WO2019220357A1

WO2019220357A1 - Composition comprising a hydrogel

Info

Publication number: WO2019220357A1
Application number: PCT/IB2019/054011
Authority: WO
Inventors: Alessandra MARRELLA; Silvia Scaglione; Maurizio AIELLO
Original assignee: React4Life S.R.L.
Priority date: 2018-05-17
Filing date: 2019-05-15
Publication date: 2019-11-21
Also published as: IT201800005459A1

Abstract

The present invention relates to a composition in the form of a hydrogel comprising viable tumour cells and a method for its preparation.

Description

COMPOSITION COMPRISING A HYDROGEL

The present invention relates to a composition in the form of a hydrogel comprising viable cancer cells and a method for its preparation.

Neuroblastoma (NB) is the most common extracranial and fourth most common tumour in children. The use of two-dimensional (2D) monolayer cultures of cancer cell lines as simple models for studying in vitro the processes underlying tumour onset and progression dates back to the l950s. Nevertheless, tumour tissues are 3D entities, similar to healthy organs, composed of cells surrounded by abundant extracellular matrix. To date, it is generally acknowledged that cultures of monolayer cell lines suffer from numerous inadequacies in the reproduction of tumour onset phenomena that actually occur in vivo, thus limiting their ability to predict the real behaviour of cancer cells (LC Kimlin, et ah, " Three-dimensional in vitro (3D) models in cancer research: an update. " Molecular carcinogenesis 52.3 (2013): 167-182).

First of all, current 2D cultures of human tumour cells fail to mimic the biology and progression of the disease, whereas 3D systems can potentially include more factors (chemical, physical and spatial conformation) capable of emulating the architectural complexity of the tumour mass, thus allowing a greater accuracy of the model and, therefore, contributing to a better modelling of the evolution phenomena of the disease.

Clinicians and researchers have in fact often highlighted contradictory results between preclinical tests (in vitro ) and clinical results (in vivo) in terms of efficiency of anti-cancer treatments. In particular, 2D models generally show a much higher response to therapeutic agents with respect to clinical observations.

During the validation process of anticancer therapies, the results obtained from preclinical cellular tests are usually translated into animal models called "xeno-transplants", which consist in the implantation in animals of tissues coming from another species (e.g. human beings); these models are recognized, to date, as the 3D environments closest to our organism, before clinical translation. Unfortunately, along with relevant ethical implications, these xenografts are commonly generated from immuno-compromised mice, i.e. without the immune system, which limit the possibility of studying the effects of the human microenvironment and its immune system on tumour growth. Furthermore, other numerous limitations of murine models are evident, for example: (i) the patient's tumour may not be successfully grafted into the mouse, (ii) the subcutaneous or orthotropic implants do not accurately reflect the tumour microenvironment, (iii), the efficiencies of tumour engraftment in animals vary significantly, depending on the type of tumour, thus not allowing the establishment of standard protocols, (iv) tumour propagation in the mouse can cause various micro-environmental changes, which may not accurately resemble the human tumour.

Various 3D models have been consequently developed, which combine the advantages of in vitro 2D models (spatial control, temporal control of the biophysical conditions, relative ease of manipulation and analysis with respect to animal models) and in vivo models which have a higher degree of accuracy.

The first 3D models used include tumour spheroids, formed by spontaneous self-aggregation of human tumour cells on a non-adhesive surface. Spheroidal tumour models must be able to replicate specific features of tumours in vivo, such as central hypoxic conditions, heterogeneity in the phenotype and gene expression and altered cellular metabolism. In particular, spheroids are aggregates of cells which allow to reproduce the original cell-cell interactions of neoplastic tissues. Their process of fabrication is called "hanging drop" and consists in the deposition of drops composed of a high concentration of cells.

Although this method is simple and cheap, the size and uniformity of the drops are difficult to control and reproduce.

In particular, these models have various limitations. They are in fact susceptible to physical disintegration during manipulation and experimentation, and the micro-environmental conditions, such as the poor presence of extracellular matrix (ECM), limit the in vitro realization of a realistic model of cell-matrix interaction. Furthermore, the mass transport limits and the release of anticancer drugs in in vivo tumours cannot be precisely reproduced due to the non-uniform secretion of the endogenous extracellular matrix by the spheroids and the absence of an exogenous matrix (biomaterial), whose mechanical rigidity can be accurately controlled and reproduced.

