WO2016146893A1 - Human tumor based extracellular matrix for cell studies in vitro - Google Patents

Abstract

The present invention provides extracellular matrix homogenate for culturing human cells in vitro comprising homogenized human leiomyoma tissue. The present invention also provides a method for producing extracellular matrix homogenate from discarded leiomyoma tissue removed from patients in routine operations.

Description

Human tumor based extracellular matrix for cell studies in vitro

FIELD OF TH E I NVENTION

The present invention is directed to the field of three-dimensional cell cultures. Particularly, the present invention provides a cell culture composition for studying human cells in vitro comprising homogenized human uterus benign tumor, leiomyoma, tissue. The present invention also provides a method for producing extracel lular matrix homogenate from discarded leiomyoma tissue removed from patients during routine surgical operations.

BACKG ROU N D OF TH E I NVENTION

Matrigel^®, the mouse Engelbreth-Holm-Swarm (EHS) tumor -derived commercial prod uct (see US 4,829,000 and Kibbey 1994), is widely used particularly for in vitro adhesion, invasion and capillary formation assays of human cancer cells. However, since the tumor matrix of rodents clearly differs from respective human tumor microenvironment matrix (TM EM) (Wolf et al. 2009) affecting most likely also cancer invasion processes, the need for an equivalent human soluble TM EM product has been recognized. Since collagen molecu les are the most abundant proteins in the extracellular matrix (ECM), gels from purified collagens have also been used to embed cells into 3 D cultures (Fusenig et al. 1983, Nystrom et al. 2005). Rat tail tendon derived type I collagen is probably the most abundant ECM mimicking matrix used in organotypic cultures. Other commercially available ECM molecules, derived from different animal species, like fibronectin (Mao & Schwarzbauer 2005), fibrin (Ho et al. 2006) and hyaluronic acid (Gu rski et al. 2009), are also available for in vitro studies.

In addition, synthetic in vitro produced ECM or peptide matrices are available from d ifferent manufacturers. However, one separate molecule, or even a mixture of them, or totally synthetic matrices, cannot properly simulate the complex effects of natural ECM, since they obviously lack e.g. the hundreds of cell-adhesion signals, cytokines or protease cleavage sites identified in natural tumor ECM. Obviously, in vivo the combination of multiple TM E (tumor microenvironment) factors are important for cell-ECM interactions during cancer progression (Hanahan & Weinberg 2011). In the literature, three publications (Abberton et al 2008, Francis et al. 2009, Ting et al. 2014) use the term myogel for an extracellular matrix material which is derived from human, mouse, rat or pig normal skeletal muscles using similar procedures as Kibbey (1994) for preparing EHS tumor extraction. The material was shown to be adipogenic (Abberton et al. 2008, Ting et al. 2014) and to support the ex vivo amplification of corneal epithelial cells (Francis et al. 2009).

US 7,727,550 describes H u Biogel, an ECM gel derived from normal human amnion tissue containing laminin, collagen types I and IV, entactin, tenascin and heparan sulfate

proteoglycan, but lacking endogenous growth factors ( EG F, TG F-a, TG F-β, FG F and PDG F) as well as M M P-2 and -9 (Yuan et al. 2008).

Translational cancer research have thus been lacking for a long time a human tissue in vitro model that mimics the natural tumor microenvironment matrix (TM EM). This need was fulfilled by an organotypic leiomyoma 3 D solid disc model described by N urmenniemi et al. 2009, which has been successfully used already in more than 15 cancer invasion studies (see e.g. Heinonen et al. 2011, Dayan et al. 2012, Bitu et al. 2013, Lee et al. 2013, Ahmed Haji Omar et al. 2014). In this disc model, the hypoxic tumor matrix provides an authentic environment including e.g. vessels, collagen fibers, laminins, glycoproteins, cytokines and proteases (Teppo et al. 2013). It is thus noted by the authors of N urmenniemi et al. 2009 that the presence of fibroblasts, smooth muscle, endothelial, and inflammatory cells in the myoma tissue is evident to enhance the invasion of oral carcinoma cells.

The authors of Salo et al. 2012 disclosed results with myoma discs according to N urmenniemi et al. 2009 (intact or extensively rinsed) that were used for analyzing the invasion depth of mobile tongue carcinoma (HSC-3) cells and the degradation of type I I I collagen (I I ICTP). It was disclosed that HSC-3 cells invaded twice as deep, but degraded six times less type I I I collagen when cultured on top of intact compared to rinsed myoma discs. The authors concluded that intact myoma tissue contained invasion-inducting and anti-apoptotic matrix metallo- proteinase-11 (M M P-11), and hypoxia-related factor lysyl oxidase (LOX) and that both M M P- 11 and LOX could be removed from the myoma with rinsing. Rinsed myoma thus was deprived of invasion inductive TM E factors. Here, the present invention provides a novel Myogel product, prepared from human uterus benign leiomyoma tumor tissue. A solution/gel of the total protein extracts was formulated and the protein contents and characteristics of Myogel were compared with Matrigel^® using a set of in vitro experiments. Based on the results, the tumor tissue solution/gel derived from human leiomyoma offers an excellent human TM EM tool for analyzing human carcinoma cells in vitro. Th is novel Myogel product can also be combined with a low melting agarose, Myogel- LMA, and provides thus an easy to use, practical method for analyzing e.g. cancer cell invasive, adhesive or migratory properties or various potential chemotherapeutic compounds, as well as to test capillary formation. SU M MARY OF TH E I NVENTION

One of the objects of the present invention is to provide a cell culture composition comprising homogenized human leiomyoma tissue.

Another object of the present invention is to provide a method of preparing extracellular matrix homogenate comprising the steps of: (a) homogenizing human leiomyoma tumor at least twice in a NaCI-buffer and discarding the soluble fraction after each homogenization;

(b) extracting the residual leiomyoma tumor in a urea buffer and stirring the extract for about 12-18 hours;

(c) separating the extract from the residual leiomyoma tumor fraction and saving the first extract while repeating step (b) with the residual leiomyoma tumor fraction and saving the second extract resulting therefrom;

(d) combining the first and second extracts from step (c) and dialyzing against a buffer comprising a sterilizing component; and

(e) further dialyzing against a buffer not comprising a sterilizing component and recovering a dialysate.

The present invention is also directed to a use of a cell culture composition comprising homogenized human leiomyoma tissue for invasive, migratory, adhesive or capillary formation cultures of human cells. BRI EF DESCRI PTION OF TH E DRAWI NGS

Figure 1. Adhesion of HSC-3 cells to Myogel and Matrigel^®. Wells coated with BSA and plain wells kept in PBS served as controls for adhesion.

Figure 2. HSC-3 cells divided and formed colonies within Myogel combined with LMA (low melting agarose). HSC-3 cells grew similarly in Myogel-LMA than in LMA only (A and B).

Figure 3. Migration and horizontal invasion of HSC-3, MDA-MB-231, PaOlc, Pa02c, Pa03c and Pa04c cells on and through Myogel and Matrigel⁹. In scratch assay HSC-3 cells migrated more efficiently on Myogel coated wells than on Matrigel^® coated wells. In plain wells their migration was the most efficient (A and B). More HSC-3 and pancreatic cancer cells invaded horizontally through Myogel than through Matrigel^® in scratch wound invasion assay (C-E).

Figure 4. Invasion of HSC-3 cells through Myogel and Matrigel⁹. HSC-3 cells invaded more efficiently through Myogel than through Matrigel^® (A). In all different Myogel batches HSC-3 cells invaded more in Myogel than in Matrigel^® (B). In Myogel and Matrigel^® mixtures HSC-3 cells invaded more when the mixture contained more Myogel (C). Invasion pattern of HSC-3 cells through Myogel and Matrigel^® (D).

