Supplementary Data

Figure 2S 4321076543210<ul style="display: flex;"><li style="flex:1">C</li><li style="flex:1">A</li></ul>--080125 CSCs 080418 CSCs + IFN-a 48 h + IFN-a 72 h + IFN-a 48 h + IFN-a 72 h <ul style="display: flex;"><li style="flex:1">MHC I </li><li style="flex:1">MHC II </li><li style="flex:1">MICA </li><li style="flex:1">MICB </li><li style="flex:1">ULBP-1 </li><li style="flex:1">ULBP-2 </li><li style="flex:1">ULBP-3 </li><li style="flex:1">ULBP-4 </li></ul><ul style="display: flex;"><li style="flex:1">MHC I </li><li style="flex:1">MHC II </li><li style="flex:1">MICA </li><li style="flex:1">MICB </li><li style="flex:1">ULBP-1 </li><li style="flex:1">ULBP-2 </li><li style="flex:1">ULBP-3 </li><li style="flex:1">ULBP-4 </li></ul>713 12 11 10 9BD--+ IFN-a 48 h + IFN-a 72 h 080125 FBS 080125 FBS 080418 FBS + IFN-a 48 h + IFN-a 72 h 6543210876543210<ul style="display: flex;"><li style="flex:1">MHC I </li><li style="flex:1">MHC II </li><li style="flex:1">MICA </li><li style="flex:1">MICB </li><li style="flex:1">ULBP-1 </li><li style="flex:1">ULBP-2 </li><li style="flex:1">ULBP-3 </li><li style="flex:1">ULBP-4 </li></ul><ul style="display: flex;"><li style="flex:1">MHC I </li><li style="flex:1">MHC II </li><li style="flex:1">MICA </li><li style="flex:1">MICB </li><li style="flex:1">ULBP-1 </li><li style="flex:1">ULBP-2 </li><li style="flex:1">ULBP-3 </li><li style="flex:1">ULBP-4 </li></ul>Molecule Molecule Supplemental Results Table 1S. Modulation by IFN-α of APM in GBM CSC and FBS tumor cell lines. Molecule * <ul style="display: flex;"><li style="flex:1">Cell line </li><li style="flex:1">IFN-α‡ </li><li style="flex:1">HLA </li><li style="flex:1">β2-m# HLA </li><li style="flex:1">LMP </li><li style="flex:1">TAP1 TAP2 </li></ul>class II <ul style="display: flex;"><li style="flex:1">A</li><li style="flex:1">A HC§ </li><li style="flex:1">2</li><li style="flex:1">7</li><li style="flex:1">10 </li></ul>080125 CSCs 080125 FBS -+-1∞ (1) 3 (65) 2 (91) 1 (2) 6 (47) 2 (61) 1 (3) 1 (2) 1 (3) 2 (81) 11 (80) 13 (99) 1 (3) 8 (88) 4 (91) 1 (2) 1 (3) 2 (68) 2 (81) 4 (63) 4 (83) 1 (3) 6 (80) 3 (67) 2 (86) 1 (3) 2 (75) 2 (99) 14 (90) 7 (97) 5 (75) 7 (100) 6 (98) 2 (90) 1 (4) 3 (87) + 080418 CSCs 080418 FBS -+-<ul style="display: flex;"><li style="flex:1">2 (51) 1 (1) </li><li style="flex:1">1 (3) 2 (47) 2 (83) 2 (54) 1 (4) 1 (2) 1 (3) </li></ul>2 (81) 3 (76) 5 (75) 2 (50) 2 (83) 3 (71) 1 (3) 2 (87) 1 (2) 1 (3) 3 (70) 2 (88) 1 (4) 3 (87) 2 (76) 1 (3) 1 (3) 1 (2) 2 (78) 7 (98) 5 (99) 2 (94) 5 (100) 3 (100) 1 (4) 2 (100) 1 (2) +<ul style="display: flex;"><li style="flex:1">070104 CSCs </li><li style="flex:1">-</li></ul>1 (2) 1 (3) 1 (3) 2 (78) 1 (3) 1 (2) 1 (3) 1 (3) 1 (2) +2 (98) 8 (100) 10 (88) 4 (89) 3 (98) 3 (94) 1 (4) 2 (86) 2 (79) * expression of APM molecules was evaluated by intracellular staining and cytofluorimetric analysis; ‡ cells were treatead or not (+/-) for 72 h with 1000 IU/ml of IFN-α; # β-2 microglobulin; § β-2 microglobulin-free HLA-A heavy chain; ∞ values are indicated as ratio between the mean of fluorescence intensity of cells stained with the selected mAb and that of the negative control; bold