Time Points Research Articles

Healing of induced tongue defects using erythropoietin hydrogel (an experimental study on rats)

BackgroundTongue is complex muscular organ that may be affected by recurrent or chronic ulcerations and malignances that require effective treatment to enhance healing and tissue regeneration. So, this study aimed to evaluate the efficiency of erythropoietin (EPO) hydrogel as an anti-inflammatory and an inducer of neovascularization during healing of induced rats’ tongue defects.MethodsThirty six rats were divided into three groups; Group I (negative control): tongues were left without ulceration and received no treatment, Group II (positive control): tongue defects were prepared on the tongues’ dorsal surfaces, measuring (5 mm × 2 mm) using a tissue punch rotary drill for standardization, and left untreated, Group III (EPO group): tongue defects were prepared as in group II, then injected circumferentially around wound margins with a single high dose of EPO hydrogel of 5000 U/kg on the day of defect preparation. Animals were euthanized on seventh and fourteenth days after treatment, tongue specimens were collected, and paraffin blocks were prepared and processed for histological assessment by hematoxylin and eosin stain and immunohistochemical evaluation of anti-iNOS and anti-VEGF followed by histomorphometrical analysis and the relevant statistical tests.ResultsAt both time points, the EPO treated group showed significantly enhanced tissue regeneration marked by the histologically better regenerated tissue with well developed, thick walled and well-organized blood vessels and significant reduction in defect depth compared to positive control group. EPO group also showed significant decrease in iNOS and significant increase in VEGF antibodies indicating its anti-inflammatory and neovascularization effects respectively.ConclusionEPO treatment can significantly accelerate regeneration and filling of tongue defects by reducing tissue inflammation and enhancing neovascularization. Therefore, EPO could be a potential therapeutic strategy for accelerating healing of tongue ulcers. However, further investigations are required to optimize the dose and unravel any potential side effects before its clinical application.

Eggplant Little Leaf-Associated Phytoplasma Detection in Seedlings under Insect-Proof Conditions

Eggplant, or brinjal, is one of the most consumed and important tropical solanaceous vegetable crops grown worldwide. Little leaf is a disease associated with the presence of phytoplasmas especially widespread in brinjal in India. To clarify the epidemiology of this disease, a verification of its transmission through seeds to seedlings and their progeny derived from symptomatic mother plants was performed. Brinjal seeds field-collected in the Dharwad district of Karnataka State, India, were sowed in a greenhouse under insect-proof conditions. DNA was extracted from seedlings and their progeny and from symptomatic plant samples collected in the field. The first- and second-generation seedlings obtained *under these conditions were tested at various time points after germination by amplification of the 16S rRNA gene of phytoplasmas. The amplicons obtained were subjected to restriction fragment length polymorphism (RFLP) analysis and sequencing for the identification of detected phytoplasmas. Ribosomal groups 16SrI, 16SrII, 16SrIII, 16SrV, 16SrVI, and 16SrXII were identified. Moreover, a number of fruits produced from the first-generation seedlings showed precocious seed germination, and the young seedlings resulted as phytoplasma-positive. The seed transmission of phytoplasmas in eggplants for two subsequent generations highlights the risk of additional sources of infection of the disease represented by asymptomatic and infected seedlings in the presence of insect vectors. The seed transmission could explain the continuous presence of epidemic outbreaks of phytoplasmas in brinjal cultivations in several cultivation areas.

Bayesian interim analysis for prospective randomized studies: reanalysis of the acute myeloid leukemia HOVON 132 clinical trial

Randomized controlled trials (RCTs) are the gold standard to establish the benefit-risk ratio of novel drugs. However, the evaluation of mature results often takes many years. We hypothesized that the addition of Bayesian inference methods at interim analysis time points might accelerate and enforce the knowledge that such trials may generate. In order to test that hypothesis, we retrospectively applied a Bayesian approach to the HOVON 132 trial, in which 800 newly diagnosed AML patients aged 18 to 65 years were randomly assigned to a “7 + 3” induction with or without lenalidomide. Five years after the first patient was recruited, the trial was negative for its primary endpoint with no difference in event-free survival (EFS) between experimental and control groups (hazard ratio [HR] 0.99, p = 0.96) in the final conventional analysis. We retrospectively simulated interim analyses after the inclusion of 150, 300, 450, and 600 patients using a Bayesian methodology to detect early lack of efficacy signals. The HR for EFS comparing the lenalidomide arm with the control treatment arm was 1.21 (95% CI 0.81–1.69), 1.05 (95% CI 0.86–1.30), 1.00 (95% CI 0.84–1.19), and 1.02 (95% CI 0.87–1.19) at interim analysis 1, 2, 3 and 4, respectively. Complete remission rates were lower in the lenalidomide arm, and early deaths more frequent. A Bayesian approach identified that the probability of a clinically relevant benefit for EFS (HR < 0.76, as assumed in the statistical analysis plan) was very low at the first interim analysis (1.2%, 0.6%, 0.4%, and 0.1%, respectively). Similar observations were made for low probabilities of any benefit regarding CR. Therefore, Bayesian analysis significantly adds to conventional methods applied for interim analysis and may thereby accelerate the performance and completion of phase III trials.

Driving and mobile phone use: Work addiction predicts hazardous but not excessive mobile phone use in a longitudinal study of young adults.

