Endothelial Cell Infiltration Research Articles

Deciphering the cellular landscape and potential targets of nasopharyngeal and oral cancers using single-cell RNA sequencing.

Cellular heterogeneity in nasopharyngeal cancer (NPC) and oral cancer remains unclear. In the current study, using single-cell RNA sequencing techniques, we investigated the cellular landscape in NPC and oral cancers. We identified a diverse range of cell types within the tumor microenvironment (TME) and variations in cell infiltration between NPC and oral cancer. In oral cancer, we observed a predominant infiltration of epithelial cells, fibroblasts, and endothelial cells (ECs), while T cells were the main infiltrating cell population in NPCs. We further classified these infiltrating cells into subclusters. Additionally, we observed complex interactions among cells that led to distinct trajectories. In particular, a unique epithelial subcluster with high expression of major histocompatibility complex class II (MHC-II) molecules was correlated with a favorable outcome and infiltration of CD4+ T cells. In addition, MHC-II+ epithelial cells inhibited mouse tumor growth and promoted T-cell infiltration. Consequently, our findings provide a deep understanding of the TME showing a significant prognostic value and therapeutic potential.

Inflammatory breast cancer microenvironment repertoire based on DNA methylation data deconvolution reveals actionable targets to enhance the treatment efficacy

BackgroundAlthough the clinical signs of inflammatory breast cancer (IBC) resemble acute inflammation, the role played by infiltrating immune and stromal cells in this aggressive disease is uncharted. The tumor microenvironment (TME) presents molecular alterations, such as epimutations, prior to morphological abnormalities. These changes affect the distribution and the intricate communication between the TME components related to cancer prognosis and therapy response. Herein, we explored the global DNA methylation profile of IBC and surrounding tissues to estimate the microenvironment cellular composition and identify epigenetically dysregulated markers.MethodsWe used the HiTIMED algorithm to deconvolve the bulk DNA methylation data of 24 IBC and six surrounding non-tumoral tissues (SNT) (GSE238092) and determine their cellular composition. The prognostic relevance of cell types infiltrating IBC and their relationship with clinicopathological variables were investigated. CD34 (endothelial cell marker) and CD68 (macrophage marker) immunofluorescence staining was evaluated in an independent set of 17 IBC and 16 non-IBC samples.ResultsWe found lower infiltration of endothelial, stromal, memory B, dendritic, and natural killer cells in IBC than in SNT samples. Higher endothelial cell (EC) and stromal cell content were related to better overall survival. EC proportions positively correlated with memory B and memory CD8+ T infiltration in IBC. Immune and EC markers exhibited distinct DNA methylation profiles between IBC and SNT samples, revealing hypermethylated regions mapped to six genes (CD40, CD34, EMCN, HLA-G, PDPN, and TEK). We identified significantly higher CD34 and CD68 protein expression in IBC compared to non-IBC.ConclusionsOur findings underscored cell subsets that distinguished patients with better survival and dysregulated markers potentially actionable through combinations of immunotherapy and epigenetic drugs.

The contribution of adiponectin to diabetic retinopathy progression: Association with the AGEs-RAGE pathway

Diabetic retinopathy (DR) is a chronic complication of diabetes. Given that adiponectin plays a key role in DR progression, this study aims to elucidate the molecular mechanisms of sDR progression related to adiponectin. First, we extracted the microarray dataset GSE60436 from the Gene Expression Omnibus (GEO) database to identify hub genes associated with DR. Pathway enrichment analysis revealed a focus on inflammation, oxidative stress, and metabolic disease pathways. Gene Set Enrichment Analysis (GSEA) identified nine significant pathways related to DR. Immune infiltration analysis indicated increased infiltration of fibroblasts and endothelial cells in DR patients. Second, at the gene level, single-cell RNA sequencing (scRNA-seq) results showed a decrease in ADIPOQ gene expression as the disease progressed in our mouse models. At the protein level, ELISA results from sera of 31 patients and 11 control subjects demonstrated significantly lower adiponectin expression in the proliferative diabetic retinopathy (PDR) group compared to controls. Our findings reveal that adiponectin is involved in the advanced glycation end products (AGEs) and receptor of advanced glycation end products (RAGE) axis, as evidenced by hub gene analysis, scRNA-seq, and ELISA. In conclusion, adiponectin acts as a central molecule in the AGEs-RAGE axis, regulated by ADIPOQ, to influence DR progression.

