Regulator of G-protein signaling 5 (RGS5) protein: a novel marker of cancer vasculature elicited and sustained by the tumor’s proangiogenic microenvironment

We previously identified regulator of G-protein signaling 5 (RGS5) among several genes expressed by tumor-derived endothelial cells (EC). In this study, we provide the first in vivo/ex vivo evidence of RGS5 protein in the vasculature of ovarian carcinoma clinical specimens and its absence in human ovaries. Consistent with this, we show higher amounts of Rgs5 transcript in EC isolated from human cancers (as opposed to normal tissues) and demonstrate that expression is sustained by a milieu of factors typical of the proangiogenic tumor environment, including vascular endothelial growth factor (VEGF) and basic fibroblast growth factor (FGF-2). Supporting these findings, we show elevated levels of Rgs5 mRNA in the stroma from strongly (as opposed to weakly) angiogenic ovarian carcinoma xenografts and accordingly, we also show more of the protein associated to the abnormal vasculature. RGS5 protein predominantly colocalizes with the endothelium expressing platelet/endothelial cell adhesion molecule-1 (PECAM-1/CD31) and to a much lesser extent with perivascular/mural cells expressing platelet-derived growth factor receptor-beta (PDGFR-β) or alpha smooth muscle actin (αSMA). To toughen the relevance of the findings, we demonstrate RGS5 in the blood vessels of other cancer models endowed with a proangiogenic environment, such as human melanoma and renal carcinoma xenografts; to the contrary, it was undetectable in the vasculature of normal mouse tissues. RGS5 expression by the cancer vasculature triggered and retained by the proangiogenic microenvironment supports its exploitation as a novel biomarker and opens the path to explore new possibilities of therapeutic intervention aimed at targeting tumor blood vessels

Long-COVID in Patients with Cancer Previously Treated with Early Anti-SARS-CoV-2 Therapies in an Out-of-Hospital Setting: A Single-Center Experience

Simple Summary This article regards the incidence of long COVID symptoms in a cohort of patients with cancer with or without previous treatment with early therapies anti-SARS-CoV-2 in an out-of-hospital setting. The enrolled patients were invited to take part in the survey by telephone at least 12 weeks after COVID-19 diagnosis in order to evaluate the incidence of long COVID symptoms. To date, no papers have focused on the oncological population managed at home with the early anti-SARS-CoV-2 therapies. The overlap between the symptoms related to the oncological disease/oncological treatment and the symptoms of long COVID is one of the main future challenges that oncologists will have to manage. The incidence of long COVID in a cohort of patients with cancer with or without previous treatment with early therapies anti-SARS-CoV-2 in an out-of-hospital setting have to be elucidated. We prospectively enrolled all patients treated for a solid tumor at the department of Medical Oncology of the Fondazione IRCCS Policlinico San Matteo with a positive SARS-CoV-2 antigen or polymerase chain reaction test from January to September 2022 (Omicron surge). Ninety-seven patients answered the survey questions by telephone at least 12 weeks after COVID-19 diagnosis in order to evaluate the incidence of long COVID symptoms. Only twelve patients (12.4%) reported long COVID. No significant difference between early therapies anti-SARS-CoV-2 31 and long COVID (p = 0.443) was seen. The female sex (p = 0.024) and diabetes mellitus (p = 0.014) are significantly associated with long COVID. No statistically significant difference between the two groups (Long COVID vs. No Long COVID) according to the time to nasal swab viral clearance (p = 0.078). The overlap between the symptoms related to the oncological disease/oncological treatment and the symptoms of long COVID is one of the main future challenges that oncologists will have to manage

Investigation of Properties of Anti-Prostate Specific Membrane Antigen-targeted Gold Nanoparticles as Drug Carriers in Tumor Therapy

