Cell Line 4T1 Research Articles

The Central THαβ Immunity Associated Cytokine: IL-10 Has a Strong Anti-Tumor Ability Toward Established Cancer Models In Vivo and Toward Cancer Cells In Vitro.

Immunotherapy is a promising new approach for cancer treatment. In this study, I propose to use the THαβ-mediated immune response for cancer treatment. The THαβ-mediated immune response is activated by IL-10 and IL-15. Thus, I used IL-10 and-15 as therapeutic agents in the 4T1 cell line, which is a mouse cell line of breast cancer, and the NXS2 cell line, which is a mouse cell line of neuroblastoma. Cells from 4T1 and NXS2 were subcutaneously inoculated in wild type BALB/c female mice and AJ mice, respectively, and administered cytokines or an antibody treatment at various dosages. My results showed that IL-10 and IL-15 administration led to reduction in tumor volume and increase in survival. However, traditional TH1 cytokine IFN-γ administration led to increase in tumor volume and decline in survival. Antibody treatment in conjunction with IL-10 was not significantly better than IL-10, due to the expression of GD2 on immune cells. Moreover, an anti-GD2 antibody inhibited the immune cells themselves. Additionally, I found that IL-10 was directly toxic to tumor cells in vitro. Thus, I conclude that the THαβ immunological pathway is a good treatment strategy for cancer.

Engineered Human Heavy-Chain Ferritin with Half-Life Extension and Tumor Targeting by PAS and RGDK Peptide Functionalization.

Ferritin, one of the most investigated protein nanocages, is considered as a promising drug carrier because of its advantageous stability and safety. However, its short half-life and undesirable tumor targeting ability has limited its usage in tumor treatment. In this work, two types of functional peptides, half-life extension peptide PAS, and tumor targeting peptide RGDK (Arg-Gly-Asp-Lys), are inserted to human heavy-chain ferritin (HFn) at C-terminal through flexible linkers with two distinct enzyme cleavable sites. Structural characterizations show both HFn and engineered HFns can assemble into nanoparticles but with different apparent hydrodynamic volumes and molecular weights. RGDK peptide enhanced the internalization efficiency of HFn and showed a significant increase of growth inhibition against 4T1 cell line in vitro. Pharmacokinetic study in vivo demonstrates PAS peptides extended ferritin half-life about 4.9 times in Sprague Dawley rats. RGDK peptides greatly enhanced drug accumulation in the tumor site rather than in other organs in biodistribution analysis. Drug loaded PAS-RGDK functionalized HFns curbed tumor growth with significantly greater efficacies in comparison with drug loaded HFn.

Down-regulation of immune checkpoints by doxorubicin and carboplatin-containing neoadjuvant regimens in a murine breast cancer model.

Objective(s): Immune checkpoint expression on tumor-infiltrating lymphocytes (TILs) has a correlation with the outcome of neoadjuvant chemotherapy (NAC) in breast cancer. However, the reciprocal effect of these regimens on the quality and quantity of immune checkpoints has hitherto not been addressed. We aimed to evaluate the impact of three NAC regimens on TILs and immune checkpoints in a murine triple-negative breast cancer model.Materials and Methods:Syngeneic model of locally-advanced breast cancer was established in immunocompetent mice using a 4T1 cell line. Tumor-bearing animals were treated with human-equivalent dosages of doxorubicin, paclitaxel, paclitaxel and carboplatin combination, and placebo. Infiltration of CD3+, CD8+, and FoxP3+ cells into the tumor was assessed by immunohistochemistry. Expression of immune checkpoints, including PD-1, CTLA-4, and TIM-3, was evaluated by real-time PCR.Results:Doxorubicin led to a significant (P<0.01) increase in the percentage of the stromal infiltrating CD3+ and CD8+ lymphocytes. Doxorubicin also suppressed significantly (P<0.05) the relative expression of PD-1 compared with the placebo. PD-1 expression was significantly (P<0.05) lower in the group treated with paclitaxel and carboplatin combination as compared with the placebo. The relative expression of TIM-3 was significantly (P<0.05) suppressed in doxorubicin-treated mice in comparison with other interventions.Conclusion:Our findings hypothesize that NAC with doxorubicin may potentiate antitumor immunity not merely by recruitment of TILs, but via down-regulation of PD-1 and TIM-3 checkpoints. Carboplatin-containing NAC may suppress PD-1 as well.

