Co-culture Of Breast Cancer Cells Research Articles

Evaluation of Tumorigenic Properties of MDA-MB-231 Cancer Stem Cells Cocultured with Telocytes and Telocyte-Derived Mitochondria Following miR-146a Inhibition.

Telocytes have some cytoplasmic extensions called telopodes, which are thought to play a role in mitochondrial transfer in intercellular communication. Besides, it is hypothesized that telocytes establish cell membrane-mediated connections with breast cancer cells in coculture and may contribute to the survival of neoplastic cell clusters together with other stromal cells. The aim of this study is to investigate the contribution of telocytes and telocyte-derived mitochondria, which have also been identified in breast tumors, to the tumor development of breast cancer stem cells (CSCs) via miR-146a-5p. The isolation/characterization of telocytes from bone marrow mononuclear cells and the isolation of mitochondria from these cells were performed, respectively. In the next step, CSCs were isolated from the MDA-MB-231 cell line and were characterized. Then, miR-146a-5p expressions of CSCs were inhibited by anti-miR-146a-5p. The epithelial-mesenchymal transition (EMT) was determined by evaluating changes in vimentin protein levels and was evaluated by analyzing BRCA1, P53, SOX2, E-cadherin, and N-cadherin gene expression changes. Our results showed that miR-146a promoted stemness and oncogenic properties in CSCs. EMT (N-cadherin, vimentin, E-cadherin) and tumorigenic markers (BRCA1, P53, SOX2) of CSCs decreased after miR-146a inhibition. Bone marrow-derived telocytes and mitochondria derived from telocytes favored the reduction of CSC aggressiveness following this inhibition.

Abstract LB317: TNBC cell co-culture with primary human lung fibroblasts identifies a paracrine-juxtacrine signaling loop promoting fibroblast activation and autophagy-dependent breast cancer cell therapy resistance

Abstract Triple negative breast cancer (TNBC) carries a poorer prognosis than other breast cancer subtypes due to high metastatic incidence, lack of molecular drug targets, and rapid development of chemoresistance. Cancer-associated fibroblasts (CAFs) are key extrinsic drivers of chemoresistance in primary TNBC tumors, but it is unknown whether resident fibroblasts in distant metastatic tissues also influence TNBC drug sensitivity. Since the lung is a top metastatic site in TNBC, we developed a breast cancer cell (BCC) - primary human donor-derived lung fibroblast (LF) co-culture model to identify therapeutic vulnerabilities and mechanisms of fibroblast-mediated extrinsic chemoresistance in lung-metastatic TNBC. We characterized the growth and chemoresistance phenotypes of BCC co-cultured with primary LFs via bioluminescence and fluorescence imaging and evaluated their transcriptional and secretory profiles using bulk and scRNAseq and multiplex cytokine analysis. Co-culture of human BCC and primary LFs resulted in BCC molecular subtype-dependent changes in cell growth and drug resistance compared with conventional monoculture. Moreover, we also observed increased LF secretion of the immunomodulatory cytokines IL-6 and CCL2 in response to both BCC co-culture and BCC-conditioned media. Transcriptomic analysis of LFs sorted from co-culture identified upregulation of pro-inflammatory and pro-fibrotic iCAF and myCAF-like gene signatures, while co-cultured TNBC cells upregulated interferon response-mediated multi-therapy resistance genes and cellular dormancy signatures. We applied this model in a high throughput screen of 846 kinase inhibitor compounds, which identified reproducible differential BCC responses to kinase inhibitors between monoculture and co-culture conditions. When co-cultured with LFs, BCC were broadly more resistant to kinase inhibitor compounds but showed increased sensitivity to inhibitors of the lipid kinase VPS34, an important initiator of autophagy. We subsequently established that autophagy is increased in co-cultured TNBC cells and contributes to LF-mediated resistance to kinase inhibitors. Moreover, we determined that while paracrine signals from BCC can promote LF activation indicated by enhanced cytokine secretion, cell-cell contact is required for LF modulation of TNBC therapy responses in co-culture. Thus, we conclude that breast cancer cell co-culture with primary lung fibroblasts is a scalable in vitro model that can facilitate therapeutic discovery and mechanistic evaluation of extrinsic modulation of therapy responses in metastatic breast cancer, and further demonstrate that BCC can promote paracrine activation of co-cultured LFs in association with juxtacrine induction of BCC autophagy and extrinsic therapy resistance. Citation Format: Marika L. Klosowski, Kathryn Cronise, Claire Stratton, Qiong Zhou, Hector Esquer, Daniel LaBarbera, Daniel Regan. TNBC cell co-culture with primary human lung fibroblasts identifies a paracrine-juxtacrine signaling loop promoting fibroblast activation and autophagy-dependent breast cancer cell therapy resistance [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 2 (Late-Breaking, Clinical Trial, and Invited Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(7_Suppl):Abstract nr LB317.

