Background Accurate biomarkers for prognosis and selection of patients most likely to benefit from neoadjuvant chemoimmunotherapy in early breast cancer (eBC) are urgently needed. The Immune Constant of Rejection (ICR) gene expression signature, a 20-gene signature reflecting cytotoxic immune activation, is associated with favorable prognosis and pathological complete response (pCR) to neoadjuvant chemotherapy (NACT) in eBC. However, ICR requires transcriptomic profiling, restricting its routine clinical implementation. We hypothesized that the biological features captured by ICR could be inferred directly from routine hematoxylin-eosin (H&E) whole-slide images (WSIs) using deep learning. Methods We developed an artificial intelligence (AI)-based deep-learning model to predict ICR class (AI-ICR model) from WSIs and tested its predictive value for survival and pCR to NACT and neoadjuvant chemoimmunotherapy (NACTIT). Our model was trained using WSIs from 2,881 TCGA non-BC multicancer samples paired with RNA-sequencing-derived ICR classification. It was externally validated in 1,087 TCGA BC samples and 104 pretreatment biopsies from two BC prospective neoadjuvant trials. We assessed its association with overall survival (OS) in TCGA and with pCR to NACT/NACTIT in the neoadjuvant cohorts (PELICAN + NEO-R-IPC). Results AI-ICR model showed a 0.917 average cross-validated AUC in the mutlicancer internal test set, and performed well in external eBC series, with 0.937 AUC in TCGA (p = 4.13E-45) and 0.739 in PELICAN + NEO-R-IPC ( p = 5.00E-03), thus demonstrating robustness across tumor types, tissue preparations, and acquisition platforms. AI-ICR-high tumors displayed longer OS than AI-ICR-low tumors with Hazards-Ratio for death of 0.29 [95%CI 0.15–0.55] in multivariate analysis ( p = 1.69E-04). They also displayed larger pCR rate than AI-ICR-low tumors, with an Odds-Ratio for pCR of 4.33 [95%CI 1.68–11.2] in multivariate analysis ( p = 2.47E-03), and this difference was stronger in the NACTIT arm (81% versus 36%) than in the NACT arm (65% versus 30%). Transcriptomics showed enrichment of immune genes and activated immune cell types in AI-ICR-high tumors. Conclusions AI-ICR enables accurate inference of a clinically validated immune gene-expression program directly from routine H&E slides and independently predicts survival and pCR to NACT/NACTIT in eBC. After further validation, it could represent a simple, inexpensive and scalable alternative to transcriptomic profiling for precision management of eBC, including routine AI-assisted immuno-oncology.

Background The high mortality rate of patients with colorectal cancer combined with the lack of nontoxic and efficient personalized treatments makes it urgent to develop new targeted therapies for this disease. B7-H3 appears to be a good target, as it is overexpressed in tumor tissue compared with normal tissue. However, B7-H3 is a molecule with ambivalent functions and is expressed by different cell types. This complexity has contributed to the delay in identifying cell subtypes that express B7-H3 and their potential role in colorectal oncogenesis.Methods In this integrated multiomics study, we used in silico bulk, single-cell, and spatial transcriptomic data to investigate the clinical and biological characteristics of tumors with high B7-H3 expression, the specific cell types expressing high levels of B7-H3, and their temporal appearance during colorectal oncogenesis.Results We found that tumors with high B7-H3 expression corresponded to tumors with a predominant stroma composed mainly of fibroblasts. Among them, two subtypes of extracellular matrix-related myofibroblastic cancer-associated fibroblasts and profibrotic pericytes specifically expressed high levels of B7-H3, the former being an independent factor for poor prognosis in patients with colorectal cancer. Finally, by examining precancerous lesions, we report that fibroblast subtypes with high levels of B7-H3 appear early during oncogenesis, especially at the inflamed stage.Conclusions We suggest that anti-B7-H3 immunotherapies might preferentially target cells from the microenvironment rather than tumor cells. This is particularly important for understanding the mode of action of the anti-B7-H3 antibody‒drug conjugate, which is currently being tested in clinical trials in several solid tumors.

