Tissue Sites Research Articles

Abstract 6764: Vascularized tumor-on-chip model for evaluating chemotherapeutic-mediated damage to adjacent healthy tissue

Abstract Introduction: Systemic chemotherapy is an effective anticancer treatment for all stages of breast cancer, with intravenous (IV) infusion being the principal method of administration. In addition to targeting cancer, systemic chemotherapy can impact the viability and function of surrounding normal tissues and vasculature. Early-discovery research tools comparing novel and existing systemic treatment strategies are urgently needed to support predictive efficacy and toxicity efforts. Microfluidic systems have the potential to fill this need while simultaneously reducing animal use. The overall goal of the study is to use an advanced microfluidic tumor-on-chip system to simultaneously evaluate the therapeutic and toxic effects of IV chemotherapy on normal and cancerous tissues. Materials and Methods: Synthetic Tumor Networks, which are comprised of primary and secondary tissue sites separated by an interconnected vasculature, were developed using in vivo images and fabricated using soft lithography. Primary vascular endothelial cells were used to establish microvasculature. A normal breast cell line (MCF 10A), or a GFP-labeled metastatic human breast cancer cell line (MDA-MB-231/GFP), were cultured in the tissue sites in a three-dimensional (3D) environment using a human-derived hydrogel. The vascular networks were perfused with endothelial cell media under physiological fluid flow conditions to establish the model. An EZH2 methyltransferase inhibitor (EZH2 inhibitor III) was delivered by IV infusion to the model tissues. Real-time monitoring of cellular growth, tumor invasion and extravasation, and cellular viability were performed using fluorescence microscopy over a two-week period. Results and Discussion: Metastatic MDA-MB-231/GFP breast cancer cells proliferated rapidly in the primary tumor site. Tumor intravasation into the vascular channels was observed in models that received no anticancer treatment, as well as extravasation into the secondary tissue site and invasion of the healthy breast tissue. Differences in tumor viability and migratory behavior were observed between untreated models or models receiving IV infusions of the EZH2 inhibitor through the vascular networks. Vascular barrier integrity and normal breast tissue viability were monitored at early (24-72 hr) and late (7-14 days) timepoints after anticancer treatment to measure short- and long-term adverse drug impacts. These results provide a unique perspective on the in vivo realism of an in vitro system for monitoring both therapeutic and toxic effects of systemic chemotherapies. Conclusions: We have developed a 3D vascularized tumor-on-chip model for monitoring the impacts of systemic chemotherapeutic agents on tumor and healthy tissues. This model can be used to investigate therapeutic and toxic effects on multiple tissue types simultaneously using real-time imaging techniques. Citation Format: Deborah Ramsey, Jenna Rosano, Cristabel Gordon, Gwen Fewell. Vascularized tumor-on-chip model for evaluating chemotherapeutic-mediated damage to adjacent healthy tissue [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 6764.

Abstract 1194: Understanding radiation and immunotherapy induced cell state shifts in the pancreatic tumor microenvironment

Abstract Pancreatic ductal adenocarcinoma (PDAC) is a lethal disease with few effective treatment options. It is characterized by a highly desmoplastic tumor microenvironment and a paucity of dendritic cells, leading to low immune cell infiltration and poor outcomes with immunotherapy treatments that are highly effective in other cancers. In a recent pilot study, metastatic PDAC patients who had progressed on chemotherapy were treated with a combination of dual immune checkpoint blockade (ICB) therapy and radiation which resulted in an 18% overall response rate (ORR) and 29% disease control rate (DCR). This led to a phase 2 study in order to further test this combination with the goal of deep correlative analysis to understand the determinants of immunotherapy response and resistance. In this follow up there was a 3.6% ORR, 10.7% DCR, progression free survival (PFS) of 2.3 months, and a single patient with complete response. We collected 36 samples from 23 patients including primary pancreas tumor tissue and liver metastases, and 13 pairs of pre and on treatment biopsies. We performed single-nucleus and paired single-cell RNA and TCR sequencing on these samples to provide comprehensive insight into the cell types and biology present in this unique tumor microenvironment. Using supervised non-negative matrix factorization we have identified both previously established and novel gene programs that co-vary between cell types. Within the various tissue sites we have identified differing proportions of basal to classical tumor cell types and a distinct shift towards the classical state post-radiation. Of particular interest is a group of interferon related gene programs present within epithelial cell subtypes, C1QC+ and MHCII+ macrophages, and CD14+/CD16+ monocytes that indicate cell type specific responses to treatment. Focusing on the T cells, we found a distinct shift towards exhausted or proliferating states on treatment and using TCR sequencing data we have identified novel and expanding clones on treatment, largely consisting of those same cell subtypes. We also collected spatial transcriptomics data on a subset of patient samples, and analysis is ongoing to further confirm the cell type relationships identified in single-cell resolution data, along with spatial localization of basal to classical tumor states. This vast multimodal dataset allows us novel insight into the radiation and dual ICB induced cellular state shifts in the tumor microenvironment. Citation Format: Milan Parikh, Ryan Park, Aparna R. Parikh, Julie L. Koenig, Leon Pappas, Moshe Sade-Feldman, Lynn Bi, Nicole Carzo, Tarin M. Grillo, Islam Baiev, Olanike Asupoto, Irena Gushterova, Tom LaSalle, Anna Gonye, Emily Blaum, David P. Ryan, David T. Ting, Theodore S. Hong, Dana Pe'er, Nir Hacohen, Arnav Mehta. Understanding radiation and immunotherapy induced cell state shifts in the pancreatic tumor microenvironment [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 1194.

