作者评肆，Evil Genius

今天一大早债查，最新的消息來了，智聯(lián)崩了瓜挽。

GDP增長了5.2%盹廷，失業(yè)率屢創(chuàng)新高，但是可支配收入增加了久橙，這些矛盾的背后俄占，只有一種解釋，公務(wù)員等編制類的崗位待遇上升了剥汤。

這一篇我們繼續(xù)我們的多組學(xué)颠放，單細(xì)胞、空間吭敢、外顯子碰凶。參考的文章在An atlas of epithelial cell states and plasticity in lung adenocarcinoma，2024年2月發(fā)表于nature(IF 64.8)鹿驼。

關(guān)于單細(xì)胞如何聯(lián)合外顯子的突變信息進(jìn)行聯(lián)合分析還需要學(xué)習(xí)學(xué)習(xí)欲低。

其中大家如果對臨床檢測或者做過臨檢的話，對KRAS這個基因應(yīng)該并不陌生畜晰，這個基因的突變導(dǎo)致多種癌癥的發(fā)生砾莱，其中在肺腺癌中KRAS的突變，早期出現(xiàn)KRAS突變通常能達(dá)到臨床治愈凄鼻，但對于中晚期肺腺癌腊瑟，出現(xiàn)KRAS突變一般不能治愈聚假，KRAS突變癌細(xì)胞表現(xiàn)出明顯的轉(zhuǎn)錄特征，分化減少和低水平的非整倍體闰非。,在胰腺癌中KRAS的G12C突變有具體的靶向藥可以治療膘格。

重復(fù)一下之前的重點，基因過量表達(dá)多發(fā)生在細(xì)胞癌變的起始階段财松，基因點突變可能是細(xì)胞癌變啟動階段的一個主要事件瘪贱，這一階段的可逆性較大；對于基因表達(dá)辆毡，單細(xì)胞技術(shù)可以提供很好的幫助菜秦，對于突變，就需要借助外顯子舶掖，對于免疫治療球昨，就需要空間、VDJ和外顯子多組學(xué)的內(nèi)容了眨攘。

文章研究條件

樣本：來自16例早期luad和47例匹配的正常肺樣本的246,102個上皮細(xì)胞（tumour-adjacent, tumour-intermediate and tumour-distant locations）褪尝。
組學(xué)：單細(xì)胞、空間(10X期犬、DSP)、外顯子避诽、蛋白組
富集上皮細(xì)胞的基因:EPCAM

背景知識龟虎，吸煙會導(dǎo)致基因突變（例如KRAS）和免疫環(huán)境轉(zhuǎn)變，這些變化會影響臨近組織的生態(tài)環(huán)境沙庐，并且與肺惡性病變和LUAD的發(fā)展密切相關(guān)鲤妥。

上皮細(xì)胞圖譜

單細(xì)胞QC之后，保留246,102個上皮細(xì)胞用于分析拱雏。通過整合推斷拷貝數(shù)變異(intercnv)棉安、聚類分布、譜系特異性基因表達(dá)和攜帶KRAS^G12D體細(xì)胞突變的reads的存在等信息铸抑，將惡性細(xì)胞(n = 17,064)與非惡性正常細(xì)胞(n = 229,038)區(qū)分開來贡耽。

上皮非惡性細(xì)胞分類：alveolar、airway and a small subset of proliferative cells鹊汛。其中Airway cells包括basal (KRT17⁺), ciliated (FOXJ1⁺) and club and secretory (SCGB1A1⁺) populations, as well as rare cell types such as ionocytes (ASCL3⁺), neuroendocrine cells (ASCL1⁺) and tuft cells (GNAT3⁺) 蒲赂。
Alveolar cells 包括alveolar type?1 (AT1) cells (AGER1⁺ETV5⁺), AT2 cells (SFTPB⁺SFTPC⁺), SCGB1A1⁺SFTPC⁺ dual-positive cells and a cluster of alveolar intermediate cells (AICs) that was closely tucked between AT1 and AT2 clusters and shared gene expression features with both major alveolar cell types。

