Activation of disseminated pancreatic cancer cells in bone marrow microenvironment using in-vital live imaging technique

Abstract

Background: Pancreatic cancer is an intractable carcinoma showing dismal prognosis due to early systemic metastasis. Even after margin-negative resection of primary pancreatic cancer, the majority of patients undergo systemic metastasis. However, the reactivation of disseminated tumor cell (DTC) from pancreatic cancer in secondary organs, especially bone marrow, is not well understood. Materials and methods: We investigated the specific biology of DTC in the bone marrow microenvironment using an in vitro model of bone marrow and in vivo models for live imaging technique. For in-vital live imaging for bone marrow, we established two unique mouse models for efficient live imaging of DTC in bone marrow: specifically, the calvarial bone marrow in skull and the dorsal transplanted femur bone marrow. Results: Well-established animal models for DTC in the bone marrow microenvironment showed that DTCs are distributed mainly in the perivascular area and also showed significant decreased cell motility in 4D imaging tracking. The dormant phenotype of DTCs were confirmed at the molecular level that p38 expression was significantly high and ERK 1/2 expression was relatively low in co-culture with mouse bone marrow derived cell in vitro. Even after constant contact with immune cells such as bone marrow neutrophils and macrophages, disseminated tumor cells showed robust viability in the bone marrow environment. Therapeutic resistance from cancer dormancy was also identified by 4D image tracking analysis obtained from our novel bone marrow imaging system in vivo. Chemotherapeutic agents caused significant damages to the in situ bone marrow component, but did not influence the viability of disseminated tumor cells in the bone marrow environment. The reactivation of disseminated tumor cells was confirmed by the intravenous injection of granulocyte-macrophage colony stimulating factor (GM-CSF). Conclusion: DTCs showed chemo-resistance constantly in the bone marrow environment and the reactivation of disseminated tumor cells in bone marrow environment can be developed after bone marrow activation by stimulation of GM-CSF. Recurrent relapse of cancer even after curative treatment including complete surgical resection with adjuvant chemotherapy for pancreatic cancer can be explained by our observation using novel in vivo bone marrow model with a live imaging system.