Murine Mammary Cells Research Articles

Tumor Receptor-Mediated Morphological Transformation and In Situ Polymerization of Diacetylene-Containing Lipidated Peptide Amphiphile on Cell Membranes for Tumor Suppression.

In situ polymerization on cell membranes can decrease cell mobility, which may inhibit tumor growth and invasion. However, the initiation of radical polymerization traditionally requires exogenous catalysts or free radical initiators, which might cause side effects in normal tissues. Herein, we synthesized a Y-type diacetylene-containing lipidated peptide amphiphile (TCDA-KFFFFK(GRGDS)-YIGSR, Y-DLPA) targeting integrins and laminin receptors on murine mammary carcinoma 4T1 cells, which underwent nanoparticle-to-nanofiber morphological transformation and in situ polymerization on cell membranes. Specifically, the polymerized Y-DLPA induced 4T1 cell apoptosis and disturbed the substance exchange and metabolism. In vitro assays demonstrated that the polymerized Y-DLPA nanofibers decreased the migration capacity of 4T1 cells, potentially suppressing tumor invasion and metastasis. When administered locally to 4T1 tumor-bearing mice, the Y-DLPA nanoparticles formed a biomimetic extracellular matrix that effectively suppressed tumor growth. This study provides an in situ polymerization strategy that can serve as an effective drug-free biomaterial with low side effects for antitumor therapy.

Syndecan-1 Plays a Role in the Pathogenesis of Sjögren's Disease by Inducing B-Cell Chemotaxis through CXCL13-Heparan Sulfate Interaction.

In Sjögren's disease (SjD), the salivary glandular epithelial cells can induce the chemotaxis of B cells by secreting B-cell chemokines such as C-X-C motif chemokine ligand 13 (CXCL13). Syndecan-1 (SDC-1) is a major transmembrane heparan sulfate proteoglycan (HSPG) predominantly expressed on epithelial cells that binds to and regulates heparan sulfate (HS)-binding molecules, including chemokines. We aimed to determine whether SDC-1 plays a role in the pathogenesis of SjD by acting on the binding of HS to B-cell chemokines. To assess changes in glandular inflammation and SDC-1 concentrations in the submandibular gland (SMG) and blood, female NOD/ShiLtJ and sex- and age-matched C57BL/10 mice were used. In the SMG of NOD/ShiLtJ mice, inflammatory responses were identified at 8 weeks of age, but increased SDC-1 concentrations in the SMG and blood were observed at 6 weeks of age, when inflammation had not yet started. As the inflammation of the SMG worsened, the SDC-1 concentrations in the SMG and blood increased. The expression of the CXCL13 and its receptor C-X-C chemokine receptor type 5 (CXCR5) began to increase in the SMG at 6 weeks of age and continued until 12 weeks of age. Immunofluorescence staining in SMG tissue and normal murine mammary gland cells confirmed the co-localization of SDC-1 and CXCL13, and SDC-1 formed a complex with CXCL13 in an immunoprecipitation assay. Furthermore, NOD/ShiLtJ mice were treated with 5 mg/kg HS intraperitoneally thrice per week for 6-10 weeks of age, and the therapeutic effects in the SMG were assessed at the end of 10 weeks of age. NOD/ShiLtJ mice treated with HS showed attenuated salivary gland inflammation with reduced B-cell infiltration, germinal center formation and CXCR5 expression. These findings suggest that SDC-1 plays a pivotal role in the pathogenesis of SjD by binding to CXCL13 through the HS chain.

Abstract PO5-24-05: The Metabolic and Epigenetic Contributions of Fibroblast Growth Factor Receptor-Mediated Breast Cancer Progression

