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Reverse of non‐small cell lung cancer drug resistance induced by cancer‐associated fibroblasts via a paracrine pathway

The tumor microenvironment orchestrates the sustained growth, metastasis and recurrence of cancer. As an indispensable component of the tumor microenvironment, cancer‐associated fibroblasts (CAF) are considered as an essential synthetic machine producing various tumor components, leading to cancer sustained stemness, drug resistance and tumor recurrence. Here, we developed a sustainable primary culture of lung cancer cells fed with lung cancer‐associated fibroblasts, resulting in enrichment and acquisition of drug resistance in cancer cells. Moreover, IGF2/AKT/Sox2/ABCB1 signaling activation in cancer cells was observed in the presence of CAF, which induces upregulation of P‐glycoprotein expression and the drug resistance of non‐small cell lung cancer cells. Our results demonstrated that CAF cells constitute a mechanism for cancer drug resistance. Thus, traditional chemotherapy combined with insulin‐like growth factor 2 (IGF2) signaling inhibitor may present an innovative therapeutic strategy for non‐small cell lung cancer therapy.

The tumor microenvironment orchestrates the sustained growth, metastasis and recurrence of cancer. As an indispensable component of the tumor microenvironment, cancer-associated fibroblasts (CAF) are considered as an essential synthetic machine producing various tumor components, leading to cancer sustained stemness, drug resistance and tumor recurrence. Here, we developed a sustainable primary culture of lung cancer cells fed with lung cancer-associated fibroblasts, resulting in enrichment and acquisition of drug resistance in cancer cells. Moreover, IGF2/AKT/Sox2/ABCB1 signaling activation in cancer cells was observed in the presence of CAF, which induces upregulation of P-glycoprotein expression and the drug resistance of non-small cell lung cancer cells. Our results demonstrated that CAF cells constitute a mechanism for cancer drug resistance. Thus, traditional chemotherapy combined with insulin-like growth factor 2 (IGF2) signaling inhibitor may present an innovative therapeutic strategy for non-small cell lung cancer therapy.

K E Y W O R D S
cancer-associated fibroblasts, drug resistance, insulin-like growth factor 2, non-small cell lung cancer, P-glycoprotein

| INTRODUCTION
Lung cancer is the most common malignancy and has a high death rate worldwide. Non-small cell lung cancer (NSCLC) accounts for 80%-85% of lung cancers. 1 The morbidity and mortality rates of NSCLC are relatively high in China, with the 5-year survival rate less than 20%, 2 which is often due to the drug resistance developed after treatment for 1 year. 3,4 Traditional anticancer strategies targeted cancer cells as a separate group, but tumor cells develop in a complex microenvironment, which is crucial for the development of drug resistance. 5 Hence, specifically targeting the tumor microenvironment and reversing drug resistance have been suggested in recent 10 years as new strategies for cancer therapy.
It is clear that the development of drug resistance does not depend solely on cancer cell autonomous defectsand is controlled by various important factors, including upregulation of ATP-binding cassette (ABC) transporter proteins, DNA damage repair, anti-apoptotic protein such as BCL-2 expression, and activation of prosurvival pathway induced by tumor microenvironments. 5 The tumor microenvironment has received recent attention as an important determinant of cancer cells drug resistance. 6 A dominant of tumor microenvironment is cancer-associated fibroblasts (CAF), 7 and many studies in recent 20 years have suggested that CAF play a prominent functional role for these cells in relation to cancer behavior. 8 Cancerassociated fibroblasts (CAF) are spindle-shaped mesenchymal cells with characteristics of smooth muscle cells and fibroblasts in tumor tissues. 9 They participate in tumor growth and metastasis in several ways, such as through secretion of various tumor growth factors 10 or matrix metalloproteinases (MMP), 11 remodeling of the tumor microenvironment, and the induction of epithelial-mesenchymal transition (EMT) progress. 12 Cancer-associated fibroblasts (CAF) also play an important role in enhancing cancer cell resistance to multiple drugs in various tumors, including breast cancer and colorectal carcinoma. 13 However, the specific mechanism of drug resistance induced by CAF remains unclear and it is still a challenge to effectively overcome the resistance and achieve better patient outcomes.
In the present study, enriched CAF were observed in tumor tissues from drug-resistant NSCLC patients. This tumor microenvironment with abundant CAF was demonstrated to facilitate the multi-drugsresistance in lung cancer. Furthermore, our studies revealed that NSCLC drug resistance was regulated in an insulinlike growth factor 2 (IGF2)/insulin-like growth factor receptor-1 (IGF-1R)/AKT/Sox2/P-GP pathway. The application of IGF-1R inhibitor could reverse the drug resistance and inhibit the growth induced by CAF. Based on our results, we found that CAF might cause NSCLC drug resistance development, and chemotherapy combined with IGF-1R inhibitor could be a potential strategy to treat NSCLC.

| Primary culture of fibroblasts and tumor cells
We cultured fibroblast cells from NSCLC tumor tissues. Briefly, after washing the samples with RPMI-1640 medium and PBS, the tissues were cut into pieces as small as possible. Then tissues were removed to another dish with DMEM (HyClone, USA) containing ACCUMAX (SIGMA, USA) medium to digest at 37°C, 5% CO 2 incubator for 2 hours, followed by filtration (BD Biosciences, San Jose, CA, USA).
After washing with PBS, cell precipitation was collected and seeded into 6-well plates in 2-mL RPMI-1640 medium supplemented with 10% FBS overnight at 37°C. The next day, the medium was replaced with fresh medium to remove the nonadherent cells and the remaining cells were collected. After 5-10 passages, we sorted the CD90positive cells with BD FACSAria III (USA) to collect fibroblasts and cultured the cells for further study. 14 We also obtained primary tumor cells from NSCLC tumor tissues. Briefly, primary tumor specimens were finely minced and then were treated with collagenase II/IV to digest for 2 hours at 37°C, 5% CO 2 incubator. After this, the suspension was filtered and the tumor cells were cultured in RPMI-1640 containing EGF (Fusheng Company, Shanghai, China) and 10% FBS (Thermo Fisher Scientific, MA, USA). The media was changed every 4-5 days until cells covered the bottom. Then we sorted the CD45RA negative cells to culture and named it as LCP1.

| Flow cytometry
It has been reported that CD90 was a marker for CAF. 15 Therefore, to detect the percent of CAF in lung cancer tumor samples, anti-CD90 human antibody (APC, eBioseicence, USA) was added to the cell suspension which was obtained from chemo-resistant and chemo-sensitive lung cancer patients' tissues. After incubating for 30 minutes at room temperature, samples were detected with a BD Accuri C6 (USA) the isotype was stained as a negative control.
Then cells were harvested at different time points (1, 2, 4 and 8 hours) to detect the intracellular doxorubicin fluorescence intensity by flow cytometry (BD Accuri C6). 16

| Viability tests
All the cell viability in this study was measured by MTT assay.
Briefly, 2 9 10 4 A549 or LCP1 cells pre-co-cultured with CAF, IGF2  2.6 | Real-time PCR Total RNA was isolated from cells using TRIZOL (Invitrogen) and cDNA was synthesized using a Transcriptor First Strand cDNA Synthesis Kit (Toyobo, Osaka, Japan). A total of 2 lg cDNA was used as the template to perform the quantitative real-time PCR for the detection of the target genes (SYBR Green real-time PCR master mixes, ThermoFisher Scientific, USA). The GAPDH was used as the internal control and 3 independent experiments were performed for each sample. The relative expression was quantified by normalizing the target gene level to the GAPDH using the DDCt method. The primer pairs used are listed in Table S1.

