Targeted Co-delivery of the Iron Chelator Deferoxamine and a HIF1Î±

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Targeted Co-Delivery of the Iron Chelator Deferoxamine and a HIF1# Inhibitor Impairs Pancreatic Tumor Growth Jiayan Lang, Xiao Zhao, Xiuchao Wang, Ying Zhao, Yiye Li, Ruifang Zhao, Keman Cheng, Yao Li, Xuexiang Han, Xiaowei Zheng, Hao Qin, Marzieh Geranpayehvaghei, Jian Shi, Gregory J. Anderson, Jihui Hao, He Ren, and Guangjun Nie ACS Nano, Just Accepted Manuscript • Publication Date (Web): 24 Jan 2019 Downloaded from http://pubs.acs.org on January 24, 2019

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Targeted Co-Delivery of the Iron Chelator Deferoxamine and a HIF1α Inhibitor Impairs Pancreatic Tumor Growth Jiayan Lang1,5,6#, Xiao Zhao1,2,3#*, Xiuchao Wang3#, Ying Zhao1, Yiye Li1, Ruifang Zhao1, Keman Cheng1, Yao Li1, Xuexiang Han1,6, Xiaowei Zheng3, Hao Qin1,6, Marzieh Geranpayehvaghei1,7, Jian Shi1, Gregory J. Anderson4, Jihui Hao3, He Ren3,8*, Guangjun Nie1,2,6*

1CAS

Key Laboratory for Biomedical Effects of Nanomaterials and Nanosafety, & CAS Center for

Excellence in Nanoscience, National Center for Nanoscience and Technology of China, 11 Beiyitiao, Zhongguancun, Beijing, 100190, China 2Center

of Materials Science and Optoelectronics Engineering, University of Chinese Academy of

Sciences, Beijing 100049, China 3Department

of Pancreatic Carcinoma, Tianjin Medical University Cancer Institute and Hospital,

National Clinical Research Center of Cancer, Key Laboratory of Cancer Prevention and Therapy, Tianjin , 300060, China 4Iron

Metabolism Laboratory, QIMR Berghofer Medical Research Institute, Brisbane, Queensland

4006, Australia 5Sino-Danish

Center for Education and Research/ Sino-Danish College of UCAS, Beijing, 100190,

China 6University

of Chinese Academy of Sciences, Beijing 100049, China

7Department

of Nanobiotechnology, Faculty of Biological Sciences, Tarbiat Modares University,

Tehran, Iran 8Affiliated

Hospital of Qingdao University, Qingdao, China

*Corresponding authors Xiao Zhao: [email protected], Tel: +86-10-82545529, Fax:+86-10-62656765 He Ren: [email protected], Tel: +86-022-23340123, Fax:+86-022-23340123 Guangjun Nie: [email protected], Tel: +86-10-82545529, Fax:+86-10-62656765 #The

authors contributed equally to this paper.

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Abstract Rapidly growing cancer cells exhibit a strong dependence on iron for their survival. Thus, iron-removing drugs, iron chelators, have potential applications in cancer treatment. Deferoxamine (DFO) is an efficient iron chelator, but its short circulation half-life and ability to induce hypoxia-inducible factor 1α (HIF1α) overexpression restricts its use as an antitumor agent. In the present study, we first found that a pattern of iron-related protein expression favoring higher intracellular iron closely correlates with shorter overall and relapse-free survival in pancreatic cancer patients. We subsequently found that a combination of DFO and the HIF1α inhibitor, Lificiguat (also named YC1), significantly enhanced the antitumor efficacy of DFO in vitro. We then employed transferrin receptor 1 (TFR1)-targeting liposomes to co-deliver DFO and YC1 to pancreatic tumors in a mouse model. The encapsulation of DFO prolonged its circulation time, improved its accumulation in tumor tissues via the enhanced permeability and retention (EPR) effect and facilitated efficient uptake by cancer cells, which express high level of TFR1. After entering the tumor cells, the encapsulated DFO and YC1 were released to elicit a synergistic antitumor effect in subcutaneous and orthotopic pancreatic cancer xenografts. In summary, our work overcame two major obstacles in DFO-based cancer treatment through a simple liposome-based drug delivery system. This nanoencapsulation and targeting paradigm lays the foundation for future application of iron chelation in cancer therapy.

Keywords: targeted delivery, deferoxamine, YC1, hypoxia-inducible factor 1, pancreatic cancer, iron metabolism

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Iron is essential for cell proliferation and cancer cells have particularly high iron requirements to support their rapid growth. Iron overload has been reported to induce carcinogenesis and to accelerate tumor growth1, 2, while iron depletion using iron chelators will suppress tumor growth3-6. However, the anti-tumor efficacy of iron chelators is not satisfactory, so this strategy has not been established as cancer therapy in the clinic. A possible reason for this is that iron chelation results in stabilization and overexpression of hypoxia inducible factor 1α (HIF1α) through the inhibition of prolyl hydroxylases, iron-dependent enzymes responsible for HIF1α degradation7-9. As an important transcriptional factor in tumor development, HIF1 (composed of HIF1α and a constitutively expressed β-subunit HIF1β) promotes cancer cell proliferation, cancer stem cell (CSC) activity and angiogenesis via the HIF1-vascular endothelial growth factor (VEGF) signaling pathway10-12. We therefore hypothesized that a HIF1 inhibitor may enhance the anti-tumor effect of iron chelation. Thus, in the present study, we have explored the potential synergistic antitumor effects of the iron chelator Deferoxamine (DFO) and Lificiguat (YC1), a HIF1α inhibitor13, 14. We chose this inhibitor for two reasons: first, there is rigorous data around the use of YC1 as a HIF1 inhibitor; second, YC1 possesses high hydrophobicity and is suitable to be encapsulated into the lipid bilayer of nanoparticles.

