Renin-Angiotensin Inhibitor, Captopril, Attenuates Growth of Patient-Derived Colorectal Liver Metastasis Organoids.

The recurrence of colorectal liver metastasis (CRLM) following liver resection is common; approximately 40% of patients will experience tumor recurrence post-surgery. Renin-angiotensin inhibitors (RASis) have been shown to attenuate the growth and progression of CRLM in pre-clinical models following liver resection. This study examined the efficacy of the RASi captopril on patient-derived colorectal liver metastasis organoids. Patient-derived organoids (PDOs) were established using fresh samples of colorectal liver metastasis from appropriately consented patients undergoing liver resection. To mimic the regenerating liver post-CRLM liver resection, PDOs were cultured under hepatocyte regeneration conditions in vitro. CRLM PDOs were established from three patients' parent tissue. CRLM PDOs and parent tissue expressed markers of colorectal cancer, CDX2 and CK20, consistently. Furthermore, CRLM PDOs treated with captopril showed a dose dependent reduction in their expansion in vitro. In conclusion, CRLM PDOs recapitulate in vivo disease and displayed a dose-dependent response to treatment with captopril. RASis may be an additional viable treatment for patients with CRLM.

Epigenetic Silencing of RIPK3 in Hepatocytes Prevents MLKL-mediated Necroptosis From Contributing to Liver Pathologies.

BACKGROUND & AIMS: Necroptosis is a highly inflammatory mode of cell death that has been implicated in causing hepatic injury including steatohepatitis/ nonalcoholic steatohepatitis (NASH); however, the evidence supporting these claims has been controversial. A comprehensive, fundamental understanding of cell death pathways involved in liver disease critically underpins rational strategies for therapeutic intervention. We sought to define the role and relevance of necroptosis in liver pathology. METHODS: Several animal models of human liver pathology, including diet-induced steatohepatitis in male mice and diverse infections in both male and female mice, were used to dissect the relevance of necroptosis in liver pathobiology. We applied necroptotic stimuli to primary mouse and human hepatocytes to measure their susceptibility to necroptosis. Paired liver biospecimens from patients with NASH, before and after intervention, were analyzed. DNA methylation sequencing was also performed to investigate the epigenetic regulation of RIPK3 expression in primary human and mouse hepatocytes. RESULTS: Identical infection kinetics and pathologic outcomes were observed in mice deficient in an essential necroptotic effector protein, MLKL, compared with control animals. Mice lacking MLKL were indistinguishable from wild-type mice when fed a high-fat diet to induce NASH. Under all conditions tested, we were unable to induce necroptosis in hepatocytes. We confirmed that a critical activator of necroptosis, RIPK3, was epigenetically silenced in mouse and human primary hepatocytes and rendered them unable to undergo necroptosis. CONCLUSIONS: We have provided compelling evidence that necroptosis is disabled in hepatocytes during homeostasis and in the pathologic conditions tested in this study.

Captopril, a Renin-Angiotensin System Inhibitor, Attenuates Tumour Progression in the Regenerating Liver Following Partial Hepatectomy.

(1) Liver regeneration following partial hepatectomy for colorectal liver metastasis (CRLM) has been linked to tumour recurrence. Inhibition of the renin−angiotensin system (RASi) attenuates CRLM growth in the non-regenerating liver. This study investigates whether RASi exerts an antitumour effect within the regenerating liver following partial hepatectomy for CRLM and examines RASi-induced changes in the tumour immune microenvironment; (2) CRLM in mice was induced via intrasplenic injection of mouse colorectal tumour cells, followed by splenectomy on Day 0. Mice were treated with RASi captopril (250 mg/kg/day), or saline (control) from Day 4 to Day 16 (endpoint) and underwent 70% partial hepatectomy on Day 7. Liver and tumour samples were characterised by flow cytometry and immunofluorescence; (3) captopril treatment reduced tumour burden in mice following partial hepatectomy (p < 0.01). Captopril treatment reduced populations of myeloid-derived suppressor cells (MDSCs) (CD11b+Ly6CHi p < 0.05, CD11b+Ly6CLo p < 0.01) and increased PD-1 expression on infiltrating hepatic tissue-resident memory (TRM)-like CD8+ (p < 0.001) and double-negative (CD4-CD8-; p < 0.001) T cells; (4) RASi reduced CRLM growth in the regenerating liver and altered immune cell composition by reducing populations of immunosuppressive MDSCs and boosting populations of PD-1+ hepatic TRMs. Thus, RASi should be explored as an adjunct therapy for patients undergoing partial hepatectomy for CRLM.