Consequently, in recent decades, new tumour models have been developed based on engineered materials (mainly hydrogels) in which tumour cells can be encapsulated within matrices (mostly polymeric), which are geometrically similar in size and shape to tumour spheroids and have chemical-physical characteristics, which make them similar to the extracellular matrix where cancer cells proliferate, migrate and arrange.

These models have provided new methods for accelerating cancer research, in particular improving the quality of preclinical cancer research, which is necessary in the development and testing of innovative anticancer therapies.

Although interest in these new hydrogel-based 3D models has increased in recent years, references in literature relating to the development of engineered materials for neuroblastoma modelling are scarce, even if neuroblastoma is a particularly aggressive type of tumour and the most common extracranial tumour in infants and the fourth in children (e.g. Yeung, P. Plos One 2015, 12, e0l44l39 and W02016004068). In particular, in the literature there are some works related to the modelling of neuroblastomas, aimed at demonstrating the in vitro differentiation of murine (and non-human) cells and the formation of a nerve extension, prodromal to the repair of damaged nerve tissue (e.g. US2011/033504, NO Dhoot et al, J. Biomed, Mater Res 2004, 2, 191-200).

The paediatric oncology sector still needs a realistic human tumour model that can be used in the preclinical phase of drug testing, as an alternative to animal testing.

One of the most critical aspects in the development of tumour models is the regulation of the biomechanical properties of the substrate where cells grow (mechano-biology). In particular, the mechanical properties have the ability to significantly affect the biological response of tumour cells in the adhesion, migration, viability phases as well as in terms of expression of typical markers (e.g. membrane receptors), crucial for the development of novel anticancer therapies, such as immunotherapies, which represent the cutting-edge frontier in this field. An erroneous design of the mechanical properties (stiffness) of the substrate and/or an incorrect choice of the polymer can significantly influence the cellular behaviour and cell fate in vitro, as the cells decode the chemistry, topography and rigidity of the substrate on which they adhere and, consequently, modify their molecular pathways (G. Pennesi, S. Scaglione, P. Giannoni, R. Quarto“ Regulatory influence of scaffolds on cell behavior: how cells decode biomaterials” Review. Curr Pharm Biotechnol 2011 Feb; 12 (2): 151-9).

Specifically, for neuroblastoma, models based on collagen hydrogels with neuroblastoma cells and stromal stem cells embedded have been recently developed (P. Yeung, et al.) Microencapsulation of neuroblastoma cells and mesenchymal stromal cells in collagen microspheres: a 3D model for cancer cell study "PloS one 10.12 (2015): e0l44l39); collagen and Matrigel™ hydrogels enriched with human neuroblastoma cells of the SH-SY5Y line have been proposed (GN Li et al. "Genomic and morphological changes of neuroblastoma cells in response to three- dimensional matrices. " Tissue engineering 13.5 (2007 ): 1035-1047; A Desai et al. "Human neuroblastoma (SH-SY5Y) cell culture and differentiation in 3-D collagen hydrogels for cell-based biosensing" Biosensors and Bioelectronics 21.8 (2006): 1483-1492), and collagen-based scaffolds where two different neuroblastoma cell lines were grown: KellyLuc and KellyCis83Luc (C. Curtin, et al. " A physiologically relevant 3D collagen-based scaffold-neuroblastoma cell system exhibits chemo sensitivity similar to orthotopic xenograft models. "Acta biomaterialia 70 (2018): 84-97).

However, these approaches, which are based on the use of polymers of animal origin (collagen and extracellular matrix gel -ECM- from Engelbreth-Holm-Swarm murine sarcoma, e.g. Matrigel™ having a Lowry protein content of 8 to 12 mg/ml, produced by Coming Life Science) have several limits such as the difficult reproducibility, due to variations among the various batches and the limited possibility of modelling; these limitations make almost impossible the modelling of some biomechanical aspects discussed above, which are crucial in the onset and progression of the neoplasia. Furthermore, collagen and extracellular matrix gel (ECM) from Engelbreth-Holm-Swarm murine sarcoma, hereafter also referred to as "ECM gel", are commonly used in the liquid state and require manipulation at low temperatures to avoid premature gelation, making difficult their handling and manipulation with viable cells and biological material.

Therefore, there is the need to develop new 3D human tumour models, and in particular neuroblastoma, having high biological accuracy in order to study microenvironmental events, which currently jeopardize the prognosis of cancer patients, and to develop new screening systems for drug testing to improve the clinical efficiency of cancer treatments.

