H1/pAIM2 nanoparticles exert anti‐tumour effects that is associated with the inflammasome activation in renal carcinoma

Abstract Renal cell carcinoma (RCC) is a high metastasis tumour with less effective treatment available currently. Absent in melanoma 2 (AIM2) as a tumour suppressor might be used as a potential therapeutic target for RCC treatment. Here, we found that AIM2 expression was significantly decreased in RCC patient specimens and renal carcinoma cell lines (786‐O and OSRC‐2). To establish a safe and effective AIM2 gene delivery system, we formed the nanoparticles consisting of a folate grafted PEI600‐CyD (H1) nanoparticle‐mediated AIM2 gene (H1/pAIM2) as an effective delivery agent. Delivery of H1/pAIM2 in renal carcinoma cells could remarkably increase the expression of AIM2, and subsequently decrease cell proliferation, migration, and invasion as well as enhance cell apoptosis. In order to evaluate the therapeutic efficacy of AIM2 in vivo, H1/pAIM2 nanoparticles were injected intratumorally into 786‐O‐xenograft mice. Administration of H1/pAIM2 nanoparticles could inhibit the tumour growth as evidenced by reduced tumour volume and weight. Furthermore, Blockade of inflammasome activation triggered by H1/pAIM2 nanoparticles using inflammasome inhibitor YVAD‐CMK abrogated the anti‐tumoral activities of H1/AIM2. These results indicated the therapeutic effect of H1/pAIM2 nanoparticles was mainly attributable to its capability to enhance the inflammasome activation. H1/AIM2 nanoparticles might act as an efficient therapeutic approach for RCC treatment.

Absent in melanoma 2 (AIM2) as a cytoplasmic DNA senor, is a family of IFN-inducible PYHIN (pyrin and HIN200 domain-containing) proteins. 7 AIM2 binding to double-stranded DNA (dsDNA) can recruit the adaptor protein apoptosis-associated speck-like protein containing a caspase-recruitment domain (CARD) (ASC) through homotypic pyrin domain (PYD) interactions. The CARD of ASC binds the CARD of pro-caspase-1 to lead to caspase-1 activation and the production of interleukin-1β (IL-1β) and IL-18 through cleavaging the pro-IL-1β and pro-IL-18. 8 The activation of AIM2 inflammasome regulating inflammatory response is well explored in innate immune cells. [7][8][9][10] Currently, AIM2 as a tumour suppressor is remarkably interest in cancer. The absence of AIM2 in melanoma promoted the cell migration, invasion and disease progression. 11 The restricted proliferation of cancer cells by over-expressing AIM2 was found in colon cancer, [12][13][14][15] breast cancer 16,17 and prostate cancer. 18 Thus, these results suggest that AIM2 plays an important role in the development and progression of cancer with decrease or deficiency of AIM2. However, AIM2 serving as a therapeutic target for RCC treatment remains largely unknown.
The efficient gene therapy depends on the ability to efficiently deliver the appropriate therapeutic materials into the target tissues or cells. 19 Currently, viral vectors including retroviruses, adenoviruses and lentiviruses as delivery system have been widely used in gene therapy. [20][21][22] However, viral vectors have limited their clinical applications for safety concern such as oncogenic potentials, immuno-recognition of the viral capsid proteins and generation of various immune responses in vivo. 23,24 Therefore, the safe and efficient gene carriers are the key to the clinical success of gene therapy. Folate-grafted PEI600-CyD (H1) as a nonviral gene delivery vehicle can effectively condense plasmid DNA to form stable functionalized nanoparticles. 25 Recent studies have shown a significant success using H1 to deliver genes in vivo. 26 More importantly, H1-based delivery system leads to neither elevate enzymes in serum nor a non-specific immune response in mice, suggesting its low toxicity. 25,26 Therefore, H1-based plasmid AIM2 (pAIM2) nanoparticles may be a therapeutic strategy for RCC treatment.
In this study, we observed that AIM2 expression was significantly decreased in RCC specimen and renal carcinoma cell lines, indicated that AIM2 might be served as a therapeutic target for RCC treatment. H1/pAIM2 nanoparticles could remarkably increase the expression of AIM2, and subsequently decrease cell proliferation, enhanced cell apoptosis and inhibit cell migration and invasion in vitro. To evaluate the therapeutic potential of AIM2 in vivo, H1/ pAIM2 nanoparticles were intratumorally administrated to 786-Oxenograft mice at the early of tumour growth. Our results indicated that administration of H1/pAIM2 could significantly ameliorate the tumour growth of renal carcinoma. The therapeutic efficacy of H1/ pAIM2 was mainly attributable to its capability to inhibit the inflammasome activation. Our findings indicated that H1/pAIM2 nanoparticles might represent a novel therapeutic approach for RCC treatment and provide a better understanding of the underlying mechanism of RCC pathogenesis.