Pancreatic cancer remains a devastating disease with limited therapeutic options. Accumulating evidence shows that cancer-associated fibroblasts (CAFs) and tumor-associated macrophages, the predominant cells in the pancreatic cancer (PDAC) tumor microenvironment, hinder antitumor immunity. However, the role of extracellular vesicles (EVs) in such a process is poorly understood. In this study, using human bone marrow-derived monocytes and PDAC tumor cells, we showed that tumor cell-derived EVs (TC-EVs) induced monocyte differentiation toward M2-like, immunosuppressive, CD200R+PD-L1+HLA-DRlo macrophages that express ALOX15B, that we identify as an independent PDAC poor-prognosis biomarker using a human PDAC metacohort. We also demonstrated that TC-EVs reprogrammed human primary PDAC CAFs, causing a fibronectin network reorganization associated with changes in extracellular matrix (ECM) composition, including alterations of WNT pathway elements such as secreted frizzled related protein-1 (SFRP1) enrichment. We also revealed that monocytes cultured on SFRP1-enriched ECM differentiated into M2-like, immunosuppressive macrophages. Last, we demonstrated that both directly and indirectly TC-EV- or SFRP1-enriched ECM-driven differentiated macrophages hindered T cell activation and subsequent antitumor activity. Our findings highlight potentially novel dual mechanisms of TC-EV-mediated crosstalk, involving ALOX15B+ macrophages and SFRP1+ CAFs, that simultaneously contribute to foster the immunosuppressive ecosystem of PDAC.

Abstract Context Immunotherapy based on immune checkpoint inhibitors (ICI) revolutionized the treatment of triple-negative (TN) breast carcinomas (BC), but remains more challenging in HR + /HER2 - BCs. Because invasive lobular carcinomas (ILC) generally exhibit low immune infiltration, ICIs were largely overlooked in this pathological type. The only clinical trial of ICIs dedicated to ILCs showed disappointing results, notably in HR + /HER2 - cases. The immune landscape of HR + /HER2 - ILCs has been poorly described. High level of tumor-infiltrating lymphocytes (TIL) was associated with worse prognosis in HR + /HER2 - ILCs. A better characterization of the immune landscape of HR + /HER2 - ILCs could clarify the poor efficiency of ICIs and the negative prognostic value of TILs, and reveal complementary targets able to increase immunotherapy efficiency. Method We comprehensively characterized the immune landscape of HR + /HER2 - ILCs, comparatively to HR + /HER2 - invasive ductal carcinomas (IDC), by applying multi-omics and multi-scale analysis (gene expression at the bulk and single-cell levels, and protein-based spatial analysis) to clinical samples. Results While the overall level of immune infiltration was comparable between both pathological types, the quality of immune infiltrate differed markedly. Comparatively to HR + /HER2 - IDCs, HR + /HER2 - ILCs were enriched in immune cells and tertiary lymphoid structures with anti-tumor potential, presented more spatial proximity between cancer cells and CD8 + cytotoxic T cells, and stronger theorical vulnerability to ICIs. However, in HR + /HER2 - ILCs, anti-tumor response was defective; CD8 + cytotoxic T cells failed to fully unleash their cytotoxic function and CD4 + helper T cells evidenced a pro-tumoral and naive phenotype. Furthermore, antigen-presenting compartment was defective, altogether embedded in a stronger immunosuppressive environment, enriched in immunoregulatory cancer-associated fibroblasts (iCAF). Conclusion This study contributes to explain the lesser efficiency of PD-1/PD-L1-based ICIs in HR + /HER2 - ILCs by comparison with HR + /HER2 - IDCs, by shedding light on a complex ecosystem where tumor cells shape a distinctive stroma that contribute to prevent anti-tumor immune response activation. Altogether, our findings further support the rationale for combining iCAF-targeting strategy with an ad hoc immunotherapy (such as an anti-VTCN1/B7-H4 antibody-drug conjugates for example). Graphical abstract Highlights WHAT IS ALREADY KNOWN ON THIS TOPIC Immune cells infiltrate both HR + /HER2 - IDC and HR + /HER2 - ILC tumors, but current ICIs are less effective in HR + /HER2 - ILCs than HR + /HER2 - IDCs. WHAT THIS STUDY ADDS The anti-tumor immune response is mobilized but not effective in HR + /HER2 - ILCs. A complex ecosystem - composed of immunoregulatory cancer-associated fibroblasts, high levels of TGFâ, prostaglandin, acidosis, and a lack of antigen-presenting cells - prevents anti-tumor CD8 + cytotoxic T cell activation in HR + /HER2 - ILCs. HOW THIS STUDY MIGHT AFFECT RESEARCH, PRACTICE, OR POLICY Targeting the PD-1/PD-L1 axis is not the appropriate therapeutic strategy for HR + /HER2 - ILCs. A more complex approach should be considered, notably those combining other immune-based strategies and iCAF targeting, which may offer a better chance to eradicate HR + /HER2 - ILC tumor cells.

