Gastrointestinal cancer-associated fibroblasts expressing Junctional Adhesion Molecule-A are amenable to infection by oncolytic reovirus

Primary cell and cell line culture

Primary fibroblasts were derived from human GI cancers and adjacent tissue according to the Code of Conduct for Responsible Use of human tissues or after written informed consent was obtained. The regional Medical Ethics Assessment Committee (METC) approved use of this material (registration number: B21.073). Primary fibroblast isolation was performed by mincing tumor tissue or adjacent normal tissue, obtained from patients with the appropriate consent, into small fragments and subsequent digestion with a mix (3:1 ratio) of collagenase (Gibco/Thermo Fisher Scientific, Leiden, The Netherlands) and dispase II (Roche, Basel, Switzerland) for 2 h at 37 °C. Subsequently, cells were expanded and tested via qPCR for the presence of general fibroblast marker expression (collagen type 1 α1 (Col1α1) and alpha smooth muscle actin (αSMA)) and absence of markers of epithelial, endothelial and immune origin (E-Cadherin, CD31 & CD45, respectively). Primary fibroblasts and the hPS1 pancreatic stellate cell (PSC) line [15] (kindly provided by H. Kocher, University of London, London, England) were cultured in Dulbecco’s modified Eagle’s medium (DMEM)/F12 (Thermo Fisher Scientific) supplemented with 8% fetal calf serum (FCS), 100 IU/mL penicillin and 100 μg/mL streptomycin (all Thermo Fisher Scientific). Murine fibroblasts from pancreatic KPC tumors were isolated and cultured as described previously [16].

Culture of primary tumor cell lines, organoids and organoid-CAF co-cultures was performed as described previously [17]. In short, suspension co-cultures were obtained by mixing patient-derived organoids (PDOs) and primary fibroblasts at ratios of 500:4000 (PDO:CAF), respectively. Single cells after trypsinization were homogenously mixed in fibroblast medium containing 1% Matrigel, plated in ultra-low attachment 96-well round-bottom plates (Corning), centrifuged (1200 RPM, 1 min) and incubated overnight (37 °C, 5% CO2). The formed mini-tumors (MTs) were subsequently pooled and transferred to ultra-low attachment 6-wells plates (Corning) after which infection with oncolytic viruses was performed.

The HT29 colorectal cancer and 911 embryonic retinoblast-derived cell lines were cultured in high-glucose DMEM (Invitrogen), supplemented with 100 IU/mL penicillin and 100 μg/mL streptomycin and 8% FCS (Invitrogen, Breda, the Netherlands).

All cells were cultured at 37 °C and 5% CO2. Mycoplasma tests were performed regularly by PCR and were negative throughout the duration of the experiments.

Lentiviral transductions and transgenic cell lines

Third-generation packaging vectors and HEK293T cells were used for the generation of lentiviral particles [18]. Lentiviral expression plasmids containing the full-length JAM-A open reading frame (ORF) (pLV-fullJAM-A) as well as a vector that lacks the cytoplasmic C-terminus (kindly provided by Diana van den Wollenberg, Dept. of Cell and Chemical Biology, LUMC) were described previously [19]. The PDZ-domain mutant was generated by DpnI-mediated site-directed mutagenesis [20] of the pLV-fullJAM-A vector by introducing a premature stop codon at position 295 (p295S>*), prior to the PDZ motif (-SFLV), using primers 5′-ggagaattcaaacagacctaatcattcctggtgtaatcc-3′ (forward) and 5′-ggattacaccaggaatgattaggtctgtttgaattctcc-3′ (reverse). Transduced cells were selected and cultured throughout the experiments with neomycin (400 µg/ml; Fisher Scientific). After transduction of hPS1 fibroblasts, membrane surface expression of the extracellular part of JAM-A was verified for all three constructs using flow cytometry.

For generation of JAM-A KO primary fibroblasts, sgRNA 5′- caccgTCGGGAGCCTGATCGCGATG-3′ (lowercase nucleotides are complementary to the BsmBI-restriction site) was cloned into BsmBI-digested pLentiCRISPRv2 [21] (Addgene: #98290), in which hSpCas9 and the guide RNA targeting JAM-A are co-expressed from the same vector. Lentiviral particles were generated and primary fibroblasts were subsequently transduced. After lentiviral transduction, genetic ablation of JAM-A was confirmed by flow cytometry. Cells were selected and cultured with 2 µg/ml of Puromycin (Sigma-Aldrich, Zwijndrecht, The Netherlands) for the duration of the experiments.

