined22. The expression values were log2 transformed. (D) HCL plot showing differential expression of the notch signalling pathway genes. Dendrogram colours light and dark blue specify core and UA samples respectively. The expression values were log2 transformed.
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f2: Gene expression profiling of the UA and Co samples.(A) Global profiling of gene expression. To visualize similarities or dissimilarities between Core (Co) and Ultrasonic aspirate (UA) samples, we used principal component analysis (PCA) of gene expression. In this 3D PCA plot each individual sample is represented with a ball of a certain colour. Principal components PC1, PC2 and PC3 were represented as axes 1, 2 and 3. While the PC1 separated spheres (right) from the fresh tissues (left) the second PC separated fresh tissue UA samples (dark blue) from the Fresh tissue Co samples (light blue) and the sphere (Sp) cultures expanded from UAs (darker shades of green) from the Sp cultures expanded from the core tissues (lighter shades of green). This was the case both for so-called “adherent” spheres and the spheres grown as “floating spheres”22. (B) Global PCA analysis according to the patient annotations. Individual sets of UA and Co samples were matched as they originated from the same patient. This was the case both for spheres (Sp) and Fresh tissues (Fr). The samples originating from core samples (Co) were clearly separated (pointing towards “north-east” from the samples originating from the ultrasound aspirates (UAs) (pointing towards “south-west”). This was the case both for fresh samples (right quadrants) and spheres (left quadrants). Each ball represents one sample. Each colour represents one patient. (C) Hierarchical clustering (HCL) plot showing subtyping of the sphere and fresh cultures according to the 12 gene signature22. Included in this analysis are: a) the new samples from this study (GSE84707) and b) several UA samples from the previous analysis22. The dendrogram colours red, blue and green specify mesenchymal, neural/proneural and classical subtypes, respectively as previously determined22. The expression values were log2 transformed. (D) HCL plot showing differential expression of the notch signalling pathway genes. Dendrogram colours light and dark blue specify core and UA samples respectively. The expression values were log2 transformed.

Mentions: To visualize the differences and compare UA to core biopsy samples we used principal component analysis (PCA). Using PCA to visualize global gene expression we could detect constellations such as: Fresh tissue (Fr) cluster (represented in two shades of blue) and Sphere culture (Sp) cluster (represented in different shades of green) that could be clearly separated from one another by the first principal component on the 3D PCA plot (Fig. 2A). Furthermore, the fresh tissue core samples (light blue) were grouped apart from the fresh UA tissue samples (dark blue) indicating significant differences in the gene expression. Also the sphere samples generated from either the UAs or the tissue cores could be separated by the second principal component on the 3D PCA plot. In conclusion, the PC1 separated spheres from fresh tissues while the PC2 roughly separated UA samples from Core-samples.


Ultrasonic Surgical Aspirate is a Reliable Source For Culturing Glioblastoma Stem Cells
Gene expression profiling of the UA and Co samples.(A) Global profiling of gene expression. To visualize similarities or dissimilarities between Core (Co) and Ultrasonic aspirate (UA) samples, we used principal component analysis (PCA) of gene expression. In this 3D PCA plot each individual sample is represented with a ball of a certain colour. Principal components PC1, PC2 and PC3 were represented as axes 1, 2 and 3. While the PC1 separated spheres (right) from the fresh tissues (left) the second PC separated fresh tissue UA samples (dark blue) from the Fresh tissue Co samples (light blue) and the sphere (Sp) cultures expanded from UAs (darker shades of green) from the Sp cultures expanded from the core tissues (lighter shades of green). This was the case both for so-called “adherent” spheres and the spheres grown as “floating spheres”22. (B) Global PCA analysis according to the patient annotations. Individual sets of UA and Co samples were matched as they originated from the same patient. This was the case both for spheres (Sp) and Fresh tissues (Fr). The samples originating from core samples (Co) were clearly separated (pointing towards “north-east” from the samples originating from the ultrasound aspirates (UAs) (pointing towards “south-west”). This was the case both for fresh samples (right quadrants) and spheres (left quadrants). Each ball represents one sample. Each colour represents one patient. (C) Hierarchical clustering (HCL) plot showing subtyping of the sphere and fresh cultures according to the 12 gene signature22. Included in this analysis are: a) the new samples from this study (GSE84707) and b) several UA samples from the previous analysis22. The dendrogram colours red, blue and green specify mesenchymal, neural/proneural and classical subtypes, respectively as previously determined22. The expression values were log2 transformed. (D) HCL plot showing differential expression of the notch signalling pathway genes. Dendrogram colours light and dark blue specify core and UA samples respectively. The expression values were log2 transformed.
© Copyright Policy - open-access
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC5015049&req=5

