Degranulation assay to evaluate NK cell natural and antibody-dependent cell-mediated cytotoxicity against A549 tumor spheroids.

Adoptive natural killer (NK) cell-based immunotherapy is a promising treatment approach in cancer that is showing notable efficacy against hematological malignancies. However, the success of NK cell immunotherapy in patients with solid tumors is limited due to several barriers, which include the immunosuppressive tumor microenvironment (TME), heterogeneity of tumor cells and poor NK cell infiltration into the tumor. Advances in 3D in vitro culture technologies have opened new avenues for the development of more physiological human cancer models that mimic important tumor features which are absent in traditional 2D studies and may be essential for the improvement of immunotherapies against solid tumors. Here, we describe a comprehensive protocol to generate tumor spheroids from the A549 lung carcinoma cell line, then establish co-cultures with NK cells to, ultimately, determine NK cell functional response with a degranulation assay, a surrogate of NK cell cytotoxicity against tumor spheroids. Additionally, we studied degranulation by stimulating NK cell antibody-dependent cell-mediated cytotoxicity (ADCC) with cetuximab, an IgG1 monoclonal antibody used in cancer therapy. Likewise, other monoclonal antibodies or combination treatments could also be studied in this 3D co-culture system, providing very valuable information to define effective combinations of therapeutic agents able to generate NK cells with high cytotoxic potential that could lead to more successful adoptive NK cell-based therapies for the treatment of solid tumors.

Successful cetuximab rechallenge in metastatic colorectal cancer: A case report.

BACKGROUND: Metastatic colorectal cancer (mCRC) treatment has been evolving and increasingly driven by tumor biology and gene expression analysis. Rechallenge with epidermal growth factor receptor (EGFR) inhibitors (anti-EGFR) represents a promising strategy for patients with RAS wild-type (RAS-wt) mCRC and circulating tumor DNA has emerged as a potential selection strategy. Herein, we report the case of a RAS-wt mCRC patient who had a successful response to cetuximab rechallenge. CASE SUMMARY: Our patient was diagnosed with stage IV RAS-wt, microsatellite-stable rectosigmoid junction adenocarcinoma. He was started on first-line treatment with FOLFIRI and cetuximab and achieved partial response, allowing for a left hepatectomy (R0), followed by post-operative chemotherapy and an anterior resection; progression-free survival (PFS) of 16 months was obtained. Due to hepatic and nodal relapse, second-line treatment with FOLFOX and bevacizumab was started with partial response; metastasectomy was performed (R0), achieving a PFS of 11 months. After a 15 months anti-EGFR-free interval, FOLFIRI and cetuximab were reintroduced upon disease progression, again with partial response and a PFS of 16 months. Following extensive hepatic relapse, cetuximab was reintroduced and a marked clinical and analytical improvement was seen, after only one cycle. RAS-wt status was confirmed on circulating tumor DNA. The patient's overall survival exceeded 5 years. CONCLUSION: Our case provides real-world data to support cetuximab rechallenge in later lines of RAS-wt mCRC treatment.

Prognostic factors and risk-stratification model of recurrent or metastatic head and neck squamous cell carcinoma treated with cetuximab containing regimen.

