The potential of organoids in toxicologic pathology: Histopathological and immunohistochemical evaluation of a mouse normal tissue-derived organoid-based carcinogenesis model.

Recently, we introduced an organoid-based chemical carcinogenesis model using mouse normal tissue-derived organoids. In the present review article, the histopathological and immunohistochemical characteristics of mouse normal tissue-derived organoids and tumors derived from these organoids after their in vitro treatment with genotoxic carcinogens and injection into nude mouse are reviewed. In organoids treated in vitro with genotoxic carcinogens, we confirmed macroscopic tumorigenicity and histopathological findings, including neoplastic characteristics, such as multilayered epithelia and/or invasion of epithelia into the surrounding interstitium. In contrast glandular/cystic structures with monolayered epithelia were clearly demarcated from the surrounding Matrigel/interstitium in the untreated control groups. In addition to macroscopic tumorigenicity, these microscopic epithelial changes, which are characteristic of the early stages of carcinogenesis, are included in the requirements for carcinogenicity-positive judgement of the organoid-based carcinogenesis model. Immunohistochemistry of cytokeratins (CKs), used to determine the origin of epithelia and distribution of extraductal invasive lesions, or oncogenic kinases, which reflect molecular activation in epithelia following chemical treatment, is helpful for accurate diagnosis and molecular evaluation in the early stages of carcinogenesis. This information improves our biological understanding of organoid-based chemical carcinogenesis models.

Establishment of Novel Genotoxicity Assay System Using Murine Normal Epithelial Tissue-Derived Organoids.

Short-/middle-term and simple prediction studies for carcinogenesis are needed for the safety assessment of chemical substances. To establish a novel genotoxicity assay with an in vivo mimicking system, we prepared murine colonic/pulmonary organoids from gpt delta mice according to the general procedure using collagenase/dispase and cultured them in a 3D environment. When the organoids were exposed to foodborne carcinogens-2-amino-1-methyl-6-phenylimidazo(4,5-b)pyridine (PhIP) and acrylamide (AA)-in the presence of metabolic activation systems, mutation frequencies (MFs) occurring in the gpt gene dose-dependently increased. Moreover, the mutation spectrum analysis indicated predominant G:C to T:A transversion with PhIP, and A:T to C:G and A:T to T:A transversion with AA. These data correspond to those of a previous study describing in vivo mutagenicity in gpt delta mice. However, organoids derived from the liver, a non-target tissue of PhIP-carcinogenesis, also demonstrated genotoxicity with a potency comparable to colonic organoids. Organoids and PhIP were directly incubated in the presence of metabolic activation systems; therefore, there was a lack of organ specificity, as observed in vivo. Additionally, PhIP-DNA adduct levels were comparable in hepatic and colonic organoids after PhIP exposure. Taken together, the organoids prepared in the present study may be helpful to predict chemical carcinogenesis.

The Unique Genetic and Histological Characteristics of DMBA-Induced Mammary Tumors in an Organoid-Based Carcinogenesis Model.

Here, we report a model system using in vitro 7,12-dimethylbenz[a]anthracene (DMBA; 0.6 µM)-treated mammary tissue-derived organoids generated from heterozygous BALB/c-Trp53 knockout mice to induce tumors after injection into the nude mouse subcutis. In parallel, a single oral dose of DMBA (50 mg/kg bodyweight) to the same murine strain induced mammary adenocarcinomas, characterized by biphasic structures differentiated into luminal and myoepithelial lineages and frequent Hras mutations at codon 61. In the present study, the genetic and histological characteristics of DMBA-induced tumors in the organoid-based model were evaluated to validate its similarities to the in vivo study. The organoid-derived tumors were low-grade adenocarcinomas composed of luminal and basal/myoepithelial cells. When the organoid-derived carcinomas were passaged to other nude mice, they partly progressed to squamous cell carcinomas (SCCs). Whole exome sequencing revealed no mutations at Hras codon 61 in the organoid-derived tumors. However, various mutations were detected in other genes such as Tusc3 and Tgfbr2, which have been reported as cancer-associated or homeostatic squamous cell genes. The most common mutational pattern observed in these genes were the G:C to T:A transversions and G:C to A:T transitions, which are not typical of the mutations caused by DMBA treatment. In conclusion, DMBA exhibited carcinogenicity in the both the ex vivo and in vivo mammary carcinogenesis models, albeit with distinct histological and genetical alterations. Further studies are needed to clarify whether organoid-based carcinogenesis models generated following chemical treatment in vitro could be applied to the clarification of the novel mode of action of chemical carcinogenesis.

