4T1 Murine Breast Cancer Model 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.

HDACs alters negatively to the tumor immune microenvironment in gynecologic cancers

The role of histone deacetylases (HDACs) in the tumor immune microenvironment of gynecologic tumors remains unexplored. We integrated data from The Cancer Genome Atlas and Human Protein Atlas to examine HDAC expression in breast, cervical, ovarian, and endometrial cancers. Elevated HDAC expression correlated with poor prognosis and highly malignant cancer subtypes. Gene Set Enrichment Analysis revealed positive associations between HDAC expression and tumor proliferation signature, while negative associations were found with tumor inflammation signature. Increased HDAC expression was linked to reduced infiltration of natural killer (NK), NKT, and CD8+ T cells, along with negative associations with the expression of PSMB10, NKG7, CCL5, CD27, HLA-DQA1, and HLA-DQB1. In a murine 4T1 breast cancer model, treatment with suberoylanilide hydroxamic acid (SAHA; HDAC inhibitor) and PD-1 antibody significantly inhibited tumor growth and infiltration of CD3+ and CD8+ T cells. Real-time polymerase chain reaction revealed upregulated expressions of Psmb10, Nkg7, Ccl5, Cd8a, Cxcr6, and Cxcl9 genes, while Ctnnb1 and Myc genes were inhibited, indicating tumor suppression and immune microenvironment activation. Our study revealed that HDACs play tumor-promoting and immunosuppressive roles in gynecologic cancers, suggesting HDAC inhibitors as potential therapeutic agents for these cancers.

Mucosal vaccination with an adenoviral vector vaccine protects against pulmonary breast cancer metastasis by robust induction of tissue-resident memory T-cells in the lung

Abstract The presence of tissue-resident memory T-cells (TRM) in solid tumors positively correlates with an improved overall prognosis and responsiveness to checkpoint inhibitor therapy. Rationally designed vaccine approaches capable of inducing antitumor TRM at cancer sites are needed to harness the full potential of localized immunity in tumor therapy. Our project aims to establish tumor antigen-specific lung TRM by mucosal vaccination with an adenoviral (Ad) vaccine to prevent and treat lung metastasis in the murine 4T1 breast cancer model. We employ Ad serotype 5 vectors encoding for 4T1 tumor-associated antigens from Mage-b and MuLV gp70 for intranasal vaccination, supplemented with an Interleukin-1β-encoding vector to specifically boost lung TRM induction. Indeed, particularly if Ad-IL-1β was co-delivered, mucosal vaccination established high numbers of tumor antigen-specific CD69 +CD103 +lung TRM as shown by phenotypic and functional analyses in combination with intravascular staining. Vaccine efficacy was evaluated in a 4T1 pseudometastasis model mimicking a prophylactic vaccination against lung metastasis in breast cancer patients. Local immunization led to a significant protection against pulmonary metastasis and improved survival compared to non-vaccinated and, notably, intramuscularly immunized mice. By using FTY720 to inhibit T-cell circulation upon tumor challenge, we confirmed that TRM are crucial contributors to the vaccine efficacy. In conclusion, induction of tumor-antigen specific lung TRM are a key point in local protection against pulmonary metastasis. Ongoing studies analyze the therapeutic use of this vaccination strategy as well as its potential in combination with immune checkpoint inhibition. Supported by grants from 'Anschubfinanzierung (ELAN) im IZKF' and 'Doktor Robert Pfleger-Stiftung'.

Abstract 3572: Oncolytic virotherapy with the HSV-1 (VC2) vaccine strain induces anti-tumor T-cell responses in the murine 4T1 breast cancer model

