Cancer-associated fibroblasts (CAFs) play an important role in cancer expansion and progression in tumor microenvironment (TME), via both direct and indirect interactions. Natural killer (NK) cells play a crucial role in anticancer immunity. We investigated the inhibitory effects of CAFs on NK cell activity. CAFs were isolated from endometrial cancer tissue, while normal endometrial fibroblasts (NEFs) were obtained from normal endometrium with no pathological abnormality. NK cells were obtained from allogenic healthy volunteers. CAFs or NEFs were co-cultured at an NK/fibroblast ratio of 1:1 with or without inserted membrane. For NK cell activity, K562 cells were cultured as target cells. NK cell-killing activity was determined by calculating the ratio of PI-positive K562 cells in the presence of NK cells co-cultured with fibroblasts versus NK cells alone. To examine whether NK cell activity was suppressed by IDO pathway, we inhibited IDO activity using the IDO inhibitor 1-MT. We demonstrated that CAFs derived from endometrial cancer induced greater suppression of the killing activity of allogenic NK cells compared with normal endometrial fibroblasts (NEFs). The suppression of NK cell activity by CAFs was inhibited when a membrane was inserted between the CAFs and NK cells, but not by 1-MT, an inhibitor of IDO. We focused on receptor-ligand interactions between CAFs and NK cell and found that cell-surface poliovirus receptor (PVR/CD155), a ligand of activating NK receptor DNAM-1, was downregulated in the CAFs compared with NEFs. To confirm whether PVR downregulation results in the decrease of NK cell-killing activity, PVR expression in NEFs was knocked down using siRNA against PVR (PVRsi). NK cell activity was suppressed by co-culture with PVR-knockdown NEFs, to a similar extent than CAF-induced suppression. CAFs showed increased suppression of NK cell-killing activity compared with NEFs, due to decreased PVR cell surface expression, a ligand of an NK activating receptor. This study demonstrated a novel mechanism of suppression of NK cell activity by CAFs in the TME.
Cancer-associated fibroblasts (CAFs) regulate not only carcinogenesis, but also the immune evasion of cancer in the tumor microenvironment (TME), which facilitates cancer cell proliferation, expansion, and metastasis (
NK cells play an important role in cancer immunity in the TME. A review by Chan
Poliovirus receptor (PVR/CD155) is a ligand of the paired NK receptors, DNAM-1 (activating) and TIGIT (inhibiting). NK cells can kill cancer cells expressing PVR via the DNAM-1-mediated activating signaling (
Considering the NK cell-mediated immune evasion mechanisms in the TME, we hypothesized that in addition to malignant cells, CAFs may also play a role in the suppression of NK cell activity in the TME. In this study, we used CAFs and normal endometrial fibroblasts (NEFs), derived from endometrial cancer and normal endometrial stroma, respectively. In the uterine endometrium, endometrial stroma is enriched in fibroblasts and surrounds the endometrial glandular epithelia, and these NEFs can be transformed to CAFs in endometrial cancer. Therefore, the use of endometrial cancer is suitable for comparison between CAFs and NEFs. In this study, we investigated the inhibitory effect of CAFs on NK cell-killing activity and the underlying mechanism.
Tumor samples were obtained from the patients with endometrial carcinoma, and normal endometrium were collected from those without pathology in uterine endometrium, undergoing surgical resection in our hospital. All women gave written informed consent and the Research Ethics Committee of the University of Tokyo approved all aspects of the study.
CAFs were isolated from the cancer tissues of endometrial cancer while NEFs were from the normal endometrium with no pathological abnormality. The tissues were minced and digested in DMEM/F12 medium, (Gibco, Japan), supplemented with 100 IU/ml penicillin, 100 μg/ml streptomycin, 1 mg/ml collagenase type I (Wako, Tokyo, Japan), and 25 ng/ml DNase, Roche Diagnostics GmbH at 37°C for 60 min, filtered with 100 and 70 μm cell strainers, BD Falcon, and centrifuged at 1,500 rpm for 5 min and washed with D-PBS, Wako. They were resuspended in DMEM/F12 with 10% FBS, 100 IU/ml penicillin, 100 μg/ml streptomycin, and cultured at 37°C in humidified 5% CO2 environment. Fibroblasts passaged for 2–8 passages were used before the experiments.
