- ข้อความ
- ประวัติศาสตร์
Fourier transform infrared microspe
Fourier transform infrared microspectroscopy reveals biochemical
changes associated with glioma stem cell differentiation
Saša Kenig a,
⁎, Diana E. Bedolla b
, Giovanni Birarda b
, Valentina Faoro a
, Elisa Mitri b
, Alessandro Vindigni a,c
,
Paola Storici a
, Lisa Vaccari b
a Structural Biology Laboratory, Elettra-Sincrotrone Trieste, Italy
b SISSI beamline, Elettra-Sincrotrone Trieste, Italy
c Edward Doisy Department of Biochemistry and Molecular Biology, St. Louis University, MO, USA
HIGHLIGHTS
• FTIR identifies spectral features that differ
in glioma stem and non-stem cells.
• Glioma stem cell proteome and phosphorylation
level differ from differentiated
cells.
• Glioma stem cell plasma membranes
are more rigid than those of differentiated
cells.
• Glycogen level of glioma stem cells is
affected by ATRA-differentiation.
GRAPHICAL ABSTRACT
article info abstract
Article history:
Received 31 July 2015
Received in revised form 22 September 2015
Accepted 22 September 2015
Available online 25 September 2015
Keywords:
Glioma
Cancer stem cells
Differentiation
Infrared microspectroscopy
Glycogen
Lipids
According to the cancer stem cell theory malignant glioma is incurable because of the presence of the cancer stem
cells — a subpopulation of cells that are resistant to therapy and cause the recurrence of a tumor after surgical resection.
Several protein markers of cancer stem cell were reported but none of those is fully reliable to grade the content
of stem cells in a tumor. Hereby we propose Fourier transform infrared (FTIR) microspectroscopy as an alternative,
labelfree, non-damaging and fast method to identify glioma stem cells based on their own spectral characteristics.
The analysis of FTIR data revealed that in NCH421k cells, a model of glioma stem cells, the relative content of lipids
is higher than in their all-trans retinoic acid-differentiated counterparts. Moreover, it has been assessed that stem
cells have more rigid cellular membranes and more phosphorylated proteins, whereas after differentiation glycogen
level increases. The ability of FTIR to estimate the content of stem cells in a heterogeneous sample, on the base of the
identified spectral markers, and to classify stem and non-stem cells into two separate populations was probed. Although
it was not possible to calculate the exact percentage of each subpopulation, we could clearly see that with the
increasing amount of differentiated cells in a sample, more hits occupy the PC space previously identified as a space
of differentiated cells. The present study is therefore an initial step towards the development of a FTIR based protocol
in clinical practice to estimate the content of stem cells in a tumor sample.
© 2015 Published by Elsevier B.V.
Biophysical Chemistry 207 (2015) 90–96
⁎ Corresponding author at: Elettra-Sincrotrone Trieste S.C.p.A. di interesse nazionale, Strada Statale 14 — km 163.5 in AREA Science Park, 34149 Basovizza, Trieste, Italy.
E-mail address: sasa.kenig@elettra.eu (S. Kenig).
http://dx.doi.org/10.1016/j.bpc.2015.09.005
0301-4622/© 2015 Published by Elsevier B.V.
Contents lists available at ScienceDirect
Biophysical Chemistry
journal homepage: http://www.elsevier.com/locate/biophyschem
1. Introduction
Gliomas are a heterogeneous group of primary brain tumors, glioblastoma
(GBM) being the most malignant one. Only 5% of glioblastoma
patients survive 5 years after diagnosis and the majority dies
within two years [1]. Standard therapy comprises maximal safe
resection, followed by radiotherapy with concomitant systemic therapy
using the alkylating agent temozolomide. However, patients
respond differently to the standard therapy due to the high heterogeneity
of the disease. The cancer stem cell (CSC) theory suggests
that only a subpopulation of tumor cells in glioblastoma is able to initiate
tumor growth and drive its development [2]. According to this
theory all standard therapies will eventually fail if CSCs are not
removed. The high content of CSCs has been correlated with worse
prognosis or increased aggressiveness in many tumor types, such
as breast [3], head and neck [4], oropharyngeal cancer [5], and
glioma [6]. Given that CSCs are resistant to classical chemo and
radiotherapy, several novel approaches to specifically target them have
been suggested. Among these, inducing differentiation of CSCs is one of
the most promising [7]. In particular, the bone morphogenetic protein 4
(BMP4) was found to induce CSC differentiation and to consequently
block proliferation and tumor growth [8]. The bone morphogenetic protein
7 variant (BMP7) [9] and all-trans retinoic acid (ATRA) had a very
similar effect [10].
Because of the central role of CSCs in tumor development and their
prognostic value, it would be important to develop methods to monitor
their presence/abundance in tumor samples [11], as well as the efficacy
of stem cell-differentiating agents. Glioma stem cells have some known
characteristics, such as ability of self-renewal, ability to give rise to different
cell types, and the expression of molecular markers, such as
nestin, CD133 and Sox2. These cells are also able to regenerate the heterogeneous
cell populations similar to the original tumor, when
transplanted to a nude mice. Most of these properties are however not
an exclusive feature of CSC and do not provide a reliable strategy to
study their abundance [12]. In the present study we suggest the use of
Fourier transform infrared (FTIR) microspectroscopy as an alternative
approach to find small differences between glioma stem cells and glioma
cells without stem cell properties, and to estimate the content of
CSCs in a heterogeneous sample.
FITR is a well-established label-free analytical methodology for the
analysis of biological samples [13]. It provides spatially resolved information
on the composition and structure of the most relevant biomacromolecules
[14], probing their vibrational modes without inducing
sample damaging [15]. In the last decades, advances in detector technology
[16] and source brightness [17] have made it possible to perform
single cell analysis, allowing the fine characterization of heterogeneous
cell populations.
The topic of cell differentiation has already been studied using FTIR
in different systems [18,19,20,21,22]. Nevertheless, each of the investigated
samples (cell type, tissue) is biochemically different: in adipose
cells there is an accumulation of triglycerides, in hepatocytes and muscle
cells there is a high level of glycogen, in brain cells typical lipid constituents
are different to other cell types and the panel of expressed
proteins is different for each. Therefore, the marker regions of infrared
spectra that could distinguish glioma stem cells from differentiated
ones would likely be different from those previously identified in
other cell types.
In this work, we report the changes in biochemical composition that
occur during the differentiation of NCH421k as a glioma stem cell
model. Using Principal Component Analysis (PCA), we identified clear
signatures of differentiation affecting proteome and lipidome cellular
profiles, but also the extent of protein phosphorylation and intracellular
glycogen level. Moreover, we tested the ability of the technique to estimate
the content of stem-like cells in heterogeneous samples demonstrating
the predictive capability of the technique and its potential for
diagnostic purposes.
2. Materials and methods
2.1. Cells and differentiation
NCH421k cells as a glioma stem cell model were purchased from CLS
cell lines service and grown as floating neurospheres in DMEM/F12
medium supplemented with 0.25% BSA, 1% ITS, 20 ng/mL epidermal
growth factor and 20 ng/mL basic fibroblast growth factor at 37 °C in
5% CO2 atmosphere (control cells from here on). Cells were routinely
passaged every 4 days. Differentiation of neurospheres was induced as
described by Campos [10] by growing them in the same medium containing
10% FBS and 10 nM ATRA for 72 h (ATRA-differentiated from
here on). To follow cell cycle phase distribution, PI staining was performed
as described elsewhere [23].
2.2. Sample preparation
From control cells (neurospheres) single cell suspension was prepared.
ATRA-differentiated cells were instead collected by trypsinization.
Each sample was washed in physiological solution and divided into two
parts, one for FTIR microspectroscopy analysis and the other for parallel
IF, WB or PCR.
2.3. PCR
RNA was isolated using Isol-RNA lysis reagent following the
manufacturer's instructions (5Prime) and 2 μg of RNA was transcribed
to cDNA using a cDNA Archive kit (Applied Biosystems). Expression
levels of GFAP, nestin, CD133 and β-actin as endogenous
control were measured by PCR (BioRad) using the SYBR Green master
mix (BioRad) and the following primer pairs (sequences selected
from primerdepot NIH): GFAP F: acagacttggtgtccaggct, R:
gagatcgccacctacaggaa; nestin F: gggagttctcagcctccag R:
ggagaaacagggcctacaga; CD133 F: gcattggcatcttctatggtt, R:
cgccttgtccttggtagtgt; and β-actin F: ccttgcacatgccggag R:
gcacagagcctcgcctt. PCR conditions were 50 °C for 2 min, 95 °C for
10 min and 45 cycles of 95 °C for 15 s and 60 °C for 1 min; the data
were analyzed by the ΔΔCt algorithm. Statistical significance
between expressions was determined by two tailed Student's t-test
and p b 0.05 was considered significant.
