DE102016104212A1 - Method for suitable therapy and detection methods in lung cancer - Google Patents

Abstract

The invention relates to a method for determining suitable therapy and / or detection methods and / or substances for or against the onset of cancer, wherein
• Migrating cancer cells are selected and enriched from patient or cell culture samples or
• exposing healthy tissue (s) to a cancer cell-producing medium (chemical / physical) and / or causing tumor cells to migrate into healthy tissue to form new pre- or neoplastic cells / clusters - as evidence of the neoplastic potential of the cells .
• exposing this "infiltrated" tissue to various therapeutic and / or detection methods and / or materials; and
• determining which treatment and / or detection method and / or substance is most suitable for the therapy and / or detection of this cancer.

Description

The invention relates to a method for determining suitable therapy and detection methods and / or substances against cancer.

State of the art

In medicine, cancer is referred to as a malignant tissue neoplasm (neoplasia) or a malignant (malignant) tumor (cancerous tumor, syn. Malignant). In the narrower sense, this means both the malignant epithelial tumors (carcinomas) and the malignant mesenchymal neoplasms (sarcomas). Furthermore, malignant hemoblastoses are referred to as cancer, such as leukemia as "blood cancer". Benign (benign) neoplasms are not cancerous; This is a tissue proliferation or mass in the body that does not form metastases. Thus, benign neoplasms are e.g. Neoplasms of body tissue due to dysregulation of cell growth.

Benign tissue neoplasms, such as papillomas and lipomas, are not referred to in the jargon as cancer, although they may degenerate, disrupting the function of vital organs. In general jargon, cancer is the collective term for body cells that grow uncontrollably, divide, displace and destroy healthy tissue. Cancer has different triggers that ultimately lead to disruption of the genetically regulated balance in terms of cell cycle (growth and division) and cell death (apoptosis) (Wikipedia).

The term cancer, according to the present invention, is to be understood as meaning any form of uncontrolled growth of the cells of the body when it is triggered spontaneously by genetic transformation, by any active substance or by external, comprehensible influences.

By way of example, the present invention relates to squamous cell and adenocarcinoma of the lung, and more particularly to non-small cell lung cancer (NSCLC, non-small cell lung carcinoma). 80% of all patients with this proven lung carcinoma have a survival rate of only 5 years.

Whenever possible, pre-operative and post-operative chemotherapy for the treatment of recurrence is commonly used today after surgical removal of the tumor. That is, chemotherapy is used here to prevent a relapse. This is to prevent possible non-distant or emigrated tumor cells from re-forming a tumor.

The progression model for this disease is the gradual transformation of non-specific metastatic and dysplastic changes into carcinoma in situ (CIS), which can lead to invasive cancer. As carcinoma in situ, the early stage of an epithelial tumor is described without invasive growth. The term metaplasia refers to the transformation of a differentiated tissue type and dysplasia to the change of cells through atypical growth processes.

Investigations of Pipinikas et al. (2014) as well as Baccelli and Trump (2014) On biopsy material, in contrast to the classical notion of metastasis formation, tumor cells migrated into normal healthy pulmonary epithelial cell layers to form new pre- and neoplastic cell clusters. Cell clusters are understood to mean cell aggregates of more than two cells, and such cells are often genetically modified. Therefore, compared to the primary tumor, there may be a different sensitivity of the cells to the applied chemotherapeutic agents. Targeted in vitro studies with a selection of cytostatic drugs could lead to successful treatment of the patient if the cell or cell cluster is positive.

The detection of such cells, also called "sleeper cells", and their selective enrichment can become the basis for individual treatment methods. Cell-based monitoring of patient material for such cells would then be the basis for developing a prediction test to estimate the risk of recurrence. Such a test could then be offered as a diagnostic service (e.g., in clinics) for targeted treatment of the subject patient with pre-tested chemotherapeutic agents. This approach would also be a way to develop new drugs to treat migrating neoplastic cells.

