p53 Dry Gene Powder Enhances Anti-cancer Effects of Chemotherapy against Malignant Pleural Mesothelioma

Dry gene powder is a novel gene-delivery system, which is inhalable and not infectious with high gene expression. Previously, we showed that the transfection of p16 INK4a or TP53 by dry gene powder resulted in growth inhibitions of lung cancer and malignant pleural mesothelioma (MPM) in vitro and in vivo. Here, we report that dry gene powder containing p53-expression-plasmid DNA enhanced the therapeutic effects of cisplatin (CDDP) against MPM even in the presence of endogenous p53. Furthermore, our results indicated that the safe transfection with a higher plasmid DNA (pDNA) concentration suppressed MPM growth independently of chemotherapeutic agents. Dry gene powder may rescue chemoresistance and provide a new therapeutic alternative for MPM patients without safety concerns over “vector doses”.


Introduction
Malignant pleural mesothelioma (MPM) develops from the pleura, and during progression, it penetrates neighboring tissues such as the peritoneum, pericardium, and lungs.MPM is intractable, and the median overall survival time ranges between 9 and 17 months, regardless of the stage (ref.1).First-line chemotherapeutic agents against MPM are cisplatin (CDDP) plus pemetrexed (PEM).Their combination led to the highest reported response rate of 21.3 ~ 41%.However, more than half of the patients remained unaffected and even those who were positively affected also acquired chemoresistance afterward (ref. 1).In addition, CDDP has severe adverse effects, one of which is dose-limiting nephrotoxicity (ref.2).The administration of the chemotherapeutic drug at heavy doses and/or for long periods is not preferable for the patient's health and their quality of life.In the case of MPM patients showing marked deterioration of the health status, the dose of CDDP has to be reduced or the administrations have to be postponed or aborted.Therefore, a new scheme to expedite the anti-cancer effects of established chemotherapy and to overcome the chemoresistance of MPM has to be developed.
As one of the reasons for the therapeutic resistance to DNA-damaging agents, high DNA repair performance has been considered (ref.3).Xu et al. reported the chemoresistance mechanism by investigating MPM specimens derived from cisplatin-resistant patients.They showed that the metastasisassociated gene 1 (MTA1) protein level was signi cantly elevated, which resulted in enhanced DNA repair ability (ref.4).P53 is a critical factor for DNA damage responses, and so that many studies performed therapeutic p53 transfection in MPM.Gene transduction of exogenous p53 with adenoviral (Ad) vectors into MPM cells has shown successful results in vivo and in vitro (ref.5,6).However, according to Li et al., the inhibitory effects were dependent on MPM cell types: NCI-H28 (IC 50 = 2.4 x 10 3 viral particles per cell), NCI-H226 (IC 50 = 5.5 x 10 3 viral particles per cell) and NCI-H2052 (IC 50 > 1x10 5 viral particles per cell).
These results suggest that for some MPM types, high viral dose administration may be required.Since high-dose administration of adenoviral vectors may pose safety risks, it is necessary to develop alternative therapeutic gene-delivery systems for MPM patients.
We originally developed an inhalable gene-delivery system, naked dry gene powder, without containing cationic lipids, polymers, or viral vectors.Among various excipients, we found that 50-kDa hyaluronic acid (HA) formed appropriate powders which promoted high gene expression in introduced cells (ref.[7][8][9][10][11]. HA is a natural glycosaminoglycan, and is ubiquitously distributed in neural, epithelial, and connective tissues.Therefore, it has been suggested that HA has bene ts as a drug carrier due to its biocompatibility and biodegradability, besides the biological advantage of the primary receptor CD44 to internalize HA (ref.12,13).CD44 is overexpressed in a variety of cancer cells including MPM (ref.14,15).Therefore, HA-anti cancer drug combinations have been considered promising therapeutics for targeting tumor cells.Clinical research and in vivo studies have proven the e cacy and safety of the drug delivery strategy with HA (ref.16,17).This delivery system exhibited desirable stability for a long period at room temperature (ref. 18).
In addition to not containing an infectious agent, it is easy to handle and suitable as gene therapy for pulmonary diseases.
In our previous report, we demonstrated that the dry gene powder inhibited the growth of lung cancer cells and MPM cells in vitro and in vivo by transfection of p16 INK4a or TP53, corresponding to the lack of genes in the tested cells (ref.19).However, in general, the frequency of TP53 deletion in MPM is not high (ref.20).Therefore, the effects of exogenous p53 transfection to MPM cells by the dry gene powder in the presence of endogenous p53 remains unknown.It is also unclear how p53-dry gene powder works with chemotherapeutic agents.
In this report, we explored the suppressive effects of the dry gene powder with exogenous p53 in the presence of CDDP or PEM on MPM cells expressing endogenous p53.

