Colon Cancer Metastasis Model Research Articles

Inhibition of Colon Cancer Xenograft Growth by Celecoxib Analog OSU-03013 Through Wnt/β-Catenin Pathway Modulation

OSU-03013 is a Celecoxib analog that dose not inhibit COX-2 expression. OSU-03013 effectively mimics the anti-tumor effects of Celecoxib, however, its exact mechanism in colon cancer (CC) is unknown. SW480 cells were injected subcutaneously into the axilla of nude mice until the volume of the transplanted tumor reached 80 mm3. SW480 cells were inoculated into the tail vein of nude mice to establish a liver metastasis model of CC. OSU-03013 was administered by gavage. The pathology of the tumor tissues was assessed using HE staining and cell apoptosis was measured using TUNEL staining. EMT markers in the tumors were determined using Western Blot, and IL-1β and TNF-α were determined using ELISA. VEGF-A and VEGFR-2 in tumor tissues were measured to assess angiogenesis using immunofluorescence. HE staining was conducted to detect the effect of OSU-03013 on liver metastasis. Western Blot assay was performed to determine the expression of Wnt2, β-catenin and GSK-3β. OSU-03013 reduced inflammatory cell infiltration and inhibited IL-1β and TNF-α production by modulating the Wnt/β-catenin pathway. OSU-03013 induced apoptosis of cancer cells in tumor tissues and possessed anti-angiogenic activity. OSU-03013 treatment reduced the number and size of metastatic nodules of cancer cells in liver tissues. OSU-03013 may inhibit tumor growth and angiogenesis through the Wnt3a/β-catenin pathway in CC nude mice.

Ursolic acid inhibits the metastasis of colon cancer by downregulating ARL4C expression.

Ursolic acid (UA), a natural pentacyclic triterpenoid, is known to exhibit various biological activities and anticancer effects. However, the underlying anticancer mechanism is not fully understood to date. The present study aimed to investigate the antimetastatic effect of UA through ADP‑ribosylation factor like GTPase 4C (ARL4C) in colon cancer. A lung metastasis model of colon cancer in nude mice was established through tail vein injection. A Cell Counting Kit‑8 assay was used to investigate the proliferation of colon cancer cells. Transwell assays were used to detect cell migration and invasion. The expression levels of proteins including ARL4C, matrix metallopeptidase 2 (MMP2), phosphorylated (p)‑AKT and p‑mTOR were measured using western blot analysis. Immunohistochemistry was used to determine the protein expression level in tissues. ARL4C ubiquitination levels were analysed using immunoprecipitation and western blotting. The results indicated that UA inhibits the metastasis of colon cancer invivo and invitro. The expression of ARL4C in human colon cancer tissue was significantly higher than that in adjacent tissues and its high expression level was associated with lymph node metastases and tumour stage. UA treatment significantly decreased ARL4C and MMP2 protein levels and inhibited the AKT/mTOR signalling pathway. Overexpression of ARL4C reversed the inhibitory effect of UA on the invasion and migration of HCT‑116 and SW480 cells, as well as the expression and secretion of MMP2 protein. In addition, UA and an AKT signalling pathway inhibitor (LY294002) induced the ubiquitination of the ARL4C protein, which was reversed by a proteasome inhibitor (MG‑132). Collectively, it was revealed in the present study that UA served as a novel solution to relieve colon cancer metastasis by inducing the ubiquitination‑mediated degradation of ARL4C by modulating the AKT signalling pathway. Thus, UA may be a promising treatment option to prolong the survival of patients with colon cancer metastasis.

Impact of PRaG Therapy on Peripheral Immune Cells of Subcutaneous Tumor Peritoneal Metastasis Model of Colon Cancer