A yolk sac tumor of the pancreas and derived xenograft model effectively responded to VIP chemotherapy.

BACKGROUND: Yolk sac tumors (YSTs) of the pancreas are extremely rare, and no drug responsiveness data are available regarding YSTs. METHODS: We report a pancreatic YST in a 70-year-old woman, and its chemotherapeutic responsiveness based on clinical records and evaluation of a patient-derived xenograft (PDX) line of the YST. RESULTS: The YST was an 11-cm, solid mass located in the pancreatic tail. Histologically, the tumor showed medullary proliferation of tumor cells, with a variety of growth patterns including microcystic/reticular, endodermal sinus, and hepatoid patterns. Immunohistochemically, the tumor cells were positive for Sall4, glypican-3, and alpha-fetoprotein. We administered VIP (etoposide, ifosfamide, cisplatin) chemotherapy for a recurrent liver tumor, and obtained complete pathological remission. A drug-response assay using the PDX line from this YST revealed that both VIP and gemcitabine effectively inhibit tumor growth. CONCLUSIONS: These results suggest that differential diagnosis of YST from adenocarcinoma is important for selecting appropriate chemotherapy.

Fatty pancreas: A possible risk factor for pancreatic cancer in animals and humans.

Obesity, type 2 diabetes mellitus (T2DM) and aging are associated with pancreatic cancer risk, but the mechanisms of pancreatic cancer development caused by these factors are not clearly understood. Syrian golden hamsters are susceptible to N-nitrosobis(2-oxopropyl)amine (BOP)-induced pancreatic carcinogenesis. Aging, BOP treatment and/or a high-fat diet cause severe and scattered fatty infiltration (FI) of the pancreas with abnormal adipokine production and promote pancreatic ductal adenocarcinoma (PDAC) development. The KK-Ay mouse, a T2DM model, also develops severe and scattered FI of the pancreas. Treatment with BOP induced significantly higher cell proliferation in the pancreatic ducts of KK-Ay mice, but not in those of ICR and C57BL/6J mice, both of which are characterized by an absence of scattered FI. Thus, we hypothesized that severely scattered FI may be involved in the susceptibility to PDAC development. Indeed, severe pancreatic FI, or fatty pancreas, is observed in humans and is associated with age, body mass index (BMI) and DM, which are risk factors for pancreatic cancer. We analyzed the degree of FI in the non-cancerous parts of PDAC and non-PDAC patients who had undergone pancreatoduodenectomy by histopathology and demonstrated that the degree of pancreatic FI in PDAC cases is significantly higher than that in non-PDAC controls. Moreover, the association with PDAC is positive, even after adjusting for BMI and the prevalence of DM. Accumulating evidence suggests that pancreatic FI is involved in PDAC development in animals and humans, and further investigations to clarify the genetic and environmental factors that cause pancreatic FI are warranted.

Activated Ductal Proliferation Induced by N-Nitrosobis (2-oxopropyl)amine in Fat-infiltrated Pancreas of KK-Ay Mice.

BACKGROUND/AIM: Our aim was to investigate whether tissue with fatty infiltration within the lobes of the pancreas (scattered FI) is sensitive to carcinogen-induced pancreatic ductal proliferation. MATERIALS AND METHODS: Seven-week-old female C57BL/6J, C57BL/6J-Ay, KK-Ay, and ICR mice were subcutaneously treated with N-nitrosobis(2-oxopropyl) amine at a dose of 80 mg/kg body weight, and the differences in damage-induced cell proliferation and their biochemical data were compared 2 days after. RESULTS: Scattered FI in the pancreas was obvious only in KK-Ay mice, which have high serum lipid, leptin and insulin levels, and cell proliferation both in pancreatic and common bile ducts was enhanced only in KK-Ay mice by the carcinogen treatment. CONCLUSION: Scattered FI in the pancreas per se can be an important factor for carcinogenesis. The genetic background causing scattered FI of the pancreas should be further investigated.

