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Fluorescence-guided surgery of prostate cancer bone metastasis Shinji Miwa, MD, PhD,a,b,c Yasunori Matsumoto, MD,b Yukihiko Hiroshima, MD, PhD,a,b Shuya Yano, MD,a,b Fuminari Uehara, MD,a,b Mako Yamamoto, PhD,a,b Yong Zhang, MD,a Hiroaki Kimura, MD, PhD,c Katsuhiro Hayashi, MD, PhD,c Norio Yamamoto, MD, PhD,c Michael Bouvet, MD,b,* Naotoshi Sugimoto, MD, PhD,d Hiroyuki Tsuchiya, MD, PhD,c and Robert M. Hoffman, PhDa,b a
AntiCancer, Inc, San Diego, California Department of Surgery, University of California, San Diego, San Diego, California c Department of Orthopedic Surgery, Kanazawa University Graduate School of Medical Sciences, Kanazawa, Ishikawa, Japan d Department of Physiology, Kanazawa University Graduate School of Medical Sciences, Kanazawa, Ishikawa, Japan b
article info
abstract
Article history:
Background: The aim of this study is to investigate the effectiveness of fluorescence-guided
Received 12 February 2014
surgery (FGS) of prostate cancer experimental skeletal metastasis.
Received in revised form
Materials and methods: Green fluorescent protein-expressing PC-3 human prostate cancer
16 March 2014
cells (PC-3-green fluorescent protein) were injected into the intramedullary cavity of the
Accepted 16 May 2014
tibia in 32 nude mice. After 2 wk, 16 of the mice underwent FGS; the other 16 mice un-
Available online xxx
derwent bright-light surgery (BLS). Half of BLS and FGS mice (8 mice in each group) received zoledronic acid (ZOL). Weekly fluorescence imaging of the mice was performed. Six weeks
Keywords:
after surgery, metastases to lung and inguinal lymph node were evaluated by fluorescence
Prostate cancer
imaging.
Bone metastasis
Results: The percentage of residual tumor after BLS and FGS was 9.9 2.2% and 0.9 0.3%,
GFP
respectively (P < 0.001). FGS reduced recurrent cancer growth compared with BLS (P
36 wk. The median overall survival increased from 16 wk in the BLS group to 31 wk in the FGS group. FGS resulted in a cure in 67% of mice (alive without evidence of tumor at >6 mo after surgery) compared with only 37% of mice that underwent BLS [17]. In the present report, we determined the advantage of FGS of red fluorescent protein-labeled experimental prostate cancer bone metastasis with and without zoledronic acid (ZOL).
2.
Materials and methods
2.1.
Cell lines and cell culture
The wild-type PC-3 cells, originally isolated from a human prostate adenocarcinoma that had metastasized to the bone, were genetically engineered to express GFP using a retrovirus expression vector [3]. PC-3-GFP cells were cultured in DMEM supplemented with 1% penicillin/streptomycin (Invitrogen) and 10% fetal bovine serum (SigmaeAldrich) at 37 C with 95% air and 5% CO2.
2.2.
Animal care
Athymic NCr nude mice (nu/nu) (AntiCancer Inc) aged 6e8 wk, were used in this study. Mice were maintained in a barrier facility on high efficiency particulate air-filtered racks. The animals were fed with autoclaved laboratory rodent diet. Animal experiments were performed in accordance with the Guide-lines for the Care and Use of Laboratory Animals under National Institutes of Health assurance number A3873-01.
2.3.
Tumor implantation
Six to eight-week-old male mice were anesthetized by a ketamine mixture (10 mL ketamine HCL, 7.6 mL xylazine, 2.4 mL
5
Fig. 5 e Time course of tumor growth in the bone visualized by fluorescent area. (A) FGS significantly reduced the recurrent tumor growth of prostate cancer cells in the bone (P [ 0.006). (B) ZOL slightly inhibited recurrent tumor growth in the bone, but not significantly. The data are expressed mean ± standard error of the mean. Statistical analysis was performed using the Student t-test. **P < 0.01, compared with BLS group.
acepromazine maleate, and 10 mL H2O) via subcutaneous injection. The left leg was sterilized with alcohol and an w5 mm midline skin incision was made just below the knee joint to expose the tibial tuberosity (Fig. 1A). PC-3-GFP cells (5 105) in 5 mL Matrigel (BD Bioscience) per mouse were injected into the Q6 intramedullary cavity of the left tibia with a 0.5 mL 28 G latexfree insulin syringe (TYCO Health Group LP). The skin was closed with a 6-0 suture. Two weeks after injection, fluorescence imaging was performed to confirm the tumor growing with GFP expression using the OV100 Small Imaging System (Olympus Corp) (Fig. 1B) [25]. Q7
2.4.
