Cristina Zanini is employed by a commercial company (EuroClone SpA) with a permanent position and contributed to the study as researcher. Alice Poli is employed by a commercial company (BioDigitalValley srl) and contributed to the study as researcher. Their specific competences are R&D product for Research laboratories, Technology transfer, Strategic Marketing and Writing and management of R&D projects. This does not alter the authors’ adherence to all the PLOS ONE policies on sharing data and materials.
Conceived and designed the experiments: CZ MF GC. Performed the experiments: CZ EE GM RS CR VP AP. Analyzed the data: CZ EE GM RS CR VP AP. Contributed reagents/materials/analysis tools: CZ GM GC AP. Wrote the paper: CZ GC MF.
Medulloblastoma (MB) is an aggressive pediatric tumor of the Central Nervous System (CNS) usually treated according to a refined risk stratification. The study of cancer stem cells (CSC) in MB is a promising approach aimed at finding new treatment strategies.
The CSC compartment was studied in three characterized MB cell lines (DAOY, UW228 and ONS-76) grown in standard adhesion as well as being grown as spheres, which enables expansion of the CSC population. MB cell lines, grown in adherence and as spheres, were subjected to morphologic analysis at the light and electron microscopic level, as well as cytofluorimetric determinations. Medullospheres (MBS) were shown to express increasingly immature features, along with the stem cells markers: CD133, Nestin and β-catenin. Proteomic analysis highlighted the differences between MB cell lines, demonstrating a unique protein profile for each cell line, and minor differences when grown as spheres. In MBS, MALDI-TOF also identified some proteins, that have been linked to tumor progression and resistance, such as Nucleophosmin (NPM). In addition, immunocytochemistry detected Sox-2 as a stemness marker of MBS, as well as confirming high NPM expression.
Culture conditioning based on low attachment flasks and specialized medium may provide new data on the staminal compartment of CNS tumors, although a proteomic profile of CSC is still elusive for MB.
Medulloblastoma (MB) is an aggressive pediatric tumor of the cerebellum with “embryonal” features and early leptomeningeal spreading.
A dramatic increase in crude survival has been associated with relevant toxicity as a result of chemotherapy and/or radiation therapy effects on the developing brain.
A wealth of new data, from the new pathological classification
A better understanding of the role of Cancer Stem Cells (CSC), (recently also referred as brain tumor-initiating cells) may be of peculiar interest in MB, a tumor with relevant molecular heterogeneity
Proteomic analysis of MB subtypes may be of interest not only to refine stratification of patients into risk categories but also to give new insights into the elusive existence of CSC.
With the present study we report our experience in culturing tumor cells derived from MB in a serum-free culture medium resulting in the formation of spheres. We applied proteomic techniques to evaluate variations in protein expression, and the possible relation to relevant modifications in biological behavior, such as aggressiveness and therapy resistance. Mass spectrometry analysis did not confirm a unique proteomic profile for CSC generated from the three cells line of MB. Only a few protein modifications were found in MBS without any strong evidence of enrichment in CSC.
MBS were prepared from established MB cell lines and expanded in serum-free medium.
(
ONS-76 formed large spheres (142.77 µm±74.07) compared to DAOY (45.70±12.65) and UW228 (42.03±7.81). Furthermore, DAOY spheres were more fragile and more susceptible to destruction during manipulations.
Since the conditions of culture for sphere formation are the same the different size of spheres is related mainly to intrinsic proprieties of each cell line when grown as spheres. Cells were successfully amplified in medullospheres during more than 10 passages with variable amounts of spheres obtained at each passage depending on cell line. All MB cell lines continuously formed MBS with successive passages after. DAOY showed a number of spheres that increased in the first four passages but decreased during subsequent passages. Conversely, UW228 and ONS-76 showed a different trend, with the number of spheres decreasing from the second passage onwards (
DAOY MBS generally show a stem-like appearance: featuring a high nucleus: cytoplasm, (N:C) ratio. The nucleus is irregularly shaped and the cytoplasm is sparsely organized. Only focal RER profiles arranged in a concentric way are visible. DAOY MB cell lines show a more represented cytoplasm with a lower N:C ratio. In the cytoplasm there are numerous RER profiles, Golgi apparatus and focal mitochondria aggregates. (
MBS from DAOY (
ONS-76 MBS confirmed many of the aspects described for DAOY MBS, such as the stem-like appearance characterized by a high N:C ratio. The ONS-76 MB cell line also shows a similar sub-microsopic aspect featuring very little cytoplasm largely occupied by a prominent nucleus with a kidney-shaped appearance (
UV228 MBS cells seem to recapitulate the ultra-structural aspects already described for DAOY MBS. Sometimes a rudimentary-organized RER is visible in cells with a high N:C. The UW228 MB cell line shows a peripheral kidney-shaped nucleus (
MB cell lines show a considerably undifferentiated phenotype. They are plastic-adherent when maintained in expansion conditions and express certain progenitor markers such as CD44, Nanog and Oct4 at varying levels.
CD133 | ||||||||||||
CD44 | 96.19 | ±5.30 | 97.41 | ±3.24 | 99.01 | ±1.36 | 95.21 | ±5.35 | 91.01 | ±1.23 | 98.81 | ±0.45 |
Oct4 | 94.06 | ±4.65 | 90.78 | ±0.78 | 0.13 | ±0.13 | 0.74 | ±0.64 | ||||
Nanog | 93.88 | ±5.22 | 90.95 | ±0.72 | 94.27 | ±4.78 | 93.33 | ±3.45 | 0.59 | ±0.91 | 1.39 | ±1.12 |
β-catenin | ||||||||||||
Nestin |
The table represents mean standard deviation of three independent experiments. Numbers in bold indicate a statistically significant difference of spheres with reference to the control adhesion conditions (*p<0,01).
