Cells Stained Research Articles

Uptake and Cellular Effects of Polymethylmethacrylate on Human Cell Lines

The usage of plastic and its decomposition products leads to their ubiquitous distribution, resulting in their uptake by all living beings, including humans. Polymethylmethacrylate (PMMA) is known as a biocompatible polymer and is used widely in medicine and dentistry, although recent findings have shown its induction of oxidative stress within cells. Worryingly, hardly any data exist investigating the uptake of PMMA particles by cells, the potential effects of these particles on cells and cell signaling pathways and their contributing factors. We assessed the uptake of PMMA beads via confocal microscopy after their incubation with HEK293, A549 and MRC5 cells. Through cell staining, we localized multiple PMMA beads within the cytosol of cells. No alterations regarding cell growth, cell morphology or cell division were found, implying no short-term toxicity towards human cells. Using a cAMP response element binding protein (CREB)-mediated reporter assay, we assessed whether internalized PMMA nanobeads alter cell signaling pathways after stimulation of the cells. CREB was chosen as a well-described transcription factor involved in various cellular processes. Our data led to the assumption that PMMA nano- and microbeads are internalized via endocytosis and end up in lysosomes within the cell cytosol. We concluded that differences regarding the surface composition of the PMMA nanobeads affect their potential to alter cell signaling. These findings emphasize the key role the surface composition plays regarding microplastics and their risks for human health, whereas the usage of medical-grade PMMA remains safe.

The 3D in vitro Adrenoid cell model recapitulates the complexity of the adrenal gland

The crosstalk between the chromaffin and adrenocortical cells is essential for the endocrine activity of the adrenal glands. This interaction is also likely important for tumorigenesis and progression of adrenocortical cancer and pheochromocytoma. We developed a unique in vitro 3D model of the whole adrenal gland called Adrenoid consisting in adrenocortical carcinoma H295R and pheochromocytoma MTT cell lines. Adrenoids showed a round compact morphology with a growth rate significantly higher compared to MTT-spheroids. Confocal analysis of differential fluorescence staining of H295R and MTT cells demonstrated that H295R organized into small clusters inside Adrenoids dispersed in a core of MTT cells. Transmission electron microscopy confirmed the strict cell–cell interaction occurring between H295R and MTT cells in Adrenoids, which displayed ultrastructural features of more functional cells compared to the single cell type monolayer cultures. Adrenoid maintenance of the dual endocrine activity was demonstrated by the expression not only of cortical and chromaffin markers (steroidogenic factor 1, and chromogranin) but also by protein detection of the main enzymes involved in steroidogenesis (steroidogenic acute regulatory protein, and CYP11B1) and in catecholamine production (tyrosine hydroxylase and phenylethanolamine N-methyltransferase). Mass spectrometry detection of steroid hormones and liquid chromatography measurement of catecholamines confirmed Adrenoid functional activity. In conclusion, Adrenoids represent an innovative in vitro 3D-model that mimics the spatial and functional complexity of the adrenal gland, thus being a useful tool to investigate the crosstalk between the two endocrine components in the pathophysiology of this endocrine organ.

Abstract CT230: Exploratory analyses of MGMT promoter methylation and PD-L1 expression from newly diagnosed glioblastoma patients in CheckMate 548 study suggest refinement in thresholds for predicting response to standard of care and nivolumab treatments

Abstract CheckMate 548 (NCT02667587, CM548) is a phase III, randomized trial evaluating first line nivolumab (NIVO) in combination with standard of care (SOC) [surgical resection followed by radiotherapy and temozolomide (TMZ)] versus placebo plus SOC in patients with newly diagnosed glioblastoma (GBM) positive for promoter methylation of O6-methylguanine DNA methyltransferase (MGMT). MGMT promoter methylation status and PD-L1 expression predict better outcomes in GBM patients treated with TMZ and in patients treated with NIVO in other indications, respectively. However, the impact of varying levels of MGMT promoter methylation and PD-L1 expression on patient outcome in GBM remains unclear. In CM548, MGMT promoter methylation levels were quantified through a central lab assay (LapCorp) and PD-L1 expression was determined by percentage of detectable membrane staining of tumor cells. MGMT methylation and PD-L1 data were prospectively generated at the time of enrolment. Our analyses showed that increasing levels of MGMT promoter methylation level were associated with longer overall survival in patients treated with SOC alone (n = 349). However, no survival benefit was observed with increasing levels of MGMT methylation in patients treated with NIVO in combination with SOC (n = 353). In addition, our data showed a non-linear relationship between PD-L1 expression and overall survival across both arms. In patients with intermediate PD-L1 expression (≥5%, <20%), longer survival was observed in the nivolumab + SOC arm compared to SOC alone. This benefit was not observed in patients with low (≥1%, <5%) and high (>20%) PD-L1 expression. Potential mechanisms driving these observed associations are currently explored using genomic data generated from patient samples. MGMT promoter methylation and PD-L1 expression are potential clinically relevant predictive biomarkers of response to treatments administered in the CM-548; however, the binary classification of positive/negative status of these markers does not fully capture their relationship with efficacy in CM548. Data generated from our exploratory analyses suggests that new, refined criteria are needed for better patient outcomes prediction. Citation Format: Yu-Han Hung, Aparna Chhibber, Michael Lim, Michael Weller, David A. Reardon, Antonio Omuro, Keith L. Ligon, Ana Lako, Ann Forslund. Exploratory analyses of MGMT promoter methylation and PD-L1 expression from newly diagnosed glioblastoma patients in CheckMate 548 study suggest refinement in thresholds for predicting response to standard of care and nivolumab treatments [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 2 (Late-Breaking, Clinical Trial, and Invited Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(7_Suppl):Abstract nr CT230.

