Evaluation of metastatic niches in distant organs after surgical removal of tumor-bearing lymph nodes

Evaluation of metastatic niches in distant organs after surgical removal of tumor-bearing lymph... Background: Surgical removal of primary tumors can promote the incidence of tumor metastasis. However, molecular mechanisms underlying this process remain unclear. Methods: We inoculated tumor cells expressing luciferase gene into subiliac lymph node (SiLN) of the MXH10/ Mo-lpr/lpr mice. The tumor-bearing SiLNs were surgically removed at a certain period of time after inoculation. Results: In vivo bioluminescence imaging system and histological staining revealed metastasis in lung, proper axillary lymph node (PALN) and liver. The lung metastasis rate in SiLN removal groups was significantly higher than in the control group using Fisher exact test. Mann-Whitney U-test indicated that the luciferase-positive tumor cells in the lung and liver were significantly higher than in the control groups. The lung samples in SiLN removal groups had strong expression of lysine oxidase (LOX). Moreover, the number of CD11b cells in the lung and liver in the SiLN removal groups was significantly increased, which was positively correlated with LOX expression level. In addition, the condition of LOX and CD11b in liver was similar to lung. In the SiLN surgical removal groups, the matrix metalloproteinase (MMP)-2 and VEGFA expression in the lung tissues was significantly higher than in the control groups; the collagen fibers per area around the pulmonary vessels was quite significantly lower and negatively correlated with the expression of MMP-2 by Spearman’s analysis. Our data indicated that the reticular fibers were deposited and disordered in the tumor tissues of the lungs in the removal groups, and the reticular fibers per area was higher than in the control groups. The tumor cells in the PALN of control groups were + + significantly higher than in the SiLN removal groups, and CD169 and CD11c cells were also higher than in the SiLN removal groups. Conclusions: Altogether, surgical removal of the tumor-bearing lymph node promoted tumor metastasis through changing the niche in lung and liver. Treatment targeting the metastatic niche might be an effective strategy to prevent tumor metastasis, thereby possibly increasing the survival and reducing the incidence of metastasis in cancer patients. Keywords: Surgical removal, Tumor-bearing lymph node, Metastatic niche, Metastasis * Correspondence: kodama@tohoku.ac.jp Jinhua Zheng and Limin Jia contributed equally to this work. Laboratory of Biomedical Engineering for Cancer, Graduate School of Biomedical Engineering, Tohoku University, 4-1 Seiryo, Aoba, Sendai, Miyagi 980-8575, Japan Biomedical Engineering Cancer Research Center, Graduate School of Biomedical Engineering, Tohoku University, 4-1 Seiryo, Aoba, Sendai, Miyagi 980-8575, Japan Full list of author information is available at the end of the article © The Author(s). 2018 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Zheng et al. BMC Cancer (2018) 18:608 Page 2 of 12 Background impact of the metastatic niche and enhancing thera- Surgical removal, radiotherapy, or angiogenesis inhibi- peutic efficacies and improving cancer prognoses. tor treatments usually have physiological impacts or Although the dissection of the primary tumor is bene- induce local trauma on the body, thereby leaving a ficial, it may disturb metastatic homeostasis [9], resulting chance for the survival of tumor cells [1–3]. As a re- in the activation and rapid growth of latent tumors in sult, the harm from the tumor has been transformed distant organ, which has been suggested in several can- from the harm caused by the primary tumor to the cer types including breast, lung and head and neck can- damage derived from metastases [4] or the harm cers [10–12]. Due to being inspired by above papers and caused by the treatment itself, but there are no effect- a model that has been proposed to explain the recur- ive prevention and treatment measures in clinical rence of breast cancer in a patient after surgical resec- practice. The current detailed mechanism of tumor tion of the tumor [13], we wonder if removal of metastasis is not very thorough, and the therapeutic tumor-bearing lymph node (LN) may accelerate the oc- regimens for metastatic tumors are very rare and in- currence of cancer metastasis. Similarly, our research efficient. Therefore, the clarification of the mechanism showed that iatrogenic induction of distant cancer me- of tumor metastasis is important for the prevention tastasis in the lung is activated after resection of a and treatment of metastatic cancer. tumor-positive lymph node in a lymph node metastasis The “seed and soil” hypothesis has long been used mouse model [14]. However, the concrete and detailed to explain tumor metastasis, i.e., cancer cells are con- mechanisms are not clear. sidered to be seeds that spread to certain places in In this study, we inoculated tumor cells directly the body and soil refers to cytokine formation and into the subiliac lymph node (SiLN) to construct a the recruitment of cells to cultivate the proper tumor lymph node metastasis mouse model and simulate microenvironment; tumor cells eventually begin to tumor metastasis to local lymph node. This in vivo plant and grow at the pre-metastatic site at the ap- metastasis model is stable, highly efficient, and repro- propriatetime[5]. Kaplan and his colleagues first ducible [14]. Unlike the research background of previ- proposed the concept of the pre-metastatic niche in ous studies in metastasis, we did not use any in vivo 2005 [6]. The primary tumor cells carefully plan the models with spontaneous metastasis, but we used an formation of the pre-metastatic niche by secreting a in vivo model with accelerated metastasis after surgi- variety of cytokines and growth factors that promote cal removal of tumors. the movement of bone marrow-derived cells (BMDCs) This study examined the colonization and growth of to gather in pre-metastatic sites. Research of the cancer cells in the proper axillary lymph node (PALN), pre-metastatic niche has become more and more lung and liver tissues after the surgical removal of in-depth and it has been taken into consideration that tumor-bearing SiLN. We also monitored the expression primary tumors affect and change the microenviron- level of tumor cell-secreted LOX, MMP-2 and VEGFA, ment of the secondary organs by promoting the and the bone marrow-derived CD11b cell number in formation of the pre-metastatic niche before the dis- the distant organs in the in vivo model. In addition, we semination of tumor cells [7, 8]. Metastasis is closely analyzed the expression of collagen fibers and reticular associated with the formation of the pre-metastatic fibers in the lung of different experimental groups to niche [7, 8]. A study by Kaplan et al. showed that evaluate if the changes of fibrous structures affect the vascular endothelial growth factor (VEGF) generated colonization and clonal growth of metastatic tumor cells by primary tumor promotes the production of fibro- at the metastatic sites. nectin and the secretion of MMP-9 to establish the This study was based on the molecular changes associ- pre-metastatic niche for invasive tumor cells [2]. ated with the formation of the metastatic niche after the Other studies have shown that primary tumors form surgical removal of tumor-bearing lymph node; we stud- tumor-derived secreted factors (TDSFs) to promote ied the mechanism of surgery-induced tumor metastasis the recruitment of BMDCs, which react with the and hope to provide new ideas and a theoretical basis to extracellular matrix of the target organ at the prevent and treat tumor metastasis. Further understand- pre-metastatic site to change the microenvironment ing of metastatic niche formation will help with the dis- of the target organs and to promote tumor cell covery of new therapeutics and lead to the radical colonization and growth, thereby greatly enhancing treatment of tumor metastasis. the rate of tumor metastasis [6–8]. Therefore, it is necessary to further understand exist- Methods ing anti-cancer therapeutic techniques and tumor metas- Experiments were carried out in accordance with pub- tasis patterns to improve therapeutic regimens in an lished guidelines and were approved by the Institutional alternative manner, with the intention of reversing the Animal Care and Use Committee of Tohoku University. Zheng et al. BMC Cancer (2018) 18:608 Page 3 of 12 Mice incubated with normal animal serum (1:10 in PBS) from MXH10/Mo-lpr/lpr (MXH10/Mo/lpr) mice (13–15 weeks which the second antibodies were obtained for 30 min at of age, 37 ± 2 g) were bred under pathogen-free conditions 37 °C. A blocking kit (414,321, Histofine Company, in the Animal Research Institute, Tohoku University [15]. Japan) was used according the instruction book during The mice in the FM3A group are female, while the mice MMP-2 staining. Afterwards, the tissue sections were in the KM group are male. washed and incubated with the following primary anti- bodies at 4 °C overnight: Rabbit polyclonal antibody to Cell lines Firefly luciferase (ab21176, 1:500, abcam), Rabbit Two types of cells lines were used: malignant fibrous polyclonal antibody to LOX (ab31238, 1:100, abcam), Rat histiocytoma-like KM-Luc/GFP cells and C3H/He anti-mouse CD11b Monoclonal Antibody (MCA711G, mouse mammary carcinoma cells (FM3A-Luc), which 1:500, AbD serotec), mouse monoclonal to MMP-2 were stably expressing the luciferase gene [16]. (ab86607, 1:200, abcam), Rabbit polyclonal to VEGFA (ab183100, 1:50, abcam), rat monoclonal antibody to F4/80 Lymph node metastasis mouse model (ab6640, 1:100, abcam), Armenian hamster monoclonal to KM-Luc/GFP (final concentration: 3.3 × 10 cells/mL) or CD11c (ab33483, 1:500, abcam). After washing in PBS, the FM3A-luc (final concentration: 3.3× 10 cells/mL) cells sections were treated with 0.3% hydrogen peroxide in were suspended in a mixture of 20 mL phosphate buff- methanol for 20 min at room temperature (RT) to ered saline (PBS) and 40 mL of 400 mg/mL Matrigel eliminate the endogenous peroxidase activity. The cor- (Collaborative Biomedical Products). The concrete pro- responding peroxidase-conjugated second antibodies (imme- cedures of injecting tumor cells into the SiLN and resec- diately used, Histofine company, Japan) were applied for tion of the SiLN see literature 14. 