DAOY and UW228 adherent cell lines do not express neuronal stemness markers such as Nestin, or the very early progenitor markers such as CD133. In adherent conditions, ONS-76 cell lines express the same neuronal markers at high levels (Nestin: 50.7±5.7 and CD133∶54.5±1.4). Β-catenin, a protein involved in the WNT signaling pathway was expressed in variable levels according to cell type; in DAOY (β-catenin: 1.09±0.21), UW228 (β-catenin: 64.8±7.7) and ONS-76 (β-catenin: 11.4±0.9).
MBS were tested for the same protein expression, and DAOY and UW228 cell lines showed a general and evident enrichment in stem cells, featuring increased levels of Nestin, β-catenin and CD133 in comparison with MB adherent cell lines. Expression is variable but marked, both for DAOY (Nestin: 82.4±9.6; β-catenin: 83.8±2.0 and CD133∶92.1±9.9) and for UW228 (Nestin: 29.7±4.7; β-catenin: 94.0±4.5 and CD133∶49.3±7.2). ONS-76 cells lines were also shown to be positive for the same neuronal stemness markers (Nestin: 89.8±3.2; β-catenin: 81.5±2.0 and CD133∶90.6±1.4). Values of positive cells for neuronal stemness markers in MB and MBS for all three cells lines are represented in
FACS analyses shows expression of progenitor markers, CD133 (
Migration capability was different for our MB adherent cell lines: ONS-76 migrated more than DAOY and DAOY migrated much more than UW228. On the other hand, all MBS cell lines showed a statistically significant decrease in invasion capability respect to adherent cell lines (
Invasion capability of MB cells (ONS-76, DAOY and UW228) and their corresponding MBS (ONS-76 S, DAOY S and UW228 S) at 24h from experimental set up using a transwell (Matrigel) migration assay. The results are representative of two different experiments. Bar charts represent the mean of migrating cells evaluated in two independent experiments. *P<0,05 ***P<0,0001 (unpaired, two tailed t-test).
To identify proteins involved in spheres formation three independent 2-DE experiments on cellular lysate from DAOY, UW228 and ONS-76 in adherent conditions versus spheres at first passage (P1) were performed.
Representative image from three independent experiments of Coomassie-stained 2-DE patterns of DAOY(
After MALDI-TOF analysis and a database search, 300 spots were characterized. Many spots were isoforms of the same protein, while others were not identified with an acceptable score. From these, 77 unique proteins were well identified: 11 proteins CALR, ENOA, G3P, GRP78, HSP7C, HSPB1, PARK7, PRDX2, TPIS, UCHL1, VIME (indicated in blue in
CALR_HUMAN | P27797 | Calreticulin | 48283 | 4,29 | 8 | 30 | 81 | Adh/Sp |
ENOA_HUMAN | Q6GMP2 | Alpha-enolase | 47481 | 7,01 | 9 | 40 | 48 | Adh/Sp |
G3P_HUMAN | P04406 | Glyceraldehyde-3-phosphate dehydrogenase | 36201 | 8,57 | 7 | 28 | 74 | Adh/Sp |
GRP78_HUMAN | P11021 | 78 kDa glucose-regulated protein | 72402 | 5,07 | 11 | 22 | 104 | Adh/Sp |
HSP7C_HUMAN | P11142 | Heat shock cognate 71 kDa protein | 71082 | 5,37 | 10 | 16 | 76 | Adh/Sp |
HSPB1_HUMAN | Q9UC36 | Heat shock protein beta-1 | 22826 | 5,98 | 8 | 34 | 132 | Adh/Sp |
PARK7_HUMAN | Q99497 | Protein DJ-1 | 20050 | 6,33 | 16 | 72 | 179 | Adh/Sp |
PRDX2_HUMAN | P32119 | Peroxiredoxin-2 | 22049 | 5,66 | 8 | 41 | 124 | Adh/Sp |
TPIS_HUMAN | P60174 | Triosephosphate isomerase | 31057 | 5,65 | 12 | 41 | 158 | Adh/Sp |
UCHL1_HUMAN | P09936 | Ubiquitin carboxyl-terminal hydrolase isozyme L1 | 25151 | 5,33 | 11 | 65 | 174 | Adh/Sp |
VIME_HUMAN | Q8N850 | Vimentin | 53676 | 5,06 | 13 | 37 | 128 | Adh/Sp |
Abb, abbreviation.
AC, accession number.
MW, molecular weight.
pI, isoelectric point.
NVM, number of matched mass values on number of total mass values searched.
C%, the sequence coverage, which is calculated as the percentage of identified sequence to the complete sequence of the matched protein.
MS, mascot score.
CC, culture condition.
