Tissue specimens and serum samples

Serum samples of 8 chemotherapy-sensitive patients and 9 chemotherapy-resistance patients were collected from the Beijing Chest Hospital. Six paraffin-embedded tissue specimens from chemotherapy-sensitive patients and six from chemotherapy-resistance patients were also obtained from Beijing Chest Hospital. All samples were confirmed via pathologic examination. Informed consent was obtained from all patients prior to specimen collection, in accordance with institutional guidelines. All protocols were approved by the Institutional Review Board and informed consent was obtained from the patients or their guardians.

Cell lines, reagents, and antibodies

The lung cancer cell lines NCI-H446, NCI-H196 were obtained from the Chinese National Infrastructure of Cell Line Resource. Cells were maintained in RPMI1640 supplemented with 10% FBS (VisTech, New Zealand). All cell lines were cultured at 37 ℃ with 5% CO2. The H446/cDDP, H446/VP-16, H446/PTX, H196/cDDP, H196/VP-16, H196/PTX chemo-resistant SCLC cell lines were established by our laboratory. H446 cells or H196 were treated with increased concentration of cisplatin, VP-16, or PTX. After 9–11 months of induction and screening, the final surviving cells showed resistance against cisplatin, VP-16 or PTX. Cisplatin and venetoclax were purchased from MedChemExpress (Cat#HY-17394, Cat#HY-15531). Hydroxychloroquine (HCQ) was purchased from MedChemExpress (Cat#HY-W031727) and Chongqing Kangle Pharmaceutical Co., Ltd (Cat#747-36-4). Etoposide (VP-16) and Paclitaxel (PTX) was purchased from meilunbio (Cat#MB1102-1 and Cat#MB1178-S). Commercially available antibodies are as follows: Anti-β-actin (HUABIO, Cat#R1207-1), anti-p62/SQSTM1 (Proteintech, Cat#18420-1-AP), anti-LC3B (HUABIO, ET1701-65), anti-casepase3 (Abcam, ab184787), anti-BCL2 (Abways, Cat#CY6717), anti-Beclin1/BECN1(Proteintech, Cat#11306-1-AP), anti-PIK3C3/VPS34 (Proteintech, Cat#12452-1-AP), anti-ATG14 (ABclonal, Cat#A7526), anti-Ago2 (CST, Cat#2897S), ANTI-FLAG (Proteintech, Cat#80010-1-RR).

Lentivirus package, plasmids construction, siRNAs, and transfection

The DNA fragment of LYPLAL1-DT was inserted into pLenti-GIII-EF1α-Luc-2 A-Puro Kan vector (Canada, ABM) and the LYPLAL1-DT shRNA fragment was ligated to the pHBLV-U6-MCS-CMV-ZsGreen-PGK-PURO vector (HANBIO, Shanghai, China). The overexpression lentivirus vector of LYPLAL1-DT, the knockdown and negative controls infected the H446, H446/cDDP and H196 cell lines for 48 h. Subsequently, cells were selected with 2 µg/mL puromycin (Invivogen, Cat#ant-pr-1) for 3 weeks. Furthermore, qRT-PCR was used to examine the overexpression and knockdown efficiency of these cells. The mRFP-GFP-LC3 lentivirus (HANBIO, Shanghai, China) was also infected into the H446 and H446/cDDP cells. The BCL2 expression vector and control vector were purchased from HANBIO and transfected into the cells by using Lipofectamine 3000 (Invitrogen, CA, USA, Cat#L3000015). For the knockdown of LYPLAL1-DT and BCL2, siRNAs or negative control siRNA were transfected via an RNAfit reagent (HANBIO, Shanghai, China), following the manufacturer’s instruction. The miR-204-5p mimics, inhibitors and negative controls were also purchased from HANBIO. The sequences of miR-204-5p and siRNAs for LYPLAL1-DT and BCL2 are listed in Table S1.

