Prolyl 3-hydroxylase 2 (P3H2) is a key enzyme involved in the architecture of the extracellular matrix (ECM). While previously shown to be regulated by VEGF-A and to play a role in angiogenesis, its function in cancer remains ambiguous. While characterized as a tumor suppressor, its precise function in colorectal cancer (CRC) progression is poorly defined. Bioinformatic analysis and patient data reveal that P3H2 transcript levels are significantly reduced in colon adenocarcinoma tissues, showing a progressive decline in metastatic lesions. Furthermore, VEGF-A exposure upregulates P3H2 transcripts in the HCT116 CRC cell line. To investigate its impact in CRC, we generated a stable HCT116 clone overexpressing P3H2. In vitro studies demonstrated that while P3H2 overexpression inhibited anchorage-independent growth, it significantly enhanced cellular invasion without altering cell proliferation. In vivo, however, P3H2-overexpressing tumors exhibited accelerated tumor growth and a statistically significant increase in lung metastases. P3H2 overexpression remodeled the tumor microenvironment (TME) by modifying its main substrate, Collagen IV, resulting in the induction of increased vessels density. Our study repositions P3H2 as a dynamic enzymatic switch within the TME. This work identifies P3H2-driven ECM remodeling as a promising therapeutic axis in advanced CRC, with particular relevance for combination strategies targeting angiogenesis.

Abstract The lymphatic system consists of an open, unidirectional network of lymphatic vessels maintaining tissue homeostasis, intestinal absorption, immunosurveillance and immunomodulation. These vessels are lined by lymphatic endothelial cells (LECs). Lymphangiogenesis, defined as sprouting of new lymphatic vessels from pre-existing vessels or embryonic veins, is the main mechanism driving the formation and expansion of lymphatic networks during embryogenesis and in adults under physiological conditions. This process involves numerous molecules, most notably VEGF-C, but also axon-guidance cues such as Netrins (Hu et al., 2024). Lymphangiogenesis shares many features with angiogenesis, which depends mainly on VEGF-A, but also Netrin-1. Netrin-1, acting through its receptor UNC5B, regulates vascular endothelial migration and branching, thereby stabilizing the vascular network (Lu et al., 2004). In our laboratory, scRNAseq analysis of murine uterine horns revealed that Netrin-1 and UNC5B are specifically expressed by LECs. The lymphatic network of uterine horns has never been investigated, nor has the role of the Netrin-1 and UNC5B in lymphangiogenesis in general. We hypothesize that Netrin-1 and UNC5B contributes to lymphatic network development by regulating LEC migration and tubulogenesis. Our preliminary in vivo data describe the expression patterns of Netrin-1 and UNC5B during lymphatic development in murine uterine horns. UNC5B and Netrin-1 knockout models are being generated to define their functional roles. In vitro studies on human dermal LECs are assessing Netrin-1 effects on migration and tubulogenesis. The lymphatic system is a key route for metastatic dissemination, notably in endometrial cancer (Donoghue et al., 2007). Uterine horns are continuously lined by the endometrium, suggesting that lymphatic vessels within uterine horns may provide a pathway for endometrial cancer cell dissemination. Although Netrin-1 is normally absent in adult tissues, it is re-expressed in tumors, where it promotes tumor progression. If Netrin-1 contributes to lymphangiogenesis in uterine horns, it may similarly modulate lymphatic vessel formation and thereby facilitate metastasis in endometrial cancer. Consistent with this, preliminary results showed that the neutralizing antibody anti-Netrin-1 in mice grafted with endometrial cancer cells significantly reduces lung metastases. The knockout models will help define how Netrin-1 and UNC5B could regulate lymphatic-dependent metastatic spread. This project aims to characterize lymphangiogenesis in uterine horns and to define the roles of Netrin-1 and UNC5B in physiological and tumor contexts. Ultimately, this work may support new therapeutic applications for the anti-Netrin-1 antibody developed in our laboratory, currently in phase II clinical trial. Citation Format: Lia Barcons, Nicolas Rama, Patrick Mehlen. Lymphangiogenesis in the uterine horns: Implication of the Netrin-1 and UNC5B signaling pathway in physiology and cancer [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 4799.

