Deep Flow Cytometry Unveils Distinct Immune Cell Subsets in Inducible T Cell Co-Stimulator Ligand (ICOSL)- and ICOS-Knockout Mice during Experimental Autoimmune Encephalomyelitis.

The inducible T cell co-stimulator ligand (ICOSL), expressed by antigen presenting cells, binds to the inducible T cell co-stimulator (ICOS) on activated T cells. Improper function of the ICOS/ICOSL pathway has been implicated in several autoimmune diseases, including multiple sclerosis (MS). Previous studies showed that ICOS-knockout (KO) mice exhibit severe experimental autoimmune encephalomyelitis (EAE), the animal model of MS, but data on ICOSL deficiency are not available. In our study, we explored the impact of both ICOS and ICOSL deficiencies on MOG35-55 -induced EAE and its associated immune cell dynamics by employing ICOSL-KO and ICOS-KO mice with a C57BL/6J background. During EAE resolution, MOG-driven cytokine levels and the immunophenotype of splenocytes were evaluated by ELISA and multiparametric flow cytometry, respectively. We found that both KO mice exhibited an overlapping and more severe EAE compared to C57BL/6J mice, corroborated by a reduction in memory/regulatory T cell subsets and interleukin (IL-)17 levels. It is noteworthy that an unsupervised analysis showed that ICOSL deficiency modifies the immune response in an original way, by affecting T central and effector memory (TCM, TEM), long-lived CD4+ TEM cells, and macrophages, compared to ICOS-KO and C57BL/6J mice, suggesting a role for other binding partners to ICOSL in EAE development, which deserves further study.

A simple and robust nanosystem for photoacoustic imaging of bladder cancer based on α5ß1-targeted gold nanorods.

BACKGROUND: Early detection and removal of bladder cancer in patients is crucial to prevent tumor recurrence and progression. Because current imaging techniques may fail to detect small lesions of in situ carcinomas, patients with bladder cancer often relapse after initial diagnosis, thereby requiring frequent follow-up and treatments. RESULTS: In an attempt to obtain a sensitive and high-resolution imaging modality for bladder cancer, we have developed a photoacoustic imaging approach based on the use of PEGylated gold nanorods (GNRs) as a contrast agent, functionalized with the peptide cyclic [CphgisoDGRG] (Iso4), a selective ligand of α5ß1 integrin expressed by bladder cancer cells. This product (called GNRs@PEG-Iso4) was produced by a simple two-step procedure based on GNRs activation with lipoic acid-polyethyleneglycol(PEG-5KDa)-maleimide and functionalization with peptide Iso4. Biochemical and biological studies showed that GNRs@PEG-Iso4 can efficiently recognize purified integrin α5ß1 and α5ß1-positive bladder cancer cells. GNRs@PEG-Iso4 was stable and did not aggregate in urine or in 5% sodium chloride, or after freeze/thaw cycles or prolonged exposure to 55 °C, and, even more importantly, do not settle after instillation into the bladder. Intravesical instillation of GNRs@PEG-Iso4 into mice bearing orthotopic MB49-Luc bladder tumors, followed by photoacoustic imaging, efficiently detected small cancer lesions. The binding to tumor lesions was competed by a neutralizing anti-α5ß1 integrin antibody; furthermore, no binding was observed to healthy bladders (α5ß1-negative), pointing to a specific targeting mechanism. CONCLUSION: GNRs@PEG-Iso4 represents a simple and robust contrast agent for photoacoustic imaging and diagnosis of small bladder cancer lesions.

Stool Microbiome Signature Associated with Response to Neoadjuvant Pembrolizumab in Patients with Muscle-invasive Bladder Cancer.