DAOY and UW228 modified their proteomic profile from adherent cells to spheres with nine new proteins identified in DAOY MBS (
ATP5H_HUMAN | O75947 | ATP synthase subunit d, mitochondrial | 18537 | 5,21 | 5 | 47 | 57 | Adh/Sp |
HNRH1_HUMAN | P31943 | Heterogeneous nuclear ribonucleoprotein H | 49484 | 5,89 | 7 | 27 | 61 | Adh/Sp |
IPYR_HUMAN | Q15181 | Inorganic pyrophosphatase | 33095 | 5,54 | 13 | 56 | 182 | Adh/Sp |
LEG1_HUMAN | P09382 | Galectin-1 | 15048 | 5,34 | 9 | 73 | 130 | Adh/Sp |
LGUL_HUMAN | Q04760 | Lactoylglutathione lyase | 20992 | 5,12 | 6 | 42 | 67 | Adh/Sp |
NDKA_HUMAN | P15531 | Nucleoside diphosphate kinase A | 17309 | 5,83 | 10 | 78 | 145 | Adh/Sp |
PDIA3_HUMAN | P30101 | Protein disulfide-isomerase A3 | 57146 | 5,98 | 12 | 32 | 134 | Adh/Sp |
PDLI1_HUMAN | O00151 | PDZ and LIM domain protein 1 | 36505 | 6,56 | 9 | 33 | 88 | Adh/Sp |
PPIA_HUMAN | Q9UC61 | Peptidyl-prolyl cis-trans isomerase A | 18229 | 7,68 | 9 | 74 | 126 | Adh/Sp |
RANG_HUMAN | P43487 | Ran-specific GTPase-activating protein | 23467 | 5,19 | 9 | 43 | 105 | Adh/Sp |
SODC_HUMAN | Q6NR85 | Superoxide dismutase [Cu-Zn] | 16154 | 5,7 | 5 | 38 | 72 | Adh/Sp |
STIP1_HUMAN | P31948 | Stress-induced-phosphoprotein 1 | 63227 | 6,4 | 9 | 25 | 80 | Adh/Sp |
TAGL2_HUMAN | P37802 | Transgelin-2 | 22548 | 8,41 | 7 | 43 | 84 | Adh/Sp |
YBOX1_HUMAN | P67809 | Nuclease-sensitive element-binding protein 1 | 35903 | 9,87 | 8 | 28 | 79 | Adh/Sp |
THIO_HUMAN | P10599 | Thioredoxin | 12015 | 4,82 | 4 | 54 | 60 | Adh/Sp |
NPM_HUMAN | Q8WTW5 | Nucleophosmin | 32726 | 4,64 | 7 | 26 | 52 | Adh/Sp |
CALL4_HUMAN | Q96GE6 | Calmodulin-like protein 4 | 21983 | 7,02 | 5 | 23 | 63 | Sp |
CNDG2_HUMAN | Q86XI2 | Condensin-2 complex subunit G2 | 130960 | 06∶43 | 7 | 20 | 48 | Sp |
GEMI5_HUMAN | Q8TEQ6 | Gem-associated protein 5 | 170821 | 6,17 | 7 | 7 | 56 | Sp |
ICAL_HUMAN | P20810 | Calpastatin | 76572 | 4,97 | 8 | 8 | 43 | Sp |
H2B1C_HUMAN | P62807 | Histone H2B type 1-C/E/F/G/I | 13898 | 10,31 | 9 | 43 | 84 | Sp |
LMNA_HUMAN | P02545 | Prelamin-A/C | 74380 | 6,57 | 8 | 15 | 63 | Sp |
PGK1_HUMAN | P00558 | Phosphoglycerate kinase 1 | 44985 | 8,3 | 10 | 28 | 105 | Sp |
POC1A_HUMAN | Q8NBT0 | POC1 centriolar protein homolog A | 45008 | 07∶31 | 10 | 22 | 46 | Sp |
TERA_HUMAN | P55072 | Transitional endoplasmic reticulum ATPase | 89950 | 5,14 | 7 | 14 | 60 | Sp |
Abb, abbreviation.
AC, accession number.
MW, molecular weight.
pI, isoelectric point.
NVM, number of matched mass values on number of total mass values searched.
C%, the sequence coverage, which is calculated as the percentage of identified sequence to the complete sequence of the matched protein.
MS, mascot score.
CC, culture condition.
ACTA_HUMAN | P62736 | Actin, aortic smooth muscle | 42381 | 5,23 | 8 | 22 | 88 | Adh/Sp |
ACTB_HUMAN | Q96HG5 | Actin, cytoplasmic 1 | 42052 | 5,29 | 10 | 21 | 60 | Adh/Sp |
ACTC_HUMAN | P68032 | Actin, alpha cardiac muscle 1 | 42334 | 5,23 | 8 | 22 | 88 | Adh/Sp |
ACTG_HUMAN | P63261 | Actin, cytoplasmic 2 | 42108 | 5,31 | 6 | 21 | 58 | Adh/Sp |
ACTH_HUMAN | P63267 | Actin, gamma-enteric smooth muscle | 42249 | 5,31 | 6 | 22 | 88 | Adh/Sp |
CALD1_HUMAN | Q05682 | Caldesmon | 93251 | 5,63 | 9 | 14 | 78 | Adh/Sp |
CD11A_HUMAN | Q9UP54 | Cell division protein kinase 11A | 91146 | 5,34 | 6 | 10 | 56 | Adh/Sp |
CRYAB_HUMAN | P02511 | Alpha-crystallin B chain | 20146 | 6,76 | 7 | 59 | 150 | Adh/Sp |
PDIA1_HUMAN | P07237 | Protein disulfide-isomerase | 57480 | 4,76 | 10 | 25 | 106 | Adh/Sp |
PDIA3_HUMAN | P30101 | Protein disulfide-isomerase A3 | 57146 | 5,98 | 9 | 18 | 84 | Adh/Sp |
PGK1_HUMAN | P00558 | Phosphoglycerate kinase 1 | 44985 | 8,3 | 10 | 37 | 108 | Adh/Sp |
PRDX1_HUMAN | Q06830 | Peroxiredoxin-1 | 22324 | 8,27 | 8 | 61 | 121 | Adh/Sp |
TNIP2_HUMAN | Q8NFZ5 | TNFAIP3-interacting protein 2 | 49240 | 6,03 | 7 | 15 | 66 | Adh/Sp |
TPM3_HUMAN | P06753 | Tropomyosin alpha-3 chain | 32856 | 4,68 | 8 | 16 | 74 | Sp |
ALBU_HUMAN | Q9P157 | Serum albumin | 71317 | 5,92 | 8 | 14 | 63 | Sp |
CALU_HUMAN | O43852 | Calumenin precursor | 34961 | 04∶46 | 15 | 11 | 49 | Sp |
CATD_HUMAN | P07339 | Cathepsin D | 45037 | 6,1 | 11 | 26 | 118 | Sp |
COF1_HUMAN | P23528 | Cofilin-1 | 18371 | 08∶26 | 12 | 22 | 55 | Sp |
LEG1_HUMAN | P09382 | Galectin-1 | 15048 | 5,34 | 5 | 34 | 61 | Sp |
MYL6_HUMAN | P60660 | Myosin light polypeptide 6 | 17090 | 4,56 | 10 | 50 | 112 | Sp |
ROA2_HUMAN | P22626 | Heterogeneous nuclear ribonucleoproteins A2/B1 | 37464 | 8,97 | 9 | 27 | 91 | Sp |
STMN1_HUMAN | P16949 | Stathmin | 17292 | 5,76 | 7 | 40 | 74 | Sp |
NPM_HUMAN | Q8WTW5 | Nucleophosmin | 32726 | 4,64 | 15 | 30 | 60 | Sp |
Abb, abbreviation.
