Mice Bearing 4T1 Breast Cancer Research Articles

Optimizing the administrated light dose during 5-ALA-mediated photodynamic therapy: Murine 4T1 breast cancer model.

Photodynamic therapy (PDT) is already used to treat many cancers, including breast cancer, the most common cancer in women worldwide. The destruction basis of this method is on produced singlet oxygen which is extremely reactive and is a major agent of tumor cell killing. The measurement of singlet oxygen produced within PDT is essential in predicting treatment outcomes and their optimization. This study aims to determine the optimal total light dose administered during PDT by calculating the singlet oxygen to facilitate the prediction of the treatment outcome in mice bearing 4T1 cell breast cancer. Monitoring the changes in photosensitizer fluorescence signals during PDT due to photobleaching can be one of the methods of determination of singlet oxygen generation in the PDT process. This study determined the oxygen singlet as a photodynamic dose from the three-dimensional Monte Carlo method and the photobleaching empirical dose constant. The photobleaching dose constant was established non-invasively by monitoring the invivo protoporphyrin IX (PpIX) fluorescence and photobleaching during PDT. The photobleaching dose constant (β) in J/cm2 was calculated using empirical fluorescence data. The invivo photobleaching dose constant of aminolevulinic acid was found to be 11.6 J/cm2 and based on this value, the optimal treatment light dose was estimated at 120 J/cm2 in mice bearing 4T1 breast cancer. It is concluded that information can be obtained regarding optimal treatment parameters by monitoring the invivo PpIX fluorescence and photobleaching during PDT.

Molecular engineering to red-shift the absorption band of AIE photosensitizers and improve their ROS generation ability.

Photodynamic therapy (PDT) with aggregation-induced emission photosensitizers (AIE-PSs) has attracted increasing attention for their enhanced fluorescence and reactive oxygen species (ROS) generation abilities upon aggregation. However, it is difficult for AIE-PSs to simultaneously achieve long-wavelength excitation (>600 nm) and high singlet oxygen quantum yield, which restricts their application in deep-tissue PDT. In this study, four novel AIE-PSs were developed by appropriate molecular engineering, and their absorption peaks shifted from 478 to 540 nm with a tail extending to 700 nm. Meanwhile, their emission peaks were also moved from 697 nm to 779 nm with a tail extending over 950 nm. Importantly, their singlet oxygen quantum yields successfully increased from 0.61 to 0.89. Moreover, TBQ, the best photosensitizer developed by us, has been successfully applied to image-guided PDT in BALB/C mice bearing 4T1 breast cancer under red light (605 ± 5 nm) irradiation, with IC50 less than 2.5 μM at a low light dose (10.8 J cm-2). The success of this molecular engineering indicates that increasing the number of acceptors is more effective at red-shifting the absorption band of AIE-PSs than increasing the number of donors, and extending the π-conjugation of acceptors will red-shift the absorption-emission band, increase the maximum molar extinction coefficient, and improve the ROS generation ability of AIE-PSs, thus providing a new strategy for the design of advanced AIE-PSs for deep-tissue PDT.

Repeated Contrast-Enhanced Micro-CT Examinations Decrease Animal Welfare and Influence Tumor Physiology.

