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.

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.

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.

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.

A Systematic Review and Meta-analysis on the Impact of Infertility on Men's General Health.

CONTEXT: Male infertility has been associated with increased morbidity and mortality. OBJECTIVE: To perform a systematic review and meta-analysis to provide the most critical evidence on the association between infertility and the risk of incident comorbidities in males. EVIDENCE ACQUISITION: A systematic review and meta-analysis was performed according to the Meta-analysis of Observational Studies in Epidemiology and Preferred Reporting Items for Systematic Reviews and Meta-analyses guidelines, and registered on PROSPERO. All published studies on infertile versus fertile men regarding overall mortality and risks of cancer, diabetes, and cardiovascular events were selected from a database search on PubMed, EMBASE, Google Scholar, and Cochrane. Forest plot and quasi-individual patient data meta-analysis were used for pooled analyses. A risk of bias was assessed using the ROBINS-E tool. EVIDENCE SYNTHESIS: Overall, an increased risk of death from any cause was found for infertile men (hazard risk [HR] 1.37, [95% confidence interval {CI} 1.04-1.81], p = 0.027), and a 30-yr survival probability of 91.0% (95% CI 89.6-92.4%) was found for infertile versus 95.9% (95% CI 95.3-96.4%) for fertile men (p < 0.001). An increased risk emerged of being diagnosed with testis cancer (relative risk [RR] 1.86 [95% CI 1.41-2.45], p < 0.001), melanoma (RR 1.30 [95% CI 1.08-1.56], p = 0.006), and prostate cancer (RR 1.66 [95% CI 1.06-2.61], p < 0.001). As well, an increased risk of diabetes (HR 1.39 [95% CI 1.09-1.71], p = 0.008), with a 30-yr probability of diabetes of 25.0% (95% CI 21.1-26.9%) for infertile versus 17.1% (95% CI 16.1-18.1%) for fertile men (p < 0.001), and an increased risk of cardiovascular events (HR 1.20 [95% CI 1.00-1.44], p = 0.049), with a probability of major cardiovascular events of 13.9% (95% CI 13.3-14.6%) for fertile versus 15.7% (95% CI 14.3-16.9%) for infertile men (p = 0.008), emerged. CONCLUSIONS: There is statistical evidence that a diagnosis of male infertility is associated with increased risks of death and incident comorbidities. Owing to the overall high risk of bias, results should be interpreted carefully. PATIENT SUMMARY: Male fertility is a proxy of general men's health and as such should be seen as an opportunity to improve preventive strategies for overall men's health beyond the immediate reproductive goals.

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.

Anti-Müllerian hormone predicts positive sperm retrieval in men with idiopathic non-obstructive azoospermia-findings from a multi-centric cross-sectional study.