Abstract The tumor microenvironment (TME) plays important roles in suppression of anti-tumor immune responses. Immune suppression is achieved by multiple mechanisms including interfering with antigen presentation and inhibition of effector T-cell tissue infiltration. Altering specific TME properties can reduce immune-suppression and improve anti-tumor immunity with abscopal effects. Oncolytic Virotherapy (OV) is a promising approach for cancer treatment. We evaluated the efficacy of the HSV-1 (VC2) vaccine strain for the treatment of breast cancer using the murine stage-4 (4T1) syngeneic and immunocompetent breast cancer mouse model. Following Intratumoral (IT) injection of VC2 in mice bearing implanted 4T1 tumors, treatment efficacy was evaluated by monitoring tumor weight, immune infiltration, and metastasis in the lung. Animals treated with VC2 via the IT route exhibited significantly reduced metastasis and improved intra-tumoral T-cell infiltration. Improved T-cell infiltration was not due to viral replication since improved parameters were observed during late times post VC2 treatment when no viral replication was detectable. The total area of metastasis in the lung was significantly reduced compared to control groups. VC2 treated animals via the IT route had significantly higher cytotoxic splenocytes, evident by the in-vitro killing of 4T1 cells, compared to the control group. This improved cytotoxic activity, intratumoral T-cell infiltration, and reduced metastasis in the lung suggest that IT administration of VC2 altered TME in a way that induced better antigen recognition, processing, and presentation resulting in improved anti-tumor immune responses with an abscopal effect in the lung. Further understanding of the mechanisms involved in the observed improved anti-tumor immune responses will help improve treatment efficacy and provide new approaches for the treatment of solid tumors. Citation Format: Rafiq Nabi, Farhana Musarrat, Vladimir Chouljenko, Konstantin Kousoulas. Oncolytic virotherapy with the HSV-1 (VC2) vaccine strain induces anti-tumor T-cell responses in the murine 4T1 breast cancer model [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 3572.

Imidazo[1,2-b]pyrazole-7-Carboxamide Derivative Induces Differentiation-Coupled Apoptosis of Immature Myeloid Cells Such as Acute Myeloid Leukemia and Myeloid-Derived Suppressor Cells.

Chemotherapy-induced differentiation of immature myeloid progenitors, such as acute myeloid leukemia (AML) cells or myeloid-derived suppressor cells (MDSCs), has remained a challenge for the clinicians. Testing our imidazo[1,2-b]pyrazole-7-carboxamide derivative on HL-60 cells, we obtained ERK phosphorylation as an early survival response to treatment followed by the increase of the percentage of the Bcl-xlbright and pAktbright cells. Following the induction of Vav1 and the AP-1 complex, a driver of cellular differentiation, FOS, JUN, JUNB, and JUND were elevated on a concentration and time-dependent manner. As a proof of granulocytic differentiation, the cells remained non-adherent, the expression of CD33 decreased; the granularity, CD11b expression, and MPO activity of HL-60 cells increased upon treatment. Finally, viability of HL-60 cells was hampered shown by the depolarization of mitochondria, activation of caspase-3, cleavage of Z-DEVD-aLUC, appearance of the sub-G1 population, and the leakage of the lactate-dehydrogenase into the supernatant. We confirmed the differentiating effect of our drug candidate on human patient-derived AML cells shown by the increase of CD11b and decrease of CD33+, CD7+, CD206+, and CD38bright cells followed apoptosis (IC50: 80 nM) after treatment ex vivo. Our compound reduced both CD11b+/Ly6C+ and CD11b+/Ly6G+ splenic MDSCs from the murine 4T1 breast cancer model ex vivo.

Site-Specific 111In-Radiolabeling of Dual-PEGylated Porous Silicon Nanoparticles and Their In Vivo Evaluation in Murine 4T1 Breast Cancer Model.

Polyethylene glycol (PEG) has been successfully used for improving circulation time of several nanomaterials but prolonging the circulation of porous silicon nanoparticles (PSi NPs) has remained challenging. Here, we report a site specific radiolabeling of dual-PEGylated thermally oxidized porous silicon (DPEG-TOPSi) NPs and investigation of influence of the PEGylation on blood circulation time of TOPSi NPs. Trans-cyclooctene conjugated DPEG-TOPSi NPs were radiolabeled through a click reaction with [111In]In-DOTA-PEG4-tetrazine (DOTA = 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid) and the particle behavior was evaluated in vivo in Balb/c mice bearing 4T1 murine breast cancer allografts. The dual-PEGylation significantly prolonged circulation of [111In]In-DPEG-TOPSi particles when compared to non-PEGylated control particles, yielding 10.8 ± 1.7% of the injected activity/g in blood at 15 min for [111In]In-DPEG-TOPSi NPs. The improved circulation time will be beneficial for the accumulation of targeted DPEG-TOPSi to tumors.