Work addiction (WA), characterized by dimensions such as overcommitment, difficulties in detachment from work, and work-life imbalance, is presumed to be associated with increased smartphone usage, even during risky activities like driving. The study investigated the connection between WA and future problematic and hazardous smartphone use, considering personality factors: anxiety, rumination, and worry. A three-wave longitudinal study (N=1,866) was conducted from March to July 2019, June to September 2020, and June to November 2021, involving a representative sample of 18-34-year-old residents in Hungary's capital. The study employed Hungarian versions of the Bergen Work Addiction Scale, Problematic Mobile Phone Use Questionnaire, Ruminative Response Scale, Anxiety subscale of the Brief Symptom Inventory 18, and Penn-State Worry Questionnaire. Additionally, author-developed questions on mobile phone use while driving were included. At baseline, those at risk for WA showed more frequent mobile phone use while driving at both time points 2 and 3 compared to the non-risk group. Path analyses revealed rumination, anxiety at time 1, and worry at time 2 as significant mediators between baseline WA and mobile phone use while driving at time 3. However, when analyzing all three mediators together, only anxiety at time 1 and worry at time 2 remained significant. This study demonstrates that WA predicts future mobile phone use while driving through mediation by anxiety and worry. Our findings add to the growing evidence highlighting the detrimental aspects of WA, emphasizing the need for improved prevention and treatment strategies.

Efficacy of bilateral catheter superficial parasternal intercostal plane blocks using programmed intermittent bolus for opioid-sparing postoperative analgesia in cardiac surgery with sternotomy: A randomized, double-blind, placebo-controlled trial

Study objectiveThis study investigated whether catheter superficial parasternal intercostal plane (SPIP) blocks, using a programmed intermittent bolus (PIB) with ropivacaine, could reduce opioid consumption while delivering enhanced analgesia for a period exceeding 48 h following cardiac surgery involving sternotomy. DesignA double-blind, prospective, randomized, placebo-controlled trial. SettingUniversity-affiliated tertiary care hospital. Patients60 patients aged 18 or older, scheduled for cardiac surgery via sternotomy. InterventionsThe patients were randomly assigned in a 1:1 ratio to either the ropivacaine or saline group. After surgery, patients received bilateral SPIP blocks for 48 h with 0.4% ropivacaine (20 mL per side) for induction, followed by bilateral SPIP catheters using PIB with 0.2% ropivacaine (8 mL/side, interspersed with a 2-h interval) or 0.9% normal saline following the same administration schedule. All patients were administered patient-controlled analgesia with hydromorphone. MeasurementsThe primary outcome was the cumulative morphine equivalent consumption during the initial 48 h after the surgery. Secondary outcomes included postoperative pain assessment using the Numeric Rating Scale (NRS) at rest and during coughing at designated intervals for three days post-extubation. Furthermore, recovery indicators and ropivacaine plasma levels were diligently documented. Main resultsCumulative morphine consumption within 48 h in ropivacaine group decreased significantly compared to saline group (25.34 ± 31.1 mg vs 76.28 ± 77.2 mg, respectively; 95% CI, −81.9 to −20.0, P = 0.002). The ropivacaine group also reported lower NRS scores at all recorded time points (P < 0.05) and a lower incidence of nausea and vomiting than the saline group (3/29 vs 12/29, respectively; P = 0.007). Additionally, the ropivacaine group showed significant improvements in ambulation (P = 0.018), respiratory exercises (P = 0.006), and self-reported analgesia satisfaction compared to the saline group (P = 0.016). ConclusionsBilateral catheter SPIP blocks using PIB with ropivacaine reduced opioid consumption over 48 h, concurrently delivering superior postoperative analgesia in adult cardiac surgery with sternotomy.

Pain trajectories and neuropathic pain symptoms following lung cancer surgery: A prospective cohort study.

Persistent postsurgical pain (PPSP) after lung cancer surgery is common and current definitions are based on evaluations at a single time point after surgery. Pain intensity and symptoms may however fluctuate and change over time, and be impacted by multiple and shifting factors. Studies of postoperative recovery patterns and transition from acute to chronic pain are needed for further investigation of preventive measures and treatments to modify unfavourable recovery paths. In this explorative study, 85 patients undergoing surgery due to either presumptive or confirmed lung cancer reported pain intensities bi-monthly for 12 months. Pain trajectories during recovery were investigated, using group-based trajectory modelling. Associations with possible risk factors for PPSP, including clinical variables and anxiety and depression score (HADS), were also explored. A trajectory model containing three 12-month pain recovery groups was computed. One group without PPSP fully recovered (50%) within two to three months. Another group with mild-intensity PPSP followed a protracted recovery trajectory (37%), while incomplete recovery was observed in the last group (13%). Acute postoperative pain and younger age were associated with a less favourable recovery trajectory. More neuropathic pain symptoms were observed in patients with incomplete recovery. Three clinically relevant recovery trajectories were identified, based on comprehensive pain tracking. Higher acute postoperative pain intensity was associated with an unfavourable pain recovery trajectory. Understanding the transition from acute to chronic postoperative pain and identifying preoperative risk factors is essential for the development of targeted treatments and the implementation of preventive measures. This study (1) identified distinct recovery trajectories based on frequent pain assessment follow-ups for 12 months after surgery and (2) evaluated risk factors for unfavourable postoperative pain recovery paths. Findings suggest that early higher postoperative pain intensity is associated with an unfavourable long-term recovery path.

Identification of Anoikis-Related Genes in Spinal Cord Injury: Bioinformatics and Experimental Validation.

Spinal cord injury (SCI) is a serious disease without effective therapeutic strategies. To identify the potential treatments for SCI, it is extremely important to explore the underlying mechanism. Current studies demonstrate that anoikis might play an important role in SCI. In this study, we aimed to identify the key anoikis-related genes (ARGs) providing therapeutic targets for SCI. The mRNA expression matrix of GSE45006 was downloaded from the Gene Expression Omnibus (GEO) database, and the ARGs were downloaded from the Molecular Signatures Database (MSigDB database). Then, the potential differentially expressed ARGs were identified. Next, correlation analysis, gene ontology (GO) enrichment analysis and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis, and protein-protein interaction (PPI) analysis were employed for the differentially expressed ARGs. Moreover, miRNA-gene networks were constructed by the hub ARGs. Finally, RNA expression of the top ten hub ARGs was validated in the SCI cell model and rat SCI model. A total of 27 common differentially expressed ARGs were identified at different time points (1, 3, 7, and 14days) following SCI. The GO and KEGG enrichment analysis of these ARGs indicated several enriched terms related to proliferation, cell cycle, and apoptotic process. The PPI results revealed that most of the ARGs interacted with each other. Ten hub ARGs were further screened, and all the 10 genes were validated in the SCI cell model. In the rat model, only seven genes were validated eventually. We identified 27 differentially expressed ARGs of the SCI through bioinformatic analysis. Seven real hub ARGs (CCND1, FN1, IGF1, MYC, STAT3, TGFB1, and TP53) were identified eventually. These results may expand our understanding of SCI and contribute to the exploration of potential SCI targets.

