WO2024218665A1

WO2024218665A1 - Method for in vitro assessment of the chemosensitivity of neoplastic cells to antineoplastic drugs and method for ex vivo diagnostics of breast neoplasm

Info

Publication number: WO2024218665A1
Application number: PCT/IB2024/053727
Authority: WO
Inventors: Dariusz PAWLAK; Anna TANKIEWICZ-KWEDLO; Krystyna PAWLAK; Bogumiła CZARTORYSKA - ARŁUKOWICZ; Leszek KOZŁOWSKI; Tomasz ŁOBACZ; Mariusz KODA
Original assignee: Bialostockie Centrum Onkologii Im M Sklodowskiej Curie W Bialymstoku; Uniwersytet Medyczny W Bialymstoku
Current assignee: Bialostockie Centrum Onkologii Im M Sklodowskiej Curie W Bialymstoku; Uniwersytet Medyczny W Bialymstoku
Priority date: 2023-04-18
Filing date: 2024-04-17
Publication date: 2024-10-24
Anticipated expiration: 2025-10-18
Also published as: PL444462A1

Abstract

The subject-matter of the invention is a method for in vitro assessment of the chemosensitivity of neoplastic cells to antineoplastic drugs before including the latter in the therapy of a specific oncology patient, referred to by the inventors as Oncochem-Test. Another subject-matter of the invention is a method for ex vivo diagnostics of breast neoplasm.

Description

Method for in vitro assessment of the chemosensitivity of neoplastic cells to antineoplastic drugs and method for ex vivo diagnostics of breast neoplasm

Breast cancer is the most frequently diagnosed neoplasm in the world according to the WHO. It is currently diagnosed in 12% of women per year. Over the past 20 years, the total number of breast neoplasms in the world has increased by almost 100%. In 2020, 2.3 million women worldwide were diagnosed with breast cancer and 685,000 deaths were reported. In Poland, there is still a steady increase in the incidence despite additional financial outlays, medical developments, as well as increased public awareness of cancer prevention. The death rate among Polish women with breast cancer is increasing every year. It is therefore crucial to develop a novel approach to enable rapid diagnostics and targeted chemotherapy as part of personalised medicine.

It is postulated that the CD44(+)CD24(-) receptor group are important prognostic markers in breast cancer. CD24 protein expression is considered a weak prognostic marker in hormone-receptor-positive breast cancer, as opposed to CD44 expression, which is a good prognostic marker in the group of patients without hormone receptors (Kim HJ, Kim MJ, Ahn SH, Son BH, Kim SB, Ahn JH, Noh WC, Gong G. Different prognostic significance of CD24 and CD44 expression in breast cancer according to hormone receptor status. Breast. 2011 Feb;20(l):78-85).

There are also intracellular markers that allow for assessment of breast neoplasm cells. The Ki-67 parameter is indicative of the aggressiveness of the neoplasm and reflects, among other things, the rate of cell division. The higher its value, the faster the neoplastic cell divides and grows, and moreover, this higher value indicates a greater number of cells dividing. On the other hand, such cells are likely to respond better to the chemotherapy used (Inwald EC, Klinkhammer-Schalke M, Hofstadter F, et al. Ki-67 is a prognostic parameter in breast cancer patients: results of a large population-based cohort of a cancer registry. Breast Cancer Res Treat. 2013;139(2):539-552). Another parameter, GATA-3, is associated with favourable pathological features of breast cancer, including absence of lymph node metastases and positive status of oestrogen receptor (ER). Levels have also been shown to be an independent prognostic marker, with low expression being prognostic of breast cancer recurrence (Voduc D, Cheang M, Nielsen T. GATA-3 expression in breast cancer has a strong association with oestrogen receptor but lacks independent prognostic value. Cancer Epidemiol Biomarkers Prev. 2008 Feb;17(2):365-73).

Another parameter is cytokeratin 18 associated with the proliferative capacity of tumour cells. Its presence in the blood is an unfavourable factor indicating a rapid relapse of the disease (Yang J, Gao S, Xu J, Zhu J. Prognostic value and clinicopathological significance of serum- and tissue-based cytokeratin 18 express level in breast cancer: a meta-analysis. Biosci Rep. 2018;38(2):BSR20171145).

Methods for predicting the response of breast neoplasm to a proposed treatment are known in the art.

Patent EP2524232B1 discloses a method for predicting response of a triple negative breast tumour to therapy with an antineoplastic drug, comprising:

(a) lysing a tumour cell obtained from the triple-negative breast tumour to produce a cellular extract;

(b) determining the expression level of VEGFR2 in the cellular extract;

(c) comparing the expression level of VEGFR2 in the cellular extract determined in step (b) to a reference expression level of VEGFR2, wherein the presence of a low level of VEGFR2 expression compared to the reference is predictive of response to therapy with the anticancer drug, wherein the anticancer drug is a combination of bevacizumab (Avastin®), carboplatin, and paclitaxel. Additionally, c-KIT, HER1, and/or IGF-1R levels are determined in the cellular extract.

