肿瘤相关成纤维细胞自噬对三阴性乳腺癌细胞转移的影响

doi:10.3877/cma.j.issn.1674-0807.2019.01.002

目的

探讨三阴性乳腺癌(TNBC)细胞和肿瘤相关成纤维细胞(CAFs)共培养环境下，CAFs自噬促进TNBC细胞转移的作用机制。

方法

(1)分离和鉴定CAFs：采用前瞻性研究方法收集2015年1月至2016年12月期间，就诊于南方医科大学珠江医院的6例TNBC患者(临床分期Ⅱ～Ⅲ期)的肿瘤组织和癌旁正常组织，从这些标本中分离CAFs和正常成纤维细胞(NFs)。进一步采用荧光抗体标记抗α平滑肌肌动蛋白(α-SMA)，并计算α-SMA阳性细胞比例，采用Western blot检测CAFs和NFs的α-SMA表达水平，以鉴定CAFs为本实验所需。(2)评估CAFs自噬水平：利用Western blot检测CAFs组、NFs组和CAFs＋3-MA组(加入自噬抑制剂3-MA的CAFs)自噬标志蛋白beclin 1和p62的表达水平。(3)明确CAFs自噬对MDA-MB-231和BT-549细胞迁移的作用：采用Transwell小室模型观察不同培养条件[分4组，包括空白组(含10% FBS的DMEM培养基，即普通培养基)、NFs组、CAFs组和CAFs＋3-MA组]下MDA-MB-231和BT-549的细胞迁移数。(4)明确CAFs自噬对MDA-MB-231和BT-549细胞发生上皮-间质转化(EMT)的影响：采用Western blot检测3种培养条件[普通培养基(空白组)、CAFs条件培养基(CAFs-CM组)和加入3-MA的CAFs条件培养基(CAFs＋3-MA-CM组)]下MDA-MB-231和BT-549 EMT相关蛋白(E-cadherin，N-cadherin, vimentin)的表达情况。正态分布的数据用±s表示，偏态分布的数据用M(P₂₅-P₇₅)表示。2组间α-SMA表达水平比较采用两独立样本非参数秩和检验，α-SMA阳性细胞比例比较采用两独立样本t检验，各组间细胞自噬标志蛋白表达水平、细胞迁移数和EMT相关蛋白表达水平的比较均采用单因素方差分析。

结果

(1)荧光显微镜下观察发现，每视野下CAFs组α-SMA阳性细胞比例显著高于NFs组[(81.11±3.95)%比(5.11±2.37)%，t＝49.49, P<0.001); Western blot检测表明，CAFs组α-SMA表达水平也高于NFs组[M(P₂₅～P₇₅)：0.98(0.95～0.98)比0.48(0.47～0.48)，Z＝2.023, P＝0.043]。(2)CAFs组、NFs组和CAFs＋3-MA组细胞beclin 1表达水平分别为0.99±0.03、0.73±0.04和0.26±0.02，p62表达水平分别为0.75±0.02、0.98±0.03和0.97±0.01，组间比较，差异均有统计学意义(F＝546.188、136.353，P均<0.001)，其中，CAFs组的beclin 1表达水平显著高于CAFs＋3-MA组(P<0.001)，p62表达水平明显低于CAFs＋3-MA组(P<0.001)。(3)CAFs组、NFs组、CAFs＋3-MA组以及空白组MDA-MB-231细胞迁移数分别为(41.67±2.78)、(23.33±2.18)、(22.00±1.76)、(18.00±2.12)个，4组比较，差异有统计学意义(F＝198.374，P<0.001)；同样，4组BT-549细胞的迁移数分别为(35.22±1.97)、(22.00±2.60)、(25.11±2.15)、(15.22±2.00)个，组间比较，差异也有统计学意义(F＝129.424，P<0.001)，并且，CAFs组的MDA-MB-231和BT-549细胞迁移数均明显高于其余3组(P<0.050)。(4)MDA-MB-231细胞分别与普通培养基(空白组)、CAFs条件培养基(CAFs-CM组)和加入3-MA的CAFs条件培养基(CAFs＋3-MA-CM组)共培养后，3组细胞的E-cadherin表达水平分别为0.79±0.03、0.54±0.02和0.87±0.04, N-cadherin表达水平分别为0.59±0.02、1.00±0.02和0.93±0.02, vimentin表达水平分别为0.62±0.03、1.01±0.01和0.89±0.09，组间比较，差异也均有统计学意义(F＝139.286、604.905、43.884，P均<0.001)。同样，BT-549细胞分别与以上3种培养基共培养后，3组细胞的E-cadherin表达水平分别为1.01±0.03、0.63±0.03和0.98±0.03, N-cadherin表达水平分别为0.58±0.01、0.94±0.04和0.95±0.03, vimentin表达水平分别为0.61±0.01、0.98±0.03和0.75±0.02，组间比较，差异也均有统计学意义(F＝210.102、184.477、217.659，P均<0.001)。