2.4. GFAP and CD133 immunofluorescent staining
To determine the percentage of differentiated cells, staining of
astrocyte-marker GFAP and stem cell marker CD133 was performed.
Staining was performed on cells in single cell suspension. Cells were
then fixed in 3.7% paraformaldehyde, blocked in 4% BSA and incubated
in anti-GFAP primary antibody (Novus Biologicals, 1:50 in 1% BSA in
PBS). For CD133 primary antibody incubation (Miltenyi, 1:11) was
done before fixation. Afterwards, cells were incubated in Alexa488
anti-rabbit (Invitrogen, Molecular Probes, 1:300) and nuclei were counterstained
with Toto3 (Invi
changes associated with glioma stem cell differentiation
Saša Kenig a,
⁎, Diana E. Bedolla b
, Giovanni Birarda b
, Valentina Faoro a
, Elisa Mitri b
, Alessandro Vindigni a,c
,
Paola Storici a
, Lisa Vaccari b
a Structural Biology Laboratory, Elettra-Sincrotrone Trieste, Italy
b SISSI beamline, Elettra-Sincrotrone Trieste, Italy
c Edward Doisy Department of Biochemistry and Molecular Biology, St. Louis University, MO, USA
HIGHLIGHTS
• FTIR identifies spectral features that differ
in glioma stem and non-stem cells.
• Glioma stem cell proteome and phosphorylation
level differ from differentiated
cells.
• Glioma stem cell plasma membranes
are more rigid than those of differentiated
cells.
• Glycogen level of glioma stem cells is
affected by ATRA-differentiation.
GRAPHICAL ABSTRACT
article info abstract
Article history:
Received 31 July 2015
Received in revised form 22 September 2015
Accepted 22 September 2015
Available online 25 September 2015
Keywords:
Glioma
Cancer stem cells
Differentiation
Infrared microspectroscopy
Glycogen
Lipids
According to the cancer stem cell theory malignant glioma is incurable because of the presence of the cancer stem
cells — a subpopulation of cells that are resistant to therapy and cause the recurrence of a tumor after surgical resection.
Several protein markers of cancer stem cell were reported but none of those is fully reliable to grade the content
of stem cells in a tumor. Hereby we propose Fourier transform infrared (FTIR) microspectroscopy as an alternative,
labelfree, non-damaging and fast method to identify glioma stem cells based on their own spectral characteristics.
The analysis of FTIR data revealed that in NCH421k cells, a model of glioma stem cells, the relative content of lipids
is higher than in their all-trans retinoic acid-differentiated counterparts. Moreover, it has been assessed that stem
cells have more rigid cellular membranes and more phosphorylated proteins, whereas after differentiation glycogen
level increases. The ability of FTIR to estimate the content of stem cells in a heterogeneous sample, on the base of the
identified spectral markers, and to classify stem and non-stem cells into two separate populations was probed. Although
it was not possible to calculate the exact percentage of each subpopulation, we could clearly see that with the
increasing amount of differentiated cells in a sample, more hits occupy the PC space previously identified as a space
of differentiated cells. The present study is therefore an initial step towards the development of a FTIR based protocol
in clinical practice to estimate the content of stem cells in a tumor sample.
© 2015 Published by Elsevier B.V.
Biophysical Chemistry 207 (2015) 90–96
⁎ Corresponding author at: Elettra-Sincrotrone Trieste S.C.p.A. di interesse nazionale, Strada Statale 14 — km 163.5 in AREA Science Park, 34149 Basovizza, Trieste, Italy.
E-mail address: sasa.kenig@elettra.eu (S. Kenig).
http://dx.doi.org/10.1016/j.bpc.2015.09.005
0301-4622/© 2015 Published by Elsevier B.V.
Contents lists available at ScienceDirect
Biophysical Chemistry
journal homepage: http://www.elsevier.com/locate/biophyschem
1. Introduction
Gliomas are a heterogeneous group of primary brain tumors, glioblastoma
(GBM) being the most malignant one. Only 5% of glioblastoma
patients survive 5 years after diagnosis and the majority dies
within two years [1]. Standard therapy comprises maximal safe
resection, followed by radiotherapy with concomitant systemic therapy
using the alkylating agent temozolomide. However, patients
respond differently to the standard therapy due to the high heterogeneity
of the disease. The cancer stem cell (CSC) theory suggests
that only a subpopulation of tumor cells in glioblastoma is able to initiate
tumor growth and drive its development [2]. According to this
theory all standard therapies will eventually fail if CSCs are not
removed. The high content of CSCs has been correlated with worse
prognosis or increased aggressiveness in many tumor types, such
as breast [3], head and neck [4], oropharyngeal cancer [5], and
glioma [6]. Given that CSCs are resistant to classical chemo and
radiotherapy, several novel approaches to specifically target them have
been suggested. Among these, inducing differentiation of CSCs is one of
the most promising [7]. In particular, the bone morphogenetic protein 4
(BMP4) was found to induce CSC differentiation and to consequently
block proliferation and tumor growth [8]. The bone morphogenetic protein
7 variant (BMP7) [9] and all-trans retinoic acid (ATRA) had a very
similar effect [10].
Because of the central role of CSCs in tumor development and their
prognostic value, it would be important to develop methods to monitor
their presence/abundance in tumor samples [11], as well as the efficacy
of stem cell-differentiating agents. Glioma stem cells have some known
characteristics, such as ability of self-renewal, ability to give rise to different
cell types, and the expression of molecular markers, such as
nestin, CD133 and Sox2. These cells are also able to regenerate the heterogeneous
cell populations similar to the original tumor, when
transplanted to a nude mice. Most of these properties are however not
an exclusive feature of CSC and do not provide a reliable strategy to
study their abundance [12]. In the present study we suggest the use of
Fourier transform infrared (FTIR) microspectroscopy as an alternative
approach to find small differences between glioma stem cells and glioma
cells without stem cell properties, and to estimate the content of
CSCs in a heterogeneous sample.
FITR is a well-established label-free analytical methodology for the
analysis of biological samples [13]. It provides spatially resolved information
on the composition and structure of the most relevant biomacromolecules
[14], probing their vibrational modes without inducing
sample damaging [15]. In the last decades, advances in detector technology
[16] and source brightness [17] have made it possible to perform
single cell analysis, allowing the fine characterization of heterogeneous
cell populations.
The topic of cell differentiation has already been studied using FTIR
in different systems [18,19,20,21,22]. Nevertheless, each of the investigated
samples (cell type, tissue) is biochemically different: in adipose
cells there is an accumulation of triglycerides, in hepatocytes and muscle
cells there is a high level of glycogen, in brain cells typical lipid constituents
are different to other cell types and the panel of expressed
proteins is different for each. Therefore, the marker regions of infrared
spectra that could distinguish glioma stem cells from differentiated
ones would likely be different from those previously identified in
other cell types.
In this work, we report the changes in biochemical composition that
occur during the differentiation of NCH421k as a glioma stem cell
model. Using Principal Component Analysis (PCA), we identified clear
signatures of differentiation affecting proteome and lipidome cellular
profiles, but also the extent of protein phosphorylation and intracellular
glycogen level. Moreover, we tested the ability of the technique to estimate
the content of stem-like cells in heterogeneous samples demonstrating
the predictive capability of the technique and its potential for
diagnostic purposes.
2. Materials and methods
2.1. Cells and differentiation
NCH421k cells as a glioma stem cell model were purchased from CLS
cell lines service and grown as floating neurospheres in DMEM/F12
medium supplemented with 0.25% BSA, 1% ITS, 20 ng/mL epidermal
growth factor and 20 ng/mL basic fibroblast growth factor at 37 °C in
5% CO2 atmosphere (control cells from here on). Cells were routinely
passaged every 4 days. Differentiation of neurospheres was induced as
described by Campos [10] by growing them in the same medium containing
10% FBS and 10 nM ATRA for 72 h (ATRA-differentiated from
here on). To follow cell cycle phase distribution, PI staining was performed
as described elsewhere [23].
2.2. Sample preparation
From control cells (neurospheres) single cell suspension was prepared.
ATRA-differentiated cells were instead collected by trypsinization.
Each sample was washed in physiological solution and divided into two
parts, one for FTIR microspectroscopy analysis and the other for parallel
IF, WB or PCR.