Correlation of BUB1 and BUB1B with the development and prognosis of endometrial cancer

This study aimed to evaluate the expression and clinical significance of budding uninhibited by benzimidazole 1 (BUB1) and BUB1 mitotic checkpoint serine/threonine kinase B (BUB1B) in endometrial carcinoma (EC). BUB1 and BUBIB expressions were evaluated by bioinformatics. Protein expression, clinical features, prognosis and immune cell infiltration were explored in 20 EC tumors. siRNA was used to evaluate BUB1 and BUBIB function in EC cells. BUB1 and BUBIB were highly expressed in 26 cancers. BUB1 was associated with overall survival (OS) in eight cancers and disease-free survival in ten; BUB1B was associated with OS in nine cancers and DFS in eleven. BUB1 and BUBIB exhibited high frequencies of gene changes (mainly mutations, > 5%) in cancer. BUB1 was negatively correlated and BUB1B was positively correlated with cancer-associated fibroblasts and endothelial cell infiltration. BUB1 and BUBIB knockdown decreased migration and invasion in EC cells. High BUB1 expression correlated with tumor malignant phenotypes (P < 0.05). High BUB1 mRNA expression reduced OS (P = 0.00036) and recurrence-free survival (P = 0.0011). High BUB1B mRNA expression reduced OS (P = 0.0024). BUB1/BUB1B correlated with activated CD8 + T and CD4 + T cell infiltration. BUB1 and BUBIB are highly expressed and correlated with clinicopathological characteristics in EC. BUB1 and BUBIB are potential prognosis markers and immunotherapy targets.

ACKR1+ Endothelial Cells Mediate Leukocyte Infiltration and Synergize with SFRP2/ASPN+ Fibroblasts to Promote Skin Fibrosis in Systemic Sclerosis.

Skin fibrosis is the most typical pathological manifestation of systemic sclerosis (SSc) and localized scleroderma (LS) with unclear etiology and few effective treatments. Though excessive collagen secretion by fibroblasts is the primary cause of skin fibrosis, many lines of evidence suggested that vascular damage was the initiating event and various cell types along with fibroblasts worked together to contribute to the pathogenesis of skin fibrosis. We sought to explore the relationships between vascular endothelial cell lesions and immune cell infiltration, along with the cell-cell interactions among various cell types within the fibrotic skin ecosystem. Single-cell RNA-seq (10x Genomics) was performed on skin biopsies of 3 healthy donors and 7 SSc patients in Chinese. The additional 3 localized scleroderma patients' data from NCBI database (GSE160536) were integrated by Harmony. CellChat package (v1.5.0) was applied to analyze cell communication network. Transwell assay and subcutaneous bleomycin (BLM) injection in mice were used to explore the role of ACKR1 on immune cell infiltration. Milo single-cell western blot was applied to show the activation of fibroblast subclusters. A total of 62,295 cells were obtained and subpopulations of stromal and immune cells were identified. Interaction network analysis revealed that multiple chemokines secreted by macrophages, pericytes, and pro-inflammatory fibroblasts could bind with Duffy antigen/receptor for chemokines (ACKR1), which is highly expressed on ACKR1+ endothelial cells of lesion skin. Transwell assay revealed that over-expressed ACKR1 in HUVEC facilitated leukocyte infiltration under the treatment of IL8. The BLM mice showed enhanced ACKR1 expression, massive immune cell infiltration, and fibrosis in skin, which could be attenuated by ACKR1 inhibition. Furthermore, infiltrated macrophages with TGFB1 or PDGFB high production could activate SFRP2/ASPN+ fibroblasts to contribute to excessive accumulation of extracellular matrix (ECM), and the SOX4-ASPN axis plays an important role in the TGF-β signaling cascade and the etiology of skin fibrosis. Our results reveal that highly expressed ACKR1 in endothelial cells of fibrotic skin tissue promotes immune cell infiltration, and SFRP2/ASPN+ fibroblasts synergize to exacerbate skin fibrosis.

Immune subtyping of ER-positive, PR-negative and HER2-negative breast cancers by immunohistochemistry: Molecular basis and clinical relevance.

