Adenocarcinoma Cell Research Articles

Biogenesis, characterization, and applications of Spirulina selenium nanoparticles

BackgroundNowadays, researchers are attracted to the phyco-synthesis of selenium nanoparticles (SeNPs) for biotechnological and medical applications as they possess many advantages such as safety, nutritional value, and easy biodegradation than gold, copper, and silver nanoparticles. Spirulina platensis is the preferred microalgae for SeNPs synthesis because it contains many compounds that increase their stability making them fit for biomedical treatments.ResultsThe biosynthesized Spirulina platensis selenium nanoparticles (SP-SeNPs) were spherical and crystalline, with a diameter of 65 nm and a net charge of -16.7 mV. Furthermore, they were surrounded by active groups responsible for stability. The DPPH radical scavenging test assessed the antioxidant efficacy of SP-SeNPs and exposed scavenging inhibition of 79.234% at a 100 µM dosage. ABTS and H2O2 radical scavenging assay is dose-dependent recording IC50 of 50.69 and 116.18 µg/ml, respectively. The antibacterial efficacy was investigated against 13 G-negative & G-positive bacteria. The study demonstrated that SP-SeNPs had antibacterial and antibiofilm efficiencies against the tested strains with MBC of 286–333 µg/ml. The highest percentages of biofilm inhibition were recorded for Bacillus subtilis and Klebsiella pneumoniae, with ratios of 78.8 and 69.9%, respectively. The prepared SP-SeNPS efficiently suppressed the tested fungi growth with MIC (350 µg/ml) and MFCs (480–950 µg/ml). Most notably, biogenic SeNPs effectively extended the clot formation period recording 170.4 S for prothrombin time (PT) and 195.6 S for the activated partial thromboplastin time (aPTT). SP-SeNPs reduced the cell viability of breast adenocarcinoma (MCF-7) and ovarian cancer (SKOV-3) cell lines with a percentage of 17.6009% and 14.9484% at a concentration of 100 ug/ml, respectively. Moreover, SP-SeNPs could effectively alleviate the inflammation in RAW 264.7 macrophages with a reduction percentage of 8.82% in Nitric oxide concentration.ConclusionThe investigation findings reveal that SP-SeNPs are a hopeful antimicrobial, anti-tumor, anticoagulant, antioxidant, and anti-inflammatory factor that can be applied in medical cures.

RNF6 inhibits lung adenocarcinoma cell proliferation by promoting cyclin D2 degradation.

The E3 ubiquitin ligase RNF6 has been widely recognized for its role in promoting tumorigenesis in multiple cancers. However, we found it is downregulated in lung adenocarcinoma (LUAD) and the molecular rationale for this discrepancy remains unclear. In the present study, we find that RNF6 but not its ΔRING inactive form inhibits LUAD cell proliferation and migration and sensitizes LUAD to chemotherapy. To understand the molecular mechanism, we utilize affinity purification/tandem mass spectrometry to analyze RNF6-interacting proteins and find that cyclin D2 (CCND2), a key regulator of the G1/S transition in the cell cycle. RNF6 physically binds to CCND2 and mediates its K48-linked polyubiquitination and subsequent degradation. However, ΔRING RNF6 fails to mediate CCND2 for ubiquitination and degradation. Moreover, Thr280 is critically important for CCND2 stability. When Thr280 is mutated, CCND2 becomes more stable and less ubiquitinated by RNF6. Furthermore, RNF6 arrests LUAD cell cycle at the G1 phase by inhibiting the CCND2/pRb signaling pathway, which is consistent with decreased cell proliferation. Lastly, RNF6 curtails the growth of LUAD xenografts in vivo, associated with decreased CCND2 expression. Therefore, RNF6 is a novel E3 ligase of CCND2 and suppresses LUAD cell proliferation. Implications: This study reveals a novel regulation on cell cycle transition in LUAD and suggests the RNF6/CCND2 axis may represent an alternative therapeutic target for the treatment of LUAD.

Combined protein and transcriptomics identifies DCTPP1 as a putative biomarkers for predicting immunotherapy responsiveness in gastric cancer patients.

This study aimed to screen the changes after overexpression of dCTP pyrophosphatase 1 (DCTPP1) in human gastric adenocarcinoma cells (AGS) cells by proteome and transcriptome sequencing. Gene Ontology and Kyoto Encyclopedia of Genes and Genomes enrichment analysis were performed to explore the functional significance of the differentially expressed DCTPP1 in gastric cancer (GC). Cell Counting Kit-8 (CCK-8) assay was used to detect the proliferation of cells. Western blot was used to detect the expression of proteins. A total of 28 genes that were significantly associated with DCTPP1 overexpression and had prognostic value were screened by Cox regression analysis. The results of gene set enrichment analysis showed that the genomes of patients with subtype A exhibited significant enrichment in pathways such as DNA repair, pyrimidine synthesis, and glucose metabolism. The tumor immune dysfunction and exclusion and The Cancer Immunome Atlas databases showed that patients with type A GC were better candidates for immunotherapy than patients with type B GC. Furthermore, the CCK-8 assay indicated significantly enhanced proliferative activity after overexpressing DCTPP1 in AGS cells, corroborating the findings from the bioinformatic analysis. The data suggest a potential association between DCTPP1 expression and both the prognosis of GC patients and the efficacy of immunotherapy. These findings offer valuable insights for the potential optimization of therapeutic strategies in gastric cancer.

