Mouse Mammary Carcinoma Research Articles

457 Semaphorin7a expression in breast cancer promotes susceptibility to immune checkpoint blockade

OBJECTIVES/GOALS: The goal of this study is to decipher the mechanisms by which breast cancers evade the immune system to facilitate metastasis through the lymphatic vasculature. We believe this is caused, in part, by semaphorin 7A (SEMA7A) as its expression is associated with increased lymphatic vessels, tumor-associated macrophages, and metastasis in breast cancer. METHODS/STUDY POPULATION: We have observed that breast cancer cells, lymphatics, and macrophages exhibit SEMA7A-dependent expression of PD-L1 in vitro, which may make SEMA7A+ tumors more sensitive to anti-PD-1/PD-L1 immunotherapies. Therefore, we utilized multiple orthotopic, immunocompetent mouse models to determine if SEMA7A expression makes tumors more susceptible to immune checkpoint blockade. E0771 and 66cl4 mouse mammary carcinoma cells were injected orthotopically into the #4 mammary fat pads. Once tumors reached 50-100 mm^3 mice were injected intraperitoneally with 250ug of anti-PD-1, anti-PD-L1, or IgG control every third day. Tumors were measured with calipers every other day and harvested for flow cytometry to determine the effect of immune checkpoint blockade on the TME in SEMA7A+ tumors. RESULTS/ANTICIPATED RESULTS: We reveal that growth of SEMA7A overexpressing (OE) tumors, but not controls, was significantly slowed with both anti-PD-1 and anti-PD-L1 treatments. Flow cytometric analysis of cells from the TME revealed increased LECs, TAMs, and PoEMs in SEMA7A+ tumors, compared to controls – all populations had higher PD-L1 expression, which was decreased with both anti-PD-1 or anti-PD-L1. We also observed a decrease in PD-L1 expression on the tumor cells with treatment. Furthermore, LEC, TAM, and PoEM presence was decreased within the TME alongside increased presence of activated CD4 and CD8 T cells. Collectively, our results suggest that SEMA7A expression in breast cancers activates a major pathway associated with immune evasion and helps to establish a pro-tumor microenvironment. DISCUSSION/SIGNIFICANCE: SEMA7A expression establishes a pro-tumor microenvironment, which can be targeted with readily available FDA approved drugs such as immune checkpoint-based therapies. Since SEMA7A+ breast cancers have high rates of metastasis, more specific treatments for these patient populations should be explored.

CEG-AgNPs Ameliorates DMBA-Induced Mammary Carcinogenicity by Alleviating Cytokines Expression.

<b>Background and Objective:</b> For more than a decade, breast cancer has been one of the most common forms of cancer among women around the world. The present article aimed to evaluate the protective activity of CEG-AgNPs against DMBA-induced mammary carcinoma. <b>Materials and Methods:</b> In this experimental study, green synthesis and characterization of CEG-AgNPs were carried as well as IC<sub>50</sub> against Mcf7 cell line and LD<sub>50</sub> on mice were evaluated. A total of 24 adult albino mice were divided into four groups six rats in each. Group I was given an equal amount of distilled water, group II was received 80 mg kg<sup></sup><sup>1</sup> b.wt., DMBA for 4 weeks, groups III and IV were treated with CEG-AgNPs (28.1 and 70.25 mg kg<sup></sup><sup>1</sup>) from the 5th week of DMBA administration for 4 weeks, respectively. <b>Results:</b> CEG-AgNPs were approximately 42.32±9.52 nm with a negative zeta potential of -17.44. It is IC<sub>50</sub> against the Mcf7 cell line and LD<sub>50</sub> is equal to 82.76 μg mL<sup></sup><sup>1</sup> and 1405 mg kg<sup></sup><sup>1</sup> b.wt., A significant normalization in plasma ALT, AST, AST and LDH as well as mammary MDA, TNF-α, IL-6, P53, SOD, GPx and GSH levels have been observed in CEG-AgNPs treated mice. Oral CEG-AgNPs administration has suppressed VEGF-C gene expression in DMBA-treated mice. <b>Conclusion:</b> The present results, biochemical, histological and MRI results showed that CEG-AgNPs have potent anticancer activity against DMBA-induced mammary carcinoma in mice by inducing the biosynthesizes of antioxidant biomarkers and suppression of cytokines gene expression.