3 and 6 days after 30 min at RT. For CD11c staining, biotin-labelled secondary inoculation of KM-Luc/GFP cells or 3 and 7 days after antibody (ab5744, 1:500, abcam) and peroxidase-conjugated inoculation of FM3A-Luc cells into SiLN, the streptavidin were applied for 30 min at 37 °C. Positive reac- tumor-bearing SiLN was surgically removed. tions were developed with diaminobenzidine (DAB). The negative control was performed except that the primary anti- In vivo bioluminescence imaging system body was replaced by PBS. Frozen sections were used for Tumor development in the SiLN and metastasis to the CD169 monoclonal staining. Rat anti mouse CD169 PALN, lungs and livers were detected using an in vivo (ab53443, 1:200, abcam) was applied at RT for 2 h. The rest bioluminescence imaging system (IVIS; Xenogen, USA) procedures are identical to those applied on paraffin [17]. This procedure was carried out separately at 6 h, sections. 3 days, 6 days and 9 days after inoculation of KM-Luc/ GFP cells into the SiLN (KM group), and at 3 days, Special histological staining 7 days, 14 days and 21 days after inoculation of Elastic-Masson (EM) staining combines elastic and tri- FM3A-Luc cells into the SiLN (FM3A group). Moreover, chrome staining techniques for demonstration and clear the IVIS was done immediately after and before surgical definition of elastic fibers of all sizes, connective tissue removal of the SiLN. The luciferase activities of the re- and nuclei in a single tissue section. Paraffin-embedded moved lungs, PALN and liver were measured ex vivo by lung tissues were carried out with silver impregnation IVIS on day 9 in KM group and day 21 in FM3A group, staining to display reticular fibers and to observe the dis- respectively. tribution of reticular fibers in lungs [18]. Tissue preparation Mice were anesthetized using an inhaled mixture of 2% Computer-aided morphological analysis isoflurane and oxygen. All the harvested samples includ- Image Pro Plus 6.0 (Media Cybernetics Inc., Rockville, ing lung, PALN and liver were fixed overnight in 18.5% MD, USA) was used to calculate the intensity and extent formaldehyde, dehydrated and embedded in paraffin. of staining for the detected molecules, the ratio of the Half of PALNs were embedded in OCT. collagen fibers around blood vessels by EM staining and the ratio of the reticular fibers by silver impregnation Immunohistochemistry staining area to the total area of the image in the lung Paraffin samples were sectioned at 3 μm thickness. tissues. Three microscopic fields (original magnification 0.01 M citrate buffer solution (pH 6.0) was used for 200×) were randomly selected. The integral optical dens- retrieval treatment at 120 °C for 5 min. Dako Target ity (IOD) of luciferase, LOX, CD11b, MMP-2, VEGFA, Retrieval Solution (S1699, Dako) was used during F4/80 CD11c, F4/80 and CD169 positive staining was calcu- immunohistochemical staining. After washing in lated and was considered as the expression level of cor- phosphate-buffered saline (PBS), the tissue sections were responding molecules. The per-area density of EM and Zheng et al. BMC Cancer (2018) 18:608 Page 4 of 12 silver positive staining was calculated to reflect the per- independent groups and Fisher exact test was used to centage of the collagen and reticular fibers. compare the incidence of metastasis (%) between groups. Spearman’s rank correlation coefficient test was used to Statistical analysis examine the correlations among the expressions of lucifer- Statistical analyses were performed with SPSS software ase, collagen fiber, MMP-2, Lox and CD11b. Continuous version 18.0 (SPSS Inc., Chicago, IL). Statistical differences data were presented as the median (IQR). Statistical differ- were analyzed using the Mann-Whitney U-test for 2 ences were considered significant when the P <0.05. Fig. 1 In vivo and ex vivo detection of metastases using in vivo bioluminescence imaging system. a KM-Luc/GFP cells were injected into the SiLN (n = 22) (KM group). The SiLN was removed 3 days (G-D3 group, n = 9), or 6 days (G-D6 group, n = 8) after tumor cell inoculation. SiLNs that were injected with KM-Luc/GFP cells but not removed were used as control (G-C group, n = 5). b FM3A-Luc cells were injected into the SiLN (n = 15) (FM3A group). The SiLN was removed 3 days (G-D3 group, n = 4), or 7 days (G-D7 group, n = 6) after tumor cell inoculation. SiLNs that were injected with FM3A-Luc cells but not removed were used as control (G-C group, n = 5). c-d The lung, PALN and liver metastasis rate in the control and SiLN removal groups. e-f The ex vivo luciferase activity of the lung in the control and SiLN removal group Zheng et al. BMC Cancer (2018) 18:608 Page 5 of 12 Fig. 2 The expression of luciferase in PALN, Lung and liver. The Immunohistochemical (IHC) staining of luciferase in PLAN (left panel), lung (middle panel) and liver (right panel) of the KM (a) and FM3A (b) groups. Bar: 100 μm Results metastasis rate in the SiLN removal group was 100%, In vivo and ex vivo detection of metastases using IVIS which was significantly higher than in the control group We established the mouse model via surgical removal of (P = 0.004). the tumor-bearing lymph node promoting tumor metas- The PALN metastasis rate was 20 and 23.53% in the tasis in distant organs. The SiLNs bearing tumor cells control and SiLN removal groups for KM group, respect- were surgically removed at 3 and 6 days (KM group) or ively, and there was no statistical significance. The PALN 7 days (FM3A group) after inoculation, respectively. The metastasis rate was 20% in both control and SiLN removal mice were observed by the IVIS (Fig. 1a, b). Lung metas- groups for FM3A group. The liver metastasis rate was tasis first appeared 9 days after inoculation in the KM 5.9% in the SiLN removal KM group, and no liver metas- control group (G-C) without the surgical removal of the tasis was found in the KM and FM3A control groups tumor-bearing SiLN and the lung metastasis rate was (Fig. 1c, d). The luciferase activity of the lung ex vivo in 20%. At 9 days after inoculation, the lung metastasis rate the SiLN removal group was significantly higher than in in the SiLN removal group (G-D3 and G-D6) was the KM and FM3A control group (Fig. 1e, f). 82.35%, which was significantly higher than in the con- trol group (P = 0.021) (Fig. 1c). Similarly, in FM3A IHC staining analysis of luciferase in PALN, lung and liver group, Fig. 1d shows the lung metastasis rate was 20% in To confirm the IVIS results, we stained sections of control group. Lung metastasis first appeared 21 days PALN, lung and liver with and without SiLN removal in after inoculation. At 21 days after inoculation, the lung KM and FM3A group for tumor cells marker luciferase. Table 1 Expression level of luciferase in PLAN, Lung and liver PALN Lung Liver IOD Median(IQR) P value IOD Median(IQR) P value IOD Median(IQR) P value a a a KM group G-C 489.79(161.99) 0.001 107.97(33.41) < 0.001 16.85(8.85) 0.254 b b b G-D3 151.54(68.71) 0.945 309.73(48.36) 0.005 20.29(9.95) 0.106 c c c G-D6 171.47(13.86) 0.002 416.96(99.09) < 0.001 30.19(7.73) 0.003 a a a FM3A group G-C 388.61(52.09) 0.016 183.76(33.56) < 0.001 12.14(1.39) 0.421 d d d G-D3 219.94(29.07) 0.009 407.27(31.04) 0.127 13.97(3.40) 0.691 c c c G-D7 127.55(34.01) 0.004 432.17(13.64) 0.002 15.26(0.91) 0.310 P < 0.05 was considered significant IOD integral optical density compared with G-D3 compared with G-D6 compared with G-C compared with G-D7 Zheng et al. BMC Cancer (2018) 18:608 Page 6 of 12 Fig. 3 The changes of metastatic niche in lung samples. The expression of LOX, CD11b, MMP-2 and VEGFA were determined using IHC staining in the KM (a) and FM3A (b) groups. Bar: 50 μm In the KM and FM control groups, tumor nests The lung metastatic niche were observed in some PALNs. In addition, scattered To investigate the influence of surgical removal of SiLN luciferase-positive tumor cells were seen in the blood on metastatic site and explore the pathophysiology of vessels and between the alveolar epithelial cells in the local tissue microenvironment in lung, we performed lung. At the same time, luciferase-positive tumor cells the immunohistochemical staining for LOX, CD11b, were seldom found in liver (Fig. 2a, b). MMP-2, and VEGFA. LOX is critical for pre-metastatic In both the KM and FM3A groups with the surgical niche formation and is essential for recruitment of removal of tumor-bearing SiLN, the IOD value of the BMDCs [19, 20]. LOX showed strong staining in both luciferase-positive tumor cells in the PALNs were sig- KM and FM3A SiLN removal groups.CD11b BMDCs nificantly lower than in the control groups (Table 1). have a variety of functions which may enhance Tumor nests of