标题: A New Strategy for Treatment of Malignant Tumor: Intra-Bone Marrow–Bone Marrow T [打印本页] 作者: 江边孤钓 时间: 2009-3-5 10:51 标题: A New Strategy for Treatment of Malignant Tumor: Intra-Bone Marrow–Bone Marrow T
a First Department of Pathology, + {1 ~$ X2 U3 |6 b$ |8 a3 {/ t& w9 N/ Y9 s
b Regeneration Research Center for Intractable Diseases,4 A$ e9 v0 v- K/ c! {
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c Center for Cancer Therapy, Kansai Medical University, Osaka, Japan- @8 C: ^8 B* c: N
( o! R ]8 f5 x9 @- \' @2 J4 t, v: @Key Words. Donor lymphocyte infusion (DLI) ? Malignant tumor ? Graft-versus-host disease (GvHD) ? Graft-versus-tumor (GvT) effect ? Intra-bone marrow–bone marrow transplantation (IBM-BMT) 3 j, ]5 ~! Y3 {# h, {: Q! d2 I# _ . K: F3 G- e* ~; P YCorrespondence: Susumu Ikehara, M.D., Ph.D., First Department of Pathology, Kansai Medical University, 10–15 Fumizonocho, Moriguchi City, Osaka, 570-8506, Japan. Telephone: 81-6-6993-9430; Fax: 81-6-6994-8283; e-mail: ikehara@takii.kmu.ac.jp+ Z" Y! t1 x- O# H2 D }" c
( h+ a: O+ t3 q8 y+ G0 X7 o8 _ABSTRACT - N" Q! N, _- F0 F* ^ ( z0 A* d! w) T" R$ p: kIt is well known that the graft-versus-leukemia reaction (GvLR) can cure patients with a variety of hematological malignancies . Recently, it has been reported that graft-versus-tumor (GvT) effects can induce partial (complete in some) remission of metastatic solid tumors, such as breast cancer and renal cell carcinoma . Based on these findings, donor lymphocyte infusion (DLI) has very recently been used for the treatment of malignant solid tumors even in humans. However, it is very difficult to completely eradicate the tumors, since extensive DLI induces graft-versus-host disease (GvHD). We have attempted to establish a new method for the treatment of malignant tumors. The method includes intra-bone marrow–bone marrow transplantation (IBM-BMT) plus DLI, since we have recently found that IBM-BMT can allow a reduction in radiation doses as a conditioning regimen and prevents GvHD in mice . Using the Meth-A cell line (BALB/c-derived fibrosarcoma), we here show that IBM-BMT plus injection of CD4 T-cell–depleted (but not CD8 T-cell–depleted) spleen cells (as DLI) can prevent GvHD but suppress the tumor growth. We also show that IBM-BMT plus extensive DLI (3 times every 2 weeks) leads to complete rejection of the tumor, although the success rate〞3/50, so far〞is not high. - G' c" V6 ^; J, U( |$ R # U3 v9 n7 C' VMATERIALS AND METHODS ; }) ?5 T! Y" T # F3 Y5 t! A% q9 zIn our preliminary experiments, we carried out conventional BMT (intravenous injection of BMCs: IV-BMT) as a control of IBM-BMT. However, the mice treated with IV-BMT plus DLI died of severe GvHD, as we previously reported . Therefore, we used only IBM-BMT in the subsequent experiments. 7 P7 ^' q, _$ A, t " w" X8 V8 S1 rSurvival Rates in Tumor-Bearing Mice Treated with IBM-BMT Plus DLI ; R: u2 q3 [3 F4 b. M4 s$ T* b* U/ {5 O
All the mice in the nontreated control group died of the tumor. The mean survival time was 40.8 days (range: 17–68 days, n = 20) after reaching a tumor size of 10 x 10 mm (Fig. 1A). When BALB/c whole spleen cells were injected into tumor-bearing BALB/c mice via the tail vein, and when BALB/c bone marrow cells were injected directly into the bone marrow cavity of the tumor-bearing BALB/c mice as a control (BALB/c group), the mean survival time was 36.75 days (range: 31–45 days, n = 4). No significant difference was observed between the control group and the BALB/c group (Fig. 1A). * a1 M* ~- l7 J) ? & w8 e0 h% S7 e; V; Y- I" SFigure 1. Mean (A) survival time and (B) survival curve in each mouse group treated with IBM-BMT plus DLI. Mice in the nontreated control group died of the tumor: The mean survival time was 40.8 days after reaching a tumor size of 1 x 1 cm. The mean survival time in the BALB/c group (BALB/c mice treated with IBM-BMT DLI ) was 36.7 days after reaching a tumor size of 1 x 1 cm and starting the treatment. The mean survival time in the whole group (BALB/c mice treated with IBM-BMT DLI ) was 22 days. The mean survival time in the CD4– group (BALB/c mice treated with IBM-BMT DLI ) was 46.41 days (longest), whereas that in the CD8– group (BALB/c mice treated with IBM-BMT DLI ) was 17.6 days (shortest). *p 8 W, X3 _1 l6 U$ R: V& O