Abstract 496: Insights into correlation between nanomechanical properties and cytokines expression during gold nanoparticle endocytosis

Abstract Gold nanoparticles for therapeutic interventions have become a widely popular source due to the ease in surface chemistry available for modification. Several studies undertaken focusses on the size, shape of nanoparticles as well as the signaling mechanisms and yet, knowledge regarding the alteration in membrane dynamics such as nanomechanical properties and cytokines involved during their uptake has not been studies before. To overcome the knowledge deficit, we employed atomic force microscopy and qRT-PCR techniques to acquire nanomechanical attributes and cytokines expressions respectively, in Panc-1 and AsPC-1 cell lines treated with surface modified gold nanoparticles for 1- and 24-hours’ time point. In nanomechanics paradigm, impetus was given on both linear attributes such as membrane stiffness, deformation, and adhesion as well as non-linear attributes such as drained Poisson’s ratio, diffusion coefficient and pore size. On the other hand, qRT-PCR technique yielded alterations in relative mRNA expressions of pro-tumorigenic cytokines such as CCL2, CXCL1, CXCL2, CXCL3, IL8, IL11, IL18 and TGFβ-1. Further, using Pearson’s correlation, we deduced the dependency between nanomechanical properties and cytokines expressions. We evaluated several criteria such as receptor dependent vs independent, PEGylated vs non-PEGylated and 1 hr vs 24hrs, to deduce correlations between cytokines and nanomechanical attributes. In Panc-1 cell line, we observed that PEGylation vs non-PEGylation of gold nanoparticles demonstrated most significant correlations, whereas in AsPC-1 cell line, receptor independent mechanism played a pivotal role in establishing significant correlations. For instance, D_C and P_D were highly correlated with CXCL1, CXCL2 and TGFβ-1 when non-PEGylated gold nanoparticles were up taken by Panc-1 cells. Similarly, D_C and P_D were highly correlated consistently with our cytokine panel except for TGFβ-1. In AsPC-1 cell line, deformation was significantly correlated with CCL2, CXCL2, CXCL3 and IL-8 whereas, D_C and P_D were significantly correlated with CCL2, CXCL1, CXCL3, IL-18 and TGFβ-1 in receptor independent scenario. This study demonstrated that during the uptake of gold nanoparticles, non-linear nanomechanical attributes are detrimental over linear attributes. Moreover, Surface modified gold nanoparticles induce varying levels of alteration in pro-tumorigenic cytokines. Taken together, this study boosts our insights into correlations describing cytokines regulating nanomechanical attributes in endocytosis paradigm. Citation Format: Tanmay Kulkarni, Enfeng Wang, Ramcharan Singh Angom, Debabrata Mukhopadhyay, Santanu Bhattacharya. Insights into correlation between nanomechanical properties and cytokines expression during gold nanoparticle endocytosis [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 496.

Abstract 4685: The identification of new therapeutic targets in mucinous ovarian carcinoma

Abstract Background: Epithelial ovarian cancer (EOC) is the second cause of death among gynaecological cancersworldwide. Mucinous ovarian carcinoma (mEOC) accounts for 3-5% of all EOCs, and when diagnosed at anadvanced stage its prognosis is very poor mainly for its limited chemosensitivity. PLK1 is a member of thewell-conserved serine/threonine kinase family, that plays a key role in the progression of mitosis, in the G2/Mcheckpoint regulation, DNA damage replication, stress response, and cell death pathways. mEOC has beenshown to be sensitive to PLK1 inhibition both with siRNA and small molecule inhibitor (onvansertib). Giventhis background and the need for new therapeutic approaches in mEOC, the purposes of this work were toidentify new therapeutic targets in mEOC using CRISPR/Cas9 lentivirus libraries and to find synthetic lethalpartners with the PLK1 inhibitor onvansertib. Methods: We used three different mucinous cell lines: MCAS,EFO27, TOV2414, overexpressing Cas9 gene. We evaluated the Cas9 expression through Western Blotanalysis, and the Cas9 activity through a Fluorescent Activated Cell Sorter- based-GFP assay. We used twodifferent lentiviral gRNAs Libraries (Bassik Library Human CRISPR Deletion Library - Apoptosis and cancer(Addgene #101926)/Drug targets, kinases, and phosphatases (Addgene #101927)). The EFO27 Cas9 cells wereused for the screening experiment. Cells were infected with the lentiviral libraries at MOI of 0.3 and put inpuromycin selection. Six days after puromycin addition (Time point 0) 30x10^6 cells were collected forsequencing. The remaining 30x10^6 cells were split in two: control cells and cells treated with a subtoxic doseof onvansertib (IC30). After 7 days (Time point 1) 30x10^6 cells for each condition were collected forsequencing. The infection of libraries was performed alone and in combination with sub-toxic onvansertibtreatment, to identify genes important for mucinous carcinoma cell survival, and those genes in syntheticlethality with onvansertib. Results: Bioinformatic analyses comparing control cells (time 1 vs time 0) andPLK1 treated and non -treated cells (time 1) allowed the generation of a gene ranking list, including genesinvolved in survival of EFO27 cells like ZC2HC1C, RPA2, POLE3, KIN, TUBG1, SMC2, CDC26, CDC42, HOXA9,TAF10, DCLRE1C, SENP1, MRPS31, COPS2; and genes in synthetic lethality with PLK1 treatment, like JUND,CARD9, BCL2L2. Validation experiments are ongoing and will be presented and discussed. Conclusion: Using a CRISPR-based approach, we identified genes that either alone or in combination with PLK inhibitioncould have therapeutic value in mEOC. Citation Format: Serena Petrella, Marika Colombo, Mirko Marabese, Chiara Grasselli, Andrea Panfili, Ilaria Craparotta, Maria Chiara Barbera, Giada Andrea Cassanmagnago, Marco Bolis, Giovanna Damia. The identification of new therapeutic targets in mucinous ovarian carcinoma [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 4685.

