恶性肿瘤的免疫治疗取得了很大进展，但许多患者并未从中受益。复杂的肿瘤内异质性（ITH）阻碍了对免疫治疗的深入探索。传统的组测序技术掩盖了肿瘤内的复杂性，防止更详细地发现ITH对治疗效果的影响。因此，我们发起了这项研究，以多组学空间水平探索ITH，并考虑到其存在寻找免疫治疗效果的预测生物标志物。利用数字空间分析（DSP）的分割策略，我们获得了肿瘤和基质区域在蛋白质组和转录组水平上的差异性信息。考虑到ITH，以不同区域的候选蛋白质组成的标记来预测免疫治疗的效果。本研究纳入了18名接受双特异性抗体（bsAb）KN046治疗的患者。同一样本的肿瘤和基质区域具有不同的特征。由11个和18个不同表达的DSP标记组成的标记，分别与治疗反应相关。此外，从基质区域确定的空间解析标记显示出更大的bsAb免疫治疗反应预测能力（曲线下面积=0.838）。随后，在接受免疫治疗的非小细胞肺癌患者的独立队列中验证了我们的基质标记。我们解读了空间水平上的ITH，首次证明基质区域的遗传信息可以更好地预测bsAb治疗的疗效。NCT03838848.©作者（或其雇主（们））2023年。在CC BY-NC下允许重新使用。不得商业再利用。由BMJ出版。

Immunotherapy for malignant tumors has made great progress, but many patients do not benefit from it. The complex intratumoral heterogeneity (ITH) hindered the in-depth exploration of immunotherapy. Conventional bulk sequencing has masked intratumor complexity, preventing a more detailed discovery of the impact of ITH on treatment efficacy. Hence, we initiated this study to explore ITH at the multi-omics spatial level and to seek prognostic biomarkers of immunotherapy efficacy considering the presence of ITH.Using the segmentation strategy of digital spatial profiling (DSP), we obtained differential information on tumor and stromal regions at the proteomic and transcriptomic levels. Based on the consideration of ITH, signatures constructed by candidate proteins in different regions were used to predict the efficacy of immunotherapy.Eighteen patients treated with a bispecific antibody (bsAb)-KN046 were enrolled in this study. The tumor and stromal areas of the same samples exhibited distinct features. Signatures consisting of 11 and 18 differentially expressed DSP markers from the tumor and stromal areas, respectively, were associated with treatment response. Furthermore, the spatially resolved signature identified from the stromal areas showed greater predictive power for bsAb immunotherapy response (area under the curve=0.838). Subsequently, our stromal signature was validated in an independent cohort of patients with non-small cell lung cancer undergoing immunotherapy.We deciphered ITH at the spatial level and demonstrated for the first time that genetic information in the stromal region can better predict the efficacy of bsAb treatment.NCT03838848.© Author(s) (or their employer(s)) 2023. Re-use permitted under CC BY-NC. No commercial re-use. See rights and permissions. Published by BMJ.