Indacaterol inhibits collective cell migration and IGDQ-mediated single cell migration in metastatic breast cancer MDA-MB-231 cells.

Metastasis is the main cause of deaths related to breast cancer. This is particular the case for triple negative breast cancer. No targeted therapies are reported as efficient until now. The extracellular matrix, in particular the fibronectin type I motif IGDQ, plays a major role in regulating cell migration prior metastasis formation. This motif interacts with specific integrins inducing their activation and the migratory signal transduction.Here, we characterized the migratory phenotype of MDA-MB-231 cells, using functionalized IGDQ-exposing surfaces, and compared it to integrin A5 and integrin B3 knock-down cells. A multiomic analysis was developed that highlighted the splicing factor SRSF6 as a putative master regulator of cell migration and of integrin intracellular trafficking. Indacaterol-induced inhibition of SRSF6 provoked: i) the inhibition of collective and IGDQ-mediated cell migration and ii) ITGA5 sequestration into endosomes and lysosomes. Upon further studies, indacaterol may be a potential therapy to prevent cell migration and reduce metastasis formation in breast cancer. Video Abstract.

A bi-directional dialog between vascular cells and monocytes/macrophages regulates tumor progression.

Cancer progression largely depends on tumor blood vessels as well on immune cell infiltration. In various tumors, vascular cells, namely endothelial cells (ECs) and pericytes, strongly regulate leukocyte infiltration into tumors and immune cell activation, hence the immune response to cancers. Recently, a lot of compelling studies unraveled the molecular mechanisms by which tumor vascular cells regulate monocyte and tumor-associated macrophage (TAM) recruitment and phenotype, and consequently tumor progression. Reciprocally, TAMs and monocytes strongly modulate tumor blood vessel and tumor lymphatic vessel formation by exerting pro-angiogenic and lymphangiogenic effects, respectively. Finally, the interaction between monocytes/TAMs and vascular cells is also impacting several steps of the spread of cancer cells throughout the body, a process called metastasis. In this review, the impact of the bi-directional dialog between blood vascular cells and monocytes/TAMs in the regulation of tumor progression is discussed. All together, these data led to the design of combinations of anti-angiogenic and immunotherapy targeting TAMs/monocyte whose effects are briefly discussed in the last part of this review.

Transmembrane (TMEM) protein family members: Poorly characterized even if essential for the metastatic process.

The majority of cancer-associated deaths are related to secondary tumor formation. This multistep process involves the migration of cancer cells to anatomically distant organs. Metastasis formation relies on cancer cell dissemination and survival in the circulatory system, as well as adaptation to the new tissue notably through genetic and/or epigenetic alterations. A large number of proteins are clearly identified to play a role in the metastatic process but the structures and modes of action of these proteins are essentially unknown or poorly described. In this review, we detail the involvement of members of the transmembrane (TMEM) protein family in the formation of metastases or in the mechanisms leading to cancer cell dissemination such as migration and extra-cellular matrix remodelling. While the phenotype associated with TMEM over or down-expression is clear, the mechanisms by which these proteins allow cancer cell spreading remain, for most of them, unclear. In parallel, the 3D structures of these proteins are presented. Moreover, we proposed that TMEM proteins could be used as prognostic markers in different types of cancers and could represent potential targets for cancer treatment. A better understanding of this heterogeneous family of poorly characterized proteins thus opens perspectives for better cancer patient care.

Could Protons and Carbon Ions Be the Silver Bullets Against Pancreatic Cancer?

Pancreatic cancer is a very aggressive cancer type associated with one of the poorest prognostics. Despite several clinical trials to combine different types of therapies, none of them resulted in significant improvements for patient survival. Pancreatic cancers demonstrate a very broad panel of resistance mechanisms due to their biological properties but also their ability to remodel the tumour microenvironment. Radiotherapy is one of the most widely used treatments against cancer but, up to now, its impact remains limited in the context of pancreatic cancer. The modern era of radiotherapy proposes new approaches with increasing conformation but also more efficient effects on tumours in the case of charged particles. In this review, we highlight the interest in using charged particles in the context of pancreatic cancer therapy and the impact of this alternative to counteract resistance mechanisms.