Tumor resection
A total of 32 mice were used in the experiments: 16 mice underwent FGS, and the other 16 mice underwent bright-light surgery (BLS) (Fig. 2). Two weeks after the implantation of the tumor cells, tumor-bearing mice were randomly assigned to the BLS or FGS group. Before resection of the metastatic tumor, mice were anesthetized by ketamine mixture, and their limbs were sterilized with alcohol. An w1.5 cm incision was made above the tumor. Resection of the metastatic bone tumor was performed using Dino-Lite digital camera
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651 652 653 654 655 656 657 658 659 660 661 662 663 664 665 666 667 668 669 670 671 672 673 Fig. 6 e Time lapse imaging of tumor growth after BLS and FGS. (For interpretation of the references to color in this figure, 674 the reader is referred to the web version of this article.) 675 676 677 (AM4113T-GFBW Dino-Lite Premier; AnMo Electronics Corp, intralesional or marginal tumor excision was performed in all 678 the mice. Postoperatively, the surgical resection bed was 679 Q8 Taiwan) under bright-light illumination for the BLS group and under fluorescence illumination through a GFP filter for the imaged with the OV100 imaging system under both standard 680 681 FGS group [26]. For metastatic bone tumor resection, bright field and fluorescence illumination to assess 682 683 684 685 686 687 688 689 690 691 692 693 694 695 696 697 698 699 700 701 702 703 704 705 706 707 708 709 710 711 712 713 714 Fig. 7 e KaplaneMeier curve for DFS. (A) KaplaneMeier curve for DFS of mice in each group. (B) KaplaneMeier curve for DFS 715 of BLS and FGS mice. FGS significantly improved DFS. 5.2.0 DTD YJSRE12751_proof 11 June 2014 1:55 am ce
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Fig. 8 e Fluorescence imaging of metastasis to the inguinal lymph node. (A) Inguinal lymph node without fluorescence (solid arrow). Bone lesion (dotted arrow) comprising GFP-expressing cancer cells was detected by fluorescence imaging. (B) Inguinal lymph node with GFP fluorescence. (For interpretation of the references to color in this figure, the reader is referred to the web version of this article.)
completeness of the surgical resection. The percentage of residual tumor was defined as follows: residual tumor ð%Þ ¼ ðpostoperative fluorescent areaÞ= ðpreoperative fluorescent areaÞ 100
2.5.
Postoperative adjuvant therapy
Half of the BLS and FGS mice (8 mice in each group) were randomly selected to undergo 4 wk of adjuvant therapy using ZOL (Novartis Pharmaceuticals). Subcutaneous injection of 120 mg/kg of ZOL or saline (vehicle) was performed for 5 d weekly for 4 wk.
2.6.
Animal imaging
Secondary metastases to lung and inguinal lymph
Eight weeks after surgical treatment, the lungs and inguinal lymph nodes were observed with the OV100 in all the mice to evaluate the effect of FGS or BLS and ZOL on secondary metastases to lung and lymph nodes.
Statistical analysis
Data showing comparisons between the two groups were assessed using the Student t-test or c2 test. Disease-free survival (DFS), which was defined as the time from the surgical resection to local recurrence, was calculated using the KaplaneMeier method and log-rank test. Differences were considered significant when P < 0.05. Data are expressed as mean standard error of the mean. Statistical analyses were performed with EZR (Saitama Medical Center, Jichi Medical University).
3.
To assess for recurrence and to follow postoperative tumor progression, weekly noninvasive whole-body imaging of the mice was performed and GFP-expressing areas were recorded every week using the iBox Scientia Small Animal Imaging System (UVP, LLC) [27].
2.7. nodes
2.8.
