Metastatic 4T1 Cells Research Articles

Dendrimer/metal-phenolic nanocomplexes encapsulating CuO2 for targeted magnetic resonance imaging and enhanced ferroptosis/cuproptosis/chemodynamic therapy by regulating the tumor microenvironment

The combination of ferroptosis, cuproptosis, and chemodynamic therapy (CDT) would be a potential strategy for tumor diagnosis and enhanced treatment. However, the therapeutic effect was severely limited by the lack of specific delivery of catalytic ions and the low Fenton reaction efficiency in tumor microenvironment (TME) with excess glutathione, limited acidity and insufficient endogenous hydrogen peroxide. In this work, p-carboxybenzenesulfonamide (BS), a carbonic anhydrase IX (CA IX) inhibitor, was modified on the surface of generation-5 poly(amidoamine) dendrimer to load copper peroxide nanoparticles, which were complexed with iron (Fe)-tannic acid (TF) networks for targeted magnetic resonance (MR) imaging and enhanced ferroptosis/cuproptosis/CDT by regulating TME. The formed CuO2@G5-BS/TF nanocomplexes with an average size of 39.4 nm could be specifically accumulated at tumor site and effectively internalized by metastatic 4T1 cells via the specific interaction between BS and CA IX over-expressed on tumor cells. Meanwhile, the inhibition of CA IX activity could not only decrease the intracellular pH to accelerate Fe3+/Cu2+ release, H2O2 self-supply and Fenton reaction, but also suppress tumor metastasis by alleviating the extracellular acidity in TME. Moreover, the reduction of Fe3+/Cu2+ by intracellular glutathione (GSH) could further amplify ROS generation and enhance CDT efficacy, and the GSH depletion could in turn inhibit GPX-4 mediated antioxidant reaction to induce ferroptosis, resulting in effective therapeutic efficacy. In vivo experimental results demonstrated that CuO2@G5-BS/TF could provide better tumor MR imaging, effectively inhibit the growth and metastasis of 4T1 breast tumors, and be metabolized without significant systemic toxicity. Thus, CuO2@G5-BS/TF nanocomplexes provided a new approach for targeted MR imaging and enhanced ferroptosis/cuproptosis/CDT of triple-negative breast cancer. Statement of significanceTaking the advantage of dendrimer and metal-phenolic system, stable CuO2@G5-BS/TF nanocomplexes with an average size of 39.4 nm were synthesized to efficiently load Fe3+ and CuO2 nanoparticles for TNBC treatment and MR imaging. CuO2@G5-BS/TF nanocomplexes could target tumor cells overexpressing CAIX via the specific binding with BS, and the inhibition of CAIX activity could not only decrease the intracellular pH to accelerate Fe3+/Cu2+ release, H2O2 self-supply and Fenton reaction, but also suppress tumor metastasis by alleviating the extracellular acidity. The reduction of Fe3+/Cu2+ by intracellular GSH could further amplify ·OH generation, and the GSH depletion could in turn inhibit GPX-4 mediated antioxidant reaction to induce ferroptosis, resulting in effective therapeutic efficacy by enhanced ferroptosis/cuproptosis/CDT via tumor microenvironment regulation.

Truncated O-glycosylation in metastatic triple-negative breast cancer reveals a gene expression signature associated with extracellular matrix and proteolysis

Breast cancer (BC) is the leading cause of death by cancer in women worldwide. Triple-negative (TN) BC constitutes aggressive and highly metastatic tumors associated with shorter overall survival of patients compared to other BC subtypes. The Tn antigen, a glycoconjugated structure resulting from an incomplete O-glycosylation process, is highly expressed in different adenocarcinomas, including BC. It also favors cancer growth, immunoregulation, and metastasis in TNBC. This work describes the differentially expressed genes (DEGs) associated with BC aggressiveness and metastasis in an incomplete O-glycosylated TNBC cell model. We studied the transcriptome of a TNBC model constituted by the metastatic murine 4T1 cell line that overexpresses the Tn antigen due to a mutation in one of the steps of the O-glycosylation pathway. We analyzed and compared the results with the parental wild-type cell line and with a Tn-negative cell clone that was poorly metastatic and less aggressive than the 4T1 parental cell line. To gain insight into the generated expression data, we performed a gene set analysis. Biological processes associated with cancer development and metastasis, immune evasion, and leukocyte recruitment were highly enriched among functional terms of DEGs. Furthermore, different highly O-glycosylated protein-coding genes, such as mmp9, ecm1 and ankyrin-2, were upregulated in 4T1/Tn+ tumor cells. The altered biological processes and DEGs that promote tumor growth, invasion and immunomodulation might explain the aggressive properties of 4T1/Tn+ tumor cells. These results support the hypothesis that incomplete O-glycosylation that leads to the expression of the Tn antigen, which might regulate activity or interaction of different molecules, promotes cancer development and immunoregulation.

Tumor-educated Gr1+CD11b+ cells drive breast cancer metastasis via OSM/IL-6/JAK-induced cancer cell plasticity.

