Growth Of 4T1 Mammary Tumors Research Articles

Antitumor effect of selenium-rich Brazil nuts and selenomethionine dietary supplementation on pre-existing 4T1 mammary tumor growth in mice.

Selenium (Se) is an essential micronutrient known to play an important role in the antioxidant system that can potentially influence tumor growth. We aimed to investigate the effects of dietary Se supplementation after detection of 4T1 mammary tumor growth in BALB/c mice. Thirty female mice received subcutaneous inoculation of 4T1 cells. After five days, all animals presenting palpable tumors were randomly assigned to three groups: a control group (Se-control) receiving a diet with adequate Se (0.15 mg/kg) and two other groups that received Se-supplemented diets (1.4 mg/kg of total Se) with either Brazilian nuts (Se-Nuts) or selenomethionine (SeMet). Data were assessed by either One or Two-way ANOVA followed by Tukey's HSD or Bonferroni's post hoc tests, respectively. Both Se-supplemented diets reduced tumor volume from the thirteenth day of feeding compared with the Se-adequate (control) diet (p < 0.05). The SeMet group presented a higher Se blood concentration (p < 0.05) than the Se-control group, with the Se-Nuts group presenting intermediate values. Selenoprotein P gene expression in the liver was higher in the Se-Nuts group than in the Se-control group (p < 0.05), while the SeMet group presented intermediate expression. Dietary Se supplementation, starting after detection of 4T1 palpable lesions, reduced tumor volume in mice.

Janus γ-Fe2O3/SiO2-based nanotheranostics for dual-modal imaging and enhanced synergistic cancer starvation/chemodynamic therapy

Multimodal cancer synergistic therapy exhibited remarkable advantages over monotherapy in producing an improved therapeutic efficacy. In this work, Janus-type γ-Fe2O3/SiO2 nanoparticles (JFSNs) are conjugated with glucose oxidase (GOx) for synergistic cancer starvation/chemodynamic therapy. The γ-Fe2O3 hemisphere of JFSNs can perform photoacoustic/T2 magnetic resonance dual-modal imaging of tumors. GOx on the surface of JFSNs catalyzes the decomposition of glucose and produces H2O2 for cancer starvation therapy. Subsequently, the γ-Fe2O3 hemisphere catalyzes the disproportionation of H2O2 to generate highly reactive hydroxyl radicals in an acidic tumor microenvironment. The close distance between GOx and JFSNs ensures adequate contact between the γ-Fe2O3 hemisphere and its substrate H2O2, thus enhancing the catalytic efficiency. This synergy of glucose depletion, biotoxic H2O2 and hydroxyl radicals significantly suppresses 4T1 mammary tumor growth with minimal adverse effects.

Quaternized chitosan-stabilized copper sulfide nanoparticles for cancer therapy

In this study, we report a smart and green strategy to synthesize copper sulfide nanoparticles (CuS-NPs) for clinically translatable cancer treatment. For the first time, the preparation of CuS-NPs was developed by taking advantage of the copper-amine complex as the copper source and sodium sulfide as the sulfide source, in which the quaternized chitosan (QCS) was used as a biotemplate and stabilizing agent. The obtained QCS/CuS-NPs composites (CuS@QCS-NPs) were spherical and stable with an average diameter of 5.6 nm, and showed strong NIR absorbance for photothermal conversion. Moreover, in vitro and in vivo cancer theranostic capability of CuS@QCS-NPs without any biomodification was evaluated. The result reveals that after intratumoral (i.t.) injection of CuS@QCS-NPs with NIR laser irradiation (808 nm, 1.5 W/cm2, 5 min), the 4T1 mammary tumor growth could be effectively suppressed comparing with the other control groups, and there was no obvious lethal toxicity to liver function, kidney function, and vital organs. Such QCS-stabilized CuS-NPs may provide an alternative for clinical application of CuS-based photothermal therapy.

Neratinib + Valproate] exposure permanently reduces ERBB1 and RAS expression in 4T1 mammary tumors and enhances M1 macrophage infiltration.