Abstract Despite recent improvements in treatment options, breast cancer remains the second leading cause of cancer-related deaths among women, with metastasis of the primary tumor accounting for most of these deaths. Signaling by fibroblast growth factor receptors (FGFRs) is active in 85% of breast cancers and results in enhanced proliferation, migration, invasion, and therapeutic resistance of tumor cells. A common feature of breast cancer is altered cholesterol metabolism including an increase in cholesterol stored intracellularly as cholesteryl esters (CEs). In breast cancer cells, high levels of CEs are associated with aggressive disease and CEs are essential for growth and migration of cancer cells. Furthermore, there is mounting evidence that modifications to metabolic pathways impact epigenetic processes including DNA methylation and histone post-translational modifications. These epigenetic changes support tumor progression and metastasis through oncogene activation and silencing of tumor suppressors. While there have been numerous studies focused on the role of metabolic and epigenetic changes in breast cancer, none have attempted to connect these altered pathways to upstream growth factor receptor pathways, such as the FGFR pathway, in cancer cells. We hypothesize that FGFR signaling promotes changes to cholesterol metabolism and epigenetic regulation which lead to breast cancer progression. To study the proposed roles of FGFR signaling, we utilized murine mammary cells of differing metastatic abilities with inducible, oncogenic FGFR1 signaling. We also included the FGFR-dependent murine mammary tumor lines 4T1 and 4T07 which demonstrate differing metastatic capabilities. Through gene expression studies, we found an FGFR-mediated increase in cholesterol metabolic processes including cholesterol storage, uptake, and synthesis. Along with this, we found an FGFR-mediated increase in CE content in our models. Interestingly, these changes were exclusive to our highly metastatic models. Additionally, we have identified FGFR-mediated changes in the expression of histone modifying enzymes in our cell lines. Many of the enzymes modified by FGFR have been previously reported to play a role in breast cancer metastasis. To further elucidate the role of enhanced cholesterol storage in breast cancer progression, we treated cells with pharmacological inhibitors of cholesterol storage. Using in vitro assays, we demonstrate that cell survival, migration, and invasion all decrease when cholesterol storage is inhibited. Again, we observe that our highly metastatic cell line models are more sensitive to this inhibition. Taken together, these data suggest a role for FGFR signaling in promoting breast cancer progression and metastasis. With further studies, this connection may inform the development of novel therapeutic strategies to combat therapeutic resistance in breast cancer. Citation Format: Jennifer Tuokkola, Lyndsay Reese, Kaylee Schwertfeger. The Metabolic and Epigenetic Contributions of Fibroblast Growth Factor Receptor-Mediated Breast Cancer Progression [abstract]. In: Proceedings of the 2023 San Antonio Breast Cancer Symposium; 2023 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2024;84(9 Suppl):Abstract nr PO5-24-05.

Combination of verteporfin-photodynamic therapy with 5-aza-2'-deoxycytidine enhances the anti-tumour immune response in triple negative breast cancer.

Triple negative breast cancer (TNBC) is a subtype of breast cancer characterised by its high tumourigenic, invasive, and immunosuppressive nature. Photodynamic therapy (PDT) is a focal therapy that uses light to activate a photosensitizing agent and induce a cytotoxic effect. 5-aza-2'-deoxycytidine (5-ADC) is a clinically approved immunomodulatory chemotherapy agent. The mechanism of the combination therapy using PDT and 5-ADC in evoking an anti-tumour response is not fully understood. The present study examined whether a single dose of 5-ADC enhances the cytotoxic and anti-tumour immune effect of low dose PDT with verteporfin as the photosensitiser in a TNBC orthotopic syngeneic murine model, using the triple negative murine mammary tumour cell line 4T1. Histopathology analysis, digital pathology and immunohistochemistry of treated tumours and distant sites were assessed. Flow cytometry of splenic and breast tissue was used to identify T cell populations. Bioinformatics were used to identify tumour immune microenvironments related to TNBC patients. Functional experiments showed that PDT was most effective when used in combination with 5-ADC to optimize its efficacy. 5-ADC/PDT combination therapy elicited a synergistic effect in vitro and was significantly more cytotoxic than monotherapies on 4T1 tumour cells. For tumour therapy, all types of treatments demonstrated histopathologically defined margins of necrosis, increased T cell expression in the spleen with absence of metastases or distant tissue destruction. Flow cytometry and digital pathology results showed significant increases in CD8 expressing cells with all treatments, whereas only the 5-ADC/PDT combination therapy showed increase in CD4 expression. Bioinformatics analysis of in silico publicly available TNBC data identified BCL3 and BCL2 as well as the following anti-tumour immune response biomarkers as significantly altered in TNBC compared to other breast cancer subtypes: GZMA, PRF1, CXCL1, CCL2, CCL4, and CCL5. Interestingly, molecular biomarker assays showed increase in anti-tumour response genes after treatment. The results showed concomitant increase in BCL3, with decrease in BCL2 expression in TNBC treatment. In addition, the treatments showed decrease in PRF1, CCL2, CCL4, and CCL5 genes with 5-ADC and 5-ADC/PDT treatment in both spleen and breast tissue, with the latter showing the most decrease. To our knowledge, this is the first study that shows which of the innate and adaptive immune biomarkers are activated during PDT related treatment of the TNBC 4T1 mouse models. The results also indicate that some of the immune response biomarkers can be used to monitor the effectiveness of PDT treatment in TNBC murine model warranting further investigation in human subjects.

A junction-dependent mechanism drives murine mammary cell intercalation for ductal elongation.

The luminal epithelium of the mammary gland is organized into monolayers; however, it originates from multilayered terminal end buds (TEBs) during development. Although apoptosis provides a plausible mechanism for cavitation of the ductal lumen, it doesn't account for ductal elongation behind TEBs. Spatial calculations in mice suggest that most TEB cells integrate into the outermost luminal layer to generate elongation. We developed a quantitative cell culture assay that models intercalation into epithelial monolayers. We found that tight junction proteins play a key role in this process. ZO-1 puncta form at the new cellular interface and resolve into a new boundary as intercalation proceeds. Deleting ZO-1 suppresses intercalation both in culture and in cells transplanted into mammary glands via intraductal injection. Cytoskeletal rearrangements at the interface are critical for intercalation. These data identify luminal cell rearrangements necessary for mammary development and suggest a mechanism for integration of cells into an existing monolayer.