A quinoline derivative exerts antineoplastic efficacy against solid tumour by inducing apoptosis and anti-angiogenesis both in vitro and in vivo.

Cancer is a heterogeneous and multicomplex disease with the highest morbidity and mortality rate. The targeting of tumour progression with drugs is a very well-established treatment strategy. Despite these, due to the failure of commonly used drugs in combating cancer, new drugs need to be screened and established for better therapeutic approach. With this rationale, the current investigation was aimed to develop quinoline compound (QC) derivatives as anti-tumour molecules. In this extended study, a series of QC analogues were subjected to anti proliferative assays through cell-based screening and evaluated its mechanism of action through apoptotic and anti-angiogenic assays. The change in cell behaviour was assessed through gene expression analysis using qRT-PCR and immunoblot analysis. Further, in vivo solid tumour model was developed and the anti-tumour potential of QC-4 was verified with gene expression studies. The results suggested that QC-4 exhibited significant cytotoxic effect, particularly against human lung adenocarcinoma cell lines and murine Ehrlich Ascites Carcinoma cells. The QC-4 induced condensation, nuclear damage and changes in membrane integrity resulted in apoptosis and neovascularisation inhibition. The modulation of apoptotic and angiogenic genes such as BAX, BAD, p53 and MMP-2 and 9 further supported the molecular cause of cytotoxicity induced by QC-4. The regression of in vivo solid tumour with extended survivability warranted the in vitro results and the gene expression patterns were additionally supportive. Overall, the QC-4 analogue exhibits the anti-neoplastic with a multi-target approach, reserving its capacity to be developed into a new class of the anticancer molecules.

A Phase 2 Study of Sotigalimab, a CD40 Agonist Antibody, Plus Concurrent Chemoradiation as Neoadjuvant Therapy for Esophageal and Gastroesophageal Junction Cancers.

Neoadjuvant chemoradiation (NCRT) followed by surgical resection represents a standard approach for patients with locally advanced esophageal/GEJ cancers. Sotigalimab is a high affinity CD40 agonist antibody capable of inducing and expanding anti-tumor immune responses by activating dendritic cells, T and B lymphocytes, NK cells, and M1 macrophages. This study examined the safety and efficacy of combining sotigalimab with NCRT in patients with esophageal or GEJ cancers. Patients with resectable (T1-3, Nx) adenocarcinoma(AC) or squamous cell carcinoma(SCC) of the esophagus or GEJ were eligible. T1N0 and cervical tumors were excluded. Study treatment: weekly carboplatin/paclitaxel with concurrent radiation 5040 cGy plus 3-4 doses of sotigalimab prior to Ivor-Lewis esophagectomy. Primary efficacy endpoint was pathologic complete response (path CR) rate. 33 patients were enrolled (AC 76%, SCC 24%; clinical stage III 67%). Ninety percent of patients received all planned doses of sotigalimab. The most common adverse events attributed to sotigalimab were nausea, fever/chills, fatigue, and cytokine release syndrome (CRS); most of these were Grade 1-2. Grade >3 CRS was observed in 3 pts (9%). Twenty-five of the 29 efficacy-evaluable patients underwent an R0 resection (87.9%), with an overall path CR rate of 37.9% (11/29). Post-tumor samples demonstrated increased infiltration and activation of dendritic cells, monocytes, and cytotoxic T cells compared to baseline. Sotigalimab combined with NCRT for esophageal or GEJ cancers was generally well tolerated and achieved path CR rates that compare favorably to historical data and are promising for this treatment strategy. NCT03165994.

Cholesterol Functionalized Nanoparticles Are Effective against Helicobacter pylori, the Gastric Bug: A Proof-of-Concept Study.

