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Background: Cardioprotective effects of N-acetyl-ser-asp-lys-pro (Ac-SDKP) have been reported in preclinical models of myocardial remodeling. However, the rapid degradation of this endogenous peptide in vivo limits its clinical use. Method: To prolong its bioavailability, Ac-SDKP was encapsulated by phosphocholine lipid bilayers (liposomes) similar to mammalian cell membranes. The physical properties of the liposome structures were assessed by dynamic light scattering and scanning electron microscopy. The uptake of Ac-SDKP by RAW 264.7 macrophages and human and murine primary cardiac fibroblasts was confirmed by fluorescence microscopy and flow cytometry. Spectrum computerized tomography and competitive enzyme-linked immunoassays were performed to measure the ex vivo cardiac biodistribution of Ac-SDKP. The biological effects of this novel synthetic compound were examined in cultured macrophages and cardiac fibroblasts and in a murine model of acute myocardial infarction induced by permanent coronary artery ligation. Results: A liposome formulation resulted in the greater uptake of Ac-SDKP than the naked peptide by cultured RAW 264.7 macrophages and cardiac fibroblasts. Liposome-delivered Ac-SDKP decreased fibroinflammatory genes in cultured cardiac fibroblasts co-treated with TGF-β1 and macrophages stimulated with LPS. Serial tissue and serum immunoassays showed the high bioavailability of Ac-SDKP in mouse myocardium and in circulation. Liposome-delivered Ac-SDKP improved cardiac function and reduced myocardial fibroinflammatory responses in mice with acute myocardial infarction. Conclusion: Encapsulation of Ac-SDKP in a cell membrane-like phospholipid bilayer enhances its plasma and tissue bioavailability and offers cardioprotection against ischemic myocardial injury. Future clinical trials can use this novel approach to test small protective endogenous peptides in myocardial remodeling.

CD45RA+ effector memory (EM) CD8+ T cell expansion was reported in Alzheimer’s disease (AD). Such cells are IL-7 receptor alpha (IL-7Rα)low EM CD8+ T cells, which expand with age and have a unique aging gene signature (i.e., IL-7Rαlow aging genes). Here we investigated whether IL-7Rαlow aging genes and previously reported AD and memory (ADM) genes overlapped with clinical significance in AD patients. RT-qPCR analysis of 40 genes, including 29 ADM, 9 top IL-7Ralow aging and 2 control genes, showed 8 differentially expressed genes between AD and cognitively normal groups; five (62.5%) of which were top IL-7Rαlow aging genes. Over-representation analysis revealed that these genes were highly present in molecular and biological pathways associated with AD. Distinct expression levels of these genes were associated with neuropsychological testing performance in 3 subgroups of dementia participants. Our findings support the possible implication of the IL-7Rαlow aging gene signature with AD.

INTRODUCTIONEffector memory (EM) CD8+ T cells have been associated with poor cognition in Alzheimer’s disease (AD). Our lab recently discovered an age-associated gene expression signature of IL-7 receptor alpha (IL-7Rα)low EM CD8+ T cells. We hypothesized that individuals with AD have altered levels of this IL-7Rαlow aging gene expression.METHODSForty genes associated with IL-7Rαlow EM CD8+ T cells, AD, or memory, were analyzed in peripheral blood of participants with normal cognition, mild cognitive impairment, and dementia by qPCR.RESULTSOf the eight genes that were found to be differentially expressed based on clinical diagnosis, 5 genes (62.5%) were IL-7Rαlow aging genes. Principal component analysis revealed 3 clusters of participants with dementia which had distinct expression levels of IL-7Rαlow aging genes and cognitive function.DISCUSSIONOur findings support the possible relationship of the IL-7Rαlow EM CD8+ T cell aging signature with cognition in individuals with dementia due to AD.

