EGF Receptor Research Articles

Crosstalk between cancer cells and macrophages promotes OSCC cell migration and invasion through a CXCL1/EGF positive feedback loop.

C-X-C motif chemokine ligand 1 (CXCL1) and epithelial growth factor (EGF) are highly secreted by oral squamous cell carcinoma (OSCC) cells and tumor-associated macrophages, respectively. Recent studies have shown that there is intricate "cross-talk" between OSCC cells and macrophages. However, the underlying mechanisms are still poorly elucidated. The expression of CXCL1 was detected by immunohistochemistry in OSCC clinical samples. CXCL1 levels were evaluatedby RT‒PCR and ELISA in an OSCC cell line and a normal epithelial cell line. The expression of EGF was determined by RT‒PCR and ELISA. The effect of EGF on the proliferation of OSCC cells was evaluated by CCK-8 and colony formation assays. The effect of EGF on the migration and invasion ability and epithelial-mesenchymal transition (EMT) of OSCC cells was determined by wound healing, Transwell, RT‒PCR, Western blot and immunofluorescence assays. The polarization of macrophages was evaluated by RT‒PCR and flow cytometry. Western blotting was used to study the molecular mechanism in OSCC. The expression of C-X-C motif chemokine ligand 1 (CXCL1) was higher in the OSCC cell line (Cal27) than in immortalized human keratinocytes (Hacat cells). CXCL1 derived from Cal27 cells upregulates the expression of epithelial growth factor (EGF) in macrophages. Paracrine stimulation mediated by EGF further facilitates the epithelial-mesenchymal transition (EMT) of Cal27 cells and initiates the upregulation of CXCL1 in a positive feedback-manner. Mechanistically, EGF signaling-induced OSCC cell invasion and migration can be ascribed to the activation of NF-κB signaling mediated by the epithelial growth factor receptor (EGFR), as determined by western blotting. OSCC cell-derived CXCL1 can stimulate the M2 polarization of macrophages and the secretion of EGF. Moreover, EGF significantly activates NF-κB signaling and promotes the migration and invasion of OSCC cells in a paracrine manner. A positive feedback loop between OSCC cells and macrophages was formed, contributing to the promotion of OSCC progression.

A Novel Role for the Phosphatidylinositol-4,5-Bisphosphate 3-Kinase Delta Isoform in Hepatocellular Proliferation

The phosphatidylinositol-4,5-bisphosphate 3-kinase delta isoform (Pik3cd), usually considered immune-specific, was unexpectedly identified as a gene potentially related to either regeneration and/or differentiation in animals lacking hepatocellular Integrin Linked Kinase (ILK). Since a specific inhibitor (Idelalisib, or CAL101) for the catalytic subunit encoded by Pik3cd (p110δ) has reported hepatotoxicity when used for treating Chronic Lymphocytic Leukemia and other lymphomas, we aimed to elucidate if there is a role for p110δ in normal liver function. To determine the effect on normal liver regeneration, partial hepatectomy (PHx) was performed using mice in which p110δ was first inhibited using CAL101. Inhibition led to over a 50% decrease in proliferating hepatocytes in the first two days after PHx. This difference correlated with phosphorylation changes in the HGF and EGF receptors (MET and EGFR, respectively) and NF-κB signaling. Ingenuity Pathway Analyses implicated C/EBPβ, HGF and the EGFR heterodimeric partner, ERBB2, as three of the top 20 regulators downstream of p110δ signaling as their pathways were suppressed in the presence of CAL101 at one day post-PHx. A regulatory role for p110δ signaling in mouse and rat hepatocytes through MET and EGFR was further verified using hepatocyte primary cultures, in the presence or absence of CAL101. Combined, this data supports a role for p110δ as a downstream regulator of normal hepatocytes when stimulated to proliferate.

Ethnicity-Based Variations in Focal Adhesion Kinase Signaling in Glioblastoma Gene Expression: A Study of the Puerto Rican Hispanic Population.