Helicobacter pylori chronic infection is the highest risk factor for the development of gastric cancer, being this Gram-negative bacterium classified as carcinogenic. The mounting resistance of H. pylori to antibiotics calls for innovative therapeutic strategies. Here, the proof-of-concept studies that support the development of a "trojan horse" therapeutic strategy based on cholesterol-grafted nanoparticles (Chol-NP) to counteract H. pylori infection are depicted. The bacterium ability to specifically recognize and bind to surface grafted cholesterol is demonstrated by its adhesion to cholesterol(Chol)-functionalized self-assembled monolayers (SAMs) on gold substrates (2D Chol-SAMs) in a concentration dependent manner, with optimal Chol-SAMs prepared with 25% Chol-polyethylene glycol (PEG)-thiol in solution (75% tetra(ethylene glycol)-thiol). These results further show that cholesterol functionalized gold nanoparticles (3D Chol-SAMs, Chol-NP) eradicate H. pylori at a minimum bactericidal concentration of 125 µg mL-1. Chol-NP kill H. pylori through internalization and membrane rupture, as observed by transmission electron microscopy (TEM). Chol-NP are cytocompatible (human gastric adenocarcinoma (AGS) cell line), non-hemolytic and innocuous to bacteria representative of the gut microbiota (Escherichia coli and Lactobacillus acidophilus). This study supports the further development of cholesterol functionalized biomaterials as an advanced and targeted treatment for H. pylori infection.

Cyto- and Genotoxicity of Selected Plant Extracts and Microbial Metabolites with Confirmed Activity Against Phytopathogens of Potato Seed (Solanum tuberosum L.)

The aim of this study was to evaluate the cytotoxicity and genotoxicity of potential biocontrol agents for use against phytopathogens of potato seed (Solanum tuberosum L.). Plant extracts from Allium sativum L., Syzygium aromaticum L. Merr. & Perry, Salvia officinalis L., and Curcuma longa L., as well as metabolites of bacteria Lactiplantibacillus plantarum KB2 LAB 03 and yeast Metschnikowia pulcherrima TK1, were investigated. The chemical characteristics of the plant extracts and the metabolic profiles of the tested microorganisms were evaluated by GC-MS. An insect cell line from Spodoptera frugiperda (Sf-9) and human cervix adenocarcinoma cells (HeLa) were used to evaluate cytotoxicity in the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. The single-cell electrophoresis assay was used to estimate DNA damage. The cytotoxicity and genotoxicity of the microbial metabolites depended on their chemical profiles and pH. The plant extracts induced stronger DNA damage in the Sf-9 cell line than in HeLa cells. The garlic (Allium sativum L.) extract showed the highest cytotoxicity against Sf-9 insect cells (IC50 41.6 mg/mL). The sage (Salvia officinalis L.) extract showed the highest cytotoxicity against HeLa cells (IC50 49.6 mg/mL). This study is the first to investigate not only the potential of these novel biocontrol agents for plant disease control, but also their safety for humans and biodiversity within the context of sustainable agriculture.

Spatially mapping the tumour immune microenvironments of non-small cell lung cancer.

Lung cancer is the leading cause of cancer-related deaths. An enhanced understanding of the immune microenvironments within these tumours may foster more precise and efficient treatment, particularly for immune-targeted therapies. The spatial architectural differences between lung adenocarcinoma (LUAD) and lung squamous cell carcinoma (LUSC) are relatively unexplored. Here, we applied imaging mass cytometry to a balanced cohort of LUAD and LUSC patients, matched for clinical factors such as age, sex, and smoking history, to analyze 204 histopathology images of tumours from 102 individuals with non-small cell lung cancer (NSCLC). By analyzing interactions and broader cellular networks, we interrogate the tumour microenvironment to understand how immune cells are spatially organized in clinically matched adenocarcinoma and squamous cell carcinoma subsets. This spatial analysis revealed distinct patterns of immune cell aggregation, particularly among macrophage populations, that correlated with patient prognosis differentially in adenocarcinoma and squamous cell carcinoma, suggesting potential new strategies for therapeutic intervention. Our findings underscore the importance of analyzing NSCLC histological subtypes separately when investigating the spatial immune landscape, as microenvironmental characteristics and cellular interactions differed by subtype. Recognizing these distinctions is essential for designing precision therapies tailored to each subtype's unique immune architecture, ultimately enhancing patient outcomes.

Inhibition of GPR68 induces ferroptosis and radiosensitivity in diverse cancer cell types

Radioresistance is thought to be a major consequence of tumor milieu acidification resulting from the Warburg effect. Previously, using ogremorphin (OGM), a small molecule inhibitor of GPR68, an extracellular proton sensing receptor, we demonstrated that GPR68 is a key pro-survival pathway in glioblastoma cells. Here, we demonstrate that GPR68 inhibition also induces ferroptosis in lung cell carcinoma (A549) and pancreatic ductal adenocarcinoma (Panc02) cells. Moreover, OGM synergized with ionizing radiation to induce lipid peroxidation, a hallmark of ferroptosis, as well as reduce colony size in 2D and 3D cell culture. GPR68 inhibition is not acutely detrimental but increases intracellular free ferrous iron, which is known to trigger reactive oxygen species (ROS) generation. In summary, GPR68 inhibition induces lipid peroxidation in cancer cells and sensitizes them to ionizing radiation in part through the mobilization of intracellular free ferrous iron. Our results suggest that GPR68 is a key mediator of cancer cell radioresistance activated by acidic tumor microenvironment.