Figure 5. Invasion of oral squamous cell carcinoma, melanoma and pancreatic cancer cells through Myogel and Matrigel⁹. HSC-3 cells invaded efficiently through Myogel and Matrigel^® mixed with agarose, 09-HSC-3 cells have a higher passage number than 13-HSC-3 cells (A). Invasion pattern of HSC-3 cells through different mixtures of Myogel, Matrigel^® and LMA (B). Oral squamous cell carcinoma cells SCC-9, LN-1 and LN-2 as well as melanoma cells SK-Mel and A2058 invaded more efficiently through Myogel-LMA than through growth factor- reduced Matrigel^® (Matrigel^®-G FR, (C). Oral squamous cell carcinoma cell lines SAS and SCC- 25, melanoma cell line Bowes and pancreatic cancer cell lines Pa02c, Pa03c and Pa04c invaded more efficiently through Myogel than through Matrigel^® (D). Figure 6. In vitro capillary tube formation assay. Photomicrographs showing the typical appearance of tubules formed by H UVECs in the Myogel-LMA, Matrigel^®-G FR and ECMatrix™ after 12, 24 and 72 hours (only for Myogel-LMA) with the original magnifications of 4x and lOx (A). The n umber of tubules formed (B), and the diameter of the tubules formed in each of the three in vitro assays (C). Figure 7. Viability of the drug-test cells (Pa02c) on top of plastic, Myogel or Matrigel^®. Pa02c cell viability was measured on different concentrations of Myogel and Matrigel^® using CellTiter-Glo^® luminescent assay.

DETAI LED DESCI PTION OF TH E PRESENT I NVENTION

The term "leiomyoma" or "myoma" refers herein to a benign smooth muscle neoplasm that is very rarely premalignant. Leiomyomas or myomas can occur in any organ but the most commonly in the uterus, small bowel and the esophagus. Leiomyomas of the uterus are common pathologic abnormalities of the female genital tract. Occurrence increases with age, and leiomyomas are found in even 50% of women older than 30 years. Consequently, large amou nts of myoma tissue are constantly removed from patients in routine operations and discarded, providing an unlimited amount of myoma tissue available for cancer research.

The term "homogenizing" refers herein to application of a homogenizer used for disrupting the structure of various types of organic material, such as tissue. Many different homogenizer models have been developed using various physical technologies for d isruption. The term

"homogenizing" may also refer to cryopulverization, repeated freezing and thawing, nitrogen decompression or to usage of glass, ceramic or steel beads suspended in aqueous media for the disruption of the sample.

The term "total protein extract" refers herein to a product of an extraction protocol which solubilizes proteins, including membrane proteins, from a tissue sample and separates them from other cell structures without denaturation of the extracted proteins.

The term "agarose" refers herein to a linear polymer extracted from seaweed and made up of the repeating unit of agarobiose. Agarose is commercially available and can be used as a gel for culturing motile cells and micro-organisms. The gel's porosity is directly related to the concentration of agarose in the medium.

The term "low melting point agarose" refers herein to agarose which is modified by hydroxyethylation and thus has lower melting and gelling temperature than standard agaroses. The melting temperature of low melting point agarose is usually under 65 degrees of Celsius, preferably 60-65 degrees of Celsius, and the gelling temperature is under 30 degrees of Celsius, preferably 26-30 degrees of Celsius.

In this invention, the term "cell culture composition" refers to extracellu lar matrix providing a two-d imensional or three-dimensional microenvironment in vitro for mammalian cells, preferably human cells, incubated or cultured on or in the matrix. Particularly, the term "cell culture composition" refers herein to extracellular matrix comprising homogenized human leiomyoma tissue (i.e. Myogel).

Cu rrently, the mouse Engelbreth-Holm-Swarm (EHS) sarcoma -derived Matrigel^® is the most commonly used tumor microenvironment matrix (TM EM) in research laboratories worldwide. However, since the Matrigel^® is non-human in origin, it contains molecules which are not present in human TM EM and therefore, lack of a human TM EM for in vitro cancer studies has been apparent. In order to solve this problem, the present invention provides a cell culture composition comprising homogenized human leiomyoma tissue for studying human cells in vitro or an extract thereof such as an extracellular matrix ( ECM) extract obtained from human leiomyoma tissue. Preferably, leiomyoma tissue for the cell culture composition or extracellular matrix is from uterus as availability of the material is relatively high.

A preferred cell culture composition is made of a total protein extract from human leiomyoma tissue. Said total protein extract can be prepared by multiple homogenization and purification methods known for a person skilled in the art, although the method described by Kibbey 1994 with modifications as explained in the Experimental Section below is the most preferred.

The cell culture composition preferably comprises agarose, since it provides improved manageability and reproducibility for human cell culture studies. For this purpose, low melting point agarose is particularly preferred. In one specific embod iment of the invention, the melting temperature of said low melting point agarose is under 65 degrees of Celsius. As described above, the gell ing temperature of said low melting point agarose is usually under 30 degrees of Celsius, preferably 26-30 degrees of Celsius, more preferably 30 degrees of Celsius.

The present invention is also directed to a method of preparing extracellular matrix homogenate as described above. The method comprises the steps of: (a) homogenizing human leiomyoma tumor at least twice in a NaCI-buffer and discarding the soluble fraction after each homogenization;

(b) extracting the residual leiomyoma tumor in a urea buffer and stirring the extract for about 12-18 hours; (c) separating the extract from the residual leiomyoma tumor fraction and saving the first extract while repeating step (b) with the residual leiomyoma tumor fraction and saving the second extract resulting therefrom;

(d) combining the first and second extracts from step (c) and dialyzing against a buffer comprising a sterilizing component such as chloroform; and (e) further dialyzing against a buffer not comprising a sterilizing component and recovering a dialysate.

Th is method may also comprise further steps of

(f) dialyzing the dialysate obtained from step (e) against a serum-free medium;

(g) recovering a dialysate from step (f). Preferably, the NaCI-buffer of step (a) is a buffer containing at least or about 3.4 M NaCI and the urea buffer of step (b) is a buffer containing at least or about 2 M urea.

More preferably, the buffer of step (a) is a 3.4 M NaCI buffer having a pH of about 7.4 and the urea buffer of step (b) is a 2 M urea buffer having a pH of about 7.4.

To prepare a gelatinous cell culture composition the method may comprise a further step of mixing the dialysate from step (g) with agarose, such as low melting point agarose, which preferably has melting temperature under 65 degrees of Celsius.

The present invention is thus directed to the use of the described cell culture composition for studying or incubating mammalian cells, preferably human cells, more preferably human cancer cells, mesenchymal stem cells, epithelial cells, fibroblasts, endothelial cells, neurons, and most preferably invasive cancer cells. The present invention is also directed to the use of the cell culture composition for invasive, migratory or adhesive cultures of human cells, or for capillary formation culture of human umbilical vein endothelial cells. Another preferred use of the invention is in drug discovery, particularly cancer drugs. Surprisingly, the described Myogel is able to simulate the tumor environmental conditions of human tissues similarly as the myoma disc disclosed by the prior art (N urmenniemi et al. 2009). The benefits of the cell culture composition of the present invention are thus evident for a person skilled in the art from the results disclosed in the Experimental Section below. For instance, the Myogel is easier to prepare and handle than the myoma disc of the prior art. Also it provides more repeatable results since extracel lular matrix can be prepared from mixture of several myomas whereas myoma disc is from tissue isolated from one patient.