FAM3C in Cancer-Associated Adipocytes Promotes Breast Cancer Cell Survival and Metastasis.

High FAM3C levels in cancer-associated adipocytes contribute to tumor-supportive niches and are tightly associated with metastatic growth, indicating that FAM3C inhibition could be beneficial for treating patients with breast cancer.

Modulation of SLFN11 induces changes in DNA Damage response in breast cancer

BackgroundLack of Schlafen family member 11 (SLFN11) expression has been recently identified as a dominant genomic determinant of response to DNA damaging agents in numerous cancer types. Thus, several strategies aimed at increasing SLFN11 are explored to restore chemosensitivity of refractory cancers. In this study, we examined various approaches to elevate SLFN11 expression in breast cancer cellular models and confirmed a corresponding increase in chemosensitivity with using the most successful efficient one. As oncogenic transcriptomic downregulation is often driven by methylation of the promotor region, we explore the demethylation effect of 5-aza-2′-deoxycytidine (decitabine), on the SLFN11 gene. Since SLFN11 has been reported as an interferon inducible gene, and interferon is secreted during an active anti-tumor immune response, we investigated the in vitro effect of IFN-γ on SLFN11 expression in breast cancer cell lines. As a secondary approach to pick up cross talk between immune cells and SLFN11 expression we used indirect co-culture of breast cancer cells with activated PBMCs and evaluated if this can drive SLFN11 upregulation. Finally, as a definitive and specific way to modulate SLFN11 expression we implemented SLFN11 dCas9 (dead CRISPR associated protein 9) systems to specifically increase or decrease SLFN11 expression.ResultsAfter confirming the previously reported correlation between methylation of SLFN11 promoter and its expression across multiple cell lines, we showed in-vitro that decitabine and IFN-γ could increase moderately the expression of SLFN11 in both BT-549 and T47D cell lines. The use of a CRISPR-dCas9 UNISAM and KRAB system could increase or decrease SLFN11 expression significantly (up to fivefold), stably and specifically in BT-549 and T47D cancer cell lines. We then used the modified cell lines to quantify the alteration in chemo sensitivity of those cells to treatment with DNA Damaging Agents (DDAs) such as Cisplatin and Epirubicin or DNA Damage Response (DDRs) drugs like Olaparib. RNAseq was used to elucidate the mechanisms of action affected by the alteration in SLFN11 expression. In cell lines with robust SLFN11 promoter methylation such as MDA-MB-231, no SLFN11 expression could be induced by any approach.ConclusionTo our knowledge this is the first report of the stable non-lethal increase of SLFN11 expression in a cancer cell line. Our results show that induction of SLFN11 expression can enhance DDA and DDR sensitivity in breast cancer cells and dCas9 systems may represent a novel approach to increase SLFN11 and achieve higher sensitivity to chemotherapeutic agents, improving outcome or decreasing required drug concentrations. SLFN11-targeting therapies might be explored pre-clinically to develop personalized approaches.

Biological differences between normal and cancer-associated fibroblasts in breast cancer

BackgroundCancer-associated fibroblasts (CAFs) constitute the primary constituents of the tumor microenvironment (TME) and exert significant influences on cancer progression. However, adequate comprehension of CAF profiles in breast cancer, as well as the precise mechanisms underlying their promotion of cancer, remains lacking. ObjectivesTo discerns the biological differences between normal fibroblasts (NFs) and CAFs in breast cancer and explore the underlying mechanism. MethodsThree pairs of CAFs and NFs were isolated from breast cancer patients of diverse subtypes who had not undergone prior radiotherapy or chemotherapy. Morphological characteristics of CAFs and NFs were assessed through optical and electron microscopy, their biological attributes were examined using cell counting kits and transwell assays, and their impact on breast cancer cells was simulated using a coculture system. Furthermore, the miRNA profiles of CAFs and NFs were sequenced via an Illumina HiSeq 2500 platform. ResultsCAFs exhibited higher growth rate and motility than NFs and a stronger potential to promote the malignancy of breast cancer cells. RNA sequencing of both NFs and CAFs revealed differentially expressed miRNAs with notable variability among distinct patients within their NFs and CAFs, while the enrichment of the target genes of differentially expressed miRNAs within both GO terms and KEGG pathways demonstrated significant similarity across patients with different profiles. ConclusionCAFs have greater malignancy and higher potential to influence the growth, migration, invasion and chemoresistance of cocultured breast cancer cells than NFs. In addition, the miRNAs that are differentially expressed in CAFs when compared to NFs display substantial variability across patients with distinct breast cancer subtypes, while the enrichment of target genes regulated by these miRNAs, within GO terms and KEGG pathways, remains remarkably consistent among patients with varying profiles.

Autocrine Factors Produced by Mesenchymal Stem Cells in Response to Cell-Cell Contact Inhibition Have Anti-Tumor Properties.

