The Influence Of Local Inflammation In Target Organs For Tumor Metastasis Of 4T1-Inoculated Mouse Model

Background and ObjectiveMetastasis, the spread of invasive carcinoma to sites distant from the primary tumor, is responsible for the majority of cancer-related deaths. Several discrete steps are discernable in the biological cascade of metastasis:loss of cellular adhesion, increased motility and invasiveness, intravasation, survival in the circulation, extravasation, exit into new tissue, and eventual colonization of a distant site. Previous observations and studies have demonstrated that tumors are endowed with the tendency of metastasis to some specific organs. One example is the predilection of colon cancer to spread to liver whereas breast, prostate, and lung carcinomas have a particular affinity to target and proliferate in bone. This phenomenon was explained by Stephen Paget with the "seed and soil" hypothesis. In 1889, Stephen Paget observed that circulating tumor cells, which mean "seed", would only seed there congenial "soil". Since then, attention has focused on explaining the dynamic adhesive and migratory capabilities intrinsic to tumor cells. In 2005, Prof. Rosandra Kaplan’s work characterizing the importance of bone marrow-derived hematopoietic progenitor cells (HPC) in initiating these early changes has opened new avenues for cancer research and chemotherapeutic targeting. VEGFR1-positive haematopoietic bone marrow progenitors initiate the pre-metastatic niche. Bone marrow-derived hematopoietic progenitor cells (HPCs) recently emerged as key players in initiating these early changes, creating a receptive microenvironment at designated sites for distant tumor growth and establishing the "Pre-Metastatic Niche". Consequently, there are more and more researchers begin to show interest in this area.Breast cancer is the most common type of cancer and the primary cause of cancer mortality in women. The majority of deaths from breast cancer are not due to the primary tumor itself, but are the result of metastasis to other organs in the body. Breast cancer can metastasize to multiple organ, especially lung, bone and liver, which are the most common involved targets. Why breast cancer is apt to "seed" the above three organs? Before Kaplan, there have been many studies trying to find a reasonable explanation. One of the classic perspective is based on intrinsic instability of cancer genomes in the form of DNA mutations, chromosomal rearrangements, and epigenetic alterations. Another explanation is from what we call the receptor and the ligand theory. Kaplan’s "Pre-Metastatic Niche" hypothesis open a new train of thought for the organic tendency of metastasis.Moreover, exploring the earliest events promoting circulating cancer cells to engraft and establish at secondary sites may expose new targets for diagnostic and therapeutic strategies and reduce the morbidity and mortality from metastatic disease. Understanding the cellular and molecular cross-talk between "seed" and "soil" may further our understanding of the factors that govern both site-specific patterning in metastasis and the phenomenon of tumor dormancy. Depending on the previous studies, we have perceived a special kind of function of micro-inflammation reaction in pre-metastatic 4T1-inoculated mouse lung. Our explorations in this research include two separated parts which are closely related on the logic:to observe the formation of pre-metastatic inflammation and find a possible reason to interpret its formation.Part IInflammation reaction arise in target organs of 4T1-inoculated mouse model during the pre-metastatic phaseobjective1. To insure the "pre-metastatic phase" of 4T1-inoculated mouse.2. To detect the inflammation reaction and the permeability change in the lung tissue of 4T1-inoculated mouse.3. To detect the inflammation reaction in the liver of 4T1-inoculated mouse.Method1. Building a 4T1-inoculated mouse model. Female BALB/c mice (6-8 week old) were used for group study. A total of 105 4T1 cells (>90% viability) were suspended in 0.1 ml of serum-free DMEM and injected into coupled abdominal mammary glands of each BALB/c mice. Isometric amounts of PBS were injected in the same way as a control.2. Insurance of the "pre-metastatic phase". One week after 4T1 inoculation, dissections were made for every 2-4 days to detect the change of target organs. RFP-4T1 cells were injected into female BALB/c mice instand of normal 4T1 cells and their lung tissues were cryosectioned and observed under fluorescence microscopy.3. Detecting the inflammation reaction by reading the HE staining.4. Permeability assay. Mice were injected i.v. with rhodamine-conjugated dextran (100 mg/kg). After 4 h, mice were injected i.v. with FITC-lectin at 5 mg/kg. Thirty minutes later, mice were anesthetized and perfused with saline. Lung tissues were cryosectioned and examined under a LSM 700 confocal microscope. All images were captured under identical laser settings.5. Detecting the expression of tight junction proteins by Western blot and PCR.6. Immunofluorescence were used to examine the changes of tight junction proteins.Result1. A natural process of developed 4Tl-inoculated mouse. One week after 4T1 cell injection, soya bean sized tumor nodules could be find symmetrically under the forth mammary gland.3 days later, they grown to a bean size, and local inflammation were found in some of the mouse lung, and the proportion soon rose to 50% at day 12. Eventually, metastatic tumors appeared after 14 days of inoculation, then most mouse lung showed inflammation reactions at day 20. RFP-4T1 cells helped us to further determine the pre-metastatic phase by observing fluorescence slice under microscope. H&E staining showed accumulated inflammation cells and increased pulmonary