Proteomic analysis of low- and high-grade human colon adenocarcinoma tissues and tissue-derived primary cell lines reveals unique biological functions of tumours and new protein biomarker candidates.

BACKGROUND: Colon cancer is the third most common cancer and second highest cause of cancer deaths worldwide. The aim of the study was to find new biomarkers for diagnosis, prognosis and therapeutic drug targets for this disease. METHODS: Four low-grade and four high-grade human colon adenocarcinoma tumours with patient-matched normal colon tissues were analysed. Additionally, tissue-derived primary cell lines were established from each tumour tissue. The cell lines were validated using DNA sequencing to confirm that they are a suitable in vitro model for colon adenocarcinoma based on conserved gene mutations. Label-free quantitation proteomics was performed to compare the proteomes of colon adenocarcinoma samples to normal colon samples, and of colon adenocarcinoma tissues to tissue-derived cell lines to find significantly differentially abundant proteins. The functions enriched within the differentially expressed proteins were assessed using STRING. Proteomics data was validated by Western blotting. RESULTS: A total of 4767 proteins were identified across all tissues, and 4711 across primary tissue-derived cell lines. Of these, 3302 proteins were detected in both the tissues and the cell lines. On average, primary cell lines shared about 70% of proteins with their parent tissue, and they retained mutations to key colon adenocarcinoma-related genes and did not diverge far genetically from their parent tissues. Colon adenocarcinoma tissues displayed upregulation of RNA processing, steroid biosynthesis and detoxification, and downregulation of cytoskeletal organisation and loss of normal muscle function. Tissue-derived cell lines exhibited increased interferon-gamma signalling and aberrant ferroptosis. Overall, 318 proteins were significantly up-regulated and 362 proteins significantly down-regulated by comparisons of high-grade with low-grade tumours and low-grade tumour with normal colon tissues from both sample types. CONCLUSIONS: The differences exhibited between tissues and cell lines highlight the additional information that can be obtained from patient-derived primary cell lines. DNA sequencing and proteomics confirmed that these cell lines can be considered suitable in vitro models of the parent tumours. Various potential biomarkers for colon adenocarcinoma initiation and progression and drug targets were identified and discussed, including seven novel markers: ACSL4, ANK2, AMER3, EXOSC1, EXOSC6, GCLM, and TFRC.

Colon adenocarcinoma-derived cells possessing stem cell function can be modulated using renin-angiotensin system inhibitors.

The cancer stem cell (CSC) concept proposes that cancer recurrence and metastasis are driven by CSCs. In this study, we investigated whether cells from colon adenocarcinoma (CA) with a CSC-like phenotype express renin-angiotensin system (RAS) components, and the effect of RAS inhibitors on CA-derived primary cell lines. Expression of RAS components was interrogated using immunohistochemical and immunofluorescence staining in 6 low-grade CA (LGCA) and 6 high-grade CA (HGCA) tissue samples and patient-matched normal colon samples. Primary cell lines derived from 4 HGCA tissues were treated with RAS inhibitors to investigate their effect on cellular metabolism, tumorsphere formation and transcription of pluripotency genes. Immunohistochemical and immunofluorescence staining showed expression of AT2R, ACE2, PRR, and cathepsins B and D by cells expressing pluripotency markers. ß-blockers and AT2R antagonists reduced cellular metabolism, pluripotency marker expression, and tumorsphere-forming capacity of CA-derived primary cell lines. This study suggests that the RAS is active in CSC-like cells in CA, and further investigation is warranted to determine whether RAS inhibition is a viable method of targeting CSCs.

Efficacy of a micronized, nanocrystal fenofibrate formulation in treatment of hyperlipidemia in dogs.