Recently, mesenchymal stem cell (MSC) therapies have been questioned as MSCs are capable of both promoting and inhibiting tumorigenesis. Both MSCs and tumor cells replicate to increase their population size; however, MSCs, but not tumor cells, stop dividing when they reach confluence due to cell-cell contact inhibition and then differentiate. We hypothesized that contact inhibition results in the production of effector molecules by confluent MSCs and these effectors are capable of suppressing tumor cell growth. To test this hypothesis, we co-cultured breast cancer cells (MDA-MB-231) with either confluent or sub-confluent bone-marrow-derived MSCs (BM-MSCs); in addition, we treated various tumor cells with conditioned media (CM) obtained from either confluent or sub-confluent BM-MSCs. The results showed that the growth of tumor cells co-cultured with confluent BM-MSCs or treated with CM obtained from confluent BM-MSCs was inhibited, and this effect was significantly stronger than that seen with tumor cells co-cultured with sub-confluent BM-MSCs or CM obtained from sub-confluent BM-MSCs. Subcutaneous tumor formation was completely prevented by the inoculation of tumor cells mixed with CM. In the future, soluble anti-tumor effectors, produced by confluent MSCs, may be used as cell-free therapeutics; this approach provides a solution to current concerns associated with cell-based therapies.

Synergistic Effect of Co-Culturing Breast Cancer Cells and Fibroblasts in the Formation of Tumoroid Clusters and Design of In Vitro 3D Models for the Testing of Anticancer Agents.

Breast cancer is still the leading cause of women's death due to relapse and metastasis. In vitro tumor models are considered reliable tools for drug screening and understanding cancer-driving mechanisms due to the possibility of mimicking tumor heterogeneity. Herein, a 3D breast cancer model (3D-BCM) is developed based on enzymatically-crosslinked silk fibroin (eSF) hydrogels. Human MCF7 breast cancer cells are encapsulated into eSF hydrogels, with and without human mammary fibroblasts. The spontaneously occurring conformational change from random coil to β-sheet is correlated with increased eSF hydrogels' stiffness over time. Moreover, mechanical properties analysis confirms that the cells can modify the stiffness of the hydrogels, mimicking the microenvironment stiffening occurring in vivo. Fibroblasts support cancer cells growth and assembly in the eSF hydrogels up to 14 days of culture. Co-cultured 3D-BCM exhibits an upregulated expression of genes related to extracellular matrix remodeling and fibroblast activation. The 3D-BCM is subjected to doxorubicin and paclitaxel treatments, showing differential drug response. Overall, these results suggest that the co-culture of breast cancer cells and fibroblasts in eSF hydrogels allow the development of a mimetic in vitro platform to study cancer progression. This opens up new research avenues to investigate novel molecular targets for anti-cancer therapy.

Macrophage-Conditioned Media Promotes Adipocyte Cancer Association, Which in Turn Stimulates Breast Cancer Proliferation and Migration.

Breast cancer is the most common cancer in women and the leading cause of female cancer deaths worldwide. Obesity causes chronic inflammation and is a risk factor for post-menopausal breast cancer and poor prognosis. Obesity triggers increased infiltration of macrophages into adipose tissue, yet little research has focused on the effects of macrophages in early stages of breast tumor development in obese patients. In this study, the effects of pro-inflammatory macrophages on breast cancer-adipocyte crosstalk were investigated. An innovative human cell co-culture system was built and used to model the paracrine interactions among adipocytes, macrophages, and breast cancer cells and how they facilitate tumor progression. The effects on cancer cells were examined using cell counts and migration assays. Quantitative reverse-transcription polymerase chain reaction was used to measure the expression levels of several cytokines and proteases to analyze adipocyte cancer association. Macrophage-conditioned media intensified the effects of breast cancer-adipocyte crosstalk. Adipocytes became delipidated and increased production of pro-inflammatory cytokines, even in the absence of cancer cells, although the expression levels were highest with all three cell components. As a result, co-cultured breast cancer cells became more aggressive, with increased proliferation and migration compared to adipocyte-breast cancer co-cultures treated with unconditioned media. A novel co-culture model was built to evaluate the crosstalk among human macrophages, adipocytes, and breast cancer cells. We found that macrophages may contribute to adipocyte inflammation and cancer association and thus promote breast cancer progression.

KDM6B regulates M2 polarization of macrophages by modulating the stability of nuclear β-catenin

Accumulating evidences suggest that the epigenetic regulation plays a pivotal role in establishing phenotype and function of tumor associated macrophages (TAMs). KDM6B is an epigenetic enzyme responsible for the H3K27me3 and reported to influence macrophage polarization. However, the underlying mechanism remains to be determined. Here, we demonstrated that inhibition of KDM6B in TAMs increased M2 polarization induced by coculture of breast cancer cells. Furthermore, we identified that KDM6B downregulation activated β-catenin/c-Myc signaling, and thus promoted the M2-like phenotype. KDM6B accelerated the intranuclear ubiquitination degradation of β-catenin, which depended on its demethylase activity. Therapeutically, our data showed that activated vitamin D analog paricalcitol upregulated the expression of KDM6B and decreased the M2 polarization, consequently protected against tumor progress in the xenograft mouse model of breast cancer. Taken together, our data reveal that epigenetic regulator KDM6B prevents M2 polarization via promoting the intranuclear degradation of β-catenin. Active vitamin D analog induces KDM6B and suppresses tumor progress, suggesting a novel therapeutic potential of epigenetic modulation for the tumor treatment.