NK cells were obtained from healthy volunteers after Ficoll-Paque gradient and negative magnetic selection, using human NK cell isolation kit from Miltenyi Biotec. NK cell purity was >95% as evaluated by flow cytometry. They were cultured in RPMI with 10% FBS, 100 IU/ml penicillin, 100 μg/ml streptomycin, at 37°C in humidified 5% CO2 atmosphere, and stimulated 1 ng/ml IL15 (R&D) for 48 h, before co-culture.
For co-culture experiments, CAFs or NEFs were seeded at 5×104 in 24-well plates with 500 μl of medium and cultured for 48 h. NK cells were added at 1×105 well, at an NK/fibroblasts ratio of 1:1, with or without 1-μm pore of cell-culture-insert. NK cells alone were also cultured in the absence of fibroblasts. After 24-h incubation, NK cells were harvested and analyzed. We confirmed that allogenic NK cells did not kill these fibroblasts during co-culture (data not shown).
K562 cells (obtained from ATCC; American Type Culture Collection, VA, USA) were cultured in RPMI-1640 supplemented with 10% FBS containing 100 U/ml penicillin and 100 μg/ml streptomycin. K562 cells were resuspended at 1–2×107 cells/ml in 0.1% FBS/PBS and added CFSE, using CFSE Cell Division Assay kit from Cayman chemical, to a final concentration of 2.5 μM CFSE staining solution, incubated cells at 37°C for 30 min. After centrifugation of cells at 300 g for 10 min, supernatant was discarded and they were resuspended into RPMI medium and incubated at 37°C for 30 min. The cells were washed with 2% FBS/PBS three times, and 2×104 of stained K562 cells in 200 μl medium were added to tubes. Harvested NK cells were diluted at 1×106 cells/ml and prepare dilution series 2×105 cells per 200 μl. After incubation the tubes at 37°C for 4 h, 5 μl of PI (propidium iodide solution, Biolegend, San Diego, CA, USA) was added for dead cell count. Percentage of PI-positive dead K562 cells (% K562 PI-positive cell) was evaluated by flow cytometry. In some experiments, allogenic NK cells were incubated for 24 h with NEFs, CAFs or no fibroblasts followed by exposure to target (K562) cells. NK cell killing activities were indicated by ratio of % K562 PI positive cells under each condition against that of NK cells only.
CAFs were grown on coverslips, then serum deprived and fixed in 4% paraformaldehyde, permeabilized with 0.1% Triton X-100, washed twice with PBS, and incubated for 60 min at room temperature with an anti-α-SMA antibody (mouse clone 1A4, ab7817, Abcam MA, USA), labeled with Alexa Flour 488 (Zenon). After incubation, the slides were washed with PBS and fixed with 4% paraformaldehyde. The cells were counterstained with 4′,6-diamidino-2-phenylindole dihydrochloride (DAPI).
CAFs and NEFs grown in chamber slides were fixed and each of them were collected and total proteins were extracted. Immunoblotted with anti-beta Actin antibody (rabbit polyclonal antibody, Abcam), and α-SMA (mouse clone 1A4, ab7817, Abcam).
Cultured cells were harvested and incubated with PerCP Cy5.5-conjugated CD90 (mouse clone Thy1, BioLegend), FITC-conjugated Vimentin (mouse clone RV202, ab8978, Abcam), PE-conjugated PVR (mouse clone SKII.4, BioLegend) or appropriate isotype control at 4°C for 30 min, washed twice, and analyzed using a BD FACS Calibur cytometer. The results were analyzed using Kaluza software.
