Contributed equally
Cancer-associated fibroblasts (CAFs) are implicated in the strong malignancy of pancreatic cancer (PC). Various CAF subtypes have different functions, and their heterogeneity likely influence the malignancy of PC. Meanwhile, it is known that senescent cells can create a tumor-promoting microenvironment by inducing a senescence-associated secretory phenotype (SASP). In the present study, the effects of individual differences in CAFs on PC malignancy were investigated with a focus on cellular senescence. First, primary cultures of CAFs from 8 PC patients were generated and co-cultured with PC cell lines. This co-culture assay showed that differences in CAFs induce differences in PC cell proliferation. It was further investigated which clinical factors affected the malignant potential of CAF and it was found that the difference of malignant potential of each CAF was marginally related to the age of original patients. Next, to verify the senescence of CAFs really affected the malignant potential of CAF, PCR array analysis of each CAF sample was performed and it was revealed that expression of genes about cellular senescence and SASP such as tumor protein p53, nuclear factor kappa B subunit 1, and IL6, are related to the malignant potential of CAFs impacting on PC proliferation. Finally, to elucidate the effect of p53-mediated cellular senescence of CAFs on malignant potential of PC, it was examined whether CAFs with the treatment of p53 inhibitor affected PC cell proliferation in co-culture assays. The treatment of CAFs with p53 inhibitor significantly suppressed PC cell proliferation. In addition, a comparison of the concentration of IL-6, a SASP cytokine, in the co-culture supernatant showed a significant decrease in the sample after p53 inhibitor treatment. In conclusion, the present results suggested that proliferation potential of PC may be related to p53-mediated cellular senescence and SASP of CAFs.
Pancreatic cancer (PC) still has a poor prognosis. Even with the latest multimodality treatment, the 5-year relative survival rate is only 11%, which is substantially worse than for other gastrointestinal cancers (
Previous studies demonstrated that the tumor stromal components, which are abundant in PC, are involved in the malignant potential (
In general, malignancy risk increases with aging (
Although several studies described the role of SASP in various type of cancers such as liver, colorectal and prostate cancer (
Tissue samples of human PC were collected with the approval (approval no. 18138-4) of the Human Ethics Review Committee of the Graduate School of Medicine of Osaka University (Osaka, Japan) and the National Institutes of Biomedical Innovation, Health and Nutrition (approval no. 201-01; Osaka, Japan). Written informed consent for sample use was obtained from all patients before surgery. All tissue samples were collected at Osaka University Hospital (Suita, Japan) from June 2019 to March 2020. Human pancreatic stellate cells, the reported origin cells of CAFs, were cultured from the tissue samples as previously described (
Primary culture of CAFs was performed using tissue samples collected from 8 PC patients. The clinical information for these 8 patients before the initiation of treatment are presented in
The co-culture assay was performed in a non-contact manner, using a Transwell with 0.4-µm pores (Corning, Inc.). First, CAFs (1.0×104 per well) were plated on the top layer and incubated overnight in DMEM containing 10% FBS. Once the CAFs had settled in the Transwell, PSN-1 cells (a human PC cell line, p53 mutant) were plated at 1.0×105 per well on the lower layer of a 24-well culture plate (Corning, Inc.). PSN-1 cells were obtained from the American Type Culture Collection. After an additional 48 h of co-culture in DMEM containing 0.5% FBS, the Transwell was removed and PSN-1 cell proliferation was evaluated.
PSN-1 cell proliferation was evaluated by fluorescence staining using Hoechst 33342 (Thermo Fisher Scientific, Inc.). Briefly, after removing the Transwell and the culture medium, diluted Hoechst 33342 was added and allowed to react for 10 min. Then the PSN-1 cells were washed three times with PBS, and the fluorescence intensity was measured using the EnSpire Multimode Plate Reader (PerkinElmer, Inc.). An assay was performed to compare the effects of CAFs from 8 different patients on the proliferation of PSN-1 cells. The fluorescence intensity ratio (FIR) was calculated based on the fluorescence intensity of the canonical CAFs (#1) showing the lowest fluorescence intensity: FIR (#X)=Fluorescence intensity (#X)/Fluorescence intensity (#1).