Polymersomes-Mediated Delivery of CSF1R Inhibitor to Tumor Associated Macrophages Promotes M2 to M1-Like Macrophage Repolarization.

The crosstalk between cancer cells and tumor associated macrophages (TAMs) within the tumor environment modulates tumor progression at all stages of cancer disease. TAMs are predominantly M2-like polarized macrophages with tumor-promoting activities. Nonetheless, they can be repolarized to tumoricidal M1-like macrophages through macrophage colony stimulating factor 1 receptor inhibition (CSF1Ri). CSF1Ri is being explored as multifaced therapeutic approach to suppress TAMs tumor-promoting functions and reduce cancer cell aggressiveness and viability. However, treatment with CSF1Ri results in significant TAMs death, thereby extinguishing the possibility of generating tumoricidal M1-like macrophages. Immunotherapy has not only improved overall patient's survival in some cancer types, but also caused frequent off-target toxicity. Approaches to balance efficacy versus toxicity are needed. Herein, a CSF1Ri-loaded polymersomes (PMs) based delivery platform is developed to promote M2-like macrophage repolarization. When testing in vitro on primary human monocyte-derived macrophages (MDMs), CSF1Ri-loaded PMs are preferentially taken up by M2-like macrophages and enhance M2 to M1-like macrophage repolarization while minimizing cytotoxicity in comparison to the free drug. When testing in a MDMs-MDA-MB-231 breast cancer cell coculture model, CSF1Ri-loaded PMs further retain their M2 to M1-like macrophages polarization capacity. This CSF1Ri-loaded PM-based platform system represents a promising tool for macrophage-based immunotherapy approaches.

Abstract P263: Interaction between CD36 and FABP4 regulates the import and metabolism of fatty acid in breast cancer cells

Abstract The breast cancer microenvironment is unique since the breast tissue within which the tumour originates comprises predominantly adipocytes. Adipocytes secrete various growth factors and cytokines that are reported to influence tumour progression by induction of epithelial-mesenchymal transition (EMT) which enhances cancer cell migration, invasion, and metastasis. Immunohistochemical analysis reveals that the invading breast cancer invades surrounding adipose tissues resulting in the delipidation of adipose tissues. To determine the molecular mechanism underlying these events, we co-cultured human adipocytes and breast cancer cells. Co-cultured cells had increased CD36 expression, with fatty acid import. Genetic ablation of CD36 attenuates adipocyte-induced EMT and stemness. Molecular screening for pathways potentially involved in adipocyte-induced CD36 expression reveals activation of the STAT3 signaling axis. Our study identifies a feedforward loop between CD36 and STAT3; where STAT3 binds to the CD36 promoter, activating its expression and CD36 upregulates STAT3 signaling. Breast cancer cells co-cultured with adipocytes actively accumulate fatty acids and undergo metabolic reprogramming, with a shift towards fatty acid oxidation. Seahorse metabolic analysis reveals that co-cultured CD36-expressing cells assumed an energetic phenotype, indicating enhanced mitochondrial respiration. Thus, breast cancer cells alter their metabolic programs, with increased mitochondrial respiration, to meet their energy needs, with the availability of fatty acids. Analysis of breast cancer patient data reveals that increased CD36 expression occurs with increased FABP4 expression. Mechanistic experiments reveal that CD36 directly interacts with FABP4 to regulate fatty acid import, transport, and metabolism in co-cultured breast cancer cells. In vivo studies in mouse models reveal that combined chemical inhibition of CD36 and FABP4 resulted in a significant reduction in tumour growth rate. The study presents an alternative approach by which adipocytes may enhance cancer progression via the transfer of free fatty acids from tumour-associated adipocytes to breast cancer cells. The uptake of free fatty acid subsequently serves as a secondary source of energy to drive breast cancer cell progression. Targeting CD36 in combination with FABP4 may have a potential for therapies targeting the breast cancer microenvironment. Citation Format: Jones Gyamfi, Junjeong Choi, Doru Kwon, JaSeung Koo. Interaction between CD36 and FABP4 regulates the import and metabolism of fatty acid in breast cancer cells [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2021 Oct 7-10. Philadelphia (PA): AACR; Mol Cancer Ther 2021;20(12 Suppl):Abstract nr P263.

The Interaction of the Senescent and Adjacent Breast Cancer Cells Promotes the Metastasis of Heterogeneous Breast Cancer Cells through Notch Signaling.

