Researchers here report on the development of a method to enable real-time visualization of the current degree of cellular senescence present in living tissues, using an improved version of existing florescence techniques. If accurate enough, this could replace the current standard approach of biopsy and staining of the sample for analysis. Note that the paper isn’t open access; you’ll have to obtain a copy from the usual underground sources. A practical method of assessing cellular senescence burden that works in live animals will be a big step forward for the field, as it should reduce the cost of many of the activities involved in the production of senolytic therapies, treatments capable of destroying senescent cells and thus reversing their contribution to the aging process. Consider the ability to accurately track the presence of senescent cells in the same animal from moment to moment across a lifetime and through varied senolytic dosages and treatments, for example, information that at present is challenging and costly to obtain.
The main purpose of cellular senescence is to prevent the proliferation of damaged or stressed cells and to trigger tissue repair. However, upon persistent damage or during aging, the dynamic process of tissue repair becomes inefficient and senescent cells tend to accumulate. This accumulation in tissues is believed to impair tissue functions and accelerate aging. It has been demonstrated that genetic ablation of senescent cells ameliorates a variety of aging-associated diseases, reverts long-term degenerative processes, and extends longevity. Inspired by these findings, strategies to prevent, replace, or remove senescent cells have become of interest. For instance, there is an increasing interest in the development of senolytic molecules able to induce apoptosis preferentially in senescent cells.
A related key issue in this field is the design of probes to accurately detect senescent cells in aged or damaged tissues. However, one of the major obstacles limiting progress in this research area is the lack of real-time methods to selectively track senescence in in vivo systems. Detection of senescent cells usually relies on the detection of senescence-associated βGal (SAβGal), and several fluorescent or chromogenic probes have been reported for the visualization of this enzymatic activity. However, these first-generation probes are usually unsuitable for in vivo imaging as they rely on chromogenic changes or on the use of classical one-photon fluorescence excitation. As an alternative, recent stimulating studies developing two-photon fluorescent probes for the visualization of βGal activity have been described. However, some of the reported probes are synthesized by using tedious multistep protocols. Another common drawback is the fact that probes are tested in cultured cells or in animal models that were not directly related to senescence.
In view of the aspects mentioned above, we report herein a novel molecular probe for the two-photon fluorogenic in vivo detection of senescence. The probe (AHGa) is based on a naphthalimide fluorophore as a signaling unit containing an L-histidine methyl ester linker and an acetylated galactose attached to one of the aromatic nitrogen atoms of the L-histidine through a hydrolyzable N-glycosidic bond. Probe AHGa is transformed into AH in senescent cells resulting in an enhanced fluorescent emission intensity. Targeting of senescent cells in vitro with AHGa was validated with the SKMEL-103 cancer cell line treated with the chemotherapeutic palbociclib to induce senescence. A remarkable fluorescence emission enhancement (ca. 10-fold) in the presence of AHGa for palbociclib-treated SK-MEL-103 (senescent) cells was observed when compared with control SK-MEL-103 cells, due to the formation of AH.
The ability of tracking senescence of probe AHGa was also studied in vivo by employing mice bearing subcutaneous tumor xenografts generated with SK-MEL-103 melanoma cells and treated with palbociclib. Tumors in palbociclib-untreated mice showed negligible fluorescence emission both in the absence or in the presence of AHGa, whereas tumors in mice treated with palbociclib and intravenously injected with AHGa showed a clear fluorescent signal. A marked emission enhancement (ca. 15-fold) in tumors treated with palbociclib compared to nontreated tumors was observed. AH fluorescence was only found in senescent tumors but not in other organs. The combination of selectivity, sensitivity, and straightforward synthesis make AHGa an efficient OFF-ON two-photon probe for the in vivo signaling of senescence.