Antigen presentation and interferon signatures in B cells driven by localized ablative cancer immunotherapy correlate with extended survival.

Rationale: B cells have emerged as key regulators in protective cancer immunity. However, the activation pathways induced in B cells during effective immunotherapy are not well understood. Methods: We used a novel localized ablative immunotherapy (LAIT), combining photothermal therapy (PTT) with intra-tumor delivery of the immunostimulant N-dihydrogalactochitosan (GC), to treat mice bearing mouse mammary tumor virus-polyoma middle tumor-antigen (MMTV-PyMT). We used single-cell RNA sequencing to compare the transcriptional changes induced by PTT, GC and PTT+GC in B cells within the tumor microenvironment (TME). Results: LAIT significantly increased survival in the tumor-bearing mice, compared to the treatment by PTT and GC alone. We found that PTT, GC and PTT+GC increased the proportion of tumor-infiltrating B cells and induced gene expression signatures associated with B cell activation. Both GC and PTT+GC elevated gene expression associated with antigen presentation, whereas GC elevated transcripts that regulate B cell activation and GTPase function and PTT+GC induced interferon response genes. Trajectory analysis, where B cells were organized according to pseudotime progression, revealed that both GC and PTT+GC induced the differentiation of B cells from a resting state towards an effector phenotype. The analyses confirmed upregulated interferon signatures in the differentiated tumor-infiltrating B cells following treatment by PTT+GC but not by GC. We also observed that breast cancer patients had significantly longer survival time if they had elevated expression of genes in B cells that were induced by PTT+GC therapy in the mouse tumors. Conclusion: Our findings show that the combination of local ablation and local application of immunostimulant initiates the activation of interferon signatures and antigen-presentation in B cells which is associated with positive clinical outcomes for breast cancer. These findings broaden our understanding of LAIT's regulatory roles in remodeling TME and shed light on the potentials of B cell activation in clinical applications.

Physical Activity Plus Energy Restriction Prevents 4T1.2 Mammary Tumor Progression, MDSC Accumulation, and an Immunosuppressive Tumor Microenvironment.

Physical activity and the prevention of weight gain decrease breast cancer incidence and improve survival. Unraveling the biological mechanisms underlying these cancer prevention effects is difficult because activity and dietary restriction are often linked. The goal of this study was to determine whether physical activity (PA), preventing weight gain via energy restriction (ER), or the combination was most effective in delaying tumor growth, reducing metastatic progression, and improving survival in the 4T1.2 mammary tumor model. Furthermore, we determined whether any of these interventions prevented the expansion of protumor immunosuppressive cells and altered the tumor microenvironment (TME). Female BALB/c mice (n = 7-20/group) were randomized to sedentary (SED) or PA wheel cages and fed ad libitum (AL) or 90% of control food intake (ER). After 8 weeks on the interventions, mice were inoculated with 5 × 104 4T1.2luc cells into the 4th mammary fat pad and continued on their respective intervention. PA+ER significantly delayed primary tumor growth (final tumor volume, 0.193 ± 0.042 vs. 0.369 ± 0.049 cm3, P < 0.001), reduced metastatic burden in the lungs (0.72 ± 0.36 vs. 16.27 ± 6.98, P = 0.054) and increased survival (median survival, 68 vs 40 days, P = 0.043) compared with SED+AL mice. PA+ER also reduced the expression level of metastatic and immunosuppressive genes and resulted in favorable changes in immune cell infiltrates in the tumor. These data suggest that both PA and ER are needed to reduce tumor growth, delay metastatic progression, and improve survival, and that this protection is associated with changes in immune-mediated mechanisms.

Endurance training slows breast tumor growth in mice by suppressing Treg cells recruitment to tumors.