So far, the cancer cells had to be isolated first in order to carry out appropriate tests with them, such as the examination of the reaction to certain chemotherapeutic agents to change the therapy concept or even the medication. The disadvantage of such an approach is that one must first examine the cells of the patient's original tumor, but not the emigrated cancer cells responsible for the recurrence.

In addition, the method of selection and accumulation of cancer cells can also be applied to degenerate cells that have arisen through chemical or physical action.

task

The establishment of such an innovative screening method for cancer patients (especially NSCLC patients), as a diagnostic service to support the pre- and postoperative chemotherapy in a recurrence is the object of the present invention.

Furthermore, the selection and accumulation of cells, which have arisen by chemical and / or physical treatment, an important task to detect the carcinogenic potential of the active medium.

Solution of the task

• • Zum einen wird gesundes Gewebe einem Medium (chemisch/physikalisch) das Krebszellen erzeugt (Induktion von Tumorzellen) ausgesetzt, um dessen krebserzeugendes Potential zu erkennen.
• • Zum anderen wird weiterhin geprüft, ob die Tumorzellen in gesundes Gewebe migrieren und dort neue prä- oder neoplastische Zellen/Zellcluster bilden.
• • Anhand dieser Zell-basierten Ansätze wird ermittelt, welche Therapie- und Erkennungsmethoden sich für das jeweilige Medium beziehungsweise für das betroffene Patientenmaterial zur Behandlung und Erkennung des vorliegenden Tumortyps am besten eignen.

To solve these tasks are two approaches:

• • On the one hand, healthy tissue is exposed to a medium (chemical / physical) that causes cancer cells (induction of tumor cells) to recognize its carcinogenic potential.
• • On the other hand, it is still being examined whether the tumor cells migrate into healthy tissue and form new pre- or neoplastic cells / cell clusters there.
• • These cell-based approaches are used to determine which therapy and detection methods are most suitable for the respective medium or patient material for the treatment and detection of the tumor type.

Cell-based monitoring of patient material for such tumor cells is the basis for the development of a prediction test to estimate the risk of recurrence. Such a test may then be offered as a diagnostic service (e.g., in clinics) for the potential improvement of chemotherapy. The pharmaceutical industry could then use the proposed approach to the development of new chemotherapeutic agents for the treatment of migrating neoplastic cells.

It has been demonstrated that the tumor cells can be selectively enriched in tissue culture in a new in vitro long-term culture procedure and can test for the efficacy of various chemotherapeutic agents or specific detection methods on them. The determination of the effective individual chemotherapy spectrum, as a service for clinics, serves to optimize a successful treatment of the affected patients. The enrichment of tumor cells in a computer-controlled long-term cultivation system (Cultex ^® LTC module) provides the opportunity to use a standardized culture method routinely used for diagnosis and treatment.

For the pharmaceutical industry, the described procedure is likely to be of great interest, especially if the development of new chemotherapeutic agents for genetically modified tumor cells is required. A new innovative application would be possible with this experimental approach, even for chemically and / or physically induced tumor cells.

Various solutions of the present invention will now be described by way of example with reference to the use of bronchial epithelial cells:
Initially, the isolation of bronchial epithelial cells from various NSCLC patients and the establishment of 2D cultures from these isolated cells. The isolation of bronchial epithelial cells or their progenitor cells takes place from clinical samples which originate both from tumor-related as well as "healthy" tissue portions (surgical material of tumor patients).

The procedure is applied using established isolation control (standard operating procedures - SAAs). As a rule, three weeks are required for the isolation, whereby the applied cell cultures are checked daily for their cell growth and progress is documented. The cell populations are then characterized (immunohistochemical characterization and genetic studies of the isolated cells).

A second process step is the creation of differentiated 3D cultures from isolated bronchial epithelial cells. From these, so-called ALI (Air Liquid Interface) cultures are produced, which grow on microporous membranes and are supplied basally via the membrane pores with nutrients. Apical they are - in analogy to the lungs - in contact with the surrounding atmosphere or respiratory air. ALI cultures means that such cells are cultured directly at the air-liquid interface. If the cell samples contain neoplastic cells or cell clones, they are usually found only in small numbers in a total population and therefore escape routine cell controls. This also applies to cell cultures that have been exposed to a chemical or physical medium.

