Gadolinium Oxide Nanoparticles Reinforce the Fractionated Radiotherapy-Induced Immune Response in Tri-Negative Breast Cancer via cGAS-STING Pathway.

Introduction: Radiotherapy is a widely recognized first-line clinical treatment for cancer, but its efficacy may be impeded by the radioresistance of advanced tumors. It is urgent to improve the sensitivity of radioresistant tumors to radiotherapy. In this work, gadolinium oxide nanocrystals (GONs) were utilized as radiosensitizers to enhance the killing effect and reinforce the immune activation of X-ray irradiation on 4T1 breast cancer cells in vitro and in vivo. Methods: 1.0 T small animal MR imaging (MRI) system was employed to trace GONs in vivo, while 225 kVp X-ray irradiation equipment was utilized for investigating the radiosensitization of GONs in 4T1 breast cancer cells in vitro and in vivo. Western blot, quantitative real-time PCR (RT-qPCR), immunohistochemistry, immunofluorescence, clonal survival assay, flow cytometry and reactive oxygen species assay were used to explore the biological mechanism of GON sensitization. Results: GONs exhibited exceptional utility as contrast agents for both in vivo and in vitro MRI imaging. Interestingly, a single dose of 8.0 Gy X-rays together with GONs failed to confer superior therapeutic effects in tumor-bearing mice, while only 3.0 Gy × 3 fractions X-rays combined with GONs exhibited effective tumor growth inhibition. Moreover, fractionated X-ray irradiation with GONs demonstrated a superior capacity to activate the cGAS-STING pathway. Discussion: Fractionated X-ray irradiation in the presence of GONs has demonstrated the most significant activation of the anti-tumor immune response by boosting the cGAS-STING pathway.

Tumor-targeted delivery of lnc antisense RNA against RCAS1 by live-attenuated tryptophan-auxotrophic Salmonella inhibited 4T1 breast tumors and metastasis in mice.

Emerging chemo- and radiotherapy resistance exacerbated the cancer risk and necessitated novel treatment strategies. Although RNA therapeutics against pro-oncogenic genes are highly effective, tumor-specific delivery remains a barrier to the implementation of this valuable tool. In this study, we report a tryptophan-auxotrophic Salmonella typhimurium strain as an onco-therapeutic delivery system with tumor-targeting ability using 4T1 mice breast-cancer model. The receptor-binding cancer antigen expressed on SiSo cell (RCAS1) is a cancer-specific protein that induces the apoptosis of peripheral lymphocytes and confers tumor immune evasion. We designed a long non-coding antisense-RNA against RCAS1 (asRCAS1) and delivered by Salmonella using a non-antibiotic, auxotrophic-selective, eukaryotic expression plasmid, pJHL204. After in vivo tumor-to-tumor passaging, the JOL2888 (ΔtrpA, ΔtrpE, Δasd + asRCAS1) strain exhibited high sustainability in tumors, but did not last in healthy organs, thereby demonstrating tumor specificity and safety. RCAS1 inhibition in the tumor was confirmed by western blotting and qPCR. In mice, JOL2888 treatment reduced tumor-associated macrophages, improved the T cell population, elicited cell-mediated immunity, and suppressed cancer-promoting genes. Consequently, the JOL2888 treatment significantly decreased the tumor volume by 80%, decreased splenomegaly by 30%, and completely arrested lung metastasis. These findings highlight the intrinsic tumor-targeting ability of tryptophan-auxotrophic Salmonella for delivering onco-therapeutic macromolecules.

Ginsenoside Rb1 stabilized and paclitaxel / protopanaxadiol co-loaded nanoparticles for synergistic treatment of breast tumor.

Ginsenosides are the major and key components for ginseng to exert its wide and beneficial therapeutic efficacy in clinic. Meanwhile, many ginsenosides and their metabolites showed in vitro an in vivo anti-tumor activity, among which ginsenoside Rb1 has attracted much attention due to its good solubility and amphipathy. In this study, the self-assembly behavior of Rb1 was investigated and the Rb1 nano-assembly could further stabilize or encapsulated hydrophobic drugs such as protopanaxadiol (PPD) and paclitaxel (PTX) to form nanoparticles, based on which, a natural nanoscale drug delivery system, ginsenoside Rb1 stabilized and PTX/PPD co-loaded nanoparticles (GPP NPs) were prepared. The resultant GPP NPs exhibited a small particle size of 126.2 nm, a narrow size distribution (PDI=0.145), and a zeta potential of -27.3 mV. PTX loading content was 11.06% with an encapsulation efficiency of 93.86%. GPP NPs were spherical and stable in normal saline, 5% glucose, PBS, plasma, or on-shelf storage for 7 days. Both PTX and PPD existed in an amorphous state in GPP NPs and were released in a sustained pattern. GPP NPs showed 10-fold higher in vitro anti-tumor activity of than PTX injections. In the in vivo experiment, GPP NPs achieved a much higher tumor inhibition rate than PTX injections (64.95% vs 43.17%, P < 0.01) and certain tumor target ability. In conclusion, GPP NPs had significantly enhanced anti-tumor efficacy and improved tumor microenvironment, thus were promising to be developed into a novel anti-tumor agent for the treatment of breast tumor.

