Orthotopic Breast Cancer Model Research Articles

Comparative colloidal stability, antitumor efficacy, and immunosuppressive effect of commercial paclitaxel nanoformulations

BackgroundStandard chemotherapy with taxanes, such as paclitaxel (PTX), remains the mainstay of systemic treatment of triple-negative breast cancer. Nanotechnology-based formulations have gradually replaced PTX injection and are widely used in China. However, no studies have compared the colloidal stability, antitumor efficacy, and safety of commercial PTX nanoformulations. Additionally, the desire to evaluate preclinical antitumor efficacy in human-derived tumor cells led to the widespread application of immunodeficient mouse models that likely contributed to the neglect of nanomedicines-immune system interactions. The present study investigated the colloidal stability, antitumor efficacy and safety, and nanomedicines-host immune system interactions of PTX nanoformulations. A further comparative analysis was performed to evaluate the clinical potential.ResultsCompared with liposome, PTX emulsion and PTX nanoparticle exhibited favorable colloidal stability. PTX emulsion was superior in inducing apoptosis and had a more pronounced inhibitory effect on 4T1-tumor spheroids compared with PTX liposome and PTX nanoparticle. Although PTX emulsion exhibited superior in vitro antitumor effect, no significant differences in the in vivo antitumor efficacy were found among the three types of PTX nanoformulations in an immunocompetent orthotopic 4T1 murine triple-negative breast cancer model. All PTX nanoformulations at maximum tolerated dose (MTD) induced lymphopenia and immunosuppression, as evidenced by the reduction of T cell subpopulations and inhibition of the dendritic cells maturation.ConclusionsThe MTD PTX nanomedicines-induced lymphopenia and immunosuppression may weaken the lymphocyte-mediated antitumor cellular immune response and partly account for the lack of differences in the in vivo antitumor outcomes of PTX nanoformulations. Understanding of what impacts PTX nanomedicines has on the immune system may be critical to improve the design and conduct of translational research of PTX nanomedicines in monotherapy or combination therapy with immunotherapy.Graphic abstract

Abstract 1559: Inhibition of integrin αvβ8 enhances immune checkpoint induced anti-tumor immunity by acting across immunologic synapse in syngeneic models of breast cancer

Abstract Introduction We explored whether integrin αvβ8 inhibition potentiates immune checkpoint blockade (ICB) in syngeneic orthotopic models of breast cancer. Integrin αvβ8 mediates cell type specific and tissue localized activation of TGFβ1/3 to regulate the immune system. For example, αvβ8 expressed on dendritic cells (DC) in the intestine has been shown to be a key mediator of tolerance, maintaining gut immunologic homeostasis. Methods Efficacy was evaluated in combination with anti-PD-1 in EMT6 and PyMT breast cancer syngeneic mouse models. A potent αvβ8 inhibitor was orally administered at 60mg/kg BID for 21 days. Anti-αvβ8 or non-isoform specific anti-TGFβ mAbs were dosed TIWx3 at 7 and 10mg/kg, respectively. Tumor volumes are presented as mean±SEM. Statistics were performed by t test, one-way ANOVA, or log-rank test. Flow cytometry and transcriptome analysis on bulk and single-cell levels were used to assess the mechanism of action in EMT6. Results A similar αvβ8 expression pattern on DC, macrophages and regulatory T cells (Treg) was observed in mouse models and human tumors. Combination of oral αvβ8 inhibitor with anti-PD-1 was efficacious in the primary ICB resistant EMT6 model and resulted in superior tumor regression during treatment (p=0.0003) and improved survival with 5/12 complete responders relative to 0/12 in anti-PD-1 alone. Across studies the αvβ8 inhibitor phenocopied the results obtained with αvβ8 and TGFβ mAbs (n=3 independent studies). Complete responders re-challenged 89 days after treatment with EMT6 or 4T1 tumors showed no EMT6 tumor growth, suggesting the combination induced long-term immunologic memory. Analysis of tumors by flow cytometry showed combination resulted in increased CD8 T cell infiltrates (p=0.0006), T cell activation (CD8+CD69+, p=0.0194) and IFN-γ expression (CD8+IFN-γ+, p=0.0021). Single cell transcriptomic analysis of lymph nodes showed that αvβ8 inhibition potentiated DC co-stimulation (Cd40, Cd83/6) and migration (Ccr7, Cxcl16, Ccl22). Moreover, combination treatment led to tumor infiltrated Treg dysfunction including downregulation of Ctla4, Il10 and Tigit, and upregulation of Ifn-γ. The observation of these anti-tolerance and pro-inflammatory signatures in DC and Treg has not been described previously. Anti-tumor efficacy was driven by immune-mediated mechanisms as confirmed by a CD8 depletion study. Efficacy was confirmed in PyMT breast cancer model. Conclusions An αvβ8 inhibitor in combination with anti-PD-1 showed efficacy in syngeneic mouse models, supported by increased T cell infiltrates and evidence of reduced tumor tolerance. These results show that an orally administered αvβ8 targeted inhibitor is a potent modulator of anti-tumor immune response acting across the immunologic synapse, and is a promising therapeutic approach to ICB refractory tumors. Citation Format: Natalia J. Reszka-Blanco, Vinod Yadav, Megan Krumpoch, Laura Cappellucci, Dan Cui, James E. Dowling, Elizabeth Gwara, Bryce Harrison, Dooyoung Lee, Fu-Yang Lin, Lia Luus, Meghan Monroy, Terence I. Moy, Eugene Nebelitsky, Qi Qiao, Andrew Sullivan, Dawn Troast, Blaise Lippa, Bruce Rogers, Adrian S. Ray. Inhibition of integrin αvβ8 enhances immune checkpoint induced anti-tumor immunity by acting across immunologic synapse in syngeneic models 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 1559.

