Laboratory Investigation最新文献

Exosomes play a role in cell communication by transporting content between cells. Here, we tested whether renal podocyte-derived exosomes affect the injury of glomerular endothelial cells in lupus nephritis (LN). We found that exosomes containing high levels of high mobility group box 1 (HMGB1) were released from podocytes in patients with LN, BALB/c mice injected with pristane (which induces lupus-like disease in mice), and cultured human renal glomerular endothelial cells (HRGECs) treated with LN plasma. In vitro, GW4869 (an inhibitor of exosome biogenesis/release) or exosome removal alleviated the injury of HRGECs induced by LN plasma. Additionally, leptomycin B or knockdown of HMGB1 in podocyte-derived exosomes reduced endothelial cell injury and the expression of tripartite motif-containing 27 (TRIM27). Knockdown or overexpression of TRIM27 attenuated or promoted the damage of HRGECs treated with LN plasma. In vivo, knockdown of HMGB1 in podocytes ameliorated the injury of glomerular endothelial cells (GECs) in a mouse model of LN. Furthermore, the injection of podocyte-derived exosomes into mice caused GEC dysfunction. In conclusion, our study revealed that podocyte-derived exosomes may mediate the injury of glomerular endothelial cells seen in LN.

Cardiovascular disease (CVD) is the leading cause of death worldwide and has been confirmed to be associated with a common oral bacterial infection-chronic apical periodontitis (CAP). However, the detailed mechanisms remain controversial. CAP can potentially alter systemic inflammation, lipid metabolism, and gut microbiota, all of which contribute to the progression of the aortic inflammatory response. This study aimed to explore the differential effects between E. faecalis and P. gingivalis-CAP on the aortic inflammatory response, which focused on changes in systemic inflammation, lipid metabolism, and gut microbiota, to explore potential mechanisms linking oral disease to CVD. Our result showed P. gingivalis-CAP could activate more serious aortic inflammatory cytokine mRNA expression (TNF-α, MCP-1, and ICAM-1) than E. faecalis-CAP by promoting higher serum inflammation (TNF-α, IL-6, IL-1α, and MCP-1) and lipid (LDL-C and TC) level. Simultaneously, there was no significant change in gut microbiota between them. Furthermore, all serum inflammatory cytokines showed substantial correlations with aortic inflammatory cytokine mRNA expression, and certain serum lipid indicators showed significant correlations, but only two gut microorganisms (Ruminococcaceae and Prevotellaceae) showed significant correlations. The combined results suggest that CAP might activate the aortic inflammatory response in association with changes in the three potential mechanisms. However, the promotion of gut microbiota might be relatively weak. Using experimental CAP induced by specific bacteria, in which bacteria are sequestered in the medullary cavity, avoids the direct influence of blood or intestinal pathways, and provides new perspectives for studying the mechanism of CVD associated with oral disease. Overall, these findings suggest that CAP may exacerbate systemic inflammation and serum lipid levels in patients with CVD, highlighting the importance of educating such patients on oral hygiene.

The tumor microenvironment (TME) plays a critical role in cancer progression and therapeutic responsiveness, with the tumor immune microenvironment (TIME) being a key modulator. In head and neck squamous cell carcinomas (HNSCC), immune cell infiltration significantly influences the response to radiotherapy (RT). A better understanding of the TIME in HNSCC could help identify patients most likely to benefit from combining RT with immunotherapy. Standardized, cost-effective methods for studying TIME in HNSCC are currently lacking. This study aims to leverage deep learning (DL) to quantify immune cell densities using immunohistochemistry (IHC) in untreated oropharyngeal squamous cell carcinoma (OPSCC) biopsies of patients scheduled for curative RT, and to assess their prognostic value. We analyzed 84 pre-treatment formalin-fixed paraffin-embedded (FFPE) tumor biopsies from OPSCC patients. Immunohistochemistry was performed for CD3, CD8, CD20, CD163, and FOXP3, and whole slide images (WSIs) were digitized for analysis using a U-Net-based DL model. Two quantification approaches were applied: a cell-counting method and an area-based method. These methods were applied to stained regions. The DL model achieved high accuracy in detecting stained cells across all biomarkers. Strong correlations were found between our DL pipeline, the HALO® Image Analysis Platform, and the open-source QuPath software for estimating immune cell densities. Our DL pipeline provided an accurate and reproducible approach for quantifying immune cells in OPSCC. The area-based method demonstrated superior prognostic value for recurrence-free survival (RFS), when compared to the cell-counting method. Elevated densities of CD3, CD8, CD20, and FOXP3 were associated with improved RFS, while CD163 showed no significant prognostic association. These results highlight the potential of DL in digital pathology for assessing TIME and predicting patient outcomes.

