Sample collection

Twenty CRC samples and matched 20 normal paracancerous tissues were collected from Harbin Medical University Affiliated Cancer Hospital from June 2019 to April 2022, and the diagnosis of CRC was verified by histological examination by pathologists. The study protocol was approved by the Institutional Review Board of Harbin Medical University Affiliated Cancer Hospital (approval No: KY2019-22) following the Declaration of Helsinki. Written informed consent was obtained from each patient.

AOM/DSS model

All animal experiments were approved by the Animal Ethical and Welfare Committee of Harbin Medical University Affiliated Cancer Hospital (approval No: KY2022-11). The animals were treated following the Guide for the Care and Use of Laboratory Animals, as established by the National Institutes of Health. All mice were kept in a suitable pathogen-free environment.

C57BL/6 mice (8 weeks, Vital River, Beijing, China) were administered intraperitoneally with a single dose of AOM (A5486, Merck KGaA, Darmstadt, Germany) at 10 mg/kg. After 7 days, the mice were given drinking water containing 2% DSS (42,867, Merck KGaA) for one week and then fed regular drinking water for two weeks (Ma et al. 2018). DSS treatments were repeated for two additional cycles. Control mice were not treated with AOM/DSS.

For lentiviral gene intervention in AOM/DSS-induced CRC mice, the mice were starved overnight before the start of each DSS cycle and injected intracolonically with short hairpin RNA (sh) COL9A1, sh KMT2D, and overexpression (oe) COL9A1-concentrated lentiviruses (designed and purchased by VectorBuilder, Guangzhou, Guangdong, China). On day 120, mice were euthanized with 150 mg/kg sodium pentobarbital (i.p.), and tumor load was assessed. Tumors larger than 1 mm were counted, measured, and weighed. Colon tissue was collected for subsequent studies.

Immunohistochemical staining (IHC)

Sections of human CRC tissues and mouse colon tissues were first deparaffinized, rehydrated, and incubated with 3% peroxide-methanol for 30 min. Nonspecific antigen binding was sealed with 10% BSA. The sections were probed with primary antibodies to KMT2D (1:200, PA5-57490, Invitrogen Inc., Carlsbad, CA, USA), ZNF460 (1:250, 25,299–1-AP, ProteinTech Group, Chicago, IL, USA), COL9A1 (1:500, 61–492, ProSci, Poway, CA, USA), Collagen I (1:500, PB9939, Wuhan Boster Biological Technology Co., Ltd., Wuhan, Hubei, China), LOX (1:100, ABT112, Merck KGaA), CD133 (1:250, NB120-16518, Novus Biological Inc., Littleton, CO, USA), LGR5 (1:200, NBP1-28,904, Novus Biological) overnight at 4 °C, After the incubation with secondary antibody (1:2000, ab97051, Abcam, Cambridge, MA, USA) at 37 °C for 50 min, the sections visualized with diaminobenzidine (D12384, Merck KGaA). Then, sections were counterstained with hematoxylin and visualized by microscopy. The stained tissue sections were evaluated by two pathologists. Staining results were assessed according to the rate of positive cell staining.

Separation of CCSC

The sgRNA targeting the last exon of LGR5 (exon 18) was constructed as LGR5 P2 A-GFP using a plasmid vector expressing GFP. The donor vector carrying the LGR5-GFP targeting construct was then electroporated into a human CRC cell line, HCT 116 (CL-0096, Procell, Wuhan, Hubei, China). Successfully transfected cells were seeded into ultra-low attachment 6-well plates and cultured in DMEM/F12 (51445 C, Merck KGaA) plus N2, B27, 20 ng/mL epidermal growth factor (EGF, PHG0313, Thermo Fisher Scientific Inc., Waltham, MA, USA) and 20 ng/mL bFGF (100-18B, Thermo Fisher). After one week, GFP+ clones were observed by fluorescence microscopy and individually hand-selected into new wells for propagation. After successive passages of sphere formation, LGR5+ CCSCs were screened from spheres by fluorescence‑activated cell sorting (FACS) for further experiments (Zhu et al. 2022).

Cell lines

Mouse colonic mucosal epithelial cells (iCell-0108a, Cellverse Bioscience Technology Co., Ltd., Shanghai, China) were cultured in mouse colonic mucosal epithelial cell-specialized medium (iCell-0108a-001b, Cellverse). Fetal human colon (FHC) cells (KL-C2079H, Kang Lang, Shanghai, China) and mouse colon tumor 26 (CT26, CL-0071, Procell) were maintained in RPMI-1640 (PM150110, Procell) medium plus 10% FBS + 1% P/S and placed in a culture environment of 37 °C, 5% CO2.

