Therapeutic efficacy of tumor-targeting Salmonella typhimurium A1-R on human colorectal cancer liver metastasis in orthotopic nude-mouse models.

Liver metastasis is the most frequent cause of death from colon and other cancers. Generally, liver metastasis is recalcitrant to treatment. The aim of this study is to determine the efficacy of tumor-targeting Salmonella typhimurium A1-R on liver metastasis in orthotopic mouse models. HT-29 human colon cancer cells expressing red fluorescent protein (RFP) were used in the present study. S. typhimurium A1-R infected HT-29 cells in a time-dependent manner, inhibiting cancer-cell proliferation in vitro. S. typhimurium A1-R promoted tumor necrosis and inhibited tumor growth in a subcutaneous tumor mouse model of HT-29-RFP. In orthotopic mouse models, S. typhimurium A1-R targeted liver metastases and significantly reduced their growth. The results of this study demonstrate the future clinical potential of S. typhimurium A1-R targeting of liver metastasis

Adjuvant treatment with tumor-targeting Salmonella typhimurium A1-R reduces recurrence and increases survival after liver metastasis resection in an orthotopic nude mouse model.

Colon cancer liver metastasis is often the lethal aspect of this disease. Well-isolated metastases are candidates for surgical resection, but recurrence is common. Better adjuvant treatment is therefore needed to reduce or prevent recurrence. In the present study, HT-29 human colon cancer cells expressing red fluorescent protein (RFP) were used to establish liver metastases in nude mice. Mice with a single liver metastasis were randomized into bright-light surgery (BLS) or the combination of BLS and adjuvant treatment with tumor-targeting S. typhimurium A1-R. Residual tumor fluorescence after BLS was clearly visualized at high magnification by fluorescence imaging. Adjuvant treatment with S. typhimurium A1-R was highly effective to increase survival and disease-free survival after BLS of liver metastasis. The results suggest the future clinical potential of adjuvant S. typhimurium A1-R treatment after liver metastasis resection

High-efficacy targeting of colon-cancer liver metastasis with Salmonella typhimurium A1-R via intra-portal-vein injection in orthotopic nude-mouse models.

Liver metastasis is the main cause of colon cancer-related death and is a recalcitrant disease. We report here the efficacy and safety of intra-portal-vein (iPV) targeting of Salmonella typhimurium A1-R on colon cancer liver metastasis in a nude-mouse orthotopic model. Nude mice with HT29 human colon cancer cells, expressing red fluorescent protein (RFP) (HT29-RFP), growing in the liver were administered S. typhimurium A1-R by either iPV (1×104 colony forming units (CFU)/100 μl) or, for comparison, intra-venous injection (iv; 5×107 CFU/100 μl). Similar amounts of bacteria were delivered to the liver with the two doses, indicating that iPV delivery is 5×103 times more efficient than iv delivery. Treatment efficacy was evaluated by tumor fluorescent area (mm2) and total fluorescence intensity. Tumor fluorescent area and fluorescence intensity highly correlated (p<0.0001). iPV treatment was more effective compared to both untreated control and iv treatment (p<0.01 and p<0.05, respectively with iPV treatment with S. typhimurium arresting metastatic growth). There were no significant differences in body weight between all groups. The results of this study suggest that S. typhimurium A1-R administered iPV has potential for peri-operative adjuvant treatment of colon cancer liver metastasis

DNA Methylation and Hydroxymethylation in Primary Colon Cancer and Synchronous Hepatic Metastasis

Colon cancer is one of the most frequent solid tumor and simultaneous diagnosis of primary colon cancer and liver metastases occurs in about one fourth of cases. The current knowledge on epigenetic signatures, especially those related to hydroxymethylation in primary cancer tissue, synchronous metastasis, and blood circulating cells is lacking. This study aimed to investigate both methylcytosine (mCyt) and hydroxymethylcytosine (hmCyt) status in the DNA of individual patients from colon cancer tissue, synchronous liver metastases, and in cancer-free colon and liver tissues and leukocytes. Patients undergoing curative surgery (n= 16) were enrolled and their laboratory and clinical history data collected. The contents of mCyt and hmCyt were determined by a liquid chromatography/mass spectrometry (LC/MS/MS) method in DNA extracted from primary colon cancer, synchronous hepatic metastatic tissues and homologous cancer-free tissues, i.e., colon and liver tissues as well as leukocytes. The mCyt and hmCyt levels were compared between cancerous and cancer-free tissues, and correlations between leukocytes and colon/liver tissues for both the mCyt and hmCyt levels were evaluated. The mCyt levels were similar in primary colon cancer and liver metastasis tissues (4.69 \ub1 0.37% vs. 4.77 \ub1 0.38%, respectively,p= 0.535), and both primary and metastatic tissues were hypomethylated compared to cancer-free colon (4.98 \ub1 0.26%). The difference in the mCyt content between cancerous and cancer-free colon tissues was significantly lower in primary colon cancer (p= 0.004), but not in liver metastasis (p= 0.148). The hmCyt content was similar in primary colon cancer compared to liver metastasis (0.035%, C.I. 0.024-0.052% versus 0.035%, C.I. 0.021-0.058%, respectively,p =0.905) and markedly depleted compared to the cancer-free colon (0.081%, C.I. 0.055-0.119%) with a statistically significant difference (p< 0.05) for both comparisons. The mCyt levels showed a borderline correlation between leukocytes and colon cancer tissue (Pearson's correlation coefficient = 0.51,p= 0.052) while no correlations were detected for the hmCyt levels. In conclusion, primary colon cancer and synchronous liver metastasis tissues showed a similar epigenetic status but were significantly hypomethylated and hypohydroxymethylated as compared to homologous cancer-free colon tissues