different sizes were observed around metastatic tumor growth. We found CD11b clusters the terminal bronchi and in the distal alveoli in lung. abundantly located in lung in KM and FM3A SiLN re- Moreover, small satellite-like metastatic foci were ob- moval groups, while clusters of CD11b cells were sel- served in some livers and scattered luciferase-positive dom observed in both control groups. The expression of tumor cells were found within the liver blood sinus MMP-2 and VEGFA indicated a role in matrix changes (Fig. 2a, b). Compared with the control group, the and angiogenesis. The high expression of MMP-2 and tumor burden in lung was significantly increased in VEGFA were also detected in our SiLN removal model all SiLN groups and there was statistical difference (Fig. 3a, b). only between G-D6 and G-C of KM groups in liver Statistical analysis showed the expression of LOX, (Table 1). CD11b, MMP-2 and VEGFA in all of the KM and FM3A Table 2 The expression level of LOX, CD11b, MMP-2 and VEGFA in lung KM group FM3A group Control SiLN removal P value Control SiLN removal P value IOD Median (IQR) Lox 159.88(74.58) 432.19(133.55) < 0.001 81.30(41.28) 527.83(72.49) 0.002 CD11b 419.05(162.82) 1457.15(228.39) < 0.001 480.03(58.28) 846.84(68.05) < 0.001 MMP-2 395.32(174.29) 1369.99(187.05) < 0.001 327.07(63.52) 2098.03(165.68) < 0.001 VEGFA 107.46(24.58) 796.19(76.57) 0.002 82.35(15.72) 1192.30(279.41) < 0.001 P < 0.05 was considered significant IOD integral optical density Zheng et al. BMC Cancer (2018) 18:608 Page 7 of 12 Table 3 Correlation among the expression level of LOX, Compared with the corresponding control group, the re- luciferase, CD11b and MMP-2 in lung of KM and FM3A SiLN ticular fibers in the lungs in all of the SiLN removal removal groups groups were obviously increased and fractured (Fig. 4a, b). KM group FM3A group The difference of collagen fiber per area was significant LOX Luciferase P = 0.001, r = 0.767 P = 0.013, r = 0.745 between the control group and the SiLN removal groups in both KM and FM3A groups. The difference of reticular LOX CD11b P = 0.025, r = 0.733 P = 0.019, r = 0.721 fiber per area was significant between the control group CD11b MMP-2 P = 0.036, r = 0.786 P = 0.022, r = 0.709 and the SiLN removal groups in both KM and FM3A P < 0.05 was considered significant; r: Spearman correlation coefficient groups (Table 4). The expression level of MMP-2 SiLN removal groups was significantly higher than in the showed a negative correlation with the collagen fibers per corresponding control group (Table 2). We also analyzed area (r = − 0.847, P = 0.016;r = − 0.748,P = 0.013;Table 5). the correlation among the LOX, CD11b, MMP-2 and lu- Moreover, the per area of collagen fibers showed a negative ciferase by Spearman’s correlation analysis. In both the correlation with the expression level of luciferase-positive KM and FM3A SiLN removal groups, the expression tumor cells in both the KM and FM3A SiLN removal level of LOX showed a positive correlation with the groups (r = − 0.865, P = 0.012;r = − 0.681, P = 0.030;Table 5), expression level of luciferase (r = 0.767, P = 0.001; r =0.745, which mean that the quantity of collagen fibers around the P = 0.013;Table 3) and CD11b (r=0.733, P =0.025; r = 0.721, bronchi and blood vessels were negatively correlated with P =0.019; Table 3), respectively. The expression level of the tumor metastatic foci burden. CD11b showed a positive correlation with the expression level of MMP-2 (r = 0.786, P = 0.036; r=0.709, P=0.022; Surgical removal of SiLN enhanced expression of LOX and Table 3). CD11b in liver We also investigated the expression of LOX and CD11b Matrix changes promotes metastatic tumor growth in the in the liver tissues (Fig. 5a, b). The situation was similar lung to the data obtained in the lungs. The expression levels MMP-2 is known for cleaving extracellular matrix. Using of LOX and CD11b in the liver in all of the KM and Elastic Masson staining and silver impregnation method, FM3A SiLN removal groups were significantly higher we observed the changes of collagen fibers and reticular fi- than in the corresponding control group (P < 0.001, bers. With EM staining, blue-green collagen fibers were Table 6). determined to be evenly located around the bronchi and blood vessels in the control group. Collagen fibers located around the bronchi and blood vessels in the SiLN removal Anti-tumor response in tumor-draining PALN groups were sparse and almost not found. Positive To observe morphological and histological changes in silver-stained reticular fibers were black. The reticular fi- PALN, we performed HE staining. Tumor cells flowed bers in the lungs in both control groups were slender, into the subcapsular sinus of the PALN 3 and 7 days loosely arranged, and interwoven into networks. after inoculation in the KM and FM control groups, Fig. 4 Location and expression of collagen fibers and reticular fibers. Location and expression of collagen fibers (left panel) and reticular fibers (right panel) were analyzed by Elastic-Masson (EM) staining and silver impregnation method in the KM (a) and FM3A (b) groups. Bar: 100 μm Zheng et al. BMC Cancer (2018) 18:608 Page 8 of 12 Table 4 Per area of collagen fibers and reticular fiber in lung cytokines or changes of tumor microenvironment [23]. To explain the presence of postoperative tumor metastasis, it is Collagen fibers Reticular fibers necessary to find the appropriate in vivo model, thereby per area (%) P value per area (%) P value a a gaining an in-depth understanding of the mechanism of KM group G-C 0.0148(0.0045) 0.008 0.0118(0.0018) 0.001 tumor removal-induced metastasis in order to develop ef- b b G-D3 0.005(0.001) 0.464 0.0209(0.0013) 0.008 fective treatment options and to improve metastatic cancer c c G-D6 0.0043(0.0021) 0.002 0.026(0.005) 0.008 prevention and treatment. a a FM3A group G-C 0.038(0.009) 0.001 0.025(0.004) 0.001 Our IVIS results showed that the surgical removal d d G-D3 0.024(0.007) 0.006 0.044(0.008) 0.019 of tumor-bearing SiLN promoted the incidence of c c lung metastasis (Fig. 1). Further, immunohistochemis- G-D7 0.007(0.003) 0.003 0.054(0.012) 0.002 try showed that luciferase-positive tumor cells were P < 0.05 was considered significant; Data are expressed as the median (IQR) compared with G-D3 sparsely spread in the control lung tissues and was compared with G-D6 significantly lower than in the SiLN removal group. compared with G-C compared with G-D7 Although there was no significant difference in the rate of PALN metastasis between the control and respectively. As time went by, tumor cells were found in tumor-bearing SiLN removal group, the number of lymphatic sinuses and tumor nests formed (Fig. 6a, b). luciferase-positive tumor cells in PALN in the surgical Lymph nodes play a key role in orchestrating im- SiLN removal group was significantly less than the mune response against tumors. An important event is control group (Fig. 2,Table 1). the presentation of tumor antigens within the The pre-metastatic niche is defined as microenviron- tumor-deriving lymph node. Macrophages and den- ment with a series of molecular and cellular changes, dritic cells are professional antigen-presentation cells. which forms a supportive and receptive pre-metastatic Macrophages can be identified according to the ex- site and the fertile “soil” to prepare for the colonization pressions of F4/80 and CD169. We did not observe of “seed”, that is metastatic tumor cells, thereby facilitat- significant changes in the numbers of F4/80 mature ing tumor cell adhesion and growth in the distant organs macrophages between KM and FM3A control groups and promoting tumor metastasis [8]. Studies have shown and SiLN removal groups, respectively. However, we that local and high-precision radiotherapy does not in- + + found that the numbers of CD11C and CD169 cells duce direct injury, but it directly kills or stimulates the in the PALNs in both the KM and FM SiLN control tissue cells compared with surgery, which also acceler- groups were significantly higher than in the corre- ates the incidence of tumor metastasis during sponding SiLN removal groups (P < 0.001, Fig. 6c, d; anti-cancer therapy [2, 24]. Previous studies have shown Table 7). that local radiotherapy causes a stronger expression of angiogenic factors than anti-angiogenic factors and Discussion disturbs the configuration imbalance of VEGFs in the An operation to treat cancer includes resection of the pri- vascular bed of the metastatic site, which then results in mary lesion, lymphadenectomy, reconstruction, and so on. outbreak growth of dormant metastatic tumors [24, 25]. To date, surgery has been considered to be the primary Adjuvant applications of the exogenous VEFGR inhibitor therapeutic regimen for most malignancies, even though cediranib after radiotherapy restored tumor inhibition, sometimes the effect of surgery is not ideal, since the surgi- and studies have shown that using angiogenesis inhibi- cal process could increase the risk of metastases of residual tors to target the VEGF pathway or knocking out the cancer cells to other organs [1, 4, 21]. Experimental data VEGF gene in mouse models with pancreatic cancer or suggest that increased surgical stress augments cancer me- glioblastoma has anti-cancer therapeutic effects by tastasis via surgical stress-induced expression of proteinases reducing the tumor volume and prolonging survival, but in the target organ of metastasis [22]. The effect of surgery the two actions change tumor phenotypes and enhance on metastasis may be attributed to a number of