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Spatial transcriptomics reveal the determinants of resistance to immunotherapy in NSCLC patients with mature TLS

By Pierre-Emmanuel Gaultier5 February 20255 February 2025

New paper in Cell Reports Medicine! Spatial transcriptomics reveal the determinants of resistance to immunotherapy in NSCLC patients with mature TLS

This translational research paper highlights the work of Florent Peyraud, MD, conducted during his PhD thesis at Explicyte under the supervision of Prof. Antoine Italiano (Bergonié & Gustave Roussy Comprehensive Cancer Centers) and Alban Bessede, PhD (Explicyte). This project was made possible thanks to a collaboration with the imCORE network (Roche/Genentech), and samples from the BIP trial (NCT02534649). The study aims to uncover why certain patients with non-small cell lung cancer (NSCLC) who exhibit mature tertiary lymphoid structures (mTLS)—a phenotype typically associated with a favorable response to immune checkpoint inhibitors (ICI)—do not respond to anti-PD1/PD-L1 immunotherapy.

Presence of mature TLS & clinical outcome in NSCLC
A comprehensive pathological analysis of 509 NSCLC patients treated with immune checkpoint inhibitors (ICI) was performed, confirming that the presence of mTLS predicts a better outcome in terms of response rate, overall survival, and median progression-free survival, independently of PD-L1 and genomic features.

Spatial transcriptomics in responding & non-responding mTLS-positive NSCLC patients
Specimens from 6 mTLS-positive patients with extreme ICI response (3 responders, 3 non-responders) were analyzed using the GeoMx Whole Transcriptome Atlas. While gene expression profiles did not differ within the TLS and tumor compartments, non-responders exhibited an enrichment in fibroblasts in the stromal compartment, with a pronounced expression of TGFβ signaling and epithelial-mesenchymal transition (EMT) pathways.

Multiplex immunohistofluorescence (mIHF) panel focusing on cancer-associated fibroblasts (CAFs) 77 m-TLS positive patient samples were then analyzed by digital pathology for the detection of tumor cells, cytotoxic CD8⁺ T cells, and FAP⁺αSMA⁺ CAF and MYH11⁺αSMA⁺ CAFs. Analysis revealed a higher stromal density of both CAF subsets in non-responders, correlating with poor clinical outcome.

Bulk transcriptomics to analyze interactions between the immune infiltrates & CAF status
Gene expression profiling of 40 mTLS-positive lung tumor samples identified that:

FAP⁺αSMA⁺ CAF-High tumors were associated with inflammatory response and T-cell exhaustion, which may explain the limited efficacy of ICI.

MYH11⁺αSMA⁺ CAF-High tumors were associated with an enrichment in regulatory T cell (Treg) gene signatures, which are known to participate in immunosuppression and be detrimental to ICI efficacy

Multiplex immunofluorescence panel focused on T cell exhaustion
Specimens of 64 mTLS positive NSCLC patients were then analyzed by digital pathology:

A panel consisting of CD8, PD1, CD39, LAG3, TIGIT, TIM3, and DAPI revealed a high density of intratumoral CD8⁺PD1⁺ T cells in the FAP⁺αSMA⁺ CAF-High tumors, with a higher expression of exhaustion markers.

A panel including CD4, CD8, CD20, FoxP3, ICOS, TIGIT and DAPI highlighted an increased presence of CD4⁺ T cells in the stroma of MYH11⁺αSMA⁺ CAF-High tumors, with an increased infiltration of regulatory CD4⁺FoxP3⁺ T cells expressing immunosuppressive markers.

Altogether, these findings suggest that the presence of FAP⁺αSMA⁺ CAFs and MYH11⁺αSMA⁺ CAFs is associated with poor outcomes in mTLS-positive NSCLC patients undergoing ICI treatment. They could serve as biomarkers to stratify patients, and could constitute targets, in combination with strategies addressing T-cell exhaustion, to enhance ICI efficacy in mTLS-positive NSCLC patients.

Interested in the assessment of TLS maturity? Learn about our histopathology services for TLS detection & scoring.

Read paper

By Pierre-Emmanuel Gaultier31 January 202531 January 2025

Open position: Biotech Engineer specialized in 3D cultures of tumor models (SM0225)

Reference: SM0225
Contract type: Full time position, permanent contract
Location: Bordeaux, France

You are passionate about the interplay between immune and tumor cells? You have experience with 3D tumor-based culture models in the perspective of testing innovative cancer immunotherapies? You are proficient in flow cytometry and multiparametric platforms for in vitro/ex-vivo analyses? Keep reading and apply!

