| BackgroundMetabolism commonly occured in organisms is a biochemical process,aiming to maintain the normal activities of life, such as cell growth, proliferation and differentiation. The metabolism of cancer cells significantly differ from those of normal cells, normal cells always dependent on glucose aerobic oxidation and fatty acids βoxidation for energy, while most cancer proliferating cells showed decreased aerobic oxidation even in the abundant presence of oxygen,they demonstrated increased glucose uptake and lactate production via glycolytic pathway, this aberrant phenomena was known as the’Warburg effect’. Pyruvate is an intermediate product of glycolytic metabolism, in cancer cells, pyruvate is mainly for anabolism including lipid synthesis, protein production and ribose synthesis instead oxidation through mitochondria tricarboxylic acid cycle.Fatty acids,obtained from dietary FAs or endogenous synthesized, are predominantly relevant energy source for most tissues, increased de novo fatty acid synthesis(FASN) as the other important metabolic hallmark of cancer is now widely accepted, which functionally related to the glycolytic metabolism. Compared to normal cells predominantly absorb exogenous fatty acids for triglycerides synthesis to maintain the sufficient energy supply. In cancer cells, they rely on product of glycolysis pyruvate to their endogenous de novo FA biosynthesis regardless of sufficient supply of exogenous lipids. Endogenous FASN accounted for all esterified FAs and more than 93% triglycerides synthesis in tumor. The different FAs metabolic pathways between normal cells and cancer cells revealed their discrepancy in FAs’exogenous uptake and endogenous utilization.Colorectal cancer(CRC) is a type of malignant tumor with high morbidity and mortality, it is the world’s fourth leading cause of cancer-related death in males and the third in females. An estimated 608,700 CRC-related deaths occurred globally in 2008. genetic alterations are often considered the major cause of CRC.While with the recent changes of environment and unbalanced dietary patterns, the morbidity rapidly increased, it gradually has the fastest incidence of malignant tumors in our country. The most effective way to cure cancer is early detection. There are more than 50% cancer be completely cured with early intervene in pre-cancerous states, the 5-year survival rate can be reach to 80-90%.In state of pre-neoplasia, there are commonly no obvious morphological and functional changes, diagnosis mainly rely on medical experience of physicians and some auxiliary examinations, such as tumor markers in serum, imaging, pathology. Colonoscopy has been regarded as the reliable tool in CRC detection and surveillance. Ordinary endoscopy in diagnosis of early gastrointestinal tumor showed apparent low accuracy. Numerous emerging endoscopic techniques such as chromoendoscopy, magnification endoscopy, endoscopic ultrasonography were developed to improve preclinical diagnosis of neoplasia and acquired higher detection rate.Chromoendoscopy can be used to observe the microscopic structure of mucosa, it benefit to endoscopic type analysis according to vascular texture in mucosa or layer below, it also has the relatively advantage on flat lesions’detection can be performed in all mucosal examination. Magnification endoscopy application based on topical staining of 0.3% Lugol iodine or 0.4% indigo carmine, it can amplify the regions of interest to 10 to 100 fold, definitely imaging the structure of gland especially the openings. Owing to its amplification effect, magnification endoscopy can assist endoscopic physicians effectively discriminate the type of disease and decide whether perform the endoscopic mucosal resection. Endoscopic ultrasonography combined endoscopy and ultrasound that can detect the infiltration depth of disease during examination, the vascular and the relationship of organs around can be also well described. Compared to other types of endoscopy, endoscopic ultrasonography has powerful advantages in pancreatic cancer diagnosis and pancreatic pseudocyst treatment.Confocal laser endomicroscopy (CLE) is a new imaging modality that combined ordinary endoscopy and confocal microscopy has been called’in vivo optical microscopy’.CLE provides in vivo magnification of the mucosa to 1000-fold,realizing the rapid cellular or sub-cellular imaging, its advantage in subsurface analysis of colonic lesions relies on fluorescent contrast agents like fluorescein sodium and acriflavine hydrochloride. Fluorescein sodium was an cheap and teratogenicity free agent. After several seconds of intravenous injection, fluorescein sodium can showed the cell morphology and arrangement from the epithelial layer to lamina propria under CLE. However, fluorescein sodium can’t imaging the cells