e12553 Background: The diversity and plasticity behind ER+/PR−/HER2− breast cancer (BC) remain underexplored. In our previous work, an immune subtype classification for ER+/PR-/HER2- BC with RNA-seq data was established to distinguish between immune-hot and -cold tumors to provide guidance for immune checkpoint inhibitor (ICI) therapy. However, considering the economic costs, the clinical application of sequencing methods is limited. This study aimed to identify biomarkers of the immune subtype classification and develop a diagnostic procedure for ER+/PR-/HER2- BC immune subtyping based on the immunohistochemistry (IHC) technique, thereby identifying candidates of ICI therapy and refining treatment strategies for ER+/PR-/HER2- BC. Methods: RNA-seq data for 411 patients diagnosed with ER+/PR-/HER2- BC were obtained from the METABRIC database (METABRIC cohort). The immune subtype classification was established by consensus clustering with 1480 immune-related genes. Biomarkers for each immune subtype were identified from differentially expressed genes and validated using logistic regression and ROC curve analysis. Validation of the immune classification was conducted using RNA-seq data from the GEO and TCGA databases. Transcriptomic gene chip sequencing was performed on surgical specimens from 15 ER+/PR-/HER2- BC patients in our center (Chip cohort) to classify the immune subtypes. Biomarkers identified were validated and a diagnostic procedure was designed using IHC techniques. Results: In the METABRIC cohort, immune subtypes including Clusters 1-5 were established. Five biomarkers including DCN, SPP1, CXCL9, STC2, and SRGAP1 were identified, with the AUC values of ROC curves being 0.90, 0.82, 0.99, 0.70, and 0.89, respectively. Notably, Cluster 3 exhibited higher infiltration of CD8+ T cells, B cells, and M1 macrophages, with a greater association with immune activation pathways, thus being defined as the immune-hot subtype. Cluster 1, also showing a moderate immune cell infiltration, was characterized by a more pronounced stromal infiltration. In the Chip cohort, IHC revealed that Cluster 3 exhibited the highest levels of CD8+ T cell and B cell infiltration as well as PD-L1 expression, suggesting a potential benefit from ICI therapy. In contrast, Cluster 1 demonstrated the most extensive infiltration of endothelial and fibroblast cells. The other subtypes were characterized by a suppressed immune microenvironment, especially in Cluster 2, where an infiltration of SPP1+ macrophages was observed. A diagnostic procedure was designed with the biomarkers using IHC techniques. Conclusions: This study validated the immune subtype classification for ER+/PR-/HER2- BC. An easy-to-use IHC-based diagnostic procedure was developed, facilitating the identification of potential ICI therapy candidates in clinical practice at a lower cost.

MOICS, a novel classier deciphering immune heterogeneity and aid precise management of clear cell renal cell carcinoma at multiomics level

ABSTRACT Recent studies have indicated that the tumor immune microenvironment plays a pivotal role in the initiation and progression of clear cell renal cell carcinoma (ccRCC). However, the characteristics and heterogeneity of tumor immunity in ccRCC, particularly at the multiomics level, remain poorly understood. We analyzed immune multiomics datasets to perform a consensus cluster analysis and validate the clustering results across multiple internal and external ccRCC datasets; and identified two distinctive immune phenotypes of ccRCC, which we named multiomics immune-based cancer subtype 1 (MOICS1) and subtype 2 (MOICS2). The former, MOICS1, is characterized by an immune-hot phenotype with poor clinical outcomes, marked by significant proliferation of CD4+ and CD8+ T cells, fibroblasts, and high levels of immune inhibitory signatures; the latter, MOICS2, exhibits an immune-cold phenotype with favorable clinical characteristics, characterized by robust immune activity and high infiltration of endothelial cells and immune stimulatory signatures. Besides, a significant negative correlation between immune infiltration and angiogenesis were identified. We further explored the mechanisms underlying these differences, revealing that negatively regulated endopeptidase activity, activated cornification, and neutrophil degranulation may promote an immune-deficient phenotype, whereas enhanced monocyte recruitment could ameliorate this deficiency. Additionally, significant differences were observed in the genomic landscapes between the subtypes: MOICS1 exhibited mutations in TTN, BAP1, SETD2, MTOR, MUC16, CSMD3, and AKAP9, while MOICS2 was characterized by notable alterations in the TGF-β pathway. Overall, our work demonstrates that multi-immune omics remodeling analysis enhances the understanding of the immune heterogeneity in ccRCC and supports precise patient management.

Abstract 2758: Stemness subtype gastric cancer accelerate hematogenous metastasis