In vitro synergistic effect of hesperidin and doxorubicin downregulates epithelial-mesenchymal transition in highly metastatic breast cancer cells.

BACKGROUND: We previously reported that in highly metastatic breast cancer cells, doxorubicin (DOX) at non-toxic concentrations promoted cell migration and invasion. Hesperidin (30, 5, 9-dihydroxy-40-methoxy-7-orutinosyl flavanone) is a flavonoid glycoside isolated from citrus/lemon plant that possesses a cytotoxic effect in several cancer cells. In this study, we investigate whether DOX efficacy is enhanced by hesperidin (Hsd) and the molecular pathway involved in highly metastatic breast cancer, 4T1. METHODS: Combined cytotoxicity of Hsd and DOX was evaluated with MTT assay and was analyzed using Chou-Talalay's method. To better understand the underlying mechanism, several factors, including apoptosis and cell cycle arrest were analyzed by flow cytometry. In addition, antimigration activity was evaluated by scratch wound healing assay, MMP-9 expression by ELISA and gelatin zymography, and Rac-1 protein level using western blot. The data on survival rate and expression level of MMP-9 and Rac-1 were obtained from Gene Expression OMNIBUS (GEO). RESULTS: Under MTT assay, Hsd showed a cytotoxic effect in a concentration-dependent manner with an IC50 value of 284 µM on 4T1 cells. Hsd synergistically enhanced the cytotoxic effect of DOX which seemed to correlate with an increase in apoptotic cell death, G2/M cell cycle arrest and blocked the migration of 4T1 cells. At 10 nM, doxorubicin induced lamellipodia formation, and increased the level of Rac-1 and metalloproteinase-9 (MMP-9) expression. Interestingly, combined treatment of DOX and Hsd dramatically downregulated the expression of MMP-9 and Rac-1. These results indicated that Hsd block the cell migration induced by DOX under in vitro studies. CONCLUSION: These findings strongly suggest that Hsd possesses a potential synergistic effect that can be developed to enhance the anticancer efficacy of DOX and reduce the risks of chemotherapy use in highly metastatic breast cancer.

Serum amyloid alpha 1-2 are not required for liver inflammation in the 4T1 murine breast cancer model.

Cancers induce the production of acute phase proteins such as serum amyloid alpha (SAA) in the liver and cause inflammation in various host organs. Despite the well-known coincidence of acute phase response and inflammation, the direct roles of SAA proteins in inflammation in the cancer context remains incompletely characterized, particularly in vivo. Here, we investigate the in vivo significance of SAA proteins in liver inflammation in the 4T1 murine breast cancer model. 4T1 cancers elevate the expression of SAA1 and SAA2, the two major murine acute phase proteins in the liver. The elevation of Saa1-2 correlates with the up-regulation of immune cell-related genes including neutrophil markers. To examine this correlation in detail, we generate mice that lack Saa1-2 and investigate immune-cell phenotypes. RNA-seq experiments reveal that deletion of Saa1-2 does not strongly affect 4T1-induced activation of immune cell-related genes in the liver. Flow cytometry experiments demonstrate the dispensable roles of SAA1-2 in cancer-dependent neutrophil infiltration to the liver. Consistently, 4T1-induced gene expression changes in bone marrow do not require Saa1-2. This study clarifies the negligible contribution of SAA1-2 proteins in liver inflammation in the 4T1 breast cancer model.

