A renal clearable uorogenic probe for in vivo detection of cellular senescence

There is a growing need for non-invasive, cheap, and versatile diagnostic methods and the development of low-cost point-of-care assays constitutes a potential solution. Here, we describe the design of a renal clearable uorogenic probe (Cy7Gal) which produces a uorogenic signal readable in the urine. The Cy7Gal probe is applied to the detection of the burden of senescence in several in vivo models. The probe is composed of a dye (Cy7) conjugated with a galactose derivative. Upon administration of the probe in vivo, the up-regulated β-galactosidase enzyme in senescent cells cleaves the O-glycosidic bond in Cy7Gal, releasing the highly emissive Cy7 dye that is renally cleared and measured in unmodied urine by an IVIS imaging system or by a uorimeter. A good correlation between the burden of senescence and emission in urine is observed. Cy7Gal probe is the rst example for the uorogenic in vivo detection of senescence in urine and may serve as a basis for the development of generalized uorogenic diagnostic platforms for the easy diagnosis in the urine of different diseases as well as for monitoring therapeutic treatments without the use of expensive equipment or trained personnel. overexpression of lysosomal β-galactosidase, using the uorogenic probe Cy7Gal. This enzyme hydrolyses in vivo the poorly-emissive Cy7Gal into the highly uorescent dye Cy7, which is renally cleared and detected in urine. We provide evidence of the ability of the probe to monitor the burden of cellular senescence in vivo BALB/cByJ mice bearing breast cancer tumour treated with senescence-inducing chemotherapy and in aged mice models. Cy7Gal probe is the rst diagnostic tool for the in vivo detection of senescence in the urine. Our ndings demonstrate that renal clearable uorogenic probes are a versatile modular tool that opens new opportunities to develop simple diagnoses in urine for a variety of diseases where the abnormal enzymatic activity is a biomarker. We anticipate that this technology can also be applied in the monitoring of therapeutic treatments. Such adaptive detection platform could be applicable in low-resource environments and might democratize access to advanced and sensitive diagnoses.


Introduction
The design and development of new cost-effective and of easy implementation diagnostic tools is an important goal in health. [1][2][3][4] However, diagnosis of many diseases in remote or less developed countries or resource-limited settings is di cult as in many cases it is necessary the use of costly imaging techniques, well-equipped laboratories, and trained personnel. [5][6][7] The design of low-cost easy-to-use diagnostic systems, able to detect target biomarkers from readily accessible bio-uids, constitutes a potential solution. 8 An approach that ful ls these characteristics is the design of probes able to be transformed by the action of certain biomarkers in cells and tissues and have a rapid renal clearance thus allowing detection in the urine. Based on this concept of renal clearable probes, some very few reports describe the use of multiplexed protease-responsive nanoparticles that release small reporter probes into the urine in response to proteolytic cleavage in disease environments. 5,9,10 In such systems, the reporters are nally detected in urine by mass spectrometry or immunoassays. This approach has also recently been elegantly exploited to detect acute kidney injury (AKI) with uorescent and chemiluminescent derivatives equipped with the (2-hydroxypropyl)-β-cyclodextrin (HPβCD) moiety that allowed a renal clearance of the probes. 11,12 A more recent example describes a nanosensor based on ultra-small renally removable nanoparticles able to recognize deregulated protease in cells. In this case, a colorimetric signal is indirectly detected by measuring the ability of excreted nanoparticles in urine to oxidize a chromogenic peroxidase substrate in the presence of hydrogen peroxide. 13 However, some of these systems still rely on the use of complex techniques, expensive analytical assays, or use nanoparticles which could result in undesired accumulation or side effects. 14 In this scenario, the design of probes able to be transformed by the action of certain biomarkers in tissues and have a rapid renal clearance allowing its detection is appealing. In this approach, the dye or uorophore, in an OFF state, is for instance hydrolysed in the presence of a certain deregulated enzyme at the site of the disease, turning ON the uorescence of the dye that is renally cleared allowing its detection in the urine. The rapid optical readout of diseases through a simple urine measurement after injection of the probe in the patient can revolutionize the eld of biosensors and it represents an attractive new diagnostic alternative to other procedures. However, such a simple idea has not been widely developed. Exploring this paradigm, and as a proof of concept, we report herein the preparation of a cyanine-7-based dye (Cy7Gal) for the in vivo detection of cellular senescence in the urine. Cellular senescence is a stress response mechanism characterized by cell cycle/proliferation arrest and complex changes in morphology, chromatin organization, secretome, and overexpression of lysosomal β-galactosidase, also referred to as senescence-associated β-galactosidase (SA-β-Gal). [15][16][17] Cellular senescence is one hallmark of aging and it has been reported that quanti cation of the global burden of senescence across tissues can be informative toward assessing biological aging. 18 Moreover, there is clear evidence that the accumulation of senescent cells is involved in the pathophysiology of many aging related diseases. [19][20][21][22] However, although the quanti cation of senescence in biopsies from different human tissues is an active area of research, there are not simple non-invasive procedures to monitor cellular senescence in vivo. 15,23 The probe we develop here (Cy7Gal) is used to detect SA-β-Gal activity in senescent cells. SA-β-Gal hydrolyses in cells the poorly-emissive Cy7Gal into the highly uorescent dye Cy7. 24 Moreover, Cy7 is quickly renally cleared allowing its detection and measurement in urine (Fig. 1b). We validate the procedure in vivo by monitoring chemotherapy-induced cellular senescence in breast tumour syngeneic model in BALB/cByJ mice and physiological senescence in aged mice.

