| Objective1. To isolate CD133(+) cells from colorectal cancer LoVo cell line using Magnetic Activated Cell-sorting System (MACS); detect whether CD133(+) LoVo cells have the characteristics of cancer stem cells (CSCs).2. To label AC133mAb with131I using Iodogen method; check the radiochemical purity, stability and immunoactivity of131I-AC133mAb.3. To target colorectal CSCs with131I-AC133mAb in vivo; make it clear whether the accumulation of tracer is consistent with CD133expression level by biodistribution, molecular biology techniques, and autoradiography (ARG).Methods1. Isolation, culture and identification of CSCsLoVo cells were prepared into single cell suspensions, and then FcR Blocking Reagent and CD133MicroBeads were added and incubated with LoVo cells for30min. After that CD133(+) cells and CD133(-) cells were isolated with MS column and LD column respectively. Sorted cells were maintained in serum-free DMEM/F12medium containing Epidermal Growth Factor (EGF) and basic Fibroblast Growth Factor (bFGF). The purity of sorted cells was investigated with anti-CD133/2-PE by Flow Cytometry and direct Immunofluorescence. The CD133expression ratio of sorted cells was analyzed with FCM, the change of CD133expression before and after adding Fetal Bovine Serum (FBS) was also compared. In vitro drug sensitivity to Fluorouracil (5-FU) was determined with Cell Counting Kit-8(CCK-8). The differences of tumor-forming ability between CD133(+) cells and CD133(-) cells were compared.2. In vitro experiments The mouse ascites method was used to produce AC133mAb. AC133mAb was purified with Protein G beads. AC133mAb was labeled with131I by Iodogen method. The131I-AC133mAb was purified using ultrafiltration membranes. The radiochemical purity of the labeled mAb was carried out by ITLC, the stability was also checked. The binding ratio of unsorted LoVo cells, CD133(+) cells and CD133(-) cells with131I-AC133mAb were checked in quadruple. The specific block study was performed with LoVo cells incubated with100fold unlabeled AC133mAb before131I-AC133mAb was added.3. In vivo imaging with SPECT/CT and biodistributionThe same amount of unsorted LoVo cells, CD133(+) cells and CD133(-) cells were subcutaneously injected in the left lower limbs of BALB/c-nu to establish xenograft tumor models. When the diameter of the tumors reached to10mm, the tumor-bearing mice were intravenously injected with14.8MBq～18.5MBq (400μCi-500μCi)131I-AC133mAb for imaging. In specific blocking models,40-fold of unlabeled AC133mAb was used to block the binding of the tracer30min before the injection of131I-AC133mAb. In vivo imaging was performed with SPECT/CT on1,3,5and7days after the tracer injected. After imaging, the mice were sacrificed, and blood, tumor, brain, thyroid, liver, spleen, kidney, stomach, small intestine, colorectal intestine, lung, heart, muscle and bone were extracted and weighed. The amount of radioactivity of each organ was counted with y-counter. The radioactivity uptakes of different tissue were expressed as percentage of the injected dose per gram (%ID/g) after correcting for radioactive decay.4. Fluorescence assay, Western blot and autoradiographyAfter imaging, the tumors, including the unsorted LoVo, CD133(+), CD133(-) and the specific blocking tumors, liver, kidney and muscle were excised. Fluorescence assay and Western blot were performed to check the CD133expression. Autoradiography (ARG) was performed on Cyclone Plus Phosphor Scanning System to check the131I-AC133mAb accumulation. Results1. The identification of isolation results by MACSAfter isolation, percentage of CD133(+) cells in sorted positive LoVo cells was81.30%±1.83%, which was significantly higher than that of the sorted negative subpopulation (0.98%±0.79%) and unsorted LoVo cells (48.23%±2.15%)(P<0.05). The results of direct IF demonstrated that LoVo cells showed much different expression of CD133. Abundant CD133expression could be seen only in part of the cells, and other cells showed few or rare expression of CD133. All the sorted CD133positive cells had high expression of CD133in membrane, while rare expression in the sorted negative cells. The results obtained from FCM and IF showed the totally different CD133expression in the CD133positive and negative cells, which suggested the successfully isolation cells for LoVo cells.2. The identification of CSCsLoVo spheres were observed in CD133(+) cells that cultured in SFM, which could be serially subcultured in vitro. When the tumor spheres were cultured in the presence of10%FBS, the spheres gradually disappeared, the cells migrated from spheres and differentiated into large and adherent cells. When cultured in SFM, the percentage of CD133(+) cells decreased slowly. After adding FBS to medium, percentage of CD133(+) cells quickly decreased to unsorted LoVo cells level. The CD133(+) cells exhibited enhanced ability of drug-resistance and tumor-forming compared to CD133(-) cells.3. Radiolabeling and cell binding assayThe radiolabeling efficiency of131I-AC133mAb was68.98%±11.95%. After purification, the radiochemical purity of131I-AC133mAb was84.36%±0.45%. The radiochemical purity decreased to69.92%±1.37%after being incubated in0.01M PBS at37℃for7days. Cell binding ratios of131I-AC133mAb to CD133(+) cells and unsorted LoVo cells increased with time, and reached the peak after2hours of incubation. The highest cell binding ratio of CD133(+) cells was70.01%±6.02%, which was significantly higher than the unsorted LoVo cells (30.52%±1.14%), CD133(-) cells (2.39%±0.26%) and the specific blocking cells (2.73%±0.25%)(P<0.05).4. In vivo imaging with SPECT/CT and biodistributionThere were obvious uptake of the radiolabelled tracer in the area of tumors and gradually accumulated with time in the CD133(+) tumor mice and unsorted LoVo tumor mice, whereas nearly no tracer could be seen in the tumor area of the CD133(-) and specific blocking tumor mice. Intense radioactivity accumulations were also detected in liver, spleen, kidney, heart and lung.The biodistribution results showed that the%ID/g of tumor in unsorted LoVo tumors and CD133(+) tumors was6.96±1.40and6.12±1.91respectively, this was significantly higher than CD133(-) tumors (1.35±0.48) and specific blocking tumors (1.61±0.44)(P<0.05). The%ID/g of liver, spleen, kidney, heart and lung was significantly lower than LoVo tumor. In contrast,131I-AC133mAb revealed high retention in blood (6.38±0.77of%ID/g).5. Fluorescence assay,Western blot and autoradiographyThe results of IF and Western blot revealed abundant CD133expression in the unsorted LoVo tumors, CD133(+) tumors and the specific blocking tumors, while rare and no expression in CD133(-) tumors and muscle, respectively. ARG results showed that there was obvious radioactivity accumulation in CD133(+) tumors and the unsorted LoVo tumors, while rare accumulation in CD133(-) tumors, liver, kidney and muscle.ConclusionCD133(+) LoVo cells have the characteristics of CSCs. The imaging agent131I-AC133mAb showed good characteristics of stability, specificity and immunoactivity. Noninvasively in vivo targeting colorectal cancer stem cells with131I-AC133mAb is feasible. |
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