Abstract Purpose: Metastatic colorectal cancer (mCRC) exhibits poor outcomes due to recurrence and resistance to chemotherapy driven by cancer stem cells (CSCs). We investigated the role of the dependence receptor ligand netrin-1 and its receptor UNC5B in CSC self-renewal and evaluated therapeutic inhibition of netrin-1 using the anti-netrin-1 antibody NP137. Experimental Procedures: Patient-derived organoids (PDOs) from colorectal liver metastases were treated with recombinant netrin-1, NP137, or controls. Self-renewal was quantified by extreme limiting dilution assays. UNC5B was silenced by CRISPR/Cas9 to determine receptor the implication of this Netrin-1 receptor. Single-cell RNA-sequencing elucidated signaling pathways affected by NP137. In vivo efficacy of NP137 alone or combined with FOLFOX chemotherapy was tested in PDO-xenografted mice, and paired tumor biopsies from an mCRC patient enrolled in an NP137 clinical trial were analyzed by RNA-seq. Results: Netrin-1 enhanced CSC self-renewal and survival, effects abolished by NP137 or UNC5B knockout. NP137 treatment triggered CSC apoptosis, an effect reversed by caspase inhibition. Single-cell transcriptomics revealed that UNC5B-positive cells secreted trefoil factor 3 (TFF3), which acted paracrinally to maintain stemness gene expression (LGR5, SOX4, SMOC2, PROM1). NP137 suppressed TFF3 and stemness transcripts in both PDOs and a treated patient’s tumor. Blocking TFF3 dimerization phenocopied NP137 activity, confirming TFF3 as a critical downstream effector. FOLFOX exposure upregulated netrin-1 and UNC5B, and combination therapy (FOLFOX + NP137) significantly reduced self-renewal and tumor growth in mice while decreasing intratumoral TFF3. Conclusions: Netrin-1 sustains mCRC CSC self-renewal through an UNC5B-dependent, TFF3-mediated paracrine survival mechanism. Pharmacologic inhibition of netrin-1 with NP137 induces CSC apoptosis and enhances chemotherapy efficacy, identifying the netrin-1/UNC5B/TFF3 axis as a promising therapeutic target to overcome stemness-driven resistance in metastatic colorectal cancer. Citation Format: Morgan Brisset, Kristina Radkova, Andrea Paradisi, Lea Stephan, Robin Wagner, Cyril Degletagne, Fabien Luiggi, Lisa Frydman, Alexander Heriot, Corina Behrenbruch, Tamara Vu, Frédéric Hollande, Patrick Mehlen. Netrin-1/UNC5B-TFF3 axis modulates cancer stem cells self-renewal and chemoresistance in metastatic colorectal cancer [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 822.

Abstract State of the art The extracellular matrix (ECM) is a complex and dynamic network composed of core structural proteins and matrisome-associated components. Under physiological conditions, ECM homeostasis contributes to the maintenance of natural tissue stiffness. However, disruption of this balance can lead to abnormal stiffening, a hallmark observed in various pathological conditions, including fibrosis and cancer. Our laboratory investigates the role of the dependence receptor ligand Netrin-1 (NTN1), a neuronal guidance protein predominantly expressed during early developmental stages. Intriguingly, NTN1 is frequently re-expressed in tumors, where its presence has been correlated with increased ECM stiffness. As such, our research aims to elucidate the role of NTN1 in ECM remodelling and stiffening. Methodology and resultsUsing atomic force microscopy (AFM) on inert Matrigel matrices, we observed a marked increase in stiffness upon supplementation with recombinant Netrin-1 (rNTN1). This stiffening effect was reversed by NP137, a monoclonal antibody targeting NTN1. Structural analyses via immunofluorescence staining for laminin-111 and rNTN1, alongside scanning electron microscopy (SEM), revealed a significant reduction in pore size in the presence of rNTN1. Complementary in silico analysis using PEPPI software indicated strong interactions between NTN1 and key ECM components such as Nidogen and Laminin. PerspectivesTo validate these predicted interactions, we performed biolayer interferometry with various ECM proteins. Furthermore, 3D cultures models of MCF10A normal mammary epithelial cells exposed to NTN1 displayed a phenotypic transition toward a more mesenchymal and invasive state. Interestingly, knockdown of NTN1 receptors did not prevent this phenotypic shift, suggesting a novel receptor-independent function for NTN1 in promoting cell invasiveness through direct modulation of ECM stiffness. Ongoing structural analyses are being conducted on fibroblast-derived matrices, with and without endogenous Netrin-1 expression, to evaluate its potential remodeling effects on collagen IV and fibronectin networks. In parallel, these matrices are utilized in functional assays to investigate how Netrin-1 ECM impact, influences cellular behaviours and transcriptional activity, using a range of experimental approaches. Citation Format: Gaëtan THIVOLLE LIOUX, Laurent Fattet, PATRICK MEHLEN, . Unraveling a new ECM stiffening function of the axon guidance molecule Netrin-1 [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 7489.