Cycling hypoxia: A key feature of the tumor microenvironment.

A compelling body of evidence indicates that most human solid tumors contain hypoxic areas. Hypoxia is the consequence not only of the chaotic proliferation of cancer cells that places them at distance from the nearest capillary but also of the abnormal structure of the new vasculature network resulting in transient blood flow. Hence two types of hypoxia are observed in tumors: chronic and cycling (intermittent) hypoxia. Most of the current work aims at understanding the role of chronic hypoxia in tumor growth, response to treatment and metastasis. Only recently, cycling hypoxia, with spatial and temporal fluctuations in oxygen levels, has emerged as another key feature of the tumor environment that triggers different responses in comparison to chronic hypoxia. Either type of hypoxia is associated with distinct effects not only in cancer cells but also in stromal cells. In particular, cycling hypoxia has been demonstrated to favor, to a higher extent than chronic hypoxia, angiogenesis, resistance to anti-cancer treatments, intratumoral inflammation and tumor metastasis. These review details these effects as well as the signaling pathway it triggers to switch on specific transcriptomic programs. Understanding the signaling pathways through which cycling hypoxia induces these processes that support the development of an aggressive cancer could convey to the emergence of promising new cancer treatments.

Unleashing Cancer Cells on Surfaces Exposing Motogenic IGDQ Peptides.

Thiolated peptides bearing the Ile-Gly-Asp (IGD) motif, a highly conserved sequence of fibronectin, are used for the preparation of anisotropic self-assembled monolayers (SAM gradients) to study the whole-population migratory behavior of metastatic breast cancer cells (MDA-MB-231 cells). Ile-Gly-Asp-Gln-(IGDQ)-exposing SAMs sustain the adhesion of MDA-MB-231 cells by triggering focal adhesion kinase phosphorylation, similarly to the analogous Gly-Arg-Gly-Asp-(GRGD)-terminating surfaces. However, the biological responses of different cell lines interfaced with the SAM gradients show that only those exposing the IGDQ sequence induce significant migration of MDA-MB-231 cells. In particular, the observed migratory behavior suggests the presence of cell subpopulations associated with a "stationary" or a "migratory" phenotype, the latter determining a considerable cell migration at the sub-cm length scale. These findings are of great importance as they suggest for the first time an active role of biological surfaces exposing the IGD motif in the multicomponent orchestration of cellular signaling involved in the metastatic progression.

Fast Targeting and Cancer Cell Uptake of Luminescent Antibody-Nanozeolite Bioconjugates.

Understanding the targeted cellular uptake of nanomaterials is an essential step to engineer and program functional and effective biomedical devices. In this respect, the targeting and ultrafast uptake of zeolite nanocrystals functionalized with Cetuximab antibodies (Ctxb) by cells overexpressing the epidermal growth factor receptor are described here. Biochemical assays show that the cellular uptake of the bioconjugate in the targeted cancer cells already begins 15 min after incubation, at a rate around tenfold faster than that observed in the negative control cells. These findings further show the role of Ctxb exposed at the surfaces of the zeolite nanocrystals in mediating the targeted and rapid cellular uptake. By using temperature and pharmacological inhibitors as modulators of the internalization pathways, the results univocally suggest a dissipative uptake mechanism of these nanomaterials, which seems to occur using different internalization pathways, according to the targeting properties of these nanocrystals. Owing to the ultrafast uptake process, harmless for the cell viability, these results further pave the way for the design of novel theranostic tools based on nanozeolites.

LET-dependent radiosensitization effects of gold nanoparticles for proton irradiation.

