Patient specimens and TAMs isolation
We have studied three cohorts of patients with HCC who had surgery at Zhongshan Hospital (ZSHS cohort). These cohorts comprise randomly selected cohort 1 with 108 formalin-fixed paraffin-embedded tissues, cohort 2, and cohort 3 with 40 and 117 fresh tissues, respectively. The ethics committee of Zhongshan Hospital has given its approval for this study’s protocol. All the participants in this research work gave their informed consent in writing. The research work described here was conducted following the provisions of the Declaration of Helsinki of 1975.
TAMs were separated from fresh HCC tumor samples. Small pieces (1–2 mm) of the tissues were made followed by 2 h digestion with 5% fetal bovine serum DMEM with collagenase VIII-DNase I (Sigma-Aldrich) at 37 °C. The released cells were filtered through cell strainers with a pore size of 70 μm. The cell suspension (1 ml) was layered on top of a Percoll (GE Healthcare) gradient, consisting of 40% Percoll (5 ml) and 60% Percoll (5 ml) at the bottom in a 15-ml tube. The tubes were then centrifuged at 2500 rpm for 20 min. The cell layer at in-between 40 and 60% Percoll layers contained TAMs.
Cell culture conditions and treatment
Hep3B and BEL7404 cell-lines (American Type Culture Collection) and were grown employing standard methods. Cell culture media were as recommended by the supplier, with 10% fetal bovine serum (FBS) supplementation and incubations were in chambers maintained in an atmosphere of 5% CO2 and 95% air, and at 37 °C.
Peripheral blood monocyte cells obtained from people with normal health were separated by Ficoll density gradient centrifugation at 450 g at 20 °C for 20 min. The 15-ml centrifuge tubes contained 5 ml of Ficoll (GE Healthcare) at the bottom and 10 ml of blood cell suspension on the top. Monocytes, which migrated in-between the layers of plasma and Ficoll, were collected. Monocytes were incubated with 100 ng/ml of PMA for 48 h to obtain human monocyte-derived macrophages (MDMs). DMEM culture medium plus 10% fetal bovine serum supplemented with and penicillin and streptomycin were used to grow MDMs. The MDMs were treated with 100 ng/ml of LPS for the induction of M1 macrophages and 20 ng/ml of IL-4 for the induction of M2 macrophages.
HCC cancer cells (3 × 105 cells) were transferred to the lower chamber of a 12-well Transwell system, while the upper chamber was added with macrophages (3 × 105 cells). The pore size of upper chamber was 0.4 μm. Following co-culture for 6 days, the macrophages were removed and after washing once with phosphate buffered saline, the HCC cells were cultured in freshly added growth medium.
Hematoxylin and eosin (H&E) and immunohistochemistry (IHC) assay
Tissue samples from ZSHS cohort 1 (n = 108) were embedded in blocks of paraffin, followed by preparation of 4-mm thick sections using a microtome and stained with H&E (Sigma-Aldrich, St. Louis, MO, USA) for histopathological investigation. For IHC staining, rehydration of paraffin-embedded tissue sections was done in sodium citrate buffer (pH 6.0), followed by heat-mediated antigen retrieval employing a microwave. Tissue sections were then transferred in to blocking buffer for 2 h followed by incubation with antibodies against CD68 (Abcam; ab955), CD163 (Abcam; ab182422), HK2 (Abcam; ab209847), GLUT1 (Abcam; ab115730) in a humidified box at 4 °C for 24 h. Tissue sections were then washed and incubations with secondary antibody (Proteintech Group, Inc., Wuhan, China; PK10006) were done. Immunodetection was performed using DAB. The IHC score is based on staining intensity on a scale of 1 to 4 (absent = 1, weak = 2, moderate = 3, or strong = 4). An average intensity score per tumor from 3 independent fields was calculated for each patient.
Transient transfection and transduction
Transfections were conducted with the Lipofectamine 3000 (Invitrogen) as per the supplier’s protocol. The microRNA mimics, microRNA inhibitors and their corresponding negative control RNAs (GenePharma) were transfected into the cells at 50 nM final concentration.
