Introduction

Colorectal cancer (CRC) remains a leading cause of cancer-related mortality, with tumor progression shaped by complex immune interactions. Eosinophils, traditionally associated with allergic responses, have emerged as key immune regulators in CRC, yet their functional roles across disease stages remain poorly understood. This study investigated eosinophil subset dynamics, transcriptional programs, and functional contributions to tumor progression using preclinical murine models and human CRC datasets.

Aims & Methods

To assess eosinophil function across CRC stages, we used a genetic mouse model of early adenoma formation (Apc^min/+) and a late-stage orthotopic CRC tumor organoid transplant model. Single-cell RNA sequencing (scRNA-seq) and functional profiling were performed to characterize eosinophil subsets, gene expression changes, and interactions with the tumor microenvironment (TME). Findings from murine models were compared to newly generated eosinophil scRNA-seq datasets derived from CRC patients.

Results

In murine models, eosinophils were enriched in early adenomas, displaying a predominantly basal phenotype. Their depletion led to increased adenoma burden, suggesting a protective role in tumor initiation. In contrast, eosinophil depletion did not affect primary tumor growth in our late-stage CRC model but strikingly exacerbated metastatic dissemination and clinical parameters. Mice treated with anti-IL-5 antibodies exhibited lower survival, an increased incidence of CRC liver metastases, as well as a higher occurrence of peritoneal dissemination – a feature associated with extremely poor prognosis in CRC patients. Transcriptomic profiling of the TME by scRNA-seq and multiparametric flow cytometry revealed the drastic expansion and differentiation of Spp1⁺ tumor associated macrophages (TAMs) in the absence of eosinophils – an immunosuppressive subset linked to tumor immune evasion and metastatic spreading. Mechanistically, we showed that eosinophils respond to tumor-derived signals, adopt an activated phenotype and prevent Spp1⁺ TAMs differentiation in vitro.
In parallel to our murine studies, we prospectively collected and profiled the transcriptomes of CRC patient specimens. Both mouse and human CRC exhibited conserved activation patterns, dynamic shifts, and gene expression programs within tumors, highlighting strong transcriptional concordance across species. In the TME, eosinophils adopted an activated profile and displayed unique tumor-associated gene signatures, distinct from those of non-tumoral adjacent tissue. These were characterized by the upregulation of multiple cytokines, chemokines, MHC-I components and tissue remodeling enzymes such as Mmp9, suggesting their active involvement in immunomodulation and tissue remodeling. Additionally, immunofluorescence analysis of CRC patient tissue microarrays revealed that eosinophil numbers decline as TNM stage progresses, while their activation status (PD-L1⁺) increases, thus mirroring the dynamic shifts observed in our murine models.

Conclusion

Our findings reveal that eosinophils undergo distinct, stage-dependent functional shifts in CRC, suppressing tumor growth in early disease while adopting immunomodulatory roles in advanced stages to limit metastatic spread. Eosinophil depletion accelerates tumor progression, highlighting their critical role in shaping the TME. Together, these findings establish a previously unrecognized eosinophil–macrophage axis in CRC progression and metastasis, offering new avenues for therapeutic interventions.

Introduction

Advanced combination treatment (ACT), defined as the use of two biologics or a biologic and a small molecule drug with distinct mechanisms of action, is increasingly adopted for refractory inflammatory bowel disease (IBD) and overlapping immune-mediated inflammatory diseases (IMIDs). Despite promising clinical results, the immunological consequences of ACT remain poorly characterized. Here, we investigated how two ACT regimens—anti-TNF + anti-integrin and anti-TNF + anti-IL-23—shape the peripheral CD3⁺ T cell landscape at single-cell resolution, with a focus on clonotype architecture, repertoire diversity, and antigen specificity.

Aims & Methods

We aim to characterize the peripheral CD3⁺ T cell transcriptional landscape and clonal architecture in patients with refractory IBD who achieved clinical response following initiation of two different ACT regimens: anti-TNF + anti-integrin α4β7 and anti-TNF + anti-IL-23. Peripheral blood mononuclear cells (PBMCs) were collected from six patients with Crohn’s disease who achieved clinical response to ACT. Four patients received anti-TNF + anti-integrin α4β7, and two received anti-TNF + anti-IL-23. CD3⁺ T cells were isolated and analyzed via single-cell RNA sequencing (scRNA-seq) coupled with paired T cell receptor (TCRα/β) sequencing. Cell state annotations were performed using supervised deconvolution against the scIBD reference atlas, and TCR repertoire metrics (diversity, clonality, evenness) were computed to assess clonal expansion dynamics.

