Syngeneic Breast Cancer Model Research Articles

Abstract B144: Lack of immunotherapy efficacy in a syngeneic bone metastasis model of triple-negative breast cancer

Abstract Background: Triple-negative breast cancer (TNBC) is an aggressive cancer with treatment options limited to standard-of-care chemotherapy. TNBC patients develop metastases in high incidence, and almost all patients have bone metastases at end-stage disease. Bone metastases are mainly osteolytic, causing severe bone loss that can be prevented by bone-targeted agents. Currently, there is no efficient cure for bone metastases. Activation of the patient’s own immune system by immunotherapies is widely studied in various cancers, but their efficacy on bone metastases is not well established. In this study, we aimed to evaluate the efficacy of immunotherapies in comparison to standard-of-care compounds in a preclinical TNBC bone metastasis model. Materials and methods: Mouse 4T1-GFP TNBC cells were inoculated intracardially to immunocompetent female Balb/c mice to model bone metastasis. Treatment of single agents of chemotherapy (cyclophosphamide, 100 mg/kg), bone-targeting agent (zoledronic acid, 0.1 mg/kg), and immunotherapies programmed cell death 1 antibody (mouse anti-PD-1, 10 mg/kg) and indoleamine-pyrrole 2,3-dioxygenase (IDO) inhibitor (epacadostat, 100 mg/kg), and the combination of anti-PD-1 and epacadostat were started on the day following the cancer cell inoculations. The study was terminated 12-13 days after the inoculations. Tumor burden was evaluated by GFP imaging and tumor-induced bone loss by X-ray imaging at sacrifice. Tumor infiltrating lymphocytes (TILs) were evaluated by immunohistochemical stainings of CD3+, CD4+ and CD8+ T cells. Results: Cyclophosphamide decreased skeletal tumor burden and the area of tumor-induced osteolytic bone lesions. Zoledronic acid decreased the area of osteolytic lesions but had no effect on skeletal tumor burden. Anti-PD-1, epacadostat and their combination had no effect on skeletal tumor burden or in the development of osteolytic lesions. Low number or no TILs were observed in the tumors growing in bone obtained from vehicle-treated mice. Conclusions: Immunotherapies did not prevent tumor growth in bone or tumor-induced bone changes in the syngeneic TNBC model. This is probably caused by the low number of TILs in the tumors growing in bone. Further studies are needed to evaluate the effects of treatment combinations and to increase the responsiveness to immunotherapies especially in bone metastases. Citation Format: Tiina E Kähkönen, Mari I Suominen, Jenni HE Mäki-Jouppila, Jussi M Halleen, Jenni Bernoulli. Lack of immunotherapy efficacy in a syngeneic bone metastasis model of triple-negative breast cancer [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference on Molecular Targets and Cancer Therapeutics; 2019 Oct 26-30; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2019;18(12 Suppl):Abstract nr B144. doi:10.1158/1535-7163.TARG-19-B144

Alterations in Wnt- and/or STAT3 signaling pathways and the immune microenvironment during metastatic progression.

Metastatic breast cancer is an extremely complex disease with limited treatment options due to the lack of information about the major characteristics of metastatic disease. There is an urgent need, therefore, to understand the changes in cellular complexity and dynamics that occur during metastatic progression. In the current study, we analyzed the cellular and molecular differences between primary tumors and paired lung metastases using a syngeneic p53-null mammary tumor model of basal-like breast cancer. Distinct subpopulations driven by the Wnt- and/or STAT3 signaling pathways were detected in vivo using a lentiviral Wnt- and STAT3 signaling reporter system. A significant increase in the overlapping populations driven by both the Wnt- and STAT3 signaling pathways was observed in the lung metastases as compared to the primary tumors. Furthermore, the overlapping populations showed a higher metastatic potential relative to the other populations and pharmacological inhibition of both signaling pathways was shown to markedly reduce the metastatic lesions in established lung metastases. An analysis of the unique molecular features of the lung metastases revealed a significant association with immune response signatures. Specifically, Foxp3 gene expression was markedly increased and elevated levels of Foxp3 + Treg cells were detected in close proximity to lung metastases. Collectively, these studies illustrate the importance of analyzing intratumoral heterogeneity, changes in population dynamics, and the immune microenvironment during metastatic progression.

Abstract 3969: TP-0903, a potent AXL receptor tyrosine kinase inhibitor, enhances the activity of anti-PD-1 therapy in a metastatic preclinical syngeneic model of breast cancer

