Ex VIVO cellular model system comprising heterogenous cells from human lung tumor for the evaluation of drug cytotoxicity in vitro and method thereof

Abstract

The invention is a model of research and methodology to explore the treatment options for a particular case of lung cancer as an adjuvant therapy after surgical removal of a lung tumor. In practice, the excised / removed tumor is usually examined in the pathology laboratory for revision of the diagnosis, but no functional testing is performed for its sensitivity to subsequent adjuvant 5 therapy. In addition, the tumor is disposed of as unnecessary waste. Our proposal is that the tumor remains should be harvested for cellular studies and, using the model and methodology developed and described herein, preliminary answers can be obtained within 2-3 weeks, predicting which chemotherapeutic agents are likely to have a stronger effect and which therapy is likely to be ineffective for an individual lung cancer patient.

Description

[0001] EX VIVO CELLULAR MODEL SYSTEM COMPRISING HETEROGENOUS CELLS FROM HUMAN LUNG TUMOR FOR THE EVALUATION OF DRUG CYTOTOXICITY IN VITRO AND

[0002] METHOD THEREOF

[0003] FIELD OF INVENTION

[0004] The technology described herein relates to drug screening and individualized cancer therapy. The invention relates to a cell culture system which is patient-specific and comprises several different patient-derived subcultures of cells. This system can be used as a model for individual screening of drug toxicity and prediction of efficacy in vivo for that patient.

[0005] BACKGROUND ART

[0006] The invention relates to a method for obtaining the drug-sensitivity profile of the lung tumor- derived cells. The invention comprises the following steps: generation of different cell cultures from the same tumor sample; treatment of the cell cultures with chemotherapeutic drugs or other substances; assessment of the responses to the treatments (viability of cells). The method for obtaining the drug-sensitivity profile of the lung tumor, provided by the invention, has the advantages over closest previous arts described in the patents (detailed below) and in non-patent publications (doi.org / 10.1016 / j.xpro.2020.100239 - “Protocol for generation of lung adenocarcinoma organoids from clinical samples”; https: / / doi.org / 10.18632 / oncotarget.4013 - “Spontaneously-forming spheroids as an in vitro cancer cell model for anticancer drug screening”). 1 . The tissue mass is not enzymatically changed by means applied in enzyme-based dissociation of tumor samples in similar protocols; 2. 3D cellular structures are obtained from the primary sample, not from the outgrowth of single / purified cells; 3. Majority of the testing is performed in 2D cell culture which is easier to process, faster and cheaper; 4. The subtypes / subcultures of tumor-composing cells are spatially separated according to their phenotypic (adhesion) properties and therefore can be assessed independently; 5. Preliminary drug sensitivity profile can be obtained within one week, and right away it may be checked on the original 3D tissue fragments (total two weeks). Additionally, a part of the cells can be cultivated and propagated in vitro during the primary evaluation, and also frozen in standard cryopreservation medium for additional tests later, if needed.

[0007] The present invention is different from the prior art patent CN106967672B (“Lung and lung cancer tissue culture method and method for constructing lung cancer mouse animal model by using same”) in that no animal model is involved, no healthy tissue is involved, no unnatural extracellular matrix, no enzymatic digestion. The present invention method allows the inclusion of various natural extracellular pieces / fragments (which pass through a cell strainer) in the 2D and 3D cell culture, as well as in small tissue fragments which stay on top of the cell strainer. The prior art CN106967672 takes months to provide the answer to the hypothesis and questions raised, it requires animal department, therefore it is hardly a candidate in clinical settings for personalized therapy of a patient.

[0008] For example, the European patent EP2420837B1 (“An in vitro method for the prognosis of progression of a cancer using CD45 as a marker") relies on a biochemical marker or its level. What authors demonstrate in their invention, is that certain level of certain biomarker manifestation can be translated into good or poor prognosis for the patient. The prior art is validated by comparing cohorts of patients in vivo. It means that the invention has been tested in clinical settings, not in in vitro / preclinical setting. This prior art does not suggest the means applying which the outcome should become favorable for an individual patient.

[0009] The patent .EP2844733B1 (“In vitro model for pathological or physiologic conditions”) discloses a model for studying liver and vascular diseases and it has proofs that hemodynamic environment (mimicking the blood flow) has a measurable effect on a certain cellular function when compared to a static condition. Therefore, the described model is closer to natural in vivo conditions than the majority of current in vitro models. The present invention is different from this prior art which does not relate to a pathological condition which is a cancer / tumor and is limited to vascular system and liver function. Moreover, it is dependent on an elastic support which is a porous membrane, coated or not with an extracellular matrix.

[0010] The patent EP3060654B1 (“In vitro model for a tumor microenvironment”) discloses an ability to test the efficacy of a chemotherapeutic drug in a personalized in vitro model. The prior art relies on mimicking tumor microenvironment, namely the blood flow and share stress and the model is constructed by plating different types of cells on a porous membrane. The model seems very versatile, but time-consuming and expensive.