Glioblastoma (GBM), an aggressive form of brain cancer, has a higher incidence in non-Hispanics when compared to the US Hispanic population. Using data from RT-PCR analysis of 21 GBM tissue from Hispanic patients in Puerto Rico, we identified significant correlations in the gene expression of focal adhesion kinase and proline-rich tyrosine kinase (PTK2 and PTK2B) with NGFR (nerve growth factor receptor), PDGFRB (platelet-derived growth factor receptor B), EGFR (epithelial growth factor receptor), and CXCR1 (C-X-C motif chemokine receptor 1). This study further explores these correlations found in gene expression while accounting for sex and ethnicity. Statistically significant (p < 0.05) correlations with an r value > ±0.7 were subsequently contrasted with mRNA expression data acquired from cBioPortal for 323 GBM specimens. Significant correlations in Puerto Rican male patients were found between PTK2 and PTK2B, NGFR, PDGFRB, EGFR, and CXCR1, which did not arise in non-Hispanic male patient data. The data for Puerto Rican female patients showed correlations in PTK2 with PTK2B, NGFR, PDGFRB, and EGFR, all of which did not appear in the data for non-Hispanic female patients. The data acquired from cBioPortal for non-Puerto Rican Hispanic patients supported the correlations found in the Puerto Rican population for both sexes. Our findings reveal distinct correlations in gene expression patterns, particularly involving PTK2, PTK2B, NGFR, PDGFRB, and EGFR among Puerto Rican Hispanic patients when compared to non-Hispanic counterparts.

Maternal Protection from Bacterial Translocation in a Murine Model of Late-Onset Neonatal Sepsis (LOS)

Abstract Late-onset neonatal sepsis (LOS), resulting from a bloodstream infection at least 72 hours after birth, is a major contributor to neonatal mortality. In a substantial portion of LOS cases, the causative pathogen is present as a member of the intestinal microbial community before dissemination, implicating intestinal translocation as a source of bloodstream infections. One potential pathway of bacterial translocation is through goblet cell-associated antigen passages, or GAPs, in colonic goblet cells. The presence of luminal EGF, obtained from breastmilk, and subsequent EGF receptor (EGFR) signaling in the intestine inhibits the formation of GAPs. We have established that disruption of EGFR signaling in pups leads to dissemination of the enteric pathogen Escherichia coli to the mesenteric lymph node (MLN). As pathogen dissemination was quickly followed by mortality, we use this as a preclinical model of LOS. Further work with this model focuses on the role of milk-derived immunoglobulins in reducing pathogen colonization and protecting from disease resulting from intestinal translocation. Through cross-fostering litters between dams immunized against E. coli and unimmunized dams, along with a comparison in disease between litters from immunized wild-type mice and IgA-/- mice, we aim to describe the protective role of mammary IgA. These experiments will also provide further implications for the role of human milk and human milk factors in protection from LOS.

Abstract 1055: Atxn2 Inhibition Enhances The Contractile Phenotype Of Vascular Smooth Muscle Cells

Introduction: Phenotype switching of vascular smooth muscle cells (VSMC) between their synthetic and differentiated contractile state is a critical player in controlling the stability of atherosclerotic plaque. Hundreds of genes associated with coronary artery disease (CAD) have been identified through Genome-Wide Association Studies (GWAS). However, the involvement of most identified genes in the pathogenesis of atherosclerosis remains unknown. ATXN2 was identified as a CAD GWAS candidate gene with unclear biological role in atherosclerosis. ATXN2 regulates the endocytic internalization of the EGF receptor, which is essential in the VSMC phenotype switching. Therefore, we aimed to investigate whether ATXN2 is involved in the VSMC phenotype switching. Methods and Results: Using immunofluorescence, we demonstrated that ATXN2 siRNA knockdown resulted in a 5-fold increase in the ratio of the contractile phenotype of human aortic VSMC that expressed a high alpha-smooth muscle cell actin content compared to controls. In addition, we found that ATXN2 inhibition reduced the VSMC proliferation and migration as assessed by the EdU cell proliferation assay and the wound healing migration assay, respectively. Next, we confirmed that ATXN2 siRNA knockdown increased the mRNA expression of contractile markers: calponin, alpha-smooth muscle cell actin, and smooth muscle protein 22 alpha using quantitative PCR. We found that the enhancing effect of VSMC contractile phenotype by ATXN2 siRNA was less pronounced in the absence of TGF-beta. Indeed, the enhancing effect of ATXN2 inhibition on the VSMC contractile phenotype was abolished by SMAD4 gene silencing, suggesting the involvement of TGF-beta signaling. Furthermore, we validated the expression signature of the ATXN2 gene in different phenotypic subsets of VSMC from human atherosclerotic plaque samples using single-cell RNA sequencing analysis. We detected that ATXN2 expression was negatively correlated with the contractile VSMC subset. In conclusion, our findings suggests ATXN2, CAD GWAS candidate gene, as a novel potential therapeutic target to enhance the VSMC contractile phenotype for stabilizing atherosclerotic plaque in coronary artery disease patients.

Can the HB-EGF/EGFR pathway restore injured neurons?