AC, accession number.
MW, molecular weight.
pI, isoelectric point.
NVM, number of matched mass values on number of total mass values searched.
C%, the sequence coverage, which is calculated as the percentage of identified sequence to the complete sequence of the matched protein.
MS, mascot score.
CC, culture condition.
ARFRP_HUMAN | Q13795 | ADP-ribosylation factor-related protein 1 | 22614 | 07∶50 | 9 | 12 | 50 | Adh/Sp |
CH60_HUMAN | P10809 | 60 kDa heat shock protein, mitochondrial | 61187 | 5,7 | 10 | 18 | 80 | Adh/Sp |
CHSTE_HUMAN | Q8NCH0 | Carbohydrate sulfotransferase 14 | 43369 | 9,55 | 5 | 25 | 62 | Adh/Sp |
FUBP1_HUMAN | Q96AE4 | Far upstream element-binding protein 1 | 67690 | 7,18 | 7 | 16 | 63 | Adh/Sp |
GSHB_HUMAN | P48637 | Glutathione synthetase | 52523 | 5,67 | 15 | 39 | 178 | Adh/Sp |
H2B1B_HUMAN | P33778 | Histone H2B type 1-B | 13942 | 10,31 | 5 | 46 | 80 | Adh/Sp |
H2B1C_HUMAN | P62807 | Histone H2B type 1-C/E/F/G/I | 13898 | 10,31 | 9 | 43 | 84 | Adh/Sp |
H2B1D_HUMAN | P58876 | Histone H2B type 1-D | 13928 | 10,31 | 9 | 43 | 84 | Adh/Sp |
H2B1H_HUMAN | Q93079 | Histone H2B type 1-H | 13884 | 10,31 | 9 | 43 | 84 | Adh/Sp |
H2B1J_HUMAN | P06899 | Histone H2B type 1-J | 13896 | 10,31 | 5 | 46 | 80 | Adh/Sp |
H2B1K_HUMAN | O60814 | Histone H2B type 1-K | 13882 | 10,31 | 6 | 54 | 80 | Adh/Sp |
H2B1L_HUMAN | Q99880 | Histone H2B type 1-L | 13944 | 10,31 | 9 | 43 | 84 | Adh/Sp |
H2B1M_HUMAN | Q99879 | Histone H2B type 1-M | 13981 | 10,31 | 9 | 43 | 84 | Adh/Sp |
H2B1N_HUMAN | Q99877 | Histone H2B type 1-N | 13914 | 10,31 | 9 | 43 | 84 | Adh/Sp |
H2B1O_HUMAN | P23527 | Histone H2B type 1-O | 13898 | 10,31 | 5 | 46 | 80 | Adh/Sp |
H2B2E_HUMAN | Q16778 | Histone H2B type 2-E | 13912 | 10,31 | 5 | 46 | 80 | Adh/Sp |
HDGF_HUMAN | P51858 | Hepatoma-derived growth factor (HDGF) | 26788 | 4,7 | 6 | 22 | 56 | Adh/Sp |
HMGB1_HUMAN | P09429 | High mobility group protein B1 | 25049 | 5,62 | 7 | 38 | 72 | Adh/Sp |
HNRH1_HUMAN | P31943 | Heterogeneous nuclear ribonucleoprotein H | 49484 | 5,89 | 13 | 42 | 150 | Adh/Sp |
HSP71_HUMAN | P08107 | Heat shock 70 kDa protein 1A/1B | 70294 | 5,48 | 8 | 18 | 82 | Adh/Sp |
ICAL_HUMAN | P20810 | Calpastatin | 76925 | 4,98 | 9 | 19 | 79 | Adh/Sp |
K1328_HUMAN | Q86T90 | Uncharacterized protein KIAA1328 | 66187 | 8,36 | 8 | 17 | 61 | Adh/Sp |
KCY_HUMAN | P30085 | UMP-CMP kinase | 22436 | 5,44 | 6 | 34 | 70 | Adh/Sp |
LMNA_HUMAN | P02545 | Prelamin-A/C | 74380 | 6,57 | 10 | 19 | 78 | Adh/Sp |
NPM_HUMAN | P06748 | Nucleophosmin | 32,575 | 4,64 | 11 | 17 | 52 | Adh/Sp |
OVCA2_HUMAN | Q8WZ82 | Ovarian cancer-associated gene 2 protein | 24,417 | 6,43 | 12 | 18 | 54 | Adh/Sp |
PDLI1_HUMAN | O00151 | PDZ and LIM domain protein 1 | 36505 | 6,56 | 13 | 39 | 148 | Adh/Sp |
PHB_HUMAN | P35232 | Prohibitin | 29843 | 5,57 | 12 | 61 | 174 | Adh/Sp |
PON1_HUMAN | P27169 | Serum paraoxonase/arylesterase 1 | 39877 | 5,08 | 7 | 24 | 66 | Adh/Sp |
PPIA_HUMAN | Q9UC61 | Peptidyl-prolyl cis-trans isomerase A | 18229 | 7,68 | 9 | 67 | 104 | Adh/Sp |
SFPQ_HUMAN | P23246 | Splicing factor, proline- and glutamine-rich | 76216 | 9,45 | 10 | 22 | 85 | Adh/Sp |
SODC_HUMAN | Q6NR85 | Superoxide dismutase [Cu-Zn] | 16154 | 5,7 | 5 | 38 | 67 | Adh/Sp |
Abb, abbreviation.
