Profiling the immunogenic cell (ICD) mechanisms induced by Nano-Pulse Stimulation (NPS) treatment in mouse B16-F10 melanoma tumors using NanoString technology 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 Results

Nano-Pulse Stimulation (NPS) is a non-thermal tumor therapy NPS Treatment Tumor cell releasing DAMPs binding to DC phagocytosing Mature T-cell Figures 1a-b. receptors on an immature DC tumor cell DC that delivers ultrashort electrical pulses (100-600ns) to tumor a. CD8+ cells. NPS opens nanopores in the membrane of the ER, NPS-induced T-cell immune 2+ allowing the efflux of Ca into the cytoplasm, causing ER response

stress and the production of ROS. These effects induce an CD4+ immunogenic (ICD)1 that both eliminates a primary T-cell tumor and inhibits the growth of a secondary re-challenge DAMPs Release – PRR Binding Adaptive (AIR) Priming Activation tumor in preclinical models2. To date, the primary mechanism b. Cell Death

of action of most known ICD inducers is ER stress and ROS PERK Ire1 IP3R Casp12 CD86 Calr Ifng Lrp1 CD28 Il2 Il1b production leading to intrinsic mitochondrial , and the Traf2 Ifna1 Casp8 Casp3 Ifnb1 eIF2α P2rx7 Nlrp3 release and translocation of damage associated molecular ASK1 CD80 Ifngr1 Casp1 Il1r1 4-6 Cd4 patterns (DAMPs) that bind to pattern recognition receptors Il6 Il6ra Atf4 JNK Casp7 Foxp3 Tlr4 Ifnar1 (PRRs) to prime the adaptive immune response. Here we Hmgb1 Tnf Il12a Il12rb1 Casp9 Ly96 Cd8a sought to profile the pathways involved in ER stress, apoptotic CHOP CytC Apaf1 Myd88 Il12b Il12rb2 Cd8b1 cell death and the immune response after NPS treatment, Figures 2a-d. using the NanoString PanCancer Immune Panel with an HeatMaps Fig. 2b DAMPs Fig. 2c Fig. 2a Intrinsic Fig. 2d T-cell additional 30 spike-in genes designed to investigate apoptotic and PRRs Presentation Apoptosis Activation cell death pathways. and AIR Priming

Expression Methods High Low 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 24hrs UT 4hrs 2hrs 24hrs UT 24hrs UT 2hrs 4hrs 24hrs UT 4hrs 2hrs 4hrs with NPS (N=9; 500 pulses, 200 ns in duration applied at 25 Immune Cells 4hrs 2hrs 2hrs kV/cm at 5 pps) or were surgically resected and harvested as Figures 3a-c. KEGG Apoptosis untreated tumor controls (G1; N=3). Tumors treated with NPS Pathways Figures 4ah. Immune Cell Profiles were harvested 2hrs (G2; N=3), 4hrs (G3; N=3) and 24hrs (G4; Innate Immune Response Adaptive Immune Response N=3) after treatment and placed into formalin for fixation Fig. 3a 2hrs followed by embedding in paraffin. mRNA was extracted and Fig. 4a - NK(CD56dim) vs TILs Fig. 4b - Macrophages vs TILs Fig. 4e – CD4+ Th1 cells vs TILs Fig. 4f – CD8+ T-cells vs TILs 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). Relative Cell Type Score (CTS) RelativeCell Type Experimental Timeline Score (CTS) RelativeCell Type UT 2hrs 4hrs 24hrs UT 2hrs 4hrs 24hrs UT 2hrs 4hrs 24hrs UT 2hrs 4hrs 24hrs Group NPS 2hrs post- 4hrs post- 24hrs post- Group N Injection Name Treatment NPS NPS NPS Fig. 4c - vs TILs Fig. 4d - DCs vs TILs Fig. 4g – CD4+ Treg cells vs TILs Fig. 4h – Exhausted CD8+ vs TILs 1 million None (Tumors Fig. 3b 1 Untreated 3 B16-F10 Removed) 4hrs cells 1 million 7.5kV; 200ns; Tumors 2 2hrs 3 B16-F10 5Hz;500p Removed cells 1 million 7.5kV; 200ns; Tumors 3 4hrs 3 B16-F10 -- 5Hz;500p Removed cells Relative Cell Type Score (CTS) RelativeCell Type

1 million Score (CTS) RelativeCell Type 7.5kV; 200ns; Tumors 4 24hrs 3 B16-F10 -- -- 4hrs 5Hz;500p Removed UT 2hrs 4hrs 24hrs UT 2hrs 24hrs UT 2hrs 4hrs 24hrs UT 2hrs 4hrs 24hrs cells Time Point

NPS Tumor Treatment Figure Captions Fig. 3c 1,3-4 NPS Treatment of a 24hrs Figures 1a-b. (a) Proposed mechanisms behind NPS treatment: Immunogenic cell death (ICD) is triggered in tumor cells followed by the release of damage- PulseTx – Pulse Generator associated molecular patterns (DAMPs) necessary for immune recognition through binding to PRRs4-6. The damaged cells are then phagocytosed by immature B16-F10 Melanoma Tumor DCs (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 underlying network 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 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)) NanoString Technology Overview References Conclusions

1Nuccitelli et al: Nano-Pulse Stimulation is a physical modality that can trigger immunogenic cell death Journal of of NanoString profiling revealed that transcripts coding for components 2017, 5(32): 1-13 previously identified as important for the mechanism of ICD, such as 2Nuccitelli et al: Nanoelectroablation of Murine Tumors Triggers a CD8-Dependent Inhibition of Secondary Tumor Growth. PLoS One 2015, 10(7):e0134364 ER stress-induced intrinsic apoptotic pathways, key DAMPs and 3Cao and Kaufman: Stress and in Cell Fate Decision and Human Disease Antioxidants and Redox PRRs, as well a number of immune mediators and cells involved in Signaling 2014, 21(3): 396-413 4Gebresmeskel and Johnston: Concepts and mechanisms underlying induced immunogenic cell death: impact on clinical priming the adaptive immune response were upregulated in tumor studies and considerations for combined therapies Oncotarget 2015, 6(39):41600-19 tissues 24-hrs after NPS treatment. We plan to continue to utilize the 5Galluzzi et al: Immunogenic cell death in cancer and infectious disease Nature Reviews Immunology February 2017, 17: 97-111 6Garg et al: Molecular and Translational Classifications of DAMPs in Immunogenic Cell Death Frontiers in Immunology 2015, 6(588):1-24 NanoString platform in future studies to help us to further understand 7Chen and Mellman: Oncology Meets Immunology: The Cancer-Immunity Cycle Immunity 2013, 39(1):1-10 the mechanisms involved in NPS-treatment of malignant tumors.