Searching for the link; mechanisms underlying liver regeneration and recurrence of colorectal liver metastasis post partial hepatectomy.

Despite excellent treatment of primary colorectal cancer, the majority of deaths occur as a result of metastasis to the liver. Recent population studies have estimated that one quarter of patients with colorectal cancer will incur synchronous or metachronous colorectal liver metastasis. However, only one quarter of these patients will be eligible for potentially curative resection. Tumor recurrence occurs in reportedly 60% of patients undergoing hepatic resection, and the majority of intrahepatic recurrence occurs within the first 6 months of surgery. The livers innate ability to restore its homeostatic size, and volume facilitates major hepatic resection that currently offers the only chance of cure to patients with extensive hepatic metastases. Experimental and clinical evidence supports the notion that following partial hepatectomy, liver regeneration (LR) paradoxically drives tumor progression and increases the risk of recurrence. It is becoming increasingly clear that the processes that drive liver organogenesis, regeneration, and tumor progression are inextricably linked. This presents a major hurdle in the management of colorectal liver metastasis and other hepatic malignancies because therapies that reduce the risk of recurrence without hampering LR are sought. The processes and pathways underlying these phenomena are multiple, complex, and cross-communicate. In this review, we will summarize the common mechanisms contributing to both LR and tumor recurrence.

The kallikrein-Kinin system modulates the progression of colorectal liver metastases in a mouse model.

BACKGROUND: The Kallikrein-Kinin System (KKS) has been found to play a role in tumor progression in several cancers. The KKS metabolic cascade depends on signalling through two cross talking receptors; bradykinin receptor 1 (B1R) and bradykinin receptor 2 (B2R). Activation of the Kinin receptor is responsible for multiple pathophysiologic functions including increase of vascular permeability and induction of host inflammatory responses that exert diverse effects on tumor growth. METHODS: B1R and B2R expression on mouse and human CRC cell lines was investigated. Changes in tumor growth and progression was assessed in male CBA mice bearing colorectal liver metastases (CRLM) following treatment with B1R or B2R blockers. In vitro cultures of human SW-480 and mouse colorectal cancer (MoCR) cell lines were examined for changes in their proliferation and migration properties following treatment with B1R or B2R blockers. RESULTS: Both colorectal cancer cell lines tested strongly positive for B1R and B2R expression. Inhibition of both receptors retarded tumor growth but only B1R blockade significantly reduced tumor load and increased tumor apoptosis. Blockade of either receptor reduced tumor vascularization in vivo and significantly inhibited proliferation and migration of colorectal cancer cells in vitro. CONCLUSION: Taken together, the present study demonstrated that kinin receptor blockade inhibited tumor growth and reduced its invading properties suggesting that KKS manipulation could be a novel target in colorectal cancer therapy.

Vascular disruptive agent OXi4503 and anti-angiogenic agent Sunitinib combination treatment prolong survival of mice with CRC liver metastasis.

