Investigation of squalene-doxorubicin distribution and interactions within single cancer cell using Raman microspectroscopy.

Intracellular distribution of doxorubicin (DOX) and its squalenoylated (SQ-DOX) nanoparticles (NPs) form in murine lung carcinoma M109 and human breast carcinoma MDA-MB-231 cells was investigated by Raman microspectroscopy. Pharmacological data showed that DOX induced higher cytotoxic effect than SQ-DOX NPs. Raman data were obtained using single-point measurements and imaging on the whole cell areas. These data showed that after DOX treatment at 1 µM, the spectral features of DOX were not detected in the M109 cell cytoplasm and nucleus. However, the intracellular distribution of SQ-DOX NPs was higher than DOX in the same conditions. In addition, SQ-DOX NPs were localized into both cell cytoplasm and nucleus. After 5 µM treatment, Raman bands of DOX at 1211 and 1241 cm-1 were detected in the nucleus. Moreover, the intensity ratio of these bands decreased, indicating DOX intercalation into DNA. However, after treatment with SQ-DOX NPs, the intensity of these Raman bands increased. Interestingly, with SQ-DOX NPs, the intensity of 1210/1241 cm-1 ratio was higher suggesting a lower fraction of intercalated DOX in DNA and higher amount of non-hydrolyzed SQ-DOX. Raman imaging data confirm this subcellular localization of these drugs in both M109 and MDA-MB-231 cells. These finding brings new insights to the cellular characterization of anticancer drugs at the molecular level, particularly in the field of nanomedicine.

Type I Collagen Aging Increases Expression and Activation of EGFR and Induces Resistance to Erlotinib in Lung Carcinoma in 3D Matrix Model.

Type I collagen is the major structural component of lung stroma. Because of its long half-life, type I collagen undergoes post-translational modifications such as glycation during aging process. These modifications have been shown to impact the structural organization of type I collagen fibers. In the present work we evaluated the impact of collagen aging on lung carcinoma cells response to erlotinib-induced cytotoxicity and apoptosis, and on Epidermal Growth Factor Receptor (EGFR) expression and phosphorylation. To this end, experiments were performed in 2D and 3D matrix models established from type I collagen extracted from adult (10 weeks-old) and old (100 weeks-old) rat's tail tendons. Our results show that old collagen induces a significant increase in EGFR expression and phosphorylation when compared to adult collagen in 3D matrix but not in 2D coating. Such modification was associated to an increase in the IC50 of erlotinib in the presence of old collagen and a lower sensitivity to drug-induced apoptosis. These data suggest that collagen aging confers resistance to the cytotoxic and apoptotic effects of therapies targeting EGFR kinase function in lung carcinoma. Moreover, our data underline the importance of the 3D matrix environment in this process.

DDR1 and MT1-MMP Expression Levels Are Determinant for Triggering BIK-Mediated Apoptosis by 3D Type I Collagen Matrix in Invasive Basal-Like Breast Carcinoma Cells.

Type I collagen is the major adhesive component in breast interstitial stroma, which represents the first barrier against tumor cell invasion after basement-membrane degradation. Among cellular receptors, type I collagen is able to activate discoidin domain receptors DDR1 and DDR2. We have previously shown that in 3D collagen matrix, DDR1 plays a key role as it promotes cell growth suppression and apoptosis through the upregulation of the pro-apoptotic mediator BIK in noninvasive luminal-like breast carcinoma cells. We have also shown that MT1-MMP is able to rescue these cells and protect them against the effects induced by collagen/DDR1/BIK axis. Our data suggested that the protective effect of MT1-MMP might be mediated through the degradation of type I collagen and/or DDR1 cleavage. Decreased DDR1 expression has been associated with the epithelial to mesenchymal transition process in breast cancer, and its overexpression in aggressive basal-like breast cancer cells reduces their invasiveness in 3D cultures and in vivo. In the present work, we propose to study the role of MT1-MMP in the resistance against collagen-induced apoptosis in basal-like breast carcinoma MDA-MB-231 cells. We aimed to investigate whether MT1-MMP depletion is able to restore apoptosis mediated by collagen/DDR1/BIK axis and to verify if such depletion is able to restore full-length DDR1 expression and phosphorylation. ShRNA strategy against MT1-MMP mRNA was able to partially restore full length DDR1 expression and phosphorylation. This was accompanied by a decrease in cell growth and an upregulation of BIK expression. This suggested that MT1-MMP expression in basal-like breast carcinoma cells, in addition to a low basal level of DDR1 expression, protects these cells against collagen-induced apoptosis via DDR1 cleavage. Since DDR1 was moderately expressed in MDA-MB-231 cells, we then investigated whether overexpression of DDR1 could be able to increase its ability to suppress cell growth and to induce apoptosis. Data showed that overexpression of DDR1 induced a decrease in cell growth and an increase in BIK expression, suggesting that moderate expression level of full length DDR1 in basal-like breast carcinoma provides them with a capacity to resist to collagen-induced cell growth suppression and apoptosis. Finally, the combined overexpression of DDR1 and depletion of MT1-MMP in MDA-MB-231 cells synergistically increased collagen-induced cell growth suppression and apoptosis to a level similar to that observed in luminal breast carcinoma. Taken together, our data suggest that during the acquisition of mesenchymal features, the low level of DDR1 expression should be considered as an important biomarker in the prognosis of basal-like breast carcinoma, conferring them a high rate of cell growth and resistance to BIK-mediated apoptosis induced by the stromal collagen.