Computed tomography (CT) imaging is considered relatively safe and is often used in preclinical research to study physiological processes. However, the sum of low-dose radiation, anesthesia, and animal handling might impact animal welfare and physiological parameters. This is particularly relevant for longitudinal studies with repeated CT examinations. Therefore, we investigated the influence of repeated native and contrast-enhanced (CE) CT on animal welfare and tumor physiology in regorafenib-treated and nontreated tumor-bearing mice. Mice bearing 4T1 breast cancer were divided into 5 groups: (1) no imaging, (2) isoflurane anesthesia only, (3) 4 mGy CT, (4) 50 mGy CT, and (5) CE-CT (iomeprol). In addition, half of each group was treated with the multikinase inhibitor regorafenib. Mice were imaged 3 times within 1 week under isoflurane anesthesia. Behavioral alterations were investigated by score sheet evaluation, rotarod test, heart rate measurements, and fecal corticosterone metabolite analysis. Tumor growth was measured daily with a caliper. Tumors were excised at the end of the experiment and histologically examined for blood vessel density, perfusion, and cell proliferation. According to the score sheet, animals showed a higher burden after anesthesia administration and in addition with CT imaging ( P < 0.001). Motor coordination was not affected by native CT, but significantly decreased after CE-CT in combination with the tumor therapy ( P < 0.001). Whereas tumor growth and blood vessel density were not influenced by anesthesia or imaging, CT-scanned animals had a higher tumor perfusion ( P < 0.001) and a lower tumor cell proliferation ( P < 0.001) for both radiation doses. The most significant difference was observed between the control and CE-CT groups. Repeated (CE-) CT imaging of anesthetized animals can lead to an impairment of animal motor coordination and, thus, welfare. Furthermore, these standard CT protocols seem to be capable of inducing alterations in tumor physiology when applied repetitively. These potential effects of native and CE-CT should be carefully considered in preclinical oncological research.

Poly(N-vinylpyrrolidone)-block-Poly(dimethylsiloxane)-block-Poly(N-vinylpyrrolidone) Triblock Copolymer Polymersomes for Delivery of PARP1 siRNA to Breast Cancers.

Nearly 20% of HER2-positive breast cancers develop resistance to HER2-targeted therapies requiring the use of advanced therapies. Silencing RNA therapy may be a powerful modality for treating resistant HER2 cancers due to its high specificity and low toxicity. However, the systemic administration of siRNAs requires a safe and efficient delivery platform because of siRNA's low stability in physiological fluids, inefficient cellular uptake, immunoreactivity, and rapid clearance. We have developed theranostic polymeric vesicles to overcome these hurdles for encapsulation and delivery of small functional molecules and PARP1 siRNA for in vivo delivery to breast cancer tumors. The 100 nm polymer vesicles were assembled from biodegradable and non-ionic poly(N-vinylpyrrolidone)14-block-poly(dimethylsiloxane)47-block-poly(N-vinylpyrrolidone)14 triblock copolymer PVPON14-PDMS47-PVPON14 using nanoprecipitation and thin-film hydration. We demonstrated that the vesicles assembled from the copolymer covalently tagged with the Cy5.5 fluorescent dye for in vivo imaging could also encapsulate the model drug with high loading efficiency (40%). The dye-loaded vesicles were accumulated in tumors after 18 h circulation in 4TR breast tumor-bearing mice via passive targeting. We found that PARP1 siRNA encapsulated into the vesicles was released intact (13%) into solution by the therapeutic ultrasound treatment as quantified by gel electrophoresis. The PARP1 siRNA-loaded polymersomes inhibited the proliferation of MDA-MB-361TR cells by 34% after 6 days of treatment by suppressing the NF-kB signaling pathway, unlike their scrambled siRNA-loaded counterparts. Finally, the treatment by PARP1 siRNA-loaded vesicles prolonged the survival of the mice bearing 4T1 breast cancer xenografts, with the 4-fold survival increase, unlike the untreated mice after 3 weeks following the treatment. These biodegradable, non-ionic PVPON14-PDMS47-PVPON14 polymeric nanovesicles capable of the efficient encapsulation and delivery of PARP1 siRNA to successfully knock down PARP1 in vivo can provide an advanced platform for the development of precision-targeted therapeutic carriers, which could help develop highly effective drug delivery nanovehicles for breast cancer gene therapy.