To fluorescently label KPC3 tumor cells and KPC-derived CAFs, they were lentivirally transduced with pLentiPGK Hygro DEST H2B-mCerulean3 (Addgene: #90234, KPC3 tumor cells) and pLentiPGK Hygro DEST H2B-mRuby2 (Addgene: #90236, CAFs). Cell selection and subsequent culture was performed using 400 µg/ml hygromycin (Gibco).

Flow cytometry

For cell surface staining, cells were harvested and washed twice with FACS buffer, consisting of PBS/0.5% bovine serum albumin (BSA, Sigma) and 0.05% sodium azide (Pharmacy LUMC, Leiden, The Netherlands). Cells were incubated with rabbit anti-human JAM-A (EPR23244-12, Abcam) at 4 °C for 45 min. Subsequently, cells were washed twice with FACS buffer and incubated with goat anti-rabbit-PE (Jackson ImmunoResearch Europe Ltd, United Kingdom) at 4 °C for 45 min. For staining of β1-integrin, a directly PE-conjugated anti-human β1-integrin (MAR4; BD biosciences, CA, USA) was used. Samples were measured on a LSR-II (BD biosciences) and data were analyzed with FlowJo v10.6.1 software (BD biosciences).

Western blot analysis

Total cell lysates were generated in RIPA buffer (50 mM Tris pH 7.5, 150 mM NaCl, 0.1% SDS, 0.5% DOC, 1% NP40), supplemented with complete mini protease inhibitor cocktail (Roche Applied Science, Penzberg, Germany). For the analysis of phosphorylated proteins, a commercial RIPA buffer (Pierce), supplemented with protease inhibitors (Roche), 50 U/mL benzonase (Santa Cruz) and sodium fluoride (Merck), was used to generate the lysates. Samples were cleared from cellular debris by centrifugation (13,000 RPM, 4 °C, 5 min). Protein concentrations were measured by a PierceTM BCA kit (Thermo Scientific, Rockford, IL, USA). Lysates were denatured by adding Laemmli sample buffer containing 20 mM DTT and heating for 5 min at 95 °C. Equal amounts of protein (10-30 µg) were separated by gel electrophoresis on 10% SDS-polyacrylamide gels, and transferred onto 0.2 μm nitrocellulose membranes using the Trans-Blot Turbo Transfer System (Bio-Rad). Membranes were blocked in TBS, supplemented with 0.1% Tween20 (TBST) and 10% milk or 10% BSA. Antibodies were diluted in TBST containing 5% milk, or in immuno booster (Takara) for the detection of phosphorylated proteins. Primary antibodies were incubated overnight at 4 °C, and secondary antibodies for 60 min at room temperature. Blots were washed with TBST.

The following antibodies were used: mouse anti-reovirus σ3 (4F2, Developmental Studies Hybridoma Bank, developed under the auspices of the NICHD and maintained by the University of Iowa, Department of Biology, Iowa City, IA, USA) [22], rabbit anti-phosphorylated MLKL (EPR9514, Abcam), rat anti-MLKL (MABC604, Merck Millipore), and mouse anti-vinculin (Sigma, V9131). Proteins were visualized using the Odyssey CLx Imaging System (LI-COR Biosciences). As a positive control for necroptosis induction, a cocktail was used that consists of TNF-alpha (InvivoGen Europe), BV6 (AbMole), and Z-VAD-FMK (Bachem AG).

RNA isolation and RT-qPCR analysis

Total RNA was isolated using the NucleoSpin RNA isolation kit (Macherey-Nagel, Düren, Germany) according to manufacturer’s instructions. cDNA was synthesized with the RevertAid First strand cDNA synthesis kit (Thermo Fisher Scientific) using 0.5–1.0 µg as RNA input. RT-qPCR was performed with SYBR Green Master mix (Bio-Rad laboratories, Nazareth, Belgium) using the iCycler Thermal Cycler and iQ5 Multicolour Real-Time PCR Detection System (Bio-Rad). Target genes were amplified using specific primers (supplementary Table 1). The ΔCt or ΔΔCt method was applied to calculate the levels of gene expression, relative to the reference gene (β-actin) or a control condition, respectively. Reovirus S4 10log copy numbers were determined using a standard curve, consisting of serial dilutions of plasmid pcDNA_S4. Copy numbers were calculated according to a described formula for rotavirus NSP3 quantification [23].

