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We previously demonstrated that quiescent cancer cells in a tumor are resistant to conventional chemotherapy as visualized with a fluorescence ubiquitination cell cycle indicator (FUCCI). We also showed that proliferating cancer cells exist in a tumor only near nascent vessels or on the tumor surface as visualized with FUCCI and green fluorescent protein (GFP)-expressing tumor vessels. In the present study, we show the relationship between cell-cycle phase and chemotherapy-induced tumor angiogenesis using in vivo FUCCI real-time imaging of the cell cycle and nestin-driven GFP to detect nascent blood vessels. We observed that chemotherapy-treated tumors, consisting of mostly of quiescent cancer cells after treatment, had much more and deeper tumor vessels than untreated tumors. These newly-vascularized cancer cells regrew rapidly after chemotherapy. In contrast, formerly quiescent cancer cells decoyed to S/G2 phase by a telomerase-dependent adenovirus did not induce tumor angiogenesis. The present results further demonstrate the importance of the cancer-cell position in the cell cycle in order that chemotherapy be effective and not have the opposite effect of stimulating tumor angiogenesis and progression.
The phase of the cell cycle is the main determinant whether a cancer cell can respond to a given drug. We previously monitored the cell cycle dynamics of cancer cells throughout a live tumor intravitally using a fluorescence ubiquitination cell cycle indicator (FUCCI) and observed that more than 80% of internal cancer cells of an established tumor are quiescent in G0/G1 phase. FUCCI imaging demonstrated that cytotoxic agents had little effect on quiescent cancer cells, which are the vast majority of an established tumor. Drug-resistant quiescent cancer cells restarted cycling after the cessation of chemotherapy as they reached the surface of the tumor. These results indicate why most drugs currently in clinical use, which target cancer cells in S/G2/M, are mostly ineffective on solid tumors.1 We have termed this phenomena tumor intrinsic chemoresistance (TIC).2 The results also suggest that drugs that target quiescent cancer cells are urgently needed.1
In the present study, we demonstrate that dormant/quiescent cancer cells induce nascent tumor vessels after chemotherapy, allowing tumors to regrow rapidly after cessation of treatment. Chemotherapy-treated tumors had much more and deeper tumor vessels than control tumors and rapidly began regrowing after cessation of treatment. This report further suggests that quiescent cancer cells can play a large role in tumor angiogenesis, progression, and drug resistance.
FUCCI-expressing MKN45 subcutaneous tumors were treated with cisplatinum (CDDP), paclitaxel (PXT), or doxorubicin (DOX) 14 d after implantation. FUCCI-expressing MKN45-derived subcutaneous tumors consisted mostly of quiescent cancer cells, 7 d after 3 cycles of chemotherapy, even increasing their percentage of quiescent cancer cells compared to control. By 21 d after treatment, the tumors had numerous cycling cancer cells at their surface that were formerly quiescent (Fig. 1).1
A tumor 7 d after implantation consisted of 15.3 ± 2.4% quiescent cancer cells and 84.7 ± 2.4% proliferating cancer cells. A tumor 14 d after implantation consisted of 52.4 ± 3.5% quiescent cancer cells and 47.6 ± 3.5% proliferating cancer cells. A tumor 21 d after implantation consisted of 67.5 ± 3.5% quiescent cancer cells and 32.5 ± 3.5% proliferating cancer cells (Fig. 2C).1
Tumors 3 d after implantation were sensitive to CDDP (Fig. 2A and 2B). In contrast, tumors 14 d after implantation were resistant to chemotherapy (Fig. 2A and 2B). Therefore, we investigated the relationship of the efficacy of chemotherapy and percentage of quiescent cancer cells in a tumor at the start of chemotherapy: 3 days, 7 days, or 14 d after implantation. The percentage of quiescent cancer cells negatively correlated with the efficacy of chemotherapy (Fig. 2C). Furthermore, the time to recurrence positively correlated with the frequency of quiescent cells (Fig. 2D).
We implanted FUCCI-expressing MKN45 cells in the flank subcutaneously in nestin-GFP transgenic nude mice, where nascent tumor vessels express GFP.3 Seven days after implantation, nascent tumor vessels reached the center of tumors (Fig. 3A). In contrast, 28 d after implantation, nascent tumor vessels reached only the surface area, where proliferating cancer cells were located (Fig. 3A). There were few nascent vessels near quiescent cancer cells (Figs. 3A and 3B).
FUCCI-expressing tumors were treated with CDDP, PTX, or DOX 14 d after implantation in nestin-GFP transgenic nude mice. Chemotherapy-treated FUCCI-expressing tumors had more nestin-GFP nascent tumor vessels compared with non-treated tumors 7 d after the last treatment of CDDP (Fig. 4B), PTX (Fig. 4C), or DOX (Fig. 4D) compared with control tumor (Fig. 4A). The number of vessels increased after treatment in the deeper areas of the tumors, which contained quiescent cells, but not in the superficial area which contained cycling cells (Fig. 4E). Moreover, nascent tumor vessels in the center area in the chemotherapy-treated tumors were longer than non-treated tumors (Fig. 4F).
