CN113736862A - Method for detecting CTHRC1 gene content in prostate cancer tissue by microdroplet digital PCR - Google Patents
Method for detecting CTHRC1 gene content in prostate cancer tissue by microdroplet digital PCR Download PDFInfo
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Abstract
The invention discloses a method for detecting CTHRC1 gene content in prostate cancer tissue by microdroplet digital PCR, belonging to the field of detection methods for CTHRC1 gene content. It comprises the following steps: (1) primer and plasmid preparation: designing a primer according to the sequence of the CTHRC1, and designing the primer aiming at the CTHRC1 conserved region through sequence alignment analysis, wherein the sequence of the 5 '→ 3' primer is F: CTTCATATGTGGCCGCCAGG; AGATGAGCCCCTGGAAAGCA is the ratio of R to R; meanwhile, a beta-actin internal reference primer is designed, and the sequence of a 5 '→ 3' primer is F: CCTGGCACCCAGCACAAT; GGGCCGGACTCGTCATAC is the ratio of R to R; constructing PUC57 plasmid by using the target gene fragment amplified by the CTHRC1 primer, wherein the plasmid contains 3070bp, the plasmid concentration is 40 ng/mu L, and the conversion is 1.19 multiplied by 1010copies/. mu.L; (2) carrying out enzyme digestion on the PUC57 plasmid; (3) and (5) detecting ddPCR. Verifying the specificity of the primer through electrophoresis, wherein the primer meets the requirement; the accuracy analysis of the ddPCR detection of CTHRC1 shows a good linear relation; ddPCR by detection of Serial dilutionsThe obtained plasmid has high sensitivity for detecting CTHRC 1.
Description
Technical Field
The invention relates to a method for detecting CTHRC1 gene content in prostate cancer tissues by microdroplet digital PCR, belonging to the technical field of detection methods for CTHRC1 gene content.
Background
Prostate cancer is one of the most common malignancies of the male urinary system, with mortality accounting for the third position of malignancy-related deaths in men. With the improvement of the living standard, the aging degree and the diagnosis technology of China, the incidence rate of the prostatic cancer is also increased year by year. At present, TPSA is still an economic and convenient diagnosis index for prostatic cancer in China, and the detection of early TPSA can reduce the fatality rate of prostatic cancer. However, the Chinese prostate cancer alliance (CPCC) research data show that the prostate puncture positive rate is about 26% at TPSA 4-10 ng/mL. Meanwhile, the TPSA level is influenced by factors such as inflammation, digital rectal diagnosis and benign prostatic hyperplasia, and false positive conditions are easy to generate, so that a new prostatic cancer diagnosis marker is urgently needed to be found in clinic, and a new index is provided for early screening of prostatic cancer.
CTHRC1 is a 28kD secreted glycosylated protein, the amino acid sequence of which is 92% homologous in human and rat. Multiple studies have shown that CTHRC1 can be involved in the regulation of multiple signaling pathways in cancer tissues and promote the invasion and metastasis of these cancers, and it has recently been demonstrated that high expression is observed in human pancreatic, breast, liver, stomach and large intestine cancers. CTHRC1 may also be used as a diagnostic marker for prostate cancer. At present, the detection method of the CTHRC1 gene content has the problems of low sensitivity and the like. Therefore, the method for detecting the content of the CTHRC1 gene in the prostate cancer tissue by microdroplet digital PCR is designed, and the detection sensitivity of the CTHRC1 gene content can be improved.
Disclosure of Invention
The technical problem to be solved by the invention is as follows: provides a method for detecting the content of the CTHRC1 gene in a prostate cancer tissue by microdroplet digital PCR, which solves the problem that the detection sensitivity of the CTHRC1 gene in the prostate cancer tissue at present needs to be further improved.
