Metastatic Breast Cancer Model Research Articles

TAMI-06. TUMOR CELL-DERIVED CYTOKINE EXPRESSION CHANGES ASSOCIATED WITH BRAIN METASTASIS IN A SYNGENEIC MOUSE MODEL OF BREAST CANCER

Abstract Breast cancer is the most common malignancy in women in the United States, and brain metastases occur in almost a third of patients with metastatic dissemination. Immunoediting is a critical component of metastatic tumor cell elimination, and tumor clones that develop immune-escape mechanisms are associated with progression and metastatic dissemination. We hypothesized that breast cancer brain metastatic cells harbor immunomodulatory cytokine expression changes that promote an immunosuppressive environment to avoid immune cell-mediated elimination. To study this, a syngeneic mouse model of metastatic breast cancer was used. A brain metastatic line derived from the 4T1 breast cancer parental cell line was created by serially selecting brain metastatic populations of cells after intracardiac injection (4T1 BrM). A gene-expression analysis using an 800-gene cancer immunology-specific microarray panel was performed comparing the 4T1 parental and 4T1 BrM lines. 4T1 BrM cells demonstrate gene expression changes promoting immunosuppression including significant upregulation of IL18 and Lgals9 (Galectin-9) and downregulation of CD40, IL2rg, CCL2, and EOMES. When compared to 4T1 parental lines, the 4T1 BrM line demonstrated decreased expression of CCL2 and increased expression of GM-CSF on a cytokine array, corresponding to results obtained from gene expression analysis. These results suggest tumor-intrinsic cytokine expression changes that may mediate an immunosuppressive environment.

Novel Nanoparticle-Based Cancer Treatment, Effectively Inhibits Lung Metastases and Improves Survival in a Murine Breast Cancer Model.

Sarah Nanoparticles (SaNPs) are unique multicore iron oxide-based nanoparticles, developed for the treatment of advanced cancer, following standard care, through the selective delivery of thermal energy to malignant cells upon exposure to an alternating magnetic field. For their therapeutic effect, SaNPs need to accumulate in the tumor. Since the potential accumulation and associated toxicity in normal tissues are an important risk consideration, biodistribution and toxicity were assessed in naïve BALB/c mice. Therapeutic efficacy and the effect on survival were investigated in the 4T1 murine model of metastatic breast cancer. Toxicity evaluation at various timepoints did not reveal any abnormal clinical signs, evidence of alterations in organ function, nor histopathologic adverse target organ toxicity, even after a follow up period of 25 weeks, confirming the safety of SaNP use. The biodistribution evaluation, following SaNP administration, indicated that SaNPs accumulate mainly in the liver and spleen. A comprehensive pharmacokinetics evaluation, demonstrated that the total percentage of SaNPs that accumulated in the blood and vital organs was ~78%, 46%, and 36% after 4, 13, and 25 weeks, respectively, suggesting a time-dependent clearance from the body. Efficacy studies in mice bearing 4T1 metastatic tumors revealed a 49.6% and 70% reduction in the number of lung metastases and their relative size, respectively, in treated vs. control mice, accompanied by a decrease in tumor cell viability in response to treatment. Moreover, SaNP treatment followed by alternating magnetic field exposure significantly improved the survival rate of treated mice compared to the controls. The median survival time was 29 ± 3.8 days in the treated group vs. 21.6 ± 4.9 days in the control, p-value 0.029. These assessments open new avenues for generating SaNPs and alternating magnetic field application as a potential novel therapeutic modality for metastatic cancer patients.

Nanoscale Coordination Polymers for Combined Chemotherapy and Photodynamic Therapy of Metastatic Cancer.

Combination therapy enhances anticancer efficacy through synergistic effects of different drugs/modalities and can potentially address the challenges in the treatment of metastatic diseases. Here we report the design of carb/pyro nanoscale coordination polymer nanoparticles that carry carboplatin (carb) in the core and the photosensitizer pyrolipid (pyro) on the shell for the treatment of metastatic triple negative breast cancer. Upon light irradiation, carb/pyro generated reactive oxygen species to cause severe cell apoptosis and early calreticulin exposure. Upon intravenous injection and local light irradiation, carb/pyro significantly regressed tumor growth in the 4T1 murine metastatic breast cancer model. When combined with an anti-CD47 antibody, carb/pyro with light irradiation completely eradicated primary and metastatic 4T1 tumors in 50% mice. The anticancer efficacy of carb/pyro was also demonstrated in the CT26 murine colorectal cancer model.