nuclei so that adverse to estimate the feature of disease. Some side effects including nausea, exanthema and even shock are inevitable. Topical administered acriflavine hydrochloride showed clear morphology of nuclei as it entered into cytoplasm and combined with acidic component of nuclei.whereas imging of acriflavine hydrochloride limited in the epithelium can’t describe the deeper situation, FDA has been limited its clinical utility for carcinogenicity. Based on confocal laser endomicroscopy’s great application prospect,some novel molecular imaging methods using CLE such as fluorophore labeled antibodies (CD31、VEGF、EGFR), fluorophore labeled peptide and fluorescent probes for metabolic features were recently investigated.Fluorescently labeled fatty acid (BODIPY-FA C22H31BF2N2O2) is a natural lipids analogue with inherently lipophilic, its biochemical features that molecular weight 404, excitation/emission Wavelength is 500/510nm can well matched to argonion laser excitation at 488 nm, BODIPY-FA has been generally applied for experimental research in lipid trafficking, allowing analysis of cellular fatty acid uptake by flow cytometry and confocal microscopy.ObjectiveCLE analysis the intracellular fluorescent signals after topical applied BODIPY-FA on mucosa in different mice models of colorectal cancer, compared the efficiency of different fluorescents in tumor detection, in addition, exploring the related mechanism of fatty acid decreased uptake in CRC.Method1.Time curve of BODIPY-FA uptake in colorectal cancer cell lines and fluorescent competition with stearic acidDifferent CRC cell lines were cultured in in 6-well plates with a density of 600,000 cells/well, then lml luM BODIPY-FA mix were added into cells, placed them in incubator at 37℃ in 5% CO2 followed by 10s,30s,lmin,2min,5min,10min incubation. When reached to targeted time points the BODIPY-FA mix was removed, cells were ended fluorescent uptake and rinsed by 0° pre-cold phosphate-buffered saline PBS, cells were digested, harvested and then immediately for FACS analysis.Different CRC cell lines were cultured in in 6-well plates with a density of 600,000 cells/well,equal volume of 0.15uM,1.5uM stearic acid and luM BODIPY-FA mix were previously blended respectively. Cells received lml above mixture for incubation with 10minute in incubator at 37℃ in 5% CO2. Then BODIPY-FA and C18:0 mix was removed, cells were ended fluorescent uptake and rinsed by 0° pre-cold phosphate-buffered saline PBS, cells were digested, harvested and then immediately for FACS analysis.2.Construction of mice models(1)AOM/DSS induced colorectal cancer6 week-old male balb/c mice were injected with 12.5 mg/kg body weight azoxymethane[AOM] intraperitoneally. After 7 days’normal drinking water,3% DSS dissolved in distilled water was performed for 7 days, later for 14 days of normal drinking water. That is successive 3% DSS drinking for 7 days then recover with normal water for 14 days. This cycle was repeated three times, with the last DSS cycle ended mice were prepared for in vivo CLE imaging or ex vivo tissue pathology, our run-in phase experiments demonstrated cancerous regions always occurred near the rectum with increasing thickness and stiffness of bowel wall macroscopically, guiding topical application of fluorescence agents.(2)APC-min mice6-7 week-old APC-min mice(purchase from Model Animal Research Center of Nanjing University,china) is a widely accepted model of intestinal tumorigenesis. Purchasing several C57 female mice and mating with APC-min mice,after received large number of offsprings and DNA analysis, screening the APC-min mice for research.mice were prepared for in vivo CLE imaging or ex vivo tissue pathology.(3) Colonic xenograft mice modelSelecting 6 week old Balb/c null mice.Human CRC cell of LOVO in logarithmic phase underwent digestion, calculation,centrifuge and harvest.In a rigid aseptic condition,106 LOVO were injected via 29g micro-syringe into the colonic serosa layer through laparotomy. Then closed the enterocoelia with commonly disinfection. Successful cells injection always showed white hump or longitudinal ridges along colon tract. Our previous experiments founded LOVO can break through muscularis and reach to the layer of mucosa or submucosa after 3-4 weeks implantation.mice were prepared for in vivo CLE imaging or ex vivo tissue pathology.3.In vivo staining protocol and observation with CLE(1).Mice were fasted 12h to maintain lumen clean, anaesthesia with 0.01g/ml pentobarbital of 6-7ul/g body weight._Exposure colon and/or small intestine of mice through laparotomy, intestinal tract were isolated and properly ligatured two length of 1cm between two sutures, allowing 100ul fluorescent agent directly injected into the lumen of different mice by a 10min staining, then got rid of sutures and rinsed staining regions by PBS. For in vivo tumo staining, fluorescent agent were divided into three groups,that is 200uM BODIPY-FA,500uM 2-NBDG and 0.02% acriflavine. mice were prepared for in vivo CLE imaging or ex vivo tissue pathology.