Mice Bearing EMT6 Tumors Research Articles

Abstract LB263: A first-in-class multimodal immunotherapy for induction of tertiary lymphoid structures as a novel therapeutic strategy for solid tumors

Abstract Immune checkpoint inhibitors (ICI) have transformed cancer treatment. However, long term disease control is often not achieved and resistance frequently occurs. Formation of organized intratumoral aggregates of lymphocytes (tertiary lymphoid structures, TLS) has been associated with response to ICI suggesting therapeutics able to induce intratumoral TLS could enhance the response to ICI treatment. TLS formation requires antigen presentation, stromal activation and immune stimulation - diverse events difficult to obtain with a single therapeutic. The enLIGHTEN™ Discovery Platform aims to create multimodal therapeutics by leveraging human biology and advanced analytics to select multigene payloads delivered by herpes simplex virus (HSV) vectors to the tumor microenvironment. Optimal payload combinations are predicted based on in silico experiments and then tested in proprietary multiplex assays used to evaluate the contribution of payload components and model the therapeutic biological effect of predicted payload combinations. Alpha-201, a replication-defective oncolytic virus, was selected from a suite of modified HSV vectors as the biological chassis due to its ability to release tumor antigens and activate pathways associated with response to ICI (e.g., MYC targets, E2F targets, G2M checkpoint; normalized enrichment scores of 2.4, 3.0, and 3.2, respectively, p<0.001). A library of single gene encoding Alpha-201 vectors was constructed, each expressing one in silico predicted payload component (e.g., IL-7, CXCL13, LTB or CCL19). The top two scoring combinations of these viral gene constructs (termed Multiplex A and Multiplex B) were next tested for their effects on TLS induction and anti-tumor efficacy in complementary in vivo models. First, the ability of HSV gene construct combinations to induce TLS was tested in murine salivary glands, a tissue highly permissive to TLS formation. As predicted, delivery of both multiplexes increased the number, size, organization and maturity of virally induced TLS. Of note, TLS were characterized by the presence of mature fibroblastic reticular cells and CCL21 expression, effects not observed with biological chassis administration alone. Second, anti-tumor efficacy was tested in EMT6 tumor-bearing mice. Both multiplexes induced tumor growth inhibition compared to vehicle control mice (Multiplex A: 39.8±10.5%, p=0.004, Multiplex B: 38.6%±12.1, p=0.011, n=8 per group). In combination with anti-PD-1 antibody therapy, multiplex treatment also increased the number of long-term survivors (>40 days) as compared to control (2/11 vs. 0/8). Characterization of intratumoral TLS induction is ongoing. In conclusion, we have demonstrated the ability of enLIGHTEN™ to design multimodal specific therapeutics, resulting in the development of a first-in-class immunotherapeutic for TLS induction in solid tumors. Citation Format: Anne R. Diers, Qiuchen Guo, John D. Christie, David Krisky, Fabrina M. Gaspal, Erin Richardson, Charlotte G. Smith, David R. Withers, Adam P. Croft, Paul P. Tak, Francesca Barone. A first-in-class multimodal immunotherapy for induction of tertiary lymphoid structures as a novel therapeutic strategy for solid tumors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 2 (Late-Breaking, Clinical Trial, and Invited Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(7_Suppl):Abstract nr LB263.

Carrier-free, pure nanodrugs based on dasatinib and 5-Fluorouracil show aggregation-induced emission and enhanced antitumor efficacy

Therapies based on small molecule targeted inhibitors usually suffer from several limitations such as poor water solubility, low oral bioavailability, rapid metabolism in vivo, and dose-limiting toxicity. The aim of this paper is to prepare a carrier-free nanodrug self-delivery system by self-assembly of dasatinib (DAS) and 5-Fluorouracil (Fu). By adjusting the ratio of DAS to Fu (10:1, 5:1, 2:1, 1:1), a series of nanoparticles (DAS/F NPs) with different particle sizes can be obtained without additional chemical modifications or carrier materials. More importantly, during self-assembly, DAS/F NPs produce aggregation-induced emission (AIE) effect. This makes DAS/F NPs suitable for bioimaging and tracking cell trafficking without any fluorescent probe labeling. DAS/F NPs (2:1) with a diameter around 150 nm was selected for the next antitumor study. The cellular uptake and cytotoxicity of DAS/F NPs were measured by EMT6 and MCF-7 cells as well as the corresponding multicellular spheroids. Finally, in vivo antitumor against EMT6 tumor-bearing mice, immunohistochemical studies and hemolysis assays demonstrated DAS/F NPs possess significant synergistic antitumor effects and excellent biocompatibility.

Abstract 4600: Phenotypic & functional diversity of tumor associated neutrophils in murine breast tumor models