STUDY QUESTION: Is it possible to identify a reliable marker of successful sperm retrieval (+SR) in men with idiopathic non-obstructive azoospermia (iNOA) undergoing microdissection testicular sperm extraction (mTESE)? SUMMARY ANSWER: A higher likelihood of +SR during mTESE is observed in men with iNOA and lower preoperative serum anti-Müllerian hormone (AMH) levels, with good predictive accuracy achieved using an AMH threshold of <4 ng/ml. WHAT IS KNOWN ALREADY: AMH has been previously linked to +SR in men with iNOA undergoing mTESE prior to ART. STUDY DESIGN, SIZE, DURATION: A multi-centre cross-sectional study was carried out with a cohort of 117 men with iNOA undergoing mTESE at three tertiary-referral centres. PARTICIPANTS/MATERIALS, SETTING, METHODS: Data from 117 consecutive white-European men with iNOA presenting for primary couple's infertility associated with a pure male factor at three centres were analysed. Descriptive statistics was applied to compare patients with negative (-SR) versus +SR at mTESE. Multivariate logistic regression models were fitted to predict +SR at mTESE, after adjusting for possible confounders. Diagnostic accuracy of the factors associated with +SR was assessed. Decision curve analyses were used to display the clinical benefit. MAIN RESULTS AND THE ROLE OF CHANCE: Overall, 60 (51.3%) men had an -SR and 57 (48.7%) had a +SR at mTESE. Patients with +SR had lower levels of baseline AMH (P = 0.005) and higher levels of estradiol (E2) (P = 0.01). At multivariate logistic regression analysis, lower levels of AMH (odds ratio: 0.79; 95% CI: 0.64-0.93, P = 0.03) were associated with +SR at mTESE, after adjusting for possible confounders (e.g. age, mean testicular volume, FSH, and E2). A threshold of AMH <4 ng/ml achieved the highest accuracy for +SR at mTESE, with an AUC of 70.3% (95% CI: 59.8-80.7). Decision curve analysis displayed the net clinical benefit of using an AMH <4 ng/ml threshold. LIMITATIONS, REASONS FOR CAUTION: There is a need for external validation in even larger cohorts, across different centres and ethnicities. Systematic reviews and meta-analysis to provide high level of evidence are lacking in the context of AMH and SR rates in men with iNOA. WIDER IMPLICATIONS OF THE FINDINGS: Current findings suggest that slightly more than one in two men with iNOA had -SR at mTESE. Overall, men with iNOA with lower levels of AMH had a significantly higher percentage of successful SR at surgery. A threshold of <4 ng/ml for circulating AMH ensured satisfactory sensitivity, specificity, and positive predictive values in the context of +SR at mTESE. STUDY FUNDING/COMPETING INTEREST(S): This work was supported by voluntary donations from the Urological Research Institute (URI). All authors declare no conflict of interest. TRIAL REGISTRATION NUMBER: N/A.

EDIT Software: A tool for the semi-automatic 3D reconstruction of bladder cancer and urinary bladder of animal models.

BACKGROUND AND OBJECTIVE: This study presents the EDIT software, a tool for the visualization of the urinary bladder anatomy in the 3D space and for its semi-automatic 3D reconstruction. METHODS: The inner bladder wall was computed by applying a Region of Interest (ROI) feedback-based active contour algorithm on the ultrasound images while the outer bladder wall was calculated by expanding the inner borders to approach the vascularization area on the photoacoustic images. The validation strategy of the proposed software was divided into two processes. Initially, the 3D automated reconstruction was performed on 6 phantom objects of different volume in order to compare the software computed volumes of the models with the true volumes of phantoms. Secondly, the in-vivo 3D reconstruction of the urinary bladder for 10 animals with orthotopic bladder cancer, which range in different stages of tumor progression was performed. RESULTS: The results showed that the minimum volume similarity of the proposed 3D reconstruction method applied on phantoms is 95.59%. It is noteworthy to mention that the EDIT software enables the user to reconstruct the 3D bladder wall with high precision, even if the bladder silhouette has been significantly deformed by the tumor. Indeed, by taking into account the dataset of the 2251 in-vivo ultrasound and photoacoustic images, the presented software performs segmentation with dice similarity 96.96% and 90.91% for the inner and the outer borders of the bladder wall, respectively. CONCLUSIONS: This study delivers the EDIT software, a novel software tool that uses ultrasound and photoacoustic images to extract different 3D components of the bladder.

Kidney function impairment in men with primary infertility: A case-control analysis.

