Tumor escape after immunotherapy: implication of HLA class I expression in melanoma and bladder tumors

Tesis Univ. Granada. Departamento de Bioquímica y Biología Molecular III e Inmunología. Leída el de 7 de abril del 201

Progressing vs regressing melanoma metastases (GSE24067)

[Organism] Homo sapiens[Experiment type] Expression profiling by array[Overall design] Two-condition experiment, Progressing vs regressing metastases. 3 progressing and 2 regressing metastases extracted from the same patient after M-VAX immunotherapy. One replicate per array.[Platforms (1)] GPL7088 CCDTM Hs_CCDTM36k - version 1[Relations] BioProject PRJNA1302311. Sample GSM592226. Title: melanoma progressing 1. Sample type: RNA. Platform ID: GPL7088. Series: GSE24067 Progressing vs regressing melanoma metastases; GSE26383 Response to immunotherapy in melanoma metastasis.Channel 1: [Source name] melanoma, progressing [Organism] Homo sapiens [Characteristics] tissue: melanoma treatment protocol: M-VAX preservation: cryopreservation patient: 1 [Extracted molecule] total RNA [Extraction protocol] Total RNA extracted using Qiagen's RNEasy Mini kit following manufacturer's instructions [Label] Cy5 [Label protocol] 0.5 µg of total RNA was primed with 0.25 ug oligo dT-T7 primer (5’ AAA CGA CGG CCA GTG AAT TGT AAT ACG ACT CAC TAT AGG CGC T(15) 3’) at 70°C for 3 min, then reverse transcribed at 42°C for 90 min in the presence of 200 U SuperScript II RTase (Invitrogen), and 1 mM dNTP. Second strand synthesis was done with the 5’ primer switch method using 0.25 ug Ts/SMARTII primers (5’ AAG CAG TGG TAA CAA CGC AGA GTA CGC GGG 3’) for priming at 65°C for 1 min, and subsequent primer extension at 75°C for 30 min using the Advantage cDNA Polymerase Mix (Clontech) and 0.2mM dNTP. Double stranded cDNA subjected to in vitro transcription at 42°C for 6h using Megascript T7 kit (Ambion). After two rounds of linear amplification, cRNA was chemically labeled using ULS Cy3/Cy5 Fluorescent Labeling Kit (Kreatech), and fragmented by Fragmentation Reagents kit (Ambion).Channel 2: [Source name] Total RNA from pooled PBMC [Organism] Homo sapiens [Characteristics] tissue: Blood preservation: cryopreservation reference: Total RNA from pooled PBMC [Extracted molecule] total RNA [Extraction protocol] Total RNA extracted using Qiagen's RNEasy Mini kit following manufacturer's instructions [Label] Cy3 [Label protocol] 0.5 µg of total RNA was primed with 0.25 ug oligo dT-T7 primer (5’ AAA CGA CGG CCA GTG AAT TGT AAT ACG ACT CAC TAT AGG CGC T(15) 3’) at 70°C for 3 min, then reverse transcribed at 42°C for 90 min in the presence of 200 U SuperScript II RTase (Invitrogen), and 1 mM dNTP. Second strand synthesis was done with the 5’ primer switch method using 0.25 ug Ts/SMARTII primers (5’ AAG CAG TGG TAA CAA CGC AGA GTA CGC GGG 3’) for priming at 65°C for 1 min, and subsequent primer extension at 75°C for 30 min using the Advantage cDNA Polymerase Mix (Clontech) and 0.2mM dNTP. Double stranded cDNA subjected to in vitro transcription at 42°C for 6h using Megascript T7 kit (Ambion). After two rounds of linear amplification, cRNA was chemically labeled using ULS Cy3/Cy5 Fluorescent Labeling Kit (Kreatech), and fragmented by Fragmentation Reagents kit (Ambion).