4T1 Murine Breast Cancer Research Articles

Intermittent fasting suppressed splenic CD205+ G-MDSC accumulation in a murine breast cancer model by attenuating cell trafficking and inducing apoptosis.

Immune‐based interventions are the most promising approach for new cancer treatments to achieve long‐term cancer‐free survival. However, the expansion of myeloid‐derived suppression cells (MDSCs) attenuates the therapeutic potential of immunotherapy. We recently showed that CD205+ granulocytic MDSCs (G‐MDSCs), but not T cells, are sensitive to glucose deficiency. Intermittent fasting (IF) may inhibit the growth of malignant cells by reducing serum glucose levels, but little is known regarding the influence of IF on MDSC expansion. Herein, we observed that IF selectively inhibited splenic accumulation of CD205+ G‐MDSCs in a 4T1 and 4T07 transplant murine breast cancer model. The efficiency of IF in suppressing tumor growth was comparable to that of docetaxel. Further examination revealed that CXCR4 expression was concentrated in CD205+ subsets of tumor‐induced G‐MDSCs. Downregulation of CXCR4 correlated with a reduction in CD205+ G‐MDSC trafficking from bone marrow to the spleen under IF treatment. In addition, ex vivo culture assays showed that glucose deficiency and 2‐deoxy‐D‐glucose (2DG) treatment selectively induced massive death of splenic CD205+ G‐MDSCs. Interestingly, 2DG emulated the phenomena of IF selectively suppressing the accumulation of CD205+ G‐MDSCs in the spleen, upregulating cleaved caspase 3 in the tumor, downregulating Ki67 in the lung, and retarding the growth of transplanted 4T1 and 4T07 murine breast tumors. These findings suggest that IF inhibited cell trafficking through the downregulation of CXCR4 and induced apoptosis by altering glucose metabolism; this, suppressed the accumulation of tumor‐induced splenic CD205+ G‐MDSCs and in turn enhanced antitumor immunity.

Submicron particle docetaxel intratumoral injection in combination with anti-mCTLA-4 into 4T1-Luc orthotopic implants reduces primary tumor and metastatic pulmonary lesions

We describe here characterization of the response of local and metastatic disease and immunomodulation following intratumoral (IT) injection of submicron particle docetaxel (SPD) administered alone or in combination with systemic antibody anti-mCTLA-4 (anti-mCTLA-4) in the metastatic 4T1-Luc2-1A4 (4T1) murine breast cancer model. In-life assessments of treatment tolerance, tumor volume (TV), and metastasis were performed (n = 10 animals/group). At study end, immune cell populations in tumor-site tissues and peripheral blood were analyzed using flow cytometry. Signs of distress typical of this aggressive tumor model occurred across all animals except for the combination treated which were asymptomatic and gained weight. TV at study end was significantly reduced in the combination group versus untreated [43% reduced (p < 0.05)] and vehicle controls [54% reduced (p < 0.0001)]. No evidence of thoracic metastasis was found in 40% of combination group animals and thoracic bioluminescence imaging (BLI) was reduced vs. untreated controls (p < 0.01). Significant elevations (p < 0.05) in CD4 + T, CD4 + helper T, Treg, and NKT cells were found in tumor and blood in SPD or combination treatment compared to controls or anti-mCTLA-4. Combination treatment increased tumor-associated CD8 + T cells (p < 0.01), peripheral B cells (p < 0.01), and tumor associated and circulating dendritic cells (DC) (p < 0.05). Tumor-associated NK cells were significantly increased in SPD ± anti-mCTLA-4 treatments (p < 0.01). Myeloid-derived suppressor cells (MDSC) were reduced in bloods in SPD ± anti-mCTLA-4 groups (p < 0.05). These data demonstrate that both SPD and anti-mCTLA-4 produce local anti-tumor effects as well as reductions in metastasis which are significantly enhanced when administered in combination.

Positron Emission Tomography Imaging of Autotaxin in Thyroid and Breast Cancer Models Using [18F]PRIMATX.

Autotaxin (ATX) is a secreted enzyme responsible for producing lysophosphatidic acid (LPA). The ATX/LPA signaling axis is typically activated in wound healing and tissue repair processes. The ATX/LPA axis is highjacked and upregulated in the progression and persistence of several chronic inflammatory diseases, including cancer. As ATX inhibitors are now progressing to clinical testing, innovative diagnostic tools such as positron emission tomography (PET) are needed to measure ATX expression in vivo accurately. The radiotracer, [18F]PRIMATX, was recently developed and tested for PET imaging of ATX in vivo in a murine melanoma model. The goal of the present work was to further validate [18F]PRIMATX as a PET imaging agent by analyzing its in vivo metabolic stability and suitability for PET imaging of ATX in models of human 8305C thyroid tumor and murine 4T1 breast cancer. [18F]PRIMATX displayed favorable metabolic stability in vivo (65% of intact radiotracer after 60 min p.i.) and provided sufficient tumor uptake profiles in both tumor models. Radiotracer uptake could be blocked by 8-12% in 8305C thyroid tumors in the presence of ATX inhibitor AE-32-NZ70 as determined by PET and ex vivo biodistribution analyses. [18F]PRIMATX also showed high brain uptake, which was reduced by 50% through the administration of ATX inhibitor AE-32-NZ70. [18F]PRIMATX is a suitable radiotracer for PET imaging of ATX in the brain and peripheral tumor tissues.