Among dependence receptors (DRs), which induce apoptosis when unbound by their cognate ligands, Kremen1 was initially reported to drive cancer cell death in the absence of DKK1. However, the precise mechanism of Kremen1-induced cell death remains unclear. In this study, we demonstrate that Kremen1 induces cell death with autophagic features, contrasting with the apoptotic process typically associated with DRs. Functional experiments using pharmacological inhibition of autophagy or genetic silencing of key autophagy effectors, confirmed this cell death process. Protein–protein proximity assays through biotin labeling identified SEC24C, a component of the COP-II complex, as a critical effector of this process. Moreover, the proximity between Kremen1, SEC24C and ATG9A after vesicular trafficking, fosters the proximity of SEC24C with ATG8, ERGIC and ATG9A, likely increasing the number of autophagosomes and vesicles leading to cell death. Given that the Kremen1/DKK1 pair is frequently altered in cancers, its aberrant induction should be monitored and may be targeted to offer an alternative strategy to treat cancers resistant to current therapies.

Introduction Radioligand therapy (RLT) has emerged as an effective therapeutic modality for the management of disseminated metastatic cancers. Current radiotheranostic strategies predominantly rely on biological vectors targeting membrane-bound, cancer-specific antigens. In this study, we identify the embryonic guidance cue netrin-1, an extracellular matrix-associated protein, as an unexpected and promising target for targeted alpha therapy (TAT). Netrin-1 is upregulated in multiple human malignancies, including metastatic breast and lung cancers, promoting tumor cell survival, proliferation and invasion. Accordingly, the monoclonal antibody NP137 was developed and has undergone initial safety and efficacy evaluation in several Phase I and II trials in patients with advanced solid tumors. Objectives We proposed that NP137 could be radiolabeled to determine its tumor accumulation and understand its pharmacodynamics. We therefore hypothesized that radiolabeling NP137 with alpha or beta emitters could overcome some of the resistance observed in monother Materials and Methods The expression of netrin-1 was evaluated in patient tumor sections by immunohistochemistry experiments. Preclinical models of mouse and human cancer cell lines were selected using immunoblots. Immuno-SPECT/CT imaging with either [111In]In-labeled NP137 or control IgG was performed at 24, 48, 72 and 96 hours in mice bearing subcutaneous netrin-1-positive tumors to assess tumor uptake and biodistribution. For therapeutic applications, NP137 underwent comprehensive characterization and its radiolabeling with Actinium-225 ([225Ac]Ac-DOTAGA-NP137) and Lutetium-177 ([177Lu]Lu-DOTA-NP137) was optimized. Efficacy studies were conducted 7 days after tumor engraftment using a single intravenous dose to assess treatment effects on tumor growth and median survival (MS) in mice bearing recalcitrant 4T1, EMT6 or H358 tumor models. Systemic toxicity was also evaluated by monitoring body weight, hematological parameters, and liver and renal function. Results Radiochemical purity >95% was achieved for all radionuclides following radiolabeling and purification. SPECT/CT imaging and ex vivo biodistribution of [111In]In-DOTAGA-NP137 in mice bearing 4T1 tumors demonstrated high tumor uptake, reaching 23.4 ± 6.9 %ID/g at 96 h post-injection. A single administration of either 15 kBq of [225Ac]Ac-DOTAGA-NP137 or 7 MBq of [177Lu]Lu-DOTA-NP137 induced significant tumor regression and significantly improved MS in disseminated 4T1, EMT6 and H358 models compared with non-radiolabeled controls, without significant body weight loss or hematological toxicity. Notably, [225Ac]Ac-DOTAGA-NP137 demonstrated superior therapeutic efficacy compared with its [177Lu]Lu-labeled counterpart in the H358 model. Conclusion These findings highlight NP137 as a promising radioimmunoconjugate for netrin-1–positive advanced solid tumors, with potential applications in both molecular imaging and RLT. Its high tumor specificity and uptake, combined with the strong therapeutic efficacy of this first alpha- and beta-emitting monoclonal antibody, support further evaluation and rapid clinical translation. Funding Acknowledgements This work was supported by the Junior Investigator grant (Jeunes Chercheuses et Jeunes Chercheurs), the Industrial Chair Program, and the LYriCAN Integrated Cancer Research Site, funded by the French National Cancer Institute.