The present invention relates to a composition comprising or consisting of: i. primary viable cells, either line cells or deriving from neuroblastoma patients; and

ii. a hydrogel comprising alginate in an aqueous solution or a polymeric mixture comprising alginate and at least one hydrophilic polymer (PI) different from alginate, wherein the alginate and the polymer (PI) are present in the hydrogel in volumetric ratios ranging from 99.9/0.1 to 1/99, wherein the hydrogel ii. is cross-linked by ionic cross-linking based on calcium ions or controlled physical cross-linking, to allow a final elasticity of the hydrogel ranging from 2 to 4,000KPa, measurable with the AFM technique.

The neuroblastoma cells are embedded within the hydrogel with a density that can vary, preferably, but without limitation, from 2 million to 6 million cells per ml.

The aqueous solution comprising alginate in the hydrogel ii. can consist of water and alginate or also comprise other components, for example, without limitations, it can be a saline solution, a physiological solution, it can comprise a buffer, for example phosphate, or other components.

The elastic modulus of the hydrogels can be suitably adjusted by varying the concentration of the polymer and of the crosslinker and reaching values ranging from E=2 kPa to E=4,000 kPa, for example from 20 to 2,000 kPa. This modularity of the elasticity characteristics enables to study the effects that the mechanical properties of hydrogels have on tumour cell migration and on tumour growth and progression.

The present invention also relates to a method for producing a composition as defined above, wherein the method comprises the following steps:

(a) forming a composition comprising water and at least one hydrophilic polymer, capable of forming a hydrogel as defined above;

(b) suspending viable cancer cells and in particular neuroblastoma cells of a first type in the polymer solution precursor of the hydrogel obtained in step (a);

(c) cross-linking the polymer in the suspension obtained in step (b) so as to obtain the hydrogel with cancer cells and in particular neuroblastoma cells incorporated therein.

The present invention also relates to the use of the composition as defined above comprising a hydrogel and neuroblastoma cells as a three-dimensional in vitro neuroblastoma model and, preferably, as a testing platform for molecules.

Unless otherwise indicated, in the context of the present invention, the percentages and quantities of a component in a mixture should refer to the weight of this component with respect to the total weight of the mixture.

Unless otherwise specified, in the context of the present invention, the indication that a composition "comprises" one or more components or substances means that other components or substances may be present in addition to that, or those, specifically indicated.

Unless otherwise specified, within the context of the present invention, a range of values indicated for a quantity, for example the weight content of a component, includes the lower limit and the upper limit of the range. For example, if the weight or volume content of a component A is indicated as "from X to Y", where X and Y are numerical values, A can be X or Y or any of the intermediate values.

The inventors have surprisingly found that it is possible to produce a three - dimensional in vitro model of human neuroblastoma, by forming a hydrogel based on at least one hydrophilic polymer and comprising viable human neuroblastoma cells embedded.

It should be pointed out that, although some studies reported in literature have used animal cells (e.g. murine NB2a) incorporated in alginate -based gels, especially for the regeneration of neuronal tissue, results obtained in a species (such as the murine one) do not allow the automatic transfer of the scientific outcome to the human species. This principle is at the basis of the failure of about 90% of drug testing in the preclinical phase, where efficacy tests are carried out in animals and the results obtained are used for selecting some drugs for subsequent testing in humans (clinical phase), which often, however, have unsuccessful outcome.

The preparation method of the hydrogel of the present invention was also validated by using human neuroblastoma cells, in order to approach the use of this hydrogel as an in vitro tumour model on which to test new drugs and new therapies.

In particular, it is appropriate to choose and modulate the characteristics of the hydrogel also considering the type of target tumour tissue to be modelled. The cells encapsulated in accordance with the invention are able to proliferate and aggregate within the hydrogel and they have the ability to express surface proteins typical of neuroblastoma cells and essential in tumour progression.

Therefore, through the composition of the invention, a complex 3D system containing a polymeric hydrogel has been obtained in vitro, where cells are capable of expressing at least one tumour protein typical and necessary for the development of anticancer therapies, such as immunotherapies (as the cells of the immune system specifically recognize this type of proteins).

