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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.

Supplementary Online Material

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.

Rosaria Di Lorenzo,1 Anita Iorio,2 Margherita Pinelli,3 Federica Magarini,3 Mattia Marchi,3 Andrea Sacchetti,3 Chiara Calogero,4 Gian Maria Galeazzi,5 Paola Ferri,5 Sergio Rovesti,5 Alessandro Minarini6 1Service of Psychiatric Diagnosis and Care, Department of Mental Health and Pathological Addictions, AUSL-Modena, Modena, Italy; 2Psychiatric Rehabilitation Technique Programme, University of Modena and Reggio Emilia, Reggio Emilia, Italy; 3School of Psychiatry, University of Modena and Reggio Emilia, Modena, Italy; 4Community Mental Health Center, Vignola (MO), Department of Mental Health and Pathological Addictions, AUSL-Modena, Modena, Italy; 5Department of Biomedical, Metabolic and Neural Sciences, University of Modena and Reggio Emilia, Modena, Italy; 6Community Mental Health Center, Vignola (MO), Department of Mental Health and Pathological Addictions, AUSL-Modena, Modena, MO, ItalyCorrespondence: Rosaria Di Lorenzo, Department of Mental Health and Pathological Addictions, AUSL-Modena, Service of Psychiatric Diagnosis and Care, via P. Giardini n. 1355, Baggiovara (MO), 41126, Italy, Email r.dilorenzo@ausl.mo.itPurpose: Antipsychotic long-acting injections (AP-LAIs) are indicated for patients affected by schizophrenia especially those with poor treatment adherence.Patients and Methods: To compare paliperidone palmitate 3-monthly (PP3M), paliperidone palmitate one-monthly (PP1M) and haloperidol decanoate (HAL-D) treatment, we enrolled 90 patients with schizophrenia treated in Mental Health Center with one of the three AP-LAIs for at least six months and followed them for another 6 months. At 6 and 12 months of treatment we administered Clinical Global Impression-Severity, Global Assessment of Functioning and World Health Organization Quality of Life-26 items (WHOQOL-BREF). At 1-year treatment, we evaluated relapses (psychiatric hospitalizations and urgent consultations), side effects and drop-outs.Results: We did not highlight any statistically significant difference among the three treatments in relapses and scale scores. Weight increase was significantly higher in PP1M and PP3M groups. Twelve patients (13.3%) discontinued AP-LAI. At 1-year AP-LAI treatment, 69% of patients rated quality of life as “good” or “very good” and 71% declared themselves to be “satisfied” or “very satisfied”.Conclusion: HAL-D, PP1M and PP3M 1-year treatments were similarly effective in preventing relapses and improving quality of life and health satisfaction. All discontinuations in the new 3-monthly antipsychotic treatment were caused by patient refusal to continue it.Keywords: antipsychotic long-acting injections, schizophrenia spectrum disorders, relapses, quality of life, health satisfaction

Trop-2 drives the recruitment of Akt to the cell membrane via its PH domain.

Supplementary Methods: Protein microarrays,Multiplex Western blotting,Confocal time-lapse microscopy, Western blotting, Immunohistochemistry, 3D structure analysis; Supplementary Table 3:Breast cancer case series clinical and pathological parameters; the file also contains legends for Supplementary Figures 1-6.

Trop-2 proteome profiling.

Purpose: Inhibition of AKT is a key target area for personalized cancer medicine. However, predictive markers of response to AKT inhibitors are lacking. Correspondingly, the AKT-dependent chain of command for tumor growth, which will mediate AKT-dependent therapeutic responses, remains unclear.Experimental Design: Proteomic profiling was utilized to identify nodal hubs of the Trop-2 cancer growth–driving network. Kinase-specific inhibitors were used to dissect Trop-2–dependent from Trop-2–independent pathways. In vitro assays, in vivo preclinical models, and case series of primary human breast cancers were utilized to define the mechanisms of Trop-2–driven growth and the mode of action of Trop-2–predicted AKT inhibitors.Results: Trop-2 and AKT expression was shown to be tightly coordinated in human breast cancers, with virtual overlap with AKT activation profiles at T308 and S473, consistent with functional interaction in vivo. AKT allosteric inhibitors were shown to only block the growth of Trop-2–expressing tumor cells, both in vitro and in preclinical models, being ineffective on Trop-2–null cells. Consistently, AKT-targeted siRNA only impacted on Trop-2–expressing cells. Lentiviral downregulation of endogenous Trop-2 abolished tumor response to AKT blockade, indicating Trop-2 as a mandatory activator of AKT.Conclusions: Our findings indicate that the expression of Trop-2 is a stringent predictor of tumor response to AKT inhibitors. They also support the identification of target-activatory pathways, as efficient predictors of response in precision cancer therapy. Clin Cancer Res; 22(16); 4197–205. ©2016 AACR.