Preparation Of Dry Gene Powders By Spray-freeze-drying (Sfd)
We prepared the dry gene powders as described previously (ref.9).In short, the sample solution (5 mL) was atomized into liquid nitrogen to generate frozen droplets at a ow rate of 5 mL/min and atomizing pressure of 150 kPa.The frozen droplets were rapidly transferred into a freeze-dryer (EYELA DRC-1100 and FDU-2100, Tokyo Rikakikai Co., Ltd., Tokyo, Japan) precooled at a shelf temperature of -40°C.After evaporation of the liquid nitrogen, freeze-drying was conducted for 16-18 h at 10°C at a vacuum level of 5 Pa for lyophilization to obtain dry gene powders.The obtained dry gene powders were stored in openmouth glass vials in a desiccator with silica gel.

Cell Culture
The mouse MPM cell line AB1 was obtained from CellBank Australia (Sydney, Australia).The human MPM derived cell line, NCI-H2052 was obtained from the American Type Culture Collection (Manassas, VA, USA).MPM cells were maintained in Roswell Park Memorial Institute 1640 (RPMI-1640) complete medium with the following preparation: RPMI-1640 medium with 2 mM L-glutamine (Gibco, Amarillo, Texas) adjusted to contain 4.5 g/L glucose (Fuji lm Wako Pure Chemical Corporation), supplemented with 10% FBS (Thermo Fisher Scienti c, Waltham, MA, U.S.) and 100 U/mL penicillin-streptomycin (Sigma-Aldrich, St. Louis, MO).MPM cells were cultured at 37°C with 5% CO 2 .The cells were split at subcon uence using trypsin/EDTA (Sigma-Aldrich) every 3-4 days.
Air-liquid Interface (Ali) Culture Cells were seeded into the insert of a Transwell → 12-well plate (Corning Inc., Corning, NY, USA) at 2.0 x 10 5 cells/well for AB1 cells or at 5.0 x 10 5 cells/well for NCI-H2052.The basal compartment was supplied with 1.5 mL RPMI1640 complete medium.After 2-day culture for AB1 or 3-day culture for NCI-H2052, the supernatant of the insert was aspirated, and basal medium was replaced with 1 mL of fresh RPMI1640 complete medium followed by the indicated conditions of chemotherapeutic agents and/or dry gene powder.CDDP or PEM was added to the medium of basal compartments.

Dry Gene Powder Treatment To Mpm Cells
We followed the protocol reported previously (ref.10).Brie y, we sprayed 0.5 mg of Luc-SFD or p53-SFD onto the apical surface of the cell layers in the ALI culture system using an administration device constructed by connecting a 1-mL syringe (Terumo Co., Tokyo, Japan) with a three-way stopcock (Top Co., Tokyo, Japan).Lipofection AB1 cells were seeded on a Transwell → 12-well plate at 2.0 x 10 5 cells/well.After 2-days culture, apical medium was aspirated and basal medium was refreshed with new RPMI1640 complete medium with or without 50 µM CDDP.For transfection per well, we prepared 5 µL of Lipofectamin2000 (Thermo Fisher Scienti c) and 2.5 µg of pDNA with 250 µL each of Opti-MEM (Thermo Fisher Scienti c) separately.These reagents were mixed and kept at room temperature for 20 min.The mixture was added to the apical compartments.Subsequently, the cells were incubated at 37°C with 5% CO 2 .