惡性細(xì)胞表現(xiàn)出標(biāo)記物（markers）的低表達(dá)或不表達(dá)刁憋，總體而言滥嘴，reduced lineage identity。惡性細(xì)胞形成14個clusters至耻，主要是患者特異性的若皱，這表明患者之間存在很強的異質(zhì)性镊叁。總體而言走触，惡性細(xì)胞表現(xiàn)出高水平的非整倍體晦譬。在吸煙狀況方面，沒有發(fā)現(xiàn)任何明顯的聚類模式饺汹』滋恚基于基因組圖譜(WES)的注釋顯示，3例KRAS突變LUADs患者的惡性細(xì)胞緊密聚集在一起兜辞。相比之下迎瞧，來自其他LUADs的惡性細(xì)胞表現(xiàn)出更分散的聚集模式。scRNA-seq分析證實逸吵，在患者特異性腫瘤clusters中存在拷貝數(shù)變異(CNV)和KRAS^G12D突變凶硅，而KRAS野生型LUADs (KW-LUADs)中不存在KRAS^G12D。

來自KM-LUADs的惡性細(xì)胞聚成一類扫皱，與EGFR突變LUADs (EM-LUADs)或MET突變LUADs (MM-LUADs)的惡性細(xì)胞明顯不同足绅。與其他LUAD相比，KM-LUADs在樣本和細(xì)胞水平上都表現(xiàn)出更多的轉(zhuǎn)錄組相似性韩脑。大多數(shù)KRAS突變惡性細(xì)胞與其他細(xì)胞分開聚類氢妈，這表明KRAS突變細(xì)胞中存在不同的轉(zhuǎn)錄程序。與先前的報道一致段多，來自KM-LUADs的惡性細(xì)胞在染色體上比來自EM-LUADs的細(xì)胞更穩(wěn)定首量。吸煙患者惡性細(xì)胞的CNV負(fù)荷明顯高于不吸煙患者。惡性細(xì)胞的分化狀態(tài)表現(xiàn)出高度的患者間異質(zhì)性进苍。也就是說加缘，無論腫瘤突變負(fù)荷如何，KM-LUAD細(xì)胞分化程度最低觉啊，正如其最高的CytoTRACE分?jǐn)?shù)所表明的那樣拣宏，其次是EM-LUADs。分化狀態(tài)存在腫瘤內(nèi)異質(zhì)性(ITH)杠人。

An atlas of epithelial cell states and plasticity in lung adenocarcinoma

• 圖例：a, Schematic overview of the experimental design and analysis workflow. Composition, composition of cell subsets; Program, transcriptional programs in malignant cells; Spatial, in situ spatial transcriptome and protein analyses; State, cellular transcriptional state. b, Proportions and average expression levels (scaled) of selected marker genes for ten normal epithelial and one malignant cell subset. NE, neuroendocrine. c, Unsupervised clustering of 17,064 malignant cells coloured by cluster identity. Top right inset shows malignant cells coloured by KRAS^G12D mutation status identified by scRNA-seq. d, Uniform manifold approximation and projection (UMAP) of malignant cells shown in c and coloured by driver mutations identified in each tumour sample using WES. e, Principal component analysis (PCA) plot of malignant cells coloured by driver mutations identified in each tumour sample by WES. f, UMAP plots of malignant cells coloured by patient identifier and grouped by driver mutation status. g, Top, UMAP of malignant cells by differentiation state inferred by CytoTRACE. Bottom, comparison of CytoTRACE scores between malignant cells from samples with different driver mutations. Boxes indicate the median?±?interquartile range; whiskers, 1.5× the interquartile range; centre line, median. n cells in each box-and-whisker (left to right): 9,135, 5,457 and 2,472. P?values were calculated using two-sided Wilcoxon rank-sum test with Benjamini–Hochberg correction. diff., differentiated. h, Per sample distribution of malignant cell CytoTRACE scores

LUAD malignant transcriptional programs

KRAS^G12D突變的惡性細(xì)胞分化減弱勋乾，這與KM-LUADs中肺泡分化(MP31)的喪失是一致的.來自患者P14的惡性細(xì)胞cluster表現(xiàn)出不同水平的CNV，其中KRAS^G12D細(xì)胞富集的細(xì)胞簇具有相對較晚的CNV事件(例如嗡善，染色體1p丟失市俊，染色體8和染色體12gain)，肺泡特征評分降低滤奈，結(jié)果與分化減弱一致摆昧。