Chemotherapy is one of the most common strategies for tumor treatment but often associated with post-therapy tumor recurrence. While chemotherapeutic drugs are known to induce tumor cell senescence, the roles and mechanisms of senescence in tumor recurrence remain unclear. In this study, we used doxorubicin to induce senescence in breast cancer cells, followed by culture of breast cancer cells with conditional media of senescent breast cancer cells (indirect co-culture) or directly with senescent breast cancer cells (direct co-culture). We showed that breast cancer cells underwent the epithelial–mesenchymal transition (EMT) to a greater extent and had stronger migration and invasion ability in the direct co-culture compared with that in the indirect co-culture model. Moreover, in the direct co-culture model, non-senescent breast cancer cells facilitated senescent breast cancer cells to escape and re-enter into the cell cycle. Meanwhile, senescent breast cancer cells regained tumor cell characteristics and underwent EMT after direct co-culture. We found that the Notch signaling was activated in both senescent and non-senescent breast cancer cells in the direct co-culture group. Notably, the EMT process of senescent and adjacent breast cancer cells was blocked upon inhibition of Notch signaling with N-[(3,5-difluorophenyl)acetyl]-l-alanyl-2-phenyl]glycine-1,1-dimethylethyl ester (DAPT) in the direct co-cultures. In addition, DAPT inhibited the lung metastasis of the co-cultured breast cancer cells in vivo. Collectively, data arising from this study suggest that both senescent and adjacent non-senescent breast cancer cells developed EMT through activating Notch signaling under conditions of intratumoral heterogeneity caused by chemotherapy, which infer the possibility that Notch inhibitors used in combination with chemotherapeutic agents may become an effective treatment strategy.

Cancer-derived exosomal miR-138-5p modulates polarization of tumor-associated macrophages through inhibition of KDM6B.

Rationale: Differential activation of macrophages correlates closely with tumor progression, and the epigenetic factor lysine demethylase 6B (KDM6B, previously named JMJD3) mediates the regulation of macrophage polarization through an unknown mechanism.Methods: We developed a suspension coculture system comprising breast cancer cells and macrophages and used RT-qPCR and western blotting to measure KDM6B expression. Bioinformatics and luciferase reporter assays were used to identify candidate microRNAs of cancer cells responsible for the downregulation of KDM6B. To determine if exosomes mediated the transfer of miR-138-5p between cancer cells to macrophages, we treated macrophages with exosomes collected from the conditioned medium of cancer cells. The effects of exosomal miR-138-5p on macrophage polarization were measured using RT-qPCR, flow cytometry, and chromatin immunoprecipitation assays. We employed a mouse model of breast cancer, metastatic to the lung, to evaluate the effects on tumor metastasis of macrophages treated with miR-138-5p-enriched exosomes. To develop a diagnostic evaluation index, the levels of exosomal miR-138-5p in samples from patients with breast cancer were compared to those of controls.Results: Coculture of breast cancer cells led to downregulation of KDM6B expression in macrophages. Cancer cell-derived exosomal miR-138-5p inhibited M1 polarization and promoted M2 polarization through inhibition of KDM6B expression in macrophages. Macrophages treated with exosomal miR-138-5p promoted lung metastasis, and the level of circulating exosomal miR-138-5p positively correlated with the progression of breast cancer.Conclusion: Our data suggest that miR-138-5p was delivered from breast cancer cells to tumor-associated macrophages via exosomes to downregulate KDM6B expression, inhibit M1 polarization, and stimulate M2 polarization. Therefore, exosomal miR-138-5p represents a promising prognostic marker and target for the treatment of breast cancer.

Nitroxide-functional PEGylated nanostars arrest cellular oxidative stress and exhibit preferential accumulation in co-cultured breast cancer cells.

The limited application of traditional antioxidants to reducing elevated levels of reactive oxygen species (ROS) is potentially due to their lack of stability and biocompatibility when tested in a biological milieu. For instance, the poor biological antioxidant performance of small molecular nitroxides arises from their limited diffusion across cell membranes and their significant side effects when applied at high doses. Herein, we describe the use of nanostructured carriers to improve the antioxidant activity of a typical nitroxide derivative, (2,2,6,6-tetramethylpiperidin-1-yl)oxyl (TEMPO). Polymers with star-shaped structures were synthesised and were further conjugated to TEMPO moieties via amide linkages. The TEMPO-loaded stars have small hydrodynamic sizes (<20 nm), and are better tolerated by cells than free TEMPO in a breast cancer-fibroblast co-culture, a system exhibiting elevated ROS levels. At a well-tolerated concentration, the polymer with the highest TEMPO-loading capacity successfully downregulated ROS production in co-cultured cells (a significant decrease of up to 50% vs. basal ROS levels), which was accompanied by a specific reduction in superoxide anion generation in the mitochondria. In contrast, the equivalent concentration of free TEMPO did not achieve the same outcome. Further investigation showed that the TEMPO-conjugated star polymers can be recycled inside the cells, thus providing longer term scavenging activity. Cell association studies demonstrated that the polymers can be taken up by both cell types in the co-culture, and are found to co-locate with the mitochondria. Interestingly the stars exhibited preferential mitochodria targeting in the co-cultured cancer cells compared to accompanying fibroblasts. The data suggest the potential of TEMPO-conjugated star polymers to arrest oxidative stress for various applications in cancer therapy.

