Use of PiggyBac Transposon System Constructed Murine Breast Cancer Model For Reporter Gene Imaging And Characterization of Metastatic Tumor Cells

The piggyBac transposon system is known to non-viral integrate exogenous genes to chromosomes of mammalian cells. For reporter gene imaging, this transposon system is believed to efficiently establish xenograft tumor model with low immunogenicity. Because tumor cells usually exhibit genomic instability, it is important to investigate if piggyBac mediated transduction of reporter genes would change tumor characteristics. In this study, reporter gene imaging mediated by the piggyBac transposon system was exploited to track the growth and dissemination of 4T1 triple-negative murine breast cancer cells in vivo, followed by ex vivo analysis of the metastatic cells expressing reporter genes. We demonstrated that several cell properties, including proliferation rate, invasion and migration rate, and mammosphere formation ability of 4T1 cells were not influenced by piggyBac transposon system. Further, we isolated the liver metastatic cells, named 4T1-3R_L cells for further analysis. Compared to parental 4T1 cells, 4T1-3R_L cells exhibited several cancer stem cells (CSC) related characteristics, including significant mammosphere formation ability, resistance to doxorubicin, high tumorigenicity potential in Balb/C mice and expression of CD44 CSC marker. We also found that 4T1-3R_L cells exhibited stronger migrated and invasive abilities, by wound healing assay and in vitro invasion assay, respectively. The cell adhesive ability of 4T1-3R_L cells was also lower than that of 4T1 cells. The microarray assay showed that several epithelial-mesenchymal transition (EMT) promoting markers, including vimentin, N-cadherin, Twist1, and Snail were up-regulated, and anti-EMT marker E-cadherin was down-regulated in 4T1-3R_L cells. Current data suggest that the piggyBac transposon system is a reliable and biocompatible tool to engineer cancer cells for tacking and characterizing tumor development in vivo and in vitro.

Self-assembled tocopherol-albumin nanoparticles with full biocompatibility for chemo-photothermal therapy against breast cancer

Background:: The combination of photothermal therapy (PTT) and chemotherapy has proven to be a promising strategy for cancer treatment. Various nanomaterials have shown great potential in combination therapy, including gold, graphene oxide, iron oxide, and other nanoparticles. However, their undefinable toxicity in vivo greatly slowed down their development for clinical applications. Objective: The present work aimed to develop a multifunctional nanoparticle for chemo-photothermal therapy composed of acknowledged biocompatible materials. Methods: A novel biocompatible nanoparticle (HIT-NPs) was self-assembled through the intrinsic interaction between D-α-tocopherol Succinate (TOS), human serum albumin (HSA) and indocyanine green (ICG). Doxorubicin (DOX) was then loaded due to the ion pairing between DOX and TOS. The feasibility of combined chemo-photothermal therapy induced by DOX-loaded HIT-NPs was carefully evaluated. Results: In vitro, HIT-NPs showed no cytotoxicity on human normal liver cells (HL-7702 cells) but obvious killing effects murine breast cancer cells (4T1 cells). The combined chemo-photothermal therapeutic effect on 4T1 cells was successfully obtained. DOX-loaded HIT-NPs could effectively accumulate in 4T1 subcutaneous tumors after intravenous injection, and the tumor temperature rapidly increased under laser exposure, indicating the feasibility of PTT in vivo. Conclusion: The self-assembled HIT-NPs could provide a promising platform for combined chemo-photothermal cancer therapy with full biocompatibility.

Antitumor Potential of Annona muricata Linn. An Edible and Medicinal Plant in Mexico: In Vitro, In Vivo, and Toxicological Studies

Annona muricata (Am) is a plant used in traditional Mexican medicine to treat cancer. In this study, ethanol extracts of Am collected in Acapulco and Tecpan from Guerrero state were evaluated orally on Balb/c mice inoculated with 4T1 cells, for cytotoxic activity (CA) on 4T1 cells, in brine shrimp lethality assay (BSLA), and for acute oral toxicity in mice. In addition, ethanol extracts were subjected to high-performance liquid chromatography (HPLC) with diode array detection. Results showed that the extracts collected in December in Acapulco (AcDe) and Tecpan (TeDe) exhibited the most significant antitumor and cytotoxic activity. In the BSLA, the most important effect was observed in the extracts from Acapulco and Tecpan collected in June (AcJu) and August (TeAg), respectively. The samples from Acapulco (AcJu, and AcAg) and Tecpan (TeJu and TeAg) showed the highest toxicity. The analysis of the extracts, AcDe and TeDe, by HPLC revealed that flavonoids, rutin, narcissin, and nicotinflorin were the major components. These findings suggest that extracts from Am collected in Acapulco and Tecpan in the month of December may be an important source to obtain flavonoid glycosides with anticancer potential specifically against breast cancer. This also supports the use of Am to treat cancer in Mexican traditional medicine.