Targeting the hERG1/β1 integrin complex in lipid rafts potentiates statins anti-cancer activity in pancreatic cancer

Plasma membrane macromolecular complexes function as signaling hubs that regulate cell behavior, which is particularly relevant in cancer. Our study provides evidence that the complex formed by the hERG1 potassium channel and the β1 subunit of integrin receptors preferentially localizes in Lipid Rafts (LRs) in Pancreatic Ductal Adenocarcinoma (PDAC) cell lines and primary samples. The complex recruits the p85 subunit of phosphatidyl-inositol-3-kinase (PI3K), activating phosphoinositide metabolism and triggering an intracellular signaling pathway centered on Akt. This pathway ultimately affects cancer cell proliferation through cyclins and p21, and cell migration through the small GTPase Rac-1 and f-actin organization. The hERG1/β1 integrin complex in LRs can be dissociated and the downstream signaling pathway can be inhibited by either disrupting LRs through methyl-beta-cyclodextrin (MβCD) or inhibiting cholesterol synthesis by statins. Treatment with a single chain bispecific antibody—scDb-hERG1-β1—specifically targeting the complex significantly potentiates the effects of both MβCD and statins on intracellular signaling. Consequently, these treatments decrease PDAC cell proliferation and motility in vitro. From a pharmacological perspective, different statins produce anti-neoplastic effects in synergy with scDb-hERG1-β1. Such combination also enhances tumor sensitivity to chemotherapeutic drugs, such as gemcitabine and oxaliplatin. The efficacy of these combination treatments depends on the amount of the hERG1/β1 integrin complex present on the plasma membrane of cancer cells. Finally, the combined treatment with statins and scDb-hERG1-β1 significantly reduces tumor growth and improves survival in vivo, in a preclinical mouse model. These results suggest that the combination of scDb-hERG1-β1 and statins represent a potential novel strategy for treating PDAC patients.

Partial Proliferating Cell Nuclear Antigen Functional Knockout Impairs Cisplatin Resistance and Clonogenic Potential in Lung Adenocarcinoma Cells

Background/Objectives: Lung cancer ranks as the leading cause of cancer-related deaths globally and is highly associated with cisplatin resistance due to both intrinsic and extrinsic mechanisms. Proliferating Cell Nuclear Antigen (PCNA) plays a critical role in molecular processes, such as DNA replication and repair, chromatin structure maintenance, and cell cycle progression. PCNA is known as a molecular marker for proliferation and an excellent inhibition target to shut down highly proliferative cells. One of the mechanisms of cisplatin resistance is the increase in DNA repair, and studies have reported an association between PCNA, lung cancer, and cisplatin treatment. The present study aimed to characterize the absence of PCNA in A549 lung adenocarcinoma cells. Methods: Employing a CRISPR/Cas9 gene-editing approach, we generated a monoclonal cell culture, termed PKO (PCNA knockout). Results: PKO cells exhibited a residual PCNA expression, significantly decreased clonogenic potential and ubiquitylation at K164 residue. IC50 assay suggested that PKO cells could not acquire cisplatin resistance when compared to PX. After cisplatin treatment, PKO cells presented impaired ubiquitylation and did not have increased STAT3 phosphorylation (Tyr705), a previously characterized mechanism of cisplatin resistance. Conclusions: We suggest that PCNA participates in cisplatin resistance in A549, partially by DNA damage tolerance through failure on PCNA monoubiquitylation, and its inhibition may be an approach to circumvent cisplatin resistance.

Supplying LSD1 with FAD in pancreatic cancer: A matter of protein-protein interaction?

Lysine-specific demethylase 1 (LSD1) is a key regulator in cancer epigenetic, and its activity is reliant on flavin adenine dinucleotide (FAD) as a cofactor. In this study, we investigated the correlation between LSD1 and FAD synthase isoform 2 (FADS2) protein levels in pancreatic ductal adenocarcinoma (PDAC) cell lines. We first assessed LSD1 protein and mRNA levels in mutant p53-expressing PANC-1 and MiaPaCa2 cells and p53-null AsPc-1cells, compared to human pancreatic ductal epithelial (HPDE) controls. Our results confirmed elevated LSD1 protein levels in PANC-1 and MiaPaCa2, but not in AsPc-1, despite mRNA overexpression across all cell lines. Similarly, FADS2 levels were significantly upregulated in PANC-1 and MiaPaCa2, but not in AsPc-1, highlighting a possible link between FADS2 expression and p53 gain-of-function mutations. These results prompted us to better investigate the functional relationship between FADS2 and LSD1 by performing in cellulo protein-protein interaction analyses. Our results indicate a direct interaction between LSD1 and FADS2, while no significant interaction was observed between LSD1 and FADS1. These findings reinforce the role of FAD synthesis and its delivery to LSD1 as critical events in cancer progression and shed light on potential implications of FADS2-LSD1 dynamics as targeted therapies in cancer.

