Organ-specific metastasis: an in vitro model for predicting the course of disease

Abstract

Introduction. The problem of organ-specific metastasis still captivates researchers. Elucidation of the patterns of this phenomenon is important not only for theoretical oncology but also for practice, since modern clinical oncology tends to individualize patient treatment tactics. Hundreds of genes are involved in the metastatic cascade, and their identification is a cumbersome study that is ineffective in a clinical setting. Therefore, the clinic needs a simple, fast, and cheap technique for determining the high and low probability of organ-specific metastasis. The technique should have sufficiently high diagnostic specificity and sensitivity.

The study’s aim is to create an in vitro model that can show the condition of a specific patient in real time and then test the model on patients who already have metastases. This will allow the risk of metastasis to be predicted and reference indicators to be found.

Materials and methods. We isolated leukocytes from 8–10 ml of the patient’s blood and brought them to a concentration of 2 million/ml. We used water-salt extracts of metastatic tumors from organs with possible tumor progression as antigens. In each paired well of the plate, 50 μl of culture medium, 100 μl of primary tumor extract or allogeneic metastatic tumors, and 100 μl of leukocyte suspension were added. 50 μl of serum IV (AB) were added to odd wells, and 50 μl of the subject’s autoserum were added to even wells. 50 μl of culture medium were added to control wells instead of tumor extract. We covered the plates with a lid and incubated them for 1.5 h at 37 °C. We then turned the plates upside down and incubated them for 30 min, allowing us to separate adherent cells from non-adherent ones. We removed the fluid containing non-adherent cells, fixed the adherent fraction with 96% ethanol, stained it with methylene blue, washed it three times with saline, and added dimexide. We estimated the amount of dye using spectrophotometric analysis or a color scale.

Results and discussion. We assessed the proposed method’s ability to predict metastases in 189 breast cancer patients with known metastases treated at the Donetsk Regional Antitumor Center from 1989 to 2002. The control group consisted of twenty donors. Fifty-one women had metastases in their bones, twenty-one to their lymph nodes, one to their skin, forty-five to their lungs, twenty-five to their pleurisy, eight to a pleurisy and lung parenchyma metastasis together, twenty-two to their liver, and sixteen to their ovaries. The diagnostic sensitivity was 94.12% for the bone antigen test on donor serum, 80.95% for lymph node antigens, 92.50% for lung parenchyma antigens, 84.0% for metastatic pleurisy antigens, 71.43% for combined lung lesions (parenchyma and metastatic pleurisy), 94.44% for liver metastasis antigens, and 93.75% for ovarian antigens. The combined diagnostic sensitivity on donor serum was 89.94%. The addition of donor serum to the test system allows for an assessment of the contribution of blood cells and large vesicles to the metastasis process.

The diagnostic sensitivity was 82.35% for the bone antigen test on autoserum, 66.67% for lymph node antigen, 71.11% for lung parenchyma antigens, 82.61% for metastatic pleurisy antigens, 87.50% for combined lung lesion (parenchyma and metastatic pleurisy), 90.91% for liver metastasis antigens, and 78.72% for ovarian antigens. The combined diagnostic sensitivity on autoserum was 78.72%. Autoserum has a lower combined diagnostic sensitivity than donor serum. The reason for this remains unknown.

Normal body cells, particularly those in the blood system, played a crucial role in realizing the metastatic cascade. Because they release lytic enzymes and NETosis, neutrophils help metastatic cells get past the tissue-hematological and hemato-tissue barriers. Platelets keep tumor cells moving; macrophages and lymphocytes help create a microenvironment; and neutrophils and lymphocytes wake up dormant metastases.

Conclusions. 1. The model we proposed for predicting organ-specific metastasis in patients with breast cancer demonstrates a relatively high diagnostic sensitivity and specificity. On donor serum, the diagnostic sensitivity is 89.94%, and on autoserum, it is 78.72%. The specificity level was 80%.

1. The test allows us to determine the number and optimal timing of chemotherapy courses for a specific patient.
2. We think that the goal of adjuvant chemotherapy is to affect both dormant micrometastases and normal cells in the blood system. These cells help the metastatic cascade and wake up dormant metastases.