Cell Count
Cells were washed with PBS and trypsinized.The cells were harvested with RPMI complete medium and counted by 0.4% trypan blue staining.A hemocytometer was used for the cell count.

Tunel Assay
Cells were harvested and xed with 4% paraformaldehyde for 10 min at room temperature.The cells were permeabilized by PBS-T (0.5% Tween-20 and 0.2% BSA in PBS).After washing cells in PBS twice, we followed the recommended protocol of the MEBSTAIN Apoptosis TUNEL Kit Direct (Medical and Biological Laboratories, Tokyo, Japan).TUNEL -positive cells were detected by LSRFortessa™ X-20 (Becton, Dickinson and Company, Franklin Lakes, NJ, U.S.) and BD Flowjo Software (Becton, Dickinson and Company).

Cell Cycle Analysis
Cell cycle analysis was performed by ow cytometry detection of DNA and BrdU contents using anti-BrdU antibody and propidium iodide (PI) staining.BrdU was added at 10 µM to the basal compartments for 1 h before the harvesting of cells.The cells were trypsinized, then washed with PBS, and xed overnight at − 20°C in 70% EtOH.The cells were treated with 2 N HCl/0.5% Triton X-100 for 30 min (NCI-H2052) or 2 h (AB1), and then 0.1 M sodium tetraborate (pH 8.0).The cells were stained with anti-BrdU antibody (MAB3424, clone AH4H7-1; Sigma-Aldrich), anti-Mouse IgG FITC (F6257-.5ML;Sigma-Aldrich).The cells were suspended in 2 µg/mL PI (Sigma-Aldrich)/PBS.PI and BrdU staining was detected by LSRFortessa™ X-20 and the DNA content was quanti ed using BD Flowjo Software.

Statistical analysis
To compare different groups of treatment, we used one-way ANOVA on ranks or two-way ANOVA on ranks followed by the Tukey-Kramer test.A difference was considered signi cant with a p-value < 0.05.

Results
The effects of p53-SFD on AB1 cell viability in combination with CDDP Firstly, we examined the mouse MPM cell line, AB1, which was established from mouse MPM caused by inoculation with asbestos (ref.21).Mouse MPM cells are a good tool for in vivo investigation in a syngeneic tumor mouse model.Wahlbuhl et al. reported that AB1 did not have a change in copy number of TP53 (ref. 22).To investigate the effects of p53-SFD, we compared the cell viability at 24 h after treatments (Fig. 1a-b).AB1 cells showed resistance to 10 and 50 µM of CDDP.At 10 µM, 1% p53-SFD was not able to change the viability compared to that of 1% Luc-SFD.However, at an elevated concentration of CDDP, at 50 µM, 1% p53-SFD signi cantly decreased the viability compared with that of 1% Luc-SFD (Fig. 1b).

The Effects Of P53-sfd On Ab1 Cell Viability In Combination With Pem
Next, the other part of the rst-line agents, PEM, was tested in AB1.The growth of AB1 cells was affected with the dose of 0.05 µg/mL in a dose dependent manner, but no synergistic effect of 1% p53-SFD was observed (Fig. 1c).

The Apoptosis Of Ab1 By P53-sfd With Cddp
Then, we conducted a TUNEL assay to explore the mechanism of decreased cell viability by p53-SFD under 50-µM CDDP treatment (Fig. 1d).The number of TUNEL-positive cells showed an increase with Luc-SFD, but CDDP did not signi cantly enhance the proportion of TUNEL-positive cells.On the other hand, p53-SFD without CDDP also increased the TUNEL-positive proportion compared with that of Luc-SFD without CDDP.The combination of p53-SFD and CDDP further increased TUNEL-positive cells to the highest rate compared with the other treatments.

