1. Obtaining scRNA-seq data from HGSOC patient samples before and after chemotherapy

Fig 1a：作者對11例卵巢癌患者治療前后的卵巢組織進(jìn)行了單細(xì)胞測序糠涛。(HGSOC: high-grade serous ovarian cancer; NACT: neoadjuvant chemotherapy)
Fig 1b：一共得到51,786個細(xì)胞梯捕，分別展示了細(xì)胞類型寄猩，患者和分組剑鞍。

2. PRIMUS identifies phenotypic groups from heterogenous scRNA-seq datasets

作者使用了一種名叫PRIMUS的整合方法，整合了不同患者的樣本啸盏，鑒定出了12個cluster。其中有3個(C3, C9, 和 C10)是患者特異的，9個是不同患者都有的维咸。隨后作者使用每個群的差異基因構(gòu)建了共表達(dá)網(wǎng)絡(luò)，鑒定出10個signature惠爽。d圖中作者對這10個signature做了功能富集癌蓖。

3. Chemotherapy affects the prevalence of proliferative and stress-associated cancer cell populations

作者比較了5個治療前后都有細(xì)胞的cluster的比例變化，發(fā)現(xiàn)c5 (proliferative DNA repair signature) 和c7 (stress-associated signature) 在治療前后有顯著變化婚肆。c5治療后下調(diào)租副，c7治療后比例上調(diào)。c5的下降提示化療殺死了增殖的細(xì)胞/使細(xì)胞周期暫時停滯较性。因此作者主要關(guān)注了c7用僧。隨后作者使用bulk RNA測序數(shù)據(jù)，計算了Stress評分赞咙，發(fā)現(xiàn)治療后也是出現(xiàn)顯著上調(diào)的责循。

4. Validation of the stress signature with RNA-ISH

隨后作者使用RNA-ISH對10個stress signature gene進(jìn)行了染色（正文放了三個代表），驗(yàn)證了前面的結(jié)果攀操。

5. Stress-associated state is subclonally enriched during chemotherapy

the preexisting stress-associated state offers a selective advantage to cancer cells during chemotherapy, explained by more inert proliferation when compared to stress-low subclones.

6. Stress-associated transcriptional profile predicts poor prognosis in HGSOC

stress signature和更差的預(yù)后相關(guān)

7. Inflammatory stroma correlates with stress-associated cancer cells

stress-associated腫瘤細(xì)胞高表達(dá)促炎因子如IL-6和TNF院仿，因此可能通過旁分泌影響周圍的細(xì)胞。
Fig 6a, b：作者首先對細(xì)胞進(jìn)行了注釋速和，得到了兩種DCs歹垫，ILCs，巨噬細(xì)胞健芭，肥大細(xì)胞县钥，NK，pDC慈迈，漿細(xì)胞若贮，T，內(nèi)皮痒留，基質(zhì)細(xì)胞和三群cancer associated fibroblasts (CAFs)谴麦。
Fig 6c：三群CAFs的marker基因和功能。
Fig 6d：CAFs與基質(zhì)細(xì)胞和內(nèi)皮細(xì)胞對分化關(guān)系伸头。
Fig 6e：三群CAFs匾效，基質(zhì)細(xì)胞和內(nèi)皮細(xì)胞治療前后的比例變化。CAF-2比例顯著增加恤磷。
Fig 6f：stromal cell的CAF2評分與stress score呈正相關(guān)面哼。
Fig 6g：Ligand-receptor analysis to probe for potential interactions revealed that, in particular, TNF and its downstream effector IL6 from stress-high cancer cells have a strong regulatory potential to induce the inflammatory phenotype of CAFs. This indicates that in NACT-treated ovarian cancer, TNF/IL6 drives the iCAF phenotype rather than IL1B, which has a leading role in promoting the iCAF phenotype in pancreatic cancer. [通過熱圖和點(diǎn)圖組合展示壓力相關(guān)癌細(xì)胞誘導(dǎo) CAF 的炎癥表型(CAF-2)的配體的活性(左)野宜、表達(dá)情況(中)和調(diào)節(jié)靶基因的潛力(右)]
Fig 6h：In response, iCAFs produce a wide array of ligands with rich regulatory potential to activate stress-associated signature within cancer cells, including both IL6 and TNF to promote a paracrine feed-forward loop. Our results suggest iCAFs as the main cell type expressing IL6, CXCL12, and LIF in the tumor milieu, wherein these ligands promote immu- nosuppressive changes, such as macrophage polarization, toward the M2 phenotype.[通過熱圖和點(diǎn)圖組合展示 CAF-2細(xì)胞中驅(qū)動壓力相關(guān)癌細(xì)胞出現(xiàn)應(yīng)激反應(yīng)的配體的活性(左)、 表達(dá)情況(中)和調(diào)節(jié)靶基因的潛力(右)]

最后兩張圖作者利用NicheNet來探索壓力相關(guān)的癌細(xì)胞與炎癥性癌癥相關(guān)的成纖維細(xì)胞（iCAF）之間的細(xì)胞間相互作用魔策。首先統(tǒng)計了兩組差異基因集：CAF2( iCAF )-vs-(CAF-1&CAF-3), 壓力相關(guān)癌細(xì)胞-vs-其他癌細(xì)胞匈子；然后選取兩組差異基因集中前200個上調(diào)基因，作為感興趣的基因集闯袒，分別對壓力相關(guān)癌細(xì)胞以及CAF-2虎敦，執(zhí)行 NicheNet 配體活性分析，獲取排名靠前潛在的配體以及對應(yīng)的配體-受體政敢、配體-靶基因相互作用信息其徙。
如圖G所示，細(xì)胞通訊分析結(jié)果顯示來自高應(yīng)激癌細(xì)胞的TNF及其下游效應(yīng)物IL6具有很強(qiáng)的調(diào)節(jié)潛力喷户，可誘導(dǎo) CAF 的炎癥表型 唾那。這表明在經(jīng)過術(shù)前化療的卵巢癌中，TNF/IL6驅(qū)動 iCAF 表型而不是IL1B（先前研究表明IL1B 在促進(jìn)胰腺癌 iCAF 表型中起主導(dǎo)作用）褪尝。作為響應(yīng)通贞，iCAF 會產(chǎn)生大量具有豐富調(diào)節(jié)潛力的配體，以激活癌細(xì)胞內(nèi)的應(yīng)激相關(guān)信號恼五，包括IL6和TNF，以促進(jìn)旁分泌前饋循環(huán)（如圖H所示）哭懈。