Animal Models Of Breast Cancer Research Articles

Abstract PO1-26-05: Animal Study Compares CAR T cell Exhaustion & Ability to Kill Low Antigen expressing breast cancer cells among three CAR constructs including one with 1XX mutations

Abstract Background: CAR T cells for the treatment of solid tumor cancers has not yet achieved the same success as CAR T cells for treatment of blood cancers. Two of the hurdles that must be cleared for effective use of CAR T cells for solid tumor cancers are: 1) CAR T cell exhaustion; and 2) failure to recognize and kill low antigen expressing cancer cells. A promising approach to overcoming CAR T cell exhaustion, referred to as “1XX” was developed in the Sadelain Lab at MSKCC. Mutation of Tyrosines to Phenylalanine in ITAMs 2 and 3, of the CD3z signaling domain, restrict signaling to ITAM 1. This slowing down of signaling has been reported to be effective at eliminating or greatly reducing CAR T cell exhaustion in blood cancers [Feucht, et al 2019; Park et al 2022; Schoutrop, et al 2023]. Purpose: To evaluate the potential of the 1XX mutations to overcome CAR T cell exhaustion and inability to kill low antigen expressing cells in an animal model of breast cancer. Specifically, we tested the ability of three different CARs to eliminate human breast tumors that expressed variable levels of the target antigen, MUC1*, and their ability to suppress tumor recurrence over approximately 100 days. Methods: All three CARs were targeted to the tumor by the same antibody fragment, huMNC2, that recognizes MUC1*, which is the transmembrane cleavage product of MUC1 that functions as a potent growth factor receptor. The CARs are: 1) huMNC2-41BB-CD3z; 2) huMNC2-CD28-CD3z; and 3) huMNC2-CD28-1XX. Heterogeneous tumors expressing different levels of target antigen were made as follows. T47D breast cancer cells were engineered to express even more of the target, MUC1*, and were also engineered to fluoresce green. T47D wild type cells were engineered to fluoresce red. Heterogenous tumors consisting of 250,000 cells were implanted sub-cu into female NSG mice bearing 90-day estrogen pellets. The tumors comprised either 70% wild-type/30% overexpressing cells, or 85% wild-type/15% overexpressing cells or 92.5% wild-type/7.5% overexpressing cells. Tumor engraftment was verified by bioluminescence at Day 6 post implantation. There was a total of 150 animals, 5 animals per group. Animals were administered a single dose of CAR T cells into the tail vein on Day 7, wherein the Effector to Target ratio was either 10:1, 5:1 or 1:1. Tumor volume was measured weekly by Luciferase/Luciferin bioluminescence on an IVIS instrument. In addition, the red versus green fluorescence of the tumor was tracked periodically as an indicator of which cells, high or low antigen expressing, were being killed. Between Day 93 and Day 96, animals were sacrificed, cells were recovered from blood and spleen, recovered tumors were weighed, tumor cells dissociated and fluorescent images were captured to determine which cells escaped CAR T cell killing. Conclusions: The CAR with the 1XX mutations in CD3z, huMNC2-CD28-1XX, was much more effective at suppressing breast tumor recurrence than either CAR with wild-type CD3z. At sacrifice, significantly more CAR T cells were recovered from huMNC2-CD28-1XX than from huMNC2-41BB-CD3z or huMNC2-CD28-CD3z. At high dose, the CARs with wild-type CD3z effectively suppressed the high antigen expressing cells, but the recurrent tumors were essentially made up of the low antigen expressing cells that had escaped CAR T cell killing. At low CAR T cell dose, the CARs with wild-type CD3z appeared to become exhausted about 40 days post treatment, when tumors began to recur. Post sacrifice analysis of the recurrent tumors showed that they were made up of both high and low antigen expressing cells. huMNC2-CD28-1XX effectively killed both the high antigen and low antigen expressing cells as evidenced by the live fluorescent imaging and the post-sacrifice analysis of residual tumor. Citation Format: Cynthia Bamdad, Andrew Stewart, Benoit Smagghe, Mark Carter, Danica Walkley, Kevin Yi, Jac-Leen Nash, Michael Nash, Trevor Grant, Gregory Riley. Animal Study Compares CAR T cell Exhaustion & Ability to Kill Low Antigen expressing breast cancer cells among three CAR constructs including one with 1XX mutations [abstract]. In: Proceedings of the 2023 San Antonio Breast Cancer Symposium; 2023 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2024;84(9 Suppl):Abstract nr PO1-26-05.