The position

We are looking for an experienced in vitro Immuno-Oncology Scientist, ready to take up with us the setup and operation of 3D tumor model cultures, relevant for the testing and development of novel immunotherapeutic strategies.

In your role, you will be the scientific-technical referent in 3D cell culture, on all of R&D projects and Sponsors’ studies. You will interact with the in vitro team members.

You will have versatile & empowering missions. You will report to the Chief Scientific Officer.

What you will find at Explicyte

Your skills & traits

Post-graduate degree (MSc/PhD) in oncology, immuno-oncology, or related fields

Expertise in 3D cell culture models and protocols (i.e. organoids, fragments, spheroids), with a minimum 3 years of experience

Expertise in multiparametric analysis methodologies, e.g. flow cytometry, kinetic high content imaging, multiparametric supernatant analyses (LEGENDplex, Olink, etc), immunohistofluoresence

Expertise in 2D cell culture protocols (immune/tumor cell co-cultures, immune cell isolation and cultivation, differentiation and polarization protocols)

Proficiency in project management: from implementation and performance to data analysis and on time rendering of deliverables. Ability to manage multiple projects simultaneously

Strong interpersonal & organizational skills

Ability to easily adapt to changing priorities and challenges

Fluency in written and oral English

Your missions & responsibilities

Define, implement, and adapt adequate protocols according to the scientific purpose

Provide draft protocols according to the scientific and experimental strategies

Implement studies - relying on undeniable organizational skills - according to their design and established schedules

Be responsible for handling and processing fresh tumor samples upon their receipt for their usability in 3D cultures

Manage and perform - diligently - in vitro and ex-vivo experiments
- Cell culture-based experiments for functional immuno-oncology assays
- 3D tumor model cultures: organoids, tumor fragments, and spheroids, etc
- Ex vivo processing of collected samples for immune or tumor cell isolation, or for further analyses (FACS, ELISA…)

Capture, analyze and compile data, including quality controls

Ensure accuracy and completeness of study documentation and provide study deliverables on time

Write internal protocols and standard operating procedures

Contribute to experimental development by implementing new methods and training other collaborators and team’s members

About us

Explicyte is a preclinical and translational contract research organization specialized in immuno-oncology. Our goal is to assist academics, biotechs, and pharmas in the discovery of novel targets and novel cancer immunotherapies.

With a team of 25, we’re a human-sized company, which brings under the same roof cell biologists, immunologists, in vivo scientists, medical oncologists, and bioinformaticians. Focused on sponsors’ projects, our activity also involves in-house R&D and external collaborations, which led to the publication of 30+ papers in high-impact-factor journals.

Based in Bordeaux, our lab is located at the Bergonié Comprehensive Cancer Center, where we work in close contact with medical oncology teams. We believe Explicyte is a place to learn, grow, and have impact in the fight against cancer.

More about us

Apply now!

By Pierre-Emmanuel Gaultier30 January 202531 January 2025

Targeting Antigen-Presenting Cells: A DC-mediated killing assay to assess cancer vaccines, oncolytic viruses and immune adjuvant-based therapies

Over the past 5 years, Explicyte has supported several cancer immunotherapy programs aimed at inducing or enhancing a specific anti-tumor immune response. Here's a quick overview of our capacities for the advancement of novel cancer vaccines (peptide-based, DC-based, DNA- or RNA-based), cytokine therapies, oncolytic virus-based therapies, immune modulators (such as STING agonists), and immune adjuvants.

**Specific Tumor Antigen Presentation: In vitro Efficacy & MoA Studies with a dedicated DC-mediated T-cell Killing Assay**

We designed a cell-based system to assess the effectiveness of compounds aimed at enhancing the proficiency of antigen-presenting cells (APC), such as dendritic cells (DC), to capture and cross-present the tumor antigens, and the subsequent priming of CD8+ cytotoxic T cells against cancer cells. The assay provides dynamic flexibility, thus fitting different typologies of test compounds, with different modes of action and treatment modalities.

CASE STUDY

Exposure of DCs to a target tumor antigen (TA) peptide enhances their antigenic presentation shown by the increase of its surrogate expression (surface (left) and intracellular (right)) using specific antibodies.

