EMT6 Tumors Research Articles

IMPDH inhibitors upregulate PD-L1 in cancer cells without impairing immune checkpoint inhibitor efficacy.

Tumor cells are characterized by rapid proliferation. In order to provide purines for DNA and RNA synthesis, inosine 5'-monophosphate dehydrogenase (IMPDH), a key enzyme in the de novo guanosine biosynthesis, is highly expressed in tumor cells. In this study we investigated whether IMPDH was involved in cancer immunoregulation. We revealed that the IMPDH inhibitors AVN944, MPA or ribavirin concentration-dependently upregulated PD-L1 expression in non-small cell lung cancer cell line NCI-H292. This effect was reproduced in other non-small cell lung cancer cell lines H460, H1299 and HCC827, colon cancer cell lines HT29, RKO and HCT116, as well as kidney cancer cell line Huh7. In NCI-H292 cells, we clarified that IMPDH inhibitors increased CD274 mRNA levels by enhancing CD274 mRNA stability. IMPDH inhibitors improved the affinity of the ARE-binding protein HuR for CD274 mRNA, thereby stabilizing CD274 mRNA. Guanosine supplementation abolished the IMPDH inhibitor-induced increase in PD-L1 expression. In CT26 and EMT6 tumor models used for ICIs based studies, we showed that despite its immunosuppressive properties, the IMPDH inhibitor mycophenolate mofetil did not reduce the clinical response of checkpoint inhibitors, representing an important clinical observation given that this class of drugs is approved for use in multiple diseases. We conclude that PD-L1 induction contributes to the immunosuppressive effect of IMPDH inhibitors. Furthermore, the IMPDH inhibitor mycophenolate mofetil does not antagonize immune checkpoint blockade.

Hainanenin-1, an oncolytic peptide, triggers immunogenic cell death via STING activation in triple-negative breast cancer

BackgroundIn triple-negative breast cancer (TNBC) therapy, insufficient tumor infiltration by lymphocytes significantly hinders the efficacy of immune checkpoint inhibitors. We have previously demonstrated that Hainanenin-1 (HN-1), a host defense peptide (HDP) identified from Hainan frog skin, induces breast cancer apoptosis and boots anti-tumor immunity via unknown mechanism.MethodsWe used in vitro experiments to observe immunogenic cell death (ICD) indicators in HN-1-treated TNBC cell lines, a mouse tumor model to verify HN-1 promotion of mice anti-tumor immune response, and an in vitro drug sensitivity test of patient-derived breast cancer cells to verify the inhibitory effect of HN-1.ResultsHN-1 induced ICD in TNBC in a process during which damage-associated molecular patterns (DAMPs) were released that could further increase the anti-tumor immune response. The secretion level of interleukin 2 (IL-2), IL-12, and interferon γ in the co-culture supernatant was increased, and dendritic cells (DCs) were activated via a co-culture with HN-1-pretreated TNBC cells. As a result, HN-1 increased the infiltration of anti-tumor immune cells (DCs and T lymphocytes) in the mouse model bearing both 4T1 and EMT6 tumors. Meanwhile, regulatory T cells and myeloid-derived suppressor cells were suppressed. In addition, HN-1 induced DNA damage, and double-strand DNA release in the cytosol was significantly enhanced, indicating that HN-1 might stimulate ICD via activation of STING pathway. The knockdown of STING inhibited HN-1-induced ICD. Of note, HN-1 exhibited inhibitory effects on patient-derived breast cancer cells under three-dimensional culture conditions.ConclusionsCollectively, our study demonstrated that HN-1 could be utilized as a potential compound that might augment immunotherapy effects in patients with TNBC.

Strong single-agent and PD-1/PD-L1 combination antitumor activity of antisense oligonucleotides suppressing Neuropilin 1 expression.

e14592 Background: While the introduction of immune checkpoint inhibitors (ICIs) is considered as a breakthrough in cancer therapy, large proportions of patients still do not respond or develop resistance, indicating a need for new targets and therapies to provide long-term therapeutic benefits. Neuropilin-1 (NRP1) is a membrane-bound multi domain protein expressed by different cell types in the tumor microenvironment. NRP1 promotes cell migration, survival, and angiogenesis via distinct domains. Moreover, it mediates immunosuppression by stabilization of regulatory T cells, attraction of immunosuppressive myeloid cells into the tumor microenvironment, and exhaustion of effector T cells. Therapeutic benefit is expected to be superior by simultaneous suppression of all functions and consequently all domains, which can be optimally achieved by suppression of its expression, for example mediated by antisense oligonucleotides (ASOs), instead of e.g. domain specific antibodies. Methods: Using our in-house bioinformatics and screening systems, we have identified potent and specific locked nucleic acid (LNA) modified ASOs specifically targeting NRP1. Mice bearing established syngeneic MC38 or EMT6 tumors were treated by intraperitoneal injection of unformulated ASO either as monotherapy or in combination with ICIs. Tumor growth and survival were determined as well as target engagement. Results: After systemic administration of different NRP1-specific ASOs we observed a potent downregulation of NRP1 expression in different cell types in tumors, including myeloid cells, T cells and non-immune cells as well as a strong reduction of soluble NRP1 plasma levels. Suppression of NRP1 correlated with both a delayed tumor growth or even complete tumor regression and prolonged survival as a monotherapy which was further improved in combination with ICIs. Particularly in an EMT6 breast cancer model, combination of NRP1 ASOs with PD-1 antibody resulted in tumor rejection and long-term survival in most animals. Rechallenge of long-term surviving mice with EMT6 tumor cells did not result in tumor growth. Conclusions: We could show that ASO-mediated suppression of NRP1 expression has strong potential as a novel therapeutic strategy, especially in combination with ICIs. Additional studies to further elucidate the mechanisms underlying the observed anti-tumor efficacy as well as IND-enabling activities are underway to progress the program towards clinical testing.

