A total of 40 female BALB⁄c mice (6–8 weeks old and weighing 20 g) were purchased from Beijing HFK Bioscience Co. Ltd., (Beijing, China) and maintained in a under a specific pathogen-free environment at the State Key Laboratory of Biotherapy (Chengdu, China) with controlled temperature (20–26°C), humidity (40–70%) and a 12-h light/dark cycle.

All animal experiments were approved by the Institutional Animal Care and Use Committee of Sichuan University (Chengdu, China). CT26, a BALB/c-derived murine colon carcinoma cell line, was purchased from the American Type Culture Collection (Manassas, VA, USA). CT26 cells were cultured in RPMI-1640 supplemented with 10% fetal bovine serum (Gibco; Thermo Fisher Scientific, Inc., Waltham, MA, USA) in a humidified atmosphere containing 5% CO₂ at 37°C.

PT-100 was purchased from Shanghai Speed Chemical Co. Ltd., (Shanghai, China). The purity was >97%. The molecular weight of PT-100 is 246.1 g/mol and its structure is shown in Fig. 1.

The flanks of the mice were shaved and subcutaneously inoculated with 1×10⁶ CT26 cells. The tumor-inoculated mice were divided into four groups: The saline vehicle group; the oxaliplatin group, which was treated with 5 mg/kg oxaliplatin (Jiangsu Hengrui Medicine Co., Ltd., Lianyungang, China) three times a week; the PT-100 group, which was treated with 20 µg PT-100 per mouse daily; and a combined oxaliplatin and PT-100 group. All drugs were dissolved in saline and administered by intraperitoneal injection. The day of tumor inoculation was defined as day 0, the treatment began on day 8 and animals were treated for 14 days. Tumor growth was monitored every 3 days by measurement of the length (L) and the width (W) of the xenograft tumors using Vernier calipers (Shanghai Taihai Measuring Tools Co., Ltd., Shanghai, China). The tumor volume was calculated using the following formula: Volume (cm³) = W²×0.5L. The xenograft experiment was performed twice with 5 mice per group in each experiment.

At the appropriate time-points, for paraffin embedding, the tumors were excised and post-fixed in 4% paraformaldehyde overnight. For cryopreservation, the tumors were excised and stored in optimum cutting temperature compound (OCT; Leica Microsystems GmbH, Wetzlar, Germany) at −20°C.

FAP expression was assessed by immunohistochemistry in the CT26 colon cancer cell-derived xenograft tumors from female BALB⁄c mice. Tissues were cut into 5-µm sections, incubated with 3% H₂O₂ at 4°C for 20 min, washed using phosphate-buffered saline (PBS) containing 3.74 g 12H₂O. Na₂HPO₄, 0.44 g NaH₂PO₄.2H2O and 7.2 g NaCl in 1l distilled water (PH 7.4) and incubated with serum at 37°C for 20 min. Subsequently, the sections were incubated with the following primary antibodies at 4°C overnight: Polyclonal anti-FAP (1:300; ab28244) (v/v), monoclonal anti-vimentin (1:500; ab92547) (v/v) and monoclonal anti-CD31 (1:50; ab7388; all Abcam, Cambridge, MA, USA) (v/v). Following washing with PBS, the sections were incubated with a biotin-streptavidin-horseradish peroxidase (HRP) detection system (SP-9000; OriGene Technologies, Inc., Beijing, China) at 37°C for 1 h, washed with PBS and subsequently incubated with 3,3′-diaminobenzidine (DAB) for 2 min and washed using PBS. The HRP-conjugated secondary antibody and the DAB were included in the detection system (SP-9000; ZSGB-Bio Origene Co, Ltd., Beijing, China), which was not diluted and was used according to the manufacturer's instructions. To assess the levels of apoptosis in CT26 tumors, in situ terminal deoxynucleotidyl transferase-mediated deoxy-UTP nick end labeling (TUNEL) staining was performed on the tumor sections using the the Deadend™ Fluorometric TUNEL system (G3250; Promega, Madison, WI, USA) according to the manufacturer's instructions.

