Radiofrequency ablation remodels the tumor microenvironment and promotes neutrophil-mediated abscopal immunomodulation in pancreatic cancer

Abstract

Pancreatic ductal adenocarcinoma (PDAC) presents a 5-year overall survival rate of 11%, despite efforts to improve clinical outcomes in the past two decades. Therapeutic resistance is a hallmark of this disease, due to its dense and suppressive tumor microenvironment (TME). Endoscopic ultrasound guided radiofrequency ablation (EUS-RFA) is a promising local ablative and potential immunomodulatory therapy for PDAC. In this study, we performed RFA in a preclinical tumor-bearing Kras^G12D; Trp53^R172H/+; Pdx1:Cre (KPC) syngeneic model, analyzed local and abscopal affects after RFA and compared our findings with resected PDAC specimens. We found that RFA reduced PDAC tumor progression in vivo and promoted strong TME remodeling. Additionally, we discovered tumor-infiltrating neutrophils determined abscopal effects. Using imaging mass cytometry, we showed that RFA elevated dendritic cell numbers in RFA-treated tumors and promoted a significant CD4⁺ and CD8⁺ T-cell abscopal response. In addition, RFA elevated levels of PD-L1 and checkpoint blockade inhibition targeting PD-L1 sustained tumor growth reduction in the context of RFA. This study indicates RFA treatment, which has been shown to increase tumor antigen shedding, promotes antitumor immunity. This is critical in PDAC where recent clinical immunotherapy trials have not resulted in substantial changes in overall survival.

SYNOPSIS

Novel immunotherapeutic strategies are needed to reduce PDAC mortality and metastatic potential. The data suggest that EUS-RFA provides an opportunity to address PDAC chemo- and immunotherapeutic resistance by stimulating the immune system and modulating the desmoplastic stroma.

INTRODUCTION

Current therapeutic approaches, including chemotherapy and radiation alone, or combined with immune checkpoint blockade (ICB) have shown little success in pancreatic ductal adenocarcinoma (PDAC), placing an imperative need for the discovery and application of innovative techniques (1, 2). Endoscopic ultrasound guided radiofrequency ablation (EUS-RFA) is an FDA-approved technique currently available for the treatment of several gastrointestinal malignancies including pancreatic cancer and has the advantage of being minimally invasive and safe (3). Studies in both animal models and patients with cancer show that RFA induces not only local burning/disruption of the tumor by heat but also generates coagulative necrosis, which releases large amounts of cellular debris that represent a source of tumor antigens that can ultimately trigger a host adaptive immune response against local and distant tumors, an effect referred to as the abscopal effect (4, 5).

METHODS

Animal Model

All animal procedures were performed in compliance with UTHealth’s CLAMC Animal Welfare Committee Review and approved on Dr. Bailey’s Animal Welfare Committee protocol. Three independent experiments were performed. 100,000 KPC cells in PBS:Matrigel (Corning^™, cat. CB-40230) mix (1:1) were injected in each of the left and right flanks of female C57BL/6mice (The Jackson Laboratory, cat. 000664). KPC cells were originally derived in the Tuveson Lab from Kras^LSL-G12D/+;Trp53^LSL-R172H/+;Pdx1-Cre mice, which develop PDAC and obtained from Chin-Yo Lin from the University of Houston in 2020. Cells were maintained at low passage and have not been reauthenticated. Cell culture was performed in DMEM media (Gibco, cat. 10566-016), with 10% FBS (Corning, cat. 35-010-CV) and 1% Penicillin Streptomycin (Gibco, cat. 15140-122) and trypsinized with Trypsin 0.25% in HBSS (Gen Clone, cat. 25-510). Cells were passaged approximately 3 times before use. No mycoplasma test was performed immediately before the experiment and no cell line manipulations were performed for this study. Tumor size was assessed twice per week with vernier caliper and calculated as (length × width × width)/2 in mm³. For the RFA treated cohort, ablated tumors were analyzed by hematoxylin and eosin (H&E) composite staining and the presence of a necrotic core, as indicative of ablation success, determined inclusion of the mice for further studies. Non-RFA treated tumors with positive abscopal effect were included for further analysis.