RT-qPCR arrays were performed using two kits: QIAGEN RT2 Profiler™ PCR Array ‘Human Cellular Senescence’ (GeneGlobe ID-PAHS-050Z) and ‘Human Cancer Inflammation and Immunity Crosstalk’ (GeneGlobe ID-PAHS-181Z) following the supplier's instructions (
Pathway enrichment analysis was performed using two databases: Kyoto Encyclopedia of Genes and Genomes (KEGG:
First, CAFs were plated and incubated overnight in DMEM containing 10% FBS. Once the CAFs had settled in the plate, the medium was changed and CAFs were cultured in DMEM containing 10% FBS for 5 days with the p53 inhibitor pifithrin-alpha (PFT-alpha) (Sigma-Aldrich; Merck KGaA), or with the same amount of DMSO as a control. A PFT-alpha concentration of 10 µM was used based on previous a previous study (
The IL-6 concentration was analyzed in the co-culture supernatant using an enzyme-linked immunosorbent assay (ELISA) kit (cat. no 3460-1A-6; Mabtech, Inc.), following the manufacturer's instructions. Co-culture supernatants were collected at the end of the 2-day co-culture in the aforementioned experiment. When performing the analysis, the co-culture supernatant was diluted 10-fold. This assay was also conducted using CAFs from three patients, #2, #7 and #8.
Numerical data are presented as mean ± standard deviation. Statistical analysis between two groups was performed using a two-tailed Student's t-test (unpaired t-test). Statistical analysis among three groups or more was performed using Dunnett's t-test, with the test level α=0.05. Correlation analysis was performed using Pearson's correlation coefficient. P<0.05 was considered to indicate a statistically significant difference. JMP software (JMP® Pro 16.2.0, SAS Institute, Inc.) was used as the software for these analyses.
To examine whether differences in CAFs affected PC cell proliferation, 8 CAF samples were primarily cultured and co-cultured with PSN-1 cells. The PSN-1 showed different proliferation ability when cultured with each CAFs from different patients (
It was examined how the results of co-culture assays were associated with the original clinical data of patients. The correlation between FIR in co-culture assays and five clinical data is demonstrated in
Thus, next we examined the correlation between TSR and FIR in six patients who received preoperative treatment to clarify the clinical impact of CAFs on the effect of preoperative treatment. As revealed in
To elucidate the senescence of CAFs really affected the malignant potential of CAFs, a PCR array was performed among the 8 CAFs by using the ‘Human Cellular Senescence’ PCR array kit (GeneGlobe ID-PAHS-050Z QUIAGEN) and ‘Human Cancer Inflammation and Immunity Crosstalk’ PCR array kit (GeneGlobe ID-PAHS-181Z QUIAGEN). First, the expression of the CAF markers, ACTA-2 and FAP, was examined simultaneously with the analysis using these kits. Fortunately, the genes analyzed in these kits included genes known to be marker of CAF, VIM. Therefore, the expression of these genes in 8 primary CAFs was investigated and it was found that the expression of these 3 genes was positive in all CAFs (
As revealed in
Based on the present results, focus was next addressed on the p53-mediated cellular senescence of CAFs. It was examined how CAFs treatment with the p53 inhibitor PFT-alpha altered the PC cell proliferation in co-culture assays. After 5 days of exposure to PFT-alpha, each CAF samples retained its spindle-like shape (
In recent years, a deeper understanding of the interactions between stromal and cancer cells in the senescent microenvironment has been achieved. Yang