Trop-2-associated functions driven by Akt-phosphorylation

Proteomic analysis of the Trop-2 signal transduction network

This video shows MTE 4-14 cells cotransfected with Akt PH domain-EGFP (green) and Trop-2-mRFP1 (red). Recruitment and colocalization of Akt PH domain-EGFP with Trop-2-mRFP1 at the cell membrane was found to be quantitative and dynamic. The time lapse covers a period of about 27 min.

Trop-2-dependent Akt signaling.

Supplemental Figure 4: Trop-2-independent cell growth reduction by inhibitors of mTOR, NF-kB and JNK; Supplementary Figure 5: Dose-response curves for signaling pathway inhibitors; Supplementary Figure 6: Correlated expression of Trop-2, phospho-T308 Akt and phospho-S473 Akt in primary breast cancer

Supplementary Figure S3. Growth analysis of endometrial cancer cells in the presence/absence of Cisplatin and IL6 pathway inhibitors.

Supplementary Table S2. Summary of serial transplantation assays of primary endometrial cancer cells.

Online Supplementary Materials and Methods

Legends to Supplementary Figures

Paneth cells (PCs), responsible for the secretion of antimicrobial peptides in the small intestine and for niche support to Lgr5+ crypt-base columnar stem cells (CBCs), have been shown to respond to inflammation by dedifferentiating into stem-like cells in order to sustain a regenerative response1,2. Therefore, PCs may represent the cells-of-origin of intestinal cancer in the context of inflammation. To test this hypothesis, we targeted Apc, Kras, and Tp53 mutations in Paneth cells by Cre-Lox technology and modelled inflammation by dextran sodium sulfate (DSS) administration. PC-specific loss of Apc resulted in multiple small intestinal tumors, whereas Kras or Tp53 mutations did not. Compound Apc and Kras mutations in PCs resulted in a striking increase in tumor multiplicity even in the absence of the inflammatory insult. By combining scRNAseq with lineage tracing to capture the conversion of PCs into bona fide tumor cells, we show that they progress through a “revival stem cell” (RSC) state characterized by high Clusterin (Clu) expression and Yap1 signaling, reminiscent of what has been previously observed upon irradiation of the mouse digestive tract3. Accordingly, comparison of PC- and Lgr5-derived murine intestinal tumors revealed differences related to Wnt signaling and inflammatory pathways which match the dichotomy of CBCs and injury-induced RSCs4 between human sporadic colon cancers and those arising in the context of inflammatory bowel diseases. Last, we show that western-style dietary habits, known to trigger a low-grade inflammation throughout the intestinal tract, underlie the analogous de-differentiation of Paneth cells and their acquisition of stem-like features. Taken together, our results show that intestinal cancer arises in the context of inflammation through the dedifferentiation of committed secretory lineages such as Paneth cells and the activation of the revival stem cell state. As such, a true quiescent stem cell identity may be hidden in fully committed and post-mitotic lineages which, upon inflammation, support the regenerative response by re-entering the cell cycle and dedifferentiating into RSCs. The chronic nature of the tissue insult in inflammatory bowel diseases and even in the context of western-style dietary habits is likely to result in the expansion of cell targets for tumor initiation and progression.

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.