The development of new modalities and protocols is of major interest to improve the outcome of cancer treatment. Given the appealing physical properties of protons and the emerging evidence of biological relevance of the use of gold nanoparticles (GNPs), the radiosensitization effects of GNPs (5 or 10 nm) have been investigated in vitro in combination with a proton beam of different linear energy transfer (LET). After the incubation with GNPs for 24 h, nanoparticles were observed in the cytoplasm of A431 cells exposed to 10 nm GNPs, and in the cytoplasm as well as the nucleus of cells exposed to 5 nm GNPs. Cell uptake of 0.05 mg ml-1 of GNPs led to 0.78 pg Au/cell and 0.30 pg Au/cell after 24 h incubation for 10 and 5 nm GNPs respectively. A marked radiosensitization effect of GNPs was observed with 25 keV µm-1 protons, but not with 10 keV µm-1 protons. This effect was more pronounced for 10 nm GNPs than for 5 nm GNPs. By using a radical scavenger, a major role of reactive oxygen species in the amplification of the death of irradiated cell was identified. All together, these results open up novel perspectives for using high-Z metallic NPs in protontherapy.

miRNA-196b inhibits cell proliferation and induces apoptosis in HepG2 cells by targeting IGF2BP1.

BACKGROUND: Tumor hypoxia is one of the features of tumor microenvironment that contributes to chemoresistance. miRNAs have recently been shown to play important roles in tumorigenesis and drug resistance. Moreover, hypoxia also regulates the expression of a series of miRNAs. However, the interaction between chemoresistance, hypoxia and miRNAs has not been explored yet. The aim of this study is to understand the mechanisms activated/inhibited by miRNAs under hypoxia that induce resistance to chemotherapy-induced apoptosis. METHODS: TaqMan low-density array was used to identify changes in miRNA expression when cells were exposed to etoposide under hypoxia or normoxia. The effects of miR-196b overexpression on apoptosis and cell proliferation were studied in HepG2 cells. miR-196b target mRNAs were identified by proteomic analysis, luciferase activity assay, RT-qPCR and western blot analysis. RESULTS: Results showed that hypoxia down-regulated miR-196b expression that was induced by etoposide. miR-196b overexpression increased the etoposide-induced apoptosis and reversed the protection of cell death observed under hypoxia. By a proteomic approach combined with bioinformatics analyses, we identified IGF2BP1 as a potential target of miR-196b. Indeed, miR-196b overexpression decreased IGF2BP1 RNA expression and protein level. The IGF2BP1 down-regulation by either miR-196b or IGF2BP1 siRNA led to an increase in apoptosis and a decrease in cell viability and proliferation in normal culture conditions. However, IGF2BP1 silencing did not modify the chemoresistance induced by hypoxia, probably because it is not the only target of miR-196b involved in the regulation of apoptosis. CONCLUSIONS: In conclusion, for the first time, we identified IGF2BP1 as a direct and functional target of miR-196b and showed that miR-196b overexpression reverses the chemoresistance induced by hypoxia. These results emphasize that the chemoresistance induced by hypoxia is a complex mechanism.

M1 and M2 macrophages derived from THP-1 cells differentially modulate the response of cancer cells to etoposide.