Background: We sought to determine whether differences in subtype distribution and differentially expressed genes exist between African Americans (AAs) and European Americans (EAs) in patients with high-risk nonmuscle-invasive bladder cancer (NMIBC). Methods: We performed a retrospective cohort study including 26 patients (14 AAs and 12 EAs) from the University of Texas Medical Branch and the Durham Veterans Affair Health Care System from 2010 to 2020 among treatment naïve, high-risk NMIBC. Profiled gene expressions were performed using the UROMOL classification system. Results: UROMOL racial subtype distributions were similar with class 2a being most common with 10 genes commonly upregulated in AAs compared to EAs including EFEMP1, S100A16, and MCL1 which are associated with progression to muscle-invasive bladder cancer, mitomycin C resistance, and bacillus Calmette-Guérin durability, respectively. We used single nuclei analysis to map the malignant cell heterogeneity in urothelial cancer which 5 distinct malignant epithelial subtypes whose presence has been associated with different therapeutic response prediction abilities. We mapped the expression of the 10 genes commonly upregulated by race as a function of the 5 malignant subtypes. This showed borderline (P = 0.056) difference among the subtypes suggesting AAs and EAs may be expected to have different therapeutic responses to treatments for bladder cancer. AAs were enriched with immune-related, inflammatory, and cellular regulation pathways compared to EAs, yet appeared to have reduced levels of the aggressive C3/CDH12 bladder tumor cell population. Conclusions: While premature, gene expression differed between AAs and EAs, supporting potential race-based etiologies for muscle-invasion, response to treatments, and transcriptome pathway regulations.

Standard chemotherapy agents, including carboplatin, have known immunogenic properties. We sought to determine how carboplatin may influence lymphocyte trafficking to tumor sites.Murine models of ovarian cancer were utilized to examine lymphocyte trafficking with common clinically used agents including carboplatin, anti-PD-1 antibody, or anti-VEGFR-2 antibody. Adhesion interactions of lymphocytes with tumor vasculature were measured using intravital microscopy, lymphocyte homing with immunohistochemistry, and treatment groups followed for overall survival.Carboplatin chemotherapy profoundly alters the tumor microenvironment to promote lymphocyte adhesive interactions with tumor vasculature and resultant improvement in lymphocyte trafficking. The measured results seen with carboplatin in the tumor microenvironment were superior to anti-PD-1 treatment or anti-VEGFR-2 which may have contributed to increased overall survival in carboplatin treated groups.These novel findings suggest a role for chemotherapeutic agents to broadly influence anti-tumor immune responses beyond the induction of immunogenic tumor cell death.

Phenotypic plasticity is a hallmark of cancer and increasingly realized as a mechanism of resistance in androgen indifferent prostate tumors. It is critical to identify mechanisms and actionable targets driving phenotypic plasticity. Here, we report that loss of tristetraprolin (TTP, geneZFP36), an RNA binding protein that regulates mRNA stability increases NF-κB activation and is associated with higher rates of aggressive disease and early recurrence in primary prostate cancer (PCa). We examined the clinical and biological impact ofZFP36loss combined withPTENloss, a known driver of PCa. Combined loss ofPTENandZFP36expression was associated with increased risk of recurrence in multiple independent primary PCa cohorts, and significantly reduced overall survival and time to progression following castration in genetically engineered mouse models.ZFP36loss alters the cell state that is driven byPTENloss, demonstrated by positive enrichment of gene sets including EMT, inflammation, TNFα/NF-κB, IL6-JAK/STAT3.ZFP36loss also induces enrichment of multiple gene sets involved in cell migration, chemotaxis, and proliferation. Use of the NF-κB inhibitor dimethylaminoparthenolide induced significant therapeutic responses in tumors withPTENandZFP36co-loss and reversed castration resistance. This work identifies a novel molecular mechanism driving phenotypic plasticity and castration resistance through loss ofZFP36expression, that can be reversed by inhibition of NF-κB activity.

