Betacoronaviruses SARS-CoV-2 and HCoV-OC43 infections in IGROV-1 cell line require aryl hydrocarbon receptor.

The emergence of novel betacoronaviruses has posed significant financial and human health burdens, necessitating the development of appropriate tools to combat future outbreaks. In this study, we have characterized a human cell line, IGROV-1, as a robust tool to detect, propagate, and titrate betacoronaviruses SARS-CoV-2 and HCoV-OC43. IGROV-1 cells can be used for serological assays, antiviral drug testing, and isolating SARS-CoV-2 variants from patient samples. Using time-course transcriptomics, we confirmed that IGROV-1 cells exhibit a robust innate immune response upon SARS-CoV-2 infection, recapitulating the response previously observed in primary human nasal epithelial cells. We performed genome-wide CRISPR knockout genetic screens in IGROV-1 cells and identified Aryl hydrocarbon receptor (AHR) as a critical host dependency factor for both SARS-CoV-2 and HCoV-OC43. Using DiMNF, a small molecule inhibitor of AHR, we observed that the drug selectively inhibits HCoV-OC43 infection but not SARS-CoV-2. Transcriptomic analysis in primary normal human bronchial epithelial cells revealed that DiMNF blocks HCoV-OC43 infection via basal activation of innate immune responses. Our findings highlight the potential of IGROV-1 cells as a valuable diagnostic and research tool to combat betacoronavirus diseases.

Therapeutic Potential of 47Sc in Comparison to 177Lu and 90Y: Preclinical Investigations.

Targeted radionuclide therapy with 177Lu- and 90Y-labeled radioconjugates is a clinically-established treatment modality for metastasized cancer. 47Sc is a therapeutic radionuclide that decays with a half-life of 3.35 days and emits medium-energy ß--particles. In this study, 47Sc was investigated, in combination with a DOTA-folate conjugate, and compared to the therapeutic properties of 177Lu-folate and 90Y-folate, respectively. In vitro, 47Sc-folate demonstrated effective reduction of folate receptor-positive ovarian tumor cell viability similar to 177Lu-folate, but 90Y-folate was more potent at equal activities due to the higher energy of emitted ß--particles. Comparable tumor growth inhibition was observed in mice that obtained the same estimated absorbed tumor dose (~21 Gy) when treated with 47Sc-folate (12.5 MBq), 177Lu-folate (10 MBq), and 90Y-folate (5 MBq), respectively. The treatment resulted in increased median survival of 39, 43, and 41 days, respectively, as compared to 26 days in untreated controls. There were no statistically significant differences among the therapeutic effects observed in treated groups. Histological assessment revealed no severe side effects two weeks after application of the radiofolates, even at double the activity used for therapy. Based on the decay properties and our results, 47Sc is likely to be comparable to 177Lu when employed for targeted radionuclide therapy. It may, therefore, have potential for clinical translation and be of particular interest in tandem with 44Sc or 43Sc as a diagnostic match, enabling the realization of radiotheragnostics in future.

Combining Albumin-Binding Properties and Interaction with Pemetrexed to Improve the Tissue Distribution of Radiofolates.