Transduction of macrophages (3 × 105 cells per well) using lentiviral particles (MOI of 100) with 5 μg/ml Polybrene was conducted in 24-well plates. The nucleotide sequences of microRNA mimics, microRNA inhibitors, shRNAs and their corresponding negative control are given in Supplementary Table S1.
Determination of glucose consumption, lactate production and ECAR
HCC cells were seeded into 6-well plates with freshly added growth medium and incubated for 24 h. Then the incubation media were collected, and kits from Biovision were employed to determine consumption of glucose and production of lactate, as detailed in the manufacturer’s brochure. Cell number was calculated with a cell counter. The seahorse extracellular Flux analyzer XF96 (Seahorse Bioscience) was employed to determine metabolic changes in cells, in vitro, as mentioned in the manufacturer’s instructions. Briefly, HCC cells with the treatments, as specified, were transferred into 96-well culture plates followed by incubation at 37 °C overnight. Then the cells were used for measurement of ECAR. After measurement of baseline concentration, glucose, oligomycin, and 2-deoxyglucose were added sequentially into each well at the indicated time points for ECAR measurement. The measured ECAR values were adjusted to the total protein content and shown as mpH/min. ECAR measurements are shown as the mean ± s.d. of experimental triplicates.
Cell proliferation analyses and apoptosis assay
Determination of cell proliferation was done with cell-counting kit-8 (Dojindo) as per the instructions of manufacturer. In 96-well plates, cells were first seeded and 10 μl of CCK8 solution was added at each time point, followed by 2 h incubation in dark. After the incubations, absorbance was measured at 450 nm.
As for apoptosis assay, cells were incubated with Adriamycin (2 μg/ml) for 48 h, and then treated with trypsin-EDTA to dissociate, followed by centrifugation, to collect the cells. Annexin V Apoptosis Detection kit (eBioscience) and flow cytometry (BD Biosciences) was used for measuring apoptosis.
Exosomes were separated from the cell culture medium by ultracentrifugation. First, the medium was centrifuged at 3000 rpm for 10 min at 4 °C. The supernatant was centrifuged at 10,000 g for 30 min at 4 °C and the cellular debris pellet was removed. Then the resulting supernatant was passed through a 0.22 μm filter, followed by ultracentrifugation at 100,000 g at 4 °C for 90 min, and the resulting pellet contained exosomes.
Exosomes were first quantified by NanoSight NS300 instrument (Malvern Instruments) and electron microscopy was used to assess their purity. Primary antibodies against Alix, CD9 and TSG101 were used for the identification of exosomes.
Equal numbers of exosomes derived from MDMs or TAMs were treated with RNaseA and TritonX-100 treatment, and then RNA was extracted with TRIzol and normalized to cel-miR-39-3p for qRT-PCR. In vitro studies were done by treating HCC cells with 50 μg/mL exosomes.
For the visualization of lncRNA and exosome co-localization, FAM-labeled lncMMPA (in vitro transcription) was introduced into exosomes by electroporation using a GenePulser Xcell electroporation system (Bio-Rad), followed by the labeling of exosomes with Dil (Beyotime Biotechnology). Hep3B cells were incubated with exosomes labeled with Dil followed by visualization using laser scanning confocal microscopy (TCS SP8, Leica).
RNA extraction, qRT-PCR, and Western blotting
Extraction of total RNA from indicated tumor tissues or cell lines was done using TRIzol reagent (Takara). Employing 1 μg of total RNA and PrimeScript RT Reagent Kit (Takara), the first-strand cDNA was synthesized. The levels of miRNA were determined by qRT-PCR with SYBR-Green ΙΙ PCR kit (Takara) and U6 snRNA was used as the reference. LncRNA and mRNA concentrations were measured employing SYBR-Green ΙΙ PCR kit (Takara). β-actin was used as the internal control. Supplementary Table S2 shows the sequences of the primers used.
