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Purpose:Metastasis remains a major hurdle in treating aggressive malignancies such as pancreatic ductal adenocarcinoma (PDAC). Improving response to treatment, therefore, requires a more detailed characterization of the cellular populations involved in controlling metastatic burden.Experimental Design:PDAC patient tissue samples were subjected to RNA sequencing analysis to identify changes in immune infiltration following radiotherapy. Genetically engineered mouse strains in combination with orthotopic tumor models of PDAC were used to characterize disease progression. Flow cytometry was used to analyze tumor infiltrating, circulating, and nodal immune populations.Results:We demonstrate that although radiotherapy increases the infiltration and activation of dendritic cells (DC), it also increases the infiltration of regulatory T cells (Treg) while failing to recruit natural killer (NK) and CD8 T cells in PDAC patient tissue samples. In murine orthotopic tumor models, we show that genetic and pharmacologic depletion of Tregs and NK cells enhances and attenuates response to radiotherapy, respectively. We further demonstrate that targeted inhibition of STAT3 on Tregs results in improved control of local and distant disease progression and enhanced NK-mediated immunosurveillance of metastasis. Moreover, combination treatment of STAT3 antisense oligonucleotide (ASO) and radiotherapy invigorated systemic immune activation and conferred a survival advantage in orthotopic and metastatic tumor models. Finally, we show the response to STAT3 ASO + radiotherapy treatment is dependent on NK and DC subsets.Conclusions:Our results suggest targeting Treg-mediated immunosuppression is a critical step in mediating a response to treatment, and identifying NK cells as not only a prognostic marker of improved survival, but also as an effector population that functions to combat metastasis.

Supplementary Figure 6 (A) Survival curve comparing patients with high expression of FLT3, the FLT3LG receptor, to patients with low expression of FLT3 expression. (B) Survival curve comparing patients with high expression of FLT3LG to patients with low expression of FLT3LG.

Supplementary Figure 5 (A) IL-15 amounts in serum of Balb/c mice treated with RT, anti-CD25, anti-asialoGM1 to deplete NK cells, and FLT3L; **** p{less than or equal to}0.0001; n=6. (B) Flow cytometric analysis of DCs MOC2 tumors after treatment with radiation therapy and anti-CD25; n=5. (C) growth curves of MOC2 tumors in individual mice treated with radiation therapy, anti-CD25, and anti-CD137.

Supplementary Figure 3 (A) MOC2 tumor volumes of mice treated with RT, anti-CD25, and pLEV vehicle control (black line); RT, anti-CD25, and pLEV FLT3L plasmid (green line), and RT + anti-CD25 (red line). n=7. (B) Flow Cytometry analysis of CD8 T cell populations in MOC2 tumors and lymph nodes (LN) of C57 BL/6 mice. MOC2 tumors were harvested 3 days after RT, NK cell depletion and FLT3L therapy; n=6.

Purpose:Natural killer (NK) cells are type I innate lymphoid cells that are known for their role in killing virally infected cells or cancer cells through direct cytotoxicity. In addition to direct tumor cell killing, NK cells are known to play fundamental roles in the tumor microenvironment through secretion of key cytokines, such as FMS-like tyrosine kinase 3 ligand (FLT3L). Although radiotherapy is the mainstay treatment in most cancers, the role of radiotherapy on NK cells is not well characterized.Experimental Design:This study combines radiation, immunotherapies, genetic mouse models, and antibody depletion experiments to identify the role of NK cells in overcoming resistance to radiotherapy in orthotopic models of head and neck squamous cell carcinoma.Results:We have found that NK cells are a crucial component in the development of an antitumor response, as depleting them removes efficacy of the previously successful combination treatment of radiotherapy, anti-CD25, and anti-CD137. However, in the absence of NK cells, the effect can be rescued through treatment with FLT3L. But neither radiotherapy with FLT3L therapy alone nor radiotherapy with anti-NKG2A yields any meaningful tumor growth delay. We also identify a role for IL2 in activating NK cells to secrete FLT3L. This activity, we show, is mediated through CD122, the intermediate affinity IL2 receptor, and can be targeted with anti-CD25 therapy.Conclusions:These findings highlight the complexity of using radio-immunotherapies to activate NK cells within the tumor microenvironment, and the importance of NK cells in activating dendritic cells for increased tumor surveillance.

Supplementary Figure 1 (A) Flow cytometry analysis of NK cell population expression of NKG2A in MOC2 tumors 3 days after RT; n=3. (B). Flow cytometry analysis of NK cell populations in MOC2 tumors 3 days after RT and anti-NKG2A therapy; n=6. (C) Flow cytometry analysis of CD4 T cell populations in MOC2 tumors 3 days after RT, anti-NKG2A therapy and NK cell depletion with NK1.1 antibody; n=6. (D) MOC2 tumor volumes of C57BL/6 mice treated with RT and isotype control (red line), anti-CD137, anti-CD25, and anti-NKG2A (blue line), anti-CD17 and antiCD25 (black line); n=7.