US8021831B2 discloses a method for comparing cell cycle profiles for determining the chemosensitivity of a breast cancer cell to a taxane, comprising: a) measuring a CDK1 activity level of a target breast cancer cell treated by a taxane in vitro, and a CDK2 activity level, a cyclin E expression level, a p21 expression level and a CDK6 expression level of the target breast cancer cell before the target breast cancer cell is treated by the taxane in vitro, to provide a cell-cycle profile; b) comparing the cell-cycle profile of the target breast cancer cell with a cell-cycle profile of a breast cancer cell that is resistant to the taxane or a cell-cycle profile of a breast cancer cell that is not resistant to the taxane for determining the chemosensitivity of the target breast cancer cell to the taxane.

Currently, chemotherapeutic treatment is selected based on an assessment of the patient's clinical condition, the result of histopathological examination of the material extracted during the biopsy and molecular tests aimed at assessing predispositions that only allow estimating the prognosis for patients. Methods to date do not make it possible to assess the response of cells isolated from a patient to a treatment that will be proposed in the near future. The claimed invention makes it possible to subsequently use the targeted chemotherapy with a high probability of efficacy, guaranteeing that the patient is cured with limited side effects resulting from the use of inappropriate chemotherapy. Another advantage of this invention is that it reduces the total treatment time for an oncology patient due to the use, from the very first stages of treatment, of antineoplastic drugs to which that patient's neoplastic cells show sensitivity.

The purpose of the invention was to develop a method for an in vitro assessment of the chemosensitivity of neoplastic cells obtained directly from the tumour, the method providing rapid management and a fully personalised approach to the oncology patient, and the use of this method as one of the steps in a method of ex vivo diagnostics for breast neoplasm. To date, no simple, low-cost and effective diagnostic and therapeutic test has been developed to assess the chemosensitivity of neoplastic cells to antineoplastic drugs in vitro prior to their inclusion in the therapy of a specific oncology patient. The method enables efficient isolation of neoplastic cells in the material from the patient in more than 90% of cases.

The method for the in vitro assessment of the chemosensitivity of neoplastic cells to antineoplastic drugs according to the invention comprises the following steps: a) the cells are incubated with at least one antineoplastic drug for 24 to 48 hours in the culture medium, b) after incubation, cell viability and cell morphology and lactate dehydrogenase (LDH) activity in the culture medium are assessed, wherein, if the cells show a statistically significant decrease in viability compared to the control sample containing cells not incubated with the antineoplastic drug and the morphology of the cells is impaired compared to the control sample containing cells not incubated with the antineoplastic drug and there is a statistically significant increase in lactate dehydrogenase (LDH) activity in the culture medium compared to the control sample containing cells not incubated with the antineoplastic drug, the neoplastic cells are considered sensitive to the antineoplastic drug.

Preferably, the antineoplastic drug is Doxorubicin and/or Hydroxycyclophosphamide and/or Cisplatin and/or Paclitaxel and/orTrastuzumab and/or Docetaxel or combinations thereof.

Preferably, in step b), cell viability is assessed by the Carmichael's method using tetrazole salt.

Preferably, the assessment of cell morphology in step b) is carried out using light microscopy, wherein, if a loss of cell adherent properties and/or a loss of cell-cell contact is observed, it should be assumed that the cell morphology is impaired.

Preferably, the increase in lactate dehydrogenase (LDH) activity in the culture medium is measured by measuring the extinction at a wavelength of 490 nm of the culture medium after 5 minutes of incubation in the presence of lithium lactate and a mixture containing phenazine methosulphate, iodonitrotetrazolium chloride and nicotinamide adenine dinucleotide at pH=8.

The method for ex vivo diagnostics of a breast neoplasm according to the invention comprises histopathological examination and/or immunohistochemistry test of the neoplastic tissue previously isolated from the patient, wherein the chemosensitivity of the neoplastic cells to antineoplastic drugs is additionally assessed in vitro by the method for the in vitro assessment of the chemosensitivity of the neoplastic cells to antineoplastic drugs according to the invention.

Preferably, the histopathological examination is performed by fixing a tissue fragment in paraffin and then slicing the fixed tissue fragment into at least two sections, the sections then being stained with haematoxylin and eosin, followed by microscopic observation of the stained sections in search of neoplastic cells. Preferably, the immunohistochemistry test is performed on a section of tissue and includes the following steps: a) exposing antigenic determinants, b) blocking non-specific reactions, c) using antibodies, d) using a detection system using diaminobenzidine (DAB), e) microscopic assessment.

Preferably, neoplastic cells are obtained from neoplastic tissue after it has been treated with type IV collagenase at a concentration of 1 mg/ml, at a temperature of 37°C in an atmosphere enriched with 5% carbon dioxide for 3 to 12 hours, preferably 8-12 hours, followed by isolation of the neoplastic cells by centrifugation, preferably at 1500 revolutions per minute at room temperature for 10 minutes, culturing the cells to an adherent line, incubating with trypsin and transferring the material to new medium, preferably DMEM/F-12 supplemented with 10% serum, penicillin (100 lU/ml), streptomycin (100 pg/ml) and amphotericin B (0.25 pg/ml).

The invention makes it possible to assess the sensitivity of breast cancer cells to the drugs used in a simple, minimally invasive, fast and accurate way and, on this basis, select the most effective treatment plan as part of personalised therapy. The proposed method provides effective elimination of neoplastic cells obtained from the patient under in vitro conditions and shows high efficiency. This outcome translates into a potential implementation of this method into routine therapy for breast cancer patients.