RFA

RFA treatment was initiated when tumors reached 200–500 mm³ using a previously described protocol (6). The non-RFA side contralateral tumor did not receive RFA treatment. Mice were observed for signs of pain or discomfort post-ablation.

H&E staining

Dry tissue slides from control, RFA and non-RFA treated tumor tissues from tumor-bearing mice and resected samples from PDAC patients were soaked in Histoclear (National Diagnostics, cat. HS-200) for 8 min and gradually rehydrated in decreasing concentrations of alcohol (Fisher Bioreagents, cat. BP28184) 100% and 95% for 4 min each. Slides were then rinsed in the following solutions for 3 min each: tap water, Millipore Sigma 65067-75 HARLECO^® Hematoxylin (cat. EM-65067-75), tap water, Epredia^™ Signature Series Clarifier^™ (Thermo Scientific, cat. 22-050-117), tap water, Scott’s Bluing Reagent (Polysciences, cat. 24605-1) and tap water. A counterstain was performed by 95% EtOH for 1 min followed by Eosin-Y stain (Richard-Allan Scientific, cat. #71311) and a final dehydration step was performed in increasing concentrations of alcohol 95% for 1 min, followed by 100% for 4 min and Histoclear for 4 min. Slides were mounted in Cytoseal XYL (Epredia, cat. 8312-4) mounting media.

Immunohistochemistry, Immunofluorescence, and ImageJ analysis

Dry tissue slides from control, RFA and non-RFA treated tumor tissues from tumor-bearing mice and resected samples from PDAC patients, when applicable, were processed and stained for IHC as previously described (6) using antibodies specific for cleaved caspase 3 (Cell Signaling, cat. #9664), Granzyme B (Abcam, cat. #ab255598), NIMPR14 (Abcam, cat. #ab2557), myeloperoxidase (MPO) (Abcam, cat. #ab208670), αSMA (Abcam, cat. #ab5694), CD31 (Abcam, cat. #ab281583) and CD8α (Cell Signaling, cat. #98941T). For immunofluorescence (IF) staining, the IHC protocol was followed without antigen retrieval, ABC reagent, DAB reagent, or counterstaining steps. Primary antibodies specific for MPO (Abcam, cat. #ab208670) and NIMPR14 (Abcam, cat. #ab2557) were used followed by anti-rat (Jackson immunology, cat. #AB2340683) and anti-rabbit (Invitrogen, cat. #31635) secondary antibodies. For collagen quantification in pancreatic sections, Masson’s trichrome staining was performed by using Abcam Trichrome Stain (Abcam, cat. ab150686) kit where the sections were deparaffinized with Histoclear and rehydrated as mentioned above. Bouin's Fluid was preheated to 60C, and sections were incubated for 60 min and then stained with Weigert's Iron Hematoxylin for 5 min, Biebrich Scarlet for 15 min, Phosphomolybdic Acid for 12 min, Aniline Blue Solution for 20 min and Acetic Acid Solution for 5 min. Sections were dehydrated with Histoclear and mounted in Cytoseal XYL (Epredia, cat. 8312-4) mounting media. For all experiments described in this report, 15–20 fields per group were quantified. Image analysis was performed using ImageJ (http://imagej.nih.gov/ij/) software (Version ImageJ 2.9.0, Java 1.8.0_172 [64-bit]).

Human Samples

Human serum samples and post resection images had been deidentified to all research personnel and were used in compliance with UT Health human subject’s research under IRB EUS-RFA Prospective study HSC-MS-18-0192 (PI: Thosani). Written informed consent was obtained for all patients enrolled in the study. Serum was collected from one Stage III PDAC patient post EUS-RFA and stored at 4°C until further processing. Two Stage I and one Stage III resected tumor samples from PDAC patients post EUS-RFA were fixed in formalin, processed, embedded, and sectioned by the Memorial Hermann TMC Pathology Core. This study was conducted in accordance with UTHealth IRB protocols abided by Helsinki Declaration.

