In vivo models
We obtained three tumor samples from patients treated at Hokkaido University Hospital. Informed consent was obtained from all subjects, and all patients were histologically diagnosed with epithelial ovarian cancer. To establish the PDX, we obtained the first and third tumors during surgery and immediately transplanted them into female NOD/Shi-scid, IL-2Rγ KO Jic mice (CLEA Japan, Inc., 6-week-old and 18–22 g). We preserved the second tumor in liquid nitrogen and transplanted it on the other day. All tumors were cut into small pieces (< 1 mm), injected subcutaneously, and left until all tumor diameters increased to approximately 10 mm. All mice were randomly assigned to each treatment group. All tumor volumes were measured under general anesthesia using isoflurane inhalation (Wako, Japan) and calculated using the following formula: volume (mm3) = most extended diameter × shortest diameter × thickness. Mice were housed in a pathogen-free environment and kept on a 12/12 h light-dark cycle at a temperature of 23°C to 25°C with unrestricted access to food and DDW water. We administered the following treatments every 3 or 4 days by two diagonal injections of 25 µL of dissolved drugs directly to the tumor edge: CCR2i (low-dose: 20 mg/kg or high-dose: 40 mg/kg in DMSO, BMS CCR2 22; TOCRIS Bioscience, Bristol, UK), and BEV (10 mg/kg in saline; Chugai, Japan). Cervical subluxation sacrifice therapies were used when the size of the tumor interfered with the mobility of the mice or when the condition of the mice deteriorated severely.
RNA isolation and qRT–PCR
Total RNA was isolated from the frozen PDXs and incubated with KF28 and RMG-I cells. After dissolving the tissues and cells in TRIzol (Invitrogen, Carlsbad, CA, USA), total RNA was extracted using the RNeasy Mini Kit (Qiagen, Hilden, Germany) following the manufacturer's instructions. A NanoDrop 2000 spectrophotometer (Applied Biosystems, Thermo Fisher Scientific Inc.) was used to quantify RNA. The mRNA expression of each target gene was normalized to that of the housekeeping gene 18S.
18S-probe (Applied Biosystems, Assay ID: Hs99999901_s1) FAM-CCATTGGAGGGCAAGTCTGGTGCCA-NFQ
CCR2A-probe (Applied Biosystems, Assay ID: Hs00174150_m1) FAM-GGAGAAGTTCAGAAGCCTTTTTCAC-NFQ
CCR2B-probe (Applied Biosystems, Assay ID: Hs00704702_s1) FAM-GGAGTGAGGGATAGTGGGGTCAGGG-NFQ
VEGF-probe (Applied Biosystems, Assay ID: Hs00900055_m1) FAM-ACATCACCATGCAGATTATGCGGAT-NFQ
TGF-β-probe (Applied Biosystems, Assay ID: Hs00998133_m1) FAM-AGTACAGCAAGGTCCTGGCCCTGTA-NFQ
CCL2-probe (Applied Biosystems, Assay ID: Hs00234140_m1) FAM-CGCTCAGCCAGATGCAATCAATGCC-NFQ
The probe was employed in a 20µL reaction with 10µL TaqMan Fast Advanced Master Mix (Applied Biosystems, Thermo Fisher Scientific Inc., Waltham, MA, USA), 1µL probe, 7µL water, and 2µL cDNA template. Data were analyzed using the StepOnePlus Real-Time PCR system (Applied Biosystems). All reactions were conducted using the following temperature profile: 50℃ for 2 min (UNG incubation), 95℃ for 20 s (polymerase activation), 40 cycles of 95℃ for 1 s (denaturation), 60℃ for 20 s (annealing, extension). Relative quantification of the target gene expression was performed using the 2-ΔΔCt method.