For histological analysis, the paraffin-embedded tumor tissues were de-paraffinized in xylene, re-hydrated in 100, 95, 85 and then 75% ethanol, immersed in PBS (pH 7.4) and stained with hematoxylin (H3136-25G) and eosin (E4009-5G; both Sigma-Aldrich, St. Louis, MO, USA), according to the manufacturer's instructions. A Leica DM 2500 microscope was used for visualization (Leica Microsystems GmbH).

On day 22, the tumors were excised and proteins were extracted using radioimmunoprecipitation assay buffer (Beyotime Institute of Biotechnology, Inc., Haimen, China) supplemented with protease inhibitor cocktail (100:1; Sigma-Aldrich). Tumor tissues were homogenized prior to western blot and PCR analysis in liquid nitrogen using a mortar and pestle. Total protein concentrations in the supernatant were determined via the Bicinchoninic Acid assay (P0013B; Beyotime Institute of Biotechnology, Inc.). A total of 30 µg protein was loaded per lane for western blot analysis. Protein samples were separated by 10% SDS-PAGE and transferred onto polyvinylidene difluoride membranes (EMD Millipore, Billerica, MA, USA). After blocking with 5% skimmed milk in TBS containing 0.1% Tween 20 (TBST) for 2 h at 37°C, the membranes were incubated with anti-FAP (1:500; 28244; Abcam) or anti-β-actin (1:1,000; sc-130657; Santa Cruz Biotechnology, Inc., Dallas, TX, USA) primary polyclonal antibodies at 4°C overnight. After washing four times with TBST for 10 min, the membranes were incubated with the goat anti-rabbit HRP-conjugated secondary antibody (1:5,000; sc-2054; Santa Cruz Biotechnology, Inc.) for 1 h at 37°C. After washing with TBST as described above, the bands were visualized using enhanced chemiluminescence reagents (WBKLS0100 EMD Millipore).

On day 22, the tumors were excised, dissected into small pieces using a scalpel, digested in collagenase digestion buffer (1% collagenase in RPMI-1640; Gibco; Thermo Fisher Scientific, Inc.) for 2 h at 37°C with agitation and the cells in the resulting suspension were counted. Cells were passed through a 70 µm cell strainer (BD Biosciences, Franklin Lakes, NJ, USA) following disaggregation A total of 1×10⁶ cells in 100 µl were stained. The cells were labeled using rat anti-mouse CD11b-allophycocyanine (1:50; cat no. 553312; BD Biosciences), hamster anti-mouse CD11c-fluorescein isothiocyanate (1:100; cat no. 553801; BD Biosciences), or rat anti-mouse F4/80-A488 (1:50; cat no. MCA497A488; Serotec, Bio-Rad Laboratories, Inc., Hercules, CA, USA) antibodies on ice for 30 min, followed by analysis by flow cytometry using FACSCalibur or LSR II flow cytometers (BD Biosciences); data were analyzed using CellQuest 6.0 software (BD Biosciences).

