Orthotopic Microinjection of Human Colon Cancer Cells in Nude Mice Induces Tumor Foci in All Clinically Relevant Metastatic Sites
2007; Elsevier BV; Volume: 170; Issue: 3 Linguagem: Inglês
10.2353/ajpath.2007.060773
ISSN1525-2191
AutoresMaría Virtudes Céspedes, Carolina Espina, Miguel Ángel García‐Cabezas, Manuel Trías, Alicia Boluda, María Teresa Gómez del Pulgar, Francesc J. Sancho, Manuel Nistal, Juan Carlos Lacal, Ramón Mangues,
Tópico(s)Cancer Genomics and Diagnostics
ResumoDespite metastasis as an important cause of death in colorectal cancer patients, current animal models of this disease are scarcely metastatic. We evaluated whether direct orthotopic cell microinjection, between the mucosa and the muscularis layers of the cecal wall of nude mice, drives tumor foci to the most relevant metastatic sites observed in humans and/or improves its yield as compared with previous methods. We injected eight animals each tested human colorectal cancer cell line (HCT-116, SW-620, and DLD-1), using a especially designed micropipette under binocular guidance, and evaluated the take rate, local growth, pattern and rate of dissemination, and survival time. Take rates were in the 75 to 88% range. Tumors showed varying degrees of mesenteric and retroperitoneal lymphatic foci (57 to 100%), hematogenous dissemination to liver (29 to 67%) and lung (29 to 100%), and peritoneal carcinomatosis (29 to 100%). Tumor staging closely correlated with animal survival. Therefore, the orthotopic cell microinjection procedure induces tumor foci in the most clinically relevant metastatic sites: colon-draining lymphatics, liver, lung, and peritoneum. The replication of the clinical pattern of dissemination makes it a good model for advanced colorectal cancer. Moreover, this procedure also enhances the rates of hematogenous and lymphatic dissemination at relevant sites, as compared with previously described methods that only partially reproduce this pattern. Despite metastasis as an important cause of death in colorectal cancer patients, current animal models of this disease are scarcely metastatic. We evaluated whether direct orthotopic cell microinjection, between the mucosa and the muscularis layers of the cecal wall of nude mice, drives tumor foci to the most relevant metastatic sites observed in humans and/or improves its yield as compared with previous methods. We injected eight animals each tested human colorectal cancer cell line (HCT-116, SW-620, and DLD-1), using a especially designed micropipette under binocular guidance, and evaluated the take rate, local growth, pattern and rate of dissemination, and survival time. Take rates were in the 75 to 88% range. Tumors showed varying degrees of mesenteric and retroperitoneal lymphatic foci (57 to 100%), hematogenous dissemination to liver (29 to 67%) and lung (29 to 100%), and peritoneal carcinomatosis (29 to 100%). Tumor staging closely correlated with animal survival. Therefore, the orthotopic cell microinjection procedure induces tumor foci in the most clinically relevant metastatic sites: colon-draining lymphatics, liver, lung, and peritoneum. The replication of the clinical pattern of dissemination makes it a good model for advanced colorectal cancer. Moreover, this procedure also enhances the rates of hematogenous and lymphatic dissemination at relevant sites, as compared with previously described methods that only partially reproduce this pattern. Colorectal cancer cases represents 15% of all cancer types. Its poor prognosis and the consequence of its metastatic spread makes colorectal cancer the second most common cause of cancer death in western countries.1Jemal A Siegel R Ward E Murray T Xu J Smigal C Thun MJ Cancer statistics, 2006.CA Cancer J Clin. 2006; 56: 106-130Crossref PubMed Scopus (5488) Google Scholar However, genetically modified mouse models of colorectal cancer are scarcely or not metastatic.2Corpet DE Pierre F Point: from animal models to prevention of colon cancer. Systematic review of chemoprevention in min mice and choice of the model system.Cancer Epidemiol Biomarkers Prev. 2003; 12: 391-400PubMed Google Scholar, 3Donehower LA Harvey M Slagle BL McArthur MJ Montgomery Jr, CA Butel JS Bradley A Mice deficient for p53 are developmentally normal but susceptible to spontaneous tumours.Nature. 