| Drug retention assay
For the drug retention assay, 2 9 10 4 A549 cells pre-co-cultured with CAF, IGF2, OSI-906, verapamil or not were seeded on 6-well chamber slides per well and treated with doxorubicin (1 lg/mL) for 8 hours. The cells were then fixed, permeabilized and stained with DAPI (Jingrong Company, Shanghai, China). Since the doxorubicin has the spontaneous fluorescence, the drug retention in nucleus was visualized using an FV1000 (Leica) laser scanning confocal microscope.

| Immunofluorescence staining and immunohistochemistry
To examine the expression of Sox2 following CAF co-culturing, A549 cells pre-treated with or without CAF were fixed and permeabilized. Then the cells were labeled with rabbit anti-Sox2 mAb (Abcam) and amplified with FITC goat anti-rabbit antibodies followed by Alexa 488 goat anti-FITC antibodies (Abcam). Nuclei were labeled with DAPI. To ensure the specificity of IF staining, primary antibodies were substituted with isotype-matched non-specific IgG in the experiment. All immunofluorescent images were captured using an FV1000 (Leica) laser scanning confocal microscope.
To examine the expression of a-SMA and IGF2 in tumor tissues, immunohistochemistry was performed in tumor tissues from chemo-sensitive and chemo-resistance patients. The tumor tissues were kept in 4% PFA overnight, then processed, embedded in paraffin, and sectioned at 4 lm. Then the sections were blocked with 5% BSA in PBS and incubated with a-SMA (1:200, Abcam) or IGF2 (1:100, Abcam) at 4°C overnight, followed by signal amplification using an ABC HRP Kit (Thermo, USA) and counter-staining with hematoxylin, dehydration with series of graded ethanol, and cleaning with xylene. A microscope (Leica, German) was used to visualize the sections.

| RNA interference
Human siRNA against Sox2 (siRNA1:5 0 -AAGGAGCACCC-3 0 and siRNA2:5 0 -GGATTATAAA-3 0 ) and control siRNA were purchased from Shanghai GenePharma. The transfection reagent and the siRNA complex were added to the A549 cells and incubated for 36 hours, followed by 24-hour IGF2 treatment. The cells were collected for further study.

| Statistical analysis
All data were presented as mean AE SEM. Graph Pad Prism 6.0 was used for data analysis (P < .05). Student t-test was used to analyze the difference between groups. Survival analysis was performed by Kaplan-Meier method and evaluated using the log-rank test. P < .05 was considered significant.  Figure S1A). We found that the chemo-resistant patients have increased fibroblasts compared to chemo-sensitive patients ( Figure 1A,B). Based on this point, we hypothesize that the accumulation of CAF in lung cancer tissues may confer the resistance of cancer cells to chemotherapy drugs. This was supported by the MTT assay, showing that pre-co-culturing with CAF ( Figure S1B) from either chemo-sensitive (CS) or chemo-resistant (CR) samples increased the cell viability in the A549 lung cancer cells with cisplatin, etoposide and vinorelbine ditartrate treatment compared with monoculture ( Figure 1C). Furthermore, we tested the primary tumor cells which were isolated from clinical NSCLC lung cancer patients' tumor tissue (labeled as LCP1 in Figure S1B) and found that pre-co-culturing with CAF from either chemo-sensitive (CS) or chemo-resistant (CR) samples could elevate the cell viability in LCP1 cells with cisplatin, etoposide and vinorelbine diatrate treatment ( Figure 1D). These results suggest that CAF may participated in the acquisition of chemotherapeutic drugs resistance in NSCLC.
3.2 | Cancer-associated fibroblasts induce the acquired chemo-resistance through the insulin-like growth factor 2/insulin-like growth factor receptor-1 paracrine pathway Next, we questioned how the CAF induced the chemo-resistance in NSCLC. It has been reported that CAF could secret cytokines or other proteins to communicate with the surrounding cells for cell growth, differentiation or migration. [18][19][20] Based on this concept, we added the conditioned medium from fibroblasts culturing with tumor cells to the A549 and LCP1 cells followed by chemotherapy drugs treatment, respectively. The MTT assay showed that the conditioned medium significantly increased the cell viability in A549 and LCP1 cells with cisplatin, etoposide and vinorelbine diatrate treatment (Figure 2A,B). This data suggests that the CAF may produce soluble  Figure 2C). Moreover, we used the recombinant IGF2 to pre-F I G U R E 2 Cancer-associated fibroblasts (CAF) induce the acquired chemo-resistance through a insulin-like growth factor 2 (IGF2)/insulin-like growth factor receptor-1 (IGF-1R) paracrine pathway. A, MTT assay of A549 cells treated with 400 nmol/L cisplatin (Cis), 150lmol/L etoposide (Eto) and 800 nmol/ L vinorelbine ditartrate (VD), respectively, with or without CAF medium (CM) pretreated (n = 3). B, MTT assay of LCP1 cells treated with Cis, Eto and VD, respectively, with or without CAF medium (CM) pretreated (n = 3). C, mRNA expression of IGF2, SDF-1, HGF, VEGFa, FGF, EGF, PDGF, CTGF, tenascin, TSP-1 and fibronectin in CAF cells co-culturing with or without LCP1 cells (n = 3). D, MTT assay of LCP1 cells treated with Cis, Eto and VD, respectively, with or without IGF2 pretreated (n = 3). E, MTT assay of LCP1 cells treated with Cis, Eto and VD, respectively, with or without CAF pre-cocultured in the presence or absence of anti-IGF2 (n = 3). F, mRNA expression of IGF-1R, VEGFR, PDGFR, HGFR, CXCR4, EGFR, SDF-1R and CTGFR in LCP1 cells with or without CAF co-cultured (n = 3). G, MTT assay of LCP1 cells treated by Cis, Eto and VD, respectively, with or without IGF2 pretreated in the presence or absence of OSI-906 (n = 3). H, A549 cells were isolated from A549-bearing nude mice with CAF, IGF2, CAF and anti-IGF2 treatment for 1 wk; the cell viability was analyzed in the addition of Cis, Eto or VD by MTT assay (n = 3). The data are presented as the means AE SEM from 3 independent experiments. *P < .05; **P < .01; ***P < .001; ns, not statistically significant treat LCP1 and A549 cells, followed by cisplatin, etoposide and vinorelbine diatrate treatment. We found that IGF2 could elevate the cell viability ( Figures 2D and S2A). It was further demonstrated in the fibroblast and tumor cell co-culturing system that the cell viability was decreased with the application of anti-IGF2 antibody ( Figures 2E and S2B). Consistently, we found that the expression of IGF2 in chemo-resistant samples was significantly higher than in chemo-sensitive samples, as evidenced by immunohistochemistry staining ( Figure S2C). The above data indicated that IGF2, indeed, could induce the drug resistance in NSCLC cells.
Numerous reports have shown that cytokines function through binding theirs corresponding receptors. 32,33 Thus, we screened for the expression of IGF-1R, VEGFR, PDGFR, HGFR, CXCR4, EGFR, SDF-1R and CTGFR in LCP1 cells pre-co-cultured with or without CAF. Consistent with the ligand expression, IGF-1R was highly expressed in LCP1 cells with CAF co-culturing ( Figure 2F). Furthermore, we used OSI-906, the inhibitor of IGF-1R, to treat the IGF2-pretreated LCP1 and A549 cells, and found that the IGF2 induced LCP1 and that A549 drug resistance was blocked (Figures 2G and S2D). This indicates that drug resistance in NSCLC cells induced by IGF2 is IGF-1R-dependent. In addition, we addressed the effects of chemotherapy on CAF and/or LCP1 cells.
We found that cisplatin, etoposide and vinorelbine diatrate increased the secretion of IGF2 by CAF only in the presence of LCP1 cells and upregulated IGF-1R in LCP1 cells only with CAF that were co-cultured ( Figure S2E,F). To further demonstrate that the IGF2 was secreted by CAF and examine the effects of CAF and IGF2 in NSCLC drug resistance, we isolated tumor cells from A549-bearing nude mice treated with CAF, IGF2 and CAF combined with IGF2 neutralizing antibody treatment and analyzed the cytotoxicity of chemotherapeutic drugs ex vivo. Consistent with the above data, CAF or IGF2 could effectively enhance the cell viability of A549 cells in vivo, while the blockade of IGF2 could reverse the drug resistance induced by CAF ( Figure 2H). We conclude that CAF could mediate the drug resistance of NSCLC cells through the IGF2/IGF-1R paracine pathway.