Since the Myogel provides a natural-like 3D environment for human cancer cells so that they show their migration characteristics, the matrix is suitable for functional irradiation studies and for other cancer study applications in vitro, e.g. angiogenesis studies, and improves the prediction of preclinical human cancer studies. Suitable cell types for such studies are, e.g. cancer cell l ines, such as carcinomas, adenocarcinomas, melanomas and sarcomas. In invasion studies, the study time in Myogel is considerably shorter than in myoma disc (a few days vs. a few weeks) and the invasion results from Myogel can be obtained by measuring absorbance, which is much faster and easier than histologic sampling from myoma disc.

Although particular embodiments have been disclosed herein in detail, this has been done by way of example for purposes of illustration only, and is not intended to be limiting with respect to the scope of the appended claims that follow. In particular, it is contemplated by the inventors that various substitutions, alterations, and modifications may be made to the invention without departing from the scope of the invention as defined by the claims. For instance, the choice of tissue disruption and protein extraction protocols and buffers as well as matrix additives are believed to be a matter of routine for a person of ordinary skill in the art with knowledge of the embodiments described herein.

Having generally described the invention above, the same will be more readily understood by reference to the following Experimental Section, which is provided by way of illustration and is not intended as limiting.

Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, suitable methods and materials are described below. The materials, methods, and examples are illustrative only and not intended to be l imiting. EXPERI M ENTAL SECTION Materials and methods

Myogel preparation

Hu man uterus leiomyoma tissue leftover pieces (after taking samples for histopathological analyses) were received from the Oulu U niversity Hospital, Department of Gynegology (see Nu rmenniemi et al. 2009) after patient's written informed consent. The ethical permission for using the myoma tissue was approved by the Ethics Committee of the Oul u U niversity

Hospital. The instructions for preparing EHS sarcoma derived Matrigel^® (BD Biosciences), were followed after the method described by Kibbey (1994), with minor modifications.

Briefly, myoma tissue, frozen with liquid nitrogen, was ground to powder with CryoMill

(Retsch), 10 g of tissue powder was suspended in 20 ml of ice cold 3.4 M, pH 7.4 NaCI buffer. After centrifugation, the pellet was homogenized in another 20 ml of the same NaCI buffer as in a previous step using T18 U ltra-Turrax (I KA^®-Werke GmbH & Co. KG). T18 U ltra-Turrax was used also in all the following homogenizations. The protein concentration in each preparation was measured using DC Protein Assay (Bio-Rad) according to manufacturer's instructions. The absorbances were read at 590 nm using Victor³V 1420 Multilabel Counter and Wallac 1420 Manager. Protein concentrations in various Myogel batches were diluted using cell culture med ia described in cell culture-section (see below) accordingly to match Matrigel^® protein concentration in every experiment. Myogel solution was stored in small (≤1 ml) aliquots at -20 °C.

Assessing the pH of Myogel and Matrigel^®

For pH comparison, Matrigel^® (BD Matrigel Matrix, BD Biosciences Cat. N umber 354234) was diluted in proportions of 1+1 (one part of Matrigel^® + one part of serum free medium), and here the same amount of total protein for Myogel was obtained by diluting it accordingly (10+6). The pH of the gels was measured in the beginning, after 17 h, and at the end of the 48 h experiment from both samples. The gels were incubated at 37 °C in a 5% CO2 humidified cell culture chamber with or without HSC-3 cells on top of the gels. Gradient SDS-PAGE of Myogel and Matrigel^® and proteomic analyses of Myogel

20 μΙ of four different Myogel batches (12, 15, 16, 17) and their mixture, as well as four different Matrigel^® (BD Matrigel Matrix, BD Biosciences Cat. N umber 354234) samples were loaded on gradient (4%, 8%, 15%) SDS-PAG E gel and ran using 15 mA for 90 min. PageRuler Plus Prestained Protein Ladder (Thermo Fisher Scientific) was ran together with the samples. Proteins were stained using Coomassie Blue and the gel was washed with elution buffer to remove excess staining. The gel was photographed over a stripping table.

For proteomic analysis, the amount of protein in four different Myogel batches (3, 4, 6, 9) was determined using Bradford assay (Bio-Rad) according to the manufacturer^'s instructions. Absorbances were measured using Biochrom Asys Expert plus Microplate Reader (Biochrom) at 595 nm. 20 μΙ of each sample was loaded on gradient (4%, 8%, 15%) SDS-PAG E gel and ran using 15 mA for 90 min. PageRuler Prestained Protein Ladder (Thermo Scientific) was ran together with the samples. Proteins were stained using Coomassie Bl ue and the gel was washed with elution buffer to remove excess staining. The gel was viewed over a stripping table and individual bands were cut and collected from the gel. After digestion (0.3 μg of trypsin was used/band) each band was resuspended with formic acid on three selected Myogel samples (3, 6, 9) and stored in -20 °C until gel digestion.

The gel digestion of selected samples for mass spectrometry was done according to

Shevchenko et al. (1996) with some modifications. Isolated bands from Myogel samples were run individually on MASS-SPEC on Orbitrack. Results were processed using Thermo Proteome Discoverer 1.4.0.288 and Discoverer Daemon 1.4 using Batch Process. The results were analyzed using Scaffold Q_3.4.5™ proteome software.

Two-dimensional gel electrophoresis (2-DE)

For the proteomic study two myoma tissue and one Matrigel^® samples were further purified by buffer exchange using an Amicon U ltra ultrafiltration unit with 10 kDa cutoff (Millipore, Billerica, MA, USA) and urea buffer (7 M urea, 2 M thiourea, 4% [w/v] CHAPS, 30 m M Tris, pH 8.5). After that the protein samples were sonicated and centrifuged. The protein amounts in the supernatants were determined in duplicate with a Bradford-based assay according to the manufacturer^'s instructions (Roti^®-Nanoquant, Carl Roth) with urea buffer as control and aliquots stored at -20 °C. For 2-DE 100 μg of the protein solution was adjusted with rehydration urea buffer (7 M urea, 2 M thiourea, 4% [w/v] CHAPS, 0.15% [w/v] DTT, 0.5% [v/v] carrier ampholytes 3-10, Complete Mini protease inhibitor cocktail [Roche]) to a final volu me of 400 μΙ. In-gel rehydration with I PG strips (pH 4-7, 18 cm, G E Healthcare) was performed overn ight. Isoelectric focusing (I EF) was carried out with the Multiphor I I system (G E Healthcare) under paraffin oil for 55 kVh. SDS-PAG E was performed overn ight in polyacrylamide gels (12.5% T, 2.6% C) with the Ettan DALT I I system (G E Healthcare) at 1-2 W per gel and 12 °C. The gels were silver stained as described earlier (Ohlmeier et al., 2008) and analyzed with the 2-D PAG E image analysis software Melanie 3.0 (GeneBio).

Zymography The amount of gelatinolytic enzymes in Myogel and Matrigel^® (BD Matrigel Matrix, BD

Biosciences Cat. N umber 354234) were detected by zymography method using fluorescently labeled gelatin (Pirila et al. 2007). Prestained low-range SDS-PAG E Standards (Bio-Rad) as well as purified control M M P-2 and M M P -9 samples were ru n in adjacent wells to the samples. After electrophoresis, gelatinases were activated by incubating the gels with zymography buffer (50 m M Tris-HCI, 5 m M CaCI₂, 1 μΜ ZnCI₂, 0.02% NaN3, pH 7.5) overnight at 37 °C. Gelatin degradation was visualized under long wave UV l ight and photographed using AlphaDigiDoc^® RT Gel Documentation System.

