Treatment of pancreatic cancer with irreversible electroporation and intratumoral CD40 antibody stimulates systemic immune responses that inhibit liver metastasis in an orthotopic model

Abstract

Background: Pancreatic cancer (PC) has a poor prognosis, and most patients present with either locally advanced or distant metastatic disease. Irreversible electroporation (IRE) is a non-thermal method of ablation used clinically in locally advanced PC, but most patients eventually develop distant recurrence. We have previously shown that IRE alone is capable of generating protective, neoantigen-specific immunity. Here, we aim to generate meaningful therapeutic immune effects by combining IRE with local (intratumoral) delivery of a CD40 agonistic antibody (CD40Ab).

Methods: KPC46 organoids were generated from a tumor-bearing male KrasLSL-G12D-p53LSL-R172H-Pdx-1-Cre (KPC) mouse. Orthotopic tumors were established in the pancreatic tail of B6/129 F1J mice via laparotomy. Mice were randomized to treatment with either sham laparotomy, IRE alone, CD40Ab alone, or IRE followed immediately by CD40Ab injection. Metastatic disease and immune infiltration in the liver were analyzed 14 days postprocedure using flow cytometry and multiplex immunofluorescence imaging with spatial analysis. Candidate neoantigens were identified by mutanome profiling of tumor tissue for ex vivo functional analyses.

Results: The combination of IRE+CD40 Ab improved median survival to greater than 35 days, significantly longer than IRE (21 days) or CD40Ab (24 days) alone (p<0.01). CD40Ab decreased metastatic disease burden, with less disease in the combination group than in the sham group or IRE alone. Immunohistochemistry of liver metastases revealed a more than twofold higher infiltration of CD8+T cells in the IRE+CD40 Ab group than in any other group (p<0.01). Multiplex immunofluorescence imaging revealed a 4-6 fold increase in the density of CD80+CD11c+ activated dendritic cells (p<0.05), which were spatially distributed throughout the tumor unlike the sham group, where they were restricted to the periphery. In contrast, CD4+FoxP3+ T-regulatory cells (p<0.05) and Ly6G+myeloid derived cells (p<0.01) were reduced and restricted to the tumor periphery in the IRE+CD40 Ab group. T-cells from the IRE+CD40 Ab group recognized significantly more peptides representing candidate neoantigens than did T-cells from the IRE or untreated control groups.

Conclusions: IRE can induce local tumor regression and neoantigen-specific immune responses. Addition of CD40Ab to IRE improved dendritic cell activation and neoantigen recognition, while generating a strong systemic antitumor T-cell response that inhibited metastatic disease progression.

Keywords: Antigen Presentation; Antigens, Neoplasm; Gastrointestinal Neoplasms; Immunotherapy; Tumor Microenvironment.

Conflict of interest statement

Competing interests: None declared.

© Author(s) (or their employer(s)) 2023. Re-use permitted under CC BY-NC. No commercial re-use. See rights and permissions. Published by BMJ.

Figures

Figure 1

Combination of IRE with agonistic CD40Ab reduces tumor growth and improves survival in subcutaneous and orthotopic organoid PC mouse models. (A) Subcutaneous KPC4580P tumor growth in C57BL/6 (n=10/group) mice implanted on one side of the flanks at 8 days before IRE (5×105 cells/mouse). Each data point represents tumor volume of a single mouse followed along the growth curve. (B) Tumor growth curves of bilateral subcutaneous KPC4580P tumors at the treated site (top) and untreated site (bottom), of n=5 mice/group, implanted 8 days before IRE, followed by IT CD40 Ab injection on day 1. Tumor volumes were measured using calipers and plotted as mean±SEM. (C) 30 000 murine PDAC organoids KPC46 were injected into the pancreas of B6/129 F1 hybrid mice 21 days before treatment. Ultrasound monitoring of orthotopic organoid tumors day 7 post-treatment showing responses to treatment. Dotted lines show the dimensions of the orthotopic tumors under sltrasound imaging. (D) Tumor volumes were measured using ultrasound and plotted as mean±SEM of n=10 mice/group. (E) Kaplan-Meier survival analysis of orthotopic organoid KPC46 tumor bearing mice post-treatment (n=20 mice/group) cumulative of three independent experiments showing significant survival benefit offered by IRE+CD40 Ab combination **p<0.01 by log rank test. (F) Tumor weights as a measure of primary tumor burden, tumors were excised from mice on euthanasia, 14 days post-treatment, each data point represents single tumor weight represented as mean±SEM. *p<0.05, **p<0.01, ***p<0.00 by one-way ANOVA with post hoc Benferroni test. ANOVA, analysis of variance; IRE, irreversible electroporation.