The technical problem to be solved by the invention is realized by adopting the following technical scheme:
the method for detecting the content of the CTHRC1 gene in the prostatic cancer tissue by microdroplet digital PCR is characterized by comprising the following steps:
(1) primer and plasmid preparation
Designing a primer according to the sequence of the CTHRC1, and designing the primer aiming at the CTHRC1 conserved region through sequence alignment analysis, wherein the sequence of the 5 '→ 3' primer is F: CTTCATATGTGGCCGCCAGG; AGATGAGCCCCTGGAAAGCA is the ratio of R to R; simultaneously designs beta-actin internal reference5 '→ 3' primer sequence F: CCTGGCACCCAGCACAAT; GGGCCGGACTCGTCATAC is the ratio of R to R; constructing PUC57 plasmid by using the target gene fragment amplified by the CTHRC1 primer, wherein the plasmid contains 3070bp, the plasmid concentration is 40 ng/mu L, and the conversion is 1.19 multiplied by 1010copies/μL；
(2) Digested pUC57 plasmid
Mixing the PUC57 plasmid containing the target gene with an EcoRI reagent according to the volume ratio, wherein the EcoRI is 10 Xbuffer, the PUC57 plasmid and ddH2Taking the enzyme digestion mixture with the total volume of 20 mu L, putting the mixture into a constant-temperature metal bath at 37 ℃ for incubation for 2h, then incubating the mixture at 65 ℃ for 20min, and inactivating EcoRI enzyme;
(3) ddPCR detection
According to the operating guide of the PCR detection instrument, the reaction system is 20 mu L, wherein 2x ddPCR Supermix Evagren is 10 mu L, the upstream primer and the downstream primer (10 mu M) are respectively 0.4 mu L, the template is 3 mu L, ddH2O6.2 μ L; using QX200TMThe droplets are generated by a droplet generator and then PX1 is usedTMHeat-sealing 96-well plate with PCR plate sealing machine, and finally using QX100TMThe PCR instrument is used for amplifying the target gene, and the reaction conditions are as follows: 5min at 95 ℃; 30s at 95 ℃, 1min at 60 ℃ and 40 cycles; 5min at 4 ℃; 5min at 90 ℃; storing at 4 deg.C; after the amplification is finished, the 96-well plate is put into a QX200TMThe amplified products were analyzed in a microdroplet reader, and the detection of fluorescence signals was scored as positive reaction, and the detection of no fluorescence was scored as negative reaction, and finally the absolute concentration of plasmids was calculated by QuantaSoft analysis software.
The invention has the beneficial effects that: verifying the specificity of the primer through electrophoresis, wherein the primer meets the requirement; the accuracy analysis of the ddPCR detection of CTHRC1 shows a good linear relation; ddPCR was performed by detecting serially diluted plasmids, which resulted in high sensitivity for detecting CTHRC 1.
Drawings
FIG. 1 is an electrophoretogram of 3 samples amplified by two pairs of primers.
FIG. 2 shows the results of 6 samples tested with the beta-actin primer.
FIG. 3 shows the results of 6 samples tested with primer No. 1.
FIG. 4 shows the result of accuracy analysis of the ddPCR detection of CTHRC 1.
FIG. 5 shows the linear range of the ddPCR detection of CTHRC 1.
FIG. 6 is 105Plasmid ddPCR assay results for copies/. mu.L.
FIG. 7 is a ROC curve for the diagnosis of prostate cancer using the combination of CTHRC1, TPSA and CTHRC1+ TPSA.
Detailed Description
In order to make the technical means, the original characteristics, the achieved purpose and the efficacy of the invention easily understood, the invention is further described with reference to the following embodiments.
Examples
1 data and method
1.1 general data
73 prostate puncture tissues of urologic inpatients in subsidiary Zhongshan Hospital at the university of Compound Dan in 2019-2020 were collected, wherein 40 prostate cancer tissues were aged 42-85 years, the mean age (70.56 + -8.42) was aged, 33 non-prostate cancer tissues were aged 49-85 years, and the mean age (68.56 + -7.67) was aged.
1.2 instruments and reagents
The electrophoresis apparatus is an energy EPS 300, and the gel imaging system is ChampGel 5000Plus, QX200TMDroplet generator, PX1TMPCR plate capper, QX100TMPCR Instrument, QX200TMThe droplet reader was purchased from Bio-Rad, USA, and the reagents and consumables were purchased from Bio-Rad, USA. EcoRI endonuclease was purchased from Shanghai Biotechnology engineering Co., Ltd.
1.3 Total RNA extraction and cDNA Synthesis
The total RNA extraction uses a column type animal tissue total RNA extraction and purification kit produced by Shanghai biological engineering Co., Ltd, the cDNA synthesis uses a first strand cDNA synthesis kit of Saimer fly, and all the operations are completed according to the reagent specification.