Combination Therapy of Bifidobacterium longum RAPO With Anti-PD-1 Treatment Enhances Anti-tumor Immune Response in Association With Gut Microbiota Modulation

ObjectivesTriple negative breast cancer (TNBC) has a poor prognosis with a high risk of metastasis and relapse, and accounts for approximately 35% of breast cancer death. Immune checkpoint inhibitors (ICI) have been in the spotlight recently as a novel treatment of TNBC due to the higher expressions of programmed cell death ligand 1 (PD-L1) and tumor-infiltrating lymphocytes in TNBC. Considering that gut microbiome is associated with immune response and ICI efficacy, we investigated whether Bifidobacterium longum RAPO supplementation would affect the efficacy of anti-PD-1 therapy against TNBC in vivo. MethodsFemale BALB/c mice bearing 4T1 breast cancer cells were randomly divided into four groups; tumor control (TC), Anti-PD-1, B. longum RAPO (RAPO), or Anti-PD-1 + RAPO (Combi). Anti-PD-1 antibody was injected i.p. 5 times at 3-day intervals and B. longum RAPO was orally administered daily from 2 days before the first injection of anti-PD-1. Spleen and tumor tissues were analyzed by flow cytometry, IHC, and qRT-PCR. Fecal samples were analyzed by 16S rRNA gene sequencing. ResultsTumor volume was reduced in the Combi group than TC and Anti-PD-1 groups on day 12. PD-L1 IHC score and PD-L1 mRNA expression were significantly increased in tumors of the Combi group. The levels of the spleen CD8/CD4 ratio and tumor NK cells were also increased in the Combi group. Compared with the Anti-PD-1 group, the pro-tumor M2 macrophages and related cytokines (IL10, Arg1) were significantly decreased, while anti-tumor M1 cytokines (IFNγ, TNFα) were elevated in tumors of the Combi group. Consistently, immunogenic cell death-associated markers were significantly higher in the Combi group. We found that the relative proportion of the genus Bifidobacterium, Lachnoclostridium, Lachnospiraceae NK4A136 group, and Clostridium sensu stricto 1, which are known to be associated with the better response to ICI therapy, were significantly increased, while the genus Mucispirillum and Dubosiella were decreased in the Combi group than the Anti-PD-1 group. ConclusionsOur data indicates that combination therapy of B. longum RAPO with anti-PD-1 enhances anti-tumor immune response in association with modulation of gut microbiota, suggesting that B. longum RAPO might be a potential candidate for pharmabiotics in ICI treatment. Funding SourcesBIFIDO CO, the National Research Foundation of Korea grant.

Divergent Effect of Tacalcitol (PRI-2191) on Th17 Cells in 4T1 Tumor Bearing Young and Old Ovariectomized Mice.

Vitamin D and its analogs are known for their role in the development of breast cancer and in immunomodulation. Our previous studies have shown the pro-metastatic effect of calcitriol and tacalcitol (PRI-2191) in young mice bearing 4T1 breast cancer and the anti-metastatic effect in aged ovariectomized (OVX) mice. Therefore, the aim of our work was to characterize Th17 cell population in young and aged OVX mice bearing 4T1 tumors treated with calcitriol and PRI-2191. The expression of genes typical for Th17 cells was examined in splenocytes, as well as splenocytes differentiated with IL-6 and TGF-β to Th17 cells (iTh17). Expression of genes encoding vitamin D receptor (Vdr) and osteopontin (Spp1) as well as the secretion of IL-17A were evaluated in iTh17 cells. PRI-2191 treatment increased the expression of Rora and Rorc transcription factors, Il17a, Il17re and Il21 in iTh17 cells from young mice. In aged OVX mice this effect was not observed. Increased expression was observed in the case of Vdr and Spp1 genes in iTh17 cells from young mice treated with PRI-2191. What is more, in young mice treated with PRI-2191 the secretion of IL-17A to the culture media by iTh17 cells was increased, whereas in aged OVX mice a significant decrease was noted. Increased expression of Spp1 in young mice treated with PRI-2191 may enhance the differentiation of Th17 cells.

Delivery of Immunotherapeutic Nanoparticles to Tumors via Enzyme-Directed Assembly.