Results
3.1. Fluorescence imaging before and after tumor resection Mouse models of skeletal metastasis of prostate cancer using bright green-fluorescent PC-3 cells were established in 32 mice (Fig. 1B). Two weeks after implantation of PC-3-GFP cells, the mice were randomly divided into BLS and FGS groups. Before the surgical resection, the fluorescent areas of BLS and FGS groups were 15.0 0.93 mm2 and 14.6 0.98 mm2, respectively (Fig. 3). There was no significant difference in preoperative tumor burden between BLS and FGS groups. A significant improvement in visualization of metastatic bone lesions enhanced distinction of tumor from surrounding
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Table 1 e Correlation between the incidence of inguinal lymph node metastases and treatment with FGS and/or ZOL. Q13
ZOL FGS
Yes, %
No, %
P value
6.3 (1/16) 18.8 (3/16)
37.5 (6/16) 25.0 (4/16)
0.033 0.669
normal bone and soft tissues in FGS mice (Fig. 4A). The significantly enhanced distinction during FGS permitted a more complete resection of tumor, resulting in a significant difference in fluorescent area of residual tumor between the mice in the two groups (Fig. 4B). Percentages of residual tumor after BLS and FGS were 9.9 2.2% and 0.9 0.3%, respectively (P < 0.001).
3.2.
Growth of residual tumor after BLS and FGS
The mice with BLS had rapid growth of tumors with GFP expression, whereas the mice with FGS had little growth of the tumors (Fig. 5). Six weeks after surgery, fluorescent areas of BLS and FGS mice were 175.3 35.8 mm2 and 48.6 22.6 mm2, respectively (P ¼ 0.005). Time-course fluorescence imaging demonstrated rapid growth of GFP-expressing areas of BLS mice after BLS (Fig. 6).
3.3.
DFS after FGS and BLS
Six weeks after surgery, DFS of the BLS-only, BLS þ ZOL, FGSonly, and FGS þ ZOL mice were 0, 12.5 (P ¼ 0.554), 25.0 (P ¼ 0.097), and 62.5% (P ¼ 0.004), respectively (Fig. 7A). DFS is defined as the time from surgical resection to local recurrence.
The combination of FGS and ZOL significantly increased DFS over the combination of BLS and ZOL (P ¼ 0.01).
3.4. Influence of ZOL and FGS on inguinal lymph node and lung metastases Six weeks after the surgery, the left inguinal lymph nodes and bilateral lungs of all mice were observed with the OV100. Metastases were detected by GFP expression (Fig. 8). Inguinal lymph node metastases were observed in four mice of BLSonly group, no mice with BLS þ ZOL, two mice in the FGSonly group, and one mouse in the FGS þ ZOL group. Although FGS showed no significant effect on lymph node metastases, ZOL significantly reduced inguinal lymph node metastases (Table 1, P ¼ 0.033). The lung metastases were observed as nodules with GFP expression (Fig. 9A and B). Lung metastases were observed in two mice in the BLS-only group, two mice in the BLS þ ZOL group, three mice in the FGS-only group, and one mouse in the FGS þ ZOL group. No significant effects of ZOL and FGS on lung metastases were found (Table 2).
4.
Discussion
In advanced prostate cancer, 70% of the patients have metastases in bone [28], and 25%e42% of the patients have metastasis in regional lymph nodes [29]. The present study demonstrates that FGS significantly reduced residual tumor after surgery and significantly improved DFS in a mouse model of prostate cancer experimental metastases in the bone. We have previously reported the effectiveness of FGS using tumor-specific fluorescent antibodies [20]. There are several tumor-specific antigens in prostate cancer such as
Fig. 9 e Fluorescence imaging of lung metastases. GFP-expressing cells enabled distinction between normal lung tissue and lung metastases. (A) Lung without metastasis. (B) Lung with metastases. (For interpretation of the references to color in this figure, the reader is referred to the web version of this article.) 5.2.0 DTD YJSRE12751_proof 11 June 2014 1:55 am ce
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Table 2 e Correlation between the incidence of lung metastases and treatment with FGS and/or ZOL.
ZOL FGS
Yes, %
No, %
P value
18.8 (3/16) 25.0 (4/16)
31.3 (5/16) 25.0 (4/16)
0.414 1.000
9
writing the article. M.B. and R.M.H. did the critical revision of the article. M.B. and R.M.H. obtained the funding.