CK2 expression gastrointestinal cancer: Exploring clinical characteristics and cancer genetics.

834 Background: The expression of protein kinase CK2 subunits (CSNK2A1, CSNK2A2, CSNK2A3, and CSNK2B) in gastrointestinal cancers has not been fully characterized. In this study, we seek to evaluate how changes in CK2 subunit expression are associated with GI cancer clinical characteristics and genetics. Methods: Data analysis was performed with data from The Cancer Genome Atlas (TCGA). R version 4.2.2 was used for analysis. We provide an analysis of several cancer cohorts looking at CK2 subunit expression, as well as cancer mutations, copy number alterations, and clinical characteristics. Results: From the TCGA datasets containing mRNA expression data, we assessed hepatocellular carcinoma (n=361), colorectal adenocarcinoma (n=339), gastric adenocarcinoma (n=306) and diffuse type gastric adenocarcinoma (n=69), pancreatic adenocarcinoma (n=169), esophageal adenocarcinoma (n=89) and squamous cell carcinoma (n=95), and cholangiocarcinoma (n=36). We explored associations between CK2 subunit expression and GI cancer-specific known driver mutations, of which we report the following significant findings (p < 0.05 for all). Mutations in TP53 were associated with increased expression of CSNK2A1 in hepatocellular carcinoma and stomach adenocarcinoma, increased CSNK2A2 in colorectal adenocarcinoma, and CSNK2B in colorectal adenocarcinoma, pancreatic adenocarcinoma, and stomach adenocarcinoma. Mutations in KRAS were associated with increased expression of CSNK2A1 and CSNK2B in pancreatic adenocarcinoma. Mutations in BRAF were associated with decreased expression of CSNK2A2 and CSNK2B in colorectal adenocarcinoma. We also noted significant changes in CSNK2 subunit expression when looking at mutations and copy number alterations in signaling pathways such as Wnt, NOTCH, PI3K, TGFβ, and more. We also explored associations between CK2 subunit expression and GI cancer clinical characteristics, of which we report the following significant findings (p < 0.05 for all). In colorectal adenocarcinoma, increased CSNK2A2 expression was associated with lymph node invasion and advanced clinical stage. In hepatocellular carcinoma, increased CSNK2A1 and CSNK2A3 expression was associated with greater tumor grade, tumor size, and more advanced clinical staging. Increased CSNK2A2 expression was associated with greater tumor size, more advanced clinical staging, and vascular invasion. Increased CS2NKB expression was associated with increased tumor grade, size, and vascular invasion. In pancreatic adenocarcinoma, increased CSNK2A3 expression was associated with a worse response to the first treatment course. Conclusions: Our results suggest that changes in protein kinase CK2 expression are associated with different cancer clinical characteristics as well as cancer-specific mutations and signaling pathways.

PLCG2, A Regulator of Lung Adenocarcinoma Proliferation and Migration Associated with Immune Infiltration.

Results from the TCGA database showed that phosphatidylinositol-specific phospholipase Cγ2 (PLCG2) expression level in Lung Adenocarcinoma (LUAD) was notably decreased compared to adjacent tissues, so we unveiled its role of LUAD. This study aims to explore the expression and clinical significance of Phosphatidyl-inositol-specific phospholipase Cγ2 (PLCG2) in lung adenocarcinoma (LUAD) cells and its role in cell proliferation and metastasis. Differential PLCG2 mRNA and protein levels between LUAD tissues and adjacent tissues were analyzed from the TCGA database, TIMER, and UALCAN database. Differentially expressed genes were screened for patients in the high and low PLCG2 mRNA expression groups by the R package as well as GSEA. The expression level of PLCG2 in LUAD cells was detected using qRT-PCR and CCK8, clone formation, Transwell, and Western blot assays. PLCG2 was lowly expressed in LUAD and did not significantly correlate with the prognosis of LUAD. PLCG2 expression levels varied significantly in terms of patients' gender, age, T, N, and pathological stage. GO/KEGG enrichment analysis showed that co-expression of PLCG2 was mainly associated with the immune response- regulating cell-surface receptors, and so on. GSEA analysis showed enrichment pathways of PLCG2-related differential gees were primarily associated with the olfactory transduction pathway, ribosome, etc. R software analysis revealed a significant correlation between PLCG2 expression and six types of immune-infiltrat-ing cells, positively correlated with immune checkpoint-related genes and negatively regulated by tumor mutational load. Overexpressing PLCG2 showed reduced LUAD cell proliferation, clone formation, cell migration and invasion, and epithelial-mesenchymal transition-associated proteins, compared with the control group. PLCG2 is lowly expressed in LUAD tissues and is involved in immune infiltration of LUAD, inhibiting LUAD cell proliferation and metastasis.