factors, in- tumor invasion and metastasis [26–28]. The discontinu- cluding immunosuppression after surgical stress, action of ation of VEGF inhibitors still enhances the tumor invasion, suggesting that this treatment increases the persist- Table 5 Correlation among per area of collagen fibers, the ence of tumor invasion. This implies that not only does the expression level of luciferase and MMP-2 in lung of KM and treatment-induced trauma lead to tumor-promoting FM3A SiLN removal groups responses, but the disappearance of the tumor itself also KM group FM3A group promotes tumor metastasis during anti-cancer therapy. For collagen fibers MMP-2 P = 0.016, r = −0.847 P = 0.013, r = − 0.748 the tumor treatment targeting a single angle, tumors can collagen fibers Luciferase P = 0.012, r = − 0.865 P = 0.030, r = − 0.681 derive responses of increased invasiveness and extended P < 0.05 was considered significant; r: Spearman correlation coefficient metastasis. Therefore, a comprehensive consideration of all Zheng et al. BMC Cancer (2018) 18:608 Page 9 of 12 Fig. 5 The changes of metastatic niche in liver samples. The expression of LOX and CD11b were observed using IHC staining in the KM (a) and FM3A (b) groups. Bar: 20 μm characteristics of the tumor microenvironment may accom- The fracture of reticular fibers in the SiLN removal groups plish a radical anti-cancer treatment. facilitated tumor cell invasion, thereby accelerating lung Studies have shown that LOX secreted from hypoxic metastasis (Fig. 4, Table 5). primary tumor cells accumulates in the pre-metastatic The above results suggested that high LOX and sites; LOX is an indispensable factor for recruiting bone MMP-2 expressions and a great number of CD11b cell marrow-derived CD11b cells (i.e., immature myeloid facilitate the formation of the metastatic niche and the progenitor cells) to metastatic sites [19, 20]. LOX ex- colonization, growth, and invasion of tumor cells in the pression in SiLN removal groups was significantly higher lung. The accumulation of LOX at the metastatic site than in the control group and was positively correlated promoted CD11b BMDC adhesion, MMP-2 production, with luciferase expression. These findings are consistent and degradation of intra-pulmonary vascular collagen. with the results of previous studies [19, 20], suggesting Moreover, the morphological changes of the extracellular that high LOX expression in the metastatic sites was matrix scaffold remodeled the local microenvironment closely associated with the colonization and metastasis and enhanced tumor cell invasion. Previous studies have of tumor cells. Moreover, the number of CD11b cells in shown that BMDC induced interstitial epithelial trans- the lung and liver of the mice in the SiLN removal formation of the disseminated tumor cells [30], secreted groups was significantly increased, which was positively chemokines to enhance metastasis and nesting of tumor correlated with LOX expressions (Fig. 3, Tables 2, 3). cells [31], and lowered in vivo immune surveillance by im- Since the activation of MMP increased the invasion of munosuppression [7, 8, 32]. The current study also BMDCs [19, 29], we detected MMP-2 expression in the showed that VEGFA expression in the surgical removal lung tissues of the surgical SiLN removal groups, which groups was significantly higher than in the control group, was interestingly higher than that in the control groups and it is known that the secretion of VEGF and other an- and it was positively correlated with CD11b expression giogenic factors promote vascular angiogenesis, thereby (Fig. 3, Tables 2, 3). possibly playing a synergistic role in promoting tumor Our results showed that the degradation of the collagen metastasis. fibers around the pulmonary vessels in the lungs of mice The current study also identified LOX, CD11b, and lu- with the surgical removal of tumor-bearing SiLN was ciferase expression in the liver and showed similar expres- quite significant and the quantity of collagen fibers was sion patterns of LOX and CD11b in the lung (Fig. 5). negatively correlated with the expression of MMP-2, lead- Interestingly, although the ex vivo IVIS results showed ing to a decrease in barrier function, thereby providing a that the liver metastasis was only 5.9% in KM SiLN re- channel for the tumor cells to invade blood vessels and moval group, immunohistochemical staining showed that establishing helpful conditions for tumor cell metastasis. luciferase-positive tumor cells were scattered in liver Table 6 The expression levels of LOX and CD11b in liver KM group FM3A group Control SiLN removal P value Control SiLN removal P value IOD Median(IQR) Lox 146.78(17.19) 318.88(53.53) < 0.001 102.35(30.71) 210.76(43.29) < 0.001 CD11b 376.03(50.23) 755.90(21.10) < 0.001 319.62(33.35) 588.03(34.53) < 0.001 IOD: integral optical density; P < 0.05 was considered significant Zheng et al. BMC Cancer (2018) 18:608 Page 10 of 12 Fig. 6 Histological changes in tumor-draining PALN. a-b HE staining of PALN in mice in KM (a) and FM3A (b) control groups. As time went by, tumor cells were found in lymphatic sinuses and tumor nests formed. c-d The expression of F4/80, CD169 and CD11c in PALN were analyzed by IHC staining. Bar: (a-c) 100 μm, (d)50 μm sinusoids in all of the SiLN removal groups (Fig. 2), which subcutaneously injected tumor cells can be transported to might be associated with tumor cell types and organotro- lymph nodes (LNs) through the lymphatic flow and were pic targeting [33], unfavorable niche formation, or the phagocytosed by CD169 macrophages in the LN subcap- short observation time in this study. Further studies will sular sinus, which then directly cross-present the dead be necessary to address the above concerns. cell-associated antigens to CD8 T cells. The current study Lymph nodes are the well-known routes for lymph- found a large luciferase-positive cell influx into the sub- atic metastasis and the primary place for anti-tumor capsular sinus in the PALN of the control group. In + + immune responses. The current study observed the addition, the number of CD169 and CD11c cells in the influx of tumor cells into the lymphoid sinus of control group was significantly higher than in the SiLN re- PALN in the control group, which resulted in tumor moval groups (Fig. 6, Table 7), suggesting the production nest formation, the retention of tumor cells, and the of anti-tumor immune responses, which, to a certain ex- reduction of tumor cell metastasis to distant organs over tent, reduced the chance of tumor cell metastasis to the time (Fig. 6). A study by Asano et al. [34] showed that distant organs, such as the lung and liver. Table 7 The expression levels of F4/80, CD169 and CD11c in tumor-draining PALN KM group FM3A group Control SiLN removal P value Control SiLN removal P value IOD Median (IQR) F4/80 8431.42(2520.87) 7726.41(824.07) 0.423 5172.4(552.41) 5028.82(1072.5) 0.356 CD169 3213.51(240.77) 1026.38(281.09) < 0.001 1394.34(489.14) 622.89(254.42) < 0.001 CD11c 368.04(65.7) 179.87(80.47) < 0.001 486.99(168.8) 173.73(74.75) < 0.001 P < 0.05 was considered significant IOD integral optical density Zheng et al. BMC Cancer (2018) 18:608 Page 11 of 12 There is growing data to support that noncoding Ethics approval and consent to participate Experiments were carried out in accordance with published guidelines and the RNAs (ncRNAs) play a significant role in tumor metas- protocol was approved by the Institutional Animal Care and Use tasis [35, 36]. A diversity of ncRNAs was demonstrated Committee of Tohoku University (Permit Number: 2016BeLMO-010, to promote proliferation and metastasis of tumor cells 2014BeA-009, 2016BeA-017). [37–39]. We preformed additional microarray detection Competing interests and indicated expression differences in both miRNAs The authors declare that they have no competing interests. and lncRNAs (data not shown) to clarify the specific mechanism associated with our findings. Our study will Publisher’sNote further demonstrate certain miRNAs and lncRNAs Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. could be pre-metastatic biomarkers, prognostic tools and potential therapeutic targets. Author details Laboratory of Biomedical Engineering for Cancer, Graduate School of Biomedical Engineering, Tohoku University, 4-1 Seiryo, Aoba, Sendai, Miyagi Conclusions 2 980-8575, Japan. Department of Anatomy, Basic Medical Science College, Our mouse model with the surgical removal of the Harbin Medical University, Harbin 150081, China. Biomedical Engineering Cancer Research Center, Graduate School of Biomedical Engineering, Tohoku tumor-bearing lymph node greatly simulated the inci- University, 4-1 Seiryo, Aoba, Sendai, Miyagi 980-8575, Japan. Department of dence of tumor metastasis after surgery in clinical prac- Oral and Maxillofacial Surgery, Tohoku University Hospital, 1-1 Seiryo, Aoba, tice [4, 21] and allowed us to observe metastatic niche Sendai, Miyagi 980-8575, Japan. changes in distant organs. Our study indicates that sur- Received: 7 July 2017 Accepted: 21 May 2018 gical removal of the tumor-bearing lymph node pro- moted tumor metastasis to lung and liver; higher References expression level of LOX and a larger number of CD11b 1. Zhang Y, Zhang N, Hoffman RM, Zhao M. 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Identification, review, and systematic cross-validation of microRNA prognostic signatures in metastatic melanoma. J Invest Dermatol. 2016; 136:245–54. http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png BMC Cancer Springer Journals