(2).Confocal imagings were obtained at fixed laser power and standardized brightness. For in vivo mice model imaging, regions were rinsed by PBS after 10 minutes staining, the confocal probe fully contact onto the surface mucosa, the observing time of each sites should be less than 20s.all confocal imagings were diagnosed by a endoscopic expert, experimenter used 29 g micro-syringe (intake Indian ink)to mark the regions correspondingly,the marked tissues underwent HE pathology and finally compared with the confocal diagnosis.4. HistochemistryFor each tissue of mice models after in vivo staining, fresh specimens were immediately embedded and frozened above the liquid nitrogen for cryosections(half for immunofluorescence and half for histology),dry cryosections for 10 to 20 minutes in room temperature.The ordinary process of HE staining is haemotoxylin staining, discrimination,backing blue, eosin staining,dehydration and mounting with neutral balsam. Fluorescent slices should stained with DAPI,a nuclear dye for 8 to 10 minutes,then mounting with fluorescence quenching agent.5. Real-time fluorescent quantitative PCRExtracted total RNA and detected their concentration,cDNA was synthesized by TAKARA reverse-transcriptase with two procedure, reverse transcription at 37°for 15 minutes and inactivated reverse transcription enzyme at 85°5s.Using TaKaRa SYBR Green Premix Ex Taq kit to subsequent Real-time PCR amplification on an LC480 System.6. Western blottingExtracted the cellular proteins, determined the protein concentration of samples by BCA assay. After electrophoresis, protein transfer to PVDF membrane,1 hour blocked by 5%BSA,first antibodies incubated overnight at 4℃ with anti-CD36 (1:400), anti-caveolin-1 (1:500),and anti-GAPDH (1:3000). Membranes were then incubated with a second antibody (1:2500) for 1 h, finally membrane-bound second antibodies were detected by enhanced chemiluminescence.7. Immunohistochemistry (IHC)Collected paired tissues from 25 patients in nan fang hospital that suffered colorectal cancer, the procedure was as followed:ordinary fixed, embedded, deparaffinized and hydrated, antigen retrieval at 95°10minutes by sodium citrate buffer (pH6.0),using SP hypersensitivity kit to block, IHC staining for caveolin-1 (1:100) and CD36 (1:100) incubation overnight at 4℃,then DAB developer to develop, haemotoxylin staining,backing blue, dehydration and mounting with neutral balsam.8.Evaluation and Statistical analysisMean fluorescence intensity of fluorescence microscopy were calculated by Image-Pro Plus 6.0, confocal imaging were analyzed by Image J. For each confocal imaging,ROIs of 60x60um was examined,select 3 different regions in the same feature of disease and calculate mean gray-scale value, all dates were show in mean ±standard error of arithmetic mean. Data were analyzed by using the statistical software package GraphPad Prism (v5.00).Student t test was used to evaluate discrepancy between two groups,one way variance analysis was used to more than two groups.p Values of<0.05 were defined statistically significant.Result1. BODIPY-FA uptake and inhibition by saturated FA in CRC cellsFACS analysis showed different states of BODIPY-FA uptake among four CRC cell lines, SW480 demonstated relatively higher BODIPY-FA fluorescence, while the signals in LOVO HCT116 were low. BODIPY-FA constantly accumulated in cytoplasm of SW480, LOVO,HCT116 within 10min, HT29 underwent decreased fluorescence signal in lOmin.this point demonstrated optimized time of BODIPY-FA staining in tumor cell lines and laid foundation for followed-up in vivo imaging.In the fluorescent competition of BODIPY-FA and saturated lipids(C18:0). FACS analysis exhibited BODIPY-FA uptake can be hampered by 0.15uM and 1.5uM saturated lipids in cell lines, SW480 showed the comparable obvious effect (Fig.1B).this experiment corroborated that BODIPY-FA was a natural lipid analogue that might share the common FAs transporter of saturated lipids(C18:0) in CRC cells, it can be used to explore the body metabolism as a substitute of free fatty acids.2. Caveolin-1 and CD36 reduced expression in human colon carcinoma tissuesRT-PCR test mRNA expression of fatty acids transport proteins.the mRNA expression of CD36 in neoplasm were lower than normal epithelium, (tumor: 0.003643±0.0006902, normal:0.07623±0.01995, P<0.005).and caveolin-1 mRNA showed significantly reduced in tumor when compared to healthy tissue (tumor: 0.1031±0.04203,normal:0.4381±0.1129, P<0.005), whereas FATP4 and FABPpm demonstrated no difference between tumor and normal mucosa.Western