The method contributes to a reduction in the side effects observed during chemotherapy, which does not always prove to be effective. The disclosed method prevents the subsequent use of a therapy to which the neoplastic cells are not sensitive, which in prospect offers a chance to use an effective chemotherapy from the first days.

The subject-matter of the present invention is explained in the embodiments in the drawing, in which: Fig. 1 shows the steps of isolation of neoplastic cells from the biopsy specimen using collagenase

Fig. 2 shows the further stages of growth and proliferation of cells forming the adherent line

Fig. 3 shows the assessment of CD24 receptor expression on cells isolated from the biopsy specimen. A. exemplary cytogram of cells isolated from patient 16, B. graph showing the percentage of cells expressing the CD24 receptor in patient 16 and 20.

Fig. 4 shows the assessment of CD44 receptor expression on cells isolated from the biopsy specimen. A - exemplary cytogram of cells isolated from patient 16, B - graph showing the percentage of cells expressing the CD44 receptor in patient 16 and 20.

Fig. 5 shows a comparison of the CD44/24 expression coefficient in patient 16 and 20.

Fig. 6 shows the results from the flow cytometer confirming that cellular heterogeneity is preserved in the material obtained from the biopsy specimen from patient 16 (A) and patient 20 (B).

Fig. 7 shows the assessment of the expression of the Ki-67 parameter in cells obtained from the biopsy specimen (A - an exemplary cytogram of cells isolated from patient 16, B - a graph showing the level of Ki-67 expression in cells in patient 16 and 20; p=0.029). C - result of immunohistochemistry test from the biopsy specimen. The immunohistochemistry test was performed in November 2021, while cytometric analysis was performed in January 2021.

Fig. 8 shows the assessment of the expression of the GATA-3 parameter in cells obtained from the biopsy specimen A - an exemplary cytogram of cells isolated from patient 16, B - a graph showing the comparison of GATA-3 expression in patient 16 and 20; p=0.018.

Fig. 9 shows the assessment of the expression of cytokeratin in cells obtained from the biopsy specimen A - an exemplary cytogram of cells isolated from patient 16, B - a graph showing the comparison of cytokeratin expression in patient 16 and 20; p=0.013. Fig. 10 shows graphs showing the assessment of viability of cells obtained from the biopsy specimen after 24 and 48 hour incubation with drugs.*** p<0.0001 vs Kon, ^AA p<0.001 vs Doc, ^AAA p<0.0001 vs. Pac.

Fig. 11 shows morphometric assessment of cells on microscopic images; A - cells of patient 16, B cells of patient 20 (xlO) after 24 h incubation with drugs administered according to the regimen described in the text.

Fig. 12 shows assessment of neoplastic cells proliferation of patient 16 (A) and patient 20 (B)

Fig. 13 shows assessment of apoptosis by flow cytometry in cells obtained from a biopsy specimen and incubated with drugs for 24 hours in patient 16 (A) and patient 20 (B).

Fig. 14 shows assessment of lactate dehydrogenase (LDH) activity in the culture medium after 24-hour incubation of neoplastic cells from patients 16 (A) and 20 (B) with drugs.

In the figures, "P" indicates gated area with maximum event counting power.

Embodiment

Starting material

The starting material to use the method according to the invention were tissue fragments (3 core needle biopsy specimens; thickness of biopsy needle 14G - 2 mm, length 19 mm) obtained from tumour biopsies from patients 16 and 20, where in the case of each patient one tissue fragment (the third biopsy specimen) was placed in a dish with sterile DMEM/F12 medium and immediately transferred to the cell culture laboratory to obtain neoplastic cells. The remaining biopsy specimens (the first and second one) were placed in 10% buffered formalin solution and transferred to the Department of Pathomorphology for routine histopathological diagnostics.

Histopathological diagnostics

The first and second biopsy specimens were delivered to the Department of Pathomorphology immediately after extraction, then fixed in a 10% buffered formalin solution, pH 7.2-7.4, at room temperature (20-25°C), in a volume lOx the tissue volume. The fixation time was 24 hrs. After the fixation step, the first and second biopsy specimens were embedded into a paraffin block. For this purpose, they were transferred to disposable plastic histopathology cassettes marked in an unambiguous and permanent way enabling identification of the patient and the examination performed. Subsequently, the cassettes containing the tissue material were placed in an automatic tissue processor, which allowed the material to be embedded into paraffin. The time (16 hours), tissue temperature (20°C), paraffin temperature (61°C) and reagent pressure (1005 mbar) were controlled in the device. When the tissue processor was finished, the biopsy specimens were transferred to metal moulds and embedded in paraffin blocks using a paraffin machine. The blocks were then cooled and cut into sections. For this purpose, microtomes were used that made it possible to cut a section of approximately 5 pm in thickness, which was placed on a basic microscope slide.

For routine histopathological diagnostics, sections were stained with haematoxylin and eosin after de-paraffining and hydration in an alcohol gradient: 30 min in an incubator at 65°C, xylene I - 3 min, xylene 11 - 3 min, xylene III - 3 min, absolute alcohol - 3 min, alcohol 96% - 2 min, alcohol 70% - 2 min, water - 2 min, eosine - 10 s, hematoxylin - 3 min, water - 4 min, alcohol 70% - 1 min, alcohol 96% - 1 min, absolute alcohol - 1 min, xylene I - 2 min, xylene II - 1 min, xylene III - 1 min. The stained and dehydrated preparations were covered with a cover slide in a permanent manner that made long-term storage possible.

