Human Breast Cancer Xenografts In Mice Research Articles

Possible pathogenic mechanisms for doxorubicin‐induced splenic atrophy in a human breast cancer xenograft mouse model

AbstractDoxorubicin‐based chemotherapy is a widely used first‐line treatment for breast cancer, yet it is associated with various side effects, including splenic atrophy. However, the pathogenic mechanisms underlying doxorubicin‐induced atrophy of the spleen remain unclear. This study investigates that doxorubicin treatment leads to splenic atrophy through several interconnected pathways involving histological changes, an inflammatory response, and apoptosis. Immunohistochemical and western blot analyses revealed reduced size of white and red pulp, decreased cellularity, amyloidosis, and fibrotic remodeling in the spleen following doxorubicin treatment. Additionally, increased secretion of pro‐inflammatory cytokines was detected using an antibody array and enzyme‐linked immunosorbent assay (ELISA), which triggers inflammation through the regulation of signal transducer and activator of transcription 3 (STAT3) and nuclear factor‐kappa B (NF‐κB) signaling pathways. Further analysis revealed that the loss of regulators and effectors of the oxidative defense system, including sirtuin (Sirt)3, Sirt5, superoxide dismutase (SOD)1, and SOD2, was implicated in the upstream regulation of caspase‐dependent cellular apoptosis. These findings provide insights on the pathogenic mechanisms underlying doxorubicin‐induced splenic atrophy and suggest that further investigation may be warranted to explore strategies for managing potential side effects in breast cancer patients treated with doxorubicin.

Preclinical Activity of 4-Demethyl-4-cholesteryloxycarbonylpenclomedine in Melanoma.

Temozolomide plays a role in treating melanoma refractory to immunomodulatory and mitogen-activated protein kinase-targeted approaches, but its efficacy is limited. 4-Demethyl-4-cholesteryloxycarbonylpenclomedine (DM-CHOC-PEN) is a polychlorinated pyridine cholesteryl carbonate. Its mechanism of action is considered to be via alkylation/adduct formation with N7-guanine. It demonstrated activity in intracranial implanted human glioma and breast cancer xenograft mouse models. The activity of DM-CHOC-PEN in melanoma models was assessed. B-16 melanoma cells were exposed to DM-CHOC-PEN at different concentrations to assess proliferation and survival. B-16 cells were implanted subcutaneously into the flank of adult female C57BL mice which were then were treated with 200 mg/kg DM-CHOC-PEN intraperitoneally daily for 5 days in the setting of palpable subcutaneous tumor. Survival was compared to mice treated with temozolomide or saline. Five mice were treated per group. In vitro, the respective half-maximal inhibitory concentrations of DM-CHOC-PEN and temozolomide were 0.5 and ≥3.0 μg/ml. Floating, heavily melanotic cells formed and these cells were separated, analyzed, and contained 10-90 ng DM-CHOC-PEN per 105 cells. The improvement in survival of mice treated with DM-CHOC-PEN or temozolomide relative to saline controls was 142% and 78%, respectively. Longer survival was seen with DM-CHOC-PEN in a C57BL murine model relative to temozolomide and saline-treated controls, supporting the development of clinical trials assessing the efficacy of DM-CHOC-PEN as treatment for metastatic melanoma.

Human Tumor Targeted Cytotoxic Mast Cells for Cancer Immunotherapy

The diversity of autologous cells being used and investigated for cancer therapy continues to increase. Mast cells (MCs) are tissue cells that contain a unique set of anti-cancer mediators and are found in and around tumors. We sought to exploit the anti-tumor mediators in MC granules to selectively target them to tumor cells using tumor specific immunoglobin E (IgE) and controllably trigger release of anti-tumor mediators upon tumor cell engagement. We used a human HER2/neu-specific IgE to arm human MCs through the high affinity IgE receptor (FcεRI). The ability of MCs to bind to and induce apoptosis of HER2/neu-positive cancer cells in vitro and in vivo was assessed. The interactions between MCs and cancer cells were investigated in real time using confocal microscopy. The mechanism of action using cytotoxic MCs was examined using gene array profiling. Genetically manipulating autologous MC to assess the effects of MC-specific mediators have on apoptosis of tumor cells was developed using siRNA. We found that HER2/neu tumor-specific IgE-sensitized MCs bound, penetrated, and killed HER2/neu-positive tumor masses in vitro. Tunneling nanotubes formed between MCs and tumor cells are described that parallel tumor cell apoptosis. In solid tumor, human breast cancer (BC) xenograft mouse models, infusion of HER2/neu IgE-sensitized human MCs co-localized to BC cells, decreased tumor burden, and prolonged overall survival without indications of toxicity. Gene microarray of tumor cells suggests a dependence on TNF and TGFβ signaling pathways leading to apoptosis. Knocking down MC-released tryptase did not affect apoptosis of cancer cells. These studies suggest MCs can be polarized from Type I hypersensitivity-mediating cells to cytotoxic cells that selectively target tumor cells and specifically triggered to release anti-tumor mediators. A strategy to investigate which MC mediators are responsible for the observed tumor killing is described so that rational decisions can be made in the future when selecting which mediators to target for deletion or those that could further polarize them to cytotoxic MC by adding other known anti-tumor agents. Using autologous human MC may provide further options for cancer therapeutics that offers a unique anti-cancer mechanism of action using tumor targeted IgE’s.

