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Context-dependent responses of Drosophila intestinal stem cells to intracellular reactive oxygen species

Reactive oxygen species (ROS) contribute to cellular redox environment and serve as signaling molecules. Excessive ROS can lead to oxidative stress that are involved in a broad spectrum of physiological and pathological conditions. Stem cells have unique ROS regulation while cancer cells frequently show a constitutive oxidative stress that is associated with the invasive phenotype. Antioxidants have been proposed to forestall tumor progression while targeted oxidants have been used to destroy tumor cells. However, the delicate beneficial range of ROS levels for stem cells and tumor cells under distinct contexts remains elusive. Here, we used Drosophila midgut intestinal stem cell (ISCs) as an in vivo model system to tackle this question. The ROS levels of ISCs remained low in comparison to that of differentiated cells and increased with ageing, which was accompanied by elevated proliferation of ISCs in aged Drosophila. Neither upregulation nor downregulation of ROS levels significantly affected ISCs, implicating an intrinsic homeostatic range of ROS in ISCs. Interestingly, we observed similar moderately elevated ROS levels in both tumor-like ISCs induced by Notch (N) depletion and extracellular matrix (ECM)-deprived ISCs induced by β-integrin (mys) depletion. Elevated ROS levels further promoted the proliferation of tumor-like ISCs while reduced ROS levels suppressed the hyperproliferation phenotype; on the other hand, further increased ROS facilitated the survival of ECM-deprived ISCs while reduced ROS exacerbated the loss of ECM-deprived ISCs. However, N- and mys-depleted ISCs, which resembled metastatic tumor cells, harbored even higher ROS levels and were subjected to more severe cell loss, which could be partially prevented by ectopic supply of antioxidant enzymes, implicating a delicate pro-surviving and proliferating range of ROS levels for ISCs. Taken together, our results revealed stem cells can differentially respond to distinct ROS levels under various conditions and suggested that the antioxidant-based intervention of stem cells and tumors should be formulated with caution according to the specific situations.


