第四章 Discussion
4.11 Future exploration
1. To apply OATP1B3 dual reporter system for in vivo cell tracking such as cell therapy and cancer cell monitoring.
2. To evaluate the efficacy of OATP1B3 in different cell lines
3. To establish a drug-drug interaction system in ASBT, NTCP, and OATP1B3 using ICG for drug selection.
4. To establish a triple reporter system (luminance, fluorescence, and MRI) using OATP1B3
5. To explore more new bio-safe fluorophores and MR contrasts for molecular imaging
6. To explore whether NTCP is a d-luciferin transporter for a further imaging application.
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Figures
Figure 1. The plasmid maps for this study. (A) GFP control plasmid. (B) pLAS5w.PtRFP-I2-Puro plasmid. (C) pWPXL-OATP1B3-ires-Puro plasmid. (D) pLAS5w.OATP1B3-new-I2-Puro plasmid. (E) pWPXL NTCP-ires-Puro plasmid. (F) RC210241L2 plasmid. (G) RC221202L2 plasmid. (G) pWPXL ASBT-ires-Puro plasmid.
(H) AS2w.FLuc.Ppuro plasmid.
Figure 2. The confirmation of the overexpression using western blotting. C:
untransduced cells. O: OATP1B3 transduced cells. N: NTCP transduced cells. A: ASBT transduced cells. V: RFP (in vector) transduced cells. Blue arrow indicates OATP1B3 band.
Figure 3. The transduction confirmed using fluorescent immunostaining. C:
untransduced cells. O: OATP1B3 transduced cells. N: NTCP transduced cells. A: ASBT transduced cells. V: RFP (in vector) transduced cells. The red color in HT-29 indicates rhodamine-phalloidin (staining actin).
Figure 4.The ICG intake ability comparison of ASBT, NTCP, and OATP1B3 in HT-1080. (A) The confocal imaging. (B) Flow cytometry. (C) The cells were treated with different time-dependent manner and the data from the multimode detection platform. C: untransduced cells. O: OATP1B3 transduced cells. N: NTCP transduced cells. A: ASBT transduced cells. N was at least 4 in each group.
Figure 5. The ICG intake and retain ability comparison of ASBT, NTCP, and OATP1B3 in HT-29. (A) The cells were treated with different time-dependent manner and the data from the multimode detection platform. (B) The data from the multimode detection platform after washing out ICG in a time-dependent manner. (C) Flow cytometry with APC-cy7-A channel for ICG detecting. (D) Flow cytometry with FITC-A channel for FITC detecting. C: untransduced cells. O: OATP1B3 transduced cells. N: NTCP transduced cells. A: ASBT transduced cells. The statistic is the comparison with control in green and blue color. The symbols in red color are the comparison between NTCP and OATP1B3 expressing cells. N was at least 4 in each group. Error bars indicate the SEM. *p: 0.05; #p: 0.01; &p: 0.001.
Figure 6. The ICG intake and retain ability comparison of NTCP and OATP1B3 in HT-29. (A) Flow cytometry. (B) IVIS. (C) The confocal imaging. C: untransduced cells.
O: OATP1B3 transduced cells. N: NTCP transduced cells.
Figure 7. The FITC intake ability comparison of ASBT, NTCP, and OATP1B3 in HT-29.
The cells were treated 100uM FITC for 10min. The images were acquired at 1 h after FITC washing out. C: untransduced cells. O: OATP1B3 transduced cells. N: NTCP transduced cells. A: ASBT transduced cells.
Figure 8. The specificity of Gd containing MR contrasts among ASBT, NTCP, and OATP1B3 in HT-1080 using T1-weighted MRI. U: untreated cells. C: untransduced cells. O: OATP1B3 transduced cells. N: NTCP transduced cells. A: ASBT transduced cells. G: GFP (in vector) transduced cells.
