Lung metastases formed by disseminated tumor cells exhibit different proliferation states

Abstract

Lung cancer is the leading cause of cancer mortality in China and worldwide, and metastasis is the main cause of patient death. Cancer cells invade and migrate from the primary tumor, enter the circulation system through intravasation, and become circulating tumor cells (CTCs). CTCs that survive in blood vessels extravasate and invade target organs to become disseminated tumor cells (DTCs). DTCs proliferate in target organs to metastasize to distant organs. The previous view was that metastasis is the final stage of cancer progression. Normal cells first transform into tumor cells and then into invasive cancer cells; thus, metastasis occurs. Therefore, the possibility of metastasis is closely related to the size of the primary tumor. This is reflected in the TNM stage (T, tumor size; N, extent of spread to regional lymph nodes; M, metastasis to distant organs), which is often referenced in clinical diagnosis. However, an increasing number of studies are currently challenging this view. A previous study showed that the metastasis of malignant tumors occurs in the early stages of cancer. When patients are diagnosed with primary cancer, dissemination occurs. CTCs already exist in the blood vessels of early-stage lung cancer patients, and in early-stage lung cancer patients, DTCs are likely to be the main source of late-stage metastasis in some cancers; they do not proliferate in target organs, so they cannot be eliminated by surgery, radiotherapy or chemotherapy. As a result, even if the lesions are removed through surgery in these patients, metastasis is still found months or years later, which affects the patient’s quality of life and reduces the patient’s survival period. These observations prompt scientists in the field of metastasis to pay more attention to the prevention and treatment of DTCs when formulating metastasis prevention strategies. To determine whether DTCs exist in different states after entering the target organ, we used a mouse lung cancer metastasis model to generate CTC-TJH-01 cells, which are circulating tumor cells derived from the peripheral blood of early-stage lung adenocarcinoma patients who extravasate into target organs and become DTCs. Then, we observed the distribution and proliferation of DTCs in the lungs. Combined with traditional Chinese medicine theory, our findings can improve clinical medication regimens and promote innovations in metastasis prevention and treatment strategies. We observed the potential distribution and proliferation status of DTCs in the lungs in a mouse lung cancer metastasis model. A lung colonization assay was performed by injecting 5 × 105 CTC-TJH-01 cells into the lateral tail vein of NOD/SCID mice, and vimentin was used as a lung tumor marker. Immunofluorescence staining was performed, and CTC-TJH-01 cells that reached the lungs through the peripheral circulation were evenly spread over 24 h. This finding showed that cancer cells can move to distant sites through the circulation, especially the lungs, which are rich in blood vessels, and stay there in the form of DTCs. However, after 12 weeks, there were only a few visible metastases in the lungs, and many tumor cells in the visible metastases were Ki67-positive. Immunohistochemistry revealed other Vimentin-positive tumor cells in the lungs, but as the number of cells decreased, the Ki67 positivity rate also decreased, and a single tumor cell was negative for Ki67. This finding shows that an unsuitable microenvironment induces DTC apoptosis, and only a very small number of DTCs mediate the formation of an immunosuppressive microenvironment and then proliferate to form metastatic lesions. In addition, DTCs that survive have different proliferation rates; some proliferate to form metastases, while others remain dormant somewhere as individuals. These metastases of different sizes that coexist in the lungs may also have different responses to radiotherapy and chemotherapy due to their different proliferation rates. This may also explain why early-stage lung cancer patients still develop metastasis after standard clinical treatment. Before disseminated tumor cells proliferate and form visible metastases, they generally cannot be detected clinically through conventional diagnostic methods or tumor biomarkers, and patients at this stage often have no clinically significant symptoms; this stage can be called the “metastasis subclinical stage”. Tian et al. proposed the pathogenesis theory of “hidden toxicity due to vital Qi deficiency” for this stage of lung cancer metastasis. According to this theory, DTCs in a dormant state are already present in the metastatic target organs of patients with early-stage lung cancer after surgery. Immunosenescence or stress mediates immune dysfunction, leading to the activation and proliferation of dormant DTCs, which in turn leads to the occurrence of clinical metastasis.

1. Introduction

Lung cancer is the leading cause of cancer mortality in China and worldwide [1,2], and metastasis is the main cause of patient death [3]. Cancer cells invade and migrate from the primary tumor, enter the circulation system through intravasation, and become circulating tumor cells (CTCs). CTCs that survive in blood vessels extravasate and invade target organs to become disseminated tumor cells (DTCs). DTCs proliferate in target organs to metastasize to distant organs [3]. The previous view was that metastasis is the final stage of cancer progression. Normal cells first transform into tumor cells and then into invasive cancer cells; thus, metastasis occurs. Therefore, the possibility of metastasis is closely related to the size of the primary tumor. This is reflected in the TNM stage (T, tumor size; N, extent of spread to regional lymph nodes; M, metastasis to distant organs), which is often referenced in clinical diagnosis.

However, an increasing number of studies are currently challenging this view. A previous study showed that the metastasis of malignant tumors occurs in the early stages of cancer. When patients are diagnosed with primary cancer, dissemination occurs. CTCs already exist in the blood vessels of early-stage lung cancer patients, and in early-stage lung cancer patients, DTCs are likely to be the main source of late-stage metastasis in some cancers [4]; they do not proliferate in target organs, so they cannot be eliminated by surgery, radiotherapy or chemotherapy. As a result, even if the lesions are removed through surgery in these patients, metastasis is still found months or years later, which affects the patient’s quality of life and reduces the patient’s survival period. These observations prompt scientists in the field of metastasis to pay more attention to the prevention and treatment of DTCs when formulating metastasis prevention strategies.