RESUMO
The complex interaction between tumor-associated macrophages (TAMs) and tumor cells through soluble factors provides essential cues for breast cancer progression. TAMs-targeted therapies have shown promising clinical therapeutical potential against cancer progression. The molecular mechanisms underlying the response to TAMs-targeted therapies depends on complex dynamics of immune cross-talk and its understanding is still incomplete. In vitro models are helpful to decipher complex responses to combined immunotherapies. In this study, we established and characterized a 3D human macrophage-ER+ PR+ HER2+ breast cancer model, referred to as macrophage-tumor spheroid (MTS). Macrophages integrated within the MTS had a mixed M2/M1 phenotype, abrogated the anti-proliferative effect of trastuzumab on tumor cells, and responded to IFNγ with increased M1-like polarization. The targeted treatment of MTS with a combined CSF1R kinase inhibitor and an activating anti-CD40 antibody increased M2 over M1 phenotype (CD163+/CD86+ and CD206+/CD86+ ratio) in time, abrogated G2/M cell cycle phase transition of cancer cells, promoted the secretion of TNF-α and reduced cancer cell viability. In comparison, combined treatment in a 2D macrophage-cancer cell co-culture model reduced M2 over M1 phenotype and decreased cancer cell viability. Our work shows that this MTS model is responsive to TAMs-targeted therapies, and may be used to study the response of ER+ PR+ HER2+ breast cancer lines to novel TAM-targeting therapies.
RESUMO
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.
Assuntos
Macrófagos , Macrófagos Associados a Tumor , Linhagem Celular Tumoral , Técnicas de Cocultura , Humanos , Imunoterapia , Macrófagos/metabolismo , Microambiente TumoralRESUMO
Cellular surface recognition and behavior are driven by a host of physical and chemical features which have been exploited to influence particle-cell interactions. Mechanical and topographical cues define the physical milieu which plays an important role in defining a range of cellular activities such as material recognition, adhesion, and migration through cytoskeletal organization and signaling. In order to elucidate the effect of local mechanical and topographical features generated by the adsorption of particles to an underlying surface on primary human monocyte-derived macrophages (MDM), a series of poly(N-isopropylacrylamide) (pNIPAM) particles with differing rigidity are self-assembled to form a defined particle-decorated surface. Assembly of particle-decorated surfaces is facilitated by modification of the underlying glass to possess a positive charge through functionalization using 3-aminopropyltriethoxysilane (APTES) or coating with poly(L-lysine) (PLL). MDMs are noted to preferentially remove particles with higher degrees of crosslinking (stiffer) than those with lower degrees of crosslinking (softer). Alterations to the surface density of particles enabled a greater area of the particle-decorated surface to be cleared. Uniquely, the impact of particle adsorption is evinced to have a direct impact on topographical recognition of the surface, suggesting a novel approach for controllably affecting cell-surface recognition and response.
Assuntos
Vidro , Macrófagos , Adsorção , Humanos , Tamanho da Partícula , Propriedades de SuperfícieRESUMO
Evaluating nanomaterial uptake and association by cells is relevant for in vitro studies related to safe-by-design approaches, nanomedicine or applications in photothermal therapy. However, standard analytical techniques are time-consuming, involve complex sample preparation or include labelling of the investigated sample system with e.g. fluorescent dyes. Here, we explore lock-in thermography to analyse and compare the association trends of epithelial cells, mesothelial cells, and macrophages exposed to gold nanoparticles and multi-walled carbon nanotubes over 24 h. The presence of nanomaterials in the cells was confirmed by dark field and transmission electron microscopy. The results obtained by lock-in thermography for gold nanoparticles were validated with inductively coupled plasma optical emission spectrometry; with data collected showing a good agreement between both techniques. Furthermore, we demonstrate the detection and quantification of carbon nanotube-cell association in a straightforward, non-destructive, and non-intrusive manner without the need to label the carbon nanotubes. Our results display the first approach in utilizing thermography to assess the carbon nanotube amount in cellular environments.