Analysis of long-lasting tumor-specific antibodies from murine tumors treated with Aliya pulsed electric fields.

e14543 Background: Pulsed electric field (PEF) tissue destruction has demonstrated meaningful systemic immune responses in mice that are superior to thermal ablation, and synergize effectively with systemic chemotherapy and checkpoint blockade (CPB) immunotherapy. Immune cell populations are increased by PEF, including tumor specific activated CD8 T-cells and plasma B-cells. Antitumor immunity can include the presence of circulating anti-cancer antibodies. However, the presence and role of circulating tumor specific antibodies induced by PEF have not been explored. Serological antibodies are often detected with the ELISA assay, where tumor-associated antigens are coated to a microtiter plate to attract the corresponding serum antibodies. Here, we quantify IgG tumor specific antibodies after PEF using an in-house ELISA. Methods: Mice bearing immune-warm (EMT6) and immune-cold (4T1) orthotopic murine breast tumors were treated with a biphasic PEF dose comparable to Aliya titrated for mouse tumors. To detect tumor specific circulating IgG1 immunoglobulin in serum, an ELISA plate was coated with EMT6 and 4T1 protein lysate, as well as with a known 4T1-expressed tumor-specific antigen (gp70). Serum draws from the treated, untreated, and naïve mice were exposed to the coated wells for 1 hour. Bound IgG were detected by anti-mouse IgG (HRP tagged) antibody added to the wells and incubated 1 hour. A TMB substrate was added and color development was allowed for 15 minutes. The optical density was quantified using a microtiter plate reader. Results: ELISA quantification of serum IgG at multiple timepoints show that 4T1 tumor-specific IgG antibodies were increased 14.3- and 2.6-fold by day 20 in the PEF-treated and untreated group survivors relative to Naïve mice, respectively, (OD=0.86 and 0.15 v. 0.06). Furthermore, IgG antibodies specific to the gp70 antigen of the 4T1 cell line were increased by 44.9- and 2.7-fold in the PEF-treated and untreated groups relative to naïve mice (OD=3.14 and 0.19 v. 0.07). In the EMT6 model, at 3 months post-treatment, only the PEF-ablated group mice survived, and showed 15.5-fold higher antibodies against EMT6 tumor antigens compared to naive mice (OD=0.62 v. 0.04). Conclusions: The specific form of PEF in this study generated tumor-specific IgG long-lived antibodies in mice for immune-warm and immune-cold tumor models, corroborating previous findings of increased murine B-cell and plasma cell populations. This effect has not been well-reported for other ablation technologies. Increases were evident by Day 20 post-PEF and persisted at least 3 months. These murine data suggest the PEF used may invoke potent, long-lived immune responses and anti-tumor immune surveillance, offering a potential approach for in situ tumor vaccination to improve metastatic disease outcomes. Future studies should determine response durability and its role in attaining distant tumor clearance.

Efficacy of IFN-γ, sCD40L, and Poly(I:C) Treated Bone Marrow-Derived Macrophages in Murine Mammary Carcinoma

ABSTRACT Introduction Here, we explored methods to generate anti-tumor bone marrow-derived macrophages (BMDM) and how delivery of the BMDM at early tumor sites could impact disease progression. Methods BMDM treated with IFN-γ, sCD40L, poly(I:C), and a combination of the three were assessed. Results Treatment with sCD40L had no significant impact on the BMDM. Treating BMDM with IFN-γ impacted IL-1β, MHC Class II, and CD80 expression. While poly(I:C) treatment had a greater impact on the BMDM than IFN-γ when assessed by the in vitro assays, the BMDM treated with poly (I:C) had mixed results in vivo where they decreased growth of the EMT6 tumor, did not impact growth of the 168 tumor, and enhanced growth of the 4T1 tumor. The combination of poly(I:C), IFN-γ, and sCD40L had the greatest impact on the BMDM in vitro and in vivo. Treatment with all three agonists resulted in increased IL-1β, TNF-α, and IL-12 expression, decreased expression of arginase and mrc, increased phagocytic activity, nitrite production, and MHC Class II and CD80 expression, and significantly impacted growth of the EMT6 and 168 murine mammary carcinoma models. Discussion Collectively, these data show that treating BMDM with poly(I:C), IFN-γ, and sCD40L generates BMDM with more consistent anti-tumor activity than BMDM generated with the individual agonists.

Abstract PO5-24-12: IRX5010, a Highly Selective RARγ Nuclear Receptor Agonist Compound Inhibits In Vivo Growth of Murine EMT-6 Triple Negative Breast Cancer and Human JIMT-1Her2+ Breast Cancer By Induction of Tumor-Infiltrating Effector Memory T-cells

