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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.

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Related in: MedlinePlus

Targeted NGS analysis showing that UA samples harbor more mutations than core biopsies in most sequenced samples.Sequencing analysis was performed on the target sequenced genes. Amplicon sequences were aligned to the human reference genome GRCh37 (hg19) in the target region of our custom gene panel using Torrent Suite™ software 5.0. SNVs calling was performed using the Variant Caller (v5.0.4.0). Three primary GBM and one secondary GBM, in addition to one normal sample (epileptic patient) were inlcuded in this analysis. Most common mutations were NF1, NOTCH1, ATRX and NF2, and more mutations were detected in UA samples that core biopsies. However, core biopsies expressed some mutations that were absent in UA samples (not more than one mutation usually). In one case, secondary GBM (T1311), core biopsy expressed more mutations than the UA. The legend at the top of the heat map is color-coded for the following variant impacts using the associated score values (unknown: 0, synonymous: 1, missense: 2, non-frameshift block substitution: 3 non-frameshift insertion: 4,nonsense: 5, stop-loss: 6, frameshift block substitution: 7, frameshift insertion: 7, frameshift deletion: 7, splice variant: 8.
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f3: Targeted NGS analysis showing that UA samples harbor more mutations than core biopsies in most sequenced samples.Sequencing analysis was performed on the target sequenced genes. Amplicon sequences were aligned to the human reference genome GRCh37 (hg19) in the target region of our custom gene panel using Torrent Suite™ software 5.0. SNVs calling was performed using the Variant Caller (v5.0.4.0). Three primary GBM and one secondary GBM, in addition to one normal sample (epileptic patient) were inlcuded in this analysis. Most common mutations were NF1, NOTCH1, ATRX and NF2, and more mutations were detected in UA samples that core biopsies. However, core biopsies expressed some mutations that were absent in UA samples (not more than one mutation usually). In one case, secondary GBM (T1311), core biopsy expressed more mutations than the UA. The legend at the top of the heat map is color-coded for the following variant impacts using the associated score values (unknown: 0, synonymous: 1, missense: 2, non-frameshift block substitution: 3 non-frameshift insertion: 4,nonsense: 5, stop-loss: 6, frameshift block substitution: 7, frameshift insertion: 7, frameshift deletion: 7, splice variant: 8.

Mentions: To check for the differences between UA and core biopsy samples on a mutational level, we performed targeted next generation sequencing (NGS). Our panel included 29 genes that were frequently reported to be mutant in adult human gliomas, pediatric gliomas and neurofibromatoses or schwannomatosis (Supplementary Table S4). We found variation in the mutational load among patients. most common mutations were (NF1, NOTCH1, ATRX, NF2). Within single patients, comparing UA to core biopsy from fresh tissue and cultured cells, there were 1-4 mutations shared among those samples, while many mutations were expressed in UA samples and absent in core biopsies, none or only one mutation was expressed in core samples and absent in UA samples (Fig. 3) and (Supplementary Table S5). For example, targeted NGS analysis of the patient T1349 show that fresh UA sample contains more mutations than core biopsy, four mutations were detected in UA sample, but not in the core biopsy. Also sphere culture derived from this sample expressed three of those four mutations, while the one derived from the core biopsy expressed only the common mutation in this patient (NF1). In total, three out of four patients that were sequenced, expressed more mutations in their UA sample compared to their core biopsy. Only the tumor core of secondary GBM (T1311) expressed more mutations than UA counterpart and the sphere culture derived from this core biopsy expressed more mutations than the one derived from the UA sample (Fig. 3) and (Supplementary Table S5).


Ultrasonic Surgical Aspirate is a Reliable Source For Culturing Glioblastoma Stem Cells
Targeted NGS analysis showing that UA samples harbor more mutations than core biopsies in most sequenced samples.Sequencing analysis was performed on the target sequenced genes. Amplicon sequences were aligned to the human reference genome GRCh37 (hg19) in the target region of our custom gene panel using Torrent Suite™ software 5.0. SNVs calling was performed using the Variant Caller (v5.0.4.0). Three primary GBM and one secondary GBM, in addition to one normal sample (epileptic patient) were inlcuded in this analysis. Most common mutations were NF1, NOTCH1, ATRX and NF2, and more mutations were detected in UA samples that core biopsies. However, core biopsies expressed some mutations that were absent in UA samples (not more than one mutation usually). In one case, secondary GBM (T1311), core biopsy expressed more mutations than the UA. The legend at the top of the heat map is color-coded for the following variant impacts using the associated score values (unknown: 0, synonymous: 1, missense: 2, non-frameshift block substitution: 3 non-frameshift insertion: 4,nonsense: 5, stop-loss: 6, frameshift block substitution: 7, frameshift insertion: 7, frameshift deletion: 7, splice variant: 8.
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f3: Targeted NGS analysis showing that UA samples harbor more mutations than core biopsies in most sequenced samples.Sequencing analysis was performed on the target sequenced genes. Amplicon sequences were aligned to the human reference genome GRCh37 (hg19) in the target region of our custom gene panel using Torrent Suite™ software 5.0. SNVs calling was performed using the Variant Caller (v5.0.4.0). Three primary GBM and one secondary GBM, in addition to one normal sample (epileptic patient) were inlcuded in this analysis. Most common mutations were NF1, NOTCH1, ATRX and NF2, and more mutations were detected in UA samples that core biopsies. However, core biopsies expressed some mutations that were absent in UA samples (not more than one mutation usually). In one case, secondary GBM (T1311), core biopsy expressed more mutations than the UA. The legend at the top of the heat map is color-coded for the following variant impacts using the associated score values (unknown: 0, synonymous: 1, missense: 2, non-frameshift block substitution: 3 non-frameshift insertion: 4,nonsense: 5, stop-loss: 6, frameshift block substitution: 7, frameshift insertion: 7, frameshift deletion: 7, splice variant: 8.
Mentions: To check for the differences between UA and core biopsy samples on a mutational level, we performed targeted next generation sequencing (NGS). Our panel included 29 genes that were frequently reported to be mutant in adult human gliomas, pediatric gliomas and neurofibromatoses or schwannomatosis (Supplementary Table S4). We found variation in the mutational load among patients. most common mutations were (NF1, NOTCH1, ATRX, NF2). Within single patients, comparing UA to core biopsy from fresh tissue and cultured cells, there were 1-4 mutations shared among those samples, while many mutations were expressed in UA samples and absent in core biopsies, none or only one mutation was expressed in core samples and absent in UA samples (Fig. 3) and (Supplementary Table S5). For example, targeted NGS analysis of the patient T1349 show that fresh UA sample contains more mutations than core biopsy, four mutations were detected in UA sample, but not in the core biopsy. Also sphere culture derived from this sample expressed three of those four mutations, while the one derived from the core biopsy expressed only the common mutation in this patient (NF1). In total, three out of four patients that were sequenced, expressed more mutations in their UA sample compared to their core biopsy. Only the tumor core of secondary GBM (T1311) expressed more mutations than UA counterpart and the sphere culture derived from this core biopsy expressed more mutations than the one derived from the UA sample (Fig. 3) and (Supplementary Table S5).

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