f2: Gene expression profiling of the UA and Co samples.(A) Global profiling of gene expression. To visualize similarities or dissimilarities between Core (Co) and Ultrasonic aspirate (UA) samples, we used principal component analysis (PCA) of gene expression. In this 3D PCA plot each individual sample is represented with a ball of a certain colour. Principal components PC1, PC2 and PC3 were represented as axes 1, 2 and 3. While the PC1 separated spheres (right) from the fresh tissues (left) the second PC separated fresh tissue UA samples (dark blue) from the Fresh tissue Co samples (light blue) and the sphere (Sp) cultures expanded from UAs (darker shades of green) from the Sp cultures expanded from the core tissues (lighter shades of green). This was the case both for so-called “adherent” spheres and the spheres grown as “floating spheres”22. (B) Global PCA analysis according to the patient annotations. Individual sets of UA and Co samples were matched as they originated from the same patient. This was the case both for spheres (Sp) and Fresh tissues (Fr). The samples originating from core samples (Co) were clearly separated (pointing towards “north-east” from the samples originating from the ultrasound aspirates (UAs) (pointing towards “south-west”). This was the case both for fresh samples (right quadrants) and spheres (left quadrants). Each ball represents one sample. Each colour represents one patient. (C) Hierarchical clustering (HCL) plot showing subtyping of the sphere and fresh cultures according to the 12 gene signature22. Included in this analysis are: a) the new samples from this study (GSE84707) and b) several UA samples from the previous analysis22. The dendrogram colours red, blue and green specify mesenchymal, neural/proneural and classical subtypes, respectively as previously determined22. The expression values were log2 transformed. (D) HCL plot showing differential expression of the notch signalling pathway genes. Dendrogram colours light and dark blue specify core and UA samples respectively. The expression values were log2 transformed.
Mentions: To visualize the differences and compare UA to core biopsy samples we used principal component analysis (PCA). Using PCA to visualize global gene expression we could detect constellations such as: Fresh tissue (Fr) cluster (represented in two shades of blue) and Sphere culture (Sp) cluster (represented in different shades of green) that could be clearly separated from one another by the first principal component on the 3D PCA plot (Fig. 2A). Furthermore, the fresh tissue core samples (light blue) were grouped apart from the fresh UA tissue samples (dark blue) indicating significant differences in the gene expression. Also the sphere samples generated from either the UAs or the tissue cores could be separated by the second principal component on the 3D PCA plot. In conclusion, the PC1 separated spheres from fresh tissues while the PC2 roughly separated UA samples from Core-samples.

View Article: PubMed Central - PubMed

ABSTRACT

Glioma stem cells (GSCs) are thought to be the source of tumor growth and therapy resistance. To understand the biology of GSCs, and target these tumors therapeutically, we need robust strategies for in vitro expansion of primary GSCs. To date, tumor core biopsies have been the main established source of GSCs. Since these samples are used for diagnostic purposes, the available tissue for cell culture and therapeutic targeting can be limited. In addition, a core biopsy is usually taken from one part of the tumor, thus would be unlikely to represent intra-tumor heterogeneity. To overcome these problems, tissue fragments from all over the tumor can be collected using an ultrasonic aspirator during surgery, thus assembling a “global tumor biopsy”. Usually, this ultrasonic aspirate (UA) sample is considered as biological waste after operations. Here, we show that UA samples offer a large and reliable source of live cells. Similar to core biopsies, UA samples enriched for GSCs that differentiated into neural lineages, showed inter-individual variation of GSC markers, and induced tumors. Molecular profiling showed that UA samples cover tumor heterogeneity better than core biopsies. These results suggest that UA samples can be used to establish large scale cultures for therapeutic applications.

No MeSH data available.


Related in: MedlinePlus