Cancer cell plasticity contributes to therapy resistance and metastasis, which represent the main causes of cancer-related death, including in breast cancer. The tumor microenvironment drives cancer cell plasticity and metastasis, and unraveling the underlying cues may provide novel strategies for managing metastatic disease. Using breast cancer experimental models and transcriptomic analyses, we show that stem cell antigen-1 positive (SCA1+) murine breast cancer cells enriched during tumor progression and metastasis had higher in vitro cancer stem cell-like properties, enhanced in vivo metastatic ability, and generated tumors rich in Gr1hiLy6G+CD11b+ cells. In turn, tumor-educated Gr1+CD11b+ (Tu-Gr1+CD11b+) cells rapidly and transiently converted low metastatic SCA1- cells into highly metastatic SCA1+ cells via secreted oncostatin M (OSM) and IL-6. JAK inhibition prevented OSM/IL-6-induced SCA1+ population enrichment, while OSM/IL-6 depletion suppressed Tu-Gr1+CD11b+-induced SCA1+ population enrichment in vitro and metastasis in vivo. Moreover, chemotherapy-selected highly metastatic 4T1 cells maintained high SCA1+ positivity through autocrine IL-6 production, and in vitro JAK inhibition blunted SCA1 positivity and metastatic capacity. Importantly, Tu-Gr1+CD11b+ cells invoked a gene signature in tumor cells predicting shorter overall survival (OS), relapse-free survival (RFS), and lung metastasis in breast cancer patients. Collectively, our data identified OSM/IL-6/JAK as a clinically relevant paracrine/autocrine axis instigating breast cancer cell plasticity and triggering metastasis.

Dual drug-loaded metal-phenolic networks for targeted magnetic resonance imaging and synergistic chemo-chemodynamic therapy of breast cancer.

The development of nanomedicines with simplified compositions and synergistic theranostic functionalities remains a great challenge. Herein, we develop a simple method to integrate both atovaquone (ATO, a mitochondrial inhibitor) and cisplatin within tannic acid (TA)-iron (Fe) networks coated with hyaluronic acid (HA) for targeted magnetic resonance (MR) imaging-guided chemo-chemodynamic synergistic therapy. The formed TFP@ATO-HA displayed good colloidal stability with a mean size of 95.5 nm, which could accumulate at tumor sites after circulation and be specifically taken up by metastatic 4T1 cells overexpressing CD44 receptors. In the tumor microenvironment, TFP@ATO-HA could release ATO/cisplatin and Fe3+ in a pH-responsive manner, deplete glutathione, and generate reactive oxygen species with endogenous H2O2 for chemodynamic therapy (CDT). Additionally, ATO could enhance chemotherapeutic efficacy by inhibiting mitochondrial respiration, relieving hypoxia, and amplifying the CDT effect by decreasing intracellular pH and elevating Fenton reaction efficiency. In vivo experiments demonstrated that TFP@ATO-HA could effectively inhibit tumor growth and suppress lung metastases without obvious systemic toxicity. Furthermore, TFP@ATO-HA exhibited a r1 relaxivity of 2.6 mM-1 s-1 and targeted MR imaging of 4T1 tumors. Dual drug-loaded metal-phenolic networks can be easily prepared and act as effective theranostic nanoplatforms for targeted MR imaging and synergistic chemo-chemodynamic therapy.

Four Birds with One Stone: A Multifunctional Polypeptide Nanocomposite to Unify Ferroptosis, Nitric Oxide, and Photothermia for Amplifying Antitumor Immunity

AbstractTumor metastasis and relapse mainly results in therapy failure and becomes a big challenge in oncology. Immunogenic cell death (ICD) of tumors mediated immunotherapy (IT) is attracting widely for solving that problem although achieving sufficient ICD and strong immune response is challenging for nanoparticles‐based cancer IT. Herein, a multifunctional polypeptide coordinate nanocomposite that possesses near infrared photothermia (PT) and responsive releases of nitric oxide (NO) and iron ions is constructed, which synergistically kills cancer cells and highly prohibits metastatic 4T1 cells invasion and migration by PT‐boosted NO release and ferroptosis (FT). Remarkably, triple FT‐NO‐PT treatment amplifies the ICD effects and outperforms combo/monotherapy FT‐PT and FT in cancer cells and tumors, which further activates dendritic cells maturation, and primed CD4+T and CD8+T cells immune responses and memory effects, playing four birds with one stone (i.e., FT‐NO‐PT‐IT). The PCSFG‐based FT‐NO‐PT not only fully eradicates 4T1 primary tumors, but also induces strong ICD, immune priming, and memory effects to reject rechallenged 4T1 tumors and inhibit malignant tumor metastasis, demonstrating synergistic amplified ICD effects with strong cell immunities and memory effects by a unified FT‐NO‐PT‐IT.

Smart Chondroitin Sulfate Micelles for Effective Targeted Delivery of Doxorubicin Against Breast Cancer Metastasis.