Immune cells in peripheral blood may be closely related to the efficacy of immune checkpoint inhibitors. T cells originally present in tumors may have limited antitumor effects, and T cells that respond to immune checkpoint inhibitors may be derived from peripheral blood. Therefore, in this study, subcutaneous tumor peritoneal metastasis model of colon cancer was constructed to reveal the changes of T cells and their subsets (CD4+T cells, CD8+T cells, CD226+T cells), MDSCs and their subsets (G-MDSCs, M-MDSCs) in peripheral blood of mice after PRaG therapy. A total of 90 male Balb/c mice aged 6-8 weeks were divided into five groups: control group, PD-1 inhibitor group, radiation group, radiation + PD-1 inhibitor group, and radiation + PD-1 inhibitor +GM-CSF (PRaG therapy) group. The subcutaneous tumor peritoneal metastasis model of colon cancer was constructed. 3×105 CT26.WT cells was inoculated subcutaneously at the right thigh root, and 5 days later, 1×105 CT26.WT cells was inoculated on the left side at the junction of the anterior superior iliac spine and the midabdominal line. The subcutaneous tumor was selected for radiotherapy of 8 Gy×3. GM-CSF (100ng, i.p.) was given on the 1st day and PD-1 inhibitor (0.25mg/kg, i.p.) was given on the 2nd day after radiotherapy with one cycle every 3 days. On day 22, the peripheral blood of mice was collected. The proportion of immune cells was detected by flow cytometry. Compared with other groups, PRaG therapy decreased the proportion of CD4+T cells and increased the proportion of CD8+T cells. Moreover, PRaG therapy increased the proportion of CD226+CD4+T cells and CD226+CD8+T cells. Finally, PRaG therapy increased the proportion of M-MDSCs and decreased the proportion of G-MDSCs. PRaG therapy can improve the immune microenvironment of peripheral blood of subcutaneous tumor peritoneal metastasis model of colon cancer in mice.

Anti-CTLA-4 and anti-PD-1 immunotherapies repress tumor progression in preclinical breast and colon model with independent regulatory T cells response

The recent development of immunotherapy represents a significant breakthrough in cancer therapy. Several immunotherapies provide robust efficacy gains in a wide variety of cancers. However, in some patients the immune checkpoint blockade remains ineffective due to poor therapeutic response and tumor relapse. An improved understanding of the mechanisms underlying tumor-immune system interactions can improve clinical management of cancer. Here, we report preclinical data evaluating two murine antibodies corresponding to recent FDA-approved antibodies for human therapy, e.g. anti-CTLA-4 and anti-PD-1. We demonstrated in two mouse syngeneic grafting models of triple negative breast or colon cancer that the two antibodies displayed an efficient anticancer activity, which is enhanced by combination treatment in the breast cancer model. We also demonstrated that CTLA-4 targeting reduced metastasis formation in the colon cancer metastasis model. In addition, using cytometry-based multiplex analysis, we showed that anti-CTLA-4 and anti-PD-1 affected the tumor immune microenvironment differently and in particular the tumor immune infiltration. This work demonstrated anti-cancer effect of CTLA-4 or PD-1 blockade on mouse colon and triple negative breast and on tumor-infiltrating immune cell subpopulations that could improve our knowledge and benefit the breast and colon cancer tumor research community.

4-Hydroxyacetophenone modulates the actomyosin cytoskeleton to reduce metastasis

Metastases are the cause of the vast majority of cancer deaths. In the metastatic process, cells migrate to the vasculature, intravasate, extravasate, and establish metastatic colonies. This pattern of spread requires the cancer cells to change shape and to navigate tissue barriers. Approaches that block this mechanical program represent new therapeutic avenues. We show that 4-hydroxyacetophenone (4-HAP) inhibits colon cancer cell adhesion, invasion, and migration in vitro and reduces the metastatic burden in an in vivo model of colon cancer metastasis to the liver. Treatment with 4-HAP activates nonmuscle myosin-2C (NM2C) (MYH14) to alter actin organization, inhibiting the mechanical program of metastasis. We identify NM2C as a specific therapeutic target. Pharmacological control of myosin isoforms is a promising approach to address metastatic disease, one that may be readily combined with other therapeutic strategies.

Resveratrol inhibits the invasion and metastasis of colon cancer through reversal of epithelial‑ mesenchymal transition via the AKT/GSK‑3β/Snail signaling pathway.

The identification of safe and effective drugs that inhibit tumor invasion and metastasis is required to improve the clinical outcome of patients with colon cancer. The present study aimed to investigate the inhibitory effects and possible mechanisms of action of resveratrol against the invasion and metastasis of colon cancer. AKT1-knockdown SW480 and SW620 colon cancer cells were used to detect the effects of resveratrol on cell invasion and metastasis, as well as changes in the expression of epithelial-mesenchymal transition (EMT) markers and serine/threonine kinase (AKT)/glycogen synthase kinase (GSK)-3β/Snail signaling pathway-related molecules in vitro. Furthermore, nude mice were inoculated with SW480 cells in the tail vein to establish an in vivo lung metastasis model of colon cancer, to investigate the effects of resveratrol on lung metastasis in colon cancer. The results revealed that resveratrol treatment and AKT1 knockdown significantly inhibited cell migration and invasion in colon cancer, and markedly increased E-cadherin expression and decreased that of N-cadherin, phospho (p)-AKT1, p-GSK-3β, and Snail in colon cancer both in vitro and in vivo. Furthermore, the effects of resveratrol were significantly weaker in the AKT1-knockdown cells. In conclusion, resveratrol may suppress the invasion and metastasis of colon cancer through reversal of EMT via the AKT/GSK-3β/Snail signaling pathway. AKT1 may therefore be a key regulator of EMT in colon cancer cells and a potential therapeutic target for this disease.