Abstract 2906: Inducing and disabling COX-2 S-nitrosylation to investigate its role in breast cancer progression

Abstract Background: We aim to assess the effect of S-nitrosylation of COX-2 on tumor attributes in models of early breast cancer. COX-2 is involved in tumor progression in many solid cancers. It is a known oncogene in murine breast cancer models, and targetable by NSAIDs. In women, its clinical correlation to breast cancer progression as well as its efficacy as a therapeutic target have yielded mixed results. These incongruities may be due, in part, to a post-translational modification, as COX-2 undergoes S-nitrosylation on Cys-526. Commercially-available antibodies selectively bind to either the S-nitrosylated (SNO-COX-2) or non-nitrosylated (nCOX-2) forms, and immunohistochemical staining of human tissue reveals that the two forms of COX-2 localize to different cellular sites in normal breast tissue and have opposite associations with breast cancer progression (PMID 33298921). Hypothesis: S-Nitrosylated and non-nitrosylated COX-2 have distinct roles in cancer progression. Approach: MCF10DCIS.com is a human breast cancer cell line chosen due to its ability to form DCIS-like (“stage 0”) lesions in culture and xenografting, which can progress to invasion when induced by signals from the tumor microenvironment (TME). Approach 1: Detect extracellular matrix (ECM) and ligand factors that upregulate SNO-COX-2, independently or in ECM/ligand pairs. We utilized a 2D microenvironment microarray (MEMA) and tested 357 TME conditions. Approach 2: Validate TME conditions in 3D culture. Approach 3: Develop a mutant MCF10DCIS.com line in which COX-2 cannot be S-nitrosylated by inducing a single disabling missense mutation at Cys-526 via CRISPR. Results: The MEMA revealed several conditions in which SNO-COX-2 was upregulated, including fibular collagens and SSP1. In 3D culture, increased SNO-COX-2 was associated with the presence of markers for EMT and an invasive growth phenotype, whereas nCOX-2 was not. Following CRISPR transformation, 59 individual MCF10DCIS.com clones were isolated and the target site of each sequenced. We identified 10 heterozygotic clones with the desired mutation, including a classical heterozygote that also retained the WT allele. Ongoing: Current studies include a second round of CRISPR to produce a homozygous mutant cell line as well as validation of the mutant protein. The resulting cell lines will model the loss of COX-2 S-nitrosylation in breast cancer cells while retaining otherwise-functional COX-2. Citation Format: Reuben J. Hoffmann, AeSoon Bensen, Elise de Wilde, James E. Korkola, Pepper Schedin. Inducing and disabling COX-2 S-nitrosylation to investigate its role in breast cancer progression [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 2906.

Abstract 2560: Genomic biomarkers of survival in patients (pts) with metastatic castrate-sensitive prostate cancer (mCSPC) undergoing androgen deprivation therapy (ADT) intensification (ADTi)