Abstract The high rate of non-responders to immune checkpoint inhibitors (ICI) represents significant challenges in the field of immune-oncology. Breast cancer has emerged as a cancer type that responds poorly to ICI. Therefore, there is an urgent need to better understand how cancer cells escape immune surveillance and resist immune attacks. Cancer cell clones with mesenchymal features seem to be less susceptible to attacks by immune cells. Hence, epithelial-to-mesenchymal transition (EMT) targeting agents such as TP-0903 are emerging as potential combination candidates to enhance immune checkpoint inhibition. AXL is overexpressed in a variety of cancers, often driving the pathogenesis and progression of disease. AXL overexpression induces cancer growth, invasion, metastasis, drug resistance, and mesenchymal characteristics. Aside from cancer cells, AXL is also expressed on several types of immune cells including macrophages and dendritic cells. Recently, it has been reported that AXL plays an important role as a negative regulator of immune response, contributing to an anti-tumor immune suppression. We have previously identified TP-0903 to be a potent small molecule inhibitor of AXL (IC50=14 nM) capable of reversing EMT. In this study, we evaluated whether TP-0903 could enhance the ICB effect in an ICB-resistant triple negative, metastatic breast cancer mouse model (4T1). In the 4T1 syngeneic model, anti-PD-1 monotherapy failed to inhibit tumor growth indicating that this tumor is resistant to anti-PD-1 treatment. The combination of TP-0903 and anti-PD-1 resulted in statistically significant tumor growth inhibition versus TP-0903 monotherapy (p<0.05). We hypothesized that the combination effect was influenced by potential CD8+ T cell depletion, and further investigated the effects of TP-0903 on immune cells in spleen and tumors. Tumor growth inhibition was found to be associated with a decrease in myeloid-derived suppressor cells (MDSC) in the spleen and an increase in infiltration and activation of dendritic cells (DCs) in the tumor. Gene expression analysis revealed that TP-0903 treatment decreased multiple immunosuppressive cytokines and chemokines including IL-6 and G-CSF in vivo. These results suggest that TP-0903 modulates the immune-suppressive tumor microenvironment (TME) to reinvigorate T cell immunity in anti-PD-1 resistant 4T1 tumors. In conclusion, AXL inhibition with TP-0903 modulates TME and enhances the anti-tumor effects of ICBs in an anti-PD-1 resistant, metastatic breast cancer mouse model. These findings support additional clinical testing of TP-0903 in combination with ICBs, which is currently being investigated in patients with advanced solid tumors in an ongoing clinical study (NCT02729298). Citation Format: Yuka Kumagai, Jun Oishi, Megumi Nakamura, Jason M. Foulks, Clifford J. Whatcott, Steven L. Warner, David J. Bearss, Masashi Goto. TP-0903, a potent AXL receptor tyrosine kinase inhibitor, enhances the activity of anti-PD-1 therapy in a metastatic preclinical syngeneic model of breast cancer [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 3969.

Abstract LB-129: Metastatic breast cancer cells promote anti-inflammatory MSC actions and release Lipocalin 2 to induce a tumor-supportive premetastatic lung microenvironment

Abstract Cancer metastasis is responsible for the vast majority of cancer-related deaths. Survival and proliferation of cancer cells within distant tissues requires that these foreign microenvironments acquire tumor-supportive characteristics. While it has been established that primary tumor cells can reprogram the molecular and cellular composition of the premetastatic niche by releasing soluble factors and extracellular vesicles, efforts to date have been limited by the use of in vivo models lacking a fully-functional immune system and/or the need for finely-tuned experiment end points to limit direct reprogramming effects of early-disseminating cells from the primary tumor. To address this need, we developed the immune-competent PyMT syngeneic murine model of breast cancer for elucidating events during premetastatic niche reprogramming in the absence of tumor cell allografting. We demonstrate that metastatic mouse PyMT breast cancer cells differentially influence the premetastatic lung and brain, selectively promoting a tumor-supportive lung microenvironment with increased CD73 expression and decreased TNFalpha expression. Using in vitro models of CD73-positive mesenchymal stem cells (MSCs) and monocytes/macrophages, we next tested whether MSCs can selectively mediate anti-inflammatory effects of metastatic breast cancer cells. Notably, conditioned media from metastatic Py230 cells reprogrammed MSCs to selectively increase anti-inflammatory CCL1 and MRC1 transcripts in monocytes/macrophages. In contrast, conditioned media from non-metastatic Py8119 cells reprogrammed MSCs to selectively increase pro-inflammatory IL23alpha and TNFalpha in monocytes/macrophages. Mining transcriptome data from Py8119 and Py230 cells revealed a lipocalin 2 (LCN2) axis that is selectively expressed in the metastatic Py230 cells and predicts poor breast cancer patient survival. Finally, we demonstrate that immunoneutralization of LCN2 in Py230 conditioned media renders it unable to reprogram the premetastatic lung microenvironment toward a tumor-supportive state. Taken together, these results establish the utility of a tumor cell-free in vivo model for interrogating breast cancer priming of the premetastatic niche, demonstrate that MSCs can mediate the anti-inflammatory state within the tumor microenvironment, and identify LCN2 as an important therapeutic target for blocking breast cancer progression. Citation Format: Francesca Sanchez, Kayla Meade, Analine Aguayo, Nathalie Nadeles, Sarkis Hamalian, Toni Uhlendorf, Lisa Banner, Jonathan A. Kelber. Metastatic breast cancer cells promote anti-inflammatory MSC actions and release Lipocalin 2 to induce a tumor-supportive premetastatic lung microenvironment [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 LB-129.

Abstract 5027: Sequencing the tumor microenvironment and myeloid derived suppressor cells to understand response to immunotherapy in primary HER2 positive breast cancer