Abstract Oncogenic progression and metastasis correlate with the acquisition of stem cell-like characteristics and the loss of differentiated phenotype. Poorly differentiated primary tumors often lead to unfavorable oncologic outcomes due to a heightened likelihood of metastasis to distant organs across various cancers. However, the relationship between stemness traits and metastasis in gastric cancer (GC) remains insufficiently explored. In this investigation, we analyzed bulk RNA-sequencing from microdissected primary tumor specimens and clinical data to pinpoint molecular subtypes linked to distant organ metastasis. Transcriptomic data from patient-derived xenograft (PDX) samples and available single-cell RNA datasets validated our bulk RNAseq findings. Comprehensive analysis highlighted the molecular traits of GC tied to hematogenous metastasis. Microdissection of primary tumor FFPE samples facilitated bulk RNA sequencing, encompassing samples from 33 metastatic and 31 metastasis-free patients, with 8 paired normal samples. Most samples contained less than 10% of tumor microenvironment (TME) components like fibroblasts, endothelial cells, and immune cells. Using non-negative matrix factorization (NMF) clustering, we achieved unsupervised subtyping of the gastric cancer gene expression matrix. Permutation tests based on subsampling indicated consistent high-level cophenetic coefficients at k=2, suggesting robust bifurcation of transcriptomic data into two clusters. One showed augmented stemness traits while the other emphasized differentiated mucosal genes, labeled as stemness and differentiated subtypes, respectively. GSEA highlighted a significant boost in the Hallmark angiogenesis score in the stemness subtype, not attributed to endothelial cell infiltration as verified by CIBERSORTx. This subtype also exhibited a marked decrease in hematogenous metastasis-free survival (HMFS) (p=0.008, log-rank test). Further, PDX samples were scrutinized. Post classification of the PDX graft expression matrix via logistic regression, we examined the endothelial cell fraction, determined by CIBERSORTx. Stemness subtype PDX grafts revealed a significant elevation in the host (mouse) endothelial cell fraction (p&amp;lt;0.05). In addition, stemness PDX patient showed significant decreased HMFS (p = 0.03). We also categorized the pseudobulk expression matrix of malignant cells into stemness or differentiated subtypes using GSE183904 scRNA cohort. scRNA sample analysis consistently presented an elevated endothelial cell fraction in the stemness GC subtype. The outcomes suggest a propensity for angiogenesis in the TME of the stemness subtype of GC. In conclusion, our findings demonstrate that the stemness subtype of GC accelerates hematogenous metastasis, as evidenced through clinical data, bulk RNAseq, PDX, and scRNAseq data. Citation Format: Seungho Lee, Jaeun Yoo, Seungbok Lee, Hyun Myong Kim, Kyoungyun Jeong, Yie-Ri Yoo, Ji-Yeon Shin, Kyoung Un Park, Hye Seung Lee, Seong-Ho Kong, Do Joong Park, Hyuk-Joon Lee, Han-Kwang Yang. Stemness subtype gastric cancer accelerate hematogenous metastasis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 2758.

Identification of CAF-related lncRNAs at the pan-cancer level represents a potential carcinogenic risk.

Cancer-associated fibroblasts (CAFs) are increasingly recognized as playing a crucial role in regulating cancer progression and metastasis. These cells can be activated by long non-coding RNAs (lncRNAs), promoting the malignant biological processes of tumor cells. Therefore, it is essential to understand the regulatory relationship between CAFs and lncRNAs in cancers. Here, we identified CAF-related lncRNAs at the pan-cancer level to systematically predict their potential regulatory functions. The identified lncRNAs were also validated using various external data at both tissue and cellular levels. This study has revealed that these CAF-related lncRNAs exhibit expression perturbations in cancers and are highly correlated with the infiltration of stromal cells, particularly fibroblasts and endothelial cells. By prioritizing a list of CAF-related lncRNAs, we can further distinguish patient subtypes that show survival and molecular differences. In addition, we have developed a web server, CAFLnc (https://46906u5t63.zicp.fun/CAFLnc/), to visualize our results. In conclusion, CAF-related lncRNAs hold great potential as a valuable resource for comprehending lncRNA functions and advancing the identification of biomarkers for cancer progression and therapeutic targets in cancer treatment.

Comprehensive molecular characteristics of clinical responses to PD-1 inhibitors plus anti-angiogenic agents (PA) in metastatic HER2-negative gastric adenocarcinoma.

279 Background: Anti-angiogensis agents were reported to enhance the efficacy of PD-1 inhibitor by inhibiting the infiltration of suppressive immune cell populations. Clinical studies indicated the synergistic anti-tumor effect of the combination of PD-1 inhibitors and anti-angiogenesis agents (PA). We aim to assess the efficacy of PA versus chemo-PA regiment in mGC, as well as the potential predictive biomarkers. Methods: A total of 55 GC tumors were analyzed using the next-generation sequencing (NGS), immunohistochemistry (IHC), and the tumor immune microenvironment (TIME) by multiplex immunofluorescence (mIHC). Clinical outcomes [Disease Control Rate (DCR), Progression-free survival (PFS) and overall survival (OS)] were compared between patients receiving PA treatment (n=32) and chemo-PA (n=23) treatment. Exploratory biomarker analyses were conducted for the prediction for the efficacy of PA/chemo-PA treatment. Results: Patients receiving PA regiment had comparable DCR (69% vs 50%, P =0.25) and PFS (median PFS: 6.53 vs 4.73 months, HR=0.70, 95%CI: 0.27-1.78, P =0.52), compared with those treated with combination of chemo-PA regiment as second or later-line treatment in mGC. Patients with HMCN1 wild-type showed significant improvements in DCR (77% vs 0%, P =0.023) and PFS (median PFS: 6.53 vs 1.17 months, P =0.0017), versus those with HMCN1 mutations in PA treatment cohort. HMCN1 mutations are associated with lower infiltration of endothelial cells and lower score of VEGF ligand-receptor interaction, according to the EPIC and pathway enrichment analysis ( P &lt; 0.05). In addition, analysis of TIME by mIHC revealed that the fraction of CD68+ tumor-associated macrophages in the tumor center was higher in responders than non-responders before treatment (median: 17.59% vs 2.45%, P=0.016). Conclusions: PA regiment and chemo-PA regiment had similar efficacy in second or later line treatment of mGC. HMCN1 mutations maybe a predictive marker for PA regiment in HER2-negative mGC, which should be further validated in a large cohort.