e16441 Background: Pancreatic ductal adenocarcinoma is one of the most lethal malignancies with a 5-year survival rate under 5%. Locally advanced pancreatic ductal adenocarcinoma (LAP) represents 30-40% of cases at the diagnosis, with an overall survival around 15 months. Optimizing chemotherapy in LAP is still a huge challenge. A concomitant inhibition of epithelial mesenchymal transition (EMT) process may potentiate chemotherapy efficacy and decrease the development of resistances. Netrin-1 is upregulated in many metastatic cancers including 60% of pancreatic cancer and promotes tumor invasiveness and metastases development through EMT induction. NP137, a first-in-class anti-Netrin-1 monoclonal antibody, has shown in phase I study the ability to inhibit EMT, potentially overcoming resistance (Cassier et al., Nature, 2023). The goal of LAP-NET1 (NCT05546853) is to evaluate the safety and efficacy of the combination of modified FOLFIRINOX with anti-Netrin-1 targeting (NP137). Methods: LAP-NET1 is a phase 1b multicentric trial studying the combination of modified FOLFIRINOX with NP-137 every 2 weeks for 12 cycles in patients naive of systemic treatment with LAP according to National Comprehensive Cancer Network criteria. A safety lead-in phase will initially enroll 3-12 patients to confirm the recommended dose of NP137 (14 or 9 mg/kg) according to a 3+3 de-escalation protocol, followed by an expansion phase of 40 patients. The primary endpoint is the proportion of patients experiencing adverse events (AEs) of any grade and grade 3/4 AEs (CTCAE v 5.0) related to the experimental treatment at 6 months. Secondary endpoints are best overall objective response according to RECIST 1.1, 12-month progression-free survival (PFS-12m), 12-month overall survival (OS-12m), surgical resection rate, quality of life (EORTC QLQ-C30), time to deterioration and ancillary outcomes based on spatial transcriptomic and classic bulk RNA sequencing. Clinical trial information: NCT05546853 .