Abstract The purpose of this study is to identify how the epigenetic modulator entinostat reprograms myeloid derived suppressor cells (MDSCs) to prime the tumor microenvironment (TME) and modulate immunotherapy response. Early trials investigating single agent ICIs, anti-CTLA-4 and anti-PD-1/PD-L1, resulted in overall response rates of 5-20% in patients with metastatic HER2 positive breast cancer but little is understood about use of these therapies in patients with early stage/ primary disease. Suboptimal and inconsistent immune responsiveness is likely the result of multiple suppressive signals within the TME that provide a formidable barrier to T cell infiltration leading to an opportunity for TME modulation to potentially improve response. We and others have shown that immunosuppressive factors can be overcome by adding the class 1 histone deacetylase inhibitor, entinostat, to ICIs to reprogram MDSCs and upregulate T cell attracting signals within the TME. We hypothesize that gene expression signatures of reprogrammed MDSCs are predictive of response with subsequent immunotherapy treatment. To test this hypothesis, we used the neu-N syngeneic mouse model of HER2 positive breast cancer. We investigated differences in survival and tumor burden in response to combinations of entinostat with anti-PD-1 and anti-CTLA-4. Having previously identified MDSCs as important targets of entinostat that modulate response to ICIs, we used methods we developed to study MDSC function. Given the broad effects of entinostat on global gene expression including that of MDSCs, we used single cell RNA-seq (scRNA-seq) on primary tumors and isolated intratumoral MDSCs to determine what is driving changes in MDSC function in primary tumors. We plan to use data obtained from scRNA-seq to develop signatures of response and identify changes in responders vs. non-responders to help develop biomarkers of response in primary disease. Our results thus far show that treatment with entinostat + ICIs improves survival, and decreases tumor burden in primary tumors. Ex-vivo studies of MDSCs demonstrate changes in immunosuppressive capabilities of MDSCs and show differences in infiltration and function of granulocytic vs. monocytic MDSCs. We expect that evaluation of the ongoing sequencing data will identify more specific immune targets within primary TME and help us understand the importance of targeting MDSCs in early disease. We predict this work will identify additional candidate targets for therapeutic testing to convert breast cancers into immune responsive tumors, and that this will facilitate improved response to ICIs that will ultimately translate into improved survival of patients with breast cancer. Citation Format: Evanthia T. Roussos Torres, Christine Rafie, Emily Davis, Luciane T. Kagohara, Elana J. Fertig, Elizabeth M. Jaffee. Sequencing the tumor microenvironment and myeloid derived suppressor cells to understand response to immunotherapy in primary HER2 positive breast cancer [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 5027.

Abstract 4553: Snail1 in primary breast tumors remotely regulates a pro-tumor immune response in the bone marrow

Abstract Systemic immunosuppression in cancer is a mechanism of immune escape allowing for cancer progression to metastasis and remains a major pathophysiologic barrier to treatment including immunotherapies. The mechanism of this phenomenon remains relatively obscured. Although immune suppressive humoral factors secreted by primary tumors are thought to be a major cause of immune escape, a major question remains whether factors critical for cancer metastasis also dually regulate systemic immunosuppression to prepare peripheral sites for the eventual encounter with metastasizing cancer cells. To that end, we report here that the master cancer epithelial-mesenchymal transition (EMT) initiating factor Snail1, whose expression is restricted to the primary tumor where it promotes the generation of invasive breast tumor-initiating cells, yet also has a direct role in creating a pro-tumor systemic immune environment to promote metastasis. These dual roles for Snail1 in breast cancer progression has not been elucidated. Using both spontaneous genetic murine model of breast cancer and a syngeneic orthotopic breast cancer model we demonstrate that Snail1 is uniquely required for metastatic spread of breast tumor cells while also directly modulates the bone marrow niche, skewing it towards a suppressive immune phenotype with an upregulation of myeloid derived suppressor cells (MDSCs) and a decrease in antigen presenting cell (APC) populations. Importantly, deletion of Snail1 specifically in primary tumors severely abrogated metastasis and simultaneously restored bone marrow immune populations to levels in non-tumor bearing animals. In vitro co-culture assays demonstrated that humoral factors from Snail1-repleted cancer cells promoted MDSC formation and suppressed APCs when compared to Snail1-deficient cancer cells. Mechanistically, Snail1 modulates systemic immunosuppression specifically through 1) upregulating the expression of GM-CSF, CXCL2, and CCL2, factors important for MDSC induction and recruitment, within the primary tumor to saturate the periphery with these factors; and 2) generating metastatic cells that may suppress immune functions within the metastatic niche. Taken together, these data demonstrate Snail1’s central role in metastatic progression in breast cancer through its direct generation of metastatic cells and suppressing the systemic immune system to increase the chance that these metastatic cells may survive the periphery. Note: This abstract was not presented at the meeting. Citation Format: Mathew Sebastian, Dongjiang Chen, Son Le, Duy Nguyen, Changwang Deng, Dan Jin, Nagheme Thomas, David Tran. Snail1 in primary breast tumors remotely regulates a pro-tumor immune response in the bone marrow [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 4553.

Abstract 2302: Natural killer cell cancer therapy in mouse model

Abstract Natural killer (NK) cells exert anti-tumor function through direct killing of tumor cells and indirect modulation of the function of other immune cells, such as T cells, dendritic cells and macrophages. However, the efficacy of adoptive NK cell transfer in cancer treatment has been limited, and only found in certain myeloid leukemia under allogeneic setting. The functional heterogeneity of NK cells and the immunosuppressive tumor microenvironment likely contribute to the limited efficacy. We establish a method to condition ex vivo expanded murine NK cells with anti-tumor activity. The conditioned NK cells are EOMES+CD127-DX5-CD49a+and CD11b+CD27+. Encountering with solid tumor cell lines in vitro enhances the production of IFN-γand triggers killing of the tumor cells by conditioned NK cells. In a syngeneic orthotopic EO771 breast cancer model, adoptive transfer of conditioned NK cells into tumor-bearing mice slows tumor progression and alters the composition and function of intra-tumor immune cells. To better imitate clinical condition, we transfer conditioned NK cells into tumor-resected mice, and found the therapy improved survival rate. We are investigating the anti-tumor mechanism of the conditioned NK cells. Citation Format: Shih-Wen Huang, Yein-Gei Lai, Jen-Qi Wu, Yae-Huei Liou, Nan-Shih Liao. Natural killer cell cancer therapy in mouse model [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 2302.

