Rationale: Intercellular tight junctions are crucial for correct regulation of the endothelial barrier. Their composition and integrity are affected in pathological contexts, such as inflammation and tumor growth. JAM-A (junctional adhesion molecule A) is a transmembrane component of tight junctions with a role in maintenance of endothelial barrier function, although how this is accomplished remains elusive. Objective: We aimed to understand the molecular mechanisms through which JAM-A expression regulates tight junction organization to control endothelial permeability, with potential implications under pathological conditions. Methods and Results: Genetic deletion of JAM-A in mice significantly increased vascular permeability. This was associated with significantly decreased expression of claudin-5 in the vasculature of various tissues, including brain and lung. We observed that C/EBP-α (CCAAT/enhancer-binding protein-α) can act as a transcription factor to trigger the expression of claudin-5 downstream of JAM-A, to thus enhance vascular barrier function. Accordingly, gain-of-function for C/EBP-α increased claudin-5 expression and decreased endothelial permeability, as measured by the passage of fluorescein isothiocyanate (FITC)-dextran through endothelial monolayers. Conversely, C/EBP-α loss-of-function showed the opposite effects of decreased claudin-5 levels and increased endothelial permeability. Mechanistically, JAM-A promoted C/EBP-α expression through suppression of β-catenin transcriptional activity, and also through activation of EPAC (exchange protein directly activated by cAMP). C/EBP-α then directly binds the promoter of claudin-5 to thereby promote its transcription. Finally, JAM-A–C/EBP-α–mediated regulation of claudin-5 was lost in blood vessels from tissue biopsies from patients with glioblastoma and ovarian cancer. Conclusions: We describe here a novel role for the transcription factor C/EBP-α that is positively modulated by JAM-A, a component of tight junctions that acts through EPAC to up-regulate the expression of claudin-5, to thus decrease endothelial permeability. Overall, these data unravel a regulatory molecular pathway through which tight junctions limit vascular permeability. This will help in the identification of further therapeutic targets for diseases associated with endothelial barrier dysfunction. Graphic An graphic abstract is available for this article.

Identifying tumor cells infiltrating normal-appearing brain tissue is critical to achieve a total glioma resection. Raman spectroscopy (RS) is an optical technique with potential for real-time glioma detection. Most RS reports are based on formalin-fixed or frozen samples, with only a few studies deployed on fresh untreated tissue. We aimed to probe RS on untreated brain biopsies exploring novel Raman bands useful in distinguishing glioma and normal brain tissue. Sixty-three fresh tissue biopsies were analyzed within few minutes after resection. A total of 3450 spectra were collected, with 1377 labelled as Healthy and 2073 as Tumor. Machine learning methods were used to classify spectra compared to the histo-pathological standard. The algorithms extracted information from 60 different Raman peaks identified as the most representative among 135 peaks screened. We were able to distinguish between tumor and healthy brain tissue with accuracy and precision of 83% and 82%, respectively. We identified 19 new Raman shifts with known biological significance. Raman spectroscopy was effective and accurate in discriminating glioma tissue from healthy brain ex-vivo in fresh samples. This study added new spectroscopic data that can contribute to further develop Raman Spectroscopy as an intraoperative tool for in-vivo glioma detection.

Isocitrate dehydrogenase (IDH) mutational status is pivotal in the management of gliomas. Patients with IDH-mutated (IDH-MUT) tumors have a better prognosis and benefit more from extended surgical resection than IDH wild-type (IDH-WT). Raman spectroscopy (RS) is a minimally invasive optical technique with great potential for intraoperative diagnosis. We evaluated the RS’s ability to characterize the IDH mutational status onto unprocessed glioma biopsies. We extracted 2073 Raman spectra from thirty-eight unprocessed samples. The classification performance was assessed using the eXtreme Gradient Boosted trees (XGB) and Support Vector Machine with Radial Basis Function kernel (RBF-SVM). Measured Raman spectra displayed differences between IDH-MUT and IDH-WT tumor tissue. From the 103 Raman shifts screened as input features, the cross-validation loop identified 52 shifts with the highest performance in the distinction of the two groups. Raman analysis showed differences in spectral features of lipids, collagen, DNA and cholesterol/phospholipids. We were able to distinguish between IDH-MUT and IDH-WT tumors with an accuracy and precision of 87%. RS is a valuable and accurate tool for characterizing the mutational status of IDH mutation in unprocessed glioma samples. This study improves RS knowledge for future personalized surgical strategy or in situ target therapies for glioma tumors.