511 Iron Oxide Nanoplatform HMC-FMX for Targeted Chemotherapeutic Treatment of Glioblastoma

INTRODUCTION: Glioblastoma multiforme (GBM), is the most common and aggressive form of primary brain tumors in adults with a high mortality rate. Despite the emergence of potent chemotherapies, clinical efficacy has been limited in part due to the blood-brain barrier (BBB). Therefore, recurrence rate is high, limiting life expectancy. Our nanoplatform is based on ferumoxytol (FMX), an FDA-approved MRI-sensitive supramagnetic iron oxide nanoparticle conjugated to heptamethine cyanine (HMC), a near infrared-flourescent ligand that specifically targets organic anion transporter polypeptides (OATP) expressed in GBM and endothelial cells. METHODS: To show the specificity of OATP-targeting by HMC-FMX nanoplatform, cells were pre-incubated with OATP inhibitors and then incubated with HMC-FMX. Fluorescent intensity and HMC uptake were assesed by flow cytometry. Cytotoxicity of the drug-loaded nanoparticles, HMC-FMX(SN38) and HMC-FMX(CPT) compared to SN38 and Camptothecin (CPT) in U87 cells was assessed by flow cytometry following cell staining with Annexin and 7-AAD. RESULTS: The HMC-FMX nanoplatform is successfully taken up by both U87 and endothelial cells. OATP inhibition significantly decreased HMC uptake (10%-30%) in comparison to control in U87 and endothelial cells. In addition, HMC-FMX(SN38) and HMC-FMX(CPT) exerted increased cytotoxic effect on U87 cells compared with SN38 and CPT alone. Furthermore, majority of apoptotic cells were observed in the HMC-positive cells in contrast to HMC-negative cells. CONCLUSIONS: HMC-FMX can encapsulate chemotherapeutic drugs, such as SN38 and CPT. HMC-FMX as a nanoplatform demonstrates specific, safe and efficient drug delivery through the BBB into GBM cells in vitro.

Chronic Administration of Methamphetamine Aggravates Atherosclerotic Vulnerable Plaques in Apolipoprotein E Knockout Mice Fed with a High-cholesterol Diet.

It has been observed previously that chronic methamphetamine (METH) administration could upregulate neuropeptide Y (NPY) expression and promote atherosclerotic formation in apolipoprotein E knockout (ApoE-/-) mice fed with a normal cholesterol or high diet and NPY might be involved in the pathogenesis of METHinduced atherogenic effects through NPY Y1 receptor pathway. Vulnerable coronary atherosclerotic plaque (VP) is a critical pathological finding responsible for the acute coronary syndrome (ACS). In this study, we explored whether METH abuse could aggravate the formation of VP in ApoE-/- mice fed with high cholesterol diet. The purpose of this study was to observe if chronic METH administration could aggravate vulnerable plaque (VP) formation in ApoE-/- mice fed with a highcholesterol diet. Male ApoE-/- mice fed with a high-cholesterol diet were intraperitoneally injected with normal saline (NS) or 8 mg/kg/day METH (M8) for 24 weeks. Body weight was monitored from baseline to 24 weeks at 2 weeks intervals. After 24 weeks of treatment, plasma lipid variables were measured. Movat's staining and immunohistochemical staining were performed on frozen sections of the aortic roots to calculate VP percentage and intraplaque hemorrhage (IPH) percentage and detect expression of NPY, vascular endothelial growth factor (VEGF), and CD31. In vitro, the expressions of Y2R, VEGF, and CD31 were detected by immunofluorescence staining in aortic endothelial cells incubated with PBS, 100μM METH, 10nmol NPY, or 100μM METH plus 10nmol NPY for 12 hours. The CD31 positive area, percentage of IPH, VP, and the expressions of NPY and VEGF were significantly increased in the M8 group than in the NS group. In vitro, the expressions of Y2R, VEGF, and CD31 were significantly increased in the METH+NPY group than in the PBS, METH, and NPY groups and these effects could be blunted by treatment with a Y2R antagonist or DPPIV inhibitor. Chronic METH administration could aggravate VP in ApoE-/- mice fed with a high-cholesterol diet, possibly through upregulating vascular NPY and VEGF expression and promoting angiogenesis and vessel rupture in atherosclerotic plaques. Our findings indicated that increased VP formation might contribute to the development of acute coronary syndrome post-chronic METH abuse by activating DPPIV/NPY/Y2R pathway.

Effect and mechanism of free total rhubarb anthraquinones on pyroptosis of J774A.1 macrophages