Abstract 586: CD4+and CD8+T cells influence 4T1.2luc mammary tumor growth and survival

Abstract The 4T1.2 syngeneic murine breast cancer model is an aggressive model of stage IV triple-negative breast cancer. Despite its significant strength, the 4T1.2 model is poorly immunogenic with few defined tumor antigens. The firefly luciferase gene, luc2, has been transfected into various tumor cell lines as a reporter for bioluminescence imaging. However, studies have shown that the luciferase (Luc) may serve as a tumor antigen and induce CD8+ cytotoxic T cell responses. The goal of the current study was to determine if luciferase expression in 4T1.2 tumor cells would enhance the immunogenicity of the tumor, and explore the role of immune effector cells in the parental 4T1.2 and luciferase-transfected 4T1.2luc tumor model. In vitro proliferation of the 4T1.2 and 4T1.2luc cells was assessed using the MTS cell proliferation assay. For in vivo experiments, female BALB/c mice were randomized into 2 groups and orthotopically injected with 5x104 4T1.2 or 4T1.2luc cells, respectively. Tumor growth was assessed until day 35 post tumor implantation. Each group was further randomized into 5 subgroups and received i.p. injections of PBS, rat IgG2b mAb, anti-CD4 mAb, anti-CD8 mAb, or anti-asialo GM1. Mice were sacrificed between day 28-35 post tumor implantation, and lung metastases were analyzed for each group. To further evaluate the immunogenicity of luciferase in the 4T1.2luc model, a separate cohort of mice were orthotopically injected with 5x104 4T1.2luc cells and sacrificed at day 35 post tumor implantation. Splenocytes were harvested and cultured in the presence of 1 or 10 µg/ml peptides representing MHC I-restricted Luc epitopes for 5 days, and cytotoxicity of the effector cells was assessed by the chromium-release assay. In vitro proliferation rates were not significantly different between 4T1.2 and 4T1.2luc tumor cells. However, 4T1.2luc tumor-bearing mice showed a significant reduction in primary tumor growth compared to 4T1.2 tumor-bearing mice. 4T1.2 primary tumor growth was not altered by the depletion of any immune cell type, while 4T1.2luc primary tumor growth was significantly increased by the depletion of CD8+ T cells, and reduced by the depletion of NK cells. Depletion of CD4+ T cells, CD8+ T cells or NK cells in 4T1.2luc tumor-bearing mice resulted in a significant reduction in median survival. Splenocytes from 4T1.2luc tumor bearing mice demonstrated Luc-specific cytotoxicity upon ex vivo restimulation with Luc peptides, and a stronger response was observed when cells were restimulated with 1 µg/ml compared to 10 µg/ml Luc peptides. These findings suggest that luciferase may serve as a tumor antigen in the 4T1.2luc tumor model, and host immune components (CD4+ and CD8+ T cells) may play a role in controlling 4T1.2luc primary tumor growth and survival. The stronger immunogenicity of 4T1.2luc conferred by luciferase will enable future immunotherapeutic research that ultimately may benefit breast cancer patients. Citation Format: Yitong Xu, Shizhao Duan, William J. Turbitt, Andrea M. Mastro, Connie J. Rogers. CD4+and CD8+T cells influence 4T1.2luc mammary tumor growth and survival [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 586.

Abstract 4147: EOS100850, an A2Areceptor antagonist with prolonged pharmacodynamic activity, mediates the generation ofspecific durable immune responses in a murine breast cancer model

Abstract We and others have shown that extracellular adenosine (Ado), acting predominantly through the A2A receptor (A2AR), mediates immunosuppression through different modes of action. These include suppression of Th1 responses and cytotoxicity as well as increasing the activity of Tregs and MDSC. We therefore developed EOS100850, a novel, non-brain penetrant and highly selective inhibitor of A2AR with sub-nanomolar Ki. As compared to other A2AR antagonist in development, EOS100850 maintain its potency in high Ado environment (>10uM). To better characterize the Ado levels found in the tumor microenvironment, we first assessed extra-cellular Ado concentrations by microdialysis. Concentrations measured in mouse and human tumors were elevated compared to normal skin and confirmed that immune cells infiltrating the tumor microenviroment are confronted with elevated suppressive Ado levels. To demonstrate and compare the ability of EOS100850 to inhibit A2AR signaling in vivo with other molecules in development, we measured the in vivo pharmacodynamic (PD) modulation of A2AR activity by investigating its ability to inhibit CREB phosphorylation (pCREB) following activation of the Ado receptor by NECA, an A2AR agonist. While EOS100850 demonstrated complete inhibition of pCREB 30 minutes after oral gavage at a dose as low as 0.1 mg/kg, other A2AR inhibitors required much higher concentrations to achieve a similar effect. Remarkably, 24 hours after dosing, more than 60% of inhibition of pCREB was still observed, at a dose of 10 mg/kg when the EOS100850 antagonist was no longer detectable in the plasma. These results demonstrate that EOS100850 has a PD activity that extends well beyond its PK, which can be explained by a long residence time. To demonstrate the in vivo antitumor efficacy of EOS100850, we used the murine syngeneic breast cancer model, EMT-6. EOS100850 administered in combination with anti-CTLA-4 mAb increased the number of complete responders (CR) and significantly reduced tumor growth compared with anti-CTLA-4 single agent treatment. Furthermore, the CR were protected against re-inoculation with EMT6 but not irrelevant CT26 tumor. Lastly, flow cytometry and RNAseq analysis, performed on tumors taken during the course of treatment, confirmed the potential of EOS100850 as single agent or in combination with anti-CTLA4 to induce a pro-inflammatory response that correlate with tumor regression. This best-in-class profile of EOS100850 A2A receptor antagonist supports its progression into clinical development. Citation Format: Romain Pirson, Anne-Catherine Michaux, Diane Jamart, Julie Preillon, Kim Frederix, Paola Basilico, Chiara Martinoli, Xavier Leroy, Gregory Driessens, Erica Houthuys, Scott Chappel, Stefano Crosignani, Reece Marillier. EOS100850, an A2Areceptor antagonist with prolonged pharmacodynamic activity, mediates the generation ofspecific durable immune responses in a murine breast cancer model [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 4147.

