Splenic Injection Model Research Articles

An Animal Model of Colorectal Cancer Liver Metastasis With a High Metastasis Rate and Clonal Dynamics.

Various animal models have been introduced into the study of liver metastasis of colorectal cancer, but they have not been compared under the same conditions. The aim of this study was to identify an optimized mouse model that showed a high rate of hepatic metastasis and expression of clonal dynamics. Athymic nude mice (n=30) were divided into two equal groups for the creation of a splenic injection model (SIM) and surgically orthotopic implantation model (SOIM) of liver metastasis of colorectal cancer using HCT116 cells. Hepatic metastasis was confirmed by gross and microscopic examinations. Expression of MET transcriptional regulator MACC1 (MACC1) in colon cancer cell lines and metastatic tumors in the group with a higher liver metastasis rate was confirmed by quantitative reverse-transcription-polymerase chain reaction. The observation time was significantly shorter for SIM than for SOIM (33.0±6.8 vs. 41.2±7.2 days, p<0.001). The rate of hepatic metastasis was significantly higher in SIM than in SOIM (76.9% vs. 38.4%, p=0.038). MACC1 was expressed in Colo201, HCT116, HT29, LS513, SW620, and WiDr cells but not in SW480 cells. All hepatic metastases in SIM mice expressed MACC1, and metastatic HCT116 cells had significantly greater expression than did the original HCT116 cells (p<0.001). With a higher rate of hepatic metastasis with clonal dynamics in a shorter observation time than the SOIM, SIM appears to be a good animal model for identifying new targets and in drug development for colorectal cancer liver metastasis. SOIM should also be considered for the study of the full steps of metastasis.

Development and optimization of orthotopic liver metastasis xenograft mouse models in uveal melanoma

BackgroundPatients with metastatic uveal melanoma (MUM) in the liver usually die within 1 year. The development of new treatments for MUM has been limited by the lack of diverse MUM cell lines and appropriate animal models. We previously reported that orthotopic xenograft mouse models established by direct injection of MUM cells into the liver were useful for the analysis associated with tumor microenvironment in the liver. However, considering that patients with UM metastasize to the liver hematogenously, direct liver injection model might not be suitable for investigation on various mechanisms of liver metastasis. Here, we aim to establish new orthotopic xenograft models via hematogenous dissemination of tumor cells to the liver, and to compare their characteristics with the hepatic injection model. We also determine if hepatic tumors could be effectively monitored with non-invasive live imaging.MethodstdtTomate-labeled, patient-derived MUM cells were injected into the liver, spleen or tail vein of immunodeficient NSG mice. Tumor growth was serially assessed with In Vivo Imaging System (IVIS) images once every week. Established hepatic tumors were evaluated with CT scan and then analyzed histologically.ResultsWe found that splenic injection could consistently establish hepatic tumors. Non-invasive imaging showed that the splenic injection model had more consistent and stronger fluorescent intensity compared to the hepatic injection model. There were no significant differences in tumor growth between splenic injection with splenectomy and without splenectomy. The splenic injection established hepatic tumors diffusely throughout the liver, while the hepatic injection of tumor cells established a single localized tumor. Long-term monitoring of tumor development showed that tumor growth, tumor distribution in the liver, and overall survival depended on the number of tumor cells injected to the spleen.ConclusionWe established a new orthotopic hepatic metastatic xenograft mouse model by splenic injection of MUM cells. The growth of orthotopic hepatic tumors could be monitored with non-invasive IVIS imaging. Moreover, we evaluated the therapeutic effect of a MEK inhibitor by using this model. Our findings suggest that our new orthotopic liver metastatic mouse model may be useful for preclinical drug screening experiments and for the analysis of liver metastasis mechanisms.