In this way, a biomaterial has been obtained, able to faithfully resemble some specific features of paediatric tumours and that can represent a reliable in vitro neuroblastoma model for the screening of drugs and anticancer therapies in the pre- clinical phases; this tumor model will allow to reduce the number of failures occurring in the earliest stages of clinical trials.

A particularly advantageous aspect of the present invention is that the expression and reduction of PVR (due to IFN-gamma conditioning) observed in this 3D model closely resembles the PVR variations that occur in vivo in the patients, but it has not been appreciated either under standard 2D culture conditions or in animal models or in other in vitro models, making this model the most suitable for testing new drugs, innovative therapies and immunotherapies.

In particular, the accuracy and reliability of the neuroblastoma model is embodied in the expression of markers typical of neoplasia and in cell proliferation. Human neuroblastoma cells must in fact express PVR, a DNAM-l ligand that is crucial for cell recognition of the immune system (R. Castriconi, et al., 'Natural killer cell-mediated killing of freshly isolated neuroblastoma cells: critical role of DNAX accessory molecule- 1 poliovirus receptor interaction" Cancer research 64.24 (2004): 9180-9184). The expression of PVR by human NB cells grown inside the hydrogel object of the present invention, which is made possible by virtue of to the specific conditions of gel elasticity, allows testing of new anticancer therapies, such as immunotherapies, which use PVR as a target ligand for cells of the immune system. Besides promoting the proliferation of human NB cells and allowing the expression of PVR, the hydrogel object of the present invention, thanks to its chemical and physical characteristics, allows an optimal diffusion of nutrients and vital gases, as revealed by the high viability of NB cells encapsulated within the hydrogels. The effective diffusion of soluble molecules within the hydrogel was also confirmed by the use of interferon-gamma (IFN-gamma), a crucial immunostimulatory cytokine released by NK activated lymphocytes and promoting the activity of different types of immune cells.

It is important to point out that immune responses must be modulated in their duration and amplitude in order to reach their effect without triggering autoimmune responses. The mechanisms that prevent these damaging events are based on the expression of immune checkpoint ligands such as PD-L1 and PD-L2.

In the tumour model object of the present invention, the conditioning of NB cells by IFN-gamma significantly increases the expression of the PD-L1 molecule and the immune checkpoint ligands B7-H3 and HLA-I, a phenomenon which had never been detected so far in 2D culture systems for some NB cells, often resistant to combined therapies, highlighting the new and unique nature of the present invention.

Furthermore, this model of NB has shown that IFN-gamma is capable of reducing the surface expression of PVR, as observed in vivo in patients at stage M who have a negative PVR on metastatic bone marrow NB cells (Castriconi, R.; Dondero, A.; Augugliaro, R.; Cantoni, C.; Carnemolla, B.; Sementa, AR; Negri, F.; Conte, R.; Corrias, MV; Moretta, L. "Identification of 4Ig-B7-H3 as a neuroblastoma-associated molecule that exerts a protective role from an NK cell- mediated lysis". Proceedings of the National Academy of Sciences 2004, 101 (34), 12640-12645).

Description of the Figures

This description is set forth below with reference to the accompanying drawings, provided for indicative purposes only and, therefore, non-limiting.

FIG. 1 Representation of the preparation process of the spherical alginate hydrogel with encapsulated human neuroblastoma cells (HTLA-230 cell line) and images representing the fluorescently labelled cells showing their complete encapsulation within the hydrogels.

FIG. 2 Fluorescence images representing the positive expression or negative expression of surface proteins characterizing human tumour after 7 days of cultivation. For each protein analyzed, images of the hydrogel are shown in transmitted light, fluorescence and the combination of the two channels. The PVR protein is normally expressed in paediatric tumours, whereas the PDL1 protein is not expressed.

FIG. 3 Fluorescence images representing the expression of surface proteins after 7 days of cultivation with and without a soluble factor (Interferon gamma - IFNg) which is released by the NKs, in order to reproduce the biochemical signals that are released by the immune system in the presence of the tumor. Fluorescence images of the hydrogel are shown for each protein analyzed. As demonstrated in in vivo patients, the PVR is down-regulated by IFN-gamma, whereas HLA-l, PDL1, PDL2 and B7H3 are up-regulated by the same soluble factor.

FIG. 4 Histogram representing the effectiveness of the drug imatinib, at different doses, on NB cells grown under 2D (cellular monolayers) and 3D conditions (alginate-based hydrogel). The effectiveness of the drug was measured in terms of percentage of viable cells compared to the initial condition. The 3D data is more realistic and comparable with clinical data with respect to the results in 2D systems.