1992; 356: 215-221Crossref PubMed Scopus (4009) Google Scholar Moreover, more metastatic cancer models, such as surgical orthotopic implantation (SOI), experimental or spontaneous metastasis assays, and orthotopic cell injection also show limitations. Thus, although SOI of human colorectal cancer in nude mice yields liver metastasis,4Hoffman RM Orthotopic metastatic mouse models for anticancer drug discovery and evaluation: a bridge to the clinic.Invest New Drugs. 1999; 17: 343-359Crossref PubMed Scopus (482) Google Scholar, 5Fu XY Besterman JM Monosov A Hoffman RM Models of human metastatic colon cancer in nude mice orthotopically constructed by using histologically intact patient specimens.Proc Natl Acad Sci USA. 1991; 88: 9345-9349Crossref PubMed Scopus (329) Google Scholar it does not generate lung metastasis, nor mesenteric or retroperitoneal lymphatic metastasis,6Rashidi B Gamagami R Sasson A Sun FX Geller J Moossa AR Hoffman RM An orthotopic mouse model of remetastasis of human colon cancer liver metastasis.Clin Cancer Res. 2000; 6: 2556-2561PubMed Google Scholar and requires the previous expansion of the tumor in subcutaneous xenografts,7Kuo TH Kubota T Watanabe M Furukawa T Teramoto T Ishibiki K Kitajima M Moossa AR Penman S Hoffman RM Liver colonization competence governs colon cancer metastasis.Proc Natl Acad Sci USA. 1995; 92: 12085-12089Crossref PubMed Scopus (84) Google Scholar, 8Guilbaud N Kraus-Berthier L Meyer-Losic F Malivet V Chacun C Jan M Tillequin F Michel S Koch M Pfeiffer B Atassi G Hickman J Pierre A Marked antitumor activity of a new potent acronycine derivative in orthotopic models of human solid tumors.Clin Cancer Res. 2001; 7: 2573-2580PubMed Google Scholar, 9Flatmark K Maelandsmo GM Martinsen M Rasmussen H Fodstad O Twelve colorectal cancer cell lines exhibit highly variable growth and metastatic capacities in an orthotopic model in nude mice.Eur J Cancer. 2004; 40: 1593-1598Abstract Full Text Full Text PDF PubMed Scopus (98) Google Scholar which may alter its growth and dissemination capacities.10Farré L Casanova I Guerrero S Trias M Capella G Mangues R Heterotopic implantation alters the regulation of apoptosis and the cell cycle and generates a new metastatic site in a human pancreatic tumor xenograft model.FASEB J. 2002; 16: 975-982Crossref PubMed Scopus (15) Google Scholar On the other hand, the experimental metastasis assay or spontaneous metastasis assay, consisting of cell injection into the tail vein or footpad, are less physiological and usually generate tumor foci only at one single site.11Giavazzi R Jessup JM Campbell DE Walker SM Fidler IJ Experimental nude mouse model of human colorectal cancer liver metastases.J Natl Cancer Inst. 1986; 77: 1303-1308PubMed Google Scholar, 12Bresalier RS Hujanen ES Raper SE Roll FJ Itzkowitz SH Martin GR Kim YS An animal model for colon cancer metastasis: establishment and characterization of murine cell lines with enhanced liver-metastasizing ability.Cancer Res. 1987; 47: 1398-1406PubMed Google Scholar, 13Garofalo A Chirivi RG Scanziani E Mayo JG Vecchi A Giavazzi R Comparative study on the metastatic behavior of human tumors in nude, beige/nude/xid and severe combined immunodeficient mice.Invasion Metastasis. 1993; 13: 82-91PubMed Google Scholar, 14Wang Y Liang X Wu S Murrell GA Doe WF Inhibition of colon cancer metastasis by a 3′-end antisense urokinase receptor mRNA in a nude mouse model.Int J Cancer. 2001; 92: 257-262Crossref PubMed Scopus (45) Google Scholar, 15Katoh M Neumaier M Nezam R Izbicki JR Schumacher U Correlation of circulating tumor cells with tumor size and metastatic load in a spontaneous lung metastasis model.Anticancer Res. 2004; 24: 1421-1425PubMed Google Scholar, 16Chen X Su Y Fingleton B Acuff H Matrisian LM Zent R Pozzi A Increased plasma MMP9 in integrin alpha1-null mice enhances lung metastasis of colon carcinoma cells.Int J Cancer. 2005; 116: 52-61Crossref PubMed Scopus (57) Google Scholar Moreover, injection of colorectal cancer cells in the ileocolic vein or in the apical lymphoid follicle12Bresalier RS Hujanen ES Raper SE Roll FJ Itzkowitz SH Martin GR Kim YS An animal model for colon cancer metastasis: establishment and characterization of murine cell lines with enhanced liver-metastasizing ability.Cancer Res. 1987; 47: 1398-1406PubMed Google Scholar, 17Goldrosen MH Murine colon adenocarcinoma: immunobiology of metastases.Cancer. 