| The insulin-like growth factor 2/insulin-like
growth factor receptor-1 signal triggers the AKT/ Sox2 pathway to induce the acquired chemoresistance As a classic downstream molecule of the insulin-like growth factor (IGF) signaling axis, AKT is involved in cell survival, cell cycle, metabolism and drug resistance. 34,35 In addition, many studies have reported that AKT is correlated to drug resistance in hepatocellular carcinoma and prostate cancer models; 36,37 therefore, we evaluated the phosphorylation of AKT in A549 cells pre-co-cultured with CAF/recombinant IGF2 or not. We found that the expression of phospho-AKT was increased in the CAF/recombinant IGF2-treated A549 cells ( Figure 3A), while adding anti-IGF2 significantly reduced the phospho-AKT level ( Figure 3B). Furthermore, adding MK-2206 38 (an inhibitor for AKT) effectively reversed the resistance of IGF2-treated A549 cells to the cisplatin, etoposide and vinorelbine diatrate ( Figure 3C). These results suggested that AKT may work as the downstream in the IGF2/IGF-1R axis in the acquisition of drug resistance in NSCLC cells.
It has been reported that Nanog, Oct4 and Sox2 are the main transcriptional factors in activating the AKT signaling pathway. [39][40][41] Thus, we analyzed the expression of Nanog, Oct4 and Sox2 in CAF-treated A549 and LCP1 cells, and we found that only the Sox2 was significantly upregulated in both A549 and LCP1 NSCLC cells after co-culturing with CAF ( Figure 3D,E). Correspondingly, the inducible expression of Sox2 by CAF and/or recombinant IGF2 was verified using western blot ( Figure 3F) and immunofluorescence ( Figure 3G), The data indicated that CAF and/or IGF2 could induce the high expression of Sox2 and its nuclear translocation. In addition, adding anti-IGF2 reversed the inducible expression of Sox2 by CAF ( Figure 3H). Furthermore, MK-2206 also markedly decreased the expression of Sox2, which was induced by IGF2 ( Figure 3I). These data suggest that the inducible expres- 3.4 | Insulin-like growth factor 2 triggers the AKT/

Sox2 pathway to elevate P glycoprotein expression
We have demonstrated that AKT/Sox2 was involved in CAF inducing the acquired chemo-resistance, but we wondered how those signals participated in the NSCLC cells survival from chemotherapy.
It has been reported that ATPase binding cassette (ABC) transporters mainly participate in drug efflux of cancer cells, leading to reduced cytotoxicity and drug resistance. Thus, we hypothesized that Sox2 might play a role in ABC gene transcription. Using the JASPAR database, we predicted that Sox2 had most putative binding sites in ABCB1 promoter ( Figure 4A). We also screen the expressions of ABC transporters, ABCA2, ABCB1, ABCB4, ABCB11, ABCC1, ABCC2 and ABCG2, and found that the gene expression of ABCB1, which encodes P-GP, has the highest upregulation (nearly 274-fold) in A549 cells pre-co-cultured with CAF compared with control cells ( Figure 4B). Moreover, the protein expression of P-GP was elevated in A549 cells with recombinant IGF2 treatment, and this upregulation was blocked when Sox2 was silenced ( Figure 4C).
These data indicated that Sox2 may mediate the P-GP expression in IGF2 induced the chemo-resistance of NSCLC cells. Furthermore, both CAF and recombinant IGF2 treatment elevated the expression of P-GP in A549 cells compared with control cells (Figure 4D), while adding anti-IGF2 or MK-2206 depressed the elevation (Figure 4E,F). These results indicate that P-GP participates in the drugresistance process in NSCLC cells, which is induced by AKT/Sox2 signal pathway. ZHANG ET AL. | 949 3.5 | Cancer-associated fibroblasts regulate chemoresistance through P-GP to decrease drug retention and increase drug efflux Because we have found that P-GP was upregulated in CAF and induced NSCLC chemo-resistance, we questioned the function of P-GP in this process. Strong evidence has been reported that P-GP may contribute to drug resistance in cancer by reducing drug uptake and enhancing drug efflux. 5,42 To investigate whether accumulated CAF-induced chemo-resistance in NSCLC tissue was associated with the drug accumulation, doxorubicin was employed for its strong fluorescence in the assessment of intracellular drug content by immunofluorescence or flow cytometry. Our data showed that preco-culturing with CAF decreased the doxorubicin retention in the nucleus of A549 cells ( Figure 5A) compared with monoculture. Correspondingly, we found that monoculture A549 cells absorbed doxorubicin in a time-dependent manner, while cells pre-co-cultured with CAF had less absorption over time ( Figure 5B). In line with the data, we observed that the drug efflux in A549 cells pre-co-cultured with CAF was higher than control A549 cells at every time point ( Figure 5C). These results indicated that CAF may decrease drug retention and increase drug efflux, leading to the anti-doxorubicin ability of A549 cells. Our previous data showed that adding recombinant IGF2 remarkably enhanced the chemo-resistance of A549 cells.
To further verify, we pre-treated A549 cells with recombinant IGF2, followed by doxorubicin treatment. And we found that rhIGF2 pretreatment reduced doxorubicin aggregation in the nucleus of A549 cells. However, adding OSI-906, an IGF-1R inhibitor, reversed the F I G U R E 3 The insulin-like growth factor 2 (IGF2)/insulin-like growth factor receptor-1 (IGF-1R) signal triggers the AKT/Sox2 pathway to induce the acquired chemo-resistance. A, Western blotting of p-AKT, total AKT and b-actin in A549 cells with or without CAF co-cultured or IGF2 treated. B, Western blotting of p-AKT, total AKT and b-actin in A549 cells with or without CAF co-cultured in the presence or absence of anti-IGF2. C, The MTT assay of A549 cells treated with Cis, Eto and VD with or without IGF2 pretreated in the presence or absence of MK-2206 (n = 3). D, The mRNA expression of Nanog, Oct4 and Sox2 in A549 cells with or without CAF co-cultured (n = 3). E, mRNA expression of Nanog, Oct4 and Sox2 in LCP1 cells with or without CAF co-cultured (n = 3). F, Western blotting of Sox2 and b-actin in A549 cells with or without CAF co-cultured or IGF2 treated. G, Immunofluorescence of Sox2 in A549 cells with or without CAF co-cultured. Red, Sox2; blue, DAPI; scale bar, 10 lm. H, Western blotting of Sox2 and b-actin in A549 cells with or without CAF co-cultured in the presence and absence of anti-IGF2. I, Western blotting of Sox2 and b-actin in A549 cells with or without IGF2 treated in the presence or absence of MK-2206. J, MTT assay in negative control (NC) or siSox2-transfected A549 cells treated with Cis, Eto and VD with or without IGF2 pretreated (n = 3). The data was presented as the means AE SEM from 3 independent experiments. *P < .05; **P < .01; ***P < .001; ns, not statistically significant doxorubicin accumulation ( Figure 5D). Similarly, we found that the doxorubicin absorption was damped but the efflux was increased in A549 cells in a time-dependent manner with IGF2 pretreatment, and the addition of IGF-1R inhibitor reversed the doxorubicin distribution ( Figure 5E,F). Next, to further verify whether P-GP was involved in the drug efflux, we used verapamil, an inhibitor of P-GP, to treat A549 cells pre-co-cultured with CAF, and followed with doxorubicin treatment; we found that the doxorubicin retention in the nucleus was recorded again ( Figure 5G). Furthermore, the cell death rate was significantly elevated in CAF co-cultured A549 cells when verapamil was added ( Figure 5H). Taken together, we conclude that CAF induced chemo-resistance by P-GP to decrease drug retention and increase drug efflux.