Nuclear Aurora kinase A triggers programmed death-ligand 1-mediated immune suppression by activating MYC transcription in triple-negative breast cancer.

BackgroundIncreasing studies have reported that oncogenes regulate components of the immune system, suggesting that this is a mechanism for tumorigenesis. Aurora kinase A (AURKA), a serine/threonine kinase, is involved in cell mitosis and is essential for tumor cell proliferation, metastasis, and drug resistance. However, the mechanism by which AURKA is involved in immune response regulation is unclear. Therefore, this study aimed to investigate the role of AURKA in immune regulation in triple‐negative breast cancer (TNBC).MethodsPeripheral blood mononuclear cells (PBMCs) were co‐cultured with TNBC cells. The xCELLigence Real‐Time Cell Analyzer‐MP system was used to detect the killing efficiency of immune cells on TNBC cells. The expression of immune effector molecules was tested by quantitative real‐time polymerase chain reaction (qRT‐PCR) to evaluate immune function. Furthermore, to validate AURKA‐regulated immune response in vivo, 4T1 murine breast cancer cell line with AURKA overexpression or downregulation was engrafted into BALB/c mice. The distribution and proportion of immune cells in tumors were further evaluated by immunohistochemistry and flow cytometry.ResultsDownregulation of AURKA in TNBC cells increased immune response by activating CD8+ T cell proliferation and activity. Nuclear rather than cytoplasmic AURKA‐derived programmed death‐ligand 1 (PD‐L1) expression was independent of its kinase activity. Mechanistic investigations showed that nuclear AURKA increased PD‐L1 expression via an MYC‐dependent pathway. PD‐L1 overexpression mostly reversed AURKA silencing‐induced expression of immune effector molecules, including interleukin‐ (IL‐2), interferon‐γ (IFN‐γ), and perforin. Moreover, AURKA expression was negatively correlated with the enrichment and activity of tumor‐infiltrating CD8+ T cells in 4T1 engrafted BALB/c mouse model.ConclusionsNuclear AURKA elevated PD‐L1 expression via an MYC‐dependent pathway and contributed to immune evasion in TNBC. Therapies targeting nuclear AURKA may restore immune responses against tumors.

Comparative colloidal stability, antitumor efficacy, and immunosuppressive effect of commercial paclitaxel nanoformulations

BackgroundStandard chemotherapy with taxanes, such as paclitaxel (PTX), remains the mainstay of systemic treatment of triple-negative breast cancer. Nanotechnology-based formulations have gradually replaced PTX injection and are widely used in China. However, no studies have compared the colloidal stability, antitumor efficacy, and safety of commercial PTX nanoformulations. Additionally, the desire to evaluate preclinical antitumor efficacy in human-derived tumor cells led to the widespread application of immunodeficient mouse models that likely contributed to the neglect of nanomedicines-immune system interactions. The present study investigated the colloidal stability, antitumor efficacy and safety, and nanomedicines-host immune system interactions of PTX nanoformulations. A further comparative analysis was performed to evaluate the clinical potential.ResultsCompared with liposome, PTX emulsion and PTX nanoparticle exhibited favorable colloidal stability. PTX emulsion was superior in inducing apoptosis and had a more pronounced inhibitory effect on 4T1-tumor spheroids compared with PTX liposome and PTX nanoparticle. Although PTX emulsion exhibited superior in vitro antitumor effect, no significant differences in the in vivo antitumor efficacy were found among the three types of PTX nanoformulations in an immunocompetent orthotopic 4T1 murine triple-negative breast cancer model. All PTX nanoformulations at maximum tolerated dose (MTD) induced lymphopenia and immunosuppression, as evidenced by the reduction of T cell subpopulations and inhibition of the dendritic cells maturation.ConclusionsThe MTD PTX nanomedicines-induced lymphopenia and immunosuppression may weaken the lymphocyte-mediated antitumor cellular immune response and partly account for the lack of differences in the in vivo antitumor outcomes of PTX nanoformulations. Understanding of what impacts PTX nanomedicines has on the immune system may be critical to improve the design and conduct of translational research of PTX nanomedicines in monotherapy or combination therapy with immunotherapy.Graphic abstract