Extracellular Metabolic Energetics Can Promote Cancer Progression

Colon cancer progression is characterized by growth of the primary tumor in the colon followed by metastasis to distant organs. The metastatic cascade involves invasion of cells from the primary tumor into the surrounding tissue, entering into and survival of cancer cells in the circulation, arrival at the end organ and finally colonization of the end organ. The liver is the primary site of colon cancer metastatic colonization, with over 70% of colon cancer patients experiencing liver metastases. Despite current standard-of-care surgical intervention and broad spectra cytotoxic chemotherapeutics, the survival rate of patients with metastatic disease is less then 5%. A greater understanding of the biology and molecular determinants of liver colonization is therefore of great importance to the scientific and clinical community. This thesis presents unbiased approaches to identify regulators of liver metastasis in colon cancer and the elucidation of the mechanisms involved. The first part of this thesis describes the identification of two microRNAs, miR- 483-5p and miR-551a as suppressors of liver metastasis by human colon cancer cells using two parallel, complementary xenograft models of colon cancer metastasis. The first approach involved a functional library-based in vivo screen of 661 microRNAs. The second approach utilized in vivo selection of liver metastatic colon cancer cell population from poorly metastatic parental population. Functional studies revealed both microRNAs to target a common downstream effector gene, Creatine Kinase Brain (CKB). CKB was found to promote metastasis and the second part of this thesis present mechanistic studies that describe CKB-mediated modulation of intra- and extracellular energetics by colon cancer cells that contributed to colon cancer cell survival in the liver microenvironment, allowing for development of macrometastases and finally liver colonization. Further investigation identified the membrane transporter SLC6a8 as an important effector of the CKB pathway and also a promoter of colon cancer metastasis. The final part of this study reveals miR-483-5p, miR-551a, CKB and SLC6a8 to be clinically relevant across multiple patient datasets and archival patient samples. MiR-483-5p and miR-551a were found to be down-regulated in liver metastases of patients relative to primary tumors, while CKB and SLC6a8 were up-regulated. In addition, proof-of-principle therapeutic experiments involving adeno associated viral delivery of the microRNAs and small molecule inhibition of CKB and SLC6a8 demonstrated the therapeutic potential of targeting this pathway in suppressing colon cancer metastasis

The Role of Host-Tumor Interactions In Liver Metastasis of Colorectal Cancer

Colon cancer is the third most frequent cancer and the second leading cause of cancer deaths in the United States. Liver metastasis is the major cause of death in colon cancer. Successful metastases depend on productive collaborations between tumor cells and host-derived cells in the tumor microenvironment, target organ environments, and cells in the hematopoietic compartment. To identify the host-tumor interactions promoting liver metastasis and their molecular and cellular mediators, an orthotopic mouse model of liver metastasis of colon cancer was established that recapitulates all stages of tumor growth and metastasis. A highly metastatic mouse carcinoma cell line CT26-FL3 was isolated from the CT26 colon adenocarcinoma cell line by in vivo selection. The CT26-FL3 cells were found to be more proficient in inducing a metastasis-promoting host environment as compared to the parental cell line. Using this mouse model, microarray analyses were utilized to determine the genetic signature of the highly metastatic CT26-FL3 cells and the genetic changes in the liver microenvironment in mice bearing tumors from CT26-FL3 cells before and during metastasis. The results showed CT26-FL3 induced immune responses and released numerous cytokines. Furthermore, Il33 and Lcn2 were selected from the genetic signature of cancer cells and liver environment respectively as target genes to verify their roles in promoting liver metastasis of colorectal cancer

Fluorescent-Antibody Targeting of Insulin-Like Growth Factor-1 Receptor Visualizes Metastatic Human Colon Cancer in Orthotopic Mouse Models.