89Zr]-Pertuzumab PET Imaging Reveals Paclitaxel Treatment Efficacy Is Positively Correlated with HER2 Expression in Human Breast Cancer Xenograft Mouse Models.

Paclitaxel (PTX) treatment efficacy varies in breast cancer, yet the underlying mechanism for variable response remains unclear. This study evaluates whether human epidermal growth factor receptor 2 (HER2) expression level utilizing advanced molecular positron emission tomography (PET) imaging is correlated with PTX treatment efficacy in preclinical mouse models of HER2+ breast cancer. HER2 positive (BT474, MDA-MB-361), or HER2 negative (MDA-MB-231) breast cancer cells were subcutaneously injected into athymic nude mice and PTX (15 mg/kg) was administrated. In vivo HER2 expression was quantified through [89Zr]-pertuzumab PET/CT imaging. PTX treatment response was quantified by [18F]-fluorodeoxyglucose ([18F]-FDG) PET/CT imaging. Spearman’s correlation, Kendall’s tau, Kolmogorov–Smirnov test, and ANOVA were used for statistical analysis. [89Zr]-pertuzumab mean standard uptake values (SUVmean) of BT474 tumors were 4.9 ± 1.5, MDA-MB-361 tumors were 1.4 ± 0.2, and MDA-MB-231 (HER2−) tumors were 1.1 ± 0.4. [18F]-FDG SUVmean changes were negatively correlated with [89Zr]-pertuzumab SUVmean (r = −0.5887, p = 0.0030). The baseline [18F]-FDG SUVmean was negatively correlated with initial [89Zr]-pertuzumab SUVmean (r = −0.6852, p = 0.0002). This study shows PTX treatment efficacy is positively correlated with HER2 expression level in human breast cancer mouse models. Molecular imaging provides a non-invasive approach to quantify biological interactions, which will help in identifying chemotherapy responders and potentially enhance clinical decision-making.

Abstract PS3-03: [89Zr]-pertuzumab pet imaging reveals paclitaxel treatment efficacy is positively correlated with her2 expression in human breast cancer xenograft mouse models

Abstract Introduction: Paclitaxel (PTX) is one of the most commonly used first-line chemotherapies in the treatment of breast cancer. However, it is reported that overall PTX response rate is between 30-60% for the treatment of metastatic breast cancer. Thus, novel discoveries of molecular mechanisms that account for ineffective response is critical in breast cancer treatment. [89Zr]-Pertuzumab (antibody that targets HER2 receptors) and 2-deoxy-2- [fluorine-18] fluoro-D-glucose ([18F]-FDG) positron emission tomography/computed tomography (PET/CT) imaging allows us to noninvasively measure the amount and heterogeneity of HER2 expression and tumor glucose metabolism. These methods are clinical translatable and can provide insight into tumor biology changes during the course of therapy. This study evaluates whether HER2 expression level is correlated with PTX treatment efficacy in controlled pre-clinical mouse models of HER2+ breast cancer. Experimental Design: BT474 (1×107), MDA-MB-361 (6×106), or MDA-MB-231 (2×106) cells were subcutaneously injected into athymic nude mice (N = 7 per cell line). When the tumor volume reached approximately 200 mm3, mice were enrolled in the study. Tumor size was measured with calipers weekly until enrolled and every 3 days after experiment started. In vivo HER2 expression level was determined by [89Zr]-Pertuzumab PET/CT imaging at one week prior to initiation of treatment and confirmed with immunohistochemistry staining. PTX (15 mg/kg) was administered via i.v. on days 0 and 3. In vivo tumor metabolism was quantified by [18F]-FDG PET/CT imaging on day 0, 3 and 6. Mean, standard deviation, sum, and frequency histogram of standard uptake value (SUV) were quantified. Tumors were harvested at day 6 for histological analysis. Hematoxylin and eosin (H&E) and cleaved caspase 3 immunohistochemistry (IHC) staining were used to determine tumor apoptosis. Pearson’s correlation and ANOVA were used as statistical analysis. Results: [89Zr]-Pertuzumab SUVmean of BT474 (HER2+) tumors were 4.9±1.5, MDA-MB-361 (HER2+) tumors were 1.4±0.2 (p<0.0001, compared with BT474), and MDA-MB-231 (HER2-) tumors were 1.1±0.4 (p<0.0001, compared with BT474). [18F]-FDG SUV sum was positively correlated with tumor volume (R2=0.1669, p=0.0250). Tumor volumes showed no significant changes during the treatment. However, normalized [18F]-FDG SUV concentration changes from day 0 to day 3 was negatively correlated with baseline [89Zr]-pertuzumab SUV concentration (R2=0.3654, p=0.05). Conclusion: Preliminary results show paclitaxel treatment efficacy is positively correlated with HER2 expression level in human breast cancer mouse models. [89Zr]-Pertuzumab PET/CT imaging quantitively measured HER2 expression level in vivo, and [18F]-FDG PET/CT imaging revealed the early signals of drug treatment efficacy. This discovery will help identify chemotherapy responders and potentially enhance clinical decision making. Acknowledgments: We thank funding sources American Cancer Society RSG-18-006-01-CCE and NIH NCI R01CA240589. Citation Format: Yun Lu, Adriana Massicano, Patrick Song, Meng Li, Suzanne Lapi, Anna Sorace. [89Zr]-pertuzumab pet imaging reveals paclitaxel treatment efficacy is positively correlated with her2 expression in human breast cancer xenograft mouse models [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 PS3-03.