AC, accession number.
MW, molecular weight.
pI, isoelectric point.
NVM, number of matched mass values on number of total mass values searched.
C%, the sequence coverage, which is calculated as the percentage of identified sequence to the complete sequence of the matched protein.
MS, mascot score.
CC, culture condition.
Calpastatin (ICAL) plays a role in neural stem cell self-renewal and differentiation. It is a protein of the calcium-dependent cysteine protease system, binds to a calmodulin- binding site (CALL4) and is also involved in the proteolysis of the amyloid precursor protein.
Poc1 is a stable basal body constituent that localizes to the basal body cartwheel, the site of new basal body assembly, and microtubule cylinder walls. Poc1 is required for stability of basal bodies, ciliary-based motility, and correct cilia formation
TMA sections were inspected after scanning with Panoramic Desk (3DHistech). A comparison on the same virtual slide of cells grown in adherence and as spheres enabled confirmation of the proteomic data on the expression of NPM that was negative in adherent UW228 cells but strongly expressed in the corresponding spheres.
Expression of CD44 and CD133 was in accordance with cytofluorimetric data but membrane staining was generally weaker in comparison to cytoplasmic or nuclear antigens.
In
Staining for Oct-4, Sox-2, beta Catenin and Nucleophosim on DAOY, ONS76 and UW228 cell lines grown as spheres are shown at 40x magnification. UW228 (second column) show smaller spheres and less intense staining for Oct-4 and Sox-2.
The networks identify all proteins (nodes) already studied in stem cell literature (
The networks identified connect all proteins (nodes) already studied in Stem Cell in the literature. The node size is proportional to the number of papers where the proteins have been studied, while the edge width is proportional to the document number where two proteins have been described together. In the network representation, the group of nodes, white colored, identify the proteins commonly detected in Adh/Sp of the cells analyzed: DAOY (
Furthermore only the proteins already identified in the Stem Cell were automatically selected by Protein Quest (PQ). So the proteins network represent a sub-group of the one experimentally analyzed. These aspect support their specific cell type expression and possibly help to further characterized their stem cell features.
The Bingo Gene Ontology was performed to easily identify all Biological Processes (BP) described by adherent vs. spheres proteins. The results are summarized in the
1,86E-05 | 7 | 25 | response to biotic stimulus | HIST1H2BC|VCP|HSPB1|PRDX2|HSPA5|HSPA8|ENO1 |
4,59E-05 | 3 | 11 | ER-nucleus signaling pathway | VCP|LMNA|HSPA5 |
7,86E-05 | 6 | 21 | negative regulation of programmed cell death | NPM1|HSPB1|GLO1|PRDX2|HSPA5|SOD1 |
1,72E-04 | 4 | 14 | nucleocytoplasmic transport | PDIA3|NPM1|LMNA|CALR |
1,76E-04 | 4 | 14 | nuclear transport | PDIA3|NPM1|LMNA|CALR |
1,76E-04 | 4 | 14 | glucose metabolic process | TPI1|PGK1|GAPDH|ENO1 |
2,70E-04 | 3 | 11 | oxygen and reactive oxygen species metabolic process | PRDX2|SOD1|PARK7 |
3,11E-04 | 5 | 18 | generation of precursor metabolites and energy | TPI1|TXN|PGK1|GAPDH|ENO1 |
4,98E-04 | 8 | 29 | regulation of signaling pathway | LGALS1|NPM1|UCHL1|PRDX2|HSPA5|SOD1|CALR|PARK7 |
5,75E-04 | 7 | 25 | homeostatic process | PDIA3|TXN|NPM1|PRDX2|SOD1|CALR|PARK7 |
6,69E-04 | 5 | 18 | regulation of protein kinase activity | NPM1|UCHL1|PRDX2|HSPA5|SOD1 |
2,73E-06 | 8 | 27 | response to biotic stimulus | ACTA2|CFL1|HSPB1|PRDX2|STMN1|HSPA5|HSPA8|ENO1 |
4,28E-06 | 4 | 13 | glucose catabolic process | TPI1|PGK1|GAPDH|ENO1 |
9,73E-06 | 4 | 13 | oxygen and reactive oxygen species metabolic process | CRYAB|PRDX2|PRDX1|PARK7 |
1,08E-05 | 7 | 23 | negative regulation of programmed cell death | CRYAB|ALB|CFL1|NPM1|HSPB1|PRDX2|HSPA5 |
3,07E-05 | 7 | 23 | cytoskeleton organization | ACTC1|CRYAB|CALD1|CFL1|NPM1|STMN1|CALR |
1,56E-04 | 5 | 17 | actin filament-based process | MYL6|ACTC1|CALD1|CFL1|CALR |
8,28E-04 | 8 | 27 | regulation of signaling pathway | LGALS1|NPM1|UCHL1|PRDX2|HSPA5|CALR|PRDX1|PARK7 |
9,00E-04 | 7 | 23 | homeostatic process | P4HB|PDIA3|NPM1|PRDX2|CALR|PRDX1|PARK7 |
7,02E-03 | 4 | 13 | regulation of protein kinase activity | NPM1|UCHL1|PRDX2|HSPA5 |
8,12E-07 | 8 | 29 | response to biotic stimulus | HIST1H2BC|HSPB1|HSPA1A|PRDX2|HSPD1|HSPA5|HSPA8|ENO1 |
3,82E-06 | 7 | 25 | negative regulation of programmed cell death | NPM1|HSPB1|HSPA1A|PRDX2|HSPD1|HSPA5|SOD1 |
1,76E-05 | 6 | 21 | cellular macromolecular complex assembly | HIST1H2BC|HIST1H2BD|HIST1H2BN|NPM1|HSPD1|CALR |
2,15E-05 | 4 | 14 | protein-DNA complex assembly | HIST1H2BC|HIST1H2BD|HIST1H2BN|NPM1 |
3,62E-05 | 4 | 14 | DNA recombination | HMGB1|PPIA|SFPQ|HSPD1 |
2,16E-04 | 3 | 11 | oxygen and reactive oxygen species metabolic process | PRDX2|SOD1|PARK7 |
4,67E-04 | 5 | 18 | regulation of protein kinase activity | NPM1|UCHL1|PRDX2|HSPA5|SOD1 |
2,24E-03 | 3 | 11 | nucleocytoplasmic transport | NPM1|LMNA|CALR |
2,28E-03 | 3 | 11 | nuclear transport | NPM1|LMNA|CALR |
2,28E-03 | 3 | 11 | glucose metabolic process | TPI1|GAPDH|ENO1 |
1,65E-03 | 7 | 25 | regulation of signaling pathway | NPM1|UCHL1|PRDX2|HSPA5|SOD1|CALR|PARK7 |
For example negative regulation of programmed cell death are commonly represented by NPM1, HSPB1, PRDX2, HSPA5 and specifically by GLO1, SOD1 for DAOY, CRYAB ALB, CFL1 for UW228 and HSPA1A, HSPD1, SOD1 for ONS-76 cells
The specific cell type BP have been also identified for DAOY such as: ER-nucleus signaling pathway p-value (p-value <10−05) nucleocytoplasmic transport (p-value<E-04) and generation of precursor metabolites and energy (p-value<E-04). Notably these BP are also enriched by spheres over expressed protein VCP, LMNA and PGK1.