Results

All patients included demonstrated clinical improvement, underscoring the immunological relevance of the findings. Single-cell transcriptomic and TCR repertoire profiling uncovered distinct treatment-specific patterns in peripheral T cell states and clonal architecture. In the anti-TNF + α4β7 group, there was marked enrichment of CD4⁺ tissue central memory T cells (CD4⁺ tissue-Tcm) and CD4⁺ naïve T cells (CD4⁺ Tn), along with greater TCR diversity, reduced clonal dominance, and enhanced repertoire evenness. In contrast, the anti-TNF + IL-23 group exhibited a marked enrichment of CD8⁺ naive T cells (CD8⁺ Tn) and a repertoire characterized by reduced diversity and pronounced oligoclonal expansion.

Conclusion

In difficult-to-treat IBD patients who achieved clinical response to ACT, distinct patterns of immunological remodeling emerged depending on the therapeutic combination. Treatment with anti-TNF + anti-integrin was associated with a diversified, polyclonal T cell repertoire consistent with a systemic rebalancing of the adaptive immune response, whereas anti-TNF + anti-IL-23 drove more focused, oligoclonal expansions indicative of selective immune activation. These divergent signatures underscore mechanistically distinct pathways of immune reprogramming and highlight the potential of T cell–based immuno-profiling to inform the rational selection of biologic combinations. Incorporating single-cell and TCR repertoire analyses into ACT trials may enable a precision medicine framework for optimizing treatment strategies in IBD.

References

Danese S, Solitano V, Jairath V, Peyrin-Biroulet L. The future of drug development for inflammatory bowel disease: the need to ACT (advanced combination treatment). Gut. 2022 Dec;71(12):2380-2387. doi: 10.1136/gutjnl-2022-327025. Epub 2022 Jun 14. PMID: 35701092.

Disclosure

Virginia Solitano has received speaker fees from Pfizer and consultancy fees from Johnson & Johnson. Vipul Jairath has received consultancy/advisory board fees from AbbVie, Alimentiv Inc., Arena pharmaceuticals, Asahi Kasei Pharma, Asieris, Astra Zeneca, Bristol Myers Squibb, Celltrion, Eli Lilly, Ferring, Flagship Pioneering, Fresenius Kabi, Galapagos, GlaxoSmithKline, Genentech, Gilead, Janssen, Merck, Mylan, Pandion, Pendopharm, Pfizer, Protagonist, Reistone Biopharma, Roche, Sandoz, Second Genome, Takeda, Teva, Topivert, Ventyx, and Vividion; and speaker’s fees from, Abbvie, Ferring, Galapagos, Janssen Pfizer Shire, Takeda, and Fresenius Kabi. Silvio Danese has received consultancy fees from AbbVie, Alimentiv, Allergan, Amgen, AstraZeneca, Athos Therapeutics, Biogen, Boehringer Ingelheim, Bristol Myers Squibb, Celgene, Celltrion, Dr Falk Pharma, Eli Lilly, Enthera, Ferring Pharmaceuticals Inc, Gilead, Hospira, Inotrem, Janssen, Johnson & Johnson, MSD, Mundipharma, Mylan, Pfizer, Roche, Sandoz, Sublimity Therapeutics, Takeda, TiGenixa, UCB Inc, and Vifor; and lecture fees from AbbVie, Amgen, Ferring Pharmaceuticals Inc, Gilead, Janssen, Mylan, Pfizer, and Takeda. The other authors have nothing to disclose.

Introduction

The BIOSTOP Trial is a prospective, multicenter, randomized, controlled, open-label study conducted in Norway to evaluate the two-year outcomes of tumor necrosis factor-alpha (anti-TNF) antibody withdrawal in patients with ulcerative colitis (UC) in sustained clinical and endoscopic remission. Prior non-randomized studies have suggested that approximately half of such patients maintain remission after anti-TNF discontinuation and that reintroduction of anti-TNF therapy is generally effective and safe in the event of relapse.

Aims & Methods

A total of 174 UC patients in confirmed clinical and endoscopic remission were enrolled across 19 gastroenterology centers between June 2017 and December 2020 and randomized 1:1 to either continue or discontinue anti-TNF therapy. Eligibility criteria included at least 12 months of first-line anti-TNF maintenance treatment, clinical remission, fecal calprotectin <200 µg/g, and endoscopic remission (Mayo endoscopic score 0 or 1). Two-year follow-up data were available for 172 patients (86 in each group). In 156 of the patients endoscopies were available for central reading (77 in the withdrawal group and 79 in the maintenance group). The primary endpoint was sustained endoscopic remission at two years.