| DISCUSSION
In our study, we found the CAF plays a crucial role in the development and maintenance of NSCLC cells' multi-drug resistance. These CAF isolated from tumor tissues with high chemotherapy resistance facilitate NSCLC cell drug resistance. The cancer cells would not be able to maintain the phenotype without the help of CAF feeders.
Previous research revealed that CAF could participate in different stages of tumor progression, including EMT, 43,44 tumor stemness maintenance 45 and metastasis, 6 through secretion of several cytokines or components that activated important signaling pathways. Our studies indicate that CAF regulate the NSCLC cell drug resistance in a paracrine manner through secretion of IGF2 and the activation of AKT/Sox2/P-GP signals in cancer cells. The IGF2 produced by CAF F I G U R E 4 Insulin-like growth factor 2 (IGF2) triggers the AKT/Sox2 pathway to elevate P-GP expression. A, Sox2 putative binding sites in ABCB1 promoter were acquired from the JASPAR database. B, mRNA expression of ABCA2, ABCB1, ABCB4, ABCB11, ABCC1, ABCC2 and ABCG2 in A549 cells with or without CAF co-cultured (n = 3). C, Western blotting of P-GP and b-actin in negative control (NC) or siSox2 transfected A549 cells with or without IGF2 treated. D, Western blotting of P-GP and b-actin in A549 cells with or without CAF co-cultured or IGF2 treated. E, Western blotting of P-GP and b-actin in A549 cells with or without CAF co-cultured in the presence or absence of anti-IGF2. F, Western blotting of P-GP and b-actin in A549 cells with or without IGF2 treated in the presence or absence of MK-2206. The data are presented as the means AE SEM from 3 independent experiments. *P < .05; **P < .01; ***P < .001; ns, not statistically significant activates the AKT pathways by binding to the membrane receptor IGF-1R in NSCLC cells to activate AKT. Then the phosphorylated AKT triggers transcription factor Sox2 nucleus translocation to bind the promoter region of the ABCB1 gene. Finally, the P-GP expression is upregulated, resulting in the development of drug resistance ( Figure 6D).
Various crucial features are believed to result in the development of multi-drug resistance, including ATP-binding cassette (ABC) transporter protein overexpression, upregulation of anti-apoptotic proteins such as BCL-XL and BCL-2, enhanced DNA damage repair and cytokines in the tumor microenvironment. 5 It was demonstrated that the tumor microenvironment plays a crucial role in tumor F I G U R E 5 Cancer-associated fibroblasts (CAF) regulate chemo-resistance through P-GP to decrease drug retention and increase drug efflux. A, The fluorescence of doxorubicin (Dox) in A549 cells with or without CAF pre-co-cultured. Red, Dox; blue, DAPI; scale bar, 10 lm. B, Mean fluorescence intensity (MFI) of Dox in A549 cells with or without CAF pre-co-cultured at different time points (n = 4). C, Released Dox in the supernatants of A549 cells with or without CAF pre-co-cultured at different time points (n = 3). D, Fluorescence of Dox in A549 cells with or without IGF2 pretreated in the presence or absence of OSI-906. Red, Dox; blue, DAPI; scale bar, 10 lm. E, MFI of Dox in A549 cells with or without IGF2 pretreated in the presence or absence of OSI-906 at different time points (n = 3). F, Released Dox in the supernatants of A549 cells with or without IGF2 pretreated in the presence or absence of OSI-906 at different time points (n = 3). G, Fluorescence of Dox in A549 cells with or without CAF pre-co-cultured in the presence or absence of verapamil. H, MTT assay of A549 cells treated with Cis, Eto and VD with or without CAF pre-co-cultured in the presence or absence of verapamil (n = 3). The data are presented as the means AE SEM from 3 independent experiments. *P < .05; **P < .01; ***P < .001; ns, not statistically significant progression, 6 54,55 or application of chemosensitizers, 56 which has been proven to be able to reverse drug resistance and inhibit tumor growth in the early stages. However, recent research revealed that the tumor microenvironment could induce drug resistance development of normal cancer cells and tumor tissues could reacquire the drug resistance under the appropriate microenvironment after chemotherapy. 5,57 Thus, solely directly targeting cancer cells may not be sufficient to reverse the cancer drug resistance; instead, blocking the interaction between CAF and cancer cells could serve as the basis of an innovative treatment to reverse the cancer drug resistance.
F I G U R E 6 Blockade insulin-like growth factor 2 (IGF2)/insulin-like growth factor receptor-1 (IGF-1R) signal relieves chemotherapy drug resistance in lung cancer. A, Mean tumor volume of subcutaneous A549 implants in PBS, Cis, OSI-906 or Cis combining OSI-906-treated nodscid mice (top, n = 6); the long-term survival of tumor-bearing mice treated with PBS, Cis, OSI-906 or Cis combining OSI-906 (bottom, n = 20). B, Mean tumor volume of subcutaneous A549 implants in PBS, Eto, OSI-906 or Eto combining OSI-906-treated nod-scid mice (top, n = 6); the long-term survival of tumor bearing mice treated with PBS, Eto, OSI-906 or Eto combining OSI-906 (bottom, n = 20). C, Mean tumor volume of subcutaneous A549 implants in PBS, VD, OSI-906 or VD combining OSI-906 treated nod-scid mice (top, n = 6); the long-term survival of tumor-bearing mice treated with PBS, VD, OSI-906 or VD combining OSI-906 (bottom, n = 20). D, Cancer-associated fibroblasts (CAF) produce IGF2 to bind IGF-1R in lung cancer cells (LCC) and activate the AKT/Sox2 pathways. Along with the Sox2 nucleus translocation, the transcription of ABCB1 gene is enhanced and the expression of P-GP is evaluated to pump out the chemotherapy drug. Using OSI-906 to block IGF2/IGF-1R signal and combining with chemotherapy drug could reverse the drug resistance of lung cancer cells. The data was presented as the means AE SEM from 3 independent experiments. *P < .05; **P < .01; ***P < .001; ns, not statistically significant It was reported that CAF in lung cancer microenvironment are associated with cancer stemness maintenance, cell proliferation and cancer metastasis. 12,58,59 However, the specific relationship between CAF and cancer cell behavior remains unclear. In our study, we further examined the precipitating factors of chemotherapy failure in NSCLC patients, and found that a mass of CAF in tumor tissues would induce the development of cancer cell drug resistance via an IGF2/IGF-1R paracrine pathway. Hence, OSI-906, an inhibitor of IGF-1R, could serve as a potential agent to block the IGF2/IGF-1R paracrine pathway induced by CAF and be combined with multiple chemotherapeutic agents to effectively reverse the drug resistance and inhibit NSCLC progression.
In summary, our data described a novel role of CAF in resistance to multiple chemotherapeutic drugs in NSCLC. In addition, CAF regulate the drug resistance in a paracrine manner through the IGF2/ IGF-1R/Sox2/P-GP pathway. The combination of a selective smallmolecule inhibitor to IGF-1R with chemotherapeutic agents will be explored as a potential therapeutic strategy for anticancer therapy.

