Model for mediastinal lymph node metastasis produced by orthotopic intrapulmonary implantation of lung cancer cells in mice

This study is designed to establish a pulmonary tumor model to investigate the biology and therapy of lung cancer in mice. Current methods for forming a solitary intrapulmonary nodule and subsequent metastasis to mediastinal lymph nodes are not well defined. Lewis lung carcinoma cell (LLC) suspensions were orthotopically introduced into the lung parenchyma of C57/BL6 mice via a limited skin incision without thoracotomy followed by direct puncture through the intercostal space. The implantation process was performed within approximately 50 sec per mouse, and the operative mortality was less than 5%. Single pulmonary nodules developed at the implanted site in 93% of animals and subsequent mediastinal lymph nodes metastasis were observed in all mice that were succeeded to form a lung nodule after intrapulmonary implantation. The size of tumor nodule and the weight of mediastinal lymph node increased in a time-dependent manner. The mean survival time of mice implanted successfully with LLC cells was 21 +/- 2 days (range; 19-24 days). Histopathological analysis revealed that no metastatic tumor was detectable in the mediastinal lymph nodes on day 11, but metastatic foci at mediastinal lymph nodes were clearly observed on days 17 and 21 after implantation. Other metastases in distant organs or lymph nodes were not observed at 21 days after the implantation. Comparative studies with intrapleural and intravenous injections of LLC cells suggest that the mediastinal lymph node metastasis by intrapulmonary implantation is due to the release of tumor cells from the primary nodule, and not due to extrapulmonary leakage of cells. An intravenous administration of CDDP on day 1 after tumor implantation tended to suppress the primary tumor nodule and significantly inhibited the lymph node metastasis. Thus, a solitary pulmonary tumor nodule model with lymph node metastasis approximates clinical lung cancer, and may provide a useful basis for lung cancer research.     

Cancer metastasis directly eradicated by targeted therapy with a modified Salmonella typhimurium

Cancer metastasis is the life‐threatening aspect of cancer and is usually resistant to standard treatment. We report here a targeted therapy strategy for cancer metastasis using a genetically‐modified strain of Salmonella typhimurium. The genetically‐modified strain of S. typhimurium is auxotrophic for the amino acids arginine and leucine. These mutations preclude growth in normal tissue but do not reduce bacterial virulence in cancer cells. The tumor‐targeting strain of S. typhimurium, termed A1‐R, and expressing green fluorescent protein (GFP), was administered to both axillary lymph and popliteal lymph node metastasis of human pancreatic cancer and fibrosarcoma, respectively, as well as lung metastasis of the fibrosarcoma in nude mice. The bacteria were delivered via a lymphatic channel to target the lymph node metastases and systemically via the tail vein to target the lung metastasis. The cancer cells expressed red fluorescent protein (RFP) in the cytoplasm and GFP in the nucleus linked to histone H2B, enabling color‐coded real‐time imaging of the bacteria targeting the metastatic tumors. After 7–21 days of treatment, the metastases were eradicated without the need of chemotherapy or any other treatment. No adverse effects were observed. This new strategy demonstrates the clinical potential of targeting and curing cancer metastasis with engineered bacteria without the need of toxic chemotherapy. J. Cell. Biochem. 106: 992–998, 2009. © 2009 Wiley‐Liss, Inc.     

Real‐time imaging of single cancer‐cell dynamics of lung metastasis

We have developed a new in vivo mouse model to image single cancer‐cell dynamics of metastasis to the lung in real‐time. Regulating airflow volume with a novel endotracheal intubation method enabled controlling lung expansion adequate for imaging of the exposed lung surface. Cancer cells expressing green fluorescent protein (GFP) in the nucleus and red fluorescent protein (RFP) in the cytoplasm were injected in the tail vein of the mouse. The right chest wall was then opened in order to image metastases on the lung surface directly. After each observation, the chest wall was sutured and the air was suctioned in order to re‐inflate the lung, in order to keep the mice alive. Observations have been carried out for up to 8 h per session and repeated up to six times per mouse thus far. The seeding and arresting of single cancer cells on the lung, accumulation of cancer‐cell emboli, cancer‐cell viability, and metastatic colony formation were imaged in real‐time. This new technology makes it possible to observe real‐time monitoring of cancer‐cell dynamics of metastasis in the lung and to identify potential metastatic stem cells. J. Cell. Biochem. 109: 58–64, 2010. © 2009 Wiley‐Liss, Inc.     