In the context of the present invention, the term "viable cell" refers to a cell capable of reproducing and giving offsprings and/or exerting at least one metabolic function such as, as a non-limiting example, the production of at least one protein.

In a preferred embodiment, in the composition in accordance with the present invention, the three-dimensional hydrogel comprises or consists of alginate incorporating neuroblastoma cells.

In one embodiment, the hydrogel is composed of alginate and one or more other hydrophilic polymers, for example alginate and ECM gel, present within the hydrogel in volumetric ratios (alginate/ECM gel) ranging from 99.9/0.1 to 1/99, preferably from 99/1 to 20/80, from 90/10 to 25/75, from 80/20 to 40/60 or from 75/25 to 50/50. The inventors have found that it is possible to properly balance the final characteristics of the hydrogel, particularly the bioactivity conferred by the protein substrate (ECM gel) with its mechanical consistency, conferred, among other things, by the alginate, which allows in vitro cells cultivation for prolonged periods of time.

The composition in accordance with the present invention offers the possibility to reproduce a reliable and stable neuroblastoma model in a simple and reproducible way without the need to work at low temperatures.

The viable cells in the composition of the present invention are preferably human cells.

In the composition in accordance with the present invention, the neuroblastoma cells are incorporated within the hydrogel with a density that can vary from 2 million to 6 million cells per ml (total volume of hydrogel).

The elastic modulus of the hydrogels can vary from E = 20 kPa to E = 4,000 kPa, modulating the density of the polymer and the concentration of calcium ions with which the polymeric solution is crosslinked. This allows the investigation of the effects that the mechanical properties of hydrogels have on tumour growth and progression. The elastic modulus was measured by using the atomic force microscopy (AFM) technique and analyzing the force-displacement curves obtained by the cantilevel on the gel surface (AM Kloxin, el al., " Mechanical properties of cellularly responsive hydrogels and their experimental determination. "Advanced materials (2010) 22.31: 3484-3494).

In a preferred embodiment, this hydrogel comprises alginate crosslinked with calcium ions.

In a preferred embodiment, the hydrogel comprises at least two hydrophilic polymers, in which at least one of these two hydrophilic polymers is alginate.

In a preferred embodiment, the hydrogel comprises alginate and another natural polymer, including collagen, extracellular matrix gel (ECM) from Engelbreth-Holm-Swarm murine sarcoma, gelatin, PEG, or other polymers of a natural or synthetic origin.

The model may be heterotypic or homotypic, i.e. it can be associated to only one type of cells or more than one, and the cultured cells can include, but they are not limited to, tumour cells and/or healthy cells. In other words, the 3D cell culture system may comprise a plurality of cells of either the same type or different types.

When two or more cell types are grown in the model, this can be used, for example, for studying cell-cell interactions between different types of cells or for the high-performance screening of drugs in a heterotypic cell environment. In an embodiment of the present invention, tissue engineering strategies can be applied in order to recreate typical cell-microenvironment interactions (e.g. 3-D cell-cell and cell-extracellular interactions, mechanical rigidity, presence of the soluble factors).

These 3D culture systems can be used for studying the physical-chemical "cross-talk" between tumour cells (e.g. neuroblastoma cells) and healthy cells (e.g. stem cells) and evaluating the importance of this mechanism on the progression of the tumour and, vice versa, on the reactivity to the anticancer therapy.

The composition, in accordance with the present invention, provides a pathologically relevant tumour microenvironment. The pathologically relevant tumour microenvironment can be used for basic research or for screening therapies (pharmacological screening), since both traditional 2D techniques and xenograft models are unsuccessful in the preclinical phase. In a preferred embodiment, the pathologically relevant tumour microenvironment in accordance with the present invention can be used for pharmacological screening, also, without limitation, with a high-throughput approach.

In one embodiment, the hydrogel is approximately spherical in shape, geometrically similar in size and shape to tumour spheroids.

This sphere can incorporate one or more types of cells.

Alginates are versatile polysaccharide polymers that can be manipulated for specific applications by controlling the molecular weight, the degradation rate and the gel formation method. Alginate hydrogels can have a variety of forms.

In one embodiment, the hydrogels are spherical in shape and are obtained by extruding a polymeric solution combined with neuroblastoma cells in a bath enriched with cross-linking agents (calcium ions).