NEFs were transfected with 100 pmol of siRNA with Lipofectamine RNAiMAX. The complexes of RNAi and 1.5 μl Lipofectamine RNAiMAX in 140 μl Opti-MEM I medium without serum. Then the complexes were added in the cultured cells at 4×104 in 24-well plates, and 500 μl of complete medium without antibiotics was gently added in each well, and incubated for 12 h at 37°C in a CO2 incubator to be ready for the next assay of gene knockdown. To knock down PVR, siRNA by R&D was used. The sequences were as follows: human PVR; sense, 5′-rCrArGrCUrAUUrCrGrGrArCUrCrCrArArATT; antisense, 5′-UUArGrGrArCUrCrCrGrArAUrArGrCUrGTT. The negative siRNA controls were obtained from Life Technologies.
Data are presented as means ± SEM. Statistical analyses were carried out using Student's t-test or Dunnett analysis using JMP software. A value of p<0.05 was considered significant. Asterisks indicate those comparisons with statistical significance (p<0.05).
To investigate the difference in effect on NK cell activity between NEFs and CAFs, we isolated fibroblasts derived from normal endometrium (NEFs) and endometrial cancer tissue (CAFs). Uterine endometrium is composed of many glands and abundant stroma. The endometrial stroma is enriched in fibroblasts, and surrounds the endometrial glandular epithelium. Endometrial cancer can transform normal fibroblasts in the stroma into CAFs. Therefore, the comparison between endometrial CAFs and NEFs was thought to be suitable for the investigation of CAF activity. Fibroblasts were isolated from cancer tissue or normal endometrium by standard isolation methods and identified by immunostaining and western blotting. Fibroblasts isolated from endometrial cancer expressed the fibroblastic markers vimentin and CD90 (
We assessed the effect of CAFs on the killing activity of NK cells to investigate the mechanism of CAF-mediated immune evasion. Assessment of NK cell activity is often performed by measuring allogenic NK cell killing activity against K562 cells (
Some studies have shown that malignant cells suppress NK cell activity via production of IDO (
To examine whether direct cell-to-cell interaction between NK cells and CAFs was required for the suppression of NK cell activity, NK cells and CAFs were cultured in a chamber with an inserted membrane separating these cells (
We next focused on the cell-surface ligands expressed on CAFs that interact with activating NK cell receptors. Several ligands of paired or activating NK receptors have been previously demonstrated to be expressed on the cell surface of target cells, including malignant cells (
To confirm whether PVR downregulation results in the decrease of NK cell-killing activity, PVR expression in NEFs was knocked down using siRNA against PVR (PVRsi) (
We demonstrated that CAFs showed increased suppression of NK cell-killing activity compared with NEFs, due to decreased PVR cell surface expression, a ligand of an NK activating receptor. In this study, human CAFs and NEFs were isolated from the stroma of endometrial cancer and normal endometrium, respectively, and their interactions with NK cells were compared. Uterine endometrium is composed of many glands and abundant stroma. The endometrial stroma is enriched in fibroblasts, and surrounds the endometrial glandular epithelium. Endometrial cancer cells can transform normal fibroblasts in the stroma into CAFs. In the cancer microenvironment, CAFs acquire cancer-specific characteristics in addition to their fibroblastic background. Therefore, we believe that the comparison between endometrial CAFs and normal endometrial stromal fibroblasts was suitable for investigation of CAF characteristics. We observed an increased α-SMA expression level in CAFs compared with NEFs, in confirmation with previous studies demonstrating the expressing of α-SMA and vimentin in active CAFs (
Many previous studies have demonstrated the suppression of NK cell activity by cancer cells by measuring the killing activity of allogenic NK cell against K562 cells (
Our data demonstrated cell-surface expression of PVR was reduced in CAFs, compared with NEFs. The MFI of PVR in CAFs was half that in NEFs. NK activating receptors include NKp30, NKp44, NKp46, NKG2D, DNAM-1, and LFA-1 (
Many studies have reported PVR involvement in NK cell-associated immune-evasion by malignant cells, and that low expression of PVR is associated with poor prognosis (
CAFs may assist malignant cells in a similar fashion to maintain the immunosuppressive microenvironment of the tumor. Additionally, immune evasion may be influenced by both CAFs and malignant cells with regard to NK cell-mediated killing activity. Therefore, soluble PVR may be used as a potential agent to activate NK cell activity in the TME. These data may provide a novel strategy for inhibiting the immune evasion system in the TME.