Therapeutic enhancement of radiation and immunomodulation by gold nanoparticles in triple negative breast cancer

ABSTRACT Gold nanoparticles (AuNPs) have been shown to enhance cancer radiotherapy (RT) gain by localizing the absorption of radiation energy in the tumor while sparing surrounding normal tissue from radiation toxicity. Previously, we showed that AuNPs enhanced RT induced DNA damage and cytotoxicity in MCF7 breast cancer cells. Interestingly, we found that cancer cells exhibited a size-dependent AuNPs intracellular localization (4 nm preferentially in the cytoplasm and 14 nm in the nucleus). We extended those studies to an in vivo model and examined the AuNPs effects on RT cytotoxicity, survival and immunomodulation of tumor microenvironment (TME) in human triple negative breast cancer (TNBC) xenograft mouse model. We also explored the significance of nanoparticle size in these AuNPs’ effects. Mice treated with RT and RT plus 4 nm or 14 nm AuNPs showed a significant tumor growth delay, compared to untreated animals, while dual RT plus AuNPs treatment exhibited additive effect compared to either RT or AuNPs treatment alone. Survival log-rank test showed significant RT enhancement with 14 nm AuNP alone; however, 4 nm AuNPs did not exhibit RT enhancement. Both sizes of AuNPs enhanced RT induced immunogenic cell death (ICD) that was coupled with significant macrophage infiltration in mice pretreated with 14 nm AuNPs. These results showing significant AuNP size-dependent RT enhancement, as evident by both tumor growth delay and overall survival, reveal additional underlying immunological mechanisms and provide a platform for studying RT multimodal approaches for TNBC that may be combined with immunotherapies, enhancing their effect.

Abstract 2775: Her2 expression level is positively correlated with paclitaxel response in human breast cancer xenograft mouse model

Abstract Introduction: The overall goal of this study is to quantify amount and heterogeneity of Her2 expression and determine if its expression affects its response to chemotherapy in breast cancer. Paclitaxel (PTX) is one of the most commonly used first-line drugs in the treatment of breast cancer. However, it is reported that overall PTX response rate is 30%-60%, as first-line treatment of metastatic breast cancer, and the main treatment failure is PTX resistance. Thus, novel discoveries of molecular mechanisms that account for ineffective response or resistance to chemotherapy is critical in breast cancer treatment. This study evaluates whether Her2 expression level is correlated with PTX treatment efficacy in a controlled pre-clinical mouse model. Experimental Design: BT474 (1 × 107) or MDA-MB-231 (2 × 106) cells were subcutaneously injected into athymic nude mice (n = 10). Mice were enrolled in the study once they achieved tumor volume of 200 mm3. Tumor size were measured with caliper weekly or every 3 days after experiment started. In vivo Her2 expression level was determined by [89Zr]-Pertuzumab (Her2 antibody) positron emission tomography/computed tomography (PET/CT) imaging at one week prior to initiation of treatment. PTX (15 mg/kg) was administrated via i.v. at day 0 and day 3. In vivo tumor metabolism was quantified by 2-deoxy-2-[fluorine-18]fluoro- D-glucose ([18F]-FDG) PET/CT imaging at day 0, day 3 and day 6. Mean, standard deviation, and frequency histogram of standard uptake value (SUV) was quantified. Tumors were harvested at day 6 for histological analysis. Pearson's correlation test was used as statistical analysis. Results: BT474 (Her2+) tumors had significantly increased [89Zr]-Pertuzumab uptake compared to MDA-MB-231 (Her2-) tumors, which indicated the specificity and accuracy of [89Zr]-Pertuzumab PET/CT imaging. [89Zr]-Pertuzumab SUV varies within BT474 tumors, and tumor growth rate was positively correlated with mean Her2 expression (R2=0.9011, p=0.001). During PTX treatment, tumor volumes did not reveal significant changes. However, [18F]-FDG SUV concentration, normalized to percent changes from day 0 to day 3, was negatively correlated with initial [89Zr]-pertuzumab SUV concentration (R2=0.3654, p=0.05). Conclusion: Preliminary results show Her2 expression level is positively correlated with chemotherapy response in mouse models of human breast cancer. This discovery will help studies of PTX resistance in breast cancer and potentially guide clinical administration of combined therapies. [89Zr]-Pertuzumab PET/CT imaging quantitively measured Her2 expression level in vivo, and [18F]-FDG PET/CT imaging revealed the early signals of drug treatment efficacy. Citation Format: Yun Lu, Adriana I. Massicano, Suzanne E. Lapi, Meng Li, Patrick N. Song, Anna G. Sorace. Her2 expression level is positively correlated with paclitaxel response in human breast cancer xenograft mouse model [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 2775.

