Blog
Aug 9, 2022 / Oncology

Seeing through tumors: Spatial transcriptomics reveal how tertiary lymphoid structures aid cancer immunotherapy

Jeanene Swanson

When it comes to immunotherapy for cancer, translational researchers still find it perplexing that some people experience positive responses to treatment, while others don’t. What makes the difference, and how can we find out? Enter single cell and spatial transcriptomics, which increasingly are being fine-tuned to probe tumors at the level necessary to see these response mechanisms in detail. 

Recently, scientists used spatial profiling technology from 10x Genomics to characterize FFPE kidney tumor samples, discovering insights into how tertiary lymphoid structures (TLSs) located within tumors may impact the architecture of the tumor microenvironment (TME) and the activation of B cells in response to immune checkpoint inhibitor (ICI) immunotherapy. In this blog, learn more about the first published study using Visium Spatial Gene Expression for FFPE to explore how TLSs play a supporting role in immunotherapy through plasma B-cell production.

Studying kidney tumors with Visium for FFPE

In mid-2021, 10x Genomics built off its existing Visium Spatial Gene Expression (Visium) platform to launch Visium Spatial Gene Expression for FFPE (Visium for FFPE). Visium allows scientists to perform whole transcriptome analysis on frozen tissues, which enables them to discover and reveal the spatial organization of cell types, states, and biomarkers. However, legacy data—especially from biobanked samples—remains inaccessible. Now, with Visium for FFPE, translational researchers can more readily study archived, biobanked samples for biomarker discovery, perform retrospective and longitudinal studies to track biological processes over time, as well as make the most of precious tissue samples by combining transcriptomics with immunofluorescence (IF) for simultaneous visualization of protein and gene expression or with H&E for morphological context.

With this in mind, we’re delighted to feature the first academic publication using the Visium for FFPE platform. In this study, scientists led by Aurélien de Reyniès, PhD, Catherine Sautès-Fridman, PhD, and Wolf Herman Fridman, PhD, of the Fridman lab at the Centre de Recherche des Cordeliers in Paris were able to spatially resolve B-cell phenotypes and localize these within tertiary lymphoid structures of kidney tumors (1). Their findings suggest a role of tertiary lymphoid structures (TLSs) in generating antibody-producing plasma cells and possibly contributing to the mechanism behind positive response to cancer immunotherapies like immune checkpoint inhibitor (ICI) therapy.

The TLS is a hotbed of activity

The TLS is a lymphoid organ that develops at sites of chronic inflammation, like tumors. The germinal center secretes memory B cells and plasma cells (PCs), which in turn produce antibodies (2). Previously published work found that response to ICI immunotherapy and survival were better predicted in multiple cancer types by the presence of B cells, PCs, and a mature TLS, including the type they studied in this paper, renal cell cancer (3, 4). However, the underlying mechanisms at the tissue, cellular, and molecular levels remained unclear.

In order to better understand the mechanism behind this interplay of structures and cells within the TME in response to immunotherapy, the Fridman group performed spatial transcriptomic profiling on 24 human clear cell renal cell carcinoma (ccRCC) samples using frozen (n=12) and FFPE sections (n=12). In TLS+ tumors, they saw that B-cell lineage genes representing all stages of development were preferentially expressed in “hot spots” within the TLS, indicating that the maturation of B cells and PCs occurs in the TLS.

Additionally, they saw the highest number of different B-cell clonotypes in TLS+ tumors. Using Visium to perform spatial B-cell receptor repertoire profiling, they were able to reveal that the diversification, selection, and expansion of B-cell clones occurred in the TLS. This analysis also found fully mature clonotypes at distance, which raised a question: how did they move outside the TLS?

Tracking down distant clonotypes

By spatially mapping gene expression to a location within the TME and/or TLS, they uncovered a 29-gene TLS+ tumor gene signature that was dominated by Ig- and B cell-associated genes. Importantly, expression of a PC-associated gene, MZB1, was high in TLS, low in the tumor area in both frozen and FFPE samples, and found in many spots at a distance from the TLS. Since a plasma cell’s main function is to secrete antibodies, the Fridman group evaluated the spatial expression of immunoglobulin genes. Overall, their data suggested that in TLS+ tumors, PCs that make IgG and IgA antibodies move along fibroblastic “tracks,” being transported and dispersed into the tumor beds.

A role for antibody-producing PCs in tumor cell death

Using multiplex IF labeling of FFPE tumor sections, the team found high numbers of antibody-producing PCs around TLS+ tumors as well as high IgG positivity. To search for potential function of these tumor-bound IgG antibodies, they evaluated tumor cell apoptosis via a cleaved caspase 3 assay. Perhaps unsurprisingly, their results showed this apoptosis marker to be significantly higher in tumors with >60% IgG staining. Essentially, TLS+ tumors exhibited not only high numbers of IgG-producing PCs but also high numbers of IgG-stained and apoptotic cancer cells, suggesting an antitumor function of these antibodies.

In a real world scenario, they evaluated ICI therapeutic response rates as a function of IgG tumoral staining. From an initial collection of 130 primary tumors, the Fridman team were able to evaluate 35 tumors specifically from Nivolumab + Ipilimumab (NI)-treated patients (both types of immunotherapy are immune checkpoint inhibitors). In NI-treated patients, there was a significant association between complete/partial response and IgG tumor cell labeling above the mean, as well as a significantly longer progression-free survival of patients with high IgG tumoral cell staining compared with low IgG tumoral cell staining.

Expanding approaches in ICI therapies

While the mechanisms behind how antibodies bind and carry out tumor cell apoptosis remains to be fully described, overall this work shines a light on where and how plasma B cells could be produced within the TME as well as how B-cell clonotypes become dispersed along fibroblastic tracks, both of which seem to have a positive effect on immunotherapy response. Additionally, Visium for FFPE was able to recover and utilize FFPE tumor samples, allowing them to analyze a broader swath of ICI-treated tissue and conclude some important functional data, one being that IgG antibodies play a role in binding tumor cells and initiating apoptosis. Altogether, this data will hopefully help guide future therapeutic approaches, including possibly spurring TLS activity in tumors or using antibodies from patients who respond positively to immunotherapy as novel therapeutics.

For more information, watch a previously recorded webinar discussing this research or visit our website to get started with Visium Spatial Gene Expression for FFPE.

References:

  1. Meylan M, et al. Tertiary lymphoid structures generate and propagate anti-tumor antibody-producing plasma cells in renal cell cancer. Immunity 55: 527–541.e5 (2022). doi: 10.1016/j.immuni.2022.02.001
  2. Bannard O & Cyster JG. Germinal centers: programmed for affinity maturation and antibody diversification. Curr Opin Immunol 45: 21–30 (2017). doi: 10.1016/j.coi.2016.12.004
  3. Vanhersecke L, et al. Mature tertiary lymphoid structures predict immune checkpoint inhibitor efficacy in solid tumors independently of PD-L1 expression. Nat Cancer 2: 794–802 (2021). doi: 10.1038/s43018-021-00232-6
  4. Fridman WH, et al. B cells and cancer: To B or not to B? J Exp Med 218(1):e20200851 (2021). doi: 10.1084/jem.20200851