Histopathological diagnostics was carried out with the use of an optical microscope enabling magnification up to 400x.

In the case cancer was identified, immunohistochemistry tests were performed to assess predictive factors such as oestrogen receptor, progesterone receptor, HER2 receptor and Ki-67 proliferation marker. The immunohistochemistry test included the following steps: exposing antigenic determinants, blocking non-specific reactions, using primary antibodies and using detection systems using diaminobenzidine (DAB).

Antibodies for breast cancer diagnostics were used as commercially available ready solutions (Roche: Oestrogen Receptor (ER) (SP1) Rabbit Monoclonal Primary Antibody, CONFIRM, Progesterone Receptor (PR) (1E2) Rabbit Monoclonal Primary Antibody, HER-2 receptor - HER-2/neu (4B5) Rabbit Monoclonal Primary Antibody, VENTANA and proliferation marker - Ki-67 (30-9) Rabbit Monoclonal Primary Antibody, CONFIRM. For each reaction, a positive control was performed, placed on the same basic slide as the tested section. The immunohistochemistry test was performed using automatic systems (Benchmark, Ventana). After the reaction, the stained and dehydrated preparations were covered with a cover slide in a permanent manner and transferred for microscopic assessment.

Obtaining and proliferation of neoplastic cells

The third biopsy specimen immediately after extraction was placed in DMEM medium heated to 37°C and supplemented with 10% serum and penicillin (100 lU/ml), streptomycin (100 pg/ml) and amphotericin B (0.25 pg/ml). Within 60 minutes, the material was delivered to the laboratory and subjected to steps to obtain neoplastic cells. Under sterile conditions, after removal of the medium, the biopsy specimen was rinsed 3 times with a sterile PBS solution. After it was cut into 4 pieces using a sterile scalpel, the tissue fragments were placed on a sterile culture plate with DMEM/F-12 medium supplemented with 10% serum, penicillin (100 lU/ml), streptomycin (100 pg/ml) and amphotericin B (0.25 pg/ml) with the addition of collagenase type IV (1 mg/ml). The slide was placed in an incubator at a temperature of 37°C in an atmosphere enriched with 5% carbon dioxide (CO2). Under the action of collagenase, neoplastic cells, which initially remained bound in the tumour tissue, were visualised after only 3-6 hours of incubation. As the collagenase action time progressed, these cells were released from the stroma and emerged in the medium. After 8-12 hours, the cell suspension was centrifuged at 1500 revolutions per minute at room temperature for 10 minutes. After the supernatant was decanted, the cell pellet was suspended in sterile medium and transferred to a sterile culture plate with DMEIVI/F-12 medium supplemented with 10% serum, penicillin (100 lU/ml), streptomycin (100 pg/ml) and amphotericin B (0.25 pg/ml). The ability of the cells to proliferate and their morphology were assessed daily using an inverted microscope (Olympus). The culture medium was changed every 3 days. Once a confluence of 80-90% was reached, the cells were passaged with 0.25% trypsin solution with EDTA and seeded into a new culture vessel at a ratio of 1:4.

After removal of collagenase from the medium, a proliferating cell colony appeared within a week, but this time varied depending on the type of neoplasm and the age of the patient. The obtained cells formed an adherent line (Fig. 1).

Verification of the presence of neoplastic cells Assessment of the expression of extracellular markers - CD24 and CD44 receptors on neoplastic cells by flow cytometry

In order to verify neoplastic cells by flow cytometry, the expression of CD24 and CD44 receptors was assessed. Breast cancer cells in their logarithmic phase of growth were used for flow cytometry analysis. To detach them from the substrate, the cells were treated with 0.25% trypsin, washed with PBS three times and then resuspended in 100 ul PBS, followed by incubation with anti-CD44- BD Horizon™ BB515 and anti-CD24-BD Horizon™ BV510 antibodies at room temperature for 40 min. The samples were then washed three times with PBS and finally resuspended in 200 pl PBS. Flow cytometry analysis was performed on BD FACSCanto II (BD Biosciences Systems). The ratio of CD44 and CD24 expression (CD44/CD24) in breast cancer cells from different patients was calculated from the percentage of CD44 and CD24 positive subpopulations in the flow cytometry analysis. Expression levels in both patients 16 and 20 were at comparable levels of 94.86±1.65% and 96.75±0.34%, respectively (Fig. 2). The expression of the CD44 receptor in patient 16 was 88.49 ±2.57%, in patient 20 it was 97.9±0.64% (Fig. 3). Comparison of CD44/24 expression coefficient showed no significant differences between patients included in the study. This coefficient was 1.09±0.05 for patient 16 and 0.99±0.01 for patient 20 (Fig. 4). Based on the cytometric analysis, it is clear that breast cancer cells were present in the material extracted from the tumour.

