Cell strains
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 Faculty, Huazhong College of Science and Expertise. Subsequently, LLC cells have been transfected with the pcDNA3-OVA plasmid (Genecreale) to acquire LLC-OVA cells. Mouse T cell hybridoma cells B3Z (#BFN608006444) have 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) have been bought from the Cellverse. Murine embryonic fibroblast cell line (NIH/3T3, #CL-0171) was bought from the Pricella. These cell strains have been cultured in DMEM (Gibco) supplemented with 10% FBS (Newzerum) at 37 °C in 5% CO2–95% air.
Mice
C57BL/6 mice have been bought from Hubei Beiente Biotechnology. They have been stored in particular pathogen-free facility within the Wuhan Laboratory Animal Heart of Tongji Medical Faculty. All animal-related procedures have been carried out in response to the Declaration of Helsinki and gained permission from the Animal Care and Use Committee of Tongji Medical Faculty ((2023) IACUC quantity 3958).
Preparation and isolation of MW-TMPs
We established an MW-assisted technique to gather TMPs from tumour cells. After 24 h of incubation below normal situations, the cell tradition medium was changed with a serum-free medium. The Petri dishes have 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 have 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 have been extracted in response to our earlier research by way of a serial centrifugation technique9. In short, 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 have been washed as soon as after which saved utilizing PBS. For drug-loaded MW-TMPs, the incubation technique was used to encapsulate MW-TMPs with small molecules, reminiscent of 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 have been ready and remoted primarily based on our former protocols10. Plentiful proteins of UV-TMPs (named as U1, U2 and U3) and MW-TMPs (named as M1, M2, M3 and M4) as Mus_musculus samples have been recognized by way of single mass spectrometry run utilizing the TMT quantitative technique. Completely, 747,975 spectrums have been generated, 19,595 peptides and three,967 proteins have been detected with 1% false discovery charge, 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 have been considered differential expression12.
Excessive-performance liquid chromatography of MW-TMPs-MTX
MW-stimulated LLC cells have 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 have been sonicated for 10 min and handed by way of a 0.2 μm water movie. As for the cumulative MTX launch evaluation by MW-TMPs, ready MW-TMPs-MTX have 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 way of the chromatographic column (Athena-C18, temperature, 30 °C; movement charge, 1.0 ml min−1) below 303 nm wavelength within the mannequin of isocratic elution. In line with 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 have been handled with both 20 μg ml−1 MW-TMPs, UV-TMPs or PBS, respectively. As well as, some LLC cells have 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 have been analysed by the WB assay (Supplementary Desk 2). HMGB1 concentrations within the cell supernatant have been measured utilizing an ELISA equipment (Bioswamp, catalogue quantity MU30043) following the producer’s pointers. CRT expression was evaluated by FCM and visualized by way 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 have 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 have been washed utilizing PBS twice to take away free dye options.
Fluorescence-labelled MW-TMPs have been added to tumour cells. At numerous incubation intervals, cell medium was eliminated and PBS was used to scrub cells twice. Cell nuclei have 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 have 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 have been visualized below bioluminescence imaging (Bruker MS FX Professional Imaging System). Excitation and emission wavelengths have been set at 600 nm and 570 nm, respectively.
Immune cell extraction and induction
Murine femurs from C57BL/6 mice (6–8 weeks) have been dissociated below sterile situations, adopted by fast centrifugation at 12,000 rpm for 1 s to isolate bone marrow cells. The cells have been handled in 1 ml pink 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, main bone marrow cells have 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 have 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 have 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 response to the producer’s directions. The remoted spleen lymphocytes have been incubated in RPMI-1640 medium supplemented with 10% FBS (Gibco), 20 ng ml−1 IL-2 (Peprotech), HEPES resolution (100×, Procell), β-mercaptoethanol (1,000×, Procell) and sodium pyruvate resolution (100×, Procell).