Abstract 3858: Peripheral CD38+ effector memory (EM) T cells are dynamic biomarkers of response to neoadjuvant immune checkpoint blockade (NeoICB) in patients (pts) with resectable dedifferentiated liposarcoma (DDLPS) and undifferentiated pleomorphic sarcoma (UPS)

Abstract Introduction: NeoICB is a promising approach for pts with DDLPS and UPS. However, biomarkers to identify which pts benefit from this treatment and understanding the mechanisms of response and resistance to ICB remains a challenge. Methods: We conducted a randomized phase 2 trial of neoICB (anti-PD1 +/- CTLA4) in pts with resectable DDLPS (n=17) and UPS (n=10). Pts with UPS received concurrent neoICB and radiation. The primary endpoint of the trial was pathological response; 3 DDLPS and 9 UPS pts were responders (R) and 14 DDLPS and 1 UPS pts were non-responders (NR). Peripheral blood mononuclear cells (PBMCs) were collected at baseline, prior to cycle 2 of neoICB (C2D1), and at the time of surgery (SURG). We performed mass cytometry (CyTOF) to identify immune cells associated with response and their dynamic changes with neoICB. We designed a custom 41-parameter panel using CD45 live cell barcoding with 31 markers for immune profiling, of which 23 were selected to detect key cell lineage and 8 to detect signaling markers. Batch correction and high dimensional data analyses were performed using CyCombine and CyTOF workflow R packages. Longitudinal comparisons between immune cell populations were conducted using Mann-Whitney U Test. Results: Of 79 available PBMC samples from 27 pts, 63 passed quality control after thawing (≥30% viability) and data clean-up (at least 10,000 events). Data was down-sampled to 5000 events for analysis. Of 19 immune cell types identified, monocytes were the most common followed by CD4+ and CD8+ T cells. There was an early increase in CD38+ effector/effector memory (EM) T cells at C2D1 in pts with DDLPS (CD8+ p=0.005, CD4+ p&amp;lt;0.001) and a similar increase observed in pts with UPS (CD8+ C2D1 p=NS, SURG p=0.034; CD4+ C2D1 p=0.024, SURG p=0.06) and these cells had higher Ki67 expression than CD38- T cells (p&amp;lt;0.005). At baseline, R pts with DDLPS had higher fractions of CD38+ EM CD8+ T cells than NR pts (p=0.044). Analysis in the UPS cohort was limited by the low number of NR pts. Higher expression of TIGIT was observed on CD38+ EM CD8+ T cells compared to CD38- counterparts at all time points. Conclusion: Peripheral CD38+ EM CD8+ T cells are associated with response to neoICB in pts with DDLPS. Longitudinal peripheral immune profiling reveals an increase in CD38+ EM CD8+ and CD4+ T cells with neoICB in pts with DDLPS and UPS. These cells are proliferative and express higher levels of TIGIT, which could be a future target for therapeutic exploration. Importantly, changes in the peripheral immune populations may precede changes in the tumor microenvironment (TME) as preliminary analyses of the TME did not identify early (C2D1) changes. TME imaging mass cytometry analyses are currently underway to associate peripheral and intratumor changes in immune populations. Citation Format: Elise F. Nassif, Hsinyi Lu, Michael Stanford, Karen Millerchip, Diana Romero, Jared Malke, Brenna Matejka, Jeffrey E. Gershenwald, Susmita Kumari, Duncan H. Mak, Angelique J. Lin, Jared K. Burks, Wei-Lien Wang, Alexander J. Lazar, Neeta Somaiah, Cara Haymaker, Christina L. Roland, Emily Z. Keung. Peripheral CD38+ effector memory (EM) T cells are dynamic biomarkers of response to neoadjuvant immune checkpoint blockade (NeoICB) in patients (pts) with resectable dedifferentiated liposarcoma (DDLPS) and undifferentiated pleomorphic sarcoma (UPS) [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 3858.

Abstract 347: Tumor necrosis factor related apoptosis inducing ligand (TRAIL)-induced cytokine production in TNBC promotes neutrophil chemotaxis and immune suppression