Figure 2

Combining IRE with CD40 agonistic activity inhibits tumor metastasis to the liver. (A) Images of liver showing differences in metastasis. Whole livers were excised from orthotopic organoid KPC46 tumor bearing B6129 F1 mice on euthanasia 14 days post-treatment under different groups (n=10/group). (B) Liver weights as a measure of metastatic burden with a significant reduction in metastasis seen with IRE+CD40 Ab combination, weights of whole liver was measured in mice surviving 14 days post procedure each data point represents single liver weight, represented as mean±SEM. (E) Manual counting of visible metastatic nodules on the liver as a measure of metastatic tumor burden (D, E) histological examination of mice liver showing metastatic tumor nodules observed using H&E. Images were acquired using Zeiss slide scanner at ×20 objective and the % liver area occupied by the tumor was calculated using QuPath plotted as mean±SEM 3 sections/mouse, 4 mice/group. (F) Untreated mice are more likely to develop bloody ascites on tumor progression compared with CD40Ab or IRE+CD40 Ab treatment. Each dot represents percentage of mice with bloody ascites/experimental group plotted as a mean of three independent experimental rounds. *p

Figure 3

Activation of antitumor immune signaling pathways observed both locally and systemically on treatment with combination of IRE and CD40Ab. (A) RNA-Seq analysis of important immunoregulatory genes in the primary pancreatic tumor between the two groups. Heat map represents fold change in the expression levels of critical genes within tumor microenvironment between sham and IRE+CD40 Ab groups (n=3/group). (B) Gene set enrichment analysis of immunological pathway related genes, represented as Log2 enrichment scores of antigen presentation pathway (Top), IFN signaling pathway (Middle) and CD40–CD40 L interaction pathway (Right) as mean±SD of n=3/group. IRE, irreversible electroporation.

Figure 4

Immune infiltration in the liver shows that the combination of IRE and CD40 Ab induces an immune mediated prevention of metastatic spread. (A) IHC for CD45 and CD8 in the liver showing increased specific staining within the metastatic tumor nodule, Images obtained using Leica Aperio slide scanner with ×20 objective. Scale bar: 100 µm. (B) Quantification of CD45 and CD8 positive cells, respectively, within the metastatic tumor nodules of the liver was performed using QuPath V.3.0, 3 mice/group including atleast five tumor nodules per group plotted as mean±SEM. (C) Flow cytometry of immune infiltrates in the metastatic site (bulk liver) performed on day 14 on whole liver (n=3/group). Decreased MDCs and Tregs with the increased infiltration of CD4+T cells cells suggest an antitumor immune activity at the distant metastatic site on treatment with IRE+CD40 Ab. (D) Flow cytometry analysis of liver showing increased presence of cross-presenting CD8+dendritic cells. Graphs plotted as mean±SEM. *p