Transcriptome analysis of heterogeneity in mouse model of metastatic breast cancer

BackgroundCancer metastasis is a complex process involving the spread of malignant cells from a primary tumor to distal organs. Understanding this cascade at a mechanistic level could provide critical new insights into the disease and potentially reveal new avenues for treatment. Transcriptome profiling of spontaneous cancer models is an attractive method to examine the dynamic changes accompanying tumor cell spread. However, such studies are complicated by the underlying heterogeneity of the cell types involved. The purpose of this study was to examine the transcriptomes of metastatic breast cancer cells using the well-established MMTV-PyMT mouse model.MethodsOrgan-derived metastatic cell lines were harvested from 10 female MMTV-PyMT mice. Cancer cells were isolated and sorted based on the expression of CD44low/EpCAMhigh or CD44high/EpCAMhigh surface markers. RNA from each cell line was extracted and sequenced using the NextSeq 500 Illumina platform. Tissue-specific genes were compared across the different metastatic and primary tumor samples. Reads were mapped to the mouse genome using STAR, and gene expression was quantified using RSEM. Single-cell RNA-seq (scRNA-seq) was performed on select samples using the ddSeq platform by BioRad and analyzed using Seurat v3.2.3. Monocle2 was used to infer pseudo-time progression.ResultsComparison of RNA sequencing data across all cell populations produced distinct gene clusters. Differential gene expression patterns related to CD44 expression, organ tropism, and immunomodulatory signatures were observed. scRNA-seq identified expression profiles based on tissue-dependent niches and clonal heterogeneity. These cohorts of data were narrowed down to identify subsets of genes with high expression and known metastatic propensity. Dot plot analyses further revealed clusters expressing cancer stem cell and cancer dormancy markers. Changes in relevant genes were investigated across pseudo-time and tissue origin using Monocle2. These data revealed transcriptomes that may contribute to sub-clonal evolution and treatment evasion during cancer progression.ConclusionsWe performed a comprehensive transcriptome analysis of tumor heterogeneity and organ tropism during breast cancer metastasis. These data add to our understanding of metastatic progression and highlight targets for breast cancer treatment. These markers could also be used to image the impact of tumor heterogeneity on metastases.

The interactions of Bcl9/Bcl9L with \u03b2-catenin and Pygopus promote breast cancer growth, invasion, and metastasis

Canonical Wnt/β-catenin signaling is an established regulator of cellular state and its critical contributions to tumor initiation, malignant tumor progression and metastasis formation have been demonstrated in various cancer types. Here, we investigated how the binding of β-catenin to the transcriptional coactivators B-cell CLL/lymphoma 9 (Bcl9) and Bcl9-Like (Bcl9L) affected mammary gland carcinogenesis in the MMTV-PyMT transgenic mouse model of metastatic breast cancer. Conditional knockout of both Bcl9 and Bcl9L resulted into tumor cell death. In contrast, disrupting the interaction of Bcl9/Bcl9L with β-catenin, either by deletion of their HD2 domains or by a point mutation in the N-terminal domain of β-catenin (D164A), diminished primary tumor growth and tumor cell proliferation and reduced tumor cell invasion and lung metastasis. In comparison, the disruption of HD1 domain-mediated binding of Bcl9/Bcl9L to Pygopus had only moderate effects. Interestingly, interfering with the β-catenin-Bcl9/Bcl9L-Pygo chain of adapters only partially impaired the transcriptional response of mammary tumor cells to Wnt3a and TGFβ treatments. Together, the results indicate that Bcl9/Bcl9L modulate but are not critically required for canonical Wnt signaling in its contribution to breast cancer growth and malignant progression, a notion consistent with the “just-right” hypothesis of Wnt-driven tumor progression.

Parsing β-catenin’s cell adhesion and Wnt signaling functions in malignant mammary tumor progression

During malignant progression, epithelial cancer cells dissolve their cell-cell adhesion and gain invasive features. By virtue of its dual function, β-catenin contributes to cadherin-mediated cell-cell adhesion, and it determines the transcriptional output of Wnt signaling: via its N terminus, it recruits the signaling coactivators Bcl9 and Pygopus, and via the C terminus, it interacts with the general transcriptional machinery. This duality confounds the simple loss-of-function analysis of Wnt signaling in cancer progression. In many cancer types including breast cancer, the functional contribution of β-catenin's transcriptional activities, as compared to its adhesion functions, to tumor progression has remained elusive. Employing the mouse mammary tumor virus (MMTV)-PyMT mouse model of metastatic breast cancer, we compared the complete elimination of β-catenin with the specific ablation of its signaling outputs in mammary tumor cells. Notably, the complete lack of β-catenin resulted in massive apoptosis of mammary tumor cells. In contrast, the loss of β-catenin's transcriptional activity resulted in a reduction of primary tumor growth, tumor invasion, and metastasis formation in vivo. These phenotypic changes were reflected by stalled cell cycle progression and diminished epithelial-mesenchymal transition (EMT) and cell migration of breast cancer cells in vitro. Transcriptome analysis revealed subsets of genes which were specifically regulated by β-catenin's transcriptional activities upon stimulation with Wnt3a or during TGF-β-induced EMT. Our results uncouple the signaling from the adhesion function of β-catenin and underline the importance of Wnt/β-catenin-dependent transcription in malignant tumor progression of breast cancer.