CONF LICT OF I NTEREST
The authors have no conflicts of interest to declare.


extfieldsofstudy: Chemistry Medicine

text:
Using tyrosinase as a tri-modality reporter gene to monitor transplanted stem cells in acute myocardial infarction

The study aimed to investigate the feasibility of noninvasive monitoring of bone marrow mesenchymal stem cells (MSCs) transduced with the tyrosinase reporter gene for acute myocardial infarction (AMI) with photoacoustic imaging (PAI), magnetic resonance imaging (MRI), and positron emission tomography (PET) in vitro and in vivo. MSCs were transduced with a lentivirus carrying a tyrosinase reporter gene. After transduction, the rate of 18F-5-fluoro-N-(2-[diethylamino]ethyl)picolinamide (18F-5-FPN) uptake was measured. PAI and MRI of stable cell lines expressing tyrosinase (TYR-MSCs) were performed in vitro. An AMI model was induced and verified. TYR-MSCs and MSCs were injected into the margins of the infarcted areas, and PAI, MRI, and PET images were acquired 1, 7, 14, 21, and 28 days after cell injection. Sham-operated models without injection were used as the control group. TYR-MSCs showed noticeably higher uptake of 18F-5-FPN and stronger signals in T1-weighted MRI and PAI than non-transduced MSCs. In vivo studies revealed prominent signals in the injected area of the infarcted myocardium on PAI/MRI/PET images, whereas no signal could be seen in rats injected with non-transduced MSCs or sham-operated rats. The uptake values of 18F-5-FPN in vivo showed a slight decrease over 28 days, whereas MRI and PAI signal intensity decreased dramatically. MSCs stably transduced with the tyrosinase reporter gene could be monitored in vivo in myocardial infarction models by PET, MRI, and PAI, providing a feasible and reliable method for checking the viability, location, and dwell time of transplanted stem cells.

Introduction
Stem cell transplantation is a new method aimed at reversing myocardial injury and improving cardiac function 1 . Unfortunately, only a minute number of these laboratory studies can be transferred to clinical practice due to limited methods to monitor the fate of bone marrow mesenchymal stem cells (MSCs) after their transplantation into injured hearts. Recent developments in imaging systems and new probes have allowed investigators to perform multimodality imaging in animal models that have employed MSC transplantation. For example, Pei et al. 2 constructed a triple-fused reporter gene that combined herpes simplex virus type 1 thymidine kinase, enhanced green fluorescent protein, and firefly luciferase to monitor stem cells using positron emission tomography (PET), fluorescence, and bioluminescence imaging. However, integrating different active groups into one entity is complicated and time-consuming. Most importantly, the deactivation of one functional group will lead to failure of the whole construction. In addition, the employed adenovirus transduction leads to transient gene expression, and gene expression levels decline quickly with time. It is necessary to obtain a simple multimodality molecular probe that is easily constructed and stably expressed.
Human tyrosinase, the key enzyme in melanin production, was first evaluated by Qin et al. 3 as a stand-alone reporter gene for in vitro and in vivo multimodality imaging, including photoacoustic imaging (PAI), magnetic resonance imaging (MRI), and PET. We first established a tyrosinase reporter gene system under the control of the Tet-on gene expression system in vitro in tumor cell lines, which were proof-of-concept studies 4 . In this study, tyrosinase was used as a multifunctional reporter gene to track stem cells transplanted in an area of myocardial infarction. After transducing the tyrosinase gene into MSCs in Sprague-Dawley (SD) rats, melanin was synthesized and subsequently absorbed light energy to achieve PAI 5 , bound with 18 F-5-fluoro-N-(2-[diethylamino]ethyl)picolinamide( 18 F-5-FPN) specifically to enable PET imaging 6 , and combined with iron for visibility on MRI 7 . The aim of this study was to establish a simple multimodality reporter gene system using tyrosinase and combining PAI, PET, and MRI to monitor the fate of MSCs after transplantation into rat models of AMI.

Materials and Methods
PET imaging tracers 18 F-5-FPN is a benzamide analog that specifically binds melanin. The synthesis process was performed according to our previously published procedures 6 . The reaction was completed in a synthesis module (GE TraceLab FX-XN, GE Healthcare, Milwaukee, WI, USA).

Construction of a recombinant lentiviral vector carrying the tyrosinase reporter gene
Tyrosinase complementary DNA was kindly provided by Dr. Zhen Cheng of Stanford University. The tyrosinase sequence was amplified and digested with AgeI/NheI, then cloned into the Ubi-MCS-3FLAG-SV40-puromycin vector, which carried puromycin-resistance genes (Shanghai Genechem Co. Ltd., Shanghai, China).