Supplementary Figure from Targeting Treg-Expressed STAT3 Enhances NK-Mediated Surveillance of Metastasis and Improves Therapeutic Response in Pancreatic Adenocarcinoma

Supplementary Figure 4 (A) Gating strategy used in flow cytometry analysis analyzing NK46+ CD3-NK cells, CD11c+ MHCII+ DCs, CD3+ CD4+ T cells, and CD3+ CD8+ T cells. (B) Flow cytometry analysis of NK cell populations in blood from WT C57BL/6 mice treated with RT, anti-CD25, and anti-CD137; n=4. (C) Flow cytometry of CD4 T cell populations in tumors from WT C57BL/6 mice treated with RT, anti-CD25, and anti-CD137 and from DEREG mice treated with RT, anti-CD25, antiCD137, and diphtheria toxin (DT); *p{less than or equal to}0.05, ** p{less than or equal to}0.01; n=6. (D) FLT3L present in serum of C57BL6 treated with radiation therapy, anti-CD25, and anti-CD137, and DEREG mice treated with radiation therapy, anti-CD25, anti-CD137, and diphtheria toxin (DT); ** p{less than or equal to}0.01; n=6

Supplementary Figure 2 (A) Flow cytometry analysis of LY2 tumors analyzing DC populations; n=6. (B) Analysis of FLT3L present in the serum of LY2 bearing mice treated with radiation therapy and isotype antibodies, radiation therapy and anti-CD25 antibody, radiation therapy and anti-CD25 antibody and anti-asialoGM1 antibody to deplete NK cells, and radiation therapy, anti-CD25 antibody, anti-asialoGM1 antibody to deplete NK cells, and FLT3L therapy; n=5.

Curative intent treatment of pancreatic adenocarcinoma (PDAC) relies on surgical resection. Modern treatment protocols focus on optimizing neoadjuvant therapy to increase resectability and improve oncologic outcomes. To elucidate differences in outcomes, we investigated the relationship between neoadjuvant chemotherapy (NAC), either with or without stereotactic body radiation therapy (SBRT), and vascular inflammation, surgical outcomes, and the resultant transcriptomic changes.Clinical data were collected from patients with borderline resectable PDAC (clinical T3-T4N0-1) that underwent NAC or NAC-SBRT followed by curative-intent resection between 2014 and 2019. Vascular structures on surgical specimens were histologically evaluated for vasculitis. RNA-seq was utilized to evaluate differential gene expression and generate enrichment maps. Multivariate analysis was used to analyze the relationship between patient characteristics and oncological outcome.In total, n=46 patients met inclusion criteria (n=12 NAC, n=34 NAC-SBRT) with a median follow up of 20.1 months. All patients underwent curative resection with 91.3% achieving R0. There was no significant difference in patterns of failure, overall survival (OS) or progression free survival between NAC and NAC-SBRT groups. Patients with vasculitis had a lower median OS compared to those without (14.5 mo vs. 28.3 mo, HR of 12.96 [95% CI 3.55-47.28], p0.001). There was no significant correlation between inflammation and surgical complications or pathologic response. Neoadjuvant therapy did not have a significant effect on development of vasculitis (OR of 1.64 for NAC-SBRT, 95% CI 0.40-8.43, p=0.52). Predictors of poor survival included perineural invasion (PNI) and high baseline CA19-9 (191 U/mL). Patients with robust CA19-9 (20% decrease) responses to neoadjuvant therapy had enrichment in immune response, chemotaxis, and cytotoxic T-cell and NK cell proliferation.Vasculitis predicts for poor survival outcomes in PDAC patients; NAC-SBRT did not increase the rate of vasculitis compared to NAC. PNI and CA19-9 remain strong prognosticators. Understanding and optimizing the immune response remains a crucial hurdle in achieving responses in pancreatic cancer.

Although IL-2 has been shown to critically mediate NK and T cell effector functions, when used in clinical trials this cytokine has failed due to toxicity and severe side effects of the systemic treatment. Our lab has investigated the use of a next-generation IL-2 compound, murine PD1-IL2v, which consists of an anti-PD-1 antibody fused to a IL2-variant with abolished binding to IL2Rα. Previous work shows this molecule results in tumor-antigen specific T cell expansion and differentiation towards immune effectors. Here, we report for the first time that radiation therapy amplifies the anti-tumor effect observed with PD1-IL2v treatment of murine orthotopic models of local tumor growth and metastasis. This anti-tumor effect is correlated with increases in proliferative and polyfunctional CD8 T cells across tumor, lymph node, and blood compartments. Furthermore, using an OVA-antigen specific orthotopic model, we found that radiation in combination with PD1-IL2v treatment enhances infiltration and activation of antigen-specific CD8 T cells, which have been shown to be critical for immunotherapy clinical success. A portion of tumor-bearing mice treated with radiation and PD1-IL2v fully eradicated their primary tumors and tumor rechallenges, which we show correlates with increased markers of memory in CD8 T cells of these mice. To demonstrate the clinical relevance of these preclinical findings, we performed RNA sequencing on paired pancreatic adenocarcinoma (PDAC) patient tumor tissue samples prior to and following radiation. We observed enhanced IL2Rβ and IL2Rγ expression as well as IL2R signaling in PDAC-patient responders to radiation therapy compared to non-responders. In conclusion, the antigen-specific, lasting memory response to preclinical PDAC models demonstrated here provides rationale for the treatment of PDAC with a radiation and PD1-IL2v combination. Citation Format: Maureen Hoen, Miles Piper, Laurel B. Darragh, Diemmy Nugyen, Jacob Gadwa, Maria Amann, Greta Durini, Michael Knitz, Benjamin Van Court, Tiffany Pham, Ross Kedl, Pablo Umana, Laura Codarri-Deak, Christian Klein, Angelo D’Alessandro, Sana D. Karam. The addition of radiation therapy to simultaneous targeting of PD-1 and IL2Rβγ enhances antigen-specific CD8 T cell anti-tumor immunity in pancreatic cancer models. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 5106.