Human amniotic epithelial cells exert anti-cancer effects through secretion of immunomodulatory exosomes

We identified here mechanism by which hAEC exert their anti-cancer effects. We showed that vaccination with live hAEC conferred effective protection against murine colon cancer and melanoma but not against breast cancer in orthotopic cancer cell inoculation model. hAEC induced strong cross-reactive antibody response to CT26 cells, but not against B16F10 and 4T1 cells. Neither heterotopic injection of tumor cells in AEC-vaccinated mice nor vaccination with hAEC lysate conferred protection against melanoma or colon cancer. Nanosized AEC-derived exosomes (ADE) induced apoptosis in CT26 cells and inhibited their proliferation. Co-administration of ADE with tumor cells substantially inhibited tumor development and increased CTL responses in vaccinated mice. Our results clearly showed that it is ADE but not the cross-reactive immune responses against tumor cells that mediate inhibitory effects of hAEC on cancer development. Our results highlighted the potential anti-cancer effects of exosomes derived from hAEC.

A Combination of Cabozantinib and Radiation Does Not Lead to an Improved Growth Control of Tumors in a Preclinical 4T1 Breast Cancer Model

The tyrosine kinase inhibitor Cabozantinib has been applied in clinical studies in combination with radiotherapy. We investigated the effect of such combination on triple-negative 4T1 cells as a metastatic breast cancer model in vitro and in vivo upon inoculation in BALB/c mice. In vitro assays indicated a potential for improved effects using the combination. Both Cabozantinib (2.5 µM) and 10 Gy of 250 kV x-rays were able to cease the growth of 4T1 cells as revealed by growth curves. In a clonogenic survival assay, the effect of Cabozantinib added on the effects of irradiation and the effectiveness of inhibiting the clonogenic survival was found to be 2 (RBE10). Additionally, cell death measurements of apoptosis plus necrosis revealed a synergistic effect when combining irradiation with Cabozantinib. Surprisingly, however, in vivo tumor growth kinetics showed no additional effect in growth control when irradiation was used together with Cabozantinib. Since both ionizing radiation and Cabozantinib are acknowledged to feature immunogenic effects, we additionally investigated the effect of the treatments on lung metastases. No difference to the control groups was found here, neither for irradiation nor Cabozantinib alone nor in combination. Yet, upon analysis of the mice’ livers, CD11b-positive cells, indicating immune suppressive myeloid derived suppressor cells were found diminished following treatment with Cabozantinib. In conclusion, despite promising in vitro controls of the combination of Cabozantinib and irradiation, tumor growth control was not increased by the combination, which was true also for the occurrence of lung metastases.

Evaluation of the immunomodulatory activity of thalidomide on tumor-associated macrophages in the 4T1 murine metastatic breast cancer model

ABSTRACT The present work evaluated the immunomodulatory effect of thalidomide (Thal) at different doses on tumor-associated macrophages (TAMs) using a mouse model of human breast cancer. Mice were inoculated with 4T1 cells in the left flank and treated with Thal once a day at concentrations of 50, 100, and 150mg/kg body weight from the 5th day until the 28th day of tumor inoculation. The tumors were sized, proliferation index and TAMs count were evaluated in primary tumors and metastatic lungs. In addition, the metastasis rate was evaluated in the lungs. Thal at 150mg/kg significantly decreased tumor growth, proliferation index, and TAMs infiltration in primary tumors. Conversely, a higher number of TAMs and lower proliferation index were observed in metastatic lungs in mice treated with 150mg/kg of Thal. Furthermore, Thal at 150mg/kg significantly decreased the metastatic nodules in the lungs. Our findings demonstrated that Thal treatment considerably decreased the primary tumor and lung metastasis in mice associated with different TAM infiltration effects in these sites.