Abstract 4806: SIRT5 inhibition causes increased oxidative stress and impairs tumor progression and metastasis

Abstract Cancer cells undergo metabolic reprogramming, producing energy through aerobic glycolysis followed by lactic acid fermentation. Consequently, targeting key metabolic enzymes has emerged as a potential avenue to selectively inhibit tumor growth. The sirtuin family of deacylases has roles in both metabolism and cancer. The mitochondrial sirtuin SIRT5 regulates protein post-translational modifications on metabolic enzymes by catalyzing the removal of succinyl, malonyl, and glutaryl moieties. SIRT5 is over-expressed in many cancers, including breast cancer. Therefore, we hypothesized that SIRT5 promotes breast cancer progression by facilitating metabolic reprogramming. SIRT5 knockdown in human cancer cells inhibited anchorage independent growth but had little effect on non-transformed cells. To examine how SIRT5 loss impacts tumorigenesis in vivo, we utilized MMTV-PyMT transgenic mice, which are prone to mammary adenocarcinoma and lung metastasis. SIRT5 knockout (KO) MMTV-PyMT mice had increased survival, decreased tumor size, and reduced lung metastases, as compared to SIRT5 wild-type (WT) MMTV-PyMT controls. SIRT5 KO cancer cells had higher levels of reactive oxygen species and displayed lower levels of important antioxidants such as NADPH and GSH. These results suggest that SIRT5 KO cells are more susceptible to oxidative stress, hinting that SIRT5 may be promoting breast cancer by mitigating ROS through one or more of its key metabolic substrates. To determine the therapeutic potential of SIRT5 inhibition, we created potent and specific small molecule SIRT5 inhibitors. Pharmacological inhibition of SIRT5 impaired mammary tumor growth in both transgenic and human breast cancer xenograft mouse models, with no apparent toxicity. Considering that Sirt5 knockout mice are generally normal, with only mild phenotypes observed, these data establish SIRT5 as a promising target for treating breast cancer. Citation Format: Irma Fernandez, Yashira N. Abril, Jun Y. Hong, Min Yang, Sushabhan Sadhukhan, Hening Lin, Robert Weiss. SIRT5 inhibition causes increased oxidative stress and impairs tumor progression and metastasis [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 4806.

Suppression of Cancer Proliferation and Metastasis by a Versatile Nanomedicine Integrating Multiple Therapeutic Mechanisms

Cancer therapeutics are varied and target diverse processes in cancer progression. Photodynamic therapy (PDT), photothermal therapy (PTT), and the inhibition of pro-cancer proteases are novel non-invasive anticancer therapeutics that attract increasing attentions for their enhanced specificities and milder systemic toxicities compared to traditional therapeutics (surgery, radio- or chemo-therapies). These modalities offer advantages to compensate for the shortcomings of their counterparts. For instance, PDT or PTT efficiently eliminates locally confined tumor cells while exhibiting no effect on metastatic tumor cells. In contrast, the inhibition of pro-cancer proteases systemically suppresses the proliferation and metastasis of cancer cells but does not eradicate existing cancer cells. To synergize these therapeutics, we hereby report a versatile nanoparticle that integrates the anticancer effects of PDT, PTT, and enzyme-inhibition. This nanoparticle (CIKP-NP) was synthesized by covalently or non-covalently modifying a photothermal nanoparticle with a photosensitizer, a pro-cancer protease inhibitor, and an albumin-binding molecule. After confirming that CIKP-NP encompasses the properties of PDT, PTT, albumin-binding, and enzyme-inhibition at the molecular level, we evaluated the anti-tumor and anti-metastatic effects of CIKP-NP at the cellular level. In addition, through a human breast cancer xenograft mouse model, we demonstrated that CIKP-NP suppressed tumor growth by PDT or PTT effect. Notably, the synergism of PDT and PTT significantly enhanced its anticancer effect. Furthermore, CIKP-NP significantly suppressed cancer metastasis in a lung metastatic mouse model. Beyond demonstrating the anti-tumor and anti-metastatic efficacy of CIKP-NP, our study also suggests a new strategy to synergize multiple anticancer therapeutics.

Dosing Time-Dependent Changes in the Anti-tumor Effect of xCT Inhibitor Erastin in Human Breast Cancer Xenograft Mice.

