Evaluation of cell metabolic adaptation in wound and tumour by Fluorescence Lifetime Imaging Microscopy

Abstract

Acidic pH occurs in acute wounds progressing to healing as consequence of a cell metabolic adaptation in response to injury-induced tissue hypoperfusion. In tumours, high metabolic rate leads to acidosis affecting cancer progression. Acidic pH affects activities of remodelling cells in vitro. the pH measurement predicts healing in pathological wounds and success of surgical treatment of burns and chronic ulcers. However, current methods are limited to skin surface or based on detection of fluorescence intensity of specific sensitive probes that suffer of microenvironment factors. Herein, we ascertained relevance in vivo of cell metabolic adaptation in skin repair by interfering with anaerobic glycolysis. Moreover, a custom-designed skin imaging chamber, 2-Photon microscopy (2PM), fluorescence lifetime imaging (FLIM) and data mapping analyses were used to correlate maps of glycolytic activity in vivo as measurement of nADH intrinsic lifetime with areas of hypoxia and acidification in models of skin injury and cancer. The method was challenged by measuring the NADH profile by interfering with anaerobic glycolysis and oxidative phosphorylation in the mitochondrial respiratory chain. Therefore, intravital NADH FLIM represents a tool for investigating cell metabolic adaptation occurring in wounds, as well as the relationship between cell metabolism and cancer. In normal skin, dermis has an extracellular neutral-alkaline pH 1,2. After tissue damage, cells involved in tissue repair undergo a metabolic adaptation resulting from low oxygen tension towards a less energy-efficient process of anaerobic metabolism that leads to microenvironment acidosis 2,3. In vitro evidence suggests a beneficial effect of acidic pH in several processes implicated in wound healing, which involve cell adhesion 4,5 , migration and proliferation 6. In a previous report 7 , we described an unprecedented role of tissue acidification in setting PTX3, a key component of the humoral arm of innate immunity, in a tissue remodelling and repair mode. In wounds, reduced oxygen tension 8 and factors downstream of cell anaerobic glycolysis, such as lactate 9,10 , effectively stimulate immune and vascular endothelial cells to release factors that support angiogenesis. Subsequent neovascularization allows restoration of nutrient delivery and oxygen, and cells use oxidative metabolism for their longer-term functions contributing to restore the wound pH to values near to neutral 2,3. While an acidic pH occurs in the inflammatory phases of acute wounds that progress on healing, chronic and highly infected wounds are characterized by abundant recruitment of neutrophils and a non-acidic pH 11,12. Chronic non-healing wounds may occur secondarily to a high alkaline pH 11,13 , and studies report a relationship between wound pH and chronic wound healing 14-16. The effect of acidic pH in the wound bed has a clinical functional relevance on the healing of chronic wounds 11. In fact, a prolonged chemical acidification of the wound bed increases the healing rate in chron...

FLIM as a Promising Tool for Cancer Diagnosis and Treatment Monitoring

“…Reproduced with permission from Ref. [ 53 ]. Copyright 2020, Springer Nature; a-iv Fluorescence lifetime maps of ex vivo human oral biopsy by ms-FLIM.…”

“…As shown in Fig. 3 a-iii, a higher NADH/NAD + ratio in tumor cells with a lower ratio in monocyte/macrophage cells can be applied for ascertaining tumor fronts, revealing that the fraction of anaerobic glycolysis in the tumor is larger than that in the surrounding monocytes or macrophages [ 53 ]. Besides, this NADH/NAD + ratio is relevant to free/bound NAD(P)H ratio based on previous researches, and the free/bound NAD(P)H ratio has been applied to melanoma progression detection [ 54 ].…”

Acid-Induced Inflammatory Cytokines in Osteoblasts: A Guided Path to Osteolysis in Bone Metastasis

“…In this case, bone inflammation that is originated and maintained by tumor-associated acidosis concurs to exacerbate OB-mediated osteolysis. This is not a completely novel concept as it has already been demonstrated in a nonmalignant disease that the inflammatory response can reduce local pH to 5.5 and that the acidic pH is itself damaging to the surrounding tissues also by stimulating new inflammatory reactions and prolonging the healing process (Morone et al, 2020). Indeed, at this point, it is to be acknowledged that acidosis does not only occur in tumors but also in several additional disease conditions, including inflammation, bone fractures (Dong et al, 2013), and intervertebral disc degeneration (Urban, 2002).…”

Automated Extraction of Skin Wound Healing Biomarkers From In Vivo Label‐Free Multiphoton Microscopy Using Convolutional Neural Networks

“…The CNNs trained in this study demonstrate a reliable, automated method to segment MPM images in a fraction of the time it takes expert users, thus providing a means for MPM to deliver rapid quantitative readouts of tissue structure and function through a non‐invasive optical biopsy. Additionally, the features and weights learned by these networks can be applied to train subsequent CNNs to quickly adapt to new features or other sources of contrast like NADH fluorescence lifetime that have been used to monitor metabolic changes in many skin wound applications [25,44‐46].…”