Osteopontin Deficiency Suppresses Intestinal Tumor Development in Apc-Deficient Min Mice.

Osteopontin (OPN) is a secreted phosphoglycoprotein, and is a transcriptional target of aberrant Wnt signaling. OPN is upregulated in human colon cancers, and is suggested to enhance cancer progression. In this study, the effect of deficiency of OPN on intestinal tumor development in Apc-deficient Min mice was investigated. At 16 weeks of age, the number of small intestinal polyps in Min/OPN(+/-) and Min/OPN(-/-) mice was lower than that of Min/OPN(+/+) mice. Colorectal tumor incidences and multiplicities in Min/OPN(+/-) and Min/OPN(-/-) mice were significantly lower than those in Min/OPN(+/+) mice, being 48% and 0.6 ± 0.8, 50% and 0.8 ± 0.9 vs. 80% and 1.6 ± 1.7, respectively. OPN expression in colorectal tumors was strongly upregulated in Min/OPN(+/+) compared to adjacent non-tumor parts, but was decreased in Min/OPN(+/-) and not detected in Min/OPN(-/-). Targets of OPN, matrix metalloproteinases (MMPs)-3, -9, and -13 were lowered by OPN deficiency. Macrophage marker F4/80 in colorectal tumors was also lowered by OPN deficiency. MMP-9 expression was observed in tumor cells and tumor-infiltrating neutrophils. These results indicate that induction of OPN by aberrant Wnt signaling could enhance colorectal tumor development in part by upregulation of MMP-3, -9, and -13 and infiltration of macrophage and neutrophils. Suppression of OPN expression could contribute to tumor prevention, but complete deficiency of OPN may cause some adverse effects.

Suppressive effects of the NADPH oxidase inhibitor apocynin on intestinal tumorigenesis in obese KK-A(y) and Apc mutant Min mice.

Obesity is a risk factor for colorectal cancer. The accumulation of abdominal fat tissue causes abundant reactive oxygen species production through the activation of NADPH oxidase due to excessive insulin stimulation. The enzyme NADPH oxidase catalyzes the production of reactive oxygen species and evokes the initiation and progression of tumorigenesis. Apocynin is an NADPH oxidase inhibitor that blocks the formation of the NADPH oxidase complex (active form). In this study, we investigated the effects of apocynin on the development of azoxymethane-induced colonic aberrant crypt foci in obese KK-A(y) mice and on the development of intestinal polyps in Apc mutant Min mice. Six-week-old KK-A(y) mice were injected with azoxymethane (200 µg/mouse once per week for 3 weeks) and given 250 mg/L apocynin or 500 mg/L apocynin in their drinking water for 7 weeks. Six-week-old Min mice were also treated with 500 mg/L apocynin for 6 weeks. Treatment with apocynin reduced the number of colorectal aberrant crypt foci in KK-A(y) mice by 21% and the number of intestinal polyps in Min mice by 40% compared with untreated mice. Both groups of mice tended to show improved oxidation of serum low-density lipoprotein and 8-oxo-2'-deoxyguanosine adducts in their adipose tissues. In addition, the inducible nitric oxide synthase mRNA levels in polyp tissues decreased. Moreover, apocynin was shown to suppress nuclear factor-κB transcriptional activity in vitro. These results suggest that apocynin and other NADPH oxidase inhibitors may be effective colorectal cancer chemopreventive agents.

Involvement of NADPH oxidases in suppression of cyclooxygenase-2 promoter-dependent transcriptional activities by sesamol.