Effects of live attenuated measles vaccine Hu191 strain on epithelial mesenchymal transition,proliferation and migration of 4T1 breast cancer cells / 中国生物制品学杂志

@#Objective To investigate the effects of live attenuated measles vaccine Hu191 strain(MV-Hu191)on epithelial mesenchymal transition(EMT),proliferation and migration of 4T1 breast cancer cells.MethodsCCK-8 and clone formation assay were used to analyze the effect of MV-Hu191 on the proliferation of 4T1 cells;The effect of MV-Hu191 on 4T1cell migration was analyzed by cell scratch test;The expression of EMT pathway proteins(MMP-2,MMP-9,E-cadherin)in4T1 cells was detected by Western blot;4T1 tumor-bearing mouse model was established in female BALB/c mice. The model mice were divided into control group(PBS),MV-Hu191(1 × 106TCID50)group and paclitaxel group(15 mg/kg),with 10 mice in each group,and injected into tumor at the dosage of 100 μL every 2 d for 5 times. At 28 d after administration,the effects of MV-Hu191 on survival time,tumorigenicity and metastasis in vivo were observed;The pathological characteristics of lung tissue and tumor tissue were observed by HE staining under microscope;The expression of EMT pathway proteins(MMP-2,MMP-9 and E-cadherin)in tumor tissue was detected by immunohistochemical staining.Results The results of in vitro experiment showed that,compared with the control group,MV-Hu191 inhibited the proliferation and migration of 4T1 cells(F = 2. 811 and 13. 535,P = 0. 001 and 0. 002,respectively),down regulated the expression of MMP-2 and MMP-9(F = 45. 433 and 9. 744,P = 0. 011 and 0. 038,respectively),and up regulated the expression of Ecadherin(F = 7. 001,P = 0. 032);The results of in vivo experiment showed that MV-Hu191 significantly prolonged the survival time of tumor-bearing mice,and decreased the tumor quality(F = 8. 301,P = 0. 003)and the number of pulmonary nodules metastasis compared with the control group(F = 33. 792,P = 0. 000);MV-Hu191 treated tumor tissue gap was small,the cells were round,and the alveolar contour was clearly visible;The expression of MMP-2 and MMP-9 in MVHu191 treated tumor tissue decreased significantly(F = 6. 705 and 9. 047,P = 0. 028 and 0. 023,respectively),while the expression of E-cadherin increased significantly(F = 3. 468,P = 0. 039).ConclusionMV-Hu191 signi-ficantly inhibits the proliferation and migration of 4T1 breast cancer cells,antagonizes the tumorigenicity and lung meta-stasis of 4T1 tumorbearing mice,and prolongs the survival time of mice. The possible mechanism of MV-Hu191 against breast cancer is closely related to the regulation of EMT pathway protein expression.

A cytotoxic survey on 2-amino-1H-imidazol based synthetic marine sponge alkaloid analogues.

Here, we describe the synthesis and biologic activity evaluation of 20 novel synthetic marine sponge alkaloid analogues with 2-amino-1H-imidazol (2-AI) core. Cytotoxicity was tested on murine 4T1 breast cancer, A549 human lung cancer, and HL-60 human myeloid leukemia cells by the resazurin assay. A total of 18 of 20 compounds showed cytotoxic effect on the cancer cell lines with different potential. Viability of healthy human fibroblasts and peripheral blood mononuclear cells upon treatment was less hampered compared to cancer cell lines supporting tumor cell specific cytotoxicity of our compounds. The most cytotoxic compounds resulted the following IC50 values 28: 2.91 µM on HL-60 cells, and 29: 3.1 µM on 4T1 cells. The A549 cells were less sensitive to the treatments with IC50 15 µM for both 28 and 29. Flow cytometry demonstrated the apoptotic effect of the most active seven compounds inducing phosphatidylserine exposure and sub-G1 fragmentation of nuclear DNA. Cell cycle arrest was also observed. Four compounds caused depolarization of the mitochondrial membrane potential as an early event of apoptosis. Two lead compounds inhibited tumor growth in vivo in the 4T1 triple negative breast cancer and A549 human lung adenocarcinoma xenograft models. Novel marine sponge alkaloid analogues are demonstrated as potential anticancer agents for further development.

Toward Exempting from Sentinel Lymph Node Biopsy in T1 Breast Cancer Patients: A Retrospective Study.