Heparin-binding epidermal growth factor-like growth factor (HB-EGF) is a transmembrane protein that, when cleaved by metalloproteases through a process called ectodomain shedding, binds to the EGF receptor (EGFR), activating downstream signaling. The HB-EGF/EGFR pathway is crucial in development and is involved in numerous pathophysiological processes. In this issue of The FEBS Journal, Sireci etal. reveal a previously unexplored function of the HB-EGF/EGFR pathway in promoting neuronal progenitor proliferation and sensory neuron regeneration in the zebrafish olfactory epithelium in response to injury.

Inhibition of neuraminidase-1 sialidase activity by interfering peptides impairs insulin receptor activity in vitro and glucose homeostasis in vivo

Neuraminidases also called sialidases are glycosidases which catalyze the removal of terminal sialic acid residues from glycoproteins, glycolipids and oligosaccharides. Mammalian Neuraminidase-1 (NEU-1) participates in regulation of cell surface receptors such as insulin receptor (IR), epithelial growth factor receptor, low density lipoprotein receptor and toll like receptor 4. At the plasma membrane, NEU-1 can be associated with the elastin-binding protein and the carboxypeptidase protective protein/cathepsin A to constitute the elastin receptor complex. In this complex, NEU-1 is essential for elastogenesis, signal transduction through this receptor and for biological effects of the elastin-derived peptides on atherosclerosis, thrombosis, insulin resistance, non-alcoholic steatohepatitis and cancers. This is why research teams are developing inhibitors targeting this sialidase. Previously, we developed interfering peptides to inhibit the dimerization and the activation of NEU-1. In this study, we investigated the effects of these peptides on IR activation in vitro and in vivo. Using cellular overexpression and endogenous expression models of NEU-1 and IR (COS-7 and HepG2 cells respectively), we have shown that interfering peptides inhibit NEU-1 dimerization and sialidase activity which results in a reduction of IR phosphorylation. These results demonstrated that NEU-1 positively regulates IR phosphorylation and activation in our conditions. In vivo, biodistribution study showed that interfering peptides are well distributed in mice. Treatment of C57Bl/6 mice during eight weeks with interfering peptides induces a hyperglycemic effect in our experimental conditions. Altogether, we report here that inhibition of NEU-1 sialidase activity by interfering peptides decreases IR activity in vitro and glucose homeostasis in vivo.

RETRACTION: Afatinib plus bevacizumab treatment for a patient with EGFR S645C-mutant NSCLC

Epithelial growth factor receptor (EGFR) tyrosine kinase inhibitors (TKIs) improve the survival outcomes of patients with advanced lung adenocarcinoma with common mutations. However, challenges persist in treating rare, uncommon EGFR mutations, such as EGFR S645C, which accounts for only 0.011% of the EGFR mutations in the Guardant360 database. Despite preclinical studies indicating its resistance to osimertinib, we present a case of using afatinib plus bevacizumab to successfully treat a patient with EGFR S645C mutation, for which an effective treatment had not been previously reported. This case report presents real-world evidence for optimal therapeutic options for patients with EGFR S645C mutation.

Co-Expression Network Analysis Unveiled lncRNA-mRNA Links Correlated to Epidermal Growth Factor Receptor-Tyrosine Kinase Inhibitor Resistance and/or Intermediate Epithelial-to-Mesenchymal Transition Phenotypes in a Human Non-Small Cell Lung Cancer Cellular Model System.

We investigated mRNA-lncRNA co-expression patterns in a cellular model system of non-small cell lung cancer (NSCLC) sensitive and resistant to the epithelial growth factor receptor (EGFR) tyrosine kinase inhibitors (TKIs) erlotinib/gefitinib. The aim of this study was to unveil insights into the complex mechanisms of NSCLC targeted therapy resistance and epithelial-to-mesenchymal transition (EMT). Genome-wide RNA expression was quantified for weighted gene co-expression network analysis (WGCNA) to correlate the expression levels of mRNAs and lncRNAs. Functional enrichment analysis and identification of lncRNAs were conducted on modules associated with the EGFR-TKI response and/or intermediate EMT phenotypes. We constructed lncRNA-mRNA co-expression networks and identified key modules and their enriched biological functions. Processes enriched in the selected modules included RHO (A, B, C) GTPase and regulatory signaling pathways, apoptosis, inflammatory and interleukin signaling pathways, cell adhesion, cell migration, cell and extracellular matrix organization, metabolism, and lipid metabolism. Interestingly, several lncRNAs, already shown to be dysregulated in cancer, are connected to a small number of mRNAs, and several lncRNAs are interlinked with each other in the co-expression network.