1980; 45: 1223-1228Crossref PubMed Scopus (38) Google Scholar, 18Schackert HK Fidler IJ Development of an animal model to study the biology of recurrent colorectal cancer originating from mesenteric lymph system metastases.Int J Cancer. 1989; 44: 177-181Crossref PubMed Scopus (48) Google Scholar limits metastases to liver and lymphatics, varying widely in their rate.We tested whether direct orthotopic cell microinjection (OCMI), between the mucosa and the muscularis externa layers of the cecal wall of nude mice, induces tumor foci in the most relevant metastatic sites observed in humans and/or improves its yield compared with previous methods. This technique required the use of an especially designed pipette, under binocular guidance. The application of this procedure to the human colorectal cancer cell lines HCT-116, SW-620, and DLD-1 yielded high tumor take and dissemination rates, replicating the metastatic spread to lymph nodes, liver, lung, and peritoneum observed in advanced human colorectal cancer.Materials and MethodsCell Lines and Micropipette ConstructionHCT-116, SW-620, and DLD-1 cell lines were purchased from the American Type Culture Collection, Rockville, MD, and maintained in RPMI or McCoy's medium with 10% fetal bovine serum in 5% CO2 at 37°C. The micropipette used for cell injection was constructed using hemocytometric capillaries (1.55 × 70 mm; Vitrex Medical, Herlev, Germany). Two series of heat and stretch timing, pulling from the edges into opposite directions, elongated the tip of the pipette to achieve a 250-μm diameter. The unmodified end of the pipette was fitted into a pediatric butterfly 25 (19.1 × 0.5 mm) (Venisystems; Johnson & Johnson, Arlington, TX) (Figure 1A). Before its use, the pipette was cleaned with 90% ethanol, 70% ethanol, and sterile water, and exposed to UV for 24 hours.Experimental DesignFour-week-old male Swiss Nu/Nu mice weighing 18 to 20 g (Charles River, Margate, Kent, UK) were used. Eight animals were injected with a cell suspension, for each of the HCT-116, SW-620, or DLD-1 colorectal cancer lines, to compare their dissemination pattern and survival time. Two additional animals per group were sacrificed 1 week after injection to analyze the distribution of the injected tumor cells within the colonic wall. Mice were housed in a sterile environment with water, bedding, and γ-ray-sterilized food. Experiments were approved by the Hospital de Sant Pau Animal Ethics Committee.OCMINude mice were anesthetized with ketamine and xylazine, exteriorizing their cecum by a laparotomy (Figure 1B). Cells (2 × 106) per cell line (HCT-116, SW-620, or DLD-1) were suspended in 50 μl of Dulbecco's modified Eagle's medium and placed into the sterile micropipette (Figure 1B). We slowly injected the cell suspension, under a binocular lens (×3 magnification), with an approximate 30° angle and its tip introduced 5 mm into the cecal wall (Figure 1, C and D). Afterward, we applied a slight pressure with a cotton stick at ∼2 mm from the injection point in the direction of the pipette axis. We pulled the pipette out and cleaned the area around the injection with 3% iodine to avoid seeding of unlikely refluxed tumor cells into the abdominal cavity. The small diameter and flexible tip of the pipette and the angular and slow rate of administration diminished resistance to the injection, limiting tissue damage and bleeding, ensuring the absence of cell reflux. After injection, the gut was returned to the abdominal cavity and closed with surgical grapes. Animals were kept until death because of their neoplastic process or until the end of the experiment (130 days).Necropsy Procedure, Tumor Grading, and StagingAt animal death, a complete necropsy procedure was performed. Cervical, thoracic, abdominal, and pelvic organs were extracted en bloc. We assessed the presence of local tumor at the injection site, measured its two largest diameters, and recorded all macroscopic tumor deposits or abnormalities in any organ. The whole block of organs was fixed with