Experimental gastric carcinogenesis in Cebus apella nonhuman primates

Abstract

The evolution of gastric carcinogenesis remains largely unknown. We established two gastric carcinogenesis models in New-World nonhuman primates. In the first model, ACP03 gastric cancer cell line was inoculated in 18 animals. In the second model, we treated 6 animals with N-methyl-nitrosourea (MNU). Animals with gastric cancer were also treated with Canova immunomodulator. Clinical, hematologic, and biochemical, including C-reactive protein, folic acid, and homocysteine, analyses were performed in this study. MYC expression and copy number was also evaluated. We observed that all animals inoculated with ACP03 developed gastric cancer on the 9(th) day though on the 14(th) day presented total tumor remission. In the second model, all animals developed pre-neoplastic lesions and five died of drug intoxication before the development of cancer. The last surviving MNU-treated animal developed intestinal-type gastric adenocarcinoma observed by endoscopy on the 940(th) day. The level of C-reactive protein level and homocysteine concentration increased while the level of folic acid decreased with the presence of tumors in ACP03-inoculated animals and MNU treatment. ACP03 inoculation also led to anemia and leukocytosis. The hematologic and biochemical results corroborate those observed in patients with gastric cancer, supporting that our in vivo models are potentially useful to study this neoplasia. In cell line inoculated animals, we detected MYC immunoreactivity, mRNA overexpression, and amplification, as previously observed in vitro. In MNU-treated animals, mRNA expression and MYC copy number increased during the sequential steps of intestinal-type gastric carcinogenesis and immunoreactivity was only observed in intestinal metaplasia and gastric cancer. Thus, MYC deregulation supports the gastric carcinogenesis process. Canova immunomodulator restored several hematologic measurements and therefore, can be applied during/after chemotherapy to increase the tolerability and duration of anticancer treatments.

Figure 1. Ultrasonography, immunohistochemistry and FISH analysis in C. apella gastric carcinogenesis models.

A) ultrasound image showing a “space” between the stomach wall where ACP03 cell line was inoculated and developed a tumor (2.5 cm); B) ultrasound image showing a tumor mass in a MNU-treated animal on the 940^th day (5 cm); C); MYC immunoreactivity in a tumor sample of a CLCA1 animal (400×); D); lack of MYC immunoreactivity in non-atrophic gastritis sample in a MNU-treated animal (400×); E) MYC immunoreactivity in intestinal metaplasia sample of a MNU-treated animal (400×); F) MYC immunoreactivity in a tumor sample of a MNU-treated animal (400×); G) lymphocytes of a healthy C. apella showing two signals for MYC probe (1000×); H) normal gastric mucosa cells of NC animal presenting two MYC signals (1000×); I) neoplastic gastric mucosa of CL animal showing MYC amplification (1000×); J) intestinal metaplasia sample of MNU-treated animal presenting 1, 2 and 3 MYC signals (1000×); K) tumor sample of MNU-treated animal showing MYC amplification (1000×). Arrow indicates the space with gastric cancer cell line; arrowhead indicates a normal gastric wall thickness; circle indicates the proliferative process.

Figure 2. Abnormal biochemical and hematologic measurements in animals of the first carcinogenesis model.

A) triglycerides; B) urea nitrogen; C) C-reactive protein; D) leukocyte; E) lymphocyte; F) erythrocyte; G) haemoglobin; H) haematocrit; I) folic acid; J) homocysteine. NC: negative control; CA: Canova group; CL: animals inoculated with ACP03 cell line; CLCA1: animals inoculated with ACP03 cell line and treated with Canova during 10 days; CLCA2: animals inoculated with ACP03 cell line and treated with Canova during 14 days. N = 6/group. * Significantly different from NC group (p<0.05) on the 14^th day. ** Significantly different from NC and CA groups (p<0.05) on the 14^th day. ^§ Significantly different from NC group (p<0.05) on the 14^th day. ^‡ Significantly different from CA group (p<0.05) on the 9^th day. ^‡‡ Significantly different from NC and CA groups (p<0.05) on the 9^th day.

Figure 3. Abnormal biochemical and hematologic measurements in animals MNU-treated and Canova-treated.

A) triglycerides; B) urea nitrogen; C) phosphorus; D) alanine aminotransferase; E) total bilirubin; F) creatinine; G) C-reactive protein; H) leukocyte; I) lymphocyte; J) neutrophil; K) erythrocyte; L) haemoglobin; M) haematocrit; N) folic acid; O) homocysteine; P) weight. N = 6 on the 0–90^th days (non-atrophic gastritis); N = 5 on the 120^th day (atrophic gastritis); N = 2 on the 300^th day (intestinal metaplasia); N = 1 on the 940^th (gastric cancer development) and 960^th (Canova treatment effect).