EGFR Mutation and FHIT Methylation: Inverse Relationship in Patients with Lung Adenocarcinoma and Tuberculosis

Objective: To investigate the genetic correlations between epithelial growth factor receptor (EGFR) mutation and FHIT methylation in patients diagnosed with lung adenocarcinoma (AC) and pulmonary tuberculosis (TB). Methods: The presence of EGFR mutations and the methylation status of the FHIT gene in patients presenting with AC and TB were analyzed. The correlation between TB status and the observed genetic and epigenetic variations was also examined. Results: Among the 90 patients included in the study, 38 exhibited EGFR mutations (14 among those with TB and 24 among those without TB), while 29 exhibited FHIT myelination (19 among those with TB and 10 among those without TB). Furthermore, the protein expression levels of EGFR and FHIT were significantly higher in patients diagnosed solely with AC compared to those presenting with both AC and TB. A robust inverse correlation was identified between TB status and the frequency of EGFR mutation (P &lt; 0.001). Moreover, significant associations were observed between TB status and FHIT methylation (P &lt; 0.01). Conclusion: The findings suggest a correlation between TB and the prevalence of EGFR mutation and FHIT methylation in the pathogenesis of AC.

Quantitative evaluation of trastuzumab deruxtecan pharmacokinetics and pharmacodynamics in mouse models of varying degrees of HER2 expression.

Trastuzumab deruxtecan (T-DXd; DS-8201; ENHERTU®) is a human epithelial growth factor receptor 2 (HER2)-directed antibody drug conjugate (ADC) with demonstrated antitumor activity against a range of tumor types. Aiming to understand the relationship between antigen expression and downstream efficacy outcomes, T-DXd was administered in tumor-bearing mice carrying NCI-N87, Capan-1, JIMT-1, and MDA-MB-468 xenografts, characterized by varying HER2 levels. Plasma pharmacokinetics (PK) of total antibody, T-DXd, and released DXd and tumor concentrations of released DXd were evaluated, in addition to monitoring γΗ2AX and pRAD50 pharmacodynamic (PD) response. A positive relationship was observed between released DXd concentrations in tumor and HER2 expression, with NCI-N87 xenografts characterized by the highest exposures compared to the remaining cell lines. γΗ2AX and pRAD50 demonstrated a sustained increase over several days occurring with a time delay relative to tumoral-released DXd concentrations. Invitro investigations of cell-based DXd disposition facilitated the characterization of DXd kinetics across tumor cells. These outputs were incorporated into a mechanistic mathematical model, utilized to describe PK/PD trends. The model captured plasma PK across dosing arms as well as tumor PK in NCI-N87, Capan-1, and MDA-MB-468 models; tumor concentrations in JIMT-1 xenografts required additional parameter adjustments reflective of complex receptor dynamics. γΗ2AX longitudinal trends were well characterized via a unified PD model implemented across xenografts demonstrating the robustness of measured PD trends. This work supports the application of a mechanistic model as a quantitative tool, reliably projecting tumor payload concentrations upon T-DXd administration, as the first step towards preclinical-to-clinical translation.

Abstract 6997: Elucidating the tumorigenic properties of alveolar epithelial type 1 cells in EGFR-mediated LUAD tumorigenesis

Abstract Originating from within the distal lung, lung adenocarcinoma (LUAD) is the most common pathologic subtype of non-small cell lung carcinoma (NSCLC) and encompasses an assortment of histologic presentations. Colocalized within the distal alveolar epithelium are two alveolar epithelial cell (AEC) subpopulations: alveolar epithelial type 1 (AT1) and alveolar epithelial type 2 (AT2) cells. We previously demonstrated that signatures unique to each AEC subpopulation were detected in LUAD tumors formed by oncogenic KRASG12D activation in mice and were associated with disparate survival outcomes, transcriptomic patterning, and histologic presentation. Epithelial growth factor receptor (EGFR) mutations are among the most frequent oncogenic drivers in thoracic oncology. Therefore, we set out to determine if EGFR oncogenic activation resulted in differential LUAD presentation and outcomes based on the cell of origin in which it was activated. To activate EGFR oncogenic mutations in AT1 or AT2 cells, we employed triple transgenic mouse models (AT1: Gramd2-CreERT2:EF1-LSL-tTA:TetO-EGFRdel20/T790 and AT2: Sftpc-CreERT2:EF1-LSL-tTA:TetO-EGFRdel20/T790), which allowed for EGFR activation in either Gramd2+ AT1 or Sftpc+ AT2 cells. Longitudinal analysis of hematoxylin and eosin-stained lungs over the course of 20 weeks was performed. Blinded pathology review demonstrated that EGFR oncogenic activation in Gramd2+ AT1 cells resulted in histologically defined LUAD with lepidic properties at early time points and solid adenocarcinoma properties at later time points. This differed from EGFR activation in Sftpc+ AT2 cells, which resulted in pre-carcinogenic atypical adenomatous hyperplasia (AAH) formation that did not progress to adenocarcinoma. While preliminary, our results suggest that cell of origin strongly influences EGFR-mediated lung oncogenesis and concomitant pathology and histologic presentation. This work may advance our understanding while informing patient stratification and treatment strategies for LUAD patients. Citation Format: Aaron M. Neely, Andrea Lanz, William Dean Wallace, Anthony W. Kim, Zea Borok, Crystal N. Marconett. Elucidating the tumorigenic properties of alveolar epithelial type 1 cells in EGFR-mediated LUAD tumorigenesis [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 6997.