In this study, J774A.1 macrophages stimulated by lipopolysaccharide(LPS) and adenosine triphosphate(ATP) were used to establish an in vitro model of pyroptosis, and the intervention mechanism of free total rhubarb anthraquinones(FTRAs) on pyroptosis was investigated. J774A.1 macrophages were cultured in vitro, and the experiment was assigned to the control group and groups with different concentrations of LPS(0.25, 0.5, and 1 μg·mL~(-1)) and ATP(1.25, 2.5, and 5 mmol·L~(-1)). An in vitro model of macrophage pyroptosis was established by detecting cell viability through CCK-8, propidium iodide(PI) apoptotic cell staining, lactate dehydrogenase(LDH), interleukin(IL)-18, and tumor necrosis factor(TNF)-α release. Then, J774A.1 macrophages were randomly divided into six groups: blank control group, LPS+ATP group, high-dose FTRA group, and low, medium, and high-dose FTRA pre-protection group. The phenotypic characteristics and key indicators of pyroptosis were detected as the basis for evaluating the effect of FTRAs on pyroptosis induced by LPS and ATP. Western blot and RT-PCR were used to detect the expression levels of protein and mRNA related to the pyroptosis pathway in caspase-1/11 and elucidate the molecular mechanism of the anti-pyroptosis effect. The results showed that the stimulation condition of 0.50 μg·mL~(-1) LPS+5.00 mmol·L~(-1) ATP was the most effective in the in vitro model of macrophage pyroptosis. FTRAs pre-protected cells for 24 h and then can increase cell viability under pyroptosis conditions, alleviate cell damage, lower the positive rate of PI staining, and reduce the release of LDH, IL-18, and TNF-α. FTRAs were able to significantly inhibit the activation of GSDMD proteins and significantly down-regulate the protein expression of the pyroptosis pathway signature molecules, TLR4, NLRP3, cleaved-caspase-1, and cleaved-caspase-11, but they had no significant effect on ASC proteins. FTRAs were also able to significantly inhibit the mRNA expression of caspase-1, caspase-11, and GSDMD. These results indicate that FTRAs have an inhibitory effect on the pyroptosis model induced by LPS and ATP and play an anti-pyroptosis effect by regulating classical and non-classical pyroptosis signaling pathways and reducing the production of inflammatory cytokines.

Water‐soluble astaxanthin and silver enriched poly(vinyl alcohol)/silk fibroin crosslinking electrospun nanofiber for wound dressing

AbstractThe aim of this work is to fabricate a novel electrospun poly(vinyl alcohol) (PVA)/silk fibroin (SF) nanofibrous membrane containing astaxanthin (ASTA) and silver (Ag) nanoparticles as potential wound dressing. Since ASTA is typically liposoluble, the bioavailability could be greatly enhanced by improving its hydrophilicity. The antioxidant performance of eletrospun membrane is verified by ABTS radical scavenging assays. The beadless fibers could be fabricated at 16 kV when PVA (12, wt%) is blended with SF (6, wt%). In addition, the membrane is carefully chemically crosslinked by comparing the efforts (i.e., mechanical strength and water resistance) of glutaraldehyde dipping and vapor treatment. The morphology of the electrospun membrane is observed by scanning electron microscopy and optical microscope. The formation of Ag nanoparticles, which provides the membranes antibacterial properties, is also confirmed by energy dispersive spectroscopy and inhibition zone test. Meanwhile, the PVA/SF/ASTA/Ag compound membrane is characterized by Fourier transform infrared spectroscopy, x‐ray diffraction, thermo gravimetric analysis, differential thermal analysis, and water contact angle. The releasing of ASTA is also measured, and kinetic data are adjusted by Higuchi models. Finally, the biocompatibility is confirmed by in vitro cell staining experiments and in vivo rat models.

Effect of Anodic Oxidation Pulse Voltage on Antibacterial Properties and Biocompatibility of Ti-Ag Alloy

For the application of titanium and titanium alloys in orthopedic implant materials, the antibacterial properties and cell biocompatibility determine whether the implant surgery is successful. In this study, a functional anodic oxidation (AO) coating was successfully prepared to modify the surface of Ti-Ag alloy. The surface characteristics of the anodized Ti-Ag alloy were analyzed using techniques such as X-ray diffraction (XRD), scanning electron microscopy (SEM), X-ray photoelectron spectroscopy (XPS), and contact angle measurements. The corrosion characteristics of Ti-Ag samples were tested by an electrochemical workstation. In addition, the antibacterial properties and cell activity were studied by the plate count method and MC3T3-E1 cell staining. The results indicate that the AO process can generate a multi-functional TiO2/Ag2O coating with a large number of block and flower-like structures on the surface of a Ti-Ag alloy. When the AO voltage of the sample is 120 V, the maximum roughness is 0.73 μm and the minimum wetting degree is 23°, which improves the biocompatibility. The corrosion test results show that AO treatment can improve the corrosion resistance of a Ti-Ag alloy. The oxidation voltage is 20 V and the coating has the best corrosion resistance. The corrosion open circuit potential (Eocp) is 107.621 mV and the corrosion current density (icorr) is 2.241 × 10−8 A·cm−2. This coating can promote ion release and show more than 99% of a strong antibacterial ability against S. aureus. The results of the compatibility evaluation by cultured cells showed that the multifunctional coating formed by the anodic oxidation process did not cause cytotoxicity and promoted the adhesion of MC3T3-E1 cells.

Decellularized human amniotic membrane scaffolds: influence on the biological behavior of dental pulp stem cells

ObjectiveThe objective of this study was to assess the characterization of human acellular amniotic membrane (HAAM) using various decellularization methods and their impact on the proliferation and differentiation of human dental pulp stem cells (DPSCs). The goal was to identify scaffold materials that are better suited for pulp regeneration.MethodsSix different decellularization methods were used to generate the amniotic membranes. The characteristics of these scaffolds were examined through hematoxylin and eosin (H&E) staining, scanning electron microscopy (SEM), and immunohistofluorescence staining (IHF). The DPSCs were isolated, cultured, and their capacity for multidirectional differentiation was verified. The third generation (P3) DPSCs, were then combined with HAAM to form the decellularized amniotic scaffold-dental pulp stem cell complex (HAAM-DPSCs complex). Subsequently, the osteogenic capacity of the HAAM-DPSCs complex was evaluated using CCK8 assay, live-dead cell staining, alizarin red and alkaline phosphatase staining, and real-time quantitative PCR (RT-PCR).ResultsOut of the assessed decellularization methods, the freeze-thaw + DNase method and the use of ionic detergent (CHAPS) showed minimal changes in structure after decellularization, making it the most effective method. The HAAM-DPSCs complexes produced using this method demonstrated enhanced biological properties, as indicated by CCK8, alizarin red, alkaline phosphatase staining, and RT-PCR.ConclusionThe HAAM prepared using the freeze-thaw + DNase method and CHAPS methods exhibited improved surface characteristics and significantly enhanced the proliferation and differentiation capacity of DPSCs when applied to them. The findings, therefore demonstrate the capacity for enhanced pulp regeneration therapy.