Radiolabeling and PET\u2013MRI microdosing of the experimental cancer therapeutic, MN-anti-miR10b, demonstrates delivery to metastatic lesions in a murine model of metastatic breast cancer

BackgroundIn our earlier work, we identified microRNA-10b (miR10b) as a master regulator of the viability of metastatic tumor cells. This knowledge allowed us to design a miR10b-targeted therapeutic consisting of an anti-miR10b antagomir conjugated to ultrasmall iron oxide nanoparticles (MN), termed MN-anti-miR10b. In mouse models of breast cancer, we demonstrated that MN-anti-miR10b caused durable regressions of established metastases with no evidence of systemic toxicity. As a first step towards translating MN-anti-miR10b for the treatment of metastatic breast cancer, we needed to determine if MN-anti-miR10b, which is so effective in mice, will also accumulate in human metastases.ResultsIn this study, we devised a method to efficiently radiolabel MN-anti-miR10b with Cu-64 (64Cu) and evaluated the pharmacokinetics and biodistribution of the radiolabeled product at two different doses: a therapeutic dose, referred to as macrodose, corresponding to 64Cu-MN-anti-miR10b co-injected with non-labeled MN-anti-miR10b, and a tracer-level dose of 64Cu-MN-anti-miR10b, referred to as microdose. In addition, we evaluated the uptake of 64Cu-MN-anti-miR10b by metastatic lesions using both in vivo and ex vivo positron emission tomography–magnetic resonance imaging (PET–MRI). A comparable distribution of the therapeutic was observed after administration of a microdose or macrodose. Uptake of the therapeutic by metastatic lymph nodes, lungs, and bone was also demonstrated by PET–MRI with a significantly higher PET signal than in the same organs devoid of metastatic lesions.ConclusionOur results demonstrate that PET–MRI following a microdose injection of the agent will accurately reflect the innate biodistribution of the therapeutic. The tools developed in the present study lay the groundwork for the clinical testing of MN-anti-miR10b and other similar therapeutics in patients with cancer.

Abstract 1187: Optimizing gemcitabine in metastatic TNBC by rational PI3K/AKT combination

Abstract Metastatic triple-negative breast cancer (TNBC) has a median survival of 13 months, and only 5 months if brain metastases are present. Advances in immunotherapy are promising, but only add weeks to a few months of progression-free survival to this guarded prognosis. Thus, there is a large unmet need for innovations that improve patient outcomes in the metastatic setting when patients are refractory to first-line treatment. Recently, we published an innovative strategy to identify multi-omic molecular correlates of drug response in TNBC. From this work, we now apply the strategy to 3D cell culture models and patient derived xenografts (PDXs) that are more representative of in vivo drug response in real patients. In brief, we have tested the dose response for a panel of 387 investigational drugs and plotted this readout against molecular characterization in 35 TNBC cell lines and PDXs. Using linear regression, we have identified statistically significant positively and negatively correlating DNA, RNA, and protein predictors of drug efficacy. From this expanded multi-omic set, we have identified a multi-omic signature for sensitivity to gemcitabine, a drug widely used in TNBC, but at present, without any a priori knowledge of who is likely to benefit. Moreover, we noted a significant correlation of response with MERIT40, an AKT substrate. MERIT40 is critical for localization of BRCA1 to facilitate DNA damage repair. MERIT40 promotes resolution of DNA damage caused by chemotherapy, and thus, based on our published work and supporting literature, PI3K/AKT inhibition is a rational synergistic partner with gemcitabine. Moreover, further supporting our hypothesis, preliminary results indicate Chou-Talalay synergy at therapeutically relevant doses when gemcitabine is combined with PI3K/AKT inhibition. In future studies, we will evaluate the ability of the gemcitabine biomarker to predict response, and the efficacy of the prioritized combinations using ex vivo patient models of metastatic breast cancer established in our laboratory. Citation Format: Nathan M. Merrill, Nathalie M. Vandecan, Athena M. Apfel, Peter J. Ulintz, Liwei Bao, Xu Cheng, Aki Morikawa, Sofia D. Merajver, Matthew B. Soellner. Optimizing gemcitabine in metastatic TNBC by rational PI3K/AKT combination [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 1187.

Abstract 1311: PET-MRI microdosing can determine the delivery of the experimental cancer therapeutic, MN-anti-miR10b, to metastatic lesions in a murine model of breast cancer