Especially under the ALI conditions, after an incubation period of about 28 days, the cells form pseudostratified, differentiated 3D constructs, i.e., the cultures now show cilia-bearing as well as mucus-producing cells, comparable to the in vivo state of organization and structure. In vivo means: the morphology and physiology of the cells is comparable to the findings in the living organism.

In the third step, the selection and enrichment of neoplastic cells / cell clusters is carried out with a long-term culture method that is especially suitable for these questions. After an incubation period of 14 days, that is, after the complete installation of the differentiated cells, the cell cultures can be brought at the optimum nutrient supply for at least another 14 days in the Cultex ^® LTC module for a computer-controlled long-term cultivation. Under such conditions, then, selection and accumulation of possible neoplastic cells occurs. This is due to the fact that normal epithelial cells do not proliferate due to their advanced differentiation, whereas omnipotent neoplastic cells, with appropriate nutrient supply, show an active division activity and accumulate in the basal and middle cell layers. For cells that have been treated with a chemical and / or physical medium, this means that one can detect a carcinogenic potential for the active medium.

A maximum of 24 ALI cultures can be accommodated in a CULTEX ^® LTC module and a maximum of 4 modules in a corresponding incubator. The use of this technique allows both the detection of migrating tumor cells as well as a targeted examination of enriched tumor cells for their sensitivity to selected chemotherapeutic agents.

The fourth and now final procedural step is the screening of chemotherapeutic agents. 3D cultures of the patient samples are now treated with the prerequisite of enrichment of neoplastic cells, based on the patient-specific treatment concept with chemotherapeutic agents. The cell cultures are finally assessed histologically and changes are documented photographically.

Such studies provide insight into whether the therapeutics used on the cells are effective, or whether a new treatment strategy needs to be developed for the patient to minimize the risk of recurrence.

Similarly, this applies to chemically / physically induced tumor cells, i. their sensitivity to appropriate detection and therapy methods can also be checked or determined here.

According to the invention, the method described should, of course, be used not only in bronchial epithelial cells, but in as far as possible all cell and tissue structures of a living being in which uncontrolled growth of cells takes place.

figure description

Further advantages, features and details of the invention will become apparent from the following description of preferred embodiments and with reference to the drawings; these show in

isolated primary bronchial epithelial cells from a patient sample (resection; ) grown for 28 and 38 days under conventional (IC: incubator cultures) and long-term culture conditions (LTC - long-term cultivation in the LTC-C module) at the air-liquid interface (ALI). Magnification: 630x.

Isolated bronchial epithelial cells of a patient sample after 23 days of cultivation at the air-liquid boundary layer (ALI) under static conditions in the incubator (manual medium change) as a prerequisite for a comparable in vivo differentiation. A. Hematoxylin / eosin stained section of culture. The upper cell layer consists of cilia-bearing and mucus-producing cells, comparable to normal bronchial epithelium of the lung. B. and C. Immunohistochemistry of cytokeratin 13 (CK13) positive cells as markers of pre-neoplastic cells. The cultures show in addition to unremarkable epithelial sections (B.) in the lower and middle cell layers CK13 positive cells. Magnification: 630x.

Histological section of 3D cultures of isolated primary bronchial epithelial cells which, after culturing in the computer-controlled long-term culture (LTC-C module with dynamic medium exchange circulation and mixing), show a selection and accumulation of pre-neoplastic cells (CK13 positive cells). Magnification: 630x.

Intermediate cell metaplasia in cell cultures of human primary bronchial epithelial cells treated with benzo (a) pyrene in non-toxic concentrations during a 43-day culture period for the first 3 weeks. The cells were immunohistochemically labeled with CK13 antibodies to demonstrate the selection and accumulation of metaplastic cells in the LTC cultures after treatment with a carcinogenic substance. Magnification: 630x.

