Modulation of Autophagy Direction to Enhance Antitumor Effect of Endoplasmic‐Reticulum‐Targeted Therapy: Left or Right?

Abstract Strategies that induce dysfunction in the endoplasmic reticulum (ER) hold great promise for anticancer therapy, but remain unsatisfactory due to the compensatory autophagy induction after ER disruption. Moreover, as autophagy can either promote or suppress cell survival, which direction of autophagy better suits ER‐targeting therapy remains controversial. Here, a targeted nanosystem is constructed, which efficiently escorts anticancer therapeutics into the ER, triggering substantial ER stress and autophagy. Concurrently, an autophagy enhancer or inhibitor is combined into the same nanoparticle, and their impacts on ER‐related activities are compared. In the orthotopic breast cancer mouse model, the autophagy enhancer increases the antimetastasis effect of ER‐targeting therapy and suppresses over 90% of cancer metastasis, while the autophagy inhibitor has a bare effect. Mechanism studies reveal that further enhancing autophagy accelerates central protein snail family transcriptional repressor 1 (SNAI1) degradation, suppressing downstream epithelial–mesenchymal transition, while inhibiting autophagy does the opposite. With the same trend, ER‐targeting therapy combined with an autophagy enhancer provokes stronger immune response and tumor inhibition than the autophagy inhibitor. Mechanism studies reveal that the autophagy enhancer elevates Ca2+ release from the ER and functions as a cascade amplifier of ER dysfunction, which accelerates Ca2+ release, resulting in immunogenic cell death (ICD) induction and eventually triggering immune responses. Together, ER‐targeting therapy benefits from the autophagy‐enhancing strategy more than the autophagy‐inhibiting strategy for antitumor and antimetastasis treatment.

Bio-Rad ChemDoc XRS System (BioRad, USA). The semi-semiquantitative results were analyzed by image J. Also, the numbers of autophagosomes in 4T1 cells were investigated by immunofluorescence. 4T1 cells were treated with ED (5 μg mL -1 ) or autophagy modulators (70 μM AP I or 10 μM AP E ) for 24 h after seeding 6 × 10 5 4T1 cells in 12-well plates overnight. After incubation with LC3B primary antibody overnight and AF647-labelled goat anti-rabbit secondary antibody for one hour, cells were washed three times by PBS, fixed by 4% polyformaldehyde, and stained with DAPI (5 µg mL −1 ) for 5 min. Cells were imaged by CLSM to observe autophagosomes.

In vitro cytotoxicity investigation:
The evaluation of the in vitro cytotoxicity of two combinations against 4T1 cells was performed using 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. To initiate the experiment, 5 × 10 3 cells of 4T1 were seeded in each well of a 96-well plate and were allowed to incubate overnight. Following this, the cells were subjected to treatment with AP I (70 μM) and AP E (10 μM) in combination with a series concentrations of ED on 4T1 cells for 24 h. A solution of MTT (200 µL, 5 mg mL -1 ) was added to each well, and subsequently incubated for a period of 4 hours. The supernatant was discarded and the remaining formazan crystals were dissolved with 150 µL of dimethyl sulfoxide (DMSO). The absorbance was measured using Varioskan Flash multimode reader at a wavelength of 490 nm (Thermo scientific, USA). The cell viability was calculated using the following formula: Cell viability = (OD 490 sample -OD 490 blank ) / (OD 490 control -OD 490 blank )×100%. IC 50 values were determined by GraphPad Prism 9.0 software.

Preparation, characterization and drug release of co-loaded nanoparticles:
Nanoparticles were synthesized using the widely employed nanoprecipitation technique. Poly-lactic-co-glycolic acid (50μl, 20 mg mL -1 ), distearoylphosphoethanolamine-poly (ethylene glycol) (20μl, 20 mg mL -1 ), and ED (100 μg, 0.14 mol), AP I (160 μg, 1.07 mol) or AP E (40 μg, 0.04 mol) were dissolved in DMSO, while soybean phospholipids (20μl, 10 mg mL -1 ) were dissolved in methanol and DMSO in a 1: 1 mixture. Deionized water was gradually combined with the resulting organic mixture, which contained polymers, lipids, and medication, for 10 minutes. Dynamic light scattering was used to measure the nanoparticles' size distribution and zeta potential (DLS). Using ultraviolet spectroscopy GENESYS 180, the amount of drug that was encapsulated (We) and the total amount of drug (Wt) were calculated (Thermo Fisher Technologies, USA). To calculate the overall weight, the nanoparticle solution was thoroughly dehydrated (Wd). The formulas EE = We/Wt × 100% and DL = We/Wd × 100% were used to compute the entrapment efficiency (EE) and drug loading capacity (DL). After that, the nanoparticles were put in a dialysis bag (MWCO 3500 Da) and incubated in 50 mL of PBS with various pH levels (pH 7.4 or 6.5). 1 mL of the solution outside the dialysis bag was taken out and replaced with an equal volume of brand-new media at each predefined time interval.