Abstract Conventional therapies, which are effective against primary cancer, oftentimes are not successful at treating metastatic cancer. In our earlier work, we identified miRNA-10b as a master regulator of the viability of metastatic tumor cells. This knowledge allowed us to design a miR-10b inhibitor, termed MN-anti-miR10b, capable of delivery to metastatic sites via intravenous injection. In mouse models of breast cancer, we demonstrated that MN-anti-miR10b, when combined with a low-dose cytostatic, caused lifelong complete regressions of established metastases with no evidence of systemic toxicity. As a first step towards translating MN-anti-miR10b for the treatment of metastatic breast cancer, we needed to determine if MN-anti-miR10b, which is so effective in mice, will also accumulate in human metastases. Towards that goal, we developed a method to radiolabel MN-anti-miR10b with Cu-64, inject a microdose of the radiolabeled MN-anti-miR10b into murine models of metastatic breast cancer, and determine the uptake of the therapeutic by the metastatic lesions using positron emission tomography-magnetic resonance imaging (PET-MRI). Specifically, we injected a microdose of 64Cu-MN-anti-miR10b into mice bearing luciferase-expressing metastatic breast adenocarcinoma and performed in vivo bioluminescence (BLI) and PET-MRI to assess uptake by the metastatic lesions. The lesions were identified by BLI, based on their luciferase expression. PET-MRI clearly demonstrated uptake of 64Cu-MN-anti-miR10b, measurable at a microdose. Ex vivo PET-MRI confirmed that the activity was associated with the metastatic lesions, as identified at necropsy. Uptake by metastatic organs was also demonstrated by scintigraphy and shown to be significantly higher than in the same organs devoid of BLI-detectable metastatic lesions. Finally, 64Cu-MN-anti-miR10b injected as a non-carrier added microdose had comparable biodistribution to that injected at a standard therapeutic dose. The value of the current study is in setting the stage for clinical PET-MRI microdosing, which would be able to answer a key question on the path to drug development without the associated cost to do a full clinical trial – the question of delivery to the target organ sites. Since the PET technique is sensitive enough to determine the concentration of radiolabeled drug with sensitivity approaching the sub-picomolar range, as little as a microgram of the radiolabeled drug is sufficient to perform a PET-MRI study in humans. In addition to proving delivery, the microdosing studies will reveal the pharmacokinetic behavior of MN-anti-miR10b which will allow one to establish dosing during therapy. Finally, in clinical trials, one can use microdosing PET-MRI to select patients for treatment, based on which patients' metastases accumulate the therapeutic. Citation Format: Marianne Le Fur, Byunghee Yoo, Nicholas Rotile, Pamela Pantazopoulos, Alana Ross, Iris Zhou, Peter Caravan, Zdravka Medarova. PET-MRI microdosing can determine the delivery of the experimental cancer therapeutic, MN-anti-miR10b, to metastatic lesions in a murine model of breast cancer [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 1311.

Abstract 2849: Shc1 as a potential metastasis driver gene of breast cancer

Abstract In the US, women diagnosed with non-metastatic breast cancer have a 5-year survival rate of approximately 98.8%. However, if women develop metastases the survival rate drops to 27.4%, highlighting the need for a better understanding of the mechanisms that regulate and drive metastasis. Somatically acquired mutations that drive metastasis were originally hypothesized by Nowell in the 1970s; however, to date few candidate metastasis driver mutations have been identified and the role of these mutations in metastasis are not well-understood. Using the PyMT genetically engineered mouse model of luminal metastatic breast cancer, previous work by our group performed whole exome sequencing on pairs of matched primary tumor and metastatic tissues from 65 mice. Analysis of this sequencing data identified recurrent metastasis-specific mutations in members of the Ras signaling pathway. One of the members was the SHC Adaptor Protein 1 (Shc1). Five PyMT animals carried different nucleotide substitutions within the same proline 561 codon that falls within the SH2 domain, suggesting an activating role in metastasis. Furthermore, changing this proline mutation to another amino acid could induce a critical change to the Shc1 protein structure and its function. To investigate the role of Shc1 in breast cancer metastasis, CRISPR was performed engineer Shc1 P561 mutations for in vitro and in vivo assays using a series of mouse mammary cancer cell lines and allograft mouse models. Preliminary evidence suggests the P561 mutations have a role in metastasis. Future experiments will address the consequences of the P561 mutation on Shc1 binding partner interactions and downstream signaling and its effects on breast cancer metastasis. Citation Format: Megan Majocha, Kent Hunter, Christina Ross. Shc1 as a potential metastasis driver gene of breast cancer [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 2849.

Docetaxel encapsulation in nanoscale assembly micelles of folate-PEG-docetaxel conjugates for targeted fighting against metastatic breast cancer in vitro and in vivo