Amphiphilic diblock copolymers are prepared by ring opening metathesis polymerization, with one block containing hydrophobic Toll-like receptor 7 (TLR7) agonists and one block containing hydrophilic peptides as substrates for matrix metalloproteinases (MMPs). A fluorescent label is incorporated into the polymer chains for in vivo imaging. Upon dialysis against aqueous solution, polymers form 15 nm spherical micelles. Subsequent exposure to MMP-9 elicits a morphological change to yield immunostimulatory microscale assemblies. The intravenous (IV) administration of the formulation to mice bearing 4T1 breast cancer tumors results in nanoparticle accumulation in tumors, reduction in primary tumor growth, and inhibition of lung metastases, as compared to saline-treated animals. Mice administered the parent immunotherapeutic small molecule (1V209) experience significantly increased plasma levels of proinflammatory cytokines IL-6, IP-10, and MCP-1 at 2 h following IV administration, whereas the nanomaterial shows no increase over saline-treated controls. These data suggest that covalently packaging low molecular weight immunotherapeutics at high weight percent loadings in enzyme-responsive nanoparticles maintains drug efficacy while decreasing immunotoxicity, providing a platform for cancer immunotherapeutic delivery.

4120Anti-tumor activity of a nanoliposomal anti-PCSK9 vaccine in BALB/c mice bearing 4T1 breast cancer

Abstract Background Although suppression of proprotein convertase subtilisin/kexin 9 (PCSK9) has been known to be an effective lipid-lowering approach, the efficacy and safety of PCSK9 inhibitors in non-cardiovascular diseases, particularly cancer, are yet unknown. Purpose The present study aimed to evaluate effect of PCSK9 inhibition on cancer behavior and endpoints in mice model of breast tumor, using a nanoliposomal anti-PCSK9 vaccine. Methods The nanoliposomal anti-PCSK9 vaccine was formulated via conjugation of immunogenic PCSK9 peptide to the surface of nanoliposome particles by using DSPE-PEG-Maleimide lipid. The prepared vaccine was adsorbed to Alum adjuvant (L-IFPTA+) and immunized subcutaneously four times with a bi-weekly interval in BALB/c mice.Two weeks after the last immunization, the vaccinated and unvaccinated mice were subcutaneously inoculated with 4T1 breast carcinoma cells into the right flank. After tumor mass was palpable, the mice were randomly divided into four groups and subjected to different treatment protocols: (1) PBS (untreated control), (2) vaccine group, (3) the combination group which involved vaccinated tumor-bearing mice who received single tail vein injection of Doxil®, and (4) Doxil® (positive control) group which involved unvaccinated tumor-bearing mice who received Doxil®. To evaluate therapeutic efficacy, mouse body weight, tumor size, and survival were monitored every other day for 60 days. Results The vaccine was found to efficiently generate specific antibodies against PCSK9 in BALB/c mice, and thereby reduce concentration of plasma PCSK9 and impede its function. Evaluation of tumor size demonstrated that time to reach endpoint (TTE) of the vaccine, combination, Doxil, and control groups was 47±10, 57±4, 60±4, 39±9 days, respectively. Rate of tumor growth in vaccine, combination, and Doxil groups was decreased by 21, 48, 53%, respectively, when compared with the control group (Figure). In the vaccine group life span was increased by 4.2%, while the survival in the combination and Doxil group was significantly lower than the control group. Figure 1. Anti-PCSK9 vaccine efficacy. Conclusions Our results reveal that PCSK9 inhibition not only exerts no harmful effects, but also can moderately improve breast cancer behavior in animal model.

Combination of intratumoural micellar paclitaxel with radiofrequency ablation: efficacy and toxicity in rodents.