Original association of ion transporters mediates the ECM-induced breast cancer cell survival: Kv10.1-Orai1-SPCA2 partnership.

In the last years it has been shown that many components of tumor microenvironment (TM) can induce cell signaling that permit to breast cancer cells (BC) to maintain their aggressiveness. Ion channels have a role in mediating TM signal; recently we have demonstrated a functional collaboration between Kv10.1 and Orai1 channels in mediating the pro-survival effect of collagen 1 on BC cells. Here we show how SPCA2 (Secretory Pathway Ca2+ ATPase) has a role in this process and is able to support survival and proliferation induced by collagen 1. By participating to an auto-sustaining loop, SPCA2 enhances membrane expression of Kv10.1 and Orai1; the activity of every component of this trio is necessary to mediate a store independent calcium entry (SICE). This SICE is fundamental to maintain both the activation of the pro-survival pathway and the membrane localization and consequently the activity of the two channels. Moreover, the three proteins and the collagen receptor DDR1 are overexpressed only in aggressive tumors tissues. In this work, we propose a novel association between SPCA2, Kv10.1 and Orai1 involved in mediating transduction signals from TM to the BC cells that can be potentially exploited in the search of novel therapeutic targets specific to tumor tissues.

Collagen type 1 promotes survival of human breast cancer cells by overexpressing Kv10.1 potassium and Orai1 calcium channels through DDR1-dependent pathway.

Collagen type 1 is among the tumor microenvironment (TM) factors, that regulates proliferation, survival, migration and invasion. Ion channels are key players in interactions between tumor cells and TM. Kv10.1 has been shown to play an essential role in breast cancer cell proliferation and migration by permitting Ca2+ influx notably via Orai1. Here, we show that human breast cancer (BC) cells growing, in culture media completely devoid of the serum and seeded on collagen 1 coating, exhibited less apoptotic rate and a decrease in Bax expression when compared to those grown on plastic. The survival conferred by collagen 1 was completely abolished by removing extracellular Ca2+ from the culture medium. In addition, Ca2+ entry was increased in collagen 1 condition along with increased Kv10.1 and Orai1 expressions. Moreover, collagen 1 was able to increase co-localization of Kv10.1 and Orai1 on the plasma membrane. Interestingly, silencing of Kv10.1 and Orai1 reduced survival and Ca2+influx without any additive effect. This calcium-dependent survival is accompanied by the activation of ERK1/2, and its pharmacological inhibition completely abolished the increase in Kv10.1 and Orai1 expressions, activities, and the cell survival induced by collagen 1. Moreover, both Kv10.1 and Orai1 knockdown reduced ERK1/2 activation but not Akt. Finally, DDR1 silencing but not ß1-integrin reduced the collagen induced survival, ERK1/2 phosphorylation and the expression of Kv10.1 and Orai1. Together these data show that the Kv10.1/Orai1 complex is involved in BC cell survival and this is dependent on collagen 1/DDR1 pathway. Therefore, they represent a checkpoint of tumor progression induced by the tumor microenvironment.

Age-related modifications of type I collagen impair DDR1-induced apoptosis in non-invasive breast carcinoma cells.