Results And Discussion
Synthesis, spectroscopic characterization, and mechanism studies The Cy7Gal probe was prepared following a two-step synthetic procedure shown in Fig. 1a. First, 2,3,4,6tetra-O-acetyl-α-D-galactopyranosyl bromide was reacted with 4-hydroxyisophthalaldehyde in anhydrous acetonitrile yielding compound 1. Then, a Knoevenagel condensation between 1 and 2,3,3-trimethyl-1-(4sulfobutyl)indolium (2) yielded the Cy7Gal probe. Besides, the Cy7 uorophore was synthesized by protecting the hydroxyl group of 4-hydroxyisophthalaldehyde with t-butyldimethylsilyl chloride followed by a Knoevenagel condensation with 2 and the subsequent deprotection of the hydroxyl group (Supplementary Information, Scheme S1). Cy7Gal and Cy7 were fully characterized by 1 H-NMR, 13 (Fig. 1c), whereas as a clear contrast, PBS (pH 7) solutions of Cy7Gal are poorly uorescent at the same excitation wavelength (Φ Cy7Gal = 0.0062) (Fig. 1c). Besides, the emission intensity of Cy7 uorophore and Cy7Gal remains unchanged in the 5-10 pH range (Supplementary Information, Figure S1). The hydrolysis of Cy7Gal in PBS (pH 7) solutions in the presence of the β-galactosidase enzyme was studied by HPLC ( Supplementary Information, Figure S2a). The obtained chromatograms show the progressive disappearance of the Cy7Gal peak with the subsequent appearance of Cy7 signal in the presence of the enzyme, whereas Cy7Gal remains stable in PBS in the absence of β-galactosidase.
Moreover, speci city and selectivity of the probe to β-galactosidase was demonstrated after incubation of Cy7Gal with different interfering species such as cations, anions, small peptides and enzymes (Supplementary Information, Figure S2b) for 0.5 h. Of all the species tested, only β-galactosidase induced a marked emission enhancement at ca. 660 nm due to the hydrolysis of Cy7Gal which generates the Cy7 uorophore. Besides, a more marked emission enhancement was observed when Cy7Gal was incubated in the presence of both esterase and β-galactosidase enzymes. This greater enhancement, when compared to that observed when only β-galactosidase was present, is ascribed to the hydrolysis of the acetate moieties in Cy7Gal by esterase and then to the rupture of the O-glycosidic bond by βgalactosidase yielding free Cy7.

Cy7Gal monitors senescence induction in mouse mammary tumour cells in vitro
In order to validate the ability of Cy7Gal to monitor senescence induction in cells, we used the 4T1 mouse mammary tumour cell line, which is considered a murine model of triple negative-breast cancer cells. 25,26 Cells were treated with palbociclib (5 µM) for two weeks. Palbociclib is a CDK4/6 inhibitor which has been reported to induce cell cycle arrest and senescence in 4T1 cells. 27  To further con rm the β-galactosidase-catalysed cleavage of Cy7Gal, we found ( Fig. 2g-2l) that a marked reduction in the emission intensity (ca. 60%) was obtained after pre-incubation of senescent 4T1 cells with D-galactose (a speci c inhibitor of β-galactosidase, 5 mM) for 0.5 h and then treated with Cy7Gal, when compared to senescent cells administered only with the probe. Besides, the response of Cy7Gal probe was demonstrated to be dependent on the increased lysosomal β-galactosidase activity in senescent cells. For this purpose, two siRNAs were used to knock-down the expression of GLB1, 19 the gene that encodes lysosomal β-galactosidase in 4T1 cells. As shown in the Supplementary Information ( Figure S3b showed a marked decrease in the emission in the red channel (ca. 54 %, Supplementary Information, Figure S3d) for cells transfected with hs.Ri.GLB1.13.3 siRNA, when compared to non-transfected cells or cells transfected with scrambled siRNA (Supplementary Information, Figure S3c). Finally, viability assays indicated that the probe was innocuous for both normal and senescent cells (Fig. 2o). Thus, Cy7Gal is an appropriate probe to monitor senescence induction in cell cultures.