Antiproliferative effects of fresh water crab hemolymph and meat extract on breast cancer cell line

Background and aims: Despite the advances in drugs, side effects of chemotherapy drugs continue to exist. Therefore, more attention has been paid to the compounds derived from medicinal herbs and aquatic organisms. This study aimed to investigate the effect of freshwater crab hemolymph and meat extract on breast cancer (BC) cell line (4T1). Methods: After isolation of freshwater crab hemolymph and meat extract, protein concentration and total antioxidant capacity were analyzed by bicinchoninic acid (BCA) and cupric reducing antioxidant capacity (CUPRAC) methods. The 4T1 cells and bone marrow mesenchymal stem cells (BMSCs) were treated with crab hemolymph (1, 2, 10 mg/mL) and meat extract (0.1, 0.2 and 1 mg/mL), and cell survival was analyzed using 3-(4, 5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay (MTT) at 48 and 72 hours. Nitric oxide (NO) secretion was measured by Griess method. Data were analyzed using one-way analysis of variance (ANOVA). Results: Protein concentration of 23.25 mg/mL was shown in crab hemolymph, and 2.3 mg/mL in meat extract. Total antioxidant capacity was reported as 1.036 µM/mL and 1.104 µM/mL in crab hemolymph and meat extract, respectively. Cell survival in the 4T1 cells was decreased in a dose- and time-dependent manner (P≤0.001). NO secretion of 4T1 cells was decreased after treatment with different concentrations of crab hemolymph and meat extract at 48 and 72 hours. Cellular growth was observed in BMSCs after treatment with different concentrations of crab hemolymph and meat extract at 48 and 72 hours. Conclusion: Since crab hemolymph and meat extract have protein and antioxidant activities, they can have anti-cancer effects on 4T1 cells.

Synthesis and characterization of Gd2O3: Er3+, Yb3+ doped with Mg2+, Li+ ions—effect on the photoluminescence and biological applications

Gd2O3:1% Er3+, 18% Yb3+, x% Mg2+ (x = 0; 2.5; 4; 5; 6; 8;10; 20; 25; 50) and Gd2O3:1% Er3+, 18% Yb3+, 2,5% Mg2+, y% Li+ (y = 0.5–2.5) nanoparticles were synthesized by homogenous precipitation method and calcined at 900 °C for 3 h in air atmosphere. Powder x-ray diffraction, scanning electron microscopy, cathodoluminescence, transmission electron microscopy, energy dispersive x-ray spectroscopy and photoluminescence techniques were employed to characterize the obtained nanoparticles. We observed a 8-fold increase in red luminescence for samples suspended in DMSO solution for 2.5% of Mg2+ doping. The x-ray analysis shows that for the concentration of 2.5% Mg, the size of the crystallites in the NPs is the largest, which is mainly responsible for the increase in the intensity of the upconversion luminescence. But the addition of Li+ ions did not improve the luminescence of the upconversion due to decreasing of crystallites size of the NPs. Synthesized nanomaterials with very effective upconverting luminescence, can act as luminescent markers in in vivo imaging. The cytotoxicity of the nanoparticles was evaluated on the 4T1 cell line for the first time.

Potent in vitro antitumor activity of B-subunit of Shiga toxin conjugated to the diphtheria toxin against breast cancer

Immunotoxins are protein-based drugs consist of a target-specific binding domain and a cytotoxic domain to eliminate target cells. Such compounds are potentially therapeutic to combat diseases such as cancer. Generally, the B-subunit of Shiga toxin (STXB) receptor, globotriaosylceramide (Gb3), is expressed in high amounts on a number of human tumors cancer cells. In this study, we evaluated a new antitumor candidate called DT389-STXB chimeric protein, which genetically fused the DT to B-subunit of Shiga-like toxin (STXB).First a chimeric protein, encoding DT389-STXB was synthesized. The optimized chimeric protein expressed in E.coli BL21 (DE3) and confirmed by anti-His Western blot analysis. T47D, SKBR3, 4T1 and MCF7 cell lines were treated separately with purified DT389-STXB recombinant protein and functional activity of DT389-STXB was analyzed by the cell enzyme-linked immunosorbentassay (ELISA), MTT, ICC, Western blot and apoptosis tests.The results indicated that the recombinant DT389-STXB fusion protein with a molecular weight of 53 kDa was successfully expressed in E.coli BL21 (DE3) and the anti-His western-blot was used to confirm the presence of the protein. The DT389-STXB fusion protein attached to T47D, SKBR3 and 4T1 cell lines with the proper affinity and induced dose-dependent cytotoxicity against GB3-expressing cancer cells in vitro.Our results showed that DT389-STXB fusion protein may be a promising candidate for antitumor therapy agent against breast cancer; however, further studies are required to explore its efficacy in vivo for therapeutic applications.