BACKGROUND: Preclinical research indicate that vascular disrupting agent (VDA) treatment induces extensive tumor death but also a systemic mobilization of bone marrow derived cells including endothelial progenitor cells (EPC) leading to revascularization and renewed growth within the residual tumor. This study investigates if combination of VDA with the anti-angiogenic agent Sunitinib increases the treatment efficacy in a colorectal liver metastases mouse model. METHODS: CBA mice with established liver metastases were given a single dose of OXi4503 at day 16 post tumor induction, a daily dose of Sunitinib starting at day 14 or day 16 post tumor induction or a combination of Sunitinib given daily from day 14 or day 16 post tumor induction in combination with a single dose of OXi4503 at day 16. Treatment was terminated at day 21 post tumor induction and its effects were assessed using stereological and immunohistochemical techniques. Long term effects were assessed in a survival study. RESULTS: Combination with long (7 day) Sunitinib treatment lead to liver toxicity but this was ameliorated in the shorter (5 day) treatment without significantly altering the effects on tumor reduction. Combination treatment resulted in significant reduction of viable tumor, reduction in tumor vasculature, reduction in tumor proliferation, increase in tumor apoptosis and prolonged mouse survival compared to control and single arm treatments. Complete tumor eradication was not achieved. Redistribution of E-cadherin and strong up regulation of ZEB1 and Vimentin were observed in the surviving tumor; indicative of epithelial to mesenchymal transition (EMT), a mechanism that could contribute to tumor resistance. CONCLUSIONS: Combination treatment significantly reduces viable tumor and prolongs animal survival. EMT in the surviving tumor may prevent total tumor eradication and could provide novel targets for a more lasting treatment.

Spatial morphological and molecular differences within solid tumors may contribute to the failure of vascular disruptive agent treatments.

BACKGROUND: Treatment of solid tumors with vascular disrupting agent OXi4503 results in over 90% tumor destruction. However, a thin rim of viable cells persists in the tumor periphery following treatment, contributing to subsequent recurrence. This study investigates inherent differences in the microenvironment of the tumor periphery that contribute to treatment resistance. METHODS: Using a murine colorectal liver metastases model, spatial morphological and molecular differences within the periphery and the center of the tumor that may account for differences in resistance to OXi4503 treatment were investigated. H&E staining and immunostaining were used to examine vessel maturity and stability, hypoxia and HIF1α levels, accumulation of immune cells, expression of proangiogenic factors/receptors (VEGF, TGF-ß, b-FGF, and AT1R) and expression of EMT markers (ZEB1, vimentin, E-cadherin and ß-catenin) in the periphery and center of established tumors. The effects of OXi4503 on tumor vessels and cell kinetics were also investigated. RESULTS: Significant differences were found between tumor periphery and central regions, including association of the periphery with mature vessels, higher accumulation of immune cells, increased growth factor expression, minimal levels of hypoxia and increased evidence of EMT. OXi4503 treatment resulted in collapse of vessels in the tumor center; however vasculature in the periphery remained patent. Similarly, tumor apoptosis and proliferation were differentially modulated between centre and periphery after treatment. CONCLUSIONS: The molecular and morphological differences between tumor periphery and center may account for the observed differential resistance to OXi4503 treatment and could provide targets for drug development to totally eliminate metastases.

SAR131675, a VEGRF3 Inhibitor, Modulates the Immune Response and Reduces the Growth of Colorectal Cancer Liver Metastasis.

Most patients with colorectal cancer (CRC) develop metastases, predominantly in the liver (CLM). Targeted therapies are being investigated to improve current CLM treatments. This study tested the effectiveness of SAR131675, a selective VEGFR-3 tyrosine kinase inhibitor, to inhibit CLM in a murine model. Following intrasplenic induction of CLM, mice were treated daily with SAR131675. Tumor growth and immune infiltrates into tumor and liver tissues were assessed at 10-, 16- and 22-days post tumor induction by stereology, IHC and flow cytometry. SAR151675 treatment significantly reduced tumor burden and F4/80+ macrophages in the liver tissues. Analysis of immune cell infiltrates in liver showed tissue that at day 22, had the proportion of CD45+ leukocytes significantly reduced, particularly myeloid cells. Analysis of myeloid cells (CD11b+ CD45+) indicated that the proportion of F4/80- Ly6Clow was significantly reduced, including a predominate PD-L1+ subset, while CD3+ T cells increased, particularly CD8+ PD1+, reflected by an increase in the CD8+:CD4+ T cell ratio. In the tumor tissue SAR11675 treatment reduced the predominant population of F4/80+ Ly6Clo and increased CD4+ T cells. These results suggest that SAR131675 alters the immune composition within tumor and the surrounding liver in the later stages of development, resulting in a less immunosuppressive environment. This immunomodulation effect may contribute to the suppression of tumor growth.