Cell lines

Hu man oral tongue squamous cell carcinoma cell lines HSC-3, SAS (both from Japan Health Sciences Foundation, Japan) and SCC-25 (American Type Cu lture Collection. ATCC) with different aggressive potential were cultured in a 1: 1 DM EM/F-12 medium (Life Technologies) supplemented with 100 U/ml penicillin (Sigma-Aldrich or Life Technologies), 100 μg/ml streptomycin (Sigma-Aldrich or Life Technologies), 250 ng/ml fungizone (Sigma-Aldrich), 50 μg/ml ascorbic acid (Sigma-Aldrich or Applichem)and 0.4 μg/ml hydrocortisone (Sigma- Ald rich) and 10% heat inactivated fetal bovine serum (FBS; Life technologies). HSC-3 cells labeled with RFP were generated by stable transduction with commercial lentiviral particles containing a non-coding control sequence (Amsbio) and selected with puromycin. They were cultured as normal HSC-3 cells. HSC-3 cells labeled with G FP were generated by stable transduction with non-silencing G I PZ lentiviral sh RNAmir control particles (pG I PZ vector contains G FP in order to track sh RNAmir expression; Thermo Fischer Open Biosystems) with puromycin (Sigma-Aldrich) selection according to manufacturer's instructions. N uclear histone-2B (H2 B)-coupled mCherry expression vector pLenti6.2V5/DEST (a gift from Dr. Cindy E. Dieteren, Department of Cell Biology, Radboud U MC, Netherland) was introduced to the HSC-3 cells with cytosolic G FP labeling using lentivirus mediated infection and selected in culture media containing 5 μg/ml blasticidin-S (Merck Millipore). HSC-3 cells expressing cytoplasmic G FP and H2B-cou pled mCherry were cultured in DM EM/F12 medium

(Gibco/Thermo Fisher Scientific), 10% heat-inactivated FBS (HyClone/Thermo Fisher

Scientific), penicillin and streptomycin (100 U/ml penicillin, 100 μβ/ΓηΙ streptomycin) (Sigma- Ald rich), 50 μg/ml L-ascorbic acid (Sigma-Aldrich) and 2 m M L-glutamine (Sigma-Aldrich) at 37 °C and 5% C0₂.

SCC-9 cell line (American Type Culture Collection, ATCC) was maintained in DM EM/F-12 med ium (Invitrogen) supplemented with 10% FBS (Cultilab), 400 ng/mL hydrocortisone, and antibiotic/antimycotic solution (Invitrogen). SCC-9 cells were labeled with ZsGreen protein and implanted subcutaneously into the footpads of the left front limb of BALB/c nude mice and LN-1 and LN-2 cell lines with increased metastatic potential were derived by in vivo selection from axillary lymph nodes with metastatic cells as described earlier (Agostini et al. 2014). Both LN-1 and LN-2 cells were maintained in culture as SCC-9.

Melanoma cell lines SK-Mel-25 and A2058 (ATCC) were maintained in RPM I medium

(Invitrogen) supplemented with 10% FBS (Cultilab) as described earlier (Seguin et al. 2012). Hu man melanoma cell line Bowes (ATCC) was cultured in DM EM medium (high glucose, Life technologies) supplemented with 100 U/ml penicillin (Life tech nologies), 100 μg/ml streptomycin (Life technologies), 250 ng/ml amphotericin B (Sigma-Aldrich), 50 μg/ml ascorbic acid (Applichem), 1 mmol/L sodium pyruvate (Sigma-Aldrich) and 10% heat- inactivated fetal bovine serum (Life technologies). Patient tumor derived xenograft pancreatic cancer cell lines PaOlc, Pa02c, Pa03c and Pa04c got from Johns Hopkins U niversity School of Medicine, US, were described earlier (Jones et al. 2008, Kim et al. 2012) and cultured in 1 : 1 DM EM/F-12 medium supplemented with 100 U/ml penicillin, 100 μg/ml streptomycin and 10% heat-inactivated fetal bovine serum (all from Life technologies). Mammary gland epithelial adenocarcinoma M DA-M B-231 cells (ATCC) were cultured in Leibovitz's L-15 medium supplemented with 10% fetal bovine serum (both from Life technologies). Normal oral gingival fibroblasts (G F) were established from the palatal gingiva mucosa biopsies and cultured in DM EM medium (high glucose, G lutaMAX™ and pyruvate) supplemented with 10% FBS, 50 U/ml penicillin, 50 μβ ιηΙ streptomycin and 2.5 μβ/ΓηΙ amphotericin B (all from Gibco). The carcinoma associated fibroblast (CAF) cell lines were generated from fragments of tongue squamous cell carcinomas by using tissue explants (Coletta et al. 1998). They were cultured in DM EM medium supplemented with 100 U/ml penicillin, 100 μg/ml streptomycin, 50 μβ/ΓηΙ ascorbic acid, 250 ng/ml fungizone, 1 mmol/L sodium pyruvate (Sigma-Aldrich) and 10% heat inactivated FBS.

Hu man umbilical vein endothelial cells (H UVEC, ATCC) were cultured in a 1: 1 mixture of DM EM/F12 medium (Invitrogen) supplemented with 10% FBS and 400 ng/ml hydrocortisone (Sigma-Aldrich).

All the cells were cultured in a humidified atmosphere of 5% CO2 at 37 °C and passaged routinely using trypsin-EDTA (Sigma-Aldrich). The media were changed in every 2-3 days. They were regularly tested and confirmed to be negative for mycoplasma infection using

MycoTrace PCR Detection Kit (PAA Laboratories GmbH).

Terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL)

An 8-wel l N unc™ Lab-Tek™ chambered slide (Thermo-Scientific) was used for the experiment. Each well was coated with either rat tail collagen type I (BD Biosciences), three different batches of Myogel and an equal (50%-50%) mixture of collagen type I and one type of Myogel . A total of 150 μΙ of coating mixtures were prepared, with a final protein

concentration of each gel mixture of 0.62 mg/ml, which were adjusted with the addition of culture medium. Two wells were not coated and were kept for positive and negative controls for the subsequent TU N EL assay. The Lab-Tek™ chamber slide was covered and placed in the incubator at 37 °C in 5% C0₂ for 2 h. Subsequently, 5 000 CAFs were added on each well and placed in the incubator overnight. On the following day, the wells were washed twice with PBS, air-dried for few min and fixed with a freshly prepared 4% (w/v) paraformaldehyde in PBS (pH 7.4) for one hour at RT. After two washes with PBS for 5 min each, the TU N EL assay was performed using a commercially available kit (In Situ Cell death Detection Kit, Roche). At the end of the assay, samples were counterstained with DAPI for 10 min at RT. After coverslip mounting with a water based medium, 100 cells were counted under confocal microscope using blue channel (405 nm) and apoptosis was detected using green channel (488 nm).