Purpose: Undifferentiated pleomorphic sarcoma (UPS) is an aggressive subtype of soft tissue sarcoma with poor outcomes, particularly in metastatic cases. The mechanisms driving metastasis in UPS remain poorly understood, limiting therapeutic advances.Experimental Design: A multi-omics approach was used to analyze paired primary and metastatic UPS tumor samples. Spatial transcriptomics, bulk RNA sequencing, and deconvolution analyses were performed to identify molecular pathways and immune microenvironment alterations associated with metastasis. Functional assays using CRISPR-Cas9 knockout (KO) UPS cell lines, alongside in vivo models, were used for functional validation experiments.Results: Transcriptomic analyses on 13 patients with UPS revealed significant upregulation of hypoxia, epithelial-mesenchymal transition, and immune-suppressive pathways in metastatic UPS. ADORA2B was identified as a key driver of these processes, with elevated expression correlating with poor disease-free survival in patients with UPS. Functional studies confirmed that ADORA2B promotes proliferation, migration, invasion, and matrix remodeling via metalloprotease regulation. In vivo, ADORA2B KO reduced primary tumor growth and metastatic dissemination in UPS models.Conclusions: This study identifies ADORA2B as a critical regulator of metastatic progression in UPS, implicating it as a promising therapeutic target. Ongoing clinical trials targeting adenosine pathways further support the translational potential of ADORA2B inhibition to disrupt metastasis and improve outcomes for patients with UPS.

Abstract Background: Pancreatic ductal adenocarcinoma (PDAC) is an aggressive cancer with a complex microenvironment in which galectins play key roles. This study evaluated galectins expression to investigate its potential as a prognostic biomarker. Methods: Transcriptomic analyses were performed in silico on a cohort of 938 individuals with primary PDAC. Laser microdissection, immunohistochemistry, and RT-qPCR were used to assess the expression profile in the tumor and stromal regions of PDAC biopsies. Serum GAL-2 levels were measured by ELISA. GAL2 expression was also evaluated during distinct stages of tumor progression using the KIC murine model. Results: To analyze the galectin transcriptomic expression profile in human PDAC, transcripts of LGALS genes were evaluated in a cohort of 938 individuals diagnosed with primary PDAC tumors. LGALS2 emerged as a key transcript, exhibiting expression levels two-fold below the median in PDAC samples. Correlation analysis of LGALS transcripts revealed a positive cluster among LGALS2, LGALS3, LGALS4, and LGALS9, and a negative association between LGALS1 and LGALS2. LGALS2 expression was downregulated in primary tumors (p = 8.76E-22) and metastatic sites (p = 2.75E-30) compared to both the control group and normal pancreatic tissue. In the KIC mouse model, a progressive reduction in LGALS2 expression was observed throughout tumor progression, from early lesions to established primary and metastatic tumors. High LGALS2 expression was associated with classical and well-differentiated epithelial PDAC subtypes, whereas low expression characterized basal-like, squamous, and mesenchymal tumors. Importantly, increased LGALS2 expression correlated with better disease-free and overall survival. Furthermore, higher LGALS2 expression was significantly associated with improved overall survival in patients treated with the FOLFIRINOX regimen (p = 4.08E-02). Additionally, low GAL-2 protein expression was observed in biopsies from patients diagnosed with PDAC. Serum GAL-2 levels were significantly lower in PDAC patients, especially those with metastasis when compared to health donors. Promoter hypomethylation (CpG island cpg25247183) was identified as a potential regulatory mechanism underlying its downregulation. Conclusions: The downregulation of LGALS2 expression across different stages of PDAC progression underscores its importance in tumor pathogenesis and suggests its potential as a prognostic and therapeutic biomarker. Citation Format: Moacyr Jesus B. Melo Rêgo, Richard Tomasini, Sophie Vasseur, Maira Pitta, Maria Clara P. Sampaio, Amanda P. Albuquerque, Pascal Finetti, Cláudio Montenegro, Michelly Cristiny Pereira, Michelle Rosa. Galectin-2 expression in the tumor microenvironment: A silent pathway of pancreatic ductal adenocarcinoma progression [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2026; Part 1 (Regular Abstracts); 2026 Apr 17-22; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2026;86(7 Suppl):Abstract nr 7916.