AICs in LUAD

AICs在AT2 - AT1細(xì)胞發(fā)育和分化過程中處于中間位置，這一結(jié)果與暴露于急性肺損傷的無癌小鼠的中間肺泡細(xì)胞相似蜒程。LUAD組織中分化程度最低的AICs的比例高于分化程度較高的AICs绅你。值得注意的是伺帘，AICs被推斷為向惡性細(xì)胞轉(zhuǎn)移，包括KRAS突變細(xì)胞忌锯，相對于EGFR突變的惡性細(xì)胞伪嫁，KRAS突變細(xì)胞的發(fā)育更晚。對AICs的進(jìn)一步分析發(fā)現(xiàn)了KRT8明顯高表達(dá)的亞群偶垮。這些KACs增加了CDKN1A张咳、CDKN2A、PLAUR和腫瘤標(biāo)志物CLDN4的表達(dá)似舵。與其他aic相比脚猾，KACs的分化程度明顯較低，發(fā)育較晚砚哗。值得注意的是龙助，KACs在軌跡推斷中轉(zhuǎn)化為KRAS突變的惡性細(xì)胞，而其他AIC與向AT1細(xì)胞的分化更密切相關(guān)蛛芥。相對于多區(qū)NL組織提鸟，LUAD中非惡性上皮細(xì)胞中KACs的比例明顯增加，且LUAD中KACs的比例明顯高于AT1仅淑、AT2或其他AIC組称勋。值得注意的是，與NL來源的KACs相比涯竟，腫瘤相關(guān)的KACs聚集在離AIC更遠(yuǎn)的地方铣缠。

為了進(jìn)一步了解KACs的重要表型和特征，通過對多個腫瘤樣本進(jìn)行空間轉(zhuǎn)錄組測序（Visium spatial transcriptomics昆禽，10X）和數(shù)字空間圖譜掃描（Digital spatial profiling），研究發(fā)現(xiàn)KACs的特征基因在mRNA水平和蛋白質(zhì)水平不僅在腫瘤細(xì)胞中升高蝇庭，有趣的是在腫瘤周圍的臨近組織中也升高醉鳖，這一發(fā)現(xiàn)支持了文章的假設(shè)：KACs可能作為一種“前體中間態(tài)細(xì)胞”，參與早期肺腺癌的發(fā)生和發(fā)展哮内〉量茫基于此，研究者在大量的獨立數(shù)據(jù)集中進(jìn)行了驗證性分析北发。首先纹因，利用TCGA肺腺癌數(shù)據(jù)集，發(fā)現(xiàn)在肺腺癌樣本中KACs信號明顯高于來自同一病人的配對癌旁樣本琳拨。其次瞭恰，在一組包含15個配對的正常肺組織，癌前病變組織狱庇，以及侵襲性肺腺癌組織的數(shù)據(jù)中惊畏，發(fā)現(xiàn)KACs的信號從正常肺組織到肺腺癌癌前病變組織再到早期肺腺癌組織的漸進(jìn)性增強恶耽。另外，他們還發(fā)現(xiàn)KACs信號與病人較差的生存預(yù)后顯著相關(guān)颜启。這些結(jié)果進(jìn)一步支持了研究者的假設(shè)偷俭。相關(guān)結(jié)果還顯示相比不含KRAS突變的肺腺癌樣本，KACs在KRAS突變型肺腺癌呈現(xiàn)出更高的表達(dá)水平缰盏，揭示了KAC和KRAS突變型肺腺癌之間的密切關(guān)系涌萤。

盡管與惡性細(xì)胞相比，KACs表現(xiàn)出較低的CNV評分口猜，但相對于AT2负溪、AT1和其他AIC, KACs表現(xiàn)出適度增加的CNV burden。KRAS^G12D存在于惡性細(xì)胞中暮的，其變異等位基因頻率(VAF)在KM-LUAD中為78%笙以。KACs存在KRAS^G12D突變，但不包括AT2冻辩、AT1或其他AIC猖腕。KRAS^G12D KACs僅在KM-LUAD的組織(主要是腫瘤)中發(fā)現(xiàn)，因此恨闪，KRAS^G12D VAF(10%)在KM-LUAD的KACs中高于所有檢測的LUAD的KACs(5%)或樣本(3%)倘感。在KM-LUAD患者(VAF為2%)NL樣本的KACs中檢測到KRAS^G12D突變。同時咙咽，在1例KM-LUAD患者的NL中檢測到其他KRAS變體(KRAS^G12C)老玛，提示有潛在的區(qū)域性癌變效應(yīng)。與此同時钧敞，KRAS^G12D型KACs的KRAS特征也比KRAS^WT型KACs顯著增加蜡豹。KRAS^WT KACs相對于其他AIC和其他AIC相對于AT2細(xì)胞的KRAS信號也增加。這一結(jié)果指向沿AT2-AIC-KAC頻譜增加的KRAS信號溉苛。來自NL或KM-LUAD而非KW-LUAD的腫瘤的KACs與其他AIC的分化程度一致且顯著低镜廉。總之愚战，研究結(jié)果將KACs描述為與人類LUAD發(fā)病機制高度相關(guān)的中間肺泡細(xì)胞亞群娇唯，特別是KM-LUAD。