Characterization of polysaccharide from Scutellaria barbata and its antagonistic effect on the migration and invasion of HT-29 colorectal cancer cells induced by TGF-β1.

In this study, we purified a water-soluble polysaccharide, SBPW3, from the whole plant of Scutellaria barbata D. Don through ethanol precipitation, deproteinization, lyophilization, dialysis and separation using a DEAE cellulose column and a Superdex 200 gel filtration chromatography column. SBPW3 is a homogeneous polysaccharide with a molecular weight of 10.2 kDa and is composed of rhamnose (2.51%), arabinose (25.68%), xylose (10.94%), mannose (12.56%), glucose (20.59%) and galactose (27.72%). FT-IR spectrum analysis of the polysaccharide showed that SBPW3 contained a pyranose ring. The effects of SBPW3 on TGF-β1-induced epithelial-mesenchymal transition (EMT) were tested in colon cancer cells. These results suggested that SBPW3 significantly suppressed TGF-β1-induced migration and invasion. Additionally, SBPW3 reduced EMT by increasing the expression of epithelial markers and by decreasing the expression of mesenchymal markers by blocking the Smad2/3 signalling pathway in colon cancer cells. Furthermore, to explore the anti-metastatic effect of SBPW3, we established a mouse model of colon cancer metastasis and found that SBPW3 significantly inhibited the metastatic dissemination of the primary tumour to the liver. These findings provide us with a potential chemotherapeutic strategy for the treatment of human colorectal cancer.

In vitro analysis of putative cancer stem cell populations and chemosensitivity in the SW480 and SW620 colon cancer metastasis model.

The cancer stem cell (CSC) theory implicates a small subpopulation of cells with stem-like properties, which is responsible for tumour initiation, development and metastasis. The unique biological and functional characteristics of CSCs, widely associated with treatment resistance, indicate an association between metastasis and stemness. It was hypothesised that metastatic cell lines may be enriched in CSCs and that this would correlate with a more resistant tumour. In the present study, the SW480 and SW620 paired cell lines derived from a colon adenocarcinoma and its lymph node metastasis, respectively were compared as an in vitro model of cancer progression. Their chemosensitivity and CSC properties were investigated. A range of in vitro assays were performed, including the side population assay, ALDEFLUOR assay, tumoursphere assay and assessment of CSC-associated surface phenotypes. It was determined that the SW480 and SW620 cells exhibited similar growth rates, although the SW480 cells were more migratory in wound healing assays on collagen and fibronectin matrices. SW480 and SW620 cells displayed similar CSC profiles, however, SW480 cells demosntrated significantly greater tumoursphere forming efficiency over SW620 cells. Tumourspheres derived from SW480 and SW620 cells also displayed differential sensitivity to 5-fluorouracil, oxaliplatin, geldanamycin and novobiocin that was not apparent when cells were grown under adherent conditions. Taken together, these results suggest that although the two cell lines have similar levels of putative CSC populations, there are differences in their biology that cannot be explained by these CSC levels. To the best of our knowledge, this is the first study to conduct a detailed analysis of the CSC populations using multiple in vitro assays in a paired cell line model. These results have clinical relevance for the understanding of the differences between primary tumours and their metastatic counterparts.

Extract of Ginkgo biloba exacerbates liver metastasis in a mouse colon cancer Xenograft model