2.3. PCR
RNA was isolated using Isol-RNA lysis reagent following the
manufacturer's instructions (5Prime) and 2 μg of RNA was transcribed
to cDNA using a cDNA Archive kit (Applied Biosystems). Expression
levels of GFAP, nestin, CD133 and β-actin as endogenous
control were measured by PCR (BioRad) using the SYBR Green master
mix (BioRad) and the following primer pairs (sequences selected
from primerdepot NIH): GFAP F: acagacttggtgtccaggct, R:
gagatcgccacctacaggaa; nestin F: gggagttctcagcctccag R:
ggagaaacagggcctacaga; CD133 F: gcattggcatcttctatggtt, R:
cgccttgtccttggtagtgt; and β-actin F: ccttgcacatgccggag R:
gcacagagcctcgcctt. PCR conditions were 50 °C for 2 min, 95 °C for
10 min and 45 cycles of 95 °C for 15 s and 60 °C for 1 min; the data
were analyzed by the ΔΔCt algorithm. Statistical significance
between expressions was determined by two tailed Student's t-test
and p b 0.05 was considered significant.
2.4. GFAP and CD133 immunofluorescent staining
To determine the percentage of differentiated cells, staining of
astrocyte-marker GFAP and stem cell marker CD133 was performed.
Staining was performed on cells in single cell suspension. Cells were
then fixed in 3.7% paraformaldehyde, blocked in 4% BSA and incubated
in anti-GFAP primary antibody (Novus Biologicals, 1:50 in 1% BSA in
PBS). For CD133 primary antibody incubation (Miltenyi, 1:11) was
done before fixation. Afterwards, cells were incubated in Alexa488
anti-rabbit (Invitrogen, Molecular Probes, 1:300) and nuclei were counterstained
with Toto3 (Invi
0/5000
Fourier transform infrared microspectroscopy reveals biochemicalchanges associated with glioma stem cell differentiationSaša Kenig a,⁎, Diana E. Bedolla b, Giovanni Birarda b, Valentina Faoro a, Elisa Mitri b, Alessandro Vindigni a,c,Paola Storici a, Lisa Vaccari ba Structural Biology Laboratory, Elettra-Sincrotrone Trieste, Italyb SISSI beamline, Elettra-Sincrotrone Trieste, Italyc Edward Doisy Department of Biochemistry and Molecular Biology, St. Louis University, MO, USAHIGHLIGHTS• FTIR identifies spectral features that differin glioma stem and non-stem cells.• Glioma stem cell proteome and phosphorylationlevel differ from differentiatedcells.• Glioma stem cell plasma membranesare more rigid than those of differentiatedcells.• Glycogen level of glioma stem cells isaffected by ATRA-differentiation.GRAPHICAL ABSTRACTarticle info abstractArticle history:Received 31 July 2015Received in revised form 22 September 2015Accepted 22 September 2015Available online 25 September 2015Keywords:GliomaCancer stem cellsDifferentiationInfrared microspectroscopyGlycogenLipidsAccording to the cancer stem cell theory malignant glioma is incurable because of the presence of the cancer stemcells — a subpopulation of cells that are resistant to therapy and cause the recurrence of a tumor after surgical resection.Several protein markers of cancer stem cell were reported but none of those is fully reliable to grade the contentof stem cells in a tumor. Hereby we propose Fourier transform infrared (FTIR) microspectroscopy as an alternative,labelfree, non-damaging and fast method to identify glioma stem cells based on their own spectral characteristics.The analysis of FTIR data revealed that in NCH421k cells, a model of glioma stem cells, the relative content of lipidsis higher than in their all-trans retinoic acid-differentiated counterparts. Moreover, it has been assessed that stemcells have more rigid cellular membranes and more phosphorylated proteins, whereas after differentiation glycogenlevel increases. The ability of FTIR to estimate the content of stem cells in a heterogeneous sample, on the base of theidentified spectral markers, and to classify stem and non-stem cells into two separate populations was probed. Althoughit was not possible to calculate the exact percentage of each subpopulation, we could clearly see that with theincreasing amount of differentiated cells in a sample, more hits occupy the PC space previously identified as a spaceof differentiated cells. The present study is therefore an initial step towards the development of a FTIR based protocolin clinical practice to estimate the content of stem cells in a tumor sample.© 2015 Published by Elsevier B.V.Biophysical Chemistry 207 (2015) 90–96⁎ Corresponding author at: Elettra-Sincrotrone Trieste S.C.p.A. di interesse nazionale, Strada Statale 14 — km 163.5 in AREA Science Park, 34149 Basovizza, Trieste, Italy.E-mail address: sasa.kenig@elettra.eu (S. Kenig).http://dx.doi.org/10.1016/j.bpc.2015.09.0050301-4622/© 2015 Published by Elsevier B.V.Contents lists available at ScienceDirectBiophysical Chemistryjournal homepage: http://www.elsevier.com/locate/biophyschem1. IntroductionGliomas are a heterogeneous group of primary brain tumors, glioblastoma(GBM) being the most malignant one. Only 5% of glioblastomapatients survive 5 years after diagnosis and the majority dieswithin two years [1]. Standard therapy comprises maximal saferesection, followed by radiotherapy with concomitant systemic therapyusing the alkylating agent temozolomide. However, patientsrespond differently to the standard therapy due to the high heterogeneityof the disease. The cancer stem cell (CSC) theory suggeststhat only a subpopulation of tumor cells in glioblastoma is able to initiatetumor growth and drive its development [2]. According to thistheory all standard therapies will eventually fail if CSCs are notremoved. The high content of CSCs has been correlated with worseprognosis or increased aggressiveness in many tumor types, suchas breast [3], head and neck [4], oropharyngeal cancer [5], andglioma [6]. Given that CSCs are resistant to classical chemo andradiotherapy, several novel approaches to specifically target them havebeen suggested. Among these, inducing differentiation of CSCs is one ofthe most promising [7]. In particular, the bone morphogenetic protein 4(BMP4) was found to induce CSC differentiation and to consequentlyblock proliferation and tumor growth [8]. The bone morphogenetic protein7 variant (BMP7) [9] and all-trans retinoic acid (ATRA) had a verysimilar effect [10].Because of the central role of CSCs in tumor development and theirprognostic value, it would be important to develop methods to monitortheir presence/abundance in tumor samples [11], as well as the efficacyof stem cell-differentiating agents. Glioma stem cells have some knowncharacteristics, such as ability of self-renewal, ability to give rise to differentcell types, and the expression of molecular markers, such asnestin, CD133 and Sox2. These cells are also able to regenerate the heterogeneouscell populations similar to the original tumor, whentransplanted to a nude mice. Most of these properties are however notan exclusive feature of CSC and do not provide a reliable strategy tostudy their abundance [12]. In the present study we suggest the use ofFourier transform infrared (FTIR) microspectroscopy as an alternativeapproach to find small differences between glioma stem cells and gliomacells without stem cell properties, and to estimate the content ofCSCs in a heterogeneous sample.FITR is a well-established label-free analytical methodology for theanalysis of biological samples [13]. It provides spatially resolved informationon the composition and structure of the most relevant biomacromolecules[14], probing their vibrational modes without inducingsample damaging [15]. In the last decades, advances in detector technology[16] and source brightness [17] have made it possible to performsingle cell analysis, allowing the fine characterization of heterogeneouscell populations.The topic of cell differentiation has already been studied using FTIRin different systems [18,19,20,21,22]. Nevertheless, each of the investigatedsamples (cell type, tissue) is biochemically different: in adiposecells there is an accumulation of triglycerides, in hepatocytes and musclecells there is a high level of glycogen, in brain cells typical lipid constituentsare different to other cell types and the panel of expressedproteins is different for each. Therefore, the marker regions of infraredspectra that could distinguish glioma stem cells from differentiatedones would likely be different from those previously identified inother cell types.In this work, we report the changes in biochemical composition thatoccur during the differentiation of NCH421k as a glioma stem cellmodel. Using Principal Component Analysis (PCA), we identified clearsignatures of differentiation affecting proteome and lipidome cellularprofiles, but also the extent of protein phosphorylation and intracellularglycogen level. Moreover, we tested the ability of the technique to estimatethe content of stem-like cells in heterogeneous samples demonstratingthe predictive capability of the technique and its potential fordiagnostic purposes.2. Materials and methods2.1. Cells and differentiationNCH421k cells as a glioma stem cell model were purchased from CLScell lines service and grown as floating neurospheres in DMEM/F12medium supplemented with 0.25% BSA, 1% ITS, 20 ng/mL epidermalgrowth factor and 20 ng/mL basic fibroblast growth factor at 37 °C in5% CO2 atmosphere (control cells from here on). Cells were routinelypassaged every 4 days. Differentiation of neurospheres was induced asdescribed by Campos [10] by growing them in the same medium containing10% FBS and 10 nM ATRA for 72 h (ATRA-differentiated fromhere on). To follow cell cycle phase distribution, PI staining was performedas described elsewhere [23].2.2. Sample preparationFrom control cells (neurospheres) single cell suspension was prepared.ATRA-differentiated cells were instead collected by trypsinization.Each sample was washed in physiological solution and divided into twoparts, one for FTIR microspectroscopy analysis and the other for parallelIF, WB or PCR.2.3. PCRRNA was isolated using Isol-RNA lysis reagent following themanufacturer's instructions (5Prime) and 2 μg of RNA was transcribedto cDNA using a cDNA Archive kit (Applied Biosystems). Expressionlevels of GFAP, nestin, CD133 and β-actin as endogenouscontrol were measured by PCR (BioRad) using the SYBR Green mastermix (BioRad) and the following primer pairs (sequences selectedfrom primerdepot NIH): GFAP F: acagacttggtgtccaggct, R:gagatcgccacctacaggaa; nestin F: gggagttctcagcctccag R:ggagaaacagggcctacaga; CD133 F: gcattggcatcttctatggtt, R:cgccttgtccttggtagtgt; and β-actin F: ccttgcacatgccggag R:gcacagagcctcgcctt. PCR conditions were 50 °C for 2 min, 95 °C for10 min and 45 cycles of 95 °C for 15 s and 60 °C for 1 min; the datawere analyzed by the ΔΔCt algorithm. Statistical significancebetween expressions was determined by two tailed Student's t-testand p b 0.05 was considered significant.2.4. GFAP and CD133 immunofluorescent stainingTo determine the percentage of differentiated cells, staining ofastrocyte-marker GFAP and stem cell marker CD133 was performed.Staining was performed on cells in single cell suspension. Cells werethen fixed in 3.7% paraformaldehyde, blocked in 4% BSA and incubatedin anti-GFAP primary antibody (Novus Biologicals, 1:50 in 1% BSA inPBS). For CD133 primary antibody incubation (Miltenyi, 1:11) wasdone before fixation. Afterwards, cells were incubated in Alexa488anti-rabbit (Invitrogen, Molecular Probes, 1:300) and nuclei were counterstainedwith Toto3 (Invi
การแปล กรุณารอสักครู่..