Abstract Background: TRAIL induces apoptosis in many preclinical cancer models including breast cancers and has been extensively studied as a potential cancer therapeutic. However, its efficacy in clinical trials is limited, suggesting an unknown modulatory mechanism responsible for lack of TRAIL activity in vivo. Here, we describe that TRAIL treatment elicits transcriptional changes in TNBC cells that alter the immune milieu. Method: We performed RNAseq of MDA-MB-231 cells treated with TRAIL for different time points, followed by validation with RT-PCR in various TNBC cells. RNAi, silencing key differentially regulated genes, along with RT-PCR, ELISA and CHIP assays, were used to validate RNAseq findings. Elucidation of the functional relevance of the outcome was supported both in vitro and in vivo by chemotaxis assay, cytotoxicity assay, RNAseq analysis of donor isolated neutrophils and intravital microscopy, CODEX analysis in TNBC xenografts from mice treated with the TRAIL respectively. Results: TRAIL treatment of the TNBC significantly induced expression of several cytokines, such as CXCLs 1, 2, 3, 8,11 and IL6, both in vitro and in vivo which are known to affect neutrophil function. Mechanistically, induction of these cytokines was predominantly mediated by death receptor 5 and caspase-8 protein, but not caspase-8 enzymatic activity. GSEA of the RNAseq data indicated that NFKB pathway was significantly enriched. Concordantly, we confirmed that both canonical NFKB1 and non-canonical NFKB2 pathways were activated by TRAIL in vitro and in vivo. However, the induction of the cytokine mRNAs was primarily dependent on the NFKB2 pathway. Neutrophils isolated from healthy human donors incubated with supernatants from TNBC cells in vitro indicated that TRAIL-induced CXCLs and IL6 significantly increased neutrophil chemotaxis. Additionally, CODEX analysis as well as intravital imaging confirmed that TRAIL treatment increases the number of neutrophils in the tumor. Preincubation of neutrophils with supernatants from TRAIL-treated TNBC significantly inhibited their cytotoxic effect against TNBCs. Further, transcriptome analysis of neutrophils incubated with either TRAIL or supernatant of TRAIL treated TNBC revealed significant enrichment of expression of inflammatory cytokine genes, immune modulating and immune checkpoint genes like PDL1. Functional studies with these neutrophils confirmed their suppressive effect on T cell function as well as the effect of TRAIL on decreased neutrophil apoptosis. Conclusion: Collectively, our study suggests the novel role of TRAIL-induced NFKB2-dependent cytokine production promoting neutrophil chemotaxis and immune suppression. This study implies that alterations in the innate immune system may modulate the effects of TRAIL on TNBC tumors. Citation Format: Manjari Kundu, Yoshimi Endo Greer, Lisa Ann Ridnour, David A Wink, Yeap S. Ng, Roberto Weigert, Stan Lipkowitz. Tumor necrosis factor related apoptosis inducing ligand (TRAIL)-induced cytokine production in TNBC promotes neutrophil chemotaxis and immune suppression [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 347.

Abstract 3897: Isolation and molecular characterization of exosomes from glioblastoma patients using a microfluidic device after ultrasound-based opening of the blood-brain barrier

Abstract Glioblastoma (GBM) is a highly aggressive tumor of the brain. Current techniques of detection like magnetic resonance imaging and tissue biopsy are inaccurate or highly invasive, requiring alternatives like liquid biopsy. Exosomes are nanosized vesicles that contain cargo, reflective of their cellular origin and have been used for early diagnosis of cancer. However, in GBM, the blood-brain barrier (BBB) leads to a paucity of pathological information in blood. This study investigates the effect of opening the BBB on circulating exosomes and their potential for early detection of GBM using a microfluidic platform (GlioExoChip). In 20 patients enrolled in a clinical trial NCT03744026, a pulsed ultrasound emitter- Sonocloud-9 was implanted into their brains to open the BBB. We show that GlioExoChip isolates GBM-specific exosomes with a high capture efficiency using phosphatidylserine and Annexin-V chemistry. Further, the amount of circulating EVs increases twofold after opening the BBB. Finally, the correlation between tumor growth rate and the exosome concentrations is evaluated longitudinally. Methods: GlioExoChip is a microfluidic platform with a polydimethylsiloxane top and a glass bottom, functionalized with neutravidin-biotinylated annexin-V. Experiments are performed with GBM cell-line derived exosomes, followed by patient plasma. Isolated exosomes are characterized using Nanoparticle tracking analysis (NTA), scanning electron microscopy, Bicinchoninic acid (BCA) assay and western blot. Statistical analyses are performed using Student’s t-test and Pearson correlation. Results: The microfluidic device GlioExoChip has a high capture efficiency of exosomes derived from GBM cell lines, i.e., 93.03±3.31% and 96.77±0.72% for GBM6 and U87, respectively. NTA and BCA showed that 15 times more exosomes were captured from patient plasma when compared with healthy. Further, western blot proved the presence of a glial protein and Flotillin-1 in exosomes isolated from patients. Exosome concentrations varied with different time points after sonication and had an average twofold increase from the BBB opening. In our longitudinal study with 8 patients, an association between the volumetric enhancement of the tumor and exosome concentrations after sonication shows that the average Pearson correlation among short progression-free survival (PFS) patients was 0.73 (avg. p=0.39), while no correlation was seen among long PFS patients (avg. r value=0.10; avg. p-value=0.54). Conclusions: A microfluidic approach was used to investigate the role of circulating exosomes using phosphatidylserine-annexin V chemistry in GBM. BBB opening via sonication leads to an increase in the average GBM-specific exosome concentrations in plasma. Thus, exosomes are promising biomarkers for early detection in GBM. Citation Format: Abha Kumari, Mark Youngblood, Andrew Gould, Yoon-Tae Kang, Li Chen, Karl Habashy, Chris Amidei, Rachel Ward, Cristal Gomez, Guillaume Bouchoux, Michael Canney, Roger Stupp, Adam M. Sonabend, Sunitha Nagrath. Isolation and molecular characterization of exosomes from glioblastoma patients using a microfluidic device after ultrasound-based opening of the blood-brain barrier [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 3897.

Abstract 119: A non-surgical method for developing a pre-clinical orthotopic mouse model for colorectal cancer