Induction of Th1Immune responses following laser ablation in a murine model of colorectal liver metastases.

BACKGROUND: Preliminary experimental studies have suggested that the in situ destruction of tumor tissue by local laser ablation (LA) may also stimulate host immunity against cancer. We investigated local and systemic induction of immune responses after laser ablation in the setting of residual tumor. METHODS: A murine colorectal cancer (CRC) liver metastasis model was used. Selected tumors of liver CRC bearing mice and livers of mice without tumor induction were treated with LA. Liver and tumor tissues from the ablation sites and from distant sites were collected at various time points following LA and changes in CD3+ T cells and Kupffer cells (F4/80 marker) infiltration and the expression of interferon gamma (IFNγ) were investigated by immunohistochemistry and ELISpot. Base line levels of CD3+ T cells and Kupffer cells were established in untreated mice. RESULTS: The presence of tumor induced significant accumulation of CD3+ T cells and Kupffer cells at the tumor-host interface, within the tumor vascular lakes and increased their baseline concentration within the liver parenchyma. LA of the liver induced accumulation of CD3+ T-cells and Kupffer cells at the site of injury and systemic induction of immune responses as discerned by the presence of IFNγ secreting splenocytes. LA of liver tumors induced significant increase of CD3+ T-cells at site of injury, within normal liver parenchyma, and the tumor-host interface of both ablated and distant tumors. In contrast Kupffer cells only accumulated in ablated tumors and the liver parenchyma but not in distant tumors. IFNγ expression increased significantly in ablated tumors and showed an increasing trend in distant tumors. CONCLUSION: Laser ablation in addition to local tumor destruction induces local and systemic Th1 type immune responses which may play a significant role in inhibiting tumor recurrence from residual micrometastases or circulating tumor cells.

Changes in growth factor levels after thermal ablation in a murine model of colorectal liver metastases.

OBJECTIVES: This study examines changes in the expression of growth factors following thermal ablation (TA) of selected colorectal cancer (CRC) liver metastases. METHODS: Using mice with established CRC liver metastases, two tumours in each animal were thermally ablated. Liver and tumour tissues were collected at various time-points (days 0, 1, 2, 3, 5 and 7) following TA treatment from the ablation site and from sites distant from ablated tumour. Changes in growth factor expression (epidermal growth factor [EGF], vascular endothelial growth factor [VEGF], hepatocyte growth factor [HGF] and transforming growth factor-ß[TGF-ß]) in comparison with baseline levels (non-ablated) were assessed by enzyme-linked immunosorbent assay (ELISA) and immunohistochemistry. RESULTS: Baseline TGF-ß and VEGF levels in the liver parenchyma of tumour-bearing mice were significantly higher than levels in naive liver parenchyma. Levels of VEGF and HGF decreased after TA treatment in all tissues. Levels of EGF decreased in ablated and distant tumour tissues, but displayed a tendency to increase in liver tissue. Levels of TGF-ß also decreased during the first 2 days following TA, but later increased in liver and tumour tissues distant from the ablation site to a level that reached significance in tumour tissue at day 7 (P < 0.001). Decreases in growth factor levels were also observed in animals that underwent laparotomy without TA treatment, which indicates that these decreases were caused by the experimental procedure. CONCLUSIONS: Tumour induces upregulation of TGF-ß and VEGF in liver parenchyma. Growth factors decreased after TA, but this appears to be the result of the experimental procedure rather than the TA itself. However, TA resulted in increased levels of TGF-ß, which may contribute to tumour recurrence.

Captopril, a Renin-Angiotensin System Inhibitor, Attenuates Features of Tumor Invasion and Down-Regulates C-Myc Expression in a Mouse Model of Colorectal Cancer Liver Metastasis.

