CD271 downregulation promotes melanoma progression and invasion in 3-dimensional models and in zebrafish

CD271 is a neurotrophin receptor variably expressed in melanoma. While contradictory data are reported on its role as a marker of tumor initiating cells, little is known on its function in tumor progression. CD271 expression was higher in spheroids derived from freshly isolated cells of primary melanomas and in primary WM115 and WM793-B cell lines, while it decreased during progression to advanced stages in cells isolated from metastatic melanomas and in metastatic WM266-4 and 1205Lu cell lines. Moreover, CD271 was scarcely detected in the highly invasive spheroids (SKMEL28 and 1205Lu). CD271, originally expressed in the epidermis of skin reconstructs, disappeared when melanoma started to invade the dermis. SKMEL8 CD271(-) cells showed greater proliferation and invasiveness in vitro, and were associated with a higher number of metastases in zebrafish, as compared to CD271(+) cells. CD271 silencing in WM115 induced a more aggressive phenotype in vitro and in vivo. On the contrary, CD271 overexpression in SKMEL28 cells reduced invasion in vitro, and CD271 overexpressing 1205Lu cells was associated with a lower percentage of metastases in zebrafish. A reduced cell-cell adhesion was also observed in absence of CD271. Taken together, these results indicate that CD271 loss is critical for melanoma progression and metastasis

The development and characterization of a human mesothelioma in vitro 3D model to investigate immunotoxin therapy.

BackgroundTumor microenvironments present significant barriers to penetration by antibodies and immunoconjugates. Tumor microenvironments, however, are difficult to study in vitro. Cells cultured as monolayers exhibit less resistance to therapy than those grown in vivo and an alternative research model more representative of the in vivo tumor is more desirable. SS1P is an immunotoxin composed of the Fv portion of a mesothelin-specific antibody fused to a bacterial toxin that is presently undergoing clinical trials in mesothelioma.Methodology/principal findingsHere, we examined how the tumor microenvironment affects the penetration and killing activity of SS1P in a new three-dimensional (3D) spheroid model cultured in vitro using the human mesothelioma cell line (NCI-H226) and two primary cell lines isolated from the ascites of malignant mesothelioma patients. Mesothelioma cells grown as monolayers or as spheroids expressed comparable levels of mesothelin; however, spheroids were at least 100 times less affected by SS1P. To understand this disparity in cytotoxicity, we made fluorescence-labeled SS1P molecules and used confocal microscopy to examine the time course of SS1P penetration within spheroids. The penetration was limited after 4 hours. Interestingly, we found a significant increase in the number of tight junctions in the core area of spheroids by electron microscopy. Expression of E-Cadherin, a protein involved in the assembly and sealing of tight junctions and highly expressed in malignant mesothelioma, was found significantly increased in spheroids as compared to monolayers. Moreover, we found that siRNA silencing and antibody inhibition targeting E-Cadherin could enhance SS1P immunotoxin therapy in vitro.Conclusion/significanceThis work is one of the first to investigate immunotoxins in 3D tumor spheroids in vitro. This initial description of an in vitro tumor model may offer a simple and more representative model of in vivo tumors and will allow for further investigations of the microenvironmental effects on drug penetration and tumor cell killing. We believe that the methods developed here may apply to the studies of other tumor-targeting antibodies and immunoconjugates in vitro

Time-lapse 3-D measurements of a glucose biosensor in multicellular spheroids by light sheet fluorescence microscopy in commercial 96-well plates

Light sheet fluorescence microscopy has previously been demonstrated on a commercially available inverted fluorescence microscope frame using the method of oblique plane microscopy (OPM). In this paper, OPM is adapted to allow time-lapse 3-D imaging of 3-D biological cultures in commercially available glass-bottomed 96-well plates using a stage-scanning OPM approach (ssOPM). Time-lapse 3-D imaging of multicellular spheroids expressing a glucose Förster resonance energy transfer (FRET) biosensor is demonstrated in 16 fields of view with image acquisition at 10 minute intervals. As a proof-of-principle, the ssOPM system is also used to acquire a dose response curve with the concentration of glucose in the culture medium being varied across 42 wells of a 96-well plate with the whole acquisition taking 9 min. The 3-D image data enable the FRET ratio to be measured as a function of distance from the surface of the spheroid. Overall, the results demonstrate the capability of the OPM system to measure spatio-temporal changes in FRET ratio in 3-D in multicellular spheroids over time in a multi-well plate format

STRENGTHEN OF DPNS FEATURES FOR THERANOSTIC APPLICATIONS AND MECHANICAL-CONTROL OF CHEMOTHERAPEUTIC EFFICACY THROUGH MODULATION OF CELL PROLIFERATION.