For the UW228 the specific BP are the protein cytoskeleton organization (p-value <10−05) and actin filament-based process (p-value <10−04). Even in this case some spheres protein are described in these processes: NMP1, CFL1, MYL6 and STMN1.
Finally also the specific BP of the ONS-76 have been identified and are related to cellular macromolecular complex assembly, protein-DNA complex assembly and DNA recombination (p-value <10−05).
To further characterized the network, the Genemania analysis was performed.
Genemania generated network represented by official symbol genes including other genes, grey colored, added at the network because strictly connected to genes analyzed.
The network generated include co-expression, co-localization, genetic interaction, physical interaction, predicted and shared protein domain
The networks of each cell type were analyzed. The most connected nodes are represented by co-expression and co-localization data (data not shown). However, in order to define other information compare to the previous analysis, only the protein-protein interactions (physical interactions) were evaluated.
In the DAOY, UW228 and ONS-76 the data of physical interactions among proteins represent respectively the 11.8%, 9.64% and 6.86% of networks generated. All the interactions have been represented in the
Protein-protein interaction are identify by pink edges. The black nodes, white edged represent the physical interactions of the common proteins differentially expressed in all DAOY, UW228 and ONS-76 adherent and sphere cell lines, while Sp overexpressed proteins are yellow edged and specific cell type DAOY(
The physical interactions were defined for common proteins differentially expressed in adherent and sphere cell lines analyzed: 9/11 of common proteins interact, comprising CARL, GAPDH, HPA5, HSPA8, PARK7, PRDX2, UCHL1 and VIM for DAOY, 7/11 including CARL, GAPDH, HSPA5, HSPA8, TPI1, HSPB1 and UCHL1 for UW228 and 3/11 corresponding to PARK7, HSPA5, HSPA8 for ONS-76. Few interactions were also identify for spheres over expressed protein: VCP, HIST1, H2BC and PGK1 in the DAOY and CTSD, CFL1, STMN1and MYL6 in UW228.
It was also possible to identify new type of interactions, represented by the proteins retrieved in the proteomic database, grey colored.
The edge weight measures relative contribute of information in the network. The physical interactions were evaluated using the default measure weighting method.
Medulloblastoma (MB), the most frequent malignant brain tumor in the pediatric age, has been studied in every aspect from pathological to molecular, in order to guide multimodal therapy to avoid as far as possible overtreatment and therapy-related permanent side effects on developing brain. The propensity of MB to leptopmeningel spreading is an important clinicopathological feature and possibly related to early appearance of invasive clone(s)
Although the CSC model has raised a large interest as an innovative approach to find new therapeutic approaches for reducing tumor resistance, the actual existence of CSC is only putative, and mainly sustained by phenotypic data and the capacity to differentiate.
The presence of markers shared with stem cells (SC) of different origins could be just a recapitulation of embryogenesis, as opposed to a true functional similarity to stemness potentiality.
Besides the issue of CSC, the comparison of protein expression in different growth conditions may give interesting insights related to growth and dissemination of tumor cells in the natural history of the disease, and possibly new data related to resistance to treatment and recurrence.
One interesting observation is the fact that, although the same growth conditions were sufficient to obtain medullospheres from the three cell lines a striking difference of size was note for ONS76. This phenomenon is observed also in neural SC form adult brain and is explained to a different sensitivity to growth factors depending on the site of origin in CNS and the concentration of cytochines in the media
The comparison of three established cell lines of MB cultured in adherence, or as medullospheres, was aimed at unravelling the influence of growth conditions on the modulation of protein expression.
The DAOY proteomic profile is extensively described in Peyrl et al. (2003)
In contradiction to what was expected, few modifications in the proteomics asset were found, and were limited to the DAOY and UW228 spheres.
No new proteins were expressed by ONS-76 cells, although quantitative modifications of expression were detected. Of particular note is the protein NPM: already present in DAOY and ONS-76 under adherence conditions, it is expressed as a new protein in UW228 spheres, and is the only protein common in all three MBS. NPM has been extensively studied in human lymphomas
Conversely, in DAOY and UW228, new proteins, not present in adherence conditions, appeared, but without any shared protein between the two lines.
Among the newly expressed proteins, Stathmin was identified, although it was exclusively expressed by UW228 spheres. This protein is described as a marker of aggressiveness.