Flexible Triboelectric Nanogenerator for Promoting the Proliferation and Migration of Human Fibroblast Cells.

Chronic wound healing is often a prolonged process with the migration and proliferation of fibroblast cells playing crucial roles. Electrical stimulation (ES) has emerged as a promising physical therapy modality to promote these key events. In this study, we address this issue by employing a triboelectric nanogenerator (TENG) as an electrical stimulator for both drug release and the stimulation of fibroblast cells. The flexible TENG with a sandwich structure was fabricated using a PCL nanofibrous layer, Kapton, and silicon rubber. The TENG could be folded to any degree and twisted, and it could return to its original shape when the force was removed. Cultured cells received ES twice and three times daily for 8 days, with a 30 min interval between sessions. By applying current in a safe range and appropriate time (twice daily), fibroblasts demonstrate an accelerated proliferation and migration rate. These observations were confirmed through cell staining. Additionally, in vitro tests demonstrated the TENG's ability to simultaneously provide ES and release vitamin C from the patch. After 2 h, the amount of released drug increased 2 times in comparison to the control group. These findings provide support for the development of a TENG for the treatment of wounds, which underlines the promise of this new technique for developing portable electric stimulation devices.

Isoliquiritigenin-infused electrospun nanofiber inhibits breast cancer proliferation and invasion through downregulation of PI3K/Akt/mTOR and MMP2/9 pathways

Mastectomy followed by chemotherapy and radiotherapy is the current treatment regimen for breast cancer (BC). However, these approaches have systemic side effects and often lead to ineffective treatment outcomes. Therefore, a novel controlled release system is required to prevent tumor recurrence. Isoliquiritigenin (ISL), a dietary flavonoid, was shown to be effective against BC in our laboratory. However, its hydrophobicity and low bioavailability limit its anti-BC efficacy. Thus, the present study was designed to fabricate electrospun ISL-nanofiber scaffolds (ISL-NF) that can inhibit breast cancer growth and migratory capacity of BC cells in vitro and in vivo. The ISL-NF was prepared using an advanced coaxial electrospinning-cryocutting method, and characterized. Various assays, including cell viability, transwell, would healing, western blotting, and staining, were employed to explore the underlying mechanisms. In vitro studies showed that ISL-NF treatment for 24 h significantly inhibited cell proliferation, and migration and improved apoptosis in various BC cell lines. ISL-NF also decreased the protein expression of PI3K, Akt, mTOR, matrix metalloproteinase (MMP)-2, and MMP-9, thereby inhibiting BC cell invasion. Consistent with the in vitro study, ISL-NF treatment decreased tumor size and weight and inhibited tumor migration in 4T1 tumor-bearing mice. Based on the study, we suggest that ISL-NF may be useful in the treatment of BC.

Abstract 2914: AI/ML-driven discovery of CTHRC1, collagen triple helix repeat-containing 1, a novel proteoglycan for stroma + tumor targeting and delivery of 4-1BB costimulation

Abstract Background: While checkpoint inhibitors have demonstrated efficacy in a number of solid tumor indications, those with high stromal presence have been difficult to treat with minimal objective responses observed. Phenomic has developed a proprietary machine learning/artificial intelligence platform to identify novel stromal targets with superior expression profiles that enable selective targeting of immune activating agents that will relieve these immunosuppressive barriers in difficult to treat indications. Methods: Using our single cell RNA Atlas, we assessed cancer-associated fibroblasts (CAFs) in several solid tumor indications for identification of novel targets, including proteoglycans. Our Atlas was also used to identify immune activating payloads whose cognate receptors were present in indications of interest. Antibodies were generated, and lead clones who demonstrated potent ligand binding and cell staining were used to generate fusion proteins to immune activating ligands. Efficacy and PD were assessed in multiple syngeneic tumor models. Results: Bioinformatic analysis identified a unique subset of pathogenic CAFs, which are TGF beta responsive, secrete several ECM proteins, and their presence tracks with poor outcome and resistance to immunotherapy in several solid tumor types. CTHRC1 was identified as a novel matrix protein highly expressed in this CAF subtype as well as tumor epithelium and is highly selective in a range of tumor types such as ovarian cancer, triple negative breast cancer, and pancreatic ductal adenocarcinoma. While secreted, CTHRC1 is complexed on the cell surface and affords the opportunity to drug this target in a variety of ways. We identified an absence of 4-1BBL expression across indications of interest, and fusion proteins were generated to deliver 4-1BBL via CTHRC1 targeting. In checkpoint-resistant tumor models, we observed significant increases in CD8 T cells and robust anti-tumor activity with anti-CTHRC1-targeted 4-1BBL. Biodistribution studies were conducted using our targeting mAb and demonstrate uptake only in sites of primary and metastatic tumors, even at doses 20-fold higher than those that achieve maximal therapeutic activity, suggesting the potential to minimize the toxicity that has been observed with other 4-1BB agonists. Conclusions: We have identified CTHRC1 as a novel proteoglycan expressed by both pathogenic CAFs and tumor cells that is highly selective for tumors, enabling the therapeutic targeting of immune activating payloads with the potential for safely delivering payloads while limiting toxicity. Given the specificity and selectivity afforded by CTHRC1 expression, ADC and CD3 engager approaches are also being pursued. These data represent novel approaches aimed at breaking down stromal barriers in tumors previously unresponsive to immunotherapies. Citation Format: Christopher Harvey, Elizabeth Koch, Amanda Hanson, Lindsey Rice, Amy Berkley, Kerry White, Reza Saberianfar, Nikolai Suslov, Sam Cooper, Michael Briskin. AI/ML-driven discovery of CTHRC1, collagen triple helix repeat-containing 1, a novel proteoglycan for stroma + tumor targeting and delivery of 4-1BB costimulation [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 2914.