Abstract 1560: Targeting RNA-binding protein HuR to improve anti-PD-1 immunotherapy response in breast cancer

Abstract Breast cancer has been considered as a less immune-sensitive disease. Patients with metastatic breast cancer have a particularly low objective response rate (ORR) to anti-PD-1/PD-L1 therapy (3%~24%). It will be valuable to improve the treatment efficacy of anti-PD-1 immunotherapy in breast cancer. Recent studies reported that exosomal PD-L1 derived from tumor cells mediated immune surveillance evasion by interacting with the PD-1 receptor on T cells and contributed to the low ORR in anti-PD-1/PD-L1 immunotherapy.The RNA-binding protein Hu antigen R (HuR) is a member of the embryonic lethal abnormal vision (ELAV) family that is overexpressed in a variety of cancers and promotes tumorigenesis by interacting with a subset of oncogenic mRNAs, including PD-L1 mRNA. HuR also plays an important role in the exosome secretion process by regulating RAB27B mRNA. In this study, we aim to identify HuR biological functions in the cancer-immune system and evaluate the combination strategy using a HuR inhibitor with an anti-PD-1 antibody in breast cancer. We show here the direct binding of HuR protein to PD-L1 mRNA in human breast cancer cell line MDA-MB-231 by Ribonucleoprotein immunoprecipitation (RNP-IP). We also show that PD-L1 protein expression level is decreased in HuR knockout clones and in exosomes derived from HuR knockout clones. HuR knockout downregulates exosome secretion with decreased size in MDA-MB-231 cells. KH-3 is a small molecule identified from several chemical libraries that disrupts the binding of HuR and its target mRNAs.To test the combination strategy using a HuR inhibitor and anti-PD-1 antibody in vivo, we established the EMT6 orthotopic mouse breast cancer model. A combination treatment with HuR inhibitor KH-3 and anti-PD-1 antibody inhibits tumor growth and prolongs survival in this breast cancer model. In conclusion, we preliminarily explored the role of HuR in the cancer-immune system, we showed that HuR may be a promising therapeutic target to overcome immune evasion and improve immunotherapy response in breast cancer. Citation Format: Qi Zhang, Xiaoqing Wu, Lan Lan, Lanjing Wei, Liang Xu. Targeting RNA-binding protein HuR to improve anti-PD-1 immunotherapy response in 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 1560.

Abstract 1766: Triggering the resolution of inflammation with agonistic anti-ChemR23 antibody dampens inflammation-driven carcinogenesis