Extrapulmonary small cell neuroendocrine carcinoma (EP-SCNC) is a rare malignancy with a poor prognosis. Most patients with EP-SCNC have metastatic disease upon presentation, and their average overall survival (OS) is less than 12 months. Our study aimed to conduct a complex analysis of EP-SCNC. One hundred and eighty-one EP-SCNC tissue samples were subjected to a complex analysis. One hundred and fifty-five tumors were pure EP-SCNC, while 26 were combined tumors. Immunohistochemistry for ASCL1, NEUROD1, YAP1, POU2F3, Rb1, p53, cyclin D1, p16, PTEN, DLL3, PD-L1, CD56, synaptophysin, chromogranin A, and INSM1 was performed, and 128 samples were analyzed molecularly using next generation sequencing (NGS), comprising DNA and RNA analysis. Detailed results on immunohistochemical and molecular analyses were provided for each primary origin of EP-SCNC separately. Median survival for the whole cohort of patients was 8.94 months. Patient age (≥ 70 years), tumor mutational burden (TMB) < 15, and TP53 and BRCA2 mutations were negative prognostic factors. High expression of ASCL-1 was associated with shorter OS, whereas high expression of YAP1 was associated with longer OS. Patients with genitourinary tumors had significantly better OS than those with gastrointestinal tract EP-SCNC tumors. Rb1 expression loss was detected more often in genitourinary tract SCNCs. In contrast, p16 overexpression was found more often in genitourinary tract SCNCs. POU2F3 expression was detected more often in combined tumors, whereas NEUROD1 was detected more often in pure EP-SCNC. Regarding "druggable markers," DLL3 was expressed in 66% of tumors and PD-L1 in 17.4%. Detailed analyses of different prognostic and predictive markers are needed to better understand EP-SCNC biology and create more personalized therapy to improve patient prognosis.

Immunotherapy has emerged as a new treatment modality in some soft tissue sarcomas, particularly for tumors associated with tertiary lymphoid structures (TLS). These structures are functional lymphoid aggregates, and their presence is indicative of an active anticancer immune response in the tumor microenvironment. The assessment of TLS as a predictive biomarker at scale on patient specimens remains challenging. While tissue microarrays could facilitate this assessment, it is unclear whether small microarray cores can represent and identify associated TLS responses. We sought to use multiplex immunohistochemistry to identify key components of TLS: T cells, B cells, and dendritic cells. The multiplex panels (CD3, CD20, CD208 and PNAd) were applied onto 80 cases both on tissue microarrays and on their cognate available full-faced sections from epithelioid sarcoma and dedifferentiated/well-differentiated liposarcoma case series. Tissue microarrays were digitally scored for the number of immune cells using the HALO image analysis platform, and cognate full-faced sections were visually evaluated for the presence of TLS. An independent validation set of soft tissue sarcomas (N=49) was stained with the CD3, CD20, and CD208, and scored by QuPath. A Combined Immune Marker (defined as the presence of more than more than 24% CD3+T cell, or 0.51% CD20+B cell, or more than 0.14% CD208+mature dendritic cell on tissue microarray core) is highly specific (100%) and moderately sensitive (61%) to predict the existence of TLS on full-faced sections. The Combined Immune Marker showed a sensitivity of 25% and specificity of 91% on the validation set. The Combined Immune Marker assessed on tissue microarrays is highly specific to infer the presence of TLS present on cognate full-faced sections. Therefore, despite the small area sampled, tissue microarrays may be utilized to assess the clinical value of TLS on datasets where specificity is critical and large sample size can mitigate low to moderate sensitivity.