Evaluation of metastatic niches in distant organs after surgical removal of tumor-bearing lymph nodes

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Biomedicine; Cancer Research; Oncology; Surgical Oncology; Health Promotion and Disease Prevention; Biomedicine, general; Medicine/Public Health, general
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Abstract

Background: Surgical removal of primary tumors can promote the incidence of tumor metastasis. However, molecular mechanisms underlying this process remain unclear. Methods: We inoculated tumor cells expressing luciferase gene into subiliac lymph node (SiLN) of the MXH10/ Mo-lpr/lpr mice. The tumor-bearing SiLNs were surgically removed at a certain period of time after inoculation. Results: In vivo bioluminescence imaging system and histological staining revealed metastasis in lung, proper axillary lymph node (PALN) and liver. The lung metastasis rate in SiLN removal groups was significantly higher than in the control group using Fisher exact test. Mann-Whitney U-test indicated that the luciferase-positive tumor cells in the lung and liver were significantly higher than in the control groups. The lung samples in SiLN removal groups had strong expression of lysine oxidase (LOX). Moreover, the number of CD11b cells in the lung and liver in the SiLN removal groups was significantly increased, which was positively correlated with LOX expression level. In addition, the condition of LOX and CD11b in liver was similar to lung. In the SiLN surgical removal groups, the matrix metalloproteinase (MMP)-2 and VEGFA expression in the lung tissues was significantly higher than in the control groups; the collagen fibers per area around the pulmonary vessels was quite significantly lower and negatively correlated with the expression of MMP-2 by Spearman’s analysis. Our data indicated that the reticular fibers were deposited and disordered in the tumor tissues of the lungs in the removal groups, and the reticular fibers per area was higher than in the control groups. The tumor cells in the PALN of control groups were + + significantly higher than in the SiLN removal groups, and CD169 and CD11c cells were also higher than in the SiLN removal groups. Conclusions: Altogether, surgical removal of the tumor-bearing lymph node promoted tumor metastasis through changing the niche in lung and liver. Treatment targeting the metastatic niche might be an effective strategy to prevent tumor metastasis, thereby possibly increasing the survival and reducing the incidence of metastasis in cancer patients. Keywords: Surgical removal, Tumor-bearing lymph node, Metastatic niche, Metastasis * Correspondence: kodama@tohoku.ac.jp Jinhua Zheng and Limin Jia contributed equally to this work. Laboratory of Biomedical Engineering for Cancer, Graduate School of Biomedical Engineering, Tohoku University, 4-1 Seiryo, Aoba, Sendai, Miyagi 980-8575, Japan Biomedical Engineering Cancer Research Center, Graduate School of Biomedical Engineering, Tohoku University, 4-1 Seiryo, Aoba, Sendai, Miyagi 980-8575, Japan Full list of author information is available at the end of the article © The Author(s). 2018 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Zheng et al. BMC Cancer (2018) 18:608 Page 2 of 12 Background impact of the metastatic niche and enhancing thera- Surgical removal, radiotherapy, or angiogenesis inhibi- peutic efficacies and improving cancer prognoses. tor treatments usually have physiological impacts or Although the dissection of the primary tumor is bene- induce local trauma on the body, thereby leaving a ficial, it may disturb metastatic homeostasis [9], resulting chance for the survival of tumor cells [1–3]. As a re- in the activation and rapid growth of latent tumors in sult, the harm from the tumor has been transformed distant organ, which has been suggested in several can- from the harm caused by the primary tumor to the cer types including breast, lung and head and neck can- damage derived from metastases [4] or the harm cers [10–12]. Due to being inspired by above papers and caused by the treatment itself, but there are no effect- a model that has been proposed to explain the recur- ive prevention and treatment measures in clinical rence of breast cancer in a patient after surgical resec- practice. The current detailed mechanism of tumor tion of the tumor [13], we wonder if removal of metastasis is not very thorough, and the therapeutic tumor-bearing lymph node (LN) may accelerate the oc- regimens for metastatic tumors are very rare and in- currence of cancer metastasis. Similarly, our research efficient. Therefore, the clarification of the mechanism showed that iatrogenic induction of distant cancer me- of tumor metastasis is important for the prevention tastasis in the lung is activated after resection of a and treatment of metastatic cancer. tumor-positive lymph node in a lymph node metastasis The “seed and soil” hypothesis has long been used mouse model [14]. However, the concrete and detailed to explain tumor metastasis, i.e., cancer cells are con- mechanisms are not clear. sidered to be seeds that spread to certain places in In this study, we inoculated tumor cells directly the body and soil refers to cytokine formation and into the subiliac lymph node (SiLN) to construct a the recruitment of cells to cultivate the proper tumor lymph node metastasis mouse model and simulate microenvironment; tumor cells eventually begin to tumor metastasis to local lymph node. This in vivo plant and grow at the pre-metastatic site at the ap- metastasis model is stable, highly efficient, and repro- propriatetime[5]. Kaplan and his colleagues first ducible [14]. Unlike the research background of previ- proposed the concept of the pre-metastatic niche in ous studies in metastasis, we did not use any in vivo 2005 [6]. The primary tumor cells carefully plan the models with spontaneous metastasis, but we used an formation of the pre-metastatic niche by secreting a in vivo model with accelerated metastasis after surgi- variety of cytokines and growth factors that promote cal removal of tumors. the movement of bone marrow-derived cells (BMDCs) This study examined the colonization and growth of to gather in pre-metastatic sites. Research of the cancer cells in the proper axillary lymph node (PALN), pre-metastatic niche has become more and more lung and liver tissues after the surgical removal of in-depth and it has been taken into consideration that tumor-bearing SiLN. We also monitored the expression primary tumors affect and change the microenviron- level of tumor cell-secreted LOX, MMP-2 and VEGFA, ment of the secondary organs by promoting the and the bone marrow-derived CD11b cell number in formation of the pre-metastatic niche before the dis- the distant organs in the in vivo model. In addition, we semination of tumor cells [7, 8]. Metastasis is closely analyzed the expression of collagen fibers and reticular associated with the formation of the pre-metastatic fibers in the lung of different experimental groups to niche [7, 8]. A study by Kaplan et al. showed that evaluate if the changes of fibrous structures affect the vascular endothelial growth factor (VEGF) generated colonization and clonal growth of metastatic tumor cells by primary tumor promotes the production of fibro- at the metastatic sites. nectin and the secretion of MMP-9 to establish the This study was based on the molecular changes associ- pre-metastatic niche for invasive tumor cells [2]. ated with the formation of the metastatic niche after the Other studies have shown that primary tumors form surgical removal of tumor-bearing lymph node; we stud- tumor-derived secreted factors (TDSFs) to promote ied the mechanism of surgery-induced tumor metastasis the recruitment of BMDCs, which react with the and hope to provide new ideas and a theoretical basis to extracellular matrix of the target organ at the prevent and treat tumor metastasis. Further understand- pre-metastatic site to change the microenvironment ing of metastatic niche formation will help with the dis- of the target organs and to promote tumor cell covery of new therapeutics and lead to the radical colonization and growth, thereby greatly enhancing treatment of tumor metastasis. the rate of tumor metastasis [6–8]. Therefore, it is necessary to further understand exist- Methods ing anti-cancer therapeutic techniques and tumor metas- Experiments were carried out in accordance with pub- tasis patterns to improve therapeutic regimens in an lished guidelines and were approved by the Institutional alternative manner, with the intention of reversing the Animal Care and Use Committee of Tohoku University. Zheng et al. BMC Cancer (2018) 18:608 Page 3 of 12 Mice incubated with normal animal serum (1:10 in PBS) from MXH10/Mo-lpr/lpr (MXH10/Mo/lpr) mice (13–15 weeks which the second antibodies were obtained for 30 min at of age, 37 ± 2 g) were bred under pathogen-free conditions 37 °C. A blocking kit (414,321, Histofine Company, in the Animal Research Institute, Tohoku University [15]. Japan) was used according the instruction book during The mice in the FM3A group are female, while the mice MMP-2 staining. Afterwards, the tissue sections were in the KM group are male. washed and incubated with the following primary anti- bodies at 4 °C overnight: Rabbit polyclonal antibody to Cell lines Firefly luciferase (ab21176, 1:500, abcam), Rabbit Two types of cells lines were used: malignant fibrous polyclonal antibody to LOX (ab31238, 1:100, abcam), Rat histiocytoma-like KM-Luc/GFP cells and C3H/He anti-mouse CD11b Monoclonal Antibody (MCA711G, mouse mammary carcinoma cells (FM3A-Luc), which 1:500, AbD serotec), mouse monoclonal to MMP-2 were stably expressing the luciferase gene [16]. (ab86607, 1:200, abcam), Rabbit polyclonal to VEGFA (ab183100, 1:50, abcam), rat monoclonal antibody to F4/80 Lymph node metastasis mouse model (ab6640, 1:100, abcam), Armenian hamster monoclonal to KM-Luc/GFP (final concentration: 3.3 × 10 cells/mL) or CD11c (ab33483, 1:500, abcam). After washing in PBS, the FM3A-luc (final concentration: 3.3× 10 cells/mL) cells sections were treated with 0.3% hydrogen peroxide in were suspended in a mixture of 20 mL phosphate buff- methanol for 20 min at room temperature (RT) to ered saline (PBS) and 40 mL of 400 mg/mL Matrigel eliminate the endogenous peroxidase activity. The cor- (Collaborative Biomedical Products). The concrete pro- responding peroxidase-conjugated second antibodies (imme- cedures of injecting tumor cells into the SiLN and resec- diately used, Histofine company, Japan) were applied for tion of the SiLN see literature 14. 