[Hybridization protocol] 6 ug of labeled, fragmented cRNA per sample was applied to microarrays in Kreatech's Hybridization buffer, and hybridized for 16 h at 42C. After hybridization, slides were washed three four times with wash solutions containg gradually decreased concentrations of SSC (1x-0x) [Scan protocol] Scanned on an Agilent DNA Microarray Scanner. Images were quantified using Agilent Feature Extraction Software (version A.9.5.3). [Description] metastasis non responding after M-VAX immunotherapy 1 [Data processing] LOWESS normalized, background subtracted data obtained from log2 of processed Red signal/processed Green signal. Agilent software was used for feature extraction.2. Sample GSM592227. Title: melanoma progressing 2. Sample type: RNA. Platform ID: GPL7088. Series (2): GSE24067 Progressing vs regressing melanoma metastases. GSE26383 Response to immunotherapy in melanoma metastasis.Channel 1 [Source name] melanoma, progressing [Organism] Homo sapiens [Characteristics] tissue: melanoma treatment protocol: M-VAX preservation: cryopreservation patient: 1 [Extracted molecule] total RNA [Extraction protocol] Total RNA extracted using Qiagen's RNEasy Mini kit following manufacturer's instructions [Label] Cy5 [Label protocol] 0.5 µg of total RNA was primed with 0.25 ug oligo dT-T7 primer (5’ AAA CGA CGG CCA GTG AAT TGT AAT ACG ACT CAC TAT AGG CGC T(15) 3’) at 70°C for 3 min, then reverse transcribed at 42°C for 90 min in the presence of 200 U SuperScript II RTase (Invitrogen), and 1 mM dNTP. Second strand synthesis was done with the 5’ primer switch method using 0.25 ug Ts/SMARTII primers (5’ AAG CAG TGG TAA CAA CGC AGA GTA CGC GGG 3’) for priming at 65°C for 1 min, and subsequent primer extension at 75°C for 30 min using the Advantage cDNA Polymerase Mix (Clontech) and 0.2mM dNTP. Double stranded cDNA subjected to in vitro transcription at 42°C for 6h using Megascript T7 kit (Ambion). After two rounds of linear amplification, cRNA was chemically labeled using ULS Cy3/Cy5 Fluorescent Labeling Kit (Kreatech), and fragmented by Fragmentation Reagents kit (Ambion).Channel 2 [Source name] Total RNA from pooled PBMC [Organism] Homo sapiens [Characteristics] tissue: Blood preservation: cryopreservation reference: Total RNA from pooled PBMC [Extracted molecule] total RNA [Extraction protocol] Total RNA extracted using Qiagen's RNEasy Mini kit following manufacturer's instructions [Label] Cy3 [Label protocol] 0.5 µg of total RNA was primed with 0.25 ug oligo dT-T7 primer (5’ AAA CGA CGG CCA GTG AAT TGT AAT ACG ACT CAC TAT AGG CGC T(15) 3’) at 70°C for 3 min, then reverse transcribed at 42°C for 90 min in the presence of 200 U SuperScript II RTase (Invitrogen), and 1 mM dNTP. Second strand synthesis was done with the 5’ primer switch method using 0.25 ug Ts/SMARTII primers (5’ AAG CAG TGG TAA CAA CGC AGA GTA CGC GGG 3’) for priming at 65°C for 1 min, and subsequent primer extension at 75°C for 30 min using the Advantage cDNA Polymerase Mix (Clontech) and 0.2mM dNTP. Double stranded cDNA subjected to in vitro transcription at 42°C for 6h using Megascript T7 kit (Ambion). After two rounds of linear amplification, cRNA was chemically labeled using ULS Cy3/Cy5 Fluorescent Labeling Kit (Kreatech), and fragmented by Fragmentation Reagents kit (Ambion).