Versican and Tumor-Associated Macrophages Promotes Tumor Progression and Metastasis in Canine and Murine Models of Breast Carcinoma.

Versican and tumor-associated macrophages (TAMs) are involved in growth and metastases in several cancers. Here, we investigated the potential role of versican, a matrix proteoglycan, and its correlation with TAMs infiltrates in different stages of two different breast cancer models: spontaneous canine mammary gland carcinomas and the murine 4T1 breast cancer model. The stromal versican expression was correlated with TAMs accumulation in tumors with an advanced stage from spontaneous canine mammary carcinoma samples. Versican expression in mice, identified in late stages of tumor progression, was associated to a high number of peri-tumoral infiltrating TAMs. Indeed, TAMs were related to a pro-inflammatory and pro-angiogenic state in the primary tumor. Furthermore, TAMs accumulation was related to versican expression in the lungs and an increased number of pulmonary metastatic nodules with pulmonary mechanical dysfunction, which was due to leukocyte influx in the airways and elevated growth factor levels in the microenvironment. Thus, we suggest that versican and TAMs as attractive targets for breast cancer therapy.

Anti-breast Cancer Enhancement of a Polysaccharide From Spore of Ganoderma lucidum With Paclitaxel: Suppression on Tumor Metabolism With Gut Microbiota Reshaping.

Increasing evidence highlights the cardinal role of gut microbiota in tumorigenesis and chemotherapy outcomes. Paclitaxel (PTX), although as a first-line chemotherapy reagent for breast cancer, still requires for improvement on its efficacy and safety due to drug resistance and adverse effects. The present work explored the enhancement of a polysaccharide derived from spore of Ganoderma lucidum (SGP) with PTX in a murine 4T1-breast cancer model. Results showed that the combination of PTX and SGP displayed an improved tumor control, in which mRNA expression of several Warburg effect-related proteins, i.e., glucose transporter 3 (Glut3), lactate dehydrogenase A (Ldha), and pyruvate dehydrogenase kinase (Pdk), and the metabolite profile of tumor was evidently altered. Flowcytometry analysis revealed that the combination treatment recovered the exhausted tumor infiltration lymphocytes (TILs) via inhibiting the expressions of immune checkpoints (PD-1 and Tim-3), while PTX alone evidently increased that of CTLA-4. 16S rRNA sequencing revealed a restoration by the combination treatment on gut microbiota dysbiosis induced by PTX, especially that Bacteroides, Ruminococcus, and other 5 genera were significantly enriched while the cancer-risk genera, Desulfovibrio and Odoribacter, were decreased. Moreover, spearman correlation analysis showed that abundance of Ruminococcus was significantly negative-associated with the amount of frucotose-6-phosphate within the tumor. Collectively, the present study suggests the clinical implication of SGP as an adjuvant candidate for PTX against breast cancer, which possibly relies on the regulation of tumor metabolism and gut microbiota.

Abstract LB-301: Inhibition of the co-inhibitory receptor KLRG1 reduces murine 4T1 breast cancer metastasis and MC38 colon cancer primary tumor growth and mortality