Identification and Validation of Stage-Associated PBMC Biomarkers in Breast Cancer Using MS-Based Proteomics.

Background: It is well-described that the transcriptome of peripheral blood mononuclear cells (PBMCs) can be altered in the context of many malignancies to allow them avoid the effective immune response, which leads to cancer invasiveness. Here, we used an MS-based strategy to discover biomarkers in the PBMCs of breast cancer (BC) patients and validated them at different stages of BC.Methods: PBMCs were isolated from the breast cancer patients and were cultured alone or co-cultured with breast cancer cell lines. The role of PBMC in the invasion property of breast cancer cells was explored. NF-kB activity was also measured in the co-cultured breast cancer cells. Identification of protein profiles in the secretome and proteome of the co-cultured PBMCs was performed using SWATH mass spectrometry. Pathway enrichment and gene ontology analyses were carried out to look for the molecular pathways correlated with the protein expression profile of PBMCs in the breast cancer patients. Quantitative real-time polymerase chain reaction (qPCR) was performed to validate the candidate genes in the PBMC fraction of the breast cancer patients at the primary and metastatic stages. In silico survival analysis was performed to assess the potential clinical biomarkers in these PBMC subtypes.Results: PBMCs could significantly increase the invasion property of the BC cells concomitant with a decrease in E-cadherin and an increase in both Vimentin and N-cadherin expression. The NF-kB activity in the BC cells significantly increased following co-culturing implying the role of PBMCs in EMT induction. Enrichment analysis showed that the differentially expressed proteins in PBMCs are mainly associated with IL-17, PI3K-Akt, and HIF-1 signaling pathway, in which a set of seven proteins including TMSB4X, HSPA4, S100A9, SRSF6, THBS1, CUL4A, and CANX were frequently expressed. Finally, in silico analysis confirmed that a gene set consisting of S100A9, SRSF6, THBS1, CUL4A, and CANX were found to provide an insight for the identification of metastasis in breast cancer patients.Conclusion: In conclusion, our study revealed that the protein expression profile in PBMCs is a reflection of the proteins expressed in the BC tissue itself; however, the abundance level is different due to the stage of cancer.

BSCI-25. THE ROLE OF THE IFNγ PATHWAY IN BREAST CANCER BRAIN METASTASIS FORMATION

In previous work, we showed the prominence of the T cell response in the formation of brain metastases of primary ER-negative breast cancers. We also showed that prior co-cultured breast cancer cells with stimulated T lymphocytes bear an overexpression of Guanylate-binding protein 1 (GBP1) and possess an increased trespassing ability through an in vitro blood-brain barrier (BBB) model. In addition, we demonstrated a predilection for metastasizing to the brain of breast cancer cells that were co-cultured with activated T cells in a mouse model. In the present work, we show that activated CD8+ cytotoxic T lymphocytes, rather than CD4+ lymphocytes, are the main cause of increasing the ability of breast cancer cells to cross the BBB. While synthetic IFNγ does not change the ability of breast cancer cells to cross the BBB, this study shows that the T lymphocyte-secreted IFNγ activates the STAT1-dependent IFNγ pathway in breast cancer cells, enabling them to cross the in vitro BBB. Direct inhibition of soluble IFNγ or blocking of the IFNγ-specific receptor in breast cancer cells significantly decreases their ability to cross the BBB. The results illustrate that IFNγ signaling pathway is one of the crucial pathways in the formation of brain metastasis of ER- breast cancer. The interference with the IFNγ pathway will develop preventive strategies against the formation of brain metastases of breast cancer.

Changes in uPA, PAI-1, and TGF-β Production during Breast Cancer Cell Interaction with Human Mesenchymal Stroma/Stem-Like Cells (MSC)

