Immunophenotyping Reveals No Significant Perturbation to PBMC Subsets When Co-cultured With Colorectal Adenocarcinoma Caco-2 Cells Exposed to X-Rays

Borsci, Giuseppina; Barbieri, Sofia; Guardamagna, Isabella; Lonati, Leonardo; Ottolenghi, A.; Ivaldi, Giovanni Battista; Liotta, M.; Fatis, Paola Tabarelli de; Baiocco, G.; Savio, Monica

doi:10.3389/fimmu.2020.01077

Cited by 8 publications

(6 citation statements)

References 24 publications

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“…Results on Caco-2 cell survival, complemented by cytological staining ( Figure 1 ), fluorescence microscopy images ( Figure 7 ) and quantification of cell death ( Figure 5 ), further add to previous findings ( 10 , 11 ) that led to the description of this cell line as “radioresistant”: in particular, in our previous works, Caco-2 viability measured with the MTT assay was found to remain as high as in the sham condition for cells irradiated with 10 Gy and followed in time up to 72 h from the exposure. The percentage of dead cells measured with the Trypan Blue assay was found to increase in a dose-dependent manner, starting from a basal condition at ~10% and reaching a maximum at around 20% at 24 h and 48 h. Results presented in this work indicate that such behavior, that can be described as “radioresistant” in terms of short-term effects, does not translate into a persistent clonogenic potential: already at 2 Gy, only ~30% of cells are able to form colonies at ~2 weeks, this survival probability decreases to ~10% at 5 Gy and few or no colonies are observed at 10 Gy.…”

Section: Discussionsupporting

confidence: 84%

“…This suggests further investigations to identify and measure some of the unknowns in Caco-2 response to radiation. Recent works with this cell line have focused on its response to different doses of X-rays from a conventional radiotherapy accelerator, with Caco-2 cells alone or co-cultured with peripheral blood mononuclear cells (PBMCs) from healthy donors ( 10 , 11 ). Doses up to 10 Gy have shown not to alter significantly Caco-2 viability (MTT assay) in a timeframe of 72 h from the irradiation.…”

Section: Introductionmentioning

confidence: 99%

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An Integrated Analysis of the Response of Colorectal Adenocarcinoma Caco-2 Cells to X-Ray Exposure

et al. 2021

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Colorectal cancer is among the three top cancer types for incidence and the second in terms of mortality, usually managed with surgery, chemotherapy and radiotherapy. In particular, radiotherapeutic concepts are crucial for the management of advanced rectal cancer, but patients’ survival remains poor, despite advances in treatment modalities. The use of well-characterized in vitro cell culture systems offers an important preclinical strategy to study mechanisms at the basis of cell response to therapeutic agents, including ionizing radiation, possibly leading to a better understanding of the in vivo response to the treatment. In this context, we present an integrated analysis of results obtained in an extensive measurement campaign of radiation effects on Caco-2 cells, derived from human colorectal adenocarcinoma. Cells were exposed to X-rays with doses up to 10 Gy from a radiotherapy accelerator. We measured a variety of endpoints at different post-irradiation times: clonogenic survival after ~ 2 weeks; cell cycle distribution, cell death, frequency of micronucleated cells and atypical mitoses, activation of matrix metalloproteases (MMPs) and of different proteins involved in DNA damage response and cell cycle regulation at earlier time points, up to 48 h post-exposure. Combined techniques of flow cytometry, immunofluorescence microscopy, gelatin zymography and western blotting were used. For selected endpoints, we also addressed the impact of the irradiation protocol, comparing results obtained when cells are plated before irradiation or first-irradiated and then re-plated. Caco-2 resistance to radiation, previously assessed up to 72 h post exposure in terms of cell viability, does not translate into a high clonogenic survival. Survival is not affected by the irradiation protocol, while endpoints measured on a shorter time frame are. Radiation mainly induces a G2-phase arrest, confirmed by associated molecular markers. The activation of death pathways is dose- and time-dependent, and correlates with a dose-dependent inhibition of MMPs. Genomic aberrations are also found to be dose-dependent. The phosphorylated forms of several proteins involved in cell cycle regulation increase following exposure; the key regulator FoxM1 appears to be downregulated, also leading to inhibition of MMP-2. A unified molecular model of the chain of events initiated by radiation is proposed to interpret all experimental results.