NRG-GI007: Secondary endpoints of safety assessment and clinical complete response (cCR) of chemoradiation with endoscopically injected oncolytic virus OBP-301 in medically inoperable esophageal cancer.

435 Background: Definitive chemoradiation (CRT) is a standard-of-care for patients (Pts) with medically inoperable esophageal cancer (EC). The NRG/RTOG 0436 study of cisplatin/paclitaxel and RT (+/- cetuximab) as definitive therapy showed that 58% of control arm Pts have locally persistent disease. OBP-301 is a conditionally-restricted, replication-competent adenovirus derived from human adenovirus type 5 that adds a human Telomerase Reverse Transcriptase gene promoter, replicating only in tumor cells to cause lysis.It may cause immunogenic cell death, enhance RT increasing radiosensitization by blocking DNA repair and improve local control. Methods: In NRG-GI007, a phase 1 study (NCT04391049), OBP-301 was added to weekly carboplatin/paclitaxel and RT (50.4 Gy/28 fxs) for medically inoperable EC Pts with pathologically proven adenocarcinoma or squamous cell carcinoma (SCC) of the esophagus or Siewert Type I/II gastroesophageal junction. Pts receive 1-2mL of intratumoral OBP-301 1×10 12 virus particles/ml via endoscopy 3 days prior to and then at days 12 and 26 of CRT. The primary endpoint was protocol-defined dose-limiting toxicity, already reported. Secondary endpoints reported here are treatment-related (definitely or probably related to any protocol treatment) AEs - all and grade 4+ non-hematologic, and cCR in the primary tumor, defined as negative EGD/biopsy 6-8 weeks post-CRT. Per the protocol design, no statistical testing is done for these endpoints. Results: Initially, 6 evaluable (eligible and started protocol treatment) Pts were enrolled from June 2020 to April 2023. As initial dose level was deemed safe, an additional 9 Pts were enrolled from August 2023 to July 2024, totaling 15 evaluable Pts for secondary endpoints. Median age was 74 years, 9 Pts (60%) with ECOG PS 1. 11 Pts (73%) had adenocarcinoma and 4 had SCC; 11 Pts had N+ disease. 14 Pts (93%) received all planned OBP-301 injections and received 50.4 Gy RT. The most common treatment-related grade 3/4 AEs were decreased neutrophil (6 Pts; 40%) and lymphocyte counts (5 Pts; 33%), expected with CRT. No grade ≥3 OBP-301-related non-hematologic AEs were reported. 2 Pts died prior to restaging neitherdefinitely or probably related to OBP-301: 1 Pt developed grade 5 respiratory failure 6 weeks after CRT (suspected RT pneumonitis), while a 2 nd Pt had an unrecognized tumor invasion of the bronchus at baseline, which worsened during week 3 of CRT, resulting in a grade 5 tracheo-esophageal fistula. 2 Pts have not yet reached time for restaging. All 11 Pts who have been restaged had a cCR, for a preliminary cCR rate of 100% (95% CI: 74.1%, 100%). Conclusions: OBP-301 + CRT is safe and the preliminary cCR rate compares favorably to the historical control from NRG/RTOG 0436. Updated safety and cCR data will be presented. A randomized study is being planned. Clinical trial information: NCT04391049 .

Zimberelimab platform study: Safety and efficacy of zimberelimab in combination with futibatinib and chemotherapy in patients with first-line advanced or metastatic esophageal carcinoma.