Specific targeted delivery and control drug release are desirable properties of a drug for tumor therapy. Nanomedicine, the science that studies the application of nanotechnology to disease treatment, might be of help. Targeted nanoparticles (NP) for their size and structure are able to enhance the accumulation in the tumor of encapsulated or linked / adsorbed molecules (gene, drug). We have therefore investigated the binding properties of gold-NPs (20 nm) conjugated to D2/B, a mAb recognizing the prostate specific membrane antigen (PMSA). PSMA for its wide distribution in prostate tumor (about 70-80% of patients are PSMA+), is an important biomarker in the management of this malignancy using targeted drugs. Gold-NPs have been synthesized by laser ablation, mixed with a reporter solution (Texas red) and treated with a thiol-PEG solution. Derivatization of PEG-COOH with EDC/Sulfo NHS has been used to covalently link the mAb to the NPs. D2/B-NP binding to LNCaP (PSMA+) cells has been assessed by cytometry. We have measured a MFI (mean fluorescence value) of 1,383 for D2/B-NP whereas the MFI of the negative control (CTRL-) was 68. The binding specificity was confirmed on PSMA– Jurkat cells (MFI of 121 and 101 for D2/B-NP and CTRL-, respectively). Binding and internalization of D2/B-NP in LNCaP cells were assayed by confocal microscopy; detection of D2/B-NP by surface-enhanced Raman scattering also revealed the selective binding of the NPs to Ag+ cells. The results of specific delivery and internalization support our idea to use mAb anti-PSMA targeted NPs to enhance the transport of toxic drugs in the tumo

PSMA-Specific CAR-Engineered T Cells Eradicate Disseminated Prostate Cancer in Preclinical Models

Immunology-based interventions have been proposed as a promising curative chance to effectively attack postoperative minimal residual disease and distant metastatic localizations of prostate tumors. We developed a chimeric antigen receptor (CAR) construct targeting the human prostate-specific membrane antigen (hPSMA), based on a novel and high affinity specific mAb. As a transfer method, we employed last-generation lentiviral vectors (LV) carrying a synthetic bidirectional promoter capable of robust and coordinated expression of the CAR molecule, and a bioluminescent reporter gene to allow the tracking of transgenic T cells after in vivo adoptive transfer. Overall, we demonstrated that CAR-expressing LV efficiently transduced short-term activated PBMC, which in turn were readily stimulated to produce cytokines and to exert a relevant cytotoxic activity by engagement with PSMA+ prostate tumor cells. Upon in vivo transfer in tumor-bearing mice, CAR-transduced T cells were capable to completely eradicate a disseminated neoplasia in the majority of treated animals, thus supporting the translation of such approach in the clinical setting

Effect of radiochemical modification on biodistribution of scFvD2B antibody fragment recognising prostate specific membrane antigen

Antibody-based reagents represent a promising strategy as clinical diagnostic tools. Prostate cancer (PCa) is the second-leading cause of death in males in the Western population. There is a presently unmet need for accurate diagnostic tool to localize and define the extent of both primary PCa and occult recurrent disease. One of the most suitable targets for PCa is the prostate-specific membrane antigen (PSMA) recognized by the monoclonal antibody D2B that we re-shaped into the single chain Fv (scFv format). Aim of this study was to evaluate in preclinical in vivo models the target specificity of scFvD2B after labelling with different radionuclides. 111In radiolabelling was performed via the chelator Bz-NOTA, and 131I radioiodination was performed using iodogen. The potential for molecular imaging and the biological behaviour of the radiolabelled scFvD2B were evaluated in mice bearing two subcutaneous PCa isogenic cell lines that differed only in PSMA expression. Biodistribution studies were performed at 3, 9, 15 and 24\u2009h after injection to determine the optimal imaging time point. A significant kidney accumulation, as percentage of injected dose of tissue (%ID/g), was observed for 111In-scFvD2B at 3\u2009h after injection (45% ID/g) and it was maintained up to 24\u2009h (26% ID/g). By contrast, kidney accumulation of 131I-scFvD2B was only marginally (0.3% ID/g at 24\u2009h). At the optimal time point defined between 15\u2009h and 24\u2009h, regardless of the radionuclide used, the scFvD2B was able to localize significantly better in the PSMA expressing tumours compared to the negative control; with 131I-scFvD2B yielding a significantly better target/background ratio compared to 111In-scFvD2B. These data suggest that, besides antigen specificity, chemical modification may affect antibody fragment biodistribution