BACKGROUND: In recent years, the addition of cetuximab to chemotherapy has improved treatment outcomes for patients with recurrent/metastatic head and neck squamous cell carcinoma (R/M HNSCC). In this study, we present the real-world survival data of R/M HNSCC patients who received cetuximab-containing regimens from thirteen medical centers in Taiwan, as well as a three-level risk stratification model for this patient population. METHODS: This study enrolled R/M HNSCC patients from thirteen medical centers in Taiwan who received cetuximab-containing regimens from January 1, 2017 to June 6, 2022. The cases were divided into a training cohort and a validation cohort based on the start of treatment. Overall survival (OS) was evaluated in both cohorts and exploratory analysis was performed to identify associated adverse clinical and laboratory factors. The results of the exploratory analysis were used to construct a three-level risk stratification prediction model for OS. RESULTS: A total of 1434 patients with R/M HNSCC were enrolled in this study and received cetuximab-containing regimens. The overall population had a median OS of 8.57 months (95% CI: 8.07 - 9.08). Multivariate analysis of the training cohort identified poor ECOG performance status, heavy alcohol consumption, and prior adjuvant CCRT or lack of prior RT as adverse prognostic factors. Comparison of laboratory data between patients with OS≦6 and OS > 6 also revealed unfavorable factors, including increased white blood cell count, decreased hemoglobin level, increased platelet count, increased absolute neutrophil count, decreased absolute lymphocyte count, and increased neutrophil-to-lymphocyte ratio. Using forward prediction, a three-level risk stratification prediction model was constructed using the variables of ECOG performance status, alcohol consumption, skin metastasis, modality of radiation therapy, hemoglobin level, and neutrophil-to-lymphocyte ratio. The median OS in the low-risk, intermediate-risk, and high-risk groups were 12.02 months (95% CI 10.44-13.61), 7.5 months (95% CI 7.33-8.17), and 4.01 months (95% CI 3.94-4.08), respectively, with a log-rank test p-value < 0.001. CONCLUSION: This study presents a three-level risk stratification model with strong prediction ability for OS in R/M HNSCC patients who received cetuximab-containing regimens. The results are based on real-world data and may provide valuable information for clinicians in treatment planning and future drug development.

A Case of Liver Injury Immediately After Initiation of Triple Therapy in a Patient With BRAF V600E Mutation-Positive Colorectal Cancer.

The combination therapy of binimetinib, encorafenib, and cetuximab ("triplet regimen") was approved in Japan in November 2020 for the second-line treatment of BRAF V600E mutation-positive colorectal cancer. In this study, we encountered a patient who developed a liver injury requiring drug withdrawal on day eight of the triplet regimen administration. The protocol for the international phase III study did not specify blood and biochemical tests on day eight. Hepatic failure occurred in 45.9% (222/484) of the patients within three months, but no specific timing of onset was reported. Since it is important to disseminate novel adverse events to the public, we report the results of this study based on a literature review.

EGFR-Targeted and NIR-Triggered Lipid-Polymer Hybrid Nanoparticles for Chemo-Photothermal Colorectal Tumor Therapy.

Background: Epidermal growth factor receptor (EGFR) is a major target for the treatment of colorectal cancer. Thus, anti-EGFR antibody conjugated lipid-polymer hybrid nanoparticles can offer a potential means of enhancing the efficacy of chemotherapeutics in EGFR overexpressing cancers. In addition, the combination of chemotherapy and photothermal therapy is a promising strategy for cancer treatment. Hence, it is highly desirable to develop a safe and effective delivery system for colorectal tumor therapy. Methods: In this study, EGFR-targeted and NIR-triggered lipid-polymer hybrid nanoparticles (abbreviated as Cet-Iri-NPs) were prepared with copolymer PPG-PEG, lipids DSPE-PEG-Mal and lecithin as carriers, CPT-11 as an anticancer chemotherapeutic agent, indocyanine green (ICG) as a photothermal agent, and cetuximab as a surface-targeting ligand. Results: In vitro analyses revealed that Cet-Iri-NPs were spherical with size of 99.88 nm, charge of 29.17 mV, drug entrapment efficiency of 51.72%, and antibody conjugation efficiency of 41.70%. Meanwhile, Cet-Iri-NPs exhibited a remarkable photothermal effect, and pH/NIR-triggered faster release of CPT-11 with near infrared (NIR) laser irradiation, which induced enhanced cytotoxicity against SW480 cells. Furthermore, the promoted tumor-growth suppression effect of Cet-Iri-NPs on SW480 tumor xenograft nude mice was achieved under NIR laser irradiation. Conclusion: These results indicate that the well-defined Cet-Iri-NPs are a promising platform for targeted colorectal cancer treatment with chemo-photothermal therapy.