Inhibition of mTORC2/RICTOR Impairs Melanoma Hepatic Metastasis

Mammalian target of rapamycin complex 2 (mTORC2) with its pivotal component rapamycin-insensitive companion of mTOR (RICTOR) is the major regulator of AKT phosphorylation and is increasingly implicated in tumor growth and progression. In cutaneous melanoma, an extremely aggressive and highly metastatic disease, RICTOR overexpression is involved in tumor development and invasiveness. Therefore, we investigated the impact of RICTOR inhibition in melanoma cells in vitro and in vivo with special emphasis on hepatic metastasis. Moreover, our study focused on the interaction of tumor cells and hepatic stellate cells (HSC) which play a crucial role in the hepatic microenvironment. In silico analysis revealed increased RICTOR expression in melanoma cells and tissues and indicated higher expression in advanced melanoma stages and metastases. In vitro, transient RICTOR knock-down via siRNA caused a significant reduction of tumor cell motility. Using a syngeneic murine splenic injection model, a significant decrease in liver metastasis burden was detected in vivo. Moreover, stimulation of melanoma cells with conditioned medium (CM) from activated HSC or hepatocyte growth factor (HGF) led to a significant induction of AKT phosphorylation and tumor cell motility. Blocking of RICTOR expression in cancer cells diminished constitutive and HGF-induced AKT phosphorylation as well as cell motility. Interestingly, RICTOR blockade also led to an abrogation of CM-induced effects on AKT phosphorylation and motility in melanoma cells. In conclusion, these results provide first evidence for a critical role of mTORC2/RICTOR in melanoma liver metastasis via cancer cell/HSC interactions.

CD47 Blockade as an Adjuvant Immunotherapy for Resectable Pancreatic Cancer.

Purpose: Patients with pancreatic ductal adenocarcinoma (PDAC) who undergo surgical resection and adjuvant chemotherapy have an expected survival of only 2 years due to disease recurrence, frequently in the liver. We investigated the role of liver macrophages in progression of PDAC micrometastases to identify adjuvant treatment strategies that could prolong survival.Experimental Design: A murine splenic injection model of hepatic micrometastatic PDAC was used with five patient-derived PDAC tumors. The impact of liver macrophages on tumor growth was assessed by (i) depleting mouse macrophages in nude mice with liposomal clodronate injection, and (ii) injecting tumor cells into nude versus NOD-scid-gamma mice. Immunohistochemistry and flow cytometry were used to measure CD47 ("don't eat me signal") expression on tumor cells and characterize macrophages in the tumor microenvironment. In vitro engulfment assays and mouse experiments were performed with CD47-blocking antibodies to assess macrophage engulfment of tumor cells, progression of micrometastases in the liver and mouse survival.Results:In vivo clodronate depletion experiments and NOD-scid-gamma mouse experiments demonstrated that liver macrophages suppress the progression of PDAC micrometastases. Five patient-derived PDAC cell lines expressed variable levels of CD47. In in vitro engulfment assays, CD47-blocking antibodies increased the efficiency of PDAC cell clearance by macrophages in a manner which correlated with CD47 receptor surface density. Treatment of mice with CD47-blocking antibodies resulted in increased time-to-progression of metastatic tumors and prolonged survival.Conclusions: These findings suggest that following surgical resection of PDAC, adjuvant immunotherapy with anti-CD47 antibody could lead to substantially improved outcomes for patients. Clin Cancer Res; 24(6); 1415-25. ©2017 AACR.

Molecular characterization of pro-metastatic functions of β4-integrin in colorectal cancer.

The β4-integrin subunit has been implicated in development and progression of several epithelial tumor types. However, its role in metastases of colorectal cancer (CRC) remains elusive. To study CRC metastasis, we generated a highly invasive, metastatic cell line MC38-LM10 (LM10) by passaging mouse CRC MC38 cells ten times, using a splenic injection model of liver metastasis. Affymetrix microarray analyses of LM10 and MC38 cell lines and their corresponding liver metastases generated a gene signature for CRC metastasis. This signature shows strong upregulation of β4-integrin in LM10 cells and corresponding metastases. Upregulation of β4-integrin in highly aggressive LM10 cells is associated with increased migration, invasion, and liver metastases. Furthermore, stable knockdown of β4-integrin in human CRC SW620 cells reduces Bcl-2 expression, increases apoptosis, and decreases invasion, tumorigenicity, and liver metastasis, thus resulting in significantly increased survival of mice (hazard ratio = 0.32, 95% confidence interval = 0.15-0.66, P<0.01). Patients with CRC tumors display higher β4-integrin levels in stages 1-4 and significantly lower survival rate. Collectively, β4-integrin plays a critical role in CRC progression, invasion, and metastasis, suggesting that it could be a potential therapeutic target for CRC patients.