Adhesion assay

Cell adhesion assay was conducted to determine how many cells bind to Myogel compared to Matrigel^® (BD Matrigel Matrix, BD Biosciences Cat. Nu mber 354234). In this assay, HSC-3 cells were cu ltured to subconfluence. Wells in 96-well plate were coated either with 100 μΙ of PBS, BSA (bovine serum albumin, 10 μg/ml, Sigma), Matrigel^® or Myogel . Matrigel^® was diluted to 1: 10 in PBS and Myogel was diluted to the same protein concentration. The wells were coated 24 h before adding the cells. At the same time, the cell cu lture medium was changed to serum-free. Next day the excess liquids were removed and the culture plates were incubated with 100 μΙ/well of 0.1% BSA for 2 h and washed with PBS. HSC-3 cells (6 000) in 100 μΙ of serum-free medium were added to each well and the wel ls were incubated at 37 °C in 5% CO2 humidified atmosphere for 2 h. The non-adherent cells were rinsed off, and the remaining cells were fixed with 10% trichloroacetic acid (TCA), stained with crystal violet and quantified using ELISA reader at 540 nm.

Ad hesion of G Fs on top of Myogel was studied using 6-wel l plates coated at 37 °C in 5% CO2 humidified atmosphere with 0.62 mg/ml Myogel diluted with DM EM without supplements. After 2 h the excess liquids were removed and 150 000 G Fs were added in their normal culture medium. The cultures were photographed with Olympus CKX41 inverted microscope after 2.5 h and 9.5 h with 20 x magnification to record the morphology of the cells.

Myogel as a supplement in soft agar colony formation -assay

For soft agar mimicking assay, one percent sterile base low melting agarose (LMA, Sea Plaque Low Melting Agarose, Lonza) melted in PBS was mixed with 10 x DM EM/F12, 100% FBS to give a final 0.8% agarose with 1 x medium, 10% FBS. Of the mixture, 0.5 ml was added to 24-well plate and let to solidify for at least 30 min in the laminar. HSC-3 (H2B-G FP) cells were trypsinized and counted. Together 10 000 cells in 50 μΙ of FBS were mixed with 50 μΙ of 10 x DM EM/F12 and 0.4 ml of 0.5% LMA (as a control, final agarose concentration 0.4%) or with 50 μΙ of 10 x DM EM/F12, 0.2 ml of 1.0% agarose and 0.2 ml Myogel (final agarose

concentration 0.4%, final Myogel protein concentration 2.2 mg/ml). Myogel was centrifuged at 4 000 rpm for 10 min prior to the procedure. Agarose mixtures were gently mixed by swirling and 0.5 ml was added on the top of LMA. The plates were incubated at 37 °C in humidified incubator for 28 days. Cells were fed twice a week with 0.25 ml normal HSC-3 med ium. After 28 days the pictures of colonies were taken with transmitted light, G FP & RFP channel in different objectives (lOx, 20x & 40x) using EVOS inverted microscope. Cells in colonies were calculated from the pictures and ImageJ software (Rasband, W.S., ImageJ, U .S. National Institutes of Health, Bethesda, Maryland, USA,) was used to measure colony area.

Scratch assay

To analyze the effects of Myogel and Matrigel^® (BD Matrigel Matrix, BD Biosciences Cat. Nu mber 354234) on the migration of HSC-3 cells, 24-well plates were coated with 0.62mg/ml Myogel or 0.62 mg/ml Matrigel^®. The coating was left to solidify for 2 h in 37 °C and were then washed twice with PBS. HSC-3 cells (90 000) were allowed to attach overnight, and were then wounded with the pipette tip, rinsed twice with PBS, post-coated for 1 h and rinsed before 1% FBS medium was added. The wounds were photographed with EVOS

photomicroscope at 0 h and 24 h after scratching. The area of the open wound was measured using ImageJ software and the results were calculated as a percentage of wound closure. In another set of experiments, scratch wound healing assay measuring migration and invasion was done with IncuCyte ZOOM^® live-cell imaging. ImageLock 96-well plates (Essen Bioscience) were coated with 300 μg/ml Myogel or Matrigel^® diluted in pre-chilled, serum-free DM EM. 50 μΙ of the dilution was added on the wells and the plates were incubated overnight at 37 °C in 5% CO2 h umidified atmosphere. Next day 20 000 -40 000 mammary gland epithelial adenocarcinoma M DA-M B-231 cel ls, oral tongue squamous cell carcinoma cells (both normal HSC-3 and G FP-labelled HSC-3 cells) and pancreatic cancer cell lines PaOlc, Pa02c, Pa03c and Pa04c were suspended into 100 μΙ of medium and seeded into the wells and incubated overnight at 37 °C in 5% CO2 humidified atmosphere. Wound Maker™ (Essen Bioscience) was used to scratch the near confluent cultures. 100 μΙ fresh appropriate culture media for each cell line was changed into the migration assay wells and the plate was placed on ice. Culture med ia was removed from the invasion assay wells and 2.4 mg/ml Myogel-LMA (0.2% LMA) or Matrigel^® (50 μΙ) was added to them. The plate was incubated 30 min at 37 °C in 5% CO2 humidified atmosphere after which 50 μΙ fresh culture med ia was added. Then both the migration and invasion assay plates were placed into the IncuCyte ZOOM™ (Essen Bioscience) for imaging at every 1 h. Transwell invasion through Myogel and Matrigel^®

To compare Myogel with Matrigel^® (BD Matrigel Matrix, BD Biosciences Cat. N u mber 354234) as an invasion assay material, experiments were carried out according to BD Biosciences instructions to coat the filter membranes with Matrigel^®. 50 μΙ of either 1+1 Matrigel^® or 10+6 Myogel diluted with serum-free medium was added on the upper chamber of

Transwell^® nylon filter membrane insert (Corning Inc.), incubated at 37 °C in 5% CO2 humidified atmosphere for 30 min after which 50 000 HSC-3 cells suspended in 100 μΙ of serum-free medium were seeded onto the upper compartment of the Transwell^® chamber. Transwell^® inserts were incubated for 12 h - 48 h at 37 °C in 5% CO2 humidified atmosphere, after which the cells were fixed in 10% TCA for 15 min, rinsed and air dried overnight. Once dry the membranes were stained with crystal violet for 20 min and the excess stain was removed by water rinsing. The uninvaded cells from the upper side of the membrane were removed by carefully sweeping with a cotton swab. Next, the membranes were removed from the inserts and placed on microscope slides and the number of invaded cells through Myogel and Matrigel^® were counted. We also tested different mixtures of Myogel and Matrigel^® (2+1, 1+1 and 1+2) in invasion assay.

Transwell Invasion through Myogel solidified with low-melting agarose (Myogel-LMA)

We used a final concentration of 0.2% agarose (Sea Plaque Low Melting Agarose (LMA), Lonza) in 3.2 mg/ml final Myogel protein concentration to solidify Myogel for invasion assays. To compare the results with Matrigel^® we diluted Matrigel^® (BD Matrigel Matrix, BD

Biosciences Cat. N umber 354234) 1+4 and used the same 0.2% agarose also with it. Serum- free cell culture medium was used for gel dilution. 50 μΙ of the mixtu re was added on the upper chamber of Transwell^® nylon filter membrane insert, incubated ½ h at RT and thereafter at 37 °C in 5% CO2 humidified atmosphere until the cells were ready to be seeded on the top of the gel. HSC-3 cells were trypsinized and counted, trypsin inhibitor instead of serum-containing medium was used to inactivate trypsin. 500 μΙ of 10% serum containing med ium was added into the lower chamber of Transwell^®, and 50 000 HSC-3 cells suspended into 100 μΙ of medium containing 0.5% lactalbumin instead of serum were seeded into the upper compartment of the Transwell^® chamber. Cells were left to invade for one to three days and the invasion was quantified by crystal violet staining and cell counting as in invasion without agarose. In another set of experiments, invasion of SCC-9, LN-1, LN-2, HSC-3, SK-Mel- 25 and A2058 cells was studied in Myogel-LMA and growth factor-reduced Matrigel^®