Growth of cancer cells is more highly dependent on various types of amino acids than that of normal cells, and thus prevention of amino acid requirement has been recognized as strategies for cancer therapies. In this study, we found that deprivation of cysteine (Cys) in culturing media prevented the growth of various types of human cancer cell lines. Cys is easily converted to cystine (Cys-Cys) in media and uptaken into cells by cystine/glutamate transporter (xCT). The incorporated Cys-Cys is decomposed into Cys, and used for synthesis of glutathione that suppresses reactive oxygen species-induced cell damage. Therefore, we examined whether a selective xCT inhibitor erastin prevented the growth of human cancer cell lines. As a result, erastin significantly prevented the proliferation of various types of human cancer cells. Among them, MDA-MB-231 breast cancer cells were identified as the most erastin-sensitive cells. To investigate the ability of erastin to prevent growth of tumor in mice, MDA-MB-231 breast cancer cells were implanted into BALB/c nude female mice kept under standardized light/dark cycle conditions. The growth of tumor implanted in mice was significantly suppressed by administration of erastin during the light phase, whereas its administration during the dark phase failed to suppress the tumor growth. The dosing time-dependency of erastin-induced cystine/cysteine deprivation was closely related to that of its anti-tumor effects. Our present findings suggest that the anti-tumor efficacy of erastin in tumor-bearing mice is improved by optimizing the dosing schedule.

A novel strategy to block mitotic progression for targeted therapy.

BackgroundBlockade of mitotic progression is an ideal approach to induce mitotic catastrophe that suppresses cancer cell expansion. Cdc20 is a critical mitotic factor governing anaphase initiation and the exit from mitosis through recruiting substrates to APC/C for degradation. Results from recent TCGA (The Cancer Genome Atlas) and pathological studies have demonstrated a pivotal oncogenic role for Cdc20-APC/C in tumor progression as well as drug resistance. Thus, deprivation of the mitotic role for Cdc20-APC/C by either inhibition of Cdc20-APC/C activity or elimination of Cdc20 protein via induced protein degradation emerges as an effective therapeutic strategy to control cancer.MethodsWe designed a proteolysis targeting chimera, called CP5V, which comprises a Cdc20 ligand and VHL binding moiety bridged by a PEG5 linker that induces Cdc20 degradation. We characterized the effect of CP5V in destroying Cdc20, arresting mitosis, and inhibiting tumor progression by measuring protein degradation, 3D structure dynamics, cell cycle control, tumor cell killing and tumor inhibition using human breast cancer xenograft mouse model.FindingsResults from our study demonstrate that CP5V can specifically degrade Cdc20 by linking Cdc20 to the VHL/VBC complex for ubiquitination followed by proteasomal degradation. Induced degradation of Cdc20 by CP5V leads to significant inhibition of breast cancer cell proliferation and resensitization of Taxol-resistant cell lines. Results based on a human breast cancer xenograft mouse model show a significant role for CP5V in suppressing breast tumor progression.InterpretationCP5V-mediated degradation of Cdc20 could be an effective therapeutic strategy for anti-mitotic therapy.

Baicalin inhibits breast cancer development via inhibiting IĸB kinase activation in vitro and in vivo.

The aim of the present study was to investigate the effect and therapeutic potential of baicalin in breast cancer. Baicalin is used to treat inflammatory diseases. The effects of baicalin were assessed in breast cancer MCF-7 and MDA-MB‑231 cells, and human breast cancer xenograft mice. Cells were treated with 0, 20 or 30µM baicalin for 48h, while xenograft mice were treated with intraperitoneal injection of 0, 100 or 200mg/kg baicalin for 30days. The results demonstrated that treatment with baicalin dose-dependently suppressed breast cancer cell invasion, migration and proliferation, and also induced G1/S-phase cell cycle arrest invitro and invivo. Baicalin alleviated inflammation injury and inhibited the secretion of tumor necrosis factor(TNF)-α and interleukin(IL)-1β, thus suppressing nuclear factor(NF)-ĸB-p65 activation via inhibition of IĸB kinase. Investigation of the mechanism underlying baicalin activity indicated that it inhibited protein expression of NF-ĸB-p65, leading to NF-ĸB‑induced increased expression of CCND1, BCL2, BIRC2 and BIRC3, thus inhibiting cell proliferation, invasion and migration and suppressing anti-apoptotic factors invitro and invivo. In addition, baicalin did not affect non-tumorigenic normal breast epithelial cells. These results indicate that baicalin may exert therapeutic effects in breast cancer.

Isomangiferin, a Novel Potent Vascular Endothelial Growth Factor Receptor 2 Kinase Inhibitor, Suppresses Breast Cancer Growth, Metastasis and Angiogenesis.