Abstract Chronic inflammation is associated with abnormal non-phlogistic clearance (efferocytosis) of apoptotic cells by macrophages and a defect of the resolution of inflammation pathways. The resolution of inflammation is an active immunological process mediated by specialized pro-resolving mediator (SPM) which target specific resolutive G-protein coupled receptors expressed by different immune cells and participate to the tissue homeostasis return after an injury. Defects in the clearance of (chemotherapy-induced) apoptotic tumor cell debris strengthens inflammation and has been associated with exacerbated tumor growth in several preclinical models. Proresolutive therapeutic approaches, such as using exogenous resolvin E1 (RvE1), the natural lipidic proresolutive ligand of the GPCR ChemR23, have been shown to dampen tumor-associated inflammation and to reduce tumor growth. Using single-cell RNA sequencing analysis, we found that ChemR23 is mainly expressed by tumor-associated macrophages in melanoma and lung cancers. Moreover, ChemR23 expression was barely expressed in non-inflamed tissues indicating a preferential expression on the inflamed or tumor site. We screened and identified an anti-ChemR23 mAb which induces RvE1-like Akt and ERK signaling in mouse and human macrophages and favors macrophage polarization towards a pro-resolutive phenotype. In vitro, the agonist antibody significantly increased the phagocytosis of apoptotic tumor primary mesothelioma cells by human tumor-polarized macrophages. In vivo, the agonist ChemR23 mAb accelerates the resolution of inflammation in an acute inflammatory model, as illustrated by a significant decrease of inflammatory macrophages and neutrophils infiltrates as compared to isotype control groups both in mice and non-human primate models. Similarly, ChemR23 triggering with the anti-ChemR23 mAb improved weight recovery, reduced diarrhea and decreased the development of colon neoplastic foci in a chronic colitis model coupled with the injection of a carcinogen agent. Furthermore, the administration of the anti-ChemR23 mAb as a monotherapy displayed some partial and complete antitumor responses in immunocompetent mouse models of subcutaneous colon carcinoma (MC38 and CT26) as well as orthotopic mesothelioma (AK7). Finally, using an orthotopic triple-negative breast cancer model (4T1), while we observed a limited impact on primary tumor growth, spontaneous metastasis development in the lung was significantly inhibited by the ChemR23 agonist mAb monotherapy. Our study reveals for the first time the therapeutic potential of triggering the proresolutive pathways using an anti-ChemR23 agonistic mAb to limit chronic inflammation in the tumor microenvironment and inhibit metastasis development. Citation Format: Vanessa Gauttier, Margot Lavy, Charlène Trilleaud, Kévin Biteau, Isabelle Girault, Lyssia Belarif, Géraldine Teppaz, Caroline Mary, Virginie Thépénier, Christophe Blanquart, Sophie Barillé-Nion, Nicolas Poirier. Triggering the resolution of inflammation with agonistic anti-ChemR23 antibody dampens inflammation-driven carcinogenesis [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 1766.

Reappraisal of anticancer nanomedicine design criteria in three types of preclinical cancer models for better clinical translation

Anticancer nanomedicines are designed to improve anticancer efficacy by increasing drug accumulation in tumors through enhanced permeability retention (EPR) effect, and to reduce toxicity by decreasing drug accumulation in normal organs through long systemic circulation. However, the inconsistent efficacy/safety of nanomedicines in cancer patients versus preclinical cancer models have provoked debate for nanomedicine design criteria. In this study, we investigate nanomedicine design criteria in three types of preclinical cancer models using five clinically used nanomedicines, which identifies the factors for better clinical translations of their observed clinical efficacy/safety compared to free drug or clinical micelle formulation. When those nanomedicines were compared with drug solution or clinical micelle formulation in breast tumors, long and short-circulating nanomedicines did not enhance tumor accumulation by EPR effect in transgenic spontaneous breast cancer model regardless of their size or composition, although they improved tumor accumulations in subcutaneous and orthotopic breast cancer models. However, when tumors were compared to normal breast tissue, nanomedicines, drug solution and clinical micelle formulation showed enhanced tumor accumulation regardless of the breast cancer models. In addition, long-circulating nanomedicines did not further increase tumor accumulation in transgenic mouse spontaneous breast cancer nor universally decrease drug accumulations in normal organs; they decreased or increased accumulation in different organs, potentially changing the clinical efficacy/safety. In contrast, short-circulating nanomedicines decreased blood concentration and altered drug distribution in normal organs, which are correlated with their clinical efficacy/safety. A reappraisal of current nanomedicine design criteria is needed to ensure consistent clinical translation for improvement of their clinical efficacy/safety in cancer patients.

NBE-002: A novel anthracycline-based antibody-drug conjugate (ADC) targeting ROR1 for the treatment of advanced solid tumors—A phase 1/2 clinical trial.