(Matrigel^®-G FR, BD Matrigel Matrix, BD Biosciences Cat. N umber 35430) diluted 1+1 with serum free medium was used without LMA and the invasion was quantified with Toluidine Bl ue -staining. Briefly, after 72 h invasion the cells were fixed with 4% formaldehyde for 1 h at RT, washed once with PBS, stained for 5-10 min in Toluidine blue solution (filtered 1%

Toluidine Blue + 1% disodium tetraborate in dd H₂0) at RT, excess dye was rinsed out with deionized water, excess gel and the cells from the upper side of the membrane were removed by using a cotton swabs and when necessary, excess dye was further removed from inside and out of the Transwell^® inserts with cotton swabs immersed in a solution of 1: 1 water/ethanol. The dye was eluted by dipping the Transwell^® inserts in a solution of 1% SDS (500 μΙ). The 1% SDS solution containing the eluted dye was transferred into a 96-well plate and the absorbance was measured at 650 nm wavelength. In a third set of experiments, 2.4 mg/ml Myogel (with 0.2% LMA) or Matrigel^® was used to study the invasion of oral squamous cell carcinoma cell lines SAS and SCC-25, melanoma cell l ine Bowes and pancreatic cancer cell lines Pa02c, Pa03c and Pa04c. The Transwell inserts were incubated for 72 h after which the invaded cells were stained with 1% Toluidine blue solution. The absorbance of the eluted dye was measured at 570 nm wavelength.

Hanging drop method

The gel from rat tail type I collagen (BD Biosciences) was prepared as described by

manufacturer's protocol, the final collagen concentration was 1.7 mg/ml. The

collagen/Matrigel^® (BD Matrigel Matrix, BD Biosciences Cat. N umber 354234) (1.5 mg/ml, 1.5 mg/ml) and collagen/Myogel (1.5 mg/ml, 4.3 mg/ml) mixtures were prepared similarly. The hanging drop technique was used to observe the cell movement in 3 D culturing condition. HSC-3 cells were washed with PBS, trypsinized and 70 000 cel ls in 10 μΙ of F12/DM EM med ium (2% FBS) were mixed with 50 μΙ of the matrix mixtu re. 20 μΙ of the cell suspension in each matrix was dropped on the 4 compartment plate. Plate was flipped around after 5 min incubation in culturing conditions and hanged for 3 h in humidified chamber in culturing conditions. Mimosine (200 μΜ) was added to the medium to synchronize the cell cycle.

Images were taken with Zeiss Axio Observer. Zl with EC Plan-Neoflaur 40x/0.75 M27 objective. Images consisting of 1024 x 1024 pixels were taken every 15 min with 19.8 μιη Z- stack volume (Ο.βμιη thickness) with Hamamatsu Camera#2 control led by Zeiss Zen Blue software (Zeiss) for 22 h.

Microarray

For microarray analysis, 90 000 HSC-3 cells transduced with RFP were seeded into uncoated or Myogel coated 6-well plates (three wells each). The next day, the cells were harvested for RNA extraction by Qiagen RNA kit. The three samples of each group (on top of plastic or Myogel coating) were pooled with an equal amount of each RNA. Affymetrix GeneChip Hu man Genome U 133 Plus 2.0 Arrays were used for microarray analysis and experimental proced ures were performed according to the Affymetrix GeneChip Expression Analysis Technical Manual. Briefly, 1 μg of total RNA was used as a template to synthesize biotinylated cRNA by means of the GeneChip 3'IVT Express kit (Affymetrix) according to the

manufacturer's instructions. The cRNA was fragmented to 35 to 200 nt prior hybridizing to Affymetrix H u man Genome U 133 Plus 2.0 arrays containing approximately 55 000 human transcripts. The array was washed and stained with streptavidin-phycoerythrin (Molecular Probes). Finally, biotinylated anti-streptavidin (Vector Laboratories Inc.) was used to amplify the staining signal and a second staining was performed with streptavidin-phycoerythrin. The arrays were scanned on GeneChip Scanner 3000. The expression data was analyzed to find genes with fold change (FC) 1.5 or more using dChip software (Li & Wong 2001). The genes with FC 1.2 or more were divided into Gene Ontology (GO) categories using DChip enrichment analysis tool.

In vitro capillary tube formation assay

96-well culture plates (BD Biosciences) were coated with Myogel with 2% low melting agarose (Myogel-LMA), Matrigel^®-G FR (BD Matrigel Matrix, Cat. N umber 35430 - BD, Lot. 3270647) or ECMatrix™ (ECMatrix - In vitro Angiogenesis Assay Kit, Cat. N umber ECM625 - Millopore, Lot. 2383502) previously thawed overnight on ice, in a total vol ume of 50 μΙ/well and allowed to solidify overnight at 37 °C. H UVEC cells were trypsinized, neutralized with DM EM/F12 with 10% FBS, washed once with PBS and resuspended in DM EM/F12 at a density of 450 000/ml, and 100 μΙ of this cell suspension was added into each well. The cells were incubated at 37 °C for 12 h . Tube formation was observed under an inverted microscope (N ikon Eclipse Ti-S, x4), photos were taken and analysed using the motic images plus 2.0 program. Three visual fields were randomly selected from each well to count the tubes, and the average value was taken for statistical analysis. Tubule perimeter was assessed by drawing a line around each tubule and measuring the line.

Drug sensitivity and resistance testing (DSRT) DSRT was performed on pancreatic cancer cells Pa02c with 132 compounds in 5

concentrations in 10-fold dilutions covering a 10 000-fold concentration range with three different conditions (no coating, Myogel and Matrigel^® (both 0.62 mg/ml) coated wells). Before the DSRT experiment, cell viability was measured on different Myogel and Matrigel^® concentrations. For coating, 8 μΙ Myogel or Matrigel^® dilutions were pipetted into the wells of 384-well plates and the plates were incubated for 2 h at 37 °C in 5% CO2 humidified atmosphere. 500 cells were suspended into 25 μΙ of med ium and seeded into the wells using Deerac LX liquid handling device (Labcyte Inc.) and incubated overnight at 37 °C in 5% CO2 humidified atmosphere. Next day, 25 μΙ drug diluted in DM EM was added into the wells using Biomek FXP -pipetting robot (Beckman Coulter). After three days incubation, cell viability was measured using CellTiter-Glo^® luminescent assay (Promega). Luminescence was detected using the PH ERAstar^® FS microplate reader (BMG Labtech) and the data were analyzed by Dotmatics Browser/Studies software (Dotmatics Ltd). Drug sensitivity score (DSS) for each drug was calculated as described (Yadav et al. 2014) and the scores were compared between different coatings. Statistical analysis

SPSS for Windows software program version 21.0 (SPSS Inc.) was used for statistical analyses. To establish the statistical significance of differences between the two independent cell culture groups, a Student's t-test or Mann-Whitney U test was used depending on the normalization of the distributions. Results

Protein content in different Myogel batches varies slightly and is different from Matrigel^®

Protein band patterns of different Myogel sample batches and the mixture of various batches looked rather similar in Coomassie Blue stained gradient SDS-PAG E gels. Only slight variation of the intensities of different size bands was visible. Overall, more bands were seen in Myogel than in Matrigel^® samples, possibly due to difference between species (human vs. mouse) and the nature of the starting material (leiomyoma vs. sarcoma).