PurposeVascular endothelial growth factor (VEGF) signal transduction mainly depends on its binding to VEGF receptor 2 (VEGFR-2). VEGF downstream signaling proteins mediate several of its effects in cancer progression, including those on tumor growth, metastasis, and blood vessel formation. The activation of VEGFR-2 signaling is a hallmark of and is considered a therapeutic target for breast cancer. Here, we report a study of the regulation of the VEGFR-2 signaling pathway by a small molecule, isomangiferin.MethodsA human breast cancer xenograft mouse model was used to investigate the efficacy of isomangiferin in vivo. The inhibitory effect of isomangiferin on breast cancer cells and the underlying mechanism were examined in vitro.ResultsIsomangiferin suppressed tumor growth in xenografts. In vitro, isomangiferin treatment inhibited cancer cell proliferation, migration, invasion, and adhesion. The effect of isomangiferin on breast cancer growth was well coordinated with its suppression of angiogenesis. A rat aortic ring assay revealed that isomangiferin significantly inhibited blood vessel formation during VEGF-induced microvessel sprouting. Furthermore, isomangiferin treatment inhibited VEGF-induced proliferation of human umbilical vein endothelial cells and the formation of capillary-like structures. Mechanistically, isomangiferin induced caspase-dependent apoptosis of breast cancer cells. Furthermore, VEGF-induced activation of the VEGFR-2 kinase pathway was down-regulated by isomangiferin.ConclusionOur findings demonstrate that isomangiferin exerts anti-breast cancer effects via the functional inhibition of VEGFR-2. Pharmaceutically targeting VEGFR-2 by isomangiferin could be an effective therapeutic strategy for breast cancer.

Effects of diffusion time on non-Gaussian diffusion and intravoxel incoherent motion (IVIM) MRI parameters in breast cancer and hepatocellular carcinoma xenograft models.

BackgroundPerfusion-related intravoxel incoherent motion (IVIM) and non-Gaussian diffusion magnetic resonance (MR) parameters are becoming important biomarkers for differentiating malignant from benign tumors without contrast agents. However, diffusion-time dependence has rarely been investigated in tumors.PurposeTo investigate the relationship between diffusion time and diffusion parameters in breast cancer and hepatocellular carcinoma xenograft mouse models.Material and MethodsDiffusion-weighted MR images (DWI) were obtained on a 7-T magnetic resonance imaging (MRI) scanner at two different diffusion times (9.6 ms and 27.6 ms) in human breast cancer (MDA-MB-231) and hepatocellular carcinoma (HepG2 and PLC/PRF/5) xenograft mouse models. Perfusion-related IVIM (fIVIM and D*) and non-Gaussian diffusion (ADC0 and K) parameters were estimated. Parametric maps of diffusion changes with the diffusion times were generated using a synthetic apparent diffusion coefficient (sADC) obtained from b = 438 and 2584 s/mm2.ResultsADC0 values significantly decreased when diffusion times were changed from 9.6 ms to 27.6 ms in MDA-MB-231, HepG2, and PLC/PRF/5 groups (P = 0.0163, 0.0351, and 0.0170, respectively). K values significantly increased in MDA-MB-231 and HepG2 groups (P < 0.0003 and = 0.0007, respectively); however, no significant difference was detected in the PLC/PRF/5 group. fIVIM values increased, although not significantly (P = 0.164–0.748). The maps of sADC changes showed that diffusion changes with the diffusion time were not homogeneous across tumor tissues.ConclusionDiffusion MR parameters in both breast cancer and HCC xenograft models were found to be diffusion time-dependent. Our results show that diffusion time is an important parameter to consider when interpreting DWI data.

Time-dependent pharmacokinetics of dexamethasone and its efficacy in human breast cancer xenograft mice: a semi-mechanism-based pharmacokinetic/pharmacodynamic model.

Dexamethasone (DEX) is the substrate of CYP3A. However, the activity of CYP3A could be induced by DEX when DEX was persistently administered, resulting in auto-induction and time-dependent pharmacokinetics (pharmacokinetics with time-dependent clearance) of DEX. In this study we investigated the pharmacokinetic profiles of DEX after single or multiple doses in human breast cancer xenograft nude mice and established a semi-mechanism-based pharmacokinetic/pharmacodynamic (PK/PD) model for characterizing the time-dependent PK of DEX as well as its anti-cancer effect. The mice were orally given a single or multiple doses (8 mg/kg) of DEX, and the plasma concentrations of DEX were assessed using LC-MS/MS. Tumor volumes were recorded daily. Based on the experimental data, a two-compartment model with first order absorption and time-dependent clearance was established, and the time-dependence of clearance was modeled by a sigmoid Emax equation. Moreover, a semi-mechanism-based PK/PD model was developed, in which the auto-induction effect of DEX on its metabolizing enzyme CYP3A was integrated and drug potency was described using an Emax equation. The PK/PD model was further used to predict the drug efficacy when the auto-induction effect was or was not considered, which further revealed the necessity of adding the auto-induction effect into the final PK/PD model. This study established a semi-mechanism-based PK/PD model for characterizing the time-dependent pharmacokinetics of DEX and its anti-cancer effect in breast cancer xenograft mice. The model may serve as a reference for DEX dose adjustments or optimization in future preclinical or clinical studies.

Heparosan-coated liposomes for drug delivery.