BackgroundMetastasis refers to the spread of a primary tumor cell from the primary site to other locations in the body and it is generally associated with the severity of a tumor. Extract of Ginkgo biloba (EGb) contains various bioactive compounds and it exerts beneficial effects including improvements in brain function and reduced risk of cardiovascular diseases. On the other hand, increased risk of thyroid and liver cancers by EGb have been reported in animals.MethodsA colon cancer metastasis model was established using intrasplenic injection of a human colon cancer cell line, SW620-luc in athymic mice to investigate the potential impact of EGb on colon cancer progression. After tumor establishment, EGb was intraperitonically injected daily for 5 wks.ResultsEGb significantly increased the rate of metastasis in mouse liver and decreased the number of necrotic and apoptotic cells in the metastatic liver when compared to the control. Meanwhile, EGb significantly induced proliferation of tumor cells in the metastatic liver, indicated by increased staining of Ki67 and H3S10p. mRNA expression of genes involved in cell cycle, metastasis, apoptosis, and oxidative stress were altered by EGb treatment in livers with tumors. Moreover, EGb activated the stress-responsive MAPK pathways in the liver with metastatic tumors.ConclusionsEGb exacerbated liver metastasis in a mouse colon cancer metastasis model. This is potentially due to the increased tumor cell proliferation involving stimulated MAPK pathways.

The synergistic antitumor effect of arsenic trioxide combined with cytotoxic T cells in pulmonary metastasis model of colon cancer.

Adoptive T cell therapy, including cytotoxic T lymphocytes (CTLs), represents a promising non-toxic anticancer strategy. The effects of this therapy can be impaired by tumor-infiltrated regulatory T cells (Tregs). Autologous murine CTLs acquired using cryopreservation exhibited a cytotoxic effect equivalent to that of conventional CTLs. The killing activity of CTLs was enhanced significantly using arsenic trioxide (ATO), accompanied by reduction in Tregs in vitro. Results using a pulmonary metastasis model of colon cancer indicated that compared with the control group, ATO group, and CTLs group, metastatic node number decreased significantly (p<0.001, p<0.001, p<0.001, respectively) and survival time was prolonged (p<0.001, p=0.669, p=0.158, respectively) in the ATO plus CTLs group. The number of infiltrated Foxp3+ Tregs decreased in the tumor center, but increased in the peri-tumor tissue. Our results indicate that this approach represents a practical protocol for acquiring autologous CTLs and a feasible strategy that uses a synergistic combination of ATO plus CTLs to treat pulmonary metastases of colon cancer.

Triptolide abrogates growth of colon cancer and induces cell cycle arrest by inhibiting transcriptional activation of E2F

Despite significant progress in diagnostics and therapeutics, over 50 thousand patients die from colorectal cancer annually. Hence, there is urgent need for new lines of treatment. Triptolide, a natural compound isolated from the Chinese herb Tripterygium wilfordii, is effective against multiple cancers. We have synthesized a water soluble analog of triptolide, named Minnelide, which is currently in phase I trial against pancreatic cancer. The aims of the current study were to evaluate whether triptolide/Minnelide is effective against colorectal cancer and to elucidate the mechanism by which triptolide induces cell death in colorectal cancer. Efficacy of Minnelide was evaluated in subcutaneous xenograft and liver metastasis model of colorectal cancer. For mechanistic studies, colon cancer cell lines HCT116 and HT29 were treated with triptolide and the effect on viability, caspase activation, annexin positivity, lactate dehydrogenase release, and cell cycle progression was evaluated. Effect of triptolide on E2F transcriptional activity, mRNA levels of E2F-dependent genes, E2F1- retinoblastoma protein (Rb) binding, and proteins levels of regulator of G1–S transition was also measured. DNA binding of E2F1 was evaluated by chromatin immunoprecipitation assay. Triptolide decreased colon cancer cell viability in a dose- and time-dependent fashion. Minnelide markedly inhibited the growth of colon cancer in the xenograft and liver metastasis model of colon cancer and more than doubles the median survival of animals with liver metastases from colon cancer. Mechanistically, we demonstrate that at low concentrations triptolide induces apoptotic cell death but at higher concentrations it induces cell cycle arrest. Our data suggest that triptolide is able to induce G1 cell cycle arrest by inhibiting transcriptional activation of E2F1. Our data also show that triptolide downregulates E2F activity by potentially modulating events downstream of DNA binding. Therefore, we conclude that Triptolide and Minnelide are effective against colon cancer in multiple pre-clinical models.