Imaging mass cytometry (IMC)

Metal-labeled antibodies were prepared according to the MaxPar antibody conjugation kit protocol (Fluidigm, cat. 201300). In brief, antibodies were obtained in carrier/protein-free buffer and then prepared according to kit instructions. After determining the percent yield by absorbance measurement at 280 nm, the metal-labeled antibodies were diluted in Candor PBS Antibody Stabilization solution (Candor Bioscience, cat. 131050) for long-term storage at 4°C. Tumor sections were baked at 60°C overnight, then dehydrated in xylene and rehydrated in a graded series of alcohol for IMC. Heat-induced epitope retrieval was conducted in a water bath at 95°C in Tris buffer with Tween20 at pH 9 for 20 minutes. After immediate cooling for 20 minutes, the sections were blocked with 3% bovine serum albumin in tris-buffered saline (TBS) for 1 hour. For staining, the sections were incubated overnight at 4°C with an antibody master mix. Samples were then washed 4 times with TBS/0.1% Tween20. For nuclear staining, the sections were stained with Fluidigm Corp Cell-ID^™ Intercalator (Fluidigm Corp, cat. NC1605684) for 5 minutes and washed twice with TBS/0.1% Tween20. Slides were air-dried and stored at 4°C for ablation.

The sections were ablated with Fluidigm Hyperion Imaging System (IMC^™, Fluidigm^™) for data acquisition. IMC data were segmented by ilastik (www.ilastik.org) and CellProfiler (http://www.cellprofiler.org). Histology topography cytometry analysis toolbox (HistoCAT, https://github.com/BodenmillerGroup/histoCAT) and R (Version 4.2.1, www.r-project.org) scripts developed in house were used to quantify cell number, generate tSNE plots, and perform neighborhood analysis. For all samples, tumor and cellular densities were averaged across 3 images per tumor, with n = 3 per group. Antibodies used in this study are listed in Supplementary Table S1 (7).

Tumor and Serum Proteome Array

The Proteome Profiler^™ Mouse Cytokine Array Panel A (ARY006, R&D Systems, Minneapolis, MN, USA) was used to measure the protein levels of cytokines and chemokines in serum and tumor tissues. For serum measurements, 50μL were used. In tumor tissues, protein concentrations were quantitated using Pierce^™ BCA Protein Assay Kit (Thermo Scientific, cat 23225) following manufacturer instructions. The array was inoculated with 200μg of protein, and samples were treated according to the product specification. Similarly, Pre and Post RFA human serum samples were used for analysis with the Proteome Profiler^™ Human Cytokine Array Kit (ARY005B, R&D Systems, Minneapolis, MN, USA), which detects human cytokines, chemokines, and acute phase proteins. Assays of human samples were run with 50μL of serum according to the manufacturer’s instructions. Proteins were detected with ChemiDoc MP Imaging System (Bio-Rad, cat. 12003154) and analyzed in Image Lab Software (Bio-Rad Laboratories, version 6.1.0 build 7). Only detected proteins were included in graphs.

Neutrophil Depletion in vivo

Mice were intraperitoneally administered 400μg anti-Ly6G (clone 1A8, BioXCell, West Lebanon, NH) at two and one day prior and three more times after RFA treatment. Depletion of Ly6G⁺ cells was confirmed by IHC staining for NIMPR14 in both RFA-treated and non-RFA treated tumors. IgG2a isotype (clone 2A3, BioXCell, West Lebanon, NH) was used as control.

CXCL13 ELISA

Mouse CXCL13/BLC/BCA-1 Quantikine ELISA Kit assay was used to determine CXCL13 levels in mouse serum, splenocytes and tumors homogenates, following the manufacturer instructions (R&D Systems, cat #MCX130).

Anti–PD-L1 ICB in vivo treatment in combination with RFA

Mice were intraperitoneally administered a total of 5 injections of 100μg αPD-L1 antibody (BIO X CELL, InVivoPlus anti-mouse PD-L1, clone B7-H1, Cat#BP0101) diluted in buffer pH 7 or vehicle (BIO X CELL, InVivoPlus rat IgG2b, Cat#BP0090) diluted in buffer pH 6.5 at the day of RFA treatment and every other day up to 10 days after RFA treatment.

Statistical analysis

Throughout the manuscript, 2-way ANOVA was used when comparing 2 variables (treatment x time); One-way ANOVA was used when comparing one variable (treatment); and unpaired Student’s t test was used when comparing 2 groups, except for the analysis of changes in tumor size Pre and Post RFA, which were analyzed as correlated data with paired Student’s t test to assess differences in each tumor before and after treatment. Multiple t test was used to analyze IMC and proteome data.

