Detection of trifluridine in tumors of patients with metastatic colorectal cancer treated with trifluridine/tipiracil.

PURPOSE: Trifluridine (FTD) is the active component of the nucleoside chemotherapeutic drug trifluridine/tipiracil (FTD/TPI), which is approved worldwide for the treatment of patients with metastatic gastrointestinal cancer. FTD exerts cytotoxic effects via its incorporation into DNA, but FTD has not been detected in the tumor specimens of patients. The purpose of this study was to detect FTD in tumors resected from metastatic colorectal cancer (mCRC) patients who were administered FTD/TPI. Another purpose was to investigate the turnover rate of FTD in tumors and bone marrow in a mouse model. METHODS: Tumors and normal tissue specimens were obtained from mCRC patients who were administered FTD/TPI or placebo at Kyushu University Hospital. Tumors and bone marrow were resected from mice with peritoneal dissemination treated with FTD/TPI. To detect and quantitate FTD incorporated into DNA, immunohistochemical staining of paraffin-embedded specimens (IHC-p staining) and slot-blot analysis of DNA purified from these tissues were performed using an anti-BrdU antibody. IHC-p staining of proliferation and apoptosis markers was also performed. RESULTS: FTD was detected in metastatic tumors obtained from mCRC patients who were administered FTD/TPI, but who had discontinued the treatment several weeks before surgery. In a peritoneal dissemination mouse model, FTD was still detected in tumors 13 days after the cessation of FTD/TPI treatment, but had disappeared from bone marrow within 6 days. CONCLUSION: These results indicate that FTD persists longer in tumors than in bone marrow, which may cause a sustained antitumor effect with tolerable hematotoxicity.

The antibodies against 5-bromo-2'-deoxyuridine specifically recognize trifluridine incorporated into DNA.

Trifluridine (FTD) is a key component of the novel oral antitumor drug TAS-102 (also named TFTD), which consists of FTD and a thymidine phosphorylase inhibitor. FTD is supposed to exert its cytotoxicity via massive misincorporation into DNA, but the underlying mechanism of FTD incorporation into DNA and its correlation with cytotoxicity are not fully understood. The present study shows that several antibodies against 5-bromo-2'-deoxyuridine (BrdU) specifically cross-react with FTD, either anchored to bovine serum albumin or incorporated into DNA. These antibodies are useful for several biological applications, such as fluorescence-activated cell sorting, fluorescent immunostaining and immunogold detection for electron microscopy. These techniques confirmed that FTD is mainly incorporated in the nucleus during S phase in a concentration-dependent manner. In addition, FTD was also detected by immunohistochemical staining in paraffin-embedded HCT-116 xenograft tumors after intraperitoneal administration of FTD. Intriguingly, FTD was hardly detected in surrounding matrices, which consisted of fibroblasts with marginal expression of the nucleoside transporter genes SLC29A1 and SLC29A2. Thus, applications using anti-BrdU antibodies will provide powerful tools to unveil the underlying mechanism of FTD action and to predict or evaluate the efficacy and adverse effects of TAS-102 clinically.

Intracellular localization of drugs in cultured tumor cells by ion microscopy and image processing.

Several drugs, containing a halogen atom, F or Br, that are being used in antiviral or anticancer therapy, were studied for their localization in cultured cells by ion microanalysis. The association allows to reduce the exposure time to define the intracellular localization of the studied element. The topography of the cells is given by the image of the polyatomic ion 26CN-. The image of the distribution of 81Br- or 19F-, coded in another color scale, can be superimposed, giving a polychromic image of the cell, thus showing the intracellular localization of the compound. MCF-7 tumor cells were cultured in the presence of pyrimidine derivatives. 5-Bromo-2'-deoxyuridine (BUdR) and 5-trifluorothymidine (F3TdR) were localized in the nucleus, 5-fluoro-2'-deoxyuridine (FUdR) in the nucleus and only in some nucleoli. The method is simple and rapid, as compared with techniques using radiolabeled compounds, or with immunocytochemical techniques. It is possible to observe two different compounds in the same cell. It could be applied to other compounds containing a halogen atom.

I. Tumor growth in mice with depressed capacity to mount inflammatory responses: possible role of macrophages.

Studies were made of the effects of various treatments on the growth in mouse feet of isografts of two methylcholanthrene-induced fibrosarcomas: C-4, of CBA/J mice, and A-2, of A/J mice. The isografts were prepared by pronase digestion of subcutaneous tumors and were injected as unseparated cell suspensions or as tumor-cell-enriched suspensions after depletion of infiltrating host inflammatory cells. The recipient mice were untreated or treated with reserpine, sublethal whole body irradiation, cyclophosphamide, or corticosteroids. Depletion of host cells from the inoculum resulted in increased growth from the same number of tumor cells. Reserpine treatment decreased the growth of both tumors, whether unseparated or tumor-cell-enriched, and whether injected into the foot or the flank. Irradiation, cyclophosphamide pretreatment, and corticosteroid pretreatment decreased the growth of normal inocula or enriched inocula or both. The effects of cyclophosphamide and corticosteroids were apparently not due to cytotoxicity to tumor cells. Normal resident peritoneal cells increased tritiated thymidine uptake by tumor cells in vitro. Sedimentation velocity separation showed the largest cells to be the most potent. It is suggested that some hot inflammatory reaction is necessary for optimal tumor growth and that murine hosts produce not only cells with antitumor effects but also cells, possibly a subpopulation of macrophages, that potentiate tumor growth.