In one embodiment, the hydrogels are cylindrical (of any size) obtained by the use of agarose moulds containing calcium ions.

In one embodiment, the present invention is directed towards the use of the composition as defined above as a three-dimensional in vitro neuroblastoma model.

In one embodiment, the present invention provides a method for the screening of anticancer drugs and/or any other drug, molecule, active ingredient, natural extract, plant or its derivatives, wherein said screening comprises putting the composition comprising or consisting of viable cells of a neuroblastoma and a hydrogel comprising water and at least one hydrophilic polymer, as defined above, in contact with at least one anticancer drug to be screened.

"Putting in contact" means, in any way known to a skilled person in the field, ensuring that a specific drug or other substance/composition to be tested can have a pharmacological interaction with the tumour cells comprised in this composition, for example, without limitation, infusing a drug in this composition or introducing the composition as defined above into a solution comprising an anticancer drug or other substance/ composition to be tested.

Models of engineered human tumours (or "biomimetic tumours") can be scaled and validated, by using standard methods, including, but not limited to, high- throughput screening of (anticancer) drugs. The method provided by the present invention overcomes the known limitations associated with conventional cell culture approaches and non-human animal models, by mimicking the typical microenvironmental conditions of human tumours.

The three-dimensional cell culture systems provided by the invention can be used for drastically reducing the costs of drug development for the pharmaceutical industry (currently estimated at $ 1.4 billion for each new drug).

An embodiment of the present invention relates to a method for producing the composition as defined above, for example for producing an in vitro 3D neuroblastoma model, wherein this method comprises at least the following steps: (a) forming a composition comprising water and at least one hydrophilic polymer as defined above, capable of forming a hydrogel;

(b) suspending viable neuroblastoma cells of a first type in the polymer solution precursor of the hydrogel obtained in step (a);

(c) cross-linking the polymer in the suspension obtained in step (b) to obtain the hydrogel embedding neuroblastoma cells incorporated.

In a preferred embodiment, the method in accordance with the present invention also foresees the embedding of a second type of cells, different rfrom the first type of embedded neuroblastoma cells, and that this second type of cells are suspended within the same composition comprising water and the at least one hydrophilic polymer in step (b) together with the neuroblastoma cells of a first type prior to cross-linking (c) of the polymer to finally obtain a hydrogel embedding a first type of cells of neuroblastoma cells and a second type of cells.

In a preferred embodiment, the hydrogel solution comprises only alginate as hydrophilic polymer.

In one embodiment, the hydrogel solution comprises alginate and another natural polymer, including collagen, extracellular matrix gel (ECM) from Engelbreth-Holm-Swarm murine sarcoma, gelatin, PEG, or other polymers.

In one embodiment, the incorporation of the cells within the hydrogel is achieved by combining the composition of step (b), comprising the cells suspended in the hydrogel solution, with a crosslinking solution based on calcium ions.

In another preferred embodiment, the hydrogel obtained in step (c) is a spheroid with a diameter ranging from 2 millimetres to 5 millimetres based on the extruder used. In particular, the starting polymer solution can have a concentration of 1% of weight of polymer respect to the total volume of the solution (w/v). In step (b), a mixture of a first suspension containing the neuroblastoma cells and a solution comprising a hydrophilic polymer of step (a) is prepared, for example with a volume ratio of 1/1 between the first suspension and the polymer solution, therefore the final concentration of the hydrogel is 0.5% of weight of polymer with respect to the total volume of the composition (w/v) obtained in step (b).

In one embodiment, the mixture containing neuroblastoma cells and the polymer solution is extruded with a needle in a solution containing calcium ions at a concentration of 0.5 M, forming spheres with a size of 2 mm in diameter.

In one embodiment, the hydrogels are cylinders (of any size) obtained by using agarose molds enriched with calcium ions, in particular the suspension of neuroblastoma cells incorporated in the polymer solution is poured into moulds produced within agarose gel. The calcium present in the agarose gels crosslinks the polymer solution forming cylindrical hydrogels of varying sizes, depending on the mold used. This technique allows the formation of hydrogels having a predefined size and shape, based on the geometry of the mould used.

In another embodiment, the starting polymer solution has a concentration of 1% by weight of polymer with respect to the total volume of the solution (w/v).

In another embodiment, the cell density is 5 x 10⁶ cells/ml in the hydrogel solution.