Candida Albicans Activated Splenocytes Promote Strong Immune Responses in a Murine Model of Breast Cancer

Background: The potential of Candida albicans to modulate antigen-presenting cells maturation has been documented in past studies. Dendritic cells are critical modulators in the orchestration of adaptive immune responses alongside myeloid subtypes, which play an important role in the presentation of antigens to T cells. The aim of this study was to evaluate the efficacy of splenocytes activated with the extract of heated 4T1 cells and the yeast form of C. albicans against breast cancer growth in vivo. Methods: 4T1 cells were subcutaneously injected into the left flanks of female BALB/c mice (n=40). At a time when palpable tumors had developed, experimental groups were immunized twice at one-week interim with either activated splenocytes with the extract of heated 4T1 or the killed preparation of yeast form of C. albicans or a combination of the two-One week after the second injection, one-half of animals (n=20) were euthanized to investigate the immune response profile. Results: Administration of activated splenocytes with the combination protocol caused a favorable survival curve and slower rates of tumor development compared to other tumor-bearing mice. Moreover, combination therapy significantly increased the secretion of IFN-γ, respiratory burst and nitric oxide production and conversely diminished the secretion of IL-4, IL-10 and TGF-β in the splenocyte population. Conclusion: Since the murine 4T1 cell line is similar to the final stage of human breast carcinoma, we postulate that activated splenocytes with the extract of heated 4T1 cells and yeast form of C. albicans can reduce tumor development in tumor-bearing mice.

The Prolactin Inducible Protein Modulates Antitumor Immune Responses and Metastasis in a Mouse Model of Triple Negative Breast Cancer.

The prolactin inducible protein (PIP) is expressed to varying degrees in more than 90% of breast cancers (BCs). Although high levels of PIP expression in BC has been shown to correlate with better prognosis and patient response to chemotherapy, some studies suggest that PIP may also play a role in metastasis. Here, we investigated the role of PIP in BC using the well-established 4T1 and E0771 mouse BC cell lines. Stable expression of PIP in both cell lines did not significantly alter their proliferation, migration, and response to anticancer drugs in vitro compared to empty vector control. To assess the effect of PIP expression on breast tumorigenesis in vivo, the 4T1 syngeneic transplantable mouse model was utilized. In immunocompetent syngeneic BALB/c mice, PIP-expressing 4T1 primary tumors displayed delayed tumor onset and reduced tumor growth, and this was associated with higher percentages of natural killer cells and reduced percentages of type 2 T-helper cells in the tumor environment. The delayed tumor onset and growth were abrogated in immunodeficient mice, suggesting that PIP-mediated modulation of primary tumor growth involves an intact immune system. Paradoxically, we also observed that PIP expression was associated with a higher number of 4T1 colonies in the lungs in both the immunocompetent and immunodeficient mice. Gene expression analysis of PIP-expressing 4T1 cells (4T1-PIP) revealed that genes associated with tumor metastasis such as CCL7, MMP3 and MMP13, were significantly upregulated in 4T1-PIP cells when compared to the empty vector control (4T1-EV) cells. Collectively, these studies strongly suggest that PIP may possess a double-edge sword effect in BC, enhancing both antitumor immunity as well as metastasis.

Improving anti-tumour efficacy of PEGylated liposomal doxorubicin by dual targeting of tumour cells and tumour endothelial cells using anti-p32 CGKRK peptide