Total RNA was extracted from the xenograft tumors using the AxyPrep™ Multisource Total RNA Miniprep kit (Axygen Biosciences, Union City, CA, USA) in accordance with the manufacturer's instructions. The PrimeScript™ RT reagent kit with gDNA Eraser (Takara Bio Inc., Otsu, Japan) was used to synthesize cDNA, and RT-qPCR analysis was performed using SsoAdvanced™ SYBR Green Supermix (Bio-Rad Laboratories, Inc.) following the manufacturer's instructions. Transcript expression was determined relative to GAPDH. The primers used were purchased from BGI-Shenzhen (Shenzhen, China) and were as follows: GAPDH, 5′-ACCCAGAAGACTGTGGATGG-3′ (forward) and 5′-TCTAGACGGCAGGTCAGGTC-3′ (reverse); TGF-β, 5′-AAGTGGGTCCATGAACCTAA-3′ (forward) and 5′-GCTACATTTACAAGAC66CAC-3′ (reverse); FGF-2, 5′-GGCTGCTGGCTTCTAAGTGT-3′ (forward) and 5′-CCGTTTTGGATCCGAGTTTA-3′ (reverse); and osteopontin, 5′-TGCACCCAGATCCTATAGCC-3′ (forward) and 5′-CTCCATCGTCATCATCATC-3′ (reverse). The 20 µl PCR system contained 2 µl genomic DNA, 2 µl dNTPs, 10 µl buffer, 0.5 µl Rox reference dye and 5 pmol of each primer. Thermal cycling was performed as follows: Denaturing at 95°C for 3 min, then 30 cycles of 95°C for 30 sec, 55°C for 30 sec and 72°C for 30 sec, with elongation at 72°C for 10 min using a CFX96 Real-Time C1000 thermocycler (Bio-Rad Laboratories, Inc.). PCR products were electrophoresed on 1% agarose gel (800669; Schwarz/Mann Biotech, Cleveland, OH, USA) at 200 mA until separation was achieved. DNA fragments were visualized using a long wave UV light box and images were captured using a Universal Hood II Gel Doc™ XR camera (1708170) and were analyzed using Image Lab software (both Bio-Rad Laboratories, Ltd.).

SPSS version 11.5 (SPSS, Inc., Chicago, IL, USA) was used for statistical analyses. Values are expressed as the mean ± standard error of the mean. One-way analysis of variance was used to assess statistical significance. Survival curves were compared using the log-rank test. P<0.05 was considered to indicate a statistically significant difference.

Chemotherapy is widely used for the treatment of colorectal cancer; however, drug resistance has become problematic due to its increasing frequency. Several mechanisms of drug resistance of cancers have been proposed (31,32); however, a consensus has not yet been reached and further elucidation is required. The present study hypothesized that CAFs may have an important role in drug resistance of cancers. To test this hypothesis, Balb/C mice (n=5 per group) were inoculated with CT26 colon carcinoma cells (day 0) and then treated with the saline vehicle or oxaliplatin for 14 days (from days 8–22). Subsequently, the mice were sacrificed via cervical dislocation and the tumor xenografts were excised, sectioned and stained using an anti-FAP and anti-vimentin antibodies. As shown in Fig. 2, treatment with oxaliplatin markedly increased the amount of FAP and vimentin, which are specific markers of CAFs, expressed in the stroma of the tumor tissues, indicating that oxaliplatin increased the accumulation of CAFs in the xenograft tumors.

In order to evaluate whether the ability of oxaliplatin to increase the accumulation of CAFs in the xenograft tumors was clinically relevant, the present study combined pharmacological inhibition of CAFs by PT-100 with the chemotherapeutic drug oxaliplatin, to which CT26 cells are partially sensitive, in a xenograft model. Mice (n=5 per group) were subcutaneously injected with CT26 cells on day 0 and then treated with saline vehicle, PT-100 alone, oxaliplatin alone, or PT-100 combined with oxaliplatin from days 8–32. As shown in Fig. 3A, PT-100 combined with oxaliplatin suppressed tumor growth more significantly than treatment with either oxaliplatin or PT-100 alone (P<0.05). PT-100 combined with oxaliplatin also significantly increased the survival of the mice compared to that in the other three treatment groups, particularly the mice treated with saline vehicle, which died after 45–60 days (Fig. 3B).

Consistent with the results shown in Fig. 2, FAP, a specific marker for CAFs, was most highly expressed in the stroma of the tumors of the mice treated with oxaliplatin alone, indicating that oxaliplatin increased the accumulation of CAFs in the xenograft tumors. However, PT-100 combined with oxaliplatin markedly reduced the expression of FAP and therefore prevented the accumulation of CAFs in the stroma of the tumor tissues. In addition, TUNEL staining was performed on the xenograft tumor sections, which revealed that PT-100 combined with oxaliplatin increased the number of apoptotic tumor cells compared with that in the three other treatment groups (Fig. 3C–F).