BACKGROUND: Infertile men have a worse overall health status than their fertile counterparts. OBJECTIVE: We aimed to (1) compare kidney function in men presenting for primary couple's infertility with that of fertile men and (2) assess kidney function impairment toward sperm quality in infertile men. MATERIALS AND METHODS: In this case-control study, 387 consecutive white-European infertile men were matched by age with 134 same-ethnicity fertile men. Complete clinical and laboratory data were available for each patient. The Chronic Kidney Disease Epidemiology Collaboration function was used for estimated glomerular filtration rate calculation. Kidney functional impairment was defined as an estimated glomerular filtration rate <90 mL/min per 1.73 m2 , according to the Kidney Disease Improving Global Outcomes criteria. Multivariable logistic regression analysis was used to (1) assess the association between kidney function impairment and infertility status and (2) investigate the association between kidney function and semen analysis abnormalities in infertile men. RESULTS: After matching, 34 (8.8%) infertile men depicted at least a mild unknown impairment of kidney function compared to only four (3%) fertile men, with four (3%) of the infertile presenting with an overt kidney function impairment (estimated glomerular filtration rate <60 mL/min per 1.73 m2 ). There were no differences in terms of age, body mass index and rate of comorbidities between the two groups (all p > 0.05). After adjusting for major confounders, infertility status was associated with a higher risk of reduced estimated glomerular filtration rate (odds ratio 3.20; 95% confidence interval 1.21-5.2; p = 0.002). Conversely, estimated glomerular filtration rate was not associated with sperm abnormalities in infertile men. CONCLUSIONS: Mild kidney function impairment was found in 9% of asymptomatic and unaware men presenting for primary couple's infertility investigation. This novel finding corroborates growing data on a significant association of male infertility with a poorer overall male health status and the need for tailored preventive strategies.

A stapled chromogranin A-derived peptide homes in on tumors that express αvß6 or αvß8 integrins.

Rationale: The αvß6- and αvß8-integrins, two cell-adhesion receptors upregulated in many tumors and involved in the activation of the latency associated peptide (LAP)/TGFß complex, represent potential targets for tumor imaging and therapy. We investigated the tumor-homing properties of a chromogranin A-derived peptide containing an RGDL motif followed by a chemically stapled alpha-helix (called "5a"), which selectively recognizes the LAP/TGFß complex-binding site of αvß6 and αvß8. Methods: Peptide 5a was labeled with IRDye 800CW (a near-infrared fluorescent dye) or with 18F-NOTA (a label for positron emission tomography (PET)); the integrin-binding properties of free peptide and conjugates were then investigated using purified αvß6/αvß8 integrins and various αvß6/αvß8 single - or double-positive cancer cells; tumor-homing, biodistribution and imaging properties of the conjugates were investigated in subcutaneous and orthotopic αvß6-positive carcinomas of the pancreas, and in mice bearing subcutaneous αvß8-positive prostate tumors. Results: In vitro studies showed that 5a can bind both integrins with high affinity and inhibits cell-mediated TGFß activation. The 5a-IRDye and 5a-NOTA conjugates could bind purified αvß6/αvß8 integrins with no loss of affinity compared to free peptide, and selectively recognized various αvß6/αvß8 single- or double-positive cancer cells, including cells from pancreatic carcinoma, melanoma, oral mucosa, bladder and prostate cancer. In vivo static and dynamic optical near-infrared and PET/CT imaging and biodistribution studies, performed in mice with subcutaneous and orthotopic αvß6-positive carcinomas of the pancreas, showed high target-specific uptake of fluorescence- and radio-labeled peptide by tumors and low non-specific uptake in other organs and tissues, except for excretory organs. Significant target-specific uptake of fluorescence-labeled peptide was also observed in mice bearing αvß8-positive prostate tumors. Conclusions: The results indicate that 5a can home to αvß6- and/or αvß8-positive tumors, suggesting that this peptide can be exploited as a ligand for delivering imaging or anticancer agents to αvß6/αvß8 single- or double-positive tumors, or as a tumor-homing inhibitor of these TGFß activators.

Gold nanorods assisted photothermal therapy of bladder cancer in mice: A computational study on the effects of gold nanorods distribution at the centre, periphery, and surface of bladder cancer.