Abstract Despite the advances in early diagnostics and therapeutics, women with metastatic breast cancer have limited treatment options. Women with TNBC, who constitute 15-20% of breast cancer patients, are often diagnosed with aggressive/metastatic disease. Advanced studies implicated immunosuppressive tumor microenvironment (TME) in aggressive/metastatic properties of TNBC subtype. Alternatively activated immature myeloid cells including tumor-associated macrophages (TAM), tumor-associated neutrophils (TAN), tumor-associated dendritic cells (TADC) and myeloid derived suppressor cells (MDSC) constitute a major component of TME. However, anti-tumorigenic microenvironment is also reported and that may have clinical relevance in early TNBC patients. Therefore, our hypothesis is that myeloid cells polarize to become immunosuppressive and infiltrate tumors and pre-metastatic niches in patients with advanced disease, while patients with early TNBCs may elicit anti-tumor immune response eliminating disseminated tumor cells (DTC). The utilization of syngeneic immunocompetent mouse models has contributed to our current understanding of immunosuppressive or immunomodulatory TME. Using these models, we have demonstrated that tumor dissemination and growth at metastatic sites is facilitated by MDSC’s. Emerging technologies; single cell RNA sequencing (scRNA-Seq), mass cytometry (CyTOF) or cellular indexing of transcriptomes and epitopes sequencing (CITE-Seq) has been powerful platforms for detailed characterization of tumors and TME compartments. We performed scRNA-Seq and CyTOF analyses of the myeloid cell populations of tumors and spleens from metastatic 4T1 and non-invasive EMT6 tumor-bearing mice. Tumors and spleens from 4T1 tumor-bearing mice exhibited a marked expansion of myeloid cell subsets that are characterized by the expression of immunosuppressive as well as progenitor markers. On the contrary, indicated tissues from EMT6 mice were enriched in NK cells, T and B lymphocytes and they were lacking immunosuppressive myeloid cell subsets. Furthermore, we identified a distinct differentiation pattern of immature myeloid cell subsets from neutrophil progenitors (NP) in 4T1 tumor-bearing mice. Using the murine TNBC models in syngeneic mice, we provide evidence that early TNBC tumors may elicit anti-tumor immune responses and thus the survival outcome in those patients is substantially increased after complete surgical resection of the primary tumors. Whereas immunosuppressive tumor microenvironment contributes to the poor overall survival in patients with advanced TNBCs. Therefore, identifying an anti-tumor immune signature in early TNBC patients may be utilized as a clinical biomarker before surgical intervention as well as improve the survival outcome. Citation Format: Hasan Korkaya, Elayne Benson, Fulya Koksalar Alkan, Justin Wilson, Tulshi Patel, Hilmi K. Alkan, Virginia McEvoy, Nika Shekastehband, Nate Francois, Huidong Shi, Catherine C. Hedrick. Phenotypic & functional diversity of tumor associated neutrophils in murine breast tumor 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 4600.

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 P2-21-09: Detailed analyses of pro-metastatic and anti-metastatic microenvironments of murine breast tumors in syngeneic mice

Abstract Despite the advances in early diagnostics and therapeutics, women with metastatic breast cancer have limited treatment options. Women with TNBC, who constitute 15-20% of breast cancer patients, are often diagnosed with aggressive/metastatic disease. Advanced studies implicated immunosuppressive tumor microenvironment (TME) in aggressive/metastatic properties of TNBC subtype. Alternatively activated immature myeloid cells including tumor-associated macrophages (TAM), tumor-associated neutrophils (TAN), tumor-associated dendritic cells (TADC) and myeloid derived suppressor cells (MDSC) constitute a major component of TME. However, anti-tumorigenic microenvironment is also reported and that may have clinical relevance in early TNBC patients. Therefore, our hypothesis is that myeloid cells polarize to become immunosuppressive and infiltrate tumors and pre-metastatic niches in patients with advanced disease, while patients with early TNBCs may elicit anti-tumor immune response eliminating disseminated tumor cells (DTC). The utilization of syngeneic immunocompetent mouse models has contributed to our current understanding of immunosuppressive or immunomodulatory TME. Using these models, we have demonstrated that tumor dissemination and growth at metastatic sites is facilitated by MDSC’s. Emerging technologies; single cell RNA sequencing (scRNA-Seq), mass cytometry (CyTOF) or cellular indexing of transcriptomes and epitopes sequencing (CITE-Seq) has been powerful platforms for detailed characterization of tumors and TME compartments. We performed scRNA-Seq and CyTOF analyses of the myeloid cell populations of tumors and spleens from metastatic 4T1 and non-invasive EMT6 tumor-bearing mice. Tumors and spleens from 4T1 tumor-bearing mice exhibited a marked expansion of myeloid cell subsets that are characterized by the expression of immunosuppressive as well as progenitor markers. On the contrary, indicated tissues from EMT6 mice were enriched in NK cells, T and B lymphocytes and they were lacking immunosuppressive myeloid cell subsets. Furthermore, we identified a distinct differentiation pattern of immature myeloid cell subsets from neutrophil progenitors (NP) in 4T1 tumor-bearing mice. Using the murine TNBC models in syngeneic mice, we provide evidence that early TNBC tumors may elicit anti-tumor immune responses and thus the survival outcome in those patients is substantially increased after complete surgical resection of the primary tumors. Whereas immunosuppressive tumor microenvironment contributes to the poor overall survival in patients with advanced TNBCs. Therefore, identifying an anti-tumor immune signature in early TNBC patients may be utilized as a clinical biomarker before surgical intervention as well as improve the survival outcome. Citation Format: Fulya Alkan, Justin D. Wilson, Nika Shekastehband, Catherine HEDRICK, Alicia Arnold, Roni Bollag, Huidong Shi, Hasan Korkaya. Detailed analyses of pro-metastatic and anti-metastatic microenvironments of murine breast tumors in syngeneic mice [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 P2-21-09.