Figure 5

Multiplex immunofluorescence imaging shows an antitumor immune microenvironment within metastatic liver nodules post treatment with IRE+CD40 Ab. (A) Representative multiplex fluorescence microscopy images on Formal-fixed paraffine-embedded (FFPE) tissue sections of mice liver showing metastatic nodes showing tumor infiltrating immune cells stained with either panel 1—CD11c:Alexa 647(red), CD80:Alexa 488(Cyan), CD40 L:Alexa 790 (yellow), PanCK:Alexa 555(Green), DAPI (blue), Scale bars set at 400 µm (low magnification sham and IRE), 200 µm (low magnification CD40 Ab and IRE+CD40 Ab), 100 µm (medium magnification) and 20 µm (high magnification). (B, C) Quantification of dendritic cell (CD11c+) and activated dendritic cell (CD80+CD11c+) infiltration per mm2 of the metastatic nodules was performed using QuPath V.3.0 software using atleast five different nodules spanning three biological replicates per group. (D) Representative multiplex immunofluorescence imaging of CD4 Alexa 647) (Red), F4/80—Alexa 555 (Green), Ly6G—Alexa 790 (Yellow), FoxP3—488 (Cyan), DAPI (Blue). Scale bars set at 20 µm). (E–H) Quantification of T regulatory cell (CD4+FoxP3+), macrophage (F4/80+) and myeloid derived Cell (MDC—Ly6G+) infiltration to the metastatic nodules was performed using QuPath V.3.0 software using atleast five different nodules spanning three biological replicates per group. The number of immune cells was normalized to the area of the nodes and presented as mean±SEM. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001; by one-way ANOVA with post hoc Benferroni multiple comparison test. ANOVA, analysis of variance; IRE, irreversible electroporation.

Figure 6

Combination of IRE and CD40 Ab triggers expansion of systemic tumor neoantigen-specific T-cell response. (A) Circos plot showing the observed mutations in the KPC46 O tumor compared with B6/129 F1 hybrid background. First level (right next to cytogenic bands): all of the somatic mutations identified by whole exome sequencing. Second level: mutations expressed by RNAseq. Third level: histogram showing the level of RNA expression. Fourth level: 59 mutations selected for peptide synthesis and ELISPOT based on high RNA expression and sequencing depth. Representative IFNγ ELISPOT from (B) peptide pools (10 pools of 8–12 peptides each) or (C) deconvoluted individual peptides of pool 2 using T cells isolated from groups of untreated tumor-bearing mice (Ctrl), IRE-treated mice (IRE) and mice treated with a combination of IRE with IT CD40 Ab (IRE+CD40 Ab) rechallenged with live tumor cells. Data represent mean±SEM values of spot forming cells/106 cells from three independent mice per group in triplicates. Representative graph of three independent experiments. *p<0.05; by two-way ANOVA with post hoc Tukey’s multiple comparison test. (D) Measure of breadth of neoantigen detection by T-cells represented as percentage of positive detections (two SD over the background IFNγ response for that mouse) for each treatment group among total available antigens for that round. Each dot represents a single round of detection of multiple pools/peptides mean±SEM. ANOVA, analysis of variance; IRE, irreversible electroporation.

Figure 7

Decreased spatial restriction of infiltrating effector immune cells in the liver observed post treatment with IRE+CD40Ab. (A) Representative heat map overlay depicting the density of CD8+T cells around tumor perimeter shows higher concentration of CD8 T cells within the tumor perimeter post treatment with IRE+CD40 Ab. (B) Heatmap of spatial analysis of CD4+FoxP3+ positive regulatory T cell (T-reg) infiltration in the liver metastases with pseudo colors ranging from blue indicating cells closest to the tumor perimeter to red indicating closest to the center of the tumor (Scale MDR=0–0.92). Images show that not only were the number of T-regs reduced on IRE+CD40 Ab treatment but the distribution of the T-regs was restricted to the periphery of the tumor post treatment. (C–G) Spatial distribution of various immune populations were calculated using QuPath. The distance from the defined tumor perimeter was calculated for each cell of interest and normalized to the size of the corresponding node. Mean Distance ratio (MDR) = (Σ(Distance in μm of each cell of interest from tumor perimeter/mean radius of the metastatic node)/Total number of cells of interest). An MDR=0 represents cells at tumor perimeter and an MDR=1, represents cells at the farthest distance from the perimeter (ie, the centroid of the tumor). MDR values for at least three different biological replicates was calculated and the data presented as mean±SEM. *p