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In contrast, when B6 whole spleen cells were used as DLI in conjunction with IBM-BMT from B6 mice (whole group), the mean survival time was 22 days (range: 15–39 days, n = 11), which was significantly shorter than that of the control group or the BALB/c group (p % P, z" L1 D3 T" w4 D$ t
" ^9 A: } G5 f3 M1 UChanges in Body Weight of Tumor-Bearing Mice Treated with IBM-BMT plus DLI4 s, S' p& e' K- ^+ V9 o
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We measured the body weight of mice treated with the various strategies almost every day, since it is well known that loss of body weight is the most reliable indicator of GvHD in mice. When mice were treated with various strategies, the mice in all the groups except for the control group lost weight until approximately 15 days (Fig. 2). It seems unlikely that this was due to GvHD, since the BALB/c group also lost weight. It is therefore conceivable that this was a side effect of radiation (5 Gy for IBM-BMT). Thereafter, the mice in the whole group and the CD8– group continued to lose weight gradually: the ranges were 2–12 g and 3–10 g, respectively. In contrast, the mice in the CD4– group and the BALB/ c group gained weight: the ranges were 2–10 g and 2–8 g, respectively. At the end point, the control group showed a sudden loss in weight due to tumor necrosis, followed by death. These results indicate that the BALB/c mice injected with CD4 T-cell–depleted B6 spleen cells did not lose as much body weight as did those injected with B6 whole spleen cells or CD8 T-cell–depleted B6 spleen cells.' ~1 [7 m' I! ^
$ ~* v; q. k: [6 \Figure 2. Changes in body weight of each mouse group treated with IBM-BMT plus DLI. As loss of body weight is the most reliable indicator of GvHD in mice, we measured the body weight of mice treated with IBM-BMT plus DLI. Mice in all groups except for the control group (nontreated group) lost weight until approximately 15 days, due to the side effect of irradiation. Thereafter, the whole group and the CD8– group both showed a continuous loss in weight. In contrast, mice in the CD4– group and the BALB/c group gained weight. Mice in the control group showed a sudden loss in weight due to tumor necrosis, followed by death. The BALB/c mice injected with CD4 T-cell–depleted B6spleencellslostmuchlessbodyweightthandidthoseinjected with CD8 T-cell–depleted B6 spleen cells. Abbreviations: DLI, donor lymphocyte infusion; GvHD, graft-versus-host disease; IBM-BMT, intra-bone marrow–bone marrow transplantation. 8 |; j0 |( |: P. e& c* B 0 |2 w* J, j) q x/ T' [It can be concluded that CD4 (but not CD8 ) T-cell–depleted spleen cells should be used as DLI to prevent GvHD.( q5 H0 ?7 c( n- Y4 M; j: N
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Effects of IBM-BMT Plus DLI on Tumor Growth2 ?# |8 f& m+ @' |6 S$ Q! I* E
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We next analyzed the effects of IBM-BMT plus DLI on the suppression of tumor growth. Some mice in the CD4– group survived longer than did those in the control group and the BALB/c group. Until 20 days after IBM-BMT plus DLI, tumor growth was suppressed in the whole group, the CD4– group, and the CD8– group, in contrast to the control group and the BALB/c group (Fig. 3). However, thereafter, the mice in the CD4– group showed much less suppression of tumor growth than those in the whole group or the CD8–group. Although the mice in the whole group and those in the CD8– group showed continuously suppressed tumor growth, the mice in these groups died earlier than those in the CD4– group (p 3 H6 t2 l+ }- s3 W4 _. Y2 @