In vivo distribution of nanoparticles:
Mice were intravenously administered either DiD or DiD@NP when tumors grew to a size of 200 mm 3 (125 μg kg -1 equivalent DiD dosage, n = 3). The IVIS Spectrum In Vivo Imaging System was used to take pictures of mice at set intervals (PerkinElmer, Lumina 3, USA). Mice were sacrificed after receiving therapy for 24 h, and the same procedures were followed to collect tumor and major organ tissues for ex vivo fluorescence imaging.

In vitro anti-metastasis effect:
Wound healing, migration assays, and invasion assays were employed to investigate the impact of anti-metastasis in vitro. To perform the wound healing experiment, 4T1 cells were seeded onto 24-well plates and subjected to treatment with various drugs with an equivalence DOX dose of 2.5 μg mL -1 and equivalence AP I dose of 70 μM or equivalence AP E dose of 70 μM for a period of 24 hours. The distance migrated was determined by measuring the wounds at 0 h and 24 h at the same scratched place, and subsequently estimated using Image J software. In the migration test, 1×10 5 4T1 cells were seeded into the top chamber of transwell inserts. hours prior to the seeding of 4T1 cells, and the invasion test was performed in a manner similar to the migration assay.

In vitro anti-metastasis mechanism investigation:
The expression of the metastasis-associated proteins (SNAI1, vimentin) and autophagy-related proteins (LC3B, p62) were measured by western blotting. The expression of E-cadherin was analyzed using flow cytometry. Briefly, 4T1 cells were seeded onto 12-well plates and subjected to treatment with various drugs with an equivalence DOX dose of 2.5 μg mL -1 and equivalence AP I dose of 70 μM or equivalence AP E dose of 70 μM for a period of 24 h. After incubation, cells were harvested and followed by fixation with 4% paraformaldehyde for 15 min. Then the cells were incubated with anti-E-cadherin antibody in 1% BSA for 30 min at 4 °C and were subsequent stained with AF647-labelled goat anti-rabbit secondary for 1 h at 4 °C, followed by flow cytometry analysis.

In vivo anti-metastasis efficacy:
4T1 cells (3 ×10 4 cells) were injected into the third mammary fat pad of BALB/c female mice on day 0. When tumor volume reached 100 cm 3 on day 7, mice were intravenously injected with saline, AP I @NPs, AP E @NPs, ED@NPs, (ED+AP I )@NPs, and (ED+AP E )@NP with an equivalence DOX dose of 5 μmol kg -1 and equivalence AP I dose of 122 μmol kg -1 or equivalence AP E dose of 17.4 μmol kg -1 every 3 days.
On day 21, tumors were sacrificed, the tumor issue and lungs of each group were collected. Lungs from mice of all groups were fixed with Bouin's solutions for 4 h and then the metastatic nodules were counted. The expression of the metastasis-associated proteins (SNAI1) and autophagy-related proteins (LC3B, p62) were measured by western blotting. Tumor issues were fixed in 4% paraformaldehyde for at least 48 h and embedded in paraffin immunohistochemistry analysis of LC3B, p62, and E-cadherin.

4T1 cells (3 ×10 4 cells) were injected into the third mammary fat pad of BALB/c
female mice on day -7. When tumor volume reached 100 cm 3 on day 0, mice were intravenously injected with saline, AP I @NPs, AP E @NPs, ED@NPs, (ED+AP I )@NPs, and (ED+AP E )@NPs with an equivalence DOX dose of 5 μmol kg -1 and equivalence AP I dose of 122 μmol kg -1 or equivalence AP E dose of 17.4 μmol kg -1 every 3 days.
The tumor size and body weight were recorded, and survival situations were recorded.
The major organs were collected and stained with H&E (hematoxylin and eosin) for safety analysis.
In vivo CD8 depletion assay: 4T1 cells (3 ×10 4 cells) were injected into the third mammary fat pad of BALB/c female mice on day -7. When tumor volume reached 100 cm 3 on day 0, mice were intravenously injected with saline, (ED+AP I )@NPs, (ED+AP E )@NPs, companied with/without CD8 depleting antibodies (100 μg per mouse in sterile saline) per 3 days, with an equivalence DOX dose of 5 μmol kg -1 and equivalence AP I dose of 122 μmol kg -1 or equivalence AP E dose of 17.4 μmol kg -1 . The tumor volumes of mice were measured every other day. On day 18, mice were sacrificed and the tumor issues were collected to weigh. Lungs were also collected and fixed with Bouin's solutions to count the metastatic nodules.