Type I collagen and DDR1 axis has been described to decrease cell proliferation and to initiate apoptosis in non-invasive breast carcinoma in three-dimensional cell culture matrices. Moreover, MT1-MMP down-regulates these effects. Here, we address the effect of type I collagen aging and MT1-MMP expression on cell proliferation suppression and induced-apoptosis in non-invasive MCF-7 and ZR-75-1 breast carcinoma. We provide evidence for a decrease in cell growth and an increase in apoptosis in the presence of adult collagen when compared to old collagen. This effect involves a differential activation of DDR1, as evidenced by a higher DDR1 phosphorylation level in adult collagen. In adult collagen, inhibition of DDR1 expression and kinase function induced an increase in cell growth to a level similar to that observed in old collagen. The impact of aging on the sensitivity of collagen to MT1-MMP has been reported recently. We used the MT1-MMP expression strategy to verify whether, by degrading adult type I collagen, it could lead to the same phenotype observed in old collagen 3D matrix. MT1-MMP overexpression abrogated the proliferation suppression and induced-apoptosis effects only in the presence of adult collagen. This suggests that differential collagen degradation by MT1-MMP induced a structural disorganization of adult collagen and inhibits DDR1 activation. This could in turn impair DDR1-induced cell growth suppression and apoptosis. Taken together, our data suggest that modifications of collagen structural organization, due to aging, contribute to the loss of the growth suppression and induced apoptosis effect of collagen in luminal breast carcinoma. MT1-MMP-dependent degradation and aging of collagen have no additive effects on these processes.

First plasma and tissue pharmacokinetic study of the YSNSG cyclopeptide, a new integrin antagonist, using microdialysis.

The YSNSG peptide is a synthetic peptide targeting αvß3 integrin. This peptide exhibits promising activity in vitro and in vivo against melanoma. To determine pharmacokinetic parameters and predictive active doses in the central nervous system (CNS) and subcutaneous tissue (SC), we conducted microdialysis coupled with pharmacokinetic modeling and Monte Carlo simulation. After a recovery period of surgical procedures, a microdialysis probe was inserted in the caudate and in subcutaneous tissue. Plasma samples and dialysates collected 5h after YSNSG intravenous administration (10mg/kg) were analyzed by UPLC-MS/MS. A nonlinear mixed-effect modeling approach implemented in Monolix® 2016R1 was performed. Model selection and evaluation were based on the usual diagnostic plot, precision and information criteria. The primary plasma and tissue pharmacokinetic parameters were comparable with those of other integrin antagonists, such as cilengitide or ATN-161. Tissue/plasma and brain/plasma area under the curve (AUC) ratio were 66.2±21.6% and 3.6±4.7%, respectively. Two models of 2-compartments with an additional microdialysis compartment, parameterized as rate constants (k for elimination, k12/k21 and k13/k31 for distribution) and volumes (central V1 and peripheral microdialysis compartment V3) with zero-order input were selected to describe the dialysate concentrations in CNS and SC. The inter-individual variability (IIV) was described by exponential terms, and residual variability was described by a combined additive and proportional error model. Individual AUC (plasma and tissues) values were derived for each animal using the Empirical-Bayes-Estimates of the individual parameters. The regimens needed to achieve an in vitro predetermined target concentration in tissues were studied by Monte Carlo simulations using Monolix® 2016R1. YSNSG pharmacokinetic parameters show promising results in terms of subcutaneous disposition. Further investigations into such processes as encapsulation and intratumoral disposition are currently being conducted.

Corrigendum: Discoidin Domain Receptors: Potential Actors and Targets in Cancer.

[This corrects the article on p. 55 in vol. 7, PMID: 27014069.].

Type I collagen aging impairs discoidin domain receptor 2-mediated tumor cell growth suppression.