Abstract 1564: NEU4-mediated desialylation ignites the oncogenic receptors for the dissemination of ovarian carcinoma

Abstract Background: Glycosylation profoundly influences the interactions between cancer cells and microenvironmental stromal cells during the peritoneal disseminated metastasis of ovarian carcinoma (OC), which is the major cause of cancer-related death. Although the characteristic cancer glycoconjugates are widely used as biomarkers for cancer diagnosis, our knowledge about cancer glycome remains quite fragmented due to the technique limitations in analyzing glycan chains with tremendous structural and functional heterogeneity. Method: Given the dysregulated cancer glycome is defined by the altered glycosylation machinery, here we performed a systematic loss-of-function screen on 498 genes involved in glycosylation for key regulators of OC dissemination. We identified neuraminidase 4 (NEU4), an enzyme capable of hydrolyzing terminal sialic acid from glycoconjugates, as a vital peritoneal dissemination-promoting modifier of OC glycome. What’s more, we also identified membrane proteins whose sialylation was able to be modified by NEU4 in OC cells by pull down and mass spectrum to investigate the functional significance of NEU4-mediated desialylation. Results: In human patients with high-grade serous OC (HGSOC), increased NEU4 was detected in the disseminated OC cells when compared with that in the primary tumor cells, which significantly correlated with the worse survival. Among three alternative splice-generated isoforms of human NEU4, we revealed that only the plasma membrane-localized NEU4 isoform 2 (NEU4-iso2) and intracellular isoform 3 promoted the peritoneal dissemination of OC by enhancing the cell motility and epithelial-mesenchymal transition. We also identified NEU4-iso2-regulated cell surface glycoproteome and found that NEU4-iso2 desialylated the epithelial growth factor receptor (EGFR), in particular at N196 residue, for the hyperactivation of EGFR and its downstream tumor-promoting signaling cascades. Conclusion: Our results provide new insights into how the OC glycome is dysregulated by NEU4 during OC progression and reveals a functionally important glycosite on EGFR for its abnormal activation in cancer. Citation Format: Jie Shi, Rui Zhou, Shuo Wang, Yuxin Liu, Baorui Tian, Yanhua Liu, YaNan Chen, Taoyu Hu, Yuhao Mu, Shufan Wang, Xintao Shao, Jie Yan, Pengpeng Qu, Wei Ding, Shuang Yang, Yi Shi, Jia Li, Longlong Wang. NEU4-mediated desialylation ignites the oncogenic receptors for the dissemination of 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 1564.

Abstract 3264: Characterization of EGFR ectodomain mutation in acquired resistance to cetuximab in colorectal cancer

Abstract Colorectal cancer (CRC) remains the second leading cause of cancer-related deaths in the United States (US). Individuals with surgically unresectable stage four CRC have a five-year overall survival rate of 13%. Targeting of the receptor tyrosine kinase (RTK), EGF receptor (EGFR) with therapeutic monoclonal antibodies (cetuximab, panitumumab) is approved for use in wild-type (WT) KRAS/NRAS/BRAF (RAS/RAF) advanced CRC. However, these antibodies as a single agent have response rates of 12-17% and expected progression-free survivals of only 3-4 months. Accordingly, resistance may be present at the outset (de novo resistance) or develop during treatment (acquired resistance). Here, we report the identification of an EGFR ectodomain missense mutation acquired in vitro in response to treatment with anti-epidermal growth factor receptor (EGFR) antibody cetuximab. Using 2D and 3D drug sensitivity assays, our results demonstrate that this EGFR ectodomain mutation in addition to being resistant to cetuximab and panitumumab, also could not be targeted by the oligoclonal antibody cocktail MM-151. To determine whether this EGFR ectodomain mutation is necessary and sufficient to impart cetuximab resistance, we engineered CRC lines to inducibly overexpress mutant EGFR and showed that this was sufficient to induce cetuximab resistance. Further structural, and functional characterization is underway for this ectodomain mutation that confers resistance to cetuximab and will be shared during the meeting. These findings necessitate the need for additional treatment strategies for a subset of subjects with mCRC who do not respond to treatment with cetuximab/panitumumab and MM-151. Citation Format: Neeraj Joshi, Ramona Graves Deal, Galina Bogatcheva, James N. Higginbotham, Marisol Ramirez, Benjamin P. Brown, Qi Liu, Jens Meiler, Bhuminder Singh. Characterization of EGFR ectodomain mutation in acquired resistance to cetuximab in 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 3264.