Luminal-like breast cancer (BC) constitutes the majority of BC subtypes, but, differently from highly aggressive triple negative BC, is poorly infiltrated by the immune system. The quality of the immune infiltrate in luminal-like BCs has been poorly studied, thereby limiting further investigation of immunotherapeutic strategies. By using high-dimensional single-cell technologies, we identify heterogeneous behavior within the tissue-resident memory CD8+ T (Trm) cells infiltrating luminal-like tumors. A subset of CD127− CD39^hi Trm cells, preferentially present in the tumor compared to the adjacent normal breast tissue or peripheral blood, retains enhanced degranulation capacity compared to the CD127+ CD39^lo Trm counterpart ex vivo, and is specifically associated with positive prognosis. Nevertheless, such prognostic benefit is lost in the presence of highly-suppressive CCR8^hi ICOS^hi IRF4+ effector Tregs. Thus, combinatorial strategies aiming at boosting Trm function and infiltration while relieving from Treg-mediated immunosuppression should be investigated to achieve proper tumor control in luminal-like BCs.

Neo-adjuvant chemotherapy (NAC) is the treatment of choice for patients with locally advanced breast cancer (BC). In luminal-like BC, the decision to administer NAC remains controversial. The purpose of this study was to describe the clinical characteristics, treatment, and oncological outcomes of luminal-like, node positive, BC patients treated with NAC, and to identify independent predictive factors for treatment. All consecutive patients with luminal-like, node positive BC who underwent NAC were retrospectively reviewed. Pathologic complete response (pCR) was defined as no invasive or in situ residual tumor in both breast and axillary nodes (ypT0N0). A total of 205 luminal-like, node positive BC patients underwent NAC. Overall, 34 (16.6%) patients showed pCR, 86 (42.0%) patients underwent breast-conserving surgery (BCS), 119 (58.0%) patients underwent mastectomy, 130 (63.4%) patients underwent axillary lymph node dissection (ALND) without prior sentinel lymph node biopsy (SLNB), and 75 (36.6%) patients underwent breast surgery plus SLNB. Pathologic CR to NAC (29.1% vs 7.6% if no pCR, odds ratio = 2.866, 95% confidence interval = 1.296-6.341, p = 0.009) was found to significantly increase the probability to receive BCS. There was no significant difference in terms of disease-free and overall survival between patients with luminal-like, node positive BC receiving BCS or mastectomy (p = 0.596, p = 0.134, respectively), and ALND or SLNB only (p = 0.661, p = 0.856, respectively). Luminal-like, node positive BC presents low pCR rates after NAC. Pre-operative chemotherapy increases the rate of BCS. Pathologic CR has emerged as an independent predictive factor for BCS. In patients with axillary pCR, SLNB is an acceptable procedure not associated with worse oncological outcomes.

Backgrounds: The majority of breast cancer (BC) patients treated with neo-adjuvant chemotherapy (NAC) achieves a pathologic partial response with different patterns of residual disease. No clear correlation between these patterns and oncological results was described. Our aims were to define the predictive factors for different patterns of residual disease and compare the outcomes between the scattered versus the circumscribed pattern. Methods: We reviewed 219 postoperative surgical specimens. Patients were divided into two groups: scattered versus circumscribed. Disease-free survival (DFS), distant DFS (DDFS), and overall survival (OS) were analyzed. Results: The scattered and circumscribed patterns were assessed in 111 (50.7%) and 108 (49.3%) patients. Two independent predictive factors for the circumscribed pattern were identified: discontinuation of NAC cycles (p = 0.011), and tumor size post-NAC >18 mm (p = 0.022). No difference was observed in terms of DFS and DDFS. Patients with the scattered pattern exhibited a statistically significant better OS. Discontinuation of NAC cycles, tumor size >18 mm, triple-negative BC, and ypN+ were associated with increased recurrence and poorer survival. Conclusions: Discontinuation of NAC cycles and tumor size are independent factors associated with patterns of residual disease. The scattered pattern presents better survival. Understanding the relationship between NAC, the residual pattern, and differences in survival outcomes offers the potential to optimize the therapeutic approaches.