Abstract 1755: Integrative genomic and transcriptomic profiles from a prospective cervical cancer study (RAIDs)

Abstract Background: Cervical cancer (CC) remains a leading cause of gynecological cancer related mortality worldwide and constitutes the third most common malignancy in women. We have previously reported a “metagene” of genomic markers in the PI3K pathway and epigenetic regulators associated with poor outcome. Immune therapy recently showed promising results yet new biomarkers for targeted therapeutic approaches in this type of cancer remain to be determined. Patients & Methods: Patients included in this study were enrolled in BioRAIDs [NCT02428842]. Whole exome sequencing (WES), shallow whole genome sequencing (sWGS) and RNA sequencing were performed on quality-controlled primary frozen tumor samples in 297, 306 and 280 patients, respectively who subsequently had received primary radio chemotherapy. Multi-omics bioinformatics analyses were performed to identify alterations representing new valid drug targets in CC. Tumor-stromal characteristics such as transcriptome deconvolution, CD8+, CD45+, CD68+ staining cells, PD-L1 expression, tumor infiltrating lymphocytes (TILs) together with the degree of tumor necrosis were assessed. Results: 273/306 were HPV positive (89%). Most frequently altered (≥10%) oncogenes were PIK3CA (30%) and TERT promoter (14%). Most frequently altered (≥10%) tumor suppressor genes were FBXW7 (14%), KMT2D (11%), KMT2C (11%) and FAT1 (11%).Microsatellite instability (MSI) was detected in 8/297 (3%) patients, homologous recombination deficiency (HRD)-high status in 8/297 (3%) patients and TMB-high (>10mut/Mb) in 32/297 (11%) patients. The most frequent mutational signatures were APOBEC, deaminase and MMR (DNA mismatch repair deficiency (74%, 28% and 11% respectively). Interestingly, FGFR3 fusions were detected in 8/297 patients (3%). Transcriptomic analyses showed 510 differentially expressed genes between HPV positive and HPV negative CC (adjusted p-value<0.01 and |log2FC|>1). No significantly differentially expressed genes were detected between FIGO 2018 stages (I/II vs III-IV). Both stages as well as tumor necrosis were highly significantly associated with progression-free survival (PFS), with tumor necrosis as an independent prognostic factor in a multivariable analysis (p<0.001).Presence or absence of CD8-positive lymphocytes, or CD68-positive cells of the myeloid cell compartment, histopathological subtype or number of TILs did not correlate with PFS. Conclusion: Our results confirm the high level of altered genes involved in chromatin remodeling with or without PI3K pathway mutations suggesting the relevance of epidrugs in CC. FGFR3 fusions and HRD tumors - although being rare (3%) - to be considered as new targeted approaches. Additionally, MSI and TMB high are of interest for immunotherapy beyond PD-L1 expression. Integrative analyses of omics and pathology data and correlation with PFS are ongoing and will be presented in the meeting. Citation Format: Maud Kamal, Solenn Barraud, Lolita Lecompte, Maral Halladjian, Emmanuelle Jeannot, Elodie Girard, Sylvain Baulande, Patricia Legoix, Fabrice Lecuru, Léa Pauly, Mathilde Saint-Ghislain, Stacy Cyrille, Célia Dupain, Constance Lamy, Olfa Chouchane-Mlik, Alexandra Oniga, Michel Mittelbronn, Christophe Le Tourneau, Aurélien Latouche, Nicolas Servant, Suzy Scholl, Ivan Bièche. Integrative genomic and transcriptomic profiles from a prospective cervical cancer study (RAIDs) [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 1755.

Abstract 3683: Prognostic value and biological correlates of LINE1 hypomethylation in oropharyngeal cancer