Of the iron chelators used clinically, DFO has seen the longest use, but the drug’s extremely short circulation half-life of approximately 20 min in humans, and 5 min in mice, limits its use in tumor therapy15, 16. When DFO is used in the treatment of iron overload, the drug must be administered by continuous subcutaneous infusion for up to 12 hours per day for 5-7 days per week, an onerous regimen which leads to suboptimal adherence to therapy by patients17. Two next generation orally administered iron chelators are also now in routine clinical use, but these agents have not been used as extensively as DFO18, 19. Another limitation of iron chelator use for tumor therapy is that systemic administration will not specifically target tumor cells. Nonetheless, DFO has the potential to become an effective cancer treatment option if its circulation time can be improved and it can be targeted to tumor site.

Using nanoparticles to load and deliver drugs has proven an effective strategy to prolong circulation time20, 21, as encapsulation can protect drugs against enzymatic hydrolysis and renal clearance22-24. 3


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Herein, we report a liposome-based drug delivery system that transports DFO and YC1 simultaneously (Figure 1A). DFO and YC1 became encapsulated into the hydrophilic and hydrophobic

layers

of

the

liposome,

respectively.

When

injected

intravenously,

the

liposome-encapsulated DFO exhibited significantly prolonged circulation half-time compared to free DFO (Figure 1A). In addition, we decorated the surface of the liposomes with the plasma glycoprotein transferrin, the major ligand for the transferrin receptor 1 (TFR1), via chemical crosslinking. After passive accumulation into tumor tissue through the enhanced permeability and retention (EPR) effect25,

26,

the nanoparticles were preferentially taken up by tumor cells which

express high concentrations of TFR1 on their surface27-29 (Figure 1A). Once inside the tumor cells, the encapsulated DFO and YC1 were released to exert a combined antitumor effect through the blockage of cancer cell proliferation, CSC activity and angiogenesis (Figure 1A).

We assessed the antitumor effects of our nanoformulation in pancreatic cancer, which is one of the most lethal malignant diseases in humans30. The therapeutic efficacy of the current first-line chemotherapeutic drug, gemcitabine, is not satisfactory, so there is an urgent need for a new strategy to improve drug performance in the treatment of pancreatic cancer31, 32. Interestingly, the abnormal iron metabolism of pancreatic cancer has received considerable attention as a therapeutic target; there have been some seminal works on the application of iron chelators in the treatment of pancreatic cancer3, 5. HIF1 also plays an important role in the pathogenesis of pancreatic cancer33. Thus, we expect our combination of a HIF1α inhibitor to block the HIF1α overexpression that accompanies iron chelation to have a potent tumor inhibitory effect.

Results and Discussion Expression of iron metabolism-related proteins and clinical outcome in pancreatic cancer To understand the role of iron in pancreatic cancer, we first determined the iron content in 18 paired clinical pancreatic cancer tissues and adjacent tissues and found that the iron content in pancreatic cancer tissues is significantly higher than that in adjacent tissues (Figure 1B). Next, we examined the expression of iron metabolism-related proteins (TFR1, ferritin heavy chain [FTH; ubiquitous iron storage protein], ferritin light chain [FTL; ubiquitous iron storage protein] and ferroportin 1 [FPN1; only known cellular iron export protein]) by immunohistochemistry (IHC) in a tissue microarray 4


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consisting of 96 human pancreatic cancer specimens (Figure S1). None of the patients from which this array was constructed received any prior anti-tumor treatment before the resection. Kaplan-Meier analysis indicates that the pancreatic cancer patients with high (+++) TFR1, FTL or FTH protein expression had significantly worse overall survival (OS) and relapse free survival (RFS) than those with negative, low or medium (-, + or ++) TFR1, FTL or FTH expression (Figure 1C and D). In contrast, the patients with high (+++) FPN1 protein expression exhibited longer OS and RFS than those with negative, low or medium (-, + or ++) FPN1 expression (Figure 1C and D). Congruent with these findings, the FTH or FTL expression positively correlates with histological grade and tumor size, and the FPN1 expression negatively correlates with histological grade and tumor size in the PDAC specimens (Table S1-S4). In addition, we found a positive relationship between the expression of the proliferation marker Ki67 and TFR1 or FTH expression, and a negative correlation between Ki67 and FPN1 expression (Table 1). These clinical data suggest that the pancreatic cancer cells possess an abnormally high level of intracellular iron which performs an important role in proliferation, and that iron chelation may be an effective therapeutic strategy to treat these tumors.

The effects of combined treatment with DFO and YC1 on pancreatic cancer cell lines Next, we evaluated the antitumor effects of the combination of DFO and YC1 on three pancreatic cancer cell lines. As expected, DFO treatment alone showed significant cytotoxicity, while co-treatment with DFO and YC1 showed a substantially greater toxicity, leading to a 2-3 fold reduction in the half maximal inhibitory concentration (IC50) of the chelator (Figure 2A). YC1 treatment also enhanced the inhibitory activity of DFO on the clonogenicity of PANC1 cells (Figure S2). We next examined the effect of DFO and/or YC1 treatments on cancer cell proliferation, using EdU to label proliferating cells. DFO treatment exhibited dose-dependent inhibitory activity on the proliferation of cancer cells, while proliferation under combined DFO and YC1 treatment was considerably lower than that with DFO alone (Figures 2B and S3). YC1 alone led to a small decrease in proliferation, but the effect was not significant. Western blot analysis revealed that DFO treatment induces HIF1α and its downstream target gene VEGF, but this effect was blocked when YC1 was present (Figure 2C). We examined the iron content in the cellular enzyme responsible for HIF1α degradation, prolyl hydroxylase 1, and found a significant decrease in the DFO treatment 5


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group, which may indicate that DFO treatment stimulates the HIF1α expression at the post-translational level in pancreatic cancer cells (Figure S4). In addition, the expression of both SOX2 and OCT4 (two pancreatic CSC-associated transcription factors) was lower following combined treatment compared to treatment with either agent alone (Figure 2C)34,

35.