Abstract 4089: Antitumor activity of eribulin in combination with anti-PD1 antibody in a mouse syngeneic breast cancer model

Abstract Eribulin is a synthetic analog of halichondrin B, which is isolated from the marine sponge natural product, as the first halichondrin class of microtubule dynamics inhibitors. Its clinical formulation is currently approved for advanced breast cancer (BC) and advanced liposarcoma in numerous countries. Eribulin shows antitumor activity through cell death induction by an antimitotic effect, and modulation of tumor microenvironment that include vascular remodeling, and reversal of EMT in some of preclinical subcutaneous xenograft models. Effects of eribulin on tumor microenvironment led us to test combination of eribulin with anti-PD1 blockades, and Phase2 clinical study of eribulin in combination with pembrolizumab for metastatic BC patients is in progress. In this study, we investigated antitumor activity of eribulin in combination with anti-PD1 mAb in preclinical mouse BC model using 4T1 cells. Because parent 4T1 cells express P-glycoprotein (Pgp) and eribulin is a substrate of Pgp, we established Pgp-knockout (Pgp-KO) mouse 4T1 BC cells to examine combination antitumor activity of eribulin with anti-PD1 antibody. We used a clone 31 of Pgp-KO 4T1 (#31) cells, which maintains similar tumor phenotypes to parent 4T1 orthotropic transplantation (OT) models, such as growth rate and formation of metastasis. Eribulin inhibited in vitro proliferation of Pgp-KO 4T1 (#31) cells with a lower concentration compared to parent 4T1 cells. Eribulin were intravenous administered at 1 mg/kg with either Q7Dx2 or Q4Dx3 in the combination with anti-PD1 mAb, 200 µg/head, Q3Dx10 in the Pgp-KO 4T1 (#31) OT model (6 mice per a treatment group). Anti-PD1 mAb did not clearly show in vivo antitumor activity. Treatments of eribulin by Q4Dx3 showed enhanced antitumor activity in the combination compared to each single treatment, and all tumors became non-palpable in the combination group. On the other hand, weekly treatments of eribulin did not enhance antitumor activity in the combination with anti-PD1 mAb, although eribulin single treatment significantly inhibited in vivo tumor growth of Pgp-KO 4T1 (#31) tumors. These preclinical results provide preclinical rationale for clinical testing of eribulin in combination with anti-PD1 blockades in patients with BC, and further preclinical analysis of MOA of combination antitumor activity of eribulin with anti-PD1 antibody is warranted. Citation Format: Taro Semba, Kimiyo Tabata, Yoichi Ozawa, Saori Watanabe Miyano, Junji Matsui, Yasuhiro Funahashi. Antitumor activity of eribulin in combination with anti-PD1 antibody in a mouse syngeneic breast cancer model [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 4089.

Abstract LB-197: NBE-002, an anthracycline-based immune-stimulatory antibody drug conjugate (iADC) targeting ROR1 for the treatment of triple-negative breast cancer

Abstract Introduction: We report on the development and preclinical validation of NBE-002, a next-generation antibody drug conjugate (ADC) for the treatment of triple-negative breast cancer (TNBC). NBE-002 consists of a humanized monoclonal antibody directed against the receptor tyrosine kinase ROR1, expressed on the surface of numerous solid cancers including TNBC, and is site-specifically conjugated to a derivative of the highly potent anthracycline PNU-159682. Results: Therapeutic efficacy of NBE-002 was evaluated in ROR1-low/-intermediate/-high patient-derived xenograft (PDX) models of TNBC, lung adenocarcinoma, ovarian carcinoma, and a variety of sarcomas. NBE-002 was found to display significant anti-tumor activity in each indication. The most pronounced effect was achieved in TNBC, where complete tumor regression was observed already at the lowest ADC dose tested (0.33 mg/kg), even in models expressing low levels of ROR1 (H-score ≤70). Since these PDX studies were performed in immune-compromised hosts, and anthracyclines are known inducers of immunogenic cell death, anti-tumor activity was also investigated in syngeneic tumor models in immune-competent hosts. Anti-tumor activity of PNU-ADCs involved activation of the immune system, as shown by evaluation of NBE-002 or a Trastuzumab-PNU conjugate (T-PNU) in ROR1- or HER2-positive syngeneic breast cancer models, respectively. Depletion of CD8 T cells severely reduced anti-tumor activity, demonstrating an important role for T cells in driving tumor regression. Furthermore, when tumor free animals were re-challenged with the same tumor, tumor growth was inhibited in the absence of any further ADC administration, indicating the development of an immunological memory. Notably, combination of ADC and checkpoint inhibition, such as α-PD1 or α-CTLA4, significantly enhanced tumor eradication following the treatment. Conclusion: Our results demonstrate that NBE-002 is a highly effective and promising targeted therapeutic for the treatment of ROR1 positive TNBC and potentially other solid tumor indications that warrants clinical development. Considering the pronounced immune-modulatory functions of the PNU payload, NBE-002 may be particularly well suited for combination therapy with immune checkpoint inhibitors. NBE-002 is currently undergoing GMP manufacturing and initiation of clinical studies is expected in mid-2020. Citation Format: Roger R. Beerli, Lorenz Waldmeier, Rémy Gébleux, Francesca Pretto, Ulf Grawunder. NBE-002, an anthracycline-based immune-stimulatory antibody drug conjugate (iADC) targeting ROR1 for the treatment of triple-negative breast cancer [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 LB-197.