Fluorescent-antibody targeting of metastatic cancer has been demonstrated by our laboratory to enable tumor visualization and effective fluorescence-guided surgery. The goal of the present study was to determine whether insulin-like growth factor-1 receptor (IGF-1R) antibodies, conjugated with bright fluorophores, could enable visualization of metastatic colon cancer in orthotopic nude mouse models. IGF-1R antibody (clone 24-31) was conjugated with 550 nm, 650 nm or PEGylated 650 nm fluorophores. Subcutaneous, orthotopic, and liver metastasis models of colon cancer in nude mice were targeted with the fluorescent IGF-1R antibodies. Western blotting confirmed the expression of IGF-1R in HT-29 and HCT 116 human colon cancer cell lines, both expressing green fluorescent protein (GFP). Labeling with fluorophore-conjugated IGF-1R antibody demonstrated fluorescent foci on the membrane of colon cancer cells. Subcutaneously- and orthotopically-transplanted HT-29-GFP and HCT 116-GFP tumors brightly fluoresced at the longer wavelengths after intravenous administration of fluorescent IGF-1R antibodies. Orthotopically-transplanted HCT 116-GFP tumors were brightly labeled by fluorescent IGF-1R antibodies such that they could be imaged non-invasively at the longer wavelengths. In an experimental liver metastasis model, IGF-1R antibodies conjugated with PEGylated 650 nm fluorophores selectively highlighted the liver metastases, which could then be non-invasively imaged. The IGF-1R fluorescent-antibody labeled liver metastases were very bright compared to the normal liver and the fluorescent-antibody label co-located with green fluorescent protein (GFP) expression of the colon cancer cells. The present study thus demonstrates that fluorophore-conjugated IGF-1R antibodies selectively visualize metastatic colon cancer and have clinical potential for improved diagnosis and fluorescence-guided surgery

Improved Resection and Outcome of Colon-Cancer Liver Metastasis with Fluorescence-Guided Surgery Using In Situ GFP Labeling with a Telomerase-Dependent Adenovirus in an Orthotopic Mouse Model.

Fluorescence-guided surgery (FGS) of cancer is an area of intense development. In the present report, we demonstrate that the telomerase-dependent green fluorescent protein (GFP)-containing adenovirus OBP-401 could label colon-cancer liver metastasis in situ in an orthotopic mouse model enabling successful FGS. OBP-401-GFP-labeled liver metastasis resulted in complete resection with FGS, in contrast, conventional bright-light surgery (BLS) did not result in complete resection of the metastasis. OBP-401-FGS reduced the recurrence rate and prolonged over-all survival compared with BLS. In conclusion, adenovirus OBP-401 is a powerful tool to label liver metastasis in situ with GFP which enables its complete resection, not possible with conventional BLS

根治切除後の結腸癌StageⅡ、StageⅢ、及び大腸癌肝転移における原発部位が与える影響

Purpose: Primary tumor location of colon cancer has been reported to affect the prognosis after curative resection. However, some reports suggested the impact was varied by tumor stage. This study analyzed the prognostic impact of the sidedness of colon cancer in stages II, III, and liver metastasis after curative resection using propensity-matched analysis. Methods: Right-sided colon cancer was defined as a tumor located from cecum to splenic flexure, while any more distal colon cancer was defined as left-sided colon cancer. Patients who underwent curative resection at Nara Medical University hospital between 2000 and 2016 were analyzed. Results: There were 110 patients with stage II, 100 patients with stage III, and 106 patients with liver metastasis. After propensity matching, 28 pairs with stage II and 32 pairs with stage III were identified. In the patients with stage II, overall survival (OS) and recurrence-free survival (RFS) were not significantly different for right- and left-sided colon cancers. In the patients with stage III, OS and RFS were significantly worse in right-sided colon cancer. In those with liver metastasis, OS of right-sided colon cancer was significantly worse than left-sided disease, while RFS was similar. Regarding metachronous liver metastasis, the difference was observed only in the patients whose primary colon cancer was stage III. In each stage, significantly higher rate of peritoneal recurrence was found in those with right-sided colon cancer. Conclusion: Sidedness of colon cancer had a significant and varied prognostic impact in patients with stage II, III, and liver metastasis after curative resection.博士（医学）・乙第1495号・令和3年3月15日Copyright © 2021 The Korean Society of Coloproctology.This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/4.0/) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited

Targeting delivery of lipocalin 2-engineered mesenchymal stem cells to colon cancer in order to inhibit liver metastasis in nude mice

One of the major obstacles in cancer therapy is the lack of anticancer agent specificity to tumor tissues. The strategy of cell-based therapy is a promising therapeutic option for cancer treatment. The specific tumor-oriented migration of mesenchymal stem cells (MSCs) makes them a useful vehicle to deliver anticancer agents. In this study, we genetically manipulated bone marrow-derived mesenchymal stem cells with their lipocalin 2 (Lcn2) in order to inhibit liver metastasis of colon cancer in nude mice. Lcn2 was successfully overexpressed in transfected MSCs. The PCR results of SRY gene confirmed the presence of MSCs in cancer liver tissue. This study showed that Lcn2-engineered MSCs (MSC-Lcn2) not only inhibited liver metastasis of colon cancer but also downregulated the expression of vascular endothelial growth factor (VEGF) in the liver. Overall, MSCs by innate tropism toward cancer cells can deliver the therapeutic agent, Lcn2, and inhibit cancer metastasis. Hence, it could be a new modality for efficient targeted delivery of anticancer agent to liver metastasis. © 2015, International Society of Oncology and BioMarkers (ISOBM)