We also

confirmed that YC1 significantly enhances the inhibitory effect of DFO on the stem cell-like properties of pancreatic cancer cells with the observation that the capacity to form tumor spheroids was significantly blocked by the combined treatment (Figure 2D, S5)36, 37.

Preparation and characterization of nanoparticles and TFR1-dependent cellular uptake in vitro To obtain the optimal ratio of DFO to YC1, we measured the cytotoxicity of drug combinations at different molar ratios (Figure 3A and Table 2) and calculated the combination index (CI). The cytotoxicity in pancreatic cancer cells was the strongest when the molar ratio of DFO:YC1 was 2:1 (the CI in AsPC1, PANC1 and T3M4 cells were 0.269, 0.337 and 0.199, respectively). We next assembled liposomal nanoparticles to encapsulate DFO and YC1 using a film-ultrasonic dispersion method, followed by surface decoration with the transferrin protein. We adjusted the drug dosages according to the encapsulation efficiencies of DFO and YC1, as determined by HPLC analysis. When we used 100 mg DFO and 5 mg YC1 in drug encapsulation, the encapsulation rate of DFO and YC1 was 21.3% and 92.8%, respectively, and a drug combination at a molar ratio of 2.13: 1 was encapsulated into the nanoparticles. Based on DLS measurements, the hydrodynamic diameters of NP, NP-DFO-YC1 and TNP-DFO-YC1 were 96.4 ± 8.1, 102.2 ± 12 and 97.7 ± 9.9 nm, respectively (Figure 3B). The zeta potential of the base NP was -22.2 ± 1.6 mV, which was slightly more negative for NP-DFO-YC1 (-23.6 ± 2.1 mV) and TNP-DFO-YC1 (-24.3 ± 1.3 mV) due to the negative charges of the drugs and transferrin (Figure 3C). The polydispersity indices (PDI) of the three nanoparticle types were all lower than 0.2 (Figure 3D). We examined the morphology of the nanoparticles by TEM to find that NP, NP-DFO-YC1 and TNP-DFO-YC1 all disperse as individual particles, with a well-deﬁned spherical structure and average diameters ranging from about 60 - 90 nm under the dehydrating conditions used for microscopy (Figure 3E). At physiological pH (7.4), the hydrophobic YC1 was released from TNP-DFO-YC1 at a lower rate compared with the hydrophilic DFO. However, at lysosomal pH (5.4), both drugs were released rapidly at a similar rate 6


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(Figure S6).

To test the targeting ability of the nanoparticles, we examined the ability of transferrin-decorated NPs to bind to TFR1 on the plasma membrane of cells in vitro. Normal human pancreatic ductal epithelial cells (HPNE) exhibit low TFR1 expression and were used as controls (Figure S7). The HPNE and cancer cells were incubated with NP or TNP loaded with the hydrophobic dye Cy5.5 (NP-Cy5.5 or TNP-Cy5.5, respectively) at 4°C. At this temperature, the nanoparticles bind to the cell membrane but will not be internalized. As shown in Figure 3F, TNP-Cy5.5 adhered to the cell membrane, but NP-Cy5.5 did not. More importantly, the amount of TNP-Cy5.5 bound to the cancer cell surface was significantly greater than that in HPNE cells (Figures 3F and S8). In addition, when the cells were pre-treated with an excess of transferrin to competitively block the TFR1 on the cell membrane, TNP-Cy5.5 was no longer able to bind to the cell membrane (Figures 3F, 3G and S8), verifying that TNP-Cy5.5 binding to the cell membrane is mediated by the interaction between transferrin and TFR1. The overlap of the Cy5.5 signal with that of FITC in cells treated with a FITC-labeled antibody against TFR1 further validated that TNP binds to the transferrin receptor (Figure S9). Finally, when PANC1 cells were incubated with TNP-Cy5.5 at 37°C and followed over time by confocal microscopy, TNP-Cy5.5 was endocytosed by the cells (Figure S10). Together, the above data demonstrate that the targeting protein, transferrin, enabled TNP to be efficiently taken up by cells that express TFR1.

Antitumor effects of different drug formulations in vitro We treated pancreatic cancer cells with different drug formulations and quantified cell viability using the CCK-8 assay (Figure 4A), clonogenicity using a clone formation assay (Figure S11) and cell proliferation using the EdU staining assay (Figures 4B and S12). The effects of TNP-DFO-YC1 were similar to those of free DFO+YC1. TNP-DFO-YC1 exhibited significantly higher inhibitory activity on cancer cell viability, clonogenicity and proliferation than TNP-DFO. Western blot analysis showed that the expression of HIF1α and VEGF increased after TNP-DFO treatment, but this was prevented by co-delivery of YC1 (Figure 4C). In addition, we observed decreased expression of SOX2 and OCT4 (CSC-related proteins) in the TNP-DFO-YC1 group, as well as a reduced ability to form tumor spheroids, compared with the TNP-DFO or TNP-YC1 groups (Figures 7


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4C, 4D and S13). Taken together, combined delivery with YC1 enhanced the antitumor effect of DFO of our nanoformulation in vitro.

