Cell traces
Murine LLC cell line (#CRL-1642) was bought from the American Kind Tradition Assortment. B16-F10 cell line was a present from J. Tao of the Division of Dermatology, Wuhan Union Hospital, Tongji Medical School, Huazhong College of Science and Expertise. Subsequently, LLC cells had been transfected with the pcDNA3-OVA plasmid (Genecreale) to acquire LLC-OVA cells. Mouse T cell hybridoma cells B3Z (#BFN608006444) had been bought from Bluefbio Biology Expertise Growth. Murine lung epithelial cells (MLE, #iCell-m036), murine colon adenocarcinoma cell line (MC38, #iCell-m032) and human bronchial epithelioid cells (BEAS-2B, #iCell-h023) had been bought from the Cellverse. Murine embryonic fibroblast cell line (NIH/3T3, #CL-0171) was bought from the Pricella. These cell traces had been cultured in DMEM (Gibco) supplemented with 10% FBS (Newzerum) at 37 °C in 5% CO2–95% air.
Mice
C57BL/6 mice had been bought from Hubei Beiente Biotechnology. They had been saved in particular pathogen-free facility within the Wuhan Laboratory Animal Heart of Tongji Medical School. All animal-related procedures have been carried out in line with the Declaration of Helsinki and gained permission from the Animal Care and Use Committee of Tongji Medical School ((2023) IACUC quantity 3958).
Preparation and isolation of MW-TMPs
We established an MW-assisted methodology to gather TMPs from tumour cells. After 24 h of incubation underneath commonplace situations, the cell tradition medium was changed with a serum-free medium. The Petri dishes had been subsequently positioned right into a home MW oven (setting situation 700 W, Midea) and subjected to MW heating for 20 s, a situation we designated as H20. To match the merchandise from completely different MW situations, a number of settings had been utilized to LLC cells: L10 (175 W, 10 s), L20 (175 W, 20 s), L30 (175 W, 30 s), H10 (700 W, 10 s), H20 (700 W, 20 s) and H30 (700 W, 30 s). After 48 h of tradition, the cell-conditioned medium from every MW remedy was collected for isolation of MW-TMPs. TMPs had been extracted in line with our earlier examine through a serial centrifugation technique9. Briefly, the medium was centrifuged at 200 × g for 10 min and a couple of,000 × g for 30 min at 4 °C to take away cells and particles, respectively. Second, pellet was obtained after 18,000 × g for 60 min at 4 °C after which resuspended in PBS. Lastly, MW-TMPs had been washed as soon as after which saved utilizing PBS. For drug-loaded MW-TMPs, the incubation methodology was used to encapsulate MW-TMPs with small molecules, akin to MTX. After MW remedy, 200 μM MTX was added into the tradition medium and the extraction of MW-TMPs-MTX adopted the aforementioned procedures.
TMT quantification proteomics
UV-TMPs had been ready and remoted based mostly on our former protocols10. Considerable proteins of UV-TMPs (named as U1, U2 and U3) and MW-TMPs (named as M1, M2, M3 and M4) as Mus_musculus samples had been recognized through single mass spectrometry run utilizing the TMT quantitative methodology. Completely, 747,975 spectrums had been generated, 19,595 peptides and three,967 proteins had been detected with 1% false discovery price, and the additional protein annotation with the assistance of a number of bioinformatic databases, together with KEGG and Gene Ontology, was carried out. For differential protein evaluation, an automatic software program, named IQuant, was utilized and proteins with 1.5-fold change and P < 0.05 had been considered differential expression12.