Abstract Background: ADTi with androgen receptor pathway inhibitors (ARPI) with or without docetaxel improves survival outcomes in pts with mCSPC. Even with ADTi, 20% of these pts progress within a year and experience poor overall survival (OS). Identifying baseline genomic biomarkers predicting poor survival outcomes in pts receiving ADTi remains an unmet need. Methods: In this IRB-approved retrospective study, pts with a new diagnosis of mCSPC who underwent comprehensive genomic profiling of primary prostate tissue or metastatic site or cell-free DNA and treated with ADTi (ADT with ARPI or docetaxel) were included. Genomic alterations with an incidence > 5% were included in the analysis. Study endpoints: progression-free survival (PFS) was defined from the start of therapy for mCSPC to progression (per PCWG-2) or death from any cause. OS was defined from the start of therapy for mCSPC to the date of death from any cause or censored at the last follow-up. A multivariable analysis using the Cox proportional hazards model was used, adjusting for age at mCSPC diagnosis, disease volume (high vs. low), Gleason score (≥ 8 vs. < 8), Log2PSA at baseline, and initial timing of metastasis (de novo vs. non de novo). A subgroup analysis by intensification treatment received (ARPI vs. docetaxel) was also performed. Missing baseline characteristics such as volume of disease, Gleason score, and Log2PSA were multiply imputed on 50 chained equations using “mice” package in R. All the analyses were done using R version 4.3.1. Results: A total of 289 pts were included in the study. The baseline characteristics of the cohort will be presented in the meeting. The alterations included in the analysis were TP53 (31.8%), TMPRSS2 (30.4%), PTEN (17%), APC (10.4%), SPOP (9%), RB1 (6.2%), BRCA2 (5.2%), and CDK12 (5.2%). In the multivariable analysis, TP53 (HR 1.87, 95% CI 1.29-2.72, p = 0.001), RB1 (HR 2.56, 95% CI 1.45-4.53, p = 0.001), PTEN (HR 1.77, 95% CI 1.15-2.72, p = 0.009), and BRCA2 (HR 2.68, 95% CI 1.47-4.89, p = 0.002) were associated with significantly shorter PFS while TP53 (HR 1.78, 95% CI 1.1-2.88, p = 0.019), RB1 (HR 4.78, 95% CI 2.53-9.02, p < 0.001), and PTEN (HR 2.37, 95% CI 1.37-4.1, p = 0.003) were associated with significantly worse OS. Results of the subgroup analysis by intensification treatment (ARPI vs. docetaxel) will also be presented in the meeting. Conclusions: In this real-world study, we identified genomic biomarkers that could be associated with shorter PFS and OS in pts with mCSPC treated with ADTi. Upon external validation, these results may aid in developing a clinical-genomic risk stratification model, patient counseling, and treatment selection. Citation Format: Arshit Narang, Georges Gebrael, Chadi Hage Chehade, Nicolas Sayegh, Yeonjung Jo, Beverly Chigarira, Nishita Tripathi, Ayana Srivastava, Clara Tandar, Benjamin L. Maughan, Umang Swami, Neeraj Agarwal. Genomic biomarkers of survival in patients (pts) with metastatic castrate-sensitive prostate cancer (mCSPC) undergoing androgen deprivation therapy (ADT) intensification (ADTi) [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 2560.

Abstract 3678: Beyond detection: AI-based classification of breast cancer invasiveness using cell-free orphan non-coding RNAs

Abstract Background: Approximately 1 in 8 women will be impacted by breast cancer in their lifetimes. Earlier detection of breast cancer through screening has improved survival. Liquid biopsies have the potential to complement existing screening methods by enabling earlier detection and differentiating invasive cancer (IBC) from ductal carcinoma in-situ (DCIS). We previously demonstrated high sensitivity and specificity for early detection of IBC by using a blood-based liquid biopsy platform to analyze a novel category of cancer-associated small RNAs, termed orphan RNAs (oncRNAs). Here, we developed a test that could not only detect the presence of cancer, but also classify the invasiveness of breast cancer. Methods: We utilized The Cancer Genome Atlas (TCGA) small RNA profiles to discover a library of 20,538 oncRNAs that were significantly enriched among 1,103 breast tumors compared to 349 controls from normal tissues spanning multiple tissue sites, limited to female samples. The diagnostic performance of these oncRNAs was assessed in an independent cohort of serum samples from 708 women, including 380 breast cancer patients (221 IBC and 159 DCIS; mean age: 58.0 ± 13.4 years) and 328 age-matched controls (mean age: 58.4 ± 13.7 years). We sequenced the small RNA content from 1 ml of serum from these patients at an average depth of 21.6 million 50-bp single-end reads. We detected 19,736 (96%) of the breast cancer-specific oncRNA library within at least one sample in this cohort. We then trained a multi-class generative AI model using 5-fold cross-validation to predict IBC, DCIS, and absence of breast cancer (IBC or DCIS). Results: Our oncRNA-based generative AI model achieved an overall AUC of 0.95 (95% CI: 0.94-0.97) for prediction of breast cancer versus cancer-free controls. At 90% specificity, overall model sensitivity is 90.0% (86.5%-92.8%). For DCIS and stage I IBC, the model has a sensitivity of 88.1% (82.0%-92.7%) and 90.4% (81.9%-95.8%) respectively, both at 90% specificity. In the second step, restricting to samples flagged as cancer, we observed an overall AUC of 0.9 (0.87-0.93) and sensitivity of 62.4% (55.3%-69.1%) at 90% specificity for discriminating against invasive breast cancer. Conclusions: We have demonstrated the potential utility of oncRNAs as the foundation for a liquid biopsy platform for sensitive and accurate early detection of breast cancer. Our liquid biopsy assay has the potential to complement standard of care by not only detecting breast cancer but also differentiating IBC from DCIS. Citation Format: Mehran Karimzadeh, Taylor B. Cavazos, Nae-Chyun Chen, Noura K. Tbeileh, David Siegel, Amir Momen-Roknabadi, Jennifer Yen, Jeremy Ku, Selina Chen, Diana Corti, Alice Huang, Dang Nguyen, Rose Hanna, Ti Lam, Seda Kilinc, Philip Murzynowski, Jieyang Wang, Xuan Zhao, Andy Pohl, Babak Behsaz, Helen Li, Lisa Fish, Kim H. Chau, Marra S. Francis, Laura J. Van't Veer, Laura J. Esserman, Patrick A. Arensdorf, Hani Goodarzi, Fereydoun Hormozdiari, Babak Alipanahi. Beyond detection: AI-based classification of breast cancer invasiveness using cell-free orphan non-coding RNAs [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 3678.