| Immunohistochemistry assay
Tissue slides were de-paraffinized and rehydrated sections were made by epitope retrieval via a heat-induced protocol. The sections were blocked with 10% BSA and incubated with human AIM2 antibody (1:100 dilution; Santa Cruz Biotechnology), secondary antibody, and then with streptavidin-peroxidase (Zhongshan Biotech, Beijing, China). The samples were developed by treatment with DAB detection kit (Zhongshan Biotech, Beijing, China) and with hematoxylin to counter stain the nuclei. Immunostaining evaluation was performed as described before. 27 2.3 | Formation of H1/pAIM2 nanoparticles H1 was synthesized according to our previously described method. 25 Briefly, Folic acid (0.053 g, 0.12 mmol) in DMSO was activated by CDI (0.02 g, 0.12 mmol) using Et3N as the catalyst under nitrogen atmosphere. The reaction mixture was added to the DMSO solution of PEI600-CyD (0.12 g) synthesized by a method reported previously, and stirred for 24 hours to obtain the crude product of H1, which was purified by dialysis in water for 3 days with dialysis tubing (MWCO, 12 kD). Pale yellow powder of H1 was obtained after lyophilization for 2 days.
Human AIM2 full length from plasmid pEFBOS-AIM2 was subcloned into pcDNA3.1 using EcoRI and BamHI. pcDNA3  To prepare H1/pAIM2 nanoparticle polyplexes, H1 and plasmid were mixed and dissolved in 5% glucose solution at an N/P ratio of 20:1 in the same volume. The nanoparticle polyplexes were stabilized for 10 minutes at the room temperature prior to tranfection or injection.

| Cell proliferation assay
The treated cells were plated into 96-well plates at a density of 3 × 10 3 cells/well, respectively, and cells were incubated 37°C for 24 hours, 48 hours and 72 hours. Ten microlitres solution of Cell Counting Kit-8 (CCK-8, Beyotime, Nantong, China) were added to each well and incubated at 37°C for 1 hour. Absorbance at 450 nm was measured on an ELX-800 spectrometer reader (Bio-Tek Instruments).

| Flow cytometry analysis
The collected cells were washed twice with PBS, and then fixed with fixation buffer (BD Biosciences) for 30 minutes at 4°C. The fixed cells were permeabilized with permeabilization solution (BD Biosciences) at room temperature for 0.5 hours.
Isotype-matched anti-human IgG served as control. FITC conjugated IgG (diluted 1:100; Biolegend) was used as secondary antibody. Then labelled cells were analysed using a BD FACSCanto II flow cytometer with FACSDiva software (BD Biosciences).

| Colony formation assay
For the colony formation assay, cells were transfected with pAIM2 or Vector, as described above. Twenty four hours later, transfected cells were trypsinized, counted and seeded in 6-well plate at a density of 1 × 10 3 cells per well. After 7 days of incubation, survival colonies were fixed with 4% paraformaldehyde for 10 minutes and stained with 0.5% of crystal violet and counted under the microscope.

| Cell apoptosis detection
Cells were trypsinized and rinsed twice with PBS at 72 hours posttreatment. Flow cytometry analysis of cell apoptosis was performed using an Annexin V-FITC/PI apoptosis detection kit (BD Biosciences) according to the manufacturer's instructions.

| Cell migration and invasion assay
Cell migration and invasion assays were performed using modified 2-chamber plates with a pore size of 8 μm. For migration assay,

| Wound healing assay
Cells treated with H1/pAIM2 or control were grown to confluency, a wound line was made by scraping a closed Pasteur pipette tip across the confluent cell layer. Then, cells were washed three times with PBS to remove detached cells and debris. The size of the wound was observed and measured after 24 hours.