Disclosure The authors reported no proprietary or commercial interest in any product mentioned or concept discussed in the article.
prostate-specific antigen [30] that could be targeted with a tumor-specific fluorescent antibody may enable FGS in patients with prostate cancer. FGS did not inhibit either lung or lymph-node metastasis. The results suggest lung and lymph node metastasis are early events of remetastasis from the bone and that FGS should be performed as early as possible on prostate cancer with bone metastasis. Prostate cancer frequently metastasizes to bone. As cancer patients live longer, bone metastasis frequency has increased. Destruction of bone by metastatic disease results initially in microfractures, which can cause intractable pain and eventually a complete fracture of the bone. Surgery is most commonly performed for an impending or existing fracture, or intractable pain. The main advantages of surgery are pain relief and restored function. Although surgery for bone metastasis is usually only a palliative treatment, patients can survive for years after surgery [31]. Patients with a solitary bone metastasis have better survival than patients with multiple bone metastases. Surgery can be considered for solitary bone metastases that are slowgrowing, in patients that have a relatively long survival prognosis. Although surgery is often carried out for metastatic bone disease, there are only a few reports on the outcomes [32]. The opportunity that FGS offers for bone metastasis, as shown in the present report in a clinically-relevant mouse model, should significantly improve outcomes when tested in clinical trials.
Q11
5.
Conclusions
Our study demonstrated that FGS inhibits recurrent tumor growth in a mouse model of prostate cancer metastasis to bone, and that ZOL has inhibitory effect on cancer metastasis to lymph nodes. The combination of FGS and ZOL has potential for patients with bone-metastatic prostate cancer.
Acknowledgment
Q12
This study was supported in part by National Cancer Institute grants CA132971 and CA142669. Dedication: This article is dedicated to the memory of A.R. Moossa, MD. Authors’ contributions: S.M., M.B., and R.M.H. contributed to the conception and design. S.M., Y.M., Y.H., S.Y., F.U., M.Y., Y.Z., H.K., K.H., N.Y., M.B., N.S., H.T., and R.M.H. did the analysis and interpretation. S.M., Y.M., Y.H., S.Y., F.U., M.Y., and Y.Z. did the data collection. S.M., M.B., and R.M.H. did the
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Available online at www.sciencedirect.com
ScienceDirect journal homepage: www.JournalofSurgicalResearch.com
Fluorescence-guided surgery of prostate cancer bone metastasis Shinji Miwa, MD, PhD,a,b,c Yasunori Matsumoto, MD,b Yukihiko Hiroshima, MD, PhD,a,b Shuya Yano, MD,a,b Fuminari Uehara, MD,a,b Mako Yamamoto, PhD,a,b Yong Zhang, MD,a Hiroaki Kimura, MD, PhD,c Katsuhiro Hayashi, MD, PhD,c Norio Yamamoto, MD, PhD,c Michael Bouvet, MD,b,* Naotoshi Sugimoto, MD, PhD,d Hiroyuki Tsuchiya, MD, PhD,c and Robert M. Hoffman, PhDa,b a
AntiCancer, Inc, San Diego, California Department of Surgery, University of California, San Diego, San Diego, California c Department of Orthopedic Surgery, Kanazawa University Graduate School of Medical Sciences, Kanazawa, Ishikawa, Japan d Department of Physiology, Kanazawa University Graduate School of Medical Sciences, Kanazawa, Ishikawa, Japan b
article info
abstract
Article history:
Background: The aim of this study is to investigate the effectiveness of fluorescence-guided
Received 12 February 2014
surgery (FGS) of prostate cancer experimental skeletal metastasis.
Received in revised form
Materials and methods: Green fluorescent protein-expressing PC-3 human prostate cancer
16 March 2014
cells (PC-3-green fluorescent protein) were injected into the intramedullary cavity of the
Accepted 16 May 2014
tibia in 32 nude mice. After 2 wk, 16 of the mice underwent FGS; the other 16 mice un-
Available online xxx
derwent bright-light surgery (BLS). Half of BLS and FGS mice (8 mice in each group) received zoledronic acid (ZOL). Weekly fluorescence imaging of the mice was performed. Six weeks
Keywords:
after surgery, metastases to lung and inguinal lymph node were evaluated by fluorescence
Prostate cancer
imaging.