Abstract 72: Bifidobacterium longum RAPO enhances efficacy of anti-PD-1 immunotherapy in a mouse model of triple-negative breast cancer

Abstract Background: Triple negative breast cancer (TNBC) represents approximately 15-20% of all breast cancers and 35% of breast cancer death. TNBC has a highly aggressive behavior with the increased risk of early metastasis and relapse and poor survival rate. Given that TNBC is associated with higher levels of tumor-infiltrating lymphocytes and programmed cell death ligand 1 (PD-L1), immune checkpoint inhibitors (ICI) are considered as the appropriate treatment of patients with TNBC. However, it has been reported that ICI monotherapy is less effective compared with combination therapy. Recent several studies suggest that gut microbiome plays a critical role in the efficacy of ICI therapy. Thus, we investigated the effects of Bifidobacterium longum RAPO supplementation on anti-PD-1 therapy against TNBC and the underlying mechanisms in vivo. Methods: The 4T1 murine metastatic breast cancer cells were injected s.c into the flank of female BALB/c mice. When tumors reached 50-150 mm3, mice were randomly assigned to one of four groups; tumor control (TC), Anti-PD-1, B. longum RAPO (RAPO), or Anti-PD-1+RAPO (n=10 per group). Anti-PD-1 antibody was injected i.p. 5 times at 3 day intervals. B. longum RAPO was orally administered daily from 2 days before the first injection of anti-PD-1. Spleen and tumor tissues were analyzed by flow cytometry, qRT-PCR, and immunohistochemistry, and fecal samples were collected for 16S rRNA gene sequencing. Results: On day 12, tumor volume was significantly inhibited in Anti-PD-1+RAPO group compared with TC and Anti-PD-1 groups. Higher levels of PD-L1 IHC score and PD-L1 mRNA expression were observed in tumor tissues of Anti-PD-1+RAPO group. Anti-PD-1+RAPO group was also associated with the increased levels of spleen NKT cells and tumor NK cells. The proportion of pro-tumor M2 macrophages and M2/M1 ratio were significantly decreased in tumor tissues of Anti-PD-1+RAPO group than those of Anti-PD-1 group. Similarly, combination therapy of Anti-PD-1 with RAPO significantly reduced mRNA expression of M2 markers such as IL10 and Arg1, while it augmented that of anti-tumor cytokines including IFNγ and TNFα compared with Anti-PD-1 treatment alone. In addition, the relative abundance of the genus Bifidobacterium was remarkably enriched in Anti-PD-1+RAPO group compared with other groups. In Anti-PD-1+RAPO group, the relative abundance of the genus Akkermansia and Lachnospiraceae NK4A136 group were significantly increased after administration for 2 weeks. Conclusions: Our data suggests that combination therapy of B. longum RAPO with anti-PD-1 modulates anti-tumor immunity and gut microbiota, contributing to the better response to anti-PD-1 immunotherapy in mice with TNBC. This study provides evidence that B. longum RAPO might be a potential candidate for immunomodulatory pharmabiotics in cancer immunotherapy. Citation Format: Hyeyoon Kim, Rira Oh, Sangjun Park, Geun Eog Ji, Myeong Soo Park, Sung-Eun Kim. Bifidobacterium longum RAPO enhances efficacy of anti-PD-1 immunotherapy in a mouse model of triple-negative breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 72.

Abstract 1696: Anti-tumor effects from simultaneous cancer and TAM targeting by 010DS-Zn

Abstract Direct clearance of immune-suppressive Tumor Associated Macrophages (TAM) is a less-explored cancer therapeutic approach. We developed a novel folate-ligand guided zinc-peptide complex 010DS-Zn for selective induction of parthanatos in folate receptor overexpressing targets that include many solid and blood cancer types, in addition to M2-like macrophages. Consistent with known parthanatos mechanism, its in vitro mode-of-action (MOA) tested against 4T1 murine breast cancer cell was characterized by specific and rapid PARP-dependent AIF translocation into nucleus that executed massive DNA fragmentation. This in vitro/ex vivo cytotoxicity was found highly consistent against the 90 blood and solid patient-derived cancer types tested. Further in vivo MOA studies against aPD-1 responder and non-responder syngenic cancer models showed 010DS-Zn to be effective against both, albeit at increased dosage in the MDSC enriched tumors. In the aPD-1 responder cancer model CT26, 010DS-Zn demonstrated robust T-cell immune response initiation, which was enhanced upon co-treatment with aPD-1 mAb. Regardless of the tumor-response in vivo, 010DS-Zn treatment produced robust TAM-reduction activity in dose-dependent manners. Collectively, 010DS-Zn presents new cancer immunotherapeutic potentials for enabling previously inaccessible 2-combination therapies for targeting 3 layers of immune-evasion: Tregs, TAM, and MDSC. Furthermore, its ability to directly clear M2-like macrophages presents opportunities-worth-investigation for treating the M2-like macrophage-driven severe SARS-CoV-2 autoimmune syndromes or its post-recovery heart-damages. Citation Format: Jinhyuk Fred Chung. Anti-tumor effects from simultaneous cancer and TAM targeting by 010DS-Zn [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1696.