1.4 primer and plasmid preparation
And designing a primer according to the sequence of the CTHRC1 in GenBank, and designing the primer aiming at the CTHRC1 conserved region through sequence alignment analysis. The 5 '→ 3' primer sequence is F: CTTCATATGTGGCCGCCAGG; rAGATGAGCCCCTGGAAAGCA. Meanwhile, a beta-actin internal reference primer is designed, and the sequence of a 5 '→ 3' primer is F: CCTGGCACCCAGCACAAT; GGGCCGGACTCGTCATAC is added. Constructing PUC57 plasmid by using the target gene fragment amplified by the CTHRC1 primer, wherein the plasmid contains 3070bp, the plasmid concentration is 40 ng/mu L, and the conversion is 1.19 multiplied by 1010copies/. mu.L. The synthesis of primers and the construction of plasmids were both accomplished by Shanghai Bioengineering Co., Ltd.
1.5 digestion of the pUC57 plasmid
The plasmid pUC57 containing the target gene was mixed with EcoRI reagent in a ratio to achieve a total volume of 20. mu.L, as shown in Table 1. The mixture was incubated in a thermostatted metal bath at 37 ℃ for 2 h. The mixture was then incubated for a further 20min at 65 ℃ to inactivate the EcoRI enzyme.
| Reagent | Volume (μ L) |
| EcoRI | 1 |
| 10× |
2 |
| PUC57plasmid | 4 |
| ddH2O | 13 |
TABLE 1 PUC57 plasmid 20. mu.L restriction system
1.6ddPCR assay
According to the instrument operating instructions, the reaction system was 20. mu.L, 2x ddPCR Supermix Evagren 10. mu.L, upstream and downstream primers (10. mu.M) 0.4. mu.L each, template 3. mu.L, ddH2O6.2. mu.L. Using QX200TMThe droplets are generated by a droplet generator and then PX1 is usedTMHeat-sealing 96-well plate with PCR plate sealing machine, and finally using QX100TMThe PCR instrument is used for amplifying the target gene, and the reaction conditions are as follows: 5min at 95 ℃; 30s at 95 ℃, 1min at 60 ℃ and 40 cycles; 5min at 4 ℃; 5min at 90 ℃; storing at 4 ℃. After the amplification is finished, the 96-well plate is put into a QX200TMThe amplification products were analyzed in a droplet reader, and detection of a fluorescent signal was scored as a positive reaction, while detection of no fluorescence was scored as a negative reaction. And finally, calculating the absolute concentration of the plasmid by QuantaSoft analysis software.
1.7 statistical treatment
Statistical analysis was performed using SPSS 20.0 software. The measured data are expressed as (Mean + -SD), and the number of copies is Log10And 4, converting and testing the skewed distribution data by using a Mann-Whitney U test. Linear regression analysis of the accuracy and linear range of ddPCR detection of CTHRC1 was performed using GraphPad Prism 8.0.1 software. CTHRC1 value for identifying prostate cancer was analyzed using a Receiver Operating Characteristic (ROC) curve. With P<A difference of 0.05 is statistically significant.
2 results
2.1 primer specificity verification
As shown in FIGS. 1 to 3, 3 specimens of the prostate cancer puncture specimens were extracted using a column-type animal tissue total RNA extraction purification kit manufactured by Biotechnology, Ltd, and then RNA of these 3 specimens was reverse-transcribed into cDNA using a Saimer fly first strand cDNA Synthesis kit. The primers No. 1 and the internal reference beta-actin primer designed by the invention are used for amplifying the cDNA of 3 samples, and then the amplification product is subjected to electrophoresis (2% gel, electrophoresis for 25min), and the electrophoresis result is shown in figure 1. The electrophoresis bands of the No. 1 primer and the internal reference beta-actin primer amplification products in the figure 1 accord with the positions of respective gene segments. Meanwhile, ddPCR is also used for verifying the two pairs of primers, the results of detecting 6 cases of prostate cancer puncture specimens by the beta-actin primer and the primer No. 1 are shown in figures 2 and 3, and the two pairs of primers can detect respective target genes and have the specificity meeting the requirements, as shown in figures 2 and 3. Therefore, these two pairs of primers were selected as primers for subsequent experiments.
