Injection Of HT-29 Cells Research Articles

Establishment of orthotopic colorectal cancer model by colonoscopy with submucosal injection in living mice

Objective To set up a living mice colonoscopy platform to establish an orthotopic model of colorectal cancer in mice under direct vision, and to observe its biological behavior such as metastasis. Methods Eighteen-week-old male C57/BL mice were anesthetized, and the intestinal lumen of the mice was examined by a self-developed living mice colonoscopy and Olympus URF-P5 ureteroscopy, respectively. The imaging effects of the two methods were compared. Human colon cancer HT-29 cells were injected into the colonic mucosa of BALB/c-nu mice under direct vision. The colonoscopy was performed on the 3rd, 7th and 15th day after the injection to observe the tumor formation in the intestinal lumen. The mice were sacrificed when the body weight decreased significantly or cachexia appeared, and then the abdominal cavity was examined including the tumor formation and metastasis. Results The self-developed living mice colonoscopy platform can provide clear vision of enteric cavity, and no mice died in the colonoscopy examination. In vivo subcutaneous injection of HT-29 cells in mice was performed with a perforation rate of 15%, a mortality rate of 33.3%, a tumor formation rate of 62.5%, an abdominal metastasis rate of 60%, a liver metastasis rate of 25%, and an abdominal wall transfer rate of 25%. Conclusion The self-developed mice colonoscopy platform can be used for the study of colorectum in living mice. The imaging effect is no less than that of Olympus URF-P5 ureteroscopy. In addition, an orthotopic colorectal cancer model can be established by this platform combing with submucosal injection technology. Key words: Living mice colonoscopy; Submucosal injection; T stage; Tumor microenvironment; Orthotopic colorectal cancer model

Scutellaria barbata D. Don inhibits colorectal cancer growth via suppression of Wnt/β-catenin signaling pathway

To investigate the effect of the ethanol extract of Scutellaria barbata D. Don (EESB) on colorectal cancer (CRC) growth and Wnt/β-catenin signaling pathway in vivo and in vitro. In vivo experiment, CRC xenograft mouse model was constructed with injection of HT-29 cells. Following xenograft implantation, twenty mice were randomly divided into EESB-treated group (n=10) and control group (n=10) by a random number table, and were given with intra-gastric administration of 2 g/kg EESB or saline, 5 days a week for 16 days, respectively. At the end of experiment, tumors were removed and weighed by electronic scales. The proliferation biomarker Ki-67 of tumor was evaluated by immunohistochemistry (IHC) assay. In vitro study, HT-29 cells were treated with 0, 0.5, 1.5, 2.5 mg/mL EESB for 24 h. At the end of the treatment, the viability and survival of HT-29 cells were determined by methylthiazolyldiphenyl-tetrazolium bromide (MTT) assay and colony formation assay, respectively. The mRNA expression of c-Myc, Survivin and adenomatous polyposis coli (APC) was examined by reverse transcription-polymerase chain reaction (RT-PCR) both in tumor tissues of CRC xenograft mice and HT-29 cells. Protein expression of c-Myc, Survivin, APC, and β-catenin as well as β-catenin phosphorylation level were evaluated by IHC assay or Western blotting. EESB significantly reduced tumor weight in CRC xenografts mice, compared with the control group (P<0.05). IHC assay showed that EESB significantly inhibited protein expression of Ki-67 in tumor tissues (P<0.05). MTT assay showed that EESB significantly reduced HT-29 cell viability in a dose-dependent manner (P<0.05). Colony formation assay showed that EESB dose-dependently decreased the survival of HT-29 cells (P<0.05). In addition, RT-PCR assay showed that EESB decreased the mRNA expression of c-Myc and Survivin and increased APC expression, both in tumor tissues of CRC xenograft mice and HT-29 cells (P<0.05). IHC assay or Western blotting showed that EESB decreased protein expression of β-catenin, c-Myc and Survivin, as well as increased APC expression and β-catenin phosphorylation in tumor tissues or HT-29 cells (P<0.05). EESB significantly reduced tumor growth in CRC xenografts mice, and inhibited the viability and survival of HT-29 cells. EESB could suppress the activation of the Wnt/β-catenin pathway, which might be one of the mechanisms whereby Scutellaria barbata D. Don exerts its anticancer activity.

Aspirin prevents colorectal cancer metastasis in mice by splitting the crosstalk between platelets and tumor cells.