Background and Objective: Sentinel lymph node biopsy (SLNB) is used to assess the status of axillary lymph node (ALN), but it causes many adverse reactions. Considering the low rate of sentinel lymph node (SLN) metastasis in T1 breast cancer, this study aims to identify the characteristics of T1 breast cancer without SLN metastasis and to select T1 breast cancer patients who avoid SLNB through constructing a nomogram. Methods: A total of 1,619 T1 breast cancer patients with SLNB in our hospital were enrolled in this study. Through univariate and multivariate logistic regression analysis, we analyzed the tumor anatomical and clinicopathological factors and constructed the Heilongjiang Medical University (HMU) nomogram. We selected the patients exempt from SLNB by using the nomogram. Results: In the training cohort of 1,000 cases, the SLN metastasis rate was 23.8%. Tumor volume, swollen axillary lymph nodes, pathological types, and molecular subtypes were found to be independent predictors for SLN metastasis in multivariate regression analysis. Distance from nipple or surface and position of tumor have no effect on SLN metastasis. A regression model based on the results of the multivariate analysis was developed to predict the risk of SLN metastasis, indicating an AUC of 0.798. It showed excellent diagnostic performance (AUC = 0.773) in the validation cohort. Conclusion: The HMU nomogram for predicting SLN metastasis incorporates four variables, including tumor volume, swollen axillary lymph nodes, pathological types, and molecular subtypes. The SLN metastasis rates of intraductal carcinoma and HER2 enriched are 2.05% and 6.67%. These patients could be included in trials investigating the SLNB exemption.

Tumor-associated macrophage-targeted photodynamic cancer therapy using a dextran sulfate-based nano-photosensitizer.

The M2-like phenotype of tumor-associated macrophages (TAMs) present in tumors promotes tumor growth and metastasis. Therefore, targeting M2-like TAMs is a potential strategy for cancer therapy. Herein, we fabricated a dextran sulfate-based nano-photosensitizer (dextran sulfate-conjugated chlorin e6, DS-Ce6) to specifically target M2-like TAMs for enhanced photodynamic therapy (PDT). DS-Ce6 was preferentially taken up by interleukin-4-derived M2 macrophages, which overexpressed scavenger receptor-A and selectively targeted macrophages in co-cultured 4T1 tumors/macrophages. The nano-photosensitizer also effectively induced the apoptosis of tumor cells in both monolayer co-culture and three-dimensional co-culture spheroids of tumors/macrophages under laser irradiation. Moreover, the nano-photosensitizer specifically targeted F4/80 and CD206 double-positive M2-like TAMs within tumor tissues. Therefore, the specifically targeted delivery of DS-Ce6 to M2-like TAMs prominently induced tumor apoptosis, leading to excellent phototherapeutic effects in 4T1 tumor-bearing mice after PDT, suggesting the potential of DS-Ce6 for specific targeting of M2-like TAMs and enhanced PDT.

Disrupting circadian rhythms promotes cancer-induced inflammation in mice.

Disruption of circadian rhythms occurs in rotating shift-work, jetlag, and in individuals with irregular sleep schedules. Circadian disruption is known to alter inflammatory responses and impair immune function. However, there is limited understanding of how circadian disruption modulates cancer-induced inflammation. Inflammation is a hallmark of cancer and is linked to worse prognosis and impaired brain function in cancer patients. Here, we investigated the effect of circadian disruption on cancer-induced inflammation in an orthotopic breast cancer model. Using a validated chronic jetlag protocol that advances the light-cycle by 8 â€‹h every 2 days to disrupt circadian rhythms, we found that circadian disruption alters cancer-induced inflammation in a tissue-specific manner, increasing inflammation in the body and brain while decreasing inflammation within the tumor tissue. Circadian disruption did not affect inflammation in mice without tumors, suggesting that the impact of circadian disruption may be particularly detrimental in the context of underlying inflammatory conditions, such as cancer. Importantly, circadian disruption did not affect tumor burden, suggesting that increased inflammation was not a result of increased cancer progression. Overall, these findings identify the importance of healthy circadian rhythms for limiting cancer-induced inflammation.

Utility of Oncotype DX score in clinical management for T1 estrogen receptor positive, HER2 negative, and lymph node negative breast cancer.

