Next-generation Trop-2–targeted therapy against advanced cancers is hampered by expression of Trop-2 in normal tissues. We discovered that Trop-2 undergoes proteolytic activation by ADAM10 in cancer cells, leading to the exposure of a previously inaccessible protein groove flanked by two N-glycosylation sites. We designed a recognition strategy for this region, to drive selective cancer vulnerability in patients. Most undiscriminating anti–Trop-2 mAbs recognize a single immunodominant epitope. Hence, we removed it by deletion mutagenesis. Cancer-specific, glycosylation-prone mAbs were selected by ELISA, bio-layer interferometry, flow cytometry, confocal microscopy for differential binding to cleaved/activated, wild-type and glycosylation site–mutagenized Trop-2. The resulting 2G10 mAb family binds Trop-2–expressing cancer cells, but not Trop-2 on normal cells. We humanized 2G10 by state-of-the-art complementarity determining region grafting/re-modeling, yielding Hu2G10. This antibody binds cancer-specific, cleaved/activated Trop-2 with Kd < 10−12 mol/L, and uncleaved/wtTrop-2 in normal cells with Kd 3.16×10−8 mol/L, thus promising an unprecedented therapeutic index in patients. In vivo, Hu2G10 ablates growth of Trop-2–expressing breast, colon, prostate cancers, but shows no evidence of systemic toxicity, paving the way for a paradigm shift in Trop-2–targeted therapy.

Efficient isolation of neurons and glia from the human enteric nervous system (ENS) is challenging because of their rare and fragile nature. Here, we describe a staining panel to enrich ENS cells from the human intestine by fluorescence‐activated cell sorting (FACS). We find that CD56/CD90/CD24 co‐expression labels ENS cells with higher specificity and resolution than previous methods. Surprisingly, neuronal (CD24, TUBB3) and glial (SOX10) selective markers appear co‐expressed by all ENS cells. We demonstrate that this contradictory staining pattern is mainly driven by neuronal fragments, either free or attached to glial cells, which are the most abundant cell types. Live neurons can be enriched by the highest CD24 and CD90 levels. By applying our protocol to isolate ENS cells for single‐cell RNA sequencing, we show that these cells can be obtained with high quality, enabling interrogation of the human ENS transcriptome. Taken together, we present a selective FACS protocol that allows enrichment and discrimination of human ENS cells, opening up new avenues to study this complex system in health and disease.

Somatic mutations in ARID1A (AT-rich interactive domain-containing protein 1A) are present in approximately 25 % of bladder cancers (BC) and are associated with poor prognosis. With a view to discover effective treatment options for ARID1A-deficient BC patients, we set out to identify targetable effectors dysregulated consequent to ARID1A deficiency. Integrative analyses of ARID1A depletion in normal organoids and data mining in publicly available datasets revealed upregulation of DNA repair and cell cycle-associated genes consequent to loss of ARID1A and identified CHEK1 (Checkpoint kinase 1) and chromosomal passenger complex member BIRC5 (Baculoviral IAP Repeat Containing 5) as therapeutically drug-able candidate molecular effectors. Ex vivo treatment of patient-derived BC tumoroids with clinically advanced small molecule inhibitors targeting CHEK1 or BIRC5 was associated with increased DNA damage signalling and apoptosis, and selectively induced cell death in tumoroids lacking ARID1A protein expression. Thus, integrating public datasets with patient-derived organoid modelling and ex-vivo drug testing can uncover key molecular effectors and mechanisms of oncogenic transformation, potentially leading to novel therapeutic strategies. Our data point to ARID1A protein expression as a suitable candidate biomarker for the selection of BC patients responsive to therapies targeting BIRC5 and CHEK1.