Abstract Recapitulating physiologically relevant tumor microenvironment is important yet a significant challenge in modeling colorectal cancer (CRC) for basic and translational studies. Among the different methods, using the cecal-wall injection technique is the most common way to create animal models of colorectal cancer (CRC). However, this approach requires complex surgery, and its unnatural placement creates a significant obstacle for developing treatments. Here, we report a non-surgical procedure for establishing orthotopic CRC mouse models, reflecting the appropriate tumor microenvironment to better model the disease development and facilitate translational studies including drug identification and validation. In this procedure, anesthesia is induced in overnight fasted study immunocompetent or immunodeficient mice while placed on a thermostat-controlled heating blanket. A specialized catheter is inserted and distended into the mouse colon to gently block the colon and ensure the localization for tumor development. Hereafter, the colonic epithelium is treated with proteases and mechanical abrasion to cause mild disruption. Following the local gut inflammation, the targeted colon region is incubated with tumor cells, leading to the adhesion of tumor cells to the colonic epithelium. Based on the animal background and the cell line used, the tumor developed within 3-4 weeks. The stages of tumor development at different time points were successfully confirmed using hematoxylin and eosin stain. Tumor microenvironment data revealed a significant increase in the immune cell infiltration specifically CD4+ T-cells and tumor-associated macrophages in the non-surgical procedure as compared to the cecal-wall injection. Furthermore, a significant increase in the PD-1 expression of the CD8+ T-cells in the cecal-wall injection as compared to the non-surgical procedure, suggests that the precise location of the tumor site may exert a substantial influence on pre-clinical therapeutic outcomes of immune-checkpoint inhibitors, thereby potentially hampering the clinical efficacy of drug treatments. Additional adaptations of this procedure may include: i) tumor development in different mouse strains, ii) local delivery of adeno-Cre in transgenic mice for CRC, and iii) delivering tumor organoids and patient-derived xenografts in immuno-deficient mice. Overall, our non-surgical procedure overcomes the technical difficulties of surgery and offers several advantages like ease of handling, cost-effectiveness, pain-free with nearly no procedure-associated mortality and adaptability to several strains. In conclusion, this procedure provides novel and promising alternative to conventional surgical orthotopic CRC mouse models for therapeutic interventions. Citation Format: Naresh Singh, Samantha Sharma, Kevin Van Der Jeught, Zhuolong Zhou, Xinna Zhang, Xiongbin Lu. A non-surgical method for developing a pre-clinical orthotopic mouse model for colorectal cancer [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 119.

Abstract 4788: Noninvasive longitudinal monitoring of residual disease in chemotherapy-treated colorectal cancer patients

Abstract Noninvasive monitoring of cancer shows great promise in assessing therapy response and improving patient outcomes. Recently, various groups have developed methods that detect circulating tumor DNA (ctDNA) in the plasma to measure minimal or molecular residual disease (MRD). Aberrant DNA methylation patterns are a hallmark of cancers, and robust signals can be detected by sensitive ctDNA assays. Here, we present a methylation-based approach for longitudinal monitoring of tumor burden that is tumor-naive, i.e., with no reliance on prior characterization of tumor molecular characteristics. We assess the effectiveness of our approach in a cohort of colorectal cancer (CRC) patients receiving chemotherapy with or without additional targeted agents. Longitudinal blood samples were collected from patients (n = 57) while on therapy, which averaged 4.9 months, for a total of 239 sample collection time points (median per patient = 5). Based on RECIST status, 16 patients were classified as complete responders (CR) at the end of treatment. We generated plasma cell free DNA-derived libraries for methylation sequencing targeting regions relevant to CRC. Next, we computed a disease burden score for each sample and related these scores to clinical response (CR vs. non-CR). We trained a classifier on an independent cohort of CRC and healthy donor plasma cfDNA samples, and used this classifier to check for residual disease at the end of treatment for each of our 57 patients. The classifier detected residual disease in only 3/16 CRs (19%) but 35/41 non-CRs (85%). Two of the non-CR patients were initially assessed as CRs at earlier time points. In both cases, our method successfully detected residual disease at or prior to the CR assessment and consistently showed residual disease in all follow up samples. This suggests improved sensitivity relative to RECIST and highlights the potential of using noninvasive blood tests for continuous monitoring of CRC patients receiving therapy. Citation Format: Alison D. Tang, Rebecca Gupte, Victoria Cheung, Tao Qing, Austin Cauwels, Emily Leff, Kimberly Walter, Ehsan Tabari, Alex Lovejoy, Jimmy C. Lin. Noninvasive longitudinal monitoring of residual disease in chemotherapy-treated colorectal cancer patients [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 4788.

Abstract 2047: Scale up and scale down approaches for screening of 3D patient-derived cell models

Abstract 3D cell culture is becoming more prevalent as scientists work to better model human physiology using in vitro systems. However, differences in handling 3D cell cultures compared to traditional 2D cell lines have limited adoption of 3D techniques in compound and cell therapy screening experiments. To facilitate use of patient-derived 3D tumoroid models, we developed protocols for both scale up and scale down of these models in Gibco™ OncoPro™ Tumoroid Culture Medium. OncoPro Tumoroid Culture Medium is a serum-free, conditioned medium-free culture medium designed to promote the growth of patient-derived 3D cancer models (tumoroids, cancer organoids). OncoPro medium is compatible with a suspension culture approach for easier handling compared to traditional embedded culture formats. We studied the growth of tumoroids in different flask sizes during suspension culture to optimize scale up for downstream assays. Tumoroid growth rates and morphologies were consistent across non-tissue culture treated flask sizes, and viable cell yield increased with surface area during scale up. In some cases, as many as 100 × 106 dissociated tumoroid cells can be recovered from a single Nunc™ TripleFlask™ after a week of growth. After scaling up, tumoroids were dissociated and seeded in multiwell plates to test multiple screening conditions (compound identity, compound concentration) in parallel. Dissociated cells were seeded in 96-well plates using both manual and automated liquid handling (Tecan Fluent 780). Due to the tendency of 3D cell models to aggregate and fall out of solution, initial seeding in a 96-well flat bottom plate resulted in high well-to-well variability. Optimization of cell seeding protocols led to standard deviations in tumoroid metabolic activity, number, and size that were comparable to manual seeding, with a coefficient of variation of less than 3% between wells for multiple tumoroid lines when automation was implemented. Generally, variability in cell seeding was lower when using the optimized, automated cell seeding protocol compared to manual methods. After seeding, cells were grown for three days to form tumoroids prior to the addition of compounds. At this time point, there was low well-to-well variability in tumoroid metabolic activity (CV&amp;lt;2%). Compounds and reagents to analyze cell health could also be added using automated liquid handling prior to analysis on plate readers or high-content imaging platforms. Citation Format: Colin Paul, Anthony Chatman, Amber Bullock, Xiaoyu Jenny Yang, Garrett Wong, Brittany Balhouse, Chris Yankaskas, Erik Willems, Matt Dallas, David Kuninger. Scale up and scale down approaches for screening of 3D patient-derived cell models [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 2047.

