Profiling the immunogenic cell death (ICD) mechanisms ......2015, 6(39):41600-19 5 Galluzzi et al:...
1
Profiling the immunogenic cell death (ICD) mechanisms induced by Nano - Pulse Stimulation (NPS) treatment in mouse B16 - F10 melanoma tumors using NanoString technology 1 Nuccitelli et al: Nano-Pulse Stimulation is a physical modality that can trigger immunogenic cell death Journal of ImmunoTherapy of Cancer 2017, 5(32): 1-13 2 Nuccitelli et al: Nanoelectroablation of Murine Tumors Triggers a CD8-Dependent Inhibition of Secondary Tumor Growth. PLoS One 2015, 10(7):e0134364 3 Cao and Kaufman: Endoplasmic Reticulum Stress and Oxidative Stress in Cell Fate Decision and Human Disease Antioxidants and Redox Signaling 2014, 21(3): 396-413 4 Gebresmeskel and Johnston: Concepts and mechanisms underlying chemotherapy induced immunogenic cell death: impact on clinical studies and considerations for combined therapies Oncotarget 2015, 6(39):41600-19 5 Galluzzi et al: Immunogenic cell death in cancer and infectious disease Nature Reviews Immunology February 2017, 17: 97-111 6 Garg et al: Molecular and Translational Classifications of DAMPs in Immunogenic Cell Death Frontiers in Immunology 2015, 6(588):1-24 7 Chen and Mellman: Oncology Meets Immunology: The Cancer-Immunity Cycle Immunity 2013, 39(1):1-10 Methods Amanda McDaniel , Snjezana Anand, Aman Alzubier, Juliette Berlin, Holly Hartman, Darrin Uecker and Richard Nuccitelli Pulse Biosciences, 3957 Point Eden Way, Hayward, CA 94545 Background Conclusions NanoString Technology Overview Group Group Name N Injection NPS Treatment 2hrs post- NPS 4hrs post- NPS 24hrs post- NPS 1 Untreated 3 1 million B16-F10 cells None (Tumors Removed) 2 2hrs 3 1 million B16-F10 cells 7.5kV; 200ns; 5Hz;500p Tumors Removed 3 4hrs 3 1 million B16-F10 cells 7.5kV; 200ns; 5Hz;500p -- Tumors Removed 4 24hrs 3 1 million B16-F10 cells 7.5kV; 200ns; 5Hz;500p -- -- Tumors Removed Experimental Timeline Results NPS Treatment References Tumor cell releasing DAMPs binding to receptors on an immature DC Fig. 3a 2hrs Immune Cells Figures 2a-d. HeatMaps NPS Tumor Treatment 24hrs UT 4hrs 2hrs 24hrs UT 2hrs 4hrs 24hrs UT 2hrs 4hrs 2hrs 4hrs Nano-Pulse Stimulation (NPS) is a non-thermal tumor therapy that delivers ultrashort electrical pulses (100-600ns) to tumor cells. NPS opens nanopores in the membrane of the ER, allowing the efflux of Ca 2+ into the cytoplasm, causing ER stress and the production of ROS. These effects induce an immunogenic cell death (ICD) 1 that both eliminates a primary tumor and inhibits the growth of a secondary re-challenge tumor in preclinical models 2 . To date, the primary mechanism of action of most known ICD inducers is ER stress and ROS production leading to intrinsic mitochondrial apoptosis, and the release and translocation of damage associated molecular patterns (DAMPs ) 4 - 6 that bind to pattern recognition receptors (PRRs) to prime the adaptive immune response. Here we sought to profile the pathways involved in ER stress, apoptotic cell death and the immune response after NPS treatment, using the NanoString PanCancer Immune Panel with an additional 30 spike-in genes designed to investigate apoptotic cell death pathways. C57/B6 albino mice (N=12) were injected intradermally with 1- million syngeneic B16-F10 melanoma cells into the left flank. When tumors reached ~5mm in diameter they were treated with NPS (N=9; 500 pulses, 200 ns in duration applied at 25 kV/cm at 5 pps) or were surgically resected and harvested as untreated tumor controls (G1; N=3). Tumors treated with NPS were harvested 2hrs (G2; N=3), 4hrs (G3; N=3) and 24hrs (G4; N=3) after treatment and placed into formalin for fixation followed by embedding in paraffin. mRNA was extracted and hybridized to bar-coded probes that correspond to 800 gene transcripts (770 PanCancer Immune Panel + 30 spike-in). Transcripts were read using the NanoString nCounter ® and