BACKGROUND AND OBJECTIVES: Gold nanorod-assisted photothermal therapy (GNR-PTT) is a cancer treatment whereby GNRs incorporated into the tumour act as photo-absorbers to elevate the thermal destruction effect. In the case of bladder, there are few possible routes to target the tumour with GNRs, namely peri/intra-tumoural injection and intravesical instillation of GNRs. These two approaches lead to different GNR distribution inside the tumour and can affect the treatment outcome. METHODOLOGY: The present study investigates the effects of heterogeneous GNR distribution in a typical setup of GNR-PTT. Three cases were considered. Case 1 considered the GNRs at the tumour centre, while Case 2 represents a hypothetical scenario where GNRs are distributed at the tumour periphery; these two cases represent intratumoural accumulation with different degree of GNR spread inside the tumour. Case 3 is achieved when GNRs target the exposed tumoural surface that is invading the bladder wall, when they are delivered by intravesical instillation. RESULTS: Results indicate that for a laser power of 0.6 W and GNR volume fraction of 0.01%, Case 2 and 3 were successful in achieving complete tumour eradication after 330 and 470 s of laser irradiation, respectively. Case 1 failed to form complete tumour damage when the GNRs are concentrated at the tumour centre but managed to produce complete tumour damage if the spread of GNRs is wider. Results from Case 2 also demonstrated a different heating profile from Case 1, suggesting that thermal ablation during GNR-PTT is dependant on the GNRs distribution inside the tumour. Case 3 shows similar results to Case 2 whereby gradual but uniform heating is observed. Cases 2 and 3 show that uniformly heating the tumour can reduce damage to the surrounding tissues. CONCLUSIONS: Different GNR distribution associated with the different methods of introducing GNRs to the bladder during GNR-PTT affect the treatment outcome of bladder cancer in mice. Insufficient spreading during intratumoural injection of GNRs can render the treatment ineffective, while administered via intravesical instillation. GNR distribution achieved through intravesical instillation present some advantages over intratumoural injection and is worthy of further exploration.

Noninvasive detection of any-stage cancer using free glycosaminoglycans.

Cancer mortality is exacerbated by late-stage diagnosis. Liquid biopsies based on genomic biomarkers can noninvasively diagnose cancers. However, validation studies have reported ~10% sensitivity to detect stage I cancer in a screening population and specific types, such as brain or genitourinary tumors, remain undetectable. We investigated urine and plasma free glycosaminoglycan profiles (GAGomes) as tumor metabolism biomarkers for multi-cancer early detection (MCED) of 14 cancer types using 2,064 samples from 1,260 cancer or healthy subjects. We observed widespread cancer-specific changes in biofluidic GAGomes recapitulated in an in vivo cancer progression model. We developed three machine learning models based on urine (Nurine = 220 cancer vs. 360 healthy) and plasma (Nplasma = 517 vs. 425) GAGomes that can detect any cancer with an area under the receiver operating characteristic curve of 0.83-0.93 with up to 62% sensitivity to stage I disease at 95% specificity. Undetected patients had a 39 to 50% lower risk of death. GAGomes predicted the putative cancer location with 89% accuracy. In a validation study on a screening-like population requiring ≥ 99% specificity, combined GAGomes predicted any cancer type with poor prognosis within 18 months with 43% sensitivity (21% in stage I; N = 121 and 49 cases). Overall, GAGomes appeared to be powerful MCED metabolic biomarkers, potentially doubling the number of stage I cancers detectable using genomic biomarkers.

Early diagnosis of bladder cancer by photoacoustic imaging of tumor-targeted gold nanorods.

Detection and removal of bladder cancer lesions at an early stage is crucial for preventing tumor relapse and progression. This study aimed to develop a new technological platform for the visualization of small and flat urothelial lesions of high-grade bladder carcinoma in situ (CIS). We found that the integrin α5ß1, overexpressed in bladder cancer cell lines, murine orthotopic bladder cancer and human bladder CIS, can be exploited as a receptor for targeted delivery of GNRs functionalized with the cyclic CphgisoDGRG peptide (Iso4). The GNRs@Chit-Iso4 was stable in urine and selectively recognized α5ß1 positive neoplastic urothelium, while low frequency ultrasound-assisted shaking of intravesically instilled GNRs@Chit-Iso4 allowed the distribution of nanoparticles across the entire volume of the bladder. Photoacoustic imaging of GNRs@Chit-Iso4 bound to tumor cells allowed for the detection of neoplastic lesions smaller than 0.5 mm that were undetectable by ultrasound imaging and bioluminescence.