Abstract 5101: Inhibition of Notch reverses immunosuppression in basal-like breast cancer

Abstract Aberrant Notch activation is a defining feature of basal-like breast cancer (BLBC), which has poor prognosis compared to other breast cancer molecular subtypes. Despite the clinical success of immune checkpoint blockade (ICB) in many malignancies including melanoma and small cell lung cancer, ICB has failed to demonstrate similar response in BLBCs, where most cases are highly infiltrated by tumor-associated macrophages (TAMs). There is increasing evidence that Notch is decisive in regulating intercellular communication in the tumor immune microenvironment (TIME). This includes the recruitment of TAMs which contribute to an immunosuppressive TIME, illuminating the potential of Notch-inhibition as adjuvant immunotherapy in BLBC. To examine the immune phenotype and therapeutic response of BLBC to combined Notch-inhibition and ICB, we employed an in vivo tumorgraft model using murine basal-like mammary tumor 4T1 cells. Briefly, tumor cells were orthotopically injected into the mammary fat pads of BALB/c mice. Using a crossover design, mice were randomly allocated to treatment with either Notch inhibitor (γ-secretase inhibitor, LY411575), anti-PD1 (RMP1-14), or control treatment for 12-days (stage-1), followed by randomization to a second 12-day period (stage 2) with the same, or one of the other treatments. Results: Despite the low response rate of BLBC to ICB alone, Notch inhibition reduced TAMs and induced responsiveness to sequential ICB. This response was characterized by increased cytotoxic T lymphocytes (GrB+, CTL) infiltration of the primary tumor. Similar results were observed when Notch-regulated cytokines (IL-1β and CCL2), crucial to TAM recruitment, were inhibited. Moreover, a more impressive therapeutic effect of sequential treatment was observed in lung metastasis, whereby TAM depletion and increased CTL infiltration were accompanied with near-complete abolition of metastases. Mechanistically, tumor cell Notch signaling upregulates a group of circulating cytokines including IL-1β and CCL2, which prime the lung for metastases. Additionally, compared to primary tumor cells, PD ligand 1 is up-regulated in lung metastases, rendering them profoundly sensitive to sequential ICB treatment. These findings highlight the potential of sequential Notch inhibition and ICB as a novel immunotherapeutic strategy in BLBC. Citation Format: Qiang Shen, Kiichi Murakami, Pamela Ohashi, Michael Reedijk. Inhibition of Notch reverses immunosuppression in basal-like breast cancer. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 5101.

Abstract 6638: Pulsed electric fields combined with anti-PD1 prolongs survival and triggers an adaptive immune response in an IO-non-responsive orthotopic mouse model

Abstract Background: Although anti-PD1 therapy is standard of care in multiple tumor types, unmet need remains for those non-responsive to immunotherapy. Pulsed Electric Field (PEF) treatment may alter the tumor micro-environment, converting an immune “cold” tumor into an immune “hot” tumor, triggering an adaptive immune response and abscopal effect. We evaluated PEF in combination with anti-PD1 in a highly aggressive orthotopic triple negative breast tumor mouse model. Methods: Murine mammary 4T1 cells (2 × 105) non-responsive to anti-PD1 therapy were orthotopically implanted in mammary fat of 48 female Balb/C mice. Once tumors were established (5 mm diameter), mice were randomized into six groups: Sham+IgG controls, aPD-1-only, PEF (1X), PEF (2X), aPD-1 + PEF (1X), and aPD-1 + PEF (2X). Groups 4 and 6 received two PEF treatments, five days apart. Anti-PD1 and isotype matched control IgG was administered (200 ug, intraperitoneal) weekly, starting 3 days after PEF treatment. Tumor volumes were recorded 3x/week and standard survival analyses were conducted per IACUC protocol. In the cohort of tumors collected 30 days after PEF, flow cytometry (FC) evaluated T-cell immune infiltrates and associated markers. Blood was collected at different time points to evaluate systemic immunological response. Tumor-specific cytotoxic lymphocytes (CTL) were isolated from tumor-infiltrating and circulating lymphocytes by gp70 MHC tetramer formation assay. Results: Single PEF reduced tumor growth compared to IgG and aPD-1-only groups by 65% and 62%, respectively. Double PEF treatment reduced tumor growth by 74% and prolonged survival. FC demonstrated circulating helper T-cells and CTL frequency was increased two weeks post-PEF compared to the control group. The tetramer detected substantial increases in antigen-specific T-cell populations in both single and double PEF treatment groups. PEF groups had significant tumor and spleen infiltration of CD8 and CD4 T-cells, including increased tetramer levels. Anti-PD1-only animals showed no meaningful reductions in tumor growth, prolonged survival, or significant changes in CD8, CD4 T cells or in the tetramer assay. Conclusions: PEF treatments prior to initiation of anti-PD1 in orthotopic 4T1 mouse model triggers a tumor-specific adaptive immune response in an immune “cold” tumor model, irrespective of anti-PD-1 therapy. PEF increases circulating and tumor-infiltrating T-cells and antigen-specific T-cells, with these effects observed after a single or two treatments with PEF. Collectively, our results demonstrate that PEF treatment prior to initiation of anti-PD1 treatment can reduce tumor growth, prolong survival, and activate an adaptive immune response in an immune “cold” tumor. These findings suggest that PEF treatment merits further evaluation in IO-non-responsive cancer patients. Citation Format: Ebtesam Nafie, Mukta Wagh, Chiara Pastori, Mo Trikha, Robert Neal. Pulsed electric fields combined with anti-PD1 prolongs survival and triggers an adaptive immune response in an IO-non-responsive orthotopic mouse model. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 6638.