Fourier transform Infrared microspectroscopy reveals Biochemical
changes associated with glioma STEM Cell differentiation
Saša Kenig a,
⁎, Diana E. Bedolla B
, B Birarda Giovanni
, a Faoro Valentina
, Elisa B Mitri
, Alessandro Vindigni a, C
,
Paola Storici a
, Lisa B Vaccari.
a Structural Biology Laboratory, Elettra-Sincrotrone Trieste, Italy
B SISSI beamline, Elettra-Sincrotrone Trieste, Italy
Edward Doisy Department of Biochemistry and Molecular C Biology, St. Louis University, MO, USA
HIGHLIGHTS
• identifies the FTIR spectral features that differ
in STEM glioma. and non-STEM cells.
• Glioma STEM Cell proteome and phosphorylation
level differ from differentiated
cells.
• Glioma STEM Cell Plasma membranes
are more rigid than those of differentiated
cells.
• Glycogen level of glioma STEM cells is
affected by ATRA-differentiation.
graphical ABSTRACT.
Article info abstract
Article History:
Received 31 July in 2015
Received in revised form 22 September the 2,015th
Accepted 22 September the 2015th
Available Online 25 September two thousand and fifteen
Keywords:
Glioma
Cancer STEM cells
Differentiation
Infrared microspectroscopy
Glycogen
Lipids
According to the Cancer STEM Cell Theory malignant glioma is incurable because of. the Presence of the Cancer STEM
cells - a subpopulation of cells that are resistant to Therapy and Cause the recurrence of a tumor after Surgical resection.
Several protein markers of Cancer STEM Cell were reported but None of those is fully Reliable to grade the content
of STEM. cells in a tumor. Hereby we Propose Fourier transform Infrared (FTIR) microspectroscopy as an Alternative,
Labelfree, non-damaging and fast method to Identify glioma STEM cells based on their own spectral characteristics.
The Analysis of FTIR Data revealed that in NCH421k cells, a Model of glioma STEM. cells, the Relative content of lipids
is higher than in their all-Trans retinoic acid-differentiated counterparts. Moreover, it has been Assessed that STEM
cells have membranes more rigid and more phosphorylated Cellular Proteins, whereas after differentiation glycogen
level increases. The ability of FTIR to Estimate the content of STEM cells in a heterogeneous sample, on the Base of the
identified spectral markers, and to classify non-STEM and STEM cells was probed Into Two Separate populations. Although
it was not possible to Calculate the percentage of each subpopulation Exact, we could See Clearly that with the
increasing amount of differentiated cells in a sample, more PC Space Hits occupy the previously identified as a Space
of differentiated cells. The present Study is therefore an Initial Step towards the Development of a Protocol based FTIR
in clinical Practice to Estimate the content of STEM cells in a tumor sample.
Published by Elsevier BV © the 2015th
Biophysical Chemistry 207 (2015th) 90-96
at ⁎ corresponding author. : Elettra-Sincrotrone interesse Nazionale di Trieste SCPA, Strada Statale 14 - km 163.5 in AREA Science Park, 34,149 Basovizza, Trieste, Italy.
E-mail address: Sasa.kenig@elettra.eu (S. Kenig).
HTTP: //. Dx.doi.org/10.1016/j.bpc.2015.09.005
0301-4,622 / 2015th Published by Elsevier BV ©
Contents lists available at ScienceDirect
Biophysical Chemistry
Journal Homepage: Http://www.elsevier.com/locate/biophyschem
1. Introduction
Gliomas are a heterogeneous Group of Brain primary tumors, glioblastoma
(GBM) being the Most malignant one. Only 5% of glioblastoma
patients Survive 5 years after diagnosis and the majority Dies
Within Two years [1]. Safe Therapy comprises standard maximal
resection, followed by radiotherapy with concomitant systemic Therapy
using the alkylating Agent temozolomide. However, patients
respond differently to the standard Therapy Due to the High heterogeneity
of the disease. The STEM Cancer Cell (CSC) Theory suggests
that only a subpopulation of tumor cells in glioblastoma is Able to initiate
tumor growth and its Drive Development [2]. According to this
Theory all standard therapies Will Fail if eventually CSCS are not
removed. High content of the CSCS has been correlated with worse
PROGNOSIS Increased aggressiveness or in many tumor types, such
as Breast [3], Head and neck [4], oropharyngeal Cancer [5], and
glioma [6]. Given that CSCS are resistant to classical Chemo and
radiotherapy, several novel approaches to specifically Target them have
been SUGGESTED. Among these, inducing differentiation of CSCS is one of
the Most promising [7]. In particular, the Bone morphogenetic protein 4
(BMP4) CSC was Found to induce differentiation and to consequently
Block proliferation and tumor growth [8]. The Bone morphogenetic protein
7 Variant (BMP7) [9] and all-Trans retinoic acid (ATRA) had a very
similar Effect [10].
Because of the Central role of CSCS in tumor Development and their
prognostic Value, it would be important to. develop methods to Monitor
their Presence / Abundance in tumor samples [11], as well as the efficacy
of STEM Cell-differentiating agents. STEM glioma cells have Known Some
characteristics, such as self-renewal ability of, ability to give rise to different
types Cell, and the Expression of molecular markers, such as
nestin, CD133 and Sox2. These cells are also Able to Regenerate the heterogeneous
populations similar to the Original Cell tumor, when
transplanted to a Nude mice. Most of these properties are however not
an Exclusive Feature of CSC and do not provide a Reliable Strategy to
Study their Abundance [12]. In the present Study we suggest the use of
Fourier transform Infrared (FTIR) microspectroscopy as an Alternative
approach to Find Small differences between glioma STEM cells and glioma
cells Without STEM Cell properties, and to Estimate the content of
CSCS in a heterogeneous sample.
Fitr is. a well-established analytical methodology for the Label-free
Analysis of Biological samples [13]. It provides spatially resolved information
on the composition and structure of the relevant Biomacromolecules Most
[14], probing their vibrational modes Without inducing
damaging sample [15]. In the last decades, Advances in Detector Technology
[16] and Source brightness [17] have Made it possible to Perform
single Cell Analysis, allowing the Fine characterization of heterogeneous
Cell populations.