TPS1108 Background: The receptor tyrosine kinase-like orphan receptor 1 (ROR1) is highly expressed during embryonic development, but is minimally present or absent on post-partum healthy tissues. ROR1 is expressed in a variety of hematological and solid tumors and is associated with aggressive cancer phenotype and poor clinical outcomes. NBE-002 is an ADC targeting ROR1, obtained by site-specific, enzymatic conjugation of the anthracycline-derivative PNU-159682, modified with a non-cleavable linker to a humanized recombinant IgG1 monoclonal antibody, based on a novel anti-human ROR1 monoclonal antibody XBR1-402 (Peng et al. (2017) J. Mol. Biol. 429: 2954-73). Direct anti-tumor activity of NBE-002 was evaluated in immunodeficient, ROR1 expression-low/-intermediate/-high PDX models of several carcinoma and sarcoma subtypes. The most pronounced anti-tumor effect was achieved in triple-negative breast cancer (TNBC), at doses as low as 0.033 mg/kg, suggesting a best-in-class therapeutic index in light of the high tolerability in preclinical toxicology models. Administration in a fully immune competent setting (EMT6/ROR1 orthotopic breast cancer model) led to a strong anti-tumor response and a long-lasting anti-tumor immune protection dependent on CD8 T cells. Methods: NBE-002-01 (NCT04441099) is a first-in-human, open-label, multi-center, phase (Ph) 1/2 study of NBE-002 in adult patients with advanced solid tumors. Ph 1 of the study consists of a Dose Escalation Cohort (DEC), utilizing an accelerated titration design, followed by a traditional 3+3 design, and an optional Safety Expansion Cohort (SEC). Ph 2 will include two parallel Expansion Cohorts (EC), enrolling patients with advanced TNBC (EC1) or other solid tumors (EC2), with Simon’s two-stage design. Key eligibility criteria include Eastern Cooperative Oncology Group performance status of 0-2 (Ph 1) or 0-1 (Ph 2), adequate organ function defined as: hemoglobin ≥9.0 g/dL, neutrophils ≥1500 /µL, platelets ≥100000/µL, aspartate and alanine aminotransferases ≤2.5x the upper limit of normal (ULN), total bilirubin ≤1.5x ULN, creatinine ≤1.5x ULN, left ventricular ejection fraction ≥50%. The primary objectives are to assess safety and tolerability and to establish the recommended dose for further development (Ph 1), and to evaluate anti-tumor activity of (Ph 2). Secondary and exploratory objectives include characterization of immunogenicity as well as pharmacokinetic and pharmacodynamic profiles. NBE-002 is given intravenously once every three weeks until disease progression, unacceptable toxicity, withdrawal of consent, or other protocol-specific criteria are met. Ph 1 dose escalation was initiated on 17 JULY 2020 and is still recruiting in the US. Ph 2 is planned to be initiated in 2022. Clinical trial information: NCT04441099 .

A small molecule LN435a targeting TGFβR1 exerts promising antitumor effects on breast cancer.

e15069 Background: Breast cancer has overtaken lung cancer as the most diagnosed cancer. Despite conventional treatment, metastases occur in 20-30% of patients, resulting in death. This study aims to screen of effective drugs by metastatic patient-derived organoid and the potential molecular mechanism. Methods: Breast Cancer patient-derived organoid (PDO) model was established from the patient who have multiple drug resistance, multiple visceral and contralateral breast metastases. The organoid morphologies was tested by hematoxylin-eosin (HE) staining and immunohistochemistry (IHC). Then, pharmacological activity assay of 2370 natural product monomer (from Selleck) was performed with organoids. we modified the structure of harmine(HM) and screened the best active drugs. Cell proliferation assay and wound healing assay were used to detect LN435a anticancer activity in vitro. Orthotopic, Metastatic Xenograft and Patient-Derived tumor Xenograft(PDX) model of Breast Cancer were used to detect LN435a anticancer activity in vivo. In order to explore the anti-cancer target of LN435a, we used RNA transcriptome and proteomics sequencing. To further validate anti-cancer targets,TGFβ receptor 1 (TGFβR1), we used real-time quantitative qPCR, western blot, lentiviral packing and biolayer interferometry assay. To investigate whether LN435a inhabition of EMT and stem cell markers, we performed flow cytometry, immunohistochemistry and fluorescence. Results: We observe that organoid morphologies typically matched the histopathology, hormone receptor status, and HER2 status of the original tumor. In the first anti-cancer drug screening, HM showes the best effect on PDO. Because HM contains β-carbine alkaloids as the structural units, we designe a series of active drugs based on this and did anticancer screening. We find LN435a as one of the lead compounds exerting anti-metastatic activity in the nanomolar range in PDO and breast cancer cells. Proteomic and biochemical studies identify TGFβR1 as the direct target of LN435a. And then it inhibits EMT and stem cell markers. In parallel, loss of TGFβR1 or pharmacological inhibition of TGFβR1 by LN435a reduces breast cancer extravasation into the lung in an experimental metastasis mouse model, which reveals an essential role of TGFβR1 in breast cancer progression. Conclusions: Altogether, LN435a is a novel inhibitor of promising anti-tumor effects on breast cancer that works by blocking TGFβ signaling pathways.

Resetting the tumor microenvironment to favor anti-tumor immunity after local ablation.