A comprehensive analysis of ribonucleotide reductase subunit M2 for carcinogenesis in pan-cancer.

Although there is evidence that ribonucleotide reductase subunit M2 (RRM2) is associated with numerous cancers, pan-cancer analysis has seldom been conducted. This study aimed to explore the potential carcinogenesis of RRM2 in pan-cancer using datasets from The Cancer Genome Atlas (TCGA). Data from the UCSC Xena database were analyzed to investigate the differential expression of RRM2 across multiple cancer types. Clinical data such as age, race, sex, tumor stage, and status were acquired to analyze the influence of RRM2 on the clinical characteristics of the patients. The role of RRM2 in the onset and progression of multiple cancers has been examined in terms of genetic changes at the molecular level, including tumor mutational burden (TMB), microsatellite instability (MSI), biological pathway changes, and the immune microenvironment. RRM2 was highly expressed in most cancers, and there was an obvious correlation between RRM2 expression and patient prognosis. RRM2 expression is associated with the infiltration of diverse immune and endothelial cells, immune checkpoints, tumor mutational burden (TMB), and microsatellite instability (MSI). Moreover, the cell cycle is involved in the functional mechanisms of RRM2. Our pan-cancer study provides a comprehensive understanding of the carcinogenesis of RRM2 in various tumors.

YAP1/Piezo1 involve in the dynamic changes of lymphatic vessels in UVR-induced photoaging progress to squamous cell carcinoma

BackgroundUV-induced cutaneous squamous cell carcinoma (cSCC) is one of the most common skin cancers. The constant alterations of the lymphatic-centered immune microenvironment are essential in transforming from photoaging to cSCC. Studying the mechanism will be beneficial for new targets exploration to the early prediction of cSCC.AimsTo investigate the dynamic changes and mechanism of the lymphatic-centered immune microenvironment in transforming from photoaging to cSCC induced by ultraviolet irradiation (UVR).MethodsTIMER2.0 was used to analyze whether YAP1/VEGFC signaling pathway is involved in lymphangiogenesis in head and neck squamous cell carcinoma (HNSCC). Meanwhile, lymphatic-centered immune microenvironments alterations and the related cumulative survival time were also analyzed. With the accumulated UVR, skin photoaging developed and gradually progressed into actinic keratosis and cSCC on SKH-1 hairless mice. The skin lymphatic-centered immune microenvironment was evaluated at the 0th, 8th, 12th, 16-18th, and 20-24th week of UVR. Skin phenotype was assessed using optical coherence tomography (OCT) and skin image. H&E and Masson’s trichrome staining evaluated epidermis and dermis. The structure of lymphatic vessels (LVs), blood vessels, and different types of T cells were evaluated by immunohistochemistry staining. The expression of Piezo1 whose deletion in adult lymphatics led to substantial valve degeneration, VE-cadherin that maintained the permeability of LVs, and YAP1 were evaluated by immunohistochemistry staining as well. Besides, the drainage function of LVs was assessed by Evans Blue assay in vivo.ResultsThe lymphatic function and immune cell infiltration underwent adaptive changes under continuous UVR. TIMER2.0 analysis indicated that VEGFC genes high expressed in HNSCC. YAP1 gene expression was positive correlated with VEGFC in HNSCC. LV density increased in human cSCC. More LVs in HNSCC were beneficial to prolong the survival time. VEGFC gene overexpression was positive correlated to CD8+T cell infiltration. More CD8A+T cells and CD8B+T cell infiltration in HNSCC extended survival time. When YAP1 gene overexpression and high infiltration of endothelial cells took place simultaneously might prolong the survival time of HNSCC patients. And high infiltration of CD8+T cells prolonged the survival time as well. In animal studies, UVR-induced eight weeks (photoaging) and 16–18 weeks (precancerous) were two turning points. The density of LVs in UV-8w was the least. When photoaged skin developed into AK lesions (UV-16-18w), LV slightly exceeded healthy skin and proliferated sharply in cSCC (UV-20-24w). YAP1 expression was almost consistent with LV but rose after the photoaging stage. The drainage of cSCC mice induced by UVR was better than that of photoaged skin and worse than that of health skin. The dynamic alterations of LVs number, Piezo1 expression, and collagen might be reasons for it. The expression of Piezo1 was in the highest point after 8 weeks of UVR, then gradually descended to the platform. The total T cells increased slowly, but the infiltration of CD4+T cells increased, and CD8+T cells decreased after eight weeks of UVR. The CD8+T cells and CD4+T cells increased sharply in UV-16-18w and UV-20-24w groups.ConclusionThe lymphatic-centered immune microenvironment underwent adaptive changes under continuous UVR via regulating YAP1/VEGFC and Piezo1. During the formation of cSCC, there are two turning points, eight weeks (photoaging) and 16–18 weeks (precancerous). YAP1, Piezo1, LVs, and immune cells constantly changed with the skin state induced by UVR. According to these changes the process of cSCC can be identified in advance and intervene timely.