Elevated TIMP-1 expression is associated with a prometastatic phenotype, disease relapse, and poor survival in neuroblastoma.

Approximately two-thirds of patients with neuroblastoma are found to have metastatic disease at time of diagnosis with frequent skeletal, lymph node, central nervous system, and liver involvement. Using a serial in vivo splenic injection model, we have isolated an aggressive subclone (BE(2)-C/LM2) from MYCN-amplified neuroblastomas that demonstrate an enhanced propensity to develop metastatic liver lesions. BE(2)-C/LM2 subclone cells demonstrate increased adherent, soft agar colony and tumorsphere growth in vitro. Components of the tumor microenvironment regulate cancer progression, via networks of cytokines and growth factors. Cytokine array analysis identified increased TIMP-1 in the plasma of mice injected with BE(2)-C/LM2 subclone cells, leading us to hypothesize that TIMP-1 may play a role in our observed prometastatic phenotype. Immunoblotting and ELISA demonstrated enhanced endogenous TIMP-1 expression in our isolated neuroblastoma subclone. Silencing endogenous TIMP-1 successfully blocked in vitro proliferation, soft agar colony formation and tumorsphere formation by BE(2)-C/LM2 cells. Stable RNA interference of endogenous TIMP-1 failed to reverse the prometastatic phenotype of our BE(2)-C/LM2 subclone in our liver metastasis model, suggesting that endogenous TIMP-1 levels may not be an essential component of this in vivo behavior. Notably, tissue microarray analysis and Kaplan-Meier by gene expression demonstrates that elevated TIMP-1 expression is correlated with increased disease relapse and mortality in patients with neuroblastoma. Taken together, our study identifies TIMP-1 as a novel soluble factor that is associated with a prometastatic phenotype in our in vivo model and adverse outcomes in patients with neuroblastoma.

Host and tumor derived MMP13 regulate extravasation and establishment of colorectal metastases in the liver.

BackgroundNon alcoholic fatty liver disease (NAFLD) is one of the most common liver diseases in the United States and worldwide. Our studies have previously shown an increase in metastatic burden in steatotic vs. normal livers using a mouse model of diet induced steatosis. In the present study we aim to identify and evaluate the molecular factors responsible for this increase in tumor burden.MethodsWe assessed changes in expression of a panel of matrix metalloproteinases (MMPs) using qRT-PCR between normal and steatotic livers and validated them with western blot analysis of protein levels. To evaluate the role of MMP13 on tumor development, we utilized a splenic injection model of liver metastasis in Wildtype and Mmp13 deficient mice, using either parental or stable Mmp13 knockdown cell lines. Further, to evaluate changes in the ability of tumor cells to extravasate we utilized whole organ confocal microscopy to identify individual tumor cells relative to the vasculature. MTT, migration and invasion assays were performed to evaluate the role of tumor derived MMP13 on hallmarks of cancer in vitro.ResultsWe found that MMP13 was significantly upregulated in the steatotic liver both in mice as well as human patients with NAFLD. We showed a decrease in metastatic tumor burden in Mmp13−/− mice compared to wildtype mice, explained in part by a reduction in the number of tumor cells extravasating from the hepatic vasculature in the Mmp13−/− mice compared to wildtype mice. Additionally, loss of tumor derived MMP13 through stable knockdown in tumor cell lines lead to decreased migratory and invasive properties in vitro and metastatic burden in vivo.ConclusionsThis study demonstrates that stromal as well as tumor derived MMP13 contribute to tumor cell extravasation and establishment of metastases in the liver microenvironment.Electronic supplementary materialThe online version of this article (doi:10.1186/s12943-014-0282-0) contains supplementary material, which is available to authorized users.