[0011] The patent EP2626414B1 (“Cancer stem cell mass and process for production thereof”) relies on the hypothesis of cancer stem cell existence and their specific cellular markers (publication date 201 1 ). Firstly, there are several theories of cancer progression / resistance, one of which is a cancer stem cell theory which attributes the development of a therapy-resistant cancer phenotype to a small group of tumor-composing cells named cancer stem cells. Other theories blame general cancer cell plasticity (epithelial-mesenchymal transition) or clonal evolution for the resistance. Secondly, cancer stem cells do not have any affirmed separate cellular markers (e.g. CD133 and CD44), making difficult / impossible to distinguish between healthy stem cells and cancer stem cells (doi: 10.1 186 / 2162-3619-2-17 - “CD133: a stem cell biomarker and beyond”; doi: 10.1 136 / mp.51 .4.191 - “The normal structure and function of CD44 and its role in neoplasia”). Moreover, cancer cells are capable of fusing with healthy cells to obtain cellular markers which did not belong to it (doi: 10.3892 / ol.2021 .12791 - “Cell fusion in cancer hallmarks: Current research status and future indications”). Authors of this prior art also sometimes call them “cancer stem cell markers”, and sometimes “reported cancer stem cell markers”. Today, the concept of “cancer stem cell markers” is becoming historical, yet scientifically unproven (Doi: 10.1371 / journal. pone.0225898 - “Do cancer stem cells exist? A pilot study combining a systematic review with the hierarchy-of-hypotheses approach”; Doi: 10.1155 / 2019 / 5189232 - “Cancer Stem Cells: From Historical Roots to a New Perspective”). The prior art is determined to purify / stratify / select only specific cells from the tumor mass, namely putative “cancer stem cells”, by removing other cells which do not meet the selected requirements. Our invention in general takes all the cells from the tumor tissue, not distinguishing to which type, category, or state they belong. The prior art model is constructed on implanting cancer tissue in NOD / SCID / gamma cnu" mice to obtain proliferating population of cancer stem cells which further can be cultured in vivo or in vitro. Moreover, the model seems to represent the natural tumor formation and hierarchical structure, but time-consuming (months) and expensive.

[0012] SUMMARY OF THE INVENTION

[0013] The essence of the present invention is defined by: 1 ) the technical problem to be solved; 2) the technical solution of the invention; 3) the advanced technical effect that the Invention allows to achieve by solving the problem.

[0014] 1 ) Current attitude towards lung cancer involves genetic profiling (a panel of known mutations in genes: EGFR, KRAS, cMET, RET, ROS1 , ALK, BRAF, etc.) and suggesting the targeted drugs according to the mutations found. No known mutations are found in up to 20% of the cases. As lung cancer usually is diagnosed late, and because there are few methods to treat lung cancer effectively, the overall survival for NSCLC patients is less than 5 years from the diagnosis. During this time, relapse and cancer cell resistance to the applied therapy emerges. As a consequence of research which demonstrated that cancer resistance originates as a multifaceted process involving tumor cellular heterogeneity, we developed an innovative model system. Without looking at mutations (genetic profiling can be performed but outside of our model), the invention relies on functional in vitro toxicity assays. Briefly, phenotypically different cells from the same tumor of a certain patient are cultured separately and / or together in vitro few days after isolation from the tumor, and various drug combinations are tested to impair their viability. Most effective drug combinations are tested once again in 3D cell culture which contains small pieces of the primary tumor. In the future, the resultbased prediction of the therapy efficacy can be suggested to treat the individual patient. Such model is different and more simple comparing to other current models which assess the overall toxicity of a drug (or their combinations) when applied on purified, proliferating cancer cells within cellular spheroids. ) The more cells are killed simultaneously, the less is ability of the cells that are resistant to the treatment to produce progenies with adaptation and / or new mutations. Having different cell subpopulations that are susceptible to different drugs or to a different level, the primary tumor samples (3D) will be tested for the combination of the drugs that exert efficacy on certain type / phenotype of cells from the tumor. The invention involves a step where a heterogenous cell population is forced to MET or EMT transitions via specific chemical and biological inducers to obtain additional and new populations of different phenotypes. Additionally, phenotypically different populations of cells (regarding their adhesion properties) are obtained by the invention. The more phenotypes are evaluated in drug toxicity screening, the better insight into the outcome of the future medical treatment can be achieved. Although clinical trial is required to finalize and validate the model described in this invention, the advanced technical effect can be prolongation of the disease-free (after surgery and adjuvant therapy), progression-free (advanced cancer) or overall survival of lung cancer patients. ) Current lung cancer in vitro / in vivo models are expensive and, more importantly, they take a lot of time. For example, personalized in vivo mouse model (patient-derived xenograft) may cost 5.000 USD per mouse to test 1 drug / combination (https: / / www.championsoncoloav.com / nsclc-xpressscreen ; https: / / www.marketsandmarkets.com / Market-Reports / patient-derived-xenoaraft-model- market-121598251 .html ), and it also would take no less than 3 months to obtain the result. Alternatively, 3D cellular spheroids / organoids in vitro are cheaper but also take a long time (at least 2-3 months; doi: 10.1038 / s41467-019-1 1867-6 - “Patient-derived lung cancer organoids as in vitro cancer models for therapeutic screening”). Other models involve construction of containers which simulate blood flow, heart beating, breathing and other conditions by applying a shear stress onto the cellular layers placed on a porous membrane. Such models, in our opinion, are well suited for research but merely applicable in clinical settings, unless miniaturized. The present invention described in this article suggests that approximately 80 drugs / combinations can be tested in less than 2 weeks from obtaining the ex vivo tumor sample. The method involves traditional 2D / 3D in vitro techniques which are low cost when compared to other known current methodologies to perform the functional testing of drug efficacy on tumor-derived cells. Importantly, enzymatic digestion that is applied routinely in other methods to obtain the cell culture as well as other cellular structures, is not involved in our methodology. It is known that proteolytic enzymes not only activate cell death- inducing signaling pathways, but also affect the phenotype of the cells, and damage them (doi.org / 10.1186 / 1423-0127-17-36 - “Trypsin-induced proteome alteration during cell subculture in mammalian cells”), therefore, it should be avoided in drug screening.

