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Methionine orchestrates the metabolism vulnerability in cisplatin resistant bladder cancer microenvironment – Cell Death & Disease

Republished By Plato
Republished By Plato

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Cell lines

Human and murine bladder cancer cells SV-HUC-1, RT4, UM-UC-3, T24, 5637, MB49 were obtained and cultured as previously described [44]. For inducing the cisplatin resistant T24, UMUC-3 and MB49 cells, cells were cultured in cisplatin containing medium with the cisplatin concentration increasing from 1 μM to 25 μM for 10 months.

Tissue microarray and patient samples

The Huashan cohort tissue microarray is composed of 90 pairs of normal and malignant tissues who received radical cystectomy with bladder cancer, between 2007-01 to 2013-01, with a 5-year follow-up [44].

The tissue microarray used in this study came from 90 cases of bladder cancer who received radical cystectomy in our center from January 2007 to January 2013. The inclusion and exclusion criteria are as follows:

  1. 1.

    Inclusion criteria:

  1. a.

    Radical cystectomy was performed at our center, and complete pathological specimens are available. The pathological specimen was confirmed as bladder cancer by two pathologists. The specimen contains tumors and normal tissues.

  2. b.

    Age: 18-80 years old

  1. 2.

    Exclusion criteria:

  1. a.

    The patient’s clinical information is incomplete.

  2. b.

    The quality of pathological samples is unqualified.

  3. c.

    Have a history of bladder perfusion therapy within 3 months before surgery or have adjuvant chemotherapy/radiation therapy within 6 months before surgery.

Written consent was obtained from patients and was approved by the Ethics Committee of Huashan Hospital (approval number KY2011-009, 2022-100). Patient sample for circRNA sequencing and expression analysis were also collected as the same procedure.

Metabolomics analysis

T24/T24-CR cells and T24-CR-LV-NC/T24-CR-LV-circARHGAP10OE cells were used for metabolomics analysis. Medium was discarded after reaching confluence, and cells were washed with PBS for 3 times. Cells were scraped in 2 mL cooled methanol and lysed to extract metabolites as previously described [44].

Proteomics analysis

20 µg extracted protein was separated through SDS-PAGE. Protein was digested with trypsin and LC-MS/MS was performed with a timsTOF-Pro spectrometry coupled to Nanoelute (Bruker). Raw protein data was searched with MaxQuant 1.6.14 software for quantitation analysis. Following annotation steps, the proteins were blasted to retrieve their KEGG identifications. Metabolomics and proteomics analysis and the integrated multi-omics analysis of this work were completed in Applied Protein Technology, Shanghai, China.

RNA-seq

RNA was extracted from five paired of benign and malignant bladder tissues with TRIzol (Invitrogen, USA). 3 mg total RNA was extracted and for further sequencing as previously described [41].

RNA FISH and immunofluorescence

Probes targeting the junction site of circARHGAP10 were synthesized (GenePharma Biotech, Guangzhou, China). IF and photographed procedure were performed as previously described [41]. All cell were fixed and labeled with DAPI and cell were photographed with the LSM700 confocal microscope (Carl Zeiss, Oberkochen).

RNase R treatment

Total RNA was extracted and treated with RNase R (Epicenter Technologies, USA). Stability of circRNA and lineal mRNA was analyzed via qRT-PCR [44].

Actinomycin D assays

2 mg/mL actinomycin D (Sigma) was added to the cells and the BCa cells were collected at indicated time points for further analysis as previously described [41, 44].

qRT-PCR

RNA was extracted and cDNA was obtained by using the PrimeScriptTMRT Reagent Kit (TaKaRa,Japan) to perform qRT-PCR on an ABI 7900HT machine (Thermo Fisher Scientific, USA). 2-ΔΔCT was used to analyze RNA expressions [44].

Vector construction and cell transfection

shRNAs sequences were designed to direct target circRNAs, MAT2A, TRIM25 or SLC7A6 were cloned and inserted into pGPU6/GFP/puromycin vector. circRNA sequence was cloned and insert into a plenti-ciR-GFP-T2A vector from IGEbio (Guangzhou, China) to construct circRNA over-expression plasmid. To overexpress MAT2A or SLC7A6, the vector pENTER with identical gene or control was obtained from Vigene (Shanghai, China). Flag-TRIM25, Flag-Trim25ΔRBD, Myc-MAT2A, HA-Ub and the K6, K11, K27, K48 and K63 mutant plasmid was purchased from Vigene (Shanghai, China). The siRNAs were purchased from GenePharma (Shanghai, China).The cell transfection were described as our previous work [43].