The pH of Myogel is neutral and more stable than the pH of Matrigel^®

At the beginning, the pH of Myogel was between 7.0 and 7.5, and after 48 h incubation the pH remained the same. The pH of Myogel with HSC-3 cel ls on top dropped slightly from 7.0- 7.5 to 6.5-7.0 (0 h and 48 h incubations, respectively), whereas in Matrigel^® the pH at the same time dropped from slightly alkali 8.0-8.5 to as low as 6.0-6.5 (Table 1). This indicates that Myogel samples are closer than Matrigel^® to normal neutral pH, and Myogel keeps the pH more stable than Matrigel^® during cancer cells culture experiments. Myogel contains partially the same proteins as Matrigel^®

We next analyzed the protein content of Myogel and compared it with the published data of Matrigel^®. For proteomic analyses, a new gradient SDS-PAG E was ran from different Myogel batches.

Two of the Myogel samples gave successful results on mass spectrometry. Altogether 765 proteins were identified in Myogel, among them 34% (259 proteins) were the same as in

Matrigel^®. Matrigel^® protein content was summarized from Vukicevic et al . (1992), Hughes et al. (2010) and Talbot & Caperna (2014). Based on the comparison, e.g. laminin, type IV collagen, heparan sulfate proteoglycans, nidogen and epidermal growth factor were found in both. As compared to Matrigel^® Myogel was lacking enactin, fibroblast growth factor, insulin- like growth factor 1, platelet-derived growth factor and nerve growth factor. Myogel had e.g. tenascin-C, collagen types XI I and XIV, etc. which were lacking in Matrigel^®. Based on zymography, Myogel contains both latent and active forms of M M P-2, whereas in Matrigel^® latent and active forms of both M M P-2 and M M P-9 were present. Among the 1030 proteins identified in Matrigel^®, 40 were characterized as RI KEN cDNA and 45 represented predicted genes. Hence with these data we cannot directly indicate the presence of these proteins in Myogel .

Myogel has much more proteins visible in 2-DE than Matrigel^®

The protein patterns of two Myogel samples looked quite similar. There were only some slight changes between the two samples (sample 16 vs sample 17 =+13 spots in 16 vs +21 spots in 17). There was a really big difference between Matrigel^® and Myogel samples; in Matrigel^® only a few spots were seen compared to Myogel.

Fibroblasts adhere on top of Myogel but only HSC-3 cells form colonies within Myogel- soft agar assay HSC-3 cells adhered significantly more to plates coated with Myogel than plates coated with BSA or plain plates kept in PBS during 2 h incubation before cell seeding. To Matrigel^® coated plates they adhered even more (Fig. 1). Adhesion experiment of G Fs on Myogel showed that after 9.5 h incubation on Myogel fibroblasts were spread and looked vital (not shown). Based on TU N EL assay we could confirm that from 98 to 99 % of the CAFs seeded on top of various batches of Myogel were alive after 24 h incubation (not shown). However, when CAFs were embedded within Myogel matrix almost all died within 21 days (not shown). Instead, HSC-3 cells stayed alive up to 28 days, divided and formed colonies within Myogel combined with LMA. The results with HSC-3 cells were rather similar using either Myogel-LMA than conventional LMA method (Fig. 2A). However, more colonies with the lowest cell number were present in LMA, while the highest cell number/colony was present in Myogel-LMA (Fig. 2B). According to nuclear mCherry expression, 92% of the cancer cells were alive in Myogel- LMA colonies, whereas 85% were alive in LMA colonies. The total average area of cell colonies in Myogel-LMA was three percentages less than in LMA (not shown).

The vertical migration of HSC-3 cells is faster on Myogel than on Matrigel^® coated wells In the scratch assay, HSC-3 cells migrated significantly more in Myogel coated wells than in wells coated with Matrigel^®. However, in uncoated wells their migration was faster than in coated wel ls (Fig. 3A and 3B). HSC-3 cells invade more efficiently through Myogel than through Matrigel^®

In order to test the use of Myogel on cancer invasion Transwell^® assays, we first tested the most aggressive oral tongue carcinoma cell line (HCS-3) invasion using Myogel and compared with the invasion done simultaneously with Matrigel^®. The HSC-3 cells invaded significantly more efficiently through Myogel than through Matrigel^® (Fig. 4A). Invasion varied slightly in different Myogel batches but HSC-3 cells invaded more in all Myogel samples than in

Matrigel^® (Fig. 4B). When Myogel and Matrigel^® were mixed, HSC-3 cells invaded more when the mixture contained more Myogel and less when the portion of Matrigel^® increased (Fig. 4C). Invasion pattern of HSC-3 cells was different in Myogel and Matrigel^® : cells invaded more even ly through the whole Transwell^® membrane area in Myogel than in Matrigel^® where they tended to go through membrane in groups (Fig. 4D).

Myogel solidified with agarose (Myogel-LMA) is suitable TME matrix for invasion assay

Since we noticed that Myogel batches vary slightly in their protein content, we wanted to make the gel mixture for invasion assays more homogenous, reliable and easier to handle by adding low-melting agarose into the Myogel mixture. We noticed that HSC-3 cells did not invade through plain agarose but they invaded through Myogel-agarose (Myogel-LMA) mixture (Fig. 5A and 5B). Interestingly, the passage n umber of HSC-3 cells seemed to affect the invasion through Myogel and Matrigel^®-mixtures; cells with higher passages invaded slightly less through mixtures containing Matrigel^® than through Myogel-containing mixtures, whereas cells with low passage invaded slightly less through mixtures containing Myogel-LMA than through Matrigel^®-agarose (Fig. 5A). Invasion pattern of HSC-3 cells through agarose- containing mixtures was similar than in plain Myogel or Matrigel^®; in Myogel-LMA mixtures they invaded more evenly through the whole Transwell^® membrane area than in Matrigel^®- containing mixtures (Fig. 5 B). In another experiment, Myogel-LMA was compared with Matrigel^®-G FR. In general, all the oral squamous cell carcinoma cell lines used for this invasion assays (HSC-3, LN-1, LN-2 and SCC-9) seemed to prefer Myogel-LMA to Matrigel^®-G FR d uring the invasion (Figure 5C ). HSC- 3 and LN-2 (in this order) seemed to have a higher potential to invade in both Myogel-LMA and Matrigel^®-G FR, compared to LN-1 and SSC-9 cell lines. The cells invading in the Myogel- LMA seemed to keep more of their morphological characteristics, like we see in monolayer culture. At the Matrigel^®-G FR they seemed to group together as they invaded the matrix, and to lose the "fusiform-starshape" characteristic of such oral carcinoma cell lines (not shown). In addition to oral carcinoma cell lines, we tested the use of Myogel-LMA on SK-Mel and A2058 melanoma cells and found also them to be able to invade Myogel-LMA more efficiently than Matrigel^®-G FR (Fig. 5C).

In the third experiment, the invasion capacity of oral squamous cell carcinoma cell lines SAS and SCC-25, melanoma cell line Bowes and pancreatic cancer cell lines Pa02c, Pa03c and Pa04c in Transwell^®s was compared between Myogel-LMA and Matrigel^®. All of the tested cell lines had a significantly higher invasion through Myogel than through Matrigel^® (Fig. 5D). In scratch wound invasion assay, more HSC-3 cells as well as pancreatic cancer cells invaded horizontally through Myogel-LMA than through Matrigel^® (Fig. 3C-3E).