Co-delivery of Pirarubicin and Vinorelbine by Micelles for the Treatment of Breast Cancer

To develop a pirarubicin (THP) and vinorelbine (VRL) codelivery nano-micellar system (T+V-CS micelles) of pirarubicin (THP) and vinorelbine (VRL) by using chondroitin sulfate-cholesterol polymers (CS-Chol) and DSPE-mPEG 2000 and to evaluate the therapeutic efficacy of the codelivery nano-micelles in breast cancer treatment. T+V-CS micelles were prepared by ultrasonic-dialysis method, and the physicochemical characterization were evaluated using multiple technological means. The anti-tumor efficacy of T+V-CS micelles in vitro was evaluated by MTT assay and cell cycle arrest analysis. Evaluation of the therapeutic effect of T+V-CS micelles in vivo was carried out on xenograft 4T1 murine breast cancer bearing BALB/c mice model. T+V-CS micelles displayed a nearly spherical shape when observed through transmission electron microscope. The particle size and polydispersity indexes (PDI) of T+V-CS micelles was (155.5±4.5) nm and 0.170±0.003 respectively, while the Zeta potential was (-23.0±0.9) mV. Meanwhile, T+V-CS micelles demonstrated high encapsulation efficiency of (81.87±2.56)% for THP and (87.54±2.82)% for VRL and a high overall drug loading efficiency of (10.20±1.20)%. In vitro and in vivo studies of the therapeutic efficacy of breast cancer showed that T+V-CS micelles had synergistic anti-tumor effect and induced increased G 2/M cell cycle arrest in 4T1 cells, which could significantly inhibit tumor growth and prolong survival compared with the therapeutic efficacy of micelles loaded with a single kind of drug or free drug solutions. The study showed that T+V-CS micelles had excellent anti-tumor effect, offering a reference to the clinical treatment of breast cancer.

Abstract 2810: Effect of clone dependent difference in the metastatic tumor microenvironment of 4T1 breast cancer on anti-PDL1 antibody delivery &amp; therapeutic efficacy

Abstract Heterogeneities in the response of tumors to immunotherapies have significantly limited clinical benefits. Understanding the specific driving forces behind the heterogeneities will facilitate a better understanding of therapeutic resistance. Research has revealed that tumors in patients and established cancer cell lines are composed of multiple clones. Therefore, we established single cell-derived clonal populations from parental, polyclonal 4T1 murine breast cancer cells. Our goal is to dissect these complexities and decipher mechanisms of tumor heterogeneities. We injected 4T1 parental, clone1, or clone 16 cells into the spleen to create multiple liver metastases. Tumor-bearing mice were treated with anti-PDL1 Ab or isotype control Ab, then the therapeutic effect on tumor size and survival were evaluated. In another set of mice, the window chamber was installed above the liver to image the tumors utilizing intravital microscopy. Also, fluorescently labeled anti-PDL1 Ab or control Ab was injected intravenously (iv) to image the accumulation of Ab into the individual tumor. Anti-PDL1 Ab therapy significantly inhibited clone 16 tumor growth and extended survival of the tumor-bearing mice compared to isotype control Ab. On the other hand, parental and clone 1 tumors did not respond to the anti-PDL1 Ab. Using IVM, we monitored individual tumor response to anti-PLD1 Ab for 7 days and correlated the response with the amount of fluorescently labeled anti-PDL1 Ab delivered to the tumor. The amount of iv injected anti-PDL1 Ab delivery was heterogeneous among the tumors originated from 4T1 parental cells. We found an inverse correlation between the amount of the Ab delivered to the tumor and tumor growth, indicating drug delivery dependent tumor response. Metastases originated from clone 16 cells accumulated more amount of the Ab with most tumors disappeared. While the amount of Ab delivered to clone 1 tumor was significantly lower than parental and clone 16 tumors, none of the clone 1 tumors responded. Thus, limited therapeutic efficacy can be attributed to the insufficient anti-PDL1 Ab delivery to the tumor. Immunohistochemical analysis of resected liver revealed that the expression of PDL1 in clone 16 tumors was significantly higher than parental and clone 1 tumor. On the other hand. The amount of immune cells (CD8 &amp; F4/80) were slightly higher in clone 16 than parental and clone 1 tumors. Finally, we compared the amount of coagulation inside the tumor by imaging fibrinogen, which can reduce therapeutics delivery to tumors. Interestingly, the clone 1 tumor showed significantly more amount of coagulation. Evaluation of heterogeneity in PDL1 expression, tumor coagulation, and the immune microenvironment in the patient's tumor shall be considered for personalizing the therapy. Citation Format: Yan Ting Liu, Thao Nguyen, Shreya Goel, Megumi Kai, Haifa Shen, Kenji Yokoi. Effect of clone dependent difference in the metastatic tumor microenvironment of 4T1 breast cancer on anti-PDL1 antibody delivery &amp; therapeutic efficacy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2810.