buffered formalin for 48 hours, except for the lung, which was perfused and dissected and paraffin-embedded. Six-micron-thick sections were stained with hematoxylin and eosin. Tumor sections at the injection site and tumor deposits in other organs were stained with the per-iodine acid shift reaction.Two general pathologists analyzed histopathologically the tumor at the injection site and at the end of the experiment. Tumors were classified into three different grades depending on necrotic extent, mitotic rate, and extent of gland-like structures.19Hamilton SR Aaltonen LA Pathology and genetics of tumours of the digestive system. World Health Organization Classification of Tumours. IARC Press, Lyons2000Google Scholar The presence of tumor cells in other organs was also recorded, especially those in which colorectal cancer foci were expected (mesenteric lymph nodes, liver, lung, and peritoneum). We calculated tumor take rate as the percentage of mice with local tumor growth with respect to the total number of injected mice. We monitored daily tumor progression by palpation and calculated the final tumor volume, using the formula: volume = (longer diameter) × (shorter diameter)2/2. Tumor growth rate was calculated dividing tumor volume by survival time.In addition, we developed a four-stage system to stage the tumor in each animal. Stage I consisted in the presence of local tumor growth. Stage II was stage I plus peritoneal carcinomatosis (tumor in the parietal peritoneum of the abdominal wall, the diaphragm, or the visceral peritoneum of the digestive tract and liver). Stage III was stage I and/or II plus mesenteric lymph node and/or pancreatic foci (mouse pancreas is intraperitoneal). Stage IV was stage I, II, or III plus hepatic and/or lung foci.Statistical AnalysisDifferences in final tumor volume between HCT-116, SW-620, or DLD-1 groups were analyzed using the Mann-Whitney test. The likelihood of survival was estimated according to the Kaplan and Meier method20Kaplan EL Meier P Nonparametric estimation from incomplete observations.J Am Stat Assoc. 1958; 53: 457-481Crossref Scopus (47684) Google Scholar and survival distributions compared using the log-rank test.21Mantel N Evaluation of survival data and two new rank order statistics arising in its consideration.Cancer Chemother Rep. 1966; 50: 163-170PubMed Google Scholar Differences in take rates or in the presence of lymphatic, hepatic, or lung foci, or carcinomatosis between groups were compared using Fisher's test. We considered the statistical differences significant at a value of P < 0.05.ResultsLocalization of the Injected CellsThe main objective of the OCMI procedure was to deposit human colorectal cancer cells close to the colonic mucosa (Figure 1), where colorectal carcinomas are initiated,22Spratt Jr, JS Spjut HJ Prevalence and prognosis of individual clinical and pathologic variables associated with colorectal carcinoma.Cancer. 1967; 20: 1976-1985Crossref PubMed Scopus (208) Google Scholar, 23Safford KL Spebar MJ Rosenthal D Review of colorectal cancer in patients under age 40 years.Am J Surg. 1981; 142: 767-769Abstract Full Text PDF PubMed Scopus (38) Google Scholar using a small caliber pipette (33-gauge) and a binocular lens to increase the precision of the injection. We histologically assessed the location of the injected cells in two animals per group, 1 week after injection, confirming their placement in the space between the mucosa and the muscularis externa layers of the cecal wall (Figure 2A). This was done safely because none of the animals showed any morbidity or died because of the procedure.Figure 2Site of injection and pattern of tumor growth and invasion. A: The site of injection, 1 week after the OCMI procedure, showed the tumor cells located between the mucosa and the muscularis externa layers of the cecal wall. B: Tumors grow, both tangentially and circumferentially, and protrude into the lumen of the large bowel obstructing it. C–E: Tumors derived from HCT-116 (C), SW-620 (D), or DLD-1 (E) cell lines are poorly differentiated adenocarcinomas with high cellularity, focal areas of necrosis, frequent mitoses, and highly atypical nuclei, showing invasion of all of the cecal layers. F: Tumor cells infiltrate the lymphatics of the cecal wall (white asterisk). A, C–F: H&E stains.