Isolation, cultivation, and identification of TYR-MSCs
All experiments were performed in accordance with protocols approved by the Animal Care and Use Committee of Huazhong University of Science and Technology, China. Rat bone marrow MSCs were isolated and purified from 4-week-old SD rats (male, 80-100 g) by combing gradient density centrifugation and adhesion separation. The surface makers CD44, CD90, CD34, and CD33 (antibodies from Abcam, Cambridge, MA, USA) were detected by flow cytometry. Transduction of tyrosinase lentiviral vectors into MSCs was performed at a multiplicity of infection of 3 8 . Antibiotic selection with 1.75 µg/mL puromycin began 72 h after transfection and lasted for 5 days to remove the MSCs that were not transduced successfully 9 . Masson-Fontana, western blot, and tyrosinase activity assays were performed to demonstrate enhanced melanin synthesis in stably transduced cell clones 10 .
In vitro cellular uptake assays Cellular uptake assays were performed on TYR-MSCs, MSCs, and TYR-MSCs + blocking. Cells were seeded in 24-well plates at a density of 2 × 10 5 cells per well. After overnight incubation, cells were washed twice with PBS. Then, 200 µL of completed DMEM-F 12 medium containing 37 kBq of 18 F-5-FPN was added to each well. After incubation with 18 F-5-FPN at 37°C for increasing intervals (30, 60, and 120 min), media were removed, and cells were washed twice with PBS and lysed with 1 N NaOH for 5 min. For the blocking study, TYR-MSCs were incubated for 1 h at 37°C with 18 F-5-FPN (37 kBq) in the presence of 100 µL of 10 −5 M standard 19 F-5-FPN. Radioactivity was measured using a gamma counter (2470, WIZARD; Per-kinElmer, Waltham, MA, USA). The uptake rate was obtained using the following equation 2 , and all the above experiments were performed three times with triplicate wells.
In vitro MRI MRI of cells was performed using a 7.0 T MRI (Varian, Palo Alto, CA, USA). Increasing numbers of cells (5 × 10 5 , 2.5 × 10 6 , and 5 × 10 6 ) were embedded in 1% agarose 11 , and T1-weighted imaging (T1WI) was performed 12 . To increase the production of melanin, the TYR-MSCs + tyrosine line was pretreated with 2 mM tyrosine for 24 h. T1WI parameters included a field of view 7.0 × 3.5 cm, matrix size of 256 × 256, section thickness of 1 mm, repetition time of 500 ms, and echo time of 11 ms. Image analysis was performed using ImageJ (NIH, Bethesda, MD, USA). The experiments were performed three times with triplicate wells.

In vitro PAI
PAI (Endra Nexus 128 Photoacoustic Imaging System, Endra Life Sciences, Ann Arbor, MI, USA) of increasing concentrations of cells, ranging from 5 × 10 4 to 1 × 10 7 cells per mL and embedded in 1% agarose, was performed. Photoacoustic signals generated by a given laser pulse in a target at a 5-mm depth and 680-nm wavelength in a phantom were detected by all transducers 13 . Data were reconstructed and drawn in Osirix after exporting the raw data in DICOM format (Pixmeo, Switzerland). The experiments were performed three times with triplicate wells.

Acute myocardial infarction animal model preparation
Adult SD rats (male, 160-200 g) were anesthetized by an intraperitoneal (ip) injection of 3% sodium pentobarbital (35 mg/kg, Merck, Darmstadt, Germany) and artificially ventilated with an animal ventilator (DW-3000, Jinyang Wanda, Beijing, China). Left thoracotomy was performed, and the left anterior descending coronary artery was permanently ligated at its origin to establish acute myocardial infarction (AMI) models 14 . The shamoperated controls underwent the same surgery except that the coronary artery was not ligated. A total of 21 AMI models and 13 control models were successfully established and used for in vivo experiments. Thirty minutes after ligation of the left anterior descending coronary artery, eight injections of TYR-MSCs or MSCs (2 × 10 6 cells per 50 μL PBS) were injected into the margin of the infarcted area. Seventy-two hours after ligation of the coronary artery, five other AMI models without transplanted TYR-MSCs or MSCs were humanely killed to perform 2,3,5-Triphenyltetrazolium chloride (TTC) (n = 3) or hematoxylin and eosin staining (n = 2). Five control models were also killed to perform TTC (n = 3) or hematoxylin and eosin staining (n = 2). Infarct size was expressed as a percentage of total left ventricular area. The mean of all slices from each heart was calculated. Left ventricular (LV) infarct size was measured in six to eight transverse sections of 1-2 mm from apex to base stain. The infract zone was identified by a white color. According to the result of TTC staining, infarction size was calculated using the following formula: 2 To perform hematoxylin and eosin staining, the hearts of the models were quickly removed and cut into six transverse slices from apex to base. Subsequently, partial 8-μm transverse slices from each section were prepared for hematoxylin and eosin staining. 18 F-FDG myocardial metabolic imaging was performed to confirm successful coronary occlusion in TYR-MSCs (n = 8), MSCs (n = 8) transplanted AMI models 24 h before 18 F-5-FPN imaging 15 . MRI, PET, and PAI were performed at five points post operation: 1, 7, 14, 21, and 28 days. Control models (n = 8) were also subjected to 18 F-5-FPN imaging, MRI, PET, and PAI at the same time points.

In vivo 18 F-FDG myocardial metabolic imaging
After fasting for 4 h, 16 AMI rats, 8 control models, and 2 normal rats were anesthetized with 2% isoflurane in oxygen and injected via the tail vein with a mean dose of 3.7 MBq of 18 F-FDG. A PET scan (Trans-PET BioCaliburn 700, Raycan Technology Co., Ltd., Suzhou, China) focused on the chest was acquired for 15 min starting 60 min after the injection.

In vivo 18 F-5-FPN animal PET imaging
The TYR-MSCs group (n = 8), MSCs group (n = 8), and control group (n = 8) were injected intravenously with 3.7 MBq of 18 F-5-FPN. After 1 h, rats were anesthetized with 2% isoflurane in oxygen and placed in the prone position. The PET scan acquisition conditions were identical to those with 18 F-FDG. This algorithm produces images consisting of 0.5 × 0.5 × 0.5 mm 3 voxels. Images were reconstructed into a 280 × 280 × 104 matrix using 3D OSEM with a pixel size of 0.5 × 0.5 mm 2 and a slice thickness of 0.5 mm. Regions of interest (ROIs) were marked manually and analyzed by AMIDE (UCLA, Los Angeles, CA, USA). Signals were expressed as a percent of injected dose per gram of tissue (%ID per g).

In vivo MRI imaging
The TYR-MSCs group (n = 8), MSCs group (n = 8), and control group (n = 8) were continuously anesthetized with 2% isoflurane in oxygen and placed in the prone position. The scanning parameters were as follows: repetition time of 48 ms; echo time of 3 ms; slice thickness of 1 mm; field of view of 4.5 × 4.5 cm; and 192 × 192 matrix. T2*-weighted images were detected as hypointensities caused by Fe 3+ -bound melanin 16 . Image analysis was performed using ImageJ.

In vivo PAI imaging
The TYR-MSCs group (n = 8), MSCs group (n = 8), and control group (n = 8) were anesthetized via ip injection of 3% sodium pentobarbital (35 mg/kg) using the same PAI system employed for the in vitro studies and the same wavelength 17 . After scanning a rat, signals were sent to ultrasonic transducers and then directed to a computer system to reconstruct two-dimensional and three-dimensional images using imaging software (OsiriX Foundation, Geneva, Switzerland) 18 .