ROS-responsive drug-releasing injectable microgels for ameliorating myocardial infarction

Despite of the recent advances in regulatory T cell (Treg) therapy, a limited number of available cells and specificity at the desired tissue site have severely compromised their efficacy. Herein, an injectable drug-releasing (MTK-TK-drug) microgel system in response to in situ stimulation by reactive oxygen species (ROS) was constructed with a coaxial capillary microfluidic system and UV curing. The spherical microgels with a size of 150 μm were obtained. The MTK-TK-drug microgels efficiently converted the pro-inflammatory Th17 cells into anti-inflammatory regulatory T cells (Treg) cells in vitro, and the ROS-scavenging materials synergistically enhanced the effect by modulating the inflammation microenvironment. Thus, the microgels significantly reduced cardiomyocyte apoptosis and decreased the inflammatory response in the early stages of post-myocardial infarction (MI) in vivo, thereby reducing fibrosis, promoting vascularization, and preserving cardiac function. Overall, our results indicate that the MTK-TK-drug microgels can attenuate the inflammatory response and improve MI therapeutic effects in vivo.

Deepm6A-MT: A deep learning-based method for identifying RNA N6-methyladenosine sites in multiple tissues

N6-methyladenosine (m6A) is the most prevalent, abundant, and conserved internal modification in the eukaryotic messenger RNA (mRNAs) and plays a crucial role in the cellular process. Although more than ten methods were developed for m6A detection over the past decades, there were rooms left to improve the predictive accuracy and the efficiency. In this paper, we proposed an improved method for predicting m6A modification sites, which was based on bi-directional gated recurrent unit (Bi-GRU) and convolutional neural networks (CNN), called Deepm6A-MT. The Deepm6A-MT has two input channels. One is to use an embedding layer followed by the Bi-GRU and then by the CNN, and another is to use one-hot encoding, dinucleotide one-hot encoding, and nucleotide chemical property codes. We trained and evaluated the Deepm6A-MT both by the 5-fold cross-validation and the independent test. The empirical tests showed that the Deepm6A-MT achieved the state of the art performance. In addition, we also conducted the cross-species and the cross-tissues tests to further verify the Deepm6A-MT for effectiveness and efficiency. Finally, for the convenience of academic research, we deployed the Deepm6A-MT to the web server, which is accessed at the URL http://www.biolscience.cn/Deepm6A-MT/.

BMSCs-driven graphite oxide-grafted-carbon fibers reinforced polyetheretherketone composites as functional implants: in vivo biosafety and osteogenesis

Mesenchymal stem cells (MSCs) are increasingly becoming a potential treatment approach for bone injuries due to the multi-lineage differentiation potential, ability to recognize damaged tissue sites and secrete bioactive factors that can enhance tissue repair. The aim of this work was to improve osteogenesis of carbon fibers reinforced polyetheretherketone (CF/PEEK) implants through bone marrow mesenchymal stem cells (BMSCs)-based therapy. Moreover, bioactive graphene oxide (GO) was introduced into CF/PEEK by grafting GO onto CF to boost the osteogenic efficiency of BMSCs. Subsequently, CF/PEEK was implanted into the symmetrical skull defect models of SD rats. Then in vivo biosafety and osteogenesis were evaluated. The results indicated that surface wettability of CF/PEEK was effectively improved by GO, which was beneficial for the adhesion of BMSCs. The pathological tissue sections stained with H&E showed no significant pathological change in the main organs including heart, liver, spleen, lung and kidney, which indicated no acute systemic toxicity. Furthermore, bone mineralization deposition rate of CF/PEEK containing GO was 2.2 times that of pure CF/PEEK. The X-ray test showed that the surface of CF/PEEK containing GO was obviously covered by more newly formed bone tissue than pure CF/PEEK after 8 weeks of implantation. This work demonstrated that GO effectively enhanced surface bioactivity of CF/PEEK and assisted BMSCs in accelerating differentiation into bone tissue, providing a feasible strategy for improving osteogenesis of PEEK and CF/PEEK.

Antibody-mediated depletion of select leukocyte subsets in blood and tissue of nonhuman primates.