531. Intravenous Delivery of Toca 511 Gene Therapy in Combination with 5-Fluorocytosine for Intratumoral Production of 5-Fluorouracil in a Colon Cancer Metastasis Model

Despite advances in screening, colorectal cancer (CRC) remains the fourth most commonly diagnosed cancer and the second leading cause of cancer death for both men and women in the US. Approximately one half of patients with CRC develop liver metastases (mCRC). The standard treatment for mCRC is 5-fluorouracil (5-FU) based combination chemotherapy. 5-FU combination chemotherapy has extended the median survival of these patients from 6 to >20 months.We are pursuing a unique investigational approach to treat cancer via in situ production of 5-FU. Toca 511 (vocimagene amiretrorepvec), a retroviral replicating vector (RRV), selectively replicates and spreads in malignant cells and encodes an optimized yeast cytosine deaminase (CD) gene. Within infected cells, the CD enzyme is expressed and converts 5-FC (flucytosine, an orally available anti-fungal drug) to the anti-cancer drug 5-FU. Both a direct cytotoxic effect and an extended immunotherapeutic effect have been reported using this approach.Toca 511, in conjunction with subsequent oral extended-release 5-fluorocytosine (Toca FC), is currently under investigation in patients with recurrent high grade glioma. In these studies, Toca 511 is delivered either intratumorally (NCT01156584), by injection into the surgical resection bed (NCT01470794), or intravenously (NCT01985256) in subjects scheduled for subsequent resection. We tested the suitability of the intravenous approach for the treatment of mCRC in a mouse syngeneic liver metastasis model. CT-26-luciferase colon carcinoma cells were delivered via intrasplenic injection producing multiple tumor foci within the liver. Intravenous delivery of RRV resulted in expression of the vector encoded transgene in tumor foci but not in adjacent normal liver tissue. Intravenous delivery of Toca 511 followed by courses of 5-FC resulted in shrinkage or elimination of tumor foci and improved survival in this model of mCRC. The data is supportive of future clinical trials of intravenous Toca 511 followed by cycles of Toca FC in metastatic CRC.

Pien Tze Huang inhibits liver metastasis by targeting TGF-β signaling in an orthotopic model of colorectal cancer

Metastasis is the leading cause of cancer-related mortality in almost all types of cancers, including colorectal cancer (CRC). Epithelial-mesenchymal transition (EMT) is a critical process during the metastatic cascade. This process may be a potential target for the diagnosis and treatment of CRC. Pien Tze Huang (PZH), a well-known traditional Chinese formula, has been demonstrated to be clinically effective in treating various types of human malignancies, including CRC. Our published data suggest that PZH can induce apoptosis, as well as inhibit cell proliferation and tumor angiogenesis, thus suppressing CRC growth invitro and invivo. We evaluated the therapeutic efficacy of PZH against CRC metastasis using a CRC liver metastasis mouse model to further explore the mechanisms underlying the antitumor action of PZH. MTT, migration, and Matrigel invasion assays were used to assess the effect of PZH on cell viability, migration and invasion. We then established an orthotopic liver metastasis modelof colon cancer using microsurgical techniques. Mice were intragastrically administered 234mg/kg/day dose of either PZH or saline for 14days. The body and tumor weights of the mice were measured after they were sacrificed. Moreover, we examined the effect of PZH inhibition on liver metastasis. Finally, EMT-related proteins and the TGF-β signaling pathway were assessed using immunohistochemical staining (IHS). The present data revealed that PZH significantly inhibited the migration and invasion of CT-26 cells in a dose-dependent manner, which affirmed the inhibitory effect of PZH on CRC cell metastasis. No significant change was observed between the invivo primary tumor growth and body weight. However, the control group had five cases of liver metastasis(5/6), whereas one case was found in the PZH group(1/6). Thus, PZH exhibited therapeutic efficacy against CRC metastasis without apparent toxicity. The inhibitory effect of PZH on EMT resulted in an increase in E-cadherin expression, as well as a decrease in N-cadherin expression. In addition, PZH significantly inhibited TGF-β, as well as the phosphorylation of Smad2/3 and Smad4 in the tumor tissues, indicating its suppressive action on TGF-β signaling. These molecular effects ultimately resulted in the inhibition of cancer cell EMT and tumor metastasis.

Abstract PR08: Targeting tumor microenvironment with selective small-molecule inhibitors of CDK8/19