Prostate Cancer Heterogeneous High-Metastatic Multi-Organ-Colonizing Chemo-Resistant Variants Selected by Serial Metastatic Passage in Nude Mice Are Highly Enriched for Multinucleate Giant Cells

In order to further understand the role of tumor heterogeneity in metastasis and chemo-resistance, high metastatic PC-3 human prostate cancer variants were selected by injecting parental PC-3 cells, expressing green fluorescent protein (GFP) in the footpad of nude mice, which then metastasize to inguinal lymph nodes. The PC-3-GFP cells which metastasized to the inguinal lymph nodes were collected and were re-injected to the footpad. After 6 such cycles, the PC-3-GFP cells collected from inguinal lymph nodes (PC-3-GFP-LN) were again injected to the footpad. PC-3-GFP-LN showed 100% metastasis to major lymph nodes (popliteal, inguinal, axillary, and cervical), and 100% metastasis to bone and lung. The percent of giant cell variants was enriched in PC-3-GFP-LN-6 compared to parental cells and increased with each cycle of selection, which in turn had increased metastasis. PC-3-GFP-LN-6 cells were resistant to 5-fluorouracil, doxorubicin and cisplatinum, compared to parental PC-3. However, PC-3-GFP-LN-6 was sensitive to the traditional Chinese medicine (TCM) herbal mixture LQ, similar to the parental cells. These results suggest that PC-3 tumors are heterogenous and that subpopulations of highly metastatic, drug-resistant cells can be step-wise selected using a mouse model of tumor progression.     

Spread of herpes simplex virus in lymph nodes after experimental infection of mice

Herpes simplex virus was frequently isolated from ipsilateral popliteal lymph nodes after percutaneous inoculation of the dorsal face of the footpad, and from ipsi- and contralateral submandibular lymph nodes after percutaneous inoculation of the cheek or the orofacial area of mice. Virus was detected only on very rare occasion in nondraining lymph nodes (inguinal or axillary) or in contralateral popliteal lymph nodes, but was frequently isolated in contralateral lumbar lymph nodes after footpad inoculation. The presence of virus in lymph nodes paralleled or followed the invasion of ipsilateral sensory ganglia and was associated with dissemination of virus in contralateral sensory ganglia after unilateral inoculation. In older mice virus was detected only occasionally in lymph nodes and dissemination of virus in contralateral sensory ganglia was generally not observed. The results suggest that lymphatic spread may contribute to dissemination of virus in contralateral sensory ganglia after unilateral inoculation of mice.     

Visualization of GFP-expressing tumors and metastasis in vivo

We have developed mouse models of metastatic cancer with genetically fluorescent tumors that can be imaged in fresh tissue, in situ, as well as externally. To achieve this capability, we have transduced the green fluorescent protein (GFP) gene, cloned from the bioluminescent jellyfish Aequorea victoria, into a series of human and rodent cancer cell lines that were selected in vitro to stably express GFP in vivo after transplantation to metastatic rodent models. Techniques were also developed for transduction of tumors by GFP in vivo. With this fluorescent tool, we detected and visualized for the first time tumors and metastasis in fresh viable tissue or in situ in host organs down to the single-cell level. GFP tumors on the colon, prostate, breast, brain, liver, lymph nodes, lung, pancreas, bone, and other organs can also be visualized externally, transcutaneously by quantitative whole-body fluorescence optical imaging. Real-time tumor and metastatic growth and angiogenesis and inhibition by representative drugs can be imaged and quantified for rapid antitumor, antimetastatic, and antiangiogenesis drug screening. The GFP-transfected tumor cells enabled a fundamental advance in the visualization of tumor growth and metastasis in real time in vivo.     