Western blot

Cells were lysed and then heated in 1×loading buffer (ROBY, Cat#RBU111-1) for 10 min at 99 ℃. The protein samples were separated using SDS-PAGE and transferred onto a nitrocellulose filter membrane (Pall, Cat#51146359). The membranes were blocked with 5% skimmed milk (BD, Cat#232100) for 1 h at room temperature and incubated with primary antibody overnight at 4 °C. Subsequently, the membranes were incubated with secondary antibody for 1 h at room temperature. Protein signals were detected using NcmECL Ultra (NCM Biotech, Cat#P10300) according to the manufacturer’s instructions.

RNA extraction and qRT- PCR

Total RNA was extracted using TRIzol (Vazyme, Cat#R401-01) and mRNA was reverse transcribed using 5X All-In-One RT MasterMix (with AccuRT Genomic DNA Removal Kit) (ABM, Cat#G492). qRT-PCR was performed using EvaGreen 2X qPCR MasterMix (ABM, Cat#MasterMix-S). mRNA level was quantified by the 2-ΔΔCt algorithm with β-actin as the normalizer gene, and miRNA expression was normalized to U6. All the primers used are listed in Table S2.

Cell viability assay

For CCK-8 assays, cells were seeded into 96-well plates at a density of 3,000 cells per well for 100 µL. After 12 h of culture, the cells were treated with chemotherapeutic drugs, venetoclax or HCQ, at the indicated concentration for 12 to 72 h. Then, 10 µL of CCK-8 (Gene-Protein-Link, Cat#P04D30X) was added to each well. The cells were then incubated at 37 ℃ for 2 h, and the optical density was measured at 450 nm using a BioTek ELx800 Microplate reader.

Combenefit analysis

Combenefit plots for the combination-dose response of venetoclax with cisplatin and HCQ were generated. H446/cDDP cells were treated with a combination of 2 µg/mL cisplatin and venetoclax (0–160 µM) or a combination of 2 µg/mL cisplatin and HCQ (0–50 µM). Cell viability was then measured by CCK-8 assay after 72 h. Synergy was mapped to drug-dose response using the Loewe model, calculated by SynergyFinder (https://synergyfinder.fimm.fi). Loewe model is based on expected response corresponding to a combination rather than the single drugs with increasing dose. Relative synergy scores are color-coded, where red indicates synergy, white indicates a lack of synergy and green indicates antagonism based on respective synergy scoring. For any doses xA, xB, …,xN of drugs A,B,…,N, Loewe synergy score, SLOEWE, is defined as: SLOEWE= EA, B,…,N- ELOEWE, where ELOEWE should satisfy: Σk = A, B,…,N(xkfk-1(ELOEWE)) = 1.

Hoechst-33342 staining for detecting cell apoptosis

Hoechst-33342 staining was performed according to previous report [32]. After cell culture and treatment with chemotherapeutic drugs, the medium was discarded, and the cells were fixed in 4% paraformaldehyde for 15 min. Subsequently, 200 µL of 100 µg/mL Hoechst-33342 (Solarbio, Cat#C0021) was added to cover the cells, and incubated for 15 min in the dark. Afterwards, cells were gently washed three times with phosphate buffer saline (PBS) to prevent the loss of apoptotic cells, and cellular morphology was observed using fluorescence microscopy. Cells showing intensely condensed nuclei and/or nuclei fragments were considered apoptotic.

Annexin V-FITC/PI apoptosis detection

After the cell treatment, both floating and adherent cells were collected together. Using Annexin V-FITC/PI Apoptosis Detection Kit (Vazyme, Nanjing, China, Cat#A211-01) following the manufacturer’s instructions, the cells were washed with PBS and resuspended in 100 µL binding buffer (1 × 105 cells per group). Next, 5 µL Annexin V-FITC and 5 µL propidium iodide staining solution were added to each tube. After mixing, cells were incubated at 37 ℃ for 10 min in the dark. Subsequently, an additional 400 µL of binding buffer was added to each tube, and the flow cytometry analysis was carried out using BD LSRFortessa flow cytometry.