Abstract Introduction Targeting of lymphocyte co-inhibitory receptors, such as CTLA-4, PD-1, Lag-3, and Tim-3, has gained intense interest for treatment of cancer. KLRG1 is also a lymphocyte co-inhibitory receptor, expressed predominantly on effector memory CD8+ T cells and NK cells, whose ligands E- and N-cadherin are differentially expressed during epithelial-mesenchymal transition. Targeting of KLRG1 in murine cancer models has not previously been published. Methods We constructed an anti-mouse KLRG1 antibody that neutralized the interaction of KLRG1 with its ligands. Anti-KLRG1 antibody was administered as a monotherapy in a murine metastatic tumor model (4T1 murine breast cancer) and as combination therapy with anti-PD-1 (clone RMP1-14) in a murine primary tumor growth model (MC38 murine colon cancer). Results In the 4T1 murine breast cancer model, anti-KLRG1 antibody reduced lung metastases compared to control antibody as measured by mean lung weights (0.28 g vs. 0.47 g; p=0.04) and nodule count (10.7 vs. 30.1; p=0.002). In the MC38 model, primary tumor growth was inhibited by anti-KLRG1 + anti-PD-1 antibody greater than anti-KLRG1 antibody or anti-PD-1 antibody or control antibody (day 13 tumor volume 582 vs. 1259 vs. 1279 vs. 1461 mm3, respectively). Combination therapy anti-KLRG1 + anti-PD-1 produced synergistic benefit significantly greater than that of anti-PD-1 alone for tumor volume (p=0.01) and survival (p=0.02; day 18 survival 60% of mice alive treated with combination therapy compared to 0% alive treated with anti-PD-1 alone). Ex vivo KLRG1 blockade with anti-human KLRG1 antibodies resulted in human CD8+ T cell proliferation and proinflammatory cytokine secretion. Conclusions Anti-KLRG1 neutralizing antibody demonstrates efficacy in inhibiting metastases in the murine 4T1 breast cancer model and reducing primary tumor volume and improving survival as synergistic combination therapy with anti-PD-1 in the murine MC38 colon cancer model. Anti-KLRG1 blockade with anti-human KLRG1 antibodies results in activation of CD8+ T cells. These data suggest that anti-KLRG1 treatment is a promising new approach for cancer immunotherapy. Citation Format: Steven A. Greenberg, Stefano V. Gulla, Evan Thompson. Inhibition of the co-inhibitory receptor KLRG1 reduces murine 4T1 breast cancer metastasis and MC38 colon cancer primary tumor growth and mortality [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr LB-301.

Abstract 5545: Heterodimeric interleukin 15 (hetIL-15) treatment decreases primary breast cancer 4T1 tumors and alleviates the metastatic burden

Abstract Introduction: In this preclinical study, we tried to model the therapeutic strategies often used in the clinic to treat breast cancer patients. We analyzed the role of hetIL 15 immunotherapy alone and in combination with chemotherapy and surgery using the 4T1, a triple-negative breast cancer cell line, murine breast cancer model. Experimental Procedures: We evaluated the therapeutic potential of combining surgery, chemotherapy (doxorubicin) and immunotherapy (hetIL-15) in the murine 4T1 breast cancer model. Cancer cells were orthotopically implanted into the mammary fat pad of Balb/c mice, and the efficacy of hetIL-15 in combination with doxorubicin on the growth of primary tumors and metastatic disease was assessed. In addition, we monitored the impact of surgery alone in the development of metastatic disease, and, finally, we evaluated the effects on the metastatic burden of the three different approaches combined. Results: hetIL-15 monotherapy and combination of doxorubicin chemotherapy with hetIL-15 immunotherapy inhibited the growth of primary tumors. Interrogating the role of these treatments on lung metastatic disease, the results were unexpected. Doxorubicin alone did not affect the numbers of the metastatic lesions in the lungs. In contrast, hetIL-15 monotherapy reduced the metastatic burden. The combination strategy did not show advantage as compared to hetIL-15 monotherapy. Surgical removal of the growing tumor also decreased metastatic burden, especially when performed at an early point. Chemotherapy with doxorubicin prior to and post-surgery had no impact on the metastatic disease. In contrast, hetIL-15 treatment strongly diminished or eliminated the metastatic burden. Conclusion: Our data strongly suggest that treatment with hetIL-15 as an adjuvant therapy after surgery, or as a neoadjuvant agent before surgery, significantly impacts metastatic disease. Citation Format: Dimitris Stellas, Konstantinos Dimas, Bethany A. Nagy, Antonio Valentin, Barbara K. Felber, George N. Pavlakis. Heterodimeric interleukin 15 (hetIL-15) treatment decreases primary breast cancer 4T1 tumors and alleviates the metastatic burden [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 5545.