BACKGROUND: Tumor associated macrophages (TAMs) are present in high density in solid tumors. TAMs share many characteristics with alternatively activated macrophages, also called M2. They have been shown to favor tumor development and a role in chemoresistance has also been suggested. Here, we investigated the effects of M2 in comparison to M1 macrophages on cancer cell sensitivity to etoposide. METHODS: We set up a model of macrophage polarization, starting from THP-1 monocytes differentiated into macrophages using PMA (Phorbol 12-myristate 13-acetate). Once differentiated (M0 macrophages), they were incubated with IL-4 and IL-13 in order to obtain M2 polarized macrophages or with IFN-gamma and LPS for classical macrophage activation (M1). To mimic the communication between cancer cells and TAMs, M0, M1 or M2 macrophages and HepG2 or A549 cancer cells were co-cultured during respectively 16 (HepG2) or 24 (A549) hours, before etoposide exposure for 24 (HepG2) or 16 (A549) hours. After the incubation, the impact of etoposide on macrophage polarization was studied and cancer cell apoptosis was assessed by western-blot for cleaved caspase-3 and cleaved PARP-1 protein, caspase activity assay and FACS analysis of Annexin V and PI staining. RESULTS: mRNA and protein expression of M1 and M2 markers confirmed the polarization of THP-1-derived macrophages, which provide a new, easy and well-characterized model of polarized human macrophages. Etoposide-induced cancer cell apoptosis was markedly reduced in the presence of THP-1 M2 macrophages, while apoptosis was increased in cells co-cultured with M1 macrophages. On the other hand, etoposide did not influence M1 or M2 polarization. CONCLUSIONS: These results evidence for the first time a clear protective effect of M2 on the contrary to M1 macrophages on etoposide-induced cancer cell apoptosis.

High TMEM45A expression is correlated to epidermal keratinization.

TMEM45A (DERP7, DNAPTP4 or FLJ10134) gene, belonging to the TMEM family encoding predicted transmembrane proteins, is highly expressed in epidermal keratinocytes. To investigate the potential involvement of TMEM45A during the differentiation and keratinization processes, its expression has been characterized in normal human keratinocytes and the protein subcellular localization has been studied in this cell type, both in vitro and in vivo. TMEM45A expression is upregulated with differentiation, either induced by cultured keratinocyte confluence or enhanced Ca(2+) concentration in medium. In vivo, TMEM45A mRNA and protein are mostly found in the granular layer of the epidermis. TMEM45A expression is linked to keratinization, as accumulation of the protein is detected in native and reconstructed epidermis as well as in thymic Hassal bodies, but not in non-keratinized stratified epithelia. At the subcellular level, co-detection with ER and Golgi markers reveals that TM protein 45A is associated with the Golgi apparatus and more specifically with the trans-Golgi/trans-Golgi network in vitro and in granular layer in vivo. The protein is neither related to lysosomes nor transported within corneodesmosin-containing lamellar bodies. These data demonstrate a strong correlation between TMEM45A expression and epidermal keratinization, indicating the relevance of this gene in this process.

Differential effect of hypoxia on etoposide-induced DNA damage response and p53 regulation in different cell types.

Among the main causes of cancer cell resistance to chemotherapy are p53 mutation and hypoxic tumor microenvironment. However, the effect of hypoxia can be very different from one cell type to the other. We studied the effect of hypoxia on the etoposide-induced cell death in two cancer cell lines, HepG2 and A549 cells. Hypoxia decreased etoposide-induced apoptosis in HepG2 cells but not in A549 cells. Here, we evidenced two pathways, known to play important roles in cancer cell resistance, that are differently affected by hypoxia in these two cell types. First, in HepG2 cells, hypoxia decreased p53 protein level and activity by acting post-transcriptionally and independently of HIF-1. The results suggest an effect of hypoxia on p53 translation. On the other hand, in A549 cells, no effect of hypoxia was observed on p53 level. Secondly, hypoxia decreased DNA damage response in HepG2 cells while this was not the case in A549 cells. Indeed, a decrease in the phosphorylation level of CHK2 and H2AX with a decrease in ATM activity was observed. Importantly, these results evidenced that hypoxia can prevent cancer cell apoptosis by acting at different levels in the cell and that these effects are strongly cell-type dependent.

Effects of the dual TP receptor antagonist and thromboxane synthase inhibitor EV-077 on human endothelial and vascular smooth muscle cells.