409 Background: Zimberelimab: AB122 (Zim) platform study is a Phase 1 a/b study with multiple cohorts designed to evaluate the safety and efficacy of Zim, an anti-PD-1 antibody. Futibatinib (Futi) is an FGFR inhibitor that covalently binds FGFR1–4; FGFR inhibitors have been reported to modulate the tumor immune microenvironment, including reducing MDSCs and inhibiting CAFs. These immunomodulatory effects are expected to enhance the efficacy of the anti-PD-1 antibodies. The safety and efficacy of the combination of Zim, Futi, and chemotherapy were evaluated in patients (pts) with first-line advanced or metastatic esophageal cancer (EC) in Japan. Methods: Key eligibility criteria included age ≥ 18 years, histologically confirmed advanced or metastatic EC with measurable lesions by RECIST v1.1 regardless of FGFR and PD-L1 status, previously untreated, adenocarcinoma or squamous cell carcinoma, and ECOG PS 0 or 1. Patients received Zim 360 mg on day 1, Futi 20 mg QD, fluorouracil 800 mg/m2 on days 1–5, and cisplatin 80 mg/m2 on day-1 of a three-week cycle. The primary endpoint was dose-limiting toxicity (DLT). The secondary endpoints included adverse events, antitumor activity, pharmacokinetics, pharmacodynamics, and pharmacogenomics. Results: As of May 10, 2024, 43 pts were enrolled. At the data cutoff date for this analysis (July 15, 2024), treatment was ongoing in 23 pts. Safety and efficacy were evaluated in 43 and 41 pts, respectively. The median age of the pts was 61 years (53–70). An ECOG PS score of 0 was observed in 31 pts. DLTs were evaluated in the first three pts, and none were reported. Common treatment-emergent adverse events (TEAEs)were hyperphosphatemia (81.4%), diarrhea (67.4%), nausea (62.8%), decreased appetite (62.8%), constipation (55.8%), anemia (53.5%), stomatitis (51.2%), neutrophil count decreased (37.2%), alanine aminotransferase increased (34.9%), and palmar-plantar erythrodysaesthesia syndrome (30.2%). Grade ≥ 3 TEAEs occurred in 31 pts (72.1%), and common events (≥ 30%) were anaemia (30.2%) and neutrophil count decreased (30.2%). The objective response rate was 70.7% (95% CI: 54.5–83.9), and the confirmed overall response rate was 58.5% (95% CI: 42.1–73.7). The disease control rate was 92.7% (95% CI: 80.1–98.5). Median duration of response was 5.6 months (95% CI: 4.1–5.6), and median progression-free survival was 4.9 months (95% CI: 3.4–6.7). Conclusions: The combination of Zim, Futi, and chemotherapy has a manageable safety profile without new safety signals and shows promising antitumor activity in pts with advanced or metastatic EC. As more than half of the enrolled pts are still undergoing treatment, these are preliminary data; follow-up is needed to evaluate the viability of this combination appropriately. Clinical trial information: NCT04999761 .

Sensitizing K-Ras G12D mutant colorectal patient-derived organoids via ferroptosis pathway.

211 Background: Colorectal cancer remains the third most fatal form of cancer for males and females combined, with K-Ras mutations present in approximately 40% of all cases. These mutations drive cancer progression and confer resistance to conventional therapies, underscoring the need for novel treatment strategies. Ferroptosis, a regulated non-apoptotic form of cell death characterized as iron-dependent lipid peroxidation, has emerged as a target for cancer therapy. A study published in 2023 found that K-Ras mutant pancreatic ductal adenocarcinoma and non-small cell lung cancer cells lack ferroptosis induced lipid peroxidation and concurrently upregulated ferroptosis suppressing protein (FSP-1). It is unknown if K-Ras mutant colorectal tumors also evade ferroptosis-induced cell death as a method of cancer progression and therapy resistance. This study aims to measure the effects of inhibiting K-Ras G12D, the most common K-Ras mutation found in CRC, on ferroptosis through a patient derived colorectal cancer organoid (PDO) model. We hypothesize that blocking K-Ras G12D using a MRTX-1133, a K-Ras G12D specific inhibitor, can induce ferroptosis and synergistically work with ferroptosis inducers. Methods: As a pilot study, two patient derived organoid lines were established. Surveillance screening and whole exome sequencing was performed to verify K-Ras mutation. CRC9T has wild-type and CRC30T has mutant K-Ras. Additionally, Columbia Pathology Core confirmed tissues used to generate organoids were moderately differentiated colorectal adenocarcinoma. CTG-3D assays were performed to measure IC50 using ferroptosis inducers RSL3, iFSP, and K-Ras inhibitor MRTX-1133. Organoids were cultured in standard gut media for 72 hours, treated, and collected after 72 hours. After treatment, organoids were imaged and stained with BODIPY-C11, a lipid peroxidation fluorescence dye. Flow cytometry was performed, and results were analyzed using FlowJo to show ferroptosis-induced lipid peroxidation in stained organoids. Results: Inhibiting K-Ras G12D and inducing ferroptosis showed a synergistic effect on ferroptosis-induced lipid peroxidation. CRC9T was more sensitive (IC50 RLS3: 2.96 uM, iFSP 3.46 uM) to ferroptosis inducers than CRC30T (IC50: RSL3: 5.68 uM, iFSP: 8.6 uM). Blocking K-Ras in was only efficacious in CRC30T (IC50: MRTX1133: 9.73 uM). Cells from the mutant line showed greatest lipid peroxidation when combining ferroptosis inducers with K-Ras G12D inhibitors in flow cytometry analysis. Conclusions: These findings demonstrate a synergistic effect when blocking K-Ras G12D and inducing ferroptosis and align with previous studies that showed a connection between ferroptosis suppression and oncogenic KRAS expression. We plan to validate these findings in other patient derived organoid model systems and to assess the effects of pan-KRAS inhibition and ferroptosis inducer on non-K-Ras G12D PDOs.