Non-viral Suicide Gene Therapy: Cytosine Deaminase Gene Directed by VEGF Promoter and 5-fluorocytosine as a Gene Directed Enzyme/prodrug System in Breast Cancer Model.

The present study investigated the potential of vascular endothelial growth factor (VEGF) promoter to derive cytosine deaminase (CD) transfected by polyamidoamine (G4-PAMAM) dendrimers to 4T1 murine breast cancer cell line as gene-directed enzyme/prodrug therapy. The VEGF promoter and cytosine deaminase gene were cloned into the pEGFP-N1vector from the genomic DNA of 4T1 and E. coli, respectively. The frequency of transfection for VEGF-CD-pEGFP-N1 and pEGFP-N1- CD treated groups was 35±3 and 36±4, respectively. MTT assay was perform to evaluate the cytotoxic effects of converted 5-flurocytosine on 4T1 cells. Also, the optimal concentration of 5-FC in 4T1 cells transfected by VEGF-CD-pEGFP-N1 plasmid was evaluated. The GFP expression of transfected 4T1 cells by VEGF-CD-pEGFP-N1were observed by fluorescent microscopy and flowcytometry. Results demonstrated that the suicide CD gene was successfully expressed in 4T1 cells determined by RT-PCR and GFP expression. A concentration of 200 μg/ml 5-FC was identified as optimal dose of prodrug. Furthermore, the CD/5-FC enzyme/prodrug system not only demonstrated toxicity on transformed 4T1 cells but also exerted a 'bystander effect' determined by MTT assay. The results showed that by 35% transfection with VEGF-CD-pEGFP-N1and CD-pEGFP-N1 plasmids, 80% and 90% inhibition of the cells growth occurred, respectively.

The Effect of Lidocaine and Bosutinib on 4T1 Murine Breast Cancer Cell Behaviour In Vitro.

Systemic lidocaine has recently emerged as a promising agent possessing numerous potentially anti-neoplastic effects. In vitro studies suggest that lidocaine may prevent metastasis by acting on the tyrosine kinase enzyme Src. Intravenous lidocaine has been reported to reduce pulmonary metastasis in vivo in a murine breast cancer model, however the beneficial effect is abolished by the Src inhibitor bosutinib. In this study we examined whether lidocaine and/or bosutinib affects 4T1 breast cancer cell activity in vitro and whether any drug interactions similar to that seen in murine models occur. 4T1 murine breast cancer cells were exposed to lidocaine and/or bosutinib. Cell viability after 1 h of exposure was measured using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. Cell migration after 24 h of exposure was measured using the Oris™ migration assay. Lidocaine and bosutinib alone or combined inhibited 4T1 cell viability and migration, but only at supratherapeutic concentrations. Bosutinib did not modulate lidocaine's effect on viability or migration at any concentration tested. Although lidocaine may inhibit 4T1 metastasis in vivo, a direct effect on 4T1 cells is not detectable in vitro at non-toxic concentrations and unlike murine model testing, no unusual interaction with bosutinib was detected. Lidocaine's anti-metastatic properties are likely to be complex and multifactorial and difficult to replicate outside of a biological host.

GSH-sensitive polymeric prodrug: Synthesis and loading with photosensitizers as nanoscale chemo-photodynamic anti-cancer nanomedicine

Precisely delivering combinational therapeutic agents has become a crucial challenge for anti-tumor treatment. In this study, a novel redox-responsive polymeric prodrug (molecular weight, MW: 93.5 kDa) was produced by reversible addition–fragmentation chain transfer (RAFT) polymerization. The amphiphilic block polymer-doxorubicin (DOX) prodrug was employed to deliver a hydrophobic photosensitizer (PS), chlorin e6 (Ce6), and the as-prepared nanoscale system [NPs(Ce6)] was investigated as a chemo-photodynamic anti-cancer agent. The glutathione (GSH)-cleavable disulfide bond was inserted into the backbone of the polymer for biodegradation inside tumor cells, and DOX conjugated onto the polymer with a disulfide bond was successfully released intracellularly. NPs(Ce6) released DOX and Ce6 with their original molecular structures and degraded into segments with low MWs of 41.2 kDa in the presence of GSH. NPs(Ce6) showed a chemo-photodynamic therapeutic effect to kill 4T1 murine breast cancer cells, which was confirmed from a collapsed cell morphology, a lifted level in the intracellular reactive oxygen species, a reduced viability and induced apoptosis. Moreover, ex vivo fluorescence images indicated that NPs(Ce6) retained in the tumor, and exhibited a remarkable in vivo anticancer efficacy. The combinational therapy showed a significantly increased tumor growth inhibition (TGI, 58.53%). Therefore, the redox-responsive, amphiphilic block polymeric prodrug could have a great potential as a chemo-photodynamic anti-cancer agent.