Liposomal encapsulation is a useful drug delivery strategy for small molecules, especially chemotherapeutic agents such as doxorubicin. Doxil® is a doxorubicin-containing liposome ("dox-liposome") that passively targets drug to tumors while reducing side effects caused by free drug permeating and poisoning healthy tissues. Polyethylene glycol (PEG) is the hydrophilic coating of Doxil® that protects the formulation from triggering the mononuclear phagocyte system (MPS). Evading the MPS prolongs dox-liposome circulation time thus increasing drug deposition at the tumor site. However, multiple doses of Doxil® sometimes activate an anti-PEG immune response that enhances liposome clearance from circulation and causes hypersensitivity, further limiting its effectiveness against disease. These side effects constrain the utility of PEG-coated liposomes in certain populations, justifying the need for investigation into alternative coatings that could improve drug delivery for better patient quality of life and outcome. We hypothesized that heparosan (HEP; [-4-GlcA-β1-4-GlcNAc-α1-]n) may serve as a PEG alternative for coating liposomes. HEP is a natural precursor to heparin biosynthesis in mammals. Also, bacteria expressing an HEP extracellular capsule during infection escape detection and are recognized as "self," not a foreign threat. By analogy, coating drug-carrying liposomes with HEP should camouflage the delivery vehicle from the MPS, extending circulation time and potentially avoiding immune-mediated clearance. In this study, we characterize the postmodification insertion of HEP-lipids into liposomes by dynamic light scattering and coarse-grain computer modeling, test HEP-lipid immunogenicity in rats, and compare the efficacy of drug delivered by HEP-coated liposomes to PEG-coated liposomes in a human breast cancer xenograft mouse model.

Synergistic inhibitory effects of an engineered antibody-like molecule ATF-Fc and trastuzumab on tumor growth and invasion in a human breast cancer xenograft mouse model.

The overexpression of the oncogene human epidermal growth factor receptor 2 (HER-2) has been associated with decreased disease-free survival and is a marker of poor prognosis of invasive breast cancer. Although the high efficacy of trastuzumab, a drug that targets the HER-2 oncogene, has been widely recognized, the efficiency of the treatment remains at ~30%. Therefore, novel effective treatments are required for patients with recurrent metastatic breast cancer. The present study aimed to investigate the effects of an engineered antibody-like molecule administered alone or in combination with trastuzumab on the tumor growth and metastasis of HER-2-positive breast cancer. Another aim was to investigate novel cancer therapies for HER-2-positive breast cancer. The engineered antibody-like molecule consists of the amino-terminal fragment (ATF) of human urokinase-type plasminogen (uPA) and is conjugated with the Fc fragment of human immunoglobulin G1 (ATF-Fc). The anti-cancer effect of ATF-Fc (alone and in combination with trastuzumab) on tumor cells and in a nude mouse tumor model was evaluated by detecting the expression of uPA, urokinase plasminogen activator receptor (uPAR) and HER-2. In vitro experiments demonstrated that specifically blocking the uPA-uPAR and HER-2 signaling pathways may effectively promote the apoptosis of breast cancer cells. Additionally, ATF-Fc-induced cell death in HER-2-positive breast cancer cells was observed in vivo. When ATF-Fc was administered in combination with trastuzumab, cell death was increased and breast cancer metastasis was reduced. The novel engineered antibody-like molecule ATF-Fc was able to inhibit HER-2-positive breast cancer cell growth and metastasis by interfering with uPA and its receptor (uPA-uPAR) system. Additionally, the antibody-like molecule exhibits a synergistic inhibitory effect when administered in combination with trastuzumab.

Abstract CT057: A single-arm, preoperative, pilot study to evaluate the safety and biological effects of orally administered reparixin in early breast cancer patients who are candidates for surgery