Sphere formation assay

BCa cells were digested and suspended as single cells in the 6-well ultra-low attachment plates, supplemented with 5 ng/mL recombinant EGF (Sangon, China). The sphere formation was observed 12 days later.

Cell proliferation assay

In CCK-8 assay, cells were digested and seeded at 1,500 cells per well into a 96-wells plate. We detected the OD450 value at various time points after seeding into the wells. For IC50 test, cells were incubated with cisplatin and the relative viability rate was calculated in Graphpad Prism 8 to calculate the IC50 value. For the colony-formation assay, cells were digested and seeded into 6-well plate and incubated for 14 days. The colonies were imaged after stained with 0.2% crystal violet.

Apoptosis and EdU assay

Apoptosis assay was performed with the apoptosis kit (MultiSciences, China) as our previous work [41]. EdU assay was performed with EdU kit (RiboBio, China) to detect DNA synthesis [44].

Transwell assays

Cells were stained by 0.2% crystal violet in a Transwell chamber (CoStar, USA). Five randomly selected sections of cells were analyzed for generating statistical analysis.

Molecular docking

We used Autodock Vina 1.2.2 to analyzed the binding affinities between proteins. The 3D coordinates of the proteins were downloaded from the PDB (http://rcsb.org/PDB). Molecular docking was visualized and performed with AutodockVina 1.2.2 (http://autodock.scripps. edu).

Co-immunoprecipitation

For co-IP experiment, relative cells were lysed with cooled supplemented with a combination of proteinase and phosphatase inhibitors, with or without RNase inhibitor. The supernatant was incubated with antibody conjunctive or antibody pre-incubated beads. The beads were washed and centrifuged with co-IP buffer for further western blot analysis [53].

RNA pull-down assay and RIP assay

Streptavidin beads (Life Technologies, USA) were treated with biotin labeled circARHGAP10 probes with oligo probes to perform RNA pull-down assay. The magnetic RIP kit (Millipore) was used to perform the RIP assay according to the instructions of the manufacture described as our previous work [43].

Western blot analysis

Protein were lysed from cells with RIPA buffer and separated with SDS-PAGE. The blots were blocked and incubated with antibodies overnight. Antibody: GLDC (Abcam, ab232989), SHMT2 (Abcam, ab180786), MAT2A (Proteintech, 55309-1-AP), MTHFR (Abcam, ab203786), SAHH (Abcam, ab151734), CD44 (Abcam, ab243894), Nanog (Abcam, ab109250), H3 (Abcam, ab1791), H3K4me3 (Abcam, ab213224), H3K9me3 (Abcam, ab8898), H3K27me3 (abcam, ab6002), H3K36me2 (Abcam, ab176921), and H3K36me3 (Abcam, ab9050), H3K79me2 (Abcam, ab3594), and H3K79me3 (Abcam, ab2621), Flag/DDDDK tag (Abcam, ab205606), Myc tag (Abcam, ab32), HA tag (Abcam, ab9110), TRIM25(Abcam, ab167154), USP5(Abcam, ab154170), USP14(Abcam, ab192618), p-STAT5(Abcam, ab32364), STAT5(Abcam, ab230670), SLC7A6(Abcam, ab235054), GAPDH (Proteintech, 60004-1-lg) was used as loading control. The signals were visualized using an ECL imaging system (CLiNX, Shanghai) as previously described [44].

Compounds and reagents

The compounds or enzymes used in this work are as follows: cisplatin (Selleck, USA), FIDAS (Selleck, USA), MeAIB (Selleck, USA), BCH (Selleck, USA), RNase A (Yeason, China), SAM (Sigma, USA), SAH (Sigma, USA) and HCY (Sigma, USA). Medium without certain amino acids were purchased from US Biological,USA. Amino acids were purchased from Sangon, China.

In vitro CD8 + T cell preparation and activation

Fresh tumor samples were obtained soon after sacrificed of the animals. Tumors were minced and digested with a combination of collagenase I (Sangon,China) and DNase I (Sangon, China). PBMCs were derived from patients with Ficoll (GE Healthcare, USA). The cells were stimulated, stained with membrane markers and applied to MACS column for further analysis.