Abstract Research Objectives and Rationale: Previously, RARγ agonism has been shown to play an essential role in CD8 T-cell-mediated immunity to infectious pathogens. However, we found no previous reports on effects of RARγ agonists on in vivo tumor growth of triple negative or Her2+ breast cancers. Here we explored whether RARγ could play a critical role in T-cell-mediated immunity in these breast cancers, using potent highly selective novel RARγ agonist compounds. Experimental methods and results: We synthesized and screened compounds for selective transactivation of RAR γ in reporter assays. Three highly potent compounds, demonstrating highly selective RARγ transactivation at less than one nanomolar concentrations, were selected for further evaluation of potential anti-cancer activities in vitro. None of the three evaluated compounds demonstrated significant inhibitory effects on in vitro growth of EMT-6 triple negative breast cancer cells at concentrations of 20-100 millimolar, indicating that the three compounds have insignificant direct inhibitory effects on EMT-6 cancer cell growth in vitro. We then examined the effects of one of the compounds in vivo in a syngeneic mouse model of EMT-6 triple negative breast cancer. Treatment with the most potent of the three compounds, IRX5010, demonstrated dose response inhibition of in vivo growth of EMT-6 tumors. Inhibition of tumor growth at day 17 relative to vehicle was 59% and 69% respectively in mice treated with IRX5010 at 10 or 25 mg/kg/day. A cohort treated with 50 mg/kg/day was sacrificed early on day 12 due to excessive weight loss and deaths of some of the animals. This cohort experienced a 73% inhibition of tumor growth relative to vehicle. Gross necropsies of animals in the 25 and 50 mg/kg/day dose groups did not show observable pathologic findings in any major organ. Flow cytometry was performed on freshly excised tumors on the day of termination of the in-life portion of the study in the 10 and 25 mg/kg/day dose groups. Tumors from mice treated with IRX5010 had increased intratumoral numbers of Total CD45+ cells; Total CD3+, Total CD4+, and Total CD8+ T-cells per mg of tumor tissue, relative to vehicle controls. IRX5010 treated mice also had increased intratumoral CD4+IL-17+ T-helper cells; and increased CD4+CD62L- and CD8+CD62L- T-effector memory T-cells per mg of tumor tissue relative to vehicle-treated control mice. We also conducted an experiment of treatment with IRX5010 in mice transplanted with human immune cells (to provide a human immune system) to examine its effects on xenografted human Her2+ JIMT-1 cancer cells. In this model, treatment with IRX5010 at 10 mg/kg/day resulted in a 17% inhibition of tumor growth relative to vehicle control over 26 days of treatment. Flow cytometric analysis showed that tumors from the IRX5010 treated mice contained increased numbers per mg of tumor tissue of intratumoral human CD8+ T-cells, most of which were CD8+ CD7- CD45RA+ terminally differentiated effector memory phenotype T-cells. Conclusions: Treatment with IRX5010, a potent and highly selective RARγ nuclear receptor agonist compound demonstrated substantial treatment effects relative to vehicle control on inhibition of growth of murine triple negative breast cancer in vivo, despite having essentially no growth inhibitory effect on these cells in vitro. Treatment of xenografted human Her2+ tumors in mice with humanized immune systems resulted in a moderate inhibition of Her2+ breast cancer growth relative to vehicle controls. In both models, treatment with IRX5010 induced tumor infiltrating T-lymphocytes of largely effector memory phenotypes, demonstrating an effect on the tumor immune microenvironment. These data identify RARγ and RARγ agonists as a new treatment target and novel treatment modality for breast cancers, especially for the highly unmet need for new and better treatments for triple negative breast cancer. Citation Format: Martin Sanders, Mary Topalovski, Vidyasagar Vuligonda. IRX5010, a Highly Selective RARγ Nuclear Receptor Agonist Compound Inhibits In Vivo Growth of Murine EMT-6 Triple Negative Breast Cancer and Human JIMT-1Her2+ Breast Cancer By Induction of Tumor-Infiltrating Effector Memory T-cells [abstract]. In: Proceedings of the 2023 San Antonio Breast Cancer Symposium; 2023 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2024;84(9 Suppl):Abstract nr PO5-24-12.

Abstract 736: ACTM-838 safely and comprehensively re-activates the immunosuppressive TME by exploiting myeloid biology intrinsic in many cancers

Abstract ACTM-838 is a genetically modified bacterial vehicle encoding a DNA plasmid with two engineered payloads, IL-15plex and constitutively active eSTING. The bacterial vehicle is a highly attenuated S. typhimurium, engineered to facilitate safe IV administration. ACTM-838 is designed to specifically enrich in the TME via auxotrophic dependency on extracellular adenosine and purine metabolites. ACTM-838 is then selectively internalized by tumor-resident myeloid cells achieving tumor-specific payload delivery. Previously, we showed ACTM-838 exhibited significant and durable anti-tumor efficacy across syngeneic tumor mouse models (EMT6 breast, MC38 colon) and MMTV-PyMT GEMM. To elucidate TME changes induced by ACTM-838, EMT6 tumors were analyzed post-treatment by scRNA-seq, which demonstrated that ACTM-838 induced significant transcriptional changes in several cell populations in the TME. Cytolytic pathways were upregulated in T and NK cells, while type I IFN and PDL1 were upregulated in myeloid populations consistent with previous bulk RNAseq analyses. To understand the tolerability and safety of ACTM-838, a GLP toxicology study was performed on naïve cynomolgus monkeys (NHP). No ACTM-838-related clinical or veterinary observations were reported at any dose level. Acute dose-dependent increases in serum cytokines were observed at 4 hours which reverted to baseline by 24 hours. ACTM-838 was cleared from blood by 24 hours post-dose with no significant shedding in urine or feces. ACTM-838 was only detected in the spleen in 2 and 1 animals at 22- and 49-days post treatment, respectively. Pre-existing ADA was observed in some animals with subsequent increases in ADA upon dosing in most animals. However, no correlation of serum ADA and blood PK levels suggested that the presence of ADA does not impact drug stability. The ACTM-838 mechanism of action requires high levels of adenosine or purine metabolites to enrich and colonize the TME as well as phagocytic internalization by tumor-intrinsic myeloid cells to achieve tumor-specific payload delivery. Myeloid cells comprise roughly half of all cells in the TME in human tumors. Bioinformatics analyses of the public patient datasets identified key tumor indications enriched in both adenosine and myeloid pathways. To further understand the spatial heterogeneity in the TME across tumors, we performed multiplex immunofluorescence on patient tumor microarrays to validate tumor types with a high prevalence of myeloid populations coupled with an active adenosine pathway, which will help guide future clinical study design. In summary, we demonstrate that ACTM-838 modulates the TME and is stable and safe in animal models. Additionally, our studies recommend the prioritization of certain tumor indications for treatment with ACTM-838 in the clinic. Citation Format: William Lu, Kyle Cron, Julie Janes, Leigh Berryman, Gurvinder Singh, Christopher Thanos, Akshata Udyavar. ACTM-838 safely and comprehensively re-activates the immunosuppressive TME by exploiting myeloid biology intrinsic in many cancers [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 736.

Abstract 1366: Dual immune checkpoint inhibition enhances the anti-tumor activity of trastuzumab deruxtecan in preclinical models

