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Predicting which NSCLC patients will benefit from immunotherapy requires more than measuring a single biomarker. Our previous work has helped show that key spatial features of the tumor microenvironment—such as mature tertiary lymphoid structures (Nature Cancer, 2021; Cell Reports Medicine, 2025) and macrophage infiltration within the tumor compartment (Journal for ImmunoTherapy of Cancer, 2022)—are closely associated with treatment outcome.

Building on these findings, we developed and validated a multiplex immunofluorescence (mIF) panel that integrates immune contexture, TLS presence and maturity, and macrophage infiltration in a single assay. First presented at the 2025 AACR Annual Meeting, this panel is designed to support translational research and early-phase trials in NSCLC.

 

Why immune contexture matters

 

Immune checkpoint inhibitors have transformed cancer treatment, but responses remain highly variable. One reason is that the anti-tumor immune response depends not only on whether immune cells are present, but also on where they are located. This is the principle behind immune contexture.

Tumors can be broadly grouped into three phenotypes based on CD8 spatial distribution:

• Infiltrated tumors contain CD8+ T cells within the tumor area.


• Excluded tumors show CD8+ T cells at the periphery, without effective penetration into the tumor parenchyma.


• Desert tumors display little to no CD8+ T-cell presence.

This spatial organization is informative because it reflects distinct mechanisms of anti-tumor immunity, immune suppression, or immune escape. Immune contexture does more than label tumors as “hot” or “cold”: it helps explain whether T cells are present, whether they can reach cancer cells, and whether the local microenvironment is permissive or suppressive.

Using a CD8/PanCK multiplex IHC panel, our AACR poster showed that infiltrated, excluded, and desert phenotypes are associated with different outcomes under immune checkpoint blockade in NSCLC. In both the discovery and validation cohorts, infiltrated tumors were associated with better responses, while immune exclusion was linked to resistance. These results reinforce the value of immune contexture as a spatial biomarker for patient selection.

 

Why combine it with TLS and M2 macrophages

 

Immune contexture is highly informative, but it does not capture the full complexity of the tumor microenvironment. To obtain a broader and more actionable spatial readout, we combined it with two additional components that are strongly linked to immunotherapy response in NSCLC: tertiary lymphoid structures (TLS) and M2-like tumor-associated macrophages.

Tertiary lymphoid structures (TLS) reflect the capacity of the tumor microenvironment to support local immune activation. By enabling antigen presentation, lymphocyte priming, and coordinated immune responses, they can favor effective anti-tumor immunity and improve sensitivity to immune checkpoint inhibitors. Their presence, proximity to the tumor, and especially their maturity, therefore provides important information on the quality of the local immune response. This is consistent with the Nature Cancer publication we co-authored in 2021, which identified mature TLS as a predictive feature of immunotherapy efficacy.

M2-like tumor-associated macrophages are major drivers of immunosuppressive signaling within the tumor microenvironment. By promoting tissue remodeling, dampening cytotoxic immune responses, and contributing to barriers against effective T-cell activity, they can support resistance to immunotherapy. Their spatial distribution within the tumor compartment therefore helps capture a distinct mechanism of immune escape that complements immune contexture analysis. This is supported by our 2022 Journal for ImmunoTherapy of Cancer publication on macrophage infiltration and immunotherapy response in NSCLC.

By integrating these three biomarkers into a single panel, we can generate a more complete spatial readout of the tumor immune landscape than with any marker alone.

 



 

A panel designed for spatial biology at scale

 

One of the strengths of this approach is that it goes beyond staining alone. The panel is paired with an automated tissue analysis workflow that includes tissue segmentation, cell segmentation, signal normalization, marker positivity scoring, machine learning-based classification, and downstream spatial analysis. This enables complex spatial information to be translated into standardized outputs with pathologist-guided interpretability and high reproducibility.

 



 

Our AACR poster also showed concordance between this broader mIF assay and immune-contexture classification derived from the CD8/PanCK multiplex IHC approach in an NSCLC immunotherapy-treated cohort (N=60). Together, these results support the robustness of the panel for translational use and its compatibility with scalable tissue analysis workflows.

 

Download our 2025 AACR poster

 

 

Applications for translational research and early-phase trials, beyond NSCLC

 

Implemented within Explicyte’s ISO 13485- and ISO 9001-certified quality framework, this integrated panel is particularly relevant for translational research and early-phase trials in precision oncology. It can support biomarker discovery, patient stratification, responder versus non-responder analyses, mechanism-of-action studies, and exploratory enrichment strategies in immunotherapy trials.

Although developed in NSCLC, the same approach may be valuable in other indications where spatial immune organization is likely to influence response, including head and neck cancer, urothelial carcinoma, melanoma, renal cell carcinoma, and selected breast or gastrointestinal tumors. Sarcoma is also a relevant setting, as illustrated by the PEMBROSARC TLS-positive soft-tissue sarcoma study published in Nature Medicine in collaboration with Institut Bergonié and Gustave Roussy.

Interested in bringing spatial immune profiling into your precision oncology program? Contact our team to discuss how this panel could support your next study.