Abstract 2055: WTX-330 is a conditionally activated IL-12 prodrug that fundamentally reprograms tumor infiltrating CD8+ T cells and drives tumor regression

Abstract Immune checkpoint inhibition (ICI) has established immunotherapy as the first line of treatment in a subset of cancers. However, there are still many patients and cancer types where ICI is not effective, representing a significant unmet clinical need for novel immunotherapies to expand the clinical options for patients. Interleukin 12 (IL-12) is a pleiotropic cytokine that drives naïve T cells towards a TH1 phenotype, activates NK cells and cytotoxic T cells, and strongly induces the expression of IFNγ by multiple cell types, making it an attractive pro-inflammatory cytokine for cancer immunotherapy. However, despite its demonstrated clinical benefits, poor pharmacokinetic properties and dose-limiting toxicities after systemic administration have greatly limited the use of IL-12 in the clinic. WTX-330 is a novel IL-12 pro-drug that is designed to harness the dysregulated protease activity of tumors to selectively deliver fully active IL-12 to the tumor microenvironment (TME). WTX-330 consists of wild-type IL-12 tethered to a high affinity antibody blockade domain and a half-life extension (HLE) domain via tumor protease-sensitive linkers. Following systemic administration, WTX-330 circulates as an inactive prodrug that is unable to bind to IL-12 receptors in normal tissues. When WTX-330 enters the tumor however, proteases found in the TME cleave the protease-sensitive linkers, releasing fully active IL-12 selectively in the tumor. WTX-330 is highly efficacious in murine syngeneic tumor models, leading to complete, CD8+ T cell dependent tumor regressions when MC38 tumor bearing mice were treated with a murine surrogate of WTX-330. Importantly, while murine WTX-330 was well tolerated by the mice, equimolar amounts of wild-type IL-12 was not, highlighting the tumor selective activity of the INDUKINE™ protein. Murine WTX-330 treatment of either MC38 or EMT6 tumor bearing mice increased the frequency of tumor infiltrating polyfunctional CD8+ T cells and activated tumor infiltrating NK cells in both models. In the EMT6 model, Geospatial NanoString analysis revealed significantly more tumor penetration by CD8+ T cells following murine WTX-330 treatment at multiple timepoints and demonstrated that systemic dosing with the IL-12 INDUKINE™ molecule resulted in IL-12 signaling by the tumor infiltrating CD8+ T cells. Furthermore, murine WTX-330 treatment fundamentally reprogrammed the metabolic program of tumor infiltrating CD8+ T cells, increasing both glycolysis and mitochondrial biogenesis, while also increasing TCR signaling and degranulation. Finally, ex vivo incubation of WTX-330 with various primary human tumors led to the release of active IL-12, while WTX-330 was stable in human serum and healthy human primary cells from various tissues. Together, these data support continued development of this innovative IL-12 therapy into clinical testing. Citation Format: Christopher J. Nirschl, Pam Alderhold, Heather R. Brodkin, Connor J. Dwyer, Daniel J. Hicklin, Nesreen Ismail, Andres Salmeron, Cynthia Seidel-Dugan, Philipp Steiner, Zoe Steuert, Jenna Sullivan, William M. Winston. WTX-330 is a conditionally activated IL-12 prodrug that fundamentally reprograms tumor infiltrating CD8+ T cells and drives tumor regression [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2055.

Abstract 2223: Understanding the pro- and anti-tumorigenic microenvironments in syngeneic mice

Abstract Despite the advances in early diagnostics and therapeutics, women with metastatic breast cancer have limited treatment options. Women with TNBC, who constitute 15-20% of breast cancer patients, are often diagnosed with aggressive/metastatic disease. Advanced studies implicated immunosuppressive tumor microenvironment (TME) in aggressive/metastatic properties of TNBC subtype. Alternatively activated immature myeloid cells including tumor-associated macrophages (TAM), tumor-associated neutrophils (TAN), tumor-associated dendritic cells (TADC) and myeloid derived suppressor cells (MDSC) constitute a major component of TME. However, anti-tumorigenic microenvironment is also reported and that may have clinical relevance in early TNBC patients. Therefore, our hypothesis is that myeloid cells polarize to become immunosuppressive and infiltrate tumors and pre-metastatic niches in patients with advanced disease, while patients with early TNBCs may elicit anti-tumor immune response eliminating disseminated tumor cells (DTC). The utilization of syngeneic immunocompetent mouse models has contributed to our current understanding of immunosuppressive or immunomodulatory TME. Using these models, we have demonstrated that tumor dissemination and growth at metastatic sites is facilitated by MDSC’s. Emerging technologies; single cell RNA sequencing (scRNAseq), mass cytometry (CyTOF) or cellular indexing of transcriptomes and epitopes sequencing (CITE-Seq) has been powerful platforms for detailed characterization of tumors and TME compartments. Our bulk gene expression data of the myeloid cell populations of tumor microenvironment, lung, spleen and BM from 4T1 tumor-bearing mice showed distinct MDSC gene signatures. When applied to publicly available scRNAseq data, lung gMDSCs from 4T1 metastatic tumor model appeared to show different trajectory of polarization than the tumor gMDSCs. Consistent with previous findings by Hedrick Lab, lung gMDSCs from 4T1 mice also express higher levels of NeP markers compared to BM and tumor gMDSCs as well as lung gMDSCs from EMT6 mice. However, analyses of immune cells from EMT6 tumor bearing mice exhibited an anti-tumor immune signature which is consistent with the clearance of the DTCs following complete resection of the primary tumors. Using the murine TNBC models in syngeneic mice, we provide evidence that early TNBC tumors may elicit anti-tumor immune responses and thus the survival outcome in those patients is substantially increased after complete surgical resection of the primary tumors. Whereas immunosuppressive tumor microenvironment contributes to the poor overall survival in patients with advanced TNBCs. Therefore, identifying an anti-tumor immune signature in early TNBC patients may be utilized as a clinical biomarker before surgical intervention as well as improve the survival outcome. Citation Format: Fulya Alkan, Raziye Piranlioglu, Eunmi Lee, Maria Ouzounova, Catherine C Hedrick, Huidong Shi, Hasan Korkaya. Understanding the pro- and anti-tumorigenic microenvironments in syngeneic mice [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2223.