2561 Background: Percutaneous tumor ablation is a non-surgical method of tumor destruction that leaves necrotic tumor debris in situ. Tumor associated antigens released after ablation have the potential to initiate a systemic anti-tumor immune response, however the hostile tumor microenvironment hinders antigen presentation and T cell activity. We hypothesized that resetting the tumor microenvironment with oral sodium bicarbonate to decrease tumor acidity and low-dose cyclophosphamide to deplete pro-tumor immune cells would improve the ability of ablation to initiate anti-tumor immunity. Methods: Tumor growth, overall survival, and metastatic burden was assessed in orthotopic tumor models of triple-negative breast cancer (67NR, 4T1, and E0771). Tumor ablation was performed on palpable tumors using percutaneous ethanol injection (PEI) with 6% ethylcellulose to improve retention in the tumor. Surgical excision was used as a negative control to test the role of in situ tumor debris. Before ablation mice were placed on 200 mM of sodium bicarbonate (SB) in their drinking water and received a single intraperitoneal injection of 200 mg/kg of cyclophosphamide (CP). Mice surviving to 60 days after tumor implant without a primary tumor or signs of metastases were considered "cured" and re-challenged with 50e5 tumor cells in the contralateral mammary pad. T cell dependance was assessed with in vivo CD8 depletions. Results: The combination of PEI+SB+CP produced a potent anti-tumor response, curing a majority of mice (5/7 of E0771, 8/12 of 67NR, 7/12 of 4T1). No mice were cured using PEI alone, SB alone, CP alone, or any combination of two therapies (0/51 of E0771, 0/73 of 67NR, 0/75 of 4T1,). Re-challenge tumor growth was hindered in mice cured with PEI+SB+CP. Mice receiving PEI+SB+CP had significantly less metastases and lived longer than mice receiving surgical excision alone or surgical excision with SB+CP. Additionally the anti-metastatic response of PEI+SB+CP was undone when CD8+ T cells were depleted. Conclusions: Here the anti-tumor response of local ablation produced by PEI was enhanced by priming the tumor with low-dose CP and oral SB in metastatic breast cancer. These results suggest that tumor ablation with CP and SB can create a T cell dependent, personalized immune response to a tumor using only low-cost, easily accessible supplies, and the host’s own tumor.

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.

Antiproliferative and antimetastatic characterization of an exo-heterocyclic androstane derivative against human breast cancer cell lines

Cancer in general, and specifically gynaecological neoplasms, represents a major public health issue worldwide. Based on the effect of sex hormones on breast tumorigenesis and prognosis, as well as on the development of breast cancer metastases, modification of the steroid skeleton is a hotspot of research for novel anticancer agents. Numerous recent studies support that minor modifications of the androstane skeleton yield potent antiproliferative and antimetastatic drug candidates. The aim of the present study was to assess the antitumor and antimetastatic properties, as well as the mechanism of action of a D-ring-modified exo-heterocyclic androstadiene derivative named 17APAD. The test compound was found to be highly selective towards human breast cancer-derived cell lines (MCF-7, T47D, MDA-MB-361, MDA-MB-231) compared to non-cancerous fibroblast cells (NIH/3T3), and exerted superior effect compared to the clinically applied reference drug cisplatin. Changes in MCF-7- and MDA-MB-231 cell morphology and membrane integrity induced by the test substance were assessed by fluorescent double staining. Cell cycle disturbances were analyzed by flow cytometry, and concentration-dependent alterations were detected on breast cancer cell lines. Mitochondrial apoptosis induced by the test compound was demonstrated by JC-1 staining. Inhibitory effects on metastasis formation, including the inhibition of migration, invasion and intravasation were investigated in 2D and 3D models. Significant anti-migratory and anti-invasive effects on MCF-7 and MDA-MB-231 cells were detected after 24 h exposure in 2D wound healing and Boyden-chamber assays. The anti-intravasative properties of 17APAD were evident after 4 h of incubation in a co-culture 3D circular chemorepellent-induced defects (CCID) assay, and the level of inhibition at concentrations ≥2 µM was comparable to that exerted by the focal adhesion kinase inhibitor defactinib. Single cell mass cytometry revealed that chemosensitive subpopulations of MDA-MB-231 cells engaged to apoptosis were less positive for EGFR, CD274, and CD326, while the percentage of cells positive for GLUT1, MCT4, Pan-Keratin, CD66(a,c,e), Galectin-3 and TMEM45A increased in response to 17APAD treatment. Finally, the novel androstane analogue 17APAD had an outstanding inhibitory effect on tumour growth in the 4T1 orthotopic murine breast cancer model in vivo after 2 weeks of intraperitoneal administration. These findings support that substitution of the androsta-5,16-diene framework with a N-containing heterocyclic moiety at C17 position yields a molecular entity rational to be considered for design and synthesis of novel, effective antitumor agents, and 17APAD is worth further investigation as a promising anticancer drug candidate.