The prothrombotic mediator thromboxane A2 is derived from arachidonic acid metabolism through the cyclooxygenase and thromboxane synthase pathways, and transduces its effect through the thromboxane prostanoid (TP) receptor. The aim of this study was to determine the effect of the TP receptor antagonist and thromboxane synthase inhibitor EV-077 on inflammatory markers in human umbilical vein endothelial cells and on human coronary artery smooth muscle cell proliferation. To this end, mRNA levels of different proinflammatory mediators were studied by real time quantitative PCR, supernatants were analyzed by enzyme immune assay, and cell proliferation was assessed using WST-1. EV-077 significantly decreased mRNA levels of ICAM-1 and PTX3 after TNFα incubation, whereas concentrations of 6-keto PGF1α in supernatants of endothelial cells incubated with TNFα were significantly increased after EV-077 treatment. Although U46619 did not alter coronary artery smooth muscle cell proliferation, this thromboxane mimetic enhanced the proliferation induced by serum, insulin and growth factors, which was significantly inhibited by EV-077. In conclusion, EV-077 inhibited TNFα-induced endothelial inflammation and reduced the enhancement of smooth muscle cell proliferation induced by a thromboxane mimetic, supporting that the thromboxane pathway may be associated with early atherosclerosis in terms of endothelial dysfunction and vascular hypertrophy.

Advances in the Mechanistic Understanding of Iron Oxide Nanoparticles' Radiosensitizing Properties.

Among the plethora of nanosystems used in the field of theranostics, iron oxide nanoparticles (IONPs) occupy a central place because of their biocompatibility and magnetic properties. In this study, we highlight the radiosensitizing effect of two IONPs formulations (namely 7 nm carboxylated IONPs and PEG5000-IONPs) on A549 lung carcinoma cells when exposed to 225 kV X-rays after 6 h, 24 h and 48 h incubation. The hypothesis that nanoparticles exhibit their radiosensitizing effect by weakening cells through the inhibition of detoxification enzymes was evidenced by thioredoxin reductase activity monitoring. In particular, a good correlation between the amplification effect at 2 Gy and the residual activity of thioredoxin reductase was observed, which is consistent with previous observations made for gold nanoparticles (NPs). This emphasizes that NP-induced radiosensitization does not result solely from physical phenomena but also results from biological events.

Development of an Orthotopic HPV16-Dependent Base of Tongue Tumor Model in MHC-Humanized Mice.

Head and neck squamous cell carcinomas (HNSCC) caused by infections with high-risk human papillomaviruses (HPV) are responsible for an increasing number of head and neck cancers, particularly in the oropharynx. Despite the significant biological differences between HPV-driven and HPV-negative HNSCC, treatment strategies are similar and not HPV targeted. HPV-driven HNSCC are known to be more sensitive to treatment, particularly to radiotherapy, which is at least partially due to HPV-induced immunogenicity. The development of novel therapeutic strategies that are specific for HPV-driven cancers requires tumor models that reflect as closely as possible the characteristics and complexity of human tumors and their response to treatment. Current HPV-positive cancer models lack one or more hallmarks of their human counterpart. This study presents the development of a new HPV16 oncoprotein-dependent tumor model in MHC-humanized mice, modeling the major biologic features of HPV-driven tumors and presenting HLA-A2-restricted HPV16 epitopes. Furthermore, this model was developed to be orthotopic (base of tongue). Thus, it also reflects the correct tumor microenvironment of HPV-driven HNSCC. The cancer cells are implanted in a manner that allows the exact control of the anatomical location of the developing tumor, thereby homogenizing tumor growth. In conclusion, the new model is suited to study HPV16-specific therapeutic vaccinations and other immunotherapies, as well as tumor-targeted interventions, such as surgery or radiotherapy, or a combination of all these modalities.

Budget-Friendly Generation, Biochemical Analyses, and Lentiviral Transduction of Patient-Derived Colon Organoids.