Folic-acid-based radioconjugates have been developed for nuclear imaging of folate receptor (FR)-positive tumors; however, high renal uptake was unfavorable in view of a therapeutic application. Previously, it was shown that pre-injection of pemetrexed (PMX) increased the tumor-to-kidney ratio of radiofolates several-fold. In this study, PMX was combined with the currently best performing radiofolate ([177Lu]cm13), which is outfitted with an albumin-binding entity. Biodistribution studies were carried out in mice bearing KB or IGROV-1 tumor xenografts, both FR-positive tumor types. SPECT/CT was performed with control mice injected with [177Lu]folate only and with mice that received PMX in addition. Control mice showed high uptake of radioactivity in KB and IGROV-1 tumor xenografts, but retention in the kidneys was also high, resulting in tumor-to-kidney ratios of ~0.85 (4 h p.i.) and ~0.60 (24 h p.i.) or ~1.17 (4 h p.i.) and ~1.11 (24 h p.i.) respectively. Pre-injection of PMX improved the tumor-to-kidney ratio to values of ~1.13 (4 h p.i.) and ~0.92 (24 h p.i.) or ~1.79 (4 h p.i.) and ~1.59 (24 h p.i.), respectively, due to reduced uptake in the kidneys. It was found that a second injection of PMX­3 h or 7 h after administration of the radiofolate­improved the tumor-to-kidney ratio further to ~1.03 and ~0.99 or ~1.78 and ~1.62 at 24 h p.i. in KB and IGROV-1 tumor-bearing mice, respectively. SPECT/CT scans readily visualized the tumor xenografts, whereas accumulation of radioactivity in the kidneys was reduced in mice that received PMX. In this study, it was shown that PMX had a positive impact in terms of reducing the kidney uptake of albumin-binding radiofolates; hence, the administration of PMX resulted in ~1.3⁻1.7-fold higher tumor-to-kidney ratios. This is, however, a rather moderate effect in comparison to the previously shown effect of PMX on conventional radiofolates (without albumin binder), which led to 5⁻6-fold increased tumor-to-kidney ratios. An explanation for this result may be the different pharmacokinetic profiles of PMX and long-circulating radiofolates, respectively. Despite the promising potential of this concept, it is believed that a clinical translation would be challenging, particularly when PMX had to be injected more than once.

Folate Receptor-Positive Gynecological Cancer Cells: In Vitro and In Vivo Characterization.

The folate receptor (FR) is expressed in a variety of gynecological cancer types. It has been widely used for tumor targeting with folic acid conjugates of diagnostic and therapeutic probes. The cervical KB tumor cells have evolved as the standard model for preclinical investigations of folate-based (radio) conjugates. In this study, a panel of FR-expressing human cancer cell lines-including cervical (HeLa, KB, KB-V1), ovarian (IGROV-1, SKOV-3, SKOV-3.ip), choriocarcinoma (JAR, BeWo) and endometrial (EFE-184) tumor cells-was investigated in vitro and for their ability to grow as xenografts in mice. FR-expression levels were compared in vitro and in vivo and the cell lines were characterized by determination of the sensitivity towards commonly-used chemotherapeutics and the expression of two additional, relevant tumor markers, HER2 and L1-CAM. It was found that, besides KB cells, its multiresistant KB-V1 subclone as well as the ovarian cancer cell lines, IGROV-1 and SKOV-3.ip, could be used as potentially more relevant preclinical models. They would allow addressing specific questions such as the therapeutic efficacy of FR-targeting agents in tumor (mouse) models of multi-resistance and in mouse models of metastases formation.

Cetuximab directly inhibits P-glycoprotein function in vitro independently of EGFR binding.

PURPOSE: Cancer chemotherapy typically combines anticancer drugs from different mechanisms of action. However, cancer cells could become resistant to chemotherapy via P-gp or other ATP binding cassette proteins. The objective of this study was to evaluate whether cetuximab, monoclonal antibody directed toward epidermal growth factor receptor, could increase intracellular concentration of conventional chemotherapy by interacting with P-gp. METHODS: Two human ovarian carcinoma (IGROV1) and two human embryonary kidney (HEK) cell lines, overexpressing or weakly expressing P-gp, were used. Their EGFR expressions were compared. Cetuximab effect on P-gp functionality was evaluated by measuring doxorubicin (P-gp fluorescent substrate) intracellular accumulation. Cetuximab ability to increase doxorubicin cytotoxicity was evaluated by MTT test. A quaternary structure model of the P-gp-Cetuximab complex was established. RESULTS: Exposure of cetuximab in therapeutic concentrations range with doxorubicin led to significant doxorubicin accumulation and reversion of doxorubicin resistance in P-gp expressing cells lines. Molecular modeling of P-gp-cetuximab interactions showed that cetuximab is able to bind P-gp extracellular part. CONCLUSIONS: Cetuximab increases a P-gp substrate intracellular accumulation in both P-gp expressing cell lines, independently of their EGFR expression. One hypothesis is that cetuximab binding on P-gp could hamper the conformational changes that occur during drugs efflux. Our results offer new possibilities of research on monoclonal antibodies influence in MDR phenomena.