The interactions of cancer cells with neighboring non-malignant cells in the microenvironment play an important role for progressive neoplastic development and metastasis. Long-term direct co-culture of human MDA-MB-231cherry breast cancer cells with benign human mesenchymal stroma/stem-like cells (MSC) MSC544GFP stably expressing mCherry and eGFP fluorescence proteins, respectively, was associated with the formation of three-dimensional (3D) tumor spheroids in vitro. The quantification of the breast tumor marker urokinase plasminogen activator (uPA) in mono-cultured MDA-MB-231 cells revealed an approximately 14-fold enhanced expression when compared to five different normal human MSC mono-cultures. Moreover, uPA levels in 3D tumor spheroids remained elevated 9.4-fold above the average of five different human MSC cultures. In contrast, the expression of the corresponding plasminogen activator inhibitor type-1 (PAI-1) declined by 2.6-fold in the breast cancer cells and was even further reduced by 3.2-fold in the MDA-MB-231cherry/MSC544GFP 3D co-culture spheroids when compared to the various MSC populations. The supportive data were obtained for the production of TGF-β1, which is an important growth factor in the regulation of tumor growth and metastasis formation. Whereas, TGF-β1 release in MDA-MB-231cherry/MSC544GFP co-cultures was elevated by 1.56-fold as compared to MSC544 mono-cultures after 24 h; this ratio further increased to 2.19-fold after 72 h. Quantitative PCR analyses in MSC544 and MDA-MB-231 cells revealed that MSC, rather than the breast cancer cells, are responsible for TGF-β1 synthesis and that TGF-β1 contributes to its own synthesis in these cells. These findings suggested potential synergistic effects in the expression/secretion of uPA, PAI-1, and TGF-β during the co-culture of breast cancer cells with MSC.

Abstract P5-07-15: Adipose-derived peptides from breast cancer patients promote the malignancy of breast cancer MCF-7 cells

Abstract Adipose stromal/stem cells (ADSCs) could regulate malignancy behaviors of breast cancer, which was due to adipose-derived cytokines in part, such as leptin and resistin. Recently, peptides have been demonstrated to be potential targets for cancer therapy. It's valuable to analyze the characters of adipose-secreted peptides systematically. In this study, we isolated primary adipocytes from adipose tissue adjacent to breast tumor and breast benign lesions respectively, and then co-cultured breast cancer cells MCF-7 with tumor adjacent adipocytes (TAAs)/breast benign adipocytes (BBAs) separately. Compared to BBAs, TAAs could promote proliferation and migration ability of MCF-7. Then we extracted and purified peptides from supernatant of TAAs and BBAs using ultrafiltration, and the effects of TAAs-derived peptides compound on breast cancer cells appeared accordance with the adipose cells. 100 peptides, which were derived from 90 protein precursors, were found to be differentially secreted between TAAs and BBAs by LC-MS/MS (p &amp;lt; 0.05). Gene Ontology (GO) and Pathway analyses appeared that these altered peptides were mainly contributed to cell adhesion, and regulation of Ras-protein signal transduction. These results showed that these differentially secreted peptides could regulate breast cancer cell malignancy, which indicated that they could be potential therapeutic targets and diagnostic bio-marker for breast cancer. Citation Format: Fu Z, Li W, Wu H, Huang X, Li J, Sun C, Tang J, Yin Y. Adipose-derived peptides from breast cancer patients promote the malignancy of breast cancer MCF-7 cells [abstract]. In: Proceedings of the 2018 San Antonio Breast Cancer Symposium; 2018 Dec 4-8; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2019;79(4 Suppl):Abstract nr P5-07-15.

Abstract A17: Bromodomain and extraterminal proteins regulate PD-L1/PD-1 signaling in breast cancer.

Abstract Introduction: Breast tumors are massively infiltrated immune cells of both lymphoid and myeloid origin that exert an anti-tumor pressure to limit cancer progression. Tumor cells can escape immune clearance by expressing several molecules that constitute the immune checkpoints. Antibodies targeting the PD-L1/PD-1 pathway are being evaluated clinically for several cancers and show early success. However, the realization that not all patients respond well to immunotherapy suggests other modalities could be combined to improve efficacy. The bromodomain and extraterminal (BET) proteins BRD2, BRD3 and BRD4 are epigenetically acting co-regulators of transcription, and are critical for cancer proliferation and metastasis. Little is known about the molecular mechanisms that regulate PD-L1 and PD-1 expression. BRD4 is known to bind directly to the PD-L1 promoter; its targeting suppresses PD-L1 expression while increasing CD8+ T cell activity to limit progression in ovarian tumor models. Hypotheses: BET proteins regulate PD-L1 and PD-L2 expression in breast tumors and PD-1 in CD8+ T cells. BET proteins are effectors of proinflammatory cytokine signaling, regulating PD-L1/PD-1 signaling in the breast tumor microenvironment. Methods: We used different cellular models of breast cancer. We also collected human peripheral blood mononuclear cells matched with mammary adipose tissue from mammoplasty patients at Boston Medical Center to assess expression of immune checkpoint proteins in the breast microenvironment. Pan-BET protein inhibition was performed by JQ1 treatment. Alternatively, BET proteins were individually and specifically depleted by siRNA. PD-1 and PD-L1 expression were determined by qRT-PCR and flow cytometry. Cytokines and chemokines present in the blood or secreted by the mammary adipose tissue were profiled by multiplexed antibody capture assay. Results: BET proteins regulate PD-L1 expression by breast cancer cells. BRD2 and BRD4 expression correlate with PD-1 in activated circulating T cells and BET inhibition ablates activation-induced PD-1 expression. Co-culture of breast cancer cells with activated circulating T cells enhances expression of immune checkpoint proteins in both compartments. BET protein inhibition rescues these phenotypes. Conditioned media from mammary adipose tissue increase PD-1 in PBMCs. Peripheral and mammary adipose tissue inflammatory signatures associate with PD-1 expression in PBMCs, suggesting that local inflammation in the breast tumor microenvironment contributes to diminishing anti-tumor immune responses. Conclusion: BET proteins regulate the PD-L1/PD-1 pathway in breast cancer. Next-generation BET protein inhibitors may combine well with PD-1 or PD-L1-targeted immunotherapies in difficult-to-treat cancers. Personalized BET profiles could inform individual patient responses to BET proteins and immune checkpoint inhibitors. Therapeutic approaches that treat the microenvironment should be leveraged to maximize efficacy of checkpoint inhibitor approaches for specific cancers. Citation Format: Guillaume Andrieu, Gerald V. Denis. Bromodomain and extraterminal proteins regulate PD-L1/PD-1 signaling in breast cancer. [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology and Immunotherapy; 2017 Oct 1-4; Boston, MA. Philadelphia (PA): AACR; Cancer Immunol Res 2018;6(9 Suppl):Abstract nr A17.