Excessive-performance liquid chromatography of MW-TMPs-MTX
MW-stimulated LLC cells had been incubated with 50 μM, 100 μM, 200 μM and 300 μM MTX for twenty-four h. MW-TMPs-MTX was collected and resuspended in ddH2O. Samples had been sonicated for 10 min and handed by means of a 0.2 μm water movie. As for the cumulative MTX launch evaluation by MW-TMPs, ready MW-TMPs-MTX had been incubated within the surroundings of pH = 7.4 and 37 °C and the stirring of 200 rpm at intervals of 4 h, 6 h, 12 h and 24 h. Then high-performance liquid chromatography was utilized to detect the drug content material in MW-TMPs. Cellular part (part A, 0.025 mol l−1 potassium dihydrogen phosphate buffer (pH 5.5); part B, acetonitrile) with the liquid samples (MTX or MW-TMPs-MTX) went by means of the chromatographic column (Athena-C18, temperature, 30 °C; movement price, 1.0 ml min−1) underneath 303 nm wavelength within the mannequin of isocratic elution. Based on the chromatograms of requirements and samples, the focus of MTX loaded in MW-TMPs-MTX was analysed. Drug loading effectivity was evaluated with the ratio of MTX package deal high quality and TMP high quality39.
Detection of the ICD biomarkers
LLC cells had been handled with both 20 μg ml−1 MW-TMPs, UV-TMPs or PBS, respectively. As well as, some LLC cells had been pretreated with the HMGB1 inhibitor (glycyrrhizic acid, #HY-N0184) earlier than MW-TMPs stimulation. The expression of canonical ICD markers, together with HMGB1, ATP and calreticulin (CRT), was detected 24 h post-stimulation. HMGB1 expression ranges had been analysed by the WB assay (Supplementary Desk 2). HMGB1 concentrations within the cell supernatant had been measured utilizing an ELISA equipment (Bioswamp, catalogue quantity MU30043) following the producer’s pointers. CRT expression was evaluated by FCM and visualized by means of immunofluorescence (Supplementary Desk 4). ATP content material within the LLC supernatant was quantified utilizing an ATP assay equipment (Beyotime, catalogue quantity S0026).
MW-TMPs fluorescence labelling and uptake in vitro and in vivo
MW-TMPs had been labelled with lipophilic membrane tracers DiI (Beyotime, #C1036), DiO (Beyotime, #C1038) or DiR (MedChemExpress, #HY-D1048) referring to the producer’s devices. After 10 min incubation in darkness at room temperature, MW-TMPs had been washed utilizing PBS twice to take away free dye options.
Fluorescence-labelled MW-TMPs had been added to tumour cells. At numerous incubation intervals, cell medium was eliminated and PBS was used to clean cells twice. Cell nuclei had been stained with DAPI after which the internalization of MW-TMPs was assessed by a fluorescence digital scanner (3DHISTECH, Pannoramic SCAN) or FCM (BD LSRFortessa X-20).
Feminine subcutaneous tumour-bearing C57BL/6 mice had been intraperitoneally injected with DiR-labelled MW-TMPs. After post-administration of 1 h, 12 h and 24 h, the presence of MW-TMPs in tumour websites and freshly eliminated very important organs had been visualized underneath bioluminescence imaging (Bruker MS FX Professional Imaging System). Excitation and emission wavelengths had been set at 600 nm and 570 nm, respectively.
Immune cell extraction and induction
Murine femurs from C57BL/6 mice (6–8 weeks) had been dissociated underneath sterile situations, adopted by fast centrifugation at 12,000 rpm for 1 s to isolate bone marrow cells. The cells had been handled in 1 ml crimson blood cell lysis buffer for 3 min after which neutralized with RPMI-1640 medium supplemented with 10% FBS. The cell suspension was then centrifuged at 1,800 rpm, 5 min at 4 °C. For induction of BMDCs, major bone marrow cells had been cultured in RPMI-1640 medium supplemented with 10% FBS, 20 ng ml−1 interleukin-4 (IL-4) (Peprotech, #214-12) and GM-CSF (Peprotech, #315-03) for 1 week. As well as, BMDMs had been induced by culturing bone marrow cells in RPMI-1640 medium supplemented with 10% FBS and 20 ng ml−1 M-CSF (Peprotech, #315-02) for 1 week.