Statistical analysis
Quantitative data are expressed as the means ± SD. Statistical analysis was performed using one-way analysis of variance and the Student's t test (SPSS 16.0 software package, SPSS Inc., Chicago, IL, USA). A 95% confidence interval was chosen to determine the significance of differences between groups. A probability value of P < 0.05 was considered statistically significant.

In vitro tyrosinase introduction and melanin expression in MSCs
MSCs were successfully transduced with lentivirus carrying the tyrosinase reporter gene and selected with puromycin. After centrifugation, TYR-MSCs were black in color, whereas non-transduced MSCs were white or light yellow (Fig. 1a). TYR-MSCs showed a specific band of~96 kDa corresponding to tyrosinase (Fig. 1b). Masson-Fontana staining demonstrated melanin deposition within TYR-MSCs (Fig. 1c). Cellular tyrosinase activity analysis showed that the absorbance at 490 nm was substantially higher in TYR-MSCs than in MSCs (0.65 ± 0.12 vs 0.11 ± 0.02; n = 3, P < 0.05).

Cellular uptake of 18 F-5-FPN
Uptake levels of 18 F-5-FPN in TYR-MSCs and MSCs at 30, 60, and 120 min are shown in Fig. 2a. The uptake value of TYR-MSCs increased from 6.32 ± 0.27% at 30 min to 7.86 ± 0.85% at 60 min, and then slightly decreased to 6.83 ± 0.24% at 120 min. The uptake values of MSCs remained at a low level (1.22 ± 0.19, 1.15 ± 0.15, 1.12 ± 0.2 at 30, 60, and 120 min, respectively) and did not show an increase with longer incubation times. The uptake value of TYR-MSCs was significantly higher than those of MSCs at all time points (P < 0.01). Administration of cold 19 F-5-FPN inhibited the binding of 18 F-5-FPN in TYR-MSCs in a concentration-dependent pattern as shown in

In vitro MRI and PAI
T1W1 MRI acquired in different concentrations of cells (Fig. 3a) revealed hyperintensity of the TYR-MSCs. The MR signals increased with increasing cell concentrations. TYR-MSCs treated with both FeCl 3 and tyrosine showed a higher signal than cells treated with FeCl 3 or tyrosine alone. MSCs produced a low signal on T1WI at all cell concentrations. The signals of MSCs treated with FeCl 3 slightly increased with an increasing number of MSCs. Fig. 3b shows quantitative analysis of MR signals in vitro in all eight groups.
As shown in Fig. 3c, the PAI signals of TYR-MSCs and TYR-MSCs treated with tyrosine increased with increasing cell concentrations. No photoacoustic signals were detected in the MSCs and MSCs treated with tyrosine, even at 5 × 10 5 cells. Fig. 3d shows quantitative analysis of the photoacoustic signals in vitro in TYR-MSCs with and without tyrosine, which shows that tyrosine enhanced the PAI signal.

Histological verification of myocardial infarction
The percent infarction of the left ventricular mass determined by TTC staining was 38.1% ± 7.3% (n = 3) in AMI animals, whereas normal rats and control models showed no area of infarction (Fig. 4a). Obvious absent uptake of 18 F-FDG in the anterior wall was noted on myocardial metabolic imaging of the AMI animals (Fig. 4b), whereas uniform uptake in the myocardium was seen in normal rats and control models. Fig. 4c shows hematoxylin and eosin staining of infarcted myocardium, normal myocardium, and the border between them. Normal myocardial fibers are arranged in neat rows with abundant capillaries. In the AMI models, the myocardiocytes are necrotic with surrounding edema and denaturation, distinct lymphocyte infiltration, myocardial fiber rupture, and vacuolar denaturation (Fig. 4c).

In vivo PET, MRI, and PAI
PET images of rats injected with TYR-MSCs and MSCs are shown in Fig. 5; the images were obtained 1 h after injection of 18 F-5-FPN. Obvious uptake of 18 F-5-FPN in the transplanted area was seen in the rats with transplanted TYR-MSCs. The signals showed a decreasing trend from 1.78 ± 0.22 %ID per g to 1.62 ± 0.13 %ID per g (n = 8) from day 1 to 28 post injection but had no significant difference. No 18 F-5-FPN uptake was seen in the MSCs group and control group.
On the first day after TYR-MSC transplantation, lowintensity MRI signals were visible in the transplanted area on T2* sequences (Fig. 6a). The area of low-intensity signals decreased over time. Signal contrast (%) of TYR-MSCs showed a tendency to increase gradually from 59.18 ± 4.9 to 80.5 ± 8.8 (n = 8), indicating that the intensity of the MRI signal decreased with time. At 28 days after transplantation, the low-intensity signals became unclear. For the MSCs group and control group, no low-intensity signals were observed.
As shown in Fig. 6b, rats transplanted with TYR-MSCs produced high photoacoustic signal intensities, whereas the MSCs group and control group did not produce photoacoustic signals. The PAI signals of TYR-MSCs were 472.6 ± 48.1, 434.8 ± 51.2, 387.3 ± 62.0, 345.9 ± 79.8, and 283.4 ± 49.2 at 1, 7, 14, 21, and 28 days post injection, respectively (n = 8).  (Fig. 7a), MRI (Fig. 7b), and PAI (Fig. 7c). The signal intensity of MRI and PAI decreased significantly with time (P < 0.05); however, there was no significant difference between the PET signals on day 1 and day 28, which implied that PET could be used to track TYR-MSCs in vivo for longer time than MRI or PAI.