All lentiviral vectors were packaged using the procedures specified by VectorBuilder. LGR5+ CCSCs were infected with sh COL9A1, sh KMT2D, oe ZNF460, oe COL9A1 lentiviruses, and their control lentiviruses in the presence of polybrene infection/transfection reagent (TR-1003, Merck KGaA). The cells were screened with 1 μg/mL puromycin for 14 d to harvest stably infected cells. Target gene expression in stable clones was verified by RT-qPCR.

CT26 cells were infected with concentrated lentiviruses containing sh COL9A1, sh KMT2D, oe COL9A1, and their control lentiviruses using polybrene infection/transfection reagent (TR-1003, Merck KGaA) for 24 h. The screened clones (as mentioned above) were collected for further culture (Zhang et al. 2020). After verification of target gene expression in infected cells by RT-qPCR, these cells were used in tumor transplantation experiments.

RNA isolation and qPCR

Total RNA was isolated from LGR5− or LGR5+ HCT 116 cells as well as CT26 using TRIzol (15596026 CN, Invitrogen). The cDNA was then synthesized by iScript Select cDNA Synthesis Kit (1,708,896, Bio-Rad Laboratories, Hercules, CA, USA). RT-qPCR was performed using Power SYBR Green PCR premix (4,367,659, Thermo Fisher). The 2−ΔΔCt method was used to calculate relative gene expression by normalizing to GAPDH. Table 1 details the primer sequences used.

Table 1 PCR primers used in this studyWestern blot

LGR5− or LGR5+ HCT 116 cells, mouse colonic mucosal epithelial cells, FHC, or CT26 cells were lysed using RIPA Buffer (R0278, Merck KGaA) plus protease and phosphatase inhibitor mixture (P1045, Beyotime, Shanghai, China). The lysate supernatant was concentrated 20-fold in an ultrafiltration tube and diluted 1:1 with RIPA buffer. An equal amount of total protein lysate was subjected to 10% SDS-PAGE and transferred to a PVDF membrane. After sealing with 5% goat serum in TBS/Tween-20 for 1 h, the membranes were probed overnight at 4 °C with primary antibodies. The antibodies are as follows: KMT2D (1:500, ABE1867, Merck KGaA), ZNF460 (1:2000, 26–545, ProSci), COL9A1 (1:1000, 61–492, ProSci), Collagen I (1:2000, ab34710, Abcam), LOX (1:1000, ABT112, Merck KGaA), CD133 (1:1000, NB120-16518, Novus Biological), LGR5 (1:100, NBP1-28,904, Novus Biologicals), β-actin (1:2000, PA1-16,889, Invitrogen). Visualization of the proteins was achieved using SuperSignal West Pico PLUS Chemiluminescent Substrate (34,580, Thermo Fisher) following incubation with secondary antibody (1:10,000, ab97051, Abcam).

Sphere formation

LGR5+ CCSCs (5,000) were plated into ultra-low attachment 6-well plates and cultured in DMEM/Ham Nutrient Mix F12 (51445 C, Merck KGaA) plus N2 (17,502,001, Gibco, Carlsbad, CA, USA), B27 (A3582801, Gibco), 20 ng/mL EGF, and 20 ng/mL bFGF. Spheres were observed and counted after one week, sphere formation rate = (number of spheres)/5000 × 100% (Zhu et al. 2022).

Polyacrylamide gels

A large coverslip was immersed in 0.1 M NaOH and air-dried. After that, 3‐aminopropyltrimethoxysilane (281,778, Merck KGaA) was spread across each coverslip and soaked in 0.5% glutaraldehyde (G5882, Merck KGaA) in PBS for 30 min. Coverslips were covered with nitrocellulose to increase polyacrylamide binding. Then, 25 μL of acrylamide/bisacrylamide mixture (containing 3% or 10% acrylamide and bisacrylamide at a concentration of 0.01% or 0.3%) was placed on the coverslips to prepare gels with a stiffness of 2.0 and 20.0 kPa, respectively, and covered with a small circular coverslip. The rest steps were conducted as previously reported (Pelham 1997).

Colony formation assays

Cells were plated at 500 cells/well into 6-well plates pre-laid with 2.0 kPa or 20.0 kPa polyacrylamide gels and incubated for 2 weeks. The medium was renewed every 3 days during colony growth. Colonies with more than 50 cells were counted under a light microscope.