A polyamidoamine (PAMAM) derivative dendrimer with high loading capacity of TLR7/8 agonist for improved cancer immunotherapy

Tumor associated macrophages (TAMs) tend to exhibit tumor-promoting M2 phenotype and contribute to the development of immunosuppressive microenvironment of solid tumors. Reprograming TAMs from M2 into tumoricidal M1 phenotype is robust for stimulating tumor immunosuppressive microenvironment (TIME). In this study, we developed a poly(amidoamine) (PAMAM) derivative dendrimer (denoted as fourth generation-N,N-diethylaminoethyl (G4-DEEA)) for efficient loading of Toll-like receptor 7 and 8 (TLR7/8) agonist (R848) to remodel the TIME for potent cancer immunotherapy. G4-DEEA exhibited a high loading capacity of R848 up to 35.9 wt% by taking advantage of its dendritic structure. The resulting formulation (designated as G4-DEEA@R848) effectively polarized M2 macrophages into M1 phenotype in vitro, and improved the maturation and activation of antigen-presenting cells. In the 4T1 orthotopic breast cancer model, G4-DEEA@R848 showed a stronger tumor inhibitory effect than free drug. The mechanistic studies suggested that G4-DEEA@R848 could significantly stimulate the TIME by repolarizing TAMs into M1 phenotype, reducing the presence of immunosuppressive myeloid cells and increasing the infiltration of tumor cytotoxic T cells. This study provides a simple but effective dendrimer-based strategy to improve the formulation of R848 for improved cancer immunotherapy.

Development of NIR-II Photoacoustic Probes Tailored for Deep-Tissue Sensing of Nitric Oxide.

Photoacoustic (PA) imaging has emerged as a reliable in vivo technique for diverse biomedical applications ranging from disease screening to analyte sensing. Most contemporary PA imaging agents employ NIR-I light (650-900 nm) to generate an ultrasound signal; however, there is significant interference from endogenous biomolecules such as hemoglobin that are PA active in this window. Transitioning to longer excitation wavelengths (i.e., NIR-II) reduces the background and facilitates the detection of low abundance targets (e.g., nitric oxide, NO). In this study, we employed a two-phase tuning approach to develop APNO-1080, a NIR-II NO-responsive probe for deep-tissue PA imaging. First, we performed Hammett and Brønsted analyses to identify a highly reactive and selective aniline-based trigger that reacts with NO via N-nitrosation chemistry. Next, we screened a panel of NIR-II platforms to identify chemical structures that have a low propensity to aggregate since this can diminish the PA signal. In a head-to-head comparison with a NIR-I analogue, APNO-1080 was 17.7-fold more sensitive in an in vitro tissue phantom assay. To evaluate the deep-tissue imaging capabilities of APNO-1080 in vivo, we performed PA imaging in an orthotopic breast cancer model and a heterotopic lung cancer model. Relative to control mice not bearing tumors, the normalized turn-on response was 1.3 ± 0.12 and 1.65 ± 0.07, respectively.

An ultrasound-guided injection method for a syngeneic orthotopic murine model of breast cancer.

Breast cancer is the most common cancer among women worldwide. For high-risk women, contrast enhanced (CE)-magnetic resonance imaging (MRI) is recommended as supplemental screening together with mammography. The development of new MRI contrast agents is an active field of research, which requires efficacy tests on appropriate preclinical pathological models. In this work, a refined method to orthotopically induce breast cancer in BALB/c mice was developed using ultrasound (US) as a guide for the precise localisation of the tumour induction site and to improve animal welfare. The method was coupled with CE-MRI to characterise the evolution of the tumoural lesion.

Damage to cardiac vasculature may be associated with breast cancer treatment-induced cardiotoxicity