[Hybridization protocol] 6 ug of labeled, fragmented cRNA per sample was applied to microarrays in Kreatech's Hybridization buffer, and hybridized for 16 h at 42C. After hybridization, slides were washed three four times with wash solutions containg gradually decreased concentrations of SSC (1x-0x) [Scan protocol] Scanned on an Agilent DNA Microarray Scanner. Images were quantified using Agilent Feature Extraction Software (version A.9.5.3). [Description] metastasis non responding after M-VAX immunotherapy 2 [Data processing] LOWESS normalized, background subtracted data obtained from log2 of processed Red signal/processed Green signal. Agilent software was used for feature extraction.3. Sample GSM592228. Title: melanoma progressing 3. Sample type: RNA. Platform ID: GPL7088. Series (2): GSE24067 Progressing vs regressing melanoma metastases; GSE26383 Response to immunotherapy in melanoma metastasisChannel 1 [Source name] melanoma, progressing [Organism] Homo sapiens [Characteristics] tissue: melanoma treatment protocol: M-VAX preservation: cryopreservation patient: 1 [Extracted molecule] total RNA [Extraction protocol] Total RNA extracted using Qiagen's RNEasy Mini kit following manufacturer's instructions [Label] Cy5 [Label protocol] 0.5 µg of total RNA was primed with 0.25 ug oligo dT-T7 primer (5’ AAA CGA CGG CCA GTG AAT TGT AAT ACG ACT CAC TAT AGG CGC T(15) 3’) at 70°C for 3 min, then reverse transcribed at 42°C for 90 min in the presence of 200 U SuperScript II RTase (Invitrogen), and 1 mM dNTP. Second strand synthesis was done with the 5’ primer switch method using 0.25 ug Ts/SMARTII primers (5’ AAG CAG TGG TAA CAA CGC AGA GTA CGC GGG 3’) for priming at 65°C for 1 min, and subsequent primer extension at 75°C for 30 min using the Advantage cDNA Polymerase Mix (Clontech) and 0.2mM dNTP. Double stranded cDNA subjected to in vitro transcription at 42°C for 6h using Megascript T7 kit (Ambion). After two rounds of linear amplification, cRNA was chemically labeled using ULS Cy3/Cy5 Fluorescent Labeling Kit (Kreatech), and fragmented by Fragmentation Reagents kit (Ambion).Channel 2 [Source name] Total RNA from pooled PBMC [Organism] Homo sapiens [Characteristics] tissue: Blood preservation: cryopreservation reference: Total RNA from pooled PBMC [Extracted molecule] total RNA [Extraction protocol] Total RNA extracted using Qiagen's RNEasy Mini kit following manufacturer's instructions [Label] Cy3 [Label protocol] 0.5 µg of total RNA was primed with 0.25 ug oligo dT-T7 primer (5’ AAA CGA CGG CCA GTG AAT TGT AAT ACG ACT CAC TAT AGG CGC T(15) 3’) at 70°C for 3 min, then reverse transcribed at 42°C for 90 min in the presence of 200 U SuperScript II RTase (Invitrogen), and 1 mM dNTP. Second strand synthesis was done with the 5’ primer switch method using 0.25 ug Ts/SMARTII primers (5’ AAG CAG TGG TAA CAA CGC AGA GTA CGC GGG 3’) for priming at 65°C for 1 min, and subsequent primer extension at 75°C for 30 min using the Advantage cDNA Polymerase Mix (Clontech) and 0.2mM dNTP. Double stranded cDNA subjected to in vitro transcription at 42°C for 6h using Megascript T7 kit (Ambion). After two rounds of linear amplification, cRNA was chemically labeled using ULS Cy3/Cy5 Fluorescent Labeling Kit (Kreatech), and fragmented by Fragmentation Reagents kit (Ambion).[Hybridization protocol] 6 ug of labeled, fragmented cRNA per sample was applied to microarrays in Kreatech's Hybridization buffer, and hybridized for 16 h at 42C. After hybridization, slides were washed three four times with wash solutions containg gradually decreased concentrations of SSC (1x-0x) [Scan protocol] Scanned on an Agilent DNA Microarray Scanner. Images were quantified using Agilent Feature Extraction Software (version A.9.5.3). [Description] metastasis non responding after M-VAX immunotherapy 3 [Data