[0015] BRIEF DESCRIPTION OF THE DRAWINGS

[0016] Fig. 1. What is shown here describes the first part of the specimen processing. Starting with at least two grams of the sample tissue, single cells (1 ) and tissue fragments (2) are obtained by passing the suspension through a cell strainer. Cells (1 ) are placed in various conditions (medium composition) and cell culture dishes for to attach and / or proliferate. Cells in different conditions are named “Series”. The method requires approximately 5-10 million cells to perform the testing in a full program. A portion of cells (1 ) can be preserved for additional time to be frozen later (3). Another portion of “series” and tissue fragments (2) are used for procedures described in other figures. Genetic profile is outside of this model system, but it may be useful if available. WT - “wild type” cells, meaning no recombinant / additional growth factors applied; HGF+TGF - hepatocyte and transforming growth factors, inducible EMT transition; FGF+EGF - fibroblast and epidermal growth factors, inducible MET transition.

[0017] Fig. 2. One part of the primary strained cells (4) is used for the separation of cells expressing one or several specific markers by using antibody-based applications. The enriched and depleted populations are obtained as described in the main text. The concentration of cells in every “variant” is assessed and equalized. Then, 1000 cells in 25 uL of corresponding medium are plated in a tissue culture-treated 384-well plate (7000). After 24 hours, the plates are treated with the drugs as shown in Fig. 6, as well as the “initial viability” of the variants is measured. After three days, terminal viability is measured.

[0018] Fig. 3. One portion of cells which have passed through a cell strainer (1 ) are resuspended in 30 mL of cell culture medium (in this figure, the medium is w / o recombinant / additional growth factors applied, WT). The cell number should preferably be 3 million. 0.5 million of cells are seeded into a specific 96-well plate (1000), constituting 5000 cells in 50 pL of medium per well. 0.4 million of cells are seeded into 384-well cell culture plate (2000), constituting 1000 cells in 25 pL of medium per well. The rest of cellular suspension is divided into two cell culture-treated flasks of 25 cm2bottom area ((100) and (200)). The second flask (200) can be used for the expansion of the cell culture (3) (Fig. 1 ).

[0019] Fig. 4. The cells which could not pass through the cell strainer (2) are collected by inverting and washing the cell strainer, after that they are resuspended in cell culture medium and distributed in two 96-well plates with flat, cell-culture treated bottom ((3000) and (4000)). Depending on the amount of tissue within the wells, every 2-3 days the medium is replaced with fresh one. Prior to the treatment, “initial viability” of every well is measured. After that, treatment with drugs and / or their combinations takes place, followed by terminal viability readout (in 3-5 days). These plates are for the final assessment of candidate drugs and their combinations.

[0020] Fig. 5. T25 flask (100) is used for the separation of various phenotypes of cells in regard to cellular adhesion properties. The “variants” (loosely-anchored, readily-detaching, quickly- attaching, slowly-attaching) are obtained as described in the main text. The concentration of cells in every “variant” is assessed and equalized. Then, 1000 cells in 25 uL of corresponding medium are plated in two tissue culture-treated 384-well plates ((5000) and (6000)). After 24 hours, the plates are treated with the drugs as shown in Fig. 7, as well as the “initial viability” of the variants is measured. After three days, terminal viability is measured.