(1) Background: Recent clinical and experimental data suggests that the liver's regenerative response following partial hepatectomy can stimulate tumor recurrence in the liver remnant. The Wnt/ß-catenin pathway plays important roles in both colorectal cancer carcinogenesis and liver regeneration. Studies have shown that the Wnt/ß-catenin pathway regulates multiple renin-angiotensin system (RAS) genes, whilst RAS inhibition (RASi) reduces tumor burden and progression. This study explores whether RASi attenuates features of tumor progression in the regenerating liver post-hepatectomy by modulating Wnt/ß-catenin signaling. (2) Methods: Male CBA mice underwent CRLM induction, followed one week later by 70% partial hepatectomy. Mice were treated daily with captopril, a RASi, at 250 mg/kg/day or vehicle control from experimental Day 4. Tumor and liver samples were analyzed for RAS and Wnt signaling markers using qRT-PCR and immunohistochemistry. (3) Results: Treatment with captopril reduced the expression of down-stream Wnt target genes, including a significant reduction in both c-myc and cyclin-D1, despite activating Wnt signaling. This was a tumor-specific response that was not elicited in corresponding liver samples. (4) Conclusions: We report for the first time decreased c-myc expression in colorectal tumors following RASi treatment in vivo. Decreased c-myc expression was accompanied by an attenuated invasive phenotype, despite increased Wnt signaling.

Immunomodulatory effects of renin-angiotensin system inhibitors on T lymphocytes in mice with colorectal liver metastases.

BACKGROUND: It is now recognized that many anticancer treatments positively modulate the antitumor immune response. Clinical and experimental studies have shown that inhibitors of the classical renin-angiotensin system (RAS) reduce tumor progression and are associated with better outcomes in patients with colorectal cancer. RAS components are expressed by most immune cells and adult hematopoietic cells, thus are potential targets for modulating tumor-infiltrating immune cells and can provide a mechanism of tumor control by the renin-angiotensin system inhibitors (RASi). AIM: To investigate the effects of the RASi captopril on tumor T lymphocyte distribution in a mouse model of colorectal liver metastases. METHODS: Liver metastases were established in a mouse model using an autologous colorectal cancer cell line. RASi (captopril 750 mg/kg) or carrier (saline) was administered to the mice daily via intraperitoneal injection, from day 1 post-tumor induction to endpoint (day 15 or 21 post-tumor induction). At the endpoint, tumor growth was determined, and lymphocyte infiltration and composition in the tumor and liver tissues were analyzed by flow cytometry and immunohistochemistry (IHC). RESULTS: Captopril significantly decreased tumor viability and impaired metastatic growth. Analysis of infiltrating T cells into liver parenchyma and tumor tissues by IHC and flow cytometry showed that captopril significantly increased the infiltration of CD3+ T cells into both tissues at day 15 following tumor induction. Phenotypical analysis of CD45+ CD3+ T cells indicated that the major contributing phenotype to this influx is a CD4 and CD8 double-negative T cell (DNT) subtype, while CD4+ T cells decreased and CD8+ T cells remained unchanged. Captopril treatment also increased the expression of checkpoint receptor PD-1 on CD8+and DNT subsets . CONCLUSION: Captopril treatment modulates the immune response by increasing the infiltration and altering the phenotypical composition of T lymphocytes and may be a contributing mechanism for tumor control.

The Hepatitis B Virus Pre-Core Protein p22 Activates Wnt Signaling.

An emerging theme for Wnt-addicted cancers is that the pathway is regulated at multiple steps via various mechanisms. Infection with hepatitis B virus (HBV) is a major risk factor for liver cancer, as is deregulated Wnt signaling, however, the interaction between these two causes is poorly understood. To investigate this interaction, we screened the effect of the various HBV proteins for their effect on Wnt/ß-catenin signaling and identified the pre-core protein p22 as a novel and potent activator of TCF/ß-catenin transcription. The effect of p22 on TCF/ß-catenin transcription was dose dependent and inhibited by dominant-negative TCF4. HBV p22 activated synthetic and native Wnt target gene promoter reporters, and TCF/ß-catenin target gene expression in vivo. Importantly, HBV p22 activated Wnt signaling on its own and in addition to Wnt or ß-catenin induced Wnt signaling. Furthermore, HBV p22 elevated TCF/ß-catenin transcription above constitutive activation in colon cancer cells due to mutations in downstream genes of the Wnt pathway, namely APC and CTNNB1. Collectively, our data identifies a previously unappreciated role for the HBV pre-core protein p22 in elevating Wnt signaling. Understanding the molecular mechanisms of p22 activity will provide insight into how Wnt signaling is fine-tuned in cancer.