Due to the high frequency and mortality of breast cancer, developing efficient targeted drug delivery systems for frightening against this malignancy is among cancer research priorities.The aim of this study was to synthesize a targeted micellar formulation of docetaxel (DTX) using DTX, folic acid (FA) and polyethylene glycol (PEG) conjugates as building blocks.In the current study, two therapeutic polymers consisting of FA-PEG-DTX and PEG-DTX conjugates were synthesized and implemented to form folate-targeted and non-targeted micelles. Dissipative particle dynamics (DPD) method was used to simulate the behavior of the nanoparticle.The anti-cancer drug, DTX was loaded in to the micelles via solvent switching method in order to increase its solubility and stability. The cytotoxicity of the targeted and non-targeted formulations was evaluated against 4T1 and CHO cell lines. In vivo therapeutic efficiency was studied using ectopic tumor model of metastatic breast cancer, 4T1, in Female BALB/c mice. The successful synthesis of therapeutic polymers, FA-PEG-DTX and PEG-DTX were confirmed implementing 1HNMR spectral analysis. The size of DTX-loaded non-targeted and targeted micelles were 176.3 ± 8.3 and 181 ± 10.1 nm with PDI of 0.23 and 0.17, respectively. Loading efficiencies of DTX in non-targeted and targeted micelles were obtained to be 85% and 82%, respectively. In vitro release study at pH = 7.4 and pH = 5.4 showed a controlled and continuous drug release for both formulations, that was faster at pH = 5.4 (100% drug release within 120 h) than at pH = 7.4 (80% drug release within 150 h). The targeted formulation showed a significant higher cytotoxicity against 4T1 breast cancer cells (high expression of folate receptor) within the range of 12.5 to 200 μg/mL in comparison with no-targeted one. However, there was no significant difference between the cytotoxicity of the targeted and non-targeted formulations against CHO cell line as low-expressed cell line. In accordance with in vitro investigation, in vivo studies verified the ideal anti-tumor efficacy of the targeted formulation compared to Taxotere and non-targeted formulation. Based on the obtained data, FA-targeted DTX-loaded nano-micelles significantly increased the therapeutic efficacy of DTX and therefore can be considered as a new potent platform for breast cancer chemotherapy.

Cyto-KIK: A phase II trial of cytoreductive surgery in kidney cancer plus immunotherapy (nivolumab) and targeted kinase inhibition (cabozantinib).

TPS4598 Background: Despite recent therapeutic advancements in metastatic renal cell carcinoma (mRCC), only 5-10% of patients will achieve a complete response (CR) to therapy. Cytoreductive nephrectomy removes a large portion of the tumor which may be a source of immunosuppression driven by tumor cell-intrinsic factors in the tumor microenvironment. A pre-clinical orthotopic mouse model of aggressive metastatic triple negative breast cancer showed that neoadjuvant anti-PD-1 checkpoint inhibition generated enhanced and sustained antitumor immune responses with improved survival compared to adjuvant therapy (Liu J et al. Cancer Discov. 2016:1382). Clinical validation of improved outcomes with neoadjuvant compared to adjuvant immune checkpoint inhibitors has been demonstrated in trials for patients with non-small cell lung cancer, advanced melanoma, and recurrent glioblastoma (Forde, P.M., et al. N Engl J Med. 2018:1976; Amaria, R.N., et al Nat Med. 2018:1649; Cloughesy T.F., et al. Nat Med 2019:477). Recent data from a phase III trial in subjects with untreated mRCC, demonstrated the superiority of combination cabozantinib and nivolumab over sunitinib and established a new standard of care for mRCC (Choueiri T.K., et al. Annals of Onc, 2020;31 (suppl; abstr 6960). We hypothesize that if tumor specific immune responses to immunotherapy are greatest prior to nephrectomy, then treatment with nivolumab (nivo) and cabozantinib (cabo) prior to cytoreductive nephrectomy will lead to maximal peripheral and intra-tumoral specific immune responses and higher rates of CR during the course of treatment. Methods: This is an open label phase II, multicenter clinical trial of combination nivo and cabo prior to cytoreductive nephrectomy in patients with mRCC (NCT04322955). 48 treatment- naïve subjects with radiological or histological diagnosis of mRCC will be enrolled with the primary endpoint of CR rate according to RECIST version 1.1. Subjects will receive cabo (40mg) daily and nivo (480mg) every 4 weeks for 12 weeks prior to nephrectomy and a 3+3 design will be used to evaluate the safety of the interval (21 or 14 days) between the discontinuation of cabo and nephrectomy. Post-operatively, subjects will resume treatment with cabo and nivo until evidence of disease progression. Secondary endpoints include median size reduction of the primary tumor, response rate, PFS, OS, and surgical outcomes using the Clavien-Dindo classification system. Tissue based assays will quantify treatment related changes in the renal tumor microenvironment through polychromatic immunofluorescence, single cell RNA sequencing of the biopsy and nephrectomy specimen, and multiplex assessment of circulating serum cytokines. Dynamic contrast-enhanced MRI will be performed in a subset of subjects to assess radiologic correlates of response. The study is currently open to enrollment. Clinical trial information: NCT04322955.

Highlights from Recent Cancer Literature

Highlights from Recent Cancer Literature

PD1 Blockade of Tumor-primed CD4 T Cells Instigates Protective Immunity Against Tumor Rechallenge.