Abstract 2053: Spatiotemporal tumor immune modulation by localized delivery of cancer therapeutics using an implantable microdevice

Abstract Mechanism of action studies are imperative to translating oncology therapeutics into the clinic informing testable hypotheses and potential combination partners. This often involves large and time-consuming mouse studies to test a small number of compounds in an attempt to show statistically significant benefit. Likewise, testing multiple compounds and combination partners in these systems is limited by time, cost, and a desire to reduce the number of animals used in biological research. To overcome these limitations, we utilized an implantable microdevice (IMD) developed by KiburMed to measure intra-tumoral drug responses and to differentiate the mechanism of action of multiple cytokine agents in parallel in the MC38 mouse model. After sizable tumor growth in mice and implantation of the IMD device with 9 different therapeutic candidates, MC38 mice were taken down at different time points (24 hour, 4 days,7 days) to assess response. Local tumor response in the study was measured by cyclical immunofluorescence for deep cellular response phenotyping with a panel of 32 markers for comprehensive immune cell phenotyping. Among the candidate murine surrogate compounds in the device, SAR445877 (anti-PD-1-IL-15 mutein) was tested as a monotherapy, as a combination partner with anti-PDL1 and compared against anti-PD1 monotherapy. With further validation of this novel platform, results from this work are aligned with previous studies (1) showing a murine surrogate for SAR445877 in combination with anti-PDL1 increases immune cell populations more than either single agent alone. These analyses show CD3, CD4, and CD8 were significantly increased in SAR’877 murine surrogate + anti-PDL1 compared to SAR’877 alone or anti-PD1 alone. In addition, T cells could infiltrate at a distance away from treatment release region, well into tumor core. Other significant changes in immune cell markers include increases in CD45+, CD11b, CD11c, CD20 and CD27. In conclusion, we have shown that multiple oncology therapeutics can be screened simultaneously in a small number of mice using an implantable microdevice and data obtained with this technology is consistent with that obtained from traditional mouse tumor experiments. (1) Preclinical characterization of SAR445877, an anti-PD-1 antibody-IL-15 mutein fusion protein with robust anti-tumor efficacy as monotherapy and in combination with PD-L1 blockade. Marie Bernardo; Yu-an Zhang; Dan Lu; Stella Martomo; Fatima Menas; Chen Zhu; Raymond Perez; Jeegar Patel; Donald Shaffer; Xiangming Li, Cancer Res (2023) 83 (7_Supplement): 2972. *This work was supported and funded by Sanofi. Citation Format: Xiangming Li, Julien Tessier, Joon Sang Lee, Seth Garren, Virna Cortez-Retamozo, Meenu Sharma, Angela Jankowski, Erik Zarazinski, Ann Fiore, Oliver Jonas, Colin Brenan, Donald Shaffer, Angela Hadjipanayis. Spatiotemporal tumor immune modulation by localized delivery of cancer therapeutics using an implantable microdevice [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 2053.

Exploring the ageing methylome in the model insect, Nasonia vitripennis

BackgroundThe ageing process is a multifaceted phenomenon marked by the gradual deterioration of cellular and organismal functions, accompanied by an elevated susceptibility to diseases. The intricate interplay between genetic and environmental factors complicates research, particularly in complex mammalian models. In this context, simple invertebrate organisms have been pivotal, but the current models lack detectable DNA methylation limiting the exploration of this critical epigenetic ageing mechanism.This study introduces Nasonia vitripennis, the jewel wasp, as an innovative invertebrate model for investigating the epigenetics of ageing. Leveraging its advantages as a model organism and possessing a functional DNA methylation system, Nasonia emerges as a valuable addition to ageing research.ResultsWhole-genome bisulfite sequencing unveiled dynamic alterations in DNA methylation, with differentially methylated CpGs between distinct time points in both male and female wasps. These changes were associated with numerous genes, enriching for functions related to telomere maintenance, histone methylation, and mRNA catabolic processes. Additionally, other CpGs were found to be variably methylated at each timepoint. Sex-specific effects on epigenetic entropy were observed, indicating differential patterns in the loss of epigenetic stability over time. Constructing an epigenetic clock containing 19 CpGs revealed a robust correlation between epigenetic age and chronological age.ConclusionsNasonia vitripennis emerges as a promising model for investigating the epigenetics of ageing, shedding light on the intricate dynamics of DNA methylation and their implications for age-related processes. This research not only expands the repertoire of ageing models but also opens avenues for deeper exploration of epigenetic mechanisms in the context of ageing.

Abstract 6942: Single cell resolution of intrapatient drug resistance mechanisms in metastatic melanoma