Abstract P6-11-08: Non-transformed breast epithelial cells show neural-like gene signature after lipid exposure

Abstract Introduction: The identification of women specifically at risk for estrogen receptor negative breast cancer (ER-BC) and the targeted treatment of this disease are significantly unmet clinical needs. To that end, we analyzed the gene expression profiles of epithelial cells from the contralateral unaffected breasts (CUBs) of BC patients and identified a lipid metabolism gene signature, which was enriched in the CUBs of women with ER-negative BC (PMID: 28263391). Subsequent experiment revealed that exposure of non-transformed breast epithelial cells to lipid results in significant changes gene expression, chromatin accessibility and histone posttranslational modifications (PMID: 35508495). Several of the upregulated genes are hallmarks of the various fates of vagal neural crest: Neural, neurogenic and mesenchymal lineages. We hypothesize that lipid exposure imparts a survival advantage of stem-like cells, that lipid-induced epigenetic changes lead to a neural crest-like transcription signature and that these genes are not expressed normally in the breast. Methods: MCF10A cells were exposed to vehicle or octanoic acid (OA) for 24 hours. Gene expression was assayed by RNA-seq and OA responsive genes were identified (PMID: 35508495). Single-cell RNA sequencing (scRNA-seq) data from 14 human reduction mammoplasties (RM) was obtained from a publicly accessible data set (PMID: 34031589). The scRNA data was clustered and identified by unsupervised clustering (Seurat, v3.4.1) using cell-type markers curated using Supplementary table 2 from (PMID: 34031589). The bulk RNA-seq data from the OA treated cells was deconvoluted to cell-lineages using Bisque. The most significant upregulated VNC neural/neuronal/mesenchymal genes from the gene expression analysis were then plotted on the lineage clusters using FeaturePlot to determine if these markers are found in the normal breast epithelium, or other cell lineages. The plots were then filtered and re-clustered to look at basal-luminal cell types only. We utilized a second resource, a web-application for snATAC-seq data from various stages of mouse mammary development developed by the Wahl lab (https://wahl-lab-salk.shinyapps.io/Mammary_snATAC/), to query these same genes. Results: Deconvolution of the bulk RNA-sequencing data revealed a transition to a pericyte transcription program following exposure to OA. Nerve growth factor (NGF) was found to be expressed in pericytes while nerve growth factor receptor (NGFR) was found within the basal epithelial cell lineage. Genes overexpressed in the VNC neural cluster and overexpressed in the OA-exposed MCF10 cells, PPP1R1C (2.39x, adj p=1.6E-5), FOXD3 (6.7x, adj p=6.7E-10), DIO3 (5.9x, adj p=3.9E-6) and MOXD3 (4.2x, adj p=1.5E-23) all evidence little to no expression in the normal breast but are observed in murine fetal mammary stem cells. Schwann cell precursor (SCP) markers, CDH19 and ROPN1, significantly upregulated in OA treated cells: 5.4x and 1.6x, respectively, exhibited low expression in luminal progenitors but in the mouse were observed in the mammary stroma. CDH19, a gene exclusive to SCPs, is expressed in stroma following murine birth. PRRX1, a key regulator of the VNC mesenchymal cell fate cluster, is 9.3-fold (p adj=4.9E-49) overexpressed in the OA treated cells, expressed strongly in pericytes and stroma and to a lesser extent in basal epithelial cells of the normal human breast and stroma of the murine mammary gland. Conclusions: Treatment of non-transformed mammary epithelial cells with lipid, specifically OA, shows significant upregulation of multiple VNC genes associated with both neural and mesenchymal fate. scRNA-seq from RM patients reveals that many of these same markers are either found in non-epithelial cell clusters or are found with low expression in luminal mammary lineages (both progenitors and mature). Citation Format: Gannon Cottone, Mariana Bustamante Eduardo, Shivangi Yadav, Seema Khan, Susan Clare. Non-transformed breast epithelial cells show neural-like gene signature after lipid exposure [abstract]. In: Proceedings of the 2022 San Antonio Breast Cancer Symposium; 2022 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2023;83(5 Suppl):Abstract nr P6-11-08.

Prevalence, Virulence, and Antibiotics Gene Profiles in Lactococcus garvieae Isolated from Cows with Clinical Mastitis in China.