2.2ddPCR detection of CTHRC1 accuracy assessment
First using ddH2O dilution of the digested pUC57 plasmid to 104,103,102,101And 100copies/. mu.L, then using ddPCR for each dilution to repeat the detection three times, finally the detection value and the theoretical value through logarithmic transformation, and then comparing. The Log value difference between the two sets of data was less than 1, while the two sets of data showed good fitness (R)20.9959) as shown in fig. 4. The accuracy of detecting CTHRC1 by ddPCR meets the experimental requirements.
2.3 evaluation of precision of the ddPCR assay CTHRC1
When verifying the batch precision of the detection of the CTHRC1 by ddPCR, a low-concentration plasmid and a high-concentration plasmid are selected, and the concentrations are 10 respectively1And 103The plasmid pUC57 was digested in copies/. mu.L, each concentration was measured 20 times in the same batch by ddPCR, and the SD, average and CV values were calculated. As can be seen from Table 2, the CV values of the plasmids with high and low concentrations in the batch precision are all less than 10%, which indicates that the batch precision of the ddPCR detection of CTHRC1 meets the requirement. In verifying the batch-to-batch precision of the ddPCR assay for CTHRC1, the plasmids at high and low concentrations were assayed 20 times in four batches, and the SD, average and CV values were calculated. The CV values of the high and low concentration plasmids were all less than 15%, as shown in Table 2, indicating that the batch-to-batch precision of the ddPCR assay for CTHRC1 was satisfactory.
TABLE 2 precision of ddPCR assay for CTHRC1
2.4 lower limit of detection of CTHRC1 in ddPCR
Since the lower limit of detection by ddPCR theory was 1 copies/. mu.L, the digested PUC57 plasmid was diluted to 100copies/. mu.L, 10 times by ddPCR as shown in Table 3, 100All the primers are detected 10 times by using the plasmid PUC57 with copies/mu L, so the lower limit of the DDPCR detection for CTHRC1 can reach 100copies/μL。
TABLE 3 lower limit validation results for ddPCR detection of CTHRC1
2.5ddPCR detection of the Linear Range of CTHRC1
The digested PUC57 plasmid was diluted to 104，103，102，101And 100Each dilution is detected for 3 times, the detection result is averaged, the average value and the theoretical value of the plasmid are subjected to numerical conversion, and then correlation analysis is carried out, and the result average value and the theoretical value of the PUC57 plasmid have good linear correlation R20.9972, as shown in fig. 5. At the same time, the dilution of 10 was also determined5Plasmid copies/. mu.L, but it was found that when the number of templates was greater than 105In copies/. mu.L, the number of positive droplets in ddPCR reached saturation, the fluorescence signal of the substrate substantially disappeared, and the detection value deviated from the theoretical value without agreement, as shown in FIG. 6. Therefore, the effective detection range of the ddPCR detection of CTHRC1 is 100-105copies/μL。
2.6ddPCR detection of CTHRC1 content in 73 specimens of prostate tissue puncture
73 clinical prostate tissue puncture specimens were tested by ddPCR and the results were analyzed using the Mann-Whitney U test and the ROC curve. The analysis result shows that the content of the CTHRC1 gene in 40 cases of the prostate cancer puncture specimens is obviously higher than that of the CTHRC1 gene in 33 cases of non-prostate cancer puncture specimens, and the difference is statistically significant (Z is-4.339, and P is less than 0.001). The results of the CTHRC1 test and the results of the serum TPSA are plotted as ROC curves, see fig. 7, and it is found that the combined test can significantly improve the area under the curve (AUC), sensitivity and specificity of the prostate cancer test, and the results are 0.847, 83.3% and 67.7%, respectively, and the detailed statistical results are shown in table 4.