Lysyl-Oxidase Dependent Extracellular Matrix Stiffness in Hodgkin Lymphomas: Mechanical and Topographical Evidence.

PURPOSE: The biochemical composition and architecture of the extracellular matrix (ECM) is known to condition development and invasiveness of neoplasms. To clarify this point, we analyzed ECM stiffness, collagen cross-linking and anisotropy in lymph nodes (LN) of Hodgkin lymphomas (HL), follicular lymphomas (FL) and diffuse large B-cell lymphomas (DLBCL), compared with non-neoplastic LN (LDN). METHODS AND RESULTS: We found increased elastic (Young's) modulus in HL and advanced FL (grade 3A) over LDN, FL grade 1-2 and DLBCL. Digital imaging evidenced larger stromal areas in HL, where increased collagen cross-linking was found; in turn, architectural modifications were documented in FL3A by scanning electron microscopy and enhanced anisotropy by polarized light microscopy. Interestingly, HL expressed high levels of lysyl oxidase (LOX), an enzyme responsible for collagen cross-linking. Using gelatin scaffolds fabricated with a low elastic modulus, comparable to that of non-neoplastic tissues, we demonstrated that HL LN-derived mesenchymal stromal cells and HL cells increased the Young's modulus of the extracellular microenvironment through the expression of LOX. Indeed, LOX inhibition by ß-aminopropionitrile prevented the gelatin stiffness increase. CONCLUSIONS: These data indicate that different mechanical, topographical and/or architectural modifications of ECM are detectable in human lymphomas and are related to their histotype and grading.

Inhibitors of ADAM10 reduce Hodgkin lymphoma cell growth in 3D microenvironments and enhance brentuximab-vedotin effect.

Shedding of ADAM10 substrates, like TNFa or CD30, can affect both anti-tumor immune response and antibody-drug-conjugate (ADC)-based immunotherapy. We have published two new ADAM10 inhibitors, LT4 and MN8 able to prevent such shedding in Hodgkin lymphoma (HL). Since tumor tissue architecture deeply influences the outcome of anti-cancer treatments, we set up a new threedimensional (3D) culture systems to verify whether ADAM10 inhibitors can contribute to, or enhance, the anti-lymphoma effects of the ADC brentuximab-vedotin (BtxVed). In order to recapitulate some aspects of lymphoma structure and architecture, we assembled two 3D culture models: mixed spheroids made of HL lymph node (LN) mesenchymal stromal cells (MSC) and Reed Sternberg/Hodgkin lymphoma cells (HL cells) or collagen scaffolds repopulated with LN-MSC and HL cells. In these 3D systems we found that: i) the ADAM10 inhibitors LT4 and MN8 reduce ATP content or glucose consumption, related to cell proliferation, increasing lactate dehydrogenase release as a cell damage hallmark; ii) these events are paralleled by mixed spheroids size reduction and inhibition of CD30 and TNFa shedding; iii) the effects observed can be reproduced in repopulated HL LN-derived matrix or collagen scaffolds; iv) ADAM10 inhibitors enhance the anti-lymphoma effect of the anti-CD30 ADC BtxVed both in conventional cultures and in repopulated scaffolds. Thus, we provide evidence for a direct and combined antilymphoma effect of ADAM10 inhibitors with BtxVed, leading to the improvement of ADC effects; this is documented in 3D models recapitulating features of the LN microenvironment, that can be proposed as a reliable tool for anti-lymphoma drug testing.

Risk of health status worsening in primary infertile men: A prospective 10-year follow-up study.