Metastasis is a major challenge in breast cancer therapy. The successful chemotherapy of breast cancer largely depends on the ability to block the metastatic process. Herein, we designed a dual-targeting and stimuli-responsive drug delivery system for targeted drug delivery against breast cancer metastasis. AS1411 aptamer-modified chondroitin sulfate A-ss-deoxycholic acid (ACSSD) was synthesized, and the unmodified CSSD was used as the control. Chemotherapeutic drug doxorubicin (DOX)-containing ACSSD (D-ACSSD) micelles were prepared by a dialysis method. The ACSSD conjugate was confirmed by Fourier transform infrared spectroscopy (FTIR), nuclear magnetic resonance (NMR), dynamic light scattering (DLS), and transmission electron microscopy (TEM). In vitro cellular uptake and cytotoxicity of D-ACSSD micelles were studied by confocal laser scanning microscopy (CLSM) and MTT assay in breast tumor cells. The inhibition capability of D-ACSSD micelles in cell migration and invasion was carried out in 4T1 cells. In vivo antitumor activity of DOX-containing micelles was investigated in metastatic 4T1-bearing Balb/c mice. D-ACSSD and DOX-loaded CSSD (D-CSSD) micelles exhibited high drug encapsulation content and reduction-responsive characteristics. D-ACSSD micelles were spherical in shape. Compared with D-CSSD, D-ACSSD showed higher cellular uptake and more potent killing activity in 4T1 and MDA-MB-231 cells. Additionally, D-ACSSD exhibited stronger inhibitory effects on the invasion and migration of highly metastatic 4T1 cells than unmodified D-CSSD. Among the DOX-containing formulations, D-ACSSD micelles presented the most effective inhibition of tumor growth and lung metastasis in orthotopic 4T1-bearing mice in vivo. It also revealed that ACSSD micelles did not exhibit obvious systemic toxicity. The smart D-ACSSD micelles could be a promising delivery system for the therapy of metastatic breast cancer.

Thioridazine combined with carboplatin results in synergistic inhibition of triple negative breast cancer by targeting cancer stem cells

Cancer stem cells (CSCs) in triple-negative breast cancer (TNBC) are closely related to tumorigenesis and metastasis. Thioridazine (THZ) is a usual phenothiazine antipsychotic drug that can destroy CSCs. We aimed to explore whether THZ could sensitize metastatic TNBC cells, especially the CSCs, to carboplatin (CBP) treatment. Metastatic TNBC cells, 4T1 cells, and tumor-bearing mice were treated with THZ and CBP as monotherapy or combination therapy. MTT, flow cytometry, electron microscopy, immunohistochemistry and western blotting were applied to assess the cell viability, apoptosis, mitochondrial morphology and the relevant protein levels, respectively. Tumor size and lung metastasis under different treatments as well as tumorigenesis of residual tumor cells from each group were monitored. THZ combined with CBP inhibited 4T1 tumor cell proliferation and induced apoptosis by inhibiting the PI3K-AKT-mTOR pathway and activating estrogen receptor stress. THZ also showed strong activity against breast CSCs, THZ combined with CBP significantly destroyed cancer cells, inhibited lung metastasis and relieved the tumor burden; Our data demonstrated that THZ can sensitize TNBC cells to CBP treatment and this combination therapy may provide a bright strategy for TNBC treatment by targeting both cancer cells and CSCs.

A comparative study of combination treatments in metastatic 4t1 cells: everolimus and 5- fluorouracil versus lithium chloride and 5-fluorouracil

Background: Combination therapy has been one of the most pioneering and strategic approaches implemented for malignancy treatment, which can intentionally influence multiple signaling pathways involved in cancer growth and progression. In the present study, the effects of 5-fluorouracil (5FU) in combination with everolimus (EVE) or lithium chloride (LiCl) were evaluated in 4T1 metastatic breast cancer cells and compared to control and each other. Methods and results: The resazurin assay, CompuSyn, flow cytometry, and real-time PCR were used to investigate cell proliferation, drug synergism, apoptosis, and gene expression. In comparison to the ternary combination of the drugs, the findings showed that cytotoxicity (p-value < 0.0001) and apoptosis (p-value < 0.0001) of two-by-two combinations increased dramatically as a consequence of the extreme synergy between 5FU and EVE or LiCl. Moreover, the hypoxia­inducible transcription factor 1-alpha (HIF-1α) and the vascular endothelial growth factor (VEGF) downregulated considerably compared to control (p-value < 0.0001) by combination therapies of EVE-5FU and 5FU-LiCl; however, only VEGF displayed significant downregulation in comparison to single therapies. Conclusion: The findings showed that the combination of 5FU-LiCl increased cell cytotoxicity and apoptosis significantly more than EVE-5FU but suggests a clinical potential for both to treat metastatic breast cancer encouraging validation of these results in pre-clinical models.