Next, to investigate how oxaliplatin induced the accumulation of CAFs, RT-qPCR analysis was performed to assess the expression of a number of cytokines which are associated with the accumulation of CAFs in the xenograft tumor tissues. CAFs are hypothesized to accumulate in the tumor microenvironment by the following major mechanisms: Resident fibroblasts transforming into CAFs, the EMT and the EndMT (8,10,11); these processes are mediated by several common cytokines, including TGF-β3, FGF-2 and osteopontin (9,12). Thus, the present study quantitatively measured the abundance these cytokines in the xenograft tumor tissues. As shown in Fig. 3G, oxaliplatin increased the expression of TGF-β3 and FGF-2 (basic FGF) by 1.79- and 2.63-fold, respectively, compared to those in the tumors of the saline vehicle-treated animals. Furthermore, the expression of TGF-β3 and FGF-2 in the tumors of animals treated with PT-100 alone or with PT-100 combined with oxaliplatin was lower than that in the saline vehicle-treated animals. Although oxaliplatin treatment did not significantly increase the expression of osteopontin mRNA, its expression was significantly decreased in the tumors of the animals treated with PT-100 only or with PT-100 combined with oxaliplatin (0.28- and 0.16-fold, respectively, of that in the saline vehicle-treated group). These results indicated that pharmacological inhibition of CAFs using PT-100 reduced the tumor expression levels of cytokines known to promote the accumulation of CAFs.

The tumor micro-environment can affect the malignant potential of the tumor; tumor-associated macrophages and dendritic cells have an important role in this process (33,34). Macrophages constitute an extremely heterogeneous population, including M2 (or alternatively activated) macrophages, which are now generally accepted to be TAMs. TAMs mostly exert pro-tumor functions by promoting tumor-cell survival, proliferation and dissemination. Tumor-associated dendritic cells (DCs) are another immune-regulatory cell population. A variety of sub-populations of tumor-associated DCs are known, among which CD11c+ DCs are able to promote tumorigenesis (35). In order to test whether CAFs interact with M2 macrophages or immature dendritic cells (imDCs) and tumor-infiltrating DCs (TIDCs), tumor cells from mice at day 22 were analyzed by flow cytometry. As shown in Fig. 4, the number of M2 macrophages (F4/80+) was highest in the saline vehicle-treated group and lowest in the xenograft tumors of animals treated with PT-100 combined with oxaliplatin; furthermore, tumors of animals treated with PT-100 combined with oxaliplatin contained significantly lower amounts of M2 macrophages compared with those in the groups treated with saline vehicle, oxaliplatin alone and PT-100 alone (P<0.01). In addition, the number of imDCs and TIDCs was highest in the saline vehicle-treated tumors, while the tumors of animals treated with PT-100 combined with oxaliplatin contained a significantly lower number of imDCs and TIDCs, as compared with the groups treated with saline vehicle, oxaliplatin only and PT-100 only (P<0.05). These results indicated that pharmacological inhibition of CAFs using PT-100 in combination with oxaliplatin significantly reduced the recruitment of M2 macrophages and CD11c+ DCs to the xenograft tumors.

Angiogenesis, a key event required for tumor progression, is dependent on ECM remodeling (1). As an important source of ECM components, CAFs are able to promote angiogenesis during tumor growth and metastasis (1,5). In order to test whether pharmacological inhibition of CAFs had any effect on angiogenesis in the tumor xenografts, immunohistochemical analysis was used to determine the density of CD31+ endothelial cells in the xenograft tumors at day 22. The tumors treated with PT-100 combined with oxaliplatin had a markedly lower density of CD31+ cells compared to that in the groups treated with saline vehicle, oxaliplatin alone and PT-100 alone (Fig. 5). These results indicated that pharmacological inhibition of CAFs using PT-100 in combination with oxaliplatin significantly reduced tumor angiogenesis.