Neoadjuvant pembrolizumab has been shown to be a valid treatment for patients affected by muscle-invasive bladder cancer (MIBC), as demonstrated in the PURE-01 clinical trial (NCT02736266). Among the tumor-extrinsic factors influencing immunotherapy efficacy, extensive data highlighted that the microbiome is a central player in immune-mediated anticancer activity. This report aimed to investigate the composition and role of stool microbiome in patients enrolled in the PURE-01 clinical trial. An orthotopic animal model of bladder cancer (MB49-Luc) was used to support some of the findings from human data. An analysis of stool microbiome before pembrolizumab was conducted for 42 patients, of whom 23 showed a pathologic response. The information in the preclinical model of orthotopic bladder cancer treated with anti-PD-1 antibody or control isotype was validated. Linear discriminant analysis effect size and linear models were used to identify the bacterial taxa enriched in either responders or nonresponders. The identified taxa were also tested for their association with event-free survival (EFS). Survival at 31 d after tumor instillation was used as the study endpoint in the preclinical model. Responders and nonresponders emerged to differ in terms of enrichment for 16 bacterial taxa. Of these, the genus Sutterella was enriched in responders, while the species Ruminococcus bromii was enriched in nonresponders. The negative impact of R. bromii on anti-PD-1 antibody activity was also observed in the preclinical model. EFS and survival of the preclinical model showed a negative role of R. bromii. We found different stool bacterial taxa associated with the response or lack of response to neoadjuvant pembrolizumab. Moreover, we provided experimental data about the negative role of R. bromii on immunotherapy response. Further studies are needed to externally validate our findings and provide mechanistic insights about the host-pathogen interactions in MIBC. PATIENT SUMMARY: Using prepembrolizumab stool samples collected from patients enrolled in the PURE-01 clinical trials, we identified some bacterial taxa that were enriched in patients who either responded or did not respond to immunotherapy. Using an animal model of bladder cancer, we gathered further evidence of the negative impact of the Ruminococcus bromii on immunotherapy efficacy. Further studies are needed to confirm the current findings and test the utility of these bacteria as predictive markers of immunotherapy response.

Topographic modification of the extracellular matrix precedes the onset of bladder cancer.

Background: Non-muscle invasive bladder cancer (NMIBC) patients are affected by a high risk of recurrence. The topography of collagen fibers represents a hallmark of the neoplastic extracellular microenvironment. Objective: Assess the topographic change associated with different stages of bladder cancer (from neoplastic lesions to bona fide tumor) and whether those changes favour the development of NMIBC. Design Setting and Participants: Seventy-one clinical samples of urothelial carcinoma at different stages were used. Topographic changes preceding tumor onset and progression were evaluated in the rat bladder cancer model induced by nitrosamine (BBN), a bladder-specific carcinogen. The preclinical model of actinic cystitis was also used in combination with BBN. Validated hematoxylin-eosin sections were used to assess the topography of collagen fibrils associated with pre-tumoral steps, NMIBC, and MIBC. Findings: Linearization of collagen fibers was higher in Cis and Ta vs. dysplastic urothelium, further increased in T1 and greatest in T2 tumors. In the BBN preclinical model, an increase in the linearization of collagen fibers was established since the beginning of inflammation, such as the onset of atypia of a non-univocal nature and dysplasia, and further increased in the presence of the tumor. Linearization of collagen fibers in the model of actinic cystitis was associated with earlier onset of BBN-induced tumor. Conclusions: The topographic modification of the extracellular microenvironment occurs during the inflammatory processes preceding and favoring the onset of bladder cancer. The topographic reconfiguration of the stroma could represent a marker for identifying and treating the non-neoplastic tissue susceptible to tumor recurrence.

Progressive alteration of murine bladder elasticity in actinic cystitis detected by Brillouin microscopy.

Bladder mechanical properties are critical for organ function and tissue homeostasis. Therefore, alterations of tissue mechanics are linked to disease onset and progression. This study aims to characterize the tissue elasticity of the murine bladder wall considering its different anatomical components, both in healthy conditions and in actinic cystitis, a state characterized by tissue fibrosis. Here, we exploit Brillouin microscopy, an emerging technique in the mechanobiology field that allows mapping tissue mechanics at the microscale, in non-contact mode and free of labeling. We show that Brillouin imaging of bladder tissues is able to recognize the different anatomical components of the bladder wall, confirmed by histopathological analysis, showing different tissue mechanical properties of the physiological bladder, as well as a significant alteration in the presence of tissue fibrosis. Our results point out the potential use of Brillouin imaging on clinically relevant samples as a complementary technique to histopathological analysis, deciphering complex mechanical alteration of each tissue layer of an organ that strongly relies on mechanical properties to perform its function.

Contrast enhanced photoacoustic detection of fibrillar collagen in the near infrared region-I.