Autophagy inhibition in breast cancer cells induces ROS-mediated MIF expression and M1 macrophage polarization

Autophagy can function as a survival mechanism for cancer cells and therefore, its inhibition is currently being explored as a therapy for different cancer types. For breast cancer, triple negative breast cancer (TNBC) is the subtype most sensitive to the inhibition of autophagy; but its inhibition has also been shown to promote ROS-dependent secretion of macrophage migration inhibitory factor (MIF), a pro-tumorigenic cytokine. In this work, we explore the role of MIF in breast cancer, the mechanism by which autophagy inhibition promotes MIF secretion and its effects on neighboring cancer cell signaling and macrophage polarization. We analyzed MIF mRNA expression levels in tumors from breast cancer patients from different subtypes and found that Luminal B, HER2 and Basal subtypes, which are associated to high proliferation, displayed high MIF levels. However, MIF expression had no prognostic relevance in any breast cancer subtype. In addition, we found that autophagy inhibition in 66cl4 TNBC cells increased intracellular Reactive Oxygen Species (ROS) levels, which increased MIF expression and secretion. MIF secreted from 66cl4 TNBC cells induced the activation of MIF-regulated pathways in syngeneic cell lines, increasing Akt phosphorylation in 4T1 cells and ERK phosphorylation in 67NR cells. Regarding MIF/ chemokine receptors, higher levels of CD74 and CXCR2 were found in TNBC tumor cell lines when compared to non-tumorigenic cells and CXCR7 was elevated in the highly metastatic 4T1 cell line. Finally, secreted MIF from autophagy deficient 66cl4 cells induced macrophage polarization towards the M1 subtype. Together, our results indicate an important role for the inhibition of autophagy in the regulation of ROS-mediated MIF gene expression and secretion, with paracrine effects on cancer cell signaling and pro-inflammatory repercussions in macrophage M1 polarization. This data should be considered when considering the inhibition of autophagy as a therapy for different types of cancer.

Bone metastatic breast cancer cells display downregulation of PKC-ζ with enhanced glutamine metabolism

Breast cancer is the most commonly diagnosed cancer among women and its metastases results in poor survival rates in patients. The ability to alter metabolism is a key attribute cancer cells use to survive within different metastatic microenvironments and cause organ failure. We hypothesized that evaluation of metabolic alterations within tumor cells could provide a better understanding of cancer metastasis. Therefore, to investigate underlying metabolic alterations during metastases, we utilized human MDA-MB-231 and mouse 4T1 models that closely mimic human breast cancer metastasis. The glycolysis and glutamine pathway-related changes were examined in bone metastatic cells by XF-24 extracellular flux analyzer and western blotting. The expression levels of genes related to metabolism were examined by PCR arrays. The MDA-MB-231 cells isolated after bone metastases showed reduced glucose uptake and glycolysis compared to parental cells, suggesting that these cells could alter metabolic requirements for survival. To understand these metabolic changes, we investigated glutamine, a common and naturally occurring non-essential amino acid. Interestingly, in reduced glucose conditions both cell lines showed dependence on glutamine for cell survival, and with glutamine withdrawal significantly increasing apoptotic cell death. Glutamine was also critical for normal cell proliferation even in the presence of high glucose concentrations. To further understand this metabolic switch in metastatic cells, we examined the genes related to metabolism and identified a more than seven-fold downregulation of protein kinase C zeta (PKC-ζ) expression levels in bone-derived MDA-MB-231 cells compared to the parental population. The PKC-ζ levels were also significantly reduced in metastatic 4T1 cells compared to non-metastatic MT1A2 cells. Since PKC-ζ deficiency promotes glutamine utilization via the serine biosynthesis pathway, we examined glutamine metabolism. The ectopic expression of PKC-ζ inhibited glutamine conversion to glutamate, while mutant PKC-ζ reversed this effect. Furthermore, the gene expression levels of enzymes involved in serine biosynthesis, phosphoserine phosphatase (PSPH), phosphoserine aminotransferase (PSAT1), and phosphoglycerate dehydrogenase (PHGDH) showed upregulation following glucose deprivation with PKC-ζ deficiency. The PHGDH upregulation was inhibited by ectopically expressing wild type but not mutated PKC-ζ in glucose-deprived conditions. Our results support the upregulation of serine biosynthesis pathway genes and downregulation of PKC-ζ as potential metabolic alterations for bone metastatic breast cancer cells.

Pygenic Acid A (PA) Sensitizes Metastatic Breast Cancer Cells to Anoikis and Inhibits Metastasis In Vivo.