Abstract 4984: Contribution of MMP13 to tumor development in the steatotic liver microenvironment

Abstract With the rising prevalence of obesity there has been a marked increase in the incidence of non alcoholic fatty liver disease (NAFLD). Epidemiologically, NAFLD has been linked to an increased risk for development of primary liver cancer (HCC), however the mechanisms involved are not known. The liver is also a frequent site of metastasis for several types of cancers. To determine the effect of steatosis (fatty liver) on tumor metastasis to the liver, we used a mouse model of diet induced steatosis coupled with the splenic injection model of metastasis to the liver and found a significant increase in the number of tumors in the steatotic livers. To evaluate what molecular changes distinguish the steatotic liver from normal liver, microarray analysis was performed and demonstrated that MMP13, a member of the Matrix Metalloproteinase family recognized to be involved in liver disease and cancer progression, is significantly upregulated in the steatotic liver compared to normal livers of mice. We evaluated MMP13 expression in human patients with NAFLD and found that MMP13 is elevated with the progression of NAFLD. We hypothesize that increased MMP13 levels in the steatotic liver contribute to a more permissive microenvironment for the establishment of liver tumors. To test this hypothesis, mice genetically deficient in MMP13 or control wildtype mice, with and without steatosis, were injected with syngeneic MC38 colon cancer cells. Examination of the two groups demonstrated a significant decrease in the number of tumors in the MMP13 null mice (P &amp;lt;0.01). Additionally, since the tumor cells themselves express MMP13, wildtype mice were injected with either shRNA control or MMP13 knockdown MC38 colon cancer cells to determine the role of tumor cell derived MMP13. Using transwell migration assay and the platypus invasion system with parental and MMP13 shRNA knockdown MC38 cells we found that loss of MMP13 decreases the invasive and migratory properties of the metastatic cancer cells in vitro. In conclusion, we found that MMP13 is elevated in the setting of steatosis and that loss of both stromal and tumor derived MMP13 lead to decreased number of metastatic foci in the liver. Tumor derived MMP13 effects tumor cell migration and invasion invitro. MMP13 may thus be a potential target to control initiation and growth of metastatic cancer of the liver. Citation Format: Alisha Maria Mendonsa, Michael N. VanSaun, Lee Gorden. Contribution of MMP13 to tumor development in the steatotic liver microenvironment. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 4984. doi:10.1158/1538-7445.AM2014-4984

Abstract 3959: MMP13 contributes to metastatic tumor growth in the steatotic liver microenvironment.

Abstract The expanding obesity epidemic has led to an escalation in the incidence of non-alcoholic fatty liver disease (NAFLD) and steatohepatitis, which are associated with an increase in incidence of primary and metastatic liver cancer. As NAFLD progresses, it is characterized by marked inflammation in the liver described as non-alcoholic steatohepatitis (NASH). The mechanisms by which NASH increases risk of liver cancer are not fully known but complete understanding of these mediators is necessary for effective therapeutic and preventive strategies. Importantly, metastasis represents the final stage in cancer progression and is the most significant cause of cancer related mortality. Using our mouse model of diet induced steatosis, coupled with a model of experimental liver metastasis, we have previously shown a significant increase in the number of metastatic foci in the steatotic liver compared to the normal livers. Microarray analysis revealed that MMP13, a member of the Matrix Metalloproteinase (MMP) family recognized to be involved in liver disease and cancer progression, is significantly upregulated in the steatotic liver compared to normal livers. Additionally, MMP13 is expressed in several cancer cell types. Emerging data suggests that MMP13 may play a key role in priming the microenvironment for the establishment of liver metastases, by modulation of cytokine profiles and recruitment of inflammatory cells. . We hypothesize that MMP13 is a key modulator of the steatotic microenvironment, and that increased MMP13 levels favor primary tumor growth as well as the establishment of metastases. To determine the role of stromal MMP13 on metastasis to the liver, we are using the experimental splenic injection model of liver metastasis with syngeneic MC38 colon cancer cells in mice genetically deficient in MMP13. Additionally, since the tumor cells themselves express MMP13, wildtype mice are injected with either shRNA control or MMP13 knockdown MC38 colon cancer cells to determine the role of tumor cell derived MMP13. Preliminary data shows localization of MMP13 expression to the hepatic stellate cells as well as CD11b positive myeloid cells from livers with NASH. Further, invitro analysis of migration and invasion ability of parental and MMP13 shRNA knockdown MC38 cells show that MMP13 promotes the invasive and migratory properties of the metastatic cancer cells themselves. MMP13 may thus be a potential mediator of inflammation in the tumor microenvironment as well as of the metastatic capacity of cancer cells to the liver. Citation Format: Alisha M. Mendonsa, Michael VanSaun, David L. Gorden. MMP13 contributes to metastatic tumor growth in the steatotic liver microenvironment. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 3959. doi:10.1158/1538-7445.AM2013-3959