BACKGROUND: Aerobic exercise has been shown to slow tumor progression in rodents and humans, but the mechanisms behind this effect are still unclear. Here we show that aerobic exercise in the form of chronic endurance training suppresses tumor recruitment of FoxP3+ Treg cells thus enhancing antitumor immune efficiency. METHODS: Adult wild-type and athymic BALB/c female mice were endurance-trained for 8 weeks. Circulating leukocytes as well as muscle and liver mtDNA copy number were compared to aged-matched concurrent sedentary controls to establish systemic effects. 4 T1 murine mammary tumor cells were injected subcutaneously to the 4th mammary pad at the end of the training period. Tumor growth and survival rates were compared, together with antitumor immune response. RESULTS: Exercised wild-type had 17% slower growth rate, 24% longer survival, and 2-fold tumor-CD+ 8/FoxP3+ ratio than sedentary controls. Exercised athymic BALB/c females showed no difference in tumor growth or survival rates when compared to sedentary controls. CONCLUSIONS: Cytotoxic T cells are a significant factor in endurance exercise-induced suppression of tumor growth. Endurance exercise enhances antitumor immune efficacy by increasing intratumoral CD8+/FoxP3+ ratio.

Impact of obesity on breast cancer recurrence and minimal residual disease.

BACKGROUND: Obesity is associated with an increased risk of breast cancer recurrence and cancer death. Recurrent cancers arise from the pool of residual tumor cells, or minimal residual disease (MRD), that survives primary treatment and persists in the host. Whether the association of obesity with recurrence risk is causal is unknown, and the impact of obesity on MRD and breast cancer recurrence has not been reported in humans or in animal models. METHODS: Doxycycline-inducible primary mammary tumors were generated in intact MMTV-rtTA;TetO-HER2/neu (MTB/TAN) mice or orthotopic recipients fed a high-fat diet (HFD; 60% kcal from fat) or a control low-fat diet (LFD; 10% kcal from fat). Following oncogene downregulation and tumor regression, mice were followed for clinical recurrence. Body weight was measured twice weekly and used to segregate HFD mice into obese (i.e., responders) and lean (i.e., nonresponders) study arms, and obesity was correlated with body fat percentage, glucose tolerance (measured using intraperitoneal glucose tolerance tests), serum biomarkers (measured by enzyme-linked immunosorbent assay), and tissue transcriptomics (assessed by RNA sequencing). MRD was quantified by droplet digital PCR. RESULTS: HFD-Obese mice weighed significantly more than HFD-Lean and LFD control mice (p < 0.001) and had increased body fat percentage (p < 0.001). Obese mice exhibited fasting hyperglycemia, hyperinsulinemia, and impaired glucose tolerance, as well as decreased serum levels of adiponectin and increased levels of leptin, resistin, and insulin-like growth factor 1. Tumor recurrence was accelerated in HFD-Obese mice compared with HFD-Lean and LFD control mice (median relapse-free survival 53.0 days vs. 87.0 days vs. 80.0 days, log-rank p < 0.001; HFD-Obese compared with HFD-Lean HR 2.52, 95% CI 1.52-4.16; HFD-Obese compared with LFD HR 2.27, 95% CI 1.42-3.63). HFD-Obese mice harbored a significantly greater number of residual tumor cells than HFD-Lean and LFD mice (12,550 ± 991 vs. 7339 ± 2182 vs. 4793 ± 1618 cells, p < 0.001). CONCLUSION: These studies provide a genetically engineered mouse model for study of the association of diet-induced obesity with breast cancer recurrence. They demonstrate that this model recapitulates physiological changes characteristic of obese patients, establish that the association between obesity and recurrence risk is causal in nature, and suggest that obesity is associated with the increased survival and persistence of residual tumor cells.

Entinostat Converts Immune-Resistant Breast and Pancreatic Cancers into Checkpoint-Responsive Tumors by Reprogramming Tumor-Infiltrating MDSCs.