[0021] Fig. 6. An example of the 384-well plate layout for the drug toxicity measurement. Rows A,B,C contain a certain marker-enriched cell population (9), while rows D,E,F contain marker- depleted cell population (10), as described in the main text. Monotherapy (columns 3-24) of single drugs at the indicated concentration may have different level of effect on these two populations of cells. The layout is suggested for the antibody-sorted cells (plate (7000)) (Fig.2). This treatment approach can highlight the effect of the specific marker on tumor-derived cell susceptibility to therapeutic treatments.

[0022] Fig. 7. An example of the 384-well plate layout for the drug toxicity measurement. Every two rows contain a phenotypically different cell type. Monotherapy (columns 3-24) represent the individual toxicity of a single drug at the indicated concentration. This layout is suggested for the “variants” (5-6-7-8) of “series” (plates (5000) and (6000)) (Fig.4). This treatment approach can highlight the effect of EMT / MET transitions on tumor-derived cell susceptibility to therapeutic treatments.

[0023] Fig. 8. An example of the 384-well plate layout for the drug toxicity measurement. Monotherapy (rows A and B) represent the individual toxicity of a single drug at the indicated concentration, except for the columns 1 and 2 (controls). Other rows represent 77 combinations of the drugs. This layout is suggested only for the “strained cells” (1 ), plate (2000) (Fig.3). Hereafter, this treatment approach has the least number of interventions into the tumor phenotype. DETAILED DESCRIPTION OF THE INVENTION

[0024] This description provides a protocol for organizing and performing drug toxicity assays on ex vivo tumor-derived cells. The predictive result of such an assay is an expression of relative viability in several 96-well and / or 384-format plates that can be handled by common-form multiwell plate readers. Relative viability in this context means the ratio of the measured viability after the treatment (3-5 days long) to the initial viability which is determined now of the treatment (time point 0).

[0025] The methodology is intended to introduce several additional aspects of tumor cell phenotypic heterogeneity / plasticity which to our knowledge are not attributed in current / disclosed methodologies of in vitro testing of ex vivo tumor-derived cells and / or organoids in evaluation of efficacy of anticancer drugs and other substances.

[0026] Viability in this context is a measurable value of biological activity which is commonly accepted as occurring only in living cells, e.g., enzymatic activity of oxidoreductases which can reduce substrates such as but not limited to MTT, resazurin, XTT, WST and similar. Alternatively, viability can be measured by the amount of intracellular ATP (adenosine triphosphate) which directly correlates with the health / viability of a cell.

[0027] A digestive enzyme in this context is a substance with proteolytic activity. It can be trypsin, dispase, collagenase, libertase, modified enzymes, their mixtures and similar. Within this invention, proteolytic digestion is applied only to prepare the cells for automated cell counting, and to provide cellular subcultures which are different in cellular adhesion characteristics. It is important to address that within the invention proteolytic enzymes do not serve to extract the cells from their native milieu.

[0028] Tumor sample in this context is restricted to lung cancer, preferably non-small cell lung cancer which accounts for approx. 70 percent of lung cancers. Other types of cancers can be adapted to fit within the methodology, but the objective is to take advantage of most used CO2 incubators which do not allow to control the O2 concentration. The concentration of oxygen is important because only skin and lung tissues in vivo are surrounded with air of standard composition (20-21% 02) while other organs, tissues and tumors reside in quite / very hypoxic conditions. Authors postulate that standard concentration of O2 (20-21% 02) is unnatural for majority of human tissues and in vitro cultivation of cells under such conditions induces oxidative stress which distorts the therapeutic in vivo relevance (doi: 10.1007 / s40495-016-0050-5 - “Oxidative Stress Under Ambient and Physiological Oxygen Tension in Tissue Culture"). Furthermore, O2 concentration control in vitro would mean additional costs. Therefore, more relevant and less costly option of the methodology is achieved with lung tissues. There are many in vitro models, including in vivo models of mouse xenografts, which are used to model the personalized therapeutic response of cancer treatment in vivo (DOI: 10.5772 / intechopen.89395 - “Recent Approaches Encompassing the Phenotypic Cell Heterogeneity for Anticancer Drug Efficacy Evaluation"). One of the limitations for many of those approaches is the timing, which sometimes may exceed the life expectancy of the patient of interest. By this invention, the timing is reduced to two-three weeks, depending on the number of available cells and the extent of profiling. The faster the prediction of the outcome is known, the sooner the suggestions for the patient’s treatment can be made.

[0029] The methodology is dependent on the size of the tumor sample. The authors presume that a minimum of two grams of cancerous tissue should be supplied for the method to achieve the goals of the testing. This condition should not be an obstacle in the case of lung cancer since lung cancer is usually diagnosed late and at the stage above 2, which means that the tumor is larger than 3-5 cm in diameter. Even after histopathological analysis or biobanking (which are outside of the invention) the remaining tissue should be sufficient to derive the required number of cancerous cells.