Solid tumors are complex biological structures which are composed of cellular and matrix components, everything being perfused by blood vessels. During tumor development, modifications of both biochemical and mechanical parameters are observed and can feedback on one another. Cancer cells constantly interact with their mechanical environment and the whole tissue is mostly confined by its surrounding. Compressive mechanical stress develops in part from cell proliferation and could eventually result in the clamping of blood vessels leading to increased interstitial fluid pressure (hydrostatic pressure). The consequent hypoperfusion poses important obstacles to drug delivery and nanomedicines. In fact, the tortuous tumor microvasculature has blood velocities up to one order of magnitude lower compared to healthy capillary networks. Moreover, the fast angiogenesis during tumor progression leads to high vascular density in solid tumors, large gaps exist between endothelial cells in tumor blood vessels, and tumor tissues show selective extravasation and retention of macromolecular drugs (Enhanced Permeation Retention \u2013 EPR \u2013 effect). These effects have served as a basis for the development of drug delivery systems which are aimed at enhancing tumor tissue targeting and drug therapeutic effectiveness. Over the last 15 years, a plethora of materials and different formulations have been proposed for the realization of nanomedicines. Yet, drug-loading efficiency, sequestration by phagocytic cells, and tumor accumulation of nanoparticle-loaded agents - nanomedicines - are sub-optimal. Starting from these considerations, during my PhD, I studied two complementary approaches: in the first two years my work was focused on implementing the characteristics of Discoidal Polymeric Nanoconstructs designed with controlled geometries and mechanical properties. In the last year, I investigated the role of mechanical stress on chemotherapeutic efficacy. More precisely, this work first reviews the use of deformable discoidal nanoconstructs (DPNs) as a novel delivery strategy for therapeutic and imaging agents. Inspired by blood cell behavior, these nanoconstructs are designed to efficiently navigate the circulatory system, minimize sequestration by phagocytic cells, and recognize the tortuous angiogenic microvasculature of neoplastic masses. In this work, the synthesis, drug loading and release, and physico-chemical characterization of DPNs were enhanced with particular emphasis on the ability to independently control size, shape, surface properties, and mechanical stiffness. Two different loading strategies were tested, namely the straightforward \u201cdirect loading\u201d and the \u201cabsorbance loading\u201d. In the former case, the agent was directly mixed with the polymeric paste to realize DPNs whereas, in the latter case, DPNs were first lyophilized and then rehydrated upon exposure to a concentrated aqueous solution of the agent. Under these two loading conditions, the encapsulation efficiencies and release profiles of three different molecules and their corresponding prodrugs were systematically assessed (1,2-Distearoyl-sn-glycero-3-phosphorylethanolamine lipid chains or 1 kDa PEG chains were directly conjugated with Cy5.5 or methotrexate and Doxorubicin). Moderately hydrophobic compounds with low molecular weight showed encapsulation efficiencies of 80%, with absorption loading (direct loading has efficiencies around 1%). The DOX-DPN showed on triple negative breast cancer cells a toxicity comparable to free DOX. Preliminary in vivo preliminary studies conducted with directly loaded Cy5-DPN demonstrated a fairly solid integration of the imaging compound with the polymer matrix of the particles. The second part of the work dissect what happens to free drugs or to drugs carried by nanovectors once they reach the tumor site. As we mention above, the elevated mechanical stress derived from tumor progression could result in blood vessels clamping with consequent reduction of drug efficacy. It is quite obvious to imagine that if the drug fails to reach the tumor it cannot act on it. Indeed, mechanical stress within the tumor site is present from the early stages of the disease. Our goal was to understand what happens when mechanical stress is not yet so large enough to fully collapse the blood vessels. Are there mechanical alterations that can affect the efficacy of a chemotherapeutic? We studied how mechanical perturbations of the tumor microenvironment could contribute to the mechanical-form of Gemcitabine drug resistance. Specifically, we developed a new in vitro strategy to mimic the mechanical compression stress induced by the stroma during tumor progression. We embedded pancreatic tumor spheroids into agarose polymeric matrix in order to demonstrate the effect of mechanical compressive stress on tumor proliferation. Then, we validated our results with other types of mechanical stresses. Finally, we investigated the therapeutic efficacy of a proliferation-based chemotherapy: Gemcitabine. Collectively, having the physical cues of cancer in mind, it can be important to cross-fertilize the fields of physical oncology and nanomedicine