Target TA-exposed DCs lead to optimized priming of CD8 T cells, as shown through the increased IFNγ levels released in CD8 T cell / DC co-cultures.

Specific TA-exposed DC-mediated priming of CD8 T cells results in the induction of an adaptive effector T cell-mediated killing towards SK-MEL-5 tumor cells (target TA-positive). Real-time monitoring of SK-MEL-5 tumor cell killing mediated by CD8 T cells, primed by either TA-exposed or unexposed DCs, where apoptosis and tumor cell count were monitored over ~4 days and analyzed. In addition, IFNγ release by CD8 T cells is shown to be increased upon their TA-exposed DC priming, compared to TA-unexposed DCs.

TAKE HOME MESSAGE: Enhancing DC-mediated tumor antigen presentation induces a specific, optimized, adaptive effector T cell-mediated killing response.

How does it work?

A step-by-step overview of our tumor antigen-specific T-cell mediated tumor killing assay:

Choice of the right tumor cell lines based on their expression of the target antigens and their representativeness of the cancer indications of interest (>100 in-house human tumor cell lines)

Isolation of PBMC-originating immune populations required (monocytes, CD8) for autologous co-cultures

Generation of monocyte-derived APC such as differentiated & mature DCs

DC - CD8 T cell co-cultures for DC-mediated priming and activation of CD8 cells

CD8 T cell - target tumor cell co-cultures to capture the tumor-targeted cytotoxic response

Adequate treatment windows in line with the expected mechanisms of action and eventual promising combination treatments that could hold potential for improving anti-tumor response

Relevant readouts to capture each cell component (DC cytokines and surface markers, CD8 cytokines and surface markers, target tumor cell apoptosis/proliferation…)

More data ? Some questions ? Contact our team !

Our capacities for the identification & Validation of Target Antigen Expression in Tumor Samples

Starting from FFPE tumor specimens, our translational team can explore and/or validate the expression of target tumor antigens across a set of cancer indications, using various platforms:

Single-Cell RNA-Seq to identify & quantify tumor antigen expression, and investigate tumor antigen heterogeneity and the immune landscape

Xenium Single-Cell Transcriptomics to explore tumor antigen patterns and tumor heterogeneity with respect to the immune microenvironment (immune infiltration, response, gene expression profiles and signatures…)

GeoMx Digital Spatial Profiling for comprehensive antigen mapping within the tumor microenvironment (TME)

Digital Pathology for precise and multiplexed analysis of antigen expression with the TME - providing a comprehensive view of the tumor antigenic profile and how it could interact with other components of the TME

Talk to a scientist from our translational team

By Pierre-Emmanuel Gaultier24 January 202524 January 2025

Open position: Single-Cell Data Scientist (DS0125)

Reference: DS0125
Contract type: Full-time position, permanent contract
Location: Bordeaux, France

You like to play with large multi-omics datasets and analyze spatial information ? You have an interest in the biological processes involved in the response to cancer immunotherapies ? Keep reading and apply!

About Explicyte

Explicyte is a preclinical and translational contract research organization specialized in immuno-oncology. Our mission is to support academics, biotechs and pharmas in the discovery of novel targets and novel cancer immunotherapies.

With a team of 25, we’re a human-sized company, which brings under the same roof cell biologists, immunologists, medical oncologists and bioinformaticians. Focused on sponsors’ projects, our activity also involves in-house R&D and external collaborations, which led to the publication of 30+ papers in high impact-factor journals.

Based in Bordeaux, our lab is located at the Bergonié Comprehensive Cancer Center, where we work in close contact with medical oncology teams. We believe Explicyte is a place to learn, grow, and have impact in the fight against cancer.

More about us

Your position

To strengthen our translational research activities and benefit our partners, we are currently looking for a trained Data Scientist with expertise in single-cell analysis.

In your role, you will be accountable for data mining & analysis in relation to sponsors’ and R&D studies. You will play with data from various biological samples (e.g. FFPE & fresh tumor samples, Peripheral Blood Mononuclear Cells, tumor immune infiltrates, etc.), captured through a range of platforms that include Chromium (10X Genomics) for single cell analysis, Geomx DSP (Nanostring) for spatial transcriptomics as well as Xenium (10X genomics) for in situ single cell analysis.

You will interact with the translational team, and will report to the Chief Technology Officer.

What you will find at Explicyte