The xenografted tumors were removed, fixed with 4% formaldehyde, embedded in paraffin, and sectioned into 4-µm-thick sections. Slides were incubated with primary antibodies against CD31 (1:1 000 dilution, Proteintech Cat# 11265-1-AP, RRID:AB_2299349), CA9 (1:400 dilution, Proteintech Cat# 11071-1-AP, RRID:AB_2066528), CCR2A (1:200 dilution, Proteintech Cat# 16153-1-AP, RRID:AB_2262945), CCR2B (1:300 dilution; Proteintech Cat# 16154-1-AP, RRID:AB_2878224). For microscopic observation, the sections were incubated with diaminobenzidine (DAB) chromogen and counterstained with hematoxylin.
Tissue clearing for PDXs
Preparation of clearing cocktails
Clearing cocktails were selected for chemical screening (26). CUBIC-L for decolorization and delipidation was prepared as a mixture of 10 wt% polyethylene glycol mono-p-isooctylphenyl ether/Triton X-100 (12967-45, Nacalai Tesque, Kyoto, Japan) and ten wt% N-butyldiethanolamine (B0725, Tokyo Chemical Industry Co., Ltd.). CUBIC-R for refractive index (RI) adjustment was prepared as a mixture of 45 wt% 2,3-dimethyl-1-phenyl-5-pyrazolone/antipyrine (D1876, Tokyo Chemical Industry Co., Ltd.), 30 wt% nicotinamide (N0078, Tokyo Chemical Industry Co., Ltd.) pH of CUBIC-R was adjusted to 9.5 by N-buthyldiethanolamine.
Tissue clearing for excised tumors
The fixed tumors were washed three times with PBS to remove PFA before clearing. For tissue decolorization and delipidation, the tumors were immersed in 50% (v/v) CUBIC-L (1:1 mixture of water and CUBIC-L), then 100% CUBIC-L with gentle shaking at 37°C for three days. CUBIC-L was refreshed when the cocktail was colored. After decolorization and delipidation, the tumors were washed thrice with PBS at room temperature. For RI adjustment, the tumors were further immersed in 50% (v/v) CUBIC-R (1:1 mixture of water and CUBIC-R) and then in CUBIC-R at room temperature with gentle shaking for one day.
Decolorized and delipidated tumors were subjected to immunostaining with diluted monoclonal anti-actin, α-smooth muscle-FITC antibody (Sigma-Aldrich Cat# F3777, RRID:AB_476977) using 1:200 dilutions in staining buffer composed of 0.5% (v/v) Triton X-100, 0.25% casein (37528, Thermo Fisher Scientific), and 0.01% sodium azide (31208-82, Nacalai Tesque) for three days at room temperature with gentle shaking. The stained tumors were washed thrice with PBS at room temperature with gentle shaking and then post-fixed with 4% PFA for one hour at room temperature before RI adjustment.
Tumor images were acquired using a light-sheet fluorescence microscope (UltraMicroscope II, Miltenyi Biotec, Germany). Images were captured using a 1.1 × objective lens. Lasers with wavelengths of 488 nm and 561 nm were used for image acquisition. The RI-matched tumors were immersed in a mixture of silicon oil HIVAC-F4 (RI = 1.555; Shin-Etsu Chemical Co., Ltd., Tokyo, Japan) and mineral oil (RI = 1.467; M8410; Sigma-Aldrich) during image acquisition. The 3D rendered images were visualized and captured using the Imaris software (version 9.6.0, Bitplane AG, Zurich, Switzerland).