To determine whether radiofrequency ablation (RFA) is more effective when combined with intratumoural injection (IT) than with intravenous injection (IV) of micelles. Balb/c mice bearing 4T1 breast cancer were used. The tumour drug accumulation and biodistribution in major organs were evaluated at different time points after IT, IV, IT+RFA and IV+RFA. Periablational drug penetration was evaluated by quantitative analysis and pathologic staining after different treatments. For long-term outcomes, mice bearing tumours were randomised into six groups (n = 7/group): the control, IV, IT, RFA alone, IV+RFA and IT+RFA groups. The end-point survival was estimated for the different treatment groups. In vivo, intratumoural drug accumulation was always much higher for IT than for IV within 48h (p< 0.001). The IT+RFA group (3084.7 ± 985.5μm) exhibited greater and deeper drug penetration than the IV+RFA group (686.3 ± 83.7μm, p< 0.001). Quantitatively, the intratumoural drug accumulation in the IT+RFA group increased approximately 4.0-fold compared with that in the IV+RFA group (p< 0.001). In addition, compared with the IT treatment, the IT+RFA treatment further reduced the drug deposition in the main organs. Survival was longer in the IT+RFA group than in the IV+RFA (p= 0.033) and RF alone (p= 0.003) groups. The use of IT+RFA achieved much deeper and greater intratumoural drug penetration and accumulation, resulting in better efficacy, and decreased the systemic toxicity of nanoparticle-delivered chemotherapy. • Association of IT+RFA achieved much deeper and greater intratumoural drug penetration than of IV+RFA, leading to better therapeutic efficacy. • Compared with IV or IT chemotherapy alone, the combination with RFA decreased toxicity, especially in the IT+RFA group.

In vivo monitoring of tumor distribution of hyaluronan polymeric micelles labeled or loaded with near-infrared fluorescence dye

Development of delivery systems which allow real-time visual inspection of tumors is critical for effective therapy. Near-infrared (NIR) fluorophores have a great potential for such an application. To overcome NIR dyes short blood circulation time and increase tumor accumulation, a NIR dye, cypate, was associated with oleyl hyaluronan, which can self-assemble into polymeric aggregates. The cypate association with oleyl hyaluronan was performed either by a covalent linkage, or physical entrapment. The two systems were compared for tumor targeting and contrast enhancement using BALB/c mice bearing 4T1 breast cancer tumors. Independently on the way of cypate association, it took more than 24 h from intravenous administration to detect NIR signal in tumors and the tumors were clearly visualized for 2 following weeks without substrate reinjection. Covalently linked cypate generated 2–3 fold stronger fluorescence signal than physically loaded cypate. This study demonstrates the potential of HA matrix to be used as carrier of contrast agents for non-invasive long-term tumor visualization.

Abstract 2005: Synergistic effect of phenyl alpha-galactosylceramide C34 and chemotherapy on 4T1-tumor bearing mouse model

Abstract Invariant natural killer T (iNKT) cells represent a unique subpopulation of innate immune cells which have immune modulatory effects on many immune effector cells such as CD4 T cells and CD8 T cells. Alpha-galactosylceramide (a-GalCer) is a well-known iNKT-stimulating glycolipid. Using a-GalCer as a template, we have generated a more potent a-GalCer analog C34, which contains two aromatic rings at the acyl chain and demonstrated its anti-cancer effect on tumor bearing mice model. It has been reported that NKT cells killed cancer cell lines treated with chemotherapy agents through TRAIL- and FasL-mediated pathways. In this study, we evaluated the anti-cancer effects of chemotherapy and C34 on tumor-bearing mouse model. First, BALB/c mice bearing 4T1 breast cancer were injected with various dose of chemotherapy agents weekly x 5 and the sub-optimal dose of docetaxel and doxorubicin was determined to be 0.5 mg/Kg. Similarly, sub-optimal dose of cisplatin and docetaxel in TC1 lung cancer-bearing C57BL/6 mice was determined to be 1 mg/Kg and 2 mg/Kg, respectively. In both tumor models, the suboptimal dose of C34 was found to be 0.1 ug per mouse. The treatment schedules of combination of chemotherapy and C34 were examined by administration of C34 at the same day (0D), three days (-3D) and one day (-1D) before chemotherapy and one day (+1D) after chemotherapy. We observed a survival benefit in mice injected with C34 one day after docetaxel treatment in 4T1-bearing mice as compared to those treated with the other schedules. Moreover, the survival of mice was significantly prolonged when C34 was injected three days after cisplatin treatment in TC1-bearing mice (median survival: 46 days for cisplatin and 55 days for cisplatin+C34, log-rank test p = 0.041). In the 4T1 tumor microenvironment, we found the number of CD3+CD8+ T cells was significantly greater in mice treated with docetaxel+C34 (23,536 +/- 901.4) than those treated with docetaxel only (2,270 +/- 1,536, t-test P &amp;lt; 0.0001). In addition, the number of PD1+ CD8 T cells was slightly higher, but did not reach statistical significance (t-test p = 0.3), in mice treated with docetaxel+C34 (23.3 +/- 8.5) than in mice treated with docetaxel only (14.6 +/- 10.1). In short, we have established an optimal treatment schedule for the combination of C34 and chemotherapy that showed synergistic anti-tumor efficacy. We also demonstrated that C34 could increase the number of CD8+ T cells in the tumor microenvironment in mice receiving chemotherapy. These results indicate that combination of C34 and chemotherapy administered with an optimal treatment schedule might have synergistic therapeutic efficacy for patients with lung cancer and breast cancer. Citation Format: Jung-Tung Hung, Jen-Chien Wu, Tsai-Hsien Hung, Jing-Rong Huang, John Yu, Alice L. Yu. Synergistic effect of phenyl alpha-galactosylceramide C34 and chemotherapy on 4T1-tumor bearing mouse model [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 2005. doi:10.1158/1538-7445.AM2017-2005

