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首先作者通過對CT26細(xì)胞進(jìn)行不同濃度的氯化鐵和鐵螯合劑DFO處理，檢測CT26細(xì)胞活力和TfR1、FPN1蛋白含量，證明Fe3+在腫瘤細(xì)胞增殖中起到了關(guān)鍵作用，且腫瘤細(xì)胞是通過TfR1-FPN1軸維持鐵穩(wěn)態(tài)。隨后，通過不同濃度鎵化合物Ga(NO3)3和硫化氫供體NaHS的處理，檢測CT26細(xì)胞活力和TfR1、FPN1蛋白含量，證明Ga3+能夠有效抑制腫瘤細(xì)胞生長，又由于Ga3+與Fe3+具有相似的性質(zhì)，所以Ga3+可以通過模仿Fe3+作為“特洛伊木馬”運(yùn)行，導(dǎo)致鐵功能障礙；而NaHS可以顯著降低TfR1，增加FPN1的表達(dá)，實現(xiàn)鐵代謝軸的重編程功能。為了整合Ga3+的鐵干擾能力和硫化氫的重組鐵代謝系統(tǒng)能力，作者采用高溫?zé)岱纸夥椒ê铣闪松锘钚訥aS?納米點。
Figure 1. lron metabolism investigation and GaSx, nanodots synthesis. (a)Scheme of iron metabolism in healthy tumor cells.(b)Relative cell viability of CT26 cells after incubation with Fe3+ solutions (the medium without FBS) for 12h. (c)Relative cell viability of CT26 cells afte incubation with DFO for 12h. (d)WB detection of iron metabolism regulatory axis-related proteins, including TfR1 and FPN1, in CT26 cells after treatment with Fe3+ solutions and DFO. (e)Relative cell viability of CT26 cells after incubation with Ga3+ solutions for 12h. (f)Relative cell viability of CT26 cells afer incubation with NaHS for 12h. (g)WB detection of iron metabolism regulatory axis-related proteins, including TfR1 and FPN1, in CT26 cells after incubation with NaHS and Ga3+ solution. (h)Scheme of H2S-mediated disruption of iron metabolism in tumor cells.
利用層浪FongCyte?進(jìn)行流式細(xì)胞術(shù)檢測，分析在不同濃度GaS?處理后細(xì)胞中BCECF的熒光信號變化，并加以免疫熒光實驗的驗證，證明GaSx能被細(xì)胞有效攝取并分解為有效成分。其次通過Western Blot檢測TfR1-FPN1的蛋白含量實驗，ICP-MS定量分析細(xì)胞內(nèi)鐵含量實驗和轉(zhuǎn)錄組測序?qū)嶒灒M(jìn)一步驗證GaS?具有干擾鐵代謝的潛力。
Figure 2. GaSx disrupted tumor cell iron metabolism. (a)Confocal images of CT26 cell endocytosis after GaSx incubation for different time. (b)Confocal images of WSP-1 fuorescence staining of CT26 cells after diferent treatments. (c)BECEF signaling detection in CT26 cells by flow cytometry after treatment with different concentrations of GaSx. (d)Statistical analysis of the BECEF signals in each group in (b). (e)WB detection of iron metabolism regulatory axis-related proteins, including TfR1 and FPN1, in CT26 cells after different treatments. (f)Determination of Ga and Fe contents in CT26 cells after GaSx treatment for different time by ICP-MS. (g)Clustering heat map of differential genes related to iron metabolism. (h)Scheme of the mechanism by which GaSx disrupted iron metabolism in tumor cells.