Abstract Blockade of immune checkpoint PD1 in cancer patients results in remarkable therapeutic benefits. Revealing mechanisms underlying this revolutionary therapy is critical to identifying biomarkers for predicting responses to this treatment and developing novel PD1 blockade combined therapeutic regimens for broader clinical efficacy. Rejuvenation of preexisting intratumoral ‘exhausted’ T cells and recruitment of peripheral antitumor T cells into tumors have been suggested to explain how PD1 blockade works, but mechanisms of action of anti-PD1 therapy remain to be fully delineated. In a clinically relevant mouse model of spontaneous metastatic breast cancer 4T1.2-Neu, we show that effective and durable anti-PD1 therapy depends on systemic immunity, including type 1 conventional dendritic cells (cDC1: CD8α+/CD103+DC), B cells, CD4 and CD8 T cells. Increased circulating and/or tumor-infiltrating IL12+CD103+DC and IL2+CD4/CD8 T cells are associated with the response to anti-PD1 therapy. PD1 blockade results in tumor-primed CD44+IL2+CD4T cells in the periphery regardless of cDC1. In the breast cancer model with adoptive cell transfer (ACT), PD1 blockade-driven CD44+CD4+T cells infiltrate into tumors and ignite protective immunity against new tumors in an IL2-dependent and tumor-specific manner. In a distinct ACT model using enforced IL2-expressing melanoma antigen specific TRP1-transgenic (Tg) CD4 T cells, IL2+TRP1-Tg CD4 T cells control the growth of PD1-resistant endogenous BP or B16 melanoma. These data suggest the prominent role of PD1 blockade on the effectiveness of tumor-primed CD4T cells in tumor-specific immunity.

Macrophages Mediate the Antitumor Effects of the Oncolytic Virus HSV1716 in Mammary Tumors.

Oncolytic viruses (OV) have been shown to activate the antitumor functions of specific immune cells like T cells. Here, we show OV can also reprogram tumor-associated macrophage (TAM) to a less immunosuppressive phenotype. Syngeneic, immunocompetent mouse models of primary breast cancer were established using PyMT-TS1, 4T1, and E0771 cell lines, and a metastatic model of breast cancer was established using the 4T1 cell line. Tumor growth and overall survival was assessed following intravenous administration of the OV, HSV1716 (a modified herpes simplex virus). Infiltration and function of various immune effector cells was assessed by NanoString, flow cytometry of dispersed tumors, and immunofluorescence analysis of tumor sections. HSV1716 administration led to marked tumor shrinkage in primary mammary tumors and a decrease in metastases. This was associated with a significant increase in the recruitment/activation of cytotoxic T cells, a reduction in the presence of regulatory T cells and the reprograming of TAMs towards a pro-inflammatory, less immunosuppressive phenotype. These findings were supported by in vitro data demonstrating that human monocyte-derived macrophages host HSV1716 replication, and that this led to immunogenic macrophage lysis. These events were dependent on macrophage expression of proliferating cell nuclear antigen (PCNA). Finally, the antitumor effect of OV was markedly diminished when TAMs were depleted using clodronate liposomes. Together, our results show that TAMs play an essential role in support of the tumoricidal effect of the OV, HSV1716-they both host viral replication via a novel, PCNA-dependent mechanism and are reprogramed to express a less immunosuppressive phenotype.

Abstract IA001: Deconvoluting the tumor microenvironment to enhance immunotherapy

Abstract Cancer evolution to metastasis (the metastatic cascade) involves not only the acquisition of mutations or epigenetic modifications in oncogenes and tumor suppressor gene but also an interplay with the surrounding tumor microenvironment (TME). The TME promotes every step of the metastatic cascade. In this TME immune cells of both the acquired and innate system are highly represented and generally these act in a pro-tumoral fashion or are inert. Much work has been performed on primary tumors and studies of metastasis have lagged behind despite the fact that ~90% of deaths from solid tumors are caused by this process. We have addressed this issue using mouse models of breast cancer lung and bone metastasis. Our studies have particularly focussed upon metastasis associated macrophages (MAMs) that are distinct from the tumor associated macrophages in primary tumors. We have shown that MAMs and their progenitors promote metastatic seeding and persistent growing in both organs. In the lung particularly we have described complex signalling pathways including the regulation of recruitment of classical monocytes to the metastatic site and stages of MAM differentiation form these monocytes. Our data show not only direct pro-tumoral effects of these cells on tumor cells such as conferring survival signals but also indirect ones via suppression of tumor cell cytotoxic T and NK cell activity (Kitamura et al., 2017; Kitamura et al., 2019; Ma et al., 2020). In parallel we have profiled TAMs in primary human breast and endometrial cancers and shown that they are different from circulating monocytes but surprisingly, are also different from each other. Further bioinformatic analysis and multiplex immunohistochemistry using breast cancer data sets shows that TAM signatures can be derived that predict poor disease specific overall survival and disease sub-classes. These TAMs have many different subtypes within a tumor and thus may play independent roles including anti-tumoral roles particularly in the face of immunotherapy. Exploiting these possibilities for therapy will be discussed (Lopez-Yrigoyen et al., 2020). Kitamura, T., Doughty-Shenton, D., Cassetta, L., Fragkogianni, S., Brownlie, D., Kato, Y., Carragher, N., and Pollard, J.W. (2017). Monocytes Differentiate to Immune Suppressive Precursors of Metastasis-Associated Macrophages in Mouse Models of Metastatic Breast Cancer. Frontiers in immunology 8, 2004. Kitamura, T., Kato, Y., Brownlie, D., Soong, D.Y.H., Sugano, G., Kippen, N., Li, J., Doughty-Shenton, D., Carragher, N., and Pollard, J.W. (2019). Mammary Tumor Cells with High Metastatic Potential Are Hypersensitive to Macrophage-Derived HGF. Cancer immunology research 7, 2052-2064. Lopez-Yrigoyen, M., Cassetta, L., and Pollard, J.W. (2020). Macrophage targeting in cancer. Ann. N. Y. Acad. Sci. Ma, R.Y., Zhang, H., Li, X.F., Zhang, C.B., Selli, C., Tagliavini, G., Lam, A.D., Prost, S., Sims, A.H., Hu, H.Y., et al. (2020). Monocyte-derived macrophages promote breast cancer bone metastasis outgrowth. J. Exp. Med. 217. Citation Format: Jeffrey W. Pollard. Deconvoluting the tumor microenvironment to enhance immunotherapy [abstract]. In: Proceedings of the AACR Virtual Special Conference on the Evolving Tumor Microenvironment in Cancer Progression: Mechanisms and Emerging Therapeutic Opportunities; in association with the Tumor Microenvironment (TME) Working Group; 2021 Jan 11-12. Philadelphia (PA): AACR; Cancer Res 2021;81(5 Suppl):Abstract nr IA001.