[0030] Also outside of the invention, validation in clinical settings should be made prior to proceeding with application of this model in personalized cancer therapy. No correlation data is currently available regarding the unique drug toxicity profile obtained by using this model / methodology with respect to clinical response to a certain treatment of a certain patient. Accuracy of the invention and its impact on overall patient survival or other clinical outcome cannot be evaluated without a dedicated Phase I clinical trial.

[0031] Abbreviations:

[0032] PBS - phosphate buffered saline;

[0033] FBS - fetal bovine serum;

[0034] IMDM - Iscove’s modified Dulbeco’s medium;

[0035] N2 - a mixture of supplements: Bovine Insulin, Human Transferrin, Putrescine, Selenite, and Progesterone;

[0036] EDTA - Ethylenediaminetetraacetic acid;

[0037] EGF, FGF, TGF, HGF - recombinant growth factors;

[0038] ATP - adenosine triphosphate.

[0039] Methodology:

[0040] 1. Tumor sample preparation:

[0041] 1.1 A piece of available tumor is diced / minced mechanically to the smallest possible fragments, using forceps, tweezers, scissors, blades and / or similar. 1 .21 / 2 of the mass from 1 .1 is separated, concentrated by centrifugation (1 min 1 ,000x g), and dispersed in a standard cryopreservation medium, making 2 aliquots for freezing at -80 deg. C or lower. (NOTE: It is advisable to store the samples for no shorter than until the patient finishes adjuvant therapy).

[0042] 1.3 Another1 / 2 of the mass from 1.1 is transferred to a grinding device / homogenizer, preferably Tissue Grinder TIGR (2022), or gentleMACS, or similar https: / / www.frontiersin.org / articles / 10.3389 / fmed.2022.721639 / full; https: / / www.ols- bio.de / products / tigr-tissue-arinder-and-dissociator ; https: / / www.miltenyibiotec.eom / US-en / products / gentlemacs-dissociator.html#130- 093-235 until the sample can be collected by using a pipette or a similar suction device.

[0043] 1 .4 The mass from 1 .3 is incubated with RBC lysis buffer (0.15 M ammonium chloride, 10 mM potassium bicarbonate, 0.1 mM EDTA, or similar) for 5-15 min, to remove red blood cells.

[0044] 1 .5 The mass from 1 .4 is filtered through a cell strainer with a pore size ranging from 70 to 150 pm, preferably 100 pm.

[0045] 1 .6 The cell strainer is washed with PBS, collecting the passing material into the same container.

[0046] 1 .7 The larger fragments from the top of the strainer are collected into other than 1 .5 / 1 .6 container by reverse washing with PBS or similar medium / solution, then concentrated by centrifugation, triturated and seeded in one or more separate 96-well microplates, applying media of different compositions. Such samples are further referenced as “tissue fragments”. To proceed with tissue fragments, see paragraph 7.

[0047] 1 .8 The container with the single cells and small aggregates which had passed through the cell strainer is centrifuged (1 min. 1 .OOOx g).

[0048] 1 .9 The pellet is washed once again with PBS and centrifuged (1 min. 1 .OOOx g).

[0049] 1.10 For cell counting, a small portion of the cells from 1.9 is suspended in a sufficient volume of digestive enzyme for 10 minutes to prepare a single-cell solution for cell counting. Preferably, 1 percent of the cells from 1.9 is suspended in 1 mL of 0.25% trypsin / EDTA solution for 10 minutes to prepare a single-cell solution for cell counting in an automated cell counting apparatus. (NOTE: suggestively, round-shaped objects with a diameter within the range of 10 - 40 pm should account for the cell number).

[0050] 1 .1 1 The pellet from 1 .9 is resuspended in media with supplements, which may include but are not limited to Advanced DMEM, DMEM-F12, IMDM, with or without FBS, N2, B27 additives. Preferably, the pellet from 1 .9 is resuspended in IMDM medium with N2 supplement. Such samples are further referenced as “strained cells”. Each composition of medium is further referred to as “initial medium”. (NOTE: the protocol design is calculated for approximately 10 million of strained cells. Based on the actual cell number, the protocol can be expanded or truncated by removing optional experiments.)

[0051] 2. Preparation for the enrichment:

[0052] 2.1 0.5 million cells obtained from step 1 .11 are diluted / concentrated in 1 -5 mL of IMDM supplemented with FBS additive. Preferably, 0.5 million cells from 1.1 1 are diluted / concentrated in 3 mL of IMDM supplemented with 10 % FBS.

[0053] 2.2 The magnetic beads-conjugated antibodies, the use of which allows to extract the XX marker-expressing cells from the suspension, are added.

[0054] 2.3 Antibodies against certain cellular surface XX markers are comprising one or several of but not limited to CD44, CD45, CD90, CD95, CD133, CD324, PD-L1 .

[0055] 2.4 By means of magnetic force, the beads with the cells are attracted to the wall of the vessel, allowing the XX marker-free cells to be removed from the vessel. The removed cell culture is named “XX-depleted cells”.

[0056] 2.5 The vessel from 2.4 is filled with the medium.

