Introduction

Radioimmunotherapy (RIT) is a major anti-cancer therapy in cancer management multimodalities. ¹⁷⁷Lu-Nimotuzumab has been in the use for radioimmunotherapy of EGFR expressing tumors. This study focuses on understanding the differential cellular and molecular mechanisms of anti-tumor effects of ¹⁷⁷Lu-Nimotuzumab on low EGFR expressing A549 and high EGFR expressing A431 tumor cells.

Materials and methods

Nimotuzumab labeled with ¹⁷⁷Lu was characterized by SE-HPLC. Specificity of ¹⁷⁷Lu-Nimotuzumab to EGFR expressed on A549 and A431 cells was confirmed by competitive assay using increasing amounts of unlabeled Nimotuzumab. Cellular responses of A549 (low EGFR) and A431 (high EGFR) in response to different doses of ¹⁷⁷Lu-Nimotuzumab were determined by Viable count assay for cellular viability, cell-cycle analysis by DNA staining, apoptotic assay for cell death, and CFSE dilution assay for cellular proliferation capacity. The number of DNA DSBs formed was determined using γ-H2AX assay with PI staining. Transcription of genes involved in DNA damage response and repair (DRR) pathways was monitored by RT-qPCR.

Results

¹⁷⁷Lu-Nimotuzumab characterized by SE-HPLC exhibited a radiochemical purity of 99.1 ± 0.6%. Cell binding competition studies with ¹⁷⁷Lu-Nimotuzumab showed specific binding of 34.3 ± 1.7% with A431 cells and 18.4 ± 1.9% with A549 cells which decreased when co-incubated with unlabeled Nimotuzumab. Cytotoxicity and DNA damage (DNA DSBs) increased with an increase in the dose of ¹⁷⁷Lu-Nimotuzumab. A549 displayed G2/M arrest while A431 showed G1 arrest. Apoptotic death was determined to be one of the modes of death of arrested A549 and A431 cells which increases with the increase in the dose of ¹⁷⁷Lu-Nimotuzumab. Loss of proliferation capacity was higher in A431 showed by CFSE staining at different doses of ¹⁷⁷Lu-Nimotuzumab. Transcription profile of most DRR genes in A431 and A549 showed a decrease in the transcription at 4 h followed by recovery at 16 h post-treatment. The degree of transcription of most DRR genes was similar, irrespective of ¹⁷⁷Lu-Nimotuzumab dose.

Conclusion

¹⁷⁷Lu-Nimotuzumab induces different cellular arrest and molecular responses in low EGFR expressing A549 and high EGFR expressing A431 tumor cells. This study would enable the development of integrative novel treatment strategies for radioimmunotherapy in low and high EGFR expressing tumors by ¹⁷⁷Lu-Nimotuzumab with therapeutic gains.

中文翻译：

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在使用不同剂量的177Lu-Nimotuzumab处理的低EGFR表达A549和高EGFR表达A431肿瘤细胞中诱导不同的细胞停滞和分子应答。

介绍

放射免疫疗法（RIT）是多种癌症治疗方法中的一种主要的抗癌疗法。¹⁷⁷ Lu-Nimotuzumab已用于表达EGFR的肿瘤的放射免疫治疗。这项研究的重点是了解¹⁷⁷ Lu-Nimotuzumab对低EGFR表达A549和高EGFR表达A431肿瘤细胞的抗肿瘤作用的不同细胞和分子机制。

材料和方法

用SE-HPLC表征用¹⁷⁷ Lu标记的尼莫妥单抗。特异性¹⁷⁷路，尼妥珠单抗对EGFR表达对A549和A431细胞，通过竞争性分析中使用未标记尼妥珠单抗的越来越多的证实。通过活计数测定细胞活力，DNA染色进行细胞周期分析，细胞死亡的凋亡分析和CFSE，确定了对不同剂量的¹⁷⁷ Lu-Nimotuzumab的应答，A549（低EGFR）和A431（高EGFR）的细胞应答稀释法检测细胞增殖能力。使用PI染色的γ-H2AX分析确定形成的DNA DSB的数量。通过RT-qPCR监测与DNA损伤反应和修复（DRR）途径有关的基因的转录。

结果

用SE-HPLC表征的¹⁷⁷ Lu-Nimotuzumab的放射化学纯度为99.1±0.6％。用^177种Lu-Nimotuzumab进行的细胞结合竞争研究表明，与A431细胞的特异性结合为34.3±1.7％，与A549细胞的特异性结合为18.4±1.9％，当与未标记的Nimotuzumab共同孵育时会降低。细胞毒性和DNA损伤（DNA DSB）随着¹⁷⁷ Lu-Nimotuzumab剂量的增加而增加。A549显示G2 / M逮捕，而A431显示G1逮捕。凋亡的死亡被确定为被逮捕的A549和A431细胞死亡的方式之一，随着¹⁷⁷ Lu-Nimotuzumab剂量的增加而增加。CFSE染色显示，在不同剂量的^177下，A431的增殖能力丧失更高尼妥珠单抗。A431和A549中大多数DRR基因的转录谱显示在4 h转录降低，然后在治疗后16 h恢复。不管¹⁷⁷ Lu-Nimotuzumab的剂量如何，大多数DRR基因的转录程度都是相似的。

结论

¹⁷⁷ Lu-Nimotuzumab在低EGFR表达的A549和高EGFR表达的A431肿瘤细胞中诱导不同的细胞停滞和分子应答。这项研究将使¹⁷⁷ Lu-Nimotuzumab在治疗低和高EGFR表达的肿瘤中进行放射免疫疗法的综合新治疗策略的开发，具有治疗意义。