The Topic of Cell differentiation has already been studied using FTIR
in different. systems [18,19,20,21,22]. Nevertheless, each of the investigated
samples (Cell Type, tissue) is biochemically different: in adipose
cells there is an accumulation of Triglycerides, in hepatocytes and Muscle
cells there is a High level of glycogen, in Brain cells Typical lipid constituents
are different to Other. Cell types and the Panel of expressed
Proteins is different for each. Therefore, the Marker Regions of Infrared
Spectra that could distinguish glioma STEM cells from differentiated
Ones would likely be different from those previously identified in
Other Cell types.
In this Work, we Report the changes in Biochemical composition that
occur during the differentiation of NCH421k as a. glioma STEM Cell
Model. Principal Component Analysis using (PCA), we identified Clear
signatures of differentiation affecting Lipidome Cellular and proteome
profiles, but also the extent of intracellular protein phosphorylation and
glycogen level. Moreover, we tested the ability of the Technique to Estimate
the content of STEM-like cells in heterogeneous samples Demonstrating
the predictive capability of the Technique and its potential for
Diagnostic purposes.
2. Materials and methods
2.1. Cells and differentiation
NCH421k cells as a glioma STEM Cell Model were purchased from CLS
Cell Lines Service and Grown as Floating Neurospheres in DMEM / F12
Medium supplemented with twelve twenty-five% BSA, 1% ITS, 20 NG / mL epidermal
growth factor and 20 NG / mL. Basic fibroblast growth factor at 37 ° C in
5% CO2 atmosphere (Control cells from here on). Routinely cells were
passaged every 4 days. Neurospheres of differentiation was induced as
described by Campos [10] by them in the Same Growing Medium containing
10% FBS and 10 nM ATRA for 72 H (ATRA-differentiated from
here on). Cell Cycle Phase Distribution to follow, PI staining was performed
as described Elsewhere [23].
2.2. Sample Preparation
From Control cells (Neurospheres) single Cell suspension was prepared.
ATRA-differentiated cells were instead Collected by Trypsinization.
Each sample was washed in physiological Solution and divided Into Two
Parts, one for FTIR microspectroscopy Analysis and the Other for parallel
IF, WB. or PCR.
2.3. PCR
RNA was isolated using Isol-RNA lysis reagent following the
manufacturer's instructions (5Prime) and 2 g of RNA was transcribed
to cDNA using a cDNA Archive Kit (Applied Biosystems). Expression
levels of GFAP, nestin, CD133 and beta-actin as endogenous
Control were measured by PCR (Biorad) using the SYBR Green Master
Mix (Biorad) and the following Primer pairs (sequences selected
from Primerdepot NIH): GFAP F: Acagacttggtgtccaggct, R. :
Gagatcgccacctacaggaa; nestin F: Gggagttctcagcctccag R:
Ggagaaacagggcctacaga; CD133 F: Gcattggcatcttctatggtt, R:
Cgccttgtccttggtagtgt; and beta-actin F: Ccttgcacatgccggag R:
Gcacagagcctcgcctt. PCR conditions were 50 ° C for 2 min, 95 ° C for
10 min and 45 ° C for 15 s Cycles of 95 and 60 ° C for 1 min; the Data
were analyzed by the algorithm ΔΔCt. Statistical significance
was determined by Expressions between Two tailed Student's T-Test
and PB 0:05 was considered significant.
2.4. GFAP immunofluorescent staining and CD133
To Determine the percentage of differentiated cells, staining of
astrocyte GFAP-Marker and STEM Cell Marker CD133 was performed.
Staining was performed on single cells in suspension Cell. Cells were
then fixed in 3.7% paraformaldehyde, Blocked in 4% BSA and incubated
in Anti-GFAP primary antibody (Novus Biologicals, one fifty in 1% BSA in
PBS). Incubation primary antibody for CD133 (Miltenyi, one eleven) was
done before Fixation. Afterwards, cells were incubated in Alexa488
Anti-Rabbit (Invitrogen, Molecular Probes, 1: 300) and nuclei were Counterstained
with Toto3 (invi.
changes associated with glioma STEM Cell differentiation
Saša Kenig a,
⁎, Diana E. Bedolla B
, B Birarda Giovanni
, a Faoro Valentina
, Elisa B Mitri
, Alessandro Vindigni a, C
,
Paola Storici a
, Lisa B Vaccari.
a Structural Biology Laboratory, Elettra-Sincrotrone Trieste, Italy
B SISSI beamline, Elettra-Sincrotrone Trieste, Italy
Edward Doisy Department of Biochemistry and Molecular C Biology, St. Louis University, MO, USA
HIGHLIGHTS
• identifies the FTIR spectral features that differ
in STEM glioma. and non-STEM cells.
• Glioma STEM Cell proteome and phosphorylation
level differ from differentiated
cells.
• Glioma STEM Cell Plasma membranes
are more rigid than those of differentiated
cells.
• Glycogen level of glioma STEM cells is
affected by ATRA-differentiation.
graphical ABSTRACT.
Article info abstract
Article History:
Received 31 July in 2015
Received in revised form 22 September the 2,015th
Accepted 22 September the 2015th
Available Online 25 September two thousand and fifteen
Keywords:
Glioma
Cancer STEM cells
Differentiation
Infrared microspectroscopy
Glycogen
Lipids
According to the Cancer STEM Cell Theory malignant glioma is incurable because of. the Presence of the Cancer STEM
cells - a subpopulation of cells that are resistant to Therapy and Cause the recurrence of a tumor after Surgical resection.
Several protein markers of Cancer STEM Cell were reported but None of those is fully Reliable to grade the content
of STEM. cells in a tumor. Hereby we Propose Fourier transform Infrared (FTIR) microspectroscopy as an Alternative,
Labelfree, non-damaging and fast method to Identify glioma STEM cells based on their own spectral characteristics.
The Analysis of FTIR Data revealed that in NCH421k cells, a Model of glioma STEM. cells, the Relative content of lipids
is higher than in their all-Trans retinoic acid-differentiated counterparts. Moreover, it has been Assessed that STEM
cells have membranes more rigid and more phosphorylated Cellular Proteins, whereas after differentiation glycogen
level increases. The ability of FTIR to Estimate the content of STEM cells in a heterogeneous sample, on the Base of the
identified spectral markers, and to classify non-STEM and STEM cells was probed Into Two Separate populations. Although
it was not possible to Calculate the percentage of each subpopulation Exact, we could See Clearly that with the
increasing amount of differentiated cells in a sample, more PC Space Hits occupy the previously identified as a Space
of differentiated cells. The present Study is therefore an Initial Step towards the Development of a Protocol based FTIR
in clinical Practice to Estimate the content of STEM cells in a tumor sample.
Published by Elsevier BV © the 2015th
Biophysical Chemistry 207 (2015th) 90-96
at ⁎ corresponding author. : Elettra-Sincrotrone interesse Nazionale di Trieste SCPA, Strada Statale 14 - km 163.5 in AREA Science Park, 34,149 Basovizza, Trieste, Italy.
E-mail address: Sasa.kenig@elettra.eu (S. Kenig).
HTTP: //. Dx.doi.org/10.1016/j.bpc.2015.09.005
0301-4,622 / 2015th Published by Elsevier BV ©
Contents lists available at ScienceDirect
Biophysical Chemistry
Journal Homepage: Http://www.elsevier.com/locate/biophyschem
1. Introduction
Gliomas are a heterogeneous Group of Brain primary tumors, glioblastoma
(GBM) being the Most malignant one. Only 5% of glioblastoma
patients Survive 5 years after diagnosis and the majority Dies
Within Two years [1]. Safe Therapy comprises standard maximal
resection, followed by radiotherapy with concomitant systemic Therapy
using the alkylating Agent temozolomide. However, patients
respond differently to the standard Therapy Due to the High heterogeneity
of the disease. The STEM Cancer Cell (CSC) Theory suggests
that only a subpopulation of tumor cells in glioblastoma is Able to initiate
tumor growth and its Drive Development [2]. According to this
Theory all standard therapies Will Fail if eventually CSCS are not
removed. High content of the CSCS has been correlated with worse
PROGNOSIS Increased aggressiveness or in many tumor types, such
as Breast [3], Head and neck [4], oropharyngeal Cancer [5], and
glioma [6]. Given that CSCS are resistant to classical Chemo and
radiotherapy, several novel approaches to specifically Target them have
been SUGGESTED. Among these, inducing differentiation of CSCS is one of
the Most promising [7]. In particular, the Bone morphogenetic protein 4
(BMP4) CSC was Found to induce differentiation and to consequently
Block proliferation and tumor growth [8]. The Bone morphogenetic protein
7 Variant (BMP7) [9] and all-Trans retinoic acid (ATRA) had a very
similar Effect [10].