| Item | AUC | Sensitivity (%) | Specificity (%) | 95% confidence interval |
| TPSA | 0.720 | 73.3 | 61.3 | 0.589-0.850 |
| CTHRC1 | 0.797 | 80.0 | 66.7 | 0.696-0.897 |
| CTHRC1+TPSA | 0.847 | 83.3 | 67.7 | 0.746-0.949 |
TABLE 4ROC Curve evaluation of statistical results for TPSA, CTHRC1 and CTHRC1+ TPSA joint detection
Discussion of 3
In recent years, the morbidity and mortality of prostate cancer in China tend to increase year by year, and the difficulty of early diagnosis is a main factor influencing the diagnosis and treatment effect. Currently, TPSA and fPSA are most widely applied clinically, but TPSA has low specificity, and is also increased in benign diseases such as prostatitis and benign prostatic hyperplasia, and fPSA has the defects of low content, short half-life, poor stability and the like, so that the generation and development mechanism of prostate cancer is deeply researched, and a molecular marker for early diagnosis of prostate cancer is discovered, so that the TPSA and fPSA has important significance for treatment and prognosis of prostate cancer. CTHRC1 is a secreted protein whose increased expression can increase the infiltration capacity and the risk of metastasis of cancer cells, thereby exacerbating the progression of clinical conditions. Studies have shown that CTHRC1 is closely associated with metastasis of a variety of solid tumors.
ddPCR is independent of an external calibration curve, can detect nucleic acid sensitively and specifically, has high tolerance to PCR inhibitors compared with qPCR, and becomes a novel molecular detection technology. The application of ddPCR is more and more extensive, and the application is more and more in cancer detection and nucleic acid quantification. ddPCR is rapidly replacing qPCR as an effective method for independent quantitative analysis of nucleic acids.
In the research, a primer is designed for a section of conserved region of the CTHRC1, a plasmid is constructed for the section of conserved region, and the constructed PUC57 plasmid is used for verifying the performance of the ddPCR technology for detecting the CTHRC 1. The experimental result shows that the detection value of the CTHRC1 detected by the ddPCR technology shows high fitting degree (R) with the theoretical value of the plasmid in terms of accuracy20.9959). In terms of precision, the intra-batch precision and the inter-batch precision are verified, and the CV values of the intra-batch precision and the inter-batch precision of the CTHRC1 detected by the ddPCR technology can reach 4.8 percent and 6.9 percent at the lowest. In terms of the lower detection limit, the lowest detection limit of the DDPCR technology for detecting the CTHRC1 template can reach 1 copies/mu L. In the aspect of verifying the linear range, the linear range of the CTHRC1 detected by the ddPCR technology is evaluated by using serial dilution PUC57 plasmid, and the result shows that the effective detection range of the method is 1-105copies/. mu.L, because when the upper limit of the CTHRC1 plasmid exceeds 105At copies/. mu.L, the droplets generated by ddPCR reach saturation, which disables the Poisson statistical method. Data are verified through methodology to prove that the ddPCR is suitable for detecting the CTHRC1 content in clinical samples of prostate cancer patients, and the method is a reliable, accurate and sensitive cancer research tool.
In order to clarify the relationship between CTHRC1 and prostate cancer, the CTHRC1 gene content was examined in 40 cases of prostate cancer puncture specimens and 33 cases of non-prostate cancer puncture specimens using ddPCR. The analysis result shows that the gene content of the CTHRC1 in the prostate cancer patient sample is obviously higher than that of the non-prostate cancer patient, the difference is statistically significant (P <0.001), and the high expression of the CTHRC1 gene content in the prostate cancer is preliminarily confirmed. Meanwhile, in order to evaluate the diagnosis value of the CTHRC1 on the prostate cancer, an ROC curve is drawn by the detection result of the CTHRC1 and the result of serum TPSA, and the result shows that compared with TPSA, the CTHRC1 has better prediction value as a judgment factor of the prostate cancer. The joint detection is an effective means for screening the prostate cancer, so that the frequency of discovering the localized prostate cancer and the asymptomatic latent prostate cancer is increased, and the prostate cancer which can be radically treated can be discovered early. Therefore, the ROC curve is used for analyzing the performance of the CTHRC1 and TPSA in the combined detection of the prostate cancer and the non-prostate cancer, and the combined detection has higher area under the curve, sensitivity and specificity than the TPSA and CTHRC1 detection alone, and the results are 0.847, 83.3% and 67.7% respectively, so that the combined detection of the CTHRC1 and the TPSA is beneficial to further improving the diagnosis capability of the prostate cancer and provides help for early treatment and improvement of prognosis of the prostate.
4 conclusion
In conclusion, the high sensitivity and high specificity of ddPCR can be used for detecting the content of the CTHRC1 gene in the tissues of the prostate cancer patients, and the abnormal expression of CTHRC1 in the prostate cancer suggests that the gene is possibly used as a screening index and a potential target for treatment of early diagnosis of the prostate cancer patients. However, the specific mechanism of action of CTHRC1 in prostate cancer development and whether it can be used as a tumor marker for clinical detection of prostate cancer still requires extensive research to be elaborated upon.