High stathmin expression has been correlated with tumor dissemination, is an important prognostic factor of medulloblastoma, and may serve as a useful marker for more intensive adjuvant therapy
GeneOntology analysis showed that all cell types have different Biological Processes (BP) (p-value <10−7–10−4) in common, some characterizing stem cell features: response to biotic stimulus, negative regulation of programmed cell death, oxygen and reactive oxygen species, metabolic and regulation of protein kinase activity
Considering all these results, it is remarkable that ONS-76 tumor cells display a more immature or primitive profile, expressing at least some SC markers even when cultured in adherence conditions, and showing only minor modifications induced by the conditioning to spheres. Ultrastructural analysis confirmed that ONS-76 MB cell line feature the undifferentiated phenotype, as evident in MBS. This aspect is in contrast with the evident proteomic changes induced in DAOY and UW228 cell lines when grown as spheres, which show undifferentiated aspects in both cell lines, i.e. the high nucleus:cytoplasmic ratio rather than minimal aspects of differentiation, such as a rudimentary RER in a fairly well represented cytoplasm, as seen in MB cell lines.
Immunohistochemistry confirmed stemness enrichment of spheres, showing expression of Sox-2, a marker of neural stem cells, and increased expression of NPM, a resistance marker.
A recent report by Wu et al. (2012)
Conversely, an impressive simplification of the proteomic asset of MB was reported in a relapsing MB after radiotherapy with the expression of some new proteins
While sphere formation may not be resolving as far as the issue of CSC, it may however be important for dissection of the adaptive modifications of tumor cells from primary or stabilized cell lines in an environmental context less permissive than adherence culture and with limited oxygen
MEM/EBSS, fetal bovine serum, sodium pyruvate, non-essential amino acids (NEAA), 2 mL-Glutamine, streptomycin and penicillin, DMEM/F12 Glutamine, RPMI, EUROMED CSC Neuronal medium, streptomycin and penicillin were obtained from EuroClone, Pero, Italy. Anti-CD44 and anti-Stathmin were purchased from Cell Signaling (Danvers, MA, USA). Anti-CD133, anti-Oct4, anti-Nanog, anti-Nestin, anti-Sox-2 mouse and goat polyclonal secondary anti-rabbit IgG H&L FITC-conjugated were purchased all from AbCam (Cambridge, UK). Transwell membrane was purchased from NeuroProbe Inc, (Gaithersburg, MD) and Matrigel from BD Bioscience (Bedford, MA). Anti- β-catenin, anti-Nucleophosmin (NPM) were purchased from Santa Cruz (Heidelberg, Germany). Fixation/Permeabilization solution was purchased from (BD). Ki67 was purchased from Ventana, Martinengo, Italy. DC Protein assay kit, acrylamide, agarose, readymade immobilized pH gradient (IPG) strip (7-cm IPG strips pH 3–10NL) were purchased from Bio-Rad (Hercules, CA, USA). Ampholine pH 3.5–10 was obtained from GE Healthcare (MI, ITALY). May-Grunwald Giemsa solution, protease inhibitors, ammonium persulfate (APS), bromophenol blue, glycerol, N,N,N’,N’-tetramethylethylene-diamine (TEMED), sodium dodecyl sulfate (SDS), TRIZMA, urea, 3-[(3-cholamidopropyl) dimethylammonio]-1-propanesulphonate (CHAPS), dithiothreitol (DTT), iodoacetamide were purchased from Sigma-Aldrich (St. Louis, MO, USA).
DAOY cells line was purchased from ATCC (United Kingdom U.K.), UW228
MB cell lines DAOY, UW228 and ONS-76 were cultured at 37°C, in 5% CO2. DAOY (3×104/ml) was cultured in MEM/EBSS supplemented with 10% heat-activated fetal bovine serum, sodium pyruvate, non-essential amino acids (NEAA), 2 mL-Glutamine, 100 g/mL streptomycin and 100 U/mL penicillin; UW228 (5×104/ml) was cultured in DMEM/F12 supplemented with 10% heat-activated fetal bovine serum, 2 mL-Glutamine, 100 g/mL streptomycin and 100 U/mL penicillin and finally ONS-76 was cultured (3×104/ml) in RPMI supplemented with 10% heat-activated fetal bovine serum, NEAA, 2 mL-Glutammina, 100 g/mL streptomycin and 100 U/mL penicillin. In order to produce MBS, DAOY, UW228 and ONS-76 were grown at confluence in adhesive condition, trypsinized, pelleted and plated (6×104/ml) in ultra-low attachment T25 Flasks (Corning Inc., NY, USA) for a further 7 days in serum-free EUROMED CSC Neuronal medium. After the first passage (P1) MBS were spun down at 1400 rpm (368 g), harvested and resuspended as a single cells suspension in the same serum-free medium at the same concentration in order to obtain subsequent passages. The spheres were identified as spherical ovoid aggregates with smooth outlines of more then 20 cells under microscope observation. For each passage, sphere suspensions from all cell lines (50 µl) were collected and counted in 96-well plates by inverted microscope (Olympus CKX41).
MB cells (DAOY, UW228 and ONS-76) and their corresponding MBS morphology were investigated with phase contrast by inverted microscopy and photographed with 40X magnification with a digital camera (Leica DFC345 FX). Dimension analysis of spheres was determined using the Panoramic Viewer (3D Histech).