4197 Background: Pancreatic ductal adenocarcinoma (PDAC) is a highly lethal malignancy characterized by aggressive tumor dissemination and resistance to therapy; processes significantly driven by the epithelial-to-mesenchymal transition (EMT). Netrin-1 is a key regulator for EMT. NP137, an anti-netrin-1 antibody, has shown to inhibit EMT in preclinical models and in a phase 1 monotherapy trial. Methods: LAPNET-01 (NCT06203821) is a single arm phase Ib clinical study to assess the combination of NP137 with mFOLFIRINOX in naive locally advanced unresectable PDAC. A safety lead-in (3–12 pts, 3+3 design, NP137 14 vs 9 mg/kg) was followed by a 40-patient expansion. Treatment consisted of NP137 + mFOLFIRINOX every 2 weeks, up to 12 cycles. The primary endpoint was safety at 6 months (all-grade and grade 3/4 adverse events [AEs], CTCAE v5.0). Secondary endpoints included objective response rate (ORR), progression-free survival (PFS) and overall survival (OS), surgical conversion rate and exploratory transcriptomic analyses. Laser capture microdissection was performed on 22 pre-treatment and 6 surgery samples to allow microbulk RNA sequencing. Immunohistochemistry was also performed on pre-treatment samples. Results: A total of 43 patients were enrolled in this trial. NP137 was well tolerated. AE occurred in 100% of patients (58% grade ≥ 3). ORR and disease control rate were 29% and 95%. Median PFS was 10.9 months (95% CI, 10.0 – 15.6) and median OS was 16.4 months (95% CI, 12.8 – NR) with 21 patients still alive at time of the data cut-off. Surgery was made possible in 23% of patients. Microbulk RNA sequencing revealed that the main pathway downregulated with the combination mFOLFIRINOX+NP137 is EMT, bringing a clinical validation of the main mechanism of action of NP137. Moreover, patients with tumors expressing high levels of the netrin-1 receptor neogenin (high-NEO1) at baseline (both at the RNA and proteic level (IHC)) demonstrated an improved outcomes compared to the low-NEO1 subgroup including longer median PFS (15.7 vs 10.2 months, p = 0.01) and longer median OS (not reached vs 16.5, p = 0.024). These results are consistent with experimental data demonstrating the implication of NEO1 in PDAC EMT and its progression. Conclusions: NP137 in combination with mFOLFIRINOX demonstrates a favorable safety profile, promising clinical activity and a mechanistically distinct mode of action supported by translational analyses. These results warrant further investigation of netrin-1 blockade in randomized trials and provide a rationale for biomarker-driven development of NP137 in PDAC. Further work is ongoing to better characterize the distribution of NEO1 in first-line unresectable PDAC. Clinical trial information: NCT06203821 .

Netrin1, a developmental cue, is a master regulator of tumour epithelial-to-mesenchymal transition (EMT)1, a mechanism that is known to drive resistance to chemotherapy2. A netrin1 antibody (NP137)3 has been shown to inhibit tumour EMT in preclinical1 and clinical4 settings. In animal models of pancreatic cancer, netrin1 and its receptor neogenin have been shown to promote tumour progression5, EMT5 and metastasis6. Here we report the results of a phase 1b study that assesses the combination of NP137 with modified FOLFIRINOX (mFOLFIRINOX) in first line patients with locally advanced pancreatic cancer (ClinicalTrials.gov: NCT05546853 ). Forty-three patients were enrolled and received mFOLFIRINOX plus NP137 every other week for up to 12 cycles. NP137 was well tolerated. Median progression-free survival (PFS) was 10.85 months (95% confidence interval, 10.03-15.61) and median overall survival was 16.43 months (95% confidence interval, 12.75-non-reached), with 21 patients remaining alive at the time of data cut-off. Post-therapy conversion surgery occurred in 23% of patients. Laser capture microdissection was performed on pre-therapeutic biopsies and surgical specimens. Microbulk RNA sequencing confirmed that the main pathway that was down-regulated with the combination of mFOLFIRINOX plus NP137 was EMT. Moreover, survival outcomes were extended for patients with tumour cells that expressed high levels of the netrin1 receptor neogenin-median PFS 15.65 months in neogenin-high versus 10.22 months in neogenin low. Our results support the idea that netrin1 blockade alleviates resistance to chemotherapy by inhibiting EMT, particularly in neogenin-high pancreatic cancer.

Small intestinal neuroendocrine tumors (siNETs) are rare bowel tumors arising from malignant enteroendocrine cells, which normally regulate digestion throughout the intestine. Though infrequent, their incidence is rising through better diagnosis, fostering research into their origin and treatment. To date, siNETs are considered to be a single entity and are clinically treated as such. Here, by performing a multi-omics analysis of siNETs, we unveil four distinct molecular groups with strong clinical relevance and provide a resource to study their origin and clinical features. Transcriptomic, genetic and DNA methylation profiles identify two groups linked to distinct enteroendocrine differentiation patterns, another with a strong immune phenotype, and the last with mesenchymal properties. This latter subtype displays the worst prognosis and resistance to treatments in line with infiltration of cancer-associated fibroblasts. These data provide insights into the origin and diversity of these rare diseases, in the hope of improving clinical research into their management.