In the present study, primary CAFs were examined and it was found that differences in the expression of cellular senescence and SASP-related genes in CAFs affected the PC cell proliferation. The advantage of the present study was that not primary PC cells but established cancer cell lines were used to examine the malignant potential of CAFs. Although the gene expression of CAFs may be influenced by cancer cells, the present study was able to assess the true malignancy of each CAF by conducting experiments using primary cultures of CAFs without cancer cells and unified cancer cell lines. In the current results, the same cancer cell grew differently by co-culture with different primary CAFs, indicating directly that different primary CAFs had different malignant potential. In addition, the malignant potential of each CAFs was significantly related to original age of the patient, thus it was considered that the malignant potential could be relevant to the senescence of CAFs and SASP. Actually, Toste
Notably, what was important for the cancer cell proliferation was not the expression levels of senescence and SASP-related genes in the presence or absence of serum stimulation, but the increase of the expression ratio of the genes with versus without serum stimulation. Serum stimulation is known to promote cell division and cause telomere shortening and replicative senescence (
Based on our co-culture assays and PCR array results, a validation experiment focusing on TP53 was further conducted, among 11 senescence- and SASP-related genes that are considered to be related to cancer cell proliferation. Focus was addressed on TP53 because p53, the nuclear protein encoded by TP53 and one of the best-known tumor suppressor genes, plays a vital role in the induction of both cellular senescence and SASP. Additionally, the specific inhibitor of p53, PFT-alpha, reportedly alters post-translational modification patterns and differentially inhibits p53 target genes (
The present study revealed that cellular senescence of CAFs and expression of SASP-related genes are involved in the malignant potential of PC by analyzing CAFs alone, but other parameters in addition to the age of patients may also influence the malignant potential of CAFs, such as the characteristics of the original cancer, and this requires further investigation. It is also very important to examine whether this cellular senescence of CAF is a change unique to CAFs or it also occurs in normal fibroblasts. The authors are also interested in the differences between normal fibroblasts and CAFs and primary culture of normal fibroblasts from normal pancreatic tissue in resected specimens has been attempted. However, normal fibroblasts were difficult to culture and the quality of the cells was unfortunately inadequate for the assay. The results may indicate that CAFs may have been affected in some way by the original cancer cells, thus in the next study it will be attempted to perform further assays to determine the differences between the original fibroblasts and CAFs. It is also considered that future study is needed to examine the effects of the cancer microenvironment on CAFs in terms of induction of cellular senescence.
In summary, the present data provided the first evidence, to the best of our knowledge, that p53-mediated cellular senescence and SASP of CAFs affect the malignant potential of PC cells. Thus, controlling cellular senescence and SASP in CAFs may be a new strategy for PC treatment.
The authors would like to thank Eiko Moriishi and Mami Ikeda (Laboratory of Immunosenescence, Center for Vaccine and Adjuvant Research, National Institutes of Biomedical Innovation, Health and Nutrition, Osaka) for sample preservation and data analyses.
The datasets generated and/or analyzed during the current study are available from the corresponding author on reasonable request.
MH and HM designed the present study. ST helped to analyze data. HA, SK, ST, YI, DY, YT, TN, KG, YD, TY and HE helped to design the study. SK and TY confirm the authenticity of all the raw data. All authors read and approved the final manuscript.
Tissue samples of human PC were collected with the approval (approval no. 18138-4) of the Human Ethics Review Committee of the Graduate School of Medicine of Osaka University (Osaka, Japan) and the National Institutes of Biomedical Innovation, Health and Nutrition, (approval no. 201-01; Osaka, Japan). Written informed consent for sample use was obtained from all patients before surgery.
Not applicable.