According to Histological sections of 3D cultures established from isolated primary bronchial epithelial cells of a resection sample are shown, where imaging part A represents the original tissue of the patient sample and imaging parts B-E the establishment.

The cultures were for this under conventional conditions in the incubator, also abbreviated IC called, and parallel under Long-term culture conditions, cultured in a Cultex ^® LTC module for 28 or 38 days, as a condition for a comparable in vivo differentiation. The enlargement of the figure has the factor 630.

By means of the above-described technology for long-term cultivation at the air-liquid boundary layer, ie ALI cultures, both normal and genetically modified cells of the respiratory tract of humans, such as e.g. Bronchial cells are bred in such a way that they have histologically as well as immunohistologically very high homology with the natural bronchial mucosa.

In the For example, an ALI culture of isolated bronchial epithelial cells after a culture period of 23 days at the air-liquid interface is shown under conventional incubator conditions.

In Figure A, a hematoxylin / eosin stained section of the culture is shown. It can be clearly seen that the upper cell layer consists of cilia-bearing and mucus-producing cells that are comparable to normal bronchial epithelium of the lung.

Figures B and C are immunohistochemical labels of cylokeratin 13, further abbreviated CK13, positive cells as markers for pre-neoplastic cells. The cultures show, in addition to inconspicuous epithelium sections according to Figure B, in the lower and middle cell layers CK13 positive cells 1 according to Figure C.

These studies with isolated bronchial epithelial cells, ie primary cells, from resection material of NSCLC patients showed that even in the area of clinically "healthy" tissue proportions prenoplastic cells are to be expected represents.

In becomes an ALI culture of in isolated bronchial described after a culture period of 23 days shown at the air-liquid interface culture under dynamic conditions in Cultex ^® LTC module. Under these conditions, there is a selection and accumulation of the preneoplastic cells, as evidenced by the massive CK13-labeled cells.

Differentiated cell cultures, ie, cilia-bearing and mucus producing cells that have been cultured from such tissue samples in the so-called long-term process in Cultex ^® LTC module, show progresses cultivation within four weeks at the air-liquid interface, the enrichment of pre-neoplastic cells in the basal and middle cell layers. These findings indicate that these patients are at increased risk of relapsing and need to be specifically hired for effective chemotherapy.

This technological and experimental approach is relevant for both basic and user-oriented research, as it involves comparable toxicological studies of drugs (eg pharmaceuticals, growth factors, cytokines) and pollutants (cigarette smoke, particles, aerosols) on specific cell populations within the epithelial cell association for risk assessment and for the preparation of starting material for cell or molecular biological follow-up examinations allowed.

According to is an intermediate cell metaplasia in cell cultures of human primary bronchial epithelial cells treated within a 43-day culture period, the first 3 weeks with a carcinogenic substance (benzo (a) pyrene) in non-toxic concentrations. The cells were immunohistochemically labeled with CK13 antibodies to demonstrate the selection and accumulation of metaplastic cells in the cultures after treatment with a carcinogenic substance. Magnification factor is also 630x.

Continue to say that such a cell model can also provide information on the carcinogenic potential of drugs. Experiments with benzo (a) pyrene, a known carcinogenic, polycyclic hydrocarbon, led to the long-term exposure of non-toxic bronchial epithelial cells to the induction of cancer cells, which can be selected and accumulated in long-term culture, analogous to the primary tumor cells.

A limitation of these cell models is the high variability between the donors and the availability of the cell cultures, since they lose their in vivo properties after a few passages. Here, an immortalized cell line (eg, CL-1548, Cultex Laboratories GmbH), which has a comparable phenotype compared to the normal parent cells, is readily available, and retains its differentiation characteristics over a long culture period, providing the unique opportunity for a reproducible and readily available tumor model establish.