The Cell Cycle Of Ab1 By P53-sfd With Cddp
We also conducted cell cycle analyses with AB1 at 16 h (Fig. 1e).Without dry gene powder treatment, CDDP increased the population of S phase and corresponding decrease of G1 phase.Luc-SFD CDDP (+) did not change the balance of each cell cycle phases compared to that of NT CDDP (+) (Fig. 1f, 1g).The G1/S and G2M/S ratios were not changed by p53-SFD CDDP (+) compared with those of Luc-SFD CDDP (+).Our TUNEL assay and cell cycle data indicated that the decreased cell viability caused by the p53-SFD and CDDP combination was mainly induced by apoptosis rather than cell cycle arrest.

The Toxicity Of Lipofection And Effects Of P53-expression Plasmid On Ab1 Cell Viability
To reveal the bene ts of the dry gene powder, we also examined cell viability using Lipofectamine2000 → for plasmid transfection (Fig. 2a).When we treated with 0.5 mg of 1% p53-SFD, we estimated that about 50% of the powder dispersed from the apparatus reached the surface of the cell layer according to a separated test with a powder containing a uorescent dye for determination (data not shown).Therefore, to transduce 2.5 µg of pDNA, we used 5 µL of Lipofectamine2000 , following the manufacturer's recommended lowest dose of reagent.
AB1 viability showed a signi cant decrease with p53/LFN CDDP (+) compared with Luc/LFN CDDP (+).The decrease was similar to that showed with p53-SFD CDDP (+), shown in Fig. 1b.However, Lipofectamine2000 → itself showed unavoidable toxicity even at the lowest dosage in the range of the manufacturer's recommended amounts.
These results showed that the growth-inhibiting effects of the p53-encoding plasmid were the same when transfected by dry gene powder or Lipofectamine2000 → ; however, dry gene powder delivered plasmid DNA with much lower toxicity compared with Lipofectamine2000 .

Dry Gene Powder With High Pdna Concentration Showed Ab1 Tumor Suppression Without Cddp
Because of the low toxicity, dry gene powder was able to deliver a larger amount of pDNA into AB1 cells.We prepared 2% p53-SFD and examined its tumor-suppressive effects (Fig. 2b).2% Luc-SFD led to a decrease of cell viability compared with that of NT.Interestingly, 2% p53-SFD led to a further decrease without CDDP compared with 2% Luc-SFD, but this was not signi cant.However, when we compared the viability of 2% p53-SFD and that of NT, there was a signi cant difference, which was nearly the same as that of 1% p53-SFD in combination with 50 µM of CDDP.These data showed that the high DNA content overcame the requirement of CDDP for the inhibition of AB1 growth.

The Effects Of P53-sfd On Nci-h2052 Cell Viability
Next, we investigated whether the effects of p53-SFD observed in AB1 cells are also applicable to the human MPM cell (Fig. 3a).NCI-H2052 cells were resistant to 50 µM CDDP.However, with CDDP, 0.5% p53-SFD reduced the cell viability compared with NT.Furthermore, the increased DNA dose of 1% p53-SFD showed a more e cient decrease of cell viability under CDDP treatment (Fig. 3b).These results led us examine p53-SFD for a higher dose of DNA, as well as AB1 cells.Similar to the result in AB1, 2% p53-SFD alone was su cient to repress NCI-H2052 growth signi cantly, compared with NT (Fig. 3c).

The Apoptosis Of Nci-h2052 By P53-sfd With Cddp
We further explored the mechanisms of growth inhibition in NCI-H2052 caused by p53-SFD and CDDP combination.The TUNEL assay showed that TUNEL-positive cells were not signi cantly increased 20 h after the treatment of the agents (Fig. 4a-c).We noted a faint rise at 16 h.Since there was no signi cant difference in TUNEL positive populations between p53-SFD and Luc-SFD with or without CDDP, we concluded that those TUNEL positive populations did not explain the reduction of the cell viability of p53-SFD CDDP (+) compared to that of Luc-SFD CDDP (+).