Cyclooxygenase-2 (COX-2) has been shown to play an important role in colon carcinogenesis. Moreover, one of the components of reduced nicotinamide adenine dinucleotide phosphate (NADPH) oxidase, NADPH oxidase 1 (NOX1), dominantly expressed in the colon, is implicated in the pathogenesis of colon cancer. We have reported that sesamol, one of the lignans in sesame seeds, suppressed COX-2 gene transcriptional activity in human colon cancer cells, and also suppressed intestinal polyp formation in Apc-mutant mice. In the present study, we investigated the involvement of NADPH oxidase in the inhibition of COX-2 transcriptional activity by sesamol. We found that several NADPH oxidase inhibitors, such as apocynin, showed suppressive effects on COX-2 transcriptional activity. Moreover, sesamol significantly suppressed NOX1 mRNA levels in a dose-dependent manner. In addition, we demonstrated that knockdown of NOX1 successfully suppressed COX-2 transcriptional activity. These results suggest that inhibition of NADPH oxidase, especially NOX1, may be involved in the mechanism of the suppression of COX-2 transcriptional activity by sesamol.

Association of pancreatic Fatty infiltration with pancreatic ductal adenocarcinoma.

OBJECTIVES: Fatty infiltration (FI) in the pancreas is positively correlated with high body mass index (BMI) or obesity, and the prevalence of diabetes mellitus (DM), which are well-known risk factors of pancreatic cancer. However, the association of FI in the pancreas with pancreatic cancer is unclear. Recently, we have shown that Syrian golden hamsters feature FI of the pancreas, the severity of which increases along with the progression of carcinogenesis induced by a chemical carcinogen. To translate the results to a clinical setting, we investigated whether FI in the pancreas is associated with pancreatic cancer in a series of patients who had undergone pancreatoduodenectomy. METHODS: In the series, we identified 102 cases with pancreatic ductal adenocarcinoma (PDAC) and 85 controls with cancers except for PDAC. The degree of FI was evaluated histopathologically from the area occupied by adipocytes in pancreas sections, and was compared between the cases and controls. RESULTS: The degree of FI in the pancreas was significantly higher in cases than in controls (median 26 vs. 15%, P<0.001) and positively associated with PDAC, even after adjustment for BMI, prevalence of DM and other confounding factors (odds ratio (OR), 6.1; P<0.001). BMI was identified as the most significantly associated factor with FI in the pancreas. CONCLUSIONS: There is a positive correlation between FI in the pancreas and pancreatic cancer.

Ay allele promotes azoxymethane-induced colorectal carcinogenesis by macrophage migration in hyperlipidemic/diabetic KK mice.

The incidence of colorectal cancer has been increasing and is associated with obesity and diabetes. We have found that type 2 diabetes model KK-Ay/TaJcl (KK-Ay) mice develop tumors within a short period after treatment with azoxymethane (AOM). However, factors that contribute to the promotion of carcinogenesis have not been clarified. Therefore, we looked at the genetic background of KK-Ay, including two genetic characteristics of KK/TaJcl (KK) mice and C57BL/6J-Ham-Ay/+ (Ay) mice, compared with other non-obese and non-diabetic mouse strains C57BL/6J and ICR, and induced colorectal premalignant lesions, aberrant crypt foci (ACF), and tumors using AOM (150 µg/mouse/week for 4 weeks and 200 µg/mouse/week for 6 weeks, respectively). The mice with a diabetes feature, KK-Ay and KK, developed significantly more ACF, 67 and 61 per mouse, respectively, whereas ICR, Ay, and C57BL/6J mice developed 42, 24, and 18 ACF/mouse, respectively, at 17 weeks of age. Serum insulin and triglyceride levels in KK-Ay and KK mice were quite high compared with other non-diabetic mouse strains. Interestingly, KK-Ay mice developed more colorectal tumors (2.7 ± 2.3 tumor/mouse) than KK mice (1.2 ± 1.1 tumor/mouse) at 25 weeks of age, in spite of similar diabetic conditions. The colon cancers that developed in both KK-Ay and KK mice showed similar activation of ß-catenin signaling. However, mRNA levels of inflammatory factors related to the activation of macrophages were significantly higher in colorectal cancer of KK-Ay mice than in KK. These data indicate that factors such as insulin resistance and dyslipidemia observed in obese and diabetic patients could be involved in susceptibility to colorectal carcinogenesis. In addition, increase of tumor-associated macrophages may play important roles in the stages of promotion of colorectal cancer.