BACKGROUND: The management of estrogen receptor positive (ER+)/HER2- and lymph node (LN) negative breast cancers can be influenced by Oncotype DX recurrence score (RS) in the USA. However, the benefit of RS in T1 tumors (≤ 1 cm) is not clear. METHODS: We retrieved 199 T1 ER+/HER2-/LN- breast cancer diagnosed between 1993 and 2016 that had undergone RS testing. The median follow-up time was 51 months. We examined the disease-free survival (DFS) and distant metastasis and their association with RS and other clinicopathologic features. RESULTS: Of the 199 cases, 40 were T1a (≤ 0.5 cm) and 159 were T1b (> 0.5 cm to 1 cm) tumors. In the 40 T1a tumors, 11 would benefit from chemotherapy by the TAILORx study results. Of these T1a tumors, 36 were Nottingham grade 1/2, 3 were grade 3, and 1 was microinvasive carcinoma; 2 (5%) had local recurrence and 1 (2.5%) had distant metastasis to the bone. The only patient with T1a tumor (Nottingham grade 3, RS = 42) and distant metastasis to bone had received adjuvant chemotherapy. In the 159 T1b tumors, 25 would benefit chemotherapy by the TAILORx results. Of the T1b tumors, 149 were Nottingham grade 1/2 and 10 were grade 3. Nine (5.7%) had local recurrence and 2 (1.3%) had distant metastasis to bone and mediastinum, respectively. The two T1b tumors with distant metastasis had a RS 20 and Nottingham grade 2, and RS 27 and Nottingham grade 3, respectively. Both patients received adjuvant chemotherapy. In multivariate analysis of the entire cohort (T1a and T1b tumors), Nottingham tumor grade and receiving chemotherapy were significantly associated with DFS. In univariate analysis of the entire cohort, Nottingham tumor grade, receiving adjuvant chemotherapy, and RS were significantly associated with distant metastasis. CONCLUSION: This study demonstrates that the metastatic rate of T1a and T1b ER+/HER2-/LN- breast cancer is very low. Patients with low grade (1 or 2), T1a ER+/HER2-/LN- breast cancer may not need RS for treatment decision-making; however, in patients with high-grade T1a or T1b ER+/HER2-/LN- breast cancer, RS analysis should be strongly considered.

Evaluation of Radiotherapy-Induced Systemic Antitumor Effects in Mice Bearing 4T1 Mouse Breast Cancer Cells.

Background: Recently, several clinical studies have reported that combination treatments of radiation therapy (RT) and immunotherapy in patients with multiple lesions can improve tumor regression at a distance from the irradiated site, known as the abscopal effect. However, when RT and immunotherapy are concurrently applied, it is hard to distinguish the pure systemic effects of RT from those of the immunotherapy drug. In this preclinical study, the authors investigated the systemic antitumor effects of RT alone according to fraction dose size and splitting schedules. Materials and Methods: 4T1 mouse breast cancer cells were implanted into the right and left sides of mammary gland fat pads of BALB/c mice, followed by irradiation with 6 Gy × 3, 8 Gy × 2, and 13 Gy × 1 fractions when the right-side tumors were palpable. Results: The different irradiation schedules produced similar antitumor effects in irradiated right-side tumors and unirradiated left-side tumors. However, 8 Gy × 2 and 13 Gy × 1 fractions exhibited better antimetastatic potential than that from irradiation using 6 Gy × 3 fractions. Furthermore, 8 Gy × 2 and 13 Gy × 1 fractions produced higher expressions of HMGB1 and lower expressions of the proinflammatory cytokines, IFN-γ, TNF-α, IL-6, and IL-1ß, from the irradiated tumor tissues. Conclusions: These findings suggest that 8 Gy × 2 and 13 Gy × 1 fractions can provide better systemic antitumor effects than 6 Gy × 3 fractions. The authors hope these results provide clues to optimize RT dose regimens to make the abscopal effect clinically more relevant in future combination treatments.

Piperine-Loaded Glycyrrhizic Acid- and PLGA-Based Nanoparticles Modified with Transferrin for Antitumor : Piperine-Loaded Glycyrrhizic Acid- and PLGA-Based Nanoparticles.

The purpose of this study was to enhance the antitumor effect of piperine by constructing the nanoparticles modified with transferrin (Tf-PIP-NPs) and evaluating their efficacy in vitro and in vivo. The Tf-PIP-NPs were prepared by the solvent evaporation method, and their properties were characterized. The effects of Tf-PIP-NPs on cytotoxicity, cell uptake, apoptosis, and mitochondrial membrane potential were evaluated in HepG2 cells, MDA-MB-231 cells, and 4T1 cells. In a 4T1 tumor-bearing mouse model, the antitumor efficacy of Tf-PIP-NPs was assessed in terms of tumor volumes, changes in body weight, HE staining, and immunohistochemical analysis. With a mean particle size of 112.2 ± 1.27 nm, the zeta potential of (- 28.0 ± 1.6 mV) Tf-PIP-NPs were rapidly internalized by tumor cells after 1 h through the transferrin receptor (TfR)-mediated endocytosis pathway, significantly inducing cellular apoptosis and mitochondrial membrane potential loss. Although Tf-PIP-NPs had no significant difference with PIP-NPs in tumor volume inhibition due to the presence of tumor microenvironment, it could significantly upregulate the expression of related pro-apoptotic proteins and induce tumor necrosis. We used the self-assembly properties of glycyrrhizic acid (GL) and polymer-PLGA to encapsulate piperine and modified with the transferrin, which provided a promising approach to improve the antitumor efficacy for anticarcinogen.