Tspan8 is expressed in breast cancer and regulates E-cadherin/catenin signalling and metastasis accompanied by increased circulating extracellular vesicles.

Tspan8 exhibits a functional role in many cancer types including pancreatic, colorectal, oesophagus carcinoma, and melanoma. We present a first study on the expression and function of Tspan8 in breast cancer. Tspan8 protein was present in the majority of human primary breast cancer lesions and metastases in the brain, bone, lung, and liver. In a syngeneic rat breast cancer model, Tspan8+ tumours formed multiple liver and spleen metastases, while Tspan8− tumours exhibited a significantly diminished ability to metastasise, indicating a role of Tspan8 in metastases. Addressing the underlying molecular mechanisms, we discovered that Tspan8 can mediate up‐regulation of E‐cadherin and down‐regulation of Twist, p120‐catenin, and β‐catenin target genes accompanied by the change of cell phenotype, resembling the mesenchymal–epithelial transition. Furthermore, Tspan8+ cells exhibited enhanced cell–cell adhesion, diminished motility, and decreased sensitivity to irradiation. As a regulator of the content and function of extracellular vesicles (EVs), Tspan8 mediated a several‐fold increase in EV number in cell culture and the circulation of tumour‐bearing animals. We observed increased protein levels of E‐cadherin and p120‐catenin in these EVs; furthermore, Tspan8 and p120‐catenin were co‐immunoprecipitated, indicating that they may interact with each other. Altogether, our findings show the presence of Tspan8 in breast cancer primary lesion and metastases and indicate its role as a regulator of cell behaviour and EV release in breast cancer. © 2019 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of Pathological Society of Great Britain and Ireland.

Epithelial-type systemic breast carcinoma cells with a restricted mesenchymal transition are a major source of metastasis.

Carcinoma cells undergo epithelial-mesenchymal transition (EMT); however, contributions of EMT heterogeneity to disease progression remain a matter of debate. Here, we addressed the EMT status of ex vivo cultured circulating and disseminated tumor cells (CTCs/DTCs) in a syngeneic mouse model of metastatic breast cancer (MBC). Epithelial-type CTCs with a restricted mesenchymal transition had the strongest lung metastases formation ability, whereas mesenchymal-type CTCs showed limited metastatic ability. EpCAM expression served as a surrogate marker to evaluate the EMT heterogeneity of clinical samples from MBC, including metastases, CTCs, and DTCs. The proportion of epithelial-type CTCs, and especially DTCs, correlated with distant metastases and poorer outcome of patients with MBC. This study fosters our understanding of EMT in metastasis and underpins heterogeneous EMT phenotypes as important parameters for tumor prognosis and treatment. We further suggest that EpCAM-dependent CTC isolation systems will underestimate CTC numbers but will quantify clinically relevant metastatic cells.

Pan-TAM Tyrosine Kinase Inhibitor BMS-777607 Enhances Anti-PD-1 mAb Efficacy in a Murine Model of Triple-Negative Breast Cancer.

Tyro3, Axl, and Mertk (TAM) represent a family of homologous tyrosine kinase receptors known for their functional role in phosphatidylserine (PS)-dependent clearance of apoptotic cells and also for their immune modulatory functions in the resolution of inflammation. Previous studies in our laboratory have shown that Gas6/PS-mediated activation of TAM receptors on tumor cells leads to subsequent upregulation of PD-L1, defining a putative PS→TAM receptor→PD-L1 inhibitory signaling axis in the cancer microenvironment that may promote tolerance. In this study, we tested combinations of TAM inhibitors and PD-1 mAbs in a syngeneic orthotopic E0771 murine triple-negative breast cancer model, whereby tumor-bearing mice were treated with pan-TAM kinase inhibitor (BMS-777607) or anti-PD-1 alone or in combination. Tyro3, Axl, and Mertk were differentially expressed on multiple cell subtypes in the tumor microenvironment. Although monotherapeutic administration of either pan-TAM kinase inhibitor (BMS-777607) or anti-PD-1 mAb therapy showed partial antitumor activity, combined treatment of BMS-777607 with anti-PD-1 significantly decreased tumor growth and incidence of lung metastasis. Moreover, combined treatment with BMS-777607 and anti-PD-1 showed increased infiltration of immune stimulatory T cells versus either monotherapy treatment alone. RNA NanoString profiling showed enhanced infiltration of antitumor effector T cells and a skewed immunogenic immune profile. Proinflammatory cytokines increased with combinational treatment. Together, these studies indicate that pan-TAM inhibitor BMS-777607 cooperates with anti-PD-1 in a syngeneic mouse model for triple-negative breast cancer and highlights the clinical potential for this combined therapy. SIGNIFICANCE: These findings show that pan-inhibition of TAM receptors in combination with anti-PD-1 may have clinical value as cancer therapeutics to promote an inflammatory tumor microenvironment and improve host antitumor immunity.