In vivo evaluation of oxidized multiwalled-carbon nanotubes-mediated hyperthermia treatment for breast cancer

Breast cancer is one of the most common types of cancer that contribute to high mortality worldwide. Hyperthermia (HT) was introduced as one of the alternative treatments to treat breast cancer but has major drawback of damaging normal adjacent cells. This study explores the integration effect of multiwalled‑carbon nanotubes (MWCNTs) in combination with hyperthermia treatment for breast cancer therapy regimes. In this study, acid-functionalized MWCNTs (ox-MWCNTs) were prepared by acid washing methods using H2SO4/HNO3 (98%/68%) with the ratio of 3:1 (ν/ν) and characterized by colloidal dispersibility test, FTIR, TGA, XRD, FESEM and EDX analysis. EMT6 tumor-bearing mice were treated with ox-MWCNTs in combination with local HT at 43 °C. The tumor progression was monitored and the influence of immune response was evaluated. Results from this study demonstrated that mice from ox-MWCNTs in combination with local HT treatment group experienced complete tumor eradication, accompanied by a significant increase in median survival of the mice. Histological and immunohistochemical analysis of tumor tissues revealed that tumor treated with combined treatment underwent cell necrosis and there was a significant reduction of proliferating cells when compared to the untreated tumor. This observation is also accompanied with an increase in Hsp70 expression in tumor treated with HT. Flow cytometry analysis of the draining lymph nodes showed an increase in dendritic cells infiltration and maturation in mice treated with combined treatment. In addition, a significant increase of tumor-infiltrated CD8+ and CD4+ T cells along with macrophages and natural killer cells was observed in tumor treated with combined treatment. Altogether, results presented in this study suggested the potential of ox-MWCNTs-mediated HT as an anticancer therapeutic agent, hence might be beneficial in the future of breast cancer treatment.

Abstract 1849: ICOS co-stimulation in combination immune checkpoint blockade and/or dose-optimized focal irradiation results in enhanced tumor control

Abstract In recent years, antibody-based immunotherapies targeting immune checkpoint proteins cytotoxic T lymphocyte antigen-4 (CTLA-4) and programmed death receptor-1 (PD-1), have revolutionized the treatment of cancer. Despite durable clinical responses in many indications, a significant number of patients fail to yield meaningful benefit, emphasizing the need to identify novel and/or complementary therapeutic interventions (e.g., alternative immune checkpoints, cytotoxic agents, etc.). Inducible T cell Co-Stimulator (ICOS/CD278) is a receptor belonging to the CD28/CTLA-4 superfamily that is predominantly expressed on T cells shortly after T cell receptor (TCR) activation. A growing body of non-clinical and clinical evidence supports the concept of antibody-mediated induction of ICOS signaling as a means to promote durable antitumor responses, particularly in collaboration with other checkpoint immunotherapies. To support ongoing and future clinical studies for feladilimab (GSK3359609), a non-depleting IgG4 ICOS agonist antibody currently being evaluated in pivotal clinical trials, we conducted a series of in vivo studies using a rodent surrogate (7E.17G9 mouse [m]IgG1; analogous non-depleting Fc) in ICOS-sensitive (EMT6 breast carcinoma) and ICOS-insensitive (CT26 colon carcinoma) tumor models. Female BALB/c mice with pre-established EMT6 tumors, exhibited partial responses to GSK'609 surrogate/tool antibody (7E.17G9 mIgG1), with ~20% (range 0-40%) of mice demonstrating tumor clearance. Concurrent administration of anti-PD-1 (RMP1-14 rat [r]IgG2a) and CTLA-4 (9D9 mIgG2b) dramatically improved tumor growth inhibition and survival. Notably, a triplet of TIM-3 blockade (RMT3-23 rIgG2a) in concert with ICOS and PD-1 mAbs further enhanced the survival of EMT6 tumor-bearing mice, suggesting complementary therapeutic mechanisms. In addition to combination with immune checkpoint blockade, we also explored the potential of ICOS co-stimulation with different focal irradiation (FRT) doses and frequencies in both EMT6 and CT26 tumor models. Intriguingly, we observed dramatic differences in response to concurrent FRT and ICOS mAb depending on the tumor model and associated FRT dose and frequency. Further, we identified a FRT dose threshold required for ICOS agonist appreciable combination responses, enabling exploration of additional immunotherapeutic combinations on top of ICOS/FRT. Collectively, these data support the combination of ICOS co-stimulation with different checkpoint immunotherapies (doublets and triplets), several of which are currently being evaluated in clinical studies. Moreover, these data demonstrate potential of ICOS co-stimulation with cytotoxic strategies, such as FRT - providing various dosing considerations and a framework future therapeutic intervention strategies. Citation Format: Jeremy D. Waight, Meixia Bi, Chris Hopson, Sapna Yadavilli, Kenneth W. Hance, Marc Ballas, Axel Hoos. ICOS co-stimulation in combination immune checkpoint blockade and/or dose-optimized focal irradiation results in enhanced tumor control [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1849.