Bone metastasis
Results: The percentage of residual tumor after BLS and FGS was 9.9 2.2% and 0.9 0.3%,
GFP
respectively (P < 0.001). FGS reduced recurrent cancer growth compared with BLS (P
36 wk. The median overall survival increased from 16 wk in the BLS group to 31 wk in the FGS group. FGS resulted in a cure in 67% of mice (alive without evidence of tumor at >6 mo after surgery) compared with only 37% of mice that underwent BLS [17]. In the present report, we determined the advantage of FGS of red fluorescent protein-labeled experimental prostate cancer bone metastasis with and without zoledronic acid (ZOL).
2.
Materials and methods
2.1.
Cell lines and cell culture
The wild-type PC-3 cells, originally isolated from a human prostate adenocarcinoma that had metastasized to the bone, were genetically engineered to express GFP using a retrovirus expression vector [3]. PC-3-GFP cells were cultured in DMEM supplemented with 1% penicillin/streptomycin (Invitrogen) and 10% fetal bovine serum (SigmaeAldrich) at 37 C with 95% air and 5% CO2.
2.2.
Animal care
Athymic NCr nude mice (nu/nu) (AntiCancer Inc) aged 6e8 wk, were used in this study. Mice were maintained in a barrier facility on high efficiency particulate air-filtered racks. The animals were fed with autoclaved laboratory rodent diet. Animal experiments were performed in accordance with the Guide-lines for the Care and Use of Laboratory Animals under National Institutes of Health assurance number A3873-01.
2.3.
Tumor implantation
Six to eight-week-old male mice were anesthetized by a ketamine mixture (10 mL ketamine HCL, 7.6 mL xylazine, 2.4 mL
5
Fig. 5 e Time course of tumor growth in the bone visualized by fluorescent area. (A) FGS significantly reduced the recurrent tumor growth of prostate cancer cells in the bone (P [ 0.006). (B) ZOL slightly inhibited recurrent tumor growth in the bone, but not significantly. The data are expressed mean ± standard error of the mean. Statistical analysis was performed using the Student t-test. **P < 0.01, compared with BLS group.
acepromazine maleate, and 10 mL H2O) via subcutaneous injection. The left leg was sterilized with alcohol and an w5 mm midline skin incision was made just below the knee joint to expose the tibial tuberosity (Fig. 1A). PC-3-GFP cells (5 105) in 5 mL Matrigel (BD Bioscience) per mouse were injected into the Q6 intramedullary cavity of the left tibia with a 0.5 mL 28 G latexfree insulin syringe (TYCO Health Group LP). The skin was closed with a 6-0 suture. Two weeks after injection, fluorescence imaging was performed to confirm the tumor growing with GFP expression using the OV100 Small Imaging System (Olympus Corp) (Fig. 1B) [25]. Q7
2.4.
Tumor resection
A total of 32 mice were used in the experiments: 16 mice underwent FGS, and the other 16 mice underwent bright-light surgery (BLS) (Fig. 2). Two weeks after the implantation of the tumor cells, tumor-bearing mice were randomly assigned to the BLS or FGS group. Before resection of the metastatic tumor, mice were anesthetized by ketamine mixture, and their limbs were sterilized with alcohol. An w1.5 cm incision was made above the tumor. Resection of the metastatic bone tumor was performed using Dino-Lite digital camera
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651 652 653 654 655 656 657 658 659 660 661 662 663 664 665 666 667 668 669 670 671 672 673 Fig. 6 e Time lapse imaging of tumor growth after BLS and FGS. (For interpretation of the references to color in this figure, 674 the reader is referred to the web version of this article.) 675 676 677 (AM4113T-GFBW Dino-Lite Premier; AnMo Electronics Corp, intralesional or marginal tumor excision was performed in all 678 the mice. Postoperatively, the surgical resection bed was 679 Q8 Taiwan) under bright-light illumination for the BLS group and under fluorescence illumination through a GFP filter for the imaged with the OV100 imaging system under both standard 680 681 FGS group [26]. For metastatic bone tumor resection, bright field and fluorescence illumination to assess 682 683 684 685 686 687 688 689 690 691 692 693 694 695 696 697 698 699 700 701 702 703 704 705 706 707 708 709 710 711 712 713 714 Fig. 7 e KaplaneMeier curve for DFS. (A) KaplaneMeier curve for DFS of mice in each group. (B) KaplaneMeier curve for DFS 715 of BLS and FGS mice. FGS significantly improved DFS. 5.2.0 DTD YJSRE12751_proof 11 June 2014 1:55 am ce
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Fig. 8 e Fluorescence imaging of metastasis to the inguinal lymph node. (A) Inguinal lymph node without fluorescence (solid arrow). Bone lesion (dotted arrow) comprising GFP-expressing cancer cells was detected by fluorescence imaging. (B) Inguinal lymph node with GFP fluorescence. (For interpretation of the references to color in this figure, the reader is referred to the web version of this article.)