| Caspase-1 activity assay
The caspase-1 activity was detected by the colorimetric caspase-1 assay kits (Beyotime Biotechnology). Briefly, the extracts were centrifuged at 12 000 g for 10 minutes, and the supernatant was collected. A volume of supernatant equivalent to 100 μg of protein were incubated with 50 mmol/L substrate (Ac-YVAD-MCA for caspase-1 proteinase), 0.05 mol/L NaCl, 2.5 mmol/L dithiothreitol and 10 mmol/L HEPES, pH 7.5, in 300 μL of the reaction mixture for 1 hour at 37°C. The fluorescence intensity of the released AMC was measured with a spectrofluorometer at 380 nm/460 nm excitation/ emission wavelength. One unit was defined as the amount of the enzyme required to release 1 μmol AMC per hour at 37°C.

| ELISA measurement of cytokines
To assess protein levels of IL-1β in culture supernatant or in homogenized tumour tissues and IL-6 in serum of mice, enzyme-linked immunosorbent assays (ELISA) were performed according to the manufacturer's instructions (eBioscience).

| Statistical analysis
Statistical analysis was performed with SPSS software (version 16.0, Armonk, NY, USA) and expressed as means ± SD. Statistical significance was evaluated using two-tailed Student's t test. Multiple comparisons were performed using one-way ANOVA. The statistical significance level was set as *P < 0.05; **P < 0.01; ***P < 0.001.

RCC patient tissues and renal cancer 786-O or OSRC-2 cell lines
To determine whether AIM2 was involved in pathogenesis of RCC, we firstly detected the expression of AIM2 in 298 specimens of RCC patients. As shown in Figure 1A and B, Immunohistochemical staining and staining scores showed that AIM2 expression was lower in RCC tissues than normal renal tissues. Furthermore, we also  Figure 1E and F). These results indicated that the decreased AIM2 expression might be involved in pathogenesis of RCC, and the increase of AIM2 expression might serve a therapeutic strategy for RCC treatment.

| H1/AIM2 inhibited renal cancer cell proliferation and promoted cell apoptosis
Our previous study has demonstrated that H1 nanoparticles were an effective delivery system for gene expression in vitro and in vivo. 26 Thus, H1/pAIM2 or control nanoparticles were prepared (Figure 2A Figure 2E and F). The apoptosis of renal cancer cells was also increased in H1/pAIM2 group in contrast to control group ( Figure 2G and H). These results indicated that H1/AIM2 could inhibit renal cancer cell proliferation and enhance cell apoptosis.  Figure 3E-H). These results indicated that H1/AIM2 could inhibit the migration and invasion of renal cancer cells.

| Administration of H1/AIM2 reduced the tumour growth in renal cancer cell-xenografted nude mice
To further evaluate the therapeutic efficacy of H1/AIM2 in RCC, the subcutaneous 786-O-xenograft model established in nude mice were intratumorally administrated with H1/pAIM2 or control. We firstly detected the expression efficiency of H1/AIM2 in tumour, and found that the higher AIM2 expression was observed in H1/pAIM2 group than control group ( Figure 4A). At day 35 after tumour inoculation, the significantly suppressed tumour growth was observed in H1/AIM2-treated group compared with control-treated group ( Figure 4B and C).
Accordingly, the volume and weight of tumour were decreased in H1/ AIM2-treated group ( Figure 4D and E). Moreover, H1/pAIM2 could not significantly affect the body weight ( Figure 4F) and elevate the level of cytokines, like IIL-6 in serum ( Figure 4G). There is a non-specific immune response evidenced by normal pathological tissue of heart and liver in H1/pAIM2-treated mice ( Figure 4H), indicated suggesting its low toxicity. Taken together, these data indicated that H1/AIM2 treatment with low toxicity could effectively prevent the aggravation of tumour.

| H1/AIM2 enhanced the inflammasome activation of AIM2 in renal cancer cells
The inflammasome is a multiprotein complex that has recently been shown to the corresponding contribution of innate immunity pathways (G). At day 2 or 7 after the initial administration, the serum were collected, the levels of IL-6 were determined by ELISA assay. (H) At day 35 after tumour inoculation, the pathology was evaluated by H&E staining of heart or liver tissues. Each experiment was performed independently at least three times and the results of one representative experiment are shown. Data are means ± SD, ***P < 0.001, ns, no significant difference in suppressing tumour growth. 15,28 Thus, we are attracted to define whether the anti-tumour effect of H1/AIM2 in RCC is mediated by AIM2 inflammasome. Our data indicated that the increased inflammasome components (AIM2/casepase-1/IL-1β) were observed in H1/ pAIM2-treated 786-O or OSRC-2 cells compared the control-treated cells ( Figure 5A-C). Equally, caspase-1 activity and IL-1β production were significantly up-regulated in these H1/pAIM2-treated cells (Figure 5D-G), indicated that AIM2 inflammasome was formed and activated. These results indicated that H1/AIM2 exerting its anti-tumour effect was associated with AIM2 inflammasome activation.  F and G). The levels of IL-1β were measured in treated renal cancer cell lines. Data are from one representative experiment of three performed and presented as the mean ± SD. The different significance was set at *P < 0.05, **P < 0.01 and ***P < 0.001