Abstract Background Trastuzumab deruxtecan (T-DXd) is an antibody-drug conjugate (ADC) composed of an anti-human epidermal growth factor receptor 2 (HER2) antibody, a cleavable tetrapeptide-based linker, and a topoisomerase I inhibitor payload. Recent clinical trials such as BEGONIA have highlighted the potential therapeutic benefit in combining T-DXd with immuno-oncology (IO) agents such as the PD-L1 inhibitor durvalumab. Here, we report preclinical findings demonstrating enhanced anti-tumor activity when T-DXd is combined with dual immune checkpoint inhibition. Methods HER2-expressing human cancer cell lines were treated in vitro with T-DXd and assessed for induction of immunogenic cell death (ICD) markers and expression of NK cell-activating ligands. Human macrophages and T cells were incubated with supernatants from treated cancer cells, and their activation status evaluated via flow cytometry. In vivo, the effects of T-DXd in combination with inhibitors of PD-L1, CTLA-4, and a monovalent bispecific anti-PD-1/TIGIT antibody - a murine surrogate of rilvegostomig - on tumor growth were evaluated in BALB/c mice bearing human HER2-low EMT6 murine mammary tumors. The anti-tumor activity of T-DXd in combination with volrustomig, a monovalent bispecific antibody targeting PD-1 and CTLA-4, was also assessed in hu-CD34+ NSG mice bearing HER2-low Caki-1 renal cell carcinoma tumors. Results In vitro, T-DXd induced ICD via DXd-mediated extracellular release of inflammatory mediators ATP and HMGB1, and cell surface exposure of calreticulin. This was associated with a greater than 2-fold increase in expression of NK cell-activating ligands, and activation of both macrophages and T cells. In immunocompetent mice bearing human HER2 expressing EMT6 tumors, T-DXd treatment drove tumor growth inhibition (53%, P <0.001) that was associated with an increase in tumoral T cells (2.6-fold, P <0.05) and increased CD8+ T cell expression of the immune checkpoints PD-1, TIGIT, and TIM-3. Tumor growth inhibition relative to vehicle-treated mice was enhanced when T-DXd was combined with inhibitors of PD-L1 (108%, P <0.001), PD-L1 plus CTLA-4 (172%, P <0.001), or a bispecific PD-1/TIGIT inhibitor (181%, P <0.001). Pharmacodynamic analysis revealed how the combination of T-DXd and PD-1/TIGIT inhibition increased tumoral NK cells (3.2-fold, P <0.001) and CD8+ T cells (3.8-fold, P <0.001) relative to vehicle. Consistent with findings in syngeneic models, the combination of T-DXd with a bispecific PD-1/CTLA-4 inhibitor also enhanced tumor growth inhibition in a humanised Caki-1 model (121%) compared to T-DXd alone (72%) (P <0.001). Conclusions These data provide insight into the clinical activity observed with T-DXd and provide scientific rationale for combination strategies with novel, bispecific IO agents targeting CTLA-4 or TIGIT in addition to the PD-1/PD-L1 axis. Citation Format: Liam Jenkins, Laura Kazlauskas, Matt Wilson, Scott A. Hammond, Theresa Proia, Jerome Mettetal. Dual immune checkpoint inhibition enhances the anti-tumor activity of trastuzumab deruxtecan in preclinical models [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 1366.

Abstract IA011: Engineered IL-12 delivery to the tumor matrix to achieve enhanced efficacy and safety in multiple advanced tumors

Abstract Background. Our goal is to create an effective and novel immunotherapy against multiple solid tumors with minimal toxicity. IL-12 is an “ideal” immune-activator, capable of activating both innate and acquired immune response. We tested more than 20 reported pro-inflammatory cytokines, and found IL-12 shows the most potent anti-tumor activity. Peripheral toxicity limits the use of systemic IL-12 therapy in the clinic. We have hypothesized that the tumor-specific drug delivery system would improve IL-12 immunotherapies. We took original approaches of protein engineering. Methods. We developed technologies to turn the tumor collagen into a drug reservoir after intravenous injections. The recombinant fusion of IL-12 to a collagen binding domain (CBD) derived from the VWF A3 domain allows for targeted IL-12 delivery into the tumor stroma. Here, we evaluated the pharmacology, safety, efficacy and the effects of CBD-IL-12 to the tumor microenvironment. Results. Intravenously-injected CBD proteins preferentially localized to the tumor, but not in other organs in mice. Single intravenous injection of CBD-IL-12 (recombinant fusion protein) eradicated most EMT6 breast cold tumors. CBD-IL-12-treated mice that were tumor-free developed systemic immunological memory, as 12 mice out of 13 rejected rechallenge with the breast tumor cells in their contralateral mammary fat pad. Single intravenous injection of CBD-IL-12 induced 67% complete remission of established B16F10 tumors. CBD fusion to IL-12 reduced the systemic toxicity, such as 50% reduction of hepatotoxicity markers and cytokine storm. CBD-IL-12 treatment increased intratumoral cytokine and immune cell numbers. In short, this tumor collagen-targeting approach improves both safety and efficacy of IL-12. We showed that the CBD can target multiple solid tumor types, because the CBD exploits the abnormality of tumor vasculature-specific pathological structure (e.g. leakiness and exposure of collagen to the bloodstream). So far, we have shown that CBD-IL-12 has significant anti-tumor efficacy in breast, lung, ovarian, liver, colon, prostate, melanoma, head and neck, pancreatic cancers and melanoma, sarcoma, and glioblastoma in syngeneic mouse models. CBD-IL-12 is effective in cold, advanced, spontaneous tumors, low-mutation burden tumors, and prevents metastasis and recurrence. CBD-IL-12 stimulated antigen-presenting cells and T cells. Also, humanized mice treated with humanized CBD-IL-12 show great anti-tumor effects in patient-derived lung and ovarian tumors. Humanized CBD-IL-12 was tested in dogs, and showed great tolerability at the therapeutic dose. Human CBD-IL-12 molecule was successfully manufactured in a clinical-grade. Conclusions. Intravenously injected CBD–IL-12 is effective to multiple difficult, immunologically cold tumors with low mutation burdens. Collagen is an attractive target because abnormally high expression of collagen is a shared nature of tumors, regardless of tumor types and patients. VWF A3 domain naturally exists in our body, minimizing the risk of immunogenicity. Citation Format: Jun Ishihara. Engineered IL-12 delivery to the tumor matrix to achieve enhanced efficacy and safety in multiple advanced tumors [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2023 Oct 11-15; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2023;22(12 Suppl):Abstract nr IA011.