Involvement of inflammatory factors in pancreatic carcinogenesis and preventive effects of anti-inflammatory agents.

Chronic inflammation is known to be a risk for many cancers, including pancreatic cancer. Heavy alcohol drinking and cigarette smoking are major causes of pancreatitis, and epidemiological studies have shown that smoking and chronic pancreatitis are risk factors for pancreatic cancer. Meanwhile, inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2) are elevated in pancreatitis and pancreatic cancer tissues in humans and in animal models. Selective inhibitors of iNOS and COX-2 suppress pancreatic cancer development in a chemical carcinogenesis model of hamsters treated with N-nitrosobis(2-oxopropyl)amine (BOP). In addition, hyperlipidemia, obesity, and type II diabetes are also suggested to be associated with chronic inflammation in the pancreas and involved in pancreatic cancer development. We have shown that a high-fat diet increased pancreatic cancer development in BOP-treated hamsters, along with aggravation of hyperlipidemia, severe fatty infiltration, and increased expression of adipokines and inflammatory factors in the pancreas. Of note, fatty pancreas has been observed in obese and/or diabetic cases in humans. Preventive effects of anti-hyperlipidemic/anti-diabetic agents on pancreatic cancer have also been shown in humans and animals. Taking this evidence into consideration, modulation of inflammatory factors by anti-inflammatory agents will provide useful data for prevention of pancreatic cancer.

Cancer chemopreventive ability of conjugated linolenic acids.

Conjugated fatty acids (CFA) have received increased interest because of their beneficial effects on human health, including preventing cancer development. Conjugated linoleic acids (CLA) are such CFA, and have been reviewed extensively for their multiple biological activities. In contrast to other types of CFAs including CLA that are found at low concentrations (less than 1%) in natural products, conjugated linolenic acids (CLN) are the only CFAs that occur in higher quantities in natural products. Some plant seeds contain a considerably high concentration of CLN (30 to 70 wt% lipid). Our research group has screened CLN from different plant seed oils to determine their cancer chemopreventive ability. This review describes the physiological functions of CLN isomers that occur in certain plant seeds. CLN are able to induce apoptosis through decrease of Bcl-2 protein in certain human cancer cell lines, increase expression of peroxisome proliferator-activated receptor (PPAR)-γ, and up-regulate gene expression of p53. Findings in our preclinical animal studies have indicated that feeding with CLN resulted in inhibition of colorectal tumorigenesis through modulation of apoptosis and expression of PPARγ and p53. In this review, we summarize chemopreventive efficacy of CLN against cancer development, especially colorectal cancer.

Enhancement of carcinogenesis and fatty infiltration in the pancreas in N-nitrosobis(2-oxopropyl)amine-treated hamsters by high-fat diet.

OBJECTIVES: Obesity is associated with increased pancreatic cancer risk, although the mechanisms have yet to be detailed. This study aimed to elucidate promotion of pancreatic cancer by obesity and hyperlipidemia. METHODS: Six-week-old female Syrian golden hamsters were treated with N-nitrosobis(2-oxopropyl)amine (BOP) and after 1 week were fed a high-fat diet (HFD) or standard diet (STD) for 6 or 17 weeks. RESULTS: Body weight and serum levels of lipids and leptin were significantly higher in the HFD than the STD group at 14 weeks of age. Pancreatic ductal adenocarcinomas developed only in the BOP + HFD group, with an incidence of 67% (P < 0.01) at 14 weeks of age. In addition, the multiplicity was 2-fold greater in the BOP + HFD group than in the BOP + STD group (P < 0.05) at 25 weeks of age. Pancreatic fatty infiltration was increased by BOP treatment and further enhanced by the HFD, correlating with progression of BOP-induced pancreatic ductal adenocarcinoma and up-regulated expression of adipocytokines and cell proliferation-related genes in the pancreas. CONCLUSIONS: High-fat diet is shown to increase serum lipid levels and enhance fatty infiltration in the pancreas with abnormal adipocytokine production, which may accelerate and enhance pancreatic cancer.