Even 21 d after the last treatment, chemotherapy-treated tumors had more nascent vessels compared with non-treated tumors. More proliferating cancer cells were located near the nascent tumor vessels in the chemotherapy-treated tumors than non-treated tumors. These results suggested that conventional chemotherapy induces tumor angiogenesis, resulting in increased tumor aggressiveness.
We previously reported viral-induced4 or bacteria-induced decoy5 of quiescent cancer cells within the tumor from G0/G1 phase to S/G2 phase, which converts them to chemosensitivity. FUCCI-expressing tumors in ND-GFP transgenic nude mice were first treated with telomerase-dependent adenovirus OBP-301. Decoyed tumors had fewer tumor vessels than non-treated tumors 21 d after the last chemotherapy, as well as 7 d after the last treatment (Fig. 5). Decoyed S/G2 phase cancer cells lost their ability to induce nascent tumor tumor vessels after chemotherapy.
The present report and our previous studies1,2,4-10 have demonstrated the contribution of quiescent cancer cells to tumor chemoresistance. The present report demonstrates that in addition to being chemoresistant, quiescent cancer cells within tumors induce angiogenesis after chemotherapy. This effect can be overcome by cell-cycle decoy by a tumor-specific adenovirus, further indicating the potential of decoy chemotherapy.1,2,4,5,10
MKN45 is a radio-resistant poorly-differentiated stomach adenocarcinoma-derived from a liver metastasis of a patient.11 The cells were grown in RPMI 1640 medium with 10% fetal bovine serum and penicillin/streptomycin.1
For cell-cycle-phase visualization, the FUCCI (fluorescent ubiquitination-based cell-cycle indicator) expression system was used.12 Plasmids expressing mKO2-hCdt1 (red/orange fluorescent protein) or mAG-hGem (green fluorescent protein) were obtained from the Medical and Biological Laboratory (City, Japan).1
Athymic nu/nu nude mice (AntiCancer, Inc.) were bred and maintained in a barrier facility under HEPA filtration and fed with autoclaved laboratory rodent diet (Teklad LM-485; Harlan).1 Nestin-driven green fluorescent protein (ND-GFP) transgenic nude mice carry the GFP gene under the control of the nestin promoter, were also bred and maintained at AntiCancer Inc.3,13-15 All animal studies were conducted in accordance with the principles and procedures outlined in the National Institute of Health Guide for the Care and Use of Animals under Assurance Number A3873-1.
All animal procedures were performed under anesthesia using s.c. administration of a ketamine mixture (10 µl ketamine HCI, 7.6 µl xylazine, 2.4 µl acepromazine maleate, and 10 µl PBS) (Henry-Schein, San Diego, CA). FUCCI-expressing MKN454 cells were harvested by brief trypsinization. Single-cell suspensions were prepared at a final concentration of 5×106 cells/50 µl. FUCCI-expressing MKN45 cells were inoculated into the flank of nude mice.
FUCCI-expressing MKN45 cells were harvested by brief trypsinization. Single-cell suspensions were prepared at a final concentration of 1×107 cells/50 µl. FUCCI-expressing MKN45 cells were implanted into the flank of ND-GFP transgenic nude mice.
Confocal laser scanning microscopy (CLSM) was performed using the FV-1000 (Olympus Corp.) with 2-laser diodes (473 nm and 559 nm). A 4× (0.20 numerical aperture immersion) objective lens and 20 × (0.95 numerical aperture immersion) objective lens (Olympus) were used. Scanning and image acquisition were controlled by Fluoview software (Olympus).16
To evaluate the in vivo antitumor efficacy of CDDP against nascent, intermediate, or established tumors, CDPP (4 mg/kg) was injected intraperitoneally into mice with a subcutaneous tumor at 3 d (for nascent tumors), 7 d (for intermediate tumors), or 14 d (established tumors) after implantation. Mice were treated every 3 d for a total of 3 times.
To evaluate the in vivo angiogenesis after chemotherapy, CDDP (4 mg/kg), PAX (5 mg/kg), or DOX (6 mg/kg) were injected intraperitoneally into ND-GFP nude mice with a subcutaneous tumor at 14 d after implantation. Mice were treated every 3 d for a total of 3 times.
To evaluate in vivo angiogenesis after viral infection, OBP-301 (1×108 PFU) was injected into a subcutaneous tumor at 14 d after inoculation. Mice were treated every 3 d for a total of 3 times.
Data are shown as means ± SD. For comparison between 2 groups, significant differences were determined using the Student's t-test. For comparison of more than 2 groups, statistical significances were determined with a one-way analysis of variance (ANOVA) followed by a Bonferroni multiple group comparison test. P-values of < 0.05 were considered significant.
S. Yano, K. Takehara, and R.M. Hoffman are unsalaried associates of AntiCancer, Inc. H. Tazawa and T. Fujiwara are consultants to Oncolys biopharma, Inc. Y Urata is CEO of Oncolys Biopharma, Inc.
This study was supported in part by grants from the Ministry of Health, Labor, and Welfare, Japan (to T. Fujiwara; No. 10103827, No. 13801426, No. 14525167) and grants from the Ministry of Education, Culture, Sports, Science and Technology, Japan (to T. Fujiwara; No. 25293283).
This paper is dedicated to the memory of A.R. Moossa, MD, and Sun Lee, MD.