Tumor cells are confronted to a type I collagen rich environment which regulates cell proliferation and invasion. Biological aging has been associated with structural changes of type I collagen. Here, we address the effect of collagen aging on cell proliferation in a three-dimensional context (3D).We provide evidence for an inhibitory effect of adult collagen, but not of the old one, on proliferation of human fibrosarcoma HT-1080 cells. This effect involves both the activation of the tyrosine kinase Discoidin Domain Receptor 2 (DDR2) and the tyrosine phosphatase SHP-2. DDR2 and SHP-2 were less activated in old collagen. DDR2 inhibition decreased SHP-2 phosphorylation in adult collagen and increased cell proliferation to a level similar to that observed in old collagen.In the presence of old collagen, a high level of JAK2 and ERK1/2 phosphorylation was observed while expression of the cell cycle negative regulator p21CIP1 was decreased. Inhibition of DDR2 kinase function also led to an increase in ERK1/2 phosphorylation and a decrease in p21CIP1 expression. Similar signaling profile was observed when DDR2 was inhibited in adult collagen. Altogether, these data suggest that biological collagen aging could increase tumor cell proliferation by reducingthe activation of the key matrix sensor DDR2.

Discoidin Domain Receptors: Potential Actors and Targets in Cancer.

The extracellular matrix critically controls cancer cell behavior by inducing several signaling pathways through cell membrane receptors. Besides conferring structural properties to tissues around the tumor, the extracellular matrix is able to regulate cell proliferation, survival, migration, and invasion. Among these receptors, the integrins family constitutes a major class of receptors that mediate cell interactions with extracellular matrix components. Twenty years ago, a new class of extracellular matrix receptors has been discovered. These tyrosine kinase receptors are the two discoidin domain receptors DDR1 and DDR2. DDR1 was first identified in the Dictyostelium discoideum and was shown to mediate cell aggregation. DDR2 shares highly conserved sequences with DDR1. Both receptors are activated upon binding to collagen, one of the most abundant proteins in extracellular matrix. While DDR2 can only be activated by fibrillar collagen, particularly types I and III, DDR1 is mostly activated by type I and IV collagens. In contrast with classical growth factor tyrosine kinase receptors which display a rapid and transient activation, DDR1 and DDR2 are unique in that they exhibit delayed and sustained receptor phosphorylation upon binding to collagen. Recent studies have reported differential expression and mutations of DDR1 and DDR2 in several cancer types and indicate clearly that these receptors have to be taken into account as new players in the different aspects of tumor progression, from non-malignant to highly malignant and invasive stages. This review will discuss the current knowledge on the role of DDR1 and DDR2 in malignant transformation, cell proliferation, epithelial to mesenchymal transition, migratory, and invasive processes, and finally the modulation of the response to chemotherapy. These new insights suggest that DDR1 and DDR2 are new potential targets in cancer therapy.

Encapsulated Ruthenium(II) Complexes in Biocompatible Poly(d,l-lactide-co-glycolide) Nanoparticles for Application in Photodynamic Therapy.

The elaboration, characterisation and efficiency of potential two-photon-excited photodynamic therapy (PDT) treatment of new poly(d,l-lactide-co-glycolide) nanoparticles loaded with ruthenium(II) complexs are presented. The materials are based on the encapsulation of RuII complexes through an all-biocompatible process. The size of the nanoparticles is around 100 nm. The internal concentration is several orders of magnitude higher than the overall concentration, which leads to a more efficient and targeted effect. The therapeutic potential for PDT of these nanoparticles has been studied in vitro on C6 glioma cells.

Bifunctional polypyridyl-Ru(II) complex grafted onto gadolinium-based nanoparticles for MR-imaging and photodynamic therapy.

The synthesis, optical properties and efficiency of new multifunctional nanoparticles as theranostic (fluorescence/MRI/PDT) agents are described. They are based on a polysiloxane network and surrounded by gadolinium(III) chelates and ruthenium(II) complexes. The size of the nanoparticles is maintained under 5 nm in order to permit their efficient elimination from the body. Their potential use as a theranostic agent (PDT/MRI) is described. The magnetic properties of the nanoparticles are studied by relaxometry (r1 = 9.21 mM(-1) s(-1) at 40 MHz; r2/r1 = 1.14) and the signal enhancement is validated by the acquisition of phantoms on a 3 T MRI imager. The therapeutic potential for photodynamic therapy of the nanoparticles has been studied in vitro on HEK293 cells and an effective quantum yield of 0.33 for (1)O2 production has been determined in deuterated water.

Etoposide encapsulation in surface-modified poly(lactide-co-glycolide) nanoparticles strongly enhances glioma antitumor efficiency.

