Background The tumor microenvironment (TME) is composed of highly heterogeneous cell types that dynamically interact with each other. The constant interaction between a tumor and its microenvironment plays a critical role in how the cancer develops, progresses, and responds to therapies. Traditional tissue-based studies of TME can be limited to a small number of target analytes, which can limit biological insights. In this work, we used the Visium CytAssist instrument and Visium CytAssist Spatial Gene and Protein Expression assay from 10x Genomics, which enables whole transcriptome gene expression and multiplexed protein expression profiling from the same formalin fixed & paraffin embedded (FFPE) tissue. Our data provides a more comprehensive understanding of cellular behavior in and around tumors, yielding new insights into disease progression and therapeutic response.

Methods Tumor FFPE tissues (breast, prostate, lung, and ovarian cancer) were spatially profiled using the Visium CytAssist instrument and Visium CytAssist Spatial Gene and Protein Expression assay. Tissues were mounted on glass slides, then Hematoxylin & Eosin (H&E) or immunofluorescence (IF) stained. Following decrosslinking, the samples were incubated with RTL and antibody-conjugated probes, then prepared for probe transfer to spatially barcoded Visium slides with 6.5 x 6.5 or 11 x 11 mm capture areas. The captured probes were collected from the slides and used to generate sequencing-ready libraries for transcriptomic and proteomic analysis with spatial context.

Results Using the CytAssist workflow, we showcase the ability to spatially resolve oncogenes and immune cells associated with multiple tumor tissues. In the prostate cancer samples, strong correlation was observed between gene and protein expression for immune cell subsets such as activated T, B, and regulatory dendritic cells. CD274 demonstrates the ability to resolve protein expression data even when gene signal is low. Breast and lung cancer samples were screened with fluorescently labeled antibodies in addition to transcriptomic profiling. In particular, PCNA and Vimentin showed the precision and accuracy of probe transfer using the Visium CytAssist Spatial Gene and Protein Expression assay. Expression of these markers map back to distinct morphological features within the samples, allowing identification of differentially expressed genes and proteins associated with those areas.

Conclusions These data highlight that Visium CytAssist can retrieve transcriptome and protein information from FFPE sections in a spatial context. Implementing Visium CytAssist Spatial Gene and Protein Expression Assay in immuno-oncology studies provides a more comprehensive understanding of clinical tissue samples and provides novel insights into architectural and cellular heterogeneity across multiple cancers.