Abstract LBA051: Importance of drug osmotic pressure and viscosity for efficient drug delivery using lymphatic drug delivery system

Abstract Metastasis is known to be the prime cause of cancer associated fatality. It is therefore imperative to treat and prevent metastasis in order to curb cancer-associated morbidity and mortality. Metastatic lymph nodes (MLNs) are known to be crucial diagnostic and prognostic indicators. Furthermore, as per the lymph node mediated hematogenous theory, due to extant communication between the lymphatic network and the venous system, the lymphatic network facilitates the passage and subsequent colonization of tumor cells to distant sites, thus, serving as a starting point for distant metastasis even during the initial stages. Treatment of MLNs, therefore, is imperative. Proposed in 2015, the Lymphatic Drug Delivery System (LDDS) is a novel and promising drug delivery system for the treatment of MLNs that overcomes the limitations of conventional chemotherapy. Unlike systemic chemotherapy, LDDS entails a direct intranodal drug administration to sentinel lymph node, post its identification using radioisotopes and fluorescent molecules, in order to treat it and its downstream lymph nodes. Previous studies have documented the superior efficacy of LDDS as a MLN management strategy as compared to conventional therapy. Higher drug delivery to target sites and prolonged retention with lower systemic toxicity, and thereby stronger anti-tumor effect have been reported to have been achieved upon drug administration via LDDS. In the present study, MXH10/Mo/lpr mice and FM3A-Luc, mouse mammary carcinoma cells expressing the firefly luciferase gene, have been utilized for the establishment of a mouse model of Lymph node metastasis (LNM) to investigate the effect of drug osmotic pressure and viscosity on therapeutic efficacy of the LDDS. Two lymph nodes, exhibiting systemic lymphadenopathy, in the subiliac and axillary region, the subiliac lymph node (SiLN) and proper axillary lymph node (PALN) have been used to model a network of interconnected human lymph nodes. Drug solution of varying osmotic pressure and viscosity were administered via LDDS to the murine model of LNM and subsequently therapeutic efficacy was monitored using the high frequency ultrasound system, VEVO and bioluminescent imaging system, IVIS, and confirmed through histology. The study revealed an optimized window of drug osmotic pressure and viscosity (π = 1000 - 2000 kPa; µ = 0.9 - 11.5 mPa·s.) where a significantly stronger anti-tumor response was observed. Venturing below or beyond the optimized range results in decreased therapeutic efficacy. The study therefore concluded that drug osmotic pressure and viscosity are important fluid parameters that require careful consideration prior to preparation of drug cocktails as they can severely impact the prognosis. The findings of this study have the potential for translation to clinics and we anticipate that these results can be replicated in clinical trials, positively altering the fate of many cancer patients. Citation Format: Radhika Mishra, Ariunbuyan Sukhbaatar, Sora Shouta, Maya Sakamoto, Shiro Mori, Tetsuya Kodama. Importance of drug osmotic pressure and viscosity for efficient drug delivery using lymphatic drug delivery system [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2021 Oct 7-10. Philadelphia (PA): AACR; Mol Cancer Ther 2021;20(12 Suppl):Abstract nr LBA051.

Diagnosis of Prostate Cancer and Prostatitis Using near Infra-Red Fluorescent AgInSe/ZnS Quantum Dots.

The link between the microbiome and cancer has led researchers to search for a potential probe for intracellular targeting of bacteria and cancer. Herein, we developed near infrared-emitting ternary AgInSe/ZnS quantum dots (QDs) for dual bacterial and cancer imaging. Briefly, water-soluble AgInSe/ZnS QDs were synthesized in a commercial kitchen pressure cooker. The as-synthesized QDs exhibited a spherical shape with a particle diameter of 4.5 ± 0.5 nm, and they were brightly fluorescent with a photoluminescence maximum at 705 nm. The QDs showed low toxicity against mouse mammary carcinoma (FM3A-Luc), mouse colon carcinoma (C26), malignant fibrous histiocytoma-like (KM-Luc/GFP) and prostate cancer cells, a greater number of accumulations in Staphylococcus aureus, and good cellular uptake in prostate cancer cells. This work is an excellent step towards using ternary QDs for diagnostic and guided therapy for prostate cancer.

MnTnHex-2-PyP5+, Coupled to Radiation, Suppresses Metastasis of 4T1 and MDA-MB-231 Breast Cancer via AKT/Snail/EMT Pathways.

Tumor migration and invasion induced by the epithelial-to-mesenchymal transition (EMT) are prerequisites for metastasis. Here, we investigated the inhibitory effect of a mimic of superoxide dismutase (SOD), cationic Mn(III) ortho-substituted N-n-hexylpyridylporphyrin (MnTnHex-2-PyP5+, MnHex) on the metastasis of breast cancer in cellular and animal models, focusing on the migration of tumor cells and the factors that modulate this behavior. Wound healing and Transwell migration assays revealed that the migration of mouse mammary carcinoma 4T1 cells was markedly reduced during the concurrent treatment of MnHex and radiation therapy (RT) compared with that of the control and RT alone. Bioluminescence imaging showed that MnHex/RT co-treatment dramatically reduced lung metastasis of 4T1 cells in mice, compared with the sham control and both single treatments. Western blotting and immunofluorescence showed that MnHex treatment of 4T1 cells reversed the RT-induced EMT via inhibiting AKT/GSK-3β/Snail pathway in vitro, thereby decreasing cell migration and invasion. Consistently, histopathological analyses of 4T1 tumors showed that MnHex/RT reduced Snail expression, blocked EMT, and in turn suppressed metastases. Again, in the human metastatic breast cancer MDA-MB-231 cell line, MnHex inhibited metastatic potential in vitro and in vivo and suppressed the RT-induced Snail expression. In addition to our previous studies showing tumor growth inhibition, this study demonstrated that MnHex carries the ability to minimize the metastatic potential of RT-treated cancers, thus overcoming their radioresistance.