Liver regeneration and tumor stimulation--a review of cytokine and angiogenic factors.

Liver resection for metastatic (colorectal carcinoma) tumors is often followed by a significant incidence of tumor recurrence. Cellular and molecular changes resulting from hepatectomy and the subsequent liver regeneration process may influence the kinetics of tumor growth and contribute to recurrence. Clinical and experimental evidence suggests that factors involved in liver regeneration may also stimulate the growth of occult tumors and the reactivation of dormant micrometastases. An understanding of the underlying changes may enable alternative strategies to minimize tumor recurrence and improve patient survival after hepatectomy.

Renin-angiotensin inhibitors reprogram tumor immune microenvironment: A comprehensive view of the influences on anti-tumor immunity.

Renin-angiotensin system inhibitors (RASi) have shown potential anti-tumor effects that may have a significant impact in cancer therapy. The components of the renin-angiotensin system (RAS) including both, conventional and alternative axis, appear to have contradictory effects on tumor biology. The mechanisms by which RASi impair tumor growth extend beyond their function of modulating tumor vasculature. The major focus of this review is to analyze other mechanisms by which RASi reprogram the tumor immune microenvironment. These involve impairing hypoxia and acidosis within the tumor stroma, regulating inflammatory signaling pathways and oxidative stress, modulating the function of the non-cellular components and immune cells, and regulating the cross-talk between kalli krein kinin system and RAS.

Advances in targeted and immunobased therapies for colorectal cancer in the genomic era.

Targeted therapies require information on specific defective signaling pathways or mutations. Advances in genomic technologies and cell biology have led to identification of new therapeutic targets associated with signal-transduction pathways. Survival times of patients with colorectal cancer (CRC) can be extended with combinations of conventional cytotoxic agents and targeted therapies. Targeting EGFR- and VEGFR-signaling systems has been the major focus for treatment of metastatic CRC. However, there are still limitations in their clinical application, and new and better drug combinations are needed. This review provides information on EGFR and VEGF inhibitors, new therapeutic agents in the pipeline targeting EGFR and VEGFR pathways, and those targeting other signal-transduction pathways, such as MET, IGF1R, MEK, PI3K, Wnt, Notch, Hedgehog, and death-receptor signaling pathways for treatment of metastatic CRC. Additionally, multitargeted approaches in combination therapies targeting negative-feedback loops, compensatory networks, and cross talk between pathways are highlighted. Then, immunobased strategies to enhance antitumor immunity using specific monoclonal antibodies, such as the immune-checkpoint inhibitors anti-CTLA4 and anti-PD1, as well as the challenges that need to be overcome for increased efficacy of targeted therapies, including drug resistance, predictive markers of response, tumor subtypes, and cancer stem cells, are covered. The review concludes with a brief insight into the applications of next-generation sequencing, expression profiling for tumor subtyping, and the exciting progress made in in silico predictive analysis in the development of a prescription strategy for cancer therapy.

Delivery of a heterologous antigen by a registered Salmonella vaccine (STM1).

STM1 is an aro A(-) attenuated mutant of Salmonella enterica serovar Typhimurium, and is a well-characterised vaccine strain available to the livestock industry for the prevention of salmonellosis in chickens. This strain has potential for heterologous antigen delivery, and here we show that the strain can be used to deliver a model antigen, ovalbumin, to immune cells in vitro and in vivo. Two plasmid constructs expressing the ovalbumin gene were utilised, one of which uses a prokaryotic promoter and the other the CMV promoter (DNA vaccine). In vitro, STM1 carrying ovalbumin-encoding plasmids was able to invade dendritic cells and stimulate a CD8(+) cell line specific for the dominant ovalbumin epitope, SIINFEKL. In vivo, spleen cells were responsive to SIINFEKL after vaccination of mice with ovalbumin-encoding plasmids in STM1, and finally, humoral responses, including IgA, were induced after vaccination.