Abstract 1415: Reduction in photoimmunotherapy-induced edema with COX-2 inhibition: Combatting clinically relevant adverse events without compromising efficacy

Abstract ASP-1929 photoimmunotherapy (PIT), an investigational drug-device combination, consists of an epithelial growth factor receptor (EGFR)-targeting drug, cetuximab, conjugated to a light-activatable dye, IRDye® 700DX, and a laser light. Localized illumination results in rapid and selective tumor necrosis in the preclinical setting, and early clinical studies demonstrate a manageable safety profile in patients with head and neck squamous cell carcinoma. In the ongoing clinical trials, however, edema is a commonly reported adverse event and laryngeal edema, requiring prophylactic tracheostomy, has been noted in some patients. Thus, a critical need exists to provide clinicians with tools to manage ASP-1929 PIT-associated edema to ensure optimal patient outcome. To evaluate edema following cancer-targeted PIT, we developed a mouse tumor model and used it to characterize the mechanism of edema development and evaluate various methods for edema reduction. Briefly, cohorts of mice bearing syngeneic LL/2 tumors engineered to express Ephrin A2 (EphA2) were administered an anti-EphA2 antibody conjugated to IR700 (conjugate) or saline control. Light was applied 24h following administration and edema was measured by caliper. In control mice, light alone did not generate edema; however, mice treated with conjugate plus light showed a light-dose dependent increase in edema volume which peaked at 6h post light delivery. We next evaluated a series of inflammatory cytokines and immune cell populations in the blood and tumor region at the onset (2h), peak (6h), and resolution (24h) of PIT-induced edema. We found a striking increase in neutrophils (500-fold greater than control mice, n=10, p&amp;lt;0.0001) in the blood at the peak of edema formation coinciding with a significant increase in IL-6 (n=5, p&amp;lt;0.001) and IL-10 (n=5, p&amp;lt;0.05), indicating the onset of a heightened inflammatory response. To combat the edema formation, we next evaluated the effect of various anti-inflammatory drugs commonly used in the clinical setting. Cohorts of mice were administered conjugate plus light in the presence or absence of steroids or the selective COX-2 inhibitor meloxicam. Results show there is no reduction in edema by steroids regardless of timing of administration. Conversely, the addition of meloxicam resulted in a significant reduction in edema at all time points and light doses evaluated: 40-50% reduction at both 2h and 6h post-PIT when administered prophylactically and 25% reduction at 6h post-PIT when administered post-light (all settings, n=10, p&amp;lt;0.0001). Importantly, we found the reduction in edema with meloxicam was not associated with a loss of therapeutic benefit based on measurement of tumor growth. In conclusion, the use of COX-2 inhibitors may be directly translated to the clinic for the benefit of patients receiving ASP-1929 PIT and should undergo further evaluation. Citation Format: Gina Ma, Myra Gordon, Jason Lapetoda, Abram Lozano, Christopher M. Amantea, Takuya Osada, Amy H. Thorne. Reduction in photoimmunotherapy-induced edema with COX-2 inhibition: Combatting clinically relevant adverse events without compromising efficacy [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 1415.