Assessment of the expression of intracellular markers

In addition, in order to confirm the presence of neoplastic cells in the isolated material, the expression of intracellular markers was assessed before incubation of the cells with the drugs. The expression of Ki-67, cytokeratin, GATA3, as well as Annexin V was assessed by flow cytometry. Flow cytometry analysis was performed on breast cancer cells in their logarithmic phase of growth. After digesting with 0.25% trypsin and washing with PBS three times, the cells were resuspended in 100 pl PBS, and then stained with anti-Ki-67-BD Horizon™ BV421, anti-GATA3-PE-CyTM7, and anti-cytoceratin-Alexa Fluor 647 at room temperature for 40 min. The samples were then washed three times with PBS and finally resuspended in 200 pl PBS. The analysis was performed on BD FACSCanto II flow cytometer (BD Biosciences Systems). The ratio of CD44 and CD24 expression (CD44/CD24) as well as Ki-67, cytokeratin and GATA3 in breast cancer cells from different patients was calculated from the percentage of cell subpopulations positive for Ki-67, GATA3 and cytokeratin in the flow cytometry analysis. In the cells obtained from the biopsy specimen, the expression of Ki-67 differed statistically significantly (p=0.0294) between patients and was 53.116.0% for patient 16 and 33.015.4% for patient 20 (Fig. 7AB). The obtained results are similar to the immunohistochemistry results performed three months earlier and amounting to 70% and 15%, respectively (Fig. 7C). The observations once again confirm the presence of neoplastic cells in the conducted in vitro tests, and furthermore, based on the similarity of the obtained results from cytometric analysis and immunohistochemistry, they indicate in vitro stability of the cell lines isolated from tumours within a period of 3 months.

The second intracellular marker assessed in vitro was GATA-3. The expression of GATA-3 in patient 16 was statistically significantly higher (p=0.018) and was 52.215.5% compared to patient 20 (32.2±4.7%) (Fig. 8).

Another intracellular marker assayed was cytokeratin, which is an insoluble structural protein of epithelial cells. The expression of cytokeratin in patient 16 was statistically significantly higher (p=0.013) and was 53.16±6.03% compared to patient 20, in which it was 31.2714.38% (Fig. 9). This shows that there were more cancer cells in the sample from patient 16.

Confirmation of cellular heterogeneity

Since the growing tumour in the patient's body is a heterogeneous formation, it is important to maintain such an environment as reliably as possible in in vitro tests. In order to assess the analysis of these conditions, the samples were subjected to cytometric analysis. It was observed that three cell lines were present in both the material from patient 16 and patient 20. The obtained results indicate that cellular heterogeneity was maintained in both cases, and therefore all in vitro tests were carried out under conditions as close as possible to those prevailing in the oncology patient's body (Fig. 6).

Assessment of cell viability after incubation with drugs used for the treatment of breast cancer

Neoplastic cells from the third biopsy specimen, which formed an adherent colony and reached a confluence of about 90%, were subjected to passaging. For this purpose, from the removal of the culture medium, the cells were rinsed 3 times with a solution of PBS without Mg²⁺ and Ca²⁺ ions. Then, 0.25% trypsin was added in the amount of 500 pl per 10 cm² plate, in order to detach the cells from the medium and seed them onto a new plate. After the cells were incubated for 7 minutes at a temperature of 37°C, 5 ml of DM EM -Fl 2 culture medium was added, everything was transferred to a sterile tube and centrifuged at 2,500 revolutions/min at room temperature. For reproducibility of results, cells from one 10 cm² plate were seeded into one 24-well plate. After seeding the cells onto a new plate, the confluence of the cells at the 'start' time was approximately 40%. On the new plate, the cells were incubated in an incubator at a temperature of 37°C for 24 and 48 hours with drugs used in breast cancer patients according to the following regimens (the volume of drug added is 50 pl per 1 ml of DMEM-F12 medium):

1. Doxorubicin (4 pM Doxorubicin-Ebewe) + (1 pM) Hydroxycyclophosphamide (active metabolite of cyclophosphamide (Endoxan))

2. Cisplatin (20 pM Cisplatin-Ebewe)

3. Paclitaxel (2 pM Paclitaxel-Ebewe)

4. Paclitaxel (2 pM Paclitaxel-Ebewe) + Trastuzumab (10 pg/ml Herceptin)

5. Docetaxel (1 pg/ml Docetaxel Accord)

6. Docetaxel (1 pg/ml Docetaxel Accord) + Trastuzumab (10 pg/ml Herceptin)

After 24 and 48 hours of incubation, cell viability was assessed using the Carmichael's method, with cell viability being determined using tetrazolium salt (MTT). In viable cells, under the influence of mitochondrial dehydrogenases, the applied stain was transforming to purple formazan. This transformation did not occur in dead cells. To assess the cytotoxic properties of the tested drugs, cells were seeded in 24-well plates and then DMEM/F-12 media supplemented with 10% serum, penicillin (100 lU/ml), streptomycin (100 pg/ml) and amphotericin B (0.25 pg/ml) were added in a volume of 1 ml. After 24 hours, the medium was changed and drugs were added according to the above regimen. The cells were incubated for 24 and 48 hours. After this time, an MTT solution with a final concentration of 2 mg/ml was added to each well and the cells were incubated for 4 hours in an incubator at a temperature of 37°C under 5% CO2 enriched atmosphere. After this time, the culture medium was removed from above the cells and 200 pl of dimethyl sulfoxide was added to each well to dissolve the resulting formazan crystals. Absorbance was read using a microplate reader (Bio- Tek Instruments) at a wavelength of 570 nm. The degree of cell viability was assessed against the control (cells treated in the same way, i.e. seeded, grown under identical conditions, except that for 24 and 48 hours they were incubated in parallel with the test culture, but without the addition of drugs), in which the viability was 100%.