Abstract 389: MMP12 and myeloid cell populations influence establishment of tumor metastases in the steatotic liver microenvironment

Abstract Introduction: Non-alcoholic fatty liver disease (NAFLD), encompassing steatosis and progression to non-alcoholic steatohepatitis (NASH) are liver disorders of increasing clinical significance. Studies in our lab have shown that hepatic steatosis establishes a permissive microenvironment for metastatic tumor seeding and tumor progression in the liver. To understand the molecular factors influencing the initial survival and growth of tumors in the steatotic liver microenvironment, we adopted a candidate approach and performed microarray analysis to compare RNA from normal vs steatotic liver samples in mice. MMP12 (macrophage metalloelastase) was identified as an important proteinase associated with the microenvironments of hepatic steatosis and steatohepatitis. MMP12 can influence immune-mediated injury response by processing latent TNF alpha and regulating neutrophil infiltration, cytokine release as well as macrophage recruitment. We hypothesize that increased MMP12 levels in the steatotic liver contribute to a more permissive microenvironment for primary tumor growth and establishment of metastases by alteration of inflammatory cell populations. Results: Realtime qPCR anlaysis verified a significant increase in MMP12 expression in murine steatotic livers compared to normal control livers. In a splenic injection model, hepatic metastases in steatotic MMP12 deficient livers were significantly reduced as compared to hepatic metastases in steatotic wildtype livers. Flow cytometric analysis demonstrated an alteration in the level of Gr-1 positive inflammatory cell populations in the steatotic livers of both wildtype and MMP12 deficient livers when compared to normal nonsteatotic livers. Although no significant changes were observed in the percentage of F4/80 positive macrophage population, using immunohistochemistry we did observe accumulation of F4/80 positive macrophages into crown-like structures within steatotic MMP12 deficient livers. To determine whether the inflammatory cells in the liver microenvironment were contributing to survival of tumor cells, we performed an in vitro co-culture assay of syngeneic MC38 tumor cells with either CD90.2 cells (pan T cells) or CD11b cells (macrophages/monocytes). Fewer numbers MC38 cells were present 3 days after co-culture with CD11b cells from MMP12 deficient mice, which was corroborated with decreased luciferase measurements from MC38-luc tagged cells. Conclusions: These results suggest a role for MMP12 in mediating the inflammatory response accompanying tumor establishment in the steatotic liver microenvironment. The molecular mechanisms by which MMP12 influences a decrease in metastatic tumor burden in the setting of fatty liver disease are currently being investigated. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 389. doi:1538-7445.AM2012-389

High Fat Diet Induced Hepatic Steatosis Establishes a Permissive Microenvironment for Colorectal Metastases and Promotes Primary Dysplasia in a Murine Model

Non-alcoholic fatty liver disease (NAFLD), which includes steatosis and its progression to non-alcoholic steatohepatitis, is a liver disorder of increasing clinical significance. Here we characterize a murine model of high fat diet-induced NAFLD with progression from liver steatosis to histological features compatible with steatohepatitis and more advanced stages of NAFLD in humans, including chronic portal inflammation, pericellular and bridging fibrosis, Mallory body formation, and bile ductular reaction. Chronic changes induced by the prolonged consumption of a high-fat diet alone culminate in the development of primary liver dysplasias. Importantly, we extend these studies to demonstrate that even the early stages of uncomplicated steatosis provide a permissive microenvironment for the growth of colon cancer cells that are metastatic to the liver. High fat diet-induced steatosis, coupled with a splenic injection model of experimental liver metastasis using syngeneic MC38 colon cancer cells, resulted in an increased number of secondary tumor nodules and metastatic burden in steatotic livers. Metastatic nodules were associated with focal peritumoral areas of infiltrating inflammatory cells and associated apoptotic cell populations. These results suggest that the modulation of specific host factors in the steatotic liver contributes to tumor progression in the microenvironment of NAFLD.