Ultrasound Doppler as an Imaging Modality for Selection of Murine 4T1 Breast Tumors for Combination Radiofrequency Hyperthermia and Chemotherapy

Noninvasive radiofrequency-induced (RF) hyperthermia has been shown to increase the perfusion of chemotherapeutics and nanomaterials through cancer tissue in ectopic and orthotopic murine tumor models. Additionally, mild hyperthermia (37°C-45°C) has previously shown a synergistic anticancer effect when used with standard-of-care chemotherapeutics such as gemcitabine and Abraxane. However, RF hyperthermia treatment schedules remain unoptimized, and the mechanisms of action of hyperthermia and how they change when treating various tumor phenotypes are poorly understood. Therefore, pretreatment screening of tumor phenotypes to identify key tumors that are predicted to respond more favorably to hyperthermia will provide useful mechanistic data and may improve therapeutic outcomes. Herein, we identify key biophysical tumor characteristics in order to predict the outcome of combinational RF and chemotherapy treatment. We demonstrate that ultrasound imaging using Doppler mode can be utilized to predict the response of combinational RF and chemotherapeutic therapy in a murine 4T1 breast cancer model.

Acid-sensitive lipidated doxorubicin prodrug entrapped in nanoemulsion impairs lung tumor metastasis in a breast cancer model.

To develop an acid-sensitive lipidated, doxorubicin (Dox) prodrug (C16-Dox) to be entrapped in lipid nanoemulsion (NE-C16-Dox) as a nanocarrier to treat breast cancer models (in vitro and in vivo). We report the efficacy of NE-C16-Dox in in vitro experiments, as well as the improved chemotherapeutic index and tumor-control efficacy compared with treatment with free Dox in an in vivo murine 4T1 breast cancer model. In addition, NE-C16-Dox allowed the use of a higher dose of Dox, acceptable biocompatibility and a significant reduction in lung metastasis. Taken together, these results indicate that NE-C16-Dox is promising for breast cancer treatment, thus creating possibilities to translate these nanotechnology concepts to clinical applications.

Gemcitabine-generated ROS promotes neutrophil chemotactic activity through CXCL8 production via PKCδ/ERK pathway in a murine 4T1 breast cancer model

Abstract Chemotherapy-induced neutropenia (CIN) is a common side effect that necessitate dose reductions during treatment of cancer patients. Preventing CIN is critical in chemotherapy because a rapid decline of neutrophil counts increase susceptibility to infection of cancer patients. In spite of its importance, the mechanism of CIN still remains unclear. This study is aimed to investigate the mechanism of CIN in an aspect of neutrophil activation by chemotaxis in gemcitabine-treated breast tumor model. We hypothesized that gemcitabine-generated ROS promotes CXCL8 or MIP-2 production by upregulating ERK pathway in PKCδ-activated intratumoral macrophages and thus neutrophil recruitment to the chemokine gradient. In 4T1 breast tumor mouse model, we found that the basal level of PKCδ activity was higher than in normal mice. The number of blood and peritoneal leukocytes was analyzed at 24h after intraperitoneal administration of gemcitabine. The neutrophil counts decreased in the blood but increased in the peritoneal cavity. In addition, intratumoral neutrophils were elevated in the gemcitabine-treated group. The mRNA levels of neutrophil attracting chemokines/receptors were increased in the peritoneal and tumoral cells. ROS inhibitors N-Acetyl-L-cysteine and Apocynin blocked gemcitabine-induced neutrophil migration, mRNA expression of the chemokines and receptors and PKCδ/ERK phosphorylation. CXCR1/2 inhibitor reparixin decreased neutrophil recruitment to the peritoneal cavity and the tumor tissue. Together, our results suggest that gemcitabine treatment promotes neutrophil chemotactic activity by increasing ROS/PKCδ/ERK/CXCL8 pathway. These findings will provide new insights for developing a therapeutic for CIN.

Abstract 4909: The oncolytic peptide LTX-315 enhances tumor-specific immune responses and tumor regression in murine 4T1 breast cancer when combined with doxorubicin