Immune-checkpoint inhibition (ICI) has revolutionized treatment in cancers that are naturally immunogenic by enabling infiltration of T cells into the tumor microenvironment (TME) and promoting cytotoxic signaling pathways. Tumors possessing complex immunosuppressive TMEs such as breast and pancreatic cancers present unique therapeutic obstacles as response rates to ICI remain low. Such tumors often recruit myeloid-derived suppressor cells (MDSCs), whose functioning prohibits both T-cell activation and infiltration. We attempted to sensitize these tumors to ICI using epigenetic modulation to target MDSC trafficking and function to foster a less immunosuppressive TME. We showed that combining a histone deacetylase inhibitor, entinostat (ENT), with anti-PD-1, anti-CTLA-4, or both significantly improved tumor-free survival in both the HER2/neu transgenic breast cancer and the Panc02 metastatic pancreatic cancer mouse models. Using flow cytometry, gene-expression profiling, and ex vivo functional assays, we characterized populations of tumor-infiltrating lymphocytes (TILs) and MDSCs, as well as their functional capabilities. We showed that addition of ENT to checkpoint inhibition led to significantly decreased suppression by granulocytic MDSCs in the TME of both tumor types. We also demonstrated an increase in activated granzyme-B-producing CD8+ T effector cells in mice treated with combination therapy. Gene-expression profiling of both MDSCs and TILs identified significant changes in immune-related pathways. In summary, addition of ENT to ICI significantly altered infiltration and function of innate immune cells, allowing for a more robust adaptive immune response. These findings provide a rationale for combination therapy in patients with immune-resistant tumors, including breast and pancreatic cancers.

Improved resection and prolonged overall survival with PD-1-IRDye800CW fluorescence probe-guided surgery and PD-1 adjuvant immunotherapy in 4T1 mouse model.

An intraoperative technique to accurately identify microscopic tumor residuals could decrease the risk of positive surgical margins. Several lines of evidence support the expression and immunotherapeutic effect of PD-1 in breast cancer. Here, we sought to develop a fluorescence-labeled PD-1 probe for in vivo breast tumor imaging and image-guided surgery. The efficacy of PD-1 monoclonal antibody (PD-1 mAb) as adjuvant immunotherapy after surgery was also assessed. PD-1-IRDye800CW was developed and examined for its application in tumor imaging and image-guided tumor resection in an immunocompetent 4T1 mouse tumor model. Fluorescence molecular imaging was performed to monitor probe biodistribution and intraoperative imaging. Bioluminescence imaging was performed to monitor tumor growth and evaluate postsurgical tumor residuals, recurrences, and metastases. The PD-1-IRDye800CW exhibited a specific signal at the tumor region compared with the IgG control. Furthermore, PD-1-IRDye800CW-guided surgery combined with PD-1 adjuvant immunotherapy inhibited tumor regrowth and microtumor metastases and thus improved survival rate. Our study demonstrates the feasibility of using PD-1-IRDye800CW for breast tumor imaging and image-guided tumor resection. Moreover, PD-1 mAb adjuvant immunotherapy reduces cancer recurrences and metastases emanating from tumor residuals.

Investigating the Antiangiogenic, Anti-drug Resistance and Apoptotic Effects of Soy Isoflavone Extract Alone or in Combination with Docetaxel on Murine 4T1 Breast Tumor Model.

BACKGROUND: One major concern in the treatment of cancer patients during chemotherapy is drug resistance. Here we investigated the effects of soy isoflavone extracts alone or in combination with Docetaxel on the drug resistance, angiogenesis, apoptosis, and tumor volume in mouse 4T1 breast tumor model. METHODS: Sixty female BALB/c mice were randomly divided into 4 groups: control, dietary soy isoflavone extract [Iso, 100 mg/kg diet (0.01%)], Docetaxel (10 mg/kg) injection, and the combination of dietary soy isoflavone extract and intravenous Docetaxel injection (Docetaxel + Iso). One week after the third injection, the breast tumors of eight mice from each group were excised to analyze NF-κBp65' vascular endothelial growth factor receptor-2 (VEGFR2) and Pgp gene and protein expressions and the other seven mice were monitored for survival rate analysis until they died. RESULTS: NF-κBp65 gene and protein expressions were significantly lower in the Docetaxel + Iso group in comparison with that of the Docetaxel group. VEGFR2 protein expression in the Docetaxel + Iso and Iso groups was significantly lower than that of the Docetaxel group. CONCLUSION: These findings may indicate that the combined use of isoflavone extracts together with chemotherapeutic agents has more efficient anti-carcinogenic effects than their individual use.