| H1/AIM2 inhibited the cell migration and invasion and exerted its anti-tumour effect through AIM2 inflammasome activation
To further validate our hypothesis that H1/AIM2 could suppress the migration and invasion of RCC by enhancing the inflammasome activation, renal cancer cells was treated with H1/pAIM2 nanoparticles followed with or without the downstream inflammatory caspase inhibitors YVAD-CMK. The results showed that the inflammasome activation was blocked in 786-O or OSRC-2 cells (Figure 6A and B) and the inhibited ability of cell proliferation, migration and invasion was prevented in Yvad-CMK-treated H1/pAIM2 group ( Figure 6C-H).  H). The invasion was detected in renal cancer cells. Data are from one representative experiment of three performed and presented as the mean ± SD. The different significance was set at **P < 0.01, and ***P < 0.001 Further study showed that after blocking the effect of inflammasome activation by administration of YVAD-CMK in vivo, the levels of capase-1 and IL-1β remarkably were reduced in tumour ( Figure 7A and B). Furthermore, the rescued malignant behaviours including tumour volume ( Figure 7C) and tumour weight ( Figure 7D) were observed in YVAD-CMK-treated H1/pAIM2 group compared with H1/pAIM2 group. These data indicated that the therapeutic efficacy of anti-tumour by H1/pAIM2 is depended on the inflammasome activation.

| DISCUSSION
Renal cell carcinoma has a worse prognosis and easily develop metastatic disease, which are usually incurable, and the median survival of metastatic RCC patients is significantly worse. 29 Currently, the treatments for RCC have evolved from conventional drugs to biologic targeting therapies, among which cytokines are the most crucial therapeutic targets. 30,31 However, the existing therapies for RCC are not satisfactory owing to the low efficacy in suppressing disease activity as well as the adverse effects in patients. In this study, H1 nanoparticles with a low toxicity were applied in the delivery system of targeting therapeutic. Absent in melanoma 2 as DNA sensor, a range of cellular defence mechanisms including cytokines secretion are triggered to activate the immune system. 32 Moreover, the down-regulated AIM2 expression is a risk factor in tumour progression. 14  The inflammasome responds to multiple danger signals, including a decrease in cytosolic potassium concentrations and an increase in cytosolic DNA levels, dying tumour cells and bacterial products. 33 H1/AIM2 may contribute to the stimulation of the inflammasome activation by recognizing dsDNA from dying tumour cells. Previous study revealed that AIM2 inflammasome in HCC cells suppressed mammalian target of rapamycin (mTOR)-S6K1 pathway, 28 and suggested that mTOR pathway might be involved in AIM2-induced downstream pathway. The activation of inflammasome has been reported to inhibit cancer cell invasion and metastasis, angiogenesis or tumorigenesis in model systems, suggesting that the defective of inflammasome in tumour cells is a tumour suppression mechanism. 34 Our data demonstrated that H1/pAIM2 expression significantly enhanced the expression of inflammasome competent (AIM2/ Caspase-1/IL-1β) in renal cancer cell lines. Accordingly, the inhibition of cell invasion and metastasis by H1/pAIM2 was blocked by inflammasome inhibitor YVAD-CMK in vitro. In vivo experiment, we observed the prevent effect of the reduced growth tumour in YVAD-CMK-treated 786-O cell-xenografted nude mice administrated with H1/pAIM2. These data indicated that the therapeutic effect of H1/pAIM2 in the inhibition of cell migration and invasion was associated with enhancing the activation of inflammasome. Taken together, AIM2 might serve as a common danger sensor in different cancer cell types, which helps the host cells to be exempt from immune inflammation, and thus maintains the intracellular homeostasis and sanctity.
In the future, we would further investigate the mechanism of AIM2 inflammasome on the side of cancer cell apoptosis.
In this study, our results suggested that H1/AIM2 nanoparticles could inhibit malignancies of renal cancer through enhancing the inflammasome pathway, which suggested an effective treatment of H1/AIM2 against renal cancer. Therefore, it indicated that therapeutic strategy by H1/AIM2 might provide a new way for manipulating renal cancer.