values indicate significant MRFI (≥ 2). The numbers in parenthesis represent the percentage of positive cells. The experiment has been repeated three times; SD for each value ≥ 0.8 ≤ 4. Table 2S . Expression of proteasome and APM-associated chaperon molecules in GBM CSC and FBS tumor cell lines. <ul style="display: flex;"><li style="flex:1">Cell line </li><li style="flex:1">Molecule * </li></ul><ul style="display: flex;"><li style="flex:1">Delta MB1 </li><li style="flex:1">Z</li><li style="flex:1">Calnexin Calreticulin ERp57 Tapasin </li></ul>0627 CSCs 7 ‡ (100) 1 (3) 1 (5) 20 (100) 0627 FBS 1 (1) 1 (2) 1 (3) 3 (99) <ul style="display: flex;"><li style="flex:1">2 (92) </li><li style="flex:1">2 (67) 2 (86) </li></ul><ul style="display: flex;"><li style="flex:1">1 (3) </li><li style="flex:1">1 (2) </li><li style="flex:1">1 (2) </li></ul>080125 CSCs 16 (85) 1 (2) 1 (2) 49 (99) 080125 FBS 1 (5) 1 (4) 1 (5) 28 (100) 2 (85) 1 (3) 1 (2) 2 (99) 3 (95) 1 (3) <ul style="display: flex;"><li style="flex:1">080418 CSCs 8 (100) 1 (5) 1 (4) 13 (100) </li><li style="flex:1">2 (99) </li></ul>1 (5) 1 (3) 1 (4) 1 (5) <ul style="display: flex;"><li style="flex:1">080418 FBS 7 (89) 1 (5) 1 (4) 16 (98) </li><li style="flex:1">3 (98) </li></ul><ul style="display: flex;"><li style="flex:1">080201 CSCs 36 (99) 3 (98) 3 (98) 55 (100) </li><li style="flex:1">10 (98) </li><li style="flex:1">3 (98) 7 (97) </li></ul><ul style="display: flex;"><li style="flex:1">080201 FBS 16 (98) 3 (98) 6 (98) 80 (100) </li><li style="flex:1">9 (98) </li><li style="flex:1">2 (97) 5 (98) </li></ul><ul style="display: flex;"><li style="flex:1">071011 CSCs 17 (98) 3 (87) 3 (75) 100 (100) 15 (98) </li><li style="flex:1">3 (87) 7 (76) </li></ul>071011 FBS 16 (100) 1 (4) 2 (87) 20 (100) 080325 CSCs 10 (98) 3 (98) 4 (98) 200 (100) 070104 CSCs 1 (3) 2 (93) 1 (4) 52 (100) 1869 EBV-B 2 (99) 3 (89) 2 (82) 100 (91) 4 (87) 5 (99) 2 (88) 2 (94) 1 (3) 2 (95) 5 (98) 1 (4) 1 (3) 2 (81) 2 (70) 2 (98) * expression of APM molecules was evaluated by immunofluorescence on permeabilized cells and cytofluorimetric analysis; ‡ values are indicated as ratio between the mean of fluorescence intensity of cells stained with the selected mAb and that of the negative control; bold values indicate significant MRFI (≥ 2). The numbers in parenthesis represent the percentage of positive cells. The experiment has been repeated three times; SD for each value ≥ 1.2 ≤ 4. Table 3S . Modulation by IFN-α of the expression of proteasome and APM-associated chaperon molecules in GBM CSC and FBS tumor cell lines. Molecule * <ul style="display: flex;"><li style="flex:1">Cell line </li><li style="flex:1">IFN-α ‡ Delta MB1 </li><li style="flex:1">Z</li><li style="flex:1">Calnexin Calreticulin ERp57 Tapasin </li></ul><ul style="display: flex;"><li style="flex:1">080125 CSCs </li><li style="flex:1">-</li></ul>+-16# (85) 1 (2) 1 (5) 49 (100) 22 (100) 1 (3) 1 (4) 73 (100) 1 (5) 1 (4) 1 (5) 28 (100) 10 (99) 1 (2) 2 (99) 30 (100) 2 (95) 2 (100) 3 (95) 3 (98) 1 (2) 2 (3) 1 (2) 2 (2) 1 (2) 1 (3) 2 (89) 1 (2) 080125 FBS +080418 CSCs 080418 FBS -+-8 (100) 1 (5) 1 (4) 13 (100) 8 (100) 1 (4) 1 (3) 13 (100) 7 (89) 1 (5) 1 (4) 16 (98) 9 (100) 1 (3) 1 (3) 26 (100) 2 (99) 1 (5) 1 (4) 3 (100) 8 (99) 1 (3) 3 (98) 1 (3) 1 (5) 1 (2) 1 (3) +3 (99) <ul style="display: flex;"><li style="flex:1">070104 CSCs </li><li style="flex:1">-</li></ul><ul style="display: flex;"><li style="flex:1">1 (3) 2 (93) 1 (4) 52 (100) </li><li style="flex:1">2 (88) </li></ul>1 (4) 1 (3) 1 (3) 1 (3) +<ul style="display: flex;"><li style="flex:1">13 (99) 1 (3) 1 (4) 27 (100) </li><li style="flex:1">3 (98) </li></ul>* expression of APM molecules was evaluated by immunofluorescence on permeabilized cells and cytofluorimetric analysis; ‡ cells were treatead or not (+/-) for 72 h with 1000 IU/ml of IFN-α; # values are indicated as ratio between the mean of fluorescence intensity of cells stained with the selected mAb and that of the negative control; bold values indicate significant MRFI (≥ 2). The numbers in parenthesis represent the percentage of positive cells. The experiment has been repeated three times; SD for each value ≥ 1 ≤ 4. Table 4S. Modulation of MHC and APM molecules by 5-Aza CdR treatment of GBM and FBS tumor cell lines <ul style="display: flex;"><li style="flex:1">Cell line </li><li style="flex:1">5-Aza-CdR * </li><li style="flex:1">Molecule ‡ </li></ul><ul style="display: flex;"><li style="flex:1">MHC II </li><li style="flex:1">MICA </li><li style="flex:1">MB1 </li><li style="flex:1">LMP10 </li><li style="flex:1">TAP1 </li></ul>080125 CSCs 080125 FBS -+-1# (2) 1 (2) 1 (2) 1 (3) 1 (4) 1 (3) 1 (2) 2 (98) 1 (3) 1 (3) 1 (3) 1 (4) 2 (80) 1 (2) 1 (3) 1 (2) 2 (88) 3 (95) 3 (98) +3 (90) 080418 CSCs 080418 FBS -+-1 (2) 1 (3) 1 (3) 1 (3) 1 (2) 1 (3) 1 (5) 1 (3) 1 (4) 1 (3) 1 (3) 1 (2) 1 (3) 6 (99) 3 (95) 4 (99) 3 (98) 6 (100) 4 (97) 6 (100) +<ul style="display: flex;"><li style="flex:1">070104 </li><li style="flex:1">-</li></ul>2 (18) 1 (3) 1 (4) 2 (93) 1 (3) 1 (2) 1 (3) 1 (3) +<ul style="display: flex;"><li style="flex:1">5 (87) </li><li style="flex:1">2 (97) </li></ul><ul style="display: flex;"><li style="flex:1">1869 EBV-B </li><li style="flex:1">-</li></ul>12 (96) 1 (3) 1 (3) <ul style="display: flex;"><li style="flex:1">3 (89) </li><li style="flex:1">3 (91) </li><li style="flex:1">4 (95) </li></ul>+<ul style="display: flex;"><li style="flex:1">20 (100) </li><li style="flex:1">5 (100) </li><li style="flex:1">4 (100) </li><li style="flex:1">8 (100) </li></ul>* 5-Aza-2'-deoxycytidine (5μM) was added to culture medium for 4 days; ‡ molecule expression was assessed by surface (MHC II and MICA) or intracellular staining (MIB, LMP10 and TAP1) of cells with specific antibodies (see Material and Methods) and cytofluorimetric analysis. # values are indicated as ratio between the mean of fluorescence intensity of cells stained with the selected mAb and that of the negative control; bold values indicate significant MRFI (≥ 2). The numbers in parenthesis represent the percentage of positive cells. The experiment has been repeated three times; SD for each value ≥ 1 ≤ 4. Table 5S. Expression of immune response inhibitory molecules by GBM CSC or FBS tumor cell lines <ul style="display: flex;"><li style="flex:1">Cell line </li><li