Assuntos
Nanopartículas Metálicas , Nanotubos de Carbono , Ouro , Macrófagos , Microscopia Eletrônica de TransmissãoRESUMO
A series of tricarbonyl manganese complexes bearing 4-ethynyl-2,2'-bipyridine and 5-ethynyl-1,10-phenanthroline α-diimine ligands were synthetized, characterized and conjugated to vitamin B12, previously used as a vector for drug delivery, to take advantage of its water solubility and specificity toward cancer cells. The compounds act as photoactivatable carbon monoxide-releasing molecules rapidly liberating on average ca. 2.3 equivalents of CO upon photo-irradiation. Complexes and conjugates were tested for their anticancer effects, both in the dark and following photo-activation, against breast cancer MCF-7, lung carcinoma A549 and colon adenocarcinoma HT29 cell lines as well as immortalized human bronchial epithelial cells 16HBE14o- as the non-carcinogenic control. Our results indicate that the light-induced cytotoxicity these molecules can be attributed to both their released CO and to their CO-depleted metal fragments including liberated ligands.
Assuntos
Monóxido de Carbono/química , Complexos de Coordenação/química , Luz , Manganês/química , Neoplasias/metabolismo , Células A549 , Monóxido de Carbono/metabolismo , Complexos de Coordenação/metabolismo , Cristalografia por Raios X/métodos , Células HT29 , Humanos , Ligantes , Células MCF-7 , Manganês/metabolismo , Neoplasias/patologia , Compostos Organometálicos/química , Compostos Organometálicos/metabolismo , Fenantrolinas/química , Fotólise , Solubilidade , Vitamina B 12/metabolismoRESUMO
Pore-forming antimicrobial peptides (AMPs) are attracting interest as cytolytic antibiotics and drug delivery agents with potential use for targeting cancer cells or multidrug-resistant pathogens. Ceratotoxin A (CtxA) is an insect-derived cytolytic AMP with 36 amino acids that is thought to protect the eggs of the medfly Ceratitis capitata against pathogens. Single channel recordings using planar lipid bilayers have shown that CtxA forms pores with well-defined conductance states resembling those of alamethicin; it also forms one of the largest pores among the group of ceratotoxins. In this work, we modified CtxA at its N-terminus with an azide group and investigated its pore-forming characteristics in planar lipid bilayer experiments. We demonstrate the possibility to target specific lipids by carrying out click reactions in-situ on lipid membranes that display a dibenzocyclooctyne (DBCO) moiety on their head group. As a result of covalent linkage of the peptides to the bilayer, pore-formation occurs at 10-fold reduced peptide concentration and with a reduced dependence on the transmembrane voltage compared to unlinked CtxA-azide peptides or native CtxA peptides.
Assuntos
Azidas/farmacologia , Proteínas de Insetos/metabolismo , Proteínas de Insetos/farmacologia , Sequência de Aminoácidos , Aminoácidos , Antibacterianos/química , Peptídeos Catiônicos Antimicrobianos/metabolismo , Peptídeos Catiônicos Antimicrobianos/farmacologia , Azidas/metabolismo , Membrana Celular/efeitos dos fármacos , Membrana Celular/metabolismo , Células HeLa , Humanos , Proteínas de Insetos/química , Células KB , Bicamadas Lipídicas/química , Metabolismo dos Lipídeos/efeitos dos fármacos , Lipídeos/químicaRESUMO
Herein we report the synthesis of a new biomaterial designed for targeted delivery of poorly water-soluble inorganic anticancer drugs, with a focus on colorectal cancer. Diatomaceous earth microparticles derived from marine microalgae were coated with vitamin B12 (cyanocobalamin) as a tumor targeting agent and loaded with the well-known anticancer agents cisplatin, 5-fluorouracil (5-FU), and a tris-tetraethyl[2,2'-bipyridine]-4,4'-diamine-ruthenium(ii) complex. The successful functionalization of the biomaterial was demonstrated by different analytical techniques and by synthesizing an organometallic fluorescein analogue of cyanocobalamin detectable by confocal laser scanning microscopy. The drug releasing properties were evaluated for all three species. We found that while cisplatin and 5-FU are rapidly lost from the material, the ruthenium complex showed an unprecedented release profile, being retained in the material up to 5 days in aqueous media but readily released in lipophilic environments as in the cell membrane. The increased adherence of the B12 coated diatoms to colorectal cancer cell line HT-29 and breast cancer cell line MCF-7 was demonstrated in vitro. In both cases, the adherence of the B12 modified diatoms was at least 3 times higher than that of the unmodified ones and was correlated with the increased transcobalamin II (TC(II)) and transcobalamin II receptor (TC(II)-R) expression of the targeted tissue. Our results suggest that this type of B12 modified diatoms could be a promising tool to achieve targeted delivery of water insoluble inorganic complexes to tumor tissues by acting as a micro-shuttle interacting with the sites of interest before delivering the drug in the vicinity of the tumor tissue.