Transwell assay

Cell migration and invasion assays were performed using Corning Transwell 24-well plates (CLS3422, Merck KGaA). LGR5+ CSCs were first pre-cultured on polyacrylamide gels of 2.0 kPa or 20.0 kPa for 48 h to form mechanical memory in response to physical and mechanical stimuli. Then, 200 μL of LGR5+ CSCs in serum-free medium was seeded in the upper chamber, and 500 μL of medium containing 10% FBS was loaded into the lower chamber. After 12 h, non-migrating/non-invasive cells attached to the surface of the upper chamber were removed. The cells were fixed, treated with 0.5% crystal violet for 4 h at 37 °C, and imaged. Transwell filter membranes were pre-coated with Matrix-Gel (C0376, Beyotime) for the invasion assay.

Immunofluorescence staining

LGR5+ CCSCs (1 × 105), which inherently contained GFP green fluorescence, were fixed in 4% paraformaldehyde for 20 min and permeabilized with 0.5% Triton X-100 for 5 min. The cells were blocked in 10% goat serum for 1 h at room temperature and probed with the anti-Ki67 (1:100, PB9026, Boster) at 4 °C overnight and with IgG (1:500, ab150083, Abcam) at room temperature for 1.5 h, followed by counter-staining with DAPI. Fluorescence signals were imaged.

HCT 116 cells were cultured at a density of 5 × 105 cells per well on sterile glass coverslips in 24-well plates, fixed using 4% paraformaldehyde, permeabilized with 0.5% Triton X-100, and sealed using goat serum. The cells were stained overnight with anti-KMT2D (1:50, 701,869, Invitrogen) and anti-H3 K4 me1 (1:100, 49–1003, Invitrogen) and with goat anti-rabbit IgG (A-11008, Invitrogen) and DAPI. The mean fluorescence intensity was quantified using Image J.

Hematoxylin–eosin (HE) staining

HE staining kits (C0105S, Beyotime) were used to assess inflammatory infiltration of mouse colon tissues. The paraffin-embedded sections of mouse colon tissue were dewaxed and hydrated, then stained with hematoxylin for 10 min, treated with differentiation solution for 30 s, and then stained with eosin for 1 min, washed with 70% ethanol, and then subjected to dehydration, clearing, sealing, and observation. The scoring system was based on a previous study (Johansson et al. 2014).

Masson's staining

The extent of collagen deposition in mouse colon tissues was assessed using a Masson’s trichrome staining kit (HT15, Merck KGaA). After dewaxing and hydration, the paraffin-embedded sections were treated with hematoxylin, Biebrich scarlet-acid fuchsin, phosphotungstic phosphomolybdic acid, and aniline blue (all for 5 min). Finally, the slides were incubated in 1% acetic acid for 2 min, dehydrated, cleared, and then visualized on the microscope. For quantification of collagen fiber deposition, images were analyzed under the microscope using Image-Pro Plus 6.0 image analysis software (Media Cybernetics, Bethesda, MD, USA).

Subcutaneous xenografts

CT26 cells (5 × 105) infected with sh Ctrl, sh COL9A1, sh KMT2D, sh KMT2D + oe Ctrl, or sh KMT2D + oe COL9A1 were suspended in 100 μL PBS and administered subcutaneously into the dorsal right ventral side of BALB/c mice (211, Vital River) (n = 6). After measuring subcutaneous tumor size in mice for the first time on the fifth day, the monitoring was performed every 4 days thereafter for 25 days. Tumor volume (V) was assessed with this formula: V(mm3) = 0.5 × length × width2. On day 25, the mice were euthanized by 150 mg/kg sodium pentobarbital (i.p.).

Isolation of tumor-infiltrating lymphocytes (TIL)

To isolate TIL, BALB/c mouse subcutaneous tumors derived from mouse CT26 cells were minced using scissors and a scalpel, and detached in Hanks'buffered salt solution (HBSS, 88,284, Thermo Scientific) containing 1 mg/ml collagenase (C5138, Merck KGaA), 0.1 mg/mL DNAse I (DN25, Merck KGaA), and 2.5 U/mL hyaluronidase (H1115000, Merck KGaA) for 2 h to prepare tumor cell suspensions. The resulting suspension was passed through a 70-μm cell filter and resuspended in PBS + 3% BSA to 1 × 106 cells/mL. Mouse TILs were purified by Mouse CD90.2 MicroBeads (130–121–278, Miltenyi Biotec, Bergisch Gladbach, Germany) and incubated under 3000 IU/mL of IL-2 (HY-P7077, MedChemExpress, Monmouth Junction, NJ, USA) for 5 days, after which the TILs were harvested for the following experiments.