For the past decade, three-dimensional (3D) culture models have been emerging as powerful tools in translational research to overcome the limitations of two-dimensional cell culture models. Thanks to their ability to recapitulate the phenotypic and molecular heterogeneity found in numerous organs, organoids have been used to model a broad range of tumors, such as colorectal cancer. Several approaches to generate organoids exist, with protocols using either pluripotent stem cells, embryonic stem cells, or organ-restricted adult stem cells found in primary tissues, such as surgical resections as starting material. The latter, so-called patient-derived organoids (PDOs), have shown their robustness in predicting patient drug responses compared to other models. Because of their origin, PDOs are natural offspring of the patient tumor or healthy surrounding tissue, and therefore, have been increasingly used to develop targeted drugs and personalized therapies. Here, we present a new protocol to generate patient-derived colon organoids (PDCOs) from tumor and healthy tissue biopsies. We emphasize budget-friendly and reproducible techniques, which are often limiting factors in this line of research that restrict the development of this 3D-culture model to a small number of laboratories worldwide. Accordingly, we describe efficient and cost-effective techniques to achieve immunoblot and high-resolution microscopy on PDCOs. Finally, a novel strategy of lentiviral transduction of PDCOs, which could be applied to all organoid models, is detailed in this article. © 2023 The Authors. Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1: Establishment of PDCOs from biopsies Basic Protocol 2: Long-term maintenance and expansion of PDCOs in BME domes Basic Protocol 3: Cryopreservation and thawing of PDCOs Basic Protocol 4: Lentiviral transduction of PDCOs Basic Protocol 5: Immunoblot and evaluation of variability between donors Basic Protocol 6: Immunofluorescence labeling and high-resolution microscopy of PDCOs Basic Protocol 7: Transcriptomic analyses of PDCOs by RT-qPCR.

TMEM45A is essential for hypoxia-induced chemoresistance in breast and liver cancer cells.

BACKGROUND: Hypoxia is a common characteristic of solid tumors associated with reduced response to radio- and chemotherapy, therefore increasing the probability of tumor recurrence. The aim of this study was to identify new mechanisms responsible for hypoxia-induced resistance in breast cancer cells. METHODS: MDA-MB-231 and HepG2 cells were incubated in the presence of taxol or etoposide respectively under normoxia and hypoxia and apoptosis was analysed. A whole transcriptome analysis was performed in order to identify genes whose expression profile was correlated with apoptosis. The effect of gene invalidation using siRNA was studied on drug-induced apoptosis. RESULTS: MDA-MB-231 cells incubated in the presence of taxol were protected from apoptosis and cell death by hypoxia. We demonstrated that TMEM45A expression was associated with taxol resistance. TMEM45A expression was increased both in MDA-MB-231 human breast cancer cells and in HepG2 human hepatoma cells in conditions where protection of cells against apoptosis induced by chemotherapeutic agents was observed, i.e. under hypoxia in the presence of taxol or etoposide. Moreover, this resistance was suppressed by siRNA-mediated silencing of TMEM45A. Kaplan Meier curve showed an association between high TMEM45A expression and poor prognostic in breast cancer patients. Finally, TMEM45 is highly expressed in normal differentiated keratinocytes both in vitro and in vivo, suggesting that this protein is involved in epithelial functions. CONCLUSION: Altogether, our results unravel a new mechanism for taxol and etoposide resistance mediated by TMEM45A. High levels of TMEM45A expression in tumors may be indicative of potential resistance to cancer therapy, making TMEM45A an interesting biomarker for resistance.

The impact of macrophages on endothelial cells is potentiated by cycling hypoxia: Enhanced tumor inflammation and metastasis.