Data availability statement

The data generated in this study are available within the article and its supplementary data files or from the corresponding author upon reasonable request.

RESULTS

RFA reduces PDAC progression in vivo and increases pro-inflammatory mediators

To determine the effects of RFA in vivo, we established a syngeneic mouse model of pancreatic cancer and performed tumor RFA in one flank (Fig. 1A). Assessment of tumor growth in successfully ablated mice revealed significant inhibition of tumor growth in RFA-treated tumors when compared to Sham-treated controls (Fig. 1B) with an associated abscopal effect in 87.5% of non-RFA treated tumors (Supplementary Figure S1A-1B) (Fig. 1C). RFA ablation did not affect body weight (Supplementary Fig. S1C).

Figure 1. RFA reduces PDAC tumor progression in vivo and increases pro-inflammatory mediators.

Tumor size was recorded 4 days before (Initial), right before (Pre) and 4 days after (Post) Sham or RFA treatment. Proteome arrays were performed in locally ablated tumors and serum of ablated mice and compared to Sham-treated mice (Control). A, Experimental design of RFA-treated mice. B, Growth curves show Control tumors (n = 8) significantly increased in size 4 days after treatment when compared to RFA-treated (n = 8) and non-RFA treated (n = 7) tumors. C, At the time of euthanization only Sham-treated tumors (n = 8) had significantly increased in size compared with pretreatment size; no difference in size was observed in RFA-treated (n = 8) and non-RFA treated (n = 7) tumors Pre and Post RFA. D, ImageJ quantification of necrosis, which was detected by H&E staining. RFA significantly increased necrosis on the RFA- and non-RFA-treated tumors compared to control Sham-treated tumors. E, Representative composite H&E staining of control, RFA, and non-RFA treated tumors showing necrotic areas inside dashed lines. F, ImageJ quantification showing RFA increased cleaved caspase 3⁺ cells in the RFA-treated and non-RFA treated tumors compared to control Sham-treated control tumors, as assessed by IHC. G, Representative IHC staining for cleaved caspase 3 in control, RFA, and non-RFA treated tumors. H, ImageJ quantification revealed RFA significantly increased the number of granzyme B⁺ cells in the RFA-treated tumors compared to controls and non-RFA treated tumors, as assessed by IHC. I, IHC staining for granzyme B in control, RFA, and non-RFA treated tumors. J, RFA-treated tumors (n = 3) presented increased expression of C5/C5a, IL-23 and CXCL12 compared to control (n = 2) tumor content. K, CXCL10, CXCL12, CXCL13 and TIMP-1 were significantly elevated in serum from RFA-treated (n = 4) mice compared to Sham-treated (n = 3) control serum. Time x Treatment comparisons were performed using Two-way ANOVA, treatment only comparisons by One-way ANOVA and proteome arrays were analyzed by multiple t test. Bar plots showing mean with SEM were used to represent data. *, P ≤ 0.05; **, P ≤ 0.01; ***, P ≤ 0.001; ****, P ≤ 0.0001; n.s., not significant. Scale bars are 50μM.

Composite images of H&E staining revealed RFA-treated and non-RFA treated tumors had significantly increased necrotic area compared to control tumors (Fig. 1D-E). IHC staining of cleaved caspase 3 and granzyme B revealed significantly increased staining in the RFA-treated side when compared to control and non-RFA treated tumors (Fig. 1F-I), suggesting differential antitumor mechanisms of RFA response when comparing local and abscopal responses. We performed a membrane-based antibody array in tumor homogenates and found that RFA significantly increased C5/C5a, IL-23 and CXCL12 expression in RFA-treated tumors compared to control tumors (Fig. 1J) and chemotactic chemokines CXCL10, CXCL12 and CXCL13 were increased in serum from RFA-treated mice compared to Sham-treated mice (Fig. 1K). CXCL13 recruits T and B cells to promote the initiation and maintenance of antitumor responses and is secreted by dendritic cells and myofibroblasts (8-10). Thus, we used a CXCL13 ELISA, which revealed RFA significantly increased CXCL13 in serum, RFA-treated tumors, non-RFA tumors and splenocytes from RFA-treated mice compared to controls (Supplementary Fig. 1D-F). These findings are consistent with the role of CXCL13 in the activation of the germinal center and adaptive immunity (11).