Characterization of circulation half-life and tumor targeting in vivo To evaluate the circulation half-lives of free and NP-encapsulated DFO, we injected TNP-DFO-YC1 into mice via the tail vein and measured the plasma DFO levels at different time intervals using HPLC. Compared to the reported circulation half-life of free DFO13-15, the circulating half-life of DFO in TNP-DFO-YC1 group was over 39 times longer (5 vs. 197 min; Figure 5A).

To assess the distribution of DFO to organs and tumors after delivery by nanoparticles, free DFO, NP-DFO-YC1 or TNP-DFO-YC1 were injected by tail vein into mice bearing subcutaneous human pancreatic tumors (T3M4 xenografts), followed by a quantitative evaluation of DFO distribution in the organs at different time-points using HPLC analysis. As shown in Figure 5B, DFO in most organs and tumor tissue in the free DFO group was below the lowest detectable threshold 1 h after injection, while DFO accumulation in all the organs and tumor tissue in the NP-DFO-YC1 and TNP-DFO-YC1

groups

was

observed

for

at

least

8

h.

Most

NP-DFO-YC1-

and

TNP-DFO-YC1-derived DFO accumulated in the liver and spleen, likely due to uptake by the reticuloendothelial system. Most importantly, significantly more DFO accumulated in the tumor tissue in the TNP-DFO-YC1 group than in the NP-DFO-YC1 group (7.6% vs. 3.9% injected dose/gram tissue 2 h after injection) (Figure 5B). To directly observe the targeting effect, we once again used NP-Cy5.5 and TNP-Cy5.5, which we injected (i.v.) into mice bearing subcutaneous T3M4 tumors. As shown in Figure 5C, there was obvious Cy5.5 accumulation in the NP-Cy5.5 and TNP-Cy5.5 groups at 4 h after tail vein injection. As expected, the signal in the TNP-Cy5.5 was much stronger than that in the NP-Cy5.5 group, indicating that the NP surface modification with transferrin endowed TNP improved tumor targeting of the nanoparticle. Importantly, we found a similar ratio of DFO:YC1 in tumor tissue at different times after injection with TNP-DFO-YC1 as that in the nanoparticles (2.13:1), which is congruent with a simultaneous delivery of the two drugs into the tumors by the nanoparticles (Figure S14). In addition, we encapsulated two fluorescent dyes, rhodamine B (RhoB) and Cy5.5 into TNP (TNP-RhoB-Cy5.5) and injected via tail vein into the mice bearing subcutaneous human pancreatic tumors (T3M4 xenografts). After 4 h, the tumor tissues were 8


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collected and sliced, and obvious co-location of the fluorescent signals of RhoB and Cy5.5 closed to the cell nucleus were observed, which indicate that most TNPs were taken up by the cancer cells (Figure S15).

Next, we examined TFR1 expression in the liver and tumor tissues after NP-DFO-YC1 or TNP-DFO-YC1 treatment. TFR1 expression levels in both the liver and tumor tissues increased at 12 h after DFO treatment, however TFR1 expression in the tumor tissue was still at a high level after 48 h (Figure 5D). In liver tissue, the TFR1 expression levels returned to the basal level seen prior to the treatment after 48 h. We administered a second injection of NP-DFO-YC1 or TNP-DFO-YC1 to the mice and analyzed the DFO accumulation in the liver and tumor tissues. Compared with the DFO distribution data after the first injection (Figure 5B), the amount of DFO in the liver shifted in both groups: In the NP-DFO-YC1 group, the amount of DFO that accumulated in the tumor tissue after the second injection was similar to that following the first injection (3.6% vs. 3.9% injected dose/gram tissue 2 h after injection; Figure 5B and 5E), while, in the TNP-DFO-YC1 group, significantly more DFO accumulated in the tumor tissue after the second injection than after the first injection (10.3% vs. 7.6% injected dose/gram tissue 2 h after injection; Figure 5B and 5E). In summary, the persistent TFR1 overexpression in cancer cells after iron chelation with DFO significantly enhanced the tumor targeting effect of TNP-DFO-YC1.

Inhibition of tumor growth in vivo Mice (nu/nu) bearing T3M4 subcutaneous xenografts were randomly divided into seven groups: control (saline), TNP, free DFO+YC1, TNP-YC1, TNP-DFO, NP-DFO-YC1 and TNP-DFO-YC1. Empty TNP exhibited no effect on tumor growth (Figure 6A). Compared to the tumors in the free DFO+YC1 group, the tumors in the NP-DFO-YC1 group grew more slowly, and growth of those in the TNP-DFO-YC1 group was reduced even further. These findings demonstrate the value of incorporating DFO and YC1 into liposomes and the advantages of decorating the liposomes with transferrin for enhancing the antitumor effects of DFO and YC1 (Figure 6A). In addition, the growth of tumors in the TNP-DFO-YC1 group was significantly slower than those in the TNP-YC1 and TNP-DFO groups, demonstrating a synergistic antitumor effect in the combination of DFO and YC1 (Figure 6A). After sacrificing the mice, the tumors were collected and weighed (Figure 6B). 9


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Consistent with the growth curve, the mean weight of the tumors in the TNP-DFO-YC1 group was the lowest among all groups (Figure 6C).