BACKGROUND: A severe male infertility factor has been associated with both lower health status and increased mortality in infertile men. OBJECTIVES: To investigate reproductive factors associated with health status impairment in infertile men over a 10-year time frame since the first clinical evaluation. MATERIALS AND METHODS: Data from 899 infertile men were analysed at baseline between 2003 and 2010. Health-significant comorbidities were scored with the Charlson Comorbidity Index. Patients were followed up yearly recording any worsening in their health status until 2019. Cox regression models were used to estimate hazard ratios and 95% confidence intervals of Charlson Comorbidity Index score increase. RESULTS: At a median follow-up of 136 months (Interquartile range: 121, 156), 85 men (9.5%) depicted an increase of their baseline Charlson Comorbidity Index score of at least one point. The most frequent reason for Charlson Comorbidity Index upgrade was cancer (34%), cardiovascular diseases (29%) and diabetes mellitus (22%). Compared to patients without a Charlson Comorbidity Index increase, patients with a Charlson Comorbidity Index increase presented with higher body mass index and follicle-stimulating hormone values, a higher rate of baseline Charlson Comorbidity Index ≥ 1 (all p < 0.01) and a greater proportion of non-obstructive azoospermia (p < 0.001). In the Cox regression model, the patient's BMI (p < 0.001), baseline Charlson Comorbidity Index ≥ 1 (p < 0.01) and azoospermia status (p = 0.001) were found to be independently associated with Charlson Comorbidity Index increases. CONCLUSIONS: Almost 10% of men presenting for primary infertility had a decrease of the overall health status already in the relatively short 10-year time frame after the first presentation. Non-obstructive azoospermic men showed the worst health status impairment and should be strictly followed-up regardless of their fertility status.

Leiomyoadenomatoid tumours of the epididymis: A new case report and review of the literature.

Benign tumours of the epididymis are rare, and the most common tumour types include adenomatoid tumours, representing more than half of all cases, and leiomyomas. Here, we reported a case of leiomyoadenomatoid tumours of the epididymis, a very rare, benign histological entity with only few cases described in the English literature, which have been reviewed and summarised. Clinically, the lesion presented as a solitary mass growing at the level of the tail of the right epididymis. After the intraoperative frozen section analysis revealed a benign adenomatoid lesion, the mass was enucleated with a conservative surgery sparing the testis. This case highlights the importance for both pathologists and urologists to be aware of these rare, but benign, tumours, to avoid misdiagnosis, especially in the setting of frozen intraoperative consultation, or primary radical surgical procedures, as radical orchiepididymectomy without frozen section consultation.

A numerical study to investigate the effects of tumour position on the treatment of bladder cancer in mice using gold nanorods assisted photothermal ablation.

Gold nanorods assisted photothermal therapy (GNR-PTT) is a new cancer treatment technique that has shown promising potential for bladder cancer treatment. The position of the bladder cancer at different locations along the bladder wall lining can potentially affect the treatment efficacy since laser is irradiated externally from the skin surface. The present study investigates the efficacy of GNR-PTT in the treatment of bladder cancer in mice for tumours growing at three different locations on the bladder, i.e., Case 1: closest to skin surface, Case 2: at the bottom half of the bladder, and Case 3: at the side of the bladder. Investigations were carried out numerically using an experimentally validated framework for optical-thermal simulations. An in-silico approach was adopted due to the flexibility in placing the tumour at a desired location along the bladder lining. Results indicate that for the treatment parameters considered (laser power 0.3 W, GNR volume fraction 0.01% v/v), only Case 1 can be used for an effective GNR-PTT. No damage to the tumour was observed in Cases 2 and 3. Analysis of the thermo-physiological responses showed that the effectiveness of GNR-PTT in treating bladder cancer depends not only on the depth of the tumour from the skin surface, but also on the type of tissue that the laser must pass through before reaching the tumour. In addition, the results are reliant on GNRs with a diameter of 10 nm and an aspect ratio of 3.8 - tuned to exhibit peak absorption for the chosen laser wavelength. Results from the present study can be used to highlight the potential for using GNR-PTT for treatment of human bladder cancer. It appears that Cases 2 and 3 suggest that GNR-PTT, where the laser passes through the skin to reach the bladder, may be unfeasible in humans. While this study shows the feasibility of using GNRs for photothermal ablation of bladder cancer, it also identifies the current limitations needed to be overcome for an effective clinical application in the bladder cancer patients.