Abstract PO2-05-08: ISM5043, a novel, selective KAT6 inhibitor for the treatment of advanced and refractory ER+ breast cancer

Abstract Breast cancer is the most commonly diagnosed cancer and the second leading cause of cancer death among women in the US. Approximately 70% of human breast tumors are estrogen receptor positive (ER+), and endocrine therapy (ET), which inhibits the estrogen signaling pathway, remains the backbone of therapy for ER+ breast cancer. However, ET resistance often develops, and patients eventually experience disease progression. This is a significant clinical challenge and highlights the urgent need for novel therapies that may help overcome the resistance. KAT6A is a histone (lysine) acetyltransferase (HAT) belonging to the MYST family. KAT6A and its paralog KAT6B play important roles in cell cycle progression, neuron stem cell maintenance and hematopoietic development via acetylation of H3K23. Molecular dysregulation of KAT6A, including amplification and fusion, has been reported in many cancers. In breast cancer, KAT6A is amplified as part of the 8p11 amplicon in about 10-15% of the ER+ breast cancer population, where it functions as an epigenetic modulator of ER expression, and is associated with a worse clinical outcome. Therefore, inhibition of KAT6A may be a promising therapy for ER+ breast cancer. Here, we evaluated ISM5043, a novel, small molecule KAT6 inhibitor for anti-tumor effects in ER+/HER2- breast cancer cell lines and animal models. ISM5043 demonstrated potent inhibitory activity against KAT6A (IC50, 8 nM) and KAT6B (IC50, 16 nM) with selectivity over KAT7 (IC50, 344 nM) and other HATs (IC50 >2000 nM). The compound showed robust, dose-dependent, anti-proliferation activity (IC50 < 10 nM) in multiple ER+/HER2- breast cancer cell lines with KAT6A amplification, in accordance with its suppression of H3K23 acetylation. Investigation of the mechanism of action revealed that treatment with ISM5043 resulted in the downregulation of the expression of ERα in these breast cancer cells. In the ZR-75-1 (ER+, HER2-) xenograft model, ISM5043 demonstrated robust and dose-dependent anti-tumor activity, as monotherapy, with Tumor Growth Inhibition (TGI) between 80~110% at doses ranging from 0.3 to 10 mg/kg, QD. ISM5043 (0.3 mg/kg, QD), in combination with tamoxifen (20 mg/kg, QD) exhibited synergistic effect against tumor growth, with a Q value of 1.25 based on Jin’s equation. Notably, ISM5043 displayed a strong anti-tumor effect (TGI of 67.7% and 83.6% at 3 and 10 mg/kg QD, respectively) as monotherapy, in a patient-derived ER+/HER2- breast cancer xenograft model representing disease progression following multiple prior lines of treatment (including palbociclib in combination with letrozole). ISM5043 also showed favorable ADME properties as well as good pharmacokinetic and safety profiles. Taken together, these data support the clinical evaluation of ISM5043 as a targeted therapy in patients with advanced and refractory ER+ breast cancer. Citation Format: Xin Cai, Xin Cheng, Luoheng Qin, Man Zhang, Junwen Qiao, Supriya Bavadekar, Sujata Rao, Feng Ren. ISM5043, a novel, selective KAT6 inhibitor for the treatment of advanced and refractory ER+ breast cancer [abstract]. In: Proceedings of the 2023 San Antonio Breast Cancer Symposium; 2023 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2024;84(9 Suppl):Abstract nr PO2-05-08.

Metformin improves the therapeutic effect of metronomic cyclophosphamide in breast cancer animal model

Metformin improves the therapeutic effect of metronomic cyclophosphamide in breast cancer animal model

Abstract P53: Inhibition of STING/Galectin-9 Axis Sensitize Anthracyclines Treatment And Induce Anti-tumor Immunity

Abstract Anthracyclines, are a class of conventional and routinely used firstline chemotherapy drugs to treat breast cancer. Immunogenic cell death (ICD) is characterized by the exposure of danger signals that promote dendritic cell maturation and antigen presentation and is often associated with the induction of cancer cell-intrinsic IFN-I signaling pathway. In addition, among multiple chemotherapeutic drugs such as doxorubicin (Dox) is well-known to promote tumor immunogenicity by interfering with DNA synthesis. Galectin-9 (Gal-9), as a member of β-galactoside-binding protein family, has been demonstrated to induce T cell death and promote immunosuppression in the tumor microenvironment. In addition, it has been shown that some interferon (IFN) and interleukin (IL) factors can induce Gal-9 expression. However, the mechanism cross-talk between anthracyclines and Gal-9 immunecheckpoint inhibitor remains unknown. Here we show that doxorubicin induces PD-L1 expression in human breast cancer cell lines and animal models. Mechanistically, doxorubicin promoted IFNβ expression, which in turn enhanced doxorubicin-induced Gal-9. Furthermore, we discovered that doxorubicin induces tumor Gal-9 via STING upregulation. The combination of doxorubicin and anti-Gal-9 therapy compared with each agent alone significantly increased the therapeutic efficacy in vivo. Clinically, a higher Gal-9 and p-STING level in the tumor region of after-treatment anthracyclines as compared to before treatment is indicated in breast cancer tumor tissue. Taken together, our study demonstrates crosstalk between anthracyclines-induced ICD and tumor-associated immunosuppression and provides a novel therapeutic strategy by combining anthracyclines and Gal-9 antibody in breast cancer. Citation Format: Xian Sun, Wei-Jan Wang, Jilu Lang, Riyao Yang, Wan-Jou Shen, Linlin Sun, Mien-Chie Hung. Inhibition of STING/Galectin-9 Axis Sensitize Anthracyclines Treatment And Induce Anti-tumor Immunity [abstract]. In: Proceedings of Frontiers in Cancer Science; 2023 Nov 6-8; Singapore. Philadelphia (PA): AACR; Cancer Res 2024;84(8_Suppl):Abstract nr P53.