We assessed the iron levels in the tumor tissue after the different treatments. DFO chelation significantly decreased the iron levels in the tumor tissue in the free DFO+YC1, TNP-DFO, NP-DFO-YC1 and TNP-DFO-YC1 groups. Since TNP more specifically accumulates in tumors, its effect was the strongest in the TNP-DFO and TNP-DFO-YC1 groups (Figure 6D). To demonstrate the ability of HIF1 inhibition to reduce tumor growth, we sectioned tumor xenografts and examined HIF1 expression using IHC. As shown in Figure 6E, following TNP-DFO treatment, HIF1 expression was higher than that in the TNP group. Consistent with this finding, the expression of VEGF (a downstream HIF1 target gene) and CD31 (a marker of vascular endothelial cells) were also increased after TNP-DFO treatment. However, combined delivery with YC1 overcame the effect DFO on HIF1 levels, leading to a decreased expression of HIF1, VEGF and CD31 in the tumor tissue (TNP-DFO-YC1 group; Figure 6E). In addition, Ki67, SOX2 and OCT4 expression in the TNP-DFO-YC1 group was the lowest among all groups (Figure 6E). These results show that the co-delivery of YC1 can efficiently diminish HIF1 gene expression in vivo, which in turn enhances the antitumor effects of DFO by blocking tumor cell proliferation, stem cell behavior and angiogenesis.

To further explore the antitumor effects in vivo, we established orthotopic pancreatic cancer models by injecting luciferase expressing PANC1 cells into the pancreatic tail of nu/nu mice. Firstly, we repeated the tumor targeting experiment in this model, and found that TNP-DFO group also exhibited a higher rate of accumulation in the tumor tissues compared with the NP-DFO group (Figure 7A). Then, at 14 days after injection of tumor cells, we initiated treatment with different drug formulations (including saline for control, Free DFO+YC1, TNP-DFO, TNP-YC1 and TNP-DFO-YC1) and continued the treatment for 21 days. Bioluminescent imaging of the mice was performed every 5 days during the treatment (Figure 7B). As shown in Figure 7C and 7D, TNP-DFO-YC1 exhibited significantly stronger inhibitory efficiency against tumor growth than Free DFO+YC1, TNP-YC1 or TNP-DFO. Although YC1 treatment did not affect the PANC1 cell proliferation in vitro (Figure 2B), TNP-YC1 can inhibit the growth of PANC1 tumor in vivo (Figure 10


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7C and 7D). They underlying mechanism may be that cancer cell proliferation is more dependent on HIF1 level in the hypoxic microenvironment in vivo compared to that in vitro38-40. At day 36, tumor tissues were collected and weighed (Figure 7E and 7F). The mean tumor mass of TNP-DFO-YC1 group was the lowest among all groups. The iron levels in the tumor tissues after different treatments were also analyzed. The lowest iron levels in the tumor tissue were in TNP-DFO-YC1 group (Figure 7G). In addition, as shown in Figure 7H, the protein expression levels were evaluated by IHC staining. Similar to subcutaneous tumor models, CD31, HIF1α and VEGF expression levels increased after TNP-DFO treatment and this up-regulation was blocked in the TNP-DFO-YC1 group. Furthermore, the relative lower expression of Ki67, SOX2 and OCT4 in the TNP-DFO-YC1 group, compared to that in the free DFO+YC1, TNP-DFO or TNP-YC1 groups, indicated the importance of the nanoparticle delivery and combination of DFO and YC1, respectively.

Toxicity evaluation in vivo Given the important role of iron in the cell metabolism and proliferation, we examined the safety of TNP-DFO-YC1 in vivo. After administration of TNP-DFO-YC1 via tail vein injection once every 2 days for 28 days, we did not observe any deleterious effects on the body weight of BALB/c mice (Figure S16A). In addition, there was no evidence of functional toxicity of the heart, liver, kidney or bone marrow (Figure S16B and S16C). No obvious damage was observed in H&E staining of the major organs in the TNP-DFO-YC1 group (Figure S16D). However, as described previously, the cancer cells need more iron for their rapid growth than the normal cells. The transient iron chelation may only affect the proliferation and function of tumor, other than the other organs. Collectively, these results are indicative that our nanoformulation at this dose is tolerable and safe in vivo.

In a permanent state of rapid proliferation, cancer cells exhibit an enhanced dependence on iron relative to most normal cells, a phenomenon referred to as iron addiction41. In the present study, we first used IHC to analyze the expression of four proteins with important roles in iron metabolism (TFR1, FTH, FTL and FPN1) in pancreatic cancers. In most cells, TFR1 is the gateway for iron uptake. The transmembrane receptor is expressed at particularly high levels in tumor cells42, 43. The iron storage protein ferritin is a heteropolymer of 24 subunits of two types (FTH and FTL) and is responsible for storing excess iron, thereby preventing it from catalyzing reactions leading to the 11


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production of reactive oxygen species. No clear pattern of expression of ferritin in tumor cells has been described. In some cases, its expression is decreased, which might be expected in a cell with high iron requirements, but in most studies, it has been found to be increased and may protect cancer cells, including pancreatic cancer cells, against the action of ROS44, 45. Various oncogenes have also been shown to exert effects on ferritin expression, and different cell types within the same tumor may even regulate their ferritin levels differently, adding confusion to the question of ferritin’s role in cancer46,

47.