[0057] 2.6 Magnetic beads are removed from the labeled cells according to manufacurer’s instructions. The resulting cell culture is called “XX marker-positive cells”.

[0058] 2.7 Depleted cells are divided in two (e.g., two 15 mL conical tubes).

[0059] 2.8 XX-positive cells are transferred to one of the tubes from 2.7 and the resulting cell culture will be referred to as “XX-enriched cells”.

[0060] 2.9 Another part of 2.4 depleted cells is supplied with appropriate amount of initial medium.

[0061] 2.10 The concentration of the cells in 2.8 and 2.9 is measured as described in 1.10 and then equalized to contain no less than 40.000 cells / mL. Preferably, the concentration of the cells should not exceed 200.000 / mL.

[0062] 2.1 1 The cells are seeded into a 384-well plate to make no less than 1000 cells in 25 pL of initial medium per every single well. An example of testing design is presented in Fig. 6.

[0063] 2.12 No less than 4 wells (technical replicates) are dedicated for each control and each treatment.

[0064] 2.13 The cells are left in the humidified CO2 incubator for 16-24 hours.

[0065] 2.14 The next day, the drugs are applied, and the initial control is measured as described below in paragraph 6.

[0066] 3. Preparation of the variants: 3.1 Strained cells are divided into no less than three parts, e.g., 50-mL centrifugal tubes, but not limited to:

[0067] 3.1.1 Series No.1 is standard / control cells which will be named as “wild-type (WT) cells”, meaning they are placed in the initial medium (e.g., IMDM+FBS+N2+B27). Preferably, Series No.1 “WT cells”, are placed in the medium IMDM supplemented with 1 % N2 and antibiotics.

[0068] 3.1 .2 Series No.2 is initial medium +EGF (10 ng / mL) +bFGF (2 ng / mL).

[0069] 3.1 .3 Series No.3 is initial medium +TGFbeta (1 ng / mL) +HGF (10 ng / mL).

[0070] 3.2 One part of cells from 3.1 .1 -3.1 .3 are seeded into 384-well plates, 1000 cells in 25 uL of the Initial medium+ the specific supplement, total 0.4 million cells in 10 mL of medium.

[0071] 3.3 At least 24 hours are required before the drugs are applied.

[0072] 3.4 Another part of cells from 3.1.1 -3.1.3 are seeded into a special 96-well plate, at least 2000 cells per well in the Initial medium+ the specific supplement. Preferably, the 96- well plate is not transparent, V-shaped (conical) bottom, low-adhesion surface, sterile, with lid, capacity of no less than 100 pL.

[0073] 3.5 The plate in 3.4 is populated with cells in the way that no more than a half of the maximal working volume of the well is filled. Preferably, 5000 cells in 50 pL of specific medium are present in every single well (total 0.5 million cells in 5 mL of specific medium).

[0074] 3.6 The rest of cells from 3.1 .1 -3.1 .3 are seeded (concentrated or not) into two cell culture flasks. Preferably, the flasks are of 25 cm2 cell growth area, with cell-culture treated surface and filter caps. The number of cells should be no less than 1 million in each. Preferably, the number of cells should be 3 million in each.

[0075] 4. Collection of non-adherent cells:

[0076] 4.1 After 20-24 hours, unattached or loosely attached cells from 3.6 are collected after vigorous shaking (2-10 sec.) of the cell culture flasks. IMPORTANT NOTE: unattached or loosely attached cells from different series (3.1 .1 -3.1 .3) can be mixed, if the number of cells is very low, in order to have at least one set of floating cells.

[0077] 4.2 One flask is filled with a fresh medium. With another it may be done the same or proceed to 5.1.

[0078] 4.3 The collected suspension of floating cells is centrifuged. Preferably, centrifugation is 5 min. 500x g. The pellet is called “loosely anchored and suspension cells”.

[0079] 4.4 Supernatant is discarded, cells are resuspended in a fresh medium and plated in a 384- well plate. Preferably, 1000 cells in 25 pL of the Initial medium+ the specific supplement should be placed in every single well of the plate.

[0080] 4.5 An example of further testing design is presented in Fig. 7. reparation of the subcultures: First passage of the attached cells from one flask in step 4.2 is realized by utilizing the time-difference trypsin (or analogous digestive enzyme) digestion, in order to stratify internally different types of cells according to their adhesive properties:

[0081] 5.1.1 a short (10-30 sec.) application of the digestive enzyme. Preferably, 0.25% trypsin / EDTA solution is applied for 10 sec.

[0082] 5.1 .2 The flask is rinsed with 1 -3 mL of PBS. The solution is discarded and replaced with 1 -3 mL of fresh PBS.

[0083] 5.1 .3 The flask is placed in the shaker for 2-10 min. Preferably, the temperature of the shaker is 37 °C.

[0084] 5.1 .4 Dissociating cells during the first 2-10 min. are collected by aspirating the whole content from the flask. The collected cells are called “readily detaching”.