The foregoing illustrates and describes the principles, general features, and advantages of the present invention. It will be understood by those skilled in the art that the present invention is not limited to the embodiments described above, and that various changes and modifications may be made without departing from the spirit and scope of the invention as defined in the appended claims. The scope of the invention is defined by the appended claims and equivalents thereof.
Claims (1)
1. The method for detecting the content of the CTHRC1 gene in the prostatic cancer tissue by microdroplet digital PCR is characterized by comprising the following steps:
(1) primer and plasmid preparation
Designing a primer according to the sequence of the CTHRC1, and designing the primer aiming at the CTHRC1 conserved region through sequence alignment analysis, wherein the sequence of the 5 '→ 3' primer is F: CTTCATATGTGGCCGCCAGG; AGATGAGCCCCTGGAAAGCA is the ratio of R to R; meanwhile, a beta-actin internal reference primer is designed, and the sequence of a 5 '→ 3' primer is F: CCTGGCACCCAGCACAAT; GGGCCGGACTCGTCATAC is the ratio of R to R; constructing PUC57 plasmid by using the target gene fragment amplified by the CTHRC1 primer, wherein the plasmid contains 3070bp, the plasmid concentration is 40 ng/mu L, and the conversion is 1.19 multiplied by 1010copies/μL；
(2) Digested pUC57 plasmid
Mixing the PUC57 plasmid containing the target gene with an EcoRI reagent according to the volume ratio, wherein the EcoRI is 10 Xbuffer, the PUC57 plasmid and ddH2Taking the enzyme digestion mixture with the total volume of 20 mu L, putting the mixture into a constant-temperature metal bath at 37 ℃ for incubation for 2h, then incubating the mixture at 65 ℃ for 20min, and inactivating EcoRI enzyme;
(3) ddPCR detection
According to the operating guide of the PCR detection instrument, the reaction system is 20 mu L, wherein 2x ddPCR Supermix Evagren is 10 mu L, the upstream primer and the downstream primer (10 mu M) are respectively 0.4 mu L, the template is 3 mu L, ddH2O6.2 μ L; generating microdroplets by using a microdroplet generator, then thermally sealing a 96-well plate by using a PCR plate sealing machine, and finally amplifying the target gene by using the PCR generator under the reaction conditions that: 5min at 95 ℃; 30s at 95 ℃, 1min at 60 ℃ and 40 cycles; 5min at 4 ℃; 5min at 90 ℃; storing at 4 deg.C; after the amplification is finished, the 96-well plate is placed into a microdroplet reader to analyze the amplification product, the detected fluorescence signal is marked as a positive reaction, the non-detected fluorescence signal is marked as a negative reaction, and finally, the absolute concentration of the plasmid is calculated through QuantaSoft analysis software.
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| US20130137584A1 (en) * | 2010-02-01 | 2013-05-30 | The Regents Of The University Of California | Novel diagnostic and therapeutic targets associated with or regulated by n-cadherin expression and/or epithelial to mesenchymal transition (emt) in prostate cancer and other malignancies |
| CN108285931A (en) * | 2018-03-30 | 2018-07-17 | 武汉大学 | A kind of the droplet type digital pcr method and kit of clinical detection HBV cccDNA |
| WO2021063972A1 (en) * | 2019-09-30 | 2021-04-08 | Fundació Institut De Recerca Biomèdica (Irb Barcelona) | Cthrc1 as biomarker for a tgfbeta-activated tumor microenvironment |
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| US20130137584A1 (en) * | 2010-02-01 | 2013-05-30 | The Regents Of The University Of California | Novel diagnostic and therapeutic targets associated with or regulated by n-cadherin expression and/or epithelial to mesenchymal transition (emt) in prostate cancer and other malignancies |
| CN108285931A (en) * | 2018-03-30 | 2018-07-17 | 武汉大学 | A kind of the droplet type digital pcr method and kit of clinical detection HBV cccDNA |
| WO2021063972A1 (en) * | 2019-09-30 | 2021-04-08 | Fundació Institut De Recerca Biomèdica (Irb Barcelona) | Cthrc1 as biomarker for a tgfbeta-activated tumor microenvironment |
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