Study protocol for Near-infrared molecular imaging for lung cancer detection and treatment during mini-invasive surgery (phase II Trial) - (the RECOGNISE study).

INTRODUCTION: To date, radical surgery remains the best curative option in patients with early-stage lung cancer. In patients with small lung lesions, video-assisted thoracic surgery (VATS) should be increasingly chosen as a fundamental alternative to thoracotomy as it is associated with less postoperative pain and better quality of life. This scenario necessarily increases the need for thoracic surgeons to implement new localization techniques. The conventional near-infrared (NIR) indocyanine green (ICG) method demonstrated a significant limitation in deep cancer recognition, principally due to its intrinsic low-depth tissue penetration. Similarly, the lymph-node sentinel approach conducted by the ICG method was demonstrated to be inefficient, mainly due to the non-specificity of the tracker and the irregular pathway of pulmonary lymph node drainage. Our study aims to evaluate the effectiveness of Cetuximab- IRDye800CW in marking lung nodules and mediastinal lymph nodes. METHODS AND ANALYSIS: This study is defined as an open-label, single-arm, single-stage phase II trial evaluating the effectiveness of Cetuximab-IRDye800CW in detecting tumors and lymph-node metastases in patients with lung cancer who are undergoing video-assisted thoracic surgery (VATS). Cetuximab is a monoclonal antibody that binds, inhibits, and degrade the EGFR. The IRDye® 800CW, an indocyanine-type NIR fluorophore, demonstrated enhanced tissue penetration compared to other NIR dyes. The combination with the clinical approved monoclonal antibody anti-epidermal growth factor EGFR Cetuximab (Cetuximab-IRDye800) has shown promising results as a specific tracker in different cancer types (i.e., brain, pancreas, head, and neck). The study's primary outcome is focused on the proportion of patients with lung nodules detected during surgery using an NIR camera. The secondary outcomes include a broad spectrum of items, including the proportion of patients with detection of unexpected cancer localization during surgery by NIR camera and the proportion of patients with negative surgical margins, the evaluation of the time spawns between the insertion of the NIR camera and the visualization of the nodule and the possible morbidity of the drug assessed during and after the drug infusion. ETHICS AND DISSEMINATION: This trial has been approved by the Ethical Committee of Azienda Ospedaliera Universitaria Città della Salute e della Scienza di Torino (Torino, Italy) and by the Italian Medicines Agency (AIFA). Findings will be written as methodology papers for conference presentations and published in peer-reviewed journals. The Azienda Ospedaliera Universitaria Città della Salute e della Scienza di Torino, the University of Torino, and the AIRC Public Engagement Divisions will help identify how best to publicize the findings.Trial registration EudraCT 202,100,645,430. CLINICALTRIALS: gov NCT06101394 (October 23, 2023).

Comprehensive Analysis of Cetuximab Critical Quality Attributes: Impact of Handling on Antigen-Antibody Binding.

BACKGROUND/OBJECTIVES: Cetuximab, formulated in Erbitux® (5 mg/mL), is a therapeutic monoclonal antibody (mAb) widely used in several cancer treatments. Currently, there is insufficient knowledge about the behavior of cetuximab with regard to the risk associated with its routine handling or unintentional mishandling in hospitals. Forced degradation studies can simulate these conditions and provide insights into the biophysical and biochemical properties of mAbs. METHODS: In this study, we conducted a deep physicochemical and functional characterization of the critical quality attributes of cetuximab in control samples and under controlled degraded conditions, including freeze-thaw cycles, heat, agitation, and light exposure. To achieve this purpose, we used a set of proper analytical techniques, including CD, IT-FS, DLS, SE/UHPLC-UV, UHPLC-MS/MS, and ELISA, to check functionality based on antigen-antibody binding. RESULTS: The results revealed that light exposure was the stress stimuli with the greatest impact on the drug product, leading to the formation of non-natural oligomers, fragmentation, and oxidation of methionine residues. Additionally, cetuximab (Erbitux®, 5 mg/mL) showed a tendency to aggregate when submitted to 60 °C for 1 h. In terms of functionality, cetuximab (Erbitux®, 5 mg/mL) samples were found to be affected when subjected to freeze-thaw cycles, 60 °C (1 h), and when exposed to light (daylight with room temperature excursion and accelerated light exposure). CONCLUSIONS: Thus, we suggest that Erbitux® (5 mg/mL) should be shielded from these environmental conditions, as they compromise both the safety and efficacy of the drug product.