analyzed with nSolver software (see below). NanoString profiling revealed that transcripts coding for components previously identified as important for the mechanism of ICD, such as ER stress-induced intrinsic apoptotic pathways, key DAMPs and PRRs, as well a number of immune mediators and cells involved in priming the adaptive immune response were upregulated in tumor tissues 24-hrs after NPS treatment. We plan to continue to utilize the NanoString platform in future studies to help us to further understand the mechanisms involved in NPS-treatment of malignant tumors. DC phagocytosing tumor cell Figures 4ah. Immune Cell Profiles Innate Immune Response Fig. 2a Intrinsic Apoptosis Fig. 2b DAMPs and PRRs Fig. 2c Antigen Presentation and AIR Priming Fig. 2d T-cell Activation Figures 1a-b. NPS-induced immune response Fig. 3b 4hrs Fig. 3c 24hrs Figures 1a-b. (a) Proposed mechanisms behind NPS treatment: Immunogenic cell death (ICD) is triggered in tumor cells 1,3-4 followed by the release of damage- associated molecular patterns (DAMPs) necessary for immune recognition through binding to PRRs 4-6 . The damaged cells are then phagocytosed by immature DCs (or other APCs) 5-7. Upon maturation, these DCs are able to prime and activate CD4+ and CD8+ T-cells 6-7 . (b) A representative schematic of the underlying network of some of the key genes involved in immunogenic cell death, priming and activating the adaptive immune response 3-7 . Figures 2a-d. Clustering heatmaps for four gene sets involved in the cell death and immune response to NPS treatment 4-7 . The overall expression levels of genes in all four of the gene sets was highest 24hrs post-NPS treatment (green = untreated; orange = 2hrs; blue = 4hrs; red = 24hrs). Figures 3a-c. Genes on panel mapped to the KEGG apoptosis pathway. Differential gene expression information is overlaid on the protein-based KEGG pathway image (red = upregulated relative to untreated; green = downregulated relative to untreated; gray = no differential expression). By 24hrs initiator caspases 9 and 12, as well as effector caspase 7 are highly upregulated. Figures 4a-h. Each figure displays the abundance of a specific immune cell type relative to the abundance of tumor infiltrating lymphocytes (TILs) and is displayed as a cell type score (CTS) for each condition. Calculation of CTS: Expression of genes that are stably expressed and specific to a cell type are averaged to obtain a raw abundance cell type score (CTS). To calculate the abundance of a cell type relative to the abundance of TILs, the raw CTS for TILs is subtracted from the raw CTS for the cell type (CTS(Cell Type) – CTS(TILs) = Relative Abundance CTS for Cell Type)) PulseTx – Pulse Generator a. b. Mature DC T-cell CD8+ T-cell CD4+ T-cell UT 2hrs 4hrs 24hrs Figures 3a-c. KEGG Apoptosis Pathways High Low Expression NPS Treatment of a B16-F10 Melanoma Tumor UT 2hrs 4hrs 24hrs Fig. 4b - Macrophages vs TILs UT 2hrs 4hrs 24hrs Fig. 4a - NK(CD56 dim ) vs TILs Relative Cell Type Score (CTS) UT 2hrs 4hrs 24hrs Fig. 4d - DCs vs TILs UT 2hrs 4hrs 24hrs Fig. 4c - Neutrophils vs TILs Relative Cell Type Score (CTS) UT 2hrs 4hrs 24hrs Fig. 4f – CD8+ T-cells vs TILs UT 2hrs 4hrs 24hrs Fig. 4h – Exhausted CD8+ vs TILs UT 2hrs 4hrs 24hrs Fig. 4e – CD4+ Th1 cells vs TILs Relative Cell Type Score (CTS) UT 2hrs 4hrs 24hrs Fig. 4g – CD4+ Treg cells vs TILs Relative Cell Type Score (CTS) Time Point Figure Captions Adaptive Immune Response Il12rb2 Cell Death DAMPs Release – PRR Binding Adaptive Immune Response (AIR) Priming T cell Activation Myd88 Ly96 Nlrp3 Il12a PERK eIF2α Ire1 Traf2 IP3R ASK1 JNK CytC Apaf1 CHOP Atf4 Lrp1 Calr Casp1 P2rx7 Tlr4 Hmgb1 Casp3 Casp7 Casp9 Casp12 Ifnar1 Ifna1 Ifnb1 Il1b Il12b Il6 Tnf CD80 CD86 CD28 Il12rb1 Il2 Il6ra Il1r1 Cd8a Cd8b1 Cd4 Foxp3 Ifng Ifngr1 Casp8
Transcript of Profiling the immunogenic cell death (ICD) mechanisms ......2015, 6(39):41600-19 5 Galluzzi et al:...