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Section: Discussionsupporting

confidence: 84%

Section: Introductionmentioning

confidence: 99%

An Integrated Analysis of the Response of Colorectal Adenocarcinoma Caco-2 Cells to X-Ray Exposure

et al. 2021

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“…The Caco-2/PBMC co-culture system ( Fig. 1 ) was developed to investigate the anti-inflammatory effects of faecal fermentation supernatants collected at time 24 h of fermentation using the test substrates, after an inflammatory stimulus represented by lipopolysaccharide (LPS) ( Borsci et al., 2020 ; Kämpfer et al., 2017 ). The transwell inserts containing the Caco-2 cells at a concentration of 1 × 10 5 cells/cm 2 were transferred to the plate containing PBMCs incubated overnight at a concentration of 2 × 10 6 cells/mL.…”

Section: Methodsmentioning

confidence: 99%

In vitro faecal fermentation of Tritordeum breads and its effect on the human gut health

Arora,

Gaudioso,

Solovyev

et al. 2024

Current Research in Microbial Sciences

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“…The synthesized materials were exposed to peripheral mononuclear cells (PBMC), and the cell viability was analyzed using the Methyl thiazolyl tetrazolium bromide (MTT) assay 21 . A healthy volunteer's whole blood was obtained in this typical experiment, and the buffy coat was meticulously extracted using the density gradient ficoll‐paque method.…”

Section: Experimental Sectionsmentioning

confidence: 99%

Influence of pH in the synthesis of calcium phosphate based nanostructures with enhanced bioactivity and pro‐angiogenic properties

2022

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In synthetic fabrication, the process parameters decide the growth nucleation, phase translation, and the evolution of morphological facets of nanostructured materials. This work demonstrates the formation of different crystallographic phases of calcium phosphate by the influence of pH from acidic to alkaline conditions and also investigated their bone regeneration, protein adsorption, and pro‐angiogenic properties. Present results illustrate that the alteration of pH is the crucial factor for the synthesis of calcium phosphate (CP) phases. The structural analysis reveals the monetite (CaHPO4) phase with a triclinic crystal system for pH 5, dual‐phase of monetite combined with hydroxyapatite at the neutral pH 7, and pure phase of hydroxyapatite (Ca10[PO4]6OH2) with hexagonal structure at pH 10. Microscopic analysis portrays the cubic and rod‐like morphologies by changing the pH values. FTIR and RAMAN spectroscopic analyses confirm the stretching, bending, and vibrational modes of dominant phosphate groups of different CP phases. Further, the biocompatibility of the prepared CP phases was examined by hemolysis assay, which showed less than 2% of lysis and enhanced cell viability. Moreover, the bioactivity study revealed rapid mineralization and a higher protein adsorption rate for the monetite CP phase (M‐CP). Subsequently, the chick embryo angiogenesis assay elucidated 33% higher neovascularization for M‐CP compared with the other two CP phases. The fabricated M‐CP nanostructure constitutes a promising candidate for biomedical applications.

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Immunophenotyping Reveals No Significant Perturbation to PBMC Subsets When Co-cultured With Colorectal Adenocarcinoma Caco-2 Cells Exposed to X-Rays

Cited by 8 publications

References 24 publications

An Integrated Analysis of the Response of Colorectal Adenocarcinoma Caco-2 Cells to X-Ray Exposure

An Integrated Analysis of the Response of Colorectal Adenocarcinoma Caco-2 Cells to X-Ray Exposure

In vitro faecal fermentation of Tritordeum breads and its effect on the human gut health

Influence of pH in the synthesis of calcium phosphate based nanostructures with enhanced bioactivity and pro‐angiogenic properties

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