There was a statistically significant reduction in the viability of cells from patient 16 after incubation with all drugs for each time interval. However, after 24 hours of incubation, this effect was most strongly observed after exposure of cells to docetaxel (Doc) (57.3% vs 100% in control; p<0.0001), doxorubicin with cyclophosphamide (Dox+Cyclo) (63.2% vs 100% in control; p<0.0001), docetaxel with trastuzumab (Doc+Tras) (64.0% vs 100% in control; p<0.0001), paclitaxel with trastuzumab (Pac+Tras) (65.2% vs 100% in control; p<0.0001), paclitaxel (Pac) (74.8% vs 100% in control; p<0.0001) (Fig. 10A).

In the case of patient 20 after 24 hours of incubation, the reduction in viability was most strongly observed after exposure of cells to docetaxel with trastuzumab (Doc+Tras (49.7% vs 100% in control; p<0.0001), docetaxel (Doc) (54.6% vs 100% in control; p<0.0001), doxorubicin with cyclophosphamide (Dox+Cyclo) (64.2% vs 100% in control; p<0.0001), paclitaxel with trastuzumab (Pac+Tras) (65.2% vs 100% in control; p<0.0001), paclitaxel (Pac) (74.1% vs 100% in control; p<0.0001) (Fig. 10B).

Assessment of the effect of drugs on the morphology of neoplastic cells

Assessment of the morphology of drug-treated neoplastic cells under visible light provides only a preliminary opportunity for cytotoxic drug selection. However, when supported by the results of other tests, it makes it possible to select the drugs that are most beneficial for the oncology patient. Incubation of cells from patients 16 (Fig. 11A) and 20 (Fig. 11B) resulted in loss of adherent properties and cel l-to-cel I contact after exposure to paclitaxel (Pac), paclitaxel with trastuzumab (Pac+Tras), docetaxel (Doc) and docetaxel with trastuzumab (Doc+Tras). On the other hand, cisplatin (Cis) or doxorubicin with cyclophosphamide (Dox+Cyclo) did not impair the morphology of the neoplastic cells.

Assessment of the apoptosis process

In order to assess the chemosensitivity of the cells, apoptosis was tested using the fluorescein isothiocyanate-labelled annexin V method (Annexin V-FITC), which forms complexes with phosphatidylserine in the presence of calcium ions. This test was carried out using a FITC Annexin V Apoptosis Detection Kit II (BD Biosciences, USA) and a BD FACSCanto II flow cytometer (BD Biosciences Systems, San Jose, CA, USA). The results were analysed using the FACSDiva program (BD Biosciences Systems, San Jose, CA, USA). Cells were incubated for 24 hours in an incubator at the temperature of 37^C in a 5% CO2 atmosphere with drugs according to the previous regimen. Two control tests, i.e. one positive and one negative, were performed to calibrate the instrument. The positive control was a standard of control cells in which apoptosis was induced by the addition of 3% formaldehyde. Three control trials were performed. The first one contained control cells and propidium iodide, the second one contained control cells and V-FITC annexin, and the third one contained control cells and propidium iodide and V-FITC annexin. After the addition of 2 pl of 3% formaldehyde, the cells were placed in the refrigerator for 10 minutes. Negative control were control cells that were not exposed to any factor. Cells from control trials and cells treated with the test compounds (1 x 10⁶ cells/ml), suspended in the buffer included in the kit, were further tested. 200 pl of the suspension was taken from each test sample and transferred to test tubes. 3 pl of V-FITC annexin and 5 pl of propidium iodide were added to each sample at room temperature and placed in a dark place for 15 minutes. The thus prepared cell suspension was analysed within one hour in a BD FACSCanto II flow cytometer using BD FacsDiva software. The results of the described experiment can be defined as follows: viable cells (double negative sample - no reaction with annexin V and PI), early apoptosis (positive sample with annexin V and negative sample with PI), late apoptosis (positive sample with annexin V and positive sample with PI) and necrosis (negative sample with annexin V and positive sample with PI).

After a 24-hour incubation of breast cancer cells with chemotherapeutics, their effect on the induction of apoptosis was assessed. It has been shown that all drugs used, as a result of their action, lead to programmed cell death. The strongest apoptosis was observed after incubation of the cells with paclitaxel (Pac) in patient 16. In this group, early apoptosis was 433.3% of control, late apoptosis was 2750.0% of control, necrosis was 66.7% of control, and viable cells were 79.7% of control. In addition to paclitaxel, in this patient it would be possible to use doxorubicin with cyclophosphamide (Dox+Cyclo) (early apoptosis 233.3% of control, late apoptosis 1650.0% of control, necrosis 166.7% of control, viable cells 90.7% of control), paclitaxel with trastuzumab (Pac+Tras) (early apoptosis 233.3% of control, late apoptosis 1850.0% of control, necrosis 133.3% of control, viable cells 90.3% of control) and docetaxel with trastuzumab (Doc+Tras) (early apoptosis 245.2% of control, late apoptosis 1650.0% of control, necrosis 166.7% of control, viable cells 90.1% of control). However, due to the high percentage of viable cells (94.9% of control), patient 16 should not be treated with cisplatin (Cis) (early apoptosis 192.9% of control, late apoptosis 600.0% of control, necrosis 133.3% of control, viable cells 90.7% of control) or docetaxel (Doc) alone (early apoptosis 178.6% of control, late apoptosis 450.0% of control, necrosis 200% of control) (Fig. 13A).