Maleimide-Functionalized Liposomes for Tumor Targeting via In Situ Binding of Endogenous Albumin

Albumin, the most abundant protein in plasma, has been widely used in drug delivery studies. Here, we developed maleimide-functionalized liposomes (Mal-Lip) that can bind to endogenous albumin to improve the tumor targeting efficiency of liposomes. Transmission electron microscopy and gel electrophoresis studies showed that albumin can bind to Mal-Lip due to the chemical coupling of the albumin thiol groups with the maleimide group. Both conventional liposomes and Mal-Lip showed minimal cytotoxicity within the tested range of lipid concentrations, indicating that the maleimide functionality did not increase the toxicity of liposomes to various cells. Mal-Lip was taken up by 4T1 cells to a greater extent than conventional liposomes, and Mal-Lip accumulated in 4T1 tumors in mice more than conventional liposomes after intravenous injection. These results suggest that the maleimide group can improve the tumor targeting efficiency of liposomes in vivo by binding to endogenous albumin in situ. However, the maleimide group also enhanced the uptake of Mal-Lip by Raw264.7 cells and shortened their time in circulation, indicating that further studies should be performed to prevent elimination of Mal-Lip by the immune system.

The Large GTPase, GBP-2, Regulates Rho Family GTPases to Inhibit Migration and Invadosome Formation in Breast Cancer Cells

Breast cancer is the most common cancer in women. Despite advances in early detection and treatment, it is predicted that over 43,000 women will die of breast cancer in 2021. To lower this number, more information about the molecular players in breast cancer are needed. Guanylate-Binding Protein-2 has been correlated with better prognosis in breast cancer. In this study, we asked if the expression of GBP-2 in breast cancer merely provided a biomarker for improved prognosis or whether it actually contributed to improving outcome. To answer this, the 4T1 model of murine breast cancer was used. 4T1 cells themselves are highly aggressive and highly metastatic, while 67NR cells, isolated from the same tumor, do not leave the primary site. The expression of GBP-2 was examined in the two cell lines and found to be inversely correlated with aggressiveness/metastasis. Proliferation, migration, and invadosome formation were analyzed after altering the expression levels of GBP-2. Our experiments show that GBP-2 does not alter the proliferation of these cells but inhibits migration and invadosome formation downstream of regulation of Rho GTPases. Together these data demonstrate that GBP-2 is responsible for cell autonomous activities that make breast cancer cells less aggressive.

MnTnHex-2-PyP5+, Coupled to Radiation, Suppresses Metastasis of 4T1 and MDA-MB-231 Breast Cancer via AKT/Snail/EMT Pathways

Tumor migration and invasion induced by the epithelial-to-mesenchymal transition (EMT) are prerequisites for metastasis. Here, we investigated the inhibitory effect of a mimic of superoxide dismutase (SOD), cationic Mn(III) ortho-substituted N-n-hexylpyridylporphyrin (MnTnHex-2-PyP5+, MnHex) on the metastasis of breast cancer in cellular and animal models, focusing on the migration of tumor cells and the factors that modulate this behavior. Wound healing and Transwell migration assays revealed that the migration of mouse mammary carcinoma 4T1 cells was markedly reduced during the concurrent treatment of MnHex and radiation therapy (RT) compared with that of the control and RT alone. Bioluminescence imaging showed that MnHex/RT co-treatment dramatically reduced lung metastasis of 4T1 cells in mice, compared with the sham control and both single treatments. Western blotting and immunofluorescence showed that MnHex treatment of 4T1 cells reversed the RT-induced EMT via inhibiting AKT/GSK-3β/Snail pathway in vitro, thereby decreasing cell migration and invasion. Consistently, histopathological analyses of 4T1 tumors showed that MnHex/RT reduced Snail expression, blocked EMT, and in turn suppressed metastases. Again, in the human metastatic breast cancer MDA-MB-231 cell line, MnHex inhibited metastatic potential in vitro and in vivo and suppressed the RT-induced Snail expression. In addition to our previous studies showing tumor growth inhibition, this study demonstrated that MnHex carries the ability to minimize the metastatic potential of RT-treated cancers, thus overcoming their radioresistance.