Neovascular Progression and Retinal Dysfunction in the Laser-Induced Choroidal Neovascularization Mouse Model.

The mouse model of laser-induced choroidal neovascularization (LI-CNV) has been widely used to study neovascular age-related macular degeneration; however, it still lacks a comprehensive characterization. Here, CNV was induced in the eyes of 12-week-old C57BL/6J male mice by argon laser irradiation. We studied the CNV lesion progression of an LI-CNV mouse cohort by using multimodal imaging (color fundus, optical coherence tomography (OCT), and fluorescence angiography, focal electroretinography features for 14 days, and related cytokines, angiogenic factors, and reactive gliosis for 5 days. CNV lesions involving the rupture of the Bruch's membrane were confirmed using funduscopy and OCT after laser photocoagulation. During the initial stage, from the CNV induction until day 7, CNV lesions presented leakage observed by using fluorescence angiography and a typical hyperreflective area with cell infiltration, subretinal leakage, and degeneration of photoreceptors observed through OCT. This correlated with decreased retinal responses to light. Moreover, inflammatory and angiogenic markers were reduced to basal levels in the first 5 days of CNV progression. In contrast, reactive gliosis and the VEGF expression in retinal sections were sustained, with infiltration of endothelial cells in the subretinal space. In the second stage, between days 7 and 14 post-induction, we observed stabilization of the CNV lesions, a hyperfluorescent area corresponding to the formation of fibrosis, and a partial rescue of retinal function. These findings suggest that the LI-CNV lesion development goes through an acute phase during the first seven days following induction, and then the CNV lesion stabilizes. According to these results, this model is suitable for screening anti-inflammatory and anti-angiogenic drugs in the early stages of LI-CNV. At the same time, it is more convenient for screening anti-fibrotic compounds in the later stages.

CACNA1C mutation as a prognosis predictor of immune checkpoint inhibitor in skin cutaneous melanoma.

Aims: There is an urgent need for appropriate biomarkers that can precisely and reliably predict immunotherapy efficacy, as immunotherapy responses can differ in skin cutaneous melanoma (SKCM) patients. Methods: In this study, univariate regression models and survival analysis were used to examine the link between calcium voltage-gated channel subunit alpha 1C (CACNA1C) mutation status and immunotherapy outcome in SKCM patients receiving immunotherapy. Mutational landscape, immunogenicity, tumor microenvironmentand pathway-enrichment analyses were also performed. Results: The CACNA1C mutation group had a better prognosis, higher immunogenicity, lower endothelial cell infiltration, significant enrichment of antitumor immune response pathwaysand significant downregulation of protumor pathways. Conclusion: CACNA1C mutation status is anticipated to be a biomarker for predicting melanoma immunotherapy effectiveness.

Abstract P1129: Directing Revascularization In Vivo With Local Controlled Release Of An Optimized Angiogenic Growth Factor Cocktail

Reduced vascular perfusion due to aging, ischemia, or injury impedes healing and tissue function in conditions like skin ulcers and myocardial infarction, motivating development of therapies to restore vasculature locally in target tissues. We hypothesize that specific combinations of signaling molecules stimulate angiogenesis and vascular remodeling in the local tissue environment to increase perfused vessel density. Using a vascular tissue engineering approach with a fractional factorial design of experiments (DOE), we evaluated in vivo angiogenesis of combinations of ten growth factors, cytokines and/or enzymes including VEGF, bFGF, Shh, PDGF, IGF-1, EGF, TGF-β1, Ang-1, MCP-1 and MMP-9 in a subcutaneous gel plug assay in the WT immune competent rat. Our initial assessment with Matrigel identified 5 factors that maximized CD31+ vessel lumen density and aSMA+ encoated vessels at 7 days, warranting further evaluation. We developed a fully defined biomaterial system with covalently bound heparin to alginate mixed with collagen type 1 as an injectable gel. This defined biomaterial prolonged protein release up to 14 days in vitro versus Matrigel and unmodified alginate (2-4 days). The defined biomaterial was used for a second round DOE to deliver high (2 μg) and low (0.5 μg) doses of VEGF, bFGF, Shh, PDGF, and/or IGF-1. The defined biomaterial alone enhanced average lumen diameter 15-fold and endothelial cell infiltration 12-fold versus Matrigel. Perfused lumen density increased 16-fold with high Shh; aSMA-positive lumen wall area increased 4-fold with high VEGF, PDGF, and IGF-1; perfused vessel size increased 3-fold with high VEGF, bFGF, and Shh; and endothelial cell recruitment increased 2-fold with high IGF-1 relative to unloaded controls after 7 days in vivo. Empirical testing of 2- to 5-factor combinations showed that cocktail potency required initiation of angiogenic sprouting by VEGF or bFGF and vascularization responses were strongest in the presence of Shh and IGF-1. In conclusion, we identified sets of highly potent cocktails and leveraged controlled release from a defined biomaterial as a tailored revascularization therapy for local angiogenesis, vascular remodeling, and tissue regeneration.