Lactococcus garvieae (L. garvieae) is a pathogenic gram-positive, catalase-negative (GPCN) bacterium that causes bovine mastitis. A total of 49 L. garvieae isolates were identified from 1441 clinical mastitis (CM) samples. The pathogenic effects of L. garvieae were studied with two infection models: bovine mammary epithelial cells cultured in vitro and murine mammary infections in vivo. The overall farm prevalence was 15.5% (13/84 farms in 9/19 provinces) and sample prevalence was 3.40% (49/1441). Post-treatment somatic cell count (SCC) post L. garvieae infection was significantly higher than the other GPCN pathogens isolated, and the bacteriological cure fraction was 41.94% (13/31) after intramammary antibiotic treatment. All L. garvieae isolates were resistant to rifaximin, 12.24% of isolates were resistant to cephalexin, and 10.20% (5/49) were multidrug-resistant (MDR). The most prevalent virulence genes were Hemolysin 1 (hly1)(100%), Hemolysin 2 (hly2) (97.96%), NADH oxidase (NADHO) (100%), Superoxide dismutase (SOD) (100%), Adhesin Pav (Pav) (100%), Adhesin PsaA (PsaA) (100%), Enolase (eno) (100%), Adhesin cluster 1(AC1) (100%), Adhesin cluster 2 (AC2) (100%), and several exopolysaccharides. L. garvieae rapidly adhered to bovine mammary epithelial cells, resulting in an elevated lactate dehydrogenase release. Edema and congestion were observed in challenged murine mammary glands and bacteria were consistently isolated at 12, 24, 48, 72, and 120 h after infection. We concluded that L. garvieae had good adaptive ability in the bovine and murine mammary cells and tissue. Given the resistance profile, penicillin and ampicillin are potential treatments for CM cases caused by L. garvieae.

PEGylation of genistein-loaded bovine serum albumin nanoparticles and its effect on in vitro cell viability and genotoxicity properties

Self-assembled bovine serum albumin nanoparticles loaded with the isoflavone genistein have shown apoptosis-mediated cytotoxicity against murine mammary adenocarcinoma F3II cells. Due to their protein nature and small particle size (13–15 nm), their parenteral administration could be affected by possible immunogenic reactions and rapid clearance from the bloodstream. To avoid these problems, PEGylation of the systems was achieved in this work by using a 30 kDa methoxy-polyethylene glycol carbonyl imidazole derivative through the reaction between the carbonyl imidazole group and the amino groups of Lys residues on the protein surface, which was confirmed by a 17% reduction in the available amino groups content measured by the o-phthaldialdehyde method. PEGylated isoforms were obtained, showing an increase of particle size from 13 to 15 nm to around 260 nm, and were purified by SEC-FPLC and characterized by SDS-PAGE, DLS and AFM techniques. The effect of PEGylation on BSAnp-Gen cytotoxicity and genotoxicity against F3II cells was evaluated in vitro by MTT assay, flow cytometry analysis and micronucleus assay. From the results, PEGylation produced an improvement of the biological properties of genistein-loaded nanoparticles in terms of cytotoxicity (lower IC50), not affecting the induction of apoptosis, decreasing the genotoxicity of the systems (less induction of micronucleus formation).

LncRNA EPR-induced METTL7A1 modulates target gene translation.

EPR is a long non-coding RNA (lncRNA) that controls cell proliferation in mammary gland cells by regulating gene transcription. Here, we report on Mettl7a1 as a direct target of EPR. We show that EPR induces Mettl7a1 transcription by rewiring three-dimensional chromatin interactions at the Mettl7a1 locus. Our data indicate that METTL7A1 contributes to EPR-dependent inhibition of TGF-β signaling. METTL7A1 is absent in tumorigenic murine mammary gland cells and its human ortholog (METTL7A) is downregulated in breast cancers. Importantly, re-expression of METTL7A1 in 4T1 tumorigenic cells attenuates their transformation potential, with the putative methyltransferase activity of METTL7A1 being dispensable for its biological functions. We found that METTL7A1 localizes in the cytoplasm whereby it interacts with factors implicated in the early steps of mRNA translation, associates with ribosomes, and affects the levels of target proteins without altering mRNA abundance. Overall, our data indicates that METTL7A1—a transcriptional target of EPR—modulates translation of select transcripts.