Abstract Background. We have recently demonstrated that LINE-1 hypomethylation is a negative prognostic factor in oropharyngeal squamous cell carcinoma (OPSCC). HPV-positive patients with LINE-1 methylation <55% showed an almost 5-fold higher risk of death than hypermethylated once, with an overall survival as poor as HPV-negative patients. Somatic TP53 mutations are a common feature in HPV-negative OPSCC, whereas their presence has been reported in only a small subset of HPV-related OPSCC. At present, little is known about the correlation between LINE-1 methylation and p53 expression status and/or TP53 mutation in OPSCC. Methods. Since 2019, an ongoing prospective study has been enrolling patients with OPSCC in nine cancer centers in Northern Italy. HPV16 E6 DNA was quantified using real-time quantitative PCR (qPCR). LINE-1 methylation was evaluated by methylation-specific qPCR. p53 expression was detected by immunohistochemical staining. TP53 exon mutations were analyzed by Next Generation Sequencing. Results. Up to date, 134 OPSCC patients have been enrolled, including cancer of the tonsil (n=74; 55.2%), base of tongue (n=47; 35.1%), and other subsites (n=13; 9.7%). HPV16 DNA was found in 54.3% of cases: 65.7% in base of tongue, 57.1% in tonsil, and 16.7% in other subsites (p<0.001). LINE-1 methylation was higher in HPV16-positive patients (median: 55.8%, interquartile rage [IQR]: 42.5-68.5) than HPV16-negative ones (median: 40.7; IQR: 23.9-59.1; p=0.003). Nevertheless, the median LINE-1 methylation was lower (50.7%) among HPV-16 OPSCC patients who relapsed than those who did not (56.0%), thus confirming the findings previously reported. However, data from the present investigation are presently not mature to purse the study aims (median follow-up: 12.3 months). The correlation between LINE-1 hypomethylation and p53 expression has also been evaluated in OPSCC patients. p53 expression was categorized into three groups (0%, 1-49%, and ≥50%) based on the overall intensity of nuclear staining of the tumor cells. p53 was considered null, overexpressed, or wild type when its nuclear staining was 0 %, ≥50%, or ranged between 1% and 49%, respectively. Of note, the highest level of LINE-1 methylation was observed in OPSCC tissues with nuclear p53 staining between 1% and 49% (57.8%), whereas OPSCC patients with p53 expression ≥50% showed a decline of LINE-1 methylation levels (40.6; p=0.026). Interestingly, LINE-1 methylation was lower in OPSCC patients harboring TP53 mutations in both HPV16-negative (24.8%) and HPV16-positive (24.0%) OPSCC patients, even if statistical significance was reached only in the HPV-negative group (p=0.015). Conclusion. Preliminary results confirm the potential of LINE-1 methylation to identify HPV16-positive patients with poor prognosis. In addition, this study suggest that TP53 mutations might influence LINE-1 methylation, irrespective of HPV status. Citation Format: Mariateresa Casarotto, Roberto Guerrieri, Paolo Boscolo-Rizzo, Monica Schiappacassi, Sara D'Andrea, Valentina Lupato, Susanna Chiocca, Marta Tagliabue, Rita De Berardinis, Anna Menegaldo, Alice Piccinato, Paola Stritoni, Mohssen Ansarin, Doriano Politi, Giuseppe Fanetti, Giorgio Giurato, Jerry Polesel, Elisabetta Fratta. Prognostic value and biological correlates of LINE1 hypomethylation in oropharyngeal 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 3683.

Abstract 3121: Therapeutic potential of a HER3 antibody-drug conjugate for the treatment of HER3-expressing cancers

Abstract To address the need for targeted therapy in the treatment of HER3-positive solid tumors, the human HER3 mAb, seribantumab, was evaluated in the context of an antibody-drug conjugate (ADC). As a proof-of-concept, seribantumab was conjugated with a cleavable valine-citrulline linker and monomethyl auristatin E (MMAE) payload via the stochastic cysteine conjugation method to yield HER3-ADC1. A drug-antibody ratio of 4 was selected for preclinical assessment in HER3-expressing models. Methods: HER3-ADC1 was evaluated in vitro and in vivo, with patritumab deruxtecan (patri-DXd) as a comparator. Binding to BT474 breast carcinoma cells (HER3 high; immunohistochemical (IHC) staining intensity 3+) was measured by flow cytometry. Internalization properties were established using seribantumab and patritumab coupled to Fab-AF488 following 4 hours incubation in SK-BR-3 breast adenocarcinoma (HER3 high; IHC 3+) and HCC1569 breast carcinoma (HER3 low; IHC 0-1+) cell lines. In vitro cytotoxicity was evaluated for HER3-ADC1, isotype-MMAE and free MMAE payload as well as patri-DXd, isotype-DXd and free deruxtecan payload in BT474 cells, SK-BR-3 cells and NCI-H446 lung carcinoma (HER3 low; IHC 0-1+) cells. In vivo anti-tumor activity was assessed for HER3-ADC1, isotype-MMAE, patri-DXd and isotype-DXd in patient derived xenograft (PDX) models of pancreatic (HER3 high; IHC 3+) and breast cancer (HER3 low; IHC 0-1+). Results: HER3-ADC1 binding to cancer cells, endocytosis, MMAE release, and inhibition of proliferation were dependent on HER3 expression. In assays investigating antibody internalization, seribantumab displayed similar internalization capacity as patritumab. For both HER3 mAbs, internalization was greater in a HER3 high than a HER3 low cell line. In cytotoxicity assays, HER3-ADC1 and patri-DXd displayed HER3 expression level-dependent cell killing. HER3-ADC1 outperformed patri-DXd in HER3 high cell lines based on in vitro cytotoxicity. HER3-ADC1 and patri-DXd were ineffective against NCI-H466 cells with low HER3 expression. In a PDX model of pancreatic cancer (HER3 high), HER3-ADC1 induced tumor regression, while isotype-MMAE had only a moderate anti-tumor effect. Both patri-DXd and isotype-DXd had moderate anti-tumor effects, indicating a lack of HER3 target dependence. In a PDX model of breast cancer (HER3 low), HER3-ADC1 and isotype-MMAE lacked anti-tumor activity; isotype-DXd had a moderate anti-tumor effect and patri-DXd had a substantial anti-tumor effect. Conclusions: HER3-ADC1 demonstrated target-dependent in vitro cytotoxicity and in vivo anti-tumor activity in a HER3-expressing pancreatic cancer PDX model. Results from in vitro and in vivo studies highlight the promising therapeutic potential of a seribantumab-based ADC for patients with HER3-expressing cancers. Additional results on the optimization and characterization of HER3-ADC1 will be presented. Citation Format: Thomas O'Hare, Jaclyn Cleveland, Valerie M. Jansen, David Dornan. Therapeutic potential of a HER3 antibody-drug conjugate for the treatment of HER3-expressing cancers [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 3121.