Induction of the MCP chemokine cluster cascade in the periphery by cancer cell-derived Ccl3.

The induction of localized pro-inflammatory niches in the periphery is instrumental in metastasis. In order to better understand how tumors engage distal sites and activate a pro-inflammatory response we utilized syngeneic breast cancers as a model and showed that soluble factors from the neoplastic epithelium activate the expression of the monocyte chemoattractive protein (MCP) chemokines of the mouse 11C cluster that include Ccl1, Ccl2, Ccl7, Ccl8, Ccl11 and Ccl12. Tissues such as the lungs and the brain, that are more prone to colonization by breast cancer cells, were more sensitive to MCP cluster chemokine induction than others such as the liver. Subsequent analyses involving chemokine arrays in breast cancer cells and media followed by functional validation assays in in vitro and in vivo identified the cytokine Ccl3 as the principle mediator of the communication between the neoplastic epithelium and the peripheral tissues in terms of MCP cluster chemokine induction. Our results show that MCP chemokines are activated in peripheral tissues of breast cancer-bearing mice, by a mechanism that involves breast cancer cell-derived Ccl3. Interference with the expression of cancer cell-derived Ccl3 may find application in the management of breast cancer metastases.

Antitumor effect of the combination of manumycin A and Immodin is associated with antiplatelet activity and increased granulocyte tumor infiltration in a 4T1 breast tumor model.

Manumycin A is a natural antibiotic isolated from Streptomyces parvulus with broad range of biological activities including antineoplastic activity in several in vitro and in vivo cancer models. Immodin [dialyzable leukocyte extract (DLE)] is a dialysate released from disintegrated blood leukocytes of healthy donors which exerts immunonormalizing effects on cell-mediated immune responses. The aim of the present study was to explore the antitumor potential of the combination of manumycin A and Immodin in an experimental breast cancer model. Experiments were carried using a 4T1 tumor-bearing BALB/c mouse model. Survival analysis, tumor growth, hematological and biochemical profiles, leukocyte differential, phagocytic activity of leukocytes and histology of the primary tumor were examined. The combination treatment suppressed the tumor growth and prolonged the survival of tumor-bearing mice, decreased the number of monocytes, plateletes and plateletcrit in peripheral blood of the tumor-bearing mice and increased the infiltration of neutrophils and eosinophils in the primary tumor. Moreover, individual therapies enhanced the phagocytic activity of monocytes and neutrophils. These findings demonstrate the antitumor effect of the combination of manumycin A and Immodin in 4T1 tumor-bearing mice associated with strong antiplatelet activity and innate immunity activation.

PTEN Loss in E-Cadherin-Deficient Mouse Mammary Epithelial Cells Rescues Apoptosis and Results in Development of Classical Invasive Lobular Carcinoma.

Invasive lobular carcinoma (ILC) is an aggressive breast cancer subtype with poor response to chemotherapy. Besides loss of E-cadherin, a hallmark of ILC, genetic inactivation of PTEN is frequently observed in patients. Through concomitant Cre-mediated inactivation of E-cadherin and PTEN in mammary epithelium, we generated a mouse model of classical ILC (CLC), the main histological ILC subtype. While loss of E-cadherin induced cell dissemination and apoptosis, additional PTEN inactivation promoted cell survival and rapid formation of invasive mammary tumors that recapitulate the histological and molecular features, estrogen receptor (ER) status, growth kinetics, metastatic behavior, and tumor microenvironment of human CLC. Combined inactivation of E-cadherin and PTEN is sufficient to cause CLC development. These CLCs showed significant tumor regression upon BEZ235-mediated inhibition of PI3K signaling. In summary, this mouse model provides important insights into CLC development and suggests inhibition of phosphatidylinositol 3-kinase (PI3K) signaling as a potential therapeutic strategy for targeting CLC.