Abstract 3414: Brd4 inhibition enhances checkpoint therapy by inducing MDSC apoptosis

Abstract Myeloid derived suppressor cells (MDSCs) antagonize antitumor immune responses, and limit the efficacy of immune based therapies for cancer. As a result, MDSCs have garnered attention as therapeutic targets. Unfortunately, there has been limited success in translating agents targeting MDSCs to the clinic. Brd4 is an epigenetic reader and is itself a therapeutic target in oncology due to its ability to regulate the expression of oncogenes such as Myc. In addition, Brd4 is known to regulate inflammatory cytokine production and innate immune responses including myeloid cell function. As a result, it was hypothesized that inhibition of Brd4 would impact MDSC function or expansion. In multiple tumor models (EMT6, 4T1, LLC, and C26) Brd4 inhibitors, both experimental (JQ1) and those in clinical development (PLX51107 and PLX2853) significantly reduced the abundance of total, PMN, and M-MDSC subsets within the tumor and spleen as measured by flow cytometry and IHC (p< 0.05). Furthermore, Nanostring gene expression analysis of EMT6 tumors identified a significant reduction in the abundance signature of neutrophils and macrophages with PLX51107 further confirming a reduction of myeloid cells in the presence of a Brd4 inhibitor (p< 0.01). The Nanostring analysis also showed a significant reduction of IL6 mRNA in PLX51107 treated tumors, and this was confirmed at the protein level by ELISA in the serum of EMT6 and 4T1 tumor bearing mice (p< 0.05). However, exogenous IL6 could not rescue MDSC levels in vivo or in vitro suggesting Brd4 inhibition might be directly affecting MDSCs. This was confirmed by showing that PLX51107 significantly inhibited the expansion of MDSCs from mouse bone marrow cells or healthy donor PBMCs cultured in IL6 and GMCSF as measured by flow cytometry (p<0.05). Furthermore, mouse and FACS sorted MDSCs from patients with melanoma or bladder cancer were found to undergo apoptosis when treated with PLX51107 as measured by flow cytometry (annexin and caspase3, p< 0.05), IF, and western blot. In addition, a pancaspase inhibitor rescued both mouse and patient derived MDSCs from apoptosis in the presence of PLX51107. Further investigation with specific caspase inhibitors showed this effect relied mostly on activation of the intrinsic apoptosis pathway and caspase9. The importance of Brd4 to MDSC survival was further confirmed using a LysMCre Brd4 floxed mouse model. Finally, depletion of MDSCs in vivo with PLX51107 significantly enhanced the efficacy of anti-PDL1 therapy as compared to either agent alone (p< 0.05 in EMT6, 4T1, and LLC tumor models). These results identify Brd4 and the TXNIP/ASK1 apoptosis pathway as novel regulators of MDSC survival, and provide evidence to further investigate Brd4 inhibitors in combination with immune based therapies for solid tumors. Citation Format: Andrew Stiff, Himanshu Savardekar, Robert Wesolowski, Megan Duggan, Luke Scarberry, Brooke Benner, Darren Wethington, Gabby Lapurga, Steven Sun, Jack Hedberg, Logan Good, William E. Carson. Brd4 inhibition enhances checkpoint therapy by inducing MDSC apoptosis [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 3414.

Abstract 2837: Dual inhibition of SHP2 and CDK4/6 leads to immunological memory and immune-mediated anti-tumor activity in a mouse syngeneic model of breast cancer

Abstract Allosteric inhibition of the phosphatase SHP2, a key signaling node in the RAS pathway, is a novel, investigational therapeutic strategy for patients bearing tumors with specific oncogenic mutations in this pathway. SHP2 also participates in signal transduction downstream of regulatory immunoreceptors and we have previously shown that inhibition of SHP2 drives anti-tumor immunity through modulation of both innate and adaptive mechanisms in preclinical models (Quintana E. et al, A5019, AACR2019). Rational combination strategies offer an opportunity to optimize the therapeutic potential of SHP2 inhibitors. For example, we have demonstrated that SHP2 inhibition has combinatorial anti-tumor effects with a CDK4/6 inhibitor in several RAS-driven human cancer cell-line derived xenograft models (Lee G. et al, A1322, AACR2019); however, these models use immunocompromised mice, making it impossible to assess an effect of the combination on anti-tumor immunity. To evaluate the impact of dual SHP2 and CDK4/6 inhibition, we tested this combination in immunocompetent EMT6 tumor-bearing mice. EMT6 is a RAS-driven model of triple-negative breast cancer, which is partially sensitive to SHP2 or CDK4/6 inhibition via cell intrinsic mechanisms. While monotherapy treatment with either RMC-4550, an allosteric inhibitor of SHP2, or abemaciclib, a CDK4/6 inhibitor, resulted in modest tumor growth inhibition, combination treatment led to complete tumor regressions in 40% (4 of 10) of the mice. Furthermore, 30% (3 of 10) of the mice remained tumor-free upon cessation of treatment and, when re-challenged with EMT6 cells tumors failed to grow demonstrating that this dual SHP2-CDK4/6 inhibition results in formation of immunological memory. Combination treatment was well tolerated in mice. Recently, CDK4/6 inhibitors have been shown to augment anti-tumor immunity via a number of mechanisms, including increases in antigen presentation by the tumor and enhancement of T cell activation. Analysis of the immune landscape in the tumor microenvironment nine days after treatment revealed that RMC-4550 increased the frequency of CD8+T cells and depleted F4/80+ macrophages in EMT6 tumors, and the combination with abemaciclib amplified these changes. Our results suggest that dual SHP2 and CDK4/6 targeting is a promising investigational strategy against tumors driven by mutations in the RAS signaling pathway that combines direct inhibition of cancer cell growth with immunomodulation to potentially achieve robust and durable anti-tumor responses. Citation Format: Nataliya Tovbis Shifrin, Grace J. Lee, Neil Omaque, Tram Nguyen, Amira Belwafa, Edward G. Lorenzana, Robert J. Nichols, Jacqueline A. Smith, Mallika Singh, Elsa Quintana. Dual inhibition of SHP2 and CDK4/6 leads to immunological memory and immune-mediated anti-tumor activity in a mouse syngeneic model of breast cancer [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 2837.