Abstract 1914: Amivantamab efficacy in wildtype EGFR NSCLC tumors correlates with levels of ligand expression

Abstract Amivantamab (RYBREVANT™) is an FDA-approved, low-fucose IgG1 bispecific antibody targeting EGF and Met receptor tyrosine kinases, with proven clinical activity against EGFR mutant non-small cell lung cancer (NSCLC). Preclinical studies demonstrated that three context-dependent mechanisms of action (MOA) contribute to amivantamab efficacy: immune cell-mediated killing by antibody-dependent cellular cytotoxicity and trogocytosis, receptor internalization and degradation, and inhibition of ligand binding to both EGF and Met receptors. In tumors lacking known cancer-driver genetic aberrations, ligand binding to wildtype EGFR (EGFRWT) and/or MetWT may activate downstream signaling pathways, inducing pro-tumorigenic processes. Among the EGFR ligands, we demonstrated that amphiregulin (AREG) is highly expressed in EGFRWT NSCLC primary tumors, with significantly higher circulating protein levels in NSCLC patients than in healthy volunteers, and induces EGFR phosphorylation, promoting downstream signaling and increasing cell proliferation. Importantly, treatment of AREG-stimulated EGFRWT cells/tumors with amivantamab, or an AREG-targeting antibody, inhibits ligand-induced signaling and cell/tumor proliferation/growth. Across 10 EGFRWT NSCLC patient-derived xenograft mouse models, amivantamab efficacy correlated with AREG RNA levels, suggesting AREG as a potential predictive marker for amivantamab activity in this population. Interestingly, in these xenograft models, amivantamab anti-tumor activity was independent of amivantamab's Fc engagement with immune cells, suggesting that, in this context, the amivantamab’s ligand-blocking function is sufficient for its efficacy. Juxtaposedly, we previously demonstrated that low fucose amivantamab, active Fc domain, was necessary for maximal anti-tumor activity against tumors with EGFR or Met activating mutations. Altogether, these results demonstrated amivantamab context-dependent MOA for efficacy. In conclusion, these data 1) highlight EGFR ligand AREG as a driver of tumor growth in some EGFRWT NSCLC models, 2) illustrate the preclinical efficacy of amivantamab in ligand-driven EGFRWT NSCLC, and 3) identify AREG as a potential predictive biomarker for amivantamab against anti-EGFRWT therapies. Citation Format: Ricardo Rivera-Soto, Benjamin Henley, Marian Pulgar, Stacey Lehman-Tenuto, Himanshu Gupta, Megan Weindorfer, Smruthi Vijayaraghavan, Tsun-Wen Yao, Sylvie Laquerre, Sheri Moores. Amivantamab efficacy in wildtype EGFR NSCLC tumors correlates with levels of ligand expression [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 1914.

Abstract 6724: Mechanistic validation of MAIT cell engagers in HUB patient-derived organoids (PDO) co-culture model

Abstract In the field of cancer immunotherapy, bispecific antibodies have emerged as a promising approach for redirecting T-cells to target and combat tumors. However, their efficacy is limited to only a subset of patients. A major challenge that hinders the development of bispecific antibody targets is the lack of preclinical models that preserve patient-specific tumor antigens and recapitulate the complex interaction between the human immune system and tumor cells. HUB Organoids technology presents a valuable platform for novel immunotherapies testing and validation as they are directly derived from patient tumor tissues, accurately reflecting patient-specific tumor antigens. BIOMUNEX Pharmaceuticals has developed the BiXAb® technology platform, a computational modeling approach that rapidly generates bispecific antibodies from various monospecific monoclonal antibodies, eliminating the need for extensive engineering. This platform includes a bivalent T cell engager specifically targeting MAIT cells and human epithelial growth factor receptor (HER2), a tumor antigen highly expressed in some colorectal cancer (CRC) patients. This innovative approach forms an efficient immunological synapse, allowing exclusive redirection of MAIT cells to directly eliminate cancer cells. In a collaborative research initiative between HUB Organoids® and BIOMUNEX Pharmaceuticals, the mechanisms by which BiXAb® induce tumor cell killing were confirmed using CRC patient-derived organoids (HUB Organoids® or PDOs) co-cultured with allogenic MAIT cells in an in vitro model. The study results depicted BiXAb® induced MAIT-cell activation in a dose-dependent manner, as detected by FACS and ELISA. Furthermore, BiXAb®-mediated MAIT-cell-specific killing of CRC PDOs was observed in a HER2-dependent manner, determined using an image-based viability assay. In conclusion, the co-culture platform of CRC PDOs-MAIT cells described here effectively captured the activation and targeted killing of MAIT cells mediated by BiXAb®, underscoring the added value of this PDO-based co-culture platform in developing novel immunotherapies. Citation Format: Yasmine Abouleila, Julie Prigent, Mayke Doorn, Claire Germain, Sylvia Boj, Simon Plyte, Carla Verissimo. Mechanistic validation of MAIT cell engagers in HUB patient-derived organoids (PDO) co-culture model [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 6724.