Understanding the immunological control of pathogens requires a detailed evaluation of the mechanistic contributions of individual cell types within the immune system. While knockout mouse models that lack certain cell types have been used to help define the role of those cells, the biological and physiological characteristics of mice do not necessarily recapitulate that of a human. To overcome some of these differences, studies often look towards nonhuman primates (NHPs) due to their close phylogenetic relationship to humans. To evaluate the immunological role of select cell types, the NHP model provides distinct advantages since NHP more closely mirror the disease manifestations and immunological characteristics of humans. However, many of the experimental manipulations routinely used in mice (e.g., gene knock-out) cannot be used with the NHP model. As an alternative, the in vivo infusion of monoclonal antibodies that target surface proteins on specific cells to either functionally inhibit or deplete cells can be a useful tool. Such depleting antibodies have been used in NHP studies to address immunological mechanisms of action. In these studies, the extent of depletion has generally been reported for blood, but not thoroughly assessed in tissues. Here, we evaluated four depleting regimens that primarily target T cells in NHP: anti-CD4, anti-CD8α, anti-CD8β, and immunotoxin-conjugated anti-CD3. We evaluated these treatments in healthy unvaccinated and IV BCG-vaccinated NHP to measure the extent that vaccine-elicited T cells - which may be activated, increased in number, or resident in specific tissues - are depleted compared to resting populations in unvaccinated NHPs. We report quantitative measurements of in vivo depletion at multiple tissue sites providing insight into the range of cell types depleted by a given mAb. While we found substantial depletion of target cell types in blood and tissue of many animals, residual cells remained, often residing within tissue. Notably, we find that animal-to-animal variation is substantial and consequently studies that use these reagents should be powered accordingly.

Serum IL-36β levels are associated with Insulin sensitivity in paediatric patients with obesity

Although the orchestrating role of Interleukin-36 cytokines in regulating inflammation at barrier tissue sites, is well established, whether they play a significant role in the settings of metabolic health and disease, has yet to be fully established. Several recent studies have demonstrated that IL-36 cytokine expression is elevated among adult patients with obesity, and can play roles in regulating both insulin sensitivity and driving inflammation. In this report, we have extended these analyses to paediatric patients and identified an association between elevated serum levels of expression of the specific Interleukin-36 subfamily member, IL-36β, among children with obesity displaying insulin sensitivity, compared to children with obesity who are insulin resistant. While these data further indicate a possible protective role for IL-36 in metabolic health, they also differ with previous findings from an adult patient cohort, where elevated levels of the related cytokine, IL-36γ, were found to occur in association with improved metabolic health. While highlighting important differences between paediatric and adult patient cohorts in the context of metabolic disease associated with obesity, these data underscore the need for a deeper mechanistic analysis of the role of IL-36 cytokines in disease.

Comparison of 2 T-Cell Receptor-γ Clonality Assays on Skin Biopsies Suspicious for Mycosis Fungoides.

PCR-based fragment analysis of the T-cell receptor (TCR) gene is used extensively in diagnostic labs to assess clonality in T-cell populations in multiple tissue sites. Of the numerous TCR assays that have been reported, studies assessing use on biopsies suspicious for mycosis fungoides specifically are lacking. We compared clonality findings from a previously run 2-tube/2-fluorochrome dye assay to a redesigned 1-tube/1-fluorochrome dye assay on formalin-fixed skin biopsies. Overall, the accuracy of the 2-tube assay was marginally better (75.7% vs. 71.4%), when using clinical history combined with histologic diagnosis as the gold standard. The 2-tube assay had better sensitivity (73.7% vs. 65.8%), while the 1-tube assay had superior specificity (93.8% vs. 87.5%). Clonality results were easier to interpret with the 1-tube assay. In nearly 19% of cases, a change of assays on the same biopsy resulted in a change of clonality interpretation. For laboratories that change TCR-γ clonality assays, follow-up biopsies for mycosis fungoides assessment may result in a change of diagnosis.

DNA methylation heterogeneity attributable to a complex tumor immune microenvironment prompts prognostic risk in glioma

ABSTRACT Gliomas are malignant tumours of the human nervous system with different World Health Organization (WHO) classifications, glioblastoma (GBM) with higher grade and are more malignant than lower-grade glioma (LGG). To dissect how the DNA methylation heterogeneity in gliomas is influenced by the complex cellular composition of the tumour immune microenvironment, we first compared the DNA methylation profiles of purified human immune cells and bulk glioma tissue, stratifying three tumour immune microenvironmental subtypes for GBM and LGG samples from The Cancer Genome Atlas (TCGA). We found that more intermediate methylation sites were enriched in glioma tumour tissues, and used the Proportion of sites with Intermediate Methylation (PIM) to compare intertumoral DNA methylation heterogeneity. A larger PIM score reflected stronger DNA methylation heterogeneity. Enhanced DNA methylation heterogeneity was associated with stronger immune cell infiltration, better survival rates, and slower tumour progression in glioma patients. We then created a Cell-type-associated DNA Methylation Heterogeneity Contribution (CMHC) score to explore the impact of different immune cell types on heterogeneous CpG site (CpG ct ) in glioma tissues. We identified eight prognosis-related CpG ct to construct a risk score: the Cell-type-associated DNA Methylation Heterogeneity Risk (CMHR) score. CMHR was positively correlated with cytotoxic T-lymphocyte infiltration (CTL), and showed better predictive performance for IDH status (AUC = 0.96) and glioma histological phenotype (AUC = 0.81). Furthermore, DNA methylation alterations of eight CpG ct might be related to drug treatments of gliomas. In conclusion, we indicated that DNA methylation heterogeneity is associated with a complex tumour immune microenvironment, glioma phenotype, and patient’s prognosis.