Nanoscopic, millisecond-lasting membrane platforms are modeled to constitute main functional signaling units. However, multiple signaling events in cell growth induction, including receptor Tyr-kinase (RTK) stimulation, calcium signaling, cytoplasmic kinase recruitment, operate over much broader space-time frames. Through dynamic confocal/super-resolution/electron-microscopy on living cells, we discovered that signaling competence is acquired through growth factor-induced coalescence of activated growth inducers (RTK, CD9, CD81, CD98, Co-029, CD316, Trop-1, Trop-2, PI3K, PI4K), and downstream effectors (Grb-2, PKCα, ERK, Akt, Src, Syk, ILK, ezrin) into confined segments of the cell membrane (‘docks’), that recursively assemble over hundreds of seconds. Average size (25±5 μm; ~80,000 individual analyses) and lifetime (222±25 sec) of docks extend by orders of magnitude previously recognized space-time frames for cell signaling. Activated/phosphorylated kinases clustered at membrane docks (99% P-Erb-B4; 91% P-Ret; 90% P-PKCα; 90% P-Src). STED super-resolution microscopy revealed up to 10 µm2 signal transducer areas of effector colocalization. Docks >1.0 µm2 accounted for ~90% of colocalizations versus ≈1% of raft-sized domains (HGF, IGF-1>>SCF/VEGF), were inhibited by Tyr kinase and cytoskeleton inhibitors, and were shown to induce cancer cell proliferation. Citation Format: Saverio Alberti, Valeria Relli, Romina Tripaldi, Pasquale Simeone, Emanuela Guerra, Andrea Sacchetti, Paolo Ciufici, Antonino Moschella, Valeria Caiolfa, Moreno Zamai, Marco Trerotola. Growth factor-induced cell membrane macroplatforms drive protein kinase signaling for transformed cell growth [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 2550.

In this paper we give a sharper condition for the blow-up of the solution to a nonlinear Schroedinger equation with free/Stark/quadratic potential by improving the well known Zakharov-Glassey's method., 16 pages

Trop-2 is a transmembrane signal transducer that is overexpressed in most human cancers, and drives malignant progression. To gain knowledge on the higher-order molecular mechanisms that drive Trop-2 signaling, we applied next-generation sequencing, proteomics, and high-resolution microscopy to models and primary cases of human colorectal cancer (CRC). We had previously shown that Trop-2 induces a Ca2+ signal. We reveal here that Trop-2 binds the cell membrane Na+/K+-ATPase, and that clustering of Trop-2 induces an intracellular Ca2+ rise followed by membrane translocation of PKCα, which in turn phosphorylates the Trop-2 cytoplasmic tail. This feed-forward signaling is promoted by the binding of Trop-2 to the PKCα membrane-anchor CD9. CRISPR-based inactivation of CD9 in CRC cells shows that CD9 is required by Trop-2 for recruiting PKCα and cofilin-1 to the cell membrane. This induces malignant progression through proteolytic cleavage of E-cadherin, remodeling of the β-actin cytoskeleton, and activation of Akt and ERK. The interaction between Trop-2 and CD9 was validated in vivo in murine models of CRC growth and invasion. Overexpression of the components of this Trop-2-driven super-complex significantly worsened disease-free and overall survival of CRC patients, supporting a pivotal relevance in CRC malignant progression. Our findings demonstrate a previously unsuspected layer of cancer growth regulation, which is dormant in normal tissues, and is activated by Trop-2 in cancer cells.

INTRODUCTION: Desmoid-type fibromatosis (DTF) is a rare, soft tissue tumour. Sorafenib, a multikinase inhibitor, has demonstrated antitumour efficacy in DTF patients. Little is known about the underlying molecular mechanisms, which are crucial to know to further optimize systemic treatments. Here we investigated the molecular effects of sorafenib exposure on DTF and stromal cells, with an emphasis on cell death mechanisms.MATERIAL AND METHODS: DTF primary cell cultures, with known CTNNB1 status, and primary stromal cell cultures, derived from DTF tissue, were exposed to clinically relevant concentrations of sorafenib in the presence or absence of inhibitors of ferroptosis, apoptosis and autophagy. Cell viability was determined after 24 and 48 h using MTT assays. Annexin V/PI staining, lipid peroxidation analysis and immunoblotting were performed to assess apoptosis, ferroptosis and autophagy.RESULTS: Exposure to sorafenib caused a significant, concentration- and time-dependent decrease in cell viability in all primary DTF and stromal cell cultures. Inhibitors of ferroptosis and apoptosis protected against sorafenib-mediated cytotoxicity implicating that both cell death mechanisms are activated. Annexin V/PI stainings and lipid peroxidation analyses confirmed induction of apoptosis and ferroptosis, respectively. Autophagy inhibition enhanced the cytotoxic effect of sorafenib and led to a stronger induction of apoptosis and ferroptosis.CONCLUSION: This study identified ferroptosis and apoptosis as mechanisms for the sorafenib induced cell death in DTF cells as well as stromal cells. Furthermore, autophagy inhibition enhanced the cytotoxic effects of sorafenib. Knowledge of the mechanisms by which sorafenib affects DTF at a cellular level may help to optimize its clinical efficacy and mitigate toxic effects.