Figure 9. Evaluation of ICG intake in vivo. (A) ICG tracking with IVIS after administrating ICG to NTCP and OATP1B3 expressing HT-29 tumor-bearing mice. (B) The average efficiency of ICG after injecting ICG to NTCP and OATP1B3 expressing HT-29 tumor-bearing mice. (C) The relative ICG intensity was the average efficiency normalized based on control tumor size to obtain relative ICG intensity. N was 3 and 4 for NTCP and OATP1B3 HT-29 tumor-bearing mice, respectively. C: control HT-29 tumor. N: NTCP expressing HT-29 tumor. O: OATP1B3 expressing HT-29 tumor. Error bars indicate the SEM. * p < 0.05, # p < 0.01, & p < 0.001. *, #, & in purple and green color indicate the control compared with OATP1B3 and NTCP expressing HT-29 tumor
cells, respectively. * in red color indicates the NTCP expressing HT-29 tumor compared with OATP1B3 expressing HT-29 tumor.
Figure 10. The biodistribution of ICG for the comparison between NTCP and OATP1B3 expressing HT-29 xenografts in IVIS. After the injection of ICG for 2 d, the mice were euthanized to observe the ICG signals using IVIS. (A) Ex vivo IVIS C: control tumor;
N: NTCP expressing HT-29 tumor; O: OATP1B3 expressing HT-29 tumor. Liv: liver;
LK: left kidney; RK: right kidney; Lu: Lung; H: heart; S: spleen; I: small intestine. (B) The quantification of the ICG intensity form NTCP and OATP1B3 expressing HT-29 tumor-bearing mice; N was 7 and 5, respectively. Error bars indicate the SEM. The symbols a, b, c, and d indicate statistically significant differences among groups. (C) NTCP immunochemistry staining in control and NTCP expressing HT-29 tumor-bearing mice. (D) OATP1B3 immunochemistry staining in control and OATP1B3 expressing HT-29 tumor-bearing mice. The nuclei were stained using hematoxylin. Scale bar: 100 µm.
Figure 11. MRI contrast-Primovist intake in vivo in MR imaging. (A) MR imaging and IVIS imaging were acquired at day 11 and day 9 after xenografting NTCP or OATP1B3 expressing HT29. (B) The relative signal intensity of the control and NTCP and OATP1B3 expressing HT-29 tumors normalized with the signal intensity of muscles in MR images. Error bars indicate the SEM. N was 8, 3, and 5 in control, NTCP expressing HT-29 tumors, and OATP1B3 expressing HT-29 tumors, respectively. (C) The relationship between tumor size and MRI signal at 1 h after Primovist injection into NTCP and OATP1B3 expressing HT-29 tumor-bearing mice. C: control HT-29 tumor. N:
NTCP expressing HT-29 tumor. O: OATP1B3 expressing HT-29 tumor. & p < 0.001.
Figure 12. The application of ICG-OATP1B3 system in vivo. (A)The OATP1B3 expressing HT-1080 tumor-bearing nude mice were observed after administering 200 mg ICG. The black- and orange-dotted circles indicate the control HT-1080 tumor and OATP1B3 expressing HT-1080tumor, respectively. (B)The relative ICG intensity normalized to the ROI of the 1 h control tumor. N was at least 4 in each group. *p, 0.05,
#p, 0.01. (C) After administering ICG for 2 d, the organs and tumors were evaluated the ICG signal through IVIS, and compared with those of the mice without ICG. K: kidney;
Liv: liver; S: spleen; H: heart; C: control HT-1080 tumor; O: OATP1B3 expressing HT-1080 tumor. (D) Quantification of ex vivo IVIS data. Error bars show the SEM. N was 3 in each group. The symbol a and b indicate the significant different groups. (E) Tumors stained for OATP1B3. Brown: OATP1B3. The nucleus was stained with hematoxylin. Scale bar: 100 μm.
Figure 13. MRI revealed cell death in OATP1B3 expressing HT-1080-bearing mouse (A) The signal intensity (blue arrows) of the cross-section shown in C. The red arrows indicate cell death. (B) Histology of control and OATP1B3 expressing HT-1080 tumors.