Crocin and Metformin suppress metastatic breast cancer progression via VEGF and MMP9 downregulations: in vitro and in vivo studies.

Metastatic breast cancer remains a serious health concern and numerous investigations recommended medicinal plants as a complementary therapy. Crocin is one of the known anticancer bio-component. Recently, the inhibitory effect of metformin has been studied on the various aspects of cancer. However, no study reported their combination effects on metastatic breast cancer. In the present study, we have assessed their anti-metastatic effects on in vitro and in vivo breast cancer models. Using MTT assay, scratch, and adhesion tests, we have evaluated the cytotoxic, anti-invasive and anti-adhesion effects of crocin and metformin on 4T1 cell line, respectively. Their protective effects and MMP9 as well as VEGF protein expression levels (Western blotting) investigated in the 4T1 murine breast cancer model. Our results showed that both crocin and metformin reduced cell viability, delayed scratch healing and inhibited the cell adhesion, in vitro. While crocin alone restored the mice's weight reduction, crocin, metformin, and their combination significantly reduced the tumor volume size and enhanced animal survival rate in murine breast cancer model, responses that were associated with VEGF and MMP9 down-regulation. These findings suggest that a combination of crocin and metformin could serve as a novel therapeutic approach to enhance the effectiveness of metastatic breast cancer therapy.

Post-transcriptional repression of circadian component CLOCK regulates cancer-stemness in murine breast cancer cells.

Disruption of the circadian clock machinery in cancer cells is implicated in tumor malignancy. Studies on cancer therapy reveal the presence of heterogeneous cells, including breast cancer stem-like cells (BCSCs), in breast tumors. BCSCs are often characterized by high aldehyde dehydrogenase (ALDH) activity, associated with the malignancy of cancers. In this study, we demonstrated the negative regulation of ALDH activity by the major circadian component CLOCK in murine breast cancer 4T1 cells. The expression of CLOCK was repressed in high-ALDH-activity 4T1, and enhancement of CLOCK expression abrogated their stemness properties, such as tumorigenicity and invasive potential. Furthermore, reduced expression of CLOCK in high-ALDH-activity 4T1 was post-transcriptionally regulated by microRNA: miR-182. Knockout of miR-182 restored the expression of CLOCK, resulted in preventing tumor growth. Our findings suggest that increased expression of CLOCK in BCSCs by targeting post-transcriptional regulation overcame stemness-related malignancy and may be a novel strategy for breast cancer treatments.

Seed- and Soil-Dependent Differences in Murine Breast Tumor Microenvironments Dictate Anti-PD-L1 IgG Delivery and Therapeutic Efficacy.

We sought to determine if Stephen Paget’s “seed and soil” hypothesis of organ-preference patterns of cancer metastasis can explain the development of heterogeneity in a tumor microenvironment (TME) as well as immunotherapeutic delivery and efficacy. We established single-cell-derived clones (clones 1 and 16) from parental 4T1 murine breast cancer cells to create orthotopic primary and liver metastasis models to deconvolute polyclonal complexity cancer cells and the difference in TME-derived heterogeneities. Tumor-bearing mice were treated with anti-PD-L1 IgG or a control antibody, and immunofluorescent imaging and quantification were then performed to evaluate the therapeutic efficacy on tumor growth, the delivery of therapy to tumors, the development of blood vessels, the expression of PD-L1, the accumulation of immune cells, and the amount of coagulation inside tumors. The quantification showed an inverse correlation between the amount of delivered therapy and therapeutic efficacy in parental-cell-derived tumors. In contrast, tumors originating from clone 16 cells accumulated a significantly greater amount of therapy and responded better than clone-1-derived tumors. This difference was greater when tumors grew in the liver than the primary site. A similar trend was found in PD-L1 expression and immune cell accumulation. However, the change in the number of blood vessels was not significant. In addition, the amount of coagulation was more abundant in clone-1-derived tumors when compared to others. Thus, our findings reconfirmed the seed- and soil-dependent differences in PD-L1 expression, therapeutic delivery, immune cell accumulation, and tumor coagulation, which can constitute a heterogeneous delivery and response of immunotherapy in polyclonal tumors growing in different organs.