Pulsed Electric Field Ablation versus Radiofrequency Thermal Ablation in Murine Breast Cancer Models: Anticancer Immune Stimulation, Tumor Response, and Abscopal Effects

PurposeTo compare the immune response and survival after size-matched radiofrequency (RF) ablation and a proprietary form of pulsed electric field (PEF) ablation in murine tumors. Material and MethodsOrthotopically inoculated EMT6 or 4T1 murine tumors received sham, RF ablation, or PEF ablation. 4T1 tumor ablations included subgroups with intraperitoneal checkpoint inhibition immunotherapy (αPD-1). Blood was collected for cytokine profiling and flow cytometry. Tumor size was measured and survival was monitored. Tumor samples were processed for histology, immunohistochemistry, flow cytometry, and cytokine profiling. Lungs were collected from 4T1-bearing mice for hematoxylin and eosin histology to assess metastatic spread and abscopal effect induced by ablation. ResultsPEF elicited distinct immunomodulatory effects, with clear differences in serum and tumor cytokine profiles compared with RF ablation, including intratumoral downregulation of vascular endothelial growth factor, hypoxia-inducible factor 1α, c-MET, interleukin-10, Ki67, and tumor necrosis factor-α (all P < .05). PEF increased innate immune activation, with enhanced recruitment of dendritic cells, M1 macrophages, and natural killer cells coupled with a reduction in M2 macrophages and myeloid-derived suppressor cells (all P < .05). Concurrently, PEF strengthened adaptive immunity compared with RF ablation, characterized by increased antigen-specific T cells and decreased regulatory T cells (all P < .05). PEF stalled tumor growth and increased survival at the end of the study (≥4× versus RFA). Finally, PEF promoted an abscopal effect of clearing metastases in the lungs, which was stronger in combination with αPD-1 than with PEF alone. ConclusionsThe proprietary form of PEF used in this study evoked a preferential immunostimulatory profile versus RF ablation thermal ablation in mice, with implications for enhancing the therapeutic effectiveness of checkpoint inhibition immunotherapy for immunotherapy-unresponsive tumors.

A single targeted gamma-ray irradiation induced an acute modulation of immune cells and related cytokines in EMT6 mouse-bearing tumour model.

A complicated interplay between radiation doses, tumour microenvironment (TME), and host immune system is linked to the active participation of immune response. The effects of single targeted 2 Gy and 8 Gy gamma-ray irradiations on the immune cell population (lymphocytes, B-cells, T-cells, neutrophils, eosinophils, and macrophages) in EMT6 mouse-bearing tumour models was investigated. The effects of both irradiation doses in early (96 hours) and acute phase (5 to 11 days) post-irradiation on immune parameters were monitored in blood circulation and TME using flow cytometry. Simultaneously, selected cytokines related to immune cells within the TME were measured using multiplex ELISA. A temporary reduction in systemic total white blood count (TWBC) resulted from an early phase (96 hours) of gamma-ray irradiation at 2 Gy and 8 Gy compared to sham control group. No difference was obtained in the acute phase. Neutrophils dominated among other immune cells in TME in sham control group. Eosinophils in TME was significantly increased after 8 Gy treatment in acute phase compared to sham control (p< 0.005). Furthermore, the increment of tumour necrosis (TNF)-α, eotaxin and interleukin (IL)-7 (p< 0.05) in both treatment groups and phases were associated with anti-tumour activities within TME by gamma-ray irradiation. The temporary changes in immune cell populations within systemic circulation and TME induced by different doses of gamma-ray irradiation correlated with suppression of several pro-tumorigenic cytokines in mouse-bearing EMT6 tumour models.

The Impact of Tumor-Associated Macrophages Found at Mammary Carcinoma Sites is Dependent Upon the Tumor Type

ABSTRACT Background Previously, we reported that tumor-associated macrophages (TAM) at early sites of mammary carcinoma showed a decrease in ATP production rate and a higher dependence on oxidative phosphorylation. Methods Since these changes can result from activation of AMP-activated protein kinase (AMPK) and glucose transporter 1 (Glut1) during metabolic stress, we investigated whether the TAM showed increased expression of ampk and glut1, as well as another indicator of metabolic stress, pkm2. Indeed, the TAM exhibited significant expression of pkm2, glut1, and ampk. Results Bone marrow-derived macrophages (BMDM) co-cultured with 4T1, EMT6, and 168 in vitro similarly showed increased expression of pkm2, glut1, and ampk. Moreover, lactate, which is expressed at significant levels by all three tumors, induced expression of these same genes in BMDM suggesting that lactate may induce a metabolic stress response in these TAM. Yet, the three different mammary carcinoma models benefited from different targeting strategies. Macrophage depletion studies revealed that the TAM contributed to growth of the EMT6 tumor and metastasis of the 4T1 tumor. Targeting the stress response with the Integrated Stress Response Inhibitor (ISRIB), which targets eIF2, impacted 168 tumor progression, and ISRIB as well as FX-11, which targets lactate dehydrogenase, impacted 4T1 tumor progression and metastasis. Conclusions Collectively, these data demonstrate that targeting TAM or metabolism at early tumor sites can impact tumor progression. However, variability in the responses underscore the fact that the impact of macrophages differs even within three different syngeneic mammary carcinoma models.

Combined PD-L1/TGFβ blockade allows expansion and differentiation of stem cell-like CD8 T cells in immune excluded tumors

TGFβ signaling is associated with non-response to immune checkpoint blockade in patients with advanced cancers, particularly in the immune-excluded phenotype. While previous work demonstrates that converting tumors from excluded to inflamed phenotypes requires attenuation of PD-L1 and TGFβ signaling, the underlying cellular mechanisms remain unclear. Here, we show that TGFβ and PD-L1 restrain intratumoral stem cell-like CD8 T cell (TSCL) expansion and replacement of progenitor-exhausted and dysfunctional CD8 T cells with non-exhausted T effector cells in the EMT6 tumor model in female mice. Upon combined TGFβ/PD-L1 blockade IFNγhi CD8 T effector cells show enhanced motility and accumulate in the tumor. Ensuing IFNγ signaling transforms myeloid, stromal, and tumor niches to yield an immune-supportive ecosystem. Blocking IFNγ abolishes the anti-PD-L1/anti-TGFβ therapy efficacy. Our data suggest that TGFβ works with PD-L1 to prevent TSCL expansion and replacement of exhausted CD8 T cells, thereby maintaining the T cell compartment in a dysfunctional state.

Pulsed electrical fields in combination with anti-PD1 and survival of mice with TNBC (EMT6) murine breast tumor.