Netrin-1 signaling is an essential prototypical neuronal guidance mechanism during embryonic development that also regulates tumor cell survival in a variety of adult cancer entities. In line with these data, a monoclonal netrin-1 blocking antibody (anti-netrin-1 mAb/NP137) has been preclinically developed and netrin-1 blockade has recently been investigated in phase 1 and 2 clinical trials in several adult cancers. Here, we investigate the role of netrin-1 in the most common malignant pediatric brain cancer, Medulloblastoma. Interestingly, we find that netrin-1 is upregulated in medulloblastoma subgroups associated with developmental dysregulation, in particular in medulloblastoma with Sonic Hedgehog (SHH) activation. First, we demonstrate that genetic deletion of netrin-1 or systemic treatment with the clinical-stage anti-netrin-1 blocking antibody significantly reduces tumor growth in vivo in various orthotopic models of SHH medulloblastomas. Second, in vitro and in vivo, we unexpectedly uncover that SHH medulloblastomas treated with an SHH-inhibitor targeting Smoothened (SMO) increase netrin-1 expression, paving the way for combinatorial therapy. In line with that, we next show that netrin-1 blockade potentiates the efficacy of SMO inhibitor therapy in vivo. Together, our data indicate that, netrin-1 blockade, used as monotherapy or in combination with SMO inhibitors, is a promising therapeutic strategy in SHH medulloblastomas.

Supplementary Figure S1: Results of CRISPR screen to identify YAP effectors

The nervous system undergoes dynamic structural remodeling to infiltrate cancerous tumors, contributing to their growth and progression. Emerging evidence indicates that neuroplasticity initiates early, with nerve terminals detecting and responding to tissue changes even during precancerous stages. Notably, dense sympathetic axon sprouting has been observed around pancreatic intraepithelial neoplasia (PanIN), a common precursor lesion to pancreatic cancer. However, the molecular signals driving this early neuroplasticity and its functional consequences remain poorly understood. Here, we identify the axon guidance molecule Netrin-1 as a key factor secreted by pancreatic cells within precursor lesions of pancreatic cancer. Netrin-1 promotes sympathetic axon growth and branching through its receptor, Deleted in Colorectal Cancer (DCC). Inhibition of Netrin-1 disrupts sympathetic axon remodeling while accelerating PanIN formation and progression, driven by increased precancerous cell proliferation. Furthermore, human pancreatic tissue analysis corroborates Netrin-1 expression in precursor lesions. These findings suggest that Netrin-1-driven sympathetic neuroplasticity plays a protective role in the precancerous microenvironment by modulating local cellular dynamics, providing insights into early cancer progression.

Supplementary Figure S3: Regulation of UNC5 family and NTN1 mRNA by YAP in YAP-off vs YAP-on cancers

Dependence receptors (DRs) induce cell death by apoptosis in the absence of their ligand. Among them, Kremen1 was first described to induce cancer cell death in the absence of its ligand, DKK1. Nevertheless, the exact mechanism of Kremen1-triggered cell death remains unexplored. In this study, we demonstrate that Kremen1 induces cell death with autophagic features, contrasting with the apoptotic process typically associated with dependence receptors. Specifically, chemical inhibition of autophagy by spautin or wortmannin or genetic inhibition of ATG9A or ATG18B effectively blocks this cell death process. Furthermore, through a proximity protein interactome analysis of Kremen1, we identified Sec24C, a component of the COP-II complex, as important effector of Kremen1-induced autophagy and cell death. Our findings reveal an interaction between Sec24C and ATG8 regulated by Kremen1, potentially underlying the increased number of autophagosomes in cancer cells, leading to their death. This correlation between Kremen1 and the induction of aberrant autophagy deserves particular attention, especially as the Kremen1/DKK1 pair is frequently altered in cancers, with higher DKK1 expression associated with poor survival outcomes. Thus, targeting this pathway may offer a potential strategy for treating cancers resistant to current therapies. ### Competing Interest Statement The authors have declared no competing interest.