Glutathione-activated DNA-Au nanomachine as targeted drug delivery platform for imaging-guided combinational cancer therapy

Multimodal therapy is a combination of different therapeutic modalities within one single platform to enhance treatment efficiency, which holds great promise in biomedical research and clinical practice. Herein, a glutathione (GSH)-activated DNA-Au nanomachine with tumor-targeting capacity has been developed for imaging-guided photodynamic/photothermal/chemo combination therapy of murine breast cancer. In this theranostic nanoplatform, G-quadruplex double-stranded DNA (G-dsDNA) containing AS1411 aptamer is served as not only the targeting unit to specifically recognize murine 4T1 breast cancer cells but also the carrier for loading photosensitizer chlorins e6 (Ce6), iron-containing porphyrin hemin and anticancer drug doxorubicin (Dox). In tumor microenvironment, the excessive GSH triggers the rupture of disulfide bridges and results in the dissociation of DNA-Au nanomachine along with the release of Dox for chemotherapy and the aggregation of AuNPs for photothermal therapy (PTT) as well as in situ imaging. Meanwhile, hemin and Ce6 delivered by G-quadruplexes alleviate the tumor hypoxia, which further promotes the generation of reactive oxygen species (ROS) for enhanced photodynamic therapy (PDT). The in vitro and in vivo assays validate that the combination of PDT/PTT/chemotherapy shows a high efficiency for targeted damage of 4T1 cells and inhibition of tumor growth. Therefore, the proposed multifunctional DNA-Au nanomachine provides a powerful and robust theranostic nanoplatform for imaging-guided combinatorial anticancer therapy.

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

Simple SummaryThis study provides a conceptual overview correlating temporal changes in the tumor immune-microenvironment (TIME) to the non-ablative pulse focused ultrasound (pFUS) in two different types of tumors. The pFUS-induced immunomodulation revealed differential anti-tumor molecular and cellular responses between the two tumor types, demonstrating tumor-type-dependent responses to identical pFUS sonication parameters. It also supports the potential use of pFUS as a possible adjuvant to ablative tumor treatment to elicit enhanced anti-tumor responses and slow tumor growth.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.

Reprograming of Tumor-Associated Macrophages in Breast Tumor-Bearing Mice under Chemotherapy by Targeting Heme Oxygenase-1.

Tumor-associated macrophages (TAMs) represent one of the most abundant components of the tumor microenvironment and play important roles in tumor development and progression. TAMs display plasticity and functional heterogeneity as reflected by distinct phenotypic subsets. TAMs with an M1 phenotype have proinflammatory and anti-tumoral properties whereas M2-like TAMs exert anti-inflammatory and pro-tumoral functions. Tumor cell debris generated during chemotherapy can stimulate primary tumor growth and recurrence. According to our previous study, phagocytic engulfment of breast tumor cell debris by TAMs attenuated chemotherapeutic efficacy through the upregulation of heme oxygenase-1 (HO-1). To verify the impact of HO-1 upregulation on the profile of macrophage polarization during cytotoxic therapy, we utilized a syngeneic murine breast cancer (4T1) model in which tumor bearing mice were treated with paclitaxel (PTX). PTX treatment markedly downregulated the surface expression of the M1 marker CD86 in infiltrated TAMs. Notably, there were significantly more cytotoxic CD8+ T cells in tumors of mice treated with PTX plus the HO-1 inhibitor, zinc protophorphyrin IX (ZnPP) than in mice treated with PTX alone. Interestingly, the tumor-inhibiting efficacy of PTX and ZnPP co-treatment was abrogated when macrophages were depleted by clodronate liposomes. Macrophage depletion also decreased the intratumoral CD8+ T cell population and downregulated the expression of Cxcl9 and Cxcl10. The expression of the M1 phenotype marker, CD86 was higher in mice injected with PTX plus ZnPP than that in mice treated with PTX alone. Conversely, the PTX-induced upregulation of the M2 marker gene, Il10 in CD11b+ myeloid cells from 4T1 tumor-bearing mice treated was dramatically reduced by the administration of the HO-1 inhibitor. Genetic ablation of HO-1 abolished the inhibitory effect of 4T1 tumor cell debris on expression of M1 marker genes, Tnf and Il12b, in LPS-stimulated BMDMs. HO-1-deficient BMDMs exposed to tumor cell debris also exhibited a diminished expression of the M2 macrophage marker, CD206. These findings, taken all together, provide strong evidence that HO-1 plays a pivotal role in the transition of tumor-inhibiting M1-like TAMs to tumor-promoting M2-like ones during chemotherapy.