Malignant peripheral nerve sheath tumors (MPNSTs) are aggressive sarcomas arising from peripheral nerves, accounting for 3% to 5% of soft tissue sarcomas. MPNSTs often recur locally, leading to poor survival. Achieving tumor-free surgical margins is essential to prevent recurrence, but current methods for determining tumor margins are limited, highlighting the need for improved biomarkers. In this study we investigated the degree to which MPNST extends into nerves adjacent to tumors. Alterations to the lipidome of MPNST and adjacent peripheral nerves were assessed using spatial lipidomics. Tissue samples from 5 patients with MPNST were analyzed, revealing alterations of the lipid profile extending into the peripheral nerves beyond what was expected based on macroscopic and histologic observations. Integration of spatial lipidomics and high-resolution accurate-mass profiling identified distinct lipid profiles associated with healthy nerves, connective tissue, and tumors. Notably, histologically normal nerves exhibited myelin degradation and infiltration of protumoral M2 macrophages, particularly near the tumor. Furthermore, aberrant osmium staining patterns and loss of H3K27me3 staining in the absence of atypia were observed in a case with tumor recurrence. This exploratory study thereby highlights the changes occurring in the nerves affected by MPNST beyond what is visible on hematoxylin and eosin staining and provides leads for further biomarker studies, including aberrant osmium staining, to assess resection margins in MPNST.

About 20% of human cancers harbor mutations of genes encoding switch/sucrose nonfermentable (SWI/SNF) complex subunits. Deficiency of subunits of the complex is present in 10% of non-small-cell lung cancers (NSCLC; SMARCA4/SMARCA2 deficient), 100% thoracic SMARCA4/A2-deficient undifferentiated tumors (TSADUDT; SMARCA4/A2 deficient), malignant rhabdoid tumor, and atypical/teratoid tumor (SMARCB1-deficient), >90% of small cell carcinoma of the ovary, hypercalcemic type (SMARCA4/SMARCA2 deficient), frequently in undifferentiated/dedifferentiated endometrial carcinoma (SMARCA4, SMARCA2, SMARCB1, and ARID1A/B deficient), 100% SMARCA4 deficient undifferentiated uterine sarcoma (SMARCA4 deficient); and in various other tumors from multifarious anatomical sites. Silencing of SWI/SNF gene expression may be genomically or epigenetically driven, causing loss of tumor suppression function or facilitating other oncogenic events. The SWI/SNF-deficient tumors share the phenotype of poor or no differentiation, often with a variable component of rhabdoid tumor cells. They present at advanced stages with poor prognosis. Rhabdoid tumor cell phenotype is a useful feature to prompt investigation for this group of tumors. In the thoracic space, the overlap in morphology, immunophenotype, genetics, and epigenetics of SMARCA4/A2-deficient NSCLC and TSADUDT appears more significant. This raises a possible nosologic relationship between TSADUDT and SMARCA4/A2-deficient NSCLC. Increased understanding of the genetics, epigenetics, and mechanisms of oncogenesis in these poor prognostic tumors, which are often resistant to conventional treatment, opens a new horizon of therapy for the tumors.

