Introduction

The study of cancer heterogeneity and its impact on immunotherapy response has been hindered by the lack of appropriate experimental approaches to investigate and model functional heterogeneity in vivo. To bridge this technological gap, we have developed a new platform that allows visualization of the spatial architecture of subclones and their microenvironmental features. 

Methods

By employing barcode lineage tracing combined with next-generation sequencing analysis, we can quantitatively evaluate spatial distribution and temporal clonal changes in vivo. Significantly, our platform enables us to investigate tumor heterogeneity under pharmacological perturbations, such as immune checkpoint blockade (ICB).

Results

Our study revealed that anti-PD1 treatment had limited effect on pancreatic tumor growth, but induced significant changes in tumor clonal architecture and immune landscape. Using our multiplexed staining technology, which employs 30 markers to infer ~40 microenvironmental features, we unveiled domains where subclones with similar ICB response tend to cluster together. Furthermore, we observed high levels of spatial heterogeneity within the tumor microenvironment.

When we coupled spatial barcode sequencing with multiplexed staining, we were able to reveal the differential microenvironment profiles of ICB-sensitive and ICB-resistant clones. To validate the microenvironmental features, we isolated treatment-naïve clones and analyzed monoclonal tumors. Surprisingly, we found that the tumors formed by resistant clones had lower CD8+ T cell infiltration, which recapitulates the phenotype in tumors formed by the parental cell mixture. Moreover, these resistant clones expressed a different secretome signature, which could be the reason why they are less immunogenic.

Conclusion

Our findings have demonstrated that subclones within tumors exhibit functional and spatial heterogeneity. Moving forward, we plan to investigate the interactions between these subclones by characterizing isolated, treatment-naïve clones and performing in vivo perturbations. This will provide a deeper understanding of the mechanisms underlying how cancer cells shape their tumor microenvironment and evade immune surveillance. Ultimately, our goal is to identify novel vulnerabilities in the tumor microenvironment and develop effective strategies to overcome resistance to immunotherapy.