Desensitization of Capsaicin-Sensitive Afferents Accelerates Early Tumor Growth via Increased Vascular Leakage in a Murine Model of Triple Negative Breast Cancer.

There is growing interest in the role of nerve-driven mechanisms in tumorigenesis and tumor growth. Capsaicin-sensitive afferents have been previously shown to possess antitumoral and immune-regulatory properties, the mechanism of which is currently poorly understood. In this study, we have assessed the role of these terminals in the triple negative 4T1 orthotopic mouse model of breast cancer. The ultrapotent capsaicin-analogue resiniferatoxin (RTX) was used for the selective, systemic desensitization of capsaicin-sensitive afferents. Growth and viability of orthotopically implanted 4T1 tumors were measured by caliper, in vivo MRI, and bioluminescence imaging, while tumor vascularity and protease enzyme activity were assessed using fluorescent in vivo imaging. The levels of the neuropeptides Calcitonin Gene-Related Peptide (CGRP), Substance P (SP), and somatostatin were measured from tumor tissue homogenates using radioimmunoassay, while tumor structure and peritumoral inflammation were evaluated by conventional use of CD31, CD45 and CD3 immunohistology. RTX-pretreated mice demonstrated facilitated tumor growth in the early phase measured using a caliper, which was coupled with increased tumor vascular leakage demonstrated using fluorescent vascular imaging. The tumor size difference dissipated by day seven. The MRI tumor volume was similar, while the intratumoral protease enzyme activity measured by fluorescence imaging was also comparable in RTX-pretreated and non-pretreated animals. Tumor viability or immunohistopathological profile was measured using CD3, CD31, and CD45 stains and did not differ significantly from the non-pretreated control group. Intratumoral somatostatin, CGRP, and SP levels were similar in both groups. Our results underscore the beneficial, antitumoral properties of capsaicin sensitive nerve terminals in this aggressive model of breast cancer, which is presumed to be due to the inhibition of tumor vascular bed disruption. The absence of any difference in intratumoral neuropeptide levels indicates non-neural sources playing a substantial part in their expression.

pH-Sensitive PEGylated Liposomal Silybin: Synthesis, In Vitro and In Vivo Anti-Tumor Evaluation.

The drug delivery systems improve the efficacy of chemotherapeutics through enhanced targeting and controlled release however, biological barriers of tumor microenvironment greatly impede the penetration of nanomedicine within the tumor. We report herein the fabrication of a PEG-detachable silybin (SLB) pH-sensitive liposome decorated with TAT-peptide. For this, Acyl hydrazide-activated PEG2000 was prepared and linked with ketone-derivatized DPPE via an acid-labile hydrazone bond to form mPEG2000-HZ-DPPE. TAT peptide was conjugated with a shorter -PEG1000-DSPE spacer and post-inserted into PEGylated liposome (DPPC: mPEG2000-DSPE: Chol). To prepare nanoliposomes (around 100 nm), first, a novel method was used to prepare SLB-Soya PC (SLB-SPC) complex, then this complex was incorporated into nanoliposomes. The pH-sensitivity and shielding effect of long PEG chain on TAT peptide was investigated using DiI liposome and FACS analysis. Pre-treatment to the lowered pH enhanced cellular association of TAT-modified pH-sensitive liposome due to the cleavage of hydrazone bond and TAT exposure. Besides, TAT-modified pH-sensitive liposomes significantly reduced cell viability compared to the plain liposome. In vivo results were very promising with pH-sensitive liposome by detaching PEG moieties upon exposure to the acidic tumor microenvironment, enhancing cellular uptake, retarding tumor growth, and prolonging the survival of 4T1 breast tumor-bearing BALB/c mice. TAT modification of pH-sensitive liposome improved cancer cell association and cytotoxicity and demonstrated potential intracellular delivery upon exposure to acidic pH. However, in in vivo studies, TAT as a targeting ligand significantly decreased the therapeutic efficacy of the formulation attributed to an inefficient tumor accumulation and higher release rate in the circulation. The results of this study indicated that pH-sensitive liposome containing SLB, which was prepared with a novel method with a significant SLB loading efficiency, is very effective in the treatment of 4T1 breast tumor-bearing BALB/c mice and merits further investigation.