completeness of the surgical resection. The percentage of residual tumor was defined as follows: residual tumor ð%Þ ¼ ðpostoperative fluorescent areaÞ= ðpreoperative fluorescent areaÞ 100
2.5.
Postoperative adjuvant therapy
Half of the BLS and FGS mice (8 mice in each group) were randomly selected to undergo 4 wk of adjuvant therapy using ZOL (Novartis Pharmaceuticals). Subcutaneous injection of 120 mg/kg of ZOL or saline (vehicle) was performed for 5 d weekly for 4 wk.
2.6.
Animal imaging
Secondary metastases to lung and inguinal lymph
Eight weeks after surgical treatment, the lungs and inguinal lymph nodes were observed with the OV100 in all the mice to evaluate the effect of FGS or BLS and ZOL on secondary metastases to lung and lymph nodes.
Statistical analysis
Data showing comparisons between the two groups were assessed using the Student t-test or c2 test. Disease-free survival (DFS), which was defined as the time from the surgical resection to local recurrence, was calculated using the KaplaneMeier method and log-rank test. Differences were considered significant when P < 0.05. Data are expressed as mean standard error of the mean. Statistical analyses were performed with EZR (Saitama Medical Center, Jichi Medical University).
3.
To assess for recurrence and to follow postoperative tumor progression, weekly noninvasive whole-body imaging of the mice was performed and GFP-expressing areas were recorded every week using the iBox Scientia Small Animal Imaging System (UVP, LLC) [27].
2.7. nodes
2.8.
Results
3.1. Fluorescence imaging before and after tumor resection Mouse models of skeletal metastasis of prostate cancer using bright green-fluorescent PC-3 cells were established in 32 mice (Fig. 1B). Two weeks after implantation of PC-3-GFP cells, the mice were randomly divided into BLS and FGS groups. Before the surgical resection, the fluorescent areas of BLS and FGS groups were 15.0 0.93 mm2 and 14.6 0.98 mm2, respectively (Fig. 3). There was no significant difference in preoperative tumor burden between BLS and FGS groups. A significant improvement in visualization of metastatic bone lesions enhanced distinction of tumor from surrounding
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Table 1 e Correlation between the incidence of inguinal lymph node metastases and treatment with FGS and/or ZOL. Q13
ZOL FGS
Yes, %
No, %
P value
6.3 (1/16) 18.8 (3/16)
37.5 (6/16) 25.0 (4/16)
0.033 0.669
normal bone and soft tissues in FGS mice (Fig. 4A). The significantly enhanced distinction during FGS permitted a more complete resection of tumor, resulting in a significant difference in fluorescent area of residual tumor between the mice in the two groups (Fig. 4B). Percentages of residual tumor after BLS and FGS were 9.9 2.2% and 0.9 0.3%, respectively (P < 0.001).
3.2.
Growth of residual tumor after BLS and FGS
The mice with BLS had rapid growth of tumors with GFP expression, whereas the mice with FGS had little growth of the tumors (Fig. 5). Six weeks after surgery, fluorescent areas of BLS and FGS mice were 175.3 35.8 mm2 and 48.6 22.6 mm2, respectively (P ¼ 0.005). Time-course fluorescence imaging demonstrated rapid growth of GFP-expressing areas of BLS mice after BLS (Fig. 6).
3.3.
DFS after FGS and BLS
Six weeks after surgery, DFS of the BLS-only, BLS þ ZOL, FGSonly, and FGS þ ZOL mice were 0, 12.5 (P ¼ 0.554), 25.0 (P ¼ 0.097), and 62.5% (P ¼ 0.004), respectively (Fig. 7A). DFS is defined as the time from surgical resection to local recurrence.
The combination of FGS and ZOL significantly increased DFS over the combination of BLS and ZOL (P ¼ 0.01).