Induction of tumor lymphangiogenesis by VEGF-C promotes breast cancer metastasis

Metastasis of breast cancer occurs primarily through the lymphatic system, and the extent of lymph node involvement is a key prognostic factor for the disease. Whereas the significance of angiogenesis for tumor progression has been well documented, the ability of tumor cells to induce the growth of lymphatic vessels (lymphangiogenesis) and the presence of intratumoral lymphatic vessels have been controversial. Using a novel marker for lymphatic endothelium, LYVE-1, we demonstrate here the occurrence of intratumoral lymphangiogenesis within human breast cancers after orthotopic transplantation onto nude mice. Vascular endothelial growth factor (VEGF)-C overexpression in breast cancer cells potently increased intratumoral lymphangiogenesis, resulting in significantly enhanced metastasis to regional lymph nodes and to lungs. The degree of tumor lymphangiogenesis was highly correlated with the extent of lymph node and lung metastases. These results establish the occurrence and biological significance of intratumoral lymphangiogenesis in breast cancer and identify VEGF-C as a molecular link between tumor lymphangiogenesis and metastasis.     

Inhibition of VEGF-C Modulates Distal Lymphatic Remodeling and Secondary Metastasis

Tumor-associated lymphatics are postulated to provide a transit route for disseminating metastatic cells. This notion is supported by preclinical findings that inhibition of pro-lymphangiogenic signaling during tumor development reduces cell spread to sentinel lymph nodes (SLNs). However, it is unclear how lymphatics downstream of SLNs contribute to metastatic spread into distal organs, or if modulating distal lymph transport impacts disease progression. Utilizing murine models of metastasis, longitudinal in vivo imaging of lymph transport, and function blocking antibodies against two VEGF family members, we provide evidence that distal lymphatics undergo disease course-dependent up-regulation of lymph transport coincidental with structural remodeling. Inhibition of VEGF-C activity with antibodies against VEGF-C or NRP2 prevented these disease-associated changes. Furthermore, utilizing a novel model of adjuvant treatment, we demonstrate that antagonism of VEGF-C or NRP2 decreases post SLN metastasis. These data support a potential therapeutic strategy for inhibiting distant metastatic dissemination via targeting tumor-associated lymphatic remodeling.     

肺癌组织中岩藻糖化糖链结构免疫组化研究

Lewis X(Le~x)、唾液酸化的Lewis X(Sialyl Lewis X,SLe~x)和唾液酸化的双岩藻糖Lewis X(Sialyl Dimeric Lewis X,SDLe~x)是细胞表面外侧带α1,3岩藻糖的糖链结构。本文用免疫组化ABC法研究了肺癌原发灶、转移灶和癌旁组织中这三种抗原结构的表达。结果发现这三种抗原在肺癌细胞表面及胞浆中均有不同程度的表达,而在肺癌癌旁组织及正常肺组织中未见表达。有转移的肺癌和(或)低分化肺癌中这三种抗原结构的表达要明显高于未发生转移和高、中分化肺癌中相同抗原结构的表达。其中以SLe~x的表达与肺癌细胞的转移能力和分化程度关系最为密切。另外,肺癌浸润转移的淋巴结中也有Le~x、SLe~x的明显表达和SDLe~x的少量表达,而未被肺癌浸润转移的淋巴结中就没有它们的表达。     

Vascular endothelial growth factor-D is a key molecule that enhances lymphatic metastasis of soft tissue sarcomas