style="flex:1">Molecule* </li></ul><ul style="display: flex;"><li style="flex:1">CTLA-4 </li><li style="flex:1">PD-1 </li><li style="flex:1">PD-L1 </li><li style="flex:1">PD-L2 </li></ul>1 (4) 070104 CSC 2‡ (69) <ul style="display: flex;"><li style="flex:1">5 (95) </li><li style="flex:1">12 (98) </li></ul>080125 CSCs 080125 FBS 2 (81) 2 (94) 10 (99) 1 (3) <ul style="display: flex;"><li style="flex:1">2 (65) </li><li style="flex:1">3 (96) </li><li style="flex:1">19 (99) </li><li style="flex:1">2 (70) </li></ul>080418 CSCs 080418 FBS <ul style="display: flex;"><li style="flex:1">2 (55) </li><li style="flex:1">2 (85) </li><li style="flex:1">7 (99) </li></ul>1 (2) <ul style="display: flex;"><li style="flex:1">1 (3) </li><li style="flex:1">1 (5) </li><li style="flex:1">1 (2) </li></ul>9 (98) 080201 CSCs 080201 FBS <ul style="display: flex;"><li style="flex:1">2 (77) </li><li style="flex:1">3 (95) </li><li style="flex:1">11 (99) </li></ul>1 (3) <ul style="display: flex;"><li style="flex:1">2 (95) </li><li style="flex:1">2 (97) </li><li style="flex:1">31 (98) </li><li style="flex:1">2 (93) </li></ul>071011 CSCs 071011 FBS <ul style="display: flex;"><li style="flex:1">2 (42) </li><li style="flex:1">2 (59) </li><li style="flex:1">4 (97) </li></ul>1 (2) <ul style="display: flex;"><li style="flex:1">1 (3) </li><li style="flex:1">1 (5) </li><li style="flex:1">1 (4) </li></ul>2 (50) 080325 CSCs SW480 col 1061 mel 2 (62) 2 (59) 2 (54) 2 (56) 2 (80) 1 (1) 4 (94) 6 (98) 6 (99) 1 (3) 1 (1) 1 (2) 1869 EBV-B 1 (1) <ul style="display: flex;"><li style="flex:1">2 (83) </li><li style="flex:1">5 (97) </li></ul>1 (2) * expression of the indicated molecules was evaluated by immunofluorescence and cytofluorimetric analysis; ‡ values are indicated as ratio between the mean of fluorescence intensity of cells stained with the selected mAb and that of the negative control (MRFI); bold values indicate significant MRFI (≥ 2). The numbers in parenthesis represent the percentage of positive cells. The experiment has been repeated three times with SD for each value ≥ 0.6 ≤ 4. SW480 col and 1061 mel, that are a colorectal and a melanoma cell lines, respectively, were used in the experiment as not GBM tumor cells. Table 6S. Genes differentially expressed between CSC and FBS, p<0.005. Genes are ranked according to fold change. Fold- Paramet UG cluster ric p- value Permutati change on p-value (CSC/FB S) <ul style="display: flex;"><li style="flex:1">Gene symbol </li><li style="flex:1">Description </li></ul>oligodendrocyte transcription factor 1 (OLIG1), mRNA. ubiquitin specific peptidase 30 (USP30), mRNA. calsequestrin 2 (cardiac muscle) (CASQ2), mRNA. OLIG1 USP30 CASQ2 <ul style="display: flex;"><li style="flex:1">Hs.56663 7.53E-05 1.00E-04 </li><li style="flex:1">2192 </li></ul>2077 1593 Hs.486434 45281 Hs.57975 4.00E-07 906 134 SPARC-like 1 (hevin) (SPARCL1), transcript variant 2, mRNA. SPARCL1 CA10 Hs.62886 <ul style="display: flex;"><li style="flex:1">46627 </li><li style="flex:1">114 </li></ul>84 996 828 carbonic anhydrase X (CA10), transcript variant 3, mRNA. ADAM metallopeptidase domain 19 (meltrin beta) (ADAM19), transcript variant 2, mRNA. 