Plasmodium infection inhibits triple negative 4T1 breast cancer potentially through induction of CD8+ T cell-mediated antitumor responses in mice

We previously reported that Plasmodium infection promotes antitumor immunity in a murine Lewis lung cancer. In this study, we investigated the effects of Plasmodium infection on the tumor inhibition and antitumor CD8+ T cell responses in a murine triple negative breast cancer (TNBCA) model. The results showed that Plasmodium infection significantly inhibited tumor growth, and increased the survival rate of the tumor-bearing mice. Both effector and memory CD8+ T cells were increased in peripheral blood and tumor-draining lymph node (DLN) in the infected mice. The co-stimulatory (CD40L, GITR and OX-40) and co-inhibitory (PD-1, CTLA-4, TIM-3, LAG3) immune checkpoints were up-regulated on CD8+ T cells in infected mice. Importantly, Py induced remarkable effects on the infiltration of CD8+ T cells in the tumor and granzym B+ CD8+ T cells in tumor-bearing mice while not in tumor-free mice. In summary, the results suggested that the effects of Plasmodium infection on murine 4T1 breast cancer might be related to the induction of CD8+ T cell-mediated antitumor immune responses. This finding may provide a novel strategy for the treatment of triple negative breast cancer.

Abstract LT011: Pharmacological blockade of platelet-CysLT1 receptor counteracts platelet protumoral action and prevents breast cancer cell metastasis to bone and lungs

Abstract Metastases are the main cause of death in cancer patients. However, few metastasis-specific targets have been exploited therapeutically. Blood platelets are well known to contribute to the poor survival of patients with advance cancers. Unfortunately, platelets are mandatory for human life due to their paramount function in hemostasis. In order to define new anti-metastasis therapies exhibiting a high specificity for the pathological tumor cell-platelet interactions without abrogating normal platelet functions we developed a high throughput assay that showed that pharmacological blockade of cysteinyl leukotriene receptor 1 (CysLT1) with two already FDA-approved drugs in asthma (Zarfirlukast and Montelukast), dose-dependently inhibited the mitogenic action of human platelets on human MDA-B02/Luc breast cancer cells. Both drugs had no impact on cell proliferation in presence of serum suggesting a specific alteration of platelet function. This effect was specific to CysLT1 as the CysLT2 inhibitor HAMI3379 remained inefficient even at high doses. All human breast cancer cells including MDA-B02/Luc cells used in this study did not express CysLT1 whereas platelets do suggesting that platelet-CysLT1 was the one contributing to the protumoral action of platelets. As described previously cysteinyl leukotriene D4 (LTD4), the strongest agonist of CysLT1, has no direct effect on platelet aggregation but instead potentiates the action of platelet agonists. We next injected MDA-B02/Luc cells into the tail artery of nude mice that received a daily treatment with Zafirlukast (0.4mg/kg per os). After 7 days bone marrow cells were collected and placed in culture in presence of puromycine for 14 day. After staining with crystal violet clones of tumor cells that colonized bone (TCB) were quantified. In these conditions Zafirlulast significantly inhibited by 90% the number of TCB. In order to confirm this anti-metastasis effect we used a syngenic animal model of spontaneous metastasis dissemination of murine 4T1 breast cancer cells after orthotopic xenograph in the mammary gland of BALB/c immunocompetent mice. Animals were treated with Zafirlukast (0.4mg/kg/day, i.p.), the common chemotherapeutic agent Paclitaxel (30mg/kg/2 days, i.p), or both drugs combined starting at day 3 post-tumor cell injections until sacrifice at day 12. Lungs were collected, minced, treated with collagenase and placed in culture for 14 days in presence of 6-Thioguanine. After staining with crystal violet clones of tumor cells that colonized the lungs (TCL) were quantified. In these conditions, the number of TCL was reduced by 88% with Zafirlulast compared with 60% with Paclitaxel. The combined treatment of Zafirlukast + Paclitaxel remarkably inhibited by 100% the number of TCL. In contrast, Zafirlukast alone or in combination with Paclitaxel had no significant effect on primary tumor growth. In conclusion we described for the first time that blockade of platelet-CysLT1 receptor altered the protumoral action of platelets and prevented breast cancer cell metastasis to bone and lungs. Citation Format: Lou Saier, Olivier Peyruchaud. Pharmacological blockade of platelet-CysLT1 receptor counteracts platelet protumoral action and prevents breast cancer cell metastasis to bone and lungs [abstract]. In: Proceedings of the AACR Virtual Special Conference on the Evolving Tumor Microenvironment in Cancer Progression: Mechanisms and Emerging Therapeutic Opportunities; in association with the Tumor Microenvironment (TME) Working Group; 2021 Jan 11-12. Philadelphia (PA): AACR; Cancer Res 2021;81(5 Suppl):Abstract nr LT011.