The aim of this study was to surface-functionalize PEGylated liposomal doxorubicin (PLD) using anti-p32 CGKRK peptide to evaluate its anti-angiogenic and anti-tumour activities. CGKRK was conjugated to DSPE-mPEG2000-maleimide and post-inserted into PLD at 25, 50, 100, 200 and 400 peptides per each liposome and characterised for their size, zeta potential, drug loading, release properties; and cell binding, cell uptake and cytotoxicity on three C26, 4T1 and human umbilical vein endothelial cell (HUVEC) cell lines. The in vitro results indicated the better efficiency of the PLD-100 (PLD with 100 CGKRK) formulation on 4T1 and HUVEC cell lines. The results of anti-tube formation and spheroid assay indicated the efficiencies of the PLD-100 formulation compared with Caelyx® in vitro. The in vivo studies indicated the higher tumour accumulation of PLD-100 formulation in comparison with Caelyx® which also implied the higher survival rates in mice treated with PLD-100 formulation. Histological evaluations demonstrated that PLD-100 had no side-effects on major organs. In conclusion, the results of this study indicated that PLD-CGKRK- could efficiently target endothelial and tumour parenchymal cells which enhance the therapeutic efficacy of PLD and merits further investigation.

Formulation and Biocompatibility of Microemulsion-Based PMBN as an Efficient System for Paclitaxel Delivery

Introduction: Paclitaxel (PTX), an effective chemotherapeutic drug, is widely used to treat several types of cancers. However, its use is limited due to poor water solubility resulted in poor bioavailability. One of the main ways to increase the efficacy and endurance of medications depends on the carrier that used for it. In the current study, the microemulsions (ME) were investigated based on Poly [2-methacryloyloxyethyl phosphorylcholine (MPC)-co-n-butyl methacrylate (BMA)-co-p-nitrophenyl-oxycrabonyl poly ethylene glycol-methacrylate (ME-ONP)] (PMBN) ability as a carrier for PTX to increase the half-life and its bioavailability. Also, the physicochemical properties of the ME system were evaluated. Materials and Methods: PMBN, tween 80, triacetin, and glycerol were used as the drug carrier, surfactant, oil phase, and co-surfactant, respectively. The hemolytic activity was characterized using the RBC hemolysis assay to evaluate the blood compatibility of the MEs. In addition, the in vitro cytotoxic effect of PMBN-PTX-ME and PTX on MCF-7, 4T1, and HFF-2 cell lines was performed. PI and Annexin-V dyes were used for cell apoptosis. Results: The conductivity of ME evaluated and the results showed 432 to 589 µS/cm. In vitro, the drug release study revealed that PTX has controlled release at different pH levels. Refractive Index (RI) and % transmittance of the MEs remained ranging from 1.43 to 1.53, and 89% to 98%, respectively. The MTT assay determined that PMBN-ME without PTX had no significant toxicity on HFF-2, MCF-7, and 4T-1 cell lines. Based on the apoptosis assay, treated cell lines with PMBN approximately remained alive. Conclusions: The results revealed that ME-based on PMBN would be a promising drug delivery system for PTX drug delivery.

Macrophages Mediate the Antitumor Effects of the Oncolytic Virus HSV1716 in Mammary Tumors.

Oncolytic viruses (OV) have been shown to activate the antitumor functions of specific immune cells like T cells. Here, we show OV can also reprogram tumor-associated macrophage (TAM) to a less immunosuppressive phenotype. Syngeneic, immunocompetent mouse models of primary breast cancer were established using PyMT-TS1, 4T1, and E0771 cell lines, and a metastatic model of breast cancer was established using the 4T1 cell line. Tumor growth and overall survival was assessed following intravenous administration of the OV, HSV1716 (a modified herpes simplex virus). Infiltration and function of various immune effector cells was assessed by NanoString, flow cytometry of dispersed tumors, and immunofluorescence analysis of tumor sections. HSV1716 administration led to marked tumor shrinkage in primary mammary tumors and a decrease in metastases. This was associated with a significant increase in the recruitment/activation of cytotoxic T cells, a reduction in the presence of regulatory T cells and the reprograming of TAMs towards a pro-inflammatory, less immunosuppressive phenotype. These findings were supported by in vitro data demonstrating that human monocyte-derived macrophages host HSV1716 replication, and that this led to immunogenic macrophage lysis. These events were dependent on macrophage expression of proliferating cell nuclear antigen (PCNA). Finally, the antitumor effect of OV was markedly diminished when TAMs were depleted using clodronate liposomes. Together, our results show that TAMs play an essential role in support of the tumoricidal effect of the OV, HSV1716-they both host viral replication via a novel, PCNA-dependent mechanism and are reprogramed to express a less immunosuppressive phenotype.

Smaller Copper Oxide Nanoparticles have More Biological Effects Versus Breast Cancer and Nosocomial Infections Bacteria.