Myogel induces efficiently the tube formation

In vitro angiogenesis assays are commonly used to assess pro- or anti-angiogenic drug properties (Donovan et al. 2001). Here, the tube formation could be quantified in all matrix- assays tested (Figure 6A). With Myogel-LMA, the tube formation was visible after 12 h, and even after 72 h (Fig. 6A) the endothelial cells were alive, unlike in Matrigel^®-G FR or

ECMatrix™ (not shown), where most of the H UVEC cells even already after 24 h were apoptotic. We found three times higher number of tubules formed in Myogel-LMA compared to ECMatrix™ (Fig. 6B). Otherwise, measu ring the diameters of the capillaries, the tubule parameters in Matrigel^®-G FR, and especially in ECMatrix™ assays, were significantly higher than in tubes formed in Myogel-LMA (Fig. 6C).

HSC-3 cells move faster in Myogel-collagen matrix than in Matrigel^®-collagen matrix in 3D culturing condition

To observe the cell behavior in 3D culture in different matrixes, we have established an optimized hanging drop method modified from the previously shown protocol (Yip & Cho

2013). HSC-3 cells seemed to move relatively similarly in pure collagen, Myogel-collagen and Matrigel^®-collagen matrices. The nuclei count was higher and varied more in all matrices than the cytosomal cell count, the count was highest in pure collagen in both. The nuclei size was about the same and rather even in all the matrices while the cell size varied more, both between the matrices and during the recording in each matrix. Both the nuclei and the cell sizes were largest in Matrigel^®-collagen matrix. The average eccentricity/roundness measured by nuclear stain was about the same and rather even in all the matrices while was highest in pure collagen and varied more when measured by cytosomal stain. In speed the result was rather similar measured by nuclear and cytosomal stain in each matrix. However, the speed was highest in Myogel-collagen matrix and lowest in Matrigel^®-collagen matrix.

Genes related to intracellular organelle and cytoskeleton organization and biogenesis change when HSC-3 cells cultured on plastic are compared on Myogel cultured ones

Approximately 751 genes were changed (FC 1.5 or more) when HSC-3 cel ls cultured on top of plastic were compared to the corresponding cells grown on top of Myogel. When the genes were divided into groups according to their biological function we found that Myogel coating affected genes that are related to intracellular organelle and cytoskeleton organization and biogenesis. Significant change in pathway analysis was found only in G 13 signaling pathway that is related to e.g. actin polymerization and reorganization (www.genmapp.org). The factors in this pathway, such as Wiskott-Aldrich syndrome-like (WASP), play important roles in cell response to extracellular stimuli by actin filament reorganization and contribute crucially to cancer cell motility (Takenawa & Miki (2001).

Myogel is suitable coating for drug testing

Viability of the cells to be used in drug testing (Pa02c) was first measured on different concentrations of Myogel and Matrigel^®. The viability of the cel ls was significantly higher in Myogel coated wells than in Matrigel^® coated ones (Fig. 7). About one third of the drugs (48) had different IC50 values against Pa02c cells when tested on top of Myogel vs plastic. 20% of the drugs were more effective in Myogel than in plastic, while 16% were more effective in plastic than in Myogel. More than one third of the drugs (56) had different IC50 values when tested on top of Matrigel^® vs plastic. 27% of the drugs were more effective in Matrigel^® than in plastic, while 16% were more effective in plastic than in Matrigel^®. Drug Sensitivity Scoring (DSS) showed that 33% of all the tested drugs were effective. Significant differences (more than 5 DSS) between non-coated vs Myogel-coated wells were fou nd in 10% of the drugs, between non-coated vs Matrigel^®-coated wells in 14% of the d rugs, and between Myogel- coated wel ls vs. Matrigel^®-coated wells in 9% of the drugs. Discussion

Myogel, and especially easy-to-use Myogel- LM A, are both well suited for in vitro cancer studies. They are in some cases superior to the Matrigel^® or to the rat tail type I collagen - based methods. Using easily obtained human uterus leiomyoma tumor tissue, which normally is wasted after histopathological analyses, to produce Myogel mixtu re, the material costs are relatively low.

Myogel offers a natural human TM E based matrix to study the behavior of various cancer cell lines and cancer drugs in vitro. In theory, this set of instruments may well be usable also in the future for personalized medicine where, after obtaining a fresh tumor tissue biopsy for analyses, the effects of drugs or chemoradiation therapies could be tested for finding the optimal treatment modality for the patients.

Table 1. The pH-measurements of Myogel and Matrigel^®

Myogel with HSC-3 Matrigel^® with HCS-3 Myogel without cells

0 h 7.0 - 7.5 8.0 - 8.5 7.0 - 7.5

17 h 7.0 - 7.5 8.0 7.0 - 7.5

48 h 6.5 - 7.0 6.0 - 6.5 7.0 - 7.5

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Claims

CLAI MS

1. A cell culture composition comprising homogenized human leiomyoma tissue.

2. The cell cu lture composition according to claim 1, wherein said leiomyoma tissue is from uterus.

3. The cell cu lture composition according to claim 1 or 2, wherein said homogenized human leiomyoma tissue is a total protein extract from human leiomyoma tissue.

4. The cell cu lture composition according to any one of claims 1-3 further comprising agarose.

5. The cell cu lture composition according to claim 4, wherein said agarose is low melting point agarose.

6. The cell cu lture composition according to claim 5, wherein the melting temperature of said low melting point agarose is under 65 degrees of Celsius.

7. Method of preparing extracellular matrix homogenate comprising the steps of:

(a) homogenizing human leiomyoma tumor at least twice in a NaCI-buffer and discarding the soluble fraction after each homogenization;

(b) extracting the residual leiomyoma tumor in a urea buffer and stirring the extract for about 12-18 hours;

(c) separating the extract from the residual leiomyoma tu mor fraction and saving the first extract while repeating step (b) with the residual leiomyoma tumor fraction and saving the second extract resulting therefrom;

(d) combining the first and second extracts from step (c) and dialyzing against a buffer comprising a sterilizing component; and

(e) further dialyzing against a buffer not comprising a sterilizing component and recovering a dialysate.

8. The method according to claim 7 comprising further steps of

(f) dialyzing the dialysate obtained from step (e) against a serum-free medium;

(g) recovering a dialysate from step (f).

9. The method according to claim 7 or 8, wherein the buffer of step (a) is a 3.4 M NaCI buffer having a pH of about 7.4.

10. The method according to claim 7 or 8, wherein the urea buffer of step (b) is a 2 M urea buffer having a pH of about 7.4.

11. The method according to claim 7 or 8, wherein said sterilizing component is chloroform.

12. The method according to claim 8 comprising a further step of mixing the dialysate from step (g) with agarose.

13. The method according to claim 12, wherein said agarose is low melting point agarose.

14. The method according to claim 13, wherein the melting temperature of said low melting point agarose is under 65 degrees of Celsius.

15. The method according to any one of claims 7-14 comprising a further step of incubating human cells with the extracellular matrix homogenate obtained in claim 7 or 8.

16. The method according to claim 15, wherein said human cancer cells are invasive cancer cells.

17. Use of the cell culture composition according to any one of claims 1-6 for invasive, migratory or adhesive cultures of human cells.

18. The use according to claim 17, wherein said human cells are human cancer cells.

19. Use of the cell culture composition according to any one of claims 1-6 for an invasive culture of human cancer cells.

20. The use according to claim 17 for capillary formation culture of human umbilical vein endothelial cells.

21. The use according to claim 17 in drug discovery.

22. The cell culture composition according to any one of claims 1-6 produced by the method according to any one claims 7-16.

23. A cell culture composition made of total protein extract from human leiomyoma tissue, wherein said total protein extract is prepared by homogenization and purification of human leiomyoma tissue.