Artificial Light at Night Reduces Anxiety-like Behavior in Female Mice with Exacerbated Mammary Tumor Growth.

Simple SummaryArtificial light at night, initially assumed to be innocuous, is associated with an increased risk for developing mood disorders, sleep disturbances, and cancer. However, the influence of ALAN on affective behavior in tumor-bearing mice has not been investigated. Here, we demonstrate that ALAN reduces the latency to tumor onset and increases terminal tumor volume. Additionally, tumor-bearing mice housed in dark nights exhibit increased anxiety-like behavior which is prevented via housing in ALAN.Artificial light at night (ALAN) is a pervasive phenomenon. Although initially assumed to be innocuous, recent research has demonstrated its deleterious effects on physiology and behavior. Exposure to ALAN is associated with disruptions to sleep/wake cycles, development of mood disorders, metabolic disorders, and cancer. However, the influence of ALAN on affective behavior in tumor-bearing mice has not been investigated. We hypothesize that exposure to ALAN accelerates mammary tumor growth and predict that ALAN exacerbates negative affective behaviors in tumor-bearing mice. Adult (>8 weeks) female C3H mice received a unilateral orthotropic injection of FM3A mouse mammary carcinoma cells (1.0 × 105 in 100 μL) into the fourth inguinal mammary gland. Nineteen days after tumor inoculation, mice were tested for sucrose preference (anhedonia-like behavior). The following day, mice were subjected to an open field test (anxiety-like behavior), followed by forced swim testing (depressive-like behavior). Regardless of tumor status, mice housed in ALAN increased body mass through the first ten days. Tumor-bearing ALAN-housed mice demonstrated reduced latency to tumor onset (day 5) and increased terminal tumor volume (day 21). Exposure to ALAN reduced sucrose preference independent of tumor status. Additionally, tumor-bearing mice housed in dark nights demonstrated significantly increased anxiety-like behavior that was normalized via housing in ALAN. Together, these data reaffirm the negative effects of ALAN on tumorigenesis and demonstrate the potential anxiolytic effect of ALAN in the presence of mammary tumors.

Abscopal Effect of Radiotherapy Enhanced with Immune Checkpoint Inhibitors of Triple Negative Breast Cancer in 4T1 Mammary Carcinoma Model.

Local radiotherapy (RT) is important to manage metastatic triple-negative breast cancer (TNBC). Although RT primarily reduces cancer cells locally, this control can be enhanced by triggering the immune system via immunotherapy. RT and immunotherapy may lead to an improved systemic effect, known as the abscopal effect. Here, we analyzed the antitumor effect of combination therapy using RT with an anti-programmed cell death-1 (PD-1) antibody in primary tumors, using poorly immunogenic metastatic mouse mammary carcinoma 4T1 model. Mice were injected subcutaneously into both flanks with 4T1 cells, and treatment was initiated 12 days later. Mice were randomly assigned to three treatment groups: (1) control (no treatment with RT or immune checkpoint inhibitor (ICI)), (2) RT alone, and (3) RT+ICI. The same RT dose was prescribed in both RT-alone and RT+ICI groups as 10Gy/fx in two fractions and delivered to only one of the two tumor burdens injected at both sides of flanks. In the RT+ICI group, 200 µg fixed dose of PD-1 antibody was intraperitoneally administered concurrently with RT. The RT and ICI combination markedly reduced tumor cell growth not only in the irradiated site but also in non-irradiated sites, a typical characteristic of the abscopal effect. This was observed only in radiation-sensitive cancer cells. Lung metastasis development was lower in RT-irradiated groups (RT-only and RT+ICI groups) than in the non-irradiated group, regardless of the radiation sensitivity of tumor cells. However, there was no additive effect of ICI on RT to control lung metastasis, as was already known regarding the abscopal effect. The combination of local RT with anti-PD-1 blockade could be a promising treatment strategy against metastatic TNBC. Further research is required to integrate our results into a clinical setting.

Bile-Acid-Appended Triazolyl Aryl Ketones: Design, Synthesis, In Vitro Anticancer Activity and Pharmacokinetics in Rats.