blotting examine the protein level of caveolin-1 and CD36. In 86% of the samples analyzed, caveolin-1 levels were reduced at an average of 1.6-fold in tumor compared to matched healthy mucosa. CD36 levels were decreased more than 4-fold (average,1.9-fold) in neoplasm compared to matched normal tissue.For immunohistochemistry, the staining of CD36 and caveolin-1 were both predominantly cytoplasmic with some nuclear staining, the staining of caveolin-1 was abundantly expressed in vascular and stroma as well. Caveolin-1 showed lower staining in neoplastic cells campared with healthy cells, P=0.0024. Compared to the neoplastic cells, CD36 staining were much stronger in non-neoplastic cells(P< 0.0001).3. Quantification of BODIPY-FA uptake ex vivo in mouse models Fluorescence microscopy analysed cytoplasmic fluorescence signal on cryosections confirmed a less BODIPY-FA uptake in neoplastic lesions showing the slight fluorescence. In AOM+DSS induced mice, a significantly decreased BODIPY-FA fluorescence signal can be observed in 87.1% tumor sites, healthy tissues showed higher BODIPY-FA uptake(tumor:0.0836±0.0057, normal:0.1281 ±0.0106, P=0.0005), in addition, inflammation always showed increased signals compared with normal epithelium(inflammation:0.145±0.0212, P=0.4639), the contrast will definitely benefit to discriminate the normal/neoplasia sites.In APC-min mice,100% adenoma lesions showed BODIPY-FA negative compared with normal mucosa (tumor:0.0648±0.018,normal:0.15± 0.0178, P=0.0092). Visualization of gland architecture and morphology of cell could be distinctly observed in BODIPY-FA staining, the microcosmic morphology can also be well described, just like another type of immunohistochemistry directly showed the state of disease.in both AOM+DSS induced and APC-min mice,2-NBDG staining demonstrated weak fluorescent signals, unclear figure of cell and no difference in cancerous and normal regions. Acriflavine was a nuclear dye with cytoplasm also slightly stained showing unsharp margin of nuclei and cytoplasm, there are no difference of fluorescence in diseased/normal regions in mice models.4. Quantification of BODIPY-FA uptake in vivo in mouse modelsIntracellular BODIPY-FA imaging using CLE in vivo showed statistical discrepancy in cancer and normal mucosa. In AOM+DSS induced mice,84.6% of tumor sites demonstrated specifically weak BODIPY-FA signal, the mean grey scale values in tumor were dramatically decreased compared to normal mucosa (tumor: 66.27±3.387,normal:121.8±8.281,P<0.0001), inflammation displayed elevated BODIPY-FA fluorescence signal versus normal epithelium(inflammation: 133.4±11.13, P=0.4077).In APC-min mice, BODIPY-FA low expression was observed in 100% adenoma lesions,the mean grey scale values in adenoma is greatly lower than normal mucosa (adenoma:56.23±4.03, normal:148.8±10.85, P<0.0001). Topical applied BODIPY-FA using CLE displayed specifically weak signal("dark areas"), disorganized cell arrangement, atypia of cell and even the ratio of nuclei/cytoplasm in the malignant lesions, these confocal imagings with high specificity and definition to tumors resembled another type of immunohistochemistry made the margins well-defined, meanwhile only tissues with biologic activity can be stained since BODIPY-FA transport across the plasma membrane by energy consumption. However, topical administered 2-NBDG using CLE manifested no identifiable morphology such as outline of gland or cytoplasmic imaging,thus have no significance to tumor diagnosis in vivo.Acriflavine can visually displayed all nucleus of ROI while unspecific to tumor admittedly.ConclusionFrom the above results, we could make the following conclusion:1.BODIPY-FA is a nature fatty acid analogue, it constantly accumulated in CRC cell lines within several minutes incubation.2.Compared to other types of fluorescent agents, BODIPY-FA staining showed relatively "dark areas"to specifically trace the neoplastic lesion.3.BODIPY-FA staining can distinctly imaging the cellular or sub-cellular morphology with high definition.4.Only topical application of BODIPY-FA was needed under confocal laser endomicroscopy with no toxic reaction, showed its safty,convenient and feasible use.5.mRNA and protein expression of Caveolin-1 and CD36 were significantly decreased in human colorectal cancer tissues, this may cause the reduced BODIPY-FA uptake in tumor.In vivo we finded that cancerous tissue uptake lower BODIPY-FA than normal mucosa. The relevant mechanism could be the decreased level of Caveolin-1 and CD36, meanwhile, CLE combined with BODIOY-FA staining realized the real time tumor tracing and cellular imaging. This research will open a new door to trace pre-neoplastic or neoplastic lesion in scientific research and clinical practice. |
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