Abstract A003: Characterizing DNA methylation patterns driving chemotherapy resistance in ovarian high-grade serous carcinoma

Abstract To characterize the role of DNA methylation (DNAm) in chemotherapy response, we analyzed whole-genome DNAm data integrated with transcriptome from 67 samples of 17 patients with ovarian high grade serous carcinoma (HGSC) belonging to the prospective, longitudinal, multi-region DECIDER trial (NCT04846933). We used a novel decomposition method, which allows separating the effects of tumor microenvironment and treatment, tissue, and patient specific changes, facilitating the integration of the heterogeneous collection of samples. We firstly characterized DNAm alterations between tissue sites. Our results show that the cancer spread from tubo-ovarian sites to metastatic deposits is accompanied by moderate changes that employ cell junctions and focal adhesions. The most drastic change in the tissue intrinsic DNAm profiles implicated upregulation of members of the ATP-binding-cassette (ABC) family of transporters, including such multidrug resistance-associated genes as ABCC2, ABCC3, and ABCC4 in omentum. We then proceeded to characterize the DNAm changes during treatment. Our results reveal that DNAm plays a distinct role in emergence, driving, and regulation of both preexisting and acquired resistance to chemotherapy. We observed that patient-specific DNAm background shows enormous intra- and intertumor heterogeneity and sustains multiple driver mechanisms to resist chemotherapy-induced cell death already prior to treatment. We also observed that neoadjuvant chemotherapy (NACT) results in emergence of resistance via acquired, tissue specific DNAm alterations in DNA damage repair, apoptosis, cellular senescence, and drug efflux. Pathway analyses show that treatment-naïve tumors from patients with poor treatment outcome have DNAm-induced upregulation of key resistance promoting signaling pathways, such as PI3K/Akt, mTOR, and Rap1 cascades. Hypoxic transcriptional program activated via HIF-1 signaling was significantly altered in poorly responding patients, accompanied by changes in extracellular matrix instigated by integrins ITGB1 and ITGA5. Tumors of patients with refractory disease demonstrated DNAm changes in genes involved in tumor-promoting chronic inflammation driven by elevation of tumor necrosis factor. NACT treatment resulted in very few changes to these DNA aberrations and no acquired mechanisms, suggesting that intrinsic DNAm patient profiles accommodate multiple drivers for tumor progression and chemoresistance. In conclusion, using a large whole-sequencing DNAm dataset we elucidated the major underlying mechanisms of acquired platinum resistance, which provide possible targets for improving platinum sensitivity. Validation of these results and the intrinsic ability of HGSC to resist platinum-based treatment is currently on-going in our recently sequenced DNAm batch consisting of 400 samples of 135 HGSC patients. Citation Format: Alexandra Lahtinen, Giovanni Marchi, Kari Lavikka, Giulia Micoli, Susanna Holmström, Elsi Pöllänen, Ilari Maarala, Daria Afenteva, Yilin Li, Taru A. Muranen, Jaana Oikkonen, Kaisa Huhtinen, Anni Virtanen, Johanna Hynninen, Antti Häkkinen, Sampsa Hautaniemi. Characterizing DNA methylation patterns driving chemotherapy resistance in ovarian high-grade serous carcinoma [abstract]. In: Proceedings of the AACR Special Conference on Ovarian Cancer; 2023 Oct 5-7; Boston, Massachusetts. Philadelphia (PA): AACR; Cancer Res 2024;84(5 Suppl_2):Abstract nr A003.

Computed tomography imaging features of hepatic perivascular epithelioid cell tumor: A report of two cases and Review of the Literature

Background: Perivascular Epithelioid Cell tumor (PEComa) is an uncommon tumor of mesenchymal origin which can arise at a variety of visceral and soft tissue sites, most frequently in the uterus whereas particularly uncommon in liver,only 8% of PEComa cases. Here, we present two pathologically proven cases of PEComa of the liver, retrospectively discuss the clinical, imaging, histological features and review the literature. Case presentation: Case 1: A 54-year-old female patient with intermittent epigastric discomfort for 2 years and aggravated for 5 days.The laboratory examinations were normal. Computed Tomography (CT) showed the lesions were located on the right lobe. The discomfort had resolved after surgery and PEComa was diagnosed after pathological examination of the surgical specimen. The patient has been followedup for 5 years without recurrence. Case 2: A 30-year-old woman was found to have a mass of liver incidentally during regular physical examination after breast cancer surgery. On contrast enhancement CT, a well demarcated mass was found. After operation, pathological diagnosis was established as malignant PEComa of the right liver. Neither primary recurrence nor metastasis was found during the 2-year follow-up. Conclusion: The CT imaging manifestations of liver PEComa have certain characteristics. When the tumor density is not uniform or contains fat, and thickening vascular shadow appears around the lesion, combined with clinical and laboratory examination, suggestive diagnosis can be made.