Molecular and immune analysis of adrenocortical carcinoma (ACC): Implications for immune checkpoint inhibition (ICI).

e16617 Background: ACC is a rare and aggressive malignancy. Response rates to ICI are low with scarce yet promising durable responses. Molecular ACC subtypes have been reported based on The Cancer Genome Atlas (TCGA). Here we report the mutational and immune landscapes of ACC obtained from a real-world patient dataset. Methods: DNA (592-gene or whole exome; N=215) and RNA sequencing (whole transcriptome; N=121) were performed for ACC specimens submitted to Caris Life Sciences (Phoenix, AZ). PD-L1+ expression was tested by IHC (SP142; ≥1%). Hierarchical agglomerative clustering (HAC) analysis was performed on a pre-defined set of immune genes. Specimens grouped into immune-high/intermediate (IH) and immune-low (IL) groups. Microenvironmental cell populations (MCP)-counter analysis was used to estimate immune cell infiltrates. Statistical significance was determined using chi-square/Fisher’s exact/Mann-Whitney U tests and adjusted for multiple comparisons (q&lt;0.05). Results: Overall, the most frequent mutations were in TP53 (37%), CTNNB1 (18.7%), ATRX (12.1%), NF1 (9.6%), and RB1 (8.75%), consistent with previous reports. Further, 6.4% of tumors were PD-L1+, 6.1% TMB-high (≥ 10 mut/MB), and 5.9% dMMR/MSI-H. 38.2% of tumors were biopsied from metastatic sites (M). Compared to M, primary (P) specimens had a higher prevalence of TP53 (43.4 vs 26.9%, p&lt;0.05) and TERT promoter mutations (11.1 vs 0%, p=0.08), and a lower prevalence of SMARCB1 (0 vs 3.7%, p=0.054) and BRCA2 (0 vs 3.8%, p=0.056) mutations. Interestingly, M lesions were enriched for HAVCR2 expression (1.3-fold, p&lt;0.05), as well as infiltration of monocytes (1.8-fold, q&lt;0.05) and endothelial cells (1.4-fold, q&lt;0.05). HAC based segregation of tumors based on immune activity was corroborated by a general enrichment of immune cell infiltrates in the IH tumors. IH tumors were enriched for common immunotherapy-related biomarkers (TMB-high, dMMR/MSI-H and PD-L1 positivity), along with a higher prevalence of TP53 and ATRX mutations, while CTNNB1 mutation and CDK4 copy number amplification were less common in IH tumors. Conclusions: Querying a large real-world ACC patient dataset, we find somatic alterations and a low prevalence of ICI-related biomarkers (&lt;10%), consistent with previous reports. Monocyte and endothelial cell infiltration in metastatic tumors suggests an immunosuppressive phenotype compared to primary tumors. Finally, identification of molecular alterations in tumors with distinct immune phenotypes may facilitate development of new therapeutic strategies in ACC. [Table: see text]

PC34. Micropuncture Induced Scaffold Vascularization and Macrophage Quantification Across Vascular Origins

PURPOSE: Vascularization is a major barrier to tissue engineering and macrophages are believed to be integral. We have developed a microsurgical approach which rapidly vascularizes an adjacent scaffold. In micropuncture (MP), the recipient blood vessel wall is precisely disrupted providing an immediate route for cell extravasation. Here, we evaluated scaffold vascularization and macrophage infiltration across vascular origins. We hypothesized that concurrent artery/vein MP results in greatest macrophage infiltration and vascularization. METHODS: MP was tested in the rat femoral artery (MPA), vein (MPV), artery and vein (MPA/V), and no MP (control). MPs were created just prior to scaffold implantation. At post-operative day (POD) 3 and 10, samples were analyzed using whole mount angiography and histology. Vascular density was computed using artificial intelligence. Endothelial cell (EC) and macrophage infiltration were quantified. RESULTS: Vascular density appeared concordant with EC infiltration with MPA/V resulting in the greatest increase. Maximum macrophage infiltration was noted in MPV at both timepoints followed by MPA/V. Both conditions hastened macrophage accumulation when compared to MPA and the control. CONCLUSION: Micropuncture creates a localized pro-angiogenic environment. Although MPA/V led to greatest vascularization, it is noteworthy that MPV is also substantially pro-angiogenic. This suggests that the vein primarily drives cellular and vascular changes. MP appears to offer a tailored microsurgical approach for angiogenic induction which may prove beneficial to reconstructive surgery and tissue engineering.