Abstract Cyclin-dependent kinase 8 (CDK8), along with its closely related paralog CDK19, are transcription-regulating kinases that, unlike some other members of the CDK family, do not regulate cell cycle progression and are not required for the growth of normal cells or most of the tumor cell types. CDK8 has been identified as an oncogene that enhances the activity of several tumor-promoting transcriptional pathways (such as TGFβ, β-catenin, HIF1A and serum factors), mediating the elongation of transcription of newly activated genes. We have previously reported the discovery of the first selective small-molecule inhibitors of CDK8/19 and their ability to block chemotherapy-induced tumor-promoting paracrine activities of both tumor and normal cells (Porter et al., PNAS 109, 13799, 2012). We now conducted chemical optimization of the original inhibitors, yielding an optimized preclinical lead compound, Senexin B. Senexin B inhibits CDK8/19 in low nanomolar range in vitro and in vivo as an ATP pocket binder, with very high target selectivity as indicated by kinome profiling. It is highly water-soluble, bioavailable, and produces no limiting toxicity upon prolonged administration in mice, at doses that yield plasma concentrations exceeding cellular IC50 by 2-3 orders of magnitude. Senexin B has been tested for efficacy in several animal models addressing different aspects of tumor growth and progression. (i) Pretreatment of tumor-free mice with Senexin B significantly inhibited the growth of triple-negative breast cancer (TNBC) cells inoculated into mice subsequently to Senexin B administration, indicating a general chemopreventive effect on the normal tissue “soil”. (ii) Senexin B potentiated the tumor-suppressive effect of doxorubicin on established TNBC xenografts; this effect was associated with the suppression of NFκB-mediated transcriptional induction of tumor-promoting cytokines. (iii) Senexin B inhibited invasive growth into the muscle layer in an orthotopic xenograft model of MDA-MB-468 TNBC cells. (iv) In a spleen-to-liver colon cancer metastasis model of syngeneic mouse CT26 tumors, Senexin B treatment of mice had the same effect as CDK8 knockdown in tumor cells: suppression of metastatic growth in the liver without a significant effect on primary tumor growth in the spleen. Taken together, these results indicate that CDK8/19 inhibition produces chemopotentiating, chemopreventive and anti-metastatic effects in different types of cancer, inhibiting tumor progression by acting both at the tumor cells (the “seed”) and the tumor microenvironment (the “soil”) of cancers. Citation Format: Donald C. Porter, Mengqian Chen, Jiaxin Liang, Vimala Kaza, Alexander Chumanevich, Serena Altilia, Elena Farmaki, Marj Pena, Gary P. Schools, Ioulia Chatzistamou, Lawrence T. Friedhoff, Mark P. Wentland, Eugenia V. Broude, Hippokratis Kiaris, Igor B. Roninson. Targeting tumor microenvironment with selective small-molecule inhibitors of CDK8/19. [abstract]. In: Abstracts: AACR Special Conference on Cellular Heterogeneity in the Tumor Microenvironment; 2014 Feb 26-Mar 1; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2015;75(1 Suppl):Abstract nr PR08. doi:10.1158/1538-7445.CHTME14-PR08

Abstract 708: Toca 511 gene therapy in combination with 5-fluorocytosine for intratumoral production of 5-fluorouracil in a colon cancer metastasis model

Abstract Despite advances in screening, colorectal cancer (CRC) remains the fourth most commonly diagnosed cancer and the second leading cause of cancer death for both men and women in the US. Approximately one half of patients with CRC develop liver metastases (mCRC). The standard treatment for mCRC is 5-fluorouracil (5-FU) based combination chemotherapy. 5-FU combination chemotherapy has extended the median survival of these patients from 6 to &amp;gt;20 months. We are pursuing a unique investigational approach to treat cancer via intratumoral production of 5-FU. Patients are first administered Toca 511 (vocimagene amiretrorepvec), a retroviral replicating vector (RRV) which selectively replicates and spreads in malignant cells and encodes an optimized yeast cytosine deaminase (CD) gene. In infected cells, the CD enzyme is expressed and converts 5-FC (flucytosine, an orally available anti-fungal drug) to the anti-cancer drug 5-FU in situ. The combination of Toca 511 with oral extended release 5-FC (Toca FC) is currently in clinical trials for recurrent High Grade Glioma (NCT01156584, NCT01470794, and NCT01985256). We tested the suitability of this approach for the treatment of mCRC in a mouse syngeneic liver metastasis model. CT-26-luciferase colon carcinoma cells were delivered via intrasplenic injection producing multiple tumor foci within the liver. Toca 511 was then delivered either by intrasplenic or intravenous injection followed by courses of 5-FC. Vector delivery to tumor was monitored by PCR. 5-FC and 5-FU were detected using LC/MS. The effect of treatment with Toca 511 and 5-FC in this mouse model of mCRC will be presented. Citation Format: Maria E. Rodriguez-Aguirre, Fernando Lopez Espinoza, Bryan Martin, Kader Yadiz, Tiffany T. Huang, Carlos E. Ibanez, Derek G. Ostertag, Noriyuki Kasahara, Harry E. Gruber, Douglas J. Jolly, Joan M. Robbins. Toca 511 gene therapy in combination with 5-fluorocytosine for intratumoral production of 5-fluorouracil in a colon cancer metastasis model. [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 708. doi:10.1158/1538-7445.AM2014-708