Abstract Background. Experimental models and retrospective clinical observations point to Cancer Stem Cells (CSC) as responsible for tumor initiation, treatment resistance, disease recurrence and metastasis. CXCR1, one of the receptors for CXCL8, was identified on breast cancer (BC) CSC. Reparixin, an investigational allosteric inhibitor of CXCR1, administered as monotherapy reduced the metastatic spread of human BC cells and the CSC content of human BC xenograft in mice (Ginestier C et al., JCI 2010).Methods. In this single arm, monotherapy trial (NCT01861054) patients were female aged ? 18 years with HER-2 negative operable BC with a clinical diameter of &amp;gt;1 cm not eligible for neoadjuvant treatment. It was planned to enrol 20 patients with ER+ and/or PgR+ and 20 patients with ER-/PgR- BC (i.e., Triple-Negative BC, TNBC). Patients received reparixin oral tablets 1000 mg 3 times daily (t.i.d.) for 21 days prior to surgery. Patients underwent core biopsies at baseline and at the completion of therapy. Primary objectives were evaluation of the effects of reparixin on CSC and tumor microenvironment and the safety of reparixin. The secondary objective was to define the pharmacokinetic (PK) profile of orally administered reparixin. Signal of Activity (SoA) was defined as a reduction by at least 20% in ALDH-1+ or CD24-/CD44+ CSC by flow cytometry (FC), with consistent reduction by immunohistochemistry (IHC).Results. 18 patients with ER+ and/or PgR+ BC and 2 with TNBC were enrolled. The trial was terminated due to slow enrolment in TNBC cohort. Safety: adverse reaction of grade ?2 (none serious) were experienced by 10/20 patients. All patients completed study treatment. PK: reparixin was rapidly absorbed (median Tmax 1h) and metabolized with median t1/2 of about 1.5 hr on both day 1 and 21. Efficacy: 12/18 and 2/2 patients in the ER+ and/or PgR+ BC group and TNBC group, respectively, had a sizeable ALDH+ and/or CD24-/CD44+ cell population at baseline and a second biopsy evaluated by FC. 11 evaluable patients achieved SoA (9/12 ER+ and/or PgR+ and 2/2 TNBC). The possibility to confirm FC results in tissue sections was hindered by very low numbers of CSC. A reduction in total CXCR1+ cells was found by FC in 7/13 evaluable patients and a reduction in p62 by IHC in 8/13 evaluable patients, suggesting target engagement and induction of autophagy.Conclusions. Enrolment of patients with TNBC in preoperative trials with single non cytotoxic agents is limited by the widespread use of neoadjuvant treatment. Reparixin 1000 mg t.i.d. for 21 consecutive days appeared to be well tolerated. SoA was achieved in 11/14 evaluable patients. The clinical relevance of a ?20% reduction of CSC following a single 21-day course of reparixin in this patient population is unknown. A randomized phase 2 study of reparixin plus paclitaxel versus paclitaxel in frontline treatment of metastatic TNBC is ongoing (NCT02370238). Citation Format: Lori J. Goldstein, Raymond P. Perez, Denise A. Yardley, Linda K. Han, James M. Reuben, Susan McCanna, Beth Butler, Pier Adelchi Ruffini, Jenny C. Chang. A single-arm, preoperative, pilot study to evaluate the safety and biological effects of orally administered reparixin in early breast cancer patients who are candidates for surgery. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr CT057.

Pharmacokinetic-pharmacodynamic modeling of the antitumor effect of TM208 and EGFR-TKI resistance in human breast cancer xenograft mice.

The novel anticancer compound TM208 is an EGFR tyrosine kinase inhibitor (EGFR-TKI). Since the development of resistance to EGFR-TKIs is a major challenge in their clinical usage, we investigated the profiles of resistance following continuous treatment with TM208 in human breast cancer xenograft mice, and identified the relationship between the tumor pEGFR levels and tumor growth inhibition. Female BALB/c nude mice were implanted with human breast cancer MCF-7 cells, and the xenograft mice received TM208 (50 or 150 mg·kg(-1)·d(-1), ig) or vehicle for 18 d. The pharmacokinetics (PK) and pharmacodynamics (PD) of TM208 were evaluated. The PK properties of TM208 were described by a one-compartment model with first-order absorption kinetics. Our study showed the inhibitory effects of TM208 on tumor pEGFR levels gradually reached a maximum effect, after which it became weaker over time, which was characterized by a combined tolerance/indirect response PD model with an estimated EC50 (55.9 μg/L), as well as three parameters ('a' of 27.2%, 'b' of 2730%, 'c' of 0.58 h(-1)) denoting the maximum, extent and rate of resistance, respectively. The relationship between the tumor pEGFR levels and tumor growth inhibition was characterized by a combined logistic tumor growth/transit compartment model with estimated parameters associated with tumor growth characteristics kng (0.282 day(-1)), drug potency kTM208 (0.0499 cm(3)/day) and the kinetics of tumor cell death k1 (0.141 day(-1)), which provided insight into drug mechanisms and behaviors. The proposed PK/PD model provides a better understanding of the pharmacological properties of TM208 in the treatment of breast cancer. Furthermore, simulation based on a tolerance model allows prediction of the occurrence of resistance.

Directed molecular assembly into a biocompatible photosensitizing nanocomplex for locoregional photodynamic therapy

Methylene blue (MB), a water-soluble cationic dye widely used in the clinic, is known to photosensitize the generation of cytotoxic singlet oxygen efficiently, and thus, has attracted interest as a potential drug for photodynamic therapy (PDT). However, its use for the in vivo PDT of cancer has been limited due to the inherently poor cell/tissue accumulation and low biological stability in the free molecular form. Here, we report a simple and biocompatible nanocomplex formulation of MB (NanoMB) that is useful for in vivo locoregional cancer treatment by PDT. NanoMB particles were constructed through the self-assembly of clinically usable molecules (MB, fatty acid and a clinically approved polymer surfactant) directed by the dual (electrostatic and hydrophobic) interactions between the ternary constituents. The nanocomplexed MB showed greatly enhanced cell internalization while keeping the photosensitization efficiency as high as free MB, leading to distinctive phototoxicity toward cancer cells. When administered to human breast cancer xenograft mice by peritumoral injection, NanoMB was capable of facile penetration into the tumor followed by cancer cell accumulation, as examined in vivo and histologically with the near-infrared fluorescence signal of MB. The quintuple PDT treatment by a combination of peritumorally injected NanoMB and selective laser irradiation suppressed the tumor volume efficaciously, demonstrating potential of NanoMB-based PDT as a biocompatible and safe method for adjuvant locoregional cancer treatment.