Abstract 6005: Generation of new oncology cell models through long-term acclimation under hypoxic and hyperbaric culture conditions

Abstract Traditional cancer cell lines used as surrogate models in vitro and in vivo have made significant contributions to cancer research and drug discovery. While cell culture and animal studies are critical steps in determining the efficacy, pharmacodynamics, and mechanism of action of novel anti-cancer drugs, it is well-known that cells grown in 2D culture systems have modified growth characteristics and different responses to chemotherapeutic drugs compared to tumors in vivo as a result of differing structural and environmental conditions that fail to replicate in vivo tumor microenvironments. AVATAR bioreactors (Xcell Biosciences, San Francisco, CA) were invented to address these limitations by providing discrete control over oxygen and hyperbaric pressure levels to better mimic the in vivo environment and allow cultured cells to be grown in conditions which mimic the human body. To better serve preclinical research needs, we generated new tumor cell models by long-term acclimating (LTA) existing cancer cells in the AVATAR incubator system using 1% O2 and 2 PSI conditions similar to the conditions expected in the tumor microenvironment (TME). Six different cancer cell lines of diverse origins including pancreas (MIA PaCa-2), breast (BT-474), colon (HCT116), prostate (LNCaP, PC-3) and melanoma (A-375) were adapted for 2-3 months with phenotypic changes recorded. Key marker changes were commonly observed in LTA cells compared to parental lines. For example, in HCT116 colorectal cancer cells, TME culture conditions induced a higher expression level of E-cadherin in both protein and mRNA levels (2.2-fold change), indicating a mesenchymal-epithelial transition of the cell after adaptation. This coincides with the observation that LTA adapted HCT116 cell bodies had a more angular cell morphology as compared to parental cells grown under conventional normoxic culture conditions. In-depth gene-expression analysis using a Nanostring tumor signaling pathway panel identified several common and tissue-specific gene and pathway changes. These included VEGFA, EGF receptor signaling pathway (EGFR, E-Cadherin, GRB7, c-Fos), TGF pathway (IGFBP3, TGFB1, TGFB2, SMAD7), and extracellular matrix signaling (LAMB3, ITGA5, ITGB3, ITGB8, PDGFA, CCND2). Current studies are focusing on drug response testing in 2D and 3D cultures based on above-mentioned gene and signaling pathway changes. Cell proliferation and migration capacity of xenograft LTA cells will be evaluated for the purposes of creating novel murine models for drug screening applications. Citation Format: Yewei Xing, Abriel Czachorowski, James Lim, Scott Wise. Generation of new oncology cell models through long-term acclimation under hypoxic and hyperbaric culture conditions [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 6005.

Evaluation of the modulating effect of epidermal growth factor receptor inhibitor cetuximab in carbon-tetrachloride induce hepatic fibrosis in rats

Liver fibrosis, developed in almost all chronic liver injuries. Epidermal growth factor receptors (EGFR) have been thought to contribute to cirrhosis and liver fibrosis. Therefore, using a rat model of carbon tetrachloride (CCl4)-induced liver fibrogenesis, we investigated the preventive effects of cetuximab, an inhibitor of the EGF receptor (EGFR). Ameliorative effects of cetuximab were examined in rats, brought on by biweekly doses of 50 mg/kg of carbon tetrachloride (CCl4). There were a total of 24 male Long Evans rats split up into four distinct groups such as control, CCl4, control+cetuximab and CCl4+cetuximab. After two weeks of treatment with cetuximab (100 μg/kg), samples of tissue and blood were taken after all the rats had been sacrificed. Plasma samples were examined for the biochemical indicators of inflammation and oxidative stress. Histological staining on liver sections was performed for morphologic pathologies, and related genes expressions analysis were done with RT-PCR in liver tissue. The findings showed that cetuximab could raise the levels of glutathione (GSH), superoxide dismutase (SOD), and catalase (CAT) and considerably lower the levels of aspartate aminotransferase (AST), alanine aminotransferase (ALT), malondialdehyde (MDA), and nitric oxide (NO). Sirius red staining and hematoxylin-eosin (H&E) displayed that cetuximab therapy reduced the inflammatory cells infiltration and enhanced fibrotic lesions. In the meantime, cetuximab therapy also dramatically reduces the expression of genes linked to inflammation in the liver tissue, including NF-кB, iNOS, IL-6, TNF-α, and TGF-β. To sum up, the anti-inflammatory, antifibrotic, and antioxidant properties of cetuximab confer curative efficacy against liver fibrosis.

Crosstalk between TGF-β and EGF receptors via direct phosphorylation.

Aristidis Moustakas discusses work from Ye-Guang Chen and colleagues (https://doi.org/10.1083/jcb.202307138) on a new mechanism by which TGF-β modulates HER2 signaling in mammary epithelia.