Abstract 4306: Bioconjugation of near infrared dyes by PEGylation improves pharmacokinetics and allows for better labeling and imaging of human gastrointestinal cancers

Abstract NIR dyes are becoming increasingly popular due to their good tissue penetration, low tissue autofluorescence and resistance to hemoglobin quenching. However, they are still limited by lower hydrophilicity and poor chemical and photo-stability in-vivo. We aimed to try to improve these properties by covalent modification of these dyes by addition of polyethylene glycol (PEG) chains. We studied 2 commercially-available NIR dyes, DyLight 650 and 750, to evaluate how PEGylation affects the pharmacokinetics, biodistribution and imaging. PEGylated and non-PEGylated DyLight 650 and 750 dyes were conjugated to a chimeric anti-CEA antibody and were injected intravenously into nude mice. Serum samples were collected at various time points to determine serum concentrations and clearance kinetics. Biodistribution was determined by tissue sonication and analyzing tissue concentration profiles over time. PEGylated dyes had significantly higher serum concentrations than non-PEGylated dyes with p=0.005 for the 650 dyes and p&amp;lt;0.001 for the 750 dyes. PEGylated dyes had significantly lower liver concentrations (p=0.03 for the 650 dyes; p=0.002 for the 750 dyes) and higher renal concentrations (p=0.003 for the 650 dyes and p&amp;lt;0.001 for the 750 dyes) compared to non-PEGylated dyes. Human pancreatic tumors subcutaneously implanted into nude mice were labeled with antibody-dye conjugates and serially imaged. Labeling with PEGylated dyes resulted in significantly brighter tumors compared to the non-PEGylated dyes (p&amp;lt;0.001 for the 650 dyes; p=0.01 for 750 dyes). In a liver metastasis model of colon cancer, PEGylated dyes showed bright labeling of metastatic tissue with good contrast between normal and malignant tissue. PEGylation of the NIR dyes DyLight 650 and 750 significantly changes their pharmacokinetics and biodistribution, allowing higher serum concentrations, longer half-life, brighter tissue labeling and decreased accumulation in lymphoid organs. This allows for high fidelity imaging of metastatic disease to any lymphoid or hemoglobin dense organ such as the liver. Citation Format: Ali A. Maawy, Yukihiko Hiroshima, George A. Luiken, Yong Zhang, Robert M. Hoffman, Michael Bouvet. Bioconjugation of near infrared dyes by PEGylation improves pharmacokinetics and allows for better labeling and imaging of human gastrointestinal cancers. [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 4306. doi:10.1158/1538-7445.AM2014-4306

A Personalized Preclinical Model to Evaluate the Metastatic Potential of Patient-Derived Colon Cancer Initiating Cells

Within the aim of advancing precision oncology, we have generated a collection of patient-derived xenografts (PDX) characterized at the molecular level, and a preclinical model of colon cancer metastasis to evaluate drug-response and tumor progression. We derived cells from 32 primary colorectal carcinomas and eight liver metastases and generated PDX annotated for their clinical data, gene expression, mutational, and histopathological traits. Six models were injected orthotopically into the cecum wall of NOD-SCID mice in order to evaluate metastasis. Three of them were treated with chemotherapy (oxaliplatin) and three with API2 to target AKT activity. Tumor growth and metastasis progression were analyzed by positron emission tomography (PET). Patient-derived cells generated tumor xenografts that recapitulated the same histopathological and genetic features as the original patients' carcinomas. We show an 87.5% tumor take rate that is one of the highest described for implanted cells derived from colorectal cancer patients. Cecal injection generated primary carcinomas and distant metastases. Oxaliplatin treatment prevented metastasis and API2 reduced tumor growth as evaluated by PET. Our improved protocol for cancer cell engraftment has allowed us to build a rapidly expanding collection of colorectal PDX, annotated for their clinical data, gene expression, mutational, and histopathological statuses. We have also established a mouse model for metastatic colon cancer with patient-derived cells in order to monitor tumor growth, metastasis evolution, and response to treatment by PET. Our PDX models could become the best preclinical approach through which to validate new biomarkers or investigate the metastatic potential and drug-response of individual patients.