[0085] 5.1 .5 Right away, not allowing the flask to dry out, a full trypsinization is performed by adding fresh solution of a digestive enzyme. Preferably, 0.5-2 mL of 0.05% trypsin / EDTA solution for 2-20 min. More particularly, 1 mL of 0.05% trypsin / EDTA solution for 10 min.

[0086] 5.1.6 Trypsinization process is stopped by neutralization with FBS and / or trypsin inhibitor containing medium (3-5 mL), after visually no adherent cells are left at the bottom of the flask. Preferably, other than FBS trypsin-neutralizing agent is used.

[0087] 5.1.7 The resulting suspension of cells is centrifuged and resuspended in respective medium from 3.1 .1 -3.1 .3.

[0088] 5.1 .8 Re-adhesion of a part of the cells in 3 mL of respective medium is allowed in 1 - 2 hours. Resulting subculture is referenced as “quickly attaching cells”.

[0089] 5.1 .9 The unattached cells from 5.1 .8 are placed separately, for example into a 15 mL tube. These cells will be referenced as “slowly attaching cells”.

[0090] 5.1 .10 The cells from 4.1 (loosely anchored), 5.1.4 (readily detaching), and 5.1.9 (slowly attaching) are counted, diluted / concentrated in respective medium to the concentration of 40.000 in 1 mL and seeded in 384-well plates.

[0091] 5.1 .1 1 Plates from 5.1 .10 and 3.2, in 24 hours after seeding, are treated with selected drugs. An example of testing design is presented in Fig. 7.

[0092] 5.1.12 Final viability of the seeded cells is measured after 70-100 hours of the treatment.

[0093] 5.1.13 Quickly attaching cells from 5.1.8 should be detached from the surface as soon as possible to join the experiment described in 5.1 .10. Without a notable delay, all the plates can be treated with the drugs as well as their viability can be measured at the same time using the same working solutions.

[0094] 5.2 Another flask from 4.2 is kept in 37 °C environment to proliferate and / or differentiate for 70-100 hours. Preferably, the flask contains 5 mL of respective medium with supplements.

[0095] 6. Drug efficacy testing:

[0096] 6.1 The drugs are diluted in a cell culture medium. Preferably, the medium is IMDM medium supplemented with 10% FBS and 1 % antibiotics.

[0097] 6.2 At / before the beginning of the experiment, negative control (medium without cells) should be assessed for the signal (it can be a whole separate plate). The obtained value should be subtracted from all future results.

[0098] 6.3 For monotherapy evaluation, an equal volume of double-concentration (2x) drug is added to the plate wells, e.g., 25 pL for the 384 well which already contains cells and 25 pL of the medium, or 100 pL for the 96 well plate; by this action, the drug becomes diluted 1 :1 and the final concentration is 1 x.

[0099] 6.4 For combinational therapy evaluation, 4x concentration of the first drug is added to the plate wells, e.g., 12.5 pL for the 384 well which already contains cells and 25 pL of the medium, or 50 pL for the 96 well plate in which the cells are within 100 pL; by this action, the drug becomes diluted 1 :2 and the intermediate concentration is 1 .33x.

[0100] 6.5 For combinational therapy evaluation, in the second step, 4x concentration of the second drug is added to the plate wells, e.g., 12.5 pL for the 384 well which already contains cells and the first drug in 37.5 pL of the medium, or 50 pL for the 96 well plate in which the cells are with the first drug in 150 pL; by this action, both drugs become diluted to the final concentration 1 x.

[0101] 6.6 Proliferation controls and initial controls are supplied with medium without drugs. NOTE: the solvent control should be incorporated in the control samples.

[0102] 6.7 Common viability assay is used to obtain the viability value. Preferably, resazurin reduction method is used.

[0103] 6.8 In case resazurin is used to measure cellular viability, it is added without removing the medium.

[0104] 6.9 In case the old medium is not removed during the measurement of cellular viability, the medium within each multiplate well is mixed / triturated to ensure that the added substance is distributed equally in all the wells.

[0105] 6.10 The plate is placed into a thermostat at 37 deg. C for 1 -4 hours for the chemical reaction to take place. Then, the wells are assayed with the help of a plate reader. In case resazurin is used to measure cellular viability, the fluorescence (excitation 530-560 nm, emission 590 nm) is measured. For the drug toxicity testing, final viability of the seeded cells is measured after 70- 100 hours from the addition of the drugs. After 70-100 hours of the treatment, steps 6.7-6.11 are done. The plate must contain at least 4 technical replicates for each treatment. Assessment of 3D cell culture viability and response to the treatments: Small pieces of tumor tissue from 1 .7 are kept in 96-well plates in 200 pL of cell culture medium with or w / o supplements. Preferably, the plate is clear, tissue culture-treated and flat bottom, the medium is as described in 3.1.1 -3.1.3. The medium in plates from 7.1 is changed every 48-96 hours. Preferably, it is replaced with a fresh one every 72 hours. The timing of 3D cell culture viability depends on the rate at which the results from steps 6.13 and 5.1.12 are obtained. Preferably, the pieces of tumor tissue reside in the plate for 120-150 hours from obtaining the sample.