Trop-2 is a transmembrane signal transducer that is overexpressed in most human cancers, and drives malignant progression. To gain knowledge on the higher-order molecular mechanisms that drive Trop-2 signaling, we applied next-generation sequencing, proteomics, and high-resolution microscopy to models and primary cases of human colorectal cancer (CRC). We had previously shown that Trop-2 induces a Ca

Phenotypic plasticity represents the most relevant hallmark of the carcinoma cell as it bestows it with the capacity of transiently altering its morphological and functional features while en route to the metastatic site. However, the study of phenotypic plasticity is hindered by the rarity of these events within primary lesions and by the lack of experimental models. Here, we identified a subpopulation of phenotypic plastic colon cancer cells: EpCAMlo cells are motile, invasive, chemo-resistant, and highly metastatic. EpCAMlo bulk and single-cell RNAseq analysis indicated (1) enhanced Wnt/β-catenin signaling, (2) a broad spectrum of degrees of epithelial to mesenchymal transition (EMT) activation including hybrid E/M states (partial EMT) with highly plastic features, and (3) high correlation with the CMS4 subtype, accounting for colon cancer cases with poor prognosis and a pronounced stromal component. Of note, a signature of genes specifically expressed in EpCAMlo cancer cells is highly predictive of overall survival in tumors other than CMS4, thus highlighting the relevance of quasi-mesenchymal tumor cells across the spectrum of colon cancers. Enhanced Wnt and the downstream EMT activation represent key events in eliciting phenotypic plasticity along the invasive front of primary colon carcinomas. Distinct sets of epithelial and mesenchymal genes define transcriptional trajectories through which state transitions arise. pEMT cells, often earmarked by the extracellular matrix glycoprotein SPARC together with nuclear ZEB1 and β-catenin along the invasive front of primary colon carcinomas, are predicted to represent the origin of these (de)differentiation routes through biologically distinct cellular states and to underlie the phenotypic plasticity of colon cancer cells.

In this talk we discuss some recent results I obtained for a class of nonlinear models in quantum mechanics. In particular we focus our attention to the nonlinear one-dimensional Schrodinger equation with a periodic potential and a Stark-type perturbation. In the limit of large periodic potential the Stark–Wannier ladders of the linear equation become a dense energy spectrum because a cascade of bifurcations of stationary solutions occurs; for a detailed treatment we refer to Sacchetti (Phys Rev E 95:062212, 2017, SIAM J Math Anal 50(6):5783–5810, 2018) where this model has been studied.

In order to identify suitable therapeutic targets for glioma anti-angiogenic therapy, the process of neovascularization mediated by circulating angiogenic cells (CACs) needs to be scrutinized. In the present study we compared the expression of neovascularization-related genes by three circulating CAC subsets (HPCs, CD34+ and KDR+ cells; internal controls: PBMCs and circulating endothelial cells) of treatment-naïve patients with glioblastoma (GBM) to those of patients undergoing reactive neovascularization (myocardial infarction (MI). CACs from umbilical cord (representing developmental neovascularization) and healthy subjects served as controls. Fluorescent activated cell sorting was used to isolate CACs, RT-PCR to determine the expression levels of a panel of 48 neovascularization-related genes, Luminex assays to measure plasma levels of 21 CAC-related circulating molecules. We found essential differences in gene expression between GBM and MI CACs. GBM CACs had a higher expression of pro-angiogenic factors (esp. KITL, CXCL12 and JAG1); growth factor and chemotactic receptors (IGF1R, TGFbR2, CXCR4 and CCR2); adhesion receptor monomers (ITGA5 and ITGA6) and matricellular factor POSTN. In addition, we found major differences in the levels of neovascularization-related plasma factors. A strong positive correlation between plasma MMP9 levels and expression of CXCR4 in the CAC subset of hematopoietic progenitor cells (HPCs) was found in GBM patients. Our findings indicate that CAC-mediated neovascularization in GBM is characterized by more efficient CAC homing to target tissue and a more potent pro-angiogenic response than in physiologic tissue repair in MI. Our findings can aid in selecting targets for therapeutic strategies acting against GBM-specific CACs.