Remote reprogramming of hepatic circadian transcriptome by breast cancer.

Cancers adversely affect organismal physiology. To date, the genes within a patient responsible for systemically spreading cancer-induced physiological disruption remain elusive. To identify host genes responsible for transmitting disruptive, cancer-driven signals, we thoroughly analyzed the transcriptome of a suite of host organs from mice bearing 4T1 breast cancer, and discovered complexly rewired patterns of circadian gene expression in the liver. Our data revealed that 7 core clock transcription factors, represented by Rev-erba and Rorg, exhibited abnormal daily expression rhythm in the liver of 4T1-bearing mice. Accordingly, expression patterns of specific set of downstream circadian genes were compromised. Osgin1, a marker for oxidative stress, was an example. Specific downstream genes, including E2f8, a transcriptional repressor that controls cellular polyploidy, displayed a striking pattern of disruption, “day-night reversal.” Meanwhile, we found that the liver of 4T1-bearing mice suffered from increased oxidative stress. The tetraploid hepatocytes population was concomitantly increased in 4T1-bearing mice, which has not been previously appreciated as a cancer-induced phenotype. In summary, the current study provides a comprehensive characterization of the 4T1-affected hepatic circadian transcriptome that possibly underlies cancer-induced physiological alteration in the liver.

Mesenchymal stem cells with Sirt1 overexpression suppress breast tumor growth via chemokine-dependent natural killer cells recruitment.

Mesenchymal stem cells (MSCs) are generally used in regenerative medicine, tissue engineering and therapy for immune disorder diseases. However, due to the immunosuppressive function of MSCs, the application of MSCs in breast cancer therapy remains limited. Sirt1 is the closest mammalian homologue of the yeast enzyme Sir2 which has an established capacity to influence yeast replicative lifespan. In this study, we demonstrated the effect of MSCs with Sirt1 overexpression (MSCs-Sirt1) in mice bearing 4T1 breast cancer and investigated the underlying mechanism. Firstly, we found that MSCs could accelerate breast tumor growth with promoted proliferation and inhibited apoptosis, whereas MSCs-Sirt1 significantly suppressed tumor growth with proliferation inhibition and apoptosis promotion. Moreover, we detected that NK cells were the prominent antitumor effectors for the MSCs-Sirt1-induced antitumor activity. Besides that, CXCL10 and IFN-γ showed the high level expression in MSCs-Sirt1 treatment group. The impulsive effect of MSCs-Sirt1 on 4T1 cells in vivo could be reversed by inhibition of CXCL10 and IFN-γ. Overall, our results suggest that MSCs-Sirt1 can effectively inhibit breast tumor growth via the recruitment of NK cells in tumor inflammatory microenvironment.