Renal clearance of Cy7 uorophore allows cell senescence burden evaluation in vivo in a 4T1 breast cancer model treated with senescence-inducing chemotherapy Once assessed the activation of Cy7Gal in 4T1 senescent cells, the probe was validated in a chemotherapy-induced senescence triple-negative breast cancer mouse model. For tumour generation, 4T1 cells were injected subcutaneously into the left mammary fat pad of young female BALB/cByJ mice. Mice were subsequently treated for 7 days with palbociclib at concentrations of 10 mg/Kg, 50 mg/Kg or 100 mg/Kg by daily oral gavage in order to induce different degrees of senescence burden in the tumours. Tumour volume was measured every two days with a calliper. Figure 3a shows that tumours from mice treated with 10 mg/Kg of palbociclib grew similarly to the untreated ones, while tumours from mice treated with 50 and 100 mg/Kg of palbociclib displayed a reduction in their size. Immunohistochemical staining of the proliferation biomarker Ki67 in autopsy samples revealed that samples from mice treated with 50 or 100 mg/Kg of palbociclib exhibited a signi cantly lower proportion of Ki67 positive nuclei than those from untreated mice or mice treated with the lowest palbociclib dose (Figs. 3b and 3c). Moreover, X-Gal staining in cryosections of mice treated or not with palbociclib revealed the speci c induction of senescence (SA-β-Gal activity) in the tumours but not in organs, such as liver or kidney ( Supplementary Information, Figure S4). This is indicative of an enhancement in cellular senescence in tumours when palbociclib treatment increases. 29 In order to test the ability of probe Cy7Gal for cellular senescence detection in vivo, BALB/cByJ mice bearing 4T1 tumours and treated with palbociclib at different concentrations (i.e. 10 to 50 and 100 mg/Kg) were anesthetized and the Cy7Gal probe was intraperitoneally (i.p.) administered (2.5 µmol). Ex vivo studies demonstrated that the Cy7Gal probe allows to detect cellular senescence in palbociclib treated tumours. In fact, ex vivo IVIS images from mice treated with Cy7Gal revealed a strong uorescence signal in tumours from mice administered with 100 mg/Kg of palbociclib, while the uorescent signal decreases as a function of the reduction in palbociclib dose (Fig. 3f). In addition, no signi cant uorescent signal in the liver, lungs, heart, spleen, or kidney from mice injected with Cy7Gal was found and no auto-uorescence was observed for palbociclib treated mice at 100 mg/Kg dose in any organ (Fig. 3g).
Interestingly, uorescence analysis in vivo using an IVIS® spectrum 15 min after Cy7Gal administration, clearly showed uorescence accumulation in the bladder suggesting a rapid renal clearance of the Cy7 uorophore ( Supplementary Information, Figure S5). Urine was, therefore, collected after mice recovered from anesthesia and analyzed by IVIS. The emission in urine was higher in mice treated with increasing amounts of palbociclib (Fig. 3d). The emission of Cy7 in the urine was also measured in a uorimeter and the amount of the uorophore determined from a calibration curve indicating a correlation between palbociclib doses and µmoles of Cy7 in urine (Fig. 3e).
To further study the renal clearance of the Cy7Gal probe, we synthesized and characterize (Supplementary Information, Scheme 2) an analogous molecule lacking the sulfonic groups (WOS-Cy7Gal) that make the Cy7Gal a more hydrophobic dye disfavouring its accumulation in bladder. 30 In a comparative experiment Cy7Gal or WOS-Cy7Gal were i.p. administered (2.5 µmol) to BALB/cByJ mice bearing 4T1 tumours induced by palbociclib (100 mg/Kg). Both probes gave a strong positive signal in the tumours and, interestingly, we detected signal accumulated in the bladder, but not the kidney of animals injected with the Cy7Gal, but not the WOS-Cy7Gal probe ( Supplementary Information, Figures  S6a and S6b). The concentrations of Cy7 and WOS-Cy7 uorophores were measured in plasma and in the excreted urine. In agreement with a renal clearance of the Cy7 uorophore, we found a detectable concentration in the urine (2.34 µmol) while the plasma concentration was signi cantly lower (0.33 µmol) ( Supplementary Information, Figure S6c). In contrast, concentrations of WOS-Cy7 uorophore were basically undetectable in plasma (0.02 µmol) and urine (0.01 µmol). These results were also corroborated by measuring the average radiance intensity of Cy7 and WOS-Cy7 in urine (Supplementary Information, Figure S6d) and indicate the crucial role of the sulfonic acid moieties in the renal clearance of the Cy7 uorophore.