The Expression Of P53 And Phosphorylation At Ser46
To con rm the expression of p53 at the protein level, we detected p53 using an antibody and owcytometry.NCI-H2052 expressed low levels of p53 as a baseline, and 50 µM of CDDP did not change the expression level (Fig. 4d).However, Luc-SFD transfection upregulated endogenous p53 with or without CDDP.p53-SFD also increased the p53 expression level, but there was no signi cant difference between Luc-SFD and p53-SFD regardless of the CDDP treatment.
To examine whether the upregulation of p53 led to the apoptosis pathway, we examined phosphorylation at Ser46 of p53, which was identi ed as an apoptotic pathway signal (ref.[23][24][25].The phosphorylation at Ser46 of p53 was not increased in any of the tested groups compared with NT CDDP (-) and NT CDDP (+) (Fig. 4e).In addition, Luc-SFD CDDP (+) and p53-SFD CDDP (+) did not show a difference in phosphorylation.Therefore, the increase of p53 expression in Luc-SFD and p53-SFD was not considered to participate in apoptosis, which was consistent with the results of the TUNEL assay.
For a better understanding, further investigations are required to clarify the detailed functional differences in p53.

The Cell Cycle Of Nci-h2052 By P53-sfd With Cddp
Then, we examined the cell cycle at 16 h as well (Fig. 4f).Treatment of 50 µM of CDDP treatment alone did not induce cell cycle change in NCI-H2052.The ratio of G1/S and G2M/S showed no signi cant difference between Luc-SFD CDDP (-) and p53-SFD CDDP (-) (Fig. 4g-h).However, p53-SFD CDDP (+) exhibited an increase both the ratio of G1/S and that of G2M/S compared with Luc-SFD CDDP (+).
Together with the data from the TUNEL assay, the results suggest that growth suppression after p53-SFD and CDDP treatment in NCI-H2052 was due to cell cycle arrest rather than apoptosis.