Stage-Specific MicroRNAs and Their Role in the Anticancer Effects of Calorie Restriction in a Rat Model of ER-Positive Luminal Breast Cancer.

MicroRNAs have emerged as ubiquitous post-transcriptional regulators that coordinate many fundamental processes within cells, including those commonly linked to cancer when dysregulated. Profiling microRNAs across stages of cancer progression provides focus as to which microRNAs are key players in cancer development and are therefore important to manipulate with interventions to delay cancer onset and progression. Calorie restriction is one of the most effective preventive interventions across many types of cancer, although its effects on microRNAs have not been well characterized. We used the dimethylbenz[a]-anthracene-induced model of luminal mammary cancer in Sprague Dawley rats to elucidate which microRNAs are linked to progression in this type of cancer and, subsequently, to study how calorie restriction affects such microRNAs. We identified eight microRNAs (miR-10a, miR-10b, miR-21, miR-124, miR-125b, miR-126, miR-145 and miR-200a) to be associated with DMBA-induced mammary tumor progression. Calorie restriction, which greatly increased tumor-free survival and decreased the overall size of tumors that did develop, significantly decreased the expression of one microRNA, miR-200a, which was positively associated with tumor progression. We further showed that inhibition of miR-200a function, mimicking the effect of calorie restriction on this microRNA, inhibited proliferation in both rat (LA7) and human (MCF7) luminal mammary cancer cell lines. These findings present, for the first time, a stage-specific profile of microRNAs in a rodent model of luminal mammary cancer. Furthermore, we have identified the regulation of miR-200a, a microRNA that is positively associated with progression in this model, as a possible mechanism contributing to the anticancer effects of calorie restriction.

Nanoreporter PET predicts the efficacy of anti-cancer nanotherapy.

The application of nanoparticle drug formulations, such as nanoliposomal doxorubicin (Doxil), is increasingly integrated in clinical cancer care. Despite nanomedicine's remarkable potential and growth over the last three decades, its clinical benefits for cancer patients vary. Here we report a non-invasive quantitative positron emission tomography (PET) nanoreporter technology that is predictive of therapeutic outcome in individual subjects. In a breast cancer mouse model, we demonstrate that co-injecting Doxil and a Zirconium-89 nanoreporter ((89)Zr-NRep) allows precise doxorubicin (DOX) quantification. Importantly, (89)Zr-NRep uptake also correlates with other types of nanoparticles' tumour accumulation. (89)Zr-NRep PET imaging reveals remarkable accumulation heterogeneity independent of tumour size. We subsequently demonstrate that mice with >25 mg kg(-1) DOX accumulation in tumours had significantly better growth inhibition and enhanced survival. This non-invasive imaging tool may be developed into a robust inclusion criterion for patients amenable to nanotherapy.

The phytoestrogenic Cyclopia extract, SM6Met, increases median tumor free survival and reduces tumor mass and volume in chemically induced rat mammary gland carcinogenesis.

SM6Met, a phytoestrogenic extract of Cyclopia subternata indigenous to the Western Cape province of South Africa, displays estrogenic attributes with potential for breast cancer chemoprevention. In this study, we report that SM6Met, in the presence of estradiol, induces a significant cell cycle G0/G1 phase arrest similar to the selective estrogen receptor modulator, tamoxifen. Furthermore, as a proof of concept, in the N-Methyl-N-nitrosourea induced rat mammary gland carcinogenesis model, SM6Met increases tumor latency by 7days and median tumor free survival by 42 days, while decreasing palpable tumor frequency by 32%, tumor mass by 40%, and tumor volume by 53%. Therefore, the current study provides proof of concept that SM6Met has definite potential as a chemopreventative agent against the development and progression of breast cancer.