processing] LOWESS normalized, background subtracted data obtained from log2 of processed Red signal/processed Green signal. Agilent software was used for feature extraction.4. Sample GSM592229. Title: melanoma regressing 1. Sample type: RNA. Platform ID: GPL7088. Series (2): GSE24067 Progressing vs regressing melanoma metastases; GSE26383 Response to immunotherapy in melanoma metastasis.Channel 1 [Source name] melanoma, regressing [Organism] Homo sapiens [Characteristics] tissue: melanoma treatment protocol: M-VAX preservation: cryopreservation patient: 1 [Extracted molecule] total RNA [Extraction protocol] Total RNA extracted using Qiagen's RNEasy Mini kit following manufacturer's instructions [Label] Cy5 [Label protocol] 0.5 µg of total RNA was primed with 0.25 ug oligo dT-T7 primer (5’ AAA CGA CGG CCA GTG AAT TGT AAT ACG ACT CAC TAT AGG CGC T(15) 3’) at 70°C for 3 min, then reverse transcribed at 42°C for 90 min in the presence of 200 U SuperScript II RTase (Invitrogen), and 1 mM dNTP. Second strand synthesis was done with the 5’ primer switch method using 0.25 ug Ts/SMARTII primers (5’ AAG CAG TGG TAA CAA CGC AGA GTA CGC GGG 3’) for priming at 65°C for 1 min, and subsequent primer extension at 75°C for 30 min using the Advantage cDNA Polymerase Mix (Clontech) and 0.2mM dNTP. Double stranded cDNA subjected to in vitro transcription at 42°C for 6h using Megascript T7 kit (Ambion). After two rounds of linear amplification, cRNA was chemically labeled using ULS Cy3/Cy5 Fluorescent Labeling Kit (Kreatech), and fragmented by Fragmentation Reagents kit (Ambion).Channel 2 [Source name] Total RNA from pooled PBMC [Organism] Homo sapiens [Characteristics] tissue: Blood preservation: cryopreservation reference: Total RNA from pooled PBMC [Extracted molecule] total RNA [Extraction protocol] Total RNA extracted using Qiagen's RNEasy Mini kit following manufacturer's instructions [Label] Cy3 [Label protocol] 0.5 µg of total RNA was primed with 0.25 ug oligo dT-T7 primer (5’ AAA CGA CGG CCA GTG AAT TGT AAT ACG ACT CAC TAT AGG CGC T(15) 3’) at 70°C for 3 min, then reverse transcribed at 42°C for 90 min in the presence of 200 U SuperScript II RTase (Invitrogen), and 1 mM dNTP. Second strand synthesis was done with the 5’ primer switch method using 0.25 ug Ts/SMARTII primers (5’ AAG CAG TGG TAA CAA CGC AGA GTA CGC GGG 3’) for priming at 65°C for 1 min, and subsequent primer extension at 75°C for 30 min using the Advantage cDNA Polymerase Mix (Clontech) and 0.2mM dNTP. Double stranded cDNA subjected to in vitro transcription at 42°C for 6h using Megascript T7 kit (Ambion). After two rounds of linear amplification, cRNA was chemically labeled using ULS Cy3/Cy5 Fluorescent Labeling Kit (Kreatech), and fragmented by Fragmentation Reagents kit (Ambion).Hybridization protocol 6 ug of labeled, fragmented cRNA per sample was applied to microarrays in Kreatech's Hybridization buffer, and hybridized for 16 h at 42C. After hybridization, slides were washed three four times with wash solutions containg gradually decreased concentrations of SSC (1x-0x) [Scan protocol] Scanned on an Agilent DNA Microarray Scanner. Images were quantified using Agilent Feature Extraction Software (version A.9.5.3). [Description] metastasis responding after M-VAX immunotherapy 1 [Data processing] LOWESS normalized, background subtracted data obtained from log2 of processed Red signal/processed Green signal. Agilent software was used for feature