Secretomes from metastatic breast cancer cells, enriched for a prognostically unfavorable LCN2 axis, induce anti-inflammatory MSC actions and a tumor-supportive premetastatic lung.

ABSTRACTCancer metastasis is responsible for the clear majority of cancer-related deaths. Survival and expansion of cancer cells at secondary sites requires that these premetastatic microenvironments be primed by primary tumor cells and their secreted factors. Efforts to date have been limited by immune-deficient in vivo models and/or the need for finely-tuned analysis time points that reduce contributions from early-disseminating cancer cells. In this regard, we developed a tumor cell-free syngeneic breast cancer model for characterizing tumor cell secretome-mediated reprogramming of premetastatic tissues. We demonstrate that secretomes from metastatic breast cancer cells differentially regulate the lung and brain, promoting a tumor-supportive lung microenvironment with both elevated CD73 expression and decreased TNFα expression. Using in vitro models of CD73-positive mesenchymal stem cells (MSCs) and macrophages/monocytes, we tested whether MSCs can mediate anti-inflammatory effects of metastatic breast cancer cells. Notably, conditioned media from metastatic Py230 cells reprogrammed the secretomes of MSCs toward an anti-inflammatory state. Mining transcriptome data from Py8119 and Py230 cells revealed a lipocalin 2 (LCN2) axis that is selectively expressed in the metastatic Py230 cells, predicts poor breast cancer patient survival and is elevated in circulating serum of mice chronically treated with conditioned media from Py230 cells. Taken together, these results establish the utility of an immune-competent tumor cell-free model for characterizing the mechanisms of breast cancer cell priming of the premetastatic niche, demonstrate that MSCs can mediate the anti-inflammatory effects of metastatic breast cancer cells and substantiate LCN2 as a promising therapeutic target for blocking breast cancer progression.

Abstract A218: Modulating glucocorticoid receptor-mediated signaling for enhancement of cancer immunotherapy

Abstract Glucocorticoids are a class of steroid hormones essential to the modulation of a number of physiologic processes, including cellular metabolism, neural development and immune system function. Functions of glucocorticoids are mediated through the glucocorticoid receptor (GR). Synthetic glucocorticoids are front-line therapies in the clinic for leukemia and lymphoma due to their potent induction of lymphocyte death. Glucocorticoids are also prescribed as adjuvant therapies to chemotherapies to alleviate adverse effects including nausea, emesis and allergic reactions. In the current regimen of immune checkpoint blockade therapies against solid tumors, glucocorticoids are incorporated to manage symptoms related to acute immune hyperactivation. However, given that glucocorticoids preferentially cause lymphocyte death, their inclusion in immune checkpoint blockade regimens which capitalize on cytotoxic T-cell-mediated killing of tumor cells is controversial. The precise effects of systematic glucocorticoids use on tumor development, and particularly, on the tumor-immune microenvironment comprised tumor-infiltrating leukocytes (TILs) remains unknown. Conversely, methods to antagonize glucocorticoid signaling could potentially boost lymphocyte number and function and potentiate cytotoxic T-cell-mediated killing of tumor cells. Presently, the treatment of “immune cold” solid tumor types remains a challenge for immunotherapy due low level of TILs and presence of immunosuppressive cells (including regulatory T-cells and myeloid-derived suppressor cells) in the tumor microenvironment. Pharmacologic modulation of GR presents a unique opportunity to regulate immune cell homeostasis and function with the goal of promoting a favorable tumor immune microenvironment to augment the effectiveness of cancer immunotherapies in solid tumors. Our research seeks to understand the impact of glucocorticoid modulation in the context of immune checkpoint blockade therapies in preclinical models, and to provide a rationale for selective pharmacologic modulation of GR to promote a favorable tumor immune microenvironment and increased efficacy of immunotherapies in solid tumors. We investigated GR activation and inhibition on the immune tumor microenvironment in murine syngeneic melanoma (B16), breast cancer (4T1) and colorectal cancer (CT26) models. Mice bearing allograft tumors were randomized into groups receiving synthetic glucocorticoids or GR inhibitors in combination with anti-PD-1 and anti-CTLA4 therapies. Following treatment, tumors were assessed for composition of intratumoral leukocyte infiltration by flow cytometry phenotyping. In the mouse breast cancer model (4T1), we found evidence supporting substantial lymphodepletion by dexamethasone (a synthetic glucocorticoid compound). Treatment with PD-1 and CTLA-4 blocking monoclonal antibodies increased lymphocyte infiltration into the tumor. Interestingly, immune checkpoint blockade therapies significantly raised the number of more CD4+ T helper cells than CD8+ cytotoxic T-cells in the tumor. Our future plan includes investigation of the effects of GR inhibition on TILs in murine solid tumor models, and utilization of RNA-sequencing on TILs for identification of genes and molecular pathways that provide opportunities to boost TIL function within the tumor. In summary, our work can provide insight into the fundamental molecular mechanisms by which the glucocorticoid receptor modulates the tumor immune microenvironment and provide novel avenues for enhancing cancer immunotherapies in solid tumors. Citation Format: Qin Tang, Myles A. Brown. Modulating glucocorticoid receptor-mediated signaling for enhancement of cancer immunotherapy [abstract]. In: Proceedings of the Fourth CRI-CIMT-EATI-AACR International Cancer Immunotherapy Conference: Translating Science into Survival; Sept 30-Oct 3, 2018; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2019;7(2 Suppl):Abstract nr A218.