BACKGROUND: Safe, effective, and readily available drug therapies are required for the management of hyperlipidemia and its associated complications in dogs. OBJECTIVES: To investigate the efficacy of a micronized, nanocrystal formulation of fenofibrate (Tricor) in the treatment of hyperlipidemia in dogs. ANIMALS: Ten client-owned dogs with primary (n = 7) and secondary (n = 3) hyperlipidemia. All dogs had hypertriglyceridemia at baseline; 3 dogs also had hypercholesterolemia. METHODS: Prospective dose-escalation study. Dogs were treated with fenofibrate orally once daily in up to 3 cycles of 21 days each. Fenofibrate dose was increased at the end of each cycle if hypertriglyceridemia persisted and adverse effects were not documented. Complete blood count, biochemistry, and urine protein:creatinine ratio were collected serially. Baseline (T0) parameters were compared to time of maximal reduction in serum triglyceride concentrations (T1) and reported as median (range). RESULTS: Triglycerides normalized in all dogs (T0 = 662 mg/dL [189-2391]; T1 = 113 mg/dL [81-132]; P = .002). Fenofibrate dose at T1 = 6.4 mg/kg PO q24h (range, 2.2-13.5). T1 was achieved at 3 (n = 4), 6 (n = 4), and 9 (n = 2) weeks. Serum cholesterol concentrations decreased in 9 of 10 dogs. Quiet demeanor and firm stools in 1 dog were the only reported adverse reactions. Fenofibrate administration resulted in a significant reduction in median alkaline phosphatase activity (P = .049). CONCLUSIONS AND CLINICAL IMPORTANCE: Over 21 to 63 days, TriCor was effective in the management of primary and secondary hyperlipidemia in dogs.

Surgical treatment of retroiliac ectopic ureters with secondary hydronephrosis and hydroureter in a dog.

CASE DESCRIPTION: An 8-month-old 41.2-kg (90.6-lb) sexually intact male Dogue de Bordeaux with urinary incontinence and signs of nausea was referred for further evaluation and treatment of bilateral hydronephrosis, hydroureter, and ectopic ureters. CLINICAL FINDINGS: Clinicopathologic analyses revealed urine specific gravity and serum concentrations of urea nitrogen and creatinine within reference limits. Abdominal ultrasonography and CT revealed unilateral abdominal cryptorchidism, ureters that bilaterally passed dorsal to and appeared compressed by the external iliac arteries (retroiliac ureters), and bilateral hydronephrosis, hydroureter, and ectopic ureters. On CT, minimal uptake of contrast medium by the right kidney indicated either a lack of renal function or ureteral obstruction. TREATMENT AND OUTCOME: The dog underwent exploratory laparotomy, right ureteronephrectomy, left neoureterocystostomy, bilateral castration, and incisional gastropexy without complication and was discharged 2 days postoperatively. Eleven days after surgery, the dog had improved but continued urinary incontinence, improved left hydronephrosis and hydroureter, and serum concentrations of urea nitrogen and creatinine within reference limits. At 24 months after surgery, the dog was reportedly clinically normal, other than having persistent urinary incontinence. CLINICAL RELEVANCE: To our knowledge, this was the first report of a dog with retroiliac ureters and compression-induced ureteral obstruction with secondary hydroureter and hydronephrosis. Retroiliac ureters should be considered as a differential diagnosis in young dogs with ureteral obstruction. Our findings indicated that a good outcome was possible for a dog with retroiliac ureters treated surgically; however, the presence of additional congenital anomalies should be considered and may alter the prognosis in dogs with retroiliac ureters.

Feline mycobacterial disease in northern California: Epidemiology, clinical features, and antimicrobial susceptibility.

BACKGROUND: Mycobacterial infections in cats are challenging to treat and incompletely described. HYPOTHESIS/OBJECTIVES: To describe the features of mycobacterial infections in cats from northern California. ANIMALS: Nineteen cats, all with nontuberculous mycobacterial (NTM) infections; 4 with Mycobacterium avium infection, 15 with rapid-growing mycobacterial (RGM) infection. METHODS: Retrospective study. Cases with positive mycobacterial culture, species identification, and susceptibility testing were included. Descriptive statistics were used. Fisher's exact test and Mann-Whitney U test were used for comparisons between M avium and RGM infections (P ≤ .05). RESULTS: Rapid-growing mycobacterial cases included Mycobacterium smegmatis (9), Mycobacterium fortuitum (4), Mycobacterium abscessus (1), and Mycobacterium thermoresistibile (1). Mycobacterium avium infections were more likely than RGM infections to be disseminated (3/4 vs 0/15; P = .004). Disease of the skin/subcutis (15/15 vs 0/4; P < .001) and outdoor access (14/15 vs 0/4; P = .001) were primary features of RGM infections. Resistance to fluoroquinolones and aminoglycosides was common among M avium isolates. A high prevalence of resistance to third- and fourth-generation cephalosporins was noted in RGM species. Death/euthanasia was noted only in M avium cases (3/4). Twelve of 15 cats with RGM infection had available follow-up; 4 of these cats achieved remission. CONCLUSIONS AND CLINICAL IMPORTANCE: The most prevalent RGM species isolated from cats from northern California are M smegmatis and M fortuitum. Susceptibility to prescribed antimicrobials does not appear to guarantee treatment success. Combination drug treatment is recommended. Repeat culture and susceptibility testing should be performed when disease is persistent/relapsing.