Antitumor Properties of Curcumin in Breast Cancer Based on Preclinical Studies: A Systematic Review

Simple SummaryNatural formulations and phytotherapies have shown promising antitumor activities. This review assesses the antitumor effects of curcumin on breast cancer. In particular, we discuss the effects of curcumin on the proliferation, viability, and apoptosis of breast cancer cell lineages and tumor volume. Studies have shown that curcumin administered at different concentrations inhibited proliferation, decreased viability, and induced apoptosis in human and animal breast cancer cells. Nanoparticle formulations of curcumin administered orally, via implant, or intraperitoneally reduced the tumor volume of human and murine mammary cells in vivo. Moreover, curcumin nanoformulations facilitate tumor growth inhibition in animal models of breast cancer. Randomized clinical trials are warranted to assess the efficacy and safety of curcumin formulations for clinical use.Breast cancer is one of the most common neoplasms among women. Anticancer strategies using natural formulations and phytotherapies are promising antitumor treatment alternatives. This review assesses the antitumor effects of curcumin on breast cancer reported in preclinical in vitro and in vivo animal models. We used five databases to search for preclinical studies published up to May 2021. The assessments included the effects of curcumin on the proliferation, viability, and apoptosis of breast cancer cell lineages and on tumor volume. In total, 60 articles met the inclusion criteria. Curcumin administered at different concentrations and via different routes of administration inhibited proliferation, decreased viability, and induced apoptosis in human and animal breast cancer cells. Nanoparticle formulations of curcumin administered orally, via implant, and intraperitoneally reduced the tumor volume of human and murine mammary cells in vivo. Moreover, curcumin nanoformulations exert positive effects on tumor growth inhibition in animal models of breast cancer. Further randomized clinical trials are warranted to assess the efficacy and safety of curcumin formulations for clinical use.

Time-restricted feeding alters the efficiency of mammary tumor growth

ABSTRACT Disruption of circadian rhythms has detrimental host consequences. Indeed, both clinical and foundational science demonstrate a clear relationship between disruption of circadian rhythms and cancer initiation and progression. Because timing of food intake can act as a zeitgeber (i.e., entrainment signal) for the circadian clock, and most individuals in the developed world have access to food at all times of the day in a “24/7” society, we sought to determine the effects of timing of food intake on mammary tumor growth. We hypothesized that restricting access to food to during the inactive phase would accelerate tumor growth. Adult female Balb/C mice received a unilateral orthotopic injection of murine mammary carcinoma 4T1 cells into the ninth inguinal mammary gland. Beginning on the day of tumor injection and continuing until the end of the experiment, mice were food restricted to their active phase (ZT12 (lights off)- ZT0 (lights on), inactive phase (ZT0 – ZT12), or had ad libitum access to food. Mice that were food restricted to their inactive phase displayed a significant increase in body mass on days 7 and 14 of tumor growth relative to active phase or ad libitum fed mice. Additionally, mice fed during their inactive phase demonstrated a 20% reduction in food consumption relative to mice fed during their active phase and a 17% reduction in food consumption relative to ab libitum fed mice. Tumor volume was not significantly different between groups. However, food restricting mice to their inactive phase increased mammary tumor growth efficiency (i.e., mg of tumor mass per gram of food intake) relative to mice fed during the active phase and approached significance (p = .06) relative to ad libitum fed mice. To determine a potential explanation for the increased tumor growth efficiency, we examined rhythms of activity and body temperature. Mice fed during the inactive phase displayed significantly disrupted daily activity and body temperature rhythms relative to both other feeding regimens. Together, these data demonstrate that improperly timed food intake can have detrimental consequences on mammary tumor growth likely via disrupted circadian rhythms.

Anti-Tumor Necrosis Factor Receptor 2 Antibody Combined With Anti-PD-L1 Therapy Exerts Robust Antitumor Effects in Breast Cancer.

Breast cancer is a leading type of malignant tumor in women; however, the immunotherapy in breast cancer is still underappreciated. In this study, we demonstrated that tumor necrosis factor receptor 2 (TNFR2) is highly expressed in both breast tumor tissue and tumor-infiltrating immunosuppressive CD4+Foxp3+ regulatory T cells (Tregs). We found that TNFR2 antagonistic antibody reduced Foxp3 expression and the proliferation of Tregs and impaired the inhibitory effect of Tregs on CD4+CD25– effector T (Teff) cells in a dose-dependent manner. The treatment of anti-TNFR2 antibody not only inhibited the proliferation of breast tumor cells in vitro but also suppressed the tumorigenesis of murine mammary carcinoma 4T1 cells in vivo. Mice recovered from tumor growth also developed 4T1-specific immunity. Furthermore, we demonstrated that anti-TNFR2 antibody in combination with anti-PD-L1 exhibited augmented antitumor effects than monotherapy. Anti-TNFR2 treatment also tended to increase the expression of proinflammatory cytokines in tumor tissues. In conclusion, our study suggests that TNFR2 antagonist could potentially offer a clinical benefit as a single agent or in combination with immune checkpoint blockade treatment for breast cancer immunotherapy.