Metastasis is the main cause of cancer-related deaths. Anoikis is a type of apoptosis caused by cell detachment, and cancer cells become anoikis resistant such that they survive during circulation and can successfully metastasize. Therefore, sensitization of cancer cells to anoikis could prevent metastasis. Here, by screening for anoikis sensitizer using natural compounds, we found that pygenic acid A (PA), a natural compound from Prunella vulgaris, not only induced apoptosis but also sensitized the metastatic triple-negative breast cancer cell lines, MDA-MB-231 cells (human) and 4T1 cells (mouse), to anoikis. Apoptosis protein array and immunoblotting analysis revealed that PA downregulated the pro-survival proteins, including cIAP1, cIAP2, and survivin, leading to cell death of both attached and suspended cells. Interestingly, PA decreased the levels of proteins associated with anoikis resistance, including p21, cyclin D1, p-STAT3, and HO-1. Ectopic expression of active STAT3 attenuated PA-induced anoikis sensitivity. Although PA activated ER stress and autophagy, as determined by increases in the levels of characteristic markers, such as IRE1α, p-elF2α, LC3B I, and LC3B II, PA treatment resulted in p62 accumulation, which could be due to PA-induced defects in autophagy flux. PA also decreased metastatic characteristics, such as cell invasion, migration, wound closure, and 3D growth. Finally, lung metastasis of luciferase-labeled 4T1 cells decreased following PA treatment in a syngeneic mouse model when compared with the control. These data suggest that PA sensitizes metastatic breast cancer cells to anoikis via multiple pathways, such as inhibition of pro-survival pathways and activation of ER stress and autophagy, leading to the inhibition of metastasis. These findings suggest that sensitization to anoikis by PA could be used as a new therapeutic strategy to control the metastasis of breast cancer.

AKT-mediated phosphorylation of TWIST1 is essential for breast cancer cell metastasis.

Previously, it was shown that human TWIST1 (basic helix-loop-helix (b-HLH) is phosphorylated by Akt kinase at S42, T121, and S123. To show in vivo effect of these phosphorylations, we created mouse TWIST1 expression vector and converted the codons of S42, T125, and S127 to unphosphorylatable alanine and phosphorylation mimicking Glutamic acid. We hypothesized that alanine mutants would inhibit the metastatic ability of 4T1 cells while glutamic acid mutants would convert nonmetastatic 67NR cells into metastatic phenotype. To confirm this hypothesis, we created metastatic 4T1 and nonmetastatic 67NR cells expressing alanine mutants and glutamic acid mutants mouse TWIST1, respectively. Then, we injected 1 × 106 67NR and 1 × 105 4T1 cells overexpressing mutants of TWIST1 into the breast tissue of BALB/c mice. At the end of the 4th week, we sacrificed the animals, determined the numbers of tumors at lungs and liver. Although 67NR cells overexpressing wild-type TWIST1 did not show any metastasis, cells overexpressing S42E and T125E mutants showed 15–30 macroscopic metastasis to liver and lungs. Parallel to this, 4T1 cells expressing S42A and T125A mutants of TWIST1 showed no macroscopic metastasis. Our results indicate that phosphorylation of S42 and T125 by AKT is essential for TWIST1-mediated tumor growth and metastasis.

Generation of multiepitope cancer vaccines based on large combinatorial libraries of survivin-derived mutant epitopes.

Immune tolerance is the main challenge in the field of cancer vaccines, so modified peptide sequences or naturally occurring mutated versions of cancer-related wild-type (WT) antigens represent a promising pathway. However, the low immunogenicity of mutation-induced neoantigens and, particularly, their incapacity to activate CD8+ T cells are generating doubts on the success of neoantigen-based cancer vaccines in clinical trials. We developed a novel vaccine approach based on a new class of vaccine immunogens, called variable epitope libraries (VELs). We used three regions of survivin (SVN), composed of 40, 49 and 51 amino acids, along with the complete SVN protein to generate the VELs as multiepitope vaccines. BALB/c mice, challenged with the aggressive and highly metastatic 4T1 cell line, were vaccinated in a therapeutic setting. We showed significant tumor growth inhibition and, most importantly, strong suppression of lung metastasis after a single immunization using VEL vaccines. We demonstrated vaccine-induced broad cellular immune responses concomitant with extensive tumor infiltration of T cells, the activation of CD107a+ IFN-γ+ T cells in the spleen and a significant increase in the number of CD3+ CD8+ Ly6C+ effector T cells. In addition, we observed the presence of interferon-γ-, granzyme B- and perforin-producing lymphocytes along with modifications in the amount of CD11b+ Ly6Cint/low Ly6G+ granulocytic myeloid-derived suppressor cells and CD4+ CD25+ FoxP3+ regulatory T cells in the lungs and tumors of mice. In summary, we showed that the VELs represent a potent new class of cancer immunotherapy and propose the application of the VEL vaccine concept as a true alternative to currently available vaccine platforms.

O,O'-diethyl-(S,S)-ethylenediamine-N,N'-di-2-(3-cyclohexyl)propanoate dihydrochloride enhances influx of effective NK and NKT cells in murine breast cancer