3 and 6 days after 30 min at RT. For CD11c staining, biotin-labelled secondary inoculation of KM-Luc/GFP cells or 3 and 7 days after antibody (ab5744, 1:500, abcam) and peroxidase-conjugated inoculation of FM3A-Luc cells into SiLN, the streptavidin were applied for 30 min at 37 °C. Positive reac- tumor-bearing SiLN was surgically removed. tions were developed with diaminobenzidine (DAB). The negative control was performed except that the primary anti- In vivo bioluminescence imaging system body was replaced by PBS. Frozen sections were used for Tumor development in the SiLN and metastasis to the CD169 monoclonal staining. Rat anti mouse CD169 PALN, lungs and livers were detected using an in vivo (ab53443, 1:200, abcam) was applied at RT for 2 h. The rest bioluminescence imaging system (IVIS; Xenogen, USA) procedures are identical to those applied on paraffin [17]. This procedure was carried out separately at 6 h, sections. 3 days, 6 days and 9 days after inoculation of KM-Luc/ GFP cells into the SiLN (KM group), and at 3 days, Special histological staining 7 days, 14 days and 21 days after inoculation of Elastic-Masson (EM) staining combines elastic and tri- FM3A-Luc cells into the SiLN (FM3A group). Moreover, chrome staining techniques for demonstration and clear the IVIS was done immediately after and before surgical definition of elastic fibers of all sizes, connective tissue removal of the SiLN. The luciferase activities of the re- and nuclei in a single tissue section. Paraffin-embedded moved lungs, PALN and liver were measured ex vivo by lung tissues were carried out with silver impregnation IVIS on day 9 in KM group and day 21 in FM3A group, staining to display reticular fibers and to observe the dis- respectively. tribution of reticular fibers in lungs [18]. Tissue preparation Mice were anesthetized using an inhaled mixture of 2% Computer-aided morphological analysis isoflurane and oxygen. All the harvested samples includ- Image Pro Plus 6.0 (Media Cybernetics Inc., Rockville, ing lung, PALN and liver were fixed overnight in 18.5% MD, USA) was used to calculate the intensity and extent formaldehyde, dehydrated and embedded in paraffin. of staining for the detected molecules, the ratio of the Half of PALNs were embedded in OCT. collagen fibers around blood vessels by EM staining and the ratio of the reticular fibers by silver impregnation Immunohistochemistry staining area to the total area of the image in the lung Paraffin samples were sectioned at 3 μm thickness. tissues. Three microscopic fields (original magnification 0.01 M citrate buffer solution (pH 6.0) was used for 200×) were randomly selected. The integral optical dens- retrieval treatment at 120 °C for 5 min. Dako Target ity (IOD) of luciferase, LOX, CD11b, MMP-2, VEGFA, Retrieval Solution (S1699, Dako) was used during F4/80 CD11c, F4/80 and CD169 positive staining was calcu- immunohistochemical staining. After washing in lated and was considered as the expression level of cor- phosphate-buffered saline (PBS), the tissue sections were responding molecules. The per-area density of EM and Zheng et al. BMC Cancer (2018) 18:608 Page 4 of 12 silver positive staining was calculated to reflect the per- independent groups and Fisher exact test was used to centage of the collagen and reticular fibers. compare the incidence of metastasis (%) between groups. Spearman’s rank correlation coefficient test was used to Statistical analysis examine the correlations among the expressions of lucifer- Statistical analyses were performed with SPSS software ase, collagen fiber, MMP-2, Lox and CD11b. Continuous version 18.0 (SPSS Inc., Chicago, IL). Statistical differences data were presented as the median (IQR). Statistical differ- were analyzed using the Mann-Whitney U-test for 2 ences were considered significant when the P <0.05. Fig. 1 In vivo and ex vivo detection of metastases using in vivo bioluminescence imaging system. a KM-Luc/GFP cells were injected into the SiLN (n = 22) (KM group). The SiLN was removed 3 days (G-D3 group, n = 9), or 6 days (G-D6 group, n = 8) after tumor cell inoculation. SiLNs that were injected with KM-Luc/GFP cells but not removed were used as control (G-C group, n = 5). b FM3A-Luc cells were injected into the SiLN (n = 15) (FM3A group). The SiLN was removed 3 days (G-D3 group, n = 4), or 7 days (G-D7 group, n = 6) after tumor cell inoculation. SiLNs that were injected with FM3A-Luc cells but not removed were used as control (G-C group, n = 5). c-d The lung, PALN and liver metastasis rate in the control and SiLN removal groups. e-f The ex vivo luciferase activity of the lung in the control and SiLN removal group Zheng et al. BMC Cancer (2018) 18:608 Page 5 of 12 Fig. 2 The expression of luciferase in PALN, Lung and liver. The Immunohistochemical (IHC) staining of luciferase in PLAN (left panel), lung (middle panel) and liver (right panel) of the KM (a) and FM3A (b) groups. Bar: 100 μm Results metastasis rate in the SiLN removal group was 100%, In vivo and ex vivo detection of metastases using IVIS which was significantly higher than in the control group We established the mouse model via surgical removal of (P = 0.004). the tumor-bearing lymph node promoting tumor metas- The PALN metastasis rate was 20 and 23.53% in the tasis in distant organs. The SiLNs bearing tumor cells control and SiLN removal groups for KM group, respect- were surgically removed at 3 and 6 days (KM group) or ively, and there was no statistical significance. The PALN 7 days (FM3A group) after inoculation, respectively. The metastasis rate was 20% in both control and SiLN removal mice were observed by the IVIS (Fig. 1a, b). Lung metas- groups for FM3A group. The liver metastasis rate was tasis first appeared 9 days after inoculation in the KM 5.9% in the SiLN removal KM group, and no liver metas- control group (G-C) without the surgical removal of the tasis was found in the KM and FM3A control groups tumor-bearing SiLN and the lung metastasis rate was (Fig. 1c, d). The luciferase activity of the lung ex vivo in 20%. At 9 days after inoculation, the lung metastasis rate the SiLN removal group was significantly higher than in in the SiLN removal group (G-D3 and G-D6) was the KM and FM3A control group (Fig. 1e, f). 82.35%, which was significantly higher than in the con- trol group (P = 0.021) (Fig. 1c). Similarly, in FM3A IHC staining analysis of luciferase in PALN, lung and liver group, Fig. 1d shows the lung metastasis rate was 20% in To confirm the IVIS results, we stained sections of control group. Lung metastasis first appeared 21 days PALN, lung and liver with and without SiLN removal in after inoculation. At 21 days after inoculation, the lung KM and FM3A group for tumor cells marker luciferase. Table 1 Expression level of luciferase in PLAN, Lung and liver PALN Lung Liver IOD Median(IQR) P value IOD Median(IQR) P value IOD Median(IQR) P value a a a KM group G-C 489.79(161.99) 0.001 107.97(33.41) < 0.001 16.85(8.85) 0.254 b b b G-D3 151.54(68.71) 0.945 309.73(48.36) 0.005 20.29(9.95) 0.106 c c c G-D6 171.47(13.86) 0.002 416.96(99.09) < 0.001 30.19(7.73) 0.003 a a a FM3A group G-C 388.61(52.09) 0.016 183.76(33.56) < 0.001 12.14(1.39) 0.421 d d d G-D3 219.94(29.07) 0.009 407.27(31.04) 0.127 13.97(3.40) 0.691 c c c G-D7 127.55(34.01) 0.004 432.17(13.64) 0.002 15.26(0.91) 0.310 P < 0.05 was considered significant IOD integral optical density compared with G-D3 compared with G-D6 compared with G-C compared with G-D7 Zheng et al. BMC Cancer (2018) 18:608 Page 6 of 12 Fig. 3 The changes of metastatic niche in lung samples. The expression of LOX, CD11b, MMP-2 and VEGFA were determined using IHC staining in the KM (a) and FM3A (b) groups. Bar: 50 μm In the KM and FM control groups, tumor nests The lung metastatic niche were observed in some PALNs. In addition, scattered To investigate the influence of surgical removal of SiLN luciferase-positive tumor cells were seen in the blood on metastatic site and explore the pathophysiology of vessels and between the alveolar epithelial cells in the local tissue microenvironment in lung, we performed lung. At the same time, luciferase-positive tumor cells the immunohistochemical staining for LOX, CD11b, were seldom found in liver (Fig. 2a, b). MMP-2, and VEGFA. LOX is critical for pre-metastatic In both the KM and FM3A groups with the surgical niche formation and is essential for recruitment of removal of tumor-bearing SiLN, the IOD value of the BMDCs [19, 20]. LOX showed strong staining in both luciferase-positive tumor cells in the PALNs were sig- KM and FM3A SiLN