RFA increases neutrophil infiltration and induces systemic TME remodeling

In tumor homogenates, IL-23, commonly secreted by neutrophils (12) (13), was significantly elevated (Fig. 1J). We assessed neutrophil abundance by IHC staining for NIMPR14 (Fig. 2A-B) and MPO (Fig. 2C-D, black arrows) and found they were significantly elevated in both RFA and non-RFA treated tumors compared to controls, especially in necrotic core regions of the RFA and non-RFA tumors. We observed colocalization between MPO and NIMPR14 indicating a number of neutrophils in RFA-treated tumors are pro-inflammatory (Supplementary Fig. S1G).

Figure 2. RFA increases neutrophil infiltration and induces systemic TME remodeling.

A, Neutrophil abundance in tumors 4 days after Sham or RFA treatment was quantified by IHC staining for NIMPR14 as number of neutrophils per field using ImageJ software. RFA and non-RFA treated tumors presented an increased neutrophil IHC staining (B) compared to control tumors. C, MPO IHC staining was used to quantify antitumor neutrophil abundance in tumors 4 days after Sham or RFA treatment. MPO was significantly abundant only in non-RFA treated tumors as shown in areas with high density of neutrophils (D, black arrows) indicating a prominent antitumor neutrophil response post RFA. E-F, αSMA IHC staining was increased in non-RFA treated tumors compared to both Sham- and RFA-treated tumors. G-H, Collagen deposition was increased in RFA and non-RFA treated tumors compared to controls. I-J, CD31⁺ cells were increased after RFA ablation in both RFA and non-RFA treated tumors compared to control tumors. Data was analyzed by One-way ANOVA. Bar plot graphs show mean with SEM. *, P ≤ 0.05; **, P ≤ 0.01; ***, P ≤ 0.001; ****, P ≤ 0.0001; n.s., not significant. Scale bars are 50μM.

The immune system orchestrates key roles in neovascularization, collagen deposition, tissue remodeling and is important for immunotherapy response (14). Non-RFA treated tumors revealed the highest expression of αSMA (Fig. 2E-F), collagen and CD31⁺ cells (Fig. 2G-J) in both RFA and non-RFA treated tumors compared to controls, indicating RFA remodels the PDAC TME in locally treated and distant non-treated sites.

Neutrophils are critical for the antitumor response on the abscopal tumor.

We used IMC to evaluate three regions from distant non-RFA treated tumors with a focus on the regions in and adjacent to the necrotic core (Fig. 3A). IMC cluster value data revealed increased abundance of Ly6G⁺CD11b⁺CD44⁺ neutrophils (Fig. 3B) and Neighborhood analysis showed neutrophils strongly colocalized with αSMA, PanCK⁺ tumor cells and PanCK⁺CD44⁺ tumor stem cells, as well as with CD11c⁺CD44⁺ dendritic cells, F480⁺CD44⁺ macrophages and CD86⁺ marker (Fig. 3C-D).

Figure 3. Neutrophils are critical for the antitumor response on the abscopal tumor.

A-B, IMC analysis of tumors 4 days after Sham or RFA treatment revealed Ly6G⁺CD11b⁺CD44⁺ neutrophils are enriched in non-RFA treated tumors. C, Neighborhood analysis identified immune cells and markers with strong neutrophil co-localization. D, Cluster and Cell Phenotype information of Neighborhood analysis of IMC data. E, Experimental design for neutrophil depletion in vivo followed by RFA. F, RFA locally ablated tumors treated with IgG2a isotype control (VEH, n = 6) or anti-Ly6G (ND, n = 8) did not show differences in tumor size right before (Pre) and 4 days after (Post) RFA ablation. In non-RFA treated tumors, anti-Ly6G (ND, n = 8) treatment revealed an increase in tumor size Post RFA treatment when compared to IgG2a isotype control (VEH, n = 6) treated tumors. G, Neutrophil depletion (ND; anti-Ly6G treated group) did not alter αSMA staining, detected by IHC, in RFA-treated tumors when compared to RFA-treated tumors with IgG2a (VEH); on the contrary, neutrophil depletion (ND) revealed non-RFA treated tumors presented a significant increase in αSMA compared to control non-neutrophil depleted (VEH) group. H, Neutrophil depletion did not alter CD31 staining, detected by IHC, in any of the groups. I, Neutrophil-depleted RFA treated tumors presented a significant reduction in CXCL13 content compared to both VEH + RFA and non-RFA treated tumors when assayed using a cytokine array. No differences were found in non-RFA treated tumors between treatments. J, Neutrophil depletion presented a trend in reducing systemic CXCL13 levels in RFA treated mice. Tumor volume was analyzed by paired Student’s t test. Tumor chemokine levels were studied by Two-way ANOVA. IHC and serum protein expression levels were analyzed by unpaired Student’s t test. Bar plots indicate mean with SEM. *, P ≤ 0.05; **, P ≤ 0.01; ****, P ≤ 0.0001; n.s., not significant.