Abstract Circulating tumor cells (CTCs) are the potential seeds of distant metastases; however, little is known about how they survive in the bloodstream. Using a large cohort of colorectal cancer (CRC) patients, we found that the pseudokinase receptor PTK7 is highly expressed in primary tumors and metastatic lesions. Consistent with previous reports, high PTK7 expression is associated with reduced disease-free survival and increased metastatic dissemination. Surprisingly, PTK7 is absent from most CTCs and undergoes a cell-autonomous ON tumor /OFF CTC /ON metastasis switch that can be recapitulated in a xenografted mouse model, in in vitro systems, and a fluidic platform. PTK7-negative cancer cells exhibit increased expression of YAP1-driven genes, senescence-like features, and enhanced resistance to hemodynamic stress following loss of cell-cell and cell-matrix adhesion. This adaptive phenotype depends on metalloproteases, notably ADAM17, whose cleavage activity remodels the CTCs surfaceome. Functionally, the PTK7 OFF CTC state confers enhanced metastatic potential in vivo , and can be pharmacologically suppressed using metalloprotease inhibitors. Collectively, our findings identify a reversible, cell-autonomous, protease-driven surfaceome remodeling program that enables metastatic adaptation during hematogenous dissemination. Highlights / statement of significance By investigating potential markers for circulating colorectal tumor cells with strong metastatic potential, we describe a reversible and cell-autonomous remodeling of the circulating tumor cell surfaceome in patients that confers resistance to anoikis and stress induced by entry into the bloodstream. One Sentence Summary The dynamic regulation of PTK7 serves as a surrogate marker for tumor cell plasticity, aggressiveness, survival in the bloodstream, and efficiency in forming metastases. Trial registration CTC colon Cohort: registered on https://ClinicalTrials.gov identifier NCT03256084 ; date of registration 2017-07-17 B-Org cohort: registered on https://ClinicalTrials.gov NCT05384184 ; date of registration 2019-06-06 Ethics statement for animal experiments Studies on animals were conducted in accordance with the current ethical standards of the European Community (Directive 2010/63/EU), the Ethics Committee for Animal Experimentation (CEEA#14) and the French Ministry of Higher Education and Research, which approved and authorized the entire procedure described in this paper (project number APAFIS #35294).

BACKGROUND AND PURPOSE:Idiopathic pulmonary fibrosis (IPF) is a fatal disease characterized by fibroblast activation and abnormal accumulation of extracellular matrix in the lungs. We previously demonstrated the importance of the heat shock protein αB-crystallin (HSPB5) in TGF-β1 profibrotic signalling, which suggests that HSPB5 could be a new therapeutic target for the treatment of IPF. The purpose of this study was thus to develop antisense oligonucleotides targeting HSPB5 and to study their effects on the development of experimental pulmonary fibrosis. EXPERIMENTAL APPROACH:Specific antisense oligonucleotides (ASO) were designed and screened in vitro, based on their ability to inhibit human and murine HSPB5 expression. The selected ASO22 was characterized in vitro in human fibroblast CCD-19Lu cells and A549 epithelial pulmonary cells, as well as in vivo using a mouse model of bleomycin-induced pulmonary fibrosis. KEY RESULTS:ASO22 was selected for its capacity to inhibit TGF-β1-induced expression of HSPB5 and additional key markers of fibrosis such as plasminogen activator inhibitor-1, collagen, fibronectin and α-smooth muscle actin in fibroblastic human CCD-19Lu cells as well as plasminogen activator inhibitor-1 and α-smooth muscle actin in pulmonary epithelial A549 cells. Intra-tracheal or intravenous administration of ASO22 in bleomycin-induced pulmonary fibrotic mice decreased HSPB5 expression and reduced fibrosis, as demonstrated by decreased pulmonary remodelling, collagen accumulation and Acta2 and Col1a1 expression. CONCLUSION AND IMPLICATIONS:Our results suggest that an antisense oligonucleotide strategy targeting HSPB5 could be of interest for the treatment of IPF.