For ultrastructural evaluation all MB cells and MBS were fixed in 2.5% glutaraldehyde in 0.1 M sodium cacodylate-buffer pH 7.3 immediately following
Three independent determinations were evaluated to determine MB cells and MBS phenotype and to calculate the average relative positivity and standard deviations for each cell line. MB cells and MBS were incubated with primary antibodies: mouse monoclonal anti-CD44 and rabbit polyclonal anti-CD133 for 30 minutes in FACS Flow (BD, Mi, Italy). Cells and spheres were subsequently incubated with goat polyclonal secondary anti-rabbit IgG H&L FITC-conjugated antibody for 30 minutes in FACS Flow. Alternatively, MB cells and MBS were fixed and permeabilized with Fixation/Permeabilization solution and incubated with rabbit polyclonal anti-Oct4, rabbit polyclonal anti-Nanog, mouse monoclonal anti-Nestin and rabbit polyclonal anti- β-catenin for 30 minutes in Perm/Wash™ buffer. Cells and spheres were subsequently incubated with goat polyclonal secondary antibody to mouse IgG H&L PE-conjugated or goat polyclonal secondary antibody to rabbit IgG H&L FITC-conjugated for 30 minutes in Perm/Wash™ buffer. For each determination at least 10,000 cells were analyzed on a Dako CyAn™-ADP. Summit® software was used for evaluation of results, including dot-plot and percentage of positive cells. The significance of differences between experimental conditions in each determination was determined using the two-tailed Student’s t test. Probability values less than 0.01 were considered significant (*p<0,01).
To assess cell migration ability in vitro, MB cells, DAOY, UW228 and ONS-76 (30,000 cells in serum-free medium), and their corresponding MBS (30,000 cells in serum-free EUROMED CSC Neuronal medium) were placed in the top chamber of a transwell migration Boyden chamber. The lower chamber was filled with 35 µl of serum (10%) containing medium. The transwell insert contained an 8-µm-pore-sized membrane with a thin layer of Matrigel. After incubation at 37°C for 24 h, cells that had not migrated to the lower chamber were removed from the upper surface of the transwell membrane with a cotton swab. Migrating cells on the lower membrane surface were fixed, stained with a May-Grunwald Giemsa solution and finally, stained cells were counted under a light microscope at 10X magnification. Two independent invasion assays were performed in duplicate. For statistical analysis data were analyzed using unpaired, two-tailed t test and the Graphpad software package for Windows (PRISM5). p<0.05 was considered to be statistically significant.
For light microscopy, cells cultured in adherence and as spheres were inglobated in fibrin mesh according to the cell-block procedure
From paraffin inclusions, tissue micro arrays (TMA) were constructed with TMA-Master (3DHistech, Euroclone, Italy) with 1.5 cores punched out from paraffin-embedded cells and spheres. Tissue sample tumors, such as medulloblastoma, and normal human tissue were also inserted in the TMA as internal controls for immunostaining.
Immunostaining was performed with Sox-2, Stathmin, β-catenin, Nestin, NPM, CD133, OCT4, Nanog, CD44 and Ki67 at the manufacturer’s suggested dilutions (or higher if necessary to avoid background staining). Antigen retrieval was induced by microwave treatment in EDTA buffer pH 8.5 for 5 minutes. Thermo Quanto (Bio-Optica, Milan, Italy) revealing kit with diaminobenzidine as chromogen was used according to the manufacturer’s instructions.
2-DE was performed using ready-made immobilized pH gradient (IPG) strips (7-cm IPG strips, pH 3–10 NL). Each sample (250 µg of protein for preparative gels) was applied onto an IPG gel by in-gel rehydration for 20 h, adding DTT 1% w/v, final concentration and Ampholine pH 3.5–10, 2% v/v, final concentration.
Isoelectric focusing was carried out in a Protean IEF cell apparatus (Bio-Rad, Segrate, Italy). Briefly, focusing for 7-cm IPG strips was started at 250 V, and the voltage was progressively increased to 4000 V until a maximum of 25000 V/h was reached. Focusing was performed at 18°C with a limit of 50 mA per strip. Subsequently, IPG strips were equilibrated by continuous shaking for 15 min in equilibration buffer 1 (6 M urea, 2% w/v SDS, 0.05 M Tris–HCl pH 8.8, 20% v/v glycerol, 1% w/v DTT) and for 12 min in equilibration buffer 2 (6 M urea, 2% w/v SDS, 0.05 M Tris–HCl pH 8.8, 20% v/v glycerol, 2.5% w/v iodoacetamide). For the second dimension, 7 cm 10% acrylamide gels were run on the Mini Protean system (Bio-Rad). Electrophoresis was performed for 30 min at 50 V and was then continued at 100 V until the Bromophenol blue front reached the lower limit of the gel. Gels were stained with colloidal Coomassie (18% v/v ethanol, 15% w/v ammonium sulfate, 2% v/v phosphoric acid, 0.2% w/v Coomassie G-250) for 48 h and destained with water.
2-DE image analysis was performed using PD-Quest software (version 7.2, Bio-Rad, Hercules, CA) according to the manufacturer’s instructions. Normalization of each individual spot was performed according to the total quantity of the valid spots in each gel, after subtraction of the background values. The spot volume was used as the analysis parameter to quantify protein expression.
Coomassie G-stained spots were excised from 2-DE preparative gels; destaining and in-gel enzymatic digestions were performed as previously described
The proteins identified in the three cell types were further analyzed by means of a web platform tool ProteinQuest (PQ) (
Image captions were extracted using the BFO Java library (
The ProteinQuest (PQ) database contains documents already tagged with biomedical dictionaries and ontologies such as proteins, drugs, cells, miRNA, diseases, bioprocess, clinical and biological techniques, etc. Each term assignment also takes into account the entire correspondent alias, so as to solve any possible disambiguation.
The biological relationship among terms identified into results can be explored by analyzing the co-occurrence of just a couple of terms.
Networks can be a representation of any relationship among terms; using PQ it is possible to generate a network from biological literature data connected by different articles.
Here the protein networks were generated by selecting the genes that are differentially expressed in the experiments described above, already associated with the stem cells in the literature. Networks connecting all proteins from analyzing stem cells described in the literature were visualized using Cytoscape
Finally the Genemania plugin
We would like to thank Dr. Radhika Srinivasan for careful editing of the completed manuscript. DAOY cells line was purchased from ATCC (United Kingdom U.K.), UW228 and ONS-76 cell lines were kindly provided by Dr. Charles G. Eberhart (John Hopkins University, Baltimora, MD) with the agreement of Dr. Mike Bobola (University of Washington, Seattle, WA).