3-hydroxy-3-methylglutarylCoenzyme A synthase 1 (soluble) (HMGCS1), transcript variant 2, mRNA. SRY (sex determining region Y)- box 8 (SOX8), mRNA. ribosomal protein S6 kinase, 90kDa, polypeptide 2 Hs.463466 18432 <ul style="display: flex;"><li style="flex:1">Hs.483944 26136 </li><li style="flex:1">ADAM19 </li></ul><ul style="display: flex;"><li style="flex:1">64 </li><li style="flex:1">750 </li></ul>HMGCS1 SOX8 Hs.397729 <ul style="display: flex;"><li style="flex:1">2109 </li><li style="flex:1">25 </li></ul>53 691 684 Hs.243678 16431 (RPS6KA2), transcript variant 2, mRNA. dipeptidyl-peptidase 4 (DPP4), mRNA. periostin, osteoblast specific factor (POSTN), transcript variant 2, mRNA. RPS6KA2 DPP4 Hs.719131 <ul style="display: flex;"><li style="flex:1">1855 </li><li style="flex:1">11 </li></ul>8 655 583 Hs.368912 25174 <ul style="display: flex;"><li style="flex:1">Hs.136348 27286 </li><li style="flex:1">POSTN </li></ul>779 57 569 <ul style="display: flex;"><li style="flex:1">553 </li><li style="flex:1">KIAA1549 KIAA1549 (KIAA1549), mRNA. Hs.605380 </li></ul>CDNA FLJ32758 fis, clone 1857 hCG_1999814 TESTI2001792 collagen, type VIII, alpha 2 (COL8A2), mRNA. Hs.666511 22226 <ul style="display: flex;"><li style="flex:1">2</li><li style="flex:1">544 </li></ul>538 506 COL8A2 EMP2 Hs.353001 43215 2117 epithelial membrane protein 2 (EMP2), mRNA. Hs.531561 <ul style="display: flex;"><li style="flex:1">2084 </li><li style="flex:1">1.00E-04 </li></ul>guanine nucleotide binding protein (G protein), gamma 7 (GNG7), mRNA. GNG7 <ul style="display: flex;"><li style="flex:1">Hs.515544 1.94E-05 1.00E-04 </li><li style="flex:1">505 </li></ul>PREDICTED: misc_RNA LOC346329 (LOC346329), miscRNA. arylsulfatase D (ARSD), transcript <ul style="display: flex;"><li style="flex:1">2.57E-05 5.00E-04 </li><li style="flex:1">484 </li></ul>473 ARSD variant 1, mRNA. Hs.528631 29984 Hs.567236 507 823 shroom family member 2 SHROOM2 (SHROOM2), mRNA. olfactory receptor, family 51, subfamily B, member 2 <ul style="display: flex;"><li style="flex:1">3.00E-04 </li><li style="flex:1">416 </li></ul>410 410 381 OR51B2 ST3GAL6 SIPA1L2 (OR51B2), mRNA. Hs.680163 44691 1307 9.00E-04 38 ST3 beta-galactoside alpha-2,3- sialyltransferase 6 (ST3GAL6), mRNA. signal-induced proliferationassociated 1 like 2 (SIPA1L2), mRNA. Hs.148716 Hs.715656 7475 1074 CD164 molecule, sialomucin (CD164), transcript variant 5, mRNA. calumenin (CALU), transcript variant 1, mRNA. CD164 CALU Hs.520313 1.10E-06 1.00E-04 Hs.718425 7871 799 381 377 paroxysmal nonkinesigenic dyskinesia (PNKD), transcript variant 2, mRNA. PNKD <ul style="display: flex;"><li style="flex:1">Hs.98475 5.75E-05 5.00E-04 </li><li style="flex:1">375 </li></ul>369 kelch domain containing 8A KLHDC8A (KLHDC8A), mRNA. Hs.10414 <ul style="display: flex;"><li style="flex:1">15658 </li><li style="flex:1">54 </li></ul>169 25 RAB11 family interacting protein RAB11FIP4 4 (class II) (RAB11FIP4), mRNA. Hs.406788 43079 chromosome 11 open reading 352 348 C11orf66 frame 66 (C11orf66), mRNA. trinucleotide repeat containing 6B (TNRC6B), transcript variant 2, mRNA.

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