removal groups.CD11b BMDCs nificantly lower than in the control groups (Table 1). have a variety of functions which may enhance Tumor nests of different sizes were observed around metastatic tumor growth. We found CD11b clusters the terminal bronchi and in the distal alveoli in lung. abundantly located in lung in KM and FM3A SiLN re- Moreover, small satellite-like metastatic foci were ob- moval groups, while clusters of CD11b cells were sel- served in some livers and scattered luciferase-positive dom observed in both control groups. The expression of tumor cells were found within the liver blood sinus MMP-2 and VEGFA indicated a role in matrix changes (Fig. 2a, b). Compared with the control group, the and angiogenesis. The high expression of MMP-2 and tumor burden in lung was significantly increased in VEGFA were also detected in our SiLN removal model all SiLN groups and there was statistical difference (Fig. 3a, b). only between G-D6 and G-C of KM groups in liver Statistical analysis showed the expression of LOX, (Table 1). CD11b, MMP-2 and VEGFA in all of the KM and FM3A Table 2 The expression level of LOX, CD11b, MMP-2 and VEGFA in lung KM group FM3A group Control SiLN removal P value Control SiLN removal P value IOD Median (IQR) Lox 159.88(74.58) 432.19(133.55) < 0.001 81.30(41.28) 527.83(72.49) 0.002 CD11b 419.05(162.82) 1457.15(228.39) < 0.001 480.03(58.28) 846.84(68.05) < 0.001 MMP-2 395.32(174.29) 1369.99(187.05) < 0.001 327.07(63.52) 2098.03(165.68) < 0.001 VEGFA 107.46(24.58) 796.19(76.57) 0.002 82.35(15.72) 1192.30(279.41) < 0.001 P < 0.05 was considered significant IOD integral optical density Zheng et al. BMC Cancer (2018) 18:608 Page 7 of 12 Table 3 Correlation among the expression level of LOX, Compared with the corresponding control group, the re- luciferase, CD11b and MMP-2 in lung of KM and FM3A SiLN ticular fibers in the lungs in all of the SiLN removal removal groups groups were obviously increased and fractured (Fig. 4a, b). KM group FM3A group The difference of collagen fiber per area was significant LOX Luciferase P = 0.001, r = 0.767 P = 0.013, r = 0.745 between the control group and the SiLN removal groups in both KM and FM3A groups. The difference of reticular LOX CD11b P = 0.025, r = 0.733 P = 0.019, r = 0.721 fiber per area was significant between the control group CD11b MMP-2 P = 0.036, r = 0.786 P = 0.022, r = 0.709 and the SiLN removal groups in both KM and FM3A P < 0.05 was considered significant; r: Spearman correlation coefficient groups (Table 4). The expression level of MMP-2 SiLN removal groups was significantly higher than in the showed a negative correlation with the collagen fibers per corresponding control group (Table 2). We also analyzed area (r = − 0.847, P = 0.016;r = − 0.748,P = 0.013;Table 5). the correlation among the LOX, CD11b, MMP-2 and lu- Moreover, the per area of collagen fibers showed a negative ciferase by Spearman’s correlation analysis. In both the correlation with the expression level of luciferase-positive KM and FM3A SiLN removal groups, the expression tumor cells in both the KM and FM3A SiLN removal level of LOX showed a positive correlation with the groups (r = − 0.865, P = 0.012;r = − 0.681, P = 0.030;Table 5), expression level of luciferase (r = 0.767, P = 0.001; r =0.745, which mean that the quantity of collagen fibers around the P = 0.013;Table 3) and CD11b (r=0.733, P =0.025; r = 0.721, bronchi and blood vessels were negatively correlated with P =0.019; Table 3), respectively. The expression level of the tumor metastatic foci burden. CD11b showed a positive correlation with the expression level of MMP-2 (r = 0.786, P = 0.036; r=0.709, P=0.022; Surgical removal of SiLN enhanced expression of LOX and Table 3). CD11b in liver We also investigated the expression of LOX and CD11b Matrix changes promotes metastatic tumor growth in the in the liver tissues (Fig. 5a, b). The situation was similar lung to the data obtained in the lungs. The expression levels MMP-2 is known for cleaving extracellular matrix. Using of LOX and CD11b in the liver in all of the KM and Elastic Masson staining and silver impregnation method, FM3A SiLN removal groups were significantly higher we observed the changes of collagen fibers and reticular fi- than in the corresponding control group (P < 0.001, bers. With EM staining, blue-green collagen fibers were Table 6). determined to be evenly located around the bronchi and blood vessels in the control group. Collagen fibers located around the bronchi and blood vessels in the SiLN removal Anti-tumor response in tumor-draining PALN groups were sparse and almost not found. Positive To observe morphological and histological changes in silver-stained reticular fibers were black. The reticular fi- PALN, we performed HE staining. Tumor cells flowed bers in the lungs in both control groups were slender, into the subcapsular sinus of the PALN 3 and 7 days loosely arranged, and interwoven into networks. after inoculation in the KM and FM control groups, Fig. 4 Location and expression of collagen fibers and reticular fibers. Location and expression of collagen fibers (left panel) and reticular fibers (right panel) were analyzed by Elastic-Masson (EM) staining and silver impregnation method in the KM (a) and FM3A (b) groups. Bar: 100 μm Zheng et al. BMC Cancer (2018) 18:608 Page 8 of 12 Table 4 Per area of collagen fibers and reticular fiber in lung cytokines or changes of tumor microenvironment [23]. To explain the presence of postoperative tumor metastasis, it is Collagen fibers Reticular fibers necessary to find the appropriate in vivo model, thereby per area (%) P value per area (%) P value a a gaining an in-depth understanding of the mechanism of KM group G-C 0.0148(0.0045) 0.008 0.0118(0.0018) 0.001 tumor removal-induced metastasis in order to develop ef- b b G-D3 0.005(0.001) 0.464 0.0209(0.0013) 0.008 fective treatment options and to improve metastatic cancer c c G-D6 0.0043(0.0021) 0.002 0.026(0.005) 0.008 prevention and treatment. a a FM3A group G-C 0.038(0.009) 0.001 0.025(0.004) 0.001 Our IVIS results showed that the surgical removal d d G-D3 0.024(0.007) 0.006 0.044(0.008) 0.019 of tumor-bearing SiLN promoted the incidence of c c lung metastasis (Fig. 1). Further, immunohistochemis- G-D7 0.007(0.003) 0.003 0.054(0.012) 0.002 try showed that luciferase-positive tumor cells were P < 0.05 was considered significant; Data are expressed as the median (IQR) compared with G-D3 sparsely spread in the control lung tissues and was compared with G-D6 significantly lower than in the SiLN removal group. compared with G-C compared with G-D7 Although there was no significant difference in the rate of PALN metastasis between the control and respectively. As time went by, tumor cells were found in tumor-bearing SiLN removal group, the number of lymphatic sinuses and tumor nests formed (Fig. 6a, b). luciferase-positive tumor cells in PALN in the surgical Lymph nodes play a key role in orchestrating im- SiLN removal group was significantly less than the mune response against tumors. An important event is control group (Fig. 2,Table 1). the presentation of tumor antigens within the The pre-metastatic niche is defined as microenviron- tumor-deriving lymph node. Macrophages and den- ment with a series of molecular and cellular changes, dritic cells are professional antigen-presentation cells. which forms a supportive and receptive pre-metastatic Macrophages can be identified according to the ex- site and the fertile “soil” to prepare for the colonization pressions of F4/80 and CD169. We did not observe of “seed”, that is metastatic tumor cells, thereby facilitat- significant changes in the numbers of F4/80 mature ing tumor cell adhesion and growth in the distant organs macrophages between KM and FM3A control groups and promoting tumor metastasis [8]. Studies have shown and SiLN removal groups, respectively. However, we that local and high-precision radiotherapy does not in- + + found that the numbers of CD11C and CD169 cells duce direct injury, but it directly kills or stimulates the in the PALNs in both the KM and FM SiLN control tissue cells compared with surgery, which also acceler- groups were significantly higher than in the corre- ates the incidence of tumor metastasis during sponding SiLN removal groups (P < 0.001, Fig. 6c, d; anti-cancer therapy [2, 24]. Previous studies have shown Table 7). that local radiotherapy causes a stronger expression of angiogenic factors than anti-angiogenic factors and Discussion disturbs the configuration imbalance of VEGFs in the An operation to treat cancer includes resection of the pri- vascular bed of the metastatic site, which then results in mary lesion, lymphadenectomy, reconstruction, and so on. outbreak growth of dormant metastatic tumors [24, 25]. To date, surgery has been considered to be the primary Adjuvant applications of the exogenous VEFGR inhibitor therapeutic regimen for most malignancies, even though cediranib after radiotherapy restored tumor inhibition, sometimes the effect of surgery is not ideal, since the surgi- and studies have shown that using angiogenesis inhibi- cal process could increase the risk of metastases of residual tors to target the VEGF pathway or knocking out the cancer cells to other organs [1, 4, 21]. Experimental data VEGF gene in mouse models with pancreatic cancer or suggest that increased surgical stress augments cancer me- glioblastoma has anti-cancer therapeutic effects by tastasis via surgical stress-induced expression of proteinases reducing the tumor volume and prolonging survival, but in the target organ of