Abstract 3341: Targeting CXCR4 via the small molecule inhibitor NMX1 as a therapeutic strategy to treat high-risk malignancies

Abstract Introduction: Cytokines are secreted by various cells of the immune system, endothelium, and stroma that play a critical role in cell survival. The activation of C-X-C chemokine receptor type 4 (CXCR4) by its ligand SDF-1 leads to signaling by the AKT and mTOR pathways. CXCR4 is highly expressed in several types of cancer and contributes to tumor growth, metastasis, and resistance to therapy. Interleukin 11 (IL-11) is a cytokine that often drives disease progression, as well as contributing to tumor immune evasion. NMX1 (NovoMedix) is a novel oral small molecule that inhibits mTOR signaling, CXCR4 expression, IL-11 secretion, and activates AMPK. In animal models of breast cancer, NMX1 effectively prevented tumor growth and metastasis, while also protecting from doxorubicin-induced cardiotoxicity. In this study, we investigated the anticancer activity of NMX1 in preclinical models of high-risk pediatric and adult malignancies. Methods: Cell viability assays using alamar blue were performed in a panel of pediatric and adult cancer cells, including leukemia, brain, breast, lung, neuroblastoma, and sarcoma. Cells were treated with increasing concentrations of NMX1 ranging from 250 nM to 16 µM for 96 hours, followed by determination of half maximal inhibitory concentrations (IC50). To validate target modulation, the level of IL-11 and CXCR4 was determined by ELISA and immunoblot, respectively. Protein kinase activity was assessed by phosphorylation levels. Induction of apoptosis was confirmed by caspase-mediated PARP cleavage. To identify effective drug combinations, NMX1-treated cells were screened with a comprehensive drug library. Synergy was evaluated in the top candidates by dose-response matrices and SynergyFinder. Safety and tolerability was tested in animal models. Results: NMX1 treatment induced cytotoxicity at micromolar concentrations in diverse types of cancer, with IC50 values ranging from 0.2-8.7 µM and 0.4-4.4 µM for leukemia and solid tumor cells, respectively. NMX1 induced cancer cell death by PARP cleavage and apoptosis. Mechanistically, CXCR4 expression, mTOR signaling, and IL-11 secretion were decreased upon treatment with NMX1, validating effective target modulation. The drug combination screen identified several candidates in a tumor type-dependent manner. Synergistic effects were observed using NMX1 combined with clinically relevant concentrations of rucaparib, linsitinib, and crizotinib, which target PARP, IGFR, and c-MET, respectively. Conclusions: Our study shows antitumor activity by NMX1 through inhibition of CXCR4, IL-11, and mTOR signaling, providing an effective therapeutic strategy for high-risk and refractory malignancies. Preclinical data on feasible drug combinations and biological correlates of activity support development of early phase clinical studies involving NMX1. Citation Format: Patrick Sipila, Son Tran, Chunfen Zhang, Laura G. Corral, Kyle W. Chan, Cathy A. Swindlehurst, Aru Narendran. Targeting CXCR4 via the small molecule inhibitor NMX1 as a therapeutic strategy to treat high-risk malignancies [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 3341.

Abstract 4149: Real-time intravital trafficking of tumor infiltrating lymphocytes in breast cancer models

Abstract Cancer immunotherapy utilizing the immunoreactive efficiency of immune cells via adoptive cell therapy (ACT) has been a novel method of cancer treatment. In tumor microenvironment, immune cell reactivity of tumor-infiltrating lymphocytes (TILs) can facilitate cancer cell attack, which finally derives efficient suppression of tumor growth. However, the conventional evaluation for cancer immunotherapy has been generally performed by ex vivo methods, providing restricted information on lymphocyte motility and immune cell dynamics within the tumor microenvironment. In vivo data with the dynamic behavior of TILs in breast cancer can provide new insight to investigate novel targets of immune checkpoint inhibitors. In this research, longitudinal intravital imaging of motility trafficking of TILs in breast cancer animal models has been performed by utilizing intravital microscopy to unveil the immunoreactive dynamics of T cell activity in early stage. In vivo analysis of TIL motility within different breast cell lines, including 4T1 (mouse breast cancer cell), MDA-MB-231 (human triple-negative breast cancer cell), and MCF7 (human breast cancer cell), respectively, has been investigated for immune responses to cancer. We’ve monitored spatiotemporal dynamics of TIL over 11 days in breast cancer xenograft mouse model generated by tumor implantation into the dorsal skinfold imaging chamber and adoptive T cell transfer by blood circulation. In the results, the adoptive immunoreactivity of TIL has been differentiated by the timepoint of adoptive T cell transfer, distance proximity of T cells in tumors, and breast cancer types. Furthermore, anti-cancer effect coordinated with dendritic cells behaviors showed induced immune responses with accumulated dendritic cells with increased TIL count over time in breast cancer model treated with various cancer therapy such as immunotherapy, radiation therapy and anti-cancer drug (anti-PD-L1), which implied a relationship between programmed death-ligand 1 expression levels and TIL/dendritic cell motility. The result demonstrates that intravital TIL motility trafficking analysis in cancer model can be an invaluable assessment method to explain the detailed cellular/molecular mechanism of immune response to cancer involved with T lymphocytes, dendritic cells, and immune checkpoint inhibitors to accelerate the efficiency of cancer immunotherapy. Citation Format: Soyeon Ahn, Kubra Akyildiz, Hyunseok Kim, Pilhan Kim. Real-time intravital trafficking of tumor infiltrating lymphocytes in breast cancer models [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 4149.