Pulsed-Focused Ultrasound Slows B16 Melanoma and 4T1 Breast Tumor Growth through Differential Tumor Microenvironmental Changes.

Focused ultrasound (FUS) has shown promise as a non-invasive treatment modality for solid malignancies. FUS targeting to tumors has been shown to initiate pro-inflammatory immune responses within the tumor microenvironment. Pulsed FUS (pFUS) can alter the expression of cytokines, chemokines, trophic factors, cell adhesion molecules, and immune cell phenotypes within tissues. Here, we investigated the molecular and immune cell effects of pFUS on murine B16 melanoma and 4T1 breast cancer flank tumors. Temporal changes following sonication were evaluated by proteomics, RNA-seq, flow-cytometry, and histological analyses. Proteomic profiling revealed molecular changes occurring over 24 h post-pFUS that were consistent with a shift toward inflamed tumor microenvironment. Over 5 days post-pFUS, tumor growth rates were significantly decreased while flow cytometric analysis revealed differences in the temporal migration of immune cells. Transcriptomic analyses following sonication identified differences in gene expression patterns between the two tumor types. Histological analyses further demonstrated reduction of proliferation marker, Ki-67 in 4T1, but not in B16 tumors, and activated cleaved-caspase 3 for apoptosis remained elevated up to 3 days post-pFUS in both tumor types. This study revealed diverse biological mechanisms following pFUS treatment and supports its use as a possible adjuvant to ablative tumor treatment to elicit enhanced anti-tumor responses and slow tumor growth.

Exosome-mediated miR-33 transfer induces M1 polarization in mouse macrophages and exerts antitumor effect in 4T1 breast cancer cell line.

Macrophages are the most abundant tumor-infiltrating immune cells. Macrophages are conventionally classified as M1 or M2 types. M2 type is the dominant phenotype of macrophages in the tumor microenvironment. M2 macrophages support different aspects of tumor development, including tumor formation, growth, and metastasis. MicroRNAs (miRNAs) have been demonstrated to regulate numerous cellular processes, including macrophage polarization. To determine whether miR-33 containing exosomes can alter macrophage polarization, we used the exosomes isolated from 4T1 breast cancer cells to deliver miR-33 mimic into IL-4 induced M2 macrophages and treated macrophages with 4T1-conditioned media. Then, we assayed the expression of M1 specific markers and the production of cytokines using real-time PCR and ELISA, respectively. Additionally, we performed MTT, migration, and invasion assays to detect the effect of miRNA-mediated macrophage repolarization on cancer cell proliferation, migration, and invasion. The results of this study showed that miR-33 containing exosomes could convert M2 to M1 phenotype as indicated by an increase in expression of M1 markers, including Irf5, Nos2, and CD86, and a decrease in M2 markers including Arg, Ym1, and CD206. Furthermore, the secretion of TNF-α and IL-1ß as M1 specific cytokines increased, while the secretion of IL-10 and TGF-ß as M2 specific cytokines decreased. Incubation of 4T1 cells with conditioned media of treated macrophages showed reduced proliferation, invasion, and migration of these cells. So, our data suggests that exosomes can be used as an efficient nanocarrier for miR-33 delivery into macrophages. Also, miR-33 is capable of inducing M1 polarization in macrophages, which is essential for suppressing tumor growth and metastasis.

The Impact of Focused Ultrasound in Two Tumor Models: Temporal Alterations in the Natural History on Tumor Microenvironment and Immune Cell Response.

Image-guided focused ultrasound (FUS) has been successfully employed as an ablative treatment for solid malignancies by exposing immune cells to tumor debris/antigens, consequently inducing an immune response within the tumor microenvironment (TME). To date, immunomodulation effects of non-ablative pulsed-FUS (pFUS) on the TME are poorly understood. In this study, the temporal differences of cytokines, chemokines, and trophic factors (CCTFs) and immune cell populations induced by pFUS were interrogated in murine B16 melanoma or 4T1 breast cancer cells subcutaneously inoculated into C57BL/6 or BALB/c mice. Natural history growth characteristics during the course of 11 days showed a progressive increase in size for both tumors, and proteomic analysis revealed a shift toward an immunosuppressive TME. With respect to tumor natural growth, pFUS applied to tumors on days 1, 5, or 9 demonstrated a decrease in the growth rate 24 h post-sonication. Flow cytometry analysis of tumors, LNs, and Sp, as well as CCTF profiles, relative DNA damage, and adaptive T-cell localization within tumors, demonstrated dynamic innate and adaptive immune-modulation following pFUS in early time points of B16 tumors and in advanced 4T1 tumors. These results provide insight into the temporal dynamics in the treatment-associated TME, which could be used to evaluate an immunomodulatory approach in different tumor types.