Identification and characterization of KACs in human LUAD

• 注：a, Pseudotime analysis of alveolar and malignant cells. b, Left, subclustering analysis of AICs. Right, proportions and average expression levels (scaled) of representative KAC marker genes. c, CytoTRACE score in KACs versus other AICs. n cells (left to right): 8,591 and 1,440. P?value was calculated using two-sided Wilcoxon rank-sum test. d, Proportion of KACs among non-malignant epithelial cells. n samples (left to right): 16, 15, 16 and 16. P?value was calculated using Kruskal–Wallis test. e, Fraction of alveolar cell subsets coloured by sample type. P?values were calculated using two-sided Fisher’s exact tests with Benjamini–Hochberg correction. f, Top, haematoxylin and eosin (H&E) staining of LUAD tumour (T), TAN displaying reactive hyperplasia of AT2 cells and uninvolved NL tissue. Bottom, digital spatial profiling showing KRT8, PanCK, CLDN4, Syto13 blue nuclear stain and composite image. Magnification, ×20. Scale bar, 200?μm. Staining was repeated four times with similar results. Dashed white lines represent the margins separating tumours and TAN regions. g, ST analysis of LUAD from patient P14 showing histologically annotated H&E-stained Visium slide (left) and spatial heatmaps (right) depicting CNV score and scaled expression of KRT8, KAC markers (b) and KRAS signature. h, Expression (top) and correlation (bottom) analyses of KAC, KRAS and alveolar signatures. n?=?1,440 (KACs), 8,593 (other AICs), 146,776 (AT2) and 25,561 (AT1). R, Spearman’s correlation coefficient. P?values were calculated using Spearman’s correlation test. i, KAC signature expression in premalignancy cohort (15 samples each). P?values were calculated using two-sided Wilcoxon signed-rank test with Benjamini–Hochberg correction. j, Fraction of KRASG12D cells in different subsets.

A KAC state is linked to mouse KM-LUAD

研究人員使用基因工程小鼠模型對一系列關(guān)于KACs的科學(xué)問題進(jìn)行了系統(tǒng)探索寂玲。他們首先在一種能更準(zhǔn)確模擬人體KRAS突變的肺腺癌發(fā)生過程的小鼠模型中進(jìn)行了實驗研究塔插。該模型在煙草致癌物（Nicotine-derived nitrosamine ketone， NNK）暴露后能夠適時地觀察到誘導(dǎo)性KRAS突變型肺部腫瘤的發(fā)生拓哟。研究人員在不同時間點采集了對照和處理組樣本并進(jìn)行了單細(xì)胞RNA測序分析想许。通過對單細(xì)胞RNA測序數(shù)據(jù)的分析證實了小鼠中存在表達(dá)特征與KACs相匹配的細(xì)胞類群，研究者稱之為小鼠KACs。通過對比在不同取樣時間點的單細(xì)胞RNA測序數(shù)據(jù)伸刃，發(fā)現(xiàn)小鼠KACs的出現(xiàn)在KRAS突變型腫瘤出現(xiàn)之前谎砾。此外，研究者還對來自小鼠模型的肺癌樣本進(jìn)行了空間轉(zhuǎn)錄組測序分析捧颅【巴迹空間轉(zhuǎn)錄組測序數(shù)據(jù)也呈現(xiàn)出和人肺腺癌樣本的空間轉(zhuǎn)錄組數(shù)據(jù)相似的特征，即小鼠腫瘤組織和腫瘤周圍鄰近組織中存在高水平的KACs特征基因的表達(dá)碉哑。

肺腺癌患者以及小鼠模型的單細(xì)胞RNA測序數(shù)據(jù)的擬時分析結(jié)果共同提示KACs可能來源于2型肺泡細(xì)胞挚币。作者使用GFP標(biāo)記工程小鼠，用來驗證小鼠KACs是否來源于2型肺泡細(xì)胞扣典。同時利用Gfp標(biāo)記的2型肺泡細(xì)胞培養(yǎng)了類器官（organoids）并進(jìn)行了多重免疫熒光染色分析妆毕， 實驗結(jié)果顯示NNK處理組的類器官樣本具有更強的KACs特征基因的表達(dá)。這些結(jié)果驗證了KACs的2型肺泡細(xì)胞來源并顯示了NNK暴露與KACs的高度相關(guān)贮尖，揭示了KACs和肺組織損傷以及腫瘤細(xì)胞生成的密切相關(guān)笛粘。