A library of bile-acid-appended triazolyl aryl ketones was synthesized and characterized by detailed spectroscopic techniques such as 1H and 13C NMR, HRMS and HPLC. All the synthesized conjugates were evaluated for their cytotoxicity at 10 µM against MCF-7 (human breast adenocarcinoma) and 4T1 (mouse mammary carcinoma) cells. In vitro cytotoxicity studies on the synthesized conjugates against MCF-7 and 4T1 cells indicated one of the conjugate 6cf to be most active against both cancer cell lines, with IC50 values of 5.71 µM and 8.71 µM, respectively, as compared to the reference drug docetaxel, possessing IC50 values of 9.46 µM and 13.85 µM, respectively. Interestingly, another compound 6af (IC50 = 2.61 µM) was found to possess pronounced anticancer activity as compared to the reference drug docetaxel (IC50 = 9.46 µM) against MCF-7. In addition, the potent compounds (6cf and 6af) were found to be non-toxic to normal human embryonic kidney cell line (HEK 293), as evident from their cell viability of greater than 86%. Compound 6cf induces higher apoptosis in comparison to 6af (46.09% vs. 33.89%) in MCF-7 cells, while similar apoptotic potential was observed for 6cf and 6af in 4T1 cells. The pharmacokinetics of 6cf in Wistar rats showed an MRT of 8.47 h with a half-life of 5.63 h. Clearly, these results suggest 6cf to be a potential candidate for the development of anticancer agents.

LyeTx I-b Peptide Attenuates Tumor Burden and Metastasis in a Mouse 4T1 Breast Cancer Model.

Cationic anticancer peptides have exhibited potent anti-proliferative and anti-inflammatory effects in neoplastic illness conditions. LyeTx I-b is a synthetic peptide derived from Lycosa erythrognatha spider venom that previously showed antibiotic activity in vitro and in vivo. This study focused on the effects of LyeTxI-b on a 4T1 mouse mammary carcinoma model. Mice with a palpable tumor in the left flank were subcutaneously or intratumorally injected with LyeTx I-b (5 mg/kg), which significantly decreased the tumor volume and metastatic nodules. Histological analyses showed a large necrotic area in treated primary tumors compared to the control. LyeTxI-b reduced tumor growth and lung metastasis in the 4T1 mouse mammary carcinoma model with no signs of toxicity in healthy or cancerous mice. The mechanism of action of LyeTx I-b on the 4T1 mouse mammary carcinoma model was evaluated in vitro and is associated with induction of apoptosis and cell proliferation inhibition. Furthermore, LyeTx I-b seems to be an efficient regulator of the 4T1 tumor microenvironment by modulating several cytokines, such as TGF-β, TNF-α, IL-1β, IL-6, and IL-10, in primary tumor and lung, spleen, and brain. LyeTx I-b also plays a role in leukocytes rolling and adhesion into spinal cord microcirculation and in the number of circulating leukocytes. These data suggest a potent antineoplastic efficacy ofLyeTx I-b.

Deletion of Col15a1 Modulates the Tumour Extracellular Matrix and Leads to Increased Tumour Growth in the MMTV-PyMT Mouse Mammary Carcinoma Model.

Basement membrane (BM) zone-associated collagen XV (ColXV) has been shown to suppress the malignancy of tumour cells, and its restin domain can inhibit angiogenesis. In human breast cancer, as well as in many other human carcinomas, ColXV is lost from the epithelial BM zone prior to tumour invasion. Here, we addressed the roles of ColXV in breast carcinogenesis using the transgenic MMTV-PyMT mouse mammary carcinoma model. We show here for the first time that the inactivation of Col15a1 in mice leads to changes in the fibrillar tumour matrix and to increased mammary tumour growth. ColXV is expressed by myoepithelial and endothelial cells in mammary tumours and is lost from the ductal BM along with the loss of the myoepithelial layer during cancer progression while persisting in blood vessels and capillaries, even in invasive tumours. However, despite the absence of anti-angiogenic restin domain, neovascularisation was reduced rather than increased in the ColXV-deficient mammary tumours compared to controls. We also show that, in robust tumour cell transplantation models or in a chemical-induced fibrosarcoma model, the inactivation of Col15a1 does not affect tumour growth or angiogenesis. In conclusion, our results support the proposed tumour suppressor function of ColXV in mammary carcinogenesis and reveal diverse roles of this collagen in different cancer types.

Different properties of mammary carcinogenesis induced by two chemical carcinogens, DMBA and PhIP, in heterozygous BALB/c Trp53 knockout mice

Chemical carcinogens, such as 7,12-dimethylbenz[a]anthracene (DMBA) and 2-amino-1-methyl-6-phenylimidazo(4,5-b)pyridine (PhIP), are known to induce mammary carcinomas in mice and rats. In the present study, the phenotypic and genotypic characteristics of carcinogen-induced mammary carcinogenesis in heterozygous BALB/c tumor protein p53 (Trp53) knockout mice were examined with reference to published data surrounding human breast cancer. A significantly accelerated induction of mammary carcinomas was observed following a single dose of DMBA (50 mg/kg of body weight at 7 weeks of age), and a modest acceleration was induced by PhIP (50 mg/kg of body weight) administered by gavage 6 times/2 weeks from 7 weeks of age. DMBA-induced mammary carcinomas were histopathologically characterized by distinct biphasic structures with luminal and myoepithelial cells, as well as a frequent estrogen receptor expression, and PhIP-induced carcinomas with solid/microacinar structures consisted of pleomorphic cells. Of note, DMBA-induced mammary carcinomas were characterized by a HRas proto-oncogene (Hras) mutation at codon 61, and gene/protein expression indicating MAPK stimulation. PhIP-induced lesions were suspected to be caused by different molecular mechanisms, including Wnt/β-catenin signaling and/or gene mutation-independent PI3K/AKT signaling activation. In conclusion, the present mouse mammary carcinogenesis models, induced by a combination of genetic and exogenous factors, may be utilized (such as the DMBA-induced model with Trp53 gene function deficiency as a model of adenomyoepithelioma, characterized by distinct biphasic cell constituents and Hras mutations), but PhIP-induced models are required to further analyze the genetic/epigenetic mechanisms promoting mammary carcinomas.