540 Background: Delivery of pulsed electric field (PEF) energy is used to destabilize cells through multiple biochemical processes. We questioned whether a specific PEF treatment type could synergize with anti-PD1 in an orthotopic Triple Negative Breast Cancer (TNBC) EMT6 mouse model. Our previous studies have demonstrated that PEF treatment induces neo-antigen presentation and triggers an innate and adaptive immune response. Anti-PD1 therapy is ineffective at blocking growth of (TNBC) EMT6 tumors that are orthotopically implanted in mice. We evaluated whether a biphasic monopolar form of PEF could synergize with anti-PD1 when it was administered intraperitoneally or intra-tumorally, and whether there was a change in immune response post treatment. Methods: Forty-eight female Balb/c mice were implanted with 200,000 EMT6 cells into the mammary fat pads. Once tumor diameter reached 5 mm they were randomized into 6 groups of 8 animals: 1) Sham/IgG; 2) intra-tumoral (IT) anti-PD-1; 3) intra-peritoneal (IP) anti-PD-1; 4) PEF + anti-PD-1(IP); 5) PEF + anti-PD-1(IT) 6) 2X PEF on day 0 and day 7 + anti-PD-1(IT administered weekly). Sham-PEF mimicked the same procedure but without PEF. Anti-PD1 was administered weekly, IT or IP at 200 µg/mouse and PEF was delivered once on day of randomization. Tumors were measured three times per week, and animal survival was monitored until tumor volume necessitated euthanasia (≥2000mm3). Blood was collected on day 14 for flow cytometric analysis of systemic immune response which included quantifying CD3 positive T cells, B cells and NK cells. Results: Anti-PD1 treatment (IT or IP) did not reduce tumor growth, nor did it prolong survival compared to Sham/IgG treated animals. All anti-PD1 alone (IT or IP) as well as Sham treated animals had to be euthanized by day 35. In contrast, PEF combined with anti-PD1 given IT and IP significantly reduced tumor growth by 83% and 70%, respectively, on Day 14 compared to anti-PD-1 only treated animals (p = 0.03). Interestingly, EMT6 bearing mice treated with double PEF + IT administered anti-PD1 survived longer compared to mice treated with single PEF + IT administered anti-PD1. On day 65, 71% of double PEF + IT administered anti-PD1 were still alive compared to 12.5% of mice treated with PEF + IT administered anti-PD1 (p = 0.002). Flow cytometry analysis on day 14 indicated an increase in circulating adaptive (T- and B-cells) and innate (NK cells) immunocytes in the PEF + anti-PD1 treated animals compared to the control. Conclusions: Collectively, these results indicate that some types of PEF ablation synergize with weekly anti-PD1 in stimulating an increase of CD3+ T cells, B cells and NK cells, reducing tumor growth and prolonging survival of tumor bearing mice. These results suggest the synergistic potential of combining Immunotherapy with some forms of PEF, which warrants further study in patients.

Mass cytometry analysis of mouse nonclassical monocyte heterogeneity in breast tumors

Abstract Monocytes have emerged as important regulators of cancer progression. Our previous studies demonstrated the importance of nonclassical Ly6C lomonocytes in regulating cancer metastasis in mouse models. Here, we aimed to study monocyte heterogeneity in two different breast cancer tumors in mice using either 4T1 or EMT6 cells. 4T1 tumors exhibit an increased ability to metastasize in the lung in a BALB/c mouse breast cancer model compared to the less aggressive EMT6 tumor model. We evaluated 35 surface markers of tumor-infiltrated monocytes and dendritic cells in tumors isolated from each model using CyTOF mass cytometry. Analysis of monocytes and dendritic cells in the tumors using the Seurat package and the FlowSom algorithm identified 12 cell subsets. Significant differences were discovered between the two tumor types in frequencies of four of 12 identified subsets. However, only one of these subsets, a nonclassical monocyte subset, was higher in the less metastatic EMT6 tumors. This subset was 5-fold higher in EMT6 tumors versus more aggressive 4T1 tumors. This Ly6C lononclassical monocyte subset showed high expression of CD169 and CX3CR1, CD86 and CD80. These differentially expressed markers suggest a functional importance in this subset in migration to the tumor and antigen presentation, both of which are anti-tumoral functions. Ongoing studies are aimed towards understanding the role of this non-classical monocyte population in regulating breast cancer metastasis. This work was supported by NIH R01 CA202987, and P01 HL136275

Abstract 6789: The oncolytic virus VET3-TGI both blocks TGF-beta signaling and activates type 2 IFN responses, resulting in potent therapeutic responses in multiple mouse models

Abstract Background: TGFB1 mediated immune resistance is one of the major mechanisms of immune suppression utilized across multiple tumor types. Immune resistance imparted by TGFB1 is mediated through its pleiotropic effects on vasculature, fibrogenesis and regulatory/effector immune cells within the tumor microenvironment. Blockade of TGFB1 (TGFBi) will likely improve response to immunotherapy. IL-12 is a cytokine that through IFNg induction, promotes type 1 inflammatory response, M1 macrophages and effector CD8 T cell response. Combining TGFB1 blockade with IL-12 may maximize therapeutic benefits through simultaneously reducing immunosuppression and enhancing anti-tumor immune response. Current studies have developed a vaccinia-based immunotherapy, combining enhanced systemic virus delivery to CXCR3 ligand rich tumors and locally expressed IL-12 and TGFBi within the tumor microenvironment, for efficient control of multiple tumor models. Methods: An oncolytic vaccinia virus expressing CXCR3, IL-12 and a TGFB1 antagonizing mini-monomer was constructed (VET3-TGI) and the expression and function of the transgenes were confirmed. Using in vivo mouse RENCA, EMT-6 and MC38 tumor models, the functionality and therapeutic efficacy of VET-TGI were tested with comparison to control virus. Post-mortem analysis was used to analyze the impact of VET3-TGI on immune/stromal/endothelial milieu of the tumors and to determine toxicity profile. Results: VET3-TGI infected cells expressed CXCR3 and showed enhanced migration to CXCR3 ligands in vitro and improved systemic delivery to tumors expressing CXCR3 ligands in vivo, even in the face of pre-existing anti-viral immunity. IL-12 expression and TGFBi blockade of TGFB1 mediated suppression of CD8 T cell proliferation were confirmed in vitro. In vivo mouse studies using EMT6, RENCA and MC38 tumor models demonstrated potent therapeutic activity, including 100% CRs, even at doses several logs below equivalent clinical doses and in multiple models. Mechanism of activity studies suggested that the therapeutic efficacy of VET3-TGI is associated with considerable modification of the tumor microenvironment. In addition, preliminary toxicity studies demonstrated the safety of VET3-TGI in mouse models. Conclusions: VET3-TGI demonstrated an ability to reduce immunosuppression and dramatically enhance antitumor immune response leading to safe and potent therapeutic activity in multiple mouse tumor models. This data led to the selection of VET3-TGI as our lead clinical candidate. A human version of the virus is currently undergoing clinical manufacture and toxicology testing. Citation Format: Ravikumar Muthuswamy, Steve Thorne. The oncolytic virus VET3-TGI both blocks TGF-beta signaling and activates type 2 IFN responses, resulting in potent therapeutic responses in multiple mouse 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 6789.