Ultrasound ablation enhances drug accumulation and survival in mammary carcinoma models.

Magnetic resonance-guided focused ultrasound (MRgFUS) facilitates noninvasive image-guided conformal thermal therapy of cancer. Yet in many scenarios, the sensitive tissues surrounding the tumor constrain the margins of ablation; therefore, augmentation of MRgFUS with chemotherapy may be required to destroy remaining tumor. Here, we used 64Cu-PET-CT, MRI, autoradiography, and fluorescence imaging to track the kinetics of long-circulating liposomes in immunocompetent mammary carcinoma-bearing FVB/n and BALB/c mice. We observed a 5-fold and 50-fold enhancement of liposome and drug concentration, respectively, within MRgFUS thermal ablation-treated tumors along with dense accumulation within the surrounding tissue rim. Ultrasound-enhanced drug accumulation was rapid and durable and greatly increased total tumor drug exposure over time. In addition, we found that the small molecule gadoteridol accumulates around and within ablated tissue. We further demonstrated that dilated vasculature, loss of vascular integrity resulting in extravasation of blood cells, stromal inflammation, and loss of cell-cell adhesion and tissue architecture all contribute to the enhanced accumulation of the liposomes and small molecule probe. The locally enhanced liposome accumulation was preserved even after a multiweek protocol of doxorubicin-loaded liposomes and partial ablation. Finally, by supplementing ablation with concurrent liposomal drug therapy, a complete and durable response was obtained using protocols for which a sub-mm rim of tumor remained after ablation.

Anti-tumor activity of folate targeted biodegradable polymer-paclitaxel conjugate micelles on EMT-6 breast cancer model.

BACKGROUND: Paclitaxel (PTX) is a first line chemotherapy drug for breast cancer. There have been few studies reported concerning the therapeutic efficacy of paclitaxel-conjugated polymeric micelles in breast cancer in vivo. METHODS: Two kinds of PTX conjugate micelles, one of which (M(PTX)) contained 25 wt.% of PTX and the other (M(FA/PTX)) contained 22.5 wt.% of PTX and 1.4 wt.% of folate (FA), were prepared for cell apoptosis and anti-tumor activity evaluation on EMT-6 mice breast cancer models in comparison with 0.9 wt.% saline (control) and equivalent PTX. Cell apoptosis was analyzed by flow cytometry. Breast tumors were examined histologically with H&E staining and immunohistochemically by examining Bax and Bcl-2 expression. The survival status of tumor-bearing mice with different treatments was also examined. RESULTS: On day 5 of the drug administration, the average tumor masses were 0.49, 0.33, 0.22, and 0.18 g for the control, PTX, M(PTX) and M(FA/PTX) groups, respectively. The inhibition rates of tumor growth calculated for the three drug groups were 32.6%, 51.6% and 62.3%, respectively. The percentage of cell apoptosis based on flow cytometry was 1.0%, 36.6%, 55.9% and 66.1%, respectively, which showed statistically significant differences (p<0.05) between three drug groups and the control group. Bcl-2 expression of PTX and M(FA/PTX) groups was lower than control group (p<0.05). Bax expression of drug groups was higher than control group (p<0.05). At an equivalent paclitaxel dose of 26.7 mg/kg, the average survival time was 33 days, 31 days, 34 days and 42 days, respectively (p<0.05). CONCLUSION: The M(FA/PTX) have better anti-tumor activity and are promising in treatment of human breast cancers.

Protective and sensitive effects of melatonin combined with adriamycin on ER+ (estrogen receptor) breast cancer.