Oncolytic viruses

The wild-type type 3 Dearing (T3D) reovirus strain R124 was isolated by plaque purification from a heterogenous T3D stock obtained from ATCC (VR-824), and propagated as described previously [24]. The jin-3 mutant reovirus was isolated from U118MG cells upon infection with wild-type T3D. The genomes of the R124 and jin-3 viruses have been fully sequenced. GenBank IDs of the R124 segments are: L1 GU991659; L2 GU991660; L3 GU991661; M1 GU991662; M2 GU991663; M3 GU991664; S1 GU991665; S2 GU991666; S3 GU991667; S4 GU991668. Ad5-Δ24 is based on human serotype 5 (Ad5) and constructed using AdEasy technology. It contains a 24-nucleotide deletion (923-946) in E1A, encoding the amino acid sequence that is implicated to be vital for binding of the tumor-suppressive Retinoblastoma protein (pRb) [25]. As a result, the virus exerts tumor-selective replication [26].

Infectious virus titers were determined by plaque assays on 911 cells, as previously described [24]. All experiments were performed using CsCl-purified virus stocks. For purification, a freeze-thaw lysate containing virus particles was incubated with 0.1% Triton (Sigma-Aldrich, Zwijndrecht, The Netherlands) and 25 units/ml Benzonase (Santa Cruz, Bio-Connect B.V. Huissen, The Netherlands) for 15 min on ice followed by 15 min at 37 °C. After two extractions with Halotec CL10 (FenS B.V. Goes, The Netherlands), the cleared lysate was loaded onto a discontinuous CsCl gradient (1.45 and 1.2 g/cm3 in phosphate-buffered saline (PBS)). After centrifugation in a SW41 rotor (Beckman Coulter, Woerden, The Netherlands) at 20,000 g for 14 h at 4 °C, the lower band containing the infectious particles was harvested and desalted in an Amicon Ultra 100 K device according to the manufacturer’s protocol (Millipore, Merck Chemicals BV, Amsterdam, the Netherlands). The CsCl-purified reoviruses were recovered in reovirus storage buffer (RSB: 10 mM Tris-HCl, pH 7.5, 150 mM NaCl, 10 mM MgCl2 • 6 H2O) and stored at 4 °C until use. The CsCl-purified adenoviruses were recovered in adenovirus storage buffer (ASB: 140 mM NaCl, 5 mM Na2HPO4.2H2O, 1.5 mM KH2PO4, 5% sucrose, pH 7.8) and stored at −80 °C until use. An aliquot was used for OD260 measurement prior to storage to calculate the amount of viral particles.

Cell viability assays

To examine the viability of cells upon oncolytic virus infection, WST-1 reagent (Roche, Woerden, The Netherlands) was employed. In 96-wells plates, cells were seeded at 5–20 × 103 cells/well (5 wells per condition) in their respective culture media. Virus infections were performed in medium containing 2% FCS. Upon exposure to reoviruses R124 or jin-3, or Ad5-Δ24 at varying MOIs (0,01–10) for 3–5 days, the WST-1 read-out was performed. In short, 5 µl of WST-1 reagent per well was supplemented with 100 µl of fresh medium and added to each well. After incubation for 1–2 h, the OD450 values were measured and the percentage of cell viability was calculated by dividing the OD450 values of the virus-treated wells by the values of the mock condition. Negative values were manually adjusted to 1% to allow for plotting on linear as well as logarithmic scales. Crystal violet staining was performed according to the manufacturer’s protocol (Sigma). Briefly, 1 × 105 cells were seeded in 12-well plates and infected with different MOIs (0.1, 1 and 10) of reovirus wildtype R124 and mutant jin-3 for 2 days. Subsequently, reovirus-infected cells were fixed in with 4% PFA (Added Pharma, Oss, The Netherlands), washed twice with PBS and stained for 15 min with crystal violet. After subsequent washing with PBS, whole wells were imaged using the Cytation Microplate Reader (Biotek, Winooski, VT, USA).