Background and Objectives:Despite promising successes in developing new drugs and pharmaceutical biotechnology, infectious diseases and cancer are still the principal causes of mortality and morbidity globally. Therefore, finding effective ways to deal with these pathogens and cancers is critical. Metal nanoparticles are one of the new strategies to combat bacteria and cancers. Methods:We examined the antimicrobial activity of 30 and 60 nm copper oxide nanoparticles (CuO-NPs) against Acinetobacter baumannii and Staphylococcus epidermidis bacteria responsible for nosocomial infections in standard and clinical strains and anti-cancer activity against 4T1 cell line as malignancy breast cancer cells. Synthesis of CuO-NPs was performed by a one-step reduction method and confirmed by DLS and TEM microscopy at 30 and 60 nm sizes. The antibacterial and anti-cancer activities of the nanoparticles were then investigated against the aforementioned bacteria and breast cancer. Results:Using disk, well, MIC, MBC methods, and viability/bacterial growth assay, 30 nm CuO NPs were found to have more antibacterial activity on standard and clinical strains than 60 nm CuO NPs. On the other hand, using MTT, apoptosis, and gene expression method, 30 nm nanoparticles were found to have more anti-cancer potential than 60 nm CuO NPs. Conclusions:Our findings implicate CuO-NPs to possess antimicrobial and anti-cancer effects and more significant potential in smaller sizes, suggesting their pharmaceutical and biomedical capacity.

Fabrication and evaluation of aptamer-conjugated paclitaxel-loaded magnetic nanoparticles for targeted therapy on breast cancer cells.

Targeted drug delivery vehicles make it possible to deliver anti-cancer drugs to the cells or tissues of interest. Aptamers are peptide or oligonucleotide molecules that can serve as targeting elements of drug carriers. In the current study, we evaluated the capacity of an aptamer-based drug carrier to deliver Paclitaxel (PTX) to cancer cells. After being synthesized, SPIONs@PTX-SYL3C aptamer was characterized using different methods, including differential light scattering (DLS), infrared spectroscopy (FTIR), Transmission electron microscopy (TEM), X-ray diffraction (XRD), Thermal gravimetric analysis (TGA), and vibrating sample magnetometer (VSM). Encapsulation efficiency (EE) and loading efficiency (LE) were also evaluated. The carrier was applied on 4T1, MCF 7, and MCF-10A breast cell lines to evaluate its drug delivery potency and specificity. EE and LE were calculated to be 77.6% and 7.76%, respectively. MTT results revealed that aptameric SPIONs@PTX was more toxic than non-aptameric SPIONs@PTX. Flowcytometry analysis and DAPI staining confirmed that SPIONs@PTX-Aptamer had higher cell internalization rate when compared to non-targeted SPIONs@PTX. Our results indicate that aptamer-conjugated SPIONs@PTX has a good capacity in recognizing its target cells and inhibiting their growth and division.

Suicide gene therapy-mediated purine nucleoside phosphorylase/fludarabine system for in vitro breast cancer model with emphasis on evaluation of vascular endothelial growth factor promoter efficacy.

In this study, a suicide gene therapy approach was optimized by a non-viral polyplex system based on pEGFP-N1 vector harboring purine nucleoside phosphorylase gene conducted by vascular endothelial growth factor promoter for an in vitro breast cancer model (4T1 cell line). The VEGF promoter and purine nucleoside phosphorylase gene were cloned into the vector from the source of 4T1 and E. coli genomic DNA, respectively. A gene construct was developed by replacing VEGF promoter instead of CMV promoter in pEGFP-N1vector. PNP gene was integrated in to the multiple cloning site of the obtained vector. On the other hand, a construct from pEGFP-N1 harboring PNP gene under the control of the original CMV promoter was developed. The transfection method using cationic polymer was optimized based on N/P ratio, cell cytotoxicity, polyplex size, zeta potential and the green fluorescent protein (GFP) expression by fluorescent microscopy and flowcytometry. Also, the effect of hypoxia condition induced by 0.5mM H2O2 on the promoter efficiency was investigated. The results showed that the performed gene delivery system is capable of the gene transfection to more than 30% of the cancer cells with both VEGF-PNP-pEGFP-N1 and PNP-pEGFP-N1 plasmids. The hypoxia condition did not show a significant effect on the VEGF promoter. But, it revealed that bystander effect can improve the efficacy of this system and reduce drug IC50 to 2 and fourfold for plasmids VEGF-PNP-pEGFP-N1 and PNP-pEGFP-N1, respectively. These results showed that the bystander effect could almost compensate the low efficiency of non-viral gene delivery systems. We suggest that the tumor-specific gene expression system mediated by the VEGF promoter can be especially useful in the present model of breast cancer gene therapy.