Autophagy and senescence facilitate the development of antiestrogen resistance in ER positive breast cancer.

Estrogen receptor positive (ER+) breast cancer is the most common breast cancer diagnosed annually in the US with endocrine-based therapy as standard-of-care for this breast cancer subtype. Endocrine therapy includes treatment with antiestrogens, such as selective estrogen receptor modulators (SERMs), selective estrogen receptor downregulators (SERDs), and aromatase inhibitors (AIs). Despite the appreciable remission achievable with these treatments, a substantial cohort of women will experience primary tumor recurrence, subsequent metastasis, and eventual death due to their disease. In these cases, the breast cancer cells have become resistant to endocrine therapy, with endocrine resistance identified as the major obstacle to the medical oncologist and patient. To combat the development of endocrine resistance, the treatment options for ER+, HER2 negative breast cancer now include CDK4/6 inhibitors used as adjuvants to antiestrogen treatment. In addition to the dysregulated activity of CDK4/6, a plethora of genetic and biochemical mechanisms have been identified that contribute to endocrine resistance. These mechanisms, which have been identified by lab-based studies utilizing appropriate cell and animal models of breast cancer, and by clinical studies in which gene expression profiles identify candidate endocrine resistance genes, are the subject of this review. In addition, we will discuss molecular targeting strategies now utilized in conjunction with endocrine therapy to combat the development of resistance or target resistant breast cancer cells. Of approaches currently being explored to improve endocrine treatment efficacy and patient outcome, two adaptive cell survival mechanisms, autophagy, and "reversible" senescence, are considered molecular targets. Autophagy and/or senescence induction have been identified in response to most antiestrogen treatments currently being used for the treatment of ER+ breast cancer and are often induced in response to CDK4/6 inhibitors. Unfortunately, effective strategies to target these cell survival pathways have not yet been successfully developed. Thus, there is an urgent need for the continued interrogation of autophagy and "reversible" senescence in clinically relevant breast cancer models with the long-term goal of identifying new molecular targets for improved treatment of ER+ breast cancer.

Cholesterol-modified sphingomyelin chimeric lipid bilayer for improved therapeutic delivery

Cholesterol (Chol) fortifies packing and reduces fluidity and permeability of the lipid bilayer in vesicles (liposomes)-mediated drug delivery. However, under the physiological environment, Chol is rapidly extracted from the lipid bilayer by biomembranes, which jeopardizes membrane stability and results in premature leakage for delivered payloads, yielding suboptimal clinic efficacy. Herein, we report a Chol-modified sphingomyelin (SM) lipid bilayer via covalently conjugating Chol to SM (SM-Chol), which retains membrane condensing ability of Chol. Systemic structure activity relationship screening demonstrates that SM-Chol with a disulfide bond and longer linker outperforms other counterparts and conventional phospholipids/Chol mixture systems on blocking Chol transfer and payload leakage, increases maximum tolerated dose of vincristine while reducing systemic toxicities, improves pharmacokinetics and tumor delivery efficiency, and enhances antitumor efficacy in SU-DHL-4 diffuse large B-cell lymphoma xenograft model in female mice. Furthermore, SM-Chol improves therapeutic delivery of structurally diversified therapeutic agents (irinotecan, doxorubicin, dexamethasone) or siRNA targeting multi-drug resistant gene (p-glycoprotein) in late-stage metastatic orthotopic KPC-Luc pancreas cancer, 4T1-Luc2 triple negative breast cancer, lung inflammation, and CT26 colorectal cancer animal models in female mice compared to respective FDA-approved nanotherapeutics or lipid compositions. Thus, SM-Chol represents a promising platform for universal and improved drug delivery.

Oncolytic Peptide-Nanoplatform Drives Oncoimmune Response and Reverses Adenosine-Induced Immunosuppressive Tumor Microenvironment.

The application of oncolytic peptides has become a powerful approach to induce complete and long-lasting remission in multiple types of carcinomas, as affirmed by the appearance of tumor-associated antigens and adenosine triphosphate (ATP) in large quantities, which jumpstarts the cancer-immunity cycle. However, the ATP breakdown product adenosine is a significant contributor to forming the immunosuppressive tumor microenvironment, which substantially weakens peptide-driven oncolytic immunotherapy. In this study, a lipid-coated micelle (CA@TLM) loaded with a stapled oncolytic peptide (PalAno) and an adenosine 2A receptor (A2AR) inhibitor (CPI-444) is devised to enact tumor-targeted oncolytic immunotherapy and to overcome adenosine-mediated immune suppression simultaneously. The CA@TLM micelle accumulates in tumors with high efficiency, and the acidic lysosomal environment prompts the rapid release of PalAno and CPI-444. Subsequently, PalAno induces swift membrane lysis of tumor cells and the release of antigenic materials. Meanwhile, CPI-444 blocks activation of the immunosuppressive adenosine-A2AR signaling pathway. This combined approach exhibit pronounced synergy at stalling tumor growth and metastasis in animal models for triple-negative breast cancer (TNBC) and melanoma, providing a novel strategy for enhanced oncolytic immunotherapy. This article is protected by copyright. All rights reserved.