Synthesis of gadolinium/iron-bimetal-phenolic coordination polymer nanoparticles for theranostic applications.

Integration of diagnostic and therapeutic components into a single coordination polymer nanoparticle is desirable for theranostic applications, but still challenging. Herein, we report the synthesis of bimetal-phenolic coordination polymer nanoparticles using gadolinium nitrate and ferrous sulphate as a metal source, and plant polyphenols (i.e., tannic acid) as an organic ligand via a metal-catechol coordination assembly process. Such coordination polymers show a tunable molar ratio of Gd/Fe and high dispersibility and stability in aqueous solution. The coordination polymers reveal composition-dependent performance for longitudinal relaxivity and photothermal conversion. The longitudinal relaxivity is positively related to the molar ratio of Gd/Fe, while the photothermal performance is negatively related to the molar ratio of Gd/Fe in the coordination polymers. The coordination polymers with an optimized molar ratio of Gd/Fe exhibit an ultra-small hydrodynamic diameter (∼23 nm), a high r1 value (9.3 mM-1 s-1) with low r2/r1 (1.26) and high photothermal conversion efficiency (η = 37%). They can be used as a contrast agent for T1-weighted magnetic resonance imaging of EMT-6 tumor bearing mice, which can effectively enhance the signals of tumors. They can also effectively suppress tumor growth via photothermal therapy. This work brings new insights for the synthesis of multifunctional coordination polymer nanoparticles and extending their potential applications in theranostics.

Abstract 724: Preclinical evaluation of TQB3728, a potent orally active IAP antagonist

Abstract Objectives: Evasion of apoptosis and avoidance of immune surveillance represent two key hallmarks of cancer. Members of the inhibitor of apoptosis (IAP) gene play important interconnecting roles in both of these characteristic pathways of tumorigenesis, providing a critical nexus in targeting of cancer. Small-molecule antagonists of the IAPs, known as Smac mimetic compounds (SMCs), are in quick clinical development for cancer therapy. We disclose here a novel IAP antagonist TQB3728 and its preclinical evaluation on tumor therapy. Methods: The biochemical activities of TQB3728 for cIAP1 BIR3, cIAP2 BIR3 and XIAP BIR3 were measured by the corresponding biochemical assays. The in vitro anti-proliferative activity was evaluated on MDA-MB-231 and EMT6 cell lines. The in vivo antitumor activity of TQB3728 was evaluated in two cell-derived xenograft (CDX) models of TNBC (MDA-MB-231) and lymphoma (Karpas 422), and one syngeneic model of TNBC (EMT6). PK/PD was assessed by western blot analysis of cIAP1 expression and cleaved caspase 3 level in the treated tumor tissues. Results: TQB3728 displayed potent biochemical activities for cIAP1 BIR3, cIAP2 BIR3 and XIAP BIR3 with IC50 of 0.7, 11.8 and 22.5 nM, respectively. The expected anti-proliferative activities were also observed in both tumor cell lines, with a IC50 of 14.3 Nm for MDA-MB-231, and IC50 of 149.5 nM for EMT6 in the presence of exogenous TNFα (1 ng/ml). TQB3728 showed in vivo antitumor activity in the MDA-MB-231 CDX model with 89.8% TGI @30 mpk QD, which is better than LCL-161 (71.9% TGI @30 mpk, QD). TQB3728 also showed good antitumor activity in vivo in the Karpas 422 lymphoma CDX model with 112.2% TGI @100 mpk Q3D. In EMT6 tumor-bearing mice, the combination of TQB3728 and anti-PD1 antibody significantly regressed the tumor growth (TGI = 106.3%), whereas monotherapies only displayed moderate activities ( 60.6% and 0.4% TGI for anti-PD1 and TQB3728, respectively). Western blot analysis of the tumor samples showed that TQB3728 promoted caspase 3 cleavage, indicating that the tumor growth inhibition could be mediated by cell apoptosis signaling. Conclusions: We have discovered a potent orally active IAP antagonist, TQB3728, which shows activity of cIAP1 inhibition and strong antitumor activity in both in vitro and in vivo preclinical tumor models. These results are highly promising and warrant moving the compound forward to clinical investigation. Citation Format: Yingchun Liu, Xiquan Zhang, Ling Yang, Xin Tian, Tiantian Dong, Charles Z. Ding, Lihong Hu, Zhaobing Xu, Yuanfeng Xia, Haijuan Tong, Wenxi Li, Lijuan Hou, Chichung Chan, Shuhui Chen. Preclinical evaluation of TQB3728, a potent orally active IAP antagonist [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 724.

Primary tumor-induced immunity eradicates disseminated tumor cells in syngeneic mouse model

Although clinically apparent metastasis is associated with late stages of cancer development, micro-metastatic dissemination may be an early event. However, the fate of these early disseminated tumor cells (DTC) remains elusive. We show that despite their capacity to disseminate into secondary organs, 4T1 tumor models develop overt metastasis while EMT6-tumor bearing mice clear DTCs shed from primary tumors as well as those introduced by intravenous (IV) injection. Following the surgical resection of primary EMT6 tumors, mice do not develop detectable metastasis and reject IV-injected tumor cells. In contrast, these cells readily grow and metastasize in immuno-deficient athymic or Rag2−/− mice, an effect mimicked by CD8+ T-cell depletion in immunocompetent mice. Furthermore, recombinant G-CSF or adoptive transfer of granulocytic-MDSCs isolated from 4T1 tumor-bearing mice, induce metastasis by suppressing CD8+ T-cells in EMT6-primed mice. Our studies support the concept of immune surveillance providing molecular insights into the immune mechanisms during tumor progression.