Background Patients with hepatocellular carcinoma (HCC) have a poor prognosis with a 5-year survival rate of only 35%. Patients with locally advanced HCC (single or multinodal tumors >3 cm) may not be candidates for curative treatments such as surgical resection, liver transplant or thermal ablation due to tumor size, poor liver function, or other medical co-morbidities. The objective of this study was to investigate the potential use of interstitial chemo-phototherapy (I-CPT) to treat locally advanced HCC with a porphyrin-phospholipid (PoP) liposomal formulation of doxorubicin (Dox) that combines the targeted delivery of doxorubicin with photodynamic therapy to improve local drug delivery. We aimed to identify a safe and effective light dose for the photoactivation of Dox-Pop in the treatment of locally advanced HCC tumors in two pre-clinical models. Methods Sprague-Dawley rats were inoculated with Morris hepatoma cells directly into the left lateral liver lobe. When the tumors reached 2-2.5 cm along the longest axis, they were treated with Dox-Pop followed by light (i.e. I-CPT). For each rat, we applied our image-based treatment planning to guide the light delivery. Following treatment, tumor response was measured through weekly contrast-enhanced magnetic resonance imaging (CE-MRI). Complete response was defined as no evidence of the tumor via MRI or pathological examination. Our image-based treatment planning was used to translate the findings in the rat study to woodchucks with 1.5-4 cm hepatitis virus induced HCC. Weekly blood samples and bi-weekly CE-MRI were performed to assess tumor response up to 70 days. Results Five out of the six rats treated with I-CPT had complete response, with three of the five surviving ≥10 weeks post treatment without tumor recurrence or progression. The other two rats with complete response died between 5-7 weeks post treatment. Death was not attributed to the original treatment. Control, untreated rats were euthanized due to tumor progression within 1-2 weeks after tumors reached 2-2.5 cm. From the rat studies, we identified that a light dose rate and dose of respectively, ≥13.4 mW/cm2 and ≥25 J/cm2 can effectively activate the Dox-Pop liposomes. Of the five woodchucks treated with I-CPT using our effective light regimen, two survived ≥ 68 days post treatment without tumor progression. Another woodchuck had a complete response, but was euthanized 55 days post treatment due to the development of a second tumor. One woodchuck had progressive disease following I-CPT. One woodchuck died unexpectedly 5 days post I-CPT. Death was attributed to high tumor necrosis. Conclusions This study demonstrated the potential use of I-CPT as a treatment option for locally advanced HCC not candidate for standard of care therapies. Ongoing studies are being conducted in woodchucks to optimize the I-CPT light settings and improve tumor response. Citation Format: Emily Oakley, Minhyung Kim, Sanjana Ghosh, Hilliard Kutscher, Jonathan Lovell, Leslie Curtin, Sandra Sexton, Alan Hutson, Gal Shafirstein. Interstitial chemo-phototherapy for treatment of locally advanced hepatocellular carcinoma: A preclinical study [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 2420.

Estrogen receptor (ER) is the most prevalent marker of breast cancer. With its ligand estrogen, ER stimulates tumor cell growth by activating a global transcriptional program. The activation involves topoisomerase 2 (TOP2), which resolves topological problems during transcription by transiently creating and re-ligating DNA double-strand breaks (DSBs). Recent studies revealed that other DNA repair proteins also participate in the repair of TOP2-induced DSBs. These noncanonical repair pathways could act as backup processes when TOP2 fails to re-ligate DSBs, but their definite roles remain elusive. Here we examined the role of tyrosyl-DNA phosphodiesterase 2 (TDP2), an enzyme that promotes the removal of abortive TOP2 complex from the 5’-end of the DNA, in the estrogen-induced transcriptome. Using TDP2-deficient ER-positive cells and mice, we showed that TDP2 regulates the expression of oncogeneMYC.MYCinduction by estrogen is a very early (1 hour) and TOP2-dependent event. Without TDP2, the induction ofMYCby estrogen became prolonged and volatile at a later time point. Bulk and single-cell RNA-seq identified oncogenesMYCandCCND1as genes in which estrogen response was regulated by TDP2. These results could imply that TDP2 plays a role in the repair of TOP2-induced DSBs in specific genomic loci and tightly regulates oncogenic gene expression.