 

https://youtu.be/bmokMO6jCHc?si=6tz9CLxEy1R_0uHo

 

Undifferentiated pleomorphic sarcoma (UPS) is one of the most common—and most aggressive—subtypes of soft tissue sarcoma (STS). Despite optimal treatment in localized stages, nearly half of patients ultimately develop metastatic disease, which drives poor outcomes.

To better understand the mechanisms underlying UPS metastasis, a team led by Prof. Antoine Italiano (Institut Bergonié, University of Bordeaux, INSERM U1312 BRIC) performed an integrated multi-omics analysis of paired primary and metastatic UPS samples, followed by functional validation in relevant patient-derived models.

Spatial biology (Explicyte)

As part of this work, Explicyte performed spatial transcriptomic profiling using the GeoMx Digital Spatial Profiler (Whole Transcriptome Atlas; >18K genes). The spatial analyses were conducted on 3 paired primary tumors and matched metastases enabling a direct comparison of tumor and microenvironmental programs across disease stages.

Key observations included:

• Upregulation of hypoxia, glycolysis, and EMT-related programs in metastases, consistent with a more aggressive, metastasis-associated biology.


• Enrichment of endothelial-cell signatures in metastatic samples, suggesting increased angiogenesis compared with primary tumors.


• Higher immune infiltration in primary tumors (including CD8+ T cells, NK cells, and memory B cells), supporting the notion of a more immune-suppressive / immune-excluded state in metastatic lesions.

To further support these findings, we confirmed a reduction in CD8+ T-cell infiltration in metastases using a multiplex immunohistofluorescence (mIHF) panel in five paired primary/metastatic cases.

Bulk RNAseq

In parallel, the team analyzed paired primary and metastatic tumor samples from 13 patients using bulk RNA-seq to capture global transcriptomic changes associated with metastatic progression. This analysis confirmed an enrichment of metastasis-associated pathways and identified 690 genes significantly altered between primary tumors and metastases. Among the top candidates, ADORA2B emerged as particularly compelling because:

• ADORA2B encodes a G protein–coupled adenosine receptor (A2B) implicated in metastatic progression in several epithelial cancers,


• ADORA2B is drug­gable, with inhibitors already being evaluated in clinical development programs.

Using public and third-party sarcoma datasets, the team further showed that ADORA2B is overexpressed in UPS and is associated with features of an immune-suppressive microenvironment in metastatic UPS—supporting ADORA2B as a promising therapeutic target in this indication.

Functional validation

To test whether ADORA2B plays a causal role in UPS aggressiveness, the authors generated ADORA2B knockout models using CRISPR-Cas9 in two patient-derived UPS cell lines.

• Transcriptomic profiling of ADORA2B-knockout cells confirmed downregulation of key pathways involved in metastatic biology.


• Functional assays demonstrated that ADORA2B loss reduces UPS cell proliferation, migration, and invasion, supporting a direct role in tumor aggressiveness.

The in vivo impact was then evaluated in Rag2-/- γc-/- mice:

• In an orthotopic model, tumors derived from ADORA2B-knockout cells showed markedly impaired growth, leading to significantly smaller, low-proliferating tumors compared with controls.


• In a forced metastasis model (tail-vein injection), ADORA2B-knockout cells produced substantially fewer metastases, translating into a striking survival benefit (100% survival in the knockout arm vs 0% in controls in this experimental setting).

Finally, the team evaluated a dual ADORA2A/ADORA2B inhibitor, M1069 (EMD Serono), currently assessed in early-phase trials. In vitro, M1069 reduced proliferation and invasion in UPS models, providing pharmacological support for the genetic findings.

Impact

Overall, this work identifies ADORA2B as a critical regulator of primary tumor growth and metastatic dissemination in UPS. It also highlights the therapeutic promise of targeting the adenosine axis, and specifically ADORA2B, as a strategy to disrupt metastatic progression and improve outcomes in this rare and aggressive cancer.

Read the paper

This research received funding from the Agence Nationale de la Recherche  (“France 2030” / ANR 21 RHUS 0010).

We're glad to announce the publication of an immunology paper led by our colleagues at ImmunoConcEpT (CNRS/Université de Bordeaux) in Science Advances: "Immune control of functional memory CD8 T cells in normal-appearing vitiligo skin."

Using single-cell and spatial transcriptomics profiling of vitiligo patient biopsies, the team demonstrates that similar CD8+ T-cell clusters infiltrate both lesional and non-lesional skin. The difference lies in the “regulatory layer”: normal-appearing skin is characterized by an enrichment in immune regulatory pathways (increased Treg infiltration and higher PD-1 expression on CD8+ T cells), consistent with tighter regulation of inflammation.

Building on the Phase 2 BARVIT trial (NCT04822584), which showed a clinical benefit of baricitinib combined with phototherapy for repigmentation, ImmunoConcEpT partnered with Explicyte to perform a paired pre/post immune profiling of skin biopsies using our automated multiplex IF workflow.

Our multiplex IF data support the proposed mechanism; after 9 months of treatment, non-lesional skin exhibits:

• reduced CD8+ T-cell infiltration
• increased PD-1 on CD8+ T cells and PD-L1 on dendritic cells
• an increased FOXP3+ / CD8+ T-cell ratio

Together, these results highlight the critical role of immune regulatory mechanisms to prevent inflammation & depigmentation in vitiligo.

> Read the article