Natural killer cells: a future star for immunotherapy of head and neck squamous cell carcinoma.

The interplay between immune components and the epithelium plays a crucial role in the development and progression of head and neck squamous cell carcinoma (HNSCC). Natural killer (NK) cells, one of the main tumor-killing immune cell populations, have received increasing attention in HNSCC immunotherapy. In this review, we explore the mechanism underlying the interplay between NK cells and HNSCC. A series of immune evasion strategies utilized by cancer cells restrict HNSCC infiltration of NK cells. Overcoming these limitations can fully exploit the antineoplastic potential of NK cells. We also investigated the tumor-killing efficacy of NK cell-based immunotherapies, immunotherapeutic strategies, and new results from clinical trials. Notably, cetuximab, the most essential component of NK cell-based immunotherapy, inhibits the epidermal growth factor receptor (EGFR) signaling pathway and activates the immune system in conjunction with NK cells, inducing innate effector functions and improving patient prognosis. In addition, we compiled information on other areas for the improvement of patient prognosis using anti-EGFR receptor-based monoclonal antibody drugs and the underlying mechanisms and prognoses of new immunotherapeutic strategies for the treatment of HNSCC.

PA-MSHA exerts potent activity against cetuximab-resistant colorectal cancer through the miR-7-5p/Akt3/Wnt-ß-catenin pathway.

Background: The prognosis and survival of individuals with cetuximab-resistant colorectal cancer (CRC) remain severely impacted by therapy for this disease. The study investigated the underlying mechanisms of Pseudomonas aeruginosa-mannose sensitive hemagglutinin (PA-MSHA), a type of therapeutic biological product approved in China, for cetuximab-resistant CRC. Methods: Cell proliferation, apoptosis, migration and invasion were detected by cell counting kit-8 (CCK-8) assay, flow cytometry, wound healing assay and transwell assay. Massively parallel sequencing of cetuximab-resistant CRC cells with PA-MSHA treatment was used to screen the differential expression profile of miRNAs. The directly target gene of miR-7-5p was revealed by dual luciferase assay. Apoptosis and invasion related proteins were detected by Western blot. Results: PA-MSHA could successfully stop the migrating and invading of cetuximab-resistant CRC cells while also inducing apoptosis. Tumor-bearing experiments in nude mice showed that PA-MSHA slowed tumor growth and lengthened mouse life. The sequencing data showed that miR-7-5p was considerably upregulated after PA-MSHA treatment. As anticipated, miR-7-5p overexpression improved PA-MSHA's anticancer properties both in vitro and in vivo. The target gene of miR-7-5p was confirmed to be Akt3 by dual luciferase assay, and Akt3 silencing undid the inhibition of PA-MSHA efficacy caused by miR-7-5p downregulation. Additionally, PA-MSHA therapy significantly reduced the activation of Wnt-ß-catenin pathway, and Akt3 expression was positively linked with several important Wnt-ß-catenin pathway genes, including Wnt and CTNNB1. Finally, we discovered that patients with CRC who had developed cetuximab resistance or disease progression had remarkably decreased serum miR-7-5p levels. Conclusions: PA-MSHA controlled the miR-7-5p/Akt3/Wnt-ß-catenin pathway to provide substantial efficacy against cetuximab-resistant CRC.

Honokiol enhances the sensitivity of cetuximab in KRASG13D mutant colorectal cancer through destroying SNX3-retromer complex.