最后，研究者構(gòu)建了能夠追蹤KACs細(xì)胞的模型小鼠湿硝，使用譜系追蹤（lineage tracing）技術(shù)回答此項研究的最終問題：KACs是否能轉(zhuǎn)化成腺癌細(xì)胞薪前。研究者使用免疫熒光染色技術(shù)分析了來自NNK暴露結(jié)束（處理組），NNK暴露后8至12周（跟進(jìn)觀察組）和腫瘤的樣本关斜。通過對比小鼠三組樣本的免疫熒光細(xì)胞計數(shù)分析結(jié)果示括，研究人員發(fā)現(xiàn)腫瘤樣本中絕大多數(shù)的細(xì)胞為2型肺泡細(xì)胞來源的，經(jīng)過KACs細(xì)胞狀態(tài)（Krt8+ Lamp3+）的細(xì)胞痢畜。此外垛膝，腫瘤樣本中Krt8（KAC的標(biāo)記基因之一）蛋白表達(dá)信號增強。譜系追蹤模型小鼠的實驗結(jié)果驗證了2型肺泡細(xì)胞來源的KACs產(chǎn)生了肺腺癌細(xì)胞丁稀。

綜合以上結(jié)果吼拥，這項大規(guī)模的多組學(xué)整合研究發(fā)現(xiàn)了KACs細(xì)胞在早期KRAS突變的肺腺癌中的重要作用，并使用轉(zhuǎn)基因工程小鼠模型系統(tǒng)性地驗證了KACs為來源于2型肺泡細(xì)胞的中間狀態(tài)細(xì)胞线衫，在2型肺泡細(xì)胞參與肺損傷修復(fù)凿可，補充1型功能性肺泡細(xì)胞的生物學(xué)過程中，參與了Kras突變型的肺腺癌細(xì)胞的產(chǎn)生過程桶雀。這項研究揭示了肺腺癌發(fā)生過程和上皮細(xì)胞可塑性的重要聯(lián)系，為肺腺癌的預(yù)防和早期干預(yù)提供了潛在的研究靶點唬复。

• 注：a, Trajectories of alveolar and malignant cells coloured by inferred pseudotime, cell differentiation status and cell type (top left to right). Distribution of inferred pseudotime (bottom left) and CytoTRACE (bottom middle) scores across the indicated cell subsets. Bottom right panel shows CytoTRACE score distribution in KACs at the two time points. b, Schematic overview showing analysis of Gprc5a^?/? mice with reporter-labelled AT2 cells (Gprc5a^?/?;Sftpc^creER/+;Rosa^Sun1GFP/+). TMX, tamoxifen. c, Fractions of AT1, AT2, KACs and KAC-like cells (KAC–KAC-like) and early tumour and AT2-like tumour cells (early–AT2-like tumour) within GFP⁺ cells from lungs of two NNK-treated and two saline-treated mice analysed at 3?months after exposure. d, IF analysis of tdT and KRT8 expression at EOE to NNK (first column; EOE) and at 8–12?weeks following NNK (follow-up after EOE) in normal-appearing regions (second column) and tumours (last two columns) of Gprc5a^?/?;Krt8-creER;Rosa^tdT/+ mice. Tamoxifen (1?mg per dose) was delivered immediately after EOE to NNK for six continuous days. Results are representative of three biological replicates per condition. Staining was performed two times with similar results. Magnification, ×20. Scale bar, 10?μm. e, Left, percentage of lung tissue areas containing tdT⁺ cells. Right, percentage of tdT⁺LAMP3⁺ cells among total tdT⁺ cells in normal-appearing regions at different time points. Error bars show the mean?±?s.d. of n biologically independent samples (left to right): 6, 6, 6, 6 and 10. P?values were calculated using Mann–Whitney U-test. f, Proposed model for alveolar plasticity, whereby a subset of AICs in the intermediate AT2-to-AT1 differentiation state are KACs and, later, acquire KRAS^G12D mutations and are implicated in KM-LUAD development from a particular region in the lung.

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