Flow cytometry

CD8 + T cells exacted from peripheral blood or cancer tissue were collected. The single-cell suspensions of CD8 + T cells were stained with surface markers with CD45 (BD Biosciences, 557833), CD3 (BD Biosciences, 552852), CD8 (BD Biosciences, 564526) and PD-1 (BD Biosciences, 561272) [44]. BD Cytofifix/Cytoperm Fixation/Permeabilization Solution Kit (BD Biosciences) was used to stain intracellular markers including IFN-g (BD Biosciences, 557643), GZMB (BD Biosciences, 560212), and IL-2(BD Biosciences, 554566). CD8 + T cells were analyzed on the BD FACS Verse Flow Cytometer as we previously described [44].

Animal study

For the subcutaneous T24-CR cell xenograft model, 4-week-old nude male athymic BALB/c mice (SLARC, Shanghai, China) were maintained in specific pathogen free culture environment. The control and methionine restriction diets were purchased from Xietong (Jiangsu, China) which control diet contained 0.86% methionine nutrition and MR diet contained 0.12% methionine nutrition in our study [30]. The mice were randomly selected into four groups. A total of 1 × 107 T24-CR-luc-LV-NC or T24-CR-luc-LV-circ cells were injected subcutaneously into nude mice fed with control or MR diets. Cisplatin (6 mg/kg) was injected intraperitoneally one week after cell implantation (one injection for every 5 days). Tumor volume was monitored every 3 days with the calculation of tumor volume = width2 × length/2. All mice were sacrificed after 6 weeks of drug treatment. The extreme limiting dilution analysis were performed with 5 × 106, 5 × 105 and 5 × 104 T24-CR cells subcutaneously injection into the nude mice to calculate the successful rate of the tumor implantation.

For the patient-derived xenograft (PDX) models, two fresh fragments of tissue of BCa patients were transplanted subcutaneously into 4-week-old NOD/SCID mice (SLARC, Shanghai, China). When the 1st generation of the xenografts reached approximately 100 mm3, the PDX tissue block were isolated and mechanically dissolved into 1 mm3 tissue blocks for the subcutaneously implantation into NOD/SCID mice for the 2nd PDX generation. The 2nd PDX generation was divided randomly into four groups. The PDX xenografts were injected with 5 nmol in vivo-grade cholesterol-conjugated si-NC or si-circARHGAP5 (RiboBio, Guangzhou, China) fed with control or MR diets. Cisplatin (6 mg/kg) was injected intraperitoneally one week after cell implantation (one injection for every 5 days). Tumor volume was monitored every 3 days with the calculation of tumor volume = (width2 × length/2). All mice were sacrificed after 6 weeks of 2nd PDX implantation.

For the lung metastasis model of T24-CR cell, 1 × 105 T24-CR-luc-LV-NC or T24-CR-luc-LV-circ cells were injected intravenously into the tails of 4-week-old nude male athymic BALB/c mice (SLARC, Shanghai, China) fed with control or MR diets. Cisplatin (6 mg/kg) was injected intraperitoneally one week after cell injection (one injection for every 5 days). After 30 days, metastasis of the tumor development was monitored via IVIS following intraperitoneal injection of 200 mg/kg luciferin.

For the subcutaneous MB49-CR cell xenograft, 4-week-old male C57BL/6 mice (SLARC, Shanghai, China). A total of 1 × 107 MB49-CR-LV-NC or MB49-CR-LV-circ cells were injected subcutaneously into mice fed with control or MR diets. Anti-PD-L1 and IgG1 (Bioxcell) (100 μg/mouse) were injected intraperitoneally one week after cell implantation (one injection for every 3 days). BCH (180 mg/kg) was injected intravenously one week after cell implantation (one injection for every 3 days). Cisplatin (6 mg/kg) was injected intraperitoneally one week after cell implantation (one injection for every 5 days). Tumor volume was monitored every 3 days with the calculation of tumor volume = (width2 × length/2). All mice were sacrificed after 6 weeks of drug treatment. All animal experiments including in this study were approved with the approve number 202212002 S.

IHC

Tumor tissue samples and mouse liver tissue were embedded in paraffin while IHC and H&E staining was performed [44, 54]. The primary antibodies were Ki-67 (Abcam, ab270650), MAT2A (Proteintech, 55309-1-AP), SLC7A6(Abcam, ab235054), CD44 (Abcam, ab243894) and CD8A (Abcam, ab217344).

Statistical analysis

The statistical analysis was analyzed with Student’s t-test or the Chi-square test in Graphpad Prism 8 [55]. K-M curve was applied for the overall survival analysis [56, 57]. We determined that p < 0.05 was statistically significant.

Reporting summary

Further information on research design is available in the Nature Research Reporting Summary linked to this article.

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