Androgen receptor signaling inhibitors (ARSIs) are standard of care for advanced prostate cancer (PCa) patients. Eventual resistance to ARSIs can include the expression of androgen receptor (AR) splice variant, AR-V7, expression as a recognized means of ligand-independent androgen signaling. We demonstrated that interleukin (IL)-6-mediated AR-V7 expression requires bone morphogenic protein (BMP) and CD105 receptor activity in both PCa and associated fibroblasts. Chromatin immunoprecipitation supported CD105-dependent ID1- and E2F-mediated expression of RBM38. Further, RNA immune precipitation demonstrated RBM38 binds the AR-cryptic exon 3 to enable AR-V7 generation. The forced expression of AR-V7 by primary prostatic fibroblasts diminished PCa sensitivity to ARSI. Conversely, downregulation of AR-V7 expression in cancer epithelia and associated fibroblasts was achieved by a CD105-neutralizing antibody, carotuximab. These compelling pre-clinical findings initiated an interventional study in PCa patients developing ARSI resistance. The combination of carotuximab and ARSI (i.e., enzalutamide or abiraterone) provided disease stabilization in four of nine assessable ARSI-refractory patients. Circulating tumor cell evaluation showed AR-V7 downregulation in the responsive subjects on combination treatment and revealed a three-gene panel that was predictive of response. The systemic antagonism of BMP/CD105 signaling can support ARSI re-sensitization in pre-clinical models and subjects that have otherwise developed resistance due to AR-V7 expression.

The effects of TEMPO-mediated oxidized cellulose nanofibril (TOCN) on the viscoelastic behavior and phase of cellulose nanocrystal (CNC) in suspension and film were investigated using polarized optical microscopy, rotational rheometry, and dynamic mechanical analysis. The sodium cation from TOCN changed the electrostatic state of CNC by screening the CNC surface charge. The volume inflation of TOCN locally increased the CNC concentration in the suspension. In turn, the CNC-CNC interactions increased the viscosity and the yield stress. Based on the experimental observation, the changing mechanisms of electrostatic state and particular interaction in the TOCN/CNC suspensions were suggested. In the hybrid film, the time dependency of complex moduli was changed owing to the different networking between CNCs and TOCNs. The CNC-CNC contacts easily collapsed by strain, while the TOCN-TOCN entanglements were slowly altered. This study provides a fundamental understanding of CNC behavior for optimizing processes and composite properties.

All-solid-state batteries (ASSBs) were fabricated with alloy-anode composite (Li3.25-Si + Li2S-P2S5), sulfide-based glass electrolyte (Li2S-P2S5), and sulfur-based composite cathode (S + Li2S-P2S5 + C). When the anode was made of only lithium silicide, voltage noise occurred under 0.5-3.7 V cut-off condition, which is out of the electrochemical stability window of the solid electrolyte (SE). This result indicates that Li-dendrites were generated by irreversible Li-ion deposition at the limited interface between the anode and the SE due to oxidative decomposition of SE. We modified the anode to the composite structure consisting of lithium silicide + sulfide SE to increase the number of the alloying reaction site. The pyrolytic graphite sheet (PGS) was placed between the anode and the current collector to facilitate electron transport. As a result, the voltage noise did not occur under the 0.5-3.7 V cut-off condition. This study indicates that increasing the number of alloying reaction sites improves the structural stability of the full cell, thereby increasing the cycle life.