Tumor imaging or therapy using cancer specific carriers combined with a radioisotope has huge potential. In radiopharmaceutical development, it is important to ensure target specificity and minimal binding to normal tissues for both tumor detection and treatment. In previous studies, we identified CAPRIN-1 as a cancer-specific antigen, which is widely expressed on the cell surface membrane in many types of solid cancers, and created TRK-950, a humanized monoclonal antibody raised against CAPRIN-1, followed by conducting clinical development. In this proof-of-concept study, we prepared radiolabeled form of TRK-950 and investigated their potential as tumor imaging or therapeutic agents. An [111In]In-DOTA-TRK-950 was prepared and administered to tumor-bearing mice, and its tumor accumulation and pharmacokinetics were evaluated with SPECT/CT imaging. Next, the anti-tumor effect of a [177Lu]Lu-DOTA-TRK-950 was evaluated. Additionally, radiolabeled TRK-950-F(ab’)2, an antibody fragment of TRK-950, was similarly evaluated for their potential. At 72 h after administration of [111In]In-DOTA-TRK-950, tumor accumulation was high at 24.8

Supplementary Figure S2: Blocking Netrin does not affect YAP levels or YAP target gene induction

Supplementary Table S1: CRISPR Screen Results

Background Metastatic colorectal cancer (mCRC) is associated with high recurrence rates and resistance to conventional treatments, largely driven by cancer stem cells (CSCs) that contribute to tumor progression and therapeutic evasion. This study aims to investigate the role of netrin-1 and its dependence receptor UNC5B in regulating CSC self-renewal in mCRC and explore their potential as therapeutic targets. Methods We used patient-derived liver metastasis organoids (PDOs) to examine the effects of netrin-1 on CSC self-renewal. The role of UNC5B was evaluated by silencing its expression using CRISPR and assessing the impact on CSC apoptosis in response to an anti-netrin-1 blocking antibody (NP137) using extreme limiting dilution assays (ELDAs). Single-cell RNA sequencing was employed to explore the molecular mechanisms behind netrin-1/UNC5B regulation of CSC fate. Clinical data from a patient with mCRC were used to validate the findings. Results Netrin-1 promoted CSC self-renewal by inhibiting apoptosis, a process reversed by NP137. UNC5B was identified as the primary receptor mediating this effect, as its silencing eliminated Netrin-1-induced self-renewal. Trefoil Factor 3 (TFF3), secreted by UNC5B-expressing cells, plays a key role in netrin-1-induced CSC self-renewal. Clinical trial data from a patient with mCRC showed a reduction in TFF3 and stemness genes expression after treatment with NP137. Furthermore, combining NP137 with FOLFOX chemotherapy enhanced cell death and inhibited tumor growth in PDO xenograft models. Conclusion This study identifies the netrin-1/UNC5B/TFF3 axis as a critical regulator of CSC self-renewal in mCRC and suggests that targeting this pathway with NP137, in combination with chemotherapy, could provide a promising therapeutic approach for mCRC patients. ### Competing Interest Statement P.M. declares to have a competing interest as founder and shareholder of Netris Pharma. National Health and Medical Research Council, , GNT1164081

Inflammation plays a crucial role in cancer progression, but the relevance of the inflammasome remains unclear. Alu RNA was the first endogenous nucleic acid shown to activate the NLRP3 (nucleotide-binding domain leucine-rich repeat containing 3) inflammasome. Here, we showed that Alu RNA can induce epithelial-to-mesenchymal transition (EMT) through NLRP3 inflammasome activation and IL-1β release in colorectal cancer (CRC) cells. Alu RNA is stored, transported and transferred to CRC cells by exosomes. Exosomal Alu RNA promotes tumorigenesis by inducing invasion, metastasis and EMT via NLRP3 inflammasome activation. Consistent with these data, we found that significantly increased Alu RNA expression correlates with the induction of NLRP3 priming in human CRC patients. Furthermore, the level of Alu RNA in circulating exosomes correlates with CRC progression in a preclinical model. These findings reveal the direct involvement of Alu RNA in cancer pathogenesis, and its presence in CRC cell-derived exosomes could be used as a noninvasive diagnostic biomarker.