Estrongen receptor positive (ER+) breast cancer (BCa)生长很大程度依赖ER signaling。对于 (ER+) 的Breast cancer patients,常规采用endocrine therapy,即通过不同方式阻断ER的signaling,因此抑制cancer生长。
目的:dissect phenotypic heterogeneity & plasticity of ER+ BCa,找到pre-adapted cells(PA cells)及其signatures
技术:scRNA-seq, live cell imaging,machine learning(使用分类器dissect)
结果:从细胞模型的确发现了PA cells;PA cells具有dormancy和mixed epithelial & mesenchymal traits;这些signatures/traits在clustered circulating tumor cells(CTC)中比single CTC高(CTC cluster比 single CTC具有更强的metastasis能力);PA cells和LTED很不同,因此只是ED过程中的一个阶段性产物,ED因此也应被视为multi-step model
Result
Absence of features of resistance in treatment-naive cells
Treatment naive cells和LTED cells很不同,体现于以下方面
gene copy number alteration (CNA) c
general transcriptome b
pathway activation d
Phenotypic heterogeneity of luminal breast cancer cells
下文找PA cells主要从CD44+的cells:CD44是基于previous knowledge,代表高细胞plasticity的marker。因此在此验证一下此gene的重要性,为下文做铺垫; 建模:MCF7 and LTED cell lines with a GFP reporter expressed under the promoter of the CD44 gene
基于paired samples(安利一下,我们lab很多年的efforts就是收集同一个病人的primary/metastasis samples,前辈做了一个简单的shiny app (现在比较卡,再过一段时间可能会升级服务器),欢迎大家访问和提议 (http://157.230.50.64:3838/apps/Paired_Mets/) ;不过unfortunately我们的dataset里面的met patients里并未发现这种上升) CD44在endocrine resistant的病人中上升,我们PI说一种可能解释为Aromatase treatment与Tamoxifen treatment引起resistance的transcriptomes会不同) a b
CD44 high cells具有plasticity,即它们在分裂后大多会lose CD44;这种现象在treatment-naive和LTED cells里都存在 c d
transcriptomically strongly biased towards features of starved cells (misclassified by random forest classifier) up b
通过上述PA cells找到的DEGs,找到一个新的marker CLDN1;only in condition of CD44 high expression, CLDN1 high cells在ED过程中具有生存优势 up d
PA cell marker genes在pathway上的特征 down a
PA cell marker genes和cell cycle related genes为negatively correlated
这一段可谓本文最神奇的地方。我们来看一看高级方法
Identification of pre-adapted cells
Two different strategies were employed to identify the pre-adapted cells.
The first one takes advantage of SWNE; a threshold was applied on the first component and the cells showing extreme values (>=0.75) were labelled as pre-adapted.The second strategy leverages random forests classifiers62. First of all, the data sets of CD44high cells in +E2 media and starved conditions (2 days) were split into training and testing sets, using 10% and 90% of the cells, (怀疑是个typo,应该是90% and 10%) respectively. The training set was then used to call the DEGs between the two conditions (+E2 vs starved), using the procedure described in the Differential expression analysis paragraph above. These DEGs were used as input features to train a random forest classifier, using the randomForest R package (v4.6-14; default parameters). This model was then used to test the remaining data. Those cells in the testing set labelled as +E2 that were showing a probability >50% of being classified as starved were considered pre-adapted.
AUCell39 (R package v1.0.0) was the used to quantify the activity of the pre-adapted signatures (and of other signatures, whenever indicated in the text) in single cells. First of all, normalised data were processed using the AUCell_buildRankings function. The resulting rankings, along with the signatures of interest, were then subject to function AUCell_calcAUC (aucMaxRank set to 5% of the number of input genes). Following inspection of the resulting distributions, thresholds were then manually set to 0.37, 0.18 and 0.32 for the signatures of pre-adapted cells either based on SWNE or random forests, or for the LTED signature (defined as those genes upregulated in LTED vs MCF7, as described in the Differential expression analysis section above).
观察到7 days of ED后, 存活的CD44 low cells和CD44 high cells有相似的transcriptomic alteration,提出假设:PA signature为acute-Endocrine therapy的bottleneck,被selected against;CD44 low cells upregulate该signature的efficiency低于CD44 high cells因而具有生存劣势(PA signature是生存的必要不充分条件)
The PA signature is enriched in clusters of CTCs
为本文的验证部分:在另一株细胞系和临床样本中进行验证。
T47D细胞中也发现了类似的PA cells (shifting on SWNE1)
PA phenotype(因显示了EMT和polarity特征),hypothesize这些特征是否在metastasis progression中有作用
Comment: 这一点比较contradictory,因为基于Fig5a,PA中EMT和apical junction都是上调的,而后者是epithelial(Epi)的特征;这里想说明CTC cluster比single cell更具有Epi特征,PA具有polarity(Epi)特征,因此CTC cluster很可能具有(实际上也是)更高的PA features,这与PA中EMT高矛盾
已知:CTC cluster contribute to >85% metastasis dissemination; single CTC有更多epithelial的feature
CTC比healthy blood中PA signature高
CTC cluster和CTC single cell相比,前者的PA signature/EMT/Cell cycle signature表达更高