To assess whether neutrophils are important in the abscopal effect, we performed an in vivo neutrophil depletion experiment in combination with RFA ablation (Fig. 3E). After tumors were established, mice were intraperitoneally administered 400μg anti-Ly6G or placebo 2 times Pre and 3 times Post RFA. Depletion of Ly6G⁺ cells was confirmed by IHC staining (Supplementary Fig. S2A). Group comparison evidenced a significant increase in tumor size only in non-RFA treated tumors from RFA-treated, neutrophil-depleted mice, indicating neutrophils are critical for the abscopal response (Fig. 3F). RFA-treated, neutrophil-depleted mice revealed increased αSMA expression only in distant non-treated tumors, indicating neutrophils may play a key role in mediating abscopal fibrosis through direct or indirect modulation of myofibroblasts (Fig. 3G; Supplementary Fig. S2B). CD31 IHC staining showed no differences between groups (Fig. 3H; Supplementary Fig. S2C). We analyzed CXCL13 levels in the serum and tumors from these mice. We observed a significant reduction in CXCL13 expression in RFA-treated, neutrophil-depleted tumors (Fig. 3I) and neutrophil depletion reduced systemic CXCL13 levels (Fig. 3J), suggesting its decrease may contribute to reduced tumor growth.

In vivo ICB therapy in combination with RFA sustains tumor progression inhibition

To determine longer-term outcomes of RFA on growth rates of RFA-treated and non-RFA tumors, we examined post RFA tumor sizes for seven days compared to Sham treated mice. Although we consistently observed significant differences in tumor size post RFA in RFA-treated and abscopal tumors at post RFA Day 4 (Post4D), when we measured tumors on Day 7 post RFA (Post 7D), the growth restraining capacity of RFA was not as prominent (Fig. 4A). To comprehensively evaluate the immune response at Post4D, when the antitumor response was most prominent, we used IMC and selected n=6 regions from Sham, RFA and non-RFA tumors (Fig. 4B). Dendritic cells (Cluster 3; CD11b⁺CD11c⁺) were significantly elevated in RFA-treated tumors compared to either Sham or non-RFA tumors (Fig. 4C-D). Myeloid-derived suppressor cells (MDSC; Cluster 2; CD11b⁺Ly6C⁺) were significantly decreased in both RFA and non-RFA tumors compared to Sham. CD4⁺ and CD8α⁺ T cells were both significantly increased in non-RFA tumors compared to RFA tumors (Cluster 10; CD4⁺) or RFA and Sham tumors (Cluster 13; CD8a⁺). These data indicate RFA promotes a systemic antitumor abscopal response driven by CD4⁺ and CD8α⁺ T cells against the non-RFA tumor.

Figure 4. In vivo ICB therapy targeting RFA-induced PD-L1 in combination with RFA sustains tumor progression inhibition.