Results

At two years, central reading of follow-up colonoscopies confirmed endoscopic remission in 64 of 77 patients (83%, 95% CI 71.8 – 93.1) in the anti-TNF withdrawal group, compared to 77 of 79 patients (96 %, 95 % CI 92.1 – 100.8) in the anti-TNF maintenance group. The non-inferiority primary endpoint of the study was thus not met. During the study period, 48% of patients in the withdrawal group and 88% in the maintenance group remained in sustained clinical and endoscopic remission without relapse. Among the 45 patients in the withdrawal group who experienced endoscopic confirmed relapse, 28 (62%) resumed anti-TNF therapy, with 26 achieving both clinical and endoscopic remission. Additionally, 10 of 13 patients with mild relapses achieved endoscopic remission with optimized non-biologic therapy. There were no difference in serious adverse events between the groups.

Conclusion

After two years, 83% of patients in the anti-TNF withdrawal group and 96% in the maintenance group were in clinical and endoscopic remission, including those who experienced flares and achieved new remission after reintroduction of anti-TNF or optimized non-biologic therapy. Nearly half of the patients maintained remission without anti-TNF, and the majority of relapses were effectively managed. These findings suggest that anti-TNF withdrawal is a safe option for selected UC patients in confirmed remission, provided they are monitored closely.
Trial Registration: ClinicalTrials.gov Identifier: NCT03011268; EudraCT Number: 2016-001409-18. Trial completion date: 31 January 2025.

Disclosure

No disclosures

Introduction

Postoperative recurrence is common in Crohn's disease following ileocecal resection, with varying management strategies employed. This study aimed to compare the efficacy of prophylactic approach (starting thiopurines or biological therapy within 6 months from the surgery) versus reactive one (starting biological therapy after evidence of disease reactivation) in preventing postoperative recurrence.

Aims & Methods

This retrospective, multicenter study included 895 adult patients who underwent ileocecal resection for Crohn's disease across 13 Italian centers (prophylactic strategy: n=196; reactive strategy: n=699). Patients were classified as high-risk (≥1 risk factor: active smoking, prior intestinal resection, penetrating disease, perianal disease, or large resection ≥50 cm) or low-risk. The primary outcome was endoscopic recurrence (Rutgeerts' score ≥i2a) at 6-12 months post-surgery. Secondary outcomes included clinical, biochemical, and radiological recurrence at various time points, as well as long-term outcomes including need for repeat surgery.

Results

The prophylactic biologic therapy group demonstrated significantly lower rates of endoscopic recurrence at 6-12 months compared to the reactive approach (51.54% vs. 64.83%, p=0.007). After adjusting for confounders, including age at surgery and previous biologic exposure, a reactive approach was associated with increased odds of endoscopic recurrence (OR 1.893, 95% CI: 1.225-2.926, p=0.004). The prophylactic strategy also showed lower rates of clinical recurrence (23.08% vs. 36.36%, p=0.002), biochemical recurrence (33.81% vs. 48.89%, p=0.004), and severe endoscopic recurrence (22.31% vs. 32.55%, p=0.028) at 6-12 months. In high-risk patients with a single risk factor, prophylaxis significantly reduced endoscopic recurrence (49.06% vs. 65.75%, p=0.033) and surgical recurrence (2.56% vs. 11.58%, p=0.017). For patients with multiple risk factors, benefits were mainly observed in clinical (29.87% vs. 45.77%, p=0.022) and biochemical recurrence (37.88% vs. 56.32%, p=0.024). In low-risk patients, prophylaxis primarily reduced severe endoscopic recurrence (10.52% vs. 34.48%, p=0.037), but not other outcomes. However, these benefits diminished over time, with no significant differences in all outcomes at 15-36 months or in long-term surgical recurrence rates as compared to patients undergoing the reactive approach in all groups (p=0.438). No significant difference was observed between the two approaches regarding the need for endoscopic dilation during follow-up (p=0.301).

Table 1: Differences in outcomes at 6-12 months between a proactive and reactive strategy approach. Abbreviations: CD: Crohn's Disease, FC: Fecal Calprotectin, HBI: Harvey-Bradshaw Index

Conclusion

A prophylactic strategy significantly reduces short-term postoperative recurrence in Crohn's disease patients following ileocecal resection, with particular benefit in high-risk patients with a single risk factor. However, the protective effects appear to diminish over time. Further research is needed to refine predictive models and validate interventions targeting high-risk populations for sustained benefits.