Discussion
The rapid growth of regenerative medicine has made cell replacement therapy possible for myocardial infarction [19][20][21] . However, numerous challenges remain in using this therapy, including tracking the location, survival, distribution, and differentiation process of transplanted cells in vivo. Multimodality molecular imaging of reporter genes is a method that allows noninvasive assessment of stem cell therapy and has the potential to diminish the shortcomings of any single imaging modality to gain more complete image 22 .
In this study, stable MSCs expressing melanin were successfully established after lentiviral transfection with the tyrosinase reporter gene. In vitro experiments showed that TYR-MSCs exhibited clear MRI and PAI signals and could specifically take up 18 F-5-FPN. After injection of Fig. 4 Verification of myocardial infarction in rat models. a TTC staining of SD rat acute myocardial infarct models (top row), normal SD rats (center row), and control models (bottom row). b Representative decay-corrected transverse (top), coronal (center), and sagittal (bottom) small animal PET images of AMI models (left row), normal rats (center row), and control models (right row) acquired at 1 h after tail vein injection of 18 F-FDG. The white arrow with no FDG uptake shows the infarct area. However, uniform uptake throughout the heart could be seen in the normal and control group. c HE staining of infarct myocardium (left), normal myocardium (center), and infarction border area (right). Scale bar = 100 µm   6 In vivo MRI and PAI imaging. a The production of melanin resulted in a dark signal in the myocardium on a T2*-weighted MR image by causing T2* shortening. b Melanin produced intensive PAI signals in TYR-MSC rats, and no signals were seen in the MSC group and control group TYR-MSCs into the infarcted myocardium, clear signals could be seen on PET, MRI, and PAI at 28 days, suggesting the stability of melanin in viable TYR-MSCs. To the best of our knowledge, this study is the first time that a stand-alone reporter gene tyrosinase has been used in multimodality monitoring of the fate of stem cells in vivo after transplantation into infarcted myocardium. Tyrosinase, when used as a multimodality reporter gene, does not require co-administration of an enzymatic substrate, and in theory, it should produce plenty of melanin to successfully perform PET, MRI, and PAI if a minimum threshold cell number is reached. Previous studies have indicated that tyrosinase expression exhibits low-level toxicity in mammalian cells 23 , and tyrosinase elicits less of an immune response than other exogenous reporter genes.
The mechanism of tri-modality imaging of the tyrosinase reporter gene has been previously discussed 4 . Although the use of tyrosinase as a tri-or bi-modality reporter gene has been validated previously by us 3, 4, 6 and other investigators 13 , the previous approaches had some limitations, such as the inability to obtain stable transduced cell lines, lack of in vivo imaging, or not using PET. Thanks to the newly synthesized probe 18 F-5-FPN, which can bind to melanin quickly with high binding capacity and high specificity, tyrosinase gene expression can be detected in vitro and in vivo through 18 F-5-FPN by specifically combining with melanin. Based on the technology of lentivirus transfection, stable cell lines were established, and the signals may last for at least 28 days.
PAI is an emerging hybrid molecular imaging tool owing to its unique property of combining optical and acoustic imaging with higher spatial resolution 24 . Melanin has strong optical absorption over a broad spectrum 25 , which allows for good tissue penetration. Meanwhile, high resolution can be maintained in PAI because the photoacoustic wave has low scattering in tissue 26 . Recently, some studies have shown that tyrosinase can be used as a reporter gene for PAI given that tyrosinase modulates melanin synthesis 27 . Märk et al. 28 transfected rat MSCs with reporter genes co-expressing tyrosinase and a fluorescent protein (mCherry) and performed photoacoustic imaging in small animal models of tissue regeneration. However, their study lacked quantitative analysis of photoacoustic signals and continuous observation in vivo. In our study, 2 × 10 6 TYR-MSCs cells implanted into the infarcted myocardium produced a strong signal on PAI, suggesting its high sensitivity. In vitro, 2.5 × 10 3 cells (5 × 10 4 cells per mL) were sufficient for imaging. Although the intensity of the PAI signal decreased over time, the signal was still visible on d 28, which means PAI is a feasible method for long-term tracking of stem cells in vivo. However, the tissue penetration depth is limited to 5 cm, increasing the difficulty of finding a lesion in deep tissue 29 . Blood flow can also produce strong photoacoustic signals, which may distort the signals produced by TYR-MSCs. PET and MRI could be used to rectify the limitations of PAI.
Among the most important properties of melanin is its ability to strongly chelate metal ions, such as Cu 2+ , Mn 2+ , and Fe 3+ , leading to shortened T1 and T2 relaxation times in vitro or in vivo on MRI 30 . The typical contrast pattern observable in melanoma shows hyperintensity on T1WI and hypointensity on T2* weighted images. Some studies have reported that microscopic particles of iron oxide bound to MSCs can be tracked in vivo using MRI 31,32 . However, the reliability of iron particle tracking of transplanted stem cells has been challenged by several studies because iron particles may be engulfed by macrophages after stem cell death 33 . Microscopic particles of iron oxide also cannot be reproduced by transplanted MSCs, which will decrease the signal duration. Amsalem et al. 34 labeled rat MSCs with superparamagnetic iron Fig. 7 Semi-quantitative analysis of in vivo multimodality imaging. a The TYR-MSC uptake value slightly decreased with time in the PET serial images. Data are expressed as the percentage administered activity (injected dose) per gram of tissue (%ID per g). b MRI signal contrast (%) of TYR-MSCs on T2* sequences showed a tendency to increase significantly. c The intensity of the PAI signal decreased obviously with time. Data are expressed as the mean ± SD oxide nanoparticles to track MSCs in vivo using MRI. After 4-week follow-up, co-staining for iron and ED1 (resident macrophage marker) showed that the ironpositive cells were cardiac macrophages. When nanoparticles were used to track stem cells, cells have to be labeled in vitro, and the signal would be diluted if cells continue to divide, leading to decreased signal intensity per cell. A stable cell line expressing tyrosinase as a reporter gene has better biocompatibility and longer imaging time than cells using microscopic particles or nanoparticles.
Recent studies have demonstrated the feasibility of PET for analyzing the fate of stem cells transplanted into the myocardium in vivo. Kang et al. 35 labeled stem cells with 18 F-FDG and injected them into patients with myocardial infarction via an intracoronary catheter after stenting of infarct-related arteries. PET/computed tomography was performed to trace the injected stem cells. However, the process of labeling was completed in vitro, and the longest tracking time lasted only 20 h after transplantation. In our previous study, 18 F-5-FPN, a benzamide analog specifically targeting melanin in vitro and in vivo with high affinity and retention, was developed. In this study, 18 F-5-FPN showed an excellent ability to track TYR-MSCs in vivo. As normal myocardial cells did not take up 18 F-5-FPN, myocardial background radiation was very low, which led to the benefit of excellent PET imaging quality. The intensity of PET signals did not show an obvious decrease with time.
Comparing the three imaging modalities, PET imaging showed clear and obvious signals until day 28, which illustrates that the stem cells were alive and produced melanin constantly. MRI signals could also be seen, but they were not as clear as PET signals, and they decreased significantly with time. PAI combines strong optical contrast and high ultrasonic resolution in a single modality, but it provides less anatomical information than MRI. Any single imaging method has some limitations. New multimodality imaging techniques have achieved great progress in the field of diagnostic imaging.
MSCs stably transduced with the tyrosinase gene produce melanin, which is the basis for multimodality imaging with PET, MRI, and PAI for assessing the viability, location, and dwell time of transplanted stem cells. Functional imaging using these cells is a future goal. Thus far, we have performed only transplanted stem cell tracking in the infarcted myocardium. Future work may incorporate quantitative analysis of tyrosinase expression in vivo and the recovery of cardiac function after stem cell transplantation. Most studies on stem cell tracking have focused on the location and duration of stem cell viability. Truly functional multimodality molecular imaging based on reporter gene technology has a promising future.

Limitations
Some limitations should be mentioned. First, although an AMI model with homogeneous infarct area was established, the results might not be representative of human MI. The location, area, and complications of human MI are highly variable. Multimodality imaging using a tyrosinase reporter gene for assessing transplanted stem cells in human MI may still have a long way to go. Second, permanent coronary artery ligation was performed for the MI model in this study, whereas ischemia/ reperfusion models are more representative and more closely resemble the physiology of MI in human than permanent ligation. Last but not the least, it will be much better to have a known marker (e.g., green fluorescent protein) to validate the viability of tyrosinase, which will also be helpful to verify the specific uptake of 18 F-5-FPN in vitro and in vivo. Despite these limitations, we confirmed that MSCs stably transduced with the tyrosinase gene produce melanin, which is the basis for multimodality imaging with PET, MRI, and PAI for assessing the viability, location, and dwell time of transplanted stem cells.