In one embodiment, one or more types of cells (mesenchymal stem cells, natural killer (NK) lymphocytes) can be dispersed or incorporated within the hydrogel.

The advantages of the present invention are multiple. In particular, the use of alginate as bulk polymer confers reproducibility and chemical-physical and structural stability to the model, features which are difficult to obtain through the use of natural polymers of animal origin, such as collagen and ECM gel. Furthermore, the alginate allows to model different aspects of the hydrogels, such as biomechanical properties, fluid absorption capacity (swelling), the bioactivity by varying its density and the concentration of calcium ions, necessary to crosslink the polymer.

Furthermore, neuroblastoma (NB) cells express specific surface proteins related to tumour growth within the hydrogels produced in accordance with the present invention. In particular, a protein called PVR (polivirus receptor) was observed by immuno-staining. It is expressed by most NB tumours, which allows the recognition of the tumour by Natural Killer (NK) cells of the immune system. PVR is therefore a promising target for anticancer therapies, since it is over expressed in neuroblastoma and activates the cytolytic activity of NKs against NB.

Furthermore, the alginate-containing hydrogels in accordance with the present invention have proved to be valid models also in terms of diffusion of cytokines, protein molecules produced by the cells of the immune system, secreted in the surrounding medium and capable of releasing biochemical signals.

In particular, the hydrogels were cultivated in the presence of the cytokine Interferon Gamma (IFN-g), a soluble factor that is released by NKs, in order to reproduce the biochemical signals, which are released by the immune system in the presence of cancer disease; the application of IFN- g caused in the 3D NB alginate- based models a clear induction of the expression of typical surface markers (PD-F1, PD-F2 and HFA-l) on the NB HTFA-230 cell line, associated with the down- regulation of the PVR, which demonstrates the accuracy and biological reliability of the model. This biological result, for the first time observed in a 3D model, is perfectly aligned with the results obtained on NB patients; furthermore, the PD-F2 and PVR expression modifications are not detected in 2D, revealing the limitations of traditional models.

Consequently, the hydrogels produced in accordance with the present invention represent models, where cells express typical markers of the neoplasia and, thus revealing their reliability as models for testing in vitro anticancer and immunological therapies, based on the enhancement of the activity of Natural Killer cells of the immune system. The following examples are provided to illustrate some embodiments of the invention, without limiting its scope.

This example shows the development of a 3D model of neuroblastoma in vitro. This biomimetic model can be used for testing drugs or new anticancer therapies in more realistic culture conditions.

Specifically, a 3D neuroblastoma model based on alginate spheres and human neuroblastoma cells (HTLA-230) was developed and characterized.

Neuroblastoma cells were incorporated in an alginate hydrogel (1% w/v) with a 1:1 v/v volumetric ratio between the starting polymer solution and cell suspension. The cell density within the hydrogel is 4 million/ml.

Using a specific fluorescence staining“Dead/ Alive kit” based on calcein and propidium iodide, the cells were observed through fluorescence microscopy to assess the cell viability during their encapsulation within the hydrogel and the procedure of hydrogel formation (figure 1).

The cell proliferation was monitored through a metabolic kitfor up to 1 week of cultivation (Presto Blue assay).

The HTLA-230 cells maintained a good level of viability during the encapsulation process, and the proliferation curve is growing, showing that the cells are capable of duplicating and proliferating within the alginate hydrogel. In order to examine the tumour neoplasia and the expression of surface proteins typical of the most aggressive forms of neuroblastoma, the hydrogels were analyzed by immuno- staining, incubating the hydrogels with a primary antibody and then with a secondary antibody conjugated with a fluorescence dye. The fluorescence images are shown in greyscale, therefore the black background represents the absence of a signal, whereas the light signal in shades of grey represents the expression of the protein.

The results show that the cells grown in alginate hydrogels after 1 week selectively express a protein called PVR (polivirus receptor) expressed by most high-risk neuroblastoma tumours and essential for the survival/invasiveness of the tumour (figure 2). The cancer cells provided with PVR are recognized and killed by the Natural Killer (NK) cells thanks to specific receptors (real biological sensors) capable to recognize the protein.

Therefore, neuroblastoma cells which express PVR can be potentially treated by immunological therapy based on the enhancement of the activity of Natural Killer cells of the immune system.

The alginate hydrogels embedding neuroblastoma cells were cultivated for 1 week in the presence of a soluble factor (Interferon Gamma), produced by the Natural Killer cells, to regulate the neoplasm and the reactivity to any anticancer immunological therapy.