FPN1 is the only known iron export protein in mammalian cells. Its expression has

consistently been found to be reduced in tumors48, 49. Indeed, multiple studies have shown diminished expression of FPN1 and overexpression of TFR1, FTH and FTL in many types of tumor relative to their normal counterparts1, 2. Our IHC data confirms the correlation of this pattern of low FPN1 and high expression of TFR1, FTH and FTL with poorer prognosis in pancreatic cancer patients. We also found a positive relationship between Ki67 expression and TFR1 or FTH expression, and a negative relationship between Ki67 and FPN1 expression. These data indicate that pancreatic cells have a high intracellular iron requirement to support their rapid proliferation and suggest that iron chelation treatment has therapeutic potential in this type of tumor. Our in vitro and in vivo data indicate that DFO is indeed able to reduce tumor proliferation and growth, especially when incorporated into a targeted nanoparticle formulation.

In addition to decreasing cell proliferation, DFO inhibited the expression of SOX2 and OCT4, CSC markers, in pancreatic cancer cells and reduced the ability of the cells to form CSC-liked spheroids. It was reported recently that CSC in glioblastoma express higher levels of TFR1 and ferritin compared to non-CSC, and that small hairpin RNA targeting of FTL and FTH significantly reduced CSC activities, including the ability of form spheroids in vitro and tumorigenesis in vivo50, suggesting an essential role for iron storage in CSC. Our data indicate that chelation of iron is a viable strategy towards a CSC-targeted therapy and could provide an adjunct to the current clinical treatment of pancreatic cancer. At present, the main treatment against pancreatic cancer is gemcitabine-based chemotherapy, which is generally believed to be ineffective against CSC51, 52.

Although many studies have described antitumor effects of iron chelators, these agents have not found clinical application in cancer treatment. In the case of DFO, an important reason for its 12


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ineffectiveness in cancer therapy is its extremely short circulation half-life15, 16, meaning that little chelator would reach the site of the tumor. Two strategies have been attempted to solve this problem. In the first, DFO has been injected directly into tumor tissue, for example via interventional arterial infusion in advanced hepatocellular carcinoma4. However, the results were not satisfactory, and only 2 in 10 patients had any response. The other approach has been to use a nanoparticle-based system to deliver DFO. Imran ul-haq et al. conjugated DFO to a hyperbranched polyglycerol (HPG)-based nanopolymer scaffold. This HPG-DFO formulation exhibited a highly prolonged half-life (2663 min) over free DFO (5 min)53. Their preparation survived considerably longer than our nanoparticle-encapsulated DFO (TNP-DFO-YC1; 197 min) in the present study. However, the HPG-DFO formulation was not tested against tumors. Furthermore, prolonging the plasma half-life of DFO per se may not be sufficient to make it an effective anti-cancer agent. Any DFO released from the nanoparticles before reaching a tumor site would still be rapidly excreted from the body. An important feature in an effective therapeutic is the ability to deliver DFO to the tumor site before it is released from the carrier. In our studies, we were able to achieve this by encapsulating DFO into a targeted liposomal nanoparticle. Liposomes are an FDA approved nanocarrier to deliver drugs, are simple to prepare, and are suitable for delivering various types of therapeutic agents. By providing DFO and YC1 to the assembly process, we successfully packaged the two drugs into liposomes. The size of the resulting NP-DFO-YC1 was about 100 nm, which can passively target tumor tissue via EPR effect25,

26.

In addition, we decorated the nanoparticles with transferrin, which enabled

TNP-DFO-YC1 to actively target tumor cells as they express high levels of TFR1 on their surface27-29. This targeting ligand TFR1 is not a random choice. After iron chelation by DFO, the TFR1 expression was stimulated through the iron regulatory protein (IRP) and iron regulatory element (IRE)54. The more need of iron, the more up-regulation of TFR1 in cancer cells may occur. This feedback regulation can further enhance the targeting efficacy of TNP in the following treatment.

HIF1 is a key factor in the survival of cancer cells in the hypoxic tumor microenvironment. This transcription factor stimulates VEGF-mediated angiogenesis and other processes55. HIF is composed of the stable HIF1 subunit and the oxygen-sensitive HIF1α subunit. HIF1α is degraded via an iron-dependent process, so iron chelation stabilizes this protein. This is clearly undesirable if DFO is 13


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to be used as an effective anti-tumor agent. To overcome this limitation, we co-delivered DFO with the HIF1α inhibitor YC1, which interferes with a broad range of HIF1-stimulated activities13, 14. We found that this DFO/YC1 combination not only blocked DFO-induced HIF1-VEGF mediated angiogenesis, but also enhanced the inhibitory effects of DFO on CSC activity and cancer cell proliferation.

Conclusions In summary, we first confirmed the potential role of iron as a therapeutic target in pancreatic cancer by analyzing the relationship between indicators of intracellular iron levels and patient outcome. Next, we employed a simple, targeted liposomal drug delivery system to co-deliver DFO and YC1. This strategy overcame two disadvantages of DFO in cancer treatment. First, the direct delivery of DFO to the tumor using transferrin-decorated nanoparticles enabled DFO to preferentially accumulate in tumor tissue as these cells express high levels of TFR1. Second, the co-delivery of DFO and YC1 enhanced the antitumor effects of DFO by inhibiting cancer cell proliferation, CSC behavior and angiogenesis. Our work lays the foundation for the further development of iron chelator-based cancer treatment.