vasculature, which exhibited a typical inflammation representation.2. Permeability changed in pre-metastatic phase. To examine the vascular integrity of the pre-metastatic lung, the pulmonary vascular permeability in the 4T1 inoculated mice and control mice were analyzed by rhodaminedextran. After injection into the tail vein, circulating dextran was eliminated with saline perfusion and the pulmonary vasculature was then represented by i.v. injected FITC-lectin. Vascular integrity was represented by the diffused rhodamine-dextran. We found that more rhodamine-dextran was released from blood vessels in 4T1 inoculated mice versus control mice, which indicated that the pulmonary vasculature was destabilized and permeability was increased.3. Decreased expressions of tight junction proteins. Tight junction integrity was evaluated by detecting the three main proteins of tight junctions:ZO-1, claudin-5, and occludin by western blotting and PCR to identify their expression in mouse lung. In addition, three other claudin proteins, claudin-1,-4, and-7, whose expression has been reported in alveolar epithelial cells, were also detected. E-cadherin, VE-cadherin, and b-catenin were tested to determine the change in the adhere junctions. Western blotting showed a down regulation of ZO-1 and occludin. The loss of both occluding and ZO-1, but not claudin-5 were significant at the mRNA level, whereas claudin-1,-4,-7, and adhere junction proteins showed no significant changes as compared to the control group. The dislocation of ZO-1 and occluding expression in the distal airway of the pre-metastatic lung were observed under fluorescence microscopy.4. Inflammation in the liver of 4T1 inoculated mouse.14 days after 4T1 cell injection, microscopic changes were found in the liver of 4T1 inoculated mouse. 16 days after 4T1 cell injection, inflammation first been observed in liver.Conclusion1. The identification of pre-metastatic phase of 4T1-inoculated mouse:day10-12 after 4T1 inoculation.2. Inflammation reaction arise in lung and liver of pre-metastatic mouse.3. Primary breast tumor induces increased pulmonary vascular permeability.4. Tight junctions are disrupted in the pre-metastatic lung.5. Liver inflammation appeared later than lung inflammation; E-selectin is positive in liver of pre-metastatic phase.Part ⅡThe Mechanism and Function of Pre-metastatic Inflammationobjective1. To detect the possible cytokines in mouse serum which are related to the pre-metastatic inflammation.2. To examine the possible relations between inflammation cytokines and disrupted tight junctions.3. To examine the possible relations between inflammation cytokines and reduced vascular permeability.4. To examine the possible relations between disrupted tight junctions and reduced vascular permeability.5. To find a possible VEGF signal pathway that induces the disruption of tight junctions.Method1. Protein arrays and ELISA were used to detect the possible cytokines in mouse serum during the pre-metastatic phase.2. Isolation and culture of mouse pulmonary vascular endothelial cells (MPVECs) and cell viability assay.3. VEGF was used to stimulate MPVECs, then Western blot and PCR was used to detect the expression of tight junction proteins. The distribution of tight junctions on MPVECs were observed by immunofluorescence.4. Transwell test was used to detect the endothelial permeability of MPVECs after the treatment of VEGF.5. Western blot was used to examine the change of VEGF pathway, before and after the treatment of inhibitors.Result1. The level of VEGF significantly increased in serum. Cytokines in mouse serum were measured by a protein array. This assay revealed a significantly increased VEGF level in the 4T1 inoculated mice, which was the preponderant result when compared to the levels of TNF-a, MCSF, and IL-6 (P<0.05). This result was confirmed by an ELISA assay.2. ZO-1 decreased in VEGF treated MPVECs. Results of both Western blot and qPCR demonstrated that the expression of ZO-1 significantly decreased in VEGF treated MPVECs. Immunofluorescence of MPVECs showed the distribution of ZO-1 was disrupted between cell-cell connections.3. Endothelial permeability increased in VEGF treated MPVECs by transwell test. For in vitro experiments, primary cultured MPVECs grown on transwell chambers were incubated with VEGF for varying times and concentrations to identify the function of VEGF on promoting pulmonary vascular permeability. We found that VEGF significantly increased cell permeability at a concentration of 10 ng/ml, and this change was statistically significant after 4 h of treatment (P<0.05).4. PKC was a possible pathway that influence the expression of ZO-1. Among various downstream pathways of VEGF, the PKC pathway has been reported in numerous studies to associate with the regulation of the tight junctions of the epithelium monolayer. To examine the effect of the PKC pathway in regulating tight junction proteins, MPVECs were incubated with the PKC inhibitor bisindolylmaleimide I. The expression of ZO-1, claudin-5, and occludin was determined by western blotting and PCR. Western blot showed that bisindolylmaleimide I effectively attenuated the influence of VEGF on the expression of ZO-1 and occludin, but not claudin-5. The PCR results demonstrated that the inhibition of phosphorylated PKC increased the mRNA level of ZO-1 and occludin but had little influence on the expression of claudin-5.Conclusion1. Inoculated 4T1 cells release abundant VEGF into the circulation of mouse during the pre-metastatic phase, leading to a relative high level of VEGF in serum.2. The high level of VEGF induces a decrease of ZO-1, which disrupt the tight junctions of endothelial cells and result in increased permeability.3. VEGF stimulation decreases the expression of ZO-1 by PKC pathway.