The authors declare that they have no competing interests.
actin alpha 2
α-smooth muscle actin
ataxia telangiectasia mutated
cancer-associated fibroblasts
cyclin-dependent kinase inhibitor 1A
cyclin-dependent kinase inhibitor 2A
Dulbecco's modified Eagle's medium
fibroblast activation protein
fetal bovine serum
fluorescence intensity ratio
glyceraldehyde-3-phosphate dehydrogenase
Kyoto encyclopedia of genes and genomes
nuclear factor kappa B subunit 1
pancreatic cancer
pifithrin
senescence-associated secretory phenotype
telomerase reverse transcriptase
tumor protein p53
tumor size ratio
vimentin
Differences in CAFs induce differences in PC cell proliferation. (A) Graphical representation of the fluorescence intensity of PC cells co-cultured with each CAF sample. Values represent the mean ± SD from three independent experiments. (B) Tabular representation of the same data. Fluorescence Intensity Ratio (#X)=Fluorescence Intensity (#X)/Fluorescence Intensity (#1). *P<0.005 and **P<0.001. CAF, cancer-associated fibroblasts; PC, pancreatic cancer.
Correlation between FIR in co-culture assays and clinical data before treatment. Graph shows the FIR on the horizontal axis, and clinical data on the vertical axis. (A) Data regarding tumor size, serum CA19-9 level, and SUV max of fluorodeoxyglucose-positron emission tomography. (B) Data regarding tumor size ratio. (C) Data regarding age of patients. The square of the correlation coefficient and the P-value are shown. *N=6 (excluding the two Lewis antigen-negative patients). #N=6 (only for patients who received preoperative chemotherapy). Each symbol legend appears at the right below. FIR, fluorescence intensity ratio; CA19-9, carbohydrate antigen.
Expression of cellular senescence- and senescence-associated secretory phenotype-related genes in CAFs is related to PC cell proliferation. (A) Top 30 genes expressed by CAFs, for which the RNA level (fold-change) strongly correlates with the FIR in co-culture assays. (B) Graphical representation of the correlation between the RNA level (fold-change) of each gene and FIR. The P-value is shown on the vertical axis, and the absolute value of the correlation coefficient on the horizontal axis. Red plot: positive correlation; Blue plot: negative correlation. (C) Pathway analysis for the top 30 genes using the KEGG and Reactome databases. Text on the left indicates the enriched pathway. The ball size indicates the number of the genes enriched, and the color indicates the level of enrichment. CAF, cancer-associated fibroblasts; PC, pancreatic cancer; FIR, fluorescence intensity ratio; KEGG, Kyoto Encyclopedia of Genes and Genomes.
Correlation between FIR in co-culture assays and RNA level of cellular senescence- and SASP-related genes. (A) Data regarding four genes associated with the p53/p21 and p16/RB pathways. (B) Data regarding seven genes associated with SASP. Graph shows FIR on the horizontal axis, and RNA level (fold-change) of each gene on the vertical axis. The square of the correlation coefficient and the P-value are shown. Each symbol legend appears at the right below. FIR, fluorescence intensity ratio; SASP, senescence-associated secretory phenotype.
Correlation between TSR and RNA level of cellular senescence- and SASP-related genes. (A) Data regarding four genes associated with the p53/p21 and p16/RB pathways. (B) Data regarding seven genes associated with SASP. Graph shows RNA level (fold-change) of each gene on the horizontal axis, and TSR on the vertical axis. The square of the correlation coefficient and the P-value are shown. Data were obtained for the six patients who received preoperative chemotherapy. Each the symbol legend appears at the right below. TSR, tumor size ratio; SASP, senescence-associated secretory phenotype.
Treatment of CAFs with PFT-alpha reduces PC cell proliferation and the IL-6 concentration in the co-culture supernatant. (A) Fluorescence intensity of PC cell lines when co-cultured with DMSO-treated CAFs or PFT-alpha-treated CAFs. (B) IL-6 concentration (pg/ml) in the co-culture supernatant following co-culture with DMSO-treated CAFs or PFT-alpha-treated CAFs. The graphs show data for 3 cases, #2, #7 and #8, respectively. *P<0.05 and **P<0.01. CAF, cancer-associated fibroblast; PFT-alpha, pifithrin-alpha; PC, pancreatic cancer.