Ramasubramanian T S, Adstamongkonkul Pichet, Scribano Christina M, Johnson Christin, Caenepeel Sean, Hrycyniak Laura C F, Vedder Lindsey, Dana Nicholas, Baltes Christian, Browning Torey, Chen Yuan-I, Dietz Thomas, Flietner Evan, Kaplewski Nicholas, Kellner Anna, Korrer Michael, Liu Chao, Marhefke Nathan, McDonnell Payton, Nasreen Amreen, Pope Victoria, Prasad Abhijeet, Richardson Jordyn, Schneider Sidney, Schultz Mikaela, Sood Chetan, Sunil Aishwarya, von Euw Erika, Wait Eric, Wargowski Ellen, Advani Pooja, Broome Barbara, Bruckbauer Antje, Godwin Andrew, Kokabi Nima, Martin Robert, Robaina Mercedes, Toia Giuseppe, Routh Joshua, Friedl Andreas, Eliceiri Kevin, Szulczewski Mike, Johnson Scott, Oliner Jon, Galon Jérôme, Capitini Christian, Mukhopadhyay Debabrata, Taube Janis, Braun David, Gierman Hinco J
Elephas Biosciences, Madison, WI.
Mayo Clinic, Rochester, MN.
bioRxiv. 2025 Jul 18:2025.07.18.663728. doi: 10.1101/2025.07.18.663728.
Immune checkpoint inhibitors (ICIs) have revolutionized cancer treatment, providing durable and even curative responses. However, most patients do not respond and current biomarkers (eg, programmed death ligand (PD-L1), mismatch repair deficiency (dMMR)/high microsatellite instability (MSI) and tumor mutational burden) lack predictive accuracy. Ex vivo profiling of patient-derived tumor fragments shows promise as a predictive biomarker but relies on substantial surgical tissue to mitigate intra-specimen heterogeneity. Innovations are needed that address these challenges, particularly where limited tissue is available in core needle biopsies (CNBs).
Live tumor fragments (LTFs) were generated from 59 human tumor resections and 31 CNBs from patients enrolled in observational clinical trials (ClinicalTrials.gov identifiers: NCT05478538, NCT05520099, NCT06349642) to assess cytokine induction following ICI treatment. LTFs were encapsulated in hydrogel and cultured ex vivo for up to 72 hours. A sequential treatment strategy that applies control and treatment within the same well was used with response to ICI or αCD3/αCD28 assessed using a multiplex secretome assay. Viability was assessed using established metabolic assays and dynamic optical coherence microscopy.
LTFs maintained viability and retained T cells responsive to stimulation throughout ex vivo culture. Multiplex immunofluorescence and immunohistochemistry showed key components of the tumor microenvironment, including relative proportions of CD4+ and CD8+ immune cell populations, were preserved. Specimens positive for PD-L1 or dMMR/MSI-high were enriched for cytokine upregulation, including T-cell response cytokines IFNγ and CXCL10, after αPD-1 treatment. To demonstrate clinical applicability of the sequential treatment strategy, CNBs from patients with lung, gastrointestinal or kidney cancer were profiled and differential cytokine induction in response to ICI treatment was observed.
The novel ex vivo platform presented is capable of detecting T-cell response to ICI treatment by using a sequential treatment strategy. This approach addresses challenges associated with cross-well heterogeneity in tissue composition and requires half as much tissue as a cross-well comparison, mitigating tissue limitations typically associated with non-surgical biopsies. Importantly, the platform is compatible with established functional assays as well as non-destructive spatial imaging, enabling researchers to characterize response to ICI longitudinally. Ongoing trials will enable clinicians to assess platform performance in predicting response to immunotherapy.
免疫检查点抑制剂（ICIs）彻底改变了癌症治疗方式，带来了持久甚至治愈性的反应。然而，大多数患者并无反应，且目前的生物标志物（如程序性死亡配体（PD-L1）、错配修复缺陷（dMMR）/高微卫星不稳定性（MSI）和肿瘤突变负荷）缺乏预测准确性。对患者来源的肿瘤片段进行体外分析显示有望成为一种预测性生物标志物，但依赖大量手术组织来减轻样本内的异质性。需要创新来应对这些挑战，特别是在核心针吸活检（CNB）中可用组织有限的情况下。
从59例人类肿瘤切除术和31例参与观察性临床试验患者的CNB中获取活肿瘤片段（LTFs）（ClinicalTrials.gov标识符：NCT05478538、NCT05520099、NCT06349642），以评估ICI治疗后的细胞因子诱导情况。LTFs被封装在水凝胶中并在体外培养长达72小时。采用在同一孔内应用对照和治疗的序贯治疗策略，并使用多重分泌组分析评估对ICI或αCD3/αCD28的反应。使用既定的代谢分析和动态光学相干显微镜评估活力。
在整个体外培养过程中，LTFs保持活力并保留了对刺激有反应的T细胞。多重免疫荧光和免疫组织化学显示肿瘤微环境的关键成分，包括CD4+和CD8+免疫细胞群体的相对比例得以保留。在αPD-1治疗后，PD-L1或dMMR/MSI高阳性的标本中细胞因子上调更为丰富，包括T细胞反应细胞因子IFNγ和CXCL1(0)。为证明序贯治疗策略的临床适用性，对肺癌、胃肠道癌或肾癌患者的CNB进行了分析，并观察到对ICI治疗的不同细胞因子诱导情况。
所展示的新型体外平台能够通过序贯治疗策略检测T细胞对ICI治疗的反应。这种方法解决了与组织组成中跨孔异质性相关的挑战，并且所需组织量仅为跨孔比较的一半，减轻了通常与非手术活检相关的组织限制(1)。重要的是，该平台与既定的功能分析以及非破坏性空间成像兼容，使研究人员能够纵向表征对ICI的反应。正在进行的试验将使临床医生能够评估该平台在预测免疫治疗反应方面的性能。