Abstract 5157: Development and evaluation of a high-throughput method for rapid detection of surface antigen expression in fixed cells

Abstract Cell surface proteins are increasingly considered in the development of novel anticancer agents, as demonstrated by recent clinical successes in the field of immuno-oncology and antibody-drug conjugates (ADCs). These conjugates specifically target antigens present on the surface of tumor cells, enabling the delivery of cytotoxic agents to specific tumor sites. During the preclinical development of such compounds, for example, when they are tested on a wide panel of cell lines, it is essential to have a rapid and reliable method for monitoring target expression and distribution. Complementing our 160 Cell line panel ProLiFiler™, we report here the development of a novel method named oncoFLOW-Profiler™ which includes steps of cell fixation, micronic storage, staining, and measurement by flow cytometry. This method is applied in a 96-well format, enabling screening of all cell lines in parallel and analysis of the expression of different targets in just one day. In this proof-of-concept study, we investigated the level of ERBB2 expression across the ProLiFiler panel of 160-cell lines in order to validate this methodology. We first compared the impact of fixation methods and sample storage on knowingly positive and negative control cell lines over a period of several months. ERBB2 measurements from the oncoFLOW-Profiler™ will be analyzed and validated by comparison with various OMICs datasets from the Cancer DataMiner platform (4HF Biotec), including RNA expression, mutational status, and protein expression levels. To determine the expression cutoff of ERBB2 amplified models, results will be analyzed according to gene copy number variation. Finally, the flow cytometry data generated will be investigated for use as a predictor of response to clinically approved ADCs targeting ERBB2: Kadcyla and Enhertu. Here we present a method that we have demonstrated is feasible for investigating cell surface receptor/antigens during preclinical phase of drug development. Our method has enabled us to successfully assess ERBB2 in biological samples and to identify models sensitive to ADCs targeting ERBB2. We will also discuss the results of ongoing experiments analyzing the applicability of this screen to intracellular targets and carbohydrate-based surface antigens, which cannot be analyzed either by transcriptomics or proteomics. Citation Format: Kaja Holtorf, Anne-Lise Peille, Daniel Feger, Vincent Vuaroqueaux, Jan Erik Ehlert, Nadine Obier. Development and evaluation of a high-throughput method for rapid detection of surface antigen expression in fixed cells [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 5157.

Abstract 2060: Automation of 3D cancer spheroid assay for compound screening

Abstract Finding efficient drug combinations to treat cancer patients is critical for therapy success. Accordingly, there is a critical need to develop methods for efficient testing drug efficacy to discover new therapeutic targets. 3D cancer models are highly valuable tools for cancer research and drug development. However, the complexity of performing 3D assays remains a hurdle for adopting these methods for compound screening. To alleviate the bottlenecks that come with labor-intensive manual protocols we developed cell culture automation methods using CellXpress.ai instrument. The instrument enables automation of 2D or 3D culture for prolonged complex workflows. The CellXpress.ai provides automated plating, passaging, media exchanges, monitoring organoids, alone with compound treatment and the end point assays with high content image analysis. In the present study we describe full automation of imaging, analysis, and cell culture process which enables scaling up complex 3D cell-based assays and compound screening. As an example of 3D protocol, we automated culture and imaging of colorectal cancer 3D spheroids formed from HCT116 cell line in U-shape low attachment plates. HCT116 cells were expanded in 2D, then spheroids were formed after automated dispensing of cell suspension into U-shape 96 or 384 plates. After 48h spheroids were treated with 12 anti-cancer compounds, at multiple concentrations for 3-5 days, then stained and imaged. Cell plating, compound additions, media exchange, and staining were performed automatically by CellXpressAI instrument. During the culture spheroids were monitored using transmitted light, with immediate analysis of phenotypic changes, including inhibition of growth or spheroid disintegration. For endpoint assay spheroids were stained with combination of Hoechst nuclear stain and viability dyes Calcein AM and EtHD, then imaged, and analyzed for multiple read-outs including spheroid size, marker intensities, and live-dead cell scoring. We observed concentration-dependent inhibition of spheroid growth and cell death in response to anti-cancer compounds and evaluated effective compound concentrations for cytotoxic or cytostatic effects. Cell culture process automation for 3D assays, powered by imaging and image analysis has a potential to bring 3D biology into another level, allowing to increase throughput and reproducibility, and enabling variety of high throughput drug discovery and precision medicine applications. Citation Format: Oksana Sirenko, Angeline Lim, Astrid Michlmayr, Zhisong Tong, Emilie Keidel, Felix Spira. Automation of 3D cancer spheroid assay for compound screening [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 2060.