The irreversible ERBB1/2/4 inhibitor neratinib has been shown in vitro to rapidly reduce the expression of ERBB1/2/4 and RAS proteins via autophagic/lysosomal degradation. We have recently demonstrated that neratinib and valproate interact to suppress the growth of 4T1 mammary tumors but had not defined whether the [neratinib + valproate] drug combination, in a mouse, had altered the biology of the 4T1 cells. Exposure of 4T1 mammary tumors to [neratinib + valproate] for three days resulted, two weeks later, in tumors that expressed less ERBB1, K-RAS, N-RAS, indoleamine-pyrrole 2,3-dioxygenase (IDO-1), ornithine decarboxylase (ODC) and had increased Class I MHCA expression. Tumors previously exposed to [neratinib + valproate] grew more slowly than those exposed to vehicle control and contained more CD8+ cells and activated NK cells. M1 but not M2 macrophage infiltration was significantly enhanced by the drug combination. In vitro exposure of 4T1 tumor cells to [neratinib + valproate] variably reduced the expression of histone deacetylases 1-11. In vivo, prior exposure of tumors to [neratinib + valproate] permanently reduced the expression of HDACs 1-3, 6 and 10. Combined knock down of HDACs 1/2/3 or of 3/10 rapidly reduced the expression IDO-1, and ODC and increased the expression of MHCA. H&E staining of normal tissues at animal nadir revealed no obvious cyto-architectural differences between control and drug-treated animals. We conclude that [neratinib + valproate] evolves 4T1 tumors to grow more slowly and to be more sensitive to checkpoint immunotherapy antibodies.

HDAC inhibitors enhance neratinib activity and when combined enhance the actions of an anti-PD-1 immunomodulatory antibody in vivo.

Patients whose NSCLC tumors become afatinib resistant presently have few effective therapeutic options to extend their survival. Afatinib resistant NSCLC cells were sensitive to clinically relevant concentrations of the irreversible pan-HER inhibitor neratinib, but not by the first generation ERBB1/2/4 inhibitor lapatinib. In multiple afatinib resistant NSCLC clones, HDAC inhibitors reduced the expression of ERBB1/3/4, but activated c-SRC, which resulted in higher total levels of ERBB1/3 phosphorylation. Neratinib also rapidly reduced the expression of ERBB1/2/3/4, c-MET and of mutant K-/N-RAS; K-RAS co-localized with phosphorylated ATG13 and with cathepsin B in vesicles. Combined exposure of cells to [neratinib + HDAC inhibitors] caused inactivation of mTORC1 and mTORC2, enhanced autophagosome and subsequently autolysosome formation, and caused an additive to greater than additive induction of cell death. Knock down of Beclin1 or ATG5 prevented HDAC inhibitors or neratinib from reducing ERBB1/3/4 and K-/N-RAS expression and reduced [neratinib + HDAC inhibitor] lethality. Neratinib and HDAC inhibitors reduced the expression of multiple HDAC proteins via autophagy that was causal in the reduced expression of PD-L1, PD-L2 and ornithine decarboxylase, and increased expression of Class I MHCA. In vivo, neratinib and HDAC inhibitors interacted to suppress the growth of 4T1 mammary tumors, an effect that was enhanced by an anti-PD-1 antibody. Our data support the premises that neratinib lethality can be enhanced by HDAC inhibitors, that neratinib may be a useful therapeutic tool in afatinib resistant NSCLC, and that [neratinib + HDAC inhibitor] exposure facilitates anti-tumor immune responses.