OBJECTIVE: This study aims to investigate the protective and sensitive effects of melatonin (MLT) in the treatment of breast cancer. MATERIALS AND METHODS: ER+ breast cancer rat model was established and then rats were randomly divided into five different groups as follows: control group, Diss group, adriamycin (ADM) group, MLT group, and MLT combined with adriamycin (M+A) group. Tumor weights and one month survival rate were compared among these groups. In addition, changes of tumor tissues and expression of E-cadherin were observed under optical microscopy or electro-microscopy. RESULTS: Tumor weights were significantly lighter in M+A group than those in ADM group (p < 0.05). Under optical and electro-microscopy, tumor cell apoptosis was obviously increased in MLT group, and tumor cell injury was more severe in M+A group than that in ADM group; additionally, expression of E-cadherin was higher in MLT group and M+A group than that in other groups. Moreover, MLT group had the highest one month survival rate (100%), there was the poorest life quality in ADM group, but the best life quality in MLT. CONCLUSION: MLT could enhance the sensitivity of tumor to ADM in vivo and improve patient's life quality.

Inhibition of Mouse Breast Tumor-Initiating Cells by Calcitriol and Dietary Vitamin D.

The anticancer actions of vitamin D and its hormonally active form, calcitriol, have been extensively documented in clinical and preclinical studies. However, the mechanisms underlying these actions have not been completely elucidated. Here, we examined the effect of dietary vitamin D and calcitriol on mouse breast tumor-initiating cells (TICs, also known as cancer stem cells). We focused on MMTV-Wnt1 mammary tumors, for which markers for isolating TICs have previously been validated. We confirmed that these tumors expressed functional vitamin D receptors and estrogen receptors (ER) and exhibited calcitriol-induced molecular responses including ER downregulation. Following orthotopic implantation of MMTV-Wnt1 mammary tumor cells into mice, calcitriol injections or a vitamin D-supplemented diet caused a striking delay in tumor appearance and growth, whereas a vitamin D-deficient diet accelerated tumor appearance and growth. Calcitriol inhibited TIC tumor spheroid formation in a dose-dependent manner in primary cultures and inhibited TIC self-renewal in secondary passages. A combination of calcitriol and ionizing radiation inhibited spheroid formation more than either treatment alone. Further, calcitriol significantly decreased TIC frequency as evaluated by in vivo limiting dilution analyses. Calcitriol inhibition of TIC spheroid formation could be overcome by the overexpression of ß-catenin, suggesting that the inhibition of Wnt/ß-catenin pathway is an important mechanism mediating the TIC inhibitory activity of calcitriol in this tumor model. Our findings indicate that vitamin D compounds target breast TICs reducing tumor-initiating activity. Our data also suggest that combining vitamin D compounds with standard therapies may enhance anticancer activity and improve therapeutic outcomes.

Antitumor effect of free rhodium (II) citrate and rhodium (II) citrate-loaded maghemite nanoparticles on mice bearing breast cancer: a systemic toxicity assay.

Breast cancer is one of the most prevalent cancer types among women. The use of magnetic fluids for specific delivery of drugs represents an attractive platform for chemotherapy. In our previous studies, it was demonstrated that maghemite nanoparticles coated with rhodium (II) citrate (Magh-Rh2Cit) induced in vitro cytotoxicity and in vivo antitumor activity, followed by intratumoral administration in breast carcinoma cells. In this study, our aim was to follow intravenous treatment to evaluate the systemic antitumor activity and toxicity induced by these formulations in Balb/c mice bearing orthotopic 4T1 breast carcinoma. Female Balb/c mice were evaluated with regard to toxicity of intravenous treatments through analyses of hemogram, serum levels of alanine aminotransferase, iron, and creatinine and liver, kidney, and lung histology. The antitumor activity of rhodium (II) citrate (Rh2Cit), Magh-Rh2Cit, and maghemite nanoparticles coated with citrate (Magh-Cit), used as control, was evaluated by tumor volume reduction, histology, and morphometric analysis. Magh-Rh2Cit and Magh-Cit promoted a significant decrease in tumor area, and no experimental groups presented hematotoxic effects or increased levels of serum ALT and creatinine. This observation was corroborated by the histopathological examination of the liver and kidney of mice. Furthermore, the presence of nanoparticles was verified in lung tissue with no morphological changes, supporting the idea that our nanoformulations did not induce toxicity effects. No studies about the systemic action of rhodium (II) citrate-loaded maghemite nanoparticles have been carried out, making this report a suitable starting point for exploring the therapeutic potential of these compounds in treating breast cancer.