[0106] 7.3.1 To evaluate the effect of the treatment, viability of the tissue fragments in every single well has to be measured individually to interpret the results correctly. NOTE: the amount of tissue components can be different among the wells in the cell culture plate. However, knowing the viability level in every separate well partially resolves the issue: every analyte will have its own initial control.

[0107] 7.3.2 Analysis of drug sensitivity data from 2.1 1 , 5.1 .11 is required to select the most appropriate drugs and their combinations to be tested in 3D.

[0108] 7.3.3 After interpreting the results from 7.3.2, the medium is changed to standard medium (IMDM+FBS), 50-100 pL per well of the 96-well plate;

[0109] 7.3.4 5-10 pL of 0.1 % aqueous solution of resazurin is introduced into each well;

[0110] 7.3.5 Follow the procedure described in 6.9-6.1 1 .

[0111] 7.3.6 The obtained fluorescence value in every individual well is uniquely specific to the mass of the tissue which is within the well.

[0112] 7.3.7 The medium is carefully aspirated and discarded (NOTE: adherent tissue mass should not detach).

[0113] 7.3.8 New standard medium is applied.

[0114] 7.3.9 Drugs and / or other supplements are added as described in 6.1 -6.6.

[0115] 7.3.10 After 3-5 days, viability of individual samples within the plate is evaluated as described in 6.7-6.9. 7.3.1 1 Final viability of each sample is adjusted according to the initial measurement, described in 7.3.6.

[0116] 7.4 Cell mass in every well of the plate from 3.5 represents heterotypical spheroids which are of the same size and contain similar number of different types of cells. There is no need to measure the initial viability of every spheroid individually.

[0117] 7.4.1 Before the measurement of the initial control, other cellular aggregates are treated with selected drugs or combinations thereof. The methodology of treatments is described in 6.3-6.6.

[0118] 7.4.2 Initial control is measured in the dedicated wells by using any available 3D- applicable 1 -step viability test. Preferably, the initial control is measured by luminesce of a substrate which is activated in the presence of ATP. NOTE: the volume of the microplate with cellular aggregates might be insufficient to measure the viability directly. Therefore, it must be considered transferring the content to another luminescence-appropriate 96-well plate with a larger volume.

[0119] 7.4.3 During the initial control measurement, the rest of the plate should be covered or in other ways protected from the contamination of the cell culture to maintain the sterility.

[0120] 7.4.4 After 3-5 days, the final viability of individual samples within the plate is evaluated by using any available 3D-applicable 1 -step viability test. Preferably, the final viability is measured by luminesce of a substrate which is activated in the presence of ATP. NOTE: the volume of the microplate with cellular aggregates might be insufficient to measure the viability directly. Therefore, it must be considered transferring the content to another luminescence-appropriate 96-well plate with a larger volume.

[0121] 7.5 Results are uploaded and analyzed by using future interface and software. Most effective drugs / combinations are rated according to the decrease in viability of different variants / tissue fragments in the provided models.

Claims

CLAIMS1. A method of screening efficacy of chemical and / or biological substances to cancer cells, by means of measuring ex vivo-derived cell viability, the method comprising at least steps of:1 ) obtaining cells from a resected solid primary tumor of a patient diagnosed with non-small cell lung cancer (NSCLC);2) fractionation of the obtained cells to form subcultures different in phenotypic characteristics from the same sample;3) a feature of aggregated cell clusters is obtained from single cells;4) application of approved drugs for the treatment of non-small cell lung cancer (NSCLC) in an in-vitro model-system comprising the components:- fragments of tissue,- primary cell cultures, along epithelial-to-mesenchymal (ETM) and / or mesenchymal-to-epithelial (MET) cell cultures,- 3D aggregates.

2. The method according to claim 1 , w h e r e i n- the step of obtaining of cells, the cells are obtained without application of proteolytic enzymes or analogous proteolytic substances.

3. The method according to claim 1 , w h e r e i n the fractionation of cells is achieved by- separating adherent and non-adherent cells by means of time-resolved cellular attachment to the surface and / or application of proteolytic enzyme or analogous proteolytic substances, and- based on the specifically selected cellular marker, this marker-enriched and / or marker- depleted populations of cells are created.

4. The method according to claim 1 , w h e r e i n the aggregation of single cells into cell clusters, under the following conditions: aggregates are formed in no longer than 24 hours; aggregates are formed due to the gravitation force; aggregates are formed in microwells with conical bottom; the surface of the microwell is low-attachment; the medium contains no natural, seminatural or artificial extracellular matrix components; one aggregate comprises from 200 to 2000 cells; all aggregates are identical in shape and size.

Images