Abstract 1664: Inhibition of 4T1 mammary tumor growth in BALB/c mice by subcutaneous and intraperitoneal injection of a 4T1 whole cell vaccine containing IL-2 and GM-CSF as adjuvants

Abstract Developing animal tumor models for human cancer vaccines creates a tool to investigate the mechanism of action, variations in formulation, dosing schedules, and combinations with other forms of cancer therapy. In this study we developed a mouse whole cell mammary cancer vaccine model with both subcutaneous and intraperitoneal injection of the vaccine. The vaccine, containing 4T1 mouse mammary cancer cells (1,000,000 cells), IL-2 (0.2 µg) and GM-CSF (0.1 µg) in a total volume of 150 µl, was injected either subcutaneously into the backs of BALB/c mice or intraperitoneally. There were six injections of the vaccine (weeks 1, 2, 3, 7, 11, 15) and one week after the last injection 100,000 4T1 cells from cell culture in a volume of 100 µl were injected into the scapular region of the mice. At 22 days post 4T1 tumor transplantation there was a 37% inhibition of the growth of the 4T1 tumor in the mice receiving the subcutaneous injection of the vaccine and a 42% inhibition of the growth of the 4T1 tumor in the mice receiving the intraperitoneal injection of the vaccine. The inhibition of 4T1 mammary tumor growth in this mouse model with subcutaneous injection of a whole cell vaccine gives further support for the previously reported efficacy in a Phase 1/2 clinical trial of our therapeutic breast cancer vaccine containing autologous and allogeneic breast cancer cells in the adjuvant setting. The inhibition of 4T1 tumor growth with intraperitoneal injection suggests a delivery method that may be applicable to ovarian cancer and cancer patients with malignant ascites. Citation Format: Jonathan F. Head, Jeffrey T. Phillips, Xianpeng Jiang, Robert L. Elliott. Inhibition of 4T1 mammary tumor growth in BALB/c mice by subcutaneous and intraperitoneal injection of a 4T1 whole cell vaccine containing IL-2 and GM-CSF as adjuvants [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 1664. doi:10.1158/1538-7445.AM2017-1664

Abstract 1439: Combination of metformin plus orlistat prevents tumor progression: novel role of the metabolic hormone fibroblast growth factor 21 (FGF21)

Abstract Caloric Restriction (CR) is known to be the only model to consistently increase the lifespan and postpone age-related diseases in all species. CR increases glycogenolysis, lipolysis with fatty acid and ketone body utilization and accelerates protein catabolism. We previously reported that CR induces metabolic and signaling changes that affect the tumor microenvironment preventing the growth of 4T1 mammary tumors and their metastases. 4T1 is a highly metastatic mouse breast cancer model which resembles breast cancer in patients. Obesity causes subclinical inflammation in adipose tissue that contributes to insulin resistance and cancer progression. Chronic inflammation predisposes to different forms of cancer and diabetes, and is correlated with increased risk of breast cancer. CR reduces insulin resistance, adiposity and inflammation and the anti-diabetic drug metformin (MET) is a CR mimetic agent. In this study, we evaluated whether the combination of MET with the anti-obesity drug orlistat (OR) enhances the CR mimetic and anti-cancer properties of MET on 4T1 mammary tumor cells. Female 8 week-old BALB/c mice received: vehicle, MET (in drinking water, 3mg/ml), OR i.p. injection (240mg/kg/day) or MET + OR. After 3 weeks on drugs, 4T1 cells (105) were injected into mice and drugs continued till the end of the experiment. Treated-mice had a reduced adiposity (p&amp;lt;0.05) without any signs of toxicity. At 30 days, MET + OR co-treatment reduced tumor volume (p&amp;lt;0.01) and displayed a range of cellular alterations such as decreased proliferative index (p&amp;lt;0.05); increased apoptotic rate (p&amp;lt;0.01) and altered intra-tumor collagen deposit. MET + OR treatment reduced total vessel length (p&amp;lt;0.01) and the number of spontaneous lung metastases (p&amp;lt;0.01). In addition, combinations of MET (1-2.5mM) + OR (2.5-5µM) decreased in vitro 4T1 mammary cell proliferation (p&amp;lt;0.01), adhesion (p&amp;lt;0.05) and migration (p&amp;lt;0.01) compared to vehicle and drugs alone. Interestingly, we found that levels of fibroblast growth factor 21 (FGF21), an endocrine factor that regulates glucose and lipid metabolism, were significantly increased with MET + OR treatment (p&amp;lt;0.05). It has been shown that increased circulating levels of FGF21 correlated with decreased obesity and increased sensitivity to insulin; but up to date the role of FGF21 in tumor progression was not elucidated. We found that treatment of 4T1 cells with MET + OR increased the secretion of FGF21 (p&amp;lt;0.05) measured by ELISA assays. To elucidate the role of FGF21; we initially demonstrated that addition of recombinant protein FGF21 (0-10nM) did not change 4T1 cell proliferation. In contrast, the reduced cell proliferation and adhesion induced by MET + OR, were abolished by the pre- and co-treatment with anti-FGF21 neutralizing antibody; indicating that FGF21 may play an important role. Our results provide a new rationale basis for the use of MET plus OR as cancer therapy. Citation Format: Shobika Sivaram, Erdene Baljinnyam, Kousaku Iwatsubo, Lydia I. Puricelli, Mariana S. De Lorenzo. Combination of metformin plus orlistat prevents tumor progression: novel role of the metabolic hormone fibroblast growth factor 21 (FGF21). [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 1439. doi:10.1158/1538-7445.AM2014-1439