We investigated whether platelets prime colon cancer cells for metastasis and whether pharmacological inhibition of platelet function may prevent it. Coculturing HT29 human colon carcinoma cells with human platelets led to the induction of mesenchymal-like cancer cells characterized by downregulation of E-cadherin and upregulation of Twist1, enhanced cell mobility and a proaggregatory action on platelets. These changes were prevented by different antiplatelet agents, aspirin[an inhibitor of cyclooxygenase(COX)-1], DG-041[an antagonist of prostaglandin(PG)E2 EP3 receptor] and ticagrelor (a P2Y12 receptor antagonist). The injection of HT29 cells, exposed to platelets in vitro, into the tail vein of humanized immunodeficient mice led to higher incidence of lung metastasis compared to the injection of untreated HT29 cells. This effect was associated with enhanced systemic biosynthesis of thromboxane(TX)A2 and PGE2 in vivo. Platelet COX-1 inhibition by aspirin administration to mice prevented the increased rate of metastasis as well as the enhanced production of TXA2 and PGE2 induced by the in vitro priming of HT29 cells by platelets. In conclusion, targeting platelet COX-1 with low-dose aspirin exerts an antimetastatic action by averting the stem cell mimicry of cancer cells associated with enhanced proaggregatory effects induced by platelet-tumor cell interactions. These effects may be shared by other antiplatelet drugs.

Abstract 2029: Insight of 3D multicellular tumor spheroids for innovative culture models of potential relevance for the screening of anti-CRC compounds

Abstract OBJECTIVE - Multicellular tumor spheroid is a well-known method for 3D in-vitro culture that better represent in-vivo condition. We exploited this system to culture human established colorectal cell lines to investigate how the presence or absence of hypoxic and/or necrotic area can influence the gene expression profile and the responsiveness to drugs compared to 2D-culture and in-vivo xenografted cells. METHODS - HT29 cell line was cultured as MCTS by hanging-drop method. At size-defined time-point spheroids were analyzed for the presence of hypoxic and necrotic areas. The expression of a panel of genes related to tumor progression, metastatic behavior, and drug resistance was evaluated by real time PCR on MCTS at different growth stages in comparison to conventional 2D cultures and xenografts generated upon injection of HT29 cells in immunodeficient mice. RESULTS - Based on the absence or presence of hypoxic and/or necrotic core, we identified three different MCTS growth stages: stage 1 (MCTS diameter size &amp;lt;200μm) defined by the absence of hypoxia/necrosis, stage 2 (300-350μm) characterized by the presence of hypoxic, but not apoptotic or necrotic areas, and stage 3 (&amp;gt;500μm) identified by the presence of both hypoxic and necrotic areas. The expression of a panel of genes, including putative cancer stem cell markers CD133, CD44, CD24 was found to be upregulated in stage 1 MCTS, as compared to 2D cultures. Most interestingly a further upregulation of these genes was detected in stage 2 and especially in stage 3. Importantly, expression levels in stage 3 were the most similar to those detected in xenografts. CONCLUSION - Our results showed that culture of CRC cell lines offer the opportunity to investigate in standardized condition different MCTS stages possibly proving an improved model of in-vivo tumor growth. The next generation sequencing and drug-responsiveness of tumor cells cultured in 2D and 3D at different spheroid maturation stages is currently being evaluated. Citation Format: Silvio Däster, Nunzia Amatruda, Diego Calabrese, Paul Zajac, Giulio C. Spagnoli, Giandomenica Iezzi, Valentina Mele, Manuele Giuseppe Muraro. Insight of 3D multicellular tumor spheroids for innovative culture models of potential relevance for the screening of anti-CRC compounds. [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 2029. doi:10.1158/1538-7445.AM2014-2029

Abstract 3834: Establishment and characterization of 3D multicellular tumor spheroids as preclinical in vitro models of human colorectal cancer.