Methods/Experimental Preparation and characterization of nanoparticles Liposomal nanoparticles (NP) were prepared using a film-ultrasonic dispersion method. Briefly, a mixture of lipids (15.6 μmol in total) consisting of lecithin (36486, Alfa Aesar, USA), cholesterol (121530,

JK Chemical,

[methoxy(polyethylene

China),

1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-

glycol)-2000]

(DSPE-mPEG,

LP-R4-039)

1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[maleimide(polyethylene

and

glycol)-2000]

(DSPE-PEG-MAL, PG2-DSML-2k, Ruixi Biological Technology Co., China) at a molar ratio of 100:50:5:1 was dissolved in 10 mL dichloromethane, and dried into a thin film at the bottom of a flask under reduced pressure using a vacuum rotary evaporator, followed by hydration with 10 mL double distilled water to form multilamellar vesicles (MLVs). The resulting MLVs were then extruded using a LipoFast mini extruder (Avestin, Canada) through a polycarbonate membrane of 0.2 μm with 5 cycles to form large unilamellar vesicles (LUVs). 14


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To encapsulate DFO (D9533, Sigma, USA) and YC1 (S7958, Selleck, USA) into NP (NP-DFO-YC1), 100 mg DFO and 5 mg YC1 were added to the hydration solution and dichloromethane, respectively. The resulting LUVs were centrifuged through a 30 kD ultrafiltration device (Millipore, USA) to remove un-encapsulated drugs. The NP-DFO-YC1 was then resuspended in 10 mL double distilled water. After demulsification with 0.5% Triton-X100, high performance liquid chromatography (HPLC) analysis was used to evaluate the encapsulation efficiencies of DFO and YC1, which were 21.3% and 92.8%, respectively. There were 1071 μg lipids, 2130 μg DFO and 464 μg YC1 in 1 mL of the final NP-DFO-YC1 preparation.

To prepare transferrin-modified NP (TNP) loaded with DFO and YC1 (TNP-DFO-YC1), 140 nmol transferrin was first thiolated using Traut’s reagent (26101, Thermo Scientific, USA, 10-fold molar excess) in phosphate buffered saline (PBS; pH 7.4) for 1 h at room temperature. A PD-10 desalting column (GE Healthcare, USA) was used to remove any unreacted Traut’s reagent. The thiolated transferrin (0.15 mM, 2 mL) and Tris(2-carboxyethyl) phosphine hydrochloride (TCEP for the reduction of sulfoxides, 312334, JK Chemical, China, 100 mM, 200 μL) were then added to 10 mL NP-DFO-YC1 for 2 h at room temperature under a N2 atmosphere with gentle stirring to generate TNP-DFO-YC1. After centrifugal ultrafiltration through a 100 kD ultrafiltration device (Millipore, USA), analysis of the protein content using the BCA assay was used to show that the transferrin coupling efficiency was 54% and there was 76 nmol transferrin in 10 mL of the final TNP-DFO-YC1 preparation. Similarly, IgG was conjugated to the surface of NP-DFO-YC1 to act as a non-specific control.

For morphology, size distribution and zeta potential measurements, transmission electron microscopy (TEM) and dynamic light scattering (DLS) analysis were performed as previously described20, 21.

Cell culture The human pancreatic cancer cell lines AsPC1, PANC1 and T3M4, as well as the normal human pancreatic ductal epithelial cell line HPNE, were cultured in Dulbecco’s modified Eagle’s medium 15


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(DMEM, 319-005-CL, WISENT, Canada), supplemented with 10% fetal bovine serum (FBS, 085-150, WISENT, Canada), at 37°C in a humidified atmosphere containing 95% air and 5% CO2. The HPNE cell line was cultured with 10 ng/ml human recombinant epidermal growth factor (EGF, AF-100-15, Peprotech, USA). All cell lines were authenticated in August 2016 through the short tandem repeat analysis method and tested negative for mycoplasma contamination.

Cell viability analysis using CCK-8 assay Cell lines were seeded into 96-well plates at a density of 2,000 cells per well. Twelve hours after seeding, the cells were treated with various drug formulations in medium containing 1% FBS for 72 h. The proportion of viable cells was evaluated using the CCK-8 assay according to the manufacturer’s instructions (CK04, Dojindo, Japan). The IC50 was calculated using SPSS software, with the formula CI = IC50a’ / IC50a + IC50b’ / IC50b; where IC50a and IC50b are the IC50 of drug a and drug b as a single drug, and IC50a’ and IC50b’ are the IC50 of drug a and drug b as a combination treatment.

Cell proliferation analysis using 5-ethynyl-2′-deoxyuridine (EdU) Tumor cells were seeded into 12-well plates at a density of 200,000 cells per well. After serum starvation in FBS-free medium overnight to force the cells into the same initial proliferation state, the cells were cultured in the absence or presence of drug in complete medium for 24 hours. The cells were then stained using the Cell-Light™ EdU Apollo®488 In Vitro Flow Cytometry Kit (C10338-3, RiboBio, China) according to the manufacturer’s instructions. The proportion of EdU positive cells was assessed by flow cytometry.

Western blot analysis Whole-cell extracts were prepared by lysing cells with RIPA buffer (R0010, Solarbio, China) and the protein concentrations were determined by the BCA method. Protein lysates (20 μg) were resolved by SDS-PAGE, and target proteins were detected with the following antibodies: anti-HIF1α mouse monoclonal antibody (ab113642, 1:1000, Abcam, UK), anti-SOX2 rabbit polyclonal antibody (ab97959, 1:1000, Abcam, UK), anti-OCT4 rabbit polyclonal antibody (ab18976, 1:500, Abcam, UK), anti-VEGF rabbit polyclonal antibody (ab46154, 1:1000, Abcam, UK) and anti-TFR1 rabbit 16


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monoclonal antibody (ab108985, 1:2000, Abcam, UK).

Patient cohort and immunohistochemistry The use of human samples was approved by the Ethics Committee of Tianjin Medical University Cancer

Institute

and

Hospital.

We

obtained

informed

consent

from

all

subjects.