RNA immunoprecipitation (RIP) assay

RIP analysis was performed using Magna RIPTM RNA-Binding Protein Immunoprecipitation Kit (Millipore, Billerica, MA, USA, Cat#17–700) following the manufacturer’s protocol. H446 and H446/cDDP cell lysates were incubated with magnetic beads conjugated with anti-BECN1, anti-PIK3C3 or Ago2 antibody (CST, USA) and IgG antibody at 4 °C for 6 h. The magnetic beads were washed 4–6 times with RIP washing buffer to remove non-specific adsorption as much as possible. The protein-RNA complex with magnetic beads were digested with 0.5 mg/mL proteinase K at 55 ℃ to purified RNA and eluted RNA from magnetic beads. Finally, the extracted RNA was reverse transcribed and analyzed by qRT-PCR.

Dual-luciferase reporter assay

The LYPLAL1-DT sequence, containing 2577 bp, and the 3’-UTR of BCL2, containing 5279 bp, were cloned and inserted into the psi-check2 plasmid. The miR-204-5p target site mutations of LYPLAL1-DT and BCL2 3’-UTR luciferase reporter plasmids were generated by HANBIO (Shanghai, China). H446 and H446/cDDP cells were seeded in a 6-well plate and co-transfected with the constructed recombinant plasmids (2 µg/well) or control plasmids along with miR-204-5p mimics. After 48 h, luciferase and Renilla luciferase activities were measured separately using the Dual-Luciferase Reporter Assay Kit, following the manufacturer’s instruction (Vazyme, Cat#DL101-01). The ratio of renilla luciferase luminescence to firefly luciferase luminescence was then calculated.

Determination of mitochondrial membrane potential

The enhanced mitochondrial membrane potential assay kit with JC-1 (Beyotime, Cat#C2003S) was used. JC-1 was diluted by adding 1 mL JC-1 dyeing buffer per 5 µL JC-1 (200×). The cells were inoculated in confocal small dish. Removed the dish after drug treatment and cleaned with PBS. Next, 1 mL of cell culture medium and 1 mL of JC-1 dyeing solution were added and incubate at 37 ºC for 20 min, and then removed the supernatant and washed the cell with JC-1 staining buffer. Finally, added 2 mL of cell culture medium and observed under laser confocal microscope. Set excitation wavelength to 490 nm and the emission wavelength to 530 nm to detect JC-1 monomers. Set excitation wavelength to 525 nm and the emission wavelength to 590 nm to detect JC-1 aggregates.

Determination of caspase activity

The activity of caspase-3 was measured using the Caspase3 Activity Detection Kit (NJJCBIO, Cat#G015-1). Briefly, cells were collected after drug treatment. Next, 50 µL of lysis buffer with 1% DTT was added for 30 min, followed by the addition of 50 µL 2×reaction mixture, 5 µL acety-Asp-Glu-Val-Asp p-nitroanilide (Ac-DEVD-pNA), and overnight incubation at 37 °C. The related caspase activity was quantified at 450 nm using a BioTek ELx800 Microplate reader.

Colony formation assay

H446/cDDP cells were seeded into 6-well plates with 200 cells per well. After 12 h of incubation at 37 °C, the cells were treated with cisplatin for 3 days every week, venetoclax and HCQ for 14 days, during which the medium was changed every 3 days. The cells were fixed with 4% paraformaldehyde fix solution (APPLYGEN, Cat#B1057) for 30 min and strained with 0.01% crystal violet (APPLYGEN, Cat#B1087). Images were obtained, and the number of colonies was counted.

Transmission electron microscopy (TEM)

Cells were collected and fixed in 2.5% glutaraldehyde for 30 min at room temperature. Subsequently, the fixed cells underwent dehydrated using a graded ethanol series. A graded series of acetone was applied to the samples, which were then embedded in Epon. The embedding blocks were sliced at a thickness of 50–70 nm for follow-up observation. Electron microscopy was performed using the HT7700 transmission electron microscope (HITACHI, Japan).

Immunofluorescence and confocal microscopy

Cells were seeded into glass bottom cell culture dishes (NEST, Cat#801001) at 2 × 105 cells per well. After treatment with the indicated chemotherapeutic drugs for a specific duration, cells were washed with PBS and fixed in 4% paraformaldehyde for 30 min. Subsequently, the cells were blocked for 1 h with 5% BSA, followed by overnight incubation with a primary antibody at 4 ℃. The next day, cells were washed with PBS three times, and then incubated with secondary antibodies for 1 h at room temperature. Finally, 1 mg/mL Hoechst-33342 was added to stain nuclei, and the cells were photographed under a fluorescence microscope.