3.4. Influence of ZOL and FGS on inguinal lymph node and lung metastases Six weeks after the surgery, the left inguinal lymph nodes and bilateral lungs of all mice were observed with the OV100. Metastases were detected by GFP expression (Fig. 8). Inguinal lymph node metastases were observed in four mice of BLSonly group, no mice with BLS þ ZOL, two mice in the FGSonly group, and one mouse in the FGS þ ZOL group. Although FGS showed no significant effect on lymph node metastases, ZOL significantly reduced inguinal lymph node metastases (Table 1, P ¼ 0.033). The lung metastases were observed as nodules with GFP expression (Fig. 9A and B). Lung metastases were observed in two mice in the BLS-only group, two mice in the BLS þ ZOL group, three mice in the FGS-only group, and one mouse in the FGS þ ZOL group. No significant effects of ZOL and FGS on lung metastases were found (Table 2).
4.
Discussion
In advanced prostate cancer, 70% of the patients have metastases in bone [28], and 25%e42% of the patients have metastasis in regional lymph nodes [29]. The present study demonstrates that FGS significantly reduced residual tumor after surgery and significantly improved DFS in a mouse model of prostate cancer experimental metastases in the bone. We have previously reported the effectiveness of FGS using tumor-specific fluorescent antibodies [20]. There are several tumor-specific antigens in prostate cancer such as
Fig. 9 e Fluorescence imaging of lung metastases. GFP-expressing cells enabled distinction between normal lung tissue and lung metastases. (A) Lung without metastasis. (B) Lung with metastases. (For interpretation of the references to color in this figure, the reader is referred to the web version of this article.) 5.2.0 DTD YJSRE12751_proof 11 June 2014 1:55 am ce
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Table 2 e Correlation between the incidence of lung metastases and treatment with FGS and/or ZOL.
ZOL FGS
Yes, %
No, %
P value
18.8 (3/16) 25.0 (4/16)
31.3 (5/16) 25.0 (4/16)
0.414 1.000
9
writing the article. M.B. and R.M.H. did the critical revision of the article. M.B. and R.M.H. obtained the funding.
Disclosure The authors reported no proprietary or commercial interest in any product mentioned or concept discussed in the article.
prostate-specific antigen [30] that could be targeted with a tumor-specific fluorescent antibody may enable FGS in patients with prostate cancer. FGS did not inhibit either lung or lymph-node metastasis. The results suggest lung and lymph node metastasis are early events of remetastasis from the bone and that FGS should be performed as early as possible on prostate cancer with bone metastasis. Prostate cancer frequently metastasizes to bone. As cancer patients live longer, bone metastasis frequency has increased. Destruction of bone by metastatic disease results initially in microfractures, which can cause intractable pain and eventually a complete fracture of the bone. Surgery is most commonly performed for an impending or existing fracture, or intractable pain. The main advantages of surgery are pain relief and restored function. Although surgery for bone metastasis is usually only a palliative treatment, patients can survive for years after surgery [31]. Patients with a solitary bone metastasis have better survival than patients with multiple bone metastases. Surgery can be considered for solitary bone metastases that are slowgrowing, in patients that have a relatively long survival prognosis. Although surgery is often carried out for metastatic bone disease, there are only a few reports on the outcomes [32]. The opportunity that FGS offers for bone metastasis, as shown in the present report in a clinically-relevant mouse model, should significantly improve outcomes when tested in clinical trials.
Q11
5.
Conclusions
Our study demonstrated that FGS inhibits recurrent tumor growth in a mouse model of prostate cancer metastasis to bone, and that ZOL has inhibitory effect on cancer metastasis to lymph nodes. The combination of FGS and ZOL has potential for patients with bone-metastatic prostate cancer.
Acknowledgment
Q12
This study was supported in part by National Cancer Institute grants CA132971 and CA142669. Dedication: This article is dedicated to the memory of A.R. Moossa, MD. Authors’ contributions: S.M., M.B., and R.M.H. contributed to the conception and design. S.M., Y.M., Y.H., S.Y., F.U., M.Y., Y.Z., H.K., K.H., N.Y., M.B., N.S., H.T., and R.M.H. did the analysis and interpretation. S.M., Y.M., Y.H., S.Y., F.U., M.Y., and Y.Z. did the data collection. S.M., M.B., and R.M.H. did the
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