ORC6, a novel prognostic biomarker, correlates with T regulatory cell infiltration in prostate adenocarcinoma: a pan-cancer analysis

BackgroundThe origin recognition complex (ORC), a six-subunit DNA-binding complex, participates in DNA replication in cancer cells. Specifically in prostate cancers, ORC participates the androgen receptor (AR) regulated genomic amplification and tumor proliferation throughout the entire cell cycle. Of note, ORC6, the smallest subunit of ORC, has been reported to be dysregulated in some types of cancers (including prostate cancer), however, its prognostic and immunological significances remain yet to be elucidated.MethodsIn the current study, we comprehensively investigated the potential prognostic and immunological role of ORC6 in 33 human tumors using multiple databases, such as TCGA, Genotype-Tissue Expression, CCLE, UCSC Xena, cBioPortal, Human Protein Atlas, GeneCards, STRING, MSigDB, TISIDB, and TIMER2 databases.ResultsORC6 expression was significantly upregulated in 29 types of cancers compared to the corresponding normal adjacent tissues. ORC6 overexpression correlated with higher stage and worse prognostic outcomes in most cancer types analyzed. Additionally, ORC6 was involved in the cell cycle pathway, DNA replication, and mismatch repair pathways in most tumor types. A negative correlation was observed between the tumor endothelial cell infiltration and ORC6 expression in almost all tumors, whereas the immune infiltration of T regulatory cell was noted to be statistically positively correlated with the expression of ORC6 in prostate cancer tissues. Furthermore, in most tumor types, immunosuppression-related genes, especially TGFBR1 and PD-L1 (CD274), exhibited a specific correlation with the expression of ORC6.ConclusionsThis comprehensive pan-cancer analysis revealed that ORC6 expression serves as a prognostic biomarker and that ORC6 is involved in the regulation of various biological pathways, the tumor microenvironment, and the immunosuppression status in several human cancers, suggesting its potential diagnostic, prognostic, and therapeutic value in pan-cancer, especially in prostate adenocarcinoma.

A pan-cancer analysis of the role of WDFY2 in human tumors

ABSTRACT WDFY2 is a protein that may provide valuable insights into the mechanisms underlying human tumors and aid in the development of novel therapies. Despite its potential importance, the role of WDFY2 in pan-cancer has not been systematically investigated. In this study, we comprehensively explored the expression pattern and function of WDFY2 across 33 cancers using various databases, including TCGA, CPTAC and GEO datasets. Our results indicate that WDFY2 is downregulated in most cancer types, including BRCA, KIRP, KICH, LUAD, KIRC, PCPG, PRAD, THCA, ACC, OV, TGCT and UCS, while it is upregulated in CESC, CHOL, COAD, HNSC, LUSC, READ, STAD and UCEC. Prognostic analyses showed that higher levels of WDFY2 were associated with worse disease outcomes in ACC, BLCA, COAD, READ, SARC, MESO and OV. WDFY2 mutations were most frequent in colorectal cancer but were not associated with disease prognosis. We also found that WDFY2 expression correlated with monocyte infiltration status in SKCM, endothelial cell infiltration in COAD, KIRC, MESO, OV and THCA, and cancer-associated fibroblast infiltration in COAD, LUAD and OV. Additionally, functional enrichment analysis revealed that WDFY2 is involved in metabolism. Overall, our comprehensive analysis sheds light on the role of WDFY2 in various cancers, providing a better understanding of its role in tumorigenesis.

The Characteristics of Tumor Microenvironment Predict Survival and Response to Immunotherapy in Adrenocortical Carcinomas.

Increasing evidence confirms that tumor microenvironment (TME) can influence tumor progression and treatment, but TME is still understudied in adrenocortical carcinoma (ACC). In this study, we first scored TME using the xCell algorithm, then defined genes associated with TME, and then used consensus unsupervised clustering analysis to construct TME-related subtypes. Meanwhile, weighted gene co-expression network analysis was used to identify modules correlated with TME-related subtypes. Ultimately, the LASSO-Cox approach was used to establish a TME-related signature. The results showed that TME-related scores in ACC may not correlate with clinical features but do promote a better overall survival. Patients were classified into two TME-related subtypes. Subtype 2 had more immune signaling features, higher expression of immune checkpoints and MHC molecules, no CTNNB1 mutations, higher infiltration of macrophages and endothelial cells, lower tumor immune dysfunction and exclusion scores, and higher immunophenoscore, suggesting that subtype 2 may be more sensitive to immunotherapy. 231 modular genes highly relevant to TME-related subtypes were identified, and a 7-gene TME-related signature that independently predicted patient prognosis was established. Our study revealed an integrated role of TME in ACC and helped to identify those patients who really responded to immunotherapy, while providing new strategies on risk management and prognosis prediction.