In contrast, a 24-hour incubation of breast cancer cells obtained from a biopsy specimen from patient 20 showed the greatest increase of apoptosis caused by doxorubicin with cyclophosphamide (Dox+Cyclo) (early apoptosis 136.7% of control, late apoptosis 153.8% of control), necrosis (900% of control). Viable cells represented only 49.9% of the control. In this patient, when selecting chemotherapy, additional drugs should be considered such as paclitaxel (Pac) (early apoptosis 151.1% of control, late apoptosis 144.2% of control, necrosis 200% of control, viable cells 48.4% of control), paclitaxel with trastuzumab (Pac+Tras) (early apoptosis 154.1% of control, late apoptosis 140.2% of control, necrosis 100% of control, viable cells 48.6% of control), doxorubicin (Dox) (early apoptosis 154.1% of control, late apoptosis 120.5% of control, necrosis 100% of control, viable cells 59.1% of control), as well as doxorubicin with cyclophosphamide (Dox+Cyclo) (early apoptosis 147.4% of control, late apoptosis 134.9% of control, necrosis 100% of control, viable cells 55.3% of control). Due to the high percentage of viable cells (74.7% of control), this patient should not be treated with cisplatin (Cis), despite the increased process of apoptosis (early apoptosis 121.5% of control, late apoptosis 125.3% of control) and necrosis (200% of control) (Fig. 13B).

Based on the tests carried out, it is clear that the assessment of apoptosis in cells obtained from the patient is useful in selecting the drugs with the strongest anti- neoplastic effect and, therefore, with the most effective anti-neoplastic effect.

Assessment of cell proliferation

In order to assess proliferation, a CFSE test was performed using a commercial BD Biosciences kit enabling the detection of multiple generations of neoplastic cells by flow cytometry. In this method, the non-fluorescent compound diffuses passively through cell membranes and then is cleaved by intracellular esterases in viable cells. The cleaved stain becomes highly fluorescent and binds covalently to the amino groups of proteins in the cells via the ester group of succinimidyl. As cells divide, the CFSE stain disperses evenly across the daughter cells. Each daughter cell retains approximately half the fluorescence intensity of its parent cell.

To this end, breast cancer cells were suspended in a 200 pl PBS solution and then subjected to staining with CFSE reagent for 15 minutes at room temperature. After this time, the cells were washed with PBS solution and then suspended in DMEM/F-12 medium. Cells prepared in this way were seeded into a 6-well plate and immediately treated with antineoplastic drugs according to an agreed regimen. After a 24-hour incubation, the cells were detached, incubated with propidium iodide and subjected to cytometric analysis.

The cytometric test showed differences in the proliferation of cells treated with a number of antineoplastic drugs. This made it possible to exclude from the patient's future therapy drugs that do not inhibit or slow down proliferation; on the other hand, it identified drugs to which the patient's neoplastic cells show sensitivity.

In the case of patient 16, there was a fourth cell generation only in the cells not exposed to drugs, which clearly indicates persistent proliferation. All the drugs used in the tests inhibited cells in the third generation, which demonstrates their potential efficacy in oncology treatment. However, the strongest effect of inhibiting proliferation was observed after exposure of cells to doxorubicin with cyclophosphamide (Dox+Cyclo) (Fig. 12A, Table 1).

Table 1.

In patient 20, the fourth generation appeared after exposure of cells to docetaxel with trastuzumab (Doc+Tras), while in the case of the control group or other antineoplastic drugs, proliferation stopped in the third generation. This indicates that the use of an immunomodulating drug, such as trastuzumab, increases the proliferation of neoplastic cells in this patient. Also, the addition of trastuzumab (Pac+T ras) to paclitaxel intensifies this effect, which is consistent with the adopted assumption (Fig. 12B, Table 2). Table 2.

Assessment of LDH activity in culture medium

Lactate dehydrogenase (LDH) is an intracellular enzyme that under physiological conditions does not get out of the cell. When a cytotoxic agent is active, there is an increase in the activity of this enzyme in the extracellular environment due to damage to the continuity of the cell membrane.

The experiment evaluated the activity of this enzyme in the extracellular environment, i.e. the culture medium, after a 24-hour incubation of cancer cells with antineoplastic drugs. To assess LDH activity, 50 pl of 200 mM TRIS, pH 8, 50 pl of 50 mM lithium lactate and 50 pl of a mixture containing 100 pl of phenazine methosulphate (by dissolving 0.9 mg in 100 pl of water), 100 pl of iodonitrotetrazolium chloride (3.3 mg dissolved in 100 pl DMSO) and 2.3 ml nicotinamide adenine dinucleotide (by adding 8.6 mg of NAD to 2.3 ml of water) were added to 50 pl of culture medium. The mixture was then incubated for 5 minutes at room temperature, after which the extinction was read at a wavelength of 490 nm.