Hydrogen-Bonding-Induced H-Aggregation of Charge-Transfer Complexes for Ultra-Efficient Second Near-Infrared Region Photothermal Conversion

Hydrogen-Bonding-Induced H-Aggregation of Charge-Transfer Complexes for Ultra-Efficient Second Near-Infrared Region Photothermal Conversion

MicroRNA-200c restoration reveals a cytokine profile to enhance M1 macrophage polarization in breast cancer

Many immune suppressive mechanisms utilized by triple negative breast cancer (TNBC) are regulated by oncogenic epithelial-to-mesenchymal transition (EMT). How TNBC EMT impacts innate immune cells is not fully understood. To determine how TNBC suppresses antitumor macrophages, we used microRNA-200c (miR-200c), a powerful repressor of EMT, to drive mesenchymal-like mouse mammary carcinoma and human TNBC cells toward a more epithelial state. MiR-200c restoration significantly decreased growth of mouse mammary carcinoma Met-1 cells in culture and in vivo. Cytokine profiling of Met-1 and human BT549 cells revealed that miR-200c upregulated cytokines, such as granulocyte-macrophage colony-stimulating factor (GM-CSF), promoted M1 antitumor macrophage polarization. Cytokines upregulated by miR-200c correlated with an epithelial gene signature and M1 macrophage polarization in BC patients and predicted a more favorable overall survival for TNBC patients. Our findings demonstrate that immunogenic cytokines (e.g., GM-CSF) are suppressed in aggressive TNBC, warranting further investigation of cytokine-based therapies to limit disease recurrence.

Maltotriose-Chlorin e6 Conjugate Linked via Tetraethyleneglycol as an Advanced Photosensitizer for Photodynamic Therapy. Synthesis and Antitumor Activities against Canine and Mouse Mammary Carcinoma Cells.

Glycoconjugated chlorins represent a promising class of compounds that meet the requirements for the third-generation photosensitizer (PS) for photodynamic therapy (PDT). We have focused on the use of glucose (Glc) to improve the performance of the PS based on the Warburg effect—a phenomenon where tumors consume higher Glc levels than normal cells. However, as a matter of fact, Glc-conjugation has a poor efficacy in hydrophilic modification; thus, the resultant PS is not suitable for intravenous injection. In this study, a Glc-based oligosaccharide, such as maltotriose (Mal3), is conjugated to chlorin e6 (Ce6). The conjugation is assisted by two additional molecular tools, such as propargyl amine and a tetraethylene glycol (TEG) derivative. This route produced the target Mal3–Ce6 conjugate linked via the TEG spacer (Mal3–TEG–Ce6), which shows the required photoabsorption properties in the physiological media. The PDT test using canine mammary carcinoma (SNP) cells suggested that the antitumor activity of Mal3–TEG–Ce6 is extremely high. Furthermore, in vitro tests against mouse mammary carcinoma (EMT6) cells have been demonstrated, providing insights into the photocytotoxicity, subcellular localization, and analysis of cell death and reactive oxygen species (ROS) generation for the PDT system with Mal3–TEG–Ce6. Both apoptosis and necrosis of the EMT6 cells occur by ROS that is generated via the photochemical reaction between Mal3–TEG–Ce6 and molecular oxygen. Consequently, Mal3–TEG–Ce6 is shown to be a PS showing the currently desired properties.

Abstract PD15-02: Cx3cl1-cx3cr1 axis in liver metastasis of triple negative breast cancer