Discussion
Exogenous p53 gene therapies in cancer have drawn highly positive expectations (ref.6, 26-28).So far, Ad vectors have been taking a leading role in gene therapy for cancer.Adeno-associated viral (AAV) vectors also have been studied extensively as a gene delivery vector.Glybera → , Luxturna , and Zolgensma were approved by EMA and/or FDA between 2012 and 2019 (ref.29).However, AAV shows different tissue-tropisms by serotypes, and without the help of an adenovirus, it is di cult to induce e cient transgene expression (ref.30,31).Although engineering to improve these characteristics of AAV has been eagerly explored, direct targeting of cancer cells is still being developed [32].Currently, the applicable virus dosage is a limiting factor for the therapeutic results in clinical research for these viral vectors (ref.[33][34][35].Deaths and toxicity were reported in both AV and AAV therapies at high doses in humans (ref. 36).Given the different susceptibilities among patients, it is important to increase options for therapeutic gene delivery.
In this study, we showed the effects of p53-SFD for CDDP therapy in MPM cells.The treatment of p53-SFD and CDDP synergistically suppressed growth of AB1 cells and NCI-H2052 cells in a dose-dependent manner (Figs.1a, 1b, 3a, 3b).It was noteworthy that p53-SFD inhibited AB1 (Fig. 2b) and NCI-H2052 (Fig. 3c) tumor growth without CDDP at 2% of the pDNA content.Raising the pDNA content more than 2% is feasible in the dry gene powder (data not shown), which might be a further advantage for gene therapy.
On the other hand, AB1 cells did not show synergistic effects of p53-SFD and PEM treatment in the range we examined, where PEM alone signi cantly reduced the cell viability (Fig. 1c).One possible explanation for the differences in the synergistic effects between CDDP and PEM might be secondary in uences on the cells other than DNA damage, such as MDM2 ubiquitination activity by CDDP (ref.37,38).
As the mechanism of tumor growth restriction, the TUNEL assay revealed that p53-SFD caused enhanced apoptosis in AB1 cells with CDDP (Fig. 1d).The increase of apoptosis by Luc-SFD might be due to proteotoxic stress (ref.39,40), which may be inducible due to the e cient gene transfection.However, the apoptosis caused by Luc-SFD was not su cient to suppress the total number of AB1 cells even with or without CDDP.
Cell cycle analyses of AB1 showed that CDDP treatment alone decreased the G1 phase and increased the S phase (Fig. 1e).It has been well-studied that transient blocking of cells in the S phase occurs prior to G2 phase arrest when DNA is damaged by cisplatin, to re-establish DNA integrity (ref.41).The tendency of the G1 increase by p53-SFD treatment compared with NT under CDDP treatment was not signi cant.Therefore, we speculate that the decrease of cell viability in p53-SFD with CDDP was due more to apoptosis rather than cell cycle arrest (Fig. 5a).
The tumor suppression mechanisms were different between AB1 and NCI-H2052.CDDP and p53-SFD treated NCI-H2052 did not show evident apoptosis under the reduction of cell viability (Fig. 4a-c).At 16 h after treatment, Luc-SFD and p53-SFD slightly increased TUNEL-positive cells, but they were small populations and not CDDP-dependent.
In addition, we found that the total p53 levels were elevated in Luc-SFD and p53-SFD with or without CDDP (Fig. 4d), but these upregulations were not accompanied with phosphorylation of Ser46 (Fig. 4e).The silence of phosphorylation at Ser46 in our results corresponded to the TUNEL results.Thus, we suspect the endogenous p53 in NCI-H2052 may be functionally de cient compared with exogenous p53.However, Usami et al. investigated exons 2-11 of TP53 to cover the entire coding frame of TP53, and concluded that NCI-H2052 had wild-type in p53 coding region (ref. 42).We need to conduct further investigations of details regarding functional differences, such as gene expression of downstream targets, post-translational modi cations, and cellular localizations.For a better understanding, it is also important to explore the impact of the protein ratio of exogenous p53 to endogenous p53 in each case of different DNA contents of p53-SFD in AB1 and NCI-H2052.
In contrast to AB1, NCI-H2052 did not change the cell cycle by CDDP.NCI-H2052 cells were led to cell cycle arrest more than apoptosis by p53-SFD under the treatment of CDDP (Fig. 5b).In the regulation of cell cycle, p53 is most often reported to be correlated with G1 arrest (ref.43,44).Whereas numerous publications suggest that p53 also works also in the context of G2 The effects of p53-SFD in combination with CDDP or PEM on AB1.No dry gene powder treatment (NT) or 1% Luc-SFD or 1% p53-SFD treatment was followed by ALI culture with (+) or without (-) CDDP (a, b, d-g) or PEM (c).(a-c) At 24-h ALI culture, relative cell viability was examined by trypan blue staining.(a) 10 μM of CDDP.(b) 50 μM of CDDP.(c) Cells were treated with 0.025 μg/mL of PEM or 0.05 μg/mL of PEM or without PEM (PEM (-)).(d) 16-h ALI culture with or without 50 mM of CDDP.Apoptotic cells were examined with the TUNEL assay.(e-g) 16-h ALI culture with or without 50 μM of CDDP followed by cell cycle analysis.(e) The ratio of cells in the G1 phase to that of in the S phase was normalized by the value in NT CDDP (-).(f) The ratio of cells in the G2/M phase to that of in the S phase was normalized by the value in NT CDDP (-).n=3, excluding of (c, d), (n=4), mean and standard error, *; p-value < 0.05, N.S.; no signi cant difference.

Figure 2 The
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Figure 3 The
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Figure 4 The
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Figure 5
Figure 5 /M arrest (ref.45,46), these differences depend on cell types in MPM (ref.6).In summary, we demonstrated a new therapeutic approach against MPM, where p53-SFD enhanced CDDP tumor suppressive effects in AB1 and NCI-H2052.The synergistic effects may help decrease doses of CDDP.Furthermore, by increasing the pDNA content, p53-SFD might overcome the chemotherapyresistance and offer a therapeutic alternative for MPM patients who cannot receive current chemotherapeutic treatments.45.Lindström MS, Bartek J, Maya-Mendoza A. p53 at the crossroad of DNA replication and ribosome biogenesis stress pathways.Cell Death Differ.2022; 29:972-982.4 .Taylor WR, Stark GR.Regulation of the G2/M transition by p53.Oncogene.2001; 20:1803-1815.
table Table 1 is available in the Supplementary Files section.