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.
การแปล กรุณารอสักครู่..