Studies on lymph node metastasis of soft tissue sarcomas are insufficient because of its rarity. In this study, we examined the expressions of vascular endothelial growth factor (VEGF)-C and VEGF-D in soft tissue sarcomas metastasized to lymph nodes. In addition, the effects of the two molecules on the barrier function of a lymphatic endothelial cell monolayer against sarcoma cells were analyzed. We examined 7 patients who had soft tissue sarcomas with lymph node metastases and who had undergone neither chemotherapy nor radiotherapy before lymphadenectomy. Immunohistochemistry revealed that 2 of 7 sarcomas that metastasized to lymph nodes expressed VEGF-C both in primary and metastatic lesions. On the other hand, VEGF-D expression was detected in 4 of 7 primary and 7 of 7 metastatic lesions, respectively. Interestingly, 3 cases that showed no VEGF-D expression at primary sites expressed VEGF-D in metastatic lesions. Recombinant VEGF-C at 10(-8) and VEGF-D at 10(-7)and 10(-8)g/ml significantly increased the random motility of lymphatic endothelial cells compared with controls. VEGF-D significantly increased the migration of sarcoma cells through lymphatic endothelial monolayers. The fact that VEGF-D induced the migration of fibrosarcomas through the lymphatic endothelial monolayer is the probable reason for the strong relationship between VEGF-D expression and lymph node metastasis in soft tissue sarcomas. The important propensities of this molecule for the increase of lymph node metastases are not only lymphangiogenesis but also down-regulation of the barrier function of lymphatic endothelial monolayers, which facilitates sarcoma cells entering the lymphatic circulation.     

CD147 depletion down-regulates matrix metalloproteinase-11, vascular endothelial growth factor-A expression and the lymphatic metastasis potential of murine hepatocarcinoma Hca-F cells

Extracellular matrix metalloproteinase inducer (EMMPRIN, CD147), which is a plasma membrane glycoprotein enriched on the surface of many malignant tumors promotes adhesion, invasion and metastasis of tumor cells. In addition, tumor-associated CD147 also induces vascular endothelial growth factors (VEGFs) expression. To investigate the possible role of CD147 in the mouse hepatocarcinoma cell line Hca-F with highly metastatic potential in the lymph nodes, we used an RNA interference (RNAi) approach to silence CD147 expression. The results showed that CD147 depletion in Hca-F cells resulted in the significantly decreased expression of matrix metalloproteinase-11 (MMP-11), VEGF-A at both mRNA and protein levels. The reduced CD147 expression also attenuated the invasive, adhesive, metastatic ability of Hca-F cells to lymph nodes both in vitro and in vivo. Our current findings reveal that the tumor biological marker CD147 functionally mediates MMP-11, VEGF-A expression and tumor lymphatic metastasis.     

Lymphatic Territories (Lymphosomes) in a Canine: An Animal Model for Investigation of Postoperative Lymphatic Alterations

Background

Lymph node dissection is often performed as a part of surgical treatment for breast cancer and malignant melanoma to prevent malignant cells from traveling via the lymphatic system. Currently little is known about postoperative lymphatic drainage pattern alterations. This knowledge may be useful for management of recurrent cancer and prevention of breast cancer related lymphedema. We mapped the complete superficial lymphatic system of a dog and used this canine model to perform preliminary studies of lymphatic architectural changes in postoperative condition.

Methods

Lymphatic territories (lymphosomes) were mapped with 4 female mongrel carcasses using an indocyanine green (ICG) fluorescent lymphography and a radiographic microinjection technique. Two live dogs were then subjected to unilateral lymph node dissection of lymph basins of the forelimb, and ICG lymphography and lymphangiogram were performed 6 months after the surgery to investigate lymphatic changes. Lymphatic patterns in the carcass were then compared with postoperative lymphatic patterns in the live dogs.

Results

Ten lymphosomes were identified, corresponding with ten lymphatic basins. Postoperative fluorescent lymphographic images and lymphangiograms in the live dogs revealed small caliber lymphatic network fulfilling gaps in the surgical area and collateral lymphatic vessels arising from the network connecting to lymph nodes in the contralateral and ipsilateral neck in one dog and the ipsilateral subclavicular vein in another dog.

Conclusion

Our canine lymphosome map allowed us to observe lymphatic collateral formations after lymph node dissection in live dogs. This canine model may help clarify our understanding of postoperative lymphatic changes in humans in future studies.     