Urinary bladder cancer accounts for 81,000 new cases per year and 4.7% of the new cancer diagnosis in the US. Even though most of the patients suffer from non-invasive lesions, around 70% will recur following surgical intervention. Chemotherapy with gemcitabine in combination with cisplatin is a common treatment option for recurrent disease, however 60% of the patients will relapse due to resistance. Gemcitabine (2’,2’-difluoro 2’-deoxycytidine) is classified as an antimetabolite, is commonly administered to BCa patients. This DNA synthesis inhibitor, is also used to treat pancreatic, breast, ovarian, and non-small cell lung cancer patients. Having lower relative toxicity, BCa patients are given gemcitabine as a single agent for intravesical therapy. Resistance mechanisms for gemcitabine have been reported to involve Hedgehog, Wnt, and Notch, signaling as potential means of cancer stem cell programming dependent on the tumor type. In examining mechanisms of resistance, we identified ketone body metabolism to be important. OXCT1, a rate-limiting enzyme in ketone metabolism, was found to be highly associated with bladder cancer death - Hoglund et al. P = 2.3x10-3 and TCGA, P = 6.5x10-4. We measured the expression of OXCT1 in RT4, UMUC3, T24 and 5637 bladder cancer cell lines and observed that expression correlated with gemcitabine resistance. The generation of gemcitabine resistance of otherwise sensitive 5637 cells (5637GR) resulted in elevation of OXCT1. Conversely, the CRISPR/Cas9 knockout of OXCT1 was able to sensitize inherently resistant UMUC3 and generated 5637GR lines. Given that OXCT1 is a rate limiting enzyme in ketone metabolism, we evaluated oxygen consumption rate finding increase metabolic activity. Furthermore we evaluated stemness through sphere forming assays (hanging drop and forced floating methods) to reveal that the OXCT1-KO lines had reduced spheres compared to their parental lines. Keeping in mind that several developmental processes were altered in 5637GR and restored to parental levels in the 5637GR-OXCT1-KO, we evaluated the transcriptomic differences in the cell types to determine the mechanism that led to the metabolic changes induced by gemcitabine resistance. master regulator analysis that revealed three key transcription factors (TFs), TBX2, SOX15 and OVOL1. Analysis of the downstream targets of the three master regulators in the TCGA BCa data (n = 408), the Peter C. Black neoadjuvant chemotherapy data (n = 134) and the Hoglund muscle invasive data (n = 308) demonstrated that OVOL1 is highly associated with recurrence. Moreover, downstream targets of OVOL1 are involved in fatty acid metabolism, survival and differentiation. These data links ketone metabolism and stemness. Additionally, it opens the door for targets in gemcitabine treatment Citation Format: Krizia Rohena Rivera, Sungyoung You, Minhyung Kim, Sandrine Billet, Johanna Hoeve-Scott, Gabrielle Gonzales, Chengqun Huang, Ashley Heard, Neil A. Bhowmick. OXCT1: Role of ketone bodies in acquired gemcitabine resistance in bladder cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 3239.

e17004 Background: Optimizing outcomes in prostate cancer (PCa) requires timely detect of metastatic progression of prostate cancer patients. We aim to optimize an extracellular vesicle (EV)-based Digital Scoring Assay for detecting PCa metastasis and noninvasive monitoring disease progression. Methods: PCa EV Digital Scoring Assay was developed by combining EV Click Chip, a nanotechnology-enabled microfluidic device for purifying PCa-derived EV and reverse-transcription droplet digital PCR for profiling disease-relevant mRNAs. A panel of 11 PCa-relevant mRNA markers were selected from The Tissue Atlas and a blood-based biomarker study. Plasma samples from 20 localized, 20 metastatic PCa patients, and serial plasma from three PCa patients were used to assess the performance of PCa EV Digital Scoring Assay with the 11-gene panel. mRNA expression of the panel was computed using weighted Z score method. ROC analysis was applied to discriminate localized from metastatic PCa. Results: After optimization, the capability of EV Click Chips (92.5 ± 5.4%) for capturing PCa-derived EVs from artificial plasma samples spiked with EVs outperformed ultracentrifugation (41.0 ± 3.2%) or precipitation methods (34.9± 2.2%; P< 0.001). Strong signals of the 11-gene panel, i.e., ACP3, FOLH1, HOXB13, KLK2, KLK3, KLK4, MSMB, RLN1, SLC45A3, STEAP2, and TMPRSS2, were detected from three PCa cells (LNCaP, C4-2B, and 22Rv1) and their derived EVs, while rare signals were detected in either WBCs or EVs purified from male healthy donor. In the clinical study, the Z scores calculated from the 11-gene panel were significantly higher in metastatic than localized PCa with an area under the ROC curve of 0.88 (95% CI: 0.78-0.98). Finally, longitudinal analyses of three PCa patients with clinical progression, response, and stable diseases, respectively, showed the Z scores can precisely reflect the disease status even when it is undetectable by imaging. Conclusions: We demonstrate a sensitive PCa EV Digital Scoring Assay to identify metastatic PCa and dynamically monitor disease states in a noninvasive manner. This assay may augment current imaging tools for timely detection of PCa progression and provide a means to better personalized care.