151. Evidence for sympathetic nervous system modulation of mammary tumor pathogenesis via tumor collagen

Our laboratory uses multiphoton laser scanning microscopy to study tumor collagen structure using an optical technique known as second harmonic generation (SHG). SHG is an endogenous optical signal produced when two excitation photons combine to produce one emission photon, “catalyzed” by a non-centrosymmetric structure such as ordered collagen triple helices. Manipulating collagen structure, as indicated by changes in SHG, also alters tumor growth and metastasis. We also study the role of sympathetic nervous system (SNS) activation and the neurotransmitter norepinephrine (NE) in breast cancer. To mimic SNS activation, mice were implanted with slow-release pellets containing desipramine (DMI), a NE uptake blocker. DMI elevated NE and its metabolite normetanephrine in spleen and in 4T1 tumors implanted orthotopically (in the mammary fat pad). Furthermore, DMI increased 4T1 mammary tumor growth, compared to placebo pellet controls. Tumor sections from DMI-implanted mice exhibited elevated SHG intensity. In these same images, fluorescent intensity generated by immunofluorescent staining with anti-collagen type 1 antibody was not altered by DMI treatment, indicating no alterations in total collagen (both SHG generating and non-SHG generating collagen). These results demonstrate that NE-induced tumor growth is associated with altered tumor collagen microstructure, and suggest that SNS activation may modulate tumor pathogenesis through a novel pathway, i.e., modulation of tumor extracellular matrix. The mechanisms linking SNS activation to collagen structure and tumor pathogenesis are currently under investigation.

Abstract 528: A tumor-stroma interaction study in red mammary tumors in green mice with and without enhanced oxygenation

Abstract Background: The tumor microenvironment is increasingly recognized as pivotal in tumor progression. Thus, by completely separating tumor cells from stromal cells it enables a thorough elucidation of gene-changes in the two compartments, and thereby offers the possibility to better understand the biological effects in tumors with and without treatment. Since tumor hypoxia is considered to be relevant for several aspects of tumor pathophysiology, including activation of signalling pathways that regulate proliferation, angiogenesis and death, our hypothesis was that a reduction in the hypoxic state of the tumor, might have an inhibitory effect on tumor growth per se. Induction of hyperoxia by hyperbaric oxygen (HBO) exposure, enhance dissolved oxygen in the plasma and thereby the pO2 in the tumor tissue. Methods: The murine mammary dsRed cell line 4T1 was implanted into the mouse mammary fat pad in eGFP expressing NOD/Scid mice. One group was exposed to repeated HBO treatment (2.5 bar, 100% O2, 3 exposures à 90 min), one to daily HBO treatment (2.5bar, 100% O2, 7 exposures à 90 min), whereas the control group was housed under normal atmosphere. Treatment effects were determined by assessment of tumor growth, tumor vascularisation, tumor cell proliferation and cell death. FACS was used to completely separate red tumor cells from green host cells, RNA was extracted and gene expression profiling performed. Results: The model enabled us to completely separate the two compartments (tumor vs stroma), as verified by confocal microscopy and gene expression profiling. Highly upregulated genes in the untreated tumor stroma, included constituents of the ECM and MMP's, as well as genes related to cell-migration and motility. Significant changes in response to treatment between the tumor and stroma was found, especially concerning genes related to cell adhesion and angiogenesis. Furthermore, changes occurring within each of these compartments were found. Tumor growth was significantly inhibited (∼ 50%, after 8 days) after both repeated and daily HBO treatment compared to controls. Repeated HBO treatment showed a significant anti-angiogenic effect, while daily HBO treatment did not. The gene expression confirmed this. Neither morphology, proliferation nor the amount of cell death was significantly changed after the two HBO treatment regimes, despite the significant effect on tumor growth. Conclusion: The present model provides a significant insight into how tumor cells communicate with the stroma, both before any treatment and after enhanced oxygenation. Furthermore, hyperoxia induced a significant inhibitory effect on the 4T1 mammary tumor growth, with a significant anti-angiogenic effect after repeated hyperoxic treatment. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 528. doi:10.1158/1538-7445.AM2011-528

