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