527 Background: Patients with high-risk non-muscle-invasive bladder cancer (NMIBC) have heterogeneous outcomes with African Americans (AAs) having worse survival than European Americans (EAs). It is unknown whether race-based biological differences contribute to this disparity. Methods: We performed a retrospective cohort study including patients from the University of Texas Medical Branch (UTMB) and the Durham VA Health Care System (DVAHCS) from 2010-2020 among treatment naïve, high-risk NMIBC. Profiled gene expressions of high-risk NMIBC by race were performed using the UROMOL classification system. Results: A total of 26 patients (14 AAs and 12 EAs) matched on age and sex were included with no significant difference in clinical stage group (CIS +/- T1 or TaHG vs. TaHG or T1, no CIS), smoking status, or progression. We found a similar racial UROMOL subtype distribution with class 2a being most common. A total of 10 genes were discovered to be commonly upregulated differentially expressed genes (up-DEGs) in AAs vs EAs. EFEMP1, which has been associated with progression to muscle-invasive bladder cancer (MIBC) in vitro, and S100A16 gene expression, which has been implicated with mitomycin C resistance in bladder cancer in vitro, was significantly more common among AAs. We used single nuclei analysis to map the malignant cell heterogeneity in urothelial cancer which five distinct malignant epithelial subtypes whose presence has been associated with different therapeutic response prediction ability. We mapped the expression of the 10 genes commonly up-DEGs by race as a function of the five malignant subtypes. This showed borderline (p = 0.056) differences among the subtypes suggesting AA and EA patients may be expected to have different therapeutic responses to treatments for BC. AAs were enriched with immune-related, inflammatory, and cellular regulation pathways compared to EAs, yet appeared to have reduced levels of the aggressive C3 bladder tumor cell population. Conclusions: In this small sample, we found similar subtype distribution among high-risk NMIBC patients according to race. However, gene expression differs by race, supporting potential novel race-based etiologies for differences in muscle-invasion, response to treatments, and transcriptome pathway regulations. Further biological studies in NMIBC molecular sub-stratification, associated treatment(s), and prognoses in a larger cohort are needed to support these hypotheses.

182 Background: There is an unmet need to develop a noninvasive assay to detect the metastatic progression of prostate cancer (PCa) patients for timely treatment. We aim to optimize an extracellular vesicle (EV)-based Digital Scoring Assay to reflect the disease status of PCa. Methods: PCa EV Digital Scoring Assay integrated a PCa-derived EV purification device (i.e., EV Click Chip) and downstream reverse-transcription droplet digital PCR for transcriptomic profiling of PCa-derived EVs. The performance of the Assay for purifying PCa-derived EVs was assessed using artificial plasma samples with spiked EVs. In parallel, a panel of 11 PCa-relevant mRNA markers were selected and validated. PCa EV Digital Scoring Assay in conjugation with the 11-gene panel was tested using plasma from 20 localized, 20 metastatic PCa patients, and serial plasma from three PCa patients. mRNA expressions were computed using weighted Z score method. ROC analysis was utilized to distinguish localized from metastatic PCa. Results: After optimization, the capability of purifying PCa-derived EVs from artificial plasma samples by EV Click Chips (92.5 ± 5.4%) outperformed ultracentrifugation or precipitation methods (41.0 ± 3.2%; 34.9± 2.2%; P < 0.001). Eleven genes, i.e., ACP3, FOLH1, HOXB13, KLK2, KLK3, KLK4, MSMB, RLN1, SLC45A3, STEAP2, and TMPRSS, were selected using The Tissue Atlas and a gene set from a blood-based study as our panel. Strong signals of the panel were detected from three PCa cells (LNCaP, C4-2B, and 22Rv1) and their derived EVs, while rare signals were detected in either WBCs or EVs purified from male healthy donor. In the clinical study, the Z scores calculated from the selected 11-gene panel, were significantly higher in 20 metastatic PCa than 20 localized PCa with an area under the ROC curve of 0.88 (95% CI = 0.78-0.98). Lastly, longitudinal analyses of three PCa patients showed that the Z scores can reflect the clinical progression, response, and stable status, even when disease is undetectable by imaging. Conclusions: We develop a sensitive PCa EV Digital Scoring Assay to distinguish metastatic PCa and reveal the dynamic disease states in a noninvasive manner. This assay holds the potential to augment current imaging tools and tests for timely detection of PCa progression and improve care for patients.