Trends (2020-2022) toward Reduced Prevalence of Postcoronavirus Disease Syndrome and Improved Quality of Life for Hospitalized Coronavirus Disease 2019 Patients with Severe Infection and Venous Thromboembolism

The coronavirus disease 2019 (COVID-19) pandemic seems to be at its end. During the first outbreak, alfa was the dominant variant, and in the two following years, delta was the dominant variant. Questions remain about the prevalence and severity of post-COVID syndrome (PCS). We compared the medium-term outcomes of a selected group of patients considered at high risk for PCS: hospitalized patients with severe COVID-19 infection who presented clinical evidence of the acute onset of venous thromboembolism. Weighted Cox regression was used to estimate the adjusted hazard ratios for the risk of early and medium-term complications and quality of life (QoL) in COVID-19 patients developing acute venous thrombo-embolism according to the period of admission to the hospital. The primary outcome was the modification of QoL at a median follow-up of 24 months in patients hospitalized for COVID-19. The secondary outcome was the modification of QoL related to COVID-19 severity. The absolute risk of mortality for hospitalized COVID-19 patients was higher during the first outbreak (risk difference, 19% [95% confidence interval [CI], 16-22%]). Patients with acute onset of thromboembolism during the first outbreak had increased mortality, hospital stay, and need for intensive care unit treatment (p < 0.01). In patients who suffered from severe COVID-19 infection and thromboembolism in the following 2 years, symptoms during follow-up were less common and milder (risk difference 45% [95% CI, 40-52%]. In total, 19 patients were alive at 24 months follow-up: 12 patients (63%) reported important physical symptoms and 10 patients (52%) relevant emotional/mental symptoms. All patients reported reduced QoL in comparison with the preinfection time; in 15 patients (79%), the reduced QoL limited significantly their social and work activities. All patients reported permanent worsening of QoL after discharge from the hospital. Comparing the three different February to April interval years (2020, 2021, and 2022), patients reported a somewhat worse perception of health condition in comparison with the preinfection time, respectively, in 100, 79, and 56% respectively. The findings of our study show reduced prevalence and severity of PCS in the last 2 years. Less virulent variants, herd immunity, and vaccination may played a significant role

CAR development and validation.

<p>(A) Anti-hPSMA CAR map. EC: extracellular domain; TM: transmembrane domain; IC: intracellular domain. (B) Cytofluorimetric profile of CAR expression in transfected 293 T cells. 293 T cells were transfected with pcDNA3.1 vector bearing CAR sequence and analysed by flow cytometry after 24 hours (dark line). Untransfected cells (grey plot) served as control. (C) CAR expression as assessed by Western blotting at 24 hours (line 1) and 7 days (line 2) post 293 T cell transfection. Negative controls were the medium alone (line 3) and untransfected 293 T cells (line 4). Line 5, molecular weight markers. (D) Linear map of the recombinant viral vector containing the minCMVPGK bidirectional promoter (22). In particular, the reporter gene (eGFP or Luciferase) is under the control of minCMV promoter, while the CAR gene is under control of hPGK promoter. (E) Transduction of Jurkat cells with LV CAR anti-hPSMA/eGFP. Co-expression of c-myc and eGFP in LV-transduced Jurkat cells, as assessed by flow cytometry. Dot plot reports the events gated on total viable cells; more than 90% of cells co-express both c-myc and eGFP. (F) Transduction of Jurkat cells with LV CAR anti-hPSMA/Luciferase. <i>Left panel</i>, c-myc expression (black) in Jurkat cells at 72 h post transduction, as assessed by flow cytometry. Grey plot represents the isotype control. <i>Right panel</i>, Assessment of luciferase activity in LV-transduced Jurkat cells (2×10⁵/well) by BLI.</p