Detection of X-Ray Damage Repair by the Immediate Versus Delayed Plating Technique is Dependent on Cell Shape and Cell Concentration

A method commonly used to measure the ability of cells to repair potentially lethal damage (PLD) is to compare immediate plating (IP) and delayed plating (DP) survival. Lower cell survival under IP conditions relative to that after DP conditions has been interpreted to indicate a higher ability of cells to repair potentially lethal damage (PLO) under DP conditions. However, this IP radiosensitization has not been observed in several cell lines and tumor models. IP conditions involve treatment of cells with trypsin and plating them into fresh growth medium. We have investigated the possibility that radiosensitization under IP conditions may be related to both the cell-shape and the nutrient concentration in growth medium (GM, MEM+15% serum). This idea predicts that the IP and DP survival of spheroids will show a response similar to the IP survival of cells in monolayers and that the IP and DP survival of crowded monolayer cells in high densities will be the same. Chinese hamster V79 cells grown in monolayers (spread cells) and spheroids (clumps of round cells) were used. The IP survival was lower than the DP survival for spread log phase monolayer cells but not for round log phase cells in spheroids. Radiosensitization of cells by fresh (as opposed to spent) growth medium was absent for high density plateau phase cells in monolayers at or above 2x106 cells/ml. However, PLO repair could be demonstrated in spheroid cells and in high density plateau phase cultures by exposing cells to hyperthermia or hypertonic saline. Comparison of immediate plating versus delayed plating survival detects PLO repair only in well spread low density monolayer cells, but not in round spheroid cells nor in dense monolayer cells at \u3e 107 cells/25 cm2 flask/5 ml medium. The absence of a difference between IP and DP cell survival does not mean that PLO repair is absent. Incorrect prediction of tumor response to radiotherapy can occur when PLO repair capacity is assayed as a ratio of DP/IP survival. More than one method must be used to measure the capacity of cells to repair their PLO

Role of tumor architecture in elicitation of effector functions of human cytotoxic T-lymphocytes recognizing melanoma associated antigens

Growth in 3D architectures has been shown to promote the resistance of cancers to treatment with drugs, cytokines, or irradiation, thereby potentially playing an important role in tumor expansion. 3D architectures might also play a role in impairing immunorecognition of cancer cells by cytotoxic T lymphocytes (CTLs) specific for tumor-associated antigens. Culture of HBL, D10 (both HLA-A*0201+, TAA+) and NA8 (HLA-A*0201+, TAA-) melanoma cell lines on poly-Hydroxyethylmethacrylate-coated plates, resulted in generation of 3D multicellular tumor spheroids (MCTS). Kinetics of cell proliferation in MCTS was significantly slower than in monolayer cultures. Following long-term culture (>10-15 days) MCTS showed highly compact and organised cell growth in outer layers, with necrotic cores. To obtain an insight into the role played by tumor architecture in the elicitation of specific gene expression patterns, we addressed gene expression profiles of NA8 melanoma cells cultured in two-dimensional monolayers (2D) or in 3D (MCTS). Oligonucleotide microarray analysis of the expression of over 20,000 genes was performed on cells cultured in standard 2D, in the presence of collagen as model of extracellular matrix (ECM), or in MCTS. Gene expression profiles of cells cultured in 2D in the presence or absence of ECM were highly similar, with more than threefold differences limited to five genes. In contrast, culture in MCTS resulted in the significant, more than threefold, upregulation of the expression of >100 transcripts, while 73 transcripts were more than threefold downregulated. In particular, genes encoding CXCL1, 2, and 3 (GRO-α, -β, and γ), IL-8, CCL20 (MIP-3α), and Angiopoietin-like 4 were significantly upregulated, whereas basic-FGF and CD49d encoding genes were significantly downregulated. Oligonucleotide chip data were validated at the gene and protein level by quantitative real-time PCR, ELISA, and cell surface staining assays. Taken together, our data indicate that structural modifications of the architecture of tumor cell cultures result in a significant upregulation of the expression of a number of genes previously shown to play a role in melanoma progression and metastatic process. Then we investigated the effects of 3D culture on the recognition of melanoma cells by antigenspecific HLA class I-restricted Cytotoxic T-Lymphocytes (CTL). IFN-γ production can be used as a surrogate marker for tumor cell immunorecognition. Co-culture of melanoma spheroids with HLA-A0201 restricted Melan-A/MART-127-35-specific CTL clones resulted in significantly defective TAA recognition by CTL as compared to 2D, as witnessed by decreased IFN-γ production and decreased Fas Ligand, perforin and granzyme B gene expression. Indeed, Melan- A/MART-1 expression, at both gene and protein levels, was significantly decreased in 3D as compared with 2D tumor cell cultures. Concomitantly, a parallel decrease of HLA class I molecule expression was also observed. Differential gene profiling studies on HBL cells showed an increased expression of genes encoding molecules involved in intercellular adhesion, such as junctional adhesion molecule 2 and cadherin-like 1 (>20- and 8-fold up-regulated, respectively) in 3D as compared with 2D cultures. We further identified a multiplicity of mechanisms potentially involved. In particular : 1) MCTS per se limit CTL capacity of recognizing HLA class I restricted antigens by reducing exposed cell surfaces. 2) Expression of melanoma differentiation antigens is down-regulated in tumor cell spheroids as compared to 2D unrelated to hypoxia or increased Oncostatin M gene expression but rather to decreased MITF gene expression. 3) Expression of HLA class I molecules is frequently down-regulated in melanoma MCTS, as compared to 2D, possibly due to decreased IRF-1 gene expression. 4) Lactate production by melanoma cells is increased in MCTS, as compared to 2D and lactate significantly inhibits TAA triggered IFN-γ production by CTL. Taken together, our data suggest that mere growth of melanoma cells in 3D architectures, in the absence of immunoselective pressure, may result in defective recognition by tumor-associated antigen-specific CTL and a constellation of mechanisms are involved in causing this impairment of immunorecognition