Because of the Central role of CSCS in tumor Development and their
prognostic Value, it would be important to. develop methods to Monitor
their Presence / Abundance in tumor samples [11], as well as the efficacy
of STEM Cell-differentiating agents. STEM glioma cells have Known Some
characteristics, such as self-renewal ability of, ability to give rise to different
types Cell, and the Expression of molecular markers, such as
nestin, CD133 and Sox2. These cells are also Able to Regenerate the heterogeneous
populations similar to the Original Cell tumor, when
transplanted to a Nude mice. Most of these properties are however not
an Exclusive Feature of CSC and do not provide a Reliable Strategy to
Study their Abundance [12]. In the present Study we suggest the use of
Fourier transform Infrared (FTIR) microspectroscopy as an Alternative
approach to Find Small differences between glioma STEM cells and glioma
cells Without STEM Cell properties, and to Estimate the content of
CSCS in a heterogeneous sample.
Fitr is. a well-established analytical methodology for the Label-free
Analysis of Biological samples [13]. It provides spatially resolved information
on the composition and structure of the relevant Biomacromolecules Most
[14], probing their vibrational modes Without inducing
damaging sample [15]. In the last decades, Advances in Detector Technology
[16] and Source brightness [17] have Made it possible to Perform
single Cell Analysis, allowing the Fine characterization of heterogeneous
Cell populations.
The Topic of Cell differentiation has already been studied using FTIR
in different. systems [18,19,20,21,22]. Nevertheless, each of the investigated
samples (Cell Type, tissue) is biochemically different: in adipose
cells there is an accumulation of Triglycerides, in hepatocytes and Muscle
cells there is a High level of glycogen, in Brain cells Typical lipid constituents
are different to Other. Cell types and the Panel of expressed
Proteins is different for each. Therefore, the Marker Regions of Infrared
Spectra that could distinguish glioma STEM cells from differentiated
Ones would likely be different from those previously identified in
Other Cell types.
In this Work, we Report the changes in Biochemical composition that
occur during the differentiation of NCH421k as a. glioma STEM Cell
Model. Principal Component Analysis using (PCA), we identified Clear
signatures of differentiation affecting Lipidome Cellular and proteome
profiles, but also the extent of intracellular protein phosphorylation and
glycogen level. Moreover, we tested the ability of the Technique to Estimate
the content of STEM-like cells in heterogeneous samples Demonstrating
the predictive capability of the Technique and its potential for
Diagnostic purposes.
2. Materials and methods
2.1. Cells and differentiation
NCH421k cells as a glioma STEM Cell Model were purchased from CLS
Cell Lines Service and Grown as Floating Neurospheres in DMEM / F12
Medium supplemented with twelve twenty-five% BSA, 1% ITS, 20 NG / mL epidermal
growth factor and 20 NG / mL. Basic fibroblast growth factor at 37 ° C in
5% CO2 atmosphere (Control cells from here on). Routinely cells were
passaged every 4 days. Neurospheres of differentiation was induced as
described by Campos [10] by them in the Same Growing Medium containing
10% FBS and 10 nM ATRA for 72 H (ATRA-differentiated from
here on). Cell Cycle Phase Distribution to follow, PI staining was performed
as described Elsewhere [23].
2.2. Sample Preparation
From Control cells (Neurospheres) single Cell suspension was prepared.
ATRA-differentiated cells were instead Collected by Trypsinization.
Each sample was washed in physiological Solution and divided Into Two
Parts, one for FTIR microspectroscopy Analysis and the Other for parallel
IF, WB. or PCR.
2.3. PCR
RNA was isolated using Isol-RNA lysis reagent following the
manufacturer's instructions (5Prime) and 2 g of RNA was transcribed
to cDNA using a cDNA Archive Kit (Applied Biosystems). Expression
levels of GFAP, nestin, CD133 and beta-actin as endogenous
Control were measured by PCR (Biorad) using the SYBR Green Master
Mix (Biorad) and the following Primer pairs (sequences selected
from Primerdepot NIH): GFAP F: Acagacttggtgtccaggct, R. :
Gagatcgccacctacaggaa; nestin F: Gggagttctcagcctccag R:
Ggagaaacagggcctacaga; CD133 F: Gcattggcatcttctatggtt, R:
Cgccttgtccttggtagtgt; and beta-actin F: Ccttgcacatgccggag R:
Gcacagagcctcgcctt. PCR conditions were 50 ° C for 2 min, 95 ° C for
10 min and 45 ° C for 15 s Cycles of 95 and 60 ° C for 1 min; the Data
were analyzed by the algorithm ΔΔCt. Statistical significance
was determined by Expressions between Two tailed Student's T-Test
and PB 0:05 was considered significant.
2.4. GFAP immunofluorescent staining and CD133
To Determine the percentage of differentiated cells, staining of
astrocyte GFAP-Marker and STEM Cell Marker CD133 was performed.
Staining was performed on single cells in suspension Cell. Cells were
then fixed in 3.7% paraformaldehyde, Blocked in 4% BSA and incubated
in Anti-GFAP primary antibody (Novus Biologicals, one fifty in 1% BSA in
PBS). Incubation primary antibody for CD133 (Miltenyi, one eleven) was
done before Fixation. Afterwards, cells were incubated in Alexa488
Anti-Rabbit (Invitrogen, Molecular Probes, 1: 300) and nuclei were Counterstained
with Toto3 (invi.
การแปล กรุณารอสักครู่..
Fourier transform infrared microspectroscopy reveals biochemical
changes associated with glioma stem cell differentiation
Sa š a. Kenig a
⁎, Diana E. Bedolla, B
, Giovanni Birarda B
, Valentina Faoro a
, Elisa Mitri B
, Alessandro Vindigni a C
Paola,,, Storici a
, Lisa Vaccari B
a Structural, Biology Laboratory Elettra-Sincrotrone Trieste Italy
, B, SISSI beamline Elettra-Sincrotrone. Trieste, the Italy
.Fourier transform infrared microspectroscopy reveals biochemical
changes associated with glioma stem cell differentiation
Sa š a. Kenig a
⁎, Diana E. Bedolla, B
, Giovanni Birarda B
, Valentina Faoro a
, Elisa Mitri B
, Alessandro Vindigni a C
Paola,,, Storici a
, Lisa Vaccari B
a Structural, Biology Laboratory Elettra-Sincrotrone Trieste Italy
, B, SISSI beamline Elettra-Sincrotrone. Trieste, the Italy
.C Edward Doisy Department of Biochemistry and Molecular Biology St. Louis,,, University MO USA HIGHLIGHTS
-
FTIR identifies. Spectral features that differ
in glioma stem and non - stem cells.
- Glioma stem cell proteome and phosphorylation
level differ. From differentiated
.
- cells Glioma stem cell plasma membranes
are more rigid than those of cells differentiated
.
.Education Glycogen level of glioma stem cells is
affected by ATRA-differentiation.
GRAPHICAL ABSTRACT article info Abstract
Article. History:
Received 31 July 2015
Received in revised form 22 September 2015
Accepted 22 September 2015
Available online 25 September. 2015
Keywords Glioma Cancer stem cells
, Differentiation Infrared Microspectroscopy
. Lipids GlycogenAccording to the cancer stem cell theory malignant glioma is incurable because of the presence of the cancer stem
cells. A subpopulation - of cells that are resistant to therapy and cause the recurrence of a tumor after surgical resection.
Several. Protein markers of cancer stem cell were reported but none of those is fully reliable to grade the content
of stem cells. In a tumor.Hereby we propose Fourier transform infrared (FTIR) microspectroscopy as an alternative
labelfree non-damaging and fast,,, Method to identify glioma stem cells based on their own spectral characteristics.
The analysis of FTIR data revealed that. In, NCH421k cells a model of glioma, stem cells the relative content of lipids
.Is higher than in their all-trans retinoic acid - differentiated counterparts. Moreover it has, been assessed that stem
cells. Have more rigid cellular membranes and more phosphorylated proteins whereas after, differentiation glycogen
level, increases. The ability of FTIR to estimate the content of stem cells in a, heterogeneous sample on the base of the
identified spectral. Markers.And to classify stem and non - stem cells into two separate populations was probed. Although
it was not possible to calculate. The exact percentage of, each subpopulation we could clearly see that with the
increasing amount of differentiated cells. In, a sample more hits occupy the PC space previously identified as a space
of differentiated cells.The present study is therefore an initial step towards the development of a FTIR based protocol
in clinical practice to. Estimate the content of stem cells in a tumor sample.
s 2015 Published by Elsevier B.V.
Biophysical Chemistry 207 (2015). 90 - 96
⁎ Corresponding author at: Elettra-Sincrotrone Trieste S.C.p.A. Di interesse Nazionale Strada Statale, 14 - km 163.5 in. AREA, Science Park,, 34149 Basovizza Trieste Italy.