Immunohistochemical study of the expression of fucosylated glycoconjugates in human lung carcinoma]

Lewis X, Sialyl Lewis X and Sialyl Dimeric Lewis X are three fucosylated glycoconjugates on cell surface. With immunohistochemical method, the expression of the three structures in the original lung cancer tissues (with or without metastasis), adjacent tissues and metastatic lesions of lung carcinoma were studied. It was found that the three antigens were expressed with different intensity on the cell surface and in the cytoplasm of the lung carcinoma cell. However, there was no or only trace expression of these antigens in the adjacent tissues of lung carcinoma and normal lung tissues. Moreover, the original lesions of lung carcinoma with metastasis and/or poor differentiation expressed higher level of the three antigens than those without metastasis and/or with well or medium differentiation. Sialyl Lewis X was considered to be more closely related to the metastatic ability and differentiation of the lung carcinoma cell than the other two antigens, Lewis X and Sialyl Dimeric Lewis X. Furthermore, in the lymph nodes with lung carcinoma cell metastasis, there were expression of the three antigens with different degree, while in those lymph nodes without lung carcinoma cell metastasis, there was no expression of the three antigens.     

Involvement of an autocrine stromal cell derived factor-1/CXCR4 system on the distant metastasis of human oral squamous cell carcinoma

We have previously shown that a stromal cell-derived factor-1 (SDF-1; CXCL12)/CXCR4 system is involved in the establishment of lymph node metastasis, but not in that of distant metastasis, in oral squamous cell carcinoma (SCC). In this study, we investigated the role of the autocrine SDF-1/CXCR4 system, with a focus on distant metastasis in oral SCC cells. The immunohistochemical staining of SDF-1 and CXCR4 using primary oral SCCs and metastatic lymph nodes showed a significantly higher number of SDF-1-positive cases among the metastatic lymph nodes than among the primary oral SCCs, which was associated with a poor survival rate among those of the former group. The forced expression of SDF-1 in B88 cells, which exhibit functional CXCR4 and lymph node metastatic potential (i.e., the autocrine SDF-1/CXCR4 system), conferred enhanced cell motility and anchorage-independent growth potential onto the cells. Orthotopic inoculation of the transfectant into nude mice was associated with an increase in the number of metastatic lymph nodes and more aggressive metastatic foci in the lymph nodes. Furthermore, the SDF-1 transfectant (i.e., the autocrine SDF-1/CXCR4 system) exhibited dramatic metastasis to the lung after i.v. inoculation, whereas the mock transfectant (i.e., the paracrine SDF-1/CXCR4 system) did not. Under the present conditions, AMD3100, a CXCR4 antagonist, significantly inhibited the lung metastasis of the SDF-1 transfectant, ameliorated body weight loss, and improved the survival rate of tumor-bearing nude mice. These results suggested that, in cases of oral SCC, the paracrine SDF-1/CXCR4 system potentiates lymph node metastasis, but distant metastasis might require the autocrine SDF-1/CXCR4 system.     

Therapeutic differentiation and maturation of lymphatic vessels after lymph node dissection and transplantation

Surgery or radiation therapy of metastatic cancer often damages lymph nodes, leading to secondary lymphedema. Here we show, using a newly established mouse model, that collecting lymphatic vessels can be regenerated and fused to lymph node transplants after lymph node removal. Treatment of lymph node-excised mice with adenovirally delivered vascular endothelial growth factor-C (VEGF-C) or VEGF-D induced robust growth of the lymphatic capillaries, which gradually underwent intrinsic remodeling, differentiation and maturation into functional collecting lymphatic vessels, including the formation of uniform endothelial cell-cell junctions and intraluminal valves. The vessels also reacquired pericyte contacts, which downregulated lymphatic capillary markers during vessel maturation. Growth factor therapy improved the outcome of lymph node transplantation, including functional reconstitution of the immunological barrier against tumor metastasis. These results show that growth factor-induced maturation of lymphatic vessels is possible in adult mice and provide a basis for future therapy of lymphedema.     