The high mortality rate of colorectal cancer (CRC) combined with the lack of non-toxic and efficient personalized treatments makes it urgent to develop new targeted therapies for this disease. B7-H3 appears to be a good target as it is overexpressed in tumor tissue compared to normal tissue. However, B7-H3 is a molecule with ambivalent functions and is expressed by different cell types. This complexity contributed to the delay in identifying cell subtypes that express B7-H3 and their potential role in colorectal oncogenesis. In this study, we used in silico bulk, single-cell, and spatial transcriptomic data to investigate the clinical and biological characteristics of tumors with high B7-H3 expression, the precise nature of cells expressing high level of B7-H3, and their temporal appearance during colorectal oncogenesis. We found that tumors with high B7-H3 expression corresponded to tumors with a predominant stroma composed mainly of fibroblasts. Among them, two subtypes of ecm-myCAF fibroblasts and pro-fibrotic pericytes specifically expressed high levels of B7-H3, the former being an independent factor for poor prognosis in CRC. Finally, by examining precancerous lesions, we report that fibroblast subtypes with high levels of B7-H3 appear early during oncogenesis, especially at the inflamed stage. We also shed light on the fact that anti-B7-H3 immunotherapies might therefore preferentially target cells from the microenvironment rather than the tumor cells. This is particularly important to understand the mode of action of the anti-B7-H3 antibody-drug conjugate, which is currently being tested in clinical trials in several solid tumors. ### Competing Interest Statement The authors have declared no competing interest. Cancéropole PACA, https://ror.org/01mwvah42 GIRCI Mediterranée

BACKGROUND:Breast cancer (BC) is a major problem of public health in western countries. The long-term survival improved thanks to therapeutic progresses and mass screening. Mass screening is based on mammography but displays limitations. Efforts are ongoing to develop accurate and minimally invasive tools for early BC detection. Analysis of liquid biopsies is a promising option, among which the ones based on thermal denaturation profiling provide a "thermodynamic signature" of disease through analysis of plasma protein denaturation profiles. We recently developed a major technical breakthrough of differential scanning calorimetry by switching to nanoDSF (Differential Scanning Fluorimetry), more easily transferrable in clinical routine. Here, we applied it for the first time to samples form BC patients. METHODS:We retrospectively applied nanoDSF to plasma samples from 176 patients collected in two prospective clinical trials and 61 healthy controls (HC). The profiles were analyzed using four artificial intelligence (AI) algorithms. Our primary objective was to test the potential of this approach to distinguish BC versus HC samples. We also assessed its ability to distinguish early versus advanced BC, and major molecular subtypes of disease. RESULTS:The four algorithms provided predictive models displaying very good performances for distinguishing patients from HC. For example, the random forest-based model displayed 96.6% accuracy in properly classifying subjects, 99.4% sensitivity, and 88.5% specificity. These performances were not dependent on the clinicopathological characteristics of BC, and compared favorably to those of mammography-based screening. For comparison, the performances of predictive models centered on the secondary objectives (early versus metastatic stage, hormone receptor (HR)-positive versus HR-negative status, and HER2-positive versus HER2-negative status) were good, but inferior, likely because of the stronger unbalance in the number of patients in each group and of more subtle differences in thermograms between patients' groups than between patients and HC. CONCLUSIONS:We reveal the potential of nanoDSF and AI applied to plasma samples to discriminate between BC patients and HC. If these results are confirmed, such approach could represent a minimally-invasive, low risk, quick and low-cost technique, which could help to improve the screening of BC.