BackgroundBreast cancer is the most common female cancer worldwide. Effective therapies including doxorubicin and trastuzumab have improved survival, but are associated with a substantial risk of cardiovascular disease. Mechanisms underlying cancer treatment-induced cardiotoxicity (CTC) are poorly understood and have largely focused on cardiomyocyte damage, although other cellular populations in the heart such as the cardiac endothelium, may play an important role in cardiac damage. We treated a breast tumor-bearing mouse model with doxorubicin and trastuzumab to investigate the role of the cardiac endothelium in the development of CTC.MethodsImmune compromised mice were inoculated in the 4th mammary fat pad with human breast cancer cells overexpressing HER2 (BT474). When tumors were palpable, mice were treated weekly with doxorubicin (5 mg/kg) and trastuzumab (4 mg/kg). The cardiac phenotype of mice was assessed by echocardiography and histological evaluation of the heart. Cardiac vascular damage was assayed by in vivo permeability assays and primary cultures of murine cardiac endothelial cells were used to assay doxorubicin toxicity in vitro.ResultsThe growth of BT474 breast tumors in Balb/c Nude mice was suppressed upon treatment with doxorubicin and trastuzumab. Mice treated for 4 months with doxorubicin and trastuzumab maintained body weights, but demonstrated an echocardiographic phenotype consistent with preserved left ventricular (LV) ejection fraction, decreased LV mass and increased filling pressures (E/e’). Histological staining with Masson’s trichrome and Picrosirius red showed extensive fibrosis and increased collagen deposition in the ventricular myocardium surrounding blood vessels of treated mice compared to untreated mice. Evans blue permeability assays demonstrated increased cardiac vasculature permeability while primary cardiac endothelial cells exposed to doxorubicin in vitro showed increased cell death as compared to lung or liver endothelial cells.ConclusionsAn orthotopic mouse model of human breast cancer in Nude mice treated with doxorubicin and trastuzumab resulted in a cardiac vascular defect accompanied by preserved LV ejection fraction, decreased LV mass, suggesting mild diastolic dysfunction and cardiac remodeling consistent with subclinical cardiotoxicity. Our data suggest that cardiac endothelium is more sensitive to doxorubicin therapy as compared to other organ endothelium and cardiac endothelial damage may correlate with breast cancer treatment-induced cardiotoxicity.

Target-Driven Design of a Coumarinyl Chalcone Scaffold Based Novel EF2 Kinase Inhibitor Suppresses Breast Cancer Growth In Vivo.

Eukaryotic elongation factor 2 kinase (eEF-2K) is an unusual alpha kinase involved in protein synthesis through phosphorylation of elongation factor 2 (EF2). eEF-2K is highly overexpressed in breast cancer, and its activity is associated with significantly shortened patient survival and proven to be a potential molecular target in breast cancer. The crystal structure of eEF-2K remains unknown, and there is no potent, safe, and effective inhibitor available for clinical applications. We designed and synthesized several generations of potential inhibitors. The effect of the inhibitors at the binding pocket of eEF-2K was analyzed after developing a 3D target model by using a domain of another α-kinase called myosin heavy-chain kinase A (MHCKA) that closely resembles eEF-2K. In silico studies showed that compounds with a coumarin-chalcone core have high predicted binding affinities for eEF-2K. Using in vitro studies in highly aggressive and invasive (MDA-MB-436, MDA-MB-231, and BT20) and noninvazive (MCF-7) breast cancer cells, we identified a lead compound that was highly effective in inhibiting eEF-2K activity at submicromolar concentrations and at inhibiting cell proliferation by induction of apoptosis with no toxicity in normal breast epithelial cells. In vivo systemic administration of the lead compound encapsulated in single lipid-based liposomal nanoparticles twice a week significantly suppressed growth of MDA-MB-231 tumors in orthotopic breast cancer models in nude mice with no observed toxicity. In conclusion, our study provides a highly potent and in vivo effective novel small-molecule eEF-2K inhibitor that may be used as a molecularly targeted therapy breast cancer or other eEF-2K-dependent tumors.

High-dimensional immunotyping of tumors grown in obese and non-obese mice.

ABSTRACTObesity is a disease characterized by chronic low-grade systemic inflammation and has been causally linked to the development of 13 cancer types. Several studies have been undertaken to determine whether tumors evolving in obese environments adapt differential interactions with immune cells and whether this can be connected to disease outcome. Most of these studies have been limited to single-cell lines and tumor models and analysis of limited immune cell populations. Given the multicellular complexity of the immune system and its dysregulation in obesity, we applied high-dimensional suspension mass cytometry to investigate how obesity affects tumor immunity. We used a 36-marker immune-focused mass cytometry panel to interrogate the immune landscape of orthotopic syngeneic mouse models of pancreatic and breast cancer. Unanchored batch correction was implemented to enable simultaneous analysis of tumor cohorts to uncover the immunotypes of each cancer model and reveal remarkably model-specific immune regulation. In the E0771 breast cancer model, we demonstrate an important link to obesity with an increase in two T-cell-suppressive cell types and a decrease in CD8 T cells.

Defects in Emerin-Nucleoskeleton Binding Disrupt Nuclear Structure and Promote Breast Cancer Cell Motility and Metastasis.