Synthesis, biological evaluation and molecular docking studies of indeno [1, 2-c] pyrazol derivatives as inhibitors of mitochondrial malate dehydrogenase 2 (MDH2)

Hypoxia inducible factor-1 (HIF-1) is a pivotal transcription factor, which is strongly correlated with the induction of angiogenesis, tumor survival, metastasis, and cell proliferation, making it a pivotal therapeutic target for solid tumor therapeutic agents. Herein, a new series of multi-functional chemical probes were designed including principal groups, viz. adamantyl and indene, at various locations of the parent compound LW6. Molecular docking studies were performed on the designed compounds and their relationship with HIF-1α and malate dehydrogenase 2 (MDH2). Inhibition of MDH2 by our compounds was expected to decrease the NADH level. Indeed, treatment of the breast cancer cell line 4T1 led to a strong reduction of the NADH concentration. The greatest reduction in NADH production in mitochondria was observed with (E)-3-(4-((3r, 5r, 7r)-adamantan-1-yl) phenoxy)-N-(5-(piperidine-1-carbonyl)-1, 4-dihydroindeno [1, 2-c] pyrazol-3-yl) acrylamide (18: IC50 = 59 nM), and has the best inhibitory potential under hypoxic conditions (MCF-7: IC50 = 57 nM). This compound also gave one of the highest docking “higher than the score obtained with LW6 in parallel (−31.63 kcal/mol) in the initial docking runs (PDB Code: 4WLO). Other related compounds with good yields were also synthesized from docking results, and all the synthesized compounds (14, 18, 22, 26, 29, 30) were evaluated in vitro on human adenocarcinoma cell lines.

Abstract PS13-28: Pre-treating TNBC with docetaxel and il-12 enhances anti-PD1 efficacy

Abstract Introduction: Amongst all breast cancers, Triple Negative Breast Cancer (TNBC) account for 15-20% of all the cases. TNBC affect younger patients, and is more prevalent in African-American women. The poor prognosis for this very aggressive tumor subtype is exacerbated by the lack of specific targeted therapy against the disease. Although TNBC initially respond very well to chemotherapy, paradoxically the disease-free survival is very short. It has been showed that TNBC have higher rates of CD8+ T-cells infiltration, and express high level of PD-L1. Together, these data provide a strong rationale for the combination of chemotherapy and immunotherapy to treat TNBC patients. In this study, we investigated the response to pre-priming the tumor with one round of docetaxel and IL-12, followed by anti-PD1 maintenance in mouse E0771 and 4T1 TNBC syngeneic models. We hypothesized that docetaxel will promote the release of neo-antigens, while IL-12 will activate immunity specific to these antigens and anti-PD1 therapy prevent the exhaustion of those T-cells. Materials/Methods: Mouse TNBC E0771 and 4T1 cell lines were injected in the mammary fat pad of C57BL/6, and Balb/c mice respectively. On day 1, the mice received a single dose (20mg/kg) of docetaxel and one intratumoral injection (1.25x109) of mAdv.IL-12, a replication defective adenoviral vector containing mIL-12 (mouse) cDNA under the transcriptional control of Rous sarcoma virus long terminal repeat (provided by Dr. Chen, HMRI). Anti-PD1 (InVivoMab anti-mouse PD-1 CD279) was administered 3 times a week (2 cycles) starting 5 days post docetaxel and Il-12 treatment. At the end of the study, IFN-gamma levels were measured from blood and tumor samples; tumor sizes were compared between treatment groups (Control/mAdv.IL-12/anti-PD1/and various Combination), as well as survival curves. The metastatic burden to the lungs (H&amp;E), as well as the apoptosis in the tumor (TUNNEL) were assessed by IHC. Results: In both 4T1 and E0771 tumor models, triple combination of docetaxel + IL-12 followed by anti-PD1 significantly reduced tumor size compared to both single agents, and double combination. In the Triple Combo group, 1 mice had lung metastasis vs all of them in the other treatment groups. IHC data indicate a higher level of TILs in the treatment groups, with a statistically significant difference in the combination groups compared to single agents. There was more apoptosis in the triple combo group as indicated by TUNNEL. Conclusion:Our preliminary data strongly supports that treating TNBC models with docetaxel and mAdv.IL-12 followed by anti-PD1 significantly slows down tumor growth, and decrease lung metastasis incidence. We are actively investigating the mechanism through which the response is achieved. Citation Format: Ann Cassany Anselme, Wei Qian, Jianying Zhou, Roberto R. Rosato, Jenny C. Chang. Pre-treating TNBC with docetaxel and il-12 enhances anti-PD1 efficacy [abstract]. In: Proceedings of the 2020 San Antonio Breast Cancer Virtual Symposium; 2020 Dec 8-11; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2021;81(4 Suppl):Abstract nr PS13-28.