• 1. Toxikologische Untersuchung von Wirk- und Schadstoffen zur Risikoabschätzung
• 2. Testung des kanzerogenen (krebserzeugenden) Potentials von Wirkstoffen (Expositionspellets) an artifizieller Bronchialschleimhaut zur lokal begrenzten Induktion transformierter Zellen.
• 3. Überprüfung der Wirksamkeit von Chemotherapeutika auf derart induzierte neoplastische Läsionen oder migrierende Tumorzellen/-cluster aus Patientenproben.
• 4. Plattform für die Entwicklung neuer Behandlungsansätze, insbesondere chemotherapeutischer Ansätze.

In particular, the long-term cultivation of such 3D cell models over several weeks leads to an accumulation of transformed cells (tumor cells) that can proliferate and spread in a fully differentiated 3D bronchial epithelium, while the epithelium itself is its phenotype maintains. This combination allows the establishment of a 3D tumor model, which can be used in the following ways for the health precaution of the consumer and patient:

• 1. Toxicological study of active substances and pollutants for risk assessment
• 2. Testing of the carcinogenic (carcinogenic) potential of active substances (exposure pellets) on artificial bronchial mucosa for the localized induction of transformed cells.
• 3. Review of the efficacy of chemotherapeutic agents on such induced neoplastic lesions or migrating tumor cells / clusters from patient samples.
• 4. Platform for the development of new treatment approaches, especially chemotherapeutic approaches.

A short explanation about the CULTEX ^® LTC module, as shown in the WO 2014/086412 A1 was described in advance. The CUTEX ^® LTC module was developed for the long-term cultivation of lung cells at the air-liquid interface. It guarantees optimal and standardized nutrient supply under dynamic conditions. Under such culture conditions, normal bronchial epithelial cells differentiate particularly well into ciliated and mucus-producing cells. They form a compact cell cluster without any further proliferative activity, while neoplastic cells accumulate through enhanced division activities in the cultures.

The selection and enrichment of tumor cells in a computer-controlled long-term cultivation system (Cultex ^® LTC module) allows such a standardized culture methods use routinely for the diagnosis to test the efficacy of various chemotherapeutic agents to the tumor cells. The determination of effective individual chemotherapy spectrum, as a service to clinics, serves to optimize for successful treatment of patients.

For the pharmaceutical industry, the described cell models would have to be of great interest, since they could be used for the development of new chemotherapeutic agents, especially in genetically modified tumor cells. LIST OF REFERENCE NUMBERS 1 positive cells

QUOTES INCLUDE IN THE DESCRIPTION

This list of the documents listed by the applicant has been generated automatically and is included solely for the better information of the reader. The list is not part of the German patent or utility model application. The DPMA assumes no liability for any errors or omissions.

Cited patent literature

• WO 2014/086412 A1 [0047]

Cited non-patent literature

• Pipinikas et al. (2014) and Baccelli and Trump (2014) [0008]

Claims (3)

 Method for determining suitable therapy and / or detection methods and / or substances for or against carcinogenesis, wherein • Migrating cancer cells are selected and enriched from patient or cell culture samples or • exposing healthy tissue or cells to a cancer cell-producing medium (chemical / physical) and / or causing tumor cells to migrate into healthy tissue to form new pre- or neoplastic cells / clusters - as evidence of the neoplastic potential of the cells . • exposing this "infiltrated" tissue to various therapeutic and / or detection methods and / or materials; and • determining which treatment and / or detection method and / or substance is most suitable for the therapy and / or detection of this cancer. A method according to claim 1, characterized in that for the controlled supply of one or more tissues in cell culture containers with a medium, eg the respiratory tract in a culture guide on an air-liquid boundary layer in particular for improving cell growth and differentiation of the cells in long-term cultures a medium circulation within of the cell culture container. A method according to claim 1 or 2, characterized in that the cells or 3D cultures are arranged on a microporous membrane, wherein • a medium level below the cell culture over a period of a minimum of 3 weeks to a predetermined reference value in a tolerance of 0.2- 0.3 mm is brought by the medium is supplied via a control device and deviations are detected and balanced • a media exchange and a medium circulation, in particular for mixing, performed.

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