Profiling the immunogenic cell death (ICD) mechanisms induced by Nano-Pulse Stimulation (NPS) treatment in mouse B16-F10 melanoma
tumors using NanoString technology
1Nuccitelli et al: Nano-Pulse Stimulation is a physical modality that can trigger immunogenic cell death Journal of ImmunoTherapy of Cancer2017, 5(32): 1-132Nuccitelli et al: Nanoelectroablation of Murine Tumors Triggers a CD8-Dependent Inhibition of Secondary Tumor Growth. PLoS One 2015,10(7):e01343643Cao and Kaufman: Endoplasmic Reticulum Stress and Oxidative Stress in Cell Fate Decision and Human Disease Antioxidants and RedoxSignaling 2014, 21(3): 396-4134Gebresmeskel and Johnston: Concepts and mechanisms underlying chemotherapy induced immunogenic cell death: impact on clinicalstudies and considerations for combined therapies Oncotarget 2015, 6(39):41600-195Galluzzi et al: Immunogenic cell death in cancer and infectious disease Nature Reviews Immunology February 2017, 17: 97-1116Garg et al: Molecular and Translational Classifications of DAMPs in Immunogenic Cell Death Frontiers in Immunology 2015, 6(588):1-247Chen and Mellman: Oncology Meets Immunology: The Cancer-Immunity Cycle Immunity 2013, 39(1):1-10
Methods
Amanda McDaniel, Snjezana Anand, Aman Alzubier, Juliette Berlin, Holly Hartman, Darrin Uecker and Richard Nuccitelli
Pulse Biosciences, 3957 Point Eden Way, Hayward, CA 94545
Background
ConclusionsNanoString Technology Overview
Group Group Name N Injection NPS
Treatment2hrs post-
NPS4hrs post-
NPS24hrs post-
NPS
1 Untreated 31 million B16-F10
cells
None (Tumors Removed)
2 2hrs 31 million B16-F10
cells
7.5kV; 200ns; 5Hz;500p
Tumors Removed
3 4hrs 31 million B16-F10
cells
7.5kV; 200ns; 5Hz;500p -- Tumors
Removed
4 24hrs 31 million B16-F10
cells
7.5kV; 200ns; 5Hz;500p -- -- Tumors
Removed
Experimental Timeline
ResultsNPS Treatment
References
Tumor cell releasing DAMPs binding to receptors on an immature DC
Fig. 3a 2hrs
Immune Cells
Figures 2a-d. HeatMaps
NPS Tumor Treatment
24hrs UT 4hrs 2hrs 24hrs UT 2hrs 4hrs 24hrs UT2hrs 4hrs 2hrs 4hrs
Nano-Pulse Stimulation (NPS) is a non-thermal tumor therapythat delivers ultrashort electrical pulses (100-600ns) to tumorcells. NPS opens nanopores in the membrane of the ER,allowing the efflux of Ca2+ into the cytoplasm, causing ERstress and the production of ROS. These effects induce animmunogenic cell death (ICD)1 that both eliminates a primarytumor and inhibits the growth of a secondary re-challengetumor in preclinical models2. To date, the primary mechanismof action of most known ICD inducers is ER stress and ROSproduction leading to intrinsic mitochondrial apoptosis, and therelease and translocation of damage associated molecularpatterns (DAMPs)4-6 that bind to pattern recognition receptors(PRRs) to prime the adaptive immune response. Here wesought to profile the pathways involved in ER stress, apoptoticcell death and the immune response after NPS treatment,using the NanoString PanCancer Immune Panel with anadditional 30 spike-in genes designed to investigate apoptoticcell death pathways.
C57/B6 albino mice (N=12) were injected intradermally with 1-million syngeneic B16-F10 melanoma cells into the left flank.When tumors reached ~5mm in diameter they were treatedwith NPS (N=9; 500 pulses, 200 ns in duration applied at 25kV/cm at 5 pps) or were surgically resected and harvested asuntreated tumor controls (G1; N=3). Tumors treated with NPSwere harvested 2hrs (G2; N=3), 4hrs (G3; N=3) and 24hrs (G4;N=3) after treatment and placed into formalin for fixationfollowed by embedding in paraffin. mRNA was extracted andhybridized to bar-coded probes that correspond to 800 genetranscripts (770 PanCancer Immune Panel + 30 spike-in).Transcripts were read using the NanoString nCounter® andanalyzed with nSolver software (see below).