Antioxidant, Anti-Inflammatory, and Cytotoxic Properties and Chemical Compositions of Filipendula palmata (Pall.) Maxim.

Filipendula palmata (Pall.) Maxim. remains unexplored and underutilized resources with a high potential to improve human health. In this study, a new ursane-type triterpenoid, namely, 2α, 3β-dihydroxyurs-12-en-28-aldehyde (compound 10), and other 23 known compounds were isolated. 5 triterpenoids (compounds 6, 8, and 10–12), 11 flavonoids (compounds 13–15 and 17–24), 6 phenolic compounds (compounds 1, 2, 4, 5, 9, and 16), 2 sterols (compounds 3 and 7) were isolated from the aqueous solution extract of the aerial parts of F. palmata. The structures of all compounds were elucidated by the use of extensive spectroscopic methods such as infrared spectroscopy (IR), high-resolution electrospray ionization mass spectrometry (HR-ESI-MS), 1H-NMR, and 13C-NMR. The solvent extractions of ethyl acetate fraction were evaluated for antioxidant activities using DPPH (2, 2-diphenyl-1-picrylhydrazyl) and ABTS+ (2, 2′-azino-bis (3-ethylbenzothiazoline-6-sulfonic acid)) methods. The anti-inflammatory effects of the compounds were evaluated in lipopolysaccharide- (LPS-) stimulated RAW 264.7 macrophages. The extract cytotoxicity on the cancer cell lines MCF-7, HeLa, 4T1, and A549 was determined by MTT assay. As a result, compounds 10, 11, and 12 exhibited better antioxidant activity compared to the other compounds. Compounds 8–24 had different inhibitory effects on the release of NO, TNF-α, and IL-6 in LPS-stimulated RAW 264.7 cells. The new compound has shown a significant inhibiting effect on cancer cells, and the cell inhibition rate increased in a dose-dependent manner. Further research to elucidate the chemical compositions and pharmacological effects of F. palmata is of major importance towards the development and foundation of clinical application of the species.

Combination Therapy of Breast Cancer by Codelivery of Doxorubicin and Survivin siRNA Using Polyethylenimine Modified Silk Fibroin Nanoparticles.

Here, polyethylenimine (PEI) modified silk fibroin nanoparticles (SFNPs) were prepared for codelivery of doxorubicin (DOX) and survivin siRNA. The prepared NPs were characterized in terms of stability and structural, functional, and physicochemical properties. Moreover, the ability of the conjugate to escape from the endosome and cellular uptake were assessed. Afterward, the in vivo therapeutic efficacy was analyzed in the mice model. The siRNA loaded PEI-SFNPs showed acceptable size, zeta potential, and stability in serum. It also effectively induced apoptosis in the 4T1 mouse mammary tumor cell line. Cellular uptake and endosomal escape analyses confirmed that PEI-SFNPs containing siRNA could escape from the endosome and accumulate in the cytoplasm of 4T1 cells. Real time-PCR indicated the significant decrease in the expression of survivin mRNA in the 4T1 cell line 48 h postincubation with siRNA loaded PEI-SFNPs. In vivo biodistribution of PEI-SFNPs confirmed higher accumulation of SFNPs in the tumor site compared with other organs. The codelivery systems remarkably reduced the growth rate of breast tumor in the mice model without any obvious weight lost. Histopathological and tunnel staining exhibited more apoptotic tumor cells in the group containing both DOX and survivin siRNA. Tumorigenic breast tissue resected from the animals after treatment with siRNA also exhibited significant suppression of survivin gene. In conclusion, the prepared drug delivery system had an acceptable potential in tumor removal, apoptosis induction in cancer cells, and therapeutic efficacy. Thus, it would be a good candidate for breast cancer therapy.