Abstract P1-17-07: Preclinical characterization of neratinib in a blood-brain barrier co-culture model: Therapeutic implications for breast cancers with brain metastases

Abstract Background: Breast cancer patients who develop brain metastases have poor prognosis/short overall survival. Human epidermal growth factor receptor 2 (HER2) +ive breast cancer have an increased propensity for brain metastases. Currently, these metastases have limited therapeutic options. The endothelial cells forming the blood-brain barrier (BBB) are highly specialized to allow precise control over substances that enter/leave the brain via the tight junction. Targeted agents that are efficacious/ able to penetrate the BBB are urgently needed. Neratinib is an orally available tyrosine kinase inhibitor that irreversibly binds and inhibits EGFR, HER2/HER4 receptor tyrosine kinases and has previously been shown to reduce the onset/delay symptomatic progression of CNS metastases in patients with advanced HER2-positive breast cancers with brain metastases (Awada 2016 JAMA Oncol; Freedman ASCO 2017 Ab 1005). Using a BBB model, this study aimed to determine if neratinib is able to pass through an endothelial barrier and demonstrate activity against human breast cancer cells lines. We also compared brain and vascular endothelial cells. Methods: The BBB was represented by an in vitro transwell model using TY09 brain endothelial and HECV venous human cell lines in co-culture with a range of HER2+ive (MDA-MB-361;BT-474) or HER2-ive (MDA-MB-231;BT-549) human breast cancer cell lines. Effects on barrier function (trans-endothelial resistance/paracellular permeability) were assessed. Dose response effects on two brain endothelial cell lines (TY09/CMECD3) by neratinib were determined using ECIS (electric cell impedence sensing). Results: Initially, we compared the effects of neratinib on a single brain endothelial cell line (TY09) in comparison to a venous endothelial cell line (HECV) in the presence/absence of EGF (20pg). We used temozolomide (TMZ) (40pmol) as a control treatment. Barrier function, assessed using trans-endothelial resistance and paracellular permeability revealed that neratinib had little effect on resistance over 1-72 hrs. When co-cultured with HER2+ive breast cancer cells, resistance was decreased at 1 hr which then returned to control levels (p&amp;lt;0.05). There was little change following treatment to the HER2-negative cells. A similar effect was also observed with TMZ. There was also a marked effect in HECV cells, but this was mostly observed in those treated with TMZ, regardless of HER2 status of the breast cancer cells in co-culture (p&amp;lt;0.04). Paracellular permeability was increased by 0.5 hr when using 10kDa (p&amp;lt;0.05) but not 40kDa dextran. Neratinib reduced the resistance of both TY09 and CMECD3 cells in a dose-dependent manner, with the same effect observed in cell attachment (p&amp;lt;0.01) and motility (0.05). Conclusions: In our BBB co-culture model, neratinib was able to pass through the barrier created by both brain and venous endothelial cells and reduce the growth of HER2+ive breast cancer cells over 72 hrs. In addition, neratinib caused marked changes in barrier function of the brain endothelial cells with concurrent effects on cell behaviour. These data demonstrate that neratinib may be an attractive therapeutic for HER2-positive breast cancers with brain metastases. Citation Format: Martin TA, Lalani AS, Avogadri Connors F, Bryce RP, Jiang WG. Preclinical characterization of neratinib in a blood-brain barrier co-culture model: Therapeutic implications for breast cancers with brain metastases [abstract]. In: Proceedings of the 2017 San Antonio Breast Cancer Symposium; 2017 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2018;78(4 Suppl):Abstract nr P1-17-07.