The Role of Microenviroment in Glioblastoma Progression and Resistance Development

El glioblastoma (GBM) es un tumor cerebral primario altamente heterogéneo, con una tasa de supervivencia muy baja. Recientemente se ha demostrado que su microentorno tumoral complejo tiene un papel esencial en la progresión del tumor y la respuesta a la terapia. Por lo tanto, es crucial identificar todos los componentes y sus interacciones, e incorporarlos en modelos in vitro utilizados para estudios sobre GBM y el desarrollo de nuevas terapias. El desarrollo de nuevas tecnologías en las últimas décadas ha asegurado el progreso en ambos campos mencionados. Diferentes técnicas multiómicas permiten una caracterización detallada de las muestras de los pacientes. Por otro lado, la evolución de las técnicas de cultivo celular y los procesos de fabricación permiten la creación de sistemas in vitro más fisiológicos que el cultivo tradicional en placas de Petri (organ on chip). El principal objetivo de esta tesis fue estudiar el papel del microentorno en la respuesta del GBM al tratamiento con temozolomida (TMZ). Se modificaron dispositivos microfluídicos, desarrollados previamente dentro del grupo, para estudiar el impacto de la concentración de oxígeno en la progresión de GBM. Se demostró que la hipoxia es esencial para la formación del núcleo necrótico y protege a las células del efecto de TMZ. Además, se mejoró el diseño del dispositivo microfluídico para permitir la creación de un sistema más avanzado y controlable. Igualmente, el cultivo de esferoides nos proporcionó un modelo valioso para los estudios de desarrollo de quimio-resistencia. Tras la aplicación de dos ciclos de tratamiento clínico con TMZ, se observó la aparición de una población de esferoides resistentes. Morfológicamente, esos esferoides eran una combinación de esferoides control y esferoides tratados, que tenían un patrón de expresión génica específico. Por último, se utilizó una nueva técnica de transcriptómica espacial para caracterizar mejor las muestras de pacientes con GBM, correlacionando su expresión génica con la ubicación histológica. Esto permitió la identificación de clusters transcriptómicos diferenciales dentro de tejidos aparentemente homogéneos, confirmando la alta heterogeneidad de este tumor, no solo en el aspecto morfológico sino también molecular. Glioblastoma (GBM) is a highly heterogeneous primary brain tumor, with a very low survival rate. It has been shown recently that the complex tumor microenvironment has an essential role in tumor progression and therapy response. Hence, it is crucial to identify all the components and their interactions, and incorporate them in in vitro models used for GBM studies and therapy development. The development of new technologies in the last decades ensured progress in both mentioned fields. Different multiomics techniques allow detailed characterization of the patient samples. On the other hand, the evolution of cell culture techniques and fabrication processes enables the creation of more physiological in vitro systems than traditional Petri dish culture (organ on chip). The main aim of this thesis was to study the role of the microenvironment in the response of GBM to temozolomide (TMZ) treatment. Microfluidic devices, previously developed within the group, were modified to study the impact of oxygen concentration on GBM progression. Hypoxia was shown to be essential for the necrotic core formation and it protected cells from the TMZ effect. Moreover, the microfluidic device design was improved to enable the creation of a more advanced and controllable system. Furthermore, spheroid culture gave us a valuable model for chemoresistance development studies. After the application of two clinical TMZ treatment cycles, the presence of a population of resistant spheroids was observed. Morphologically, those spheroids were a combination of control and treated spheroids, and they had a specific gene expression pattern. Last but not least, a new spatial transcriptomics technique was used to characterize better GBM patient samples correlating their gene expression with the histological location. It enabled the identification of differential transcriptomic clusters within apparently homogeneous tissues, confirming the high heterogeneity of this tumor, not only in a morphological aspect but also molecularly.<br /