Possible role of stromal-cell-derived factor-1/CXCR4 signaling on lymph node metastasis of oral squamous cell carcinoma

We examined the role of chemokine signaling on the lymph node metastasis of oral squamous cell carcinoma (SCC) using lymph node metastatic (HNt and B88) and nonmetastatic oral SCC cells. Of 13 kinds of chemokine receptors examined, only CXCR4 expression was up-regulated in HNt and B88 cells. CXCR4 ligand, stromal-cell-derived factor-1alpha (SDF-1alpha; CXCL12), induced characteristic calcium fluxes and chemotaxis only in CXCR4-expressing cells. CXCR4 expression in metastatic cancer tissue was significantly higher than that in nonmetastatic cancer tissue or normal gingiva. Although SDF-1alpha was undetectable in either oral SCC or normal epithelial cells, submandibular lymph nodes expressed the SDF-1alpha protein, mainly in the stromal cells, but occasionally in metastatic cancer cells. The conditioned medium from lymphatic stromal cells promoted the chemotaxis of B88 cells, which was blocked by the CXCR4 neutralization. SDF-1alpha rapidly activated extracellular signal-regulated kinase (ERK)1/2 and Akt/protein kinase B (PKB), and their synthetic inhibitors attenuated the chemotaxis by SDF-1alpha. SDF-1alpha also activated Src family kinases (SFKs), and its inhibitor PP1 diminished the SDF-1alpha-induced chemotaxis and activation of both ERK1/2 and Akt/PKB. These results indicate that SDF-1/CXCR4 signaling may be involved in the establishment of lymph node metastasis in oral SCC via activation of both ERK1/2 and Akt/PKB induced by SFKs.     

Imaging the efficacy of UVC irradiation on superficial brain tumors and metastasis in live mice at the subcellular level

The effect of UVC irradiation was investigated on a model of brain cancer and a model of experimental brain metastasis. For the brain cancer model, brain cancer cells were injected stereotactically into the brain. For the brain metastasis model, lung cancer cells were injected intra‐carotidally or stereotactically. The U87 human glioma cell line was used for the brain cancer model, and the Lewis lung carcinoma (LLC) was used for the experimental brain metastasis model. Both cancer cell types were labeled with GFP in the nucleus and RFP in the cytoplasm. A craniotomy open window was used to image single cancer cells in the brain. This double labeling of the cancer cells with GFP and RFP enabled apoptosis of single cells to be imaged at the subcellular level through the craniotomy open window. UVC irradiation, beamed through the craniotomy open window, induced apoptosis in the cancer cells. UVC irradiation was effective on LLC and significantly extended survival of the mice with experimental brain metastasis. In contrast, the U87 glioma was relatively resistant to UVC irradiation. The results of this study suggest the use of UVC for treatment of superficial brain cancer or metastasis. J. Cell. Biochem. 114: 428–434, 2013. © 2012 Wiley Periodicals, Inc.     

Spatiotemporal quantification of metastatic tumour cell growth and distribution in lymph nodes by whole-mount tissue 3D imaging

Lymph nodes (LNs) are a common site of metastasis in many solid cancers. Tumour cells can migrate to LNs for further metastatic colonization of distant organs, indicating poor prognosis and requiring different clinical interventions. The histopathological diagnostic methods currently used to detect clinical lymph node metastasis (LNM) have limitations, such as incomplete visualization. To obtain a complete picture of metastatic LNs on the spatial and temporal scales, we used ultimate 3D imaging of solvent-cleared organs (uDISCO) and 3D rapid immunostaining. MC38 cells labelled with EGFP were injected into the left footpads of C57BL/6 mice. Draining lymph nodes (DLNs) harvested from these mice were cleared using the uDISCO method. Metastatic colorectal cancer (CRC) cells in various regions of DLNs from mice at different time points were quantified using 3D imaging of whole-mount tissue. Several stages of tumour cell growth and distribution in LNs were identified: 1) invasion of lymphatic vessels (LVs) and blood vessels (BVs); 2) dispersion outside LVs and BVs for proliferation and expansion; and 3) re-entry into BVs and efferent lymphatic vessels (ELVs) for further distant metastasis. Moreover, these data demonstrated that mouse fibroblast cells (MFCs) could not only promote LNM of tumour cells but also metastasize to LNs together with tumour cells, thus providing a “soil” for tumour cell colonization. In conclusion, 3D imaging of whole-mount tissue and spatiotemporal analysis of LNM may collectively constitute an auxiliary method to improve the accuracy of clinical LNM detection.