A mechanistic insight into the anti-metastatic role of the prostate specific antigen.

AIM: Since its discovery Prostate Specific Antigen (PSA), also referred to as kallikrein-3 (KLK3), has been used as standard circulating biomarker for prostate cancer (PCa). However, its specificity remains not adequate and its mechanism of action still elusive. Therefore, deciphering PSA role throughout PCa-pathobiology would be relevant in improving both cancer diagnosis and outcome prediction. We investigated the possible role played by PSA on/in the tumor microenvironment and over the first steps of cancer invasion. METHODS: Fresh PCa-specimens and cell lines were used for ex-vivo/in-vitro invasion assays and assessment of prostate tissue-PSA (tPSA), type 1 collagen (COL1A1) and ß1-integrin expression. Tissue Cancer Genome Atlas (TCGA) and Decipher® datasets were considered to estimate tPSA clinical relevance. RESULTS: A more precise, inverse, correspondence between tPSA and clinical/pathological parameters was found than for circulating PSA. KLK3 combined with Gleason grade and pathologic stage, better predicted cancer-related mortality. Consistently, we demonstrated that PSA inhibits prostate extracellular-matrix (ECM) invasion by PCa cells. As for the mechanism of action, we provided novel information that PSA is able to cleave COL1A1, a main component of the ECM. Finally, ß1-integrin, a crucial COL1A1 transducing-receptor involved in tumor adhesion/invasion, resulted to be downregulated in PCa specimens with higher levels of tPSA. CONCLUSIONS: By interfering with type 1 collagen and its downstream targets, PSA may hamper adhesion and path of the cancer cells through ECM and their migration ability, thus explaining the inverse correlation highlighted between prostate tPSA levels and clinically significant disease.

Is There a Detrimental Effect of Antibiotic Therapy in Patients with Muscle-invasive Bladder Cancer Treated with Neoadjuvant Pembrolizumab?

In locally advanced and metastatic malignancies, antibiotic (ATB) therapy has a negative effect on immunotherapy efficacy. Therefore, we aimed to evaluate whether ATB therapy and use of specific ATB classes with concomitant neoadjuvant pembrolizumab affected pathologic complete response (ypT0N0) and relapse-free survival (RFS) for patients with clinical T2-4N0M0 bladder cancer enrolled in the PURE-01 study. Of the 149 patients evaluated, 48 (32%) received any concomitant ATB therapy. The ATB class most commonly administered was fluoroquinolones (16 patients; 33%). In the ATB cohort, seven patients (15%) achieved ypT0N0 status, compared to 50 (50%; p < 0.001) in the untreated group. Moreover, ATB use was negatively associated with ypT0N0 status (odds ratio 0.18, 95% confidence interval [CI] 0.05-0.48; p = 0.001). The 24-mo RFS rate was 63% (95% CI 48-83%) in the ATB group versus 90% (95% CI 83-97) in the untreated group. We found that ATB use was associated with a higher recurrence rate (hazard ratio [HR] 2.64, 95% CI 1.08-6.50; p = 0.03). Exploratory analyses showed that fluoroquinolone use was associated with a higher recurrence rate (HR 3.28, 95% CI 1.12-9.60; p = 0.03). Our study revealed an association between ATB use and neoadjuvant immunotherapy efficacy in an intention-to-cure population, highlighting the need for future studies to better investigate this relationship. PATIENT SUMMARY: The efficacy of immunotherapy for cancer is influenced by several patient and tumor factors, including the use of antibiotics. We found that antibiotics taken at the same time as immunotherapy drugs were associated with lower rates of complete response and of recurrence-free survival among patients with muscle-invasive bladder cancer. These findings need to be confirmed in future studies.