GaS?干擾鐵代謝導(dǎo)致線粒體膜電位喪失、功能障礙，促使Bax表達(dá)上調(diào)、細(xì)胞色素 c(Cyt C)釋放，激活caspase-3依賴的凋亡途徑，同時誘導(dǎo)Endo G和AIF等釋放，觸發(fā)caspase-3非依賴的凋亡途徑，造成 DNA 損傷。GaS?對腫瘤細(xì)胞有濃度依賴性細(xì)胞毒性，對正常細(xì)胞毒性低，凋亡抑制劑可降低其細(xì)胞毒性。作者通過Annexin V - FITC 檢測，使用層浪FongCyte?進(jìn)行數(shù)據(jù)采集，結(jié)果顯示經(jīng)硫氫化鈉和Ga(NO3)3處理后，凋亡細(xì)胞數(shù)量均有所增加，但與對照組相比，GaS?處理導(dǎo)致凋亡細(xì)胞數(shù)量顯著增加，證實GaS?誘導(dǎo)細(xì)胞凋亡能力強(qiáng)。
Figure 3. GaSx induced apoptosis in tumor cells. (j) Flow cytometric analysis of CT26 cell apoptosis after different treatments. (k) Scheme of the mechanism of GaSx killing tumor cells.
GaS?誘導(dǎo)類凋亡促使免疫相關(guān)細(xì)胞變化，如導(dǎo)致核蛋白高遷移率族蛋白 B1(HMGB1) 從細(xì)胞核釋放，鈣網(wǎng)蛋白（CRT）從內(nèi)質(zhì)網(wǎng)腔轉(zhuǎn)移到細(xì)胞表面，二者作為危險信號啟動炎癥反應(yīng)和免疫激活。通過檢測發(fā)現(xiàn)，GaS?處理后 CT26 細(xì)胞中HMGB1在細(xì)胞核的綠色熒光信號呈劑量依賴性減少，CRT在細(xì)胞膜的綠色熒光信號劑量依賴性增加，表明GaS?加速了損傷相關(guān)分子模式（DAMPs）釋放，增強(qiáng)了免疫反應(yīng)。DC細(xì)胞在DAMPs刺激下成熟，通過層浪FongCyte?檢測CD80和CD86陽性比例，發(fā)現(xiàn)與GaS?處理細(xì)胞上清孵育后，成熟DCs比例以濃度依賴方式增加，表明GaS?可激活免疫反應(yīng)，通過誘導(dǎo)類凋亡發(fā)揮作用，具有克服凋亡抵抗和刺激免疫反應(yīng)的優(yōu)勢，有望與化療和免疫療法協(xié)同作用。
Figure 4. GaSx induced paraptosis in tumor cells. (j-k) Proportion of mature DCs after incubating DCs and supernatants from CT26 cells treated with different concentrations of GaSx. (l) Scheme of GaSx-induced paraptosis in tumor cells to activate the immune response.
GaSx的抗腫瘤效果
與αPD-1聯(lián)合使用
可顯著增強(qiáng)抗腫瘤免疫反應(yīng)
由于GaS?在體外具有破壞鐵代謝和誘導(dǎo)細(xì)胞凋亡的能力，作者進(jìn)一步驗證了GaS?的體內(nèi)抗腫瘤作用。結(jié)果顯示GaS?顯著抑制腫瘤生長，延長小鼠中位生存期，且對小鼠體重和主要器官無明顯影響，組織學(xué)分析證實其腫瘤殺傷效果，免疫組化和免疫熒光染色表明其可調(diào)節(jié)腫瘤細(xì)胞鐵代謝相關(guān)蛋白表達(dá)。
GaS?可誘導(dǎo)腫瘤細(xì)胞釋放DAMPs，促進(jìn)DC細(xì)胞成熟，增強(qiáng)CD4?和CD8?T細(xì)胞浸潤，使腫瘤相關(guān)巨噬細(xì)胞表型由抑制M2表型向炎癥M1表型轉(zhuǎn)變，減少調(diào)節(jié)性T細(xì)胞（Tregs）和髓源性抑制細(xì)胞（MDSCs），增加促炎細(xì)胞因子如白細(xì)胞介素-6（IL-6）、白細(xì)胞介素-12p70（IL-12p70）和腫瘤壞死因子（TNF-α），與αPD-1聯(lián)合使用可顯著抑制腫瘤進(jìn)展，增強(qiáng)抗腫瘤免疫反應(yīng)。這些結(jié)果證實了GaS?在提高臨床免疫治療療效方面的潛力。一方面，GaS?破壞鐵代謝，觸發(fā)異常分泌和DAMPs釋放。另一方面，它通過改變瘤內(nèi)鐵分布誘導(dǎo)巨噬細(xì)胞M1極化，并通過H2S介導(dǎo)的信號調(diào)控抑制MDSCs和treg的浸潤。這種協(xié)同的方法顯著增強(qiáng)腫瘤的免疫原性，逆轉(zhuǎn)免疫抑制的腫瘤微環(huán)境，有效地克服了免疫治療的障礙。