LOX, but not LOXL2, promotes bone metastasis formation and bone destruction in triple-negative breast cancer

The primary function of the lysyl oxidase (LOX) family, including LOX and its paralogue LOX-like (LOXL)-2, is to catalyze the covalent crosslinking of collagen and elastin in the extracellular matrix. LOX and LOXL2 are also facilitating breast cancer invasion and metastatic spread to visceral organs (lungs, liver) in vivo. Conversely, the contribution of LOX and LOXL2 to breast cancer bone metastasis remains scant. Here, using gene overexpression or silencing strategies, we investigated the role of LOX and LOXL2 on the formation of metastatic osteolytic lesions in animal models of triple negative breast cancer. In vivo, the extent of radiographic metastatic osteolytic lesions in animals injected with LOX-overexpressing [LOX(+)] tumor cells was 3-fold higher than that observed in animals bearing tumors silenced for LOX [LOX(−)]. By contrast, the extent of osteolytic lesions between LOXL2(+) and LOXL2(−) tumor-bearing animals did not differ, and was comparable to that observed with LOX(−) tumor-bearing animals. In situ, TRAP staining of bone tissue sections from the hind limbs of LOX(+) tumor-bearing animals was substantially increased compared to LOX(−), LOXL2(+) and LOXL2(−)-tumor-bearing animals, which was indicative of enhanced active-osteoclast resorption. In vitro, tumor-secreted LOX increased osteoclast differentiation induced by RANKL, whereas LOXL2 seemed to counteract LOX’s pro-osteoclastic activity. Furthermore, LOX (but not LOXL2) overexpression in tumor cells induced a robust production of IL-6, the latter being a pro-osteoclastic cytokine. Based on these findings, we propose a model in which LOX and IL-6 secreted from tumor cells act in concert to enhance osteoclast-mediated bone resorption that, in turn, promotes metastatic bone destruction in vivo.

Detection of metabolic adaptation in a triple-negative breast cancer animal model with [18F]choline-PET imaging as a surrogate for drug resistance.

Test the feasibility of an image-based method to identify taxane resistance in mouse bearing triple-negative breast cancer (TNBC) tumor xenografts. Xenograft tumor-bearing mice from paclitaxel-sensitive and paclitaxel-resistant TNBC cells (MDA-MD-346) were generated by orthotopic injection into female NOD-SCID mice. When tumors reached 100-150 mm3, mice were scanned using [18F]choline PET/CT. Tumors were collected and sliced for autoradiography and immunofluorescence analysis. Quantitative data was analyzed accordingly. From fifteen mice scanned, five had taxane-sensitive cell line tumors of which two underwent taxol-based treatment. From the remaining 10 mice with taxane-resistant cell line tumors, four underwent taxol-based treatment. Only 13 mice had the tumor sample analyzed histologically. When normalized to the blood pool, both cell lines showed differences in metabolic uptake before and after treatment. Treated and untreated taxane-sensitive and taxane-resistant cell lines have different metabolic properties that could be leveraged before the start of chemotherapy.

Integration of an LPAR1 Antagonist into Liposomes Enhances Their Internalization and Tumor Accumulation in an Animal Model of Human Metastatic Breast Cancer.

Lysophosphatidic acid receptor 1 (LPAR1) is elevated in breast cancer. The deregulation of LPAR1, including the function and level of expression, is linked to cancer initiation, progression, and metastasis. LPAR1 antagonists, AM095 or Ki16425, may be effective therapeutic molecules, yet their limited water solubility hinders in vivo delivery. In this study, we report on the synthesis of two liposomal formulations incorporating AM095 or Ki16425, embedded within the lipid bilayer, as targeted nanocarriers for metastatic breast cancer (MBC). The data show that the Ki16425 liposomal formulation exhibited a 50% increase in internalization by MBC mouse epithelial cells (4T1) and a 100% increase in tumor accumulation in a mouse model of MBC compared with that of a blank liposomal formulation (control). At the same time, normal mouse epithelial cells (EpH-4Ev) internalized the Ki16425 liposomal formulation 25% lesser than the control formulation. Molecular dynamics simulations show that the integration of AM095 or Ki16425 modified the physical and mechanical properties of the lipid bilayer, making it more flexible in these liposomal formulations compared with liposomes without drug. The incorporation of an LPAR1 antagonist within a liposomal drug delivery system represents a viable therapeutic approach for targeting the LPA-LPAR1 axis, which may hinder the progression of MBC.