IF/FISH

Fluorescence-conjugated probes, designed and purchased from GenePharma (Shanghai, China), were used for IF/FISH. Cells treated with chemotherapeutic drugs were hybridized to fluorescence-conjugated probes according to the manufacturer’s instructions (GenePharma, Shanghai, China). The probe sequences were as follows: LYPLAL1-DT, 5′-GGGTTTCGTCGAGGCTTGGGTCGG-3′. Anti-BECN1, anti-PIK3C3 and secondary antibodies were added for immunofluorescence. The samples were counterstained with Hoechst and observed by confocal microscopy.

Autophagic flux analysis

To analyze autophagic flux, cells were stably transfected with mRFP-GFP-LC3 Lentivirus (HANBIO, Shanghai, China) following the manufacturer’s instructions. Subsequently, the cells were treated with chemotherapy drugs for the designated time, and the expression levels of GFP and mRFP were observed using confocal fluorescence microscopy.

Co-immunoprecipitation (Co-IP)

For Co-IP, cells were lysed in RIPA buffer on ice for 30 min, and the supernatants were collected by centrifugation at 4 °C and 15,000 g for 15 min. Discard the precipitate, the cleared protein lysates were then incubated overnight at 4 °C with magnetic beads bound with primary antibody. The following day, the beads were collected, washed 5 times with 0.5 mL washing buffer and SDS loading buffer was added. The samples were denatured at 99 °C for 10 min. Finally, the supernatants were collected, stored at -80 °C, or immediately analyzed by Western blot.

Immunohistochemistry (IHC)

For immunohistochemical study, after dehydration and antigen repair, tissue sections were blocked using 10% BSA, then incubated overnight at 4 °C with primary antibodies (1: 200 dilution) against BCL2 and BECN1. Subsequently, the sections were incubated with a secondary antibody (1: 200 dilution) at room temperature for 1 h. IHC staining was visualized using the DAB Immunohistochemistry Color Development Kit (ZSGB-BIO, Cat#ZLI-9019) according to the manufacturer’s instructions. After washing 3 times and counterstaining with hematoxylin, slides were dehydrated with sequential ethanol washes and sealed. The expression of BCL2 and p62 was analyzed and evaluated using optical microscopy.

Subcutaneous xenograft experiments and patient-derived xenograft studies

For subcutaneous xenograft studies, H446 cells (stably overexpressing LYPLAL1-DT or an empty vector) or H446/cDDP cells (2 × 106 / 200 µL) were inoculated subcutaneously into 4- to 6-week-old NTG male mice (SiPeiFu, Beijing, China). For patient-derived xenograft studies, NTG mice were obtained from IDMO (Beijing, China). Cryopreserved PDX tumors (No.A7391, No.21031902, No.20090907, No.19111114) were implanted subcutaneously into the mice. Subsequently, four replicates were randomly selected per group for the following experiments. Mice were treated with cDDP, PTX, VP-16, venetoclax and HCQ after the tumor volume reaches 50–100 mm3. cDDP (1.5 mg/kg), VP-16 (5 mg/kg) and PTX (5 mg/kg) were administered via intraperitoneal injection (i.p.) once a week. Venetoclax (50 mg/kg) was administered by oral gavage (i.g.) and HCQ (15 mg/kg) was given by intraperitoneal injection (i.p.) every 2 days. Treatment study continued for 2–3 weeks. The lengths and widths of tumors were measured every two days until the endpoint. Tumor volume was calculated as height×width×width×0.5. The studies were approved by the Committee on the Ethics of Animal Experiments of Capital Medical University (No. AEEI-2019-089).

Statistical analysis

Graphing and Statistical analysis were performed using Graphpad Prism. Data were presented as mean ± SEM, and statistical significance was determined by Student’s t-test (t-test) or two-way ANOVA, as indicated in the figure legend. The sample size (n) is also reported in the figure legend for each experiment, with ‘n’ representing the number of identically treated replicates. All tests were two-tailed, and P values < 0.05 were considered statistically significant.