Anticancer effects and antimetastatic mechanisms of novel indirubin derivatives

In this current study, we are investigating the influence of 6-bromo-indirubin-3’oxime (6BIO) and 7-bromo-indirubin-3’oxime (7BIO) on induction of apoptosis, cell proliferation, and anti-migratory effects in the well characterized pancreatic carcinoma L3.6pl and breast carcinoma Skbr3 tumor cell lines and characterize underlying mechanisms. 6BIO and 7BIO at doses of 10 µM were shown to significantly reduce the proliferation and viability as well as induce apoptosis in both cell lines. In addition, 6BIO, but not 7BIO, significantly reduced the migration of both cell lines, nearly halting them completely in the Skbr3 wound healing assay at sub-apoptotic doses (3 µM). Chemotaxis was dramatically disrupted and tumor cells significantly lost their ability to invade through membranes or MatrigelTM layers in response to chemoattractants. An increase of the phosphorylation site S785 of beta1 integrin is seen upon 6BIO which has been linked to decreased motility of carcinoma cells. Additionally, adhesion of Skbr3 tumor cells to fibronectin was reduced by 6BIO stimulation. The effects of 6BIO can be attributed to its reduction of the T308 phosphorylation site of Akt, most likely through its direct inhibition of PDK1, ultimately causing long term alterations to the actin cytoskeleton. Erk, FAK and Rac1 levels are unaffected, but cycling of these signaling molecules appears to be disrupted upon treatment. Finally 6BIO reduced the metabolic capabilities of Skbr3 spheroids at low doses, caused the dissolution of spheroid structures at higher doses and significantly blocked the migration of Skbr3 spheroids. Taken together, the results of this study strongly suggest that the indirubin derivative 6BIO operates by inhibiting different mechanisms in human tumor cells to exert their potent anti-tumor efficacy

Nano-Electrochemical Characterization of a 3D Bioprinted Cervical Tumor Model

Current cancer research is limited by the availability of reliable in vivo and in vitro models that are able to reproduce the fundamental hallmarks of cancer. Animal experimentation is of paramount importance in the progress of research, but it is becoming more evident that it has several limitations due to the numerous differences between animal tissues and real, in vivo human tissues. 3D bioprinting techniques have become an attractive tool for many basic and applied research fields. Concerning cancer, this technology has enabled the development of three-dimensional in vitro tumor models that recreate the characteristics of real tissues and look extremely promising for studying cancer cell biology. As 3D bioprinting is a relatively recently developed technique, there is still a lack of characterization of the chemical cellular microenvironment of 3D bioprinted constructs. In this work, we fabricated a cervical tumor model obtained by 3D bioprinting of HeLa cells in an alginate-based matrix. Characterization of the spheroid population obtained as a function of culturing time was performed by phase-contrast and confocal fluorescence microscopies. Scanning electrochemical microscopy and platinum nanoelectrodes were employed to characterize oxygen concentrations - a fundamental characteristic of the cellular microenvironment - with a high spatial resolution within the 3D bioprinted cervical tumor model; we also demonstrated that the diffusion of a molecular model of drugs in the 3D bioprinted construct, in which the spheroids were embedded, could be measured quantitatively over time using scanning electrochemical microscop