To evaluate the immune-stimulating capability of tumour cells handled with MW-TMPs, LLC tumour cells have been handled with LLC-derived MW-TMPs for twenty-four h. These ensuing LLC cell pellets have been then co-cultured with BMDCs for an additional 24 h. Subsequently, these pretreated BMDCs have 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 resolution) have been subcutaneously inoculated into the unilateral posterior flanks of C57BL/6 mice (6 weeks). After roughly 1 week of commentary, they have been randomized into a number of teams (n = 6). The tumour quantity and physique weight have been recorded each different day till the top of the intervention. For lung orthotopic tumour fashions, 6 × 105 LLC-LUC cells have been injected into the uncovered lung tissue of mice. After 3 days, the orthotopic tumour fashions have 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 shaped 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 have 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 charge (TGI %) was used: TGI (%) = (1-mean quantityremedy/imply quantitymanagement) × 100%. In line with 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 research.
scRNA sequencing
When the intervention was accomplished, contemporary subcutaneous tumours have been saved in magnetic-activated cell sorting tissue storage resolution (#130100008) and cell suspensions have been ready by tissue homogenate. The one-cell suspension was added to the single-cell sorting honeycomb plate and extreme magnetic beads with barcodes have 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 have 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’ have been used to standardize and scale every gene after which linear dimensional discount was carried out in response to the PCA rating. Uniform manifold approximation and projection (UMAP) methods have 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
In line with 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 features. 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 response to signature gene units (Supplementary Desk 8).
Cell-cell interplay and pathway enrichment evaluation
We used the traditional ‘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, primarily based on cell gene expression. After 1,000 pairing assessments, ligand–receptor pairs with vital enrichment and P-value significance have 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 have 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. In short, single-cell suspension was collected by way of grind, digestion, filtration and lysis of pink 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) have 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 have been acquired on the finish of intervention and washed with PBS earlier than they have been fastened and embedded in paraffin. Then the sections underwent dewaxing, hydration, antigen retrieval and quenching of endogenous peroxidases. The tissues have 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 have been stained with DAPI. Lastly, the sections have been scanned utilizing a fluorescence microscope scanner.
Acquisition and remedy of human MPE samples
Human MPE samples have been obtained from lung most cancers sufferers present process thoracentesis to alleviate fluid accumulation at Wuhan Union Hospital, Tongji Medical Faculty, Huazhong College of Science and Expertise. The research protocol was authorized by the moral committee of Tongji Medical Faculty, Huazhong College of Science and Expertise and all MPE samples have been collected with sufferers’ knowledgeable consent ((2019) IEC (S1041)).
Roughly 500 ml of contemporary MPE samples per affected person have 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 inside the pleural effusion. A listing of the anti-human antibodies used is summarized in Supplementary Desk 3. All antibodies for human FCM have 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 shaped within the wells to characterize a human stable TME for additional immunomodulation experiments with numerous TMP sorts. The shaped 3D multicellular spheroids have been handled with numerous TMPs for six days, and spheroid sizes have been recorded. On the finish level of commentary, we immobilized 3D multicellular spheroids with 4% paraformaldehyde and stained them with DAPI for the next imaging. As well as, cytotoxicity assays have been carried out utilizing Stay/Lifeless detection kits on the multicellular spheroids to guage their viability on the finish of the experimental commentary.
The remaining cells underwent remedy with pink blood cell lysis buffer to take away erythrocytes, adopted by washing twice with PBS. Tumour cells have 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 have 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 inside the MPE, Dil-labelled MPs have 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 have been fastened for additional immunofluorescence.
To additional discover the impression of UV-TMPs and MW-TMPs on immune cells, they have been launched to further 12-well plates. Upon 24 h of incubation, FCM was utilized to guage cell proportion and purposeful adjustments 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 by way of 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 have been processed into single-cell suspensions utilizing mechanical and enzymatic digestion. These cells have been subsequently labelled with Vybrant CM-DiI (Thermo Fisher Scientific). In parallel, peripheral blood samples from the sufferers have been collected to isolate peripheral blood mononuclear cells (PBMCs), which have been activated in vitro and stained with DiO fluorescent dye (Thermo Fisher Scientific). The labelled tumour cells and PBMCs have been combined 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 have been all reared in fish-rearing water at 35 °C. Concerning 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 have been chosen below the microscope and randomly divided into 3 teams for tradition in 6-well plates with a 3 ml effectively quantity, with 10 zebrafish per group. A549-MW-TMPs or A549-UV-TMPs have 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 below a fluorescence microscope. The pictures have been processed utilizing NIS-Components D 3.20 superior image-processing software program to guage the antitumour development efficacy of TMPs.
Statistical evaluation
The experiments within the research have been repeated independently not less than thrice. Statistical evaluation was carried out utilizing the software program GraphPad 8.0. All information have been proven as imply values ± s.d. Important comparability between two teams was decided by way of unpaired two-tailed Pupil’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 obtainable within the Nature Portfolio Reporting Abstract linked to this text.