In vivo cell senescence burden evaluation in naturally aged BALB/cByJ mice Aging is a universal physiological state with a progressive functional decline, which is accompanied by the development of age-related diseases. 31 Senescence has been associated with age-dependent organismal changes, and the progressive increase of senescent cells with time is known to contribute to the functional impairment of different organs. 32 In fact, strong correlations have been reported between aging and certain phenotypes such as mitochondria dysfunction, 33 epigenetic changes, 34 and an increase in cellular senescence. 35 In this scenario, tools to measuring easily the pace of aging are of interest. 36 Regarding this matter, we tested the ability of probe Cy7Gal to evaluate the burden of senescence in healthy old and young individuals. For this purpose, 2 and 14 months old BALB/cJyB mice were i.p. injected with the Cy7Gal probe. IVIS images of the anesthetized mice 15 min post Cy7Gal injection revealed uorescence accumulation in the bladder of 14 months old mice ( Supplementary  Information, Figure S7). Urine collected in an Eppendorf tube after recovery from anesthesia was analyzed by IVIS (Fig. 4a). A stronger uorescent signal (ca. 5.4 fold) in urine from old mice treated with Cy7Gal was observed when compared with the uorescence in the urine of young mice also administered with the probe. The amount in µmol of Cy7 in the urine was also calculated and a signi cantly large amount of Cy7 in urine (8.1 fold) from old animals was found compared with young individuals (Fig. 4b).
Moreover, 2 and 14 months old control mice not treated with Cy7Gal showed negligible uorescence in urine ( Fig. 4a and 4b). A simple mass balance of the amount of Cy7Gal injected and that of Cy7 in urine allows calculating that, on average, 49% of injected Cy7Gal was excreted in urine as Cy7 in old mice whereas for young mice this was only 6.1% (Fig. 4b). These differences are in agreement with a larger burden of cellular senescence in old animals. 37 Then, mice were sacri ced and the bladder, brain and lungs were studied by IVIS imaging (Fig. 4c (i), (ii) and (iii) respectively). Control mice not treated with Cy7Gal showed negligible uorescence in these organs at any age. In contrast, quanti cation of the emission intensity in IVIS images revealed an increase of 5.2 in the bladder of aged mice. We also observed increases of 2.3 and 3-fold for brain and lungs respectively in aged mice when compared to young animals (Fig. 4d (i)

, (ii) and (iii) respectively). This observation is in agreement with increases in
cell senescence incidence in lungs reported with aging. 38, 39 In the brain, SA-β-gal activity is not a speci c marker of neuronal senescence, as many healthy neurons have large lysosomal compartments with increased levels of β-galactosidase. 40 Interestingly, however, the results suggest the capacity of the probe to permeate the blood-brain barrier.
In vivo senescence burden evaluation in a senescence-accelerated mouse model In addition to natural aging, we next decided to test the probe in a strain of accelerated senescence mice (SAM). Inbreeding of AKR/J mice and selection for the early appearance of features such as hair loss, skin coarseness, and short life span, led to the isolation of senescence-prone (P) and senescenceresistant (R) series of mice which were crossed separately to establish the inbred SAMP and SAMR strains. Relative to their genetic background-controls (SAMR1 mice), SAMP8 mice exhibit several traits that are known to occur during aging at earlier physiological ages and, therefore, constitute a suitable model to test aging phenotypes. 41 The SAMP8 model has been widely used in aging research to study immune dysfunction, 42 osteoporosis 43 or brain atrophy. 44 Because the phenotypic age-related differences between SAMP8 and SAMR1 mice begin to be evident after approximately 6 months of age, 45-47 2 and 14 months old SAMR1 and SAMP8 mice were i.p. injected with the Cy7Gal probe. As in the models above, urine was collected after mouse recovery from anesthesia and analyzed by IVIS (Fig. 5a).