Effects of Stryphnodendron adstringens extracts on murine 4T1 tumor line

Breast cancer appears as the main gynecological cancer and presents high morbidity and mortality. Because most diagnoses are made belatedly, it is necessary to seek therapeutic options that aim for advanced stages of the disease. This study aims to evaluate the antitumoral action of Stryphnodendron adstringens fruit extracts on 4T1 murine mammary carcinoma cell culture. The inhibitory potential of S. adstringens fruit extract on the metalloproteinases (MMPs) 2 and 9 was evaluated through zymography. From these results, MTT assays were performed to evaluate the extracts’ effects on the murine mammary carcinoma 4T1 line cell viability. From the crude extract, the following extracts were obtained: hydroalcoholic (SAFCEA), hexane (SAFCEB), chloroform (SAFCEC), and ethyl acetate (SAFCED). Lastly, the migration of the cells treated with extracts SAFCEA and SAFCED was verified. The hydroalcoholic extract (SAFCEA) was the most efficient in inhibiting gelatinases. During the phytochemical study, it was noted that alkaloids were present in all partitions. The 50 % growth inhibition (IC50) concentrations found were: 40.1 μg/mL (SAFCEA) and 70.14 μg/mL (SAFCED). After the cellular cytotoxicity assay, cell morphology was altered by treatment with the selected partitions (SAFCEA and SAFCED), obtaining morphology consistent with apoptosis. The results demonstrate that S. adstringens extracts exhibit the inhibitory activity of MMP-2 and MMP-9 as well as cytotoxicity toward 4T1 tumor cells. These findings indicate that follow-up studies of the partitions from S. adstringens may lead to the development of novel chemotherapeutics for oncological treatments.

Abstract 2649: Isolation and characterization of novel murine mammary cell line with dormant phenotype and identification of new potential targets by mass spectrometry

Abstract Breast cancer is the first cause of death from female cancer. After treatments against the primary tumor, a few cells can prevail in a dormant state for a long period, becoming minimal residual disease. These cells can resume growth and establish as metastasis, which are responsible for 90% of deaths from cancer. Patients with breast cancer diagnosed at early stage with small tumours have a 25-30% chance of recurrence after 10-15 years after treatment. These new growth often entails chemo and radio resistance and there is not response to conventional treatment. For all these reasons, there is a clear need for the development of novel therapies against these cells in a dormant state. The main limitation on the study of these dormant cells is the lack of relevant in vitro an in vivo models. In this work, we isolated and characterized two novel cell lines F3II-TP and F3II-NM. F3II-TP represents tumours with high proliferation rate whereas F3II-NM represents a dormant cell phenotype. The comparison between these cell lines is expected to yield new molecular targets for the design of new drugs that point dormant cells. The study began with the isolation and establishment of new tumour cell lines. After inoculation of the F3II murine mammary carcinoma cell line, we established cell populations in vitro, one from the primary tumour and another from a spontaneous metastatic nodule, F3II TP and F3II NM cell lines respectively. First, we analysed the expression of EMT markers such as E-cadherin, N-cadherin, Vimentin, Pan-Cytoqueratin and β-catenin by immunofluorescence. In in vitro assays, F3II NM presented major doubling time, major adhesion capacity and lower clonogenic potential. In addition, the expression of the dormancy related genes such as NR2F1 and BHLHE41 was analysed by qPCR, and we found that F3II-NM showed higher levels of transcript than F3II-TP. In addition, F3II NM showed lower chemo sensibility to cisplatin and in an orthotopic model of BALB/c mice, presented a longer latency time and lower tumour growth rate in comparison to F3II-TP. Taking all these together, F3II NM present a dormant phenotype and F3II TP a proliferative one. Finally, analysis and comparison of the cell lines using LFQ-Mass spectrometry revealed 256 differentially expressed proteins between the lines studied. Overexpressed genes of survival paths were identified in F3II-NM, such as DDX42, COX-2, ATGB4 involved in the inhibition of apoptosis, promotion of tumour recurrence via SOX-2 and therapy resistance, respectively. To sum up, we established a new interesting dormant tumour cell model, F3II NM that could help to a better understanding of the behaviour and survival of dormant cells. Moreover, we identified interesting protein targets that encourage the possibility of design new drugs that point and eliminate dormant cells Citation Format: Johanna E. Sidabra, Carla S. Capobianco, Maria F. Gottardo, Daniel F. Alonso, Hernan G. Farina. Isolation and characterization of novel murine mammary cell line with dormant phenotype and identification of new potential targets by mass spectrometry [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 2649.

ADAR1-Dependent RNA Editing Promotes MET and iPSC Reprogramming by Alleviating ER Stress.

RNA editing of adenosine to inosine (A to I) is catalyzed by ADAR1 and dramatically alters the cellular transcriptome, although its functional roles in somatic cell reprogramming are largely unexplored. Here, we show that loss of ADAR1-mediated A-to-I editing disrupts mesenchymal-to-epithelial transition (MET) during induced pluripotent stem cell (iPSC) reprogramming and impedes acquisition of induced pluripotency. Using chemical and genetic approaches, we show that absence of ADAR1-dependent RNA editinginducesaberrant innate immune responses through the double-stranded RNA (dsRNA) sensor MDA5, unleashing endoplasmic reticulum (ER) stress and hindering epithelial fate acquisition. We found that A-to-I editing impedes MDA5 sensing and sequestration of dsRNAs encoding membrane proteins, which promote ER homeostasis by activating the PERK-dependent unfolded protein response pathway to consequently facilitate MET. This study therefore establishes a critical role for ADAR1 and its A-to-I editing activity duringcell fate transitions and delineates a key regulatory layer underlying MET to control efficient reprogramming.