Extraction of protein was from the HCC cells was done using RIPA buffer, and determined using the BCA Protein Assay Kit (Beyotime Biotechnology). Separation of extracted proteins (40 μg per lane) was done by SDS-polyacrylamide gel electrophoresis and the separated proteins were transferred to polyvinylidenedifluoride (PVDF) membranes (Bio-Rad). The PVDF membranes were blocked using 5% non-fat milk for 1 h and primary antibodies against GLUT1 (Biorbyt, St Louis, MO, USA; orb157188), HK2 (Abcam; ab227198), LDHA (Abcam; ab52488), ALDH1A3 (Proteintech; 29,373–1-AP), β-actin (Abcam; ab8226), Alix (Abcam; ab88743), CD9 (Abcam; ab236630) and TSG101 (Abcam; ab125011) were used. Secondary antibodies were labeled with Peroxidase. The ECL detection system (Thermo) was used for visualization.
Luciferase reporter assay
Co-transfection of either pGLO, pGLO-lncMMPA, or pGLO-lncMMPA mut (miR-548 s binding site), or pGLO-ALDH1A3, or pGLO-ALDH1A3 mut (miR-548 s binding site) together with miR-548 s mimics or its negative control was done in Hep3B cells. Renilla luciferase activity was employed as the reference for normalizing the relative luciferase activity. A Dual-Luciferase Reporter Assay System (Promega) was used for determining the firefly luciferase and Renilla luciferase activities, according to the supplier’s instructions.
HCC cells were co-transfected pSL-MS2, pSL-MS2-lncMMPA, or pSL-MS2- lncMMPA mut (miR-548 s binding site) with pMS2-FLAG. Cells were collected 48 h post-transfection, to conduct RNA immunoprecipitation (RIP) experiments. The RNA-protein complexes were immunoprecipitated using a 3xFLAG antibody (Sigma). The RNA fraction separated by RIP was analyzed by qRT-PCR.
In vivo xenograft model
To elucidate the in vivo effect of lncMMPA on tumor growth, male BALB/c nude mice of 4-weeks age (Shanghai SLAC Laboratory Animal Co., Ltd.) were orthotopically injected with Hep3B cells into the right flank (n = 6 per group), subcutaneously, to create the HCC xenograft model. Seven days following inoculation, 30 μl of PBS suspension of exosomes (10 μg) prepared from MDMs with the indicated treatment was injected intratumorally every 3 days. For miR-548 s mimics treatment, 50 μl of OPTI-MEM containing miR-548 s mimics (100 pmol) and Lipofectamine 3000 (Thermofisher) mixture was injected intratumorally every 3 days. Tumor volume (mm3) was assessed using the following formula: Tumor volume (mm3) = longer diameter x shorter diameter2/2. After fixing in polyformaldehyde, xenografts were embedded in paraffin and processed for immunofluorescence staining for Ki67. The animal experiments were approved by the Zhongshan Hospital Animal Care and Use Committee.
For Ki-67 staining, paraffin-embedded samples were blocked with 1% bovine serum albumin in PBS for 30 min, and incubated with Ki-67 antibody (Abcam; ab243878). The secondary antibody used was Alexa Fluor® 488-conjugated goat anti-rabbit IgG. DAPI (C1002; Beyotime Biotechnology) was used to visualize cell nuclei. Slides were observed with a confocal microscopy (TCS SP8, Leica). Five images of each sample were used to count and calculate the percent of ki-67 positive cells.
Mann-Whitney U test in GraphPad Prism was used to analyze the results. Data are shown as mean ± SEM. The single cell data was analyzed by the Python-based algorithm SCANPY (version 1.7.2). Uniform Manifold Approximation and Projection (UMAP) was run for visualization. Violin plots for marker genes were generated utilizing the stacked violin function as executed in SCANPY. To evaluate the differential genes between the conditions, we computed Cohen’s d statistic to estimate the magnitude of changes in gene expression within a cell population. Statistical analyses were conducted using R (v4.0.1). The type of statistical method employed for analysis is indicated in the text during the description of corresponding results and details for the test are elaborated in the legends of the relevant figures. Statistical significance was considered when P-value < 0.05.