Fibrillar collagen accumulation emerges as a promising biomarker in several diseases, such as desmoplastic tumors and unstable atherosclerotic plaque. Gold nanorods (GNRs) hold great potential as contrast agents in high-resolution, biomedically safe, and non-invasive photoacoustic imaging (PAI). This study presents the design and characterization of a specialized imaging tool which exploits GNR assisted targeted photoacoustic imaging that is tailored for the identification of fibrillar collagen. In addition to the photoacoustic characterization of collagen in the NIR 1 and 2 regions, we demonstrate the detailed steps of conjugating a decoy to GNRs. This study serves as a proof of concept, that demonstrates that conjugated collagenase-1 (MMP-1) generates a distinct and collagen-specific photoacoustic signal, facilitating real-time visualization in the wavelength range of 700-970 nm (NIR I). As most of the reported studies utilized the endogenous contrast of collagen in the NIR II wavelength that has major limitations to perform in vivo deep tissue imaging, the approach that we are proposing is unique and it highlights the promise of MMP-1 decoy-functionalized GNRs as novel contrast agents for photoacoustic imaging of collagen in the NIR 1 region. To our knowledge this is the first time functionalized GNRs are optimized for the detection of fibrillar collagen and utilized in the field of non-invasive photoacoustic imaging that can facilitate a better prognosis of desmoplastic tumors and broken atherosclerotic plaques.

Extracellular vesicles from human plasma for biomarkers discovery: Impact of anticoagulants and isolation techniques.

Extracellular vesicles (EVs) isolated from plasma are increasingly recognized as promising circulating biomarkers for disease discovery and progression, as well as for therapeutic drug delivery. The scientific community underlined the necessity of standard operative procedures for the isolation and storage of the EVs to ensure robust results. The understanding of the impact of the pre-analytical variables is still limited and some considerations about plasma anticoagulants and isolation methods are necessary. Therefore, we performed a comparison study between EVs isolated by ultracentrifugation and by affinity substrate separation from plasma EDTA and sodium citrate. The EVs were characterized by Nano Tracking Analysis, Western Blot, cytofluorimetric analysis of surface markers, and lipidomic analysis. While anticoagulants did not significantly alter any of the analyzed parameters, the isolation methods influenced EVs size, purity, surface markers expression and lipidomic profile. Compared to ultracentrifugation, affinity substrate separation yielded bigger particles highly enriched in tetraspanins (CD9, CD63, CD81), fatty acids and glycerolipids, with a predominant LDL- and vLDL-like contamination. Herein, we highlighted that the isolation method should be carefully evaluated prior to study design and the need of standardized operative procedures for EVs isolation and application to biomarkers discovery.

Worse Disease Prognosis Is Associated to an Increase of Platelet-Derived Extracellular Vesicles in Hospitalized SARS-CoV-2 Patients.

Platelet-derived extracellular vesicles (PLT-EVs), the most abundant circulating EVs, have been found to be increased in several human diseases, including viral infections. Recently, we documented that PLT-EV counts are higher in SARS-CoV-2+ patients, enrolled during the first two waves of COVID-19, occurred in Italy last year, and we suggested PLT-EVs as a biomarker of SARS-CoV-2 infection. The present study is aimed at testing the ability of PLT-EV levels, measured at hospital admission and within one week of hospitalization, to predict patient's outcome. We applied an easy, fast, and reliable method, based on flow cytometry, for the detection of PLT-EVs in unmanipulated blood samples. In a cohort of SARS-CoV-2 patients, enrolled during the third wave of COVID-19 in Italy, we confirmed that PLT-EV counts are higher in comparison to healthy controls. Moreover, their number is not affected by prehospitalization treatment neither with heparin nor with steroids that are recommended by WHO guidelines. Noteworthy, we identified two pattern of patients, those who increased their PTL-EV level during first week and those reducing it. The former group representented more compromised patients, with higher 4C score, and unfavorable outcome. In conclusion, our new findings would suggest that a worse evolution of the disease is linked with increasing PLT-EV levels in the week after hospital admission.

Causal contributors to tissue stiffness and clinical relevance in urology.

Mechanomedicine is an emerging field focused on characterizing mechanical changes in cells and tissues coupled with a specific disease. Understanding the mechanical cues that drive disease progression, and whether tissue stiffening can precede disease development, is crucial in order to define new mechanical biomarkers to improve and develop diagnostic and prognostic tools. Classically known stromal regulators, such as fibroblasts, and more recently acknowledged factors such as the microbiome and extracellular vesicles, play a crucial role in modifications to the stroma and extracellular matrix (ECM). These modifications ultimately lead to an alteration of the mechanical properties (stiffness) of the tissue, contributing to disease onset and progression. We describe here classic and emerging mediators of ECM remodeling, and discuss state-of-the-art studies characterizing mechanical fingerprints of urological diseases, showing a general trend between increased tissue stiffness and severity of disease. Finally, we point to the clinical potential of tissue stiffness as a diagnostic and prognostic factor in the urological field, as well as a possible target for new innovative drugs.