Background/Aim. O,O'-diethyl-(S,S)-ethylenediamine- N,N'-di-2-(3-cyclohexyl)propanoate dihydrochloride (DEEDCP) has been found to possess promising cytotoxic activity against various tumor cell lines. Also, DE-EDCP reduces tumor progression by several mechanisms such as triggering tumor cell death and inhibition of cell proliferation. The aim of present study was to further evaluate antitumor activity of DE-EDCP by investigating effects on migratory potential of tumor cells and anti-tumor immune response. Methods. Migratory potential of DE-EDCP was evaluated by scratch wound assay. Female BALB/c mice were inoculated with 4T1 breast cancer cells and treatment with DE-EDCP started five days following orthotopic tumor implantation. The frequency and phenotype of tumorinfiltrating natural killer (NK) and natural killer T (NKT) cells were analyzed by flow cytometry. Results. DE-EDCP inhibited migratory potential of highly metastatic 4T1 cells. DE-EDCP facilitated accumulation of CD3+CD49+ NKT cells and CD3-CD49+ NK cells in tumor microenvironment. DE-EDCP treatment led to significant decrement of tumor infiltrating anergic NKT cells expressing cytotoxic Tlymphocyte- associated protein 4 (CTLA-4), killer cell lectin like receptor G1 (KLRG-1) and programmed cell death protein- 1 (PD-1). Mice given DE-EDCP had significantly increased percentages of tumoricidal fas ligand (FasL) positive NK cells. Conclusion. DE-EDCP inhibits murine breast cancer progression through direct effects on tumor cells and by facilitating anti-tumor immunity. DE-EDCP enhances accumulation, promotes tumoricidal phenotype and maintenances responsiveness of NK and NKT cells in 4T1 murine breast cancer.

Cytosolic Phospholipase A2 Alpha Regulates TLR Signaling and Migration in Metastatic 4T1 Cells.

Metastatic disease is the leading cause of death in breast cancer patients. Disrupting the cancer cell’s ability to migrate may be a strategy for hindering metastasis. Cytosolic phospholipase A2 α (cPLA2α), along with downstream proinflammatory and promigratory metabolites, has been implicated in several aspects of tumorigenesis, as well as metastasis, in various types of cancer. In this study, we aim to characterize the response to reduced cPLA2α activity in metastatic versus non-metastatic cells. We employ an isogenic murine cell line pair displaying metastatic (4T1) and non-metastatic (67NR) phenotype to investigate the role of cPLA2α on migration. Furthermore, we elucidate the effect of reduced cPLA2α activity on global gene expression in the metastatic cell line. Enzyme inhibition is achieved by using a competitive pharmacological inhibitor, cPLA2α inhibitor X (CIX). Our data show that 4T1 expresses significantly higher cPLA2α levels as compared to 67NR, and the two cell lines show different sensitivity to the CIX treatment with regards to metabolism and proliferation. Inhibition of cPLA2α at nontoxic concentrations attenuates migration of highly metastatic 4T1 cells, but not non-metastatic 67NR cells. Gene expression analysis indicates that processes such as interferon type I (IFN-I) signaling and cell cycle regulation are key processes regulated by cPLA2a in metastatic 4T1 cells, supporting the findings from the biological assays. This study demonstrates that two isogenic cancer cell lines with different metastatic potential respond differently to reduced cPLA2α activity. In conclusion, we argue that cPLA2α is a potential therapeutic target in cancer and that enzyme inhibition may inhibit metastasis through an anti-migratory mechanism, possibly involving Toll-like receptor signaling and type I interferons.

Highly Integrated Nanoplatform Based on an E-Selectin-Targeting Strategy for Metastatic Breast Cancer Treatment.

Therapeutic goals for metastatic breast cancer, including shrinkage of established metastasis and suppression of movement of tumor cells, are often hard to achieve and remain the main obstacles restricting the antimetastatic efficacy of targeted drug delivery systems (TDDSs). Herein, we proposed an E-selectin-targeting nanoplatform for the systemic treatment of metastatic breast cancer. Versatile functions, including killing the circulating tumor cells, shrinking the established lesions, as well as inhibiting the movement of tumor cells, were integrated into doxorubicin-loaded sialic acid-dextran-octadecanoic acid (SDO) micelles (SDD). The prepared SDD micelles could not only inhibit lung and liver metastasis in the orthotopic 4T1 tumors model, but also decrease the metastatic lesions in the metastatic 4T1 cell model, resulting in 27.33% reduced number of metastatic nodules when compared to those without sialic acid modification. It was found that the good antimetastatic effect of SDD was only partially attributed to its ability on removing metastatic cells and metastases. Most importantly, the blank SDO micelles left in the lesion could further inhibit the cell migration and cell-cell binding. These results suggest that SA-driven TDDS has the potential for specific targeting and effective treatment of cancer metastasis.

Abstract 2907: Exosome secretion is an inheritable property of cancer cells: Single-cell profiling of exosome secretion