A novel anti-HER2 anthracycline-based antibody-drug conjugate induces adaptive anti-tumor immunity and potentiates PD-1 blockade in breast cancer

Increasing evidence suggests that antibody-drug conjugates (ADCs) can enhance anti-tumor immunity and improve clinical outcome. Here, we elucidate the therapeutic efficacy and immune-mediated mechanisms of a novel HER2-targeting ADC bearing a potent anthracycline derivate as payload (T-PNU) in a human HER2-expressing syngeneic breast cancer model resistant to trastuzumab and ado-trastuzumab emtansine. Mechanistically, the anthracycline component of the novel ADC induced immunogenic cell death leading to exposure and secretion of danger-associated molecular signals. RNA sequencing derived immunogenomic signatures and TCRβ clonotype analysis of tumor-infiltrating lymphocytes revealed a prominent role of the adaptive immune system in the regulation of T-PNU mediated anti-cancer activity. Depletion of CD8 T cells severely reduced T-PNU efficacy, thus confirming the role of cytotoxic T cells as drivers of the T-PNU mediated anti-tumor immune response. Furthermore, T-PNU therapy promoted immunological memory formation in tumor-bearing animals protecting those from tumor rechallenge. Finally, the combination of T-PNU and checkpoint inhibition, such as α-PD1, significantly enhanced tumor eradication following the treatment. In summary, a novel PNU-armed, HER2-targeting ADC elicited long-lasting immune protection in a murine orthotopic breast cancer model resistant to other HER2-directed therapies. Our findings delineate the therapeutic potential of this novel ADC payload and support its clinical development for breast cancer patients and potentially other HER2 expressing malignancies.

Multiwalled Carbon Nanotubes for Combination Therapy: a Biodistribution and Efficacy Pilot Study.

A drug delivery system (DDS) for combined therapy, based on a short oxidized multiwalled carbon nanotube, is reported. It was prepared exploiting a synthetic approach which allowed loading of two drugs, doxorubicin and metformin, the targeting agent biotin and a radiolabeling tag, to enable labeling with Ga-68 or Cu-64 in order to perform an extensive biodistribution study by PET/CT. The DDS biodistribution profile changes with different administration methods. Once administered at therapeutic doses, the DDS showed a marginal beneficial effect on 4T1 tumor bearing mice, a syngeneic and orthotopic model of triple negative breast cancer, with survival extended by 1 week and 2 days in 20% of the mice. This is encouraging given the aggressiveness of the 4T1 tumor. Furthermore our DDS was well tolerated, ruling out concerns regarding the toxicity of carbon nanotubes.

Choline Kinase Alpha Inhibition by EB-3D Triggers Cellular Senescence, Reduces Tumor Growth and Metastatic Dissemination in Breast Cancer.

Choline kinase (ChoK) is the first enzyme of the Kennedy pathway leading to the biosynthesis of phosphatidylcholine (PtdCho), the most abundant phospholipid in eukaryotic cell membranes. EB-3D is a novel choline kinase α1 (ChoKα1) inhibitor with potent antiproliferative activity against a panel of several cancer cell lines. ChoKα1 is particularly overexpressed and hyperactivated in aggressive breast cancer. By NMR analysis, we demonstrated that EB-3D is able to reduce the synthesis of phosphocholine, and using flow cytometry, immunoblotting, and q-RT-PCR as well as proliferation and invasion assays, we proved that EB-3D strongly impairs breast cancer cell proliferation, migration, and invasion. EB-3D induces senescence in breast cancer cell lines through the activation of the metabolic sensor AMPK and the subsequent dephosphorylation of mTORC1 downstream targets, such as p70S6K, S6 ribosomal protein, and 4E-BP1. Moreover, EB-3D strongly synergizes with drugs commonly used for breast cancer treatment. The antitumorigenic potential of EB-3D was evaluated in vivo in the syngeneic orthotopic E0771 mouse model of breast cancer, where it induces a significant reduction of the tumor mass at low doses. In addition, EB-3D showed an antimetastatic effect in experimental and spontaneous metastasis models. Altogether, our results indicate that EB-3D could be a promising new anticancer agent to improve aggressive breast cancer treatment protocols.

Unique Photochemo-Immuno-Nanoplatform against Orthotopic Xenograft Oral Cancer and Metastatic Syngeneic Breast Cancer.

Sophisticated self-assembly may endow materials with a variety of unique functions that are highly desirable for therapeutic nanoplatform. Herein, we report the coassembly of two structurally defined telodendrimers, each comprised of hydrophilic linear PEG and hydrophobic cholic acid cluster as a basic amphiphilic molecular subunit. One telodendrimer has four added indocyanine green derivatives, leading to excellent photothermal properties; the other telodendrimer has four sulfhydryl groups designed for efficient intersubunit cross-linking, contributing to superior stability during circulation. The coassembled nanoparticle (CPCI-NP) possesses superior photothermal conversion efficiency as well as efficient encapsulation and controlled release of cytotoxic molecules and immunomodulatory agents. CPCI-NP loaded with doxorubicin has proven to be a highly efficacious combination photothermal/chemotherapeutic nanoplatform against orthotopic OSC-3 oral cancer xenograft model. When loaded with imiquimod, a potent small molecule immunostimulant, CPCI-NP is found to be highly effective against 4T1 syngeneic murine breast cancer model, particularly when photothermal/immuno-therapy is given in combination with PD-1 checkpoint blockade antibody. Such triple therapy not only eradicates the light-irradiated primary tumors, but also activates systemic antitumor immunoactivity, causing tumor death at light-unexposed distant tumor sites. This coassembled multifunctional, versatile, and easily scalable photothermal immuno-nanoplatform shows great promise for clinical translation.