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Take Rates, Local Tumor Growth, and HistopathologyWe observed high tumor take rates for all cell lines (Table 1). HCT-116 or SW-620 cells generated tumors in six of eight (75%) animals, and DLD-1 cells generated tumors in seven of eight (88%) animals. Next, we evaluated the capacity of the OCMI procedure to replicate the histopathological appearance and clinical behavior of colorectal cancer in humans. Most HCT-116, SW-620, or DLD-1 tumors invaded the normal cecum in the tangential and transverse (circumferential growth) directions, yielding tumors that protruded into the cecal lumen (Figure 2B).Table 1Local Tumor Growth and Dissemination Pattern in Animals Bearing Tumors Derived from OCMI-Implanted Human Colorectal Cancer Cell LinesLocal tumorDissemination site [mice (%)]†Mice with tumor foci at the particular distant (far away from the injection) site/total number of mice with local tumor growth per group.Cell lineMice (n)Take rate [mice (%)]*Mice with local tumor growth/total number of injected mice per group.Final tumor volume (mean ± SE) (cm3)Growth rate (cm3/day)LymphaticHepaticLungCarcinomatosisHCT-11686/8 (75)4.4 ± 0.1‡Statistically significant differences at P = 0.043 or at§P = 0.047 (Mann-Whitney test).0.856/6 (100)4/6 (67)¶Statistically significant differences at P = 0.030 or at3/6 (50)6/6 (100)∥P = 0.016 (Fisher's test).SW-62086/8 (75)3.9 ± 0.2§P = 0.047 (Mann-Whitney test).0.386/6 (100)0/6 (0)¶Statistically significant differences at P = 0.030 or at1/6 (17)5/6 (83)DLD-187/8 (88)3.7 ± 0.3‡Statistically significant differences at P = 0.043 or at0.254/7 (57)2/7 (29)2/7 (29)2/7 (29)∥P = 0.016 (Fisher's test).* Mice with local tumor growth/total number of injected mice per group.† Mice with tumor foci at the particular distant (far away from the injection) site/total number of mice with local tumor growth per group.‡ Statistically significant differences at P = 0.043 or at§ P = 0.047 (Mann-Whitney test).¶ Statistically significant differences at P = 0.030 or at∥ P = 0.016 (Fisher's test). Open table in a new tab The three cell lines gave rise to poorly differentiated adenocarcinomas. They were highly cellular, composed of atypical cells with pleomorphic nuclei, arranged mainly in sheets and solid nests, and forming diffuse fronts that invaded the normal colon. Only occasional glandular lumens were seen [Figure 2, C (HCT-116), D (SW-620), and E (DLD-1)]. Many necrotic areas were also found. The mitotic count was always higher than five mitoses per 10 high-power fields. The tumor cells invaded all of the cecal layers and were also found inside the lumen of lymphatic vessels of the cecal wall (Figure 2F).Tumor volume and growth rates showed differences among groups. Mean tumor volumes in DLD-1 (3.7 ± 0.3 cm3) and SW-620 (3.9 ± 0.2 cm3) groups were similar; however, the mean HCT-116 tumor volume was significantly (P < 0.05) higher (4.4 ± 0.1 cm3) (Table 1). In addition, the HCT-116 tumor registered the highest growth rate (0.85 cm3/day), SW-620 growth rate was intermediate (0.38 cm3/day), and DLD-1 grew slowly (0.25 cm3/day) (Table 1). There was no relation between tumor take rate and growth rate in the studied groups.Tumor Dissemination Sites and Tumor StagingWe also evaluated the usefulness of the OCMI procedure to study lymphatic and hematogenous dissemination and carcinomatosis. Mesenteric lymphatic foci occurred in almost all of the animals, with the highest frequencies in the HCT-116 or SW-620 tumor-bearing mice (six of six, 100%) and the lowest in DLD-1 (four of seven, 57%) (Table 1). Tumor foci were located in the mesenteric lymph nodes draining the cecal area (Figure 3A) or in retroperitoneal lymph nodes. Microscopically, the affected nodes showed a rim of lymphocytes under the capsule (Figure 3B). Tumor foci were also frequent in pancreatic lymphatics.Figure 3Lymphatic, hematogenous, and peritoneal tumor spread in nude mice bearing tumors derived from OCMI-implanted cell lines A: Out of the local tumor (black asterisk), tumor cells spread to the mesenteric lymph nodes (white arrow). B: Microscopically, tumor foci in the mesenteric lymph nodes