Abstract Decades of research on exosomes, nano-sized vesicles secreted by cells, have revealed novel roles of these vesicles in the formation of pre-metastasis niches that enhances the migration of tumor cells to those sites. Paradoxically, more recent work has suggested that these tumor-derived exosomes can also have an immunostimulatory role, depending on the model studied. The rate of secretion of exosomes by single cells is likely heterogeneous, and a deep profiling of the secretion capacity of individual tumor cells has been largely unexplored. We have developed a high-throughput single-cell methodology to quantify the dynamic secretion of exosomes from single cells using a modified immunoassay and sought to define: (a) heterogeneity among individual cells within the tumor cell population, and (b) the nature of the cells secreting exosomes within a tumor cell population. In order to investigate these, we chose to study models of triple negative breast cancer: the metastatic tumor lines, 4T1 (mouse) and MDA-MB-231(human); and the non-metastatic lines 67NR (mouse) and MCF7 (human). Our method revealed that MDA-MB-231 single cells secreted exosomes at a rate, ~2 fold higher than MCF7 cells. Surprisingly, the non-metastatic 67NR cells showed a higher secretion rate (~1.5 fold) than metastatic 4T1 cells. Next, we performed single-cell RNA-seq on 67NR and 4T1 single cells. Consistent with our single-cell exosomal profiling results, 67NR cells were significantly (p-value < 0.01) enriched for genes correlated to exosome formation in comparison to the 4T1 cells. Next, in order to study exosomes of single cells, we isolated single cells using an automated micromanipulator which allowed us to establish monoclonal cell lines. Measurement of these monoclonal cells showed that secretor cells of 67NR secrete exosomes with ~2 fold higher rate than non-secretor cells. Since our in vitro results clearly demonstrated that the secretor clonal cell lines had a higher frequency of exosome secreting single cells, we sought to define the in vivo relevance of these results. Consistent with the hypothesis that the exosomes from 67NR are immune-stimulatory, injection of the secretor clones into Balb/c mice led to lack of primary tumor formation (2/15 mice had tumors) whereas the injection of the non-secretor clones led to tumor formation in 7/15 mice. In aggregate, our results show that the higher rate of secretion of exosomes from non-metastatic cells can facilitate tumor rejection in vivo. We are currently performing in vitro studies with the 67NR and MDA-MB-231 exosomes to study their impact on immune cells. Citation Format: Mohsen Fathi, Robiya Joseph, Melisa Martinez-Paniagua, Jay R Adolacion, Xingyue An, Ankit Mahendra, Konrad Gabrusiewicz, Sujash Chatterjee, Sendurai A. Mani, Navin Varadarajan. Exosome secretion is an inheritable property of cancer cells: Single-cell profiling of exosome secretion [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 2907.

Editor's Note: Growth Inhibitory and Antimetastatic Effect of Green Tea Polyphenols on Metastasis-Specific Mouse Mammary Carcinoma 4T1 Cells In vitro and In vivo Systems.

Purpose: Breast cancer is the second leading cause of cancer-related deaths among females. Dietary habits may have a role in breast cancer risk and prevention as well. Here, we examined the effect of green tea polyphenols (GTP) on growth and metastasis of highly metastatic mouse mammary carcinoma 4T1 cells in vitro and in vivo systems. Experimental Design: 4T1 cells were treated with (−)-epigallocatechin-3-gallate (EGCG), and the effect was determined on cellular proliferation, induction of apoptosis, proapoptosis, and antiapoptotic proteins of Bcl-2 family, and caspase 3 and poly(ADP-ribose) polymerase activation following 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide, flow cytometry, and Western blot analysis. Anticarcinogenic and antimetastatic effect of GTP in 4T1 cells was assessed in immunocompetent BALB/c mice. Results: Treatment of 4T1 cells with EGCG resulted in inhibition of cell proliferation, induction of apoptosis in dose- and time-dependent manner. The increase in apoptosis was accompanied with decrease in the protein expression of Bcl-2 concomitantly increase in Bax, cytochrome c release, Apaf-1, and cleavage of caspase 3 and PARP proteins. Treatment of EGCG-rich GTP in drinking water to 4T1 cells bearing BALB/c mice resulted in reduction of tumor growth accompanied with increase in Bax/Bcl-2 ratio, reduction in proliferating cell nuclear antigen and activation of caspase 3 in tumors. Metastasis of tumor cells to lungs was inhibited and survival period of animals was increased after green tea treatment. Conclusion: This study suggests that GTP have the ability to prevent the development of breast cancer and its metastasis; however, further in vivo studies are required to identify the molecular targets.

Abstract B23: Intravital optical imaging of tumor vascular oxygenation and metabolism in murine breast cancer xenografts of varying metastatic potential