The expression of surface proteins relating to tumour growth in the presence of interferon gamma was analyzed by immuno-staining (figure 3). The soluble factor was able to perfuse the hydrogels and showed a key role in the induction/suppression of the expression of typical surface markers on the neuroblastoma cell line HTLA-230 (figure 3), showing that the alginate-based hydrogels can potentially represent valid models, where immunotherapies based on Natural Killer cells can be tested.

Alternatively, the alginate hydrogels embedding neuroblastoma cells embedded were cultivated to test the effectiveness of a drug, imatinib (trade-name Gleevec®) and thus validate its reliability. In particular, three different doses of drug were used and the percentage of viable NB cells with respect to the cells initially present in the gel was monitored and quantized before treatment (figure 4); as a control, drug treatment was also performed on the same cells grown in monolayer (2D). Whereas in the 2D control the efficiency of the drug is overestimated, the results obtained in 3D were comparable with the data obtained in the clinical phase (F. Morandi et al. “Updated clinical and biological information from the two-stage phase II study of imatinib mesylate in subjects with relapsed/refractory neuroblastoma "Oncolmmunology Vol7, 2018 Issue 9), demonstrating that alginate -based hydrogels represent convincing and reliable tumour models for predicting in vitro the effectiveness of an anticancer drug.

Claims

1. A composition comprising or consisting of:

i. primary viable cells, either line cells or deriving from neuroblastoma patients; and

ii. a hydrogel comprising alginate in aqueous solution or a polymeric mixture comprising alginate and at least one hydrophilic polymer (PI) different from alginate, wherein the alginate and the polymer (PI) are present in the hydrogel in volumetric ratios ranging from 99.9/0.1 to 1/99,

wherein the hydrogel ii. is cross-linked by ionic cross-linking based on calcium ions or controlled physical cross-linking, to allow a final elasticity of the hydrogel ranging from 2 to 4,000KPa, measurable with the AFM technique.

2. The composition in accordance with claim 1, wherein said polymeric mixture comprises at least one hydrophilic polymer (PI) selected from collagen, extracellular matrix (ECM) gel from Engelbreth-Holm-Swarm murine sarcoma and PEG.

3. The composition in accordance with at least one of the previous claims, wherein the viable cells are human cells.

4. The composition in accordance with at least one of the previous claims, wherein ii. is a hydrogel comprising water and a mixture comprising alginate and a hydrophilic polymer (PI), different from alginate, in volume ratios ranging from 75/25 to 50/50.

5. The composition in accordance with at least one of the previous claims, which further comprises viable cells of a second type, different from neuroblastoma cells.

6. The composition in accordance with at least one of the previous claims, wherein the hydrogel is spherical in shape to mimic the three-dimensional biological structure of the tumour.

7. A method for preparing the composition in accordance with any of the previous claims, wherein the method comprises the following steps:

(a) forming a composition comprising water and alginate, optionally in a mixture with at least one hydrophilic polymer capable of forming a hydrogel;

(c) cross-linking the polymer in the suspension obtained in step (b) to obtain the hydrogel embedding the neuroblastoma cells .

8. The method in accordance with claim 7, further comprising the use of a type of cells different from the first type of embedded neuroblastoma cells and where the cells of the second type are suspended within the same composition comprising water and alginate, optionally mixed with the at least one hydrophilic polymer in step (b) together with the first type of neuroblastoma cells prior to cross-linking (c) of the polymer to obtain a hydrogel embedding neuroblastoma cells and a second type of cells.

9. Use of the composition in accordance with any of claims 1-6 as a three- dimensional in vitro model of neuroblastoma.

10. Use of the composition in accordance with claim 9, as an in vitro three- dimensional model of neuroblastoma for the screening of anticancer drugs and/or any other drug, molecule, active ingredient, natural extract, plant or derivatives thereof.

11. A method for screening anticancer drugs and/or any other drug, molecule, active ingredient, natural extract, plant or derivatives thereof, wherein said screening comprises contacting at least one anticancer drug and/or any other drug, molecule, active ingredient, natural extract, plant or derivatives subject to the screening with the composition comprising or consisting of viable neuroblastoma cells and of a hydrogel comprising water and alginate, optionally in a composition with at least one hydrophilic polymer, in accordance with at least one of claims 1-6.