Natural killer T cell immunotherapy combined with oncolytic vesicular stomatitis virus or reovirus treatments differentially increases survival in mouse models of ovarian and breast cancer metastasis

BackgroundOncolytic viruses reduce tumor burden in animal models and have generated promising results in clinical trials. However, it is likely that oncolytic viruses will be more effective when used in...

Phase II trial of cytoreductive surgery in kidney cancer plus immunotherapy (nivolumab) and targeted kinase inhibition (cabozantinib) (Cyto-KIK).

TPS371 Background: Despite recent therapeutic advancements in metastatic renal cell carcinoma (mRCC), only 5-10% of patients will achieve a complete response (CR) to therapy. Cytoreductive nephrectomy removes a large portion of the tumor which may be a source of immunosuppression driven by tumor cell-intrinsic factors in the tumor microenvironment. A pre-clinical orthotopic mouse model of aggressive metastatic triple negative breast cancer showed that neoadjuvant anti-PD-1 checkpoint inhibition generated enhanced and sustained antitumor immune responses with improved survival compared to adjuvant therapy (Liu J et al. Cancer Discov. 2016:1382). Clinical validation of improved outcomes with neoadjuvant compared to adjuvant immune checkpoint inhibitors has been demonstrated in trials for patients with non-small cell lung cancer, advanced melanoma, and recurrent glioblastoma (Forde, P.M., et al. N Engl J Med. 2018:1976; Amaria, R.N., et al Nat Med. 2018:1649; Cloughesy T.F., et al. Nat Med 2019:477). Recent data from a phase III trial in subjects with untreated mRCC, demonstrated the superiority of combination cabozantinib and nivolumab over sunitinib and established a new standard of care for mRCC (Choueiri T.K., et al. Annals of Onc, 2020;31 (suppl; abstr 6960). We hypothesize that if tumor specific immune responses to immunotherapy are greatest prior to nephrectomy, then treatment with nivolumab (nivo) and cabozantinib (cabo) prior to cytoreductive nephrectomy will lead to maximal peripheral and intra-tumoral specific immune responses and higher rates of CR during the course of treatment. Methods: This is an open label phase II, multicenter clinical trial of combination nivo and cabo prior to cytoreductive nephrectomy in patients with mRCC (NCT04322955). 48 treatment- naïve subjects with radiological or histological diagnosis of mRCC will be enrolled with the primary endpoint of CR rate according to RECIST version 1.1. Subjects will receive cabo (40mg) daily and nivo (480mg) every 4 weeks for 12 weeks prior to nephrectomy and a 3+3 design will be used to evaluate the safety of the interval (21 or 14 days) between the discontinuation of cabo and nephrectomy. Post-operatively, subjects will resume treatment with cabo and nivo until evidence of disease progression. Secondary endpoints include median size reduction of the primary tumor, response rate, PFS, OS, and surgical outcomes using the Clavien-Dindo classification system. Tissue based assays will quantify treatment related changes in the renal tumor microenvironment through polychromatic immunofluorescence, single cell RNA sequencing of the biopsy and nephrectomy specimen, and multiplex assessment of circulating serum cytokines. Dynamic contrast-enhanced MRI will be performed in a subset of subjects to assess radiologic correlates of response. The study is currently open to enrollment. Clinical trial information: AAAS6927 .

Mevalonate Pathway Inhibition Slows Breast Cancer Metastasis via Reduced N-glycosylation Abundance and Branching.