Nuclear envelope proteins play an important role in regulating nuclear size and structure in cancer. Altered expression of nuclear lamins are found in many cancers and its expression is correlated with better clinical outcomes. The nucleus is the largest organelle in the cell with a diameter between 10 and 20 μm. Nuclear size significantly impacts cell migration. Nuclear structural changes are predicted to impact cancer metastasis by regulating cancer cell migration. Here we show emerin regulates nuclear structure in invasive breast cancer cells to impact cancer metastasis. Invasive breast cancer cells had 40% to 50% less emerin than control cells, which resulted in decreased nuclear size. Overexpression of GFP-emerin in invasive breast cancer cells rescued nuclear size and inhibited migration through 3.0 and 8.0 μm pores. Mutational analysis showed emerin binding to nucleoskeletal proteins was important for its regulation of nuclear structure, migration, and invasion. Importantly, emerin expression inhibited lung metastasis by 91% in orthotopic mouse models of breast cancer. Emerin nucleoskeleton-binding mutants failed to inhibit metastasis. These results support a model whereby emerin binding to the nucleoskeleton regulates nuclear structure to impact metastasis. In this model, emerin plays a central role in metastatic transformation, because decreased emerin expression during transformation causes the nuclear structural defects required for increased cell migration, intravasation, and extravasation. IMPLICATIONS: Modulating emerin expression and function represents new targets for therapeutic interventions of metastasis, because increased emerin expression rescued cancer metastasis.

Over-Expression of Centromere Protein U Participates in the Malignant Neoplastic Progression of Breast Cancer.

The expression of Centromere Protein U (CENP-U) is closely related to tumor malignancy. Till now, the role of CENP-U in the malignant progression of breast cancer remains unclear. In this study, we found that CENP-U protein was highly expressed in the primary invasive breast cancer tissues compared to the paired adjacent histologically normal tissues and ductal carcinoma in situ (DCIS) tissues. After CENP-U was knocked down, the proliferation and colony-forming abilities of breast cancer cells were significantly suppressed, whereas the portion of apoptotic cells was increased. Meanwhile, the PI3K/AKT/NF-κB pathway was significantly inhibited. In vivo studies showed that, the inhibition of CENP-U repressed the tumor growth in orthotopic breast cancer models. Therefore, our study demonstrated that the CENP-U might act as an oncogene and promote breast cancer progression via activation of the PI3K/AKT/NF-κB pathway, which suggests a promising direction for targeting therapy in breast cancer.

Combining losartan with radiotherapy increases tumor control and inhibits lung metastases from a HER2/neu-positive orthotopic breast cancer model

BackgroundPatients with metastatic HER2/neu-positive (HER2/neu +) breast cancer (BC) often experience treatment resistance, disease recurrences and metastases. Thus, new approaches for improving the treatment of HER2/neu + BC to prevent metastatic dissemination are urgently needed. Our previous studies have shown that losartan, an angiotensin receptor blocker, increases tumor perfusion and decreases hypoxia in a number of tumor models. Hypoxia reduces the efficacy of radiation and increases metastases. We therefore hypothesized that by modifying tumor stroma and increasing oxygenation, losartan will improve the outcome of radiotherapy and inhibit disease progression in a highly metastatic HER2/neu + murine BC model.MethodsWe established a metastatic HER2/neu + murine BC line (MCa-M3C) and used it to generate mammary fat pad isografts in syngeneic female FVB/N mice. Starting on day 3 after orthotopic tumor implantation, we administered a 7-day losartan treatment (40 mg/kg BW, gavage daily); or a 7-day losartan treatment followed by 20 Gy single dose local irradiation (S-IR) on day 10 (tumor size ~ 100 mm3), or 20 Gy local fractionated (5 × 4 Gy daily) irradiation (F-IR) on days 10–14. We analyzed tumor-growth delay (TGD), development of spontaneous lung metastases, animal survival, tumor vascular density, and tumor hypoxia.ResultsTreatments with S-IR, F-IR, Losartan + S-IR, or Losartan + F-IR resulted in a significantly increased TGD (8–16 days) in MCa-M3C tumors versus controls. However, the combination of Losartan + S-IR and Losartan + F-IR further enhanced tumor response to radiation alone by increasing TGD an additional 5 to 8 days for both single and fractionated dose irradiation (P < 0.01), decreasing lung metastasis (Losartan + IR vs. Control, P < 0.025), and increasing animal survival (Losartan + IR vs. Control, P = 0.0303). In addition, losartan treatment significantly increased tumor vascularity (P = 0.0314) and decreased pimonidazole positive (hypoxic) area (P = 0.0002).ConclusionsCombining losartan with local irradiation significantly enhanced tumor response, at least in part via reduced tumor hypoxia presumably due to increased tumor perfusion. Our findings suggest that combining losartan with radiotherapy is a potential new treatment strategy for local control and inhibiting metastasis in HER2 + BC.

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 .