UDC 616.233-006.6-076.5 Cell-based diagnostic of bronchial carcinoma: case study of a diagnostic value of tests of circulating tumor cells and organoid culture

Diagnostic based on analysis of living tumor cells is frequently used in oncology. Circulating tumor cells in the blood and cells obtained from a tumor biopsy are used to access their carcinogenic properties and subsequently to predict possible development of the disease. Here we report use of these two tests to assess aggressiveness and metastatic potential of a bronchial adenocarcinoma. The circulating tumor cells test was negative, no circulating cells was observed. It indicates that there was no metastatic spread. However, test with the surgery biopsy showed presence of aggressive cellular clones. The tumor cells from the biopsy proliferated and spread from the cultured tissue. Moreover, the tumor cells formed colonies of cells which lost contact inhibition. This is an indication of aggressive carcinogenic features of the cells in tumor organoids. Combination of both tests showed that the local tumor had an aggressive phenotype, but no detectable spreading of cells. Therefore, these tests support a management plan with removal the primary tumor and regular monitoring, without need of an extensive chemotherapy.


Introduction
Tests of living cells obtained from a tumor allow evaluation of the cellular tumorigenic behavior and responsiveness to drugs (1,2). Current standard of cancer diagnostics includes histopathological and markers tests with a tumor biopsy and body fl uids, e.g. the blood, which are performed with samples containing fi xed and non-living cells. Histopathological tests with sections of a tumor biopsy provide snapshots of a tumor status. Markers in the tumor or in the blood may show correlations with stages of tumorigenesis, but the confi dence of prediction of development of a tumor is relatively low. To access tumor physiology and responsiveness in dynamics, УДК 616.233-006. 6 Діагностика на основі аналізу живих пухлинних клітин все частіше використовується в онкології. Циркулюючі пухлинні клітини в крові та клітини отримані з біопсії пухлини, використовують для аналізу їхньої фізіології, і в подальшому для прогнозування можливого розвитку захворювання. В поданому тут клінічному спостереженні, ми повідомляємо про використання цих двох тестів для оцінки агресивності та метастатичного потенціалу бронхіальної аденокарциноми. Тест на циркулюючі пухлинні клітини був негативним, циркулюючих клітин не спостерігалося. Це свідчить про відсутність агресивного метастатичного поширення. Однак тест з хірургічною біопсією виявив наявність агресивних клітинних клонів. Пухлинні клітини біопсії проліферували активно та мігрували з культивованих пухлинних органоїдів. Пухлинні клітини утворювали колонії клітин, які втратили контактне інгібування. Це засвідчує наявність в біопсії агресивних ракових клітин. Поєднання обох тестів виявило, що первинна пухлина в легенях мала агресивний фенотип, але активного метастазування не виявлено. Використання цих двох тестів -інформативне для планування лікування пацієнта без потреби застосовувати агресивну хіміотерапію. Surgery biopsy provides access to the living tumor cells in the tumor. There have been many successful trials with using tumor cells for prognostic purpose. The tumor cells are obtained by dissociation of the tumor tissues with collagenase or other proteases (7). This technique is well-developed and ensures high rate of establishing cultures of tumor cells. However, responses of tumor cells in a culture of separated cells to anti-cancer drugs may signifi cantly diff er from the outputs of application of the same drugs in treatment of a patient. That is explained by changes in cellular physiology during establishment of the cell culture. Another reason is a dissociation of the tumor cells from their matrix and lost cell-cell contacts already at the fi rst step of culture preparation. Organ culture has been developed to preserve 3-dimensional features of a tumor, e.g. stroma and cellcell interactions. Organ culture approach shows better correlation of cellular behavior with clinical outputs. This is explained by preservation of three-dimensional structure of cells, cell-cell contacts and presence of tumor matrix and stroma (8)(9)(10). Therefore, organ culture is a method of fi rst choice for testing living tumor cells.
Carcinogenic properties of cells defi ne their proliferation rate, resistance to cell death, invasiveness, ability to form a tumor and ability to metastasize (11,12). These carcinogenic properties are crucial for diagnosis and prognosis of the disease. When living tumor cells are tested for their carcinogenic properties, diagnostic and prognostic values are high, if not even higher than traditional snapshot markers. For example, detection of aggressive cell clones is a sign of aggressiveness of the tumor. If tumor cells do not show aggressive behavior and maintain low proliferation rate and cell-cell interactions typical for nontumorigenic cells, that may indicate that the tumor is not aggressive.
In this case report, we describe application of two tests with living cells to access metastatic potential and aggressiveness of the tumor cells.

Materials and methods
Cell culture medium DMEM (certifi ed for human cells culture), fetal bovine serum (toxins and virus-free, heat inactivated) and penicillin and streptomycin antibiotics were obtained from Gibco (Thermo Fisher Scientifi c, Sedeer, Qatar). Cell culture plastic, e.g. cell culture fl asks, 24-and 96-wells plates, pipettes, were also obtained from Gibco (Thermo Fisher Scientifi c, Sedeer, Qatar).