Flow cytometry

Excised BALB/c mouse subcutaneous tumors derived from CT26 cells were sectioned into small pieces and dissociated using the Tumor Dissociation Kit (130–096–730, Miltenyi Biotec). For the isolation of mouse CD8+ T cells from tumors, Dynabeads FlowComp Mouse CD8 Kit (11462D, Invitrogen) was used. Antibodies against APC-CD3 (17–0031–82, Invitrogen), FITC-CD8 (11–0081–82, Invitrogen), FITC-CD19 (11–0193-82, Invitrogen), PE-Ki67 (12–5698-82, Invitrogen), APC-granzyme b (Gzmb, 17–8898-82, Invitrogen), PE-Perforin (Prf1, 12–9392-82, Invitrogen), APC-interferon-gamma (IFN-γ, 17–7311-82, Invitrogen) were used.

NK cells were labeled using CD3 and CD49b-FITC (ab272259, Abcam). CD11c+ cells were isolated from mouse tumors using CD11c MicroBeads UltraPure (130–125–835, Miltenyi) and stained with MHCII-FITC (11–5321-82, Invitrogen), CD103-PE-Cy7 (121,425, BioLegend), XCR1-PE (148,203, BioLegend), and IRF8-APC (17–9852-82, Invitrogen).

Co-culture and functional analysis of CT26 and CD8 +T/NK cells

Spleen CD8+ T lymphocytes from BALB/c mice were isolated using the Dynabeads FlowComp Mouse CD8 Kit. CD8+ T cells were pre-stimulated with 200 U/mL IL-2, Dynabeads Mouse T Activator CD3/CD28 (11452D, Gibco) for 48 h before the experiment. Afterwards, CD8+ T cells were co-cultured with CT26 cells in a 5:1 ratio for 48 h.

Primary NK cells were isolated from mouse spleens using the EasySep Mouse NK Cell Isolation Kit (19,855, NovoBiotechnology Co., Ltd., Beijing, China) and co-cultured with CT26 cells at a 5:1 ratio for 24 h. Cells were subsequently collected for analysis of NK cell functionality (IFN-γ+ NK cells) and toxicity to tumor cells.

Cell viability of CT26 cells was analyzed at 0, 24, 48, and 72 h using the CCK-8 (C0005, TargetMol, Boston, MA, USA).

ChIP assays

ChIP assays were conducted using a ChIP kit (17–295, Merck KGaA). HCT 116 cells were cross-linked with 1% formaldehyde, lysed, sonicated to generate DNA fragments (an average length of 500 bp), and subjected to IP using specific antibodies. ZNF460 antibody, KMT2D antibody (1:10, ABE1867, Merck KGaA), H3 K4 me1 antibody (1:10, NB21-1021, Novus Biological), and isotype control (ab171870, Abcam) were used in this assay. The magnetic beads were washed after precipitation, and the DNA was eluted, purified, and subjected to qPCR using promoter- and enhancer-specific primers for COL9A1.

Luciferase reporter assays

Luciferase reporter gene vectors were purchased from Promega (Madison, WI, USA). The pGL3-basic (firefly luciferase, Fluc) pRL-TK (Renilla luciferase, Rluc) dual-luciferase reporter gene kit (YanjiangBio, Shanghai, China) was used, and the COL9A1 promoter sequence was subcloned into the dual luciferase reporter gene vector. Next, 500 ng of reporter plasmid was co-transfected into HCT 116 cells for 24 h. The relative Fluc/Rluc activity was calculated.

Plasmid construction and cell transfection

Two single-stranded sgRNA primers with complementary sequences were annealed for 20 min at 90 °C in an annealing buffer consisting of 50 mM Tris–Cl (pH = 7.5–8.0), 250 mM NaCl, and 5 mM EDTA and cloned into the epiCRISPR plasmid (135,960, Addgene, Watertown, MA, USA). HCT 116 cells were cultured in 6-well plates at 5 × 105 cells/well, and sgRNA plasmids were transfected using Lipofectamine 3000 (L3000001, Invitrogen) to construct KMT2D-LCD1delete (sgRNA A-B) versus KMT2D-LCD2delete (sgRNA C-D) cells, and cells transfected with sgVector plasmid were used as controls. The sgRNA primers for KMT2D are listed in Table 2.

Statistical analysis

For statistical analyses in the study, GraphPad Prism 8.0.2 (GraphPad, San Diego, CA, USA) was utilized. The data is shown as mean ± SEM. For comparisons between any two groups, paired or unpaired t-tests were utilized. For multiple group comparisons, ANOVA (one-way or two-way when appropriate) was performed, with Tukey’s post hoc test. p < 0.05 indicates a statistically significant difference between the data.