showed a rim of lymphocytes (white asterisk) compressed against the capsule by tumor cells (black asterisk). C: Representative foci of hematogenous dissemination to the liver, observed in the HCT-116 cell line. D: Tumor cells at the lung were found inside peribronchial vessels. E: Tumor cells invading the muscle of the diaphragm from its peritoneal surface. B–E: H&E stains.View Large Image Figure ViewerDownload Hi-res image Download (PPT)We also studied the presence of liver or lung tumor foci because these are the most common sites for blood-borne metastases in human colorectal cancer.24De Vita V Cancer: Principles and Practice of Oncology. ed 7. Lippincott, Philadelphia2005Google Scholar We observed liver foci in the HCT-116 (four of six, 67%) and DLD-1 (two of seven, 29%) tumor-bearing animals (Table 1), with tumor cells within and around blood vessels invading the liver parenchyma (Figure 3C). However, tumors derived from the SW-620 cell line did not generate foci in the liver. Tumor microfoci in the lung (Figure 3D) were detected in 50% (three of six) of the animals bearing HCT-116 tumors, 29% (two of seven) of the DLD-1 mice, and 17% (one of six) of the SW-620 mice (Table 1). In addition, tumor deposits infiltrating the surface of the visceral and parietal peritoneum (Figure 3E) were found in six of six (100%) HCT-116 mice, five of six (83%) SW-620 mice, and two of seven (29%) DLD-1 mice (Table 1). The three different cell lines varied widely in their capacity of inducing different disease stages (Table 2). Whereas the HCT-116 cell line yielded mainly stage IV tumors (67%), most of the SW-620 tumors were at stage III (67%). In addition, whereas no early tumors were recorded in the HCT-116 or SW-620 cell lines, most of the DLD-1 tumors were at stage I of the disease (42%) (Table 2).Table 2Tumor Staging and Survival Time in Animals Bearing Tumors Derived from OCMI-Implanted Human Colorectal Cancer Cell LinesTumor staging (% of mice)Cell lineStage IStage IIStage IIIStage IVSurvival time (days, mean ± SE)HCT-1160/6 (0%)0/6 (0%)2/6 (33%)4/6 (67%)39 ± 4†P = 0.00116 (log-rank test).SW-6200/6 (0%)0/6 (0%)5/6 (67%)1/6 (17%)73 ± 10*P = 0.0002 or at†P = 0.00116 (log-rank test).DLD-13/7 (42%)0/7 (0%)2/7 (29%)2/7 (29%)110 ± 7*P = 0.0002 or at†P = 0.00116 (log-rank test).Statistically significant differences at* P = 0.0002 or at† P = 0.00116 (log-rank test). Open table in a new tab Correlation between Tumor Staging and Animal SurvivalSurvival time differed widely (4 to 16 weeks range) among groups (P = 0.032) (Table 2 and Figure 4); it was the shortest in HCT-116 tumor-bearing mice (39 ± 4 days); SW-620 mice showed intermediate survival times (73 ± 10 days), and DLD-1 mice had the longest survival (110 ± 7 days). Cell lines varied widely in their capacity to induce hematogenous or lymphatic dissemination. As described in Table 1, Table 2, there was a clear correlation between staging and animal survival in the three studied groups. Thus, the HCT-116 cell line gave rise to highly aggressive and disseminating tumors, which reached stage IV in most cases (67%) and caused death in only 39 ± 4 days. The SW-620 cell line gave rise mostly (67%) to stage III tumors, causing death in 73 ± 10 days. Finally, the DLD-1 cell line gave rise mostly (42%) to stage I tumors, which caused death only after 110 ± 7 days (see Figure 4).Figure 4Survival curves of animals from the three studied groups. Survival time was the shortest in HCT-116 tumor-bearing mice, intermediate in SW-620 animals, and the longest in DLD-1 mice. Cumulative survival values were estimated using the Kaplan and Meier method and plotted versus time after injection. The log-rank test showed statistically significant differences in survival among groups (see also Table 2).View Large Image Figure ViewerDownload Hi-res image Download (PPT)DiscussionThe OCMI procedure—the injection of human colorectal cancer cell lines between the mucosa and the muscularis externa layers of the cecal wall of nude mice—generates models in which the dissemination pattern closely replicates all relevant metastatic sites observed in humans and enhances their dissemination rate compared with previous methods. We chose to assess the HCT-116,25Tsuiji H Hayashi M Wynn DM Irimura T Expression of mucin-associated sulfo-Lea carbohydrate epitopes on human colon carcinoma cells.Jpn J Cancer Res. 1998; 89: 1267-1275Crossref PubMed Scopus (16) Google Scholar, 26de Albuquerque Garcia Redondo P Nakamura CV de Souza W Morgado-Diaz JA Differential expression of sialic acid and N-acetylgalactosamine residues on the cell surface of intestinal epithelial cells according to normal or metastatic potential.J Histochem Cytochem. 