Immunosuppressive effects of sMIC abrogate immunotherapy efficacy in a mouse model of breast cancer

Abstract Expression of the NKG2D activating receptor on CD8+ T cells and NK cells is thought to play an important role in detection and clearance of tumor cells. The soluble version of an NKG2D ligand in humans, MHC I chain-related molecule (sMIC), has been correlated with poor prognosis in multiple human cancers and hypothesized to serve as a tumor immune evasion strategy. Yet, it is unclear whether sMIC impairs the activity of T cell directed immunotherapies including checkpoint inhibitors. We utilized a syngeneic mouse model of breast cancer, EMT6, as well as EMT6 engineered to express high levels of sMIC to examine the impact of sMIC on responses to anti-PDL1/anti-TIGIT combination immunotherapy. Following treatment with anti-PDL1/anti-TIGIT combination immunotherapy, WT EMT6 tumors were rejected and tumor associated T cells and NK cells isolated from these tumors showed increased IFNγ secretion. In contrast, mice bearing EMT6 tumors expressing sMIC and treated with the same regimen were unable to reject tumors. Consistent with this observation, CD8+ and NK cell TILs from EMT6. sMIC tumors showed decreased ability to produce IFNγ compared to those from WT EMT6 tumors. Taken together these results suggest the importance of targeting the MIC-NKG2D pathway to further improve combination immunotherapy efficacy in patients.

Combination Therapy with NHS-muIL12 and Avelumab (anti-PD-L1) Enhances Antitumor Efficacy in Preclinical Cancer Models

Purpose: To determine whether combination therapy with NHS-muIL12 and the anti-programmed death ligand 1 (PD-L1) antibody avelumab can enhance antitumor efficacy in preclinical models relative to monotherapies.Experimental Design: BALB/c mice bearing orthotopic EMT-6 mammary tumors and μMt- mice bearing subcutaneous MC38 tumors were treated with NHS-muIL12, avelumab, or combination therapy; tumor growth and survival were assessed. Tumor recurrence following remission and rechallenge was evaluated in EMT-6 tumor-bearing mice. Immune cell populations within spleen and tumors were evaluated by FACS and IHC. Immune gene expression in tumor tissue was profiled by NanoString® assay and plasma cytokine levels were determined by multiplex cytokine assay. The frequency of tumor antigen-reactive IFNγ-producing CD8+ T cells was evaluated by ELISpot assay.Results: NHS-muIL12 and avelumab combination therapy enhanced antitumor efficacy relative to either monotherapy in both tumor models. Most EMT-6 tumor-bearing mice treated with combination therapy had complete tumor regression. Combination therapy also induced the generation of tumor-specific immune memory, as demonstrated by protection against tumor rechallenge and induction of effector and memory T cells. Combination therapy enhanced cytotoxic NK and CD8+ T-cell proliferation and T-bet expression, whereas NHS-muIL12 monotherapy induced CD8+ T-cell infiltration into the tumor. Combination therapy also enhanced plasma cytokine levels and stimulated expression of a greater number of innate and adaptive immune genes compared with either monotherapy.Conclusions: These data indicate that combination therapy with NHS-muIL12 and avelumab increased antitumor efficacy in preclinical models, and suggest that combining NHS-IL12 and avelumab may be a promising approach to treating patients with solid tumors. Clin Cancer Res; 23(19); 5869-80. ©2017 AACR.

Abstract 5807: Disseminated tumor cell clearance by the immune system

Abstract The classical model of metastasis suggests that the tumor cell dissemination occurs late in tumor development, however accumulating evidence coming from mouse studies and clinical data provide striking evidence that tumor cells start to disseminate during the initial steps of tumor development. However, the dissemination from the primary area does not always result in metastasis. Due to the non-permissive nature of microenvironment in distant sites, these early disseminated tumor cells might be cleared or maintained in a non-proliferative/dormant state. The mechanism by which some early disseminated tumor cells colonize and generate metastatic growth while some remain dormant is not well known. In order to understand the underlying factors that may contribute to the metastatic growth, we performed time course experiments by utilizing murine mammary tumors (4T1 as metastatic and EMT6 as less metastatic) in a syngeneic mouse model. Luciferase expressing 4T1 or EMT6 tumor cells were orthotopically implanted into the fat pads and tumor cell dissemination was analyzed over 3-week time points. We determined that both 4T1 and EMT tumors disseminated as early as one to two-week post implantation, however only 4T1 tumor develop metastasis in distant organs. Moreover, we also resected primary tumors 1, 2 and 3-week post implantation of EMT6-Luci or 4T1-Luci tumors and observed distant metastasis via optical imaging of luciferase expression in live animals. Although the majority of 4T1 tumor-bearing mice (>80%) develop pulmonary metastasis when 4T1 tumors resected 2 and 3 weeks post-implantation only 10% of mice develop metastasis when primary tumor resected one-week post implantation. In contrast, EMT6 tumors following resection only relapsed in the primary tumor site but failed to develop metastasis. Furthermore, EMT6 tumor-bearing mice efficiently cleared tail vein injected EMT6-luci cells in the lungs. We investigated the possible mechanism by which EMT6 tumor-bearing mice clears disseminated tumor cells in the lung. We provide evidence that pulmonary infiltrated mMDSCs mediate tumor cell killing via secretion of high levels of cytotoxic granules, granzyme A, granzyme B, perforin. This was confirmed by mouse transcriptome and qPCR analyses as well as biochemistry using in vivo samples and in vitro co-culture samples. Our studies provide a new paradigm in the understanding of the fate of disseminated tumor cells in secondary organs and the role of the immune system in this process. Citation Format: Raziye Piranlioglu, Eun Mi Lee, Maria Ouzunova, Ali S. Arbab, Paulo C. Rodriguez, Asm L. Iskander, Hasan Korkaya. Disseminated tumor cell clearance by the immune system [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 5807. doi:10.1158/1538-7445.AM2017-5807