E - mail address: sasa.kenig@elettra.eu (S. Kenig).
http: / / dx.doi.org / 10.1016 / j.bpc.2015.09.005
0301-4622 / © 2015 Published. By Elsevier B.V.
Contents lists available at ScienceDirect
Biophysical Chemistry journal homepage: http: / / www.elsevier.com / locate / biophyschem
1.? Introduction
Gliomas are a heterogeneous group of primary, brain tumors glioblastoma
.(GBM) being the most malignant one. Only 5% of glioblastoma
patients survive 5 years after diagnosis and the majority dies
within. Two years [1]. Standard therapy comprises maximal safe
resection followed by, radiotherapy with concomitant systemic therapy
using. The alkylating agent temozolomide. However patients
respond, differently to the standard therapy due to the high heterogeneity
.Of the disease. The cancer stem cell (CSC) theory suggests
that only a subpopulation of tumor cells in glioblastoma is. Able to initiate
tumor growth and drive its development [2]. According to this
theory all standard therapies will eventually. Fail if CSCs are not
removed. The high content of CSCs has been correlated with worse
prognosis or increased aggressiveness. In many, tumor types such
.As breast [3], head and neck [4], [], oropharyngeal cancer 5 and
glioma [6]. Given that CSCs are resistant to classical. Chemo and
radiotherapy several novel, approaches to specifically target them have
been suggested. Among these inducing,, Differentiation of CSCs is one of
the most promising []. In, 7 particular the bone morphogenetic protein 4
.Their presence / abundance in tumor samples [11], as well as the efficacy
of stem cell - differentiating agents. Glioma stem. Cells have some known
characteristics such as, ability, of self-renewal ability to give rise to different
cell types and,, The expression of, molecular markers such as
nestin CD133 and, Sox2. These cells are also able to regenerate the heterogeneous
.(BMP4) was found to induce CSC differentiation and to consequently
block proliferation and tumor growth [8]. The bone morphogenetic. Protein
7 variant (BMP7) [] and 9 all-trans retinoic acid (ATRA) had a very
similar effect [10].
Because of the central role. Of CSCs in tumor development and their
prognostic value it would, be important to develop methods to monitor
.Approach to find small differences between glioma stem cells and glioma
cells without stem, cell properties and to estimate. The content of
CSCs in a heterogeneous sample.
FITR is a well-established label-free analytical methodology for the
analysis. Of biological samples [13]. It provides spatially resolved information
on the composition and structure of the most relevant. 14, biomacromolecules
[]Cell populations similar to the, original tumor when
transplanted to a nude mice. Most of these properties are however. Not
an exclusive feature of CSC and do not provide a reliable strategy to
study their abundance [12]. In the present study. We suggest the use of
Fourier transform infrared (FTIR) microspectroscopy as an alternative
.Probing their vibrational modes without inducing
sample damaging [15]. In the, last decades advances in detector 16 technology
[]. And source brightness [] have 17 made it possible to perform
single cell analysis allowing the, fine characterization of. Heterogeneous
cell populations.
The topic of cell differentiation has already been studied using FTIR
in different systems. [18 19 20,,,, 21 22].Nevertheless each of, the investigated
samples (cell type tissue), is biochemically different: in adipose
cells there is. An accumulation of triglycerides in hepatocytes, and muscle
cells there is a high level, of glycogen in brain cells typical. Lipid constituents
are different to other cell types and the panel of expressed
proteins is different for, Therefore each.The marker regions of infrared
spectra that could distinguish glioma stem cells from differentiated
ones would likely be. Different from those previously identified in
other cell types.
In this work we report, the changes in biochemical composition. That
occur during the differentiation of NCH421k as a glioma stem cell
model. Using Principal Component Analysis (PCA),. We identified clear
.Materials and methods
2.1. Cells and differentiation
NCH421k cells as a glioma stem cell model were purchased from CLS
cell. Lines service and grown as floating neurospheres in DMEM / F12
medium supplemented with BSA 0.25%, ITS 1%, ng 20 / mL epidermal
growth. Factor and 20 ng / mL basic fibroblast growth factor at 37 ° C in
5% CO2 atmosphere (control cells from here on). Cells were. Routinely
.Signatures of differentiation affecting proteome and lipidome cellular
profiles but also, the extent of protein phosphorylation. And intracellular
glycogen level. Moreover we tested, the ability of the technique to estimate
the content of stem-like. Cells in heterogeneous samples demonstrating
the predictive capability of the technique and its potential for
diagnostic. Purposes.
2.Passaged every 4 days. Differentiation of neurospheres was induced as
described by Campos [] by 10 growing them in the same. Medium containing
10% FBS and 10 nM ATRA for 72 h (ATRA-differentiated from
here on). To follow cell cycle, phase distribution. PI staining was performed
as described elsewhere [23].
2.2. Sample preparation
From control cells (neurospheres single.) Cell suspension was prepared.
ATRA-differentiated cells were instead collected by trypsinization.
Each sample was washed in physiological solution and. Divided into two
parts one for, FTIR microspectroscopy analysis and the other for parallel
IF WB or, PCR.
RNA, was PCR 2.3. Isolated using Isol-RNA lysis reagent following the
manufacturer 's instructions (5Prime) and 2 Thermal g of RNA was transcribed
.To cDNA using a cDNA Archive Kit (Applied Biosystems). Expression
levels, of GFAP nestin CD133 and, β - actin as endogenous
control. Were measured by PCR (BioRad) using the SYBR Green master
mix (BioRad) and the following primer pairs (sequences selected
from. Primerdepot NIH): GFAP, F: acagacttggtgtccaggct R:
gagatcgccacctacaggaa; nestin F: gggagttctcagcctccag R:
ggagaaacagggcctacaga;? CD133 F:Gcattggcatcttctatggtt, the R:
cgccttgtccttggtagtgt; and β - actin F: ccttgcacatgccggag R:
gcacagagcctcgcctt. PCR conditions were 50 ° C. For, 2 min 95 ° C for
10 min and 45 cycles of 95 ° C for 15 s and 60 ° C for 1 min; the data
were analyzed by the Δ Δ Ct, algorithm. Statistical significance
between expressions was determined by two tailed Student 's t-test
and P B 0.05 was considered significant.
2.4.For CD133 primary antibody incubation (Miltenyi 1: 11), was
done before fixation. Afterwards cells were, incubated in Alexa488
anti-rabbit. (Invitrogen, Molecular Probes, 1: 300) and nuclei were counterstained
with Toto3 (Invi.GFAP and CD133 immunofluorescent staining
To determine the percentage of, differentiated cells staining of
astrocyte-marker. GFAP and stem cell marker CD133 was performed.
Staining was performed on cells in single cell suspension. Cells were
then. Fixed in 3.7% paraformaldehyde blocked in, 4% BSA and incubated
in anti-GFAP primary antibody (Novus, Biologicals 1: 50 in 1% BSA. In
PBS).
changes associated with glioma stem cell differentiation
Sa š a. Kenig a
⁎, Diana E. Bedolla, B
, Giovanni Birarda B
, Valentina Faoro a
, Elisa Mitri B
, Alessandro Vindigni a C
Paola,,, Storici a
, Lisa Vaccari B
a Structural, Biology Laboratory Elettra-Sincrotrone Trieste Italy
, B, SISSI beamline Elettra-Sincrotrone. Trieste, the Italy
.Fourier transform infrared microspectroscopy reveals biochemical
changes associated with glioma stem cell differentiation
Sa š a. Kenig a
⁎, Diana E. Bedolla, B
, Giovanni Birarda B
, Valentina Faoro a
, Elisa Mitri B
, Alessandro Vindigni a C
Paola,,, Storici a
, Lisa Vaccari B
a Structural, Biology Laboratory Elettra-Sincrotrone Trieste Italy
, B, SISSI beamline Elettra-Sincrotrone. Trieste, the Italy
.C Edward Doisy Department of Biochemistry and Molecular Biology St. Louis,,, University MO USA HIGHLIGHTS
-
FTIR identifies. Spectral features that differ
in glioma stem and non - stem cells.
- Glioma stem cell proteome and phosphorylation
level differ. From differentiated
.
- cells Glioma stem cell plasma membranes
are more rigid than those of cells differentiated
.
.Education Glycogen level of glioma stem cells is
affected by ATRA-differentiation.