Comparative Analysis of Metabolome and Transcriptome in Different Tissue Sites of Aquilaria sinensis (Lour.) Gilg.

The resinous stem of Aquilaria sinensis (Lour.) Gilg is the sole legally authorized source of agarwood in China. However, whether other tissue parts can be potential substitutes for agarwood requires further investigation. In this study, we conducted metabolic analysis and transcriptome sequencing of six distinct tissues (root, stem, leaf, seed, husk, and callus) of A. sinensis to investigate the variations in metabolite distribution characteristics and transcriptome data across different tissues. A total of 331 differential metabolites were identified by chromatography-mass spectrometry (GC-MS), of which 22.96% were terpenoids. The differentially expressed genes (DEGs) in RNA sequencing were enriched in sesquiterpene synthesis via the mevalonate pathway. The present study establishes a solid foundation for exploring potential alternatives to agarwood.

Comparing the effects of pulsed and radiofrequency catheter ablation on quality of life, anxiety, and depression of patients with paroxysmal supraventricular tachycardia: a single-center, randomized, single-blind, standard-controlled trial

BackgroundRadiofrequency catheter ablation (RFCA) may lead to decreased quality of life (QOL) and increased anxiety and depression in patients with paroxysmal supraventricular tachycardia (PSVT), possibly due to the lack of selectivity of the ablation tissue and the long ablation time. In recent years, pulsed field ablation (PFA) has been used for the first time in China to treat PSVT patients because of its ability to ablate abnormal tissue sites in a precise and transient manner. This study was conducted to compare the effects of PFA and RFCA on QOL and psychological symptoms of PSVT patients.MethodsWe have designed a single-center, randomized, single-blind, standard-controlled trial. A total of 50 participants who met the eligibility criteria would be randomly allocated into the PFA group or RFCA group in a 1:1 ratio. All participants were assessed using the 36-Item Short-Form Health Survey (SF-36) and the Hospital Anxiety and Depression Scale (HADS) at pre-procedure (T0), post-procedure (T1), and 3 months post-procedure (T2). The SPSS 21.0 software was used to analyze the data through Wilcoxon and Fisher’s exact tests and repeated measures ANOVA.ResultsTwenty-five in the PFA group and 24 in the RFCA group completed the trial. SF-36: (1) Between-group comparison: At T1, PFA group had significantly higher SF-36 scores on physiological function (PF) and general health (GH) than RFCA group, with a treatment difference of 5.61 points and 18.51 points(P < 0.05). (2) Within-group comparison: We found that in the PFA and RFCA groups, T2 showed significant improvement in the remaining 6 subscales of the SF-36 scale compared to T1 and T0 (P < 0.05), except for body pain (BP) and social function (SF) scores. HADS: (1) Between-group comparison: no significant difference (P > 0.05). (2) Within-group comparison: The HADS scores of the PFA and RFCA groups were statistically significant at T2 compared to T0 and T1 (P < 0.05).ConclusionsOur study provided new and meaningful evidence that PFA was effective in significantly improving QOL and decreasing anxiety and depression in PFA patients.Trial registrationChinese Clinical Trial Registry, ChiCTR2200060272.

Multilayer nanodrug delivery system with spatiotemporal drug release improves tumor microenvironment for synergistic anticancer therapy

The inflammatory response is one of the general symptoms that accompany tumorigenesis, the pro-inflammatory factors cyclooxygenase-2 (COX-2) and COX-2-derived prostaglandin-2 (PGE-2) in the inflammatory environment surrounding tumors possess promoting tumor development, metastasis and angiogenesis effects. In addition, the hypoxic environment of tumors severely limits the effectiveness of photodynamic therapy (PDT). In this study, a universal extracellular-intracellular ‘on-demand’ release nanomedicine DOX@PDA-ICG@MnO2@GN-CEL was developed for the combined fight against malignant tumors using a spatiotemporal controlled gelatin coated polydopamine (PDA@GN) as the carrier and loaded with the chemotherapeutic drug doxorubicin (DOX), the photosensitizer indocyanine green (ICG), the PDT enhancer MnO2 and the anti-inflammatory drug celecoxib (CEL) individually. Our results showed that DOX@PDA-ICG@MnO2@GN-CEL could release CEL extracellularly by matrix metalloproteinase-2 response and inhibit the COX-2/PGE-2 pathway, reduce chemotherapy resistance and attenuate the concurrent inflammation. After entering the tumor cells, the remaining DOX@PDA-ICG@MnO2 released DOX, ICG and MnO2 intracellularly through PDA acid response. MnO2 promoted the degradation of endogenous H2O2 to generate oxygen under acidic conditions to alleviate the tumor hypoxic environment, enhance PDT triggered by ICG. PDA and ICG exhibited photothermal therapy synergistically, and DOX exerted chemotherapy with reduced chemotherapy resistance. The dual responsive drug release switch enabled the chemotherapeutic, photothermal, photodynamic and anti-inflammatory drugs precisely acted on different sites of tumor tissues and realized a promising multimodal combination therapy.

