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首先作者通过对CT26细胞进行不同浓度的氯化铁和铁螯合剂DFO处理，检测CT26细胞活力和TfR1、FPN1蛋白含量，证明Fe3+在肿瘤细胞增殖中起到了关键作用，且肿瘤细胞是通过TfR1-FPN1轴维持铁稳态。随后，通过不同浓度镓化合物Ga(NO3)3和硫化氢供体NaHS的处理，检测CT26细胞活力和TfR1、FPN1蛋白含量，证明Ga3+能够有效抑制肿瘤细胞生长，又由于Ga3+与Fe3+具有相似的性质，所以Ga3+可以通过模仿Fe3+作为“特洛伊木马”运行，导致铁功能障碍；而NaHS可以显著降低TfR1，增加FPN1的表达，实现铁代谢轴的重编程功能。为了整合Ga3+的铁干扰能力和硫化氢的重组铁代谢系统能力，作者采用高温热分解方法合成了生物活性GaSₓ纳米点。
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.
利用层浪FongCyte™进行流式细胞术检测，分析在不同浓度GaSₓ处理后细胞中BCECF的荧光信号变化，并加以免疫荧光实验的验证，证明GaSx能被细胞有效摄取并分解为有效成分。其次通过Western Blot检测TfR1-FPN1的蛋白含量实验，ICP-MS定量分析细胞内铁含量实验和转录组测序实验，进一步验证GaSₓ具有干扰铁代谢的潜力。
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.
GaSₓ干扰铁代谢导致线粒体膜电位丧失、功能障碍，促使Bax表达上调、细胞色素 c(Cyt C)释放，激活caspase-3依赖的凋亡途径，同时诱导Endo G和AIF等释放，触发caspase-3非依赖的凋亡途径，造成 DNA 损伤。GaSₓ对肿瘤细胞有浓度依赖性细胞毒性，对正常细胞毒性低，凋亡抑制剂可降低其细胞毒性。作者通过Annexin V - FITC 检测，使用层浪FongCyte™进行数据采集，结果显示经硫氢化钠和Ga(NO3)3处理后，凋亡细胞数量均有所增加，但与对照组相比，GaSₓ处理导致凋亡细胞数量显著增加，证实GaSₓ诱导细胞凋亡能力强。
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.
GaSₓ诱导类凋亡促使免疫相关细胞变化，如导致核蛋白高迁移率族蛋白 B1(HMGB1) 从细胞核释放，钙网蛋白（CRT）从内质网腔转移到细胞表面，二者作为危险信号启动炎症反应和免疫激活。通过检测发现，GaSₓ处理后 CT26 细胞中HMGB1在细胞核的绿色荧光信号呈剂量依赖性减少，CRT在细胞膜的绿色荧光信号剂量依赖性增加，表明GaSₓ加速了损伤相关分子模式（DAMPs）释放，增强了免疫反应。DC细胞在DAMPs刺激下成熟，通过层浪FongCyte™检测CD80和CD86阳性比例，发现与GaSₓ处理细胞上清孵育后，成熟DCs比例以浓度依赖方式增加，表明GaSₓ可激活免疫反应，通过诱导类凋亡发挥作用，具有克服凋亡抵抗和刺激免疫反应的优势，有望与化疗和免疫疗法协同作用。
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的抗肿瘤效果
与αPD-1联合使用
可显著增强抗肿瘤免疫反应
由于GaSₓ在体外具有破坏铁代谢和诱导细胞凋亡的能力，作者进一步验证了GaSₓ的体内抗肿瘤作用。结果显示GaSₓ显著抑制肿瘤生长，延长小鼠中位生存期，且对小鼠体重和主要器官无明显影响，组织学分析证实其肿瘤杀伤效果，免疫组化和免疫荧光染色表明其可调节肿瘤细胞铁代谢相关蛋白表达。
GaSₓ可诱导肿瘤细胞释放DAMPs，促进DC细胞成熟，增强CD4⁺和CD8⁺T细胞浸润，使肿瘤相关巨噬细胞表型由抑制M2表型向炎症M1表型转变，减少调节性T细胞（Tregs）和髓源性抑制细胞（MDSCs），增加促炎细胞因子如白细胞介素-6（IL-6）、白细胞介素-12p70（IL-12p70）和肿瘤坏死因子（TNF-α），与αPD-1联合使用可显著抑制肿瘤进展，增强抗肿瘤免疫反应。这些结果证实了GaSₓ在提高临床免疫治疗疗效方面的潜力。一方面，GaSₓ破坏铁代谢，触发异常分泌和DAMPs释放。另一方面，它通过改变瘤内铁分布诱导巨噬细胞M1极化，并通过H2S介导的信号调控抑制MDSCs和treg的浸润。这种协同的方法显著增强肿瘤的免疫原性，逆转免疫抑制的肿瘤微环境，有效地克服了免疫治疗的障碍。
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).
GaSₓ具有 “重编程” 和 “干扰” 铁代谢途径的双重功能，能够诱导肿瘤细胞发生线粒体功能障碍和内质网应激，进而触发细胞凋亡-类凋亡混合途径，抑制肿瘤增殖。并且GaSₓ诱导的铁代谢失调显著增加了肿瘤细胞对化疗和免疫检查点阻断（ICB）治疗的敏感性。这一特性为提高现有临床治疗方法的疗效提供了可能，为肿瘤治疗带来了新的思路和策略。
在该研究中，流式技术发挥了重要作用，研究中涉及的免疫表型以及细胞功能检测皆采用层浪 FongCyte™流式细胞仪。FongCyte™具有激光器和检测器双温控设计，可实时进行温度调节，保证仪器性能及实验结果稳定；支持定量吸入和持续上吸两种上样模式，内嵌自动进样器，满足各种实验需求；可提供多达 14 色参数分析。该研究涉及多种样本和panel检测， FongCyte™的独特设计不仅为其提供客观且准确的流式数据，还为研究者提供智能化维护，提高实验效率。