extraction.5. Sample GSM592230. Title: melanoma regressing 2. Sample type: RNA. Platform ID: GPL7088. Series (2): GSE24067 Progressing vs regressing melanoma metastases; GSE26383 Response to immunotherapy in melanoma metastasis.Channel 1 [Source name] melanoma, regressing [Organism] Homo sapiens [Characteristics] tissue: melanoma treatment protocol: M-VAX preservation: cryopreservation patient: 1 [Extracted molecule] total RNA [Extraction protocol] Total RNA extracted using Qiagen's RNEasy Mini kit following manufacturer's instructions [Label] Cy5 [Label protocol] 0.5 µg of total RNA was primed with 0.25 ug oligo dT-T7 primer (5’ AAA CGA CGG CCA GTG AAT TGT AAT ACG ACT CAC TAT AGG CGC T(15) 3’) at 70°C for 3 min, then reverse transcribed at 42°C for 90 min in the presence of 200 U SuperScript II RTase (Invitrogen), and 1 mM dNTP. Second strand synthesis was done with the 5’ primer switch method using 0.25 ug Ts/SMARTII primers (5’ AAG CAG TGG TAA CAA CGC AGA GTA CGC GGG 3’) for priming at 65°C for 1 min, and subsequent primer extension at 75°C for 30 min using the Advantage cDNA Polymerase Mix (Clontech) and 0.2mM dNTP. Double stranded cDNA subjected to in vitro transcription at 42°C for 6h using Megascript T7 kit (Ambion). After two rounds of linear amplification, cRNA was chemically labeled using ULS Cy3/Cy5 Fluorescent Labeling Kit (Kreatech), and fragmented by Fragmentation Reagents kit (Ambion).Channel 2 [Source name] Total RNA from pooled PBMC [Organism] Homo sapiens [Characteristics] tissue: Blood preservation: cryopreservation reference: Total RNA from pooled PBMC [Extracted molecule] total RNA [Extraction protocol] Total RNA extracted using Qiagen's RNEasy Mini kit following manufacturer's instructions [Label] Cy3 [Label protocol] 0.5 µg of total RNA was primed with 0.25 ug oligo dT-T7 primer (5’ AAA CGA CGG CCA GTG AAT TGT AAT ACG ACT CAC TAT AGG CGC T(15) 3’) at 70°C for 3 min, then reverse transcribed at 42°C for 90 min in the presence of 200 U SuperScript II RTase (Invitrogen), and 1 mM dNTP. Second strand synthesis was done with the 5’ primer switch method using 0.25 ug Ts/SMARTII primers (5’ AAG CAG TGG TAA CAA CGC AGA GTA CGC GGG 3’) for priming at 65°C for 1 min, and subsequent primer extension at 75°C for 30 min using the Advantage cDNA Polymerase Mix (Clontech) and 0.2mM dNTP. Double stranded cDNA subjected to in vitro transcription at 42°C for 6h using Megascript T7 kit (Ambion). After two rounds of linear amplification, cRNA was chemically labeled using ULS Cy3/Cy5 Fluorescent Labeling Kit (Kreatech), and fragmented by Fragmentation Reagents kit (Ambion).[Hybridization protocol] 6 ug of labeled, fragmented cRNA per sample was applied to microarrays in Kreatech's Hybridization buffer, and hybridized for 16 h at 42C. After hybridization, slides were washed three four times with wash solutions containg gradually decreased concentrations of SSC (1x-0x) [Scan protocol] Scanned on an Agilent DNA Microarray Scanner. Images were quantified using Agilent Feature Extraction Software (version A.9.5.3). [Description] metastasis responding after M-VAX immunotherapy 2 [Data processing] LOWESS normalized, background subtracted data obtained from log2 of processed Red signal/processed Green signal. Agilent software was used for feature extraction.We documented the transcriptional pattern of 3 progressing and 2 regressing synchronous melanoma metastases from the same patient following M-VAX treatment