In patient 16, the highest LDH activity was observed after exposure of cells to paclitaxel (Pac). Other drugs with potentially high cytotoxicity are also doxorubicin with cyclophosphamide (Dox+Cyclo), paclitaxel with trastuzumab (Pac+Tras) and docetaxel with trastuzumab (Doc+Tras) (Fig. 14A).

In patient 20, on the other hand, the greatest cytotoxicity was observed after incubation of cells with paclitaxel and trastuzumab (Pac+Tras) and cisplatin alone (Cis). Another therapeutic option, but with a slightly weaker cytotoxic effect, is the use of paclitaxel (Pac) in monotherapy (Fig. 14B). The results of the assay of LDH activity in the culture medium make it possible to select the drugs with the greatest cytotoxicity against each patient's neoplastic cells.

The conclusion from the tests is that an MTT assay, morphological assessment of neoplastic cells and LDH activity in the culture medium after exposure of the neoplastic cells to the drugs is sufficient to select effective drugs. This approach will reduce the time of the test and its cost.

On the basis of these tests, it is possible to select the drug(s) to which patients' neoplastic cells show the greatest sensitivity. Both in the MTT test, in the microscopic assessment and in the assessment of LDH activity, the greatest antineoplastic activity was shown by Doc+Tras in patient 16 and Pac and Pac+Tras in patient 20.

Table 3. Criteria for drug selection based on chemosensitivity assessment.

Drug names in bold indicate their high cytotoxic efficacy against breast cancer cells.

Both patients received chemotherapy according to the chemosensitivity observed in vitro.

References:

1. Inwald EC, Klinkhammer-Schalke M, Hofstadter F, et al. Ki-67 is a prognostic parameter in breast cancer patients: results of a large population-based cohort of a cancer registry. Breast Cancer Res Treat. 2013;139(2):539-552 Yang J, Gao S, Xu J, Zhu J. Prognostic value and clinicopathological significance of serum- and tissue-based cytokeratin 18 express level in breast cancer: a meta-analysis. Biosci Rep. 2018;38(2):BSR20171145

Claims

1. The method for in vitro assessment of the chemosensitivity of neoplastic cells to antineoplastic drugs characterised in that it comprises the following steps: a) the cells are incubated with at least one antineoplastic drug for 24 to 48 hours in the culture medium, b) after incubation, cell viability and cell morphology and lactate dehydrogenase (LDH) activity in the culture medium are assessed, wherein, if the cells show a statistically significant decrease in viability compared to the control sample containing cells not incubated with the antineoplastic drug and the morphology of the cells is impaired compared to the control sample containing cells not incubated with the antineoplastic drug and there is a statistically significant increase in lactate dehydrogenase (LDH) activity in the culture medium compared to the control sample containing cells not incubated with the antineoplastic drug, the neoplastic cells are considered sensitive to the antineoplastic drug.

2. The method of claim 1, characterised in that the antineoplastic drug is Doxorubicin and/or Hydroxycyclophosphamide and/or Cisplatin and/or Paclitaxel and/or Trastuzumab and/or Docetaxel or combinations thereof.

3. The method of claim 1, characterised in that in step b), cell viability is assessed by the Carmichael's method using tetrazole salt.

4. The method of claim 1, characterised in that the assessment of cell morphology in step b) is carried out using light microscopy, wherein, if a loss of cell adherent properties and/or a loss of cell-cell contact is observed, it should be assumed that the cell morphology is impaired.

5. The method of claim 1, characterised in that the increase in lactate dehydrogenase (LDH) activity in the culture medium is measured by measuring the extinction at a wavelength of 490 nm of the culture medium after 5 minutes of incubation in the presence of lithium lactate and a mixture containing phenazine methosulphate, iodonitrotetrazolium chloride and nicotinamide adenine dinucleotide at pH=8.

6. The method for ex vivo diagnostics of a breast neoplasm comprising histopathological examination and/or immunohistochemistry test of the neoplastic tissue previously isolated from the patient, characterised in that the chemosensitivity of the neoplastic cells to antineoplastic drugs is additionally assessed in vitro by the method as defined in one of claims from 1 to 5.

7. Method for ex vivo diagnostics of breast neoplasm according to claim 6 characterised in that the histopathological examination is performed by fixing a tissue fragment in paraffin and then slicing the fixed tissue fragment into at least two sections which are then stained with haematoxylin and eosin, followed by microscopic observation of the stained sections in search of neoplastic cells.

8. Method for ex vivo diagnostics of breast neoplasm according to claim 6 or 7 characterised in that the immunohistochemistry test is performed on a section of tissue and includes the following steps: a) exposing antigenic determinants, b) blocking non-specific reactions, c) using antibodies, d) using a detection system using diaminobenzidine (DAB), e) microscopic assessment.

9. The method of claim 6 characterised in that the neoplastic cells are obtained from neoplastic tissue after it has been treated with type IV collagenase at a concentration of 1 mg/ml, at a temperature of 37°C in an atmosphere enriched with 5% carbon dioxide for 3 to 12 hours, preferably 8-12 hours, followed by isolation of the neoplastic cells by centrifugation, preferably at 1500 revolutions per minute at room temperature for 10 minutes, culturing the cells to an adherent line, incubating with trypsin and transferring the material to new medium, preferably DMEM/F-12 supplemented with 10% serum, penicillin (100 lU/ml), streptomycin (100 pg/ml) and amphotericin B (0.25 pg/ml).