The amount of Cy7 in the urine was also determined from a calibration curve (Fig. 5b). Even though urine collection from SAMP8 and SAMR1 was more challenging than in the BALB/cByJ mice models describe above due to the general lower amount of urine collected, the results still clearly show a stronger uorescent signal in the urine (due to a larger amount of Cy7) in old mice treated with the probe when compared with the emission in urine from young mice. Thus, 14-month-old SAMR1 mice excreted approximately 2.0-fold µmols of Cy7 uorophore (after Cy7Gal hydrolysis) compared to young SAMR1 mice, while a 3.1-fold Cy7 amount increase in urine is observed for SAMP8 mice aged 14 months with respect to young animals. Moreover, a larger Cy7 quantity (1.7 fold) in urine is found for 14 months old SAMP8 when compared with 14 months old SAMR1 mice in agreement with the reported large burden of cell senescence in SAMP8 animals.
Cy7Gal is a sensitive and speci c probe for senescence burden evaluation regardless of the murine model Finally, to determine the accuracy of the Cy7Gal probe as a tool for evaluation of senescence we divided mice in those with a low (LS) and high (HS) burden of senescence regardless of the murine models used. Mice in the rst group include 2 months old BALB/cJyB, SAMR1 and SAMP8 mice, and BALB/cByJ mice bearing 4T1 tumours (not treated with palbociclib), whereas the second group includes 14 months old BALB/cJyB, SAMR1 and SAMP8 mice, and BALB/cByJ mice bearing 4T1 tumours treated with 50 and 100 mg/Kg of palbociclib (Fig. 5c). The plot revealed a mean urinary signal increase of ca. 4.8 fold in the HS group relative to LS mice. Besides the rate of true positives and false positives (one-speci city) by receiver operating characteristic (ROC) curve shows that the probe discriminated well HS and LS groups with an area under the curve of 0.95 (Fig. 5d, P < 0.0001).

Conclusions
Precision medicine aims to guide health care decisions toward the most effective treatment for a certain patient to improve care quality. 48 Regarding this, the development of new cost-effective and easy implementation diagnostic tools is an important goal. 49 One attractive approach in this area is to detect biomarkers from readily accessible bio uids using detection systems as simple as possible that are inexpensive and do not require trained personnel. [50][51][52] For instance, tools that can detect the activity of a certain enzyme in vivo with a simple readout can be of importance for the diagnosis of certain diseases. 53 An approach that ful lls these characteristics is the design of uorogenic probes (in an OFF state) able to be transformed by the action of certain biomarkers (i.e an overexpressed enzyme) in cells and tissues to give a nal product (in an ON highly emissive state) that has a rapid renal clearance thus allowing its ready detection in the urine using a simple uorimeter. As an example towards the development of renal clearable uorogenic probes we report herein the case of detection of cellular senescence, based on the detection of overexpression of lysosomal β-galactosidase, using the uorogenic probe Cy7Gal. This enzyme hydrolyses in vivo the poorly-emissive Cy7Gal into the highly uorescent dye Cy7, which is renally cleared and detected in urine. We provide evidence of the ability of the probe to monitor the burden of cellular senescence in vivo BALB/cByJ mice bearing breast cancer tumour treated with senescence-inducing chemotherapy and in aged mice models. Cy7Gal probe is the rst diagnostic tool for the in vivo detection of senescence in the urine. Our ndings demonstrate that renal clearable uorogenic probes are a versatile modular tool that opens new opportunities to develop simple diagnoses in urine for a variety of diseases where the abnormal enzymatic activity is a biomarker. We anticipate that this technology can also be applied in the monitoring of therapeutic treatments. Such adaptive detection platform could be applicable in low-resource environments and might democratize access to advanced and sensitive diagnoses.  CO2 at 37 °C for 2 h; next, cellular nuclei were stained with Hoechst 33342 and then uorescence images were acquired by using a confocal microscope (Leica TCS SP8 AOBS). Representative images from repeated experiments (n = 3) are shown. (g-l) Confocal images of control 4T1 (g-i) and 4T1 cells treated with palbociclib (j-l) in the absence (g,j), in the presence of 20 µM of Cy7Gal probe (h,k), and pre-treated with D-galactose (a speci c inhibitor of β-galactosidase) and then with 20 µM of Cy7Gal probe (i,l). Cells were seeded in a clear glass 6-well plate and, after 24 h, cells were pre-incubated or not with D-galactose for 30 min and treated with Cy7Gal in DMEM (10% FBS, 0.1% DMSO) in 20% O2 and 5% CO2 at 37 °C for 2 h, next, cellular nucleus were stained with Hoechst 33342, nally confocal images were acquired by

Supplementary Files
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