Integrated functional and spatial profiling of tumour immune responses induced by immunotherapy: the iPROFILER platform

Abstract

Background: Cancer immunotherapy elicits functional activation and changes in immune cell distribution in cancer. Tumour heterogeneity is a reason for treatment failure but is difficult to capture in experimental settings. This proof-of-principle study describes the integrated functional and digital spatial profiling platform iPROFILER to capture in-situ immune activation patterns with high precision. Materials and methods: iPROFILER combines an algorithm-based image analysis approach for spatial profiling with functional analyses of patient-derived tumour fragments (PDTFs). This study utilized a folate receptor 1 (FOLR1)xCD3 bispecific antibody in dual-affinity re-targeting (DART) format as a tool for inducing T-cell responses in patient tumour samples, and an in-depth investigation of the immune perturbations induced in the tumour microenvironment was performed. Results: Ex-vivo DART stimulation induces upregulation of multiple activation markers in CD4þ and CD8þ T-cell populations and secretion of pro-inflammatory cytokines in FOLR1-positive tumour specimens. This response was reduced or absent in tissue samples that did not express FOLR1. Immunological responses were driven by a strong induction of interferon gamma (IFNg) and IFNg-induced chemokines suggestive of activation of cytotoxic or Th1-like T cells. Ex-vivo DART treatment led to a numerical increase in effector T cells and an upregulation of immune activation markers in the tumour microenvironment as captured by digital image analysis. Analysis of immune activation in tumour and stromal regions further supported the potential of the platform to measure local differences in cell-type-specific activation patterns. Conclusions: iPROFILER effectively combines functional and spatial readouts to investigate immune responses ex vivo in human tumour samples.

Deletion of SNX9 alleviates CD8 T cell exhaustion for effective cellular cancer immunotherapy

“…Interestingly, one attempt to impose an exhaustion state in human T cells utilized repetitive stimulation ex vivo with peptide-loaded human dendritic cells, which resulted in a reduction in cytokine secretion, but not degranulation or killing capacity 26 . Recently, ex vivo models have been developed using human tumor explants, which preserve many important features of the tumor microenvironment and mimic patient response 27,28 . These patient-derived cultures, however, are short-lived with a scarce infiltration of tumor-specific T cells, which hampers extensive screening approaches.…”

Protocol for ex vivo culture of patient-derived tumor fragments

“…For examples, please refer to. 1 , 8 Before starting, prepare the following materials ( Figure 3 A): One 96-well round bottom plate per tumor. Nylon mesh filters, 150 μm.…”

“…As an example, for assessing the efficacy of a bispecific T cell engager, three PDTFs per condition were sufficient as this compound activates all intratumoral T cells independent of their specificity, and not only a fraction of tumor-specific T cells like anti-PD-1. 8 Regardless, heterogeneity across conditions and between experiments can still occur and controls should be taken along (see above).…”