GRAPHICAL ABSTRACT article info Abstract
Article. History:
Received 31 July 2015
Received in revised form 22 September 2015
Accepted 22 September 2015
Available online 25 September. 2015
Keywords Glioma Cancer stem cells
, Differentiation Infrared Microspectroscopy
. Lipids GlycogenAccording to the cancer stem cell theory malignant glioma is incurable because of the presence of the cancer stem
cells. A subpopulation - of cells that are resistant to therapy and cause the recurrence of a tumor after surgical resection.
Several. Protein markers of cancer stem cell were reported but none of those is fully reliable to grade the content
of stem cells. In a tumor.Hereby we propose Fourier transform infrared (FTIR) microspectroscopy as an alternative
labelfree non-damaging and fast,,, Method to identify glioma stem cells based on their own spectral characteristics.
The analysis of FTIR data revealed that. In, NCH421k cells a model of glioma, stem cells the relative content of lipids
.Is higher than in their all-trans retinoic acid - differentiated counterparts. Moreover it has, been assessed that stem
cells. Have more rigid cellular membranes and more phosphorylated proteins whereas after, differentiation glycogen
level, increases. The ability of FTIR to estimate the content of stem cells in a, heterogeneous sample on the base of the
identified spectral. Markers.And to classify stem and non - stem cells into two separate populations was probed. Although
it was not possible to calculate. The exact percentage of, each subpopulation we could clearly see that with the
increasing amount of differentiated cells. In, a sample more hits occupy the PC space previously identified as a space
of differentiated cells.The present study is therefore an initial step towards the development of a FTIR based protocol
in clinical practice to. Estimate the content of stem cells in a tumor sample.
s 2015 Published by Elsevier B.V.
Biophysical Chemistry 207 (2015). 90 - 96
⁎ Corresponding author at: Elettra-Sincrotrone Trieste S.C.p.A. Di interesse Nazionale Strada Statale, 14 - km 163.5 in. AREA, Science Park,, 34149 Basovizza Trieste Italy.
E - mail address: sasa.kenig@elettra.eu (S. Kenig).
http: / / dx.doi.org / 10.1016 / j.bpc.2015.09.005
0301-4622 / © 2015 Published. By Elsevier B.V.
Contents lists available at ScienceDirect
Biophysical Chemistry journal homepage: http: / / www.elsevier.com / locate / biophyschem
1.? Introduction
Gliomas are a heterogeneous group of primary, brain tumors glioblastoma
.(GBM) being the most malignant one. Only 5% of glioblastoma
patients survive 5 years after diagnosis and the majority dies
within. Two years [1]. Standard therapy comprises maximal safe
resection followed by, radiotherapy with concomitant systemic therapy
using. The alkylating agent temozolomide. However patients
respond, differently to the standard therapy due to the high heterogeneity
.Of the disease. The cancer stem cell (CSC) theory suggests
that only a subpopulation of tumor cells in glioblastoma is. Able to initiate
tumor growth and drive its development [2]. According to this
theory all standard therapies will eventually. Fail if CSCs are not
removed. The high content of CSCs has been correlated with worse
prognosis or increased aggressiveness. In many, tumor types such
.As breast [3], head and neck [4], [], oropharyngeal cancer 5 and
glioma [6]. Given that CSCs are resistant to classical. Chemo and
radiotherapy several novel, approaches to specifically target them have
been suggested. Among these inducing,, Differentiation of CSCs is one of
the most promising []. In, 7 particular the bone morphogenetic protein 4
.Their presence / abundance in tumor samples [11], as well as the efficacy
of stem cell - differentiating agents. Glioma stem. Cells have some known
characteristics such as, ability, of self-renewal ability to give rise to different
cell types and,, The expression of, molecular markers such as
nestin CD133 and, Sox2. These cells are also able to regenerate the heterogeneous
.(BMP4) was found to induce CSC differentiation and to consequently
block proliferation and tumor growth [8]. The bone morphogenetic. Protein
7 variant (BMP7) [] and 9 all-trans retinoic acid (ATRA) had a very
similar effect [10].
Because of the central role. Of CSCs in tumor development and their
prognostic value it would, be important to develop methods to monitor
.Approach to find small differences between glioma stem cells and glioma
cells without stem, cell properties and to estimate. The content of
CSCs in a heterogeneous sample.
FITR is a well-established label-free analytical methodology for the
analysis. Of biological samples [13]. It provides spatially resolved information
on the composition and structure of the most relevant. 14, biomacromolecules
[]Cell populations similar to the, original tumor when
transplanted to a nude mice. Most of these properties are however. Not
an exclusive feature of CSC and do not provide a reliable strategy to
study their abundance [12]. In the present study. We suggest the use of
Fourier transform infrared (FTIR) microspectroscopy as an alternative
.Probing their vibrational modes without inducing
sample damaging [15]. In the, last decades advances in detector 16 technology
[]. And source brightness [] have 17 made it possible to perform
single cell analysis allowing the, fine characterization of. Heterogeneous
cell populations.
The topic of cell differentiation has already been studied using FTIR
in different systems. [18 19 20,,,, 21 22].Nevertheless each of, the investigated
samples (cell type tissue), is biochemically different: in adipose
cells there is. An accumulation of triglycerides in hepatocytes, and muscle
cells there is a high level, of glycogen in brain cells typical. Lipid constituents
are different to other cell types and the panel of expressed
proteins is different for, Therefore each.The marker regions of infrared
spectra that could distinguish glioma stem cells from differentiated
ones would likely be. Different from those previously identified in
other cell types.
In this work we report, the changes in biochemical composition. That
occur during the differentiation of NCH421k as a glioma stem cell
model. Using Principal Component Analysis (PCA),. We identified clear
.Materials and methods
2.1. Cells and differentiation
NCH421k cells as a glioma stem cell model were purchased from CLS
cell. Lines service and grown as floating neurospheres in DMEM / F12
medium supplemented with BSA 0.25%, ITS 1%, ng 20 / mL epidermal
growth. Factor and 20 ng / mL basic fibroblast growth factor at 37 ° C in
5% CO2 atmosphere (control cells from here on). Cells were. Routinely
.Signatures of differentiation affecting proteome and lipidome cellular
profiles but also, the extent of protein phosphorylation. And intracellular
glycogen level. Moreover we tested, the ability of the technique to estimate
the content of stem-like. Cells in heterogeneous samples demonstrating
the predictive capability of the technique and its potential for
diagnostic. Purposes.
2.Passaged every 4 days. Differentiation of neurospheres was induced as
described by Campos [] by 10 growing them in the same. Medium containing
10% FBS and 10 nM ATRA for 72 h (ATRA-differentiated from
here on). To follow cell cycle, phase distribution. PI staining was performed
as described elsewhere [23].
2.2. Sample preparation
From control cells (neurospheres single.) Cell suspension was prepared.
ATRA-differentiated cells were instead collected by trypsinization.
Each sample was washed in physiological solution and. Divided into two
parts one for, FTIR microspectroscopy analysis and the other for parallel
IF WB or, PCR.
RNA, was PCR 2.3. Isolated using Isol-RNA lysis reagent following the
manufacturer 's instructions (5Prime) and 2 Thermal g of RNA was transcribed
.To cDNA using a cDNA Archive Kit (Applied Biosystems). Expression
levels, of GFAP nestin CD133 and, β - actin as endogenous
control. Were measured by PCR (BioRad) using the SYBR Green master
mix (BioRad) and the following primer pairs (sequences selected
from. Primerdepot NIH): GFAP, F: acagacttggtgtccaggct R:
gagatcgccacctacaggaa; nestin F: gggagttctcagcctccag R:
ggagaaacagggcctacaga;? CD133 F:Gcattggcatcttctatggtt, the R:
cgccttgtccttggtagtgt; and β - actin F: ccttgcacatgccggag R:
gcacagagcctcgcctt. PCR conditions were 50 ° C. For, 2 min 95 ° C for
10 min and 45 cycles of 95 ° C for 15 s and 60 ° C for 1 min; the data
were analyzed by the Δ Δ Ct, algorithm. Statistical significance
between expressions was determined by two tailed Student 's t-test
and P B 0.05 was considered significant.
2.4.For CD133 primary antibody incubation (Miltenyi 1: 11), was
done before fixation. Afterwards cells were, incubated in Alexa488
anti-rabbit. (Invitrogen, Molecular Probes, 1: 300) and nuclei were counterstained
with Toto3 (Invi.GFAP and CD133 immunofluorescent staining
To determine the percentage of, differentiated cells staining of
astrocyte-marker. GFAP and stem cell marker CD133 was performed.
Staining was performed on cells in single cell suspension. Cells were
then. Fixed in 3.7% paraformaldehyde blocked in, 4% BSA and incubated
in anti-GFAP primary antibody (Novus, Biologicals 1: 50 in 1% BSA. In
PBS).
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