The excessive production of extracellular matrix (ECM) and the metabolic adaptations in pancreatic ductal adenocarcinoma (PDAC) contribute individually to enhanced chemoresistance, marked tumor progression, and dismal patient survival. However, ECM-driven metabolic alterations that promote chemoresistance in PDAC are so far unexplored. Here, we use in vitro-generated ECM bioscaffolds that recapitulate cell-ECM interactions and induce broad metabolic alterations in PDAC cells. High-throughput integration of multiomics datasets coupled with metabolic tracing showed that the ECM enhances the generation of guanylates in PDAC cells, the accumulation of which alleviates oxaliplatin-induced DNA damage, and boosts PDAC cell proliferation. These events are guided by the guanosine monophosphate (GMP)-producing enzymes inosine monophosphate dehydrogenase (IMPDH) and guanosine monophosphate synthase (GMPS), the expression of which correlated with that of matrisomal and DNA repair genes in samples from patients with PDAC. We propose that targeting ECM-driven metabolic processes, such as the enhanced IMPDH activity, may be an effective therapeutic approach for patients with PDAC to bypass the negative side effects of direct targeting of the ECM itself.

BACKGROUND:Tumor-associated macrophages (TAMs) are key promoters of inflammatory breast cancer (IBC), the most aggressive form of breast cancer. The receptor tyrosine kinase AXL is highly expressed in various cancer types, including IBC, but its role in TAMs remains unexplored. METHODS:We examined the effects of AXL inhibitor TP-0903 on tumor growth and tumor microenvironment (TME) component M2 macrophages (CD206+) in IBC and triple-negative breast cancer mouse models using flow cytometry and immunohistochemical staining. Additionally, we knocked out AXL expression in human THP-1 monocytes and evaluated the effect of AXL signaling on immunosuppressive M2 macrophage polarization and IBC cell growth and migration. We then investigated the underlying mechanisms through RNA sequencing analysis. Last, we performed CIBERSORT deconvolution to analyze the association between AXL expression and tumor-infiltrating immune cell types in tumor samples from the Inflammatory Breast Cancer International Consortium. RESULTS:We found that inhibiting the AXL pathway significantly reduced IBC tumor growth and decreased CD206+ macrophage populations within tumors. Mechanistically, our in vitro data showed that AXL promoted M2 macrophage polarization and enhanced the secretion of immunosuppressive chemokines, including CCL20, CCL26, and epiregulin, via the transcription factor STAT6 and thereby accelerated IBC cell growth and migration. RNA sequencing analysis further indicated that AXL signaling in immunosuppressive M2 macrophages regulated the expression of molecules and cytokines, contributing to an immunosuppressive TME in IBC. Moreover, high AXL expression was correlated with larger populations of immunosuppressive immune cells but smaller populations of immunoactive immune cells in tissues from patients with IBC. CONCLUSIONS:AXL signaling promotes IBC growth by inducing M2 macrophage polarization and driving the secretion of immunosuppressive molecules and cytokines via STAT6 signaling, thereby contributing to an immunosuppressive TME. Collectively, these findings highlight the potential of targeting AXL signaling as a novel therapeutic approach for IBC that warrants further investigation in clinical trials.

The tubulin tyrosine ligase–like (TTLL) family comprises enzymes catalyzing posttranslational modifications of tubulin, including glutamylation and glycylation. We previously described a critical role for the monoglycylase TTLL3 in colon. Here, we identified TTLL6 as the predominant polyglutamylase in the colon, specifically expressed in epithelial cells of distal and transverse segments. TTLL6 expression decreases during CRC progression, correlating with poor patient prognosis. Deletion of Ttll6 in mice resulted in elongated colonic crypts, expansion of stem and transit-amplifying compartments, and increased numbers of differentiated epithelial cells. Moreover, Ttll6 -deficient mice showed an elevated susceptibility to chemically induced colon carcinogenesis. Notably, we identified the nucleic acid–binding protein PurA as a novel TTLL6 substrate with both proteins mutually required for nuclear localization. Consistently, both nuclear polyglutamylation and PurA were present in the bottom compartment of control colons, but reduced in Ttll6 -deficient colons. These findings reveal a TTLL6-PurA axis being critical in maintaining colonic homeostasis.