Smad7 induces hepatic metastasis in colorectal cancer

Although Smad signalling is known to play a tumour suppressor role, it has been shown to play a prometastatic function also in breast cancer and melanoma metastasis to bone. In contrast, mutation or reduced level of Smad4 in colorectal cancer is directly correlated to poor survival and increased metastasis. However, the functional role of Smad signalling in metastasis of colorectal cancer has not been elucidated. We previously reported that overexpression of Smad7 in colon adenocarcinoma (FET) cells induces tumorigenicity by blocking TGF-β-induced growth inhibition and apoptosis. Here, we have observed that abrogation of Smad signalling by Smad7 induces liver metastasis in a splenic injection model. Polymerase chain reaction with genomic DNA from liver metastases indicates that cells expressing Smad7 migrated to the liver. Increased expression of TGF-β type II receptor in liver metastases is associated with phosphorylation and nuclear accumulation of Smad2. Immunohistochemical analyses have suggested poorly differentiated spindle cell morphology and higher cell proliferation in Smad7-induced liver metastases. Interestingly, we have observed increased expression and junctional staining of Claudin-1, Claudin-4 and E-cadherin in liver metastases. Therefore, this report demonstrates, for the first time, that blockade of TGF-β/Smad pathway in colon cancer cells induces metastasis, thus supporting an important role of Smad signalling in inhibiting colon cancer metastasis.

Synergistic effect of a granulocyte-macrophage colony-stimulating factor-transduced tumor vaccine and systemic interleukin-2 in the treatment of murine colorectal cancer hepatic metastases.

Granulocyte-macrophage colony-stimulating factor-transduced tumor cell vaccines are less effective against cancer as the interval between metastasis and the initial vaccination increases. Hepatic metastases were generated in BALB/c mice by using a syngeneic colorectal cancer line (CT26) with a splenic injection model. Irradiated CT26 cells transduced to secrete granulocyte-macrophage colony-stimulating factor were used as vaccine. Treatment groups received vaccine, systemic interleukin (IL-2), or both. Livers were examined for gross metastases 21 days after tumor challenge. Splenocytes were analyzed for in vitro activity against CT26 by using an enzyme-linked immunospot assay and a cytotoxic T lymphocyte assay. Eighty-eight percent of mice treated with vaccines and IL-2 were tumor free on day 21 (P </=.001 vs. control). Treatment with vaccines or IL-2 alone did not result in a significant treatment effect. Splenocytes from mice treated with both vaccines and IL-2 showed greater CT26 lysis than splenocytes from mice treated with vaccines alone at effector:target ratios of 100, 30, and 10 (P <.05 for all). More splenocytes from these mice released interferon-gamma in response to stimulation with the CT26 tumor antigen AH1 compared with mice treated with vaccines alone (P =.05). Systemic IL-2 augments tumor vaccine efficacy in the treatment of microscopic murine colorectal hepatic metastases.

In situ cancer vaccination with a replication-conditional HSV for the treatment of liver metastasis of colon cancer.

In this study, we investigated the therapeutic efficacy of a replication-conditional mutant HSV, G207, for the treatment of liver metastasis of colon carcinoma. Three liver metastasis models in syngeneic BALB/c mice were developed: (i) splenic injection, (ii) splenic and subcutaneous (s.c.) injection, and (iii) orthotopic implantation of CT26 colon carcinoma. In the splenic injection model, G207 was injected into the established splenic tumor on day 7. In the splenic and s.c. injection model, G207 were injected into the established s.c. tumor on days 5 and 8. In the orthotopic implantation model, a piece of CT26 tumor tissue was transplanted onto the wall of the cecum and G207 was injected in the established cecum tumor on day 7. On day 21 or 28, animals were sacrificed and liver metastases were evaluated. In all three models in immunocompetent mice, liver metastases were significantly reduced by intratumoral inoculation with G207 compared to the control. In athymic mice, however, there was no significant therapeutic effect of intratumoral inoculation with G207 on liver metastases. Tumor-specific cytotoxic T-lymphocyte responses were induced in mice treated with G207 in the orthotopic implantation model. These results suggest that intratumoral inoculation of G207, as an in situ cancer vaccine, can be an effective approach against liver metastasis of colon cancer and the efficacy involves tumor-specific T-cell responses.