Anticancer Effects of Date Seed Oil- Loaded Niosomes and Gemcitabine Combination in MCF-7 Human Breast Cancer Cells

Gemcitabine (GEM) is one of the most widely used chemotherapeutic medications for treating various solid tumuors. Niosomes are a novel drug delivery system, self-assembled vesicular nanocarriers and composed of several surfactants and various lipids. Date seed Oil (DSO) contain a high percentage of phenolic and flavonoid compounds that making them important for food and pharmaceutical formulations. In this study, we prepared GEM loaded into anionic niosomes (GEM-niosomes) to target breast tumor cells. The niosomes were prepared with cholesterol (CHOL), span 60, date seed oil and chloroform using the thin film hydration method. GEM-niosomes were fully characterized for their physiochemical properties and evaluated for their cytotoxicity. The targeted niosomes were 100±10nm, the loaded niosomes were in the range of 125±15. GEM-fully loaded DSO-niosomes with drug entrapment percentage (EE%) 82% using high- performance liquid chromatography (HPLC) were developed. The prepared targeted GEM-niosomes showed good stability over 4 weeks at -7ºC and better drug loading. Our targeted GEM-niosomes showed stronger activity against MCF-7 cell by approximately 10 folds compared with the free drug. Taken together, the combination of GEM and “DSO- loaded niosomes” may be of great importance for developing new treatments following in vivo investigations with breast cancer animal models. Future research should explore the in vitro and the in vivo cytotoxicity of this combination on more aggressive breast cancer ( BC).

Leptin/lipopolysaccharide-treated dendritic cell vaccine improved cellular immune responses in an animal model of breast cancer

Purpose In dendritic cells (DCs), leptin as an immune-regulating hormone, increases the IL-12 generation whereas it reduces the IL-10 production, thus contributing to TH1 cell differentiation. Using a murine model of breast cancer (BC), we evaluated the impacts of the Leptin and/or lipopolysaccharide (LPS)-treated DC vaccine on various T-cell-related immunological markers. Materials and methods Tumors were established in mice by subcutaneously injecting 7 × 105 4T1 cells into the right flank. Mice received the DC vaccines pretreated with Leptin, LPS, and both Leptin/LPS, on days 12 and 19 following tumor induction. The animals were sacrificed on day 26 and after that the frequency of the splenic cytotoxic T lymphocytes (CTLs) and TH1 cells; interferon gamma (IFN-γ), interleukin 12 (IL-12) and tumor growth factor beta (TGF-β) generation by tumor lysate-stimulated spleen cells, and the mRNA expression of T-bet, FOXP3 and Granzyme B in the tumors were measured with flow cytometry, ELISA and real-time PCR methods, respectively. Results Leptin/LPS-treated mDC group was more efficient in blunting tumor growth (p = .0002), increasing survival rate (p = .001), and preventing metastasis in comparison with the untreated tumor-bearing mice (UT-control). In comparison to the UT-control group, treatment with Leptin/LPS-treated mDC also significantly increased the splenic frequencies of CTLs (p < .001) and TH1 cells (p < .01); promoted the production of IFN-γ (p < .0001) and IL-12 (p < .001) by splenocytes; enhanced the T-bet (p < .05) and Granzyme B (p < .001) expression, whereas decreased the TGF-β and FOXP3 expression (p < .05). Conclusion Compared to the Leptin-treated mDC and LPS-treated mDC vaccines, the Leptin/LPS-treated mDC vaccine was more effective in inhibiting BC development and boosting immune responses against tumor.

Safety and efficacy of P2Et extract from Caesalpinia spinosa in breast cancer patients: study protocol for a randomized double blind phase II clinical trial (CS003-BC)

BackgroundChemotherapy in breast cancer is effective but can generate significant toxicity and lead to tumor resistance. Joint treatment with standardized plant extracts can be an alternative to improve the response and allow an effective activation of the antitumor immune response that favors recovery in the short and long term. The P2Et extract of Caesalpinia spinosa presents antitumor activity in cells and animal models of breast cancer, improves the tumor microenvironment, and induces activation of the specific immune response against the tumor and is synergistic when used together with anthracyclines, which makes it a good candidate for evaluation in patients.MethodsConducted at a single center, this phase II study is a randomized, double-blind, placebo-controlled trial aimed at assessing the safety and efficacy of P2Et extract in patients diagnosed with stage II and III breast cancer, who are eligible for neoadjuvant treatment. The study aims to determine the safety profile at the previously established optimal biological dose from phase I trial while investigating various efficacy outcomes. These outcomes include improvements in quality of life, immunomodulation, metabolic profile, microbiome, as well as clinical indicators such as tumor reduction, disease-free survival, and pathological response, assessed at different stages of the treatment regimen.DiscussionTreatment with the P2Et extract in breast cancer patients is hypothesized to enhance overall well-being, positively influencing their quality of life, while also triggering an antitumor immune response and enhancing immune infiltration. These combined effects have the potential to contribute to improved long-term survival outcomes for patients receiving the phytomedicine alongside neoadjuvant chemotherapy treatment.Trial registrationThis trial was registered in the US National Library of Medicine with identifier NCT05007444. First Registered August 16th, 2021. Last Updated: August 9th, 2022.