Abstract 6673: Tumor microbial burden is regulated by mast cells and determines immunotherapy responses

Abstract Immune checkpoint inhibitors (ICI) have notably improved overall survival rates across various cancers, particularly in cases of mismatch repair deficient cancer (MMRd). However, numerous factors influence patient response following ICI treatment. Beyond genetic factors, mutations, and levels of immune cell infiltration, several studies highlight the correlation between the gut microbiome and treatment response rates. Furthermore, specific bacterial compositions have been observed to shift during therapeutic interventions. Notably, studies have demonstrated that patients who used antibiotics before and during ICI treatment exhibited lower progression-free survival and overall survival, serving as evidence for this association. Our previous investigations elucidated the impact of distinct tumor microbial burdens on modulating immune activation. Consequently, our hypothesis posits the substantive involvement of the interplay between tumor microbial burden and the immune system response to ICI treatment. We evaluated the presence of intra-tumoral microbes within Colorectal cancers (CRC) by examining TCGA data. Patients with elevated microbial levels demonstrated heightened immune activity and better survival rates. Using cybersport analysis, we validated increased mast cell infiltration in CRC patients with abundant microbes. Quantification involved assessing bacterial burden through 16s rRNA via FISH staining and mast cell counts using tryptase via IHC staining. Reducing bacterial load in MC38 cancer model, achieved by locally administering antibiotics, led to a diminished immune response when PD-1 was introduced to these mice. Cross-referencing TCGA data, we identified upregulated genes in CRC patients with high microbial burden and downregulated genes in mice receiving local antibiotics after MC38 cell implantation. The common genes were associated with immune responses, suggesting a strong correlation between bacterial burden and immune activity in CRCs. In conclusion, the high burden of bacteria in CRC improves immune response to ICI and increases the infiltration of mast cells, which all contribute to better survival rates. Citation Format: Hajar Rajaei, Jeong-Hoon Jang, Haoyue Liu, Erika Y. Faraoni, Olivia Le Roux, Vidhi Chandra, Virginia Tahan, Le Li, Erick Riquelme, James Robert White, Luis A. Diaz Jr., Florencia McAllister. Tumor microbial burden is regulated by mast cells and determines immunotherapy responses [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 6673.

Abstract 2970: Anti-diabetic drug, semaglutide, prevents doxorubicin-induced endothelial cell toxicity

Abstract Doxorubicin (Dox) is part of the anthracycline drug family and is known for its broad and effective use as a chemotherapeutic agent. It is commonly used to treat solid tumors and malignancies such as leukemia, breast, lung, and ovarian cancers. Unfortunately, its use in treating advanced cancers is limited due to its cardiotoxic side effects. Therefore, various adjuvant approaches are required to increase Dox's efficacy and to reduce unwanted toxicities including cardiotoxicity. Semaglutideis a glucagon-like peptide-1 (GLP-1) receptor agonist commonly used to treat type-2 diabetes. In his study, the effect of Semaglutide in preventing Dox-induced endothelial cell toxicity was investigated. Our data indicate that Semaglutide prevents Dox-induced cell death in human umbilical vein endothelial cells (HUVEC) in a dose- and time-dependent manner. Similarly, live and dead cell staining assay also confirms Semaglutide prevents Dox-induced endothelial cell death. Semaglutide also prevented the Dox-induced apoptosis of HUVECs. It also prevented the Dox-induced reactive oxygen species and activation of caspase-3. Further, Semaglutide regulated the expression of various anti- and pro-apoptotic and inflammatory factors induced by Dox. Inconclusion, our current results suggests that the anti-diabetic drug, Semaglutide, could prevent Dox-induced endothelial cell death by regulating the expression of various pro-apoptotic, anti-apoptotic and inflammatory markers. Thus, our data indicate that Semaglutide could be used as a potential adjuvant drug to reduce the adverse effects of anthracycline chemotherapeutic drugs. Citation Format: Sandra Bakhit, Chelsea Amaefuna, Kota Ramana. Anti-diabetic drug, semaglutide, prevents doxorubicin-induced endothelial cell toxicity [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 2970.

Abstract 662: Vitamin B1 derivative, fursultiamine, prevents lung cancer cells growth

Abstract Lung cancer is the leading cause of cancer death in men and the second leading cause of cancer death in women worldwide. Cigarette smoke-induced oxidative and inflammatory responses are the major risk factors for the development of lung cancer. Several compounds have been tested for their efficacy in preventing lung cancer. However, very few compounds have gone through clinical studies. We hypothesize that with its potent anti-oxidative and anti-inflammatory actions, fursultiamine, a disulfide lipid-soluble derivative of vitamin B1 (thiamine), could prevent lung cancer growth and spread. Our results indicate that treatment of non-small cell lung cancer cells (A549 cells) with fursultiamine prevents A549 cells death in a dose-dependent manner. Similarly, live and dead cell staining assay also confirms that fursultiamine prevents proliferation of A549 cells. Further, fursultiamine prevents A549 cell proliferation by inducing the apoptotic cell death. It also promoted the activation of caspase-3 and cleavage of PARP protein. Fursultiamine also prevented reactive oxygen species in A549 cells. Further, fursultiamine regulated the expression of various anti-apoptotic (Bcl-2, Bcl-x) and pro-apoptotic (Cleaved Caspase-3), survival (Survivin) and anti-survival factors (Clusterin, p-Rad17) in A549 cells. We next planned to examine the effect of fursultiamine on lung cancer growth in vivo using athymic nude mice injected with A549 cells subcutaneously without or with a diet containing fursultiamine. In conclusion, our in vitro results suggest that vitamin B1 derivative, fursultiamine, by promoting the apoptotic pathways prevents lung cancer growth and has potential for further development as a chemoprevention drug. Citation Format: Noah Boekweg, Ryan Powers, Kota Ramana. Vitamin B1 derivative, fursultiamine, prevents lung cancer cells growth [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 662.