结论

CAFs可促进TNBC细胞MDA-MB-231和BT-549的迁移和EMT过程，且该作用与CAFs自噬相关。

关键词: 乳腺肿瘤, 成纤维细胞, 自噬

Objective

To clarify the effect of cancer-associated fibroblasts (CAFs ) autophagy on metastasis of triple-negative breast cancer (TNBC) cells co-cultured with CAFs.

Methods

(1) Purification and identification of CAFs. CAFs and normal fibroblasts (NFs) were prospectively obtained from the cancer tissue and normal breast tissue of six patients with TNBC at stage Ⅱ or Ⅲ, who were treated in Zhujiang Hospital of Southern Medical University from January 2015 to December 2016.α-smooth muscle actin (α-SMA) was labeled by fluorescent antibody and the proportion of α-SMA positive cells was calculated. The expression of α-SMA in CAFs and NFs was determined by Western blot, respectively, to identify CAFs used for this experiment. (2) Evaluation of CAFs autophagy. Autophagic biomarkers beclin 1 and p62 were measured by Western blot in CAFs group, NFs group and CAFs group pretreated with 3-methyladenine (CAFs+ 3-MA group), respectively. (3) Identifying the migration of MDA-MB-231 and BT-549 cells caused by CAFs autophagy. The migration of MDA-MB-231 or BT-549 cells was detected by the Transwell chamber under different conditions (medium/NFs/CAFs/CAFs+ 3-MA). (4) Clarifying the effect of CAFs autophagy on epithelial-mesenchymal transition (EMT) in MDA-MB-231 or BT-549 cells. The EMT-related proteins E-cadherin, N-cadherin and vimentin were detected by Western blot in MDA-MB-231 or BT-549 cells cultured in different conditions (medium, CAFs-CM or CAFs+ 3-MA-CM). The data of normal distribution were expressed by ±s and the data of skewed distribution were expressed by M(P₂₅-P₇₅). Nonparametric rank-sum test of two independent samples was used to compare α-SMA expression, t test of two independent samples was used to compare the percentage of α-SMA-positive cells, and single-factor analysis of variance was used to compare the autophagic protein expression, cell migration rate and EMT-related protein expression among groups.

Results

(1) α-SMA-positive cells were observed under a fluorescence microscope. The result showed that the percentage of α-SMA-positive cells in CAFs was significantly higher than that in NFs [(81.11±3.95)% vs(5.11±2.37)%, t=49.49, P<0.001)]. Western blot analysis indicated that the expression of α-SMA in CAFs and NFs was 0.98(0.95-0.98) and 0.48 (0.47-0.48), respectively, suggesting a significant difference (Z=2.023, P=0.043). (2) The expression of beclin 1 was 0.99±0.03, 0.73±0.04 and 0.26±0.02 in CAFs group, NFs group and CAFs+ 3-MA group, respectively, indicating a significant difference (F=546.188, P<0.001). The expression of beclin 1 in CAFs group was significantly higher than that in CAFs+ 3-MA group (P<0.001). The expression of p62 was 0.75±0.02, 0.98±0.03 and 0.97±0.01 in CAFs group, NFs group and CAFs+ 3-MA group, respectively, indicating a significant difference (F=136.353, P<0.001). The expression of p62 expression in CAFs group was significantly lower than that in CAFs+ 3-MA group(P<0.001). (3)The number of migrated MDA-MB-231 cells was 41.67±2.78, 23.33±2.18, 22.00±1.76 and 18.00±2.12 in CAFs group, NFs group, CAFs+ 3-MA group and medium group, respectively, indicating a significant difference(F=198.374, P<0.001). The number of migrated BT-549 cells was 35.22±1.97, 22.00±2.60, 25.11±2.15 and 15.22±2.00 in CAFs group, NFs group, CAFs+ 3-MA group and medium group, respectively, indicating a significant difference(F=129.424, P<0.001). The number of migrated MDA-MB-231 or BT-549 cells in CAFs group was significantly higher than that in other three groups (P<0.050). (4) When MDA-MB-231 cells were cultured in medium, CAFs-CM or CAFs+ 3-MA-CM, Western blot analysis showed that E-cadherin expression was 0.79±0.03, 0.54±0.02 and 0.87±0.04 in 3 groups, respectively, indicating a significant difference(F=139.286, P<0.001); N-cadherin expression was 0.59±0.02, 1.00±0.02 and 0.93±0.02 in 3 groups, respectively, indicating a significant difference(F=604.905, P<0.001); vimentin expression was 0.62±0.03, 1.01±0.01 and 0.89±0.09 in 3 groups, respectively, indicating a significant difference(F=43.884, P<0.001). When BT-549 cells was cultured in medium, CAFs-CM and CAFs+ 3-MA-CM, E-cadherin expression was 1.01±0.03, 0.63±0.03 and 0.98±0.03 in 3 groups, respectively, indicating a significant difference(F=210.102, P<0.001); N-cadherin expression was 0.58±0.01, 0.94±0.04 and 0.95±0.03 in 3 groups, respectively, indicating a significant difference(F=184.477, P<0.001), vimentin expression was 0.61±0.01, 0.98±0.03 and 0.75±0.02 in 3 groups, respectively, indicating a significant difference(F=217.659, P<0.001).