Abstract Triple negative breast cancer (TNBC) is a breast cancer subtype with increased risk of distant metastasis, especially to visceral organs. However, the detailed molecular mechanism underlying the visceral metastasis of TNBC is unknown. We used three patient-derived xenograft models derived from TNBC tumors with different metastatic capacities. PDX1 and PDX2 model developed distant metastasis in only lung and liver, respectively. In contrast, PDX3 tumor metastasized to both lung and liver. RNA sequencing of the primary tumor and distant metastatic sites of these three PDX models was performed. The gene expression profiles of the primary tumors of the three models showed minimally overlapping genes. However, the microenvironment gene expression profiles, measured by using the sequencing reads aligned to mouse genome, showed distinct difference between the models. Among the differentially expressed genes, we identified Cx3cr1 as a significantly upregulated gene in the liver microenvironment of the PDX models that metastasized to liver. Next, we used 4T1 syngeneic mouse mammary carcinoma model to validate the Cx3cr1 in the mouse liver tissues. Interestingly, the Cx3cr1 up-regulation occurred during the pre-metastatic period, and Cx3cl1, the ligand of the Cx3cr1, was also up-regulated in the liver tissue prior to the development of metastasis suggesting the Cx3cr1 regulates the formation of pre-metastatic niche. Cx3cl1, the ligand of the Cx3cr1, was also up-regulated in the liver tissue prior to the development of metastasis. These data suggest that Cx3cl1 and Cx3cr1-mediated tumor-microenvironment interaction is critical in developing liver metastasis in TNBC. Cx3cl1 increased the invasion and migration of Raw264.7 monocyte/macrophage cells and this effect was abrogated by the Cx3cr1 silencing in Raw264.7 cells or treating Cx3cl1 neutralizing antibody. Pathway analysis of the RNA sequencing data showed that genes involved in extracellular matrix remodeling was significantly dysregulated in the liver tissues including Lysyl Oxidase (Lox). Cx3cl1 treatment in Raw264.7 cells resulted in increased expression of Lox and MMP9 mRNA in three-dimensional culture system. The Raw264.7 cells caused increased invasion of 4T1 cells in vitro. In conclusion, our data suggest that Cx3cl1-Cx3cr1 axis plays critical role during the liver metastasis of TNBC. Citation Format: Woohang Heo, Hye-Youn Son, Ju hee Kim, Kyu Jin Lee, Myeonghee Jeon, Songbin Li, Jeehee Jang, Dong-Young Noh, Wonsik Han, Hyeong-Gon Moon. Cx3cl1-cx3cr1 axis in liver metastasis of 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 PD15-02.

Abstract PS3-07: Molecular imaging of hypoxia and granzyme B alterations during combination treatment with immunotherapy in triple negative breast cancer

Abstract Background: Although a portion of triple negative breast cancer (TNBC) is sensitive to chemotherapeutic treatment with agents such as paclitaxel (PTX), patients have a high risk of recurrence and short overall and progression-free survival. Clinical trials for TNBC using immune checkpoint inhibitors such as anti-programmed cell death 1 (PD1) have met some success; however, only a small percentage of patients have a positive response to immunotherapy and there is risk for severe side effects. Therefore, there is a need for improvements in monitoring and predicting patient-specific response. The goal of this study is to use non-invasive visualization of the tumor microenvironment to collect longitudinal information that can be used to study tumor response to immunotherapy. By applying positron emission tomography (PET) imaging techniques to investigate changes in the tumor microenvironment (hypoxia and immune cell activation), we seek to predict early response to immunotherapy and better identify which treatments will be effective for individual tumors in TNBC. Methods: A TNBC mouse mammary carcinoma cell line, 4T1, was transduced with CMV-luciferase and 2x105 cells were injected into the third mammary fat pad of 5-6 week old female Balb/c mice (N=28). Mice in cohort 1 received: PTX (10 mg/kg), anti-PD1 (200 µg), both, or vehicle control (saline) (n=4/group). Mice in cohort 2 received: anti-PD1 (n=5), combination PTX/anti-PD1 (n=4), or vehicle control (saline; n=3). Treatments were administered intraperitoneally on days 0, 2, and 5 for cohort 1 (n=16) who underwent granzyme B-PET imaging ([68Ga]-NOTA-GZP-PET) and on days 0, 2, 5, and 8 for cohort 2 (n=12) who underwent hypoxia imaging with [18F]-fluoromisonidazole (FMISO)-PET imaging. Bioluminescence (BLI) imaging and caliper measurements were performed to track tumor size changes at multiple timepoints and tumors were collected for histological validation on day 20. Mean standard uptake value (SUVmean) was calculated as percent of day 0, and statistical analyses were performed with unpaired t-tests and Wilcoxon-rank sum tests. Results: Voxel analysis of GZP-PET images revealed an 42.9% increase in T cell activation of TNBC tumors treated with single-agent PTX compared to anti-PD1 alone on day 3 (p=0.08). FMISO-PET revealed that tumors treated with anti-PD1 alone had lower hypoxic fraction compared to control group tumors (p=0.10) and tumors treated with combination PTX/anti-PD1 (p=0.17) on day 3. BLI data showed that treatment with PTX and anti-PD1 significantly decreased viability signal between days 3 and 6 for cohort 1 (p=0.04). Non-responders to treatment had a significantly higher tumor volume compared to responders starting on day 6 (p<0.05). SUVmean, indicating T cell activation, was significantly higher for responders compared to non-responders on days 3 and 6 (p<0.05). There was a signifciant decrease in hypoxia, as measured thorugh FMISO-PET SUVmean on day 6 for responders compared to non-responders (p=0.04). Conclusion: It is known that the tumor microenvironment can impact neovascularization, tumor cell invasion, tumor oxygenation and growth. Results from this study show that noninvasive PET imaging can provide data on changes in T cell activation and hypoxia in response to chemotherapeutic treatment and immunotherapy in TNBC. Utilizing clinically-translatable advanced imaging strategies to understand the biologically distinct features of the TNBC tumor microenvironment can aid in personalizing anti-cancer therapies. Acknowledgements: We thank the American Cancer Society for RSG-18-006-01-CCE, NIH NCI R01 CA240589, and NIH P30CA013148. Citation Format: Tiara S. Napier, Chanelle L. Hunter, Patrick N. Song, Ben M. Larimer, Anna G. Sorace. Molecular imaging of hypoxia and granzyme B alterations during combination treatment with immunotherapy in 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 PS3-07.