Zingerone improves the immune responses in an animal model of breast cancer.

The potent anti-tumorigenic effects were attributed to ginger and there are some reports regarding the anti-cancer and immunomodulatory properties ginger-derived components. This study aimed to investigate the effects of zingerone on some immune-related parameters in an animal model of breast cancer. The breast cancer was established in female BALB/c mice using a carcinogenic 4T1 cell line. At day 10 after cancer induction, tumor-bearing mice were divided into five groups and treated intraperitoneal (daily from days 11-30) with saline or zingerone (at doses 10, 20, 50 and 100mg/kg/day). The mice were sacrificed on day 31 and the number of splenic Th1- and Treg cells, the expression of IFN-γ and TGF-β in the blood mononuclear cells, the antibody production against sheep red blood cell (SRBC) were determined using flow cytometry, real time-PCR and a standard hemagglutination assay, respectively. Zingerone at doses 50 and 100mg/kg enhanced the number of splenic Th1 cells (p<0.03 and 0.007, respectively); at doses 10, 20, 50 and 100mg/kg reduced the number of splenic Treg cells (p<0.02, 0.01, and 0.01, respectively), at doses 50 and 100mg/kg enhanced the expression of IFN-γ (p<0.03), at doses 50 and 100mg/kg reduced the expression of TGF-β, at doses 50mg/kg reduced the titer of anti-SRBC antibody (p<0.05). Zingerone improve the T cell-mediated and antibody responses in a mouse model of breast cancer. Theimmunotherapeutic potentials of zingerone in cancers need more considerations.

Abstract PO065: Single cell transcriptomics of triple negative breast cancer allografts following chemotherapy treatment reveals increased T cell abundance in doxorubicin-sensitive tumors

Abstract The contribution of stromal cells on drug response in primary tumors remains unclear. To determine how individual cells within the stroma of triple negative breast cancer (TNBC) allografts respond to chemotherapy, we used single cell sequencing to profile cells present in murine tumors with or without exposure to doxorubicin. Doxorubicin is a chemotherapeutic agent commonly used to treat TNBC by inhibiting cancer cell proliferation through intercalation of DNA and preventing topoisomerase II activity. In this study, murine TNBC 4T1 cell line was utilized to generate allograft tumors in immunocompetent BALB/c mice. Syngeneic 4T1 tumors exhibit a range of responsiveness to doxorubicin treatment. Tumor growth rates were monitored throughout the chemotherapeutic regiment then stratified into sensitive or resistant response. Cellular composition and behavior were then analyzed via single cell RNA sequencing (scRNA-seq) and flow cytometry 8 days post-doxorubicin chemotherapeutic administration mimicking clinical treatment. ScRNA-seq revealed decreases in tumor infiltrating lymphocytes after doxorubicin exposure. Furthermore, an increase in T cell abundance was discovered in tumors sensitive to doxorubicin treatment. This finding was further supported by flow cytometric analysis showing a tumor specific increase in all, CD4+, and CD8+ T cell populations relative to resistant tumors. Additionally, T-cell differentiation, exhaustion, and activation states were further examined from scRNA-seq data providing insights into functional properties of the tumor residing T cell populations undergoing chemotherapeutic treatment. Future work will focus on analyzing prognostic value of specific T-cell populations in disease regression following doxorubicin treatment. This study received funding from LLNL LDRD grant 19-SI-003. This work was conducted under the auspices of the USDOE by LLNL (DE-AC52-07NA27344). Citation Format: Nicholas R. Hum, Aimy Sebastian, Sean F. Gilmore, David M. Gravano, Naiomy D. Rios-Arce, Kelly A. Martin, Elizabeth K. Wheeler, Matthew A. Coleman, Gabriela G. Loots. Single cell transcriptomics of triple negative breast cancer allografts following chemotherapy treatment reveals increased T cell abundance in doxorubicin-sensitive tumors [abstract]. In: Abstracts: AACR Virtual Special Conference: Tumor Immunology and Immunotherapy; 2020 Oct 19-20. Philadelphia (PA): AACR; Cancer Immunol Res 2021;9(2 Suppl):Abstract nr PO065.