Synthesis, Photophysical Properties, and Biological Evaluation of trans-Bisthioglycosylated Tetrakis(fluorophenyl)chlorin for Photodynamic Therapy.

trans-Bisthioglycosylated tetrakis(fluorophenyl)chlorin (7) was designed as a powerful photodynamic therapy (PDT) photosensitizer based on the findings of our systematic studies. We show here that the trans-bisthioglycosylated structure of 7 enhanced its uptake by HeLa cells and that the chlorin ring of 7 increased the efficiency of reactive oxygen species generation under the standard condition of our photocytotoxicity test. The versatility of 7 in PDT treatment was established using weakly metastatic B16F1 melanoma cells, metastatic 4T1 breast cancer cells, the RGK-1 gastric carcinoma mucosal cell line, and three human glioblastoma cell lines (U87, U251, and T98G). The pharmacokinetics of 7 in mice bearing 4T1 breast cancer cells showed a high tumor-to-skin concentration ratio (approximately 60) at 24 h after intraperitoneal injection. The PDT efficacy of 7 in vivo was approximately 250-times higher than that of mono-l-aspartyl chlorin e6 (9) in mice bearing 4T1 breast cancer cells.

A neutral polydisulfide containing Gd(III) DOTA monoamide as a redox-sensitive biodegradable macromolecular MRI contrast agent.

This work aims to develop safe and effective gadolinium (III)-based biodegradable macromolecular MRI contrast agents for blood pool and cancer imaging. A neutral polydisulfide containing macrocyclic Gd-DOTA monoamide (GOLS) was synthesized and characterized. In addition to studying the in vitro degradation of GOLS, its kinetic stability was also investigated in an in vivo model. The efficacy of GOLS for contrast-enhanced MRI was examined with female BALB/c mice bearing 4T1 breast cancer xenografts. The pharmacokinetics, biodistribution, and metabolism of GOLS were also determined in mice. GOLS has an apparent molecular weight of 23.0 kDa with T1 relaxivities of 7.20 mM(-1) s(-1) per Gd at 1.5 T, and 6.62 mM(-1) s(-1) at 7.0 T. GOLS had high kinetic inertness against transmetallation with Zn(2+) ions, and its polymer backbone was readily cleaved by L-cysteine. The agent showed improved efficacy for blood pool and tumor MR imaging. The structural effect on biodistribution and in vivo chelation stability was assessed by comparing GOLS with Gd(HP-DO3A), a negatively charged polydisulfide containing Gd-DOTA monoamide GODC, and a polydisulfide containing Gd-DTPA-bisamide (GDCC). GOLS showed high in vivo chelation stability and minimal tissue deposition of gadolinium. The biodegradable macromolecular contrast agent GOLS is a promising polymeric contrast agent for clinical MR cardiovascular imaging and cancer imaging.

Transfer of the IL-37b gene elicits anti-tumor responses in mice bearing 4T1 breast cancer

IL-37b has shown anti-cancer activities in addition to its anti-inflammatory properties. In this study, we investigated the effects of IL-37b on breast carcinoma growth in mice and to determine the involvement of T cell activation in the effects. IL-37b gene was transferred into mouse breast carcinoma cell line 4T1 (4T1-IL37b cells), the expression of secretory IL-37b by the cells was detected, and the effects of IL-37b expression on the cell proliferation in vitro was evaluated. After injection of 4T1 cells or 4T1-IL37b cells into immunocompetent BALB/c mice, immunodeficient BALB/c nude mice and NOD-SCID mice, the tumor growth and survival rate were measured. The proliferation of T cells in vitro was also detected. IL-37b was detected in the supernatants of 4T1-IL37b cells with a concentration of 12.02 ± 0.875 ng/mL. IL-37b expression did not affect 4T1 cell proliferation in vitro. BALB/c mice inoculated with 4T1-IL37b cells showed significant retardation of tumor growth. BALB/c mice inoculated with both 4T1 cells and mitomycin C-treated 4T1-IL37b cells also showed significant retardation of tumor growth. But the anti-cancer activity of IL-37b was abrogated in BALB/c nude mice and NOD-SCID mice inoculated with 4T1-IL37b cells. Recombinant IL-37b slightly promoted CD4(+) T cell proliferation without affecting CD8(+) T cell proliferation. IL-37b exerts anti-4T1 breast carcinoma effects in vivo by modulating the tumor microenvironment and influencing T cell activation.