Figure 5. Immunological evaluation of tumors from GaSx-treated mice and the combination therapy with GaSxandαPD-1 antibody. (a)Scheme of the immuno-evaluation of tumors and TDLNs after different treatments (G1:Control, G2:NaHS, G3:Ga(NO3)3, and G4:GaSx). (b)Mature DCs in tumors after different treatments in (a). (c)CD8+T cells infitration in tumors after different treatments in (a). (d)CD4+T cells infitration in mouse tumors after various treatments in (a). (e)Proportion of M2 cells intumors after various treatments in (a). (f)Proportion of M1 cells in mouse tumors after various treatments in (a). (g)Tregs infitration in mouse tumors after various treatments in (a). (h)Proportion of MDSCs in mouse tumors after various treatments in (a). (i)Mature DCs in TDLNs after different treatments in (d). (j?l)Pro-infammatory cytokine levels (j) IL-6, (k) IL-12p70, and (l) TNF-αin tumors from the mice after various treatments in (a). (m?o)Pro-infammatory cytokine levels (m) IL-6, (n) IL-12p70, and (o) TNF-αin the TDLNs of the mice after various treatments are shown in (a). (p)Scheme ofGaSx andαPD-1 combination therapy. (q)Tumor volume of the mice in each group (n=5) in (p). (r)Tumor weights ofthe mice after 14 days of different treatments in (p). (s)Photographs of tumors after 14 days of different treatments in (p). (t)Tumorvolume of mice after different treatments in (p). (u)H&E images and CD4+CD8+ stained tumors after 14 days of different treatments in (p).
GaS?具有 “重編程” 和 “干擾” 鐵代謝途徑的雙重功能，能夠誘導(dǎo)腫瘤細(xì)胞發(fā)生線粒體功能障礙和內(nèi)質(zhì)網(wǎng)應(yīng)激，進(jìn)而觸發(fā)細(xì)胞凋亡-類凋亡混合途徑，抑制腫瘤增殖。并且GaS?誘導(dǎo)的鐵代謝失調(diào)顯著增加了腫瘤細(xì)胞對化療和免疫檢查點阻斷（ICB）治療的敏感性。這一特性為提高現(xiàn)有臨床治療方法的療效提供了可能，為腫瘤治療帶來了新的思路和策略。
在該研究中，流式技術(shù)發(fā)揮了重要作用，研究中涉及的免疫表型以及細(xì)胞功能檢測皆采用層浪 FongCyte?流式細(xì)胞儀。FongCyte?具有激光器和檢測器雙溫控設(shè)計，可實時進(jìn)行溫度調(diào)節(jié)，保證儀器性能及實驗結(jié)果穩(wěn)定；支持定量吸入和持續(xù)上吸兩種上樣模式，內(nèi)嵌自動進(jìn)樣器，滿足各種實驗需求；可提供多達(dá) 14 色參數(shù)分析。該研究涉及多種樣本和panel檢測， FongCyte?的獨特設(shè)計不僅為其提供客觀且準(zhǔn)確的流式數(shù)據(jù)，還為研究者提供智能化維護(hù)，提高實驗效率。
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