metastasis [22]. The effect of surgery the two actions change tumor phenotypes and enhance on metastasis may be attributed to a number of factors, in- tumor invasion and metastasis [26–28]. The discontinu- cluding immunosuppression after surgical stress, action of ation of VEGF inhibitors still enhances the tumor invasion, suggesting that this treatment increases the persist- Table 5 Correlation among per area of collagen fibers, the ence of tumor invasion. This implies that not only does the expression level of luciferase and MMP-2 in lung of KM and treatment-induced trauma lead to tumor-promoting FM3A SiLN removal groups responses, but the disappearance of the tumor itself also KM group FM3A group promotes tumor metastasis during anti-cancer therapy. For collagen fibers MMP-2 P = 0.016, r = −0.847 P = 0.013, r = − 0.748 the tumor treatment targeting a single angle, tumors can collagen fibers Luciferase P = 0.012, r = − 0.865 P = 0.030, r = − 0.681 derive responses of increased invasiveness and extended P < 0.05 was considered significant; r: Spearman correlation coefficient metastasis. Therefore, a comprehensive consideration of all Zheng et al. BMC Cancer (2018) 18:608 Page 9 of 12 Fig. 5 The changes of metastatic niche in liver samples. The expression of LOX and CD11b were observed using IHC staining in the KM (a) and FM3A (b) groups. Bar: 20 μm characteristics of the tumor microenvironment may accom- The fracture of reticular fibers in the SiLN removal groups plish a radical anti-cancer treatment. facilitated tumor cell invasion, thereby accelerating lung Studies have shown that LOX secreted from hypoxic metastasis (Fig. 4, Table 5). primary tumor cells accumulates in the pre-metastatic The above results suggested that high LOX and sites; LOX is an indispensable factor for recruiting bone MMP-2 expressions and a great number of CD11b cell marrow-derived CD11b cells (i.e., immature myeloid facilitate the formation of the metastatic niche and the progenitor cells) to metastatic sites [19, 20]. LOX ex- colonization, growth, and invasion of tumor cells in the pression in SiLN removal groups was significantly higher lung. The accumulation of LOX at the metastatic site than in the control group and was positively correlated promoted CD11b BMDC adhesion, MMP-2 production, with luciferase expression. These findings are consistent and degradation of intra-pulmonary vascular collagen. with the results of previous studies [19, 20], suggesting Moreover, the morphological changes of the extracellular that high LOX expression in the metastatic sites was matrix scaffold remodeled the local microenvironment closely associated with the colonization and metastasis and enhanced tumor cell invasion. Previous studies have of tumor cells. Moreover, the number of CD11b cells in shown that BMDC induced interstitial epithelial trans- the lung and liver of the mice in the SiLN removal formation of the disseminated tumor cells [30], secreted groups was significantly increased, which was positively chemokines to enhance metastasis and nesting of tumor correlated with LOX expressions (Fig. 3, Tables 2, 3). cells [31], and lowered in vivo immune surveillance by im- Since the activation of MMP increased the invasion of munosuppression [7, 8, 32]. The current study also BMDCs [19, 29], we detected MMP-2 expression in the showed that VEGFA expression in the surgical removal lung tissues of the surgical SiLN removal groups, which groups was significantly higher than in the control group, was interestingly higher than that in the control groups and it is known that the secretion of VEGF and other an- and it was positively correlated with CD11b expression giogenic factors promote vascular angiogenesis, thereby (Fig. 3, Tables 2, 3). possibly playing a synergistic role in promoting tumor Our results showed that the degradation of the collagen metastasis. fibers around the pulmonary vessels in the lungs of mice The current study also identified LOX, CD11b, and lu- with the surgical removal of tumor-bearing SiLN was ciferase expression in the liver and showed similar expres- quite significant and the quantity of collagen fibers was sion patterns of LOX and CD11b in the lung (Fig. 5). negatively correlated with the expression of MMP-2, lead- Interestingly, although the ex vivo IVIS results showed ing to a decrease in barrier function, thereby providing a that the liver metastasis was only 5.9% in KM SiLN re- channel for the tumor cells to invade blood vessels and moval group, immunohistochemical staining showed that establishing helpful conditions for tumor cell metastasis. luciferase-positive tumor cells were scattered in liver Table 6 The expression levels of LOX and CD11b in liver KM group FM3A group Control SiLN removal P value Control SiLN removal P value IOD Median(IQR) Lox 146.78(17.19) 318.88(53.53) < 0.001 102.35(30.71) 210.76(43.29) < 0.001 CD11b 376.03(50.23) 755.90(21.10) < 0.001 319.62(33.35) 588.03(34.53) < 0.001 IOD: integral optical density; P < 0.05 was considered significant Zheng et al. BMC Cancer (2018) 18:608 Page 10 of 12 Fig. 6 Histological changes in tumor-draining PALN. a-b HE staining of PALN in mice in KM (a) and FM3A (b) control groups. As time went by, tumor cells were found in lymphatic sinuses and tumor nests formed. c-d The expression of F4/80, CD169 and CD11c in PALN were analyzed by IHC staining. Bar: (a-c) 100 μm, (d)50 μm sinusoids in all of the SiLN removal groups (Fig. 2), which subcutaneously injected tumor cells can be transported to might be associated with tumor cell types and organotro- lymph nodes (LNs) through the lymphatic flow and were pic targeting [33], unfavorable niche formation, or the phagocytosed by CD169 macrophages in the LN subcap- short observation time in this study. Further studies will sular sinus, which then directly cross-present the dead be necessary to address the above concerns. cell-associated antigens to CD8 T cells. The current study Lymph nodes are the well-known routes for lymph- found a large luciferase-positive cell influx into the sub- atic metastasis and the primary place for anti-tumor capsular sinus in the PALN of the control group. In + + immune responses. The current study observed the addition, the number of CD169 and CD11c cells in the influx of tumor cells into the lymphoid sinus of control group was significantly higher than in the SiLN re- PALN in the control group, which resulted in tumor moval groups (Fig. 6, Table 7), suggesting the production nest formation, the retention of tumor cells, and the of anti-tumor immune responses, which, to a certain ex- reduction of tumor cell metastasis to distant organs over tent, reduced the chance of tumor cell metastasis to the time (Fig. 6). A study by Asano et al. [34] showed that distant organs, such as the lung and liver. Table 7 The expression levels of F4/80, CD169 and CD11c in tumor-draining PALN KM group FM3A group Control SiLN removal P value Control SiLN removal P value IOD Median (IQR) F4/80 8431.42(2520.87) 7726.41(824.07) 0.423 5172.4(552.41) 5028.82(1072.5) 0.356 CD169 3213.51(240.77) 1026.38(281.09) < 0.001 1394.34(489.14) 622.89(254.42) < 0.001 CD11c 368.04(65.7) 179.87(80.47) < 0.001 486.99(168.8) 173.73(74.75) < 0.001 P < 0.05 was considered significant IOD integral optical density Zheng et al. BMC Cancer (2018) 18:608 Page 11 of 12 There is growing data to support that noncoding Ethics approval and consent to participate Experiments were carried out in accordance with published guidelines and the RNAs (ncRNAs) play a significant role in tumor metas- protocol was approved by the Institutional Animal Care and Use tasis [35, 36]. A diversity of ncRNAs was demonstrated Committee of Tohoku University (Permit Number: 2016BeLMO-010, to promote proliferation and metastasis of tumor cells 2014BeA-009, 2016BeA-017). [37–39]. We preformed additional microarray detection Competing interests and indicated expression differences in both miRNAs The authors declare that they have no competing interests. and lncRNAs (data not shown) to clarify the specific mechanism associated with our findings. Our study will Publisher’sNote further demonstrate certain miRNAs and lncRNAs Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. could be pre-metastatic biomarkers, prognostic tools and potential therapeutic targets. Author details Laboratory of Biomedical Engineering for Cancer, Graduate School of Biomedical Engineering, Tohoku University, 4-1 Seiryo, Aoba, Sendai, Miyagi Conclusions 2 980-8575, Japan. Department of Anatomy, Basic Medical Science College, Our mouse model with the surgical removal of the Harbin Medical University, Harbin 150081, China. Biomedical Engineering Cancer Research Center, Graduate School of Biomedical Engineering, Tohoku tumor-bearing lymph node greatly simulated the inci- University, 4-1 Seiryo, Aoba, Sendai, Miyagi 980-8575, Japan. Department of dence of tumor metastasis after surgery in clinical prac- Oral and Maxillofacial Surgery, Tohoku University Hospital, 1-1 Seiryo, Aoba, tice [4, 21] and allowed us to observe metastatic niche Sendai, Miyagi 980-8575, Japan. changes in distant organs. Our study indicates that sur- Received: 7 July 2017 Accepted: 21 May 2018 gical removal of the tumor-bearing lymph node pro- moted tumor metastasis to lung and liver; higher References expression level of LOX and a larger number of CD11b 1. Zhang Y, Zhang N, Hoffman RM, Zhao M. 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Journal

BMC CancerSpringer Journals

Published: May 30, 2018

References

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