Colon adenocarcinoma-derived cells that express induced-pluripotent stem cell markers possess stem cell function.

AIMS: Much work has been done to find markers of cancer stem cells (CSCs) that distinguish them from the tumor bulk cells and normal cells. Recent CSC research has applied the induced pluripotent stem cell (iPSC) concept. In this study, we investigated the expression of a panel of iPSC markers in primary colon adenocarcinoma (CA)-derived cell lines. MATERIALS AND METHODS: Expression of iPSC markers by CA-derived primary cell lines was interrogated using immunocytochemistry, western blotting and RT-qPCR. The stem cell function of these cells was then assessed in vitro using differentiation and tumorsphere assays. RESULTS: Expression of iPSC markers OCT4, SOX2, NANOG, KLF4 and c-MYC was more widespread in high-grade CA (HGCA) cell lines than low-grade CA (LGCA) cell lines, as demonstrated by western blotting and RT-qPCR. These cells could be induced to differentiate down the three embryonic lineages. Cells derived from HGCA were more capable of forming tumorspheres than those derived from LGCA. EpCAM sorting revealed that a population enriched for EpCAMHigh cells formed larger tumorspheres than EpCAMLow cells. Pluripotency markers, SSEA4 and TRA-1-60, were co-expressed by a small subpopulation of cells that also co-expressed SOX2 in 75% and OCT4 in 50% of the cell lines. CONCLUSIONS: CA-derived primary cell lines contain tumorsphere-forming cells which express key pluripotency genes and can differentiate down 3 embryonic lineages, suggesting a pluripotent CSC-like phenotype. There appear to be two iPSC-like subpopulations, one with high EpCAM expression which forms larger tumorspheres than another with low EpCAM expression. Furthermore, these cells can be characterized based on iPSC marker expression, as we have previously demonstrated in the original CA tumor tissues.

Cancer stem cell subpopulations in primary colon adenocarcinoma.

AIMS: The cancer stem cell concept proposes that tumor growth and recurrence is driven by a small population of cancer stem cells (CSCs). In this study we investigated the expression of induced-pluripotent stem cell (iPSC) markers and their localization in primary low-grade adenocarcinoma (LGCA) and high-grade adenocarcinoma (HGCA) and their patient-matched normal colon samples. MATERIALS AND METHODS: Transcription and translation of iPSC markers OCT4, SOX2, NANOG, KLF4 and c-MYC were investigated using immunohistochemical (IHC) staining, RT-qPCR and in-situ hybridization (ISH). RESULTS: All five iPSC markers were detected at the transcriptional and translational levels. Protein abundance was found to be correlated with tumor grade. Based on their protein expression within the tumors, two sub-populations of cells were identified: a NANOG+/OCT4- epithelial subpopulation and an OCT4+/NANOG- stromal subpopulation. All cases were accurately graded based on four pieces of iPSC marker-related data. CONCLUSIONS: This study suggests the presence of two putative sub-populations of CSCs: a NANOG+/OCT4- epithelial subpopulation and an OCT4+/NANOG- stromal subpopulation. Normal colon, LGCA and HGCA could be accurately distinguished from one another using iPSC marker expression. Once validated, novel combinations of iPSC markers may provide diagnostic and prognostic value to help guide patient management.

Cancer stem cells in colorectal cancer: a review.

Colorectal cancer (CRC) is the second most common cancer in women and the third most common in men. Adenocarcinoma accounts for 90% of CRC cases. There has been accumulating evidence in support of the cancer stem cell (CSC) concept of cancer which proposes that CSCs are central in the initiation of cancer. CSCs have been the focus of study in a range of cancers, including CRC. This has led to the identification and understanding of genes involved in the induction and maintenance of pluripotency of stem cells, and markers for CSCs, including those investigated specifically in CRC. Knowledge of the expression pattern of CSCs in CRC has been increasing in recent years, revealing a heterogeneous population of cells within CRC ranging from pluripotent to differentiated cells, with overlapping and sometimes unique combinations of markers. This review summarises current literature on the understanding of CSCs in CRC, including evidence of the presence of CSC subpopulations, and the stem cell markers currently used to identify and localise these CSC subpopulations. Future research into this field may lead to improved methods for early detection of CRC, novel therapy and monitoring of treatment for CRC and other cancer types.