The mechanism of E3 ubiquitin ligase HERC1 regulating ferroptosis in lung adenocarcinoma cells

The mechanism of E3 ubiquitin ligase HERC1 regulating ferroptosis in lung adenocarcinoma cells

Anticancer effect of a single-chain variable fragment against pro-matrix metalloproteinase-7 in colon cancer.

Disrupting the interaction between matrix metalloproteinase-7 (MMP-7) and syndecan-2 (SDC-2) can yield anticancer effects in colon cancer cells. Here, a single-chain variable fragment (scFv) targeting the pro-domain of MMP-7 was generated as a potential candidate anticancer agent. Among the generated scFvs, those designated 1B7 and 1C3 showed the strongest abilities to inhibit the ability of MMP-7 pro-domain to directly interact with SDC-2 in vitro and decrease the cancer activities of human HT29 colon adenocarcinoma cells. Consistently, 1B7 and 1C3 inhibited the cell-surface localization of pro-MMP-7, reduced the gelatinolytic activity of MMP-7, and suppressed the cancer activities of metastatic HCT116 human colon carcinoma cells. Notably, 1B7 inhibited the primary tumor growth and lung metastasis of CT26 mouse colon cancer cells in a mouse model. Compared to 1B7, the 1B7-Fc fusion antibody showed better anti-tumorigenic activity against HCT116 cells in culture and a syngeneic mouse model. Together, these data suggest that 1B7-Fc exerts anticancer effects by interfering with the interaction of MMP-7 and SDC-2 and could be a promising therapeutic antibody for colon cancer.

Effects of targeting CTMP on immunogenicity of MSI-L colon adenocarcinoma cells by metabolism and cell cycle modulation.

246 Background: Regimen of metastatic colon cancer (mCOAD) is moving towards combination of immune checkpoint inhibitors (ICBs) and targeted therapy, however ICBs are rarely effective in MSI-L metastatic colon cancers, limiting options for backline treatments, new targeted therapy is on demand to improve immunogenicity of MSI-L mCOAD. As targeting fatty acid metabolism or cell cycle has shown potential in improving the efficacy of ICBs, our team focused on CTMP, a thioesterase (ACOT) family member, mainly modulates fatty acid β oxidation, meanwhile it binds to phosphorylation sites of AKT thus intervenes cell cycle, currently its role on immunogenicity of COAD is not clear. Methods: Correlation between CTMP and OS in MSI-L colon cancer patients was assessed by KM plotter in GEO database.11 Clinical samples of MSI-L colon cancer, who received a third-line treatment regimen of anti-PD-1 and fruquintinib, were collected then applied in immunofluorescence and western blot analyses to evaluate CTMP and MHC-I expression. In vitro, Lentiviral transfection of targeted genes were stably established,then applied in RNA-seq and metabolic assays. Proteomic analysis and co-IP were applied to explore CTMP-modulating pathway. In vitro, IFNγ was intratumorally injected. Results: KM curve showed CTMP was negatively correlated with OS in MSI-L COAD in GEO database. In clinical samples, lower CTMP expression and higher MHC-I expression were observed in partial response (PR) than progressive disease (PD) group. In vitro, CTMP-OE reduced MHC-I, increased AKT phosphorylation and immune checkpoints (PD-L1, IDO-1), promoted clone formation. Seahorse assay showed increased capacity of glycolysis and oxidative phosphorylation in CTMP-OE than vector. RNA-seq showed CTMP-OE reduced MHC-I augmentation under IFN-γ treatment, meanwhile glucose metabolism genes, ACOT family ,immune checkpoints were also up-regulated, indicating ACOT family related metabolism promotion and immunogenicity suppression of COAD. Proteomic analysis and co-IP assay indicated CTMP interacts REV7, a key mitotic regulatory protein. Enzyme activity assay showed REV7 negatively regulates CDK1. Furthermore, REV7-OE alone is capable to induce MHC I augmentation and G2 arrest, accordingly CTMP-KD enhanced REV7's negative modulation on CDK1,contributing to G2 arrest, that also augmented MHC I . In homologous mice tumor-bearing model, tumor shrinkage was observed in CTMP-KD/ REV7-OE group. CTMP-KD/ REV7-OE increases MHC I expression,CD8+ T cell infiltration under IFN γ intra-tumoral injection. Conclusions: In summary, our studies suggested that CTMP regulates the metabolism of colorectal cancer and intervenes MHC-I molecules expression, and targeting CTMP is potential in suppressing tumor cell cycle and promoting immunogenicity through its modulation on REV7.