Conclusion

CAFs can promote the migration and EMT in MDA-MB-231 and BT-549 cells, which may be related to the autophagy of CAFs.

Key words: Breast neoplasms, Fibroblasts, Autophagy

图1 采用荧光显微镜观察CAFs组与NFs组细胞的α-SMA表达情况(×200)　a～c图分别所示CAFs组细胞质α-SMA表达水平、细胞核DAPI染色情况以及两者的融合图像；d～f图分别所示NFs组细胞质α-SMA表达水平、细胞核DAPI染色情况以及两者的融合图像

图2 Western blot检测6例乳腺癌组织标本中CAFs和NFs的α-SMA蛋白表达水平

图3 采用Western blot分别检测NFs组、CAFs组和CAFs＋3-MA组细胞beclin 1和p62表达水平

图4 采用普通光学显微镜观察并计数MDA-MB-231和BT-549细胞迁移数(×200)　a～d图分别所示空白组、NFs组、CAFs组和CAFs＋3-MA组MDA-MB-231细胞的迁移情况；e～h图分别所示空白组、NFs组、CAFs组和CAFs＋3-MA组BT-549细胞的迁移情况

表1 各组MDA-MB-231细胞上皮-间质转化相关蛋白的表达情况

组别	E-cadherin^a	N-cadherin^a	vimentin^a
空白组	0.79±0.03	0.59±0.02	0.62±0.03
CAFs-CM组	0.54±0.02	1.00±0.02	1.01±0.01
CAFs＋3-MA-CM组	0.87±0.04	0.93±0.02	0.89±0.09
F值	139.286	604.905	43.884
P值	<0.001	<0.001	<0.001

表1 各组MDA-MB-231细胞上皮-间质转化相关蛋白的表达情况

组别	E-cadherin^a	N-cadherin^a	vimentin^a
空白组	0.79±0.03	0.59±0.02	0.62±0.03
CAFs-CM组	0.54±0.02	1.00±0.02	1.01±0.01
CAFs＋3-MA-CM组	0.87±0.04	0.93±0.02	0.89±0.09
F值	139.286	604.905	43.884
P值	<0.001	<0.001	<0.001

图5 采用Western blot检测各组细胞中E-cadherin、N-cadherin和vimentin表达水平　a图所示MDA-MB-231细胞的目的蛋白和内参蛋白表达情况；b图所示BT-549细胞的目的蛋白和内参蛋白表达情况

表2 各组BT-549细胞上皮-间质转化相关蛋白的表达情况

组别	E-cadherin	N-cadherin	vimentin^c
空白组	1.01±0.03	0.58±0.01^b	0.61±0.01
CAFs-CM组	0.63±0.03^a	0.94±0.04	0.98±0.03
CAFs＋3-MA-CM组	0.98±0.03	0.95±0.03	0.75±0.02
F值	210.102	184.477	217.659
P值	<0.001	<0.001	<0.001

表2 各组BT-549细胞上皮-间质转化相关蛋白的表达情况

组别	E-cadherin	N-cadherin	vimentin^c
空白组	1.01±0.03	0.58±0.01^b	0.61±0.01
CAFs-CM组	0.63±0.03^a	0.94±0.04	0.98±0.03
CAFs＋3-MA-CM组	0.98±0.03	0.95±0.03	0.75±0.02
F值	210.102	184.477	217.659
P值	<0.001	<0.001	<0.001

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