Rationale : the proto-oncogene KRAS is frequently mutated in colorectal cancer (CRC), leading to inherent resistance against monoclonal antibodies targeting the epidermal growth factor receptor (EGFR), such as cetuximab. Therefore, addressing the primary resistance and expanding the indications for target therapy have become critical challenges. Methods : the screening of a natural product library against KRAS mutant CRC cells was conducted, leading to the discovery of a small molecule compound that sensitive to the KRASG13D mutation site. The anti-tumor activity of this small molecule compound in combination with cetuximab was evaluated using the KRASG13D mutant CRC models both in vivo and in vitro. This evaluation includes an examination of its effects on cell proliferation, viability, apoptosis, cell cycle progression, and tumor growth. Furthermore, RNA sequencing, western blot analysis, immunofluorescence, real-time quantitative PCR, and pull-down assays were employed to explore the molecular mechanisms underlying the synergistic anti-tumor effect of this small molecule compound in combination with cetuximab. Results : our study screened 882 compounds in KRAS mutant CRC cells and identified honokiol, a small molecule compound that exhibits specific sensitivity to KRASG13D mutant CRC cells. Furthermore, we revealed that the synergistic augmentation of cetuximab's sensitivity in vivo and in vitro models of KRASG13D mutant CRC in combination with honokiol. Mechanistically, honokiol suppresses SNX3-retromer mediated trafficking, thereby impeding lysosomal proteolytic capacity and inhibiting autophagy and macropinocytosis fluxes. Moreover, honokiol inhibits the conversion of RAS GDP to RAS GTP, heightening the susceptibility of KRASG13D CRC mutant cells to cetuximab. Conclusions : honokiol enhances the sensitivity of cetuximab by destroying SNX3 retromer in KRASG13D mutant CRC preclinical model. These findings present a promising strategy for expanding the indications of target therapy in KRAS mutant colorectal cancer patients.

Risk Factor Analysis for Anti-epidermal Growth Factor Receptor Monoclonal Antibody-induced Problematic Skin Toxicities in Patients With Liver Metastatic Colorectal Cancer.

BACKGROUND/AIM: We previously reported that patients with metastatic colorectal cancer (mCRC) and baseline liver metastasis are at a higher risk of developing grade ≥2 overall skin toxicities when treated with anti-epidermal growth factor receptor (EGFR) monoclonal antibody. This study aimed to identify additional factors associated with skin toxicities induced by anti-EGFR treatment in patients with liver metastatic CRC. PATIENTS AND METHODS: Patients with liver metastatic CRC who initially received anti-EGFR monoclonal antibody-containing treatment (n=77) were retrospectively assessed. The primary endpoint was to identify the factor(s) responsible for the development of grade ≥2 overall skin toxicities. Additionally, factors for grade ≥2 rash and paronychia were evaluated. RESULTS: The incidence of grade ≥2 overall skin symptoms, rash, and paronychia was 62.3%, 31.2%, and 28.6%, respectively. Multivariate Cox proportional hazard regression analyses revealed that age <65 years and anemia were independent baseline risk factors for grade ≥2 overall skin toxicities (adjusted hazard ratio 2.09, 95% confidence interval=1.10-3.97, p=0.02 for age; 2.36, 1.20-4.61, p=0.01 for anemia). In contrast, combination prophylaxis using systemic minocycline and corticosteroid ointment was a preventive factor (0.47, 0.25-0.88, p=0.02). Males and age <65 years were baseline risk factors for grade ≥2 rash, and combination prophylaxis was identified as a preventive factor. No factors were identified for paronychia. CONCLUSION: Age <65 years and anemia were identified as independent baseline risk factors. Additionally, combination prophylaxis was found to be a preventive factor against anti-EGFR monoclonal antibody-induced grade ≥2 overall skin toxicities in patients with liver metastatic CRC.

In silico evaluation of the role of Fab glycosylation in cetuximab antibody dynamics.