Aberrant N-glycan Golgi remodeling and metabolism are associated with epithelial-mesenchymal transition (EMT) and metastasis in patients with breast cancer. Despite this association, the N-glycosylation pathway has not been successfully targeted in cancer. Here, we show that inhibition of the mevalonate pathway with fluvastatin, a clinically approved drug, reduces both N-glycosylation and N-glycan-branching, essential components of the EMT program and tumor metastasis. This indicates novel cross-talk between N-glycosylation at the endoplasmic reticulum (ER) and N-glycan remodeling at the Golgi. Consistent with this cooperative model between the two spatially separated levels of protein N-glycosylation, fluvastatin-induced tumor cell death was enhanced by loss of Golgi-associated N-acetylglucosaminyltransferases MGAT1 or MGAT5. In a mouse model of postsurgical metastatic breast cancer, adjuvant fluvastatin treatment reduced metastatic burden and improved overall survival. Collectively, these data support the immediate repurposing of fluvastatin as an adjuvant therapeutic to combat metastatic recurrence in breast cancer by targeting protein N-glycosylation at both the ER and Golgi. SIGNIFICANCE: These findings show that metastatic breast cancer cells depend on the fluvastatin-sensitive mevalonate pathway to support protein N-glycosylation, warranting immediate clinical testing of fluvastatin as an adjuvant therapy for breast cancer.

Abstract PS11-38: Ginsenoside rg3 enantiomers in a defined ratio as a novel treatment for metastatic triple negative breast cancer

Abstract Background and Aims: Chemotherapy is the main treatment for metastatic triple negative breast cancer (mTNBC) but many patients develop resistance and progress to metastatic disease, with a 5-year survival rate of 11%. Therefore, more effective and less toxic treatments are required. 20(S)-ginsenoside Rg3 (SRg3) and 20(R)-ginsenoside Rg3 (RRg3) are two pharmacologically active enantiomers extracted from Panax ginseng. In our studies, we have shown that these two enantiomers have stereoselective activities in inhibition of proliferation, migration and invasion of triple-negative breast cancer (TNBC) cells, and hence, could be considered as two separate drugs that can be combined in a synergistic dose (S+R). Based on the inhibitory effects of single enantiomers on loop formation of human umbilical vein endothelial cells (HUVEC), we have optimized the concentrations of Rg3 enantiomers to be combined that have shown time- and dose-dependent inhibition of loop formation and migration of human and mouse endothelial cell lines. The aim of the current study was to show the mechanisms of S+R in inhibition of angiogenesis in vitro and its efficacy in inhibiting metastasis in vivo. Methodology: Molecular docking was used to study the Rg3- vascular endothelial growth factor receptor (VEGFR) 2 interaction, and the VEGF bioassay kit (Promega) was used to show this interaction in vitro. ELISA, quantitative-PCR and western blot (WB) were used to study the expression of transcript and protein levels of signalling molecules. Two TNBC cell lines (MDA-MB-231 and HCC1143) were cultured as mammospheres and the effects of S+R on formation of mammospheres, viability (trypan blue assay and flow cytometric analysis of apoptosis or cell cycle arrest) and expression of CD44+ cells (flow cytometry) were studied. Expression microarrays were used to determine the effect of the treatment on the PI3K signalling pathway in MDA-MB-231. Nod Scid gamma mice were used to develop a murine model of metastatic breast cancer model. Luciferase-tagged MDA-MB-231 cells were injected into the 4th mammary fat pad of the mice and upon primary tumour establishment, treatment started. Tumour growth and metastasis was monitored using IVIS Spectrum. Statistical analysis was performed using ANOVA (Prism v8). Results: According to molecular docking, binding score of Rg3-VEGFR2 was -9.0 cal/mol. The VEGF bioassay showed that Rg3 is an allosteric modulator of VEGFR2 receptor. In HUVEC, S+R treatment decreased the expression of VEGF (ELISA) and its receptor VEGFR2. It also decreased the expression of pAKT:AKT, aquaporin 1 (AQP1) and pFAK:FAK (WB). These proteins are involved in cell survival, migration, invasion and proliferation. In breast cancer cell lines, S+R decreased the expression of AQP1 and decreased mammosphere formation efficiency, not by decreasing cell viability but by decreasing the expression of CD44+ stem cell marker. In expression arrays, the treatment decreased the expression of BTK, a tyrosine kinase promoting cell death escape; increased the expression of both CASP9, promoting apoptosis, and INPPD5, favouring inhibition of metastasis. In vivo experiments showed that S+R lead to tumour shrinkage (p < .0001), decreased body burden of tumour (p < .0001) and decreased number of metastases (p = .002). Conclusion: These results support the anti-cancer effects of the combination of S+R for the treatment of mTNBC. Citation Format: Maryam Nakhjavani, Eric Smith, Timothy Price, Amanda R Townsend, Jennifer E Hardingham. Ginsenoside rg3 enantiomers in a defined ratio as a novel treatment for metastatic triple negative breast cancer [abstract]. In: Proceedings of the 2020 San Antonio Breast Cancer Virtual Symposium; 2020 Dec 8-11; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2021;81(4 Suppl):Abstract nr PS11-38.