2004; 52: 629-640Crossref PubMed Scopus (38) Google Scholar SW-620,27Hewitt RE McMarlin A Kleiner D Wersto R Martin P Tsokos M Stamp GW Stetler-Stevenson WG Validation of a model of colon cancer progression.J Pathol. 2000; 192: 446-454Crossref PubMed Scopus (185) Google Scholar and DLD-125Tsuiji H Hayashi M Wynn DM Irimura T Expression of mucin-associated sulfo-Lea carbohydrate epitopes on human colon carcinoma cells.Jpn J Cancer Res. 1998; 89: 1267-1275Crossref PubMed Scopus (16) Google Scholar, 28Honda K Yamada T Hayashida Y Idogawa M Sato S Hasegawa F Ino Y Ono M Hirohashi S Actinin-4 increases cell motility and promotes lymph node metastasis of colorectal cancer.Gastroenterology. 2005; 128: 51-62Abstract Full Text Full Text PDF PubMed Scopus (168) Google Scholar cell lines because of the previous characterization of their capacity to disseminate.Replication of Human Colorectal Cancer Stages, Dissemination Pattern, and AggressivenessAs observed in humans, tumors generated using the OCMI procedure show circumferential growth, finally protruding into the lumen.24De Vita V Cancer: Principles and Practice of Oncology. ed 7. Lippincott, Philadelphia2005Google Scholar They also replicate the pattern of lymphatic spread, disseminating to the mesenteric lymph nodes draining the cecal area and retroperitoneal lymph nodes. Moreover, hematogenous dissemination involves the liver and the lung in the HCT-116- and DLD-1-derived tumors. Only SW-620 tumors did not generate tumor foci in the liver, most likely because this cell line does not express EGFR,27Hewitt RE McMarlin A Kleiner D Wersto R Martin P Tsokos M Stamp GW Stetler-Stevenson WG Validation of a model of colon cancer progression.J Pathol. 2000; 192: 446-454Crossref PubMed Scopus (185) Google Scholar, 29Murphy LD Valverius EM Tsokos M Mickley LA Rosen N Bates SE Modulation of EGF receptor expression by differentiating agents in human colon carcinoma cell lines.Cancer Commun. 1990; 2: 345-355Crossref PubMed Scopus (28) Google Scholar a requirement for colorectal cancer metastasis in the liver.30Radinsky R Risin S Fan D Dong Z Bielenberg D Bucana CD Fidler IJ Level and function of epidermal growth factor receptor predict the metastatic potential of human colon carcinoma cells.Clin Cancer Res. 1995; 1: 19-31PubMed Google Scholar In addition, we observed a clear correlation between tumor staging and animal survival in the three studied groups, with HCT-116 being the most aggressive and DLD-1 the least aggressive, whereas tumor grading did not relate to survival. These results are consistent with the highly significant prognostic value of the tumor staging systems, and the lower prognostic value of tumor grade, in human colorectal cancer.24De Vita V Cancer: Principles and Practice of Oncology. ed 7. Lippincott, Philadelphia2005Google ScholarThe OCMI Model Improves Previous Colorectal Cancer ModelsOCMI generates significantly more tumor foci in organs distant from the injection site than transgenic, knockout, or knockin mice generate metastatic tumor foci. In addition, genetically modified mice show tumor dissemination at significantly longer time periods (1.5 to 2 years), develop tumors in the small rather than in the large bowel, and show secondary mutations different from these found in humans.31Kobaek-Larsen M Thorup I Diederichsen A Fenger C Hoitinga MR Review of colorectal cancer and its metastases in rodent models: comparative aspects with those in humans.Comp Med. 2000; 50: 16-26PubMed Google Scholar These differences could be related to a distinct transformation capacity between human and mouse cells32Hamad NM Elconin JH Karnoub AE Bai W Rich JN Abraham RT Der CJ Counter CM Distinct requirements for Ras oncogenesis in human versus mouse cells.Genes Dev. 2002; 16: 2045-2057Cr
Referência(s)