Caspase (apoptosis) assays

In 96-wells plates, hPS1 cells were seeded at 1 × 104 cells/well in triplicate. Reovirus R124 or jin-3 infections were performed at a MOI of 10. To measure caspase 3/7 activity, a Caspase-Glo 3/7 assay (Promega, Leiden, The Netherlands) or CellEvent Caspase 3/7 Green Detection assay (Invitrogen, Breda, The Netherlands) was performed according to the manufacturer’s protocol. The Caspase-Glo 3/7 read-outs were performed at 48 h post-infection on a PerkinElmer’s VictorX3 (PerkinElmer, Groningen, The Netherlands) multilabel plate reader. The fold changes over the mock were calculated by dividing the values of the virus-treated wells over the values of the mock-exposed wells. The CellEvent Caspase 3/7 Green Detection assay was measured on a Cytation Microplate Reader (Biotek) at 500 nm excitation and 530 nm emission at intervals of 30 min throughout the course of the experiment.

Immunohistochemical and immunofluorescent staining

Four-micrometers sections were deparaffinized and processed for immunohistochemistry (IHC) or immunofluorescent (IF) staining. For IHC, sections were blocked in 0.3% hydrogen peroxidase (H2O2, Merck, Darmstadt, Germany) in methanol for 20 min. Next, IHC and IF slides were rehydrated, and antigen retrieval was performed by boiling in 0.01 M sodium citrate (pH 6.0) for 10 min. Slides were washed and incubated with mouse anti-sigma 3 (4F2, Developmental Studies Hybridoma Bank, Iowa, IA, USA), mouse-anti JAM-A (2E3-1C8, Abnova, Taipei, Taiwan), mouse anti-pan-cytokeratin (PKC-26, Sigma) and/or rabbit anti-human vimentin (D21H3, Cell signaling, Leiden, The Netherlands) antibodies diluted in 1% BSA/PBS overnight at room temperature in a humidified box. The next day, slides were incubated with appropriate biotinylated secondary antibodies (Agilent technologies, CA, USA) or anti-mouse-Alexa 488 (Thermo Fisher Scientific) and anti-Rabbit-alexa 568 (Thermo Fisher Scientific). For immunofluorescent staining, slides were mounted with ProLong™ Gold Antifade Mountant (Thermo Fisher Scientific) including DAPI. For IHC, slides were incubated with Vectastain complex (Vector Laboratories, CA, USA) at room temperature for 30 min. Staining was visualized with the Dako Liquid DAB+ Substrate Chromogen kit (Agilent Technologies) for 10 min. Nuclei were counterstained with Mayer’s Hematoxylin (Merck) and slides were rinsed in tap water, dehydrated, and mounted using Entellan (Merck). Images for IHC were obtained with an Olympus BX51 Light Microscope equipped with an Olympus DP25 camera. For IF, images were taken with a Leica DMi8 or Leica DM6B microscope (Leica). All images were analyzed using Fiji software.

Animal experiments

All performed animal procedures were approved by the Central Authority for Scientific Procedures on Animals. For all experiments, 8–12 weeks old C57BL/6JIco mice of both genders were used. Sample sizes were determined based on variation observed in previous experiments. Mice were distributed over groups with equal tumor volumes and were not randomized. Treatment was not blinded. Subcutaneous injection of KPC3-luc2 (1,0 x 105/mouse) in the flank was performed at day 0 after which tumor size was followed up by caliper measurements. When tumor size reached between 50–200 mm3 (approximately day 13), 109 PFU reovirus or solvent control (reovirus storage buffer) in control animals was injected intratumorally. Mice were sacrificed at a predefined tumor volume of 1500 mm3 or when weight loss exceeded 20% of baseline body weight. Subsequently, subcutaneous tumors were explanted and processed for histology.

Statistical analysis

Data are presented as means ± standard deviation from representative experiments of independent replicates. Unpaired Student t-tests were used to compare 2 groups. Differences between more than 2 groups were measured using 1-way analysis of variance (ANOVA) or 2-way ANOVA, depending on the amount of variables, and corrected for multiple testing. The correlation between JAM-A expression and cell viability was investigated using the Pearson correlation coefficient. All analyses were performed using GraphPad Prism software (San Diego, CA, USA). P values of 0.05 or less were considered statistically significant.

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