Introduction: N-glycosylation is a post-translational modification that is highly important for the development of monoclonal antibodies (mAbs), as it regulates their biological activity, particularly in terms of immune effector functions. While typically added at the Fc level, approximately 15-25% of circulating antibodies exhibit glycosylation in the Fab domains as well. To the best of our knowledge, cetuximab (Erbitux®) is the only therapeutic antibody presenting Fab glycosylation approved world-wide targeting the epidermal growth factor receptor for the treatment of metastatic-colorectal and head and neck cancers. Additionally, it can trigger antibody-dependent cell cytotoxicity (ADCC), a response that typically is influenced by N-glycosylation at Fc level. However, the role of Fab glycosylation in cetuximab remains poorly understood. Hence, this study aims to investigate the structural role of Fab glycosylation on the conformational behavior of cetuximab. Methods: The study was performed in silico via accelerated molecular dynamics simulations. The commercial cetuximab was compared to its form without Fab glycosylation and structural descriptors were evaluated to establish conformational differences. Results: The results clearly show a correlation between the Fab glycosylation and structural descriptors that may modulate the conformational freedom of the antibody, potentially affecting Fc effector functions, and suggesting a negative role of Fab glycosylation on the interaction with FcγRIIIa. Conclusion: Fab glycosylation of cetuximab is the most critical challenge for biosimilar development, but the differences highlighted in this work with respect to its aglycosylated form can improve the knowledge and represent also a great opportunity to develop novel strategies of biotherapeutics.

BRAF + EGFR +/- MEK inhibitors after immune checkpoint inhibitors in BRAF V600E mutated and deficient mismatch repair or microsatellite instability high metastatic colorectal cancer.

BACKGROUND: Immune checkpoint inhibitors (ICIs) are the guideline endorsed first choice for patients with deficient mismatch repair or microsatellite instability high (dMMR/MSI-H) mCRC, however a significant proportion experience primary or secondary resistance. BRAF V600E mutated (BRAFm) and dMMR/MSI-H mCRC can be treated with BRAF + EGFR inhibitors but specific data on the efficacy after progression to ICIs are missing. METHODS: We collected consecutive patients with BRAFm dMMR/MSI-H mCRC treated from 2017 to 2024 with a combination of BRAFi+EGFRi+/-MEKi, after disease progression on ICIs. A control cohort of BRAFm pMMR/MSS mCRC patients treated with encorafenib+cetuximab+/-binimetinib from 2nd line was used. RESULTS: dMMR/MSI-H (n = 50) BRAFm mCRC patients were more often > 70-year-old, with right-sided primary tumors, without liver but more lymphnode metastases than pMMR/MSS (n = 170). They were treated more frequently beyond 2nd line and 45 % were primary progressors to ICIs. Lower ORR (18 % versus 32 %, p = 0.09) and DCR (60 % versus 73 %, p = 0.11) was seen without reaching significance in dMMR/MSI-H as compared to pMMR/MSS patients. After a median follow-up of 14.04 months, no differences in PFS (median 5.13 versus 4.50 months, HR 0.83, 95 %CI: 0.57-1.20, p = 0.31) and OS (median 10.75 versus 9.11 months, HR 0.89, 95 %CI: 0.59-1.32, p = 0.55) were observed. CONCLUSIONS: Our results show that BRAFm dMMR/MSI-H mCRC patients benefit from BRAFi+EGFRi+/-MEKi after progression under ICIs. Despite lower ORR and DCR, the outcome is not different from that observed in pMMR/MSS BRAFm CRC and is in line with the results of the BEACON registration trial.

A two-stage maintenance trial of cetuximab-based treatment in RAS and BRAF wild-type unresectable metastatic colorectal cancer: a retrospective real-world study.