Acetyl-cinobufagin suppresses triple-negative breast cancer progression by inhibiting the STAT3 pathway.

The incidence of breast cancer (BC) worldwide has increased substantially in recent years. Epithelial-mesenchymal transition (EMT) refers to a crucial event impacting tumor heterogeneity. Although cinobufagin acts as an effective anticancer agent, the clinical use of cinobufagin is limited due to its strong toxicity. Acetyl-cinobufagin, a pre-drug of cinobufagin, was developed and prepared with greater efficacy and lower toxicity. A heterograft mouse model using triple negative breast cancer (TNBC) cell lines, was used to evaluate the potency of acetyl-cinobufagin. Signal transducer and stimulator of transcription 3 (STAT3)/EMT involvement was investigated by gene knockout experiments using siRNA and Western blot analysis. Acetyl-cinobufagin inhibited proliferation, migration, and cell cycle S/G2 transition and promoted apoptosis in TNBC cells in vitro. In general, IL6 triggered the phosphorylation of the transcription factor STAT3 thereby activating the STAT3 pathway and inducing EMT. Mechanistically, acetyl-cinobufagin suppressed the phosphorylation of the transcription factor STAT3 and blocked the interleukin (IL6)-triggered translocation of STAT3 to the cell nucleus. In addition, acetyl-cinobufagin suppressed EMT in TNBC by inhibiting the STAT3 pathway. Experiments in an animal model of breast cancer clearly showed that acetyl-cinobufagin was able to reduce tumor growth. The findings of this study support the potential clinical use of acetyl-cinobufagin as a STAT3 inhibitor in TNBC adjuvant therapy.

The Role of Mitochondrial Dysfunction in Cytotoxic Effects of Solanum nigrum Water Extract on MCF-7 and MDA-MB-231 Breast Cancer Cells.

Recent studies suggest that numerous naturally occurring agents have the potential to kill cancer cells via mitochondrial dysfunction. Solanum nigrum is a herb widely used in alternative medical systems. This study aimed to investigate the cytotoxic effect of Solanum nigrum water extract (SNWE) against Michigan Cancer Foundation-7 (MCF-7) and MD Anderson-Metastatic Breast Cancer-231 (MDA-MB-231) cells. We used an MTT reduction assay for cytotoxicity analysis. To explore the mode of action, the cellular adenosine triphosphate (ATP) levels and mitochondrial membrane potential were analyzed using a colorimetric ATP assay and Rhodamine-123 fluorescent staining, respectively, during SNWE treatment for 72 h. The cytotoxic effect was significant in both cell lines, with IC50 values of 4.26 µg/mL and 5.30 µg/mL in MCF-7 and MDA-MB-231 cells, respectively. The 24, 48, and 72 h treatments of 100 µg/mL SNWE showed 0.85 ± 0.07, 0.38 ± 0.1, and 0.20 ± 0.1 nM ATP in MCF-7 cells and 0.94 ± 0.07, 0.84 ± 0.2 and 0.46 ± 0.2 nM in MDA-MB-231 cells, respectively. The SNWE treatment altered the mitochondrial membrane potential (ΔΨm) in a concentration-dependent manner in both the breast cancer cell lines, to 29.6 ± 4.1% in MCF-7 and 28.7 ± 4.17% in MDA-MB-231 cells, when compared with healthy mitochondria (100% ΔΨm). The cytotoxic effects of Solanum nigrum against breast cancer cells are associated with energy metabolism. Additional studies are warranted to test the anticancer effect of Solanum nigrum using an animal model of breast cancer.

In Vivo Electron Paramagnetic Resonance Molecular Profiling of Tumor Microenvironment upon Tumor Progression to Malignancy in an Animal Model of Breast Cancer.

Hypoxia and acidosis are recognized tumor microenvironment (TME) biomarkers of cancer progression. Alterations in cancer redox status and metabolism are also associated with elevated levels of intracellular glutathione (GSH) and interstitial inorganic phosphate (Pi). This study aims to evaluate the capability of these biomarkers to discriminate between stages and inform on a switch to malignancy. These studies were performed using MMTV-PyMT( +) female transgenic mice that spontaneously develop breast cancer and emulate human tumor staging. In vivo assessment of oxygen concentration (pO2), extracellular acidity (pHe), Pi, and GSH was performed using L-band electron paramagnetic resonance spectroscopy and multifunctional trityl and GSH-sensitive nitroxide probes. Profiling of the TME showed significant deviation of measured biomarkers upon tumor progression from pre-malignancy (pre-S4) to the malignant stage (S4). For the combined marker, HOP: (pHe × pO2)/Pi, a value > 186 indicated that the tumors were pre-malignant in 85% of the mammary glands analyzed, and when < 186, they were malignant 42% of the time. For GSH, a value < 3mM indicated that the tumors were pre-malignant 74% of the time, and when > 3mM, they were malignant 80% of the time. The only marker that markedly deviated as early as stage 1 (S1) from its value in pre-S1 was elevated Pi, followed by a decrease of pHe and pO2 and increase in GSH at later stages. Molecular TME profiling informs on alteration of tumor redox and metabolism during tumor staging. Early elevation of interstitial Pi at S1 may reflect tumor metabolic alterations that demand elevated phosphorus supply in accordance with the high rate growth hypothesis. These metabolic changes are supported by the following decrease of pHe due to a high tumor reliance on glycolysis and increase of intracellular GSH, a major intracellular redox buffer. The appreciable decrease in TME pO2 was observed only at malignant S4, apparently as a consequence of tumor mass growth and corresponding decrease in perfusion efficacy and increase in oxygen consumption as the tumor cells proliferate.