An improved syngeneic orthotopic murine model of human breast cancer progression.

Breast cancer drug development costs nearly $610 million and 37 months in preclinical mouse model trials with minimal success rates. Despite these inefficiencies, there are still no consensus breast cancer preclinical models. Murine mammary adenocarcinoma 4T1-luc2 cells were implanted subcutaneous (SQ) or orthotopically percutaneous (OP) injection in the area of the nipple, or surgically into the chest 2nd mammary fat pad under direct vision (ODV) in Balb/c immunocompetent mice. Tumor progression was followed by in vivo bioluminescence and direct measurements, pathology and survival determined, and tumor gene expression analyzed by genome-wide microarrays. ODV produced less variable-sized tumors and was a reliable method of implantation. ODV implantation into the chest 2nd mammary pad rather than into the abdominal 4th mammary pad, the most common implantation site, better mimicked human breast cancer progression pattern, which correlated with bioluminescent tumor burden and survival. Compared to SQ, ODV produced tumors that differentially expressed genes whose interaction networks are of importance in cancer research. qPCR validation of 10 specific target genes of interest in ongoing clinical trials demonstrated significant differences in expression. ODV implantation into the chest 2nd mammary pad provides the most reliable model that mimics human breast cancer compared from subcutaneous implantation that produces tumors with different genome expression profiles of clinical significance. Increased understanding of the limitations of the different preclinical models in use will help guide new investigations and may improve the efficiency of breast cancer drug development .

Modulatory effects of meloxicam on cardiotoxicity and antitumor activity of doxorubicin in mice.

PURPOSE: This study was undertaken to assess the possible modulatory effects and mechanisms of meloxicam, a cyclooxygenase-2 inhibitor, on the antitumor activity and cardiotoxicity of doxorubicin in a mice model of mammary carcinoma. METHODS: Solid tumor mass was developed in female albino mice using Ehrlich carcinoma cells. Forty mice-bearing tumor were divided randomly into four groups for treatment: with saline, meloxicam 10 mg/kg, doxorubicin 5 mg/kg and meloxicam 1 h ahead of doxorubicin, twice weekly for 2 weeks. Tumor volume was followed up and cardiac protective utility was estimated via measuring heart and serum parameters. RESULTS: Meloxicam expressed a non-significant increase in doxorubicin antitumor activity. Conversely, meloxicam significantly (p < 0.01) mitigated doxorubicin-induced elevation of serum cardiac enzymes [creatine kinase, lactate dehydrogenase and troponin-I]; cardiac lipid peroxidations marker; cardiac active caspase-3 content; and cardiac prostaglandin E2 content. Meloxicam significantly abrogated doxorubicin-induced disturbance in heart histology and relative heart weight to body weight. Meloxicam normalized doxorubicin-induced suppression in heart antioxidant enzymes activities and gene expressions [superoxide dismutase, glutathione peroxidase (GSH-Px) and catalase], and heart GSH content. In addition, meloxicam ameliorated doxorubicin-induced disturbance in phase II metabolizing enzyme, cardiac quinone reductase (QR), at activity level and mRNA expression. CONCLUSION: Meloxicam protects heart against doxorubicin toxicity without affecting its antitumor activity against solid mammary cancer model in mice. This protective effect is attributed to antioxidant effect, antiradical effect, antiinflammatory action, antiapoptotic effect and induction of QR enzyme.