Background: To investigate the effectiveness and safety of maintenance regimens based on cetuximab, we conducted a real-world, single-arm, retrospective study at a single center. Methods: In Fujian Medical University Union Hospital, patients with unresectable metastatic colorectal cancer (mCRC) who received cetuximab-based maintenance therapy between December 2020 and December 2021 were included. All patients had RAS and BRAF wild-type. The maintenance regimen consisted of 6-12 cycles of cetuximab plus irinotecan (Phase 1) and cetuximab (Phase 2). Patients could receive reintroduction therapy in case of disease progression during Phase 2. Progression-free survival (PFS), overall survival (OS), and safety data were collected. Results: According to the inclusion and exclusion criteria of the study, a total of 108 subjects who received maintenance therapy were included- 51 experienced disease progression during Phase 1, with PFS (1) of 7.3 months. Among the 52 patients who entered Phase 2, 17 were still in this phase at the end of follow-up, with PFS (2) of 10.1 months. In Phase 2, 35 patients experienced disease progression, of whom 24 received reintroduction therapy, with PFS (3) of 6.7 months. The overall PFS (total) during the maintenance period was 11.9 months, and the OS was 39.2 months. Grade III or higher adverse events were 4.6% during Phase 1 and 0% during Phase 2. Conclusion: Innovative cetuximab-based maintenance therapy showed a trend toward improving the prognosis of mCRC patients with RAS and BRAF wild-type, while the toxic side effects of maintenance therapy were manageable. Clinical trial registration: https://www.chictr.org.cn, identifier ChiCTR2000040940.

Analysis of translesion polymerases in colorectal cancer cells following cetuximab treatment: A network perspective.

INTRODUCTION: Adaptive mutagenesis observed in colorectal cancer (CRC) cells upon exposure to EGFR inhibitors contributes to the development of resistance and recurrence. Multiple investigations have indicated a parallel between cancer cells and bacteria in terms of exhibiting adaptive mutagenesis. This phenomenon entails a transient and coordinated escalation of error-prone translesion synthesis polymerases (TLS polymerases), resulting in mutagenesis of a magnitude sufficient to drive the selection of resistant phenotypes. METHODS: In this study, we conducted a comprehensive pan-transcriptome analysis of the regulatory framework within CRC cells, with the objective of identifying potential transcriptome modules encompassing certain translesion polymerases and the associated transcription factors (TFs) that govern them. Our sampling strategy involved the collection of transcriptomic data from tumors treated with cetuximab, an EGFR inhibitor, untreated CRC tumors, and colorectal-derived cell lines, resulting in a diverse dataset. Subsequently, we identified co-regulated modules using weighted correlation network analysis with a minKMEtostay threshold set at 0.5 to minimize false-positive module identifications and mapped the modules to STRING annotations. Furthermore, we explored the putative TFs influencing these modules using KBoost, a kernel PCA regression model. RESULTS: Our analysis did not reveal a distinct transcriptional profile specific to cetuximab treatment. Moreover, we elucidated co-expression modules housing genes, for example, POLK, POLI, POLQ, REV1, POLN, and POLM. Specifically, POLK, POLI, and POLQ were assigned to the "blue" module, which also encompassed critical DNA damage response enzymes, for example. BRCA1, BRCA2, MSH6, and MSH2. To delineate the transcriptional control of this module, we investigated associated TFs, highlighting the roles of prominent cancer-associated TFs, such as CENPA, HNF1A, and E2F7. CONCLUSION: We found that translesion polymerases are co-regulated with DNA mismatch repair and cell cycle-associated factors. We did not, however, identified any networks specific to cetuximab treatment indicating that the response to EGFR inhibitors relates to a general stress response mechanism.

Anti-EGFR immunoliposomes for cabazitaxel delivery: From formulation development to in vivo evaluation in prostate cancer xenograft model.