Isolation of neurons and glia from the enteric nervous system (ENS) enables ex vivo studies, including the analysis of genomic and transcriptomic profiles. While we previously reported a fluorescence-activated cell sorting (FACS)-based isolation protocol for human ENS cells, no equivalent exists for mice. As directly applying the human protocol to mouse tissue resulted in low recovery of live ENS cells, we optimized tissue dissociation using mouse colons. A 30 min Liberase-based digestion showed optimal recovery of viable ENS cells, with CD56 and CD24 emerging as the most reliable markers to select and subdivide these cells. ENS’ identity was further validated by FACS, using neuronal (TUBB3) and glial (SOX10) markers and reverse transcriptase quantitative PCR on sorted fractions. Overall, the mouse ENS expression profile significantly overlapped with the human one, showing that current dissociation protocols yield a mixed population of enteric neurons and glia. Nonetheless, using the imaging flow cytometer BD S8 FACS Discover and ELAVL4 as a neuronal soma-associated marker, we observed enrichment of neurons in a CD56/CD24^TIP population. In conclusion, we present here a protocol for high-purity FACS-based isolation of viable mouse ENS cells, suitable for downstream applications.

Purpose: Anti-programmed death ligand-1 monoclonal antibody (anti-PD-L1 mAb) therapy has demonstrated notable clinical success. However, efficacy is often limited by transient interactions at the tumor-immune interface, limiting sustained immune activation. To address this, we hypothesized that liposomal delivery of anti-PD-L1 mAbs could enhance therapeutic efficacy. Nanosystems may increase binding avidity and prolong cell-surface retention, and enable multivalent antibody presentation through conjugation of multiple antibodies onto a single liposome. Methods: In this study, we engineered multivalent anti-PD-L1 liposomes (anti-PD-L1 LPs) by conjugating FDA-approved anti-PD-L1 mAbs (atezolizumab) at varying densities onto a clinically relevant liposomal formulation. The biophysical properties, cellular interactions, and therapeutic potential of anti-PD-L1 LPs were evaluated using two melanoma cell line models (BLM and MZ2Mel43), employing multiple analytical techniques. Results: Our results demonstrated that high-density anti-PD-L1 LPs exhibited superior binding avidity, prolonged membrane retention, and reduced non-specific cellular interaction with PD-L1-expressing cells compared to both low-density and non-targeted LPs. Additionally, in 3D tumor spheroid models, high-density anti-PD-L1 LPs showed deeper penetration, suggesting improved tissue accessibility compared with low-density formulations. Moreover, compared to free antibodies, anti-PD-L1 LPs displayed a higher association rate (k_on) and a significantly lower dissociation rate (k_off), resulting in an overall improved (lower) dissociation constant (K_D). Functional assays confirmed that anti-PD-L1 LPs achieved superior PD-L1 blockade compared to free antibodies. Importantly, in co-cultures of human peripheral blood mononuclear cells and tumor cells, anti-PD-L1 LPs maintained immunomodulatory activity comparable to free anti-PD-L1 antibodies. Conclusion: This study highlights the critical role of ligand density in enhancing binding strength, tumor retention, and tissue penetration of the anti-PD-L1 LP system. Our nanosystem offers a promising improvement over conventional anti-PD-L1 mAbs, supporting the broader application of this modular liposomal platform to other therapeutic antibodies in melanoma and other solid tumors. Plain Language Summary: Current limitations and our approach Cancer immunotherapy drugs like anti-PD-L1 antibodies help the immune system fight cancer, but they often do not stay in tumors long enough to work effectively. To improve this, we developed nanoscale lipid-based carriers, like tiny fat bubbles (called liposomes) densely coated with anti-PD-L1 antibodies. These engineered liposomes bind to tumors more effectively, stay longer, and deliver treatment more precisely than regular antibodies. Key discoveries We discovered that packing more drugs onto each tiny carrier makes them stick to tumors better and last longer. Also, how we deliver these drugs matters too—the carrier-delivered versions bind to tumors more effectively than regular antibody drugs. Importantly, in controlled lab tests with cells, these engineered antibodies work just as powerfully as regular immunotherapy—proving we have created a delivery system without sacrificing cancer-fighting strength. Conclusion and potential benefits This research introduces an innovative approach to cancer immunotherapy delivery using antibody-coated liposomes. By enhancing tumor targeting and retention while minimizing accumulation in healthy tissues, this method could improve treatment effectiveness while reducing side effects. The platform offers three key advantages: patients may require fewer treatment sessions with more durable results, clinicians gain enhanced options for treating melanoma and solid tumors, and researchers can extend this adaptable platform to other cancer therapies. Future directions Our immediate focus will be on validating these findings in animal models and expanding the technology to other immunotherapies. The use of clinically approved components positions this approach for potential rapid translation to human trials, pending successful preclinical results.