Genome-wide investigation of SQUAMOSA promoter binding protein-like genes in Liriodendron and functional characterization of LcSPL2.

The plant-specific SQUAMOSA promoter-binding protein-like (SPL) transcription factors play a pivotal role in various developmental processes, including leaf morphogenesis and vegetative to reproductive phase transition. Liriodendron chinense and Liriodendron tulipifera are widely used in landscaping due to their tulip-like flowers and peculiar leaves. However, the SPL gene family in Liriodendron has not been identified and systematically characterized. We systematically identified and characterized the SPL family members in Liriodendron, including phylogeny, gene structure and syntenic analyses. Subsequently, we quantified the expression patterns of LcSPLs across various tissue sites through transcription-quantitative polymerase chain reaction (RT-qPCR) assays and identified the target gene, LcSPL2. Finally, we characterized the functions of LcSPL2 via ectopic transformation. Altogether, 17 LcSPL and 18 LtSPL genes were genome-widely identified in L. chinense and L. tulipifera, respectively. All the 35 SPLs were grouped into 9 clades. Both species had three SPL gene pairs arising from segmental duplication events, and the LcSPLs displayed high collinearity with the L. tulipifera genome. RT-qPCR assays showed that SPL genes were differentially expressed in different tissues, especially. Because LcSPL2 is highly expressed in pistils and leaves, it was selected to describe the SPL gene family of L. chinense by ectopic expression. We showed that overexpression of LcSPL2 in Arabidopsis thaliana resulted in earlier flowering and fewer rosette leaves. Moreover, we observed that overexpression of LcSPL2 in A. thaliana up-regulated the expression levels of four genes related to flower development. This study identified SPL genes in Liriodendron and characterized the function of LcSPL2 in advancing flower development.

Epidemiology and Clinical Outcomes of Fusobacterium Infections: A Six-Year Retrospective Study.

Background and Objectives: Anaerobic bacteria like Fusobacterium can lead to severe and life-threatening infections. The inherent complexities in the isolation of these bacteria may result in diagnostic and therapeutic delays, thereby escalating both morbidity and mortality rates. We aimed to examine data from patients with infections due to Fusobacterium to gain insights into the epidemiology and clinical outcomes of patients with these infections. Methods and Results: We conducted a retrospective analysis of clinical data from a cohort of patients with cultures positive for Fusobacterium species at a tertiary care medical center in the United States. Between 2009 and 2015, we identified 96 patients with cultures positive for Fusobacterium. Patients could be categorized into three groups based on the site of primary infection. Patients with head and neck infections constituted 37% (n 36). Patients with infections of other soft tissue sites accounted for 38.5% (n 37). Patients with anaerobic bacteremia due to Fusobacterium formed 24% (n 23) of the cohort. Surgical intervention coupled with antibiotic therapy emerged as cornerstones of management for patients with head and neck or other soft tissue infections, who generally exhibited more favorable outcomes. Patients with bacteremia were older, more likely to have malignancy, and had a high mortality rate. When speciation was available, Fusobacterium necrophorum was the most frequently isolated species. Conclusions: Our retrospective analysis of epidemiology and clinical outcomes of Fusobacterium infections revealed three distinct cohorts. Patients with head, neck, or soft tissue infections had better outcomes than those with bacteremia. Our findings highlight the importance of employing management strategies based on infection site and underlying comorbidities in patients with Fusobacterium infections. Further research is needed to investigate the optimal therapeutic strategies and identify prognostic indicators to improve clinical outcomes for these complex infections.

Detection of oral mucosal lesions by the fluorescence spectroscopy and classification of cancerous stages by support vector machine.

Detection of oral mucosal lesions has been performed by an in-house developed fluorescence-based portable device in the present study. A laser diode of 405 nm wavelength and a UV-visible spectrometer are utilized in the portable device as excitation and detection sources. At the 405 nm excitation wavelength, the flavin adenine dinucleotide (FAD) band at 500 nm and three porphyrin bands at 634, 676, and 703 nm are observed in the fluorescence spectrum of the oral cavity tissue. We have conducted this clinical study on a total of 189 tissue sites of 36 oral squamous cell carcinoma (OSCC) patients, 18 dysplastic (precancerous) patients, and 34 volunteers. Analysis of the fluorescence data has been performed by using the principal component analysis (PCA) method and support vector machine (SVM) classifier. PCA is applied first in the spectral data to reduce the dimension, and then classification among the three groups has been executed by employing the SVM. The SVM classifier includes linear, radial basis function (RBF), polynomial, and sigmoid kernels, and their classification efficacies are computed. Linear and RBF kernels on the testing data sets differentiated OSCC and dysplasia to normal with an accuracy of 100% and OSCC to dysplasia with an accuracy of 95% and 97%, respectively. Polynomial and sigmoid kernels showed less accuracy values among the groups ranging from 48 to 88% and 51 to 100%, respectively. The result indicates that fluorescence spectroscopy and the SVM classifier can help to identify early oral mucosal lesions with significant high accuracy.