Photodynamic Therapy of Breast Cancer in Animal Models and Their Potential Use in Clinical Trials-Role of the Photosensitizers: A Review.

In this article, we reviewed the use of photodynamic therapy (PDT) for breast cancer (BC) in animal models. These in vivo models imitate the cancer disease progression, aid diagnosis, as well as create opportunities to assess treatment during the approval process for the new drug. BC ranks first among women's cancers. Nowadays, there are many diagnostic methods and therapy options for BC but the majority of them have severe side effects. This article discusses the advantages and some disadvantages of the use of small and large animals used for BC models. A literature review showed that the majority of studies have used large animal models, and recently there has been more interest in developing BC in small animal models. BC cell lines such as MCF-7, BT-474, MDA-MB-231, and 4T1 are commercially available for two-dimensional and three-dimensional in vitro cell cultures and subcutaneous models. The purpose of this article is to discuss the performance of PDT in animal models and its further clinical implications. PDT is known to be a non-invasive therapy, which uses monochromatic light and energy to excite photosensitizers (PSs) for the generation of reactive oxygen species as the required factors. Herein, we discuss the use of five photosensitizers in BC models such as chlorin e6 (Ce6), methylene blue, indocyanine green, 5-aminolevulinic acid, and meta-tetra(hydroxyphenyl)chlorin. The database PubMed and Scopus were searched for keywords: 'photodynamic therapy', 'breast cancer', 'animal model', 'clinical studies', and 'photosensitizer(s)'. The PDT search results in animal experiments and its effect on a living organism indicate the possibility of its application in clinical trials on women with local and disseminated BC. The availability and accessibility of small and large BC animal models enable the progress and trial of cancer drugs for innovative technologies and new diagnostics and treatments.

The Amelia-1 study: A phase 1b/2 trial of evexomostat (SDX-7320) plus fulvestrant and alpelisib in patients with advanced breast cancer at risk for alpelisib-induced hyperglycemia.

TPS1129 Background: Alpelisib, a selective PI3K p110a inhibitor, was approved for breast cancer patients with PIK3CA mutations. An on-target toxicity of alpelisib is hyperglycemia and patients with baseline insulin resistance/elevated HbA1c are at greater risk of developing grade 3,4 hyperglycemia after receiving alpelisib. Restoring insulin sensitivity and reducing systemic insulin levels improved the efficacy of alpelisib in animal models of breast cancer. Evexomostat is a polymer-drug conjugate of a novel small molecule methionine aminopeptidase 2 (MetAP2) inhibitor. In normal mice, it reduced alpelisib-induced hyperglycemia/hyperinsulinemia and in an animal model of breast cancer synergized with alpelisib to regress tumor growth. In a Phase 1 trial, evexomostat improved insulin resistance in patients with elevated baseline insulin, reduced angiogenic factors in patients with elevated baseline levels, increased the insulin-sensitizing adipokine adiponectin and showed anti-metastatic effects in patients with advanced solid tumors. Methods: This is a phase 1b/2, open-label, single-arm pilot study (NCT05455619) in postmenopausal women with HR+, HER2- metastatic breast cancer with a PIK3CA mutation who progressed following treatment with endocrine therapy plus a CDK4/6 inhibitor and who are at risk for hyperglycemia (HbA1c between 5.5 and 6.4%inclusive). The primary goal is to determine the safety of evexomostat plus alpelisib and fulvestrant (the ‘triplet therapy’), including the severity and number of hyperglycemic events, as well as anti-tumor benefit. The starting dose of evexomostat in the triplet therapy is 36 mg/m2 (n=6; one dose below the monotherapy MTD of 49 mg/m2). Once the MTD of evexomostat in the triplet therapy has been defined, up to 20 patients will be enrolled at that dose. An additional 20 patients may be enrolled to further characterize the safety and anti-tumor effect of the triplet therapy (up to 52 patients). This trial is open to accrual at multiple sites in the USA. Safety analysis includes the type, frequency, and severity of treatment-emergent adverse events (TEAEs) per NCI CTCAE, v5, and the number and proportion of patients with grade 3 or 4 hyperglycemia during the first 2 cycles of therapy, with an estimate of the exact upper one-sided 97.5% confidence bound. Efficacy analyses include objective response rate (ORR, consisting of complete response (CR) and partial response (PR)). The number of patients without disease progression six months from the start of the triplet therapy will be assessed. The CBR of CRs, PRs plus stable disease ≥24 weeks from C1D1 will be calculated. Overall survival data will be summarized as available. QoL will be analyzed according to functional scores and recommendations in the EORTC scoring manual. ECOG performance status and change from baseline will be summarized. Clinical trial information: NCT05455619 .