Etoposide (VP-16) is a hydrophobic anticancer agent inhibiting Topoisomerase II, commonly used in pediatric brain chemotherapeutic schemes as mildly toxic. Unfortunately, despite its appropriate solubilization in vehicle solvents, its poor bioavailability and limited passage of the blood-brain barrier concur to disappointing results requiring the development of new delivery system forms. In this study, etoposide formulated as a parenteral injectable solution (Teva®) was loaded into all-biocompatible poly(lactide-co-glycolide) (PLGA) or PLGA/P188-blended nanoparticles (size 110-130 nm) using a fully biocompatible nanoprecipitation technique. The presence of coprecipitated P188 on encapsulation efficacies and in vitro drug release was investigated. Drug encapsulation was determined using HPLC. Inflammatory response was checked by FACS analysis on human monocytes. Cytotoxic activity of the various simple (Teva®) or double (Teva®-loaded NPs) formulations was studied on the murine C6 and F98 cell lines. Obtained results suggest that, although noninflammatory neither nontoxic by themselves, the use of PLGA and PLGA/P188 nanoencapsulations over pre-existing etoposide formulation could induce a greatly improved cytotoxic activity. This approach demonstrated a promising perspective for parenteral delivery of VP16 and potential development of a therapeutic entity.

Impact of carbamylation and glycation of collagen type I on migration of HT1080 human fibrosarcoma cells.

Collagen type I is an abundant component of the extracellular matrix and due to its longevity, constitutes a prominent target of non-enzymatic post-translational in vivo modifications such as carbamylation and glycation. These protein modifications involved in aging, renal diseases and diabetes, are linked to elevated cancer risk. In this in vitro study, we investigated the impact of carbamylated and glycated collagen type I on the migratory behavior of the highly invasive HT1080 human fibrosarcoma cells. The proliferation of HT1080 on modified collagens did not differ from that on native form. The glycated collagen delayed the cell adhesion time whereas the carbamylated one had no effect. The migration ability of HT1080 was studied by quantifying single cell speed using videomicroscopy. Glycation strongly inhibited mean cell speed by 47% whereas carbamylation moderately affected it by 12%. In addition, the influence of these collagen modifications on actin and vinculin organization was studied. On the glycated collagen, 63% of cells revealed a dramatic loss of actin stress fibers vs. 28% on the carbamylated one. In these cells, disorganized F-actin was accompanied with a disturbance of vinculin and both proteins were localized at the rim of the cells. Concerning the focal adhesion kinase (FAK) expression, glycated collagen only induced a significant inhibition. Whereas, both collagen modifications provoked a differential inhibition of FAK phosphorylation state by 25% for carbamylation and 60% for glycation. In conclusion, our data suggest that, in vivo, collagen glycation and carbamylation may affect tumor cell metastasis. This suggestion is supported by clinical studies reporting less aggressive tumors in diabetic or uremic patients. Consequently, the impact of such post-translational modifications has to be taken into account in order to better understand the link between aging, diabetes or uremia and cancer progression.

3D collagen type I matrix inhibits the antimigratory effect of doxorubicin.

BACKGROUND: The cell microenvironment, especially extracellular matrix proteins, plays an important role in tumor cell response to chemotherapeutic drugs. The present study was designed to investigate whether this microenvironment can influence the antimigratory effect of an anthracycline drug, doxorubicin, when tumor cells are grown in a matrix of type I collagen, a three-dimensional (3D) context which simulates a natural microenvironment. METHODS: To this purpose, we studied the migratory parameters, the integrin expression, and the activation state of focal adhesion kinase (FAK) and GTPase RhoA involved in the formation of focal adhesions and cell movement. These parameters were evaluated at non toxic concentrations which did not affect HT1080 cell proliferation. RESULTS: We show that while doxorubicin decreased cell migration properties by 70% in conventional two-dimensional (2D) culture, this effect was completely abolished in a 3D one. Regarding the impact of doxorubicin on the focal adhesion complexes, unlike in 2D systems, the data indicated that the drug neither affected beta1 integrin expression nor the state of phosphorylation of FAK and RhoA. CONCLUSION: This study suggests the lack of antiinvasive effect of doxorubicin in a 3D environment which is generally considered to better mimic the phenotypic behaviour of cells in vivo. Consistent with the previously shown resistance to the cytotoxic effect in a 3D context, our results highlight the importance of the matrix configuration on the tumor cell response to antiinvasive drugs.