Comparison of two syngeneic orthotopic murine models of pancreatic adenocarcinoma.

BACKGROUND: Pancreatic adenocarcinoma has an extremely poor prognosis. The use of appropriate in vivo models is essential in devising methods to improve treatment outcomes. METHODS: A pancreatic adenocarcinoma model based on tumor injection into the pancreatic head was compared with a pancreatic tail injection model in C57/BL6 mice. The murine pancreatic adenocarcinoma cell line PAN02, dispersed in Matrigel™, was used for tumor induction. RESULTS: Tumors developed in all animals in both models. Tumor size was more consistent within the pancreatic tail group at 20 days following induction, with no evidence of metastatic disease. Animals in the pancreatic head injection group showed signs of reduced health by 20 days following injection and developed jaundice. Microscopic liver metastases were noted in some of these animals at this time point. The overall survival of animals at 40 days following tumor induction was significantly lower in the pancreatic head injection group (0% vs. 35%; p < .001). Multiple liver metastases were noted in five of 10 (50%) animals in the head injection group, without evidence of peritoneal metastases. In the pancreatic tail injection group, 18 of 20 (90%) animals had multiple peritoneal metastases, and nine of 20 (45%) animals had evidence of isolated liver deposits. Tumors in both regions of the pancreas had similar histologic characteristics, with a dense fibrotic stroma at the interface between the tumor and the normal pancreas. CONCLUSION: Pancreatic head and tail orthotopic cancer models produce consistent tumors, but the patterns of tumor spread and survival differ according to the site of injection.

Treatment with the vascular disruptive agent OXi4503 induces an immediate and widespread epithelial to mesenchymal transition in the surviving tumor.

Epithelial to mesenchymal transition (EMT) is considered an important mechanism in tumor resistance to drug treatments; however, in vivo observation of this process has been limited. In this study we demonstrated an immediate and widespread EMT involving all surviving tumor cells following treatment of a mouse model of colorectal liver metastases with the vascular disruptive agent OXi4503. EMT was characterized by significant downregulation of E-cadherin, relocation and nuclear accumulation of ß-catenin as well as significant upregulation of ZEB1 and vimentin. Concomitantly, significant temporal upregulation in hypoxia and the pro-angiogenic growth factors hypoxia-inducible factor 1-alpha, hepatocyte growth factor, vascular endothelial growth factor and transforming growth factor-beta were seen within the surviving tumor. The process of EMT was transient and by 5 days after treatment tumor cell reversion to epithelial morphology was evident. This reversal, termed mesenchymal to epithelial transition (MET) is a process implicated in the development of new metastases but has not been observed in vivo histologically. Similar EMT changes were observed in response to other antitumor treatments including chemotherapy, thermal ablation, and antiangiogenic treatments in our mouse colorectal metastasis model and in a murine orthotopic breast cancer model after OXi4503 treatment. These results suggest that EMT may be an early mechanism adopted by tumors in response to injury and hypoxic stress, such that inhibition of EMT in combination with other therapies could play a significant role in future cancer therapy.

Vaccination with in vitro grown whole tumor cells induces strong immune responses and retards tumor growth in a murine model of colorectal liver metastases.

In vitro adaptation of a murine colorectal cell line (MoCR) rendered it less aggressive and more immunogenic than the in vivo passaged parent tumor. Vaccination of syngeneic mice with the in vitro cultured tumor cells was shown to induce immune responses and protection against tumor challenge, thus overcoming the need for antigen selection and adjuvants. A syngeneic murine model of colorectal cancer (CRC) liver metastasis was used. In a prophylactic setting mice vaccinated with in vitro cultured tumor cells produced strong cellular immune responses and significant inhibition of tumor growth, compared to sham vaccinated controls. In a therapeutic setting however, vaccination exacerbated tumor growth, suggesting that the presence of tumor subverts the course of the immune response. The mechanisms of this subversion need to be investigated and counteracted for successful immunotherapy.