Abstract 2799: Pharmacodynamic biomarkers for TGFBR1 inhibition in oncology

Abstract Transforming growth factor-beta (TGF-ß) is a master regulatory cytokine with pleotropic effects important in tumor development. TGF-ß1 binds TGF-ß receptor 2, which recruits and phosphorylates TGF-ß receptor 1 (TGFBR1). TGFBR1 in turn phosphorylates SMAD2 and SMAD3. Phosphorylated SMAD2/3 (pSMAD2/3) complexes with SMAD4 to translocate to the nucleus and enact transcriptional programs. In late-stage tumors, TGF-ß signaling is oncogenic, promoting growth, invasion, immune suppression, tissue remodeling, and epithelial to mesenchymal transition (EMT). In an effort to counter the oncogenic effects of TGF-ß signaling, Sumitomo Pharma Oncology, Inc. is developing TP-6379, an investigational, orally available, small molecule, selective TGFBR1 inhibitor. TP-6379 has a biochemical IC50 of 1.07 nM and has been shown to reduce pSMAD2 in Rhabdomyosarcoma cells with an IC50 of 108 nM. A dose dependent increase of TP-6379 was observed in plasma and tumors of A549 tumor bearing athymic nude mice (10, 50, 75, and 100 mg/kg PO). In another study using the same mouse model, pSMAD2 has shown an inverse correlation with pharmacokinetics showing &amp;gt; 90% inhibition for up to 4 hours (75 mg/kg PO). Pharmacodynamic (PD) biomarkers are critical for determining biological activity, but procuring serial tumor biopsies for PD analysis is challenging for many tumor types. We hypothesize that pSMAD2 can be measured in non-tumor tissues as a surrogate for activity within the tumor. Surrogate tissues, such as peripheral blood mononuclear cells (PBMCs), skin punches, and even circulating tumor cells (CTCs) are much less invasive and can be sampled over multiple timepoints. We observed a measurable decrease of PD biomarkers pSMAD2/3 in PBMCs and skin after treatment with TP-6379 using a bead-based immunoassay (Luminex). Healthy human donor PBMCs treated ex vivo for 2 hours with up to 1 µM TP-6379 showed a 96% reduction in pSMAD2/3 signal. Although TP-6379 was still active in reducing pSMAD2 in PBMCs when treated in whole blood for 2 hours, it required around 30-fold more drug to achieve a similar effect, possibly due to serum protein binding. Skin samples from in vivo dosed tumor bearing mice also show significant inhibition of pSMAD2/3, which correlated with significant pSMAD2/3 inhibition in tumors from both EMT6 and 4T1 syngeneic triple-negative breast cancer mouse tumor models. We confirmed that the Luminex assay is sensitive enough to reliably measure pSMAD2/3 in healthy human doner skin punches down to 3 mm in diameter. Lastly, CTCs from breast cancer patients’ whole blood treated ex vivo for 24 hours with as low as 1 µM TP-6379 showed a decrease in pSMAD2 and EMT marker SNAI1 by as much as 96% and 86%, respectively, when measured by immunofluorescence. In summary, our research has shown that pSMAD2/3 and EMT marker SNAI1 are valid PD biomarkers for TP-6379 therapy. We propose that these biomarkers would be best measured clinically using PBMCs, CTCs and/or skin punches. Citation Format: Richard E. Heinz, Curtis A. Allred, David A. Kircher, Yuta Matsumura, Bettina Franz, Zhizhou Ye, Jinny Lee, Clifford J. Whatcott, Jason M. Foulks, Adam Siddiqui, Steven L. Warner. Pharmacodynamic biomarkers for TGFBR1 inhibition in oncology [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 2799.

Fe/MOF based platform for NIR laser induced efficient PDT/PTT of cancer.

Introduction: Photodynamic therapy (PDT) and photothermal therapy (PTT) are widely used in the treatment of tumors. However, their application in the treatment of clinical tumors is limited by the complexity and irreversible hypoxia environment generated by tumor tissues. To overcome this limitation, a nanoparticle composed of indocyanine green (ICG) and Fe-MOF-5 was developed. Methods: We prepared F-I@FM5 and measured its morphology, particle size, and stability. Its enzyme like ability and optical effect was verified. Then we used MTT, staining and flow cytometry to evaluated the anti-tumor effect on EMT-6 cells in vitro. Finally, the anti-tumor effect in vivo has been studied on EMT-6 tumor bearing mice. Results: For the composite nanoparticle, we confirmed that Fe-MOF-5 has the best nanozyme activity. In addition, it has excellent photothermal conversion efficiency and generates reactive oxygen species (ROS) under near-infrared light irradiation (808nm). The composite nanoparticle showed good tumor inhibition effect in vitro and in vivo, which was superior to the free ICG or Fe-MOF-5 alone. Besides, there was no obvious cytotoxicity in major organs within the effective therapeutic concentration. Discussion: Fe-MOF-5 has the function of simulating catalase, which can promote the decomposition of excessive H2O2 in the tumor microenvironment and produce oxygen to improve the hypoxic environment. The improvement of tumor hypoxia can enhance the efficacy of PDT and PTT. This research not only provides an efficient and stable anti-tumor nano platform, but also has broad application prospects in the field of tumor therapy, and provides a new idea for the application of MOF as an important carrier material in the field of photodynamic therapy.