In conclusion, this is the first report to demonstrate that CAF-mediated suppression of NK killing activity is due to downregulation of PVR cell-surface expression in CAFs. We discovered that CAFs suppressed NK cells function via a receptor-ligand interaction, aiding cancer progression. Soluble PVR may be used as a potential agent to activate NK cell activity in the TME. These data may provide a novel strategy for inhibiting the immune evasion system in the TME.
The authors would like to thank Dr Terufumi Yokoyama for expert advice on experimental methodologies. This study was supported by a grant-in-aid from the Ministry of Health, Labour and Welfare of Japan (KK) and by a cancer research grant from the Ministry of Education, Culture, Sports, Science and Technology (K.K., K.A. and K.T.) of Japan.
cancer-associated fibroblasts
tumor microenvironment
poliovirus receptor
normal endometrial fibroblasts
indoleamine 2,3-dioxygenase
Difference of marker expression between NEFs and CAFs. Fibroblasts were collected from endometrial cancer or normal endometrial tissues. (A) Flow cytometry revealed CAFs expressed Vimentin and CD90 which were major marker of fibroblast. (B) Immunocytochemistry showed that CAFs expressed α-SMA, well-known as CAF makers (green, α-SMA; blue, DAPI). (C) Expression level of α-SMA in CAFs was assessed by western blotting comparing with NEFs.
Suppression of NK cell killing activity by CAFs. NK cells co-cultured with CAFs were assessed for killing assay changing E:T (NK cell: K562 cell) ratio. Percentage of dead K562 cell (K562 PI) was increased with increase of E:T ratio in either NK cell alone or NK cell co-cultured with CAFs. The killing activity of NK cells co-cultured with CAFs was significantly reduced, to less than one third the level of NK cells alone.
Comparison in NK cell activity between NK cell co-cultured with NEFs and CAFs. Comparison in NK cell activity between NK cell co-cultured with NEFs and CAFs was made by the killing assay. The killing activity of NK cells with CAFs was significantly decreased with one third of that of NK cells only. The difference in the NK cell activity between NK cells with CAFs and NEFs was also significant. NS, not significant. Asterisk indicates p-value of <0.05.
Decreased NK cell activity by CAFs was not rescued by indoleamine 2,3-dioxygenase inhibitor. IDO activity was blocked by 1-MT, an inhibitor of IDO, to examine whether IDO was involved in suppression of NK cell activity. The suppression of NK cell activity by CAFs was not rescued by 1-MT treatment. NS, not significant. Asterisk indicates p-value of <0.05.
Cell-to-cell interaction was critical for the decreased NK cell activity by CAFs. NK cells and CAFs were cultured in a chamber with insert membrane to separate these cells. The suppression of NK cell activity by CAFs was completely canceled by blockage of cell-to-cell interaction using insert membrane. Asterisk indicates p-value of <0.05.
Cell-surface expression of PVR/CD155 in CAFs and NEFs. Expression of poliovirus receptor (PVR/CD155), a ligand of paired NK receptors (DNAM-1 and TIGIT), differed between CAFs and NEFs. Flow cytometry analysis revealed that PVR expression was clearly decreased in CAFs compared with NEFs. Asterisk indicates p-value of <0.05.
Knock-down of PVR/CD155 in NEFs provided the downregulation of NK cell activity. (A) PVR expression in NEFs was knocked down by transduction of siRNA for PVR (PVRsi) into NEFs. (B) NK cell killing activity of NK cells co-cultured with PVRsi-transduced NEFs was decreased to approximately one third of that with control si-transduced NEFs. Asterisk indicates p-value of <0.05.