Dendritic cells cross-presenting tumor-derived antigens are eliminated by invariant natural killer T (iNKT) cells, impairing development of anti-tumor immunity to a mouse carcinoma

Meeting abstracts iNKT cells are CD1d-restricted T cells that are rapidly activated in response to antigen and play a key role in promoting adaptive anti-tumor immunity. Surprisingly, we found that mice bearing 4T1 breast cancer CD8+ T cells inhibited spontaneous metastases in the absence of iNKT

The in vivo antitumor activity of LHRH targeted methotrexate–human serum albumin nanoparticles in 4T1 tumor-bearing Balb/c mice

The use of targeted drug delivery systems is a growing trend in cancer treatment to decrease the adverse effect of anti-cancer drugs. In this study, we sought to conjugate methotrexate–human serum albumin nanoparticles (MTX–HSA NPs) with luteinizing-hormone releasing hormone (LHRH). The LHRH was intended to target LHRH receptors overexpressed on the several types of tumors. The expression of LHRH receptors on the 4T1 breast cancer cells was confirmed by FITC conjugated LHRH receptor antibody using fluorescence microscopy. Female Balb/c mice bearing 4T1 breast cancer tumor were treated with a single i.v. injection of free MTX, non-targeted MTX–HSA NPs and LHRH targeted MTX–HSA NPs. LHRH targeted MTX–HSA nanoparticles showed stronger anti-tumor activity in vivo. By 7 days after treatment, average tumor volume in the LHRH targeted MTX–HSA NPs treated group decreased to 8.67% of the initial tumor volume when the number of attached LHRH molecules on MTX–HSA NPs was the highest, while the average tumor volume in non-targeted MTX–HSA NPs treated mice grew rapidly and reached 250.7% of the initial tumor volume 7 days after the treatment. LHRH targeted MTX–HSA NPs could significantly extend the survival time of tumor bearing mice compared with the non-targeted MTX–HSA NPs and free MTX formulations.

Quercetin greatly improved therapeutic index of doxorubicin against 4T1 breast cancer by its opposing effects on HIF-1α in tumor and normal cells

The anthracycline antibiotic doxorubicin (DOX) has been used successfully for treating various types of cancers. However, the therapeutic efficacy of DOX was greatly restricted by its cumulative dose-related cardiotoxicity and common side effects such as bone marrow and immune suppression. Quercetin had better cardioprotective and hepatoprotective activities. The present study was to observe whether quercetin could improve therapeutic index of DOX and explore its mechanisms. Effects of quercetin on doxorubicin (DOX)-induced cytotoxicity were investigated in 4T1 cells and murine spleen cells by methylthiazoletetrazolium assay, flow cytometry and single cell gel electrophoresis. Influences of quercetin on therapeutic efficacy and systemic toxicity of DOX were evaluated in BALB/c mice with 4T1 breast cancer. Hypoxia-inducible factor-1 alpha (HIF-1α) in tumor and normal cells was examined to explore mechanisms of quercetin by Western blot and enzyme-linked immunosorbent assay. In vitro, quercetin at dose less than 100 μM had only slight effects on cell viability and DOX-induced cytotoxicity in 4T1 cells under normoxia, but it could reverse 4T1 cell resistance to DOX under hypoxia and protect spleen cells against DOX-induced cytotoxicity. In vivo, quercetin suppressed tumor growth and prolonged survival in BALB/c mice bearing 4T1 breast cancer. Importantly, quercetin enhanced therapeutic efficacy of DOX and simultaneously reduced DOX-induced toxic side effects. Further study showed that quercetin suppressed intratumoral HIF-1α in a hypoxia-dependent way but increased its accumulation in normal cells. HIF-1α siRNA abolished effects of quercetin on both tumor and normal cells. These results suggested that quercetin could improve therapeutic index of DOX by its opposing effects on HIF-1α in tumor and normal cells, and was a promising candidate as anticancer agents.