In vitro ER targeting efficiency:
In vitro ER dysfunction effect: In order to investigate the effects of ER damage, the cells were seeded on 12-well plates with 6 × 10 5 4T1 cells and grown for 24 h. Following this, cells were treated with various drugs with an equivalence DOX dose of 5 μg mL -1 and equivalence AP I dose of 70 μM or equivalence AP E dose of 70 μM for a period of 24 h. Then the cells were incubated with primary antibodies against CHOP, GRP78, and elf2 in 1% BSA for 1 h at 4 °C, and were subsequent stained with AF647-labelled goat anti-rabbit secondary for 1 h at 4 °C, followed by flow cytometry analysis. For the research of cellular Ca 2+ levels, 6 × 10 5 4T1 cells were seeded on 12-well plates overnight, subjected to various treatments (with an equivalence DOX dose of 5 μg mL -1 and equivalence AP I dose of 70 μM or equivalence AP E dose of 70 μM) for 12 h, stained with Fluo-4 AM (Cat No. 40704ES50; Yeasen, Shanghai, China) for 1 h, then incubated with medium for 30 min, and then subjected to flow cytometry analysis.

In vitro and in vivo ICD induction effect:
6 × 10 5 4T1 cells were seeded on 12-well plates, and the cells were cultured for 24 hours. Following this, the cells were treated with various drugs with an equivalence DOX dose of 5 μg mL -1 and equivalence AP I dose of 70 μM or equivalence AP E dose of 70 μM for a period of 24 h. Cells were harvested and washed with PBS, and incubated with anti-CRT primary antibody in 1% BSA for 1 h at 4 °C. Then Alexa Fluor647-labeled goat-anti-rabbit secondary antibody was added, the surface CRT on cells was detected by flow cytometry. To quantify in vitro ATP secretion and HMGB1 release, 6 × 10 5 4T1 cells were seeded on 12-well plates, and the cells were cultured for 24 h. Following this, the cells were treated with various drugs with an equivalence DOX dose of 5 μg mL -1 and equivalence AP I dose of 70 μM or equivalence AP E dose of 70 μM for a period of 24 h. Cell supernatants were collected and were and tested via ATP Elisa kits. The cellular and extracellular HMGB1 levels in treated cells were determined by western blotting. As for in vivo ICD induction capability, the 4T1 cells (3 ×104 cells) were injected into the third mammary fat pad of BALB/c female mice and were intravenously injected with saline, AP I @NP, AP E @NP, ED@NP, (ED+AP I )@NP, and (ED+AP E )@NP with an equivalence DOX dose of 5 μmol kg -1 and equivalence AP I dose of 122 μmol kg -1 or equivalence AP E dose of 17.4 μmol kg -1 every 3 days when tumor volume reached 100 cm 3 . After various days, the tumors in each groups were collected and the tumor interstitial fluid was obtained by grinding the tumor tissues. The same method as in vitro assays was used to determine CRT levels in tumor issues. The tumor interstitial fluid were collected to investigate ATP and HMGB1 levels by ATP Elisa kits and HMGB1 Elisa kits.

In vivo immune status investigation:
4T1 cells (3 ×10 4 cells) were injected into the third mammary fat pad of BALB/c female mice on day 0. When tumor volume reached 100 cm 3 on day 7, mice were intravenously injected with saline, AP I @NP, AP E @NP, ED@NP, (ED+AP I )@NP, and (ED+AP E )@NP with an equivalence DOX dose of 5 μmol kg -1 and equivalence AP I dose of 122 μmol kg -1 or equivalence AP E dose of 17.4 μmol kg -1 every 3 days.
The mice were sacrificed on day 14 and the tumor issues were collected. To assess ICD induction, cells in tumor issues were stained with anti-CD45-PerCP/Cy5.5 and anti-CRT-antibodies for 1 hour at 4 °C. The cells were then washed and stained for 45 minutes with a second Alexa Fluor 647-conjugated secondary antibody. The cells were then cleaned before being analyzed by flow cytometry. To assess the infiltration of T lymphocytes in the tumor issues, cells in tumor issues were treated with anti-CD16/32 antibodies at 4 °C for 20 minutes, then stained with anti-CD3-FITC, anti-CD8-APC, and anti-CD4-PerCP/Cy5 antibodies to measure cytotoxic T lymphocytes (CD3 + CD4 -CD8 + ) and T effector cells (CD3 + CD4 + CD8 -) in tumor issues.
The cells were cleaned before being subjected to a flow cytometry analysis.

Statistical analysis:
Results were presented as mean ± standard deviations (SD). Statistical analysis was calculated by one-way ANOVA analysis using SPSS 22.0 software. P value <0.05 was recognized as statistically significant. Figure S1: (1)   Data was presented as mean ± SD. n = 5.