Understanding carcinogenesis for fighting oral cancer.

Oral cancer is one of the major global threats to public health. Oral cancer development is a tobacco-related multistep and multifocal process involving field cancerization and carcinogenesis. The rationale for molecular-targeted prevention of oral cancer is promising. Biomarkers of genomic instability, including aneuploidy and allelic imbalance, are able to measure the cancer risk of oral premalignancies. Understanding of the biology of oral carcinogenesis will give us important advances for detecting high-risk patients, monitoring preventive interventions, assessing cancer risk, and pharmacogenomics. In addition, novel chemopreventive agents based on molecular mechanisms and targets against oral cancers will be derived from research using appropriate animal carcinogenesis models. New approaches, such as interventions with molecular-targeted agents and agent combinations in high-risk oral individuals, are undoubtedly needed to reduce the devastating worldwide consequences of oral malignancy.

Biomarkers for colorectal cancer.

Colorectal cancer (CRC) is the third most common epithelial malignancy in the world. Since CRC develops slowly from removable precancerous lesions, detection of the lesion at an early stage by regular health examinations can reduce the incidence and mortality of this malignancy. Colonoscopy significantly improves the detection rate of CRC, but the examination is expensive and inconvenient. Therefore, we need novel biomarkers that are non-invasive to enable us to detect CRC quite early. A number of validation studies have been conducted to evaluate genetic, epigenetic or protein markers for identification in the stool and/or serum. Currently, the fecal occult blood test is the most widely used method of screening for CRC. However, advances in genomics and proteomics will lead to the discovery of novel non-invasive biomarkers.

Increased expression of inducible nitric oxide synthase (iNOS) in N-nitrosobis(2-oxopropyl)amine-induced hamster pancreatic carcinogenesis and prevention of cancer development by ONO-1714, an iNOS inhibitor.

Elevated protein expression of inducible nitric oxide synthase (iNOS) has been observed in human pancreatic cancers and therefore, iNOS may play important roles in pancreatic carcinogenesis. This was examined in the present study, using an experimental model with N-nitrosobis(2-oxopropyl)amine (BOP)-treated hamsters. Reverse transcription-polymerase chain reaction analysis demonstrated iNOS expression in a hamster pancreatic cancer cell line as well as in human pancreatic cancer cell lines. Immunohistochemical analysis revealed increased expression of iNOS protein in atypical hyperplasia and ductal adenocarcinomas of the pancreas in BOP-treated hamsters. In addition, iNOS expression was also observed in macrophages and islet cells in pancreatic tissue surrounding tumors. In order to assess the role of iNOS expression in carcinogenesis in the pancreas, the effects of ONO-1714 [(1S, 5S, 6R, 7R)-7-chloro-3-imino-5-methyl-2-azabicyclo[4.1.0]heptane], an iNOS inhibitor, on hamster pancreatic ductal carcinogenesis were investigated. Female Syrian golden hamsters were treated with BOP at 10 mg/kg body wt, four times for 1 week, and 1 week after the last carcinogen treatment, ONO-1714 was administered at doses of 100 and 200 p.p.m. in the diet for 15 weeks. The incidences and multiplicities of atypical hyperplasia and invasive adenocarcinoma and total adenocarcinomas (non-invasive and invasive adenocarcinomas) in the pancreas were significantly lowered by treatment with 200 p.p.m. ONO-1714. Treatment with 100 p.p.m. ONO-1714 also significantly decreased the multiplicities of invasive and total adenocarcinomas. Moreover, treatment with 200 p.p.m. ONO-1714 reduced the number of BOP-induced cholangiocellular tumors. These results suggest that iNOS plays roles in promoting pancreatic carcinogenesis in both early and late stages in hamsters.