Lymphocytes had been extracted from the spleens of 6–8-week-old C57BL/6 mice utilizing the mouse CD3+ T cell isolation equipment (Vazyme, catalogue quantity CS101) in line with the producer’s directions. The remoted spleen lymphocytes had been incubated in RPMI-1640 medium supplemented with 10% FBS (Gibco), 20 ng ml−1 IL-2 (Peprotech), HEPES answer (100×, Procell), β-mercaptoethanol (1,000×, Procell) and sodium pyruvate answer (100×, Procell).
To evaluate the immune-stimulating capability of tumour cells handled with MW-TMPs, LLC tumour cells had been handled with LLC-derived MW-TMPs for twenty-four h. These ensuing LLC cell pellets had been then co-cultured with BMDCs for an additional 24 h. Subsequently, these pretreated BMDCs had been co-cultured with both B3Z cell line or remoted CD3+ T cell for 48 h, permitting for the analysis of the proportion of mature DCs, CD4+ T, CD8+ T cells, in addition to the secretion of IFNγ (#1210002, Dakewe) and IL-2 (#1210203, Dakewe).
Subcutaneous and orthotopic tumour-bearing mannequin and in vivo remedy
LLC cells (5 × 105 cells suspended in 100 μl DMEM answer) had been subcutaneously inoculated into the unilateral posterior flanks of C57BL/6 mice (6 weeks). After roughly 1 week of remark, they had been randomized into a number of teams (n = 6). The tumour quantity and physique weight had been recorded each different day till the top of the intervention. For lung orthotopic tumour fashions, 6 × 105 LLC-LUC cells had been injected into the uncovered lung tissue of mice. After 3 days, the orthotopic tumour fashions had been randomly categorised into three teams and provides particular intervention. The expansion of orthotopic tumour was monitored by utilizing the IVIS Spectrum imaging system to detect the spontaneous luminescence fashioned after d-luciferase binds to the substrate. The remedy (100 μl PBS, 3 μg g−1 MPs, 5 μg g−1 anti-PD-L1 antibody) was administrated intraperitoneally as soon as each 2 days. Mice had been sacrificed after the fifth intervention. Subcutaneous or bare tumour quantity was estimated as follows: quantitySubcutaneous tumour = size × width2 × 0.5; quantityBare tumour = size × width × peak × 0.5. To estimate the antitumour effectivity, the system about tumour development inhibition price (TGI %) was used: TGI (%) = (1-mean quantityremedy/imply quantitymanagement) × 100%. Based on the ethics committee’s laws, the maximal tumour burden permitted is 2,000 mm3. We affirm that the maximal tumour burden was not exceeded through the examine.
scRNA sequencing
When the intervention was accomplished, recent subcutaneous tumours had been saved in magnetic-activated cell sorting tissue storage answer (#130100008) and cell suspensions had been ready by tissue homogenate. The one-cell suspension was added to the single-cell sorting honeycomb plate and extreme magnetic beads with barcodes had been put into the plate. Through the part of cell lysis, the RNA was labelled as bounding to the barcode. Subsequently, reverse transcription of RNA and cDNA synthesis had been carried out to acquire cDNA library for subsequent sequencing. Paired-end sequencing was carried out within the NovaSeq 6000 sequencing platform to get the transcriptome outcomes. BD Rhapsody Evaluation pipeline was utilized to determine the UMI sequence and the cell tag sequence alignment to the reference genome (GRCm39). After eradicating low-quality cells, the normalization strategies ‘LogNormalize’ and ‘ScaleData’ had been used to standardize and scale every gene after which linear dimensional discount was carried out in line with the PCA rating. Uniform manifold approximation and projection (UMAP) strategies had been chosen to visualise the cell clusters (the visualized PCA dimension = 38). The uncooked scRNA sequencing datasets have been deposited within the NCBI database (GSE289959).