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Figure 3. Changes in mean tumor size of recipient BALB/c mice after IBM-BMT plus DLI. Tumor size was measured almost every day, and tumor size is represented every 5 days in this figure. Until 20 days after IBM-BMT plus DLI, the tumor growth was suppressed in the whole group, the CD4– group, and the CD8– group, in contrast to the control group and the BALB/c group. However, thereafter, the mice in the CD4– group showed much less suppression of tumor growth than those in the whole group or the CD8– group. Although the mice in the whole group and the mice in the CD8–group showed continuously suppressed tumor growth, the mice in these groups died earlier than those in the CD4–group (p % m4 V6 T' F: S% d* x4 o 1 ^2 g9 M7 d1 ?These results suggest that the best GvT effect (without undesirable side effects such as GvHD) results from injecting CD4 T-cell–depleted spleen cells as DLI.* |' [+ o5 j* U4 c, X+ |
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Complete Rejection of Tumor by IBM-BMT Plus Repeated DLI( q+ y, O. J$ p2 ?/ N
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Based on the above findings, we attempted to completely reject the tumor by repeated DLI plus IBM-BMT. We injected B6 whole spleen cells into the recipient BALB/c mice three times every 2 weeks. The body weight in both mice (no. 1 and no. 2 in Fig. 4A) remained almost unchanged. The tumor size increased gradually at the beginning, but thereafter decreased slowly but steadily (Fig. 4B); 45 days after the first DLI plus IBM-BMT, the tumor had in some cases (3 out of 50) completely disappeared (Fig. 4C). We confirmed that this was due to rejection based on immunological reaction, since we could not transplant Meth-A again, even when high doses (> 2 x 108) of Meth-A cells were injected into the mice that had previously rejected Meth-A.' x; }. j7 j$ N( o, \
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Figure 4. Changes in (A) body weight and (B) tumor size of the tumor-eradicated BALB/c mice. Repeated donor lymphocyte infusion (DLI) plus intra-bone marrow–bone marrow transplantation (IBM-BMT) were carried out: B6 whole spleen cells wereinjectedintotherecipientBALB/cmicethreetimesevery2 weeks. The body weight in both mice (no. 1 and no. 2) remained almost unchanged (A). The tumor size increased gradually at the beginning, but thereafter decreased slowly but steadily (B); 45 days after the first DLI plus IBM-BMT, the tumor had completely disappeared. (C): The left pictures are no. 1 mouse in (A) and (B), and the right pictures are no. 2 mouse in (A) and (B). ) ~% E- M9 Y! \9 d$ F' T 8 T$ G. D) {$ _' h# I; }4 aDISCUSSION , b% c) [2 G4 s$ [/ C9 W8 G; N3 h; L
We thank Mrs. M. Shinkawa, Ms. S. Miura, and Ms. Y. Tokuyama for their expert technical assistance, and Mr. Hilary Eastwick-Field and Ms. K. Ando for their help in the preparation of the manuscript. This work was supported by grants from Gakunai Zyosei in Kansai Medical University; a grant from the Haiteku Research Center of the Ministry of Education; a grant from Millennium of the Ministry of Education, Culture, Sports, Science and Technology; grants-in-aid for scientific research (B) 11470062 and (Hoga) 16659107; grants-in-aid for scientific research on priority areas (A) 10181225 and (A) 1162221 and Health and Labor Science research grants (Research on Human Genome, Tissue Engineering Food Biotechnology); a grant from the Science Frontier program of the Ministry of Education, Culture, Sports, Science and Technology; a grant from the the 21st Century COE Program of the Ministry of Education, Culture, Sports, Science and Technology; a grant from the Department of Transplantation for Regeneration Therapy (sponsored by Otsuka Pharmaceutical Company, Ltd.); a grant from Molecular Medical Science Institute, Otsuka Pharmaceutical Co., Ltd.; and a grant from Japan Immunoresearch Laboratories Co., Ltd. (JIMRO). ( E7 s/ ? K) r$ Q: R, ~ : r( H+ F0 J# k# B! YREFERENCES+ {0 ?6 O9 F9 M, Z$ k4 h
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