NanoString profiling revealed that transcripts coding for componentspreviously identified as important for the mechanism of ICD, such asER stress-induced intrinsic apoptotic pathways, key DAMPs andPRRs, as well a number of immune mediators and cells involved inpriming the adaptive immune response were upregulated in tumortissues 24-hrs after NPS treatment. We plan to continue to utilize theNanoString platform in future studies to help us to further understandthe mechanisms involved in NPS-treatment of malignant tumors.
DC phagocytosing tumor cell
Figures 4ah. Immune Cell Profiles
Innate Immune Response
Fig. 2a Intrinsic Apoptosis
Fig. 2b DAMPs and PRRs
Fig. 2c Antigen Presentation and AIR Priming
Fig. 2d T-cell Activation
Figures 1a-b. NPS-induced immune response
Fig. 3b 4hrs
Fig. 3c 24hrs Figures 1a-b. (a) Proposed mechanisms behind NPS treatment: Immunogenic cell death (ICD) is triggered in tumor cells1,3-4 followed by the release of damage-
associated molecular patterns (DAMPs) necessary for immune recognition through binding to PRRs4-6. The damaged cells are then phagocytosed by immatureDCs (or other APCs)5-7. Upon maturation, these DCs are able to prime and activate CD4+ and CD8+ T-cells6-7. (b) A representative schematic of the underlyingnetwork of some of the key genes involved in immunogenic cell death, priming and activating the adaptive immune response3-7.
Figures 2a-d. Clustering heatmaps for four gene sets involved in the cell death and immune response to NPS treatment4-7. The overall expression levels of genesin all four of the gene sets was highest 24hrs post-NPS treatment (green = untreated; orange = 2hrs; blue = 4hrs; red = 24hrs).
Figures 3a-c. Genes on panel mapped to the KEGG apoptosis pathway. Differential gene expression information is overlaid on the protein-based KEGG pathwayimage (red = upregulated relative to untreated; green = downregulated relative to untreated; gray = no differential expression). By 24hrs initiator caspases 9 and12, as well as effector caspase 7 are highly upregulated.
Figures 4a-h. Each figure displays the abundance of a specific immune cell type relative to the abundance of tumor infiltrating lymphocytes (TILs) and is displayedas a cell type score (CTS) for each condition. Calculation of CTS: Expression of genes that are stably expressed and specific to a cell type are averaged to obtaina raw abundance cell type score (CTS). To calculate the abundance of a cell type relative to the abundance of TILs, the raw CTS for TILs is subtracted from theraw CTS for the cell type (CTS(Cell Type) – CTS(TILs) = Relative Abundance CTS for Cell Type))
PulseTx – Pulse Generator
a.
b.
Mature DC
T-cell
CD8+ T-cell
CD4+ T-cell
UT 2hrs4hrs24hrs
Figures 3a-c. KEGG Apoptosis Pathways
High
Low
Expression
NPS Treatment of a B16-F10 Melanoma Tumor
UT 2hrs 4hrs 24hrs
Fig. 4b - Macrophages vs TILs
UT 2hrs 4hrs 24hrs
Fig. 4a - NK(CD56dim) vs TILs
Rel
ativ
e C
ell T
ype
Scor
e (C
TS)
UT 2hrs 4hrs 24hrs
Fig. 4d - DCs vs TILs
UT 2hrs 4hrs 24hrs
Fig. 4c - Neutrophils vs TILs
Rel
ativ
e C
ell T
ype
Scor
e (C
TS)
UT 2hrs 4hrs 24hrs
Fig. 4f – CD8+ T-cells vs TILs
UT 2hrs 4hrs 24hrs
Fig. 4h – Exhausted CD8+ vs TILs
UT 2hrs 4hrs 24hrs
Fig. 4e – CD4+ Th1 cells vs TILs
Rel
ativ
e C
ell T
ype
Scor
e (C
TS)
UT 2hrs 4hrs 24hrs
Fig. 4g – CD4+ Treg cells vs TILs
Rel
ativ
e C
ell T
ype
Scor
e (C
TS)
Time Point
Figure Captions
Adaptive Immune Response
Il12rb2
Cell Death DAMPs Release – PRR Binding Adaptive Immune Response (AIR) Priming T cell Activation
Myd88Ly96
Nlrp3
Il12a
PERK
eIF2α
Ire1
Traf2
IP3R
ASK1
JNK
CytC Apaf1CHOP
Atf4
Lrp1Calr
Casp1P2rx7
Tlr4Hmgb1
Casp3
Casp7
Casp9
Casp12
Ifnar1
Ifna1Ifnb1
Il1b
Il12b
Il6
Tnf
CD80
CD86CD28
Il12rb1
Il2
Il6ra
Il1r1
Cd8a Cd8b1
Cd4Foxp3
Ifng
Ifngr1Casp8