CTCs test
PubTrans CTCs test developed at Oranta CancerDiagnostics AB was used. In brief, 8 ml A B C D

Organ culture test
Biopsy was delivered to the lab after surgery, and was split in small fragments of size less than 5 mm in any dimension. The biopsy contained signifi cant areas of cell death/ necrosis and fragmented easily into small pieces. The biopsy sample was left in a cell culture medium; DMEM with 10% of fetal bovine serum, antibiotics and an additive PBT03™ to promote tumor cells growth. Status of cells, their morphology, growth and death were monitored every second day, and documented. Analysis of cells was performed with use of inverted microscope Leica DHi-1, and images were taken with the camera Leica MC179 HD.

Results and discussion
Clinical data and standard diagnostic tests showed presence of a bronchial neoplasia in the upper-left lobe of size 15 x 10 mm. The patient is a male, 35-years old. Decision was made to remove surgically this neoplasia; the whole upper-left lobe was removed. Five lymphnodes were also removed. Biopsy was taken for histological examination at the Olbio laboratory (Kyiv, Ukraine). Histopathological examination showed presence of trabecular structure of the tumor. There were observed mitotic cells and nodes of the cells separated by thin layers of fi brous stroma. Invasiveness of the tumor cells in the bronchial wall was observed. Fibrotic areas with vascularization and infi ltrations of macrophages were observed. At the time of the tests, no information was available about histological type, e.g. lepidic pattern or expression of mucine.
CTCs test showed presence of only few cells attached to the substrate after 3 days of culture. During these 3 days, erythrocytes, platelets and most of white blood cells were washed away from the culture. Culturing was continued for next two weeks. During that time most of the white blood cells and occasional MSC, endothelial cells and fi broblasts would be identifi ed. As these cells are normal cells, they would not proliferate. Only cells with enhanced proliferation capacity, which is typical for tumorigenic cells, would be proliferating in the culture. Figure 1 shows images of cells detected with the CTCs test in the blood of the patient (Fig. 1, A and B).
An image in panel C shows an example of CTCs detection in the blood of another patient with a metastatic and aggressive cancer. Note diff erences in numbers and morphology of detected CTCs of this patient ( Fig. 1 A and B) and of the patient with an aggressive cancer (Fig. 1C). Result of the CTCs test indicate that there was no presence of tumor cells in the blood. This result allows a conclusion that the cancer of this patient is not metastatic.
Test with cells obtained from the biopsy showed presence of highly proliferating cells. There were observed clones of cells which lost contact inhibition. These cells formed colonies on the top of cellular monolayer (Fig. 2). Clusters of tumor cells were observed already in the fi rst 3 days (Fig.2 A). After 1 week of culture, some cells showed mesenchymal morphology and started to spread and migrate from the clusters (Fig. 2 B and C). Proliferation rate of the cells was high, with an estimate of a doubling time to be less than 24 hrs. This is comparable with the proliferation rates of human tumor cells obtained from lung (A549), breast (MCF7, MDA-MB-231, ZR75-1), liver (HepG2) and kidney (ACHN, 786-0) tumors. The fast growing cells formed a monolayer, with the most of cells confi ned in this monolayer. However, there were observed multiple clones of cells which lost contact inhibition and initiated growth of colony-like structures (Fig. 2 D). For 2 fl asks of the cultured cells, i.e. surface area of 25 cm2, and estimated number of tumor cells between 50,000 and 100,000 cells, there were observed up to 500 clones with lost contact inhibition. We noted that as soon as cells were reaching a dense confl uent, clones were detected. The cloning coeffi cient was estimated as 1.0% to 5.0%. Loss of contact inhibition and formation of substrate-independent colonies in combination with high proliferation rate are typical for highly aggressive tumor cells. Cloning coeffi cient in the range of 10% is considered as typical for aggressive tumors (13). With the mutation rates in tumor cells estimated as 10 -6 to 10 -7 per base pair (14), the observed clonal frequency is in agreement with the genetic variability of the cells. Therefore, the results of the biopsy test suggest that the resected tumor contained clones of aggressive tumor cells.
The CTCs and biopsy tests contributed to assessment of the bronchial carcinoma as a nonmetastatic but with clones of aggressive cells.
With time, the aggressive clones could overtake the growth of the tumor and initiate invasiveness and metastases. Therefore, a combination of these two tests provided better diagnostic value that a single test would. Our results recommend to perform both tests in all cases when the blood and a biopsy may be obtained.

Conclusion
CTCs and biopsy tests showed diagnostic and prognostic values for the patient, in assessment of aggressiveness of the tumor cells. Notably that the disease was not aggressive and metastatic but the primary tumor contained clones of aggressive cells. This case reports supports use of both tests for prediction of development of the disease.