Abstract The oncolytic effect of LTX-315 involves perturbation of the plasma membrane and distortion of the mitochondrial membrane with subsequent release of DAMPs (Damage-Associated Molecular Pattern molecules) such as ATP, cytochrome C and HMGB1 (1,2). Also multidomain proteins from the BCL-2 family seem to be partially involved in LTX-315 mediated killing (3). In addition, LTX-315 induces the hallmarks of immunogenic cell death (4). Preclinical studies have demonstrated that LTX-315 is able to induce complete tumour regression after intra-tumoral treatment (5,6). In this study the effect of combining intratumoral administration of LTX-315 and systemic treatment with Doxorubicin was investigated in the murine 4T1 breast cancer model. The combination treatment was investigated both in s.c. tumors and in tumors established in the mammary fat pad. The 4T1 cell were labeled with mcherry reporter gene in order to assess tumor growth by whole body imaging. LTX-315 (1 mg in 50 ìl) was injected intratumorally for three consecutive days. Doxorubicin (8mg/kg) was injected into the tail vein as a single dose together with the first injection of LTX-315. Tumor growth was assessed by caliper measurements and whole body luminescence. The combination of local administration of LTX-315 and systemic Doxorubicin, demonstrated significant synergy in both s.c. tumors and in tumors growing in the mammary fat pad. Furthermore, the combination induced a systemic tumour specific immune response since tumors did not develop in cured animals when re-challenged with tumor cells. The tolerable safety profile and novel mode of action of LTX-315 indicate a rationale for combining LTX-315 with other immunotherapies, targeted agents, and chemotherapy.

ADAMTS-18 in the host tissues exerts little effect on breast tumor progress in a murine 4T1 breast cancer model

BackgroundIn this study, we aimed to identify a novel extracellular proteinase ADAMTS-18 that could be a potential tumor suppressor gene.ResultsWe successfully constructed Adamts-18 knockout mice with BALB / c background. RT-PCR analysis showed syngeneic mammary tumor cell line 4 T1 per se has weakly endogenous ADAMTS-18 expression. Orthotopic inoculation of 4 T1 cells within the mammary fat pad of host mice, we found no significant difference in tumor growth and metastasis between Adamts-18 knockout mice and widetype control.ConclusionsWe did not confirm that ADAMTS-18 in the host tissues is relevant for breast tumor progress in a murine 4 T1 breast cancer model.

Synergistic antitumor activity of camptothecin–doxorubicin combinations and their conjugates with hyaluronic acid

Combinations of topoisomerase inhibitors I and II have been found to synergistically inhibit cancer cell growth in vitro, yet clinical studies of these types of combinations have not progressed beyond phase II trials. The results of clinical combinations of topoisomerase (top) I and II inhibitors typically fall within one of two categories: little to no improvement in therapeutic efficacy, or augmented toxicity compared to the single drug counterparts. Hence, despite the promising activity of top I and II inhibitor combinations in vitro, their clinical applicability has not been realized. Here, we report the use of polymer–drug conjugates as a means to co-deliver synergistic doses of top I and II inhibitors camptothecin (CPT) and doxorubicin (DOX) to tumors in vivo in a 4T1 breast cancer model. At specific molar ratios, DOX and CPT were found to be among the most synergistic combinations reported to date, with combination indices between 0.01 and 0.1. The identified optimal ratios were controllably conjugated to hyaluronic acid, and elicited significant tumor reduction of murine 4T1 breast cancer model when administered intravenously. This study elucidates a method to identify synergistic drug combinations and translate them to in vivo by preserving the synergistic ratio via conjugation to a carrier polymer, thus opening a promising approach to translate drug combinations to clinically viable treatment regimens.

Berberine enhances tumor necrosis factor-related apoptosis-inducing ligand-mediated apoptosis in breast cancer

Berberine (BBR) has been used for the treatment of bacterial and fungal infections and also for cancer-associated symptoms such as diarrhea. Furthermore, it has been reported that BBR may have direct antitumor effects. Although evidence supports the theory that tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL) is a promising candidate for treating cancer, its usage may be limited due to the resistance to the TRAIL-induced apoptosis of cancer cells. In the present study, the effect of BBR on TRAIL-induced antitumor effects was investigated in vitro using recombinant TRAIL and in vivo using a 4T1 murine breast cancer model in combination with anti-DR5 (death-inducing TRAIL receptor) monoclonal antibody therapy. BBR sensitized human breast cancer cell lines to TRAIL-mediated apoptosis in vitro. The combination of BBR and recombinant TRAIL significantly activated caspase-3 and PARP cleavage in TRAIL-resistant MDA-MB-468 cells. Furthermore, BBR in combination with TRAIL more effectively induced apoptosis compared with coptisine (COP), which is structurally related to BBR. In a murine 4T1 breast cancer model, BBR treatment enhanced the efficacy of anti-DR5 antibody therapy against primary tumor growth and lung metastasis. Thus, BBR may become a new adjuvant for overcoming the resistance of cancer cells to TRAIL/DR5-mediated therapy.