Local Targeted Therapy of Liver Metastasis from Colon Cancer by Galactosylated Liposome Encapsulated with Doxorubicin

Since regional drug administration enables to maintain a high drug concentration within tumors, we compared the plasma concentration and biodistribution of doxorubicin (Dox) from drug-loaded conventional liposomes by local or systemic administration. The results demonstrated that drug concentration was substantially improved in liver as well as a decrease in blood and other organs by spleen injection mimicking portal vein perfusion (regional administration). To further investigate the targeted therapeutic effect of galactosylated liposome encapsulated doxorubicin (Dox) by regional administration, liver targeting liposomes were prepared by incorporating galactosylated-DPPE to conventional liposomes. Liposome uptake and targeting were verified in vitro and in vivo by fluorescence microscopy and xenogen IVIS imaging system, respectively. The results showed that galactose targeted liposomes presented a stronger specific cell uptake by human hepatocellular carcinoma HepG2 cells compared to the non-targeted liposomes. In vivo fluorescence imaging showed that the intra-hepatic deposition of conventional and galactosylated liposomes via spleen injection was more than that via tail vein administration, and galactosylated liposomes had higher fluorescent intensity over conventional liposomes in the liver post spleen administration. The anti-tumor effect of various drug administration routes for both liposomal formulations was evaluated using a murine liver metastasis model of colon cancer. The results indicated that tumor progression in the liver and mesenteric lymph nodes was significantly suppressed by Dox-loaded galactosylated liposomes via spleen injection, while no significance was observed in non-targeted formulations. Our data indicated that local perfusion of galactosylated liposomal doxorubicin had a great promise for the treatment of liver metastasis from colon cancer.

Genistein represses invasion in an in vitro colon cancer metastasis model by transcriptionally upregulating metastasis repressor NDRG1

Cancer metastasis accounts for most cancer deaths. Genistein, a biologically active soy isoflavone, has been indicated as an inhibitor of cancer progression. In this study, we tested our hypothesis that genistein can repress colon cancer metastasis in vitro by modifying the expression of metastasis suppressor NDRG1. Cell models SW480 and SW620 which are poorly and highly metastatic human colon cancer cell lines respectively, were used. Genistein treatment reduced the proliferation and wound healing ability of both cell lines. Interestingly, genistein specifically increased the adhesion of the metastatic cell line SW620 and inhibited the migration of SW620. Moreover, genistein increased the mRNA expression of NDRG1 in both cell lines. The low NDRG1 gene expression in SW620 compared to that in SW480 is associated with altered chromatin configuration of the gene. In conclusion, genistein treatment increased expression of several genes responsible for metastasis repression in SW620. Future research will focus on illustrating the epigenetic mechanisms underlining the metastasis inhibiting effects by genistein.Grant Funding Source : University of Illinois at Urbana‐Champaign

Lunasin potentiates the effect of oxaliplatin preventing outgrowth of colon cancer metastasis, binds to α 5β 1 integrin and suppresses FAK/ERK/NF-κB signaling

The effect of lunasin on colon cancer metastasis was studied using three human colon cancer cell lines in vitro and a liver metastasis model of colon cancer in vivo. Lunasin bound with α 5β 1 integrin and internalized into the nucleus of KM12L4 human colon cancer cells. Lunasin (10 μM) inhibited the activation of focal adhesion kinase (FAK) by 28%, 39% and 60% in RKO, HCT-116 and KM12L4 human colon cancer cells, respectively. Lunasin caused an increase in the expression of the inhibitor of kappa B alpha (IκB-α), a decrease in nuclear p50 NF-κB and a reduction in the migration of cancer cells. Lunasin (4 mg/kg bw) inhibited metastasis and potentiated the effect of oxaliplatin by reducing the expression of proliferating cell nuclear antigen. Liver metastatic nodules were reduced from 28 (PBS) to 14 (lunasin, P = 0.047) while combination of lunasin and oxaliplatin to 5 ( P = 0.004). The tumor burden was reduced from 0.13 (PBS) to 0.10 (lunasin, P = 0.039) to 0.04 (lunasin + oxaliplatin, P < 0.0001). Moreover, lunasin potentiated the effect of oxaliplatin in modifying expression of proteins involved in apoptosis and metastasis including Bax, Bcl-2, IKK-α and p-p65. Lunasin inhibited metastasis of human colon cancer cells by direct binding with α 5β 1 integrin suppressing FAK/ERK/NF-κB signaling, and potentiated the effect of oxaliplatin in preventing the outgrowth of metastasis.