Abstract Objective: Multicellular tumor spheroid is a well-known method for three-dimensional in-vitro culture that better represent in-vivo condition. We exploited this system to culture human established colorectal cell lines to investigate how the presence or absence of hypoxic and/or necrotic area, due to oxygen and nutrient gradients, can influence the gene expression profile and the responsiveness to drugs compared to 2D culture and in vivo xenografted cells. Methods: HT29 cell line was cultured as MCTS by hanging drop method. At size-defined time point spheroids were collected and analyzed for the presence of hypoxic and necrotic areas. The expression of a panel of genes related to tumor progression, metastatic behavior, and drug resistance was evaluated by real time PCR on MCTS at different growth stages in comparison to conventional 2D cultures and xenografts generated upon injection of HT29 cells in immunodeficient mice. Results: Based on the absence or presence of hypoxic and/or necrotic core, we identified three different MCTS growth stages: stage 1 (MCTS diameter size &amp;lt;200μm) defined by the absence of hypoxia/necrosis, stage 2 (300-350μm) characterized by the presence of hypoxic, but not apoptotic or necrotic areas, and stage 3 (&amp;gt;500μm) identified by the presence of both hypoxic and necrotic areas. The expression of a panel of genes, including KRAS, p53, and putative cancer stem cell markers CD133, CD44, CD24 was found to be upregulated in stage 1 MCTS, as compared to 2D cultures. Most interestingly a further upregulation of these genes was detected in stage 2 and especially in stage 3. Importantly expression levels in stage 3 were the most similar to those detected in xenografts. Conclusion: Our results showed that culture of established CRC cell lines offer the opportunity to investigate in standardized condition different MCTS stages possibly proving an improved model of in-vivo tumor growth. The drug-responsiveness of tumor cells cultured in 2D and 3D at different spheroid maturation stages is currently being assessed. Citation Format: Silvio Daester, Nunzia Amatruda, Diego Calabrese, Paul Zajac, Giulio Cesare Spagnoli, Giandomenica Iezzi, Valentina Mele, Manuele Giuseppe Muraro. Establishment and characterization of 3D multicellular tumor spheroids as preclinical in vitro models of human colorectal cancer. [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 3834. doi:10.1158/1538-7445.AM2013-3834

Anticancer Effects of IP6 in a Human Colon Carcinoma Cell Line in Nude Mice Xenografts

Purpose: Inositol hexaphosphate (IP6) is a naturally occurring polyphosphorylated carbohydrate that has been shown to suppress the growth of epithelial cancer. Because IP6 is a dietary phytochemical present in cereals, soy, legumens, and fiber-rich foods, we evaluated the efficacy of IP6 against colon cancer formation. Methods: HT-29 cells were injected into nude mice. The animals were fed a normal diet (group 1), a low IP6 diet (group 2), and a high IP6 diet (group 3) for 4 wk. Body weight, tumor volume, tumor growth rate, growth inhibition rate, and therapeutic ratio were monitored after injection of HT-29 cells. Results: HT-29-cell human-colon-carcinoma xenograft mice treated with IP6 showed a significant reduction in tumor growth rate, irrespective of the IP6 dose compared to normal diet group. Compared with the control group, group 3 showed a significant reduction (45%) in tumor volume. In the therapeutic ratio gain profiles, IP6 diet groups showed a significant alteration of therapeutic ratio when compared with the normal diet group (0% vs. 11%, P=0.014). In the body weight gain profiles, group 3 showed a significant reduction of body weight compared with the other two groups (20.25 g vs. 21.6 g, 21.7 g, P=0.009). Groups 1 and 2 showed similar changes in body weight. Tumor xenografts from IP6-fed mice showed significantly decreased cancer formation and growth, but increased toxicity was noted for high doses of IP6. Conclusion: These results indicate that in the future, IP6 could be an effective chemopreventive or chemotherapeutic agent for use in the treatment of colon cancer.

1H MRS markers of tumour growth in intrasplenic tumours and liver metastasis induced by injection of HT-29 cells in nude mice spleen.

We have characterized, by in vitro magnetic resonance spectroscopy (MRS), the metabolite pattern of perchloric acid (PCA) extracts of intrasplenic tumours and hepatic metastasis, produced by intra-spleen injection of the human colorectal carcinoma cell line HT-29 and its metastatic variant HT-29 MMM into nude mice. Our aim was to gain further understanding of colorectal tumour metabolism as a basis for future in vivo studies of human colon cancer by 1H MRS. Metabolite PCA extract analysis showed a good reproduction of the spectral pattern observed in human primary colon tumours, while they were very different from the spectral pattern of the host tissues (spleen and liver). The main differences between host and tumour tissues involved taurine, phosphocholine (PC), phosphoethanolamine (PE), creatine, glycogen and glucose. Creatine is the most promising marker to follow tumour growth because of its practical absence in the nude mice host tissues. Detection of variable levels of this compound and of taurine in hepatic foci in man, are suggested as possible diagnostic markers. No correlation could be found between spectral pattern differences and the different ability to metastasize of the two HT-29 cell lines used. Furthermore, indirect evidence for a functional link between taurine and myo-inositol in colon tumour cells is presented. In summary, our data suggest that the nude mice model may be a suitable system for the MRS study of the changes taking place in host tissues upon tumour progression.