Abstract 3992: Immune regulation of disseminated tumor cell clearance versus metastatic growth

Abstract Metastatic disease is the end stage of extremely inefficient processes that entail overcoming multiple barriers including anti-tumor immunity. Although evidences from preclinical and clinical settings suggest that dissemination of malignant cells is an early process, majority of disseminated cells either eliminated or remain dormant in distant organs, while very few cells eventually develop successful metastasis. Therefore, it is widely accepted that dynamic and reversible tumor cell plasticity is required for metastasis, however, in vivo steps and molecular mechanisms are poorly elucidated. We provide evidence that monocytic and granulocytic subsets of myeloid derived suppressor cells (m-MDS, g-MDSC) infiltrated in primary 4T1 tumor and distant organs with different time kinetics regulate spatiotemporal tumor plasticity. Using co-culture experiments and syngeneic mouse models of murine 4T1 (metastatic) tumor, we demonstrated that tumor infiltrated m-MDSCs facilitate tumor cell dissemination from the primary site by inducing the EMT/CSC phenotype. In contrast, g-MDSCs infiltrated in the lungs support metastatic growth by reverting the EMT/CSC phenotype and thus promoting tumor cell proliferation. In contrast, less invasive EMT6 tumors fail to induce efficient pulmonary infiltration of g-MDSCs and results in clearance of disseminated tumor cells in the lungs. Gene expression analyses of tumors and MDCS subsets in primary tumor site and distant organs at different time points reveal mechanistic temporal regulation of in vivo tumor plasticity by m-MDSC and g-MDSC subsets in 4T1 tumor-bearing mice. However, the lung microenvironment of EMT6 tumor-bearing mice display a gene expression signature of anti-tumor immunity which predict better survival in breast cancer patients. In our functional studies, we demonstrate that EMT6 tumor-bearing animals efficiently eliminate disseminated tumor cells in the lungs. Furthermore, g-MDSCs isolated from 4T1-tumor bearing animals significantly enhance metastatic growth of already disseminated tumor cells. Consistent with the “seed and soil” hypothesis, our studies provide a molecular mechanism by which the immune system regulate spatiotemporal tumor plasticity and generation of permissive or anti-tumorigenic microenvironment in distant organs determining the fate of disseminated tumor cells. Citation Format: Hasan Korkaya, Eunmi Lee, Raziye Piranioglu, Maria Ouzounova, Abdeljabar El-Andaloussi, Iskander Asm, Ahmet K. Korkaya, Gang Zhou, Ali Arbab, Paulo Rodriguez. Immune regulation of disseminated tumor cell clearance versus metastatic growth [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 3992. doi:10.1158/1538-7445.AM2017-3992

Redshifted Cherenkov Radiation for in vivo Imaging: Coupling Cherenkov Radiation Energy Transfer to multiple F\xf6rster Resonance Energy Transfers

Cherenkov Radiation (CR), this blue glow seen in nuclear reactors, is an optical light originating from energetic β-emitter radionuclides. CR emitter 90Y triggers a cascade of energy transfers in the presence of a mixed population of fluorophores (which each other match their respective absorption and emission maxima): Cherenkov Radiation Energy Transfer (CRET) first, followed by multiple Förster Resonance Energy transfers (FRET): CRET ratios were calculated to give a rough estimate of the transfer efficiency. While CR is blue-weighted (300–500 nm), such cascades of Energy Transfers allowed to get a) fluorescence emission up to 710 nm, which is beyond the main CR window and within the near-infrared (NIR) window where biological tissues are most transparent, b) to amplify this emission and boost the radiance on that window: EMT6-tumor bearing mice injected with both a radionuclide and a mixture of fluorophores having a good spectral overlap, were shown to have nearly a two-fold radiance boost (measured on a NIR window centered on the emission wavelength of the last fluorophore in the Energy Transfer cascade) compared to a tumor injected with the radionuclide only. Some CR embarked light source could be converted into a near-infrared radiation, where biological tissues are most transparent.

Synthesis of novel PEG-modified nitroimidazole derivatives via “hot-click” reaction and their biological evaluation as potential PET imaging agent for tumors

Ten novel PEG-modified nitroimidazole derivatives labeled with 18F were synthesized via “hot-click” reaction and evaluated as PET tumor tracers. The radiolabeling method was convenient and efficient, all compounds displayed high radiochemical purity (>95%), high yield (30–50%) and good stability in saline and human serum. The biodistribution studies in EMT-6 tumor-bearing mice demonstrated their tumor uptake values at 60 min postinjection were 1.46–2.99%ID/g, which were lower than [18F]FMISO (5.43%ID/g), their tumor-to-liver ratios were 0.73–1.07, higher than [18F]FMISO (0.69). In vitro MCF-7 cell uptake studies showed their uptake values have no significant difference between hypoxic and aerobic cells.