Liposomes functionalized with monoclonal antibodies offer targeted therapy for cancer, boasting advantages like sustained drug release, enhanced stability, passive accumulation in tumors, and interaction with overexpressed receptors on cancer cells. This study aimed to develop and characterize anti-EGFR immunoliposomes loaded with cabazitaxel and assess their properties against prostate cancer in vitro and in vivo. Using a Box-Behnken design, a formulation with soy phosphatidylcholine, 10% cholesterol, and a 1:20 drug-lipid ratio yielded nanometric particle size, low polydispersity and high drug encapsulation. Immunoliposomes were conjugated with cetuximab through DSPE-PEG-Maleimide lipid anchor. Characterization confirmed intact antibody structure and interaction with EGFR receptor following conjugation. Cabazitaxel was dispersed within the liposomes in the amorphous state, confirmed by solid-state analyses. In vitro release studies showed slower cabazitaxel release from immunoliposomes. Immunoliposomes had enhanced cabazitaxel cytotoxicity in EGFR-overexpressing DU145 cells without affecting non-tumor L929 cells. Cetuximab played an important role to improve cellular uptake in a time-dependent fashion in EGFR-overexpressing prostate cancer cells. In vivo, immunoliposomes led to significant tumor regression, improved survival, and reduced weight loss in xenograft mice. While cabazitaxel induced leukopenia, consistent with clinical findings, histological analysis revealed no evident toxicity. In conclusion, the immunoliposomes displayed suitable physicochemical properties for cabazitaxel delivery, exhibited cytotoxicity against EGFR-expressing prostate cancer cells, with high cell uptake, and induced significant tumor regression in vivo, with manageable systemic toxicity.

Targeted delivery of FAK siRNA by engineered exosomes to reverse cetuximab resistance via activating paraptosis in colon cancer.

BACKGROUND: Cetuximab is extensively used in the treatment of metastatic colorectal cancer (mCRC). However, resistance poses a significant challenge to successful therapy. Recently, paraptosis, a non-classical programmed cell death, has garnered increased attention for its potential application value in antitumor treatments. We aimed to identify the essential pathways and signaling molecules involved in paraptosis inhibition and select them as therapeutic targets in cetuximab resistance. Additionally, engineered exosome technology is used as a drug delivery system with both targeted and effector properties. RESULTS: By comparing the differential expression of paraptosis-related genes between drug-resistant colon cancer cells and sensitive cells, it was observed that the paraptosis level induced by cetuximab was significantly downregulated in drug-resistant cells. Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis identified the focal adhesion kinase (FAK) signaling pathway as a key pathway involved in the suppression of paraptosis. The biological function of FAK in cetuximab-resistant cells was investigated through cell morphology observation, CCK-8 assay, colony formation assay, RT-qPCR, Western Blot, and loss-of-function experiments. The results showed that the FAK signaling pathway was significantly upregulated in cetuximab-resistant colon cancer cells, and siRNA interference targeting FAK could notably inhibit cell proliferation while upregulating the paraptosis level. Based on this, engineered colon cancer cells targeted and FAK siRNA loaded exosomes (CT-Exo-siFAK1) were constructed. In vitro experiments, CT-Exo-siFAK1 could effectively activate paraptosis and inhibit the proliferation of drug-resistant colon cancer cells. In vivo experiments also confirmed that CT-Exo-siFAK1 significantly suppressed tumor growth and metastasis while upregulating the paraptosis level. CONCLUSION: This study suggests that FAK signaling pathway-mediated inhibition of paraptosis levels is crucial in the sensitivity of cetuximab targeted therapy in colon cancer, and the use of engineered exosomes to deliver FAK siRNA may be an effective strategy to reverse cetuximab resistance.

Alpha-Gal Syndrome: An Underrated Serious Disease and a Potential Future Challenge.

Over the past decades, red meat allergy, also known as mammalian meat allergy, which manifests differently from classic food allergies, has been reported in different countries and regions, including China. The allergen of this disease is not a protein but an oligosaccharide: galactose-α-1,3-galactose, i.e., alpha-gal or α-gal. Therefore, this clinical syndrome is also called α-gal syndrome (AGS). It clinically manifests as delayed anaphylaxis, i.e., patients generally develop allergic symptoms 2-6 h after ingesting red meat. This clinical manifestation is believed to be related to sensitization to α-gal after tick bites. Sensitized individuals may also develop anaphylaxis after ingesting food and medicine or being exposed to medical equipment containing α-gal, such as cetuximab and gelatin. Here, the literature on AGS is reviewed for a better understanding of its pathogenesis, clinical diagnosis, and treatment.