Vitamin C-driven epirubicin loading into liposomes

The encapsulation of anticancer drugs in a liposome structure protects the drug during circulation and increases drug accumulation in the cancer tissue and antitumor activity while decreasing drug toxicity. This paper presents a new method of active drug loading based on a vitamin C pH/ion gradient. Formulations were characterized in terms of the following parameters: optimal external pH, time and drug-to-lipid ratio for the purpose of remote loading, and in vitro stability. In the case of the selected drug, epirubicin (EPI), its coencapsulation increases its anticancer activity through a possibly synergistic effect previously reported by other groups for a free nonencapsulated drug/vitamin C cocktail. The method also has another advantage over other remote-loading methods: it allows faster drug release through liposome destabilization at the tumor site, thanks to the very good solubility of the EPI vitamin C salt, as seen on cryogenic transmission electron microscopy images. This influences the drug-release process and increases the anticancer activity of the liposome formulation. The liposomes are characterized as stable, with very good pharmacokinetics (half-life 18.6 hours). The antitumor activity toward MCF-7 and 4T-1 breast cancer cells was higher in the case of EPI loaded via our gradient than via an ammonium sulfate gradient. Finally, the EPI liposomal formulation and the free drug were tested using the murine 4T-1 breast cancer model. The antitumor activity of the encapsulated drug was confirmed (tumor-growth inhibition over 40% from day 16 until the end of the experiment), and the free drug was shown to have no anticancer activity at the tested dose.

Modification of a Popular Syngeneic Murine Mammary Tumor Model for Immunotherapy Studies

To facilitate the evaluation of immunotherapeutic intervention against malignant diseases, it is desirable to have a syngeneic tumor model that closely resembles the growth pattern of human tumors. Murine 4T1 breast cancer model is known for its metastatic properties that mimic its human counterpart. However, a drawback of this model is the lack of an identified tumor antigen to function as a therapeutic target for immunologic intervention. We used the piggyBac transposon system to stably transduce a tumor antigen, the human epidermal growth factor receptor 2 gene (HER2), into this tumor cell. In vitro characterization shows that the newly established cells have a similar growth pattern as the parental line. In vivo evaluation shows that host immune response was generated against the HER2 tumor antigen, despite the high homology between HER2 and its murine counterpart (neu gene). When implanted into immune-deficient mice, the HER2-expressing 4T1 cells readily formed sizable tumors, indicating that these cells are useful for evaluating the therapeutic effect of adoptively transferred cytotoxic T cells that are specifically raised or modified to target the HER2 tumor antigen.

Inhibition of metastasis in a murine 4T1 breast cancer model by liposomes preventing tumor cell-platelet interactions

The interaction between circulating tumor cells and blood components, mainly platelets, plays an important role during metastasis. In this study, we prepared liposomes containing the platelet aggregation inhibitor Cilostazol (Cil-L). The objective of this study was to investigate the effect of this Cil-L on platelet aggregation and complex formation with murine 4T1 breast cancer cells in vitro and to determine their anti-metastatic potency in a spontaneous metastasis model of 4T1 breast cancer. Cil-L significantly inhibited the aggregation of platelets by up to 78% and completely abolished the complex formation of 4T1 tumor cells in the presence of activated platelets in vitro. Intravenous (i.v.) injection of Cil-L into mice significantly reduced the aggregability of mouse platelets by 60% measured ex vivo. To gain deeper insight into the mode of metastasis formation in a spontaneous metastasis model, 4T1 breast cancer cells were transplanted into the mammary fad pad of mice and metastasis to the mouse lungs was investigated with regard to tumor cell settlement and metastatic growth. We could demonstrate that the formation of pulmonary metastases was significantly reduced by 55% when mice were treated intravenously with 100 nmol Cil-L 6 h before tumor cell inoculation and then daily for 2 weeks. We conclude that Cil-L reduced metastasis by restricting the aggregability of mouse platelets, which probably prevents the interaction between circulating 4T1 tumor cells and platelets, making the Cil-L a useful tool for the inhibition of breast cancer metastasis in mice.