Western blot cell lysates were produced by lysing cells in RIPA buffer (10 mM Tris (pH 7.4), 150 mM NaCl, 1% Triton X-100, 1% sodium deoxycholate, 5 mM EDTA, 10% glycerol, 0.1% sodium dodecyl sulfate (SDS)) We collected the supernatant following homogenization with a Polytron homogenizer and centrifugation at 15 000 g for 10 min at 4°C. Protein concentrations in whole-cell lysates were determined using the Protein Assay Dye Reagent Concentrate (Bio-Rad, USA), and then equal protein amounts (20µg) were heated to 95°C for 10 min in SDS sample buffer (25 mL glycerol, 31.2 mL Tris buffer, 7.5 mL SDS, a dash of bromophenol blue/100 mL). Proteins were separated by 15% SDS sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to PVDF membranes (Millipore, Billerica, MA, USA). Membranes were blocked for 1 h at room temperature in 5% non-fat dry milk in TBS-T buffer (0.1% Tween-20 in Tris-buffered saline). They were then incubated with primary antibodies against CCR2A (1:1 000 dilution, Proteintech Cat# 16153-1-AP, RRID:AB_2262945), and CCR2B (1:1 000 dilution; Proteintech Cat# 16154-1-AP, RRID:AB_2878224), p44/42 MAPK (Erk1/2, 1:1 000 dilution, Cell Signaling Technology Cat# 9102, RRID:AB_330744), phosphor-p44/42 MAPK (Erk1/2 Thr202/Tyr204, 1:1 000 dilution, Cell Signaling Technology Cat# 9101, RRID:AB_331646), and anti-actin (1:1 000 dilution, Millipore Cat# MAB1501, RRID:AB_2223041) in 5% milk/TBS-T or 5%BSA/TBS-T overnight at 4°C. The membranes were washed with TBS-T buffer at least thrice for ten minutes after shaking. Membranes were incubated at room temperature for 1.5 hours with a horseradish peroxide-conjugated secondary antibody at a dilution of 1:5 000 in 5% milk/TBS-T. Following washing, signals on the membrane were generated using ECL reagent (Amersham, GE Healthcare, UK) and then photographed using the ChemiDoc system (Bio-Rad, United States). The SuperSignal West Femto Maximum Sensitivity Substrate (Thermo Fisher Scientific, Inc.) was used to visualize the bands.
Dr. Yoshihiro Kikuchi kindly provided human KF28 (RRID: CVCL_V533) ovarian cancer cells (27). RMG-I human ovarian clear cell carcinoma and HUVEC human endothelial cell lines were obtained from the JCRB Cell Bank (Osaka, Japan; RRID: CVCL_1662) and the Riken Cell Bank (Tsukuba, Japan; RRID: CVCL_2959). KF28 cells were cultured in DMEM (Sigma-Aldrich, MO, USA), RMG-1 cells were cultured in Ham's F12 medium (Sigma-Aldrich), and HUVECs were cultured in EBM-2 medium (Lonza, Walkersville, MD, USA). All media were prepared with 2mM L-glutamine (Lonza, Belgium), 10% FBS (Sigma, St. Louis, Missouri, United States), and 100 U/mL penicillin/streptomycin (Lonza, Belgium) and incubated at 37°C in a 5% CO2 atmosphere.
The MTT assay for cytotoxicity was performed according to the manufacturer's instructions using Cell Proliferation Kit I (Roche, Mannheim, Germany). In summary, 7 500 cancer cells were plated per well in 96-well plates in 100µL of culture medium with reagents. We increased the concentration of CCR2i 10-fold from 10 to 10 000 nM. An equivalent amount of DMSO was used as control. The absorbance of the formazan product was measured at 550 nm wavelength using a microplate reader.
HUVECs were seeded on Corning Matrigel 24-Well Plate 8.0 micron (BioCoat, MA, USA) and cultured for 72 h at 37°C in a 5% CO2 atmosphere. Invading HUVECs (20 × 103 cells/well) were counted, and KF28 (50 × 103 cells/well) or RMG-I (50 × 103 cells/well) cells were introduced into the lower chamber with 500µL of 1% FBS medium with or without CCR2i (1 × 103 nM) as a chemoattractant. Invasive cells that had migrated through the membrane to the bottom surface were counted in three distinct fields at a magnification of 100 using a light microscope (ZEISS, Axiocam 506 Color). Each experiment was conducted in triplicate.
Statistical analyses were performed using JMP Pro 16 statistical software package (SAS Institute, Cary, NC, USA). Continuous variables were compared using Student's t-test if normally distributed. Differences in variables that were not normally distributed were compared using the nonparametric Mann–Whitney U test. Microvessel density and CCR2B mRNA expression are presented as box plots, and other data are presented as mean ± SD.
All statistical tests were two-sided, and P-values less than 0.05 were considered significant.