Abstract PD2-06: Implications of Heterogeneity in Breast Tumor Cell MHC-I Expression on Immunity and Therapeutic Resistance

Abstract Background Immune checkpoint inhibitors (ICIs) targeting the PD-1/L1 axis are approved in early-stage treatment for triple-negative breast cancer (TNBC), but only a fraction of patients benefit. Tumor expressed antigens bound to major histocompatibility complex-I (MHC-I) are required for CD8-mediated tumoricidal activity, and thus, response to anti-PD-1/L1 targeted ICI. However, many breast tumors downregulate, or heterogeneously express, MHC-I, making them less susceptible to ICIs. Tumor cells downregulating MHC-I may be effectively targeted by natural killer (NK) cells due to ‘missing self’ signals. However, this heterogeneity in MHC-I expression is poorly modeled in most preclinical studies, limiting our understanding of how to overcome ICI resistance in the context of heterogeneous MHC-I expression, as is often observed clinically. Objective We aimed to 1) quantitatively delineate how intratumoral heterogeneity in MHC class I expression affects immune responses and immunotherapy outcomes in mouse models and 2) determine whether targeting inhibitory signals on NK cells can overcome ICI resistance in MHC-I heterogenous TNBC. Methods We performed quantitative immunofluorescence for MHC-I, CD8, CD56, and pan-cytokeratin on breast cancer tumors from diverse subtypes (n=314) to obtain single-cell-resolution MHC-I expression and spatial information of tumor and immune cells. Fluorescence intensity and spatial analysis were processed to output individual tumor/stromal cell MHC-I expression and the composition of the local tumor microenvironment. To determine the functional effect of MHC-I heterogeneity in vivo, we generated a CRISPR-guided B2m knockout (B2m-null) in a murine orthotopic model (EMT6). We then combined MHC-I-proficient and MHC-I-deficient isogenic lines at various ratios to model how populations of MHC-I loss affected the immune microenvironment. We also assessed a second, intrinsically heterogenous (MHC-I expression) TNBC model E0771. To evaluate changes in the microenvironment, we used flow cytometry and an immune NanoString panel to evaluate gene expression patterns in tumor cells and infiltrating immune cells. Results TNBC patients had the highest MHC-I expression level across tumor cells, but also the highest variability and probability of demonstrating bimodal MHC-I expression (consisting of high and low/absent cells within a single tumor). ER+ tumors were unimodally low. Using spatial analysis, we identified that heterogenous MHC-I tumors had significantly higher levels of infiltrating NK cells(Paired T-Test: p=0.03). In murine models, even 10% or less of MHC-I-null (B2m-null) EMT6 cells in the tumor injection resulted in acquisition of ICI resistance. Interestingly, heterogeneity in expression of MHC-I resulted in a substantial infiltration by NKG2A+ NK cells compared to MHC-I high or -low tumors (Student T-Test: p=.002). Activation of these infiltrating NK cells via anti-NKG2A and anti-PD-L1 combination treatment restored complete responses in heterogeneous EMT6 tumors, and significantly extended survival in both E0771 (Mantel-Cox: p&amp;lt; 0.0001) and EMT6 models (Mantel-Cox: &amp;lt; 0.0001). Additionally, anti-NKG2A and anti-PDL1 combination treatment improved complete response in the heterogenous MHC-I EMT6 model to 30% and in the parental EMT-6 tumors to 70%. Conclusion Combined therapy with anti-NKG2A (targeting NK cells) and anti-PD-L1 (targeting CD8+ T cells) can restore immunotherapy responses and overcome resistance due to lack of MHC-I expression in tumor cell subpopulations. Citation Format: Brandie C. Taylor, Xiaopeng Sun, Justin Balko, Paula Gonzalez-Ericsson, Melinda Sanders. Implications of Heterogeneity in Breast Tumor Cell MHC-I Expression on Immunity and Therapeutic Resistance [abstract]. In: Proceedings of the 2022 San Antonio Breast Cancer Symposium; 2022 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2023;83(5 Suppl):Abstract nr PD2-06.

Abstract No. 7 ▪ FEATURED ABSTRACT Pulsed Electric Field (PEF) Ablation Invokes Stronger Immune Cytokine Profile and Tumor Response than Radiofrequency Thermal Ablation for Matched Ablation Volumes

Focal tumor ablation may cause secondary immunogenic upregulation that enhances local and distant tumor responses, with profiles that are unique to each modality., Pulsed electric fields (PEFs) use short duration-high voltage electrical pulses to destabilize and kill cells through various biochemical processes. Because it does not rely on thermal effects PEF does not damage interstitial proteins, fostering improved antigen integrity and immune signaling which may lead to lesion resolution. This study compared whether cell death and subsequent interstitial effects caused by PEF and radiofrequency ablation (RFA) result in differentiated primary tumor responses. Thirty female Balb/c mice, divided into sham, PEF, and RFA treatment groups (n = 10/group) were transplanted with EMT6 cells in the left mammary fat pad. Eight days after challenge (d = 0), mice in the PEF and RFA groups received treatment with doses selected to achieve similar subtotal ablation volumes. Three mice from each group were euthanized at day 4 for H&E analysis of ablation zone. The remainder were survived to 10-days post-ablation to monitor tumor growth. Serum was collected on Days -1, +1, +4 and +10 for cytokine analysis. Four-day histology demonstrated comparable subtotal ablation areas between PEF and RFA treatment. While RFA-treated tumors presented immune cells accumulating at the tumor-treatment zone boundary, PEF-treated tumors showed immune cell infiltration throughout the tumor. Further, PEF treatment slowed tumor growth compared with RFA or sham treatments, with day 10 average tumor volumes of 335, 791, and 857 mm3 for PEF, RFA, and sham group, respectively. Ingenuity Pathway Analysis (IPA) of 32 serum cytokines revealed most notable differences between groups on day 4 post-ablation. The PEF-treated group had a significant decrease in the levels of CSF1, CSF2, CSF3, CCL2, IL1β, IL6, IL13, and TNF, and an increase in the levels of IL4. These changes suggest that PEF is associated to 1) decreased accumulation of myeloid derived suppressor cells (MDSCs), 2) decreased the proliferation of tumor-associated macrophages (TAMs), 3) decreased proliferation, survival and invasion of tumor cells. Conversely, these changes were all the opposite for the RFA group. PEF treatment may cause a stronger EMT6 tumor response in mice compared with RFA treatment. These data suggest that PEF may have a greater local and distant tumor response benefit versus RFA thermal ablation. Further studies should explore potential abscopal effects.