TUNEL staining showed dead cells in brow color. Green arrows indicate dead cells. Bar:
2.5 mm. (C) MRI results acquired before and after Primovist injection. IVIS 48 h after ICG administration. Red arrows indicate dead cells.
Figure 14. Functional validation of OATP1B3 expression and the examination of cellular function in vitro. (A) MRI of vector control and OATP1B3 expressing PANC-1 treated with Primovist. (B) ICP‐MS for detecting Gd contents (one‐tailed t-test, *p <
0.05) N was 3 in each group. (C) MMP. N was 4 in each group. (D) MTT. N was at least 7 in each group. (E) ROS. N was 4 in each group. The MTT and ROS were normalized to the mean of the values of blank controls and compared between groups using analysis of variance. B: untransduced PANC-1. V: RFP (in vector) tranduced PANC-1. O:
OATP1B3 transduced PANC-1. P: positive control.
Figure 15. The morphology and hormone-producing abilities of control and OATP1B3 expressing PANC‐1 after induced differentiation. (A) The sphere formation was tracked at day 0, 4 and 7. (B) Insulin (Ins) and glucagon (GCG) cDNA expression *p < 0.05, #p
< 0.01, N was at least 3 in each group. (C) Immunofluorescence of insulin. (D) Immunofluorescence of glucagon. B: untransduced PANC-1. V: RFP (in vector) transduced PANC-1. O: OATP1B3‐transduced PANC-1.
Figure 16. The application of OATP1B3 as MR reporter gene in islet-like cells xenografting animal model. (A) TI-weighted MR imaging. (B) OATP1B3 in brown color identified in the xenograft by immunohistochemical staining. The nucleus was
stained using hematoxylin. (C) The quantification of MRI signal intensity from (A). (D) The Primovist retain ability between control and OATP1B3 expressing PANC-1 traced in a time-dependent manner after 2 h of Primovist treatment. *p < 0.05, #p < 0.01, &p <
0.001, N = 6 (control at 0, 2, and 30 h), N = 2 (control and OATP1B3 at 3 and 22 h), N
= 4 (the other samples).
Figure 17. Drug-screening platform in vitro (A) The ICG signal was visualized using IVIS. (B) The ICG intensity detected using IVIS in NTCP expressing HT-29. (C) The ICG intensity detected using the multimode detection platform in NTCP expressing HT-29. N was at least 6 in each group. Relative ICG intensity was normalized with the cell number acquired from MTT. C: control HT-29 cells; N: NTCP-expressing HT-29 cells; U: untreated; I: 5 μM ICG; Rif: 100 μM rifampicin; CsA: 0.5, 2, and 10 μM cyclosporin A; Emo: 0.5, 2, and 10 μM emodin; Ery: 0.5, 2, and 10 μM erythrosin B; Pri:
1.25 mM Primovist. The statistic was the comparison with ICG treatment. Error bars indicate the SEM. *p: 0.05; #p: 0.01; &p: 0.001.
Figure 18. OATP1B3 involving in the transportation of d-luciferin. (A) The RT-PCR. (B) The intensity of luminance. C: control HT-1080. FLuc: Luciferase positive HT-1080.
FLuc-O: Luciferase and OATP1B3 double-positive HT-1080. U: untreated group. L: 94 µM d-Luciferin. L+R: 94 µM d-Luciferin and 100 µM rifampicin. N was 6 in each group. Error bars indicate the SEM. #p: 0.01; &p: 0.001.
Table
Table 1. The list of primers for Q-PCR
Gene 5’ to 3’
human-beta actin F CAT GTA CGT TGC TAT CCA GGC
human-beta actin R CTC CTT AAT GTC ACG CAC GAT
OATP1B3 F GTC ATT GGC TTT GCA CTG GG
OATP1B3 R GAC ACA AGG AAC CCA AGC CA
human-Ins F TAC CTA GTG TGC GGG GAA CG
human-Ins R CTG CGG GCT GCG TCT AGT TG
human-GCG F CAT TTA CTT TGT GGC TGG AT
human-GCG R CGC TTG TCC TCG TTC ATC TG