IL-15/IL-15Rα Heterodimeric Complex as Cancer Immunotherapy in Murine Breast Cancer Models.

Interleukin 15 (IL-15) has been evaluated as a potential treatment for solid tumors in clinical trials, but the effectiveness of systemic IL-15 administration as a monotherapy has not been realized. IL-15 receptor alpha (IL-15Rα) can stabilize IL-15 and enhance its bioactivity. The goal of this study was to examine the activity of IL-15/IL-15Rα complex (IL-15cx) to CD8+ T cells and evaluate its potential efficacy in murine breast cancer models. The antitumor efficacy was studied in mouse mammary carcinoma models (Her2/neu transgenic and 4T1-luc mammary cancers) treated with systemic recombinant protein with/without the depletion of myeloid-derived suppressor cells or intra-tumoral gene electrotransfer (GET). IL-15cx shows superior in vivo bioactivity to expand CD8 T cells in comparison to an equimolar single chain IL-15. T-bet is partially involved in CD8 T cell expansion ex vivo and in vivo due to IL-15 or IL-15cx. Intraperitoneal administration of IL-15cx results in a moderate inhibition of breast cancer growth that is associated with an increase in the frequency of cytotoxic CD8 T cells and the improvement of their function. The depletion of myeloid-derived suppressor cells (MDSCs) has no impact on mouse breast cancer growth. IL-15cx treatment diminishes MDSCs in murine tumors. However, it also antagonizes the effects of anti-Gr-1 depleting antibodies. Intratumoral GET with plasmid IL-15/IL-15Rα leads to a long-term survival benefit in 4T1 mammary carcinoma model. An early increase of local cytotoxic cells correlates with GET treatment and an increase of long-term memory T cells results from animals with complete tumor regression. Systemic and local administration of IL-15cx shows two distinct therapeutic responses, a moderate tumor growth inhibition or heterogeneous tumor regressions with survival improvement. Further studies are warranted to improve the efficacy of IL-15cx as an immunotherapy for breast cancer.

Estrogen Receptor α Mediates Doxorubicin Sensitivity in Breast Cancer Cells by Regulating E-Cadherin

Anthracyclines resistance is commonly seen in patients with estrogen receptor α (ERα) positive breast cancer. Epithelial-mesenchymal transition (EMT), which is characterized with the loss of epithelial cell polarity, cell adhesion and acquisition of new invasive property, is considered as one of the mechanisms of chemotherapy-induced drug resistance. In order to identify factors that associated with doxorubicin resistance, we performed in vitro and in vivo experiments using human and mouse breast cancer cell lines with different ERα status. Cell survival experiments revealed that ERα-positive cells (MCF-7 and MCF-7/ADR cell lines), were less sensitive to doxorubicin than ERα-negative (MDA-MB-231, MDA-MB-468) cells, and mouse mammary carcinoma cells (4T-1). The expression of E-cadherin reduced in low-invasive ERα-positive MCF-7 cells after treatment with doxorubicin, indicating epithelial mesenchymal transition. In contrast, the expression of E-cadherin was upregulated in high-invasive ERα-negative cells, showing mesenchymal-epithelial transition (MET). Moreover, it was found that the growth inhibition of 4T-1 cells by doxorubicin was positively correlated with the expression of E-cadherin. In a mouse breast cancer xenograft model, E-cadherin was overexpressed in the primary tumor tissues of the doxorubicin-treated mice. In ERα-positive MCF-7 cells, doxorubicin treatment upregulated the expression of EMT-related transcription factors Snail and Twist, that regulate the expression of E-cadherin. Following overexpression of ERα in ERα-negative cells (MDA-MB-231 and MDA-MB-468), doxorubicin enhanced the upregulation of Snail and Twist, decreased expression of E-cadherin, and decreased the sensitivity of cells to doxorubicin. In contrast, inhibition of ERα activity increased the sensitivity to doxorubicin in ERα-positive MCF-7 cells. These data suggest that the regulation of Snail and/or Twist varies depends on different ERα status. Therefore, doxorubicin combined with anti-estrogen receptor α therapy could improve the treatment efficacy of doxorubicin in ERα-positive breast cancer.