Immunohistochemical (IHC) staining of a pancreatic cancer tissue microarray containing 96 pancreatic cancer patient specimens was performed using an anti-TFR1 rabbit monoclonal antibody (ab108985, 1:250, Abcam, UK), an anti-Ki67 rabbit monoclonal antibody (ab92742, 1:500, Abcam, UK), an anti- FTL rabbit polyclonal antibody (ab69090, 1:250, Abcam, UK), an anti-FTH rabbit monoclonal antibody (ab75972, 1:200, Abcam, UK) and an anti-FPN1 rabbit polyclonal antibody (ab78066, 1:250, Abcam, UK) to determine the levels of protein expression. All patients with a pathologically confirmed diagnosis of pancreatic ductal adenocarcinoma received a radical resection. The patients received at least three cycles of gemcitabine-based chemotherapy after the operation. Gemcitabine was delivered by a 30-min intravenous infusion at a dose of 1000 mg per square meter of body surface area weekly for two weeks followed by one week intervals, then for two weeks in a subsequent three-week course. Patients with at least one of the following conditions were excluded: (1) patients who received neoadjuvant chemotherapy, chemoradiotherapy or non-gemcitabine-based chemotherapy; (2) patients with macroscopically incomplete resection; (3) patients with a history of another major cancer; (4) patients who died within one month after the operation or due to non-cancer related causes. IHC slides were independently graded by two pathologists, who were blinded to patient outcomes. Discordant cases were assessed by a third pathologist, and a consensus was reached. Nuclear staining for Ki67 was regarded as positive. Membrane and cytoplasmic staining for TFR1 and FPN1 were regarded as positive. Cytoplasmic staining for FTL and FTH were regarded as positive. Immunoreactivity for TFR1, FPN1, FTL and FTH was scored semi-quantitatively according to the intensity and extent of tumor cell staining33. Intensity of staining was scored as (0 = negative; 1 = low; 2 = medium; 3 = high). The extent of staining was scored as 0 = 0% of the cancer cells stained; 1 = 1–25% of the cancer cells stained; 2 = 26–50% of the cancer cells stained; 3 = 51–100% of the cancer cells stained. Five random fields were observed under a light microscope. The final score was determined by multiplying the scores of intensity with the extent of staining, to give a score ranging from 0–9. Final scores of less than 1 were considered 17


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negative (-), 1–2 as low staining (+), 3–6 as medium staining (++) and 9 as high staining (+++). Immunoreactivity for Ki67 was scored according to the percentage of tumor cells with positive nuclear staining: negative staining (-) = 0% to 25% of cancer cells stained; positive staining (+) = 26% to 100% of cancer cells stained.

Cancer stem cell-related activity Cancer stem cell (CSC) activity was assessed using a spheroid formation assay. The cells were plated on Ultra-Low Attachment Surface 24 well-plates (3473, Corning, USA) in serum-free DMEM medium supplemented with B27 (1:50, 17504044, Invitrogen, USA), EGF (20 ng/mL, AF-100-15, Peprotech, USA) and bFGF (10 ng/mL, 100-18B, Peprotech, USA) at a density of 1000 cells/well, and cultured for 21 days. Then, the numbers of spheroids which contain more than 10 cancer cells were measured.

Effects of nanoparticles on established tumors in vivo All animals were obtained from Vital River Laboratory Animal Technology Co., China. The animal experimental procedures were approved by the Ethics Committee of Tianjin Medical University Cancer Institute and Hospital.

Subcutaneous pancreatic cancer xenografts were formed by injecting 5×106 T3M4 cells into the right flank of female, 6-week old nu/nu mice (5 mice/group). When the tumor volume reached approximately 100 mm3, the animals received tail vein injections of the various drug formulations (containing 31.95 mg/kg DFO and/or 6.96 mg/kg YC1) every 2 days for 31 days. During the treatment, the tumor sizes were measured and tumor volumes were calculated using the formula V = (1/2)ab2, where a is the tumor’s long axis and b is the short axis. Mice with tumor implants were euthanized 31 days after drug treatment, and the tumor xenografts were excised and weighed. The iron in the tumor tissues was measured using an iron assay kit (ab83366, Abcam, USA).

For orthotopic pancreatic cancer xenografts, 1×106 luciferase expressing PANC1 cells (PANC1-luc) were injected surgically into the tail of the pancreas of nu/nu mice. Treatments began 14 days after injection (n = 5 per group). Bioluminescent imaging of the mice was performed every 5 days. 18


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Different drug formulations (containing 31.95 mg/kg DFO and/or 6.96 mg/kg YC1) were injected intravenously (tail vein) every 2 days for 21 days. Mice with tumor implants were euthanized after 21 days of treatment, and the tumor xenografts were excised and weighed. The iron in the tumor tissues was measured using an iron assay kit (ab83366, Abcam, USA). For bioluminescent imaging, D-luciferin potassium salt (15 mg/mL, 10 μL/g/mouse) was injected into the abdominal cavity. After 10 min, mice were imaged using an IVIS Spectrum biophotonic imager (PerkinElmer, USA).

Statistical analysis Kaplan-Meier curves were used to analyze the survival of patients, and the log-rank test was used to obtain a P-value for the divergence in the Kaplan-Meier curves. Except for the clinical analysis and in vivo experiments, at least 3 independent experiments were performed. Data are expressed as the mean ± s.d. Statistical analysis was performed using SPSS version 18.0. Differences between two groups were analyzed by two-sided Student t-tests. A P-value of

Targeted Co-delivery of the Iron Chelator Deferoxamine and a HIF1Î±

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