Abstract Intrapatient heterogeneity among different metastatic sites contributes to immunotherapy (ICi) and targeted therapy resistance. Determining the evolution of metastatic melanoma lesions that develop within the same patient could help elucidate adaptive mechanisms that drive tumor progression during therapy. To assess how metastatic melanoma persists through immunotherapy and targeted inhibitor treatments, we utilized samples from a single patient who had received anti-CTLA4, BRAFi/MEKi, and BRAFi/MEKi+CDK4/6i treatments during the LOGIC2 clinical trial. Eight short-term cultures were established from different metastatic site biopsies at chronological time points throughout the patient’s treatment and subsequent post-mortem resection of the bone, ovary, and brain metastatic sites. While the initial treatment naïve metastatic tumor in the breast was BRAFV600E mutant, NRASQ61 mutations were acquired after treatment with CDK4/6i in the bone and ovary tumors. Tumors that persist through treatment and colonize distant organs adapt to and rely on organ-specific microenvironments. To assess treatment- and organ-specific adaptations, single-cell RNA sequencing was performed on seven of the short-term cultures to identify subpopulations that persist through each treatment. We analyzed samples and subpopulations of each culture to determine differences in their transcriptional cell states. The regulon transcriptional network of each sample and subpopulation was evaluated to determine site- and treatment-specific transcriptional regulation differences. In addition, we identified the treatment naïve culture is split into two major subpopulations by therapy resistance features which are further made up of six distinct subpopulations with heterogeneous regulatory networks. We determined the longitudinal evolution of subpopulations in response to treatment and organ site to evaluate how the pressure of therapies and the microenvironment influence metastatic progression. We further evaluated site-specific resistance mechanisms and detected increased HER3 expression in the melanoma brain metastasis (MBM) sample. Treatment with Neuregulin 1, a ligand for the HER3 receptor that is abundant in brain tissue, partially rescued the growth of MBM cells and restored MAPK and AKT-mTOR signaling in the presence of BRAFi/MEKi. Targeting the NRG1-HER3 signaling axis could have therapeutic potential in melanoma patients with active brain metastases. Additionally, we evaluated treatment-specific resistance mechanisms and identified high Brevican (BCAN) expression in an early emerging subpopulation that arises after ICi in this patient and in others. BCAN is implicated in both ICi- and cross-resistance. We will functionally investigate whether targeting BCAN sensitizes cells to ICi. Future studies aim to functionally examine novel site- and treatment-specific cross patient targets. Citation Format: Haley P. Wilson, Glenn L. Mersky, Jelan I. Haj, Jessica Teh, Phil F. Cheng, Signe Caksa, Casey D. Stefanski, Vivian Chua, Claudia Capparelli, Mitch P. Levesque, Reinhard Dummer, Timothy J. Purwin, Andrew E. Aplin. Single cell resolution of intrapatient drug resistance mechanisms in metastatic melanoma [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 6942.

Abstract 265: Reconstructing the early steps of breast tumor initiation

Abstract Breast cancer screening programs, detect not only the early stages of the disease but also various preneoplastic lesions which are very difficult to predict if and when they may develop into cancer. This uncertainty can lead to over-diagnosis, over-treatment and stress for millions of screened women. As consequence, discovering reliable biomarkers of this evolution could make it possible to monitor these patients appropriately and propose an effective prevention strategy. To meet this challenge, we urgently need to better understand the mechanisms that regulate human mammary homeostasis during early steps of tumor initiation. The hierarchy and the composition of human mammary epithelium, composed of stem, myoepithelial, luminal progenitors and mature luminal cells, are complex. This complexity raises the question of whether a given mammary epithelial cell is equally likely to be the cell-of-origin of breast cancer. Interestingly, increasing evidences show that disruption of lineage integrity can be a precursor of breast tumor initiation, and the basal and the luminal lineages respond differently to a given oncogenic stress and may each follow their own rule towards malignant transformation. These results strongly suggest that each lineage may have its own pliancy to be transformed by a given oncogene, hijacking the inter-tumoral heterogeneity observed in breast cancers. The objectives of this study are to develop a model to reconstruct the early steps of breast tumor initiation and understand the interplay between the initiating oncogenic event and the cell-of-origin. To answer these questions, we used a human mammary epithelial cell lines (HME) that maintains a cellular hierarchy with mammary stem cells (MaSC) and luminal cells (LC). Upon oncogenic expression of HRASG12V or c-Myc, we observed different modifications of cell homeostasis, with altered proportions of the lineages populations in the cell line. Depending on the oncogenic stress induced, we also demonstrated a modification in the morphology and the cellular composition of HME organoids, as well as in the capacity to form tumors. HRASG12V induces increase of MaSC compartment, formation of more branched organoids and generate aggressive basal-like tumors. Conversely, c-MYC leads to an almost loss of MaSC and formation of more proliferative unbranched organoid and generate tumors with limited growth. To address more precisely how cell-of-origin and lineage plasticity influence malignant transformation, we are developing for the first time a lineage tracing model in human mammary epithelium with HME. Moreover, a single cell RNA-seq was performed at different early time points during HME cellular differentiation in homeostasis and upon oncogenic stress to decipher the molecular mechanisms that underpin the very earliest steps of breast tumor initiation. Citation Format: Anaïs Grandon, Shuheng Lin, Martin Castagné, Julien Wicinski, Olivier Rosnet, Christophe Ginestier, Emmanuelle Charafe-Jauffret. Reconstructing the early steps of breast tumor initiation [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 265.

Neurocognitive effects a combined polyphenolic-rich herbal extract in healthy middle-aged adults – a randomised, double-blind, placebo-controlled study

ABSTRACT Objective: This study assessed whether polyphenolic rich supplement containing Bacopa monnieri (BM: 300 mg), Panax quinquefolius ginseng (PQ: 100 mg) and whole coffee fruit extract (WCFE: 100 mg) could enhance cognitive performance, affect and cerebral-cortical activation over 28-days of intervention. Method: A randomised, double-blind, placebo-controlled, between-group study of 52 healthy adults between 35 and 65 years (M = 50.20, SD = 9.37) was conducted. Measures of cognition, affect and brain activity were measured at three time points: baseline, 28 days post intervention and 14 days post washout. At each time point, haemodynamic response in the prefrontal cortex (PFC) was measured using functional near-infrared spectroscopy (fNIRS), and serum brain-derived neurotrophic factor (BDNF). Results: The polyphenolic-rich supplement reliably improved positive affect and delayed recall compared to placebo following 28 days of supplementation. For the brain, those in the active condition showed greater PFC activation on performance of the 2-back tasks post supplementation compared to placebo (p < .05, d = 0.6). Discussion: This is the first report of a 28-day supplement intervention and 2-week follow-up study to assess changes in affect, cognition, cerebral haemodynamic response and BDNF in healthy middle-aged adults. The potential synergistic effects of polyphenolic compounds on neurocognitive function in middle-aged adults through emotional-cognitive processing and cognitive reserve are important for promoting brain and cognitive health.