Pseudotime trajectory evaluation and calculation of signature rating
Based on differential expression genes throughout cells, monocle package deal (model 2.26.0) was used for analysing the trajectory processing of DCs, CD8+ T cells and TAMs to deduce their dynamic differentiation and capabilities. To quantify the operate of immune cells, together with DCs, CD8+ T cells and NK cells, the ‘AllModuleScore’ operate (Seurat model 4.4.0) was used to calculate the enrichment scores in line with signature gene units (Supplementary Desk 8).
Cell-cell interplay and pathway enrichment evaluation
We used the basic ‘CellChat’ package deal (model 1.6.1) to foretell and analyse the energy of interactions between completely different cell sorts, primarily together with tumour cells and numerous immune cells, in addition to DC subpopulations and T cells, based mostly on cell gene expression. After 1,000 pairing assessments, ligand–receptor pairs with important enrichment and P-value significance had been extracted (Supplementary Desk 7). On the idea of the differential expression genes amongst handled and untreated teams by performing ‘FindAllMarkers’, KEGG and hallmark pathway evaluation had been investigated to estimate the differential actions of cells evaluating teams.
Distribution of tumour-infiltrating immunocytes
The detection of immune cells in tumour tissues utilizing FCM was carried out as beforehand described10. Briefly, single-cell suspension was collected by means of grind, digestion, filtration and lysis of crimson blood cells. Cells may have stimulation by PMA (0.1 μg ml−1, Solarbio, P6741-1 mg) and ionomycin (1 μg ml−1, Aladdin, 1139530-1 mg) earlier than analysis of concentrations of Gzmb, IFNγ and IL-4. Suspension was blocked by anti-mouse CD16/32 antibody. Subsequent, a number of fluorescent antibodies (Supplementary Desk 3) had been stained in darkness for 30 min at 4 °C. Lastly, FCM instrument (Beckman Coulter, DxFLEX) was used to analyse the infiltration of immune cells within the TME.
Tissue in situ immunofluorescence
Tissues of tumours, spleens and lymph nodes had been acquired on the finish of intervention and washed with PBS earlier than they had been mounted and embedded in paraffin. Then the sections underwent dewaxing, hydration, antigen retrieval and quenching of endogenous peroxidases. The tissues had been blocked with 2% bovine serum albumin (BSA) after which incubated with fluorescence-coupled antibodies (Supplementary Desk 4) in a single day at 4 °C. Subsequent, nuclei in tissues had been stained with DAPI. Lastly, the sections had been scanned utilizing a fluorescence microscope scanner.
Acquisition and remedy of human MPE samples
Human MPE samples had been obtained from lung most cancers sufferers present process thoracentesis to alleviate fluid accumulation at Wuhan Union Hospital, Tongji Medical School, Huazhong College of Science and Expertise. The examine protocol was authorised by the moral committee of Tongji Medical School, Huazhong College of Science and Expertise and all MPE samples had been collected with sufferers’ knowledgeable consent ((2019) IEC (S1041)).
Roughly 500 ml of recent MPE samples per affected person had been subjected to centrifugation at 1,000 × g for 15 min, leading to separation into supernatants and cell pellets. A portion of the cell pellets was instantly labelled with FCM antibodies to characterize the mobile composition throughout the pleural effusion. A listing of the anti-human antibodies used is summarized in Supplementary Desk 3. All antibodies for human FCM had been used at a 1:100 dilution. One other portion of the cell pellets was seeded into agarose gel-coated 96-microwell plates for the next formation of 3D multicellular spheroids. After 5 days, we used 3D multicellular spheroids that fashioned within the wells to characterize a human stable TME for additional immunomodulation experiments with numerous TMP sorts. The fashioned 3D multicellular spheroids had been handled with numerous TMPs for six days, and spheroid sizes had been recorded. On the finish level of remark, we immobilized 3D multicellular spheroids with 4% paraformaldehyde and stained them with DAPI for the next imaging. As well as, cytotoxicity assays had been carried out utilizing Dwell/Useless detection kits on the multicellular spheroids to guage their viability on the finish of the experimental remark.