Abstract About 90% of breast cancer patients die due to metastatic spread and not due to the primary tumor. Therefore, several patients receive neoadjuvant chemotherapy to eliminate the risk associated with developing metastases even if they may be at a lower risk of developing metastases. Current clinical guidelines laid out by the American Society for Clinical Oncology (ASCO) recommend consideration of the Oncotype DX score to determine risk of recurrence so that only patients classified as high-risk receive chemotherapy. In addition to this genetic test, there is a need to develop more cost-effective prognostic indicators that can reduce the number of patients receiving life-altering chemotherapy for early-stage breast cancers with very low recurrence risk. Recent studies have shown that primary, circulating, and metastasized cancer cells adopt different metabolic profiles to fuel their survival and growth. Furthermore, highly invasive primary cancer cells may favor mitochondrial oxidative metabolism to efficiently generate ATP and promote a migratory phenotype. Optical imaging of the endogenous fluorescent molecules nicotinamide adenine dinucleotide (NADH) and flavin adenine dinucleotide (FAD) provides a nondestructive and label-free method for determining cell metabolism. The optical redox ratio (FAD/FAD+NADH) is sensitive to the balance between glycolysis and oxidative phosphorylation (OXPHOS). We previously demonstrated that a short duration of hypoxia-reoxygenation stress (1 hour of hypoxia followed by 1 hour of reoxygenation) led to metastatic potential-dependent changes in optical redox ratio in a panel of isogenic murine breast cancer cell lines (4T1, 4T07, 168FARN, 67NR). Specifically, highly metastatic 4T1 cells were found to switch to mitochondrial oxidative metabolism whereas nonmetastatic 168FARN cells were highly glycolytic and did not exhibit any changes post-stress. We further determined that deletion of TWIST, a key promoter of metastasis, in the 4T1 cell line caused these cells to respond in the same way as 168FARN to hypoxic stress. The objective of this study was to investigate these metabolic profiles in vivo and determine the relationship between tumor oxygenation and metabolism in tumor xenografts grown from the aforementioned cell lines. Tumors were grown in dorsal skinfold window chambers by injecting approx. 10-20,000 cancer cells. Imaging and quantification of vascular oxygenation was accomplished by acquiring transillumination images at wavelengths corresponding to absorption by hemoglobin (520-620 nm). Glucose uptake was imaged 60 minutes after intravenous injection of a fluorescent glucose analog called 2-NBDG. Our data show higher vascular oxygenation and lower glucose uptake in primary tumors with higher metastatic potential compared with nonmetastatic tumors, suggesting that tumors with high metastatic potential display decreased glycolysis and increased oxidative phosphorylation. Our preliminary results indicate that optical imaging of tumor bioenergetics could shed light on the metastatic tendencies of primary breast tumors. Citation Format: Narasimhan Rajaram, Raisa Rasul. Intravital optical imaging of tumor vascular oxygenation and metabolism in murine breast cancer xenografts of varying metastatic potential [abstract]. In: Proceedings of the AACR Special Conference: Advances in Breast Cancer Research; 2017 Oct 7-10; Hollywood, CA. Philadelphia (PA): AACR; Mol Cancer Res 2018;16(8_Suppl):Abstract nr B23.

Abstract 5459: Autophagy and senescence as possible mechanisms leading to proliferative recovery and escape from treatment-induced tumor dormancy

Abstract Despite the ability of chemotherapy to eliminate the majority of tumor cells, some are able to escape cell death and proliferate. In this study we evaluated mechanisms of autophagy and senescence, that in theory allow cells to escape apoptosis and might be responsible for cancer recurrence. Autophagy and senescence, either alone or in concert, may result in temporary growth arrest followed by proliferative recovery. With regard to autophagy, its function may be either cytoprotective, where autophagy inhibition results in sensitization to therapy or nonprotective, where autophagy inhibition does not alter sensitivity to chemotherapy and/or ionizing radiation. To determine the mode of autophagy in murine metastatic carcinoma 4T1 cell lines, autophagy was inhibited pharmacologically using chloroquine, and by genetic silencing of the Atg5 autophagy associated protein. Cells were treated with 1 μM Adriamycin, stained with Acridine Orange to assess autophagy by fluorescent microscopy (autophagosomes appear orange-red) and autophagy quantified by Flow Cytometry. Induction and blockade of autophagy by chloroquine was confirmed by Western blotting for the appearance of LC3B and degradation of p62. Senescence was monitored by β-galactosidase staining and quantified by Flow Cytometry based on C12FDG staining; senescence was confirmed based on the induction of p21 and p16. Adriamycin (doxorubicin) exposure resulted in breast tumor cell death as well as prolonged growth arrest; some of the arrested cells eventually recovered and formed colonies. After exposure to doxorubicin, 4T-1 cells were sorted based on intensity of β-galactosidase staining and increased size (senescent cells experience changes in size and morphology). β-galactosidase positive cells were plated and monitored over time during which some of the cells recovered the capacity to proliferate. In separate studies with mouse mammary carcinoma cells, radiation also promoted cell death and prolonged growth arrest from which some cells were able to recover proliferative capacity. Our data suggests that although chemotherapy and radiation induce prolonged growth arrest and senescence, these features are not permanent and cells are able to escape and re-emerge from the senescent state to generate proliferating daughter cells. As pharmacologic inhibition of autophagy did not result in increased sensitivity to doxorubicin in 4T1 cells (or to radiation in the mouse mammary tumor cells) or interfere with proliferative recovery, we postulate that inhibition of senescence associated pathways may block proliferative recovery and/or promote tumor cell killing in response to chemotherapy and/or radiation. Studies in progress are focused on modulation of c-myc and miR34 levels and the IL1/6/8 signaling axis as potential strategies for interference with senescence and suppression of residual (dormant) tumor growth and cancer recurrence. Citation Format: Liliya Tyutyunyk, Theresa Thekkudan, Tareq Saleh, David A. Gewirtz. Autophagy and senescence as possible mechanisms leading to proliferative recovery and escape from treatment-induced tumor dormancy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 5459. doi:10.1158/1538-7445.AM2017-5459