Cycling hypoxia (cyH), neo-angiogenesis, and tumor-associated macrophages are key features of the tumor microenvironment. In this study, we demonstrate that cyH potentiates the induction by unpolarized and M1-like macrophages of endothelial inflammatory phenotype and adhesiveness for monocytes and cancer cells. This process triggers a positive feedback loop sustaining tumor inflammation. This work opens the door for innovative therapeutic strategies to treat tumor inflammation and metastasis. In cancers, the interaction between macrophages and endothelial cells (ECs) regulates tumor inflammation and metastasis. These cells are both affected by cycling hypoxia (cyH), also called intermittent hypoxia, a feature of the tumor microenvironment. cyH is also known to favor tumor inflammation and metastasis. Nonetheless, the potential impact of cyH on the dialog between macrophages and ECs is still unknown. In this work, the effects of unpolarized, M1-like, and M2-like macrophages exposed to normoxia, chronic hypoxia (chH), and cyH on endothelial adhesion molecule expression, pro-inflammatory gene expression, and EC adhesiveness for monocytes and cancer cells were investigated. cyH increased the ability of unpolarized and M1-like macrophages to induce EC inflammation and to increase the expression of the EC endothelial adhesion molecule ICAM1, respectively. Unpolarized, M1-like, and M2-like macrophages were all able to promote EC adhesive properties toward cancer cells. Furthermore, the ability of macrophages (mostly M1-like) to shift EC phenotype toward one allowing cancer cell and monocyte adhesion onto ECs was potentiated by cyH. These effects were specific to cyH because they were not observed with chH. Together, these results show that cyH amplifies the effects of macrophages on ECs, which may promote tumor inflammation and metastasis.

Taking Advantage of the Senescence-Promoting Effect of Olaparib after X-ray and Proton Irradiation Using the Senolytic Drug, ABT-263.

Radiotherapy (RT) is a key component of cancer treatment. Although improvements have been made over the years, radioresistance remains a challenge. For this reason, a better understanding of cell fates in response to RT could improve therapeutic options to enhance cell death and reduce adverse effects. Here, we showed that combining RT (photons and protons) to noncytotoxic concentration of PARP inhibitor, Olaparib, induced a cell line-dependent senescence-like phenotype. The senescent cells were characterized by morphological changes, an increase in p21 mRNA expression as well as an increase in senescence-associated ß-galactosidase activity. We demonstrated that these senescent cells could be specifically targeted by Navitoclax (ABT-263), a Bcl-2 family inhibitor. This senolytic drug led to significant cell death when combined with RT and Olaparib, while limited cytotoxicity was observed when used alone. These results demonstrate that a combination of RT with PARP inhibition and senolytics could be a promising therapeutic approach for cancer patients.

IGDQ motogenic peptide gradient induces directional cell migration through integrin (αv)ß3 activation in MDA-MB-231 metastatic breast cancer cells.

In the context of breast cancer metastasis study, we have shown in an in vitro model of cell migration that IGDQ-exposing (IsoLeu-Gly-Asp-Glutamine type I Fibronectin motif) monolayers (SAMs) on gold sustain the adhesion of breast cancer MDA-MB-231 cells by triggering Focal Adhesion Kinase and integrin activation. Such tunable scaffolds are used to mimic the tumor extracellular environment, inducing and controlling cell migration. The observed migratory behavior induced by the IGDQ-bearing peptide gradient along the surface allows to separate cell subpopulations with a "stationary" or "migratory" phenotype. In this work, we knocked down the integrins α5(ß1) and (αv)ß since they are already known to be implicated in cell migration. To this aim, a whole proteomic analysis was performed in beta 3 integrin (ITGB3) or alpha 5 integrin (ITGA5) knock-down MDA-MB-231 cells, in order to highlight the pathways implied in the integrin-dependent cell migration. Our results showed that i) ITGB3 depletion influenced ITGA5 mRNA expression, ii) ITGB3 and ITGA5 were both necessary for IGDQ-mediated directional single cell migration and iii) integrin (αv)ß3 was activated by IGDQ fibronectin type I motif. Finally, the proteomic analysis suggested that co-regulation of recycling transport of ITGB3 by ITGA5 is potentially necessary for directional IGDQ-mediated cell migration.