The remaining cells underwent remedy with crimson blood cell lysis buffer to take away erythrocytes, adopted by washing twice with PBS. Tumour cells had been then remoted by magnetic-activated cell sorting utilizing magnetic beads conjugated with human Epcam microbeads (Miltenyi Biotec) following the producer’s directions. The sorted tumour cells had been subjected to both UV or MW stimulation for the preparation of UV-TMPs and MW-TMPs. Concurrently, a smaller fraction of the whole-cell pallets was seeded into completely different 12-well plates for analyses of cell uptake of MW-TMPs and immune activation detection.
To evaluate mobile uptake capability of TMPs by numerous cell sorts throughout the MPE, Dil-labelled MPs had been incubated with MPE pallets within the 12-well plates. After a 24 h incubation interval, FCM was used to quantify the phagocytosis ranges of Dil-MW-TMPs by particular person cell sorts. Alternatively, some cell pellets had been mounted for additional immunofluorescence.
To additional discover the impression of UV-TMPs and MW-TMPs on immune cells, they had been launched to further 12-well plates. Upon 24 h of incubation, FCM was utilized to guage cell proportion and useful modifications within the MPEs from every affected person. Concurrently, the cell supernatant was collected to measure ATP concentrations utilizing an ELISA equipment. One other portion of the cells was lysed and denatured to facilitate the detection of CRT expression through WB.
Zebrafish PDX fashions
To determine the PDX fashions in zebrafish, lung most cancers tumour tissues obtained from the affected person through the surgical procedure had been processed into single-cell suspensions utilizing mechanical and enzymatic digestion. These cells had been subsequently labelled with Vybrant CM-DiI (Thermo Fisher Scientific). In parallel, peripheral blood samples from the sufferers had been collected to isolate peripheral blood mononuclear cells (PBMCs), which had been activated in vitro and stained with DiO fluorescent dye (Thermo Fisher Scientific). The labelled tumour cells and PBMCs had been blended in a 1:1 ratio and microinjected into the yolk sacs of two days post-fertilization (dpf) wild-type AB zebrafish from Hunter Biotech, at a density of 300 cells per fish. Then the zebrafish had been all reared in fish-rearing water at 35 °C. Relating to the water high quality, 200 mg of immediate sea salt was added to every 1 l of reverse osmosis water, with {the electrical} conductivity starting from 450 μS cm−1 to 550 μS cm−1; the pH worth was between 6.5 and eight.5; and the hardness was 50–100 mg l−1 CaCO3. The license quantity for the utilization of laboratory animals is SYXK (Zhejiang) 2022-0004.
At 3 dpf, zebrafish displaying constant tumour cell expression had been chosen underneath the microscope and randomly divided into 3 teams for tradition in 6-well plates with a 3 ml properly quantity, with 10 zebrafish per group. A549-MW-TMPs or A549-UV-TMPs had been administered into the yolk sacs of zebrafish fashions, alongside a mannequin management group. Two days after the intervention, fluorescence depth was assessed by photographing 10 zebrafish per group underneath a fluorescence microscope. The photographs had been processed utilizing NIS-Parts D 3.20 superior image-processing software program to guage the antitumour development efficacy of TMPs.
Statistical evaluation
The experiments within the examine had been repeated independently at the least thrice. Statistical evaluation was carried out utilizing the software program GraphPad 8.0. All information had been proven as imply values ± s.d. Important comparability between two teams was decided by means of unpaired two-tailed Scholar’s t-test. For greater than two teams, one-way evaluation of variance (ANOVA) with additional a number of comparisons utilizing post-Turkey’s a number of comparability take a look at was carried out. P < 0.05 indicated statistical significance.
Reporting abstract
Additional data on analysis design is accessible within the Nature Portfolio Reporting Abstract linked to this text.