A, Sham treated tumors (n=5) continued to grow throughout the study. RFA treatment (n=6) inhibited tumor growth progression 4 days after treatment (Post 4D); however, 7 days after RFA (Post 7D), the growth restraining capacity was not as prominent. B, TSNE plots for IMC analysis of Sham, RFA or non-RFA treated tumors at Post 4D. C, Cluster and Cell Phenotype information of TNE plots and Neighborhood analysis of IMC data. D, Cluster cell density of IMC data showed dendritic cells (Cluster 3; CD11b⁺CD11c⁺) were significantly elevated in RFA treated tumors compared to Sham and non-RFA treated tumors. Myeloid derived suppressor cells (MDSC; Cluster 2; CD11b⁺Ly6C⁺) were significantly decreased in both RFA and non-RFA tumors compared to Sham. CD4⁺ and CD8α⁺ T cells were both significantly increased in non-RFA tumors compared to RFA (Cluster 10; CD4⁺) or RFA and Sham (Cluster 13; CD8α⁺). E, IMC analysis revealed RFA treated tumors significantly increased cluster cell density levels of PD-L1 compared to Sham tumors (Cluster 16). F, Neighborhood analysis determined PD-L1 spatially localized closely with PanCK⁺ and PanCK⁺αSMA⁺ cells (Clusters 22 and 23) in RFA and non-RFA tumors. G, Experimental design of in vivo RFA + anti–PD-L1 ICB combination therapy. H, In vivo RFA + anti–PD-L1 combination therapy (n=6) restrained tumor growth on both the RFA and non-RFA tumors 7 days after RFA (Post 7D) and continued to restrain the growth tumors compared to mice treated with an isotype control antibody (n=7) up to 10 days (Post 10D) after RFA. Data was analyzed by Two-way ANOVA and represented by bar plots with mean and SEM. *, P ≤ 0.05; **, P ≤ 0.01; ***, P ≤ 0.001; ****, P ≤ 0.0001; n.s., not significant.

IMC analyses also revealed RFA-treated tumors significantly increased cluster density levels of PD-L1 compared to Sham tumors (Cluster 16). PD-L1 was elevated in non-RFA tumors, although the trend was not significant compared to Sham treated tumors (Fig. 4E). Spatial analysis in RFA-treated tumors revealed PD-L1 localizes closely with PanCK⁺ and PanCK⁺αSMA⁺ cells (Fig. 4F) (Clusters 22 and 23, respectively).

To test the hypothesis that PD-L1 expression post RFA was important for relapse of the post RFA response, we repeated the experiment and began treating mice with anti–PD-L1 compared to isotype control antibodies at the time of RFA treatment (Fig. 4G). Treatment with anti–PD-L1 restrained tumor growth on both the RFA and non-RFA tumors on Day 7 and Day 10 post RFA. This effect was not observed when correlated tumor growth was analyzed within each tumor in mice treated with an isotype control antibody (Fig. 4H).

RFA tumor microenvironment and immune modulation in human pancreatic tumors

We examined the impact of EUS-RFA in three patients with PDAC who had received RFA ablation as previously described (15). Stage I and locally advanced Stage III pancreatic cancer resected tumors presented a necrotic area with limited epithelial cells in the ablated region, residual tumor foci and normal pancreas (Supplementary Fig. S3A-B). We analyzed MPO, CD31⁺ cells and granzyme B in the locally advanced Stage III resected tumor and observed a significant presence of MPO (Supplementary Fig. S3C top panel), CD31⁺ cells (Middle panel) and Granzyme B (Bottom panel) in ablated and distant areas. We performed a proteome profile array using PDAC patient serum Pre and Post EUS-RFA ablation and found increased levels of CCL5, CD40, C5/C5a, ICAM, MIF and SERPIN (Supplementary Fig. S3D). The results in human pancreatic tumors after EUS-RFA show a strong concordance with our preclinical findings.

DISCUSSION

Together, the studies presented herein, provide evidence that RFA-induced local and abscopal effects are capable of restraining tumor progression and inducing immune and stromal modulations that can be leveraged to improve therapeutic response. Our findings show ICB therapy targeting RFA-induced PD-L1 in combination with RFA sustained inhibition of tumor progression in both local and abscopal tumors. Prospective clinical trials are needed to better evaluate RFA effects in metastatic disease and assess whether RFA improves the therapeutic outcome of chemotherapy or ICB at distant sites. Our data are in line with previous studies that have shown radiation therapy modifies the abscopal action of tumor-infiltrating neutrophils by polarizing them to an antitumor phenotype (16). Future preclinical experiments evaluating RFA responses using multiple treatments including ICB will determine if these combination strategies can be further considered for clinical stages based on their ability to potentially restrain tumor growth and promote survival for PDAC patients, especially in patients with locally advanced or metastatic disease where innovative therapeutic options are needed to improve survival.