Tumor-secreted PGE2 Inhibits CCL5 Production in Activated Macrophages through cAMP/PKA Signaling Pathway

One of the major characteristics of tumors is their ability to evade immunosurveillance through altering the properties and functions of host stromal and/or immune cells. CCL5 has been shown to play important roles in T cell proliferation, IFN-γ, and IL-2 production, which promotes the differentiation and proliferation of Th1 cells important for immune defense against intracellular infection. In this study we found that tumor-bearing mice were more susceptible to bacterial infection and showed reduced CCL5 levels in serum during endotoxic shock. Our data further demonstrated that the soluble factors secreted by mammary gland tumor cells but not normal mammary gland epithelial cells inhibited CCL5 expression in macrophages in response to LPS, but not to TNF-α stimulation. The inhibitory effect of tumor-secreted molecules on LPS-induced CCL5 expression was regulated at the post-transcriptional level. Blocking PGE(2) synthesis by NS398 or through the use of PGE(2) receptor antagonists AH-6809 (EP2 antagonist) and AH-23848 (EP4 antagonist) completely reversed the inhibitory effect of tumor-conditioned medium (TCM) on LPS-induced CCL5 expression. Moreover, PGE(2) and the cAMP analog forskolin could mimic tumor-mediated CCL5 inhibition, and the inhibitory effects of TCM, PGE(2), and cAMP analog on LPS-induced CCL5 expression could be completely reversed by the PKA inhibitor H89. Furthermore, blocking PGE(2) synthesis in vivo led to partial recovery of CCL5 production during endotoxic shock. Taken together, our data indicate that PGE(2) secreted from breast cancer cells suppresses CCL5 secretion in LPS-activated macrophages through a cAMP/PKA signaling pathway, which may result in suppression of host immune responses against subsequent bacterial infection.

Anticancer activity of grape and grape skin extracts alone and combined with green tea infusions

Grapes and grape extracts were compared for inhibition of a growth-related and cancer-specific form of cell surface NADH oxidase with protein disulfide-thiol interchange activity designated tNOX from human cervical carcinoma (HeLa) cells and growth of HeLa and mouse mammary 4T1 cells in culture and transplanted tumors in mice. Grapes and grape extracts of several varieties had activity. With an extracted grape preparation provided by the California Table Grape Commission, an active fraction was eluted with methanol from a Diaion HP-20 column after removal of inactive water-soluble materials. Grape skins were a much more potent source than either grape pulp, juice or seeds. Ethanol extracts of the ground freeze-dried pomace was an excellent source. The grape extracts interacted, often synergistically, with decaffeinated green tea extracts both in the inhibition of tNOX activity and in the inhibition of cancer cell growth. Intratumoral injections of a 25:1 mixture of a green tea extract plus ground freeze-dried pomace was nearly as effective as standard synergistic green tea– Capsicum mixtures in inhibiting growth of 4T1 mammary tumors in situ in mice.