Tumor cell nuclear size (NS) indicates malignant potential in breast cancer; however, its clinical significance in esophageal squamous cell carcinoma (ESCC) is unknown. Artificial intelligence (AI) can quantitatively evaluate histopathological findings. The aim was to measure NS in ESCC using AI and elucidate its clinical significance. We investigated the relationship between NS assessed by AI and prognosis in 138 patients with ESCC who underwent curative esophagectomy. Hematoxylin and eosin-stained slides from the deepest tumor sections were digitized. Using HALO-AI DenseNet v2, we created a deep-learning classifier that identified tumor cells with an NS area >20 μm². Median NS was 40.14 μm², which was used to divide patients into NS-high and NS-low groups (n = 69 per group). Five-year overall survival (OS) and relapse-free survival rates were significantly lower in the NS-high group (43.2% and 39.6%) than in the NS-low group (67.7% and 49.6%). Multivariate analysis showed that greater tumor depth and NS-high status (hazard ratio: 1.79; P = .032) were independent risk factors for OS. In 77 cases with neoadjuvant chemotherapy, increased tumor depth and NS-high status (hazard ratio: 1.99; P = .048) were independent prognostic factors for unfavorable OS. Compared with the NS-low group, the NS-high group had significantly higher anisokaryosis, higher Ki-67 expression as calculated by AI analysis of immunostaining, and higher NS heterogeneity as examined by equidividing the tumors into square tiles. In conclusion, NS assessed by AI is a simple and useful prognostic factor for ESCC.

Acute kidney frailty (AKF) is a condition of increased susceptibility to acute kidney injury (AKI), an abrupt impairment of renal excretory function potentially leading to severe complications. Prevention of AKI relies on the recognition of risk factors contributing to AKF. At the population level, dehydration constitutes a predisposing factor for AKI. However, renal frailty may be context-specific, with variations among patients in the types of damage and the distinct pathological mechanisms. In this regard, we studied the combined effect of dehydration with other factors on renal homeostasis, such as increasing age and hypertension. AKF status was studied in rats bearing risk factors individually and in combination and was evaluated as the level of AKI induced by a triggering dose of cisplatin, which is known to be mildly nephrotoxic for young, healthy rats. AKI was assessed through parameters of renal function (including creatinine, urea, creatinine clearance, proteinuria, and fractional excretion of sodium) and histopathology of renal tissue specimens. The hydration status was measured by bioelectric impedance and other techniques. Water deprivation induces a dehydration state characterized by reductions in body weight and urinary flow and increases in hematocrit and plasma and urine osmolality. Bioelectric impedance showed a net loss of body water after water deprivation with no relevant changes in body mass distribution. Dehydration is not sufficient to predispose young control rats to intrinsic AKI. However, the combination of dehydration with advanced age or hypertension induces AKF evidenced by a magnified response of renal dysfunction (reduced filtration and tubular function) and tubular necrosis caused by low-dose cisplatin treatment. This study highlights the relevance of addressing AKF as a premorbid condition providing prophylactic opportunities and shows that dehydration differentially predisposes to prerenal and intrinsic AKI.

The success of immune checkpoint inhibitors (ICI) in melanoma therapy has catalyzed the introduction of ICI in increasingly early stages of the disease. This exposes many patients with a lower risk of relapse to the risk of protracted adverse events, highlighting the need for biomarkers guiding the use of ICI. Already many years ago, brisk infiltration of primary melanomas by lymphocytes has been linked to improved patient outcome, but controversial findings due to a high variability in classification systems have been described CD8+ T cells have been identified as a primary mediator of antitumor immunity in patients treated with ICI. As CD8+ T cells require the presentation of antigens via MHC-I on target cells, downregulation and loss of MHC-I have been observed as resistance mechanisms to ICI. In this study, we revisit the role of MHC-I expression and CD8+ T-cell infiltration in melanoma evolution using a cohort of advanced primary and matched metastatic melanomas by using an automated immunohistochemistry and digital pathology workflow. Our results show that downregulation of MHC-I expression is a frequent event in advanced primary melanomas that is associated with decreased CD8+ T-cell infiltration and an early metastatic spread to sentinel lymph nodes. Furthermore, MHC-I downregulation and decreased infiltration with CD8+ T cells are also associated with resistance to ICI. Our results suggest that analyses of MHC-I expression and CD8+ T-cell infiltration patterns could serve as future biomarkers to guide the decision to treat patients in early stages of melanoma with ICI.