Radiation-induced abnormal centrosome amplification and mitotic catastrophe in human cervical tumor HeLa cells and murine mammary tumor EMT6 cells.

Mitotic catastrophe is a form of cell death linked to aberrant mitosis caused by improper or uncoordinated mitotic progression. Abnormal centrosome amplification and mitotic catastrophe occur simultaneously, and some cells with amplified centrosomes enter aberrant mitosis, but it is not clear whether abnormal centrosome amplification triggers mitotic catastrophe. Here, to investigate whether radiation-induced abnormal centrosome amplification is essential for induction of radiation-induced mitotic catastrophe, centrinone-B, a highly selective inhibitor of polo-like kinase 4, was utilized to inhibit centrosome amplification, since polo-like kinase 4 is an essential kinase in centrosome duplication. When human cervical tumor HeLa cells and murine mammary tumor EMT6 cells were irradiated with 2.5 Gy of X-rays, cells with morphological features of mitotic catastrophe and the number of cells having >2 centrosomes increased in both cell lines. Although centrinone-B significantly inhibited radiation-induced abnormal centrosome amplification in both cell lines, such treatment did not change cell growth and significantly enhanced mitotic catastrophe in HeLa cells exposed to X-rays. In contrast, inhibition of centrosome amplification reduced cell growth and mitotic catastrophe in EMT6 cells exposed to X-rays. These results indicated that the role of radiation-induced abnormal centrosome amplification in radiation-induced mitotic catastrophe changes, depending on the cell type.

HIV-1 Reverse Transcriptase Promotes Tumor Growth and Metastasis Formation via ROS-Dependent Upregulation of Twist.

HIV-induced immune suppression results in the high prevalence of HIV/AIDS-associated malignancies including Kaposi sarcoma, non-Hodgkin lymphoma, and cervical cancer. HIV-infected people are also at an increased risk of “non-AIDS-defining” malignancies not directly linked to immune suppression but associated with viral infections. Their incidence is increasing despite successful antiretroviral therapy. The mechanism behind this phenomenon remains unclear. Here, we obtained daughter clones of murine mammary gland adenocarcinoma 4T1luc2 cells expressing consensus reverse transcriptase of HIV-1 subtype A FSU_A strain (RT_A) with and without primary mutations of drug resistance. In in vitro tests, mutations of resistance to nucleoside inhibitors K65R/M184V reduced the polymerase, and to nonnucleoside inhibitors K103N/G190S, the RNase H activities of RT_A. Expression of these RT_A variants in 4T1luc2 cells led to increased production of the reactive oxygen species (ROS), lipid peroxidation, enhanced cell motility in the wound healing assay, and upregulation of expression of Vimentin and Twist. These properties, particularly, the expression of Twist, correlated with the levels of expression RT_A and/or the production of ROS. When implanted into syngeneic BALB/C mice, 4T1luc2 cells expressing nonmutated RT_A demonstrated enhanced rate of tumor growth and increased metastatic activity, dependent on the level of expression of RT_A and Twist. No enhancement was observed for the clones expressing mutated RT_A variants. Plausible mechanisms are discussed involving differential interactions of mutated and nonmutated RTs with its cellular partners involved in the regulation of ROS. This study establishes links between the expression of HIV-1 RT, production of ROS, induction of EMT, and enhanced propagation of RT-expressing tumor cells. Such scenario can be proposed as one of the mechanisms of HIV-induced/enhanced carcinogenesis not associated with immune suppression.

Inferring signalling dynamics by integrating interventional with observational data.

MotivationIn order to infer a cell signalling network, we generally need interventional data from perturbation experiments. If the perturbation experiments are time-resolved, then signal progression through the network can be inferred. However, such designs are infeasible for large signalling networks, where it is more common to have steady-state perturbation data on the one hand, and a non-interventional time series on the other. Such was the design in a recent experiment investigating the coordination of epithelial–mesenchymal transition (EMT) in murine mammary gland cells. We aimed to infer the underlying signalling network of transcription factors and microRNAs coordinating EMT, as well as the signal progression during EMT.ResultsIn the context of nested effects models, we developed a method for integrating perturbation data with a non-interventional time series. We applied the model to RNA sequencing data obtained from an EMT experiment. Part of the network inferred from RNA interference was validated experimentally using luciferase reporter assays. Our model extension is formulated as an integer linear programme, which can be solved efficiently using heuristic algorithms. This extension allowed us to infer the signal progression through the network during an EMT time course, and thereby assess when each regulator is necessary for EMT to advance.Availability and implementationR package at https://github.com/cbg-ethz/timeseriesNEM. The RNA sequencing data and microscopy images can be explored through a Shiny app at https://emt.bsse.ethz.ch.Supplementary information Supplementary data are available at Bioinformatics online.