Rhus coriaria L. (Sumac) Demonstrates Oncostatic Activity in the Therapeutic and Preventive Model of Breast Carcinoma.

Comprehensive scientific data provide evidence that isolated phytochemicals or whole plant foods may beneficially modify carcinogenesis. The aim of this study was to evaluate the oncostatic activities of Rhus coriaria L. (sumac) using animal models (rat and mouse), and cell lines of breast carcinoma. R. coriaria (as a powder) was administered through the diet at two concentrations (low dose: 0.1% (w/w) and high dose: 1 % (w/w)) for the duration of the experiment in a syngeneic 4T1 mouse and chemically-induced rat mammary carcinoma models. After autopsy, histopathological and molecular analyses of tumor samples in rodents were performed. Moreover, in vitro analyses using MCF-7 and MDA-MB-231 cells were conducted. The dominant metabolites present in tested R. coriaria methanolic extract were glycosides of gallic acid (possible gallotannins). In the mouse model, R. coriaria at a higher dose (1%) significantly decreased tumor volume by 27% when compared to controls. In addition, treated tumors showed significant dose-dependent decrease in mitotic activity index by 36.5% and 51% in comparison with the control group. In the chemoprevention study using rats, R. coriaria at a higher dose significantly reduced the tumor incidence by 20% and in lower dose non-significantly reduced tumor frequency by 29% when compared to controls. Evaluations of the mechanism of oncostatic action using valid clinical markers demonstrated several positive alterations in rat tumor cells after the treatment with R. coriaria. In this regard, histopathological analysis of treated tumor specimens showed robust dose-dependent decrease in the ratio of high-/low-grade carcinomas by 66% and 73% compared to controls. In treated rat carcinomas, we found significant caspase-3, Bax, and Bax/Bcl-2 expression increases; on the other side, a significant down-regulation of Bcl-2, Ki67, CD24, ALDH1, and EpCam expressions and MDA levels. When compared to control specimens, evaluation of epigenetic alterations in rat tumor cells in vivo showed significant dose-dependent decrease in lysine methylation status of H3K4m3 and H3K9m3 and dose-dependent increase in lysine acetylation in H4K16ac levels (H4K20m3 was not changed) in treated groups. However, only in lower dose of sumac were significant decreases in the expression of oncogenic miR210 and increase of tumor-suppressive miR145 (miR21, miR22, and miR155 were not changed) observed. Finally, only in lower sumac dose, significant decreases in methylation status of three out of five gene promoters–ATM, PTEN, and TIMP3 (PITX2 and RASSF1 promoters were not changed). In vitro evaluations using methanolic extract of R. coriaria showed significant anticancer efficacy in MCF-7 and MDA-MB-231 cells (using Resazurin, cell cycle, annexin V/PI, caspase-3/7, Bcl-2, PARP, and mitochondrial membrane potential analyses). In conclusion, sumac demonstrated significant oncostatic activities in rodent models of breast carcinoma that were validated by mechanistic studies in vivo and in vitro.

Metabolism and pharmacokinetics of a novel polyphenol fatty acid ester phloridzin docosahexaenoate in Balb/c female mice

Flavonoids are known to undergo phase II metabolism and produce metabolites with similar or stronger biological effects compared to the parent flavonoids. However, the limited cellular uptake and bioavailability restrict their clinical use. We synthesized phloridzin docosahexaenoate (PZ-DHA), a novel fatty acid ester of polyphenol, through an acylation reaction with the aim of increasing the cellular availability and stability of the parent biomolecules, phloridzin (PZ) and docosahexaenoic acid (DHA). Here, we report metabolites and pharmacokinetic parameters of PZ-DHA, determined using ultra-high-performance liquid chromatography-electrospray ionization tandem mass spectrometry. PZ-DHA was taken-up by human (MDA-MB-231, MDA-MB-468, and MCF-7) and mouse (4T1) mammary carcinoma and human non-malignant mammary epithelial cells (MCF-10A) in cellular uptake assays. Our results suggested that the acylation improves the cellular uptake of PZ and stability of DHA within cells. In mouse hepatic microsomal assays, two major glucuronides of PZ-DHA, PZ-DHA-4-O-glucuronide and PZ-DHA-4′-O-glucuronide (MW = 923.02 g/mol), were detected. One tri-methylated- (4,4′,6′-O-trimethyl-PZ-DHA) (MW = 788.88 g/mol) and one di-sulphated- (PZ-DHA-4,4′-O-disulphide) PZ-DHA metabolite (MW = 906.20 g/mol) were also identified. Intraperitoneal injections of PZ-DHA (100 mg/kg) into Balb/c female mice was rapidly absorbed with a serum Cmax and Tmax of 23.7 µM and 60 min, respectively, and rapidly eliminated (t1/2 = 28.7 min). PZ-DHA and its metabolites are readily distributed throughout the body (Vd = 57 mL) into many organs. We identified in vitro and in vivo metabolites of PZ-DHA, which could be tested for potential use to treat diseases such as cancer in multiple organ systems.