Effect of Panax Notoginseng Saponins on Breast Cancer Cell Line 4T1 in Tumor-bearing Mice Through MEKK1/SEK1/JNK1/AP-1 Pathway / 中国实验方剂学杂志

Objective:To explore the potential mechanisms of Panax Notoginseng Saponins （PNS） on growth inhibition of breast cancer cell line 4T1 in tumor-bearing mice by investigating the mitogen-activated protein kinase kinase kinase 1 （MEKK1）/stress activated protein kinase （SAPK）/extracellular regulated protein kinases （Erk） Kinase （SEK1）/c-Jun N-terminal kinase 1 （JNK1）/activator protein-1 （AP-1） signaling pathways. Method:The 4T1 breast cancer mice model was established. Forty-eight mice with successful modeled and randomly divided into the low， medium and high-dose PNS groups （10， 20， 40 mg·kg-1） and the model control group （12 mice in each group）. The PNS groups received intraperitoneal injection with dosage of 10 mL·kg-1， while the controlled group was given the same dosage of saline. After administration with PNS for 28 days， tumor tissues were isolated， weighed， sliced and homogenized. Tumor cell apoptosis was detected by TdT mediated-dUTP nick end labeling （TUNEL） staining. The mRNA expressions of MEKK1， SEK1， JNK1 and AP-1 in tumor tissue were detected by Real-time polymerase chain reaction（Real-time PCR）. The protein expressions of MEKK1， SEK1， JNK1 and AP-1 in tumor tissue were detected by immunofluorescence staining and Western blot. Result:Compared with model group， the tumor weights of medium-dose and high-dose PNS groups were decreased significantly （P<0.05）. TUNEL staining showed that the number of apoptotic tumor cells increased with the rise of dosage of PNS （P<0.05）. The medium-dose and high-dose PNS groups showed a significant increase in the mRNA expressions of MEKK1， SEK1， JNK1 and AP-1 as well as the protein expressions of MEKK1， SEK1， JNK1 and AP-1 in tumor tissues （P<0.05）， with statistically significant differences （P<0.05）. Conclusion:PNS could inhibit the tumor growth of breast cancer cell line 4T1 in tumor-bearing mice， which may be related to the activation of MEKK1/SEK1/JNK1/AP-1 signaling pathways.

A Nomogram Predicting Lymph Node Metastasis in T1 Breast Cancer based on the Surveillance, Epidemiology, and End Results Program.

Background: Patients with early stage breast cancer with lymph nodes metastasis were proven to have more aggressive biologically phenotypes. This study aimed to build a nomogram to predict lymph node metastasis in patients with T1 breast cancer. Methods: We identified female patients with T1 breast cancer diagnosed between 2010 and 2014 in the Surveillance, Epidemiology and End Results database. The patients were randomized into training and validation sets. Univariate and multivariate logistic regressions were carried out to assess the relationships between lymph node metastasis and clinicopathological characteristics. A nomogram was developed and validated by a calibration curve and receptor operating characteristic curve analysis. Result: Age, race, tumour size, tumour primary site, pathological grade, oestrogen receptor (ER) status, progesterone receptor (PR) status and human epidermal growth factor receptor 2 (HER2) status were independent predictive factors of positive lymph node metastasis in T1 breast cancer. Increasing age, tumour size and pathological grade were positively correlated with the risk of lymph node metastasis. We developed a nomogram to predict lymph node metastasis and further validated it in a validation set, with areas under the receiver operating characteristic curves of 0.733 and 0.741 in the training and validation sets, respectively. Conclusions: A better understanding of the clinicopathological characteristics of T1 breast cancer patients might important for assessing their lymph node status. The nomogram developed here, if further validated in other large cohorts, might provide additional information regarding lymph node metastasis. Together with sentinel lymph node biopsy, this nomogram can help comprehensively predict lymph node metastasis.