Introduction
Reactive oxygen species (ROS) are proposed to play a key role in regulating stem cell homeostasis. A low intracellular concentration of reactive oxygen species (ROS) is emerging as a critical condition for stemness and pluripotency in several self-renewing tissues [1][2][3][4][5][6]. A moderate increase in ROS can promote cell proliferation and differentiation [7][8][9], whereas excessive amounts of ROS can cause oxidative damage to lipids, proteins and DNA [10]. However, the delicate beneficial range of ROS levels for stem cells to survive and proliferate remains elusive.
Elevated oxidative status has been found in cancer cells compared with their normal counterparts, which contribute to carcinogenesis [11][12][13]. Animal studies using either gene knockout or transgenic overexpression approaches demonstrated a potential role for antioxidant molecules as tumor suppressors [14][15][16]. If the levels of ROS reaches a certain threshold, ROS may exert a cytotoxic effect [17]. Accordingly, antioxidant supplement could actually be beneficial for tumor cells and stimulate tumor growth [18]. However, little is known about the impact of distinct endogenous ROS levels on tumor cells. Despite the advances in clinical and preclinical trials of cancer therapy, tumor metastasis is still a key target for any anti-cancer strategies and the leading cause of death of cancer patients. Tumor metastasis is a complicated progress including EMT, migration, invasion of the tumor cells and angiogenesis around the tumor lesion. It is worth noting that redox alterations in different states of cancer progression are also very complicated. Recently, the involvement of ROS signaling in tumor metastasis was highlighted [19]. The increased ROS stress in cancer cells has a pivotal role in the acquisition of epithelial-mesenchymal transition and metastasis [20,21]. However, ROS levels in different stages of cancer progression remains largely unknown. Understanding the redox alteration precisely may help in the battle against cancer.
The adult Drosophila midgut is advantageous for studies of stem cell behavior, tissue homeostasis, aging, and cancer [22][23][24][25] as it has similarities in genetic regulation and cellular composition with mammalian digestive systems [26,27]. The Drosophila midgut is maintained by multipotent intestine stem cells (ISCs) that give rise to enteroblasts (EBs), which in turn differentiate into either enterocytes (ECs) or enteroendocrine cells (EEs) [28,29]. Establishment and maintenance of the Drosophila gut epithelium require strict control of ISC proliferation and differentiation. Disruption of this cellular homeostasis can cause abnormal gut functionalities, such as tumor growth [30,31]. Overall, these features provide a unique opportunity to combine the power of Drosophila genetics and the simplicity of this adult homeostatic tissue to study the role of ROS under physiological and pathological conditions. Notch (N) is essential for lineage differentiation and disruption of N in ISCs results in accumulation of ISC-like cells, which eventually develop into tumor-like masses [28,32]. Based on this, we generated ISC tumor model by suppressing Notch signaling [25]. By using in vivo imaging, we observed tumor formation, wherein intracellular ROS levels in ISCs were moderately increased. Meanwhile, ectopic providing oxidant scavenging enzymes could limit tumor growth in this model, suggesting that intracellular redox balance of ISCs influence the rate of stem cell proliferation.
Genes relevant to EMT and metastasis include integrin [20], which is the main cellular adhesion receptor implicated in nearly every step of cancer progression from primary tumor development to metastasis. Altered integrin expression is frequently detected in tumors [33]. In the Drosophila intestine, βPS integrin subunits (encoded by the gene myospheroid, mys) are critical for both maintenance and proliferation of ISCs Pronounced gstD:GFP signal in ISCs in comparison to neighboring differentiated EC cells. Intestines from flies carrying the gstD:: GFP reporter and esg-Gal4, UAS-RFP (ISC/EB marker; red). RFP (C ′ ) and GFP (C ′′ ) staining are shown separately. Note colocalization of RFP and GFP expression (white arrowheads point to the colocalization), indicating that CncC activity is present in ISCs. Scale bars, 20 μm. (D) Elevated ROS levels in ISCs of older fruit flies. The fluorescence intensity of the DHE channel was calculated using Metamorph software. Relative DHE fluorescence in ISCs was normalized to DHE fluorescence in adjacent ECs. Quantifications of relative DHE fluorescence in intestines of 7-and 21day-old flies are shown (average and SEM; Student's t-test and Mann-Whitney test. *, P < 0.05; **, p < 0.01; ***, p < 0.001; ****, P < 0.0001 here and for all figures). (E) Increased proliferative capacity of ISCs in older fruit flies. Amounts of pH3 + cells in intestines of 7-and 21-day-old flies are shown (average and SEM; Student's t-test and Mann-Whitney test). (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.) (Lin et al.). We showed that mys-depleted ISCs had the similarly moderate elevation of ROS levels as N-depleted ISCs, which was accompanied by ISCs loss. Furthermore, we depleted both N and mys to mimic ECM-deprived tumor model. Strikingly, tumor cells that surviving without their natural ECM niches, exhibited the extraordinarily high ROS levels, and antioxidant treatments increased the surviving rate. Taken together, our results implicate that ROS plays a key role in the regulation of ISC, and demonstrated that there are distinct ROS levels under different cellular states and differential responses of stem cells or tumor cells to antioxidant treatments in different contexts.

TARGET system and temperature shifting
The TARGET system was used for temporal and spatial control of transgene expression in adult flies. Flies were reared at 18 • C. Progeny were allowed to emerge and then shifted to new vials with fresh food 1-2 days after eclosion and kept in the restrictive temperature of 29 • C incubators before dissection unless specified.

In vivo detection of ROS via DHE and MitoSOX TM
ROS levels were detected in live tissue as described (Owusu-Ansah et al., 2008). Guts were dissected and handled throughout in PBS medium. After incubation in 30 mM DHE (Invitrogen)/MitoSOX TM for 7/ 10 min in the dark at 25 • C/37 • C, guts were washed three times with PBS medium and mounted. Images were captured immediately via confocal microscopy (488/543 nm excitation, 510-540/550-610 nm detection, fixed laser power, gain and offset settings). Z-series spanning the intestinal epithelium of posterior midgut were collected and single confocal sections were used to measure signal intensities with the region measure in Metamorph software. ISCs were identified by their esgGFP expression, their size, and their basal location within the intestinal epithelium. Where possible, only isolated esgGFP + cells were measured.

Statistics
Statistical analyses were carried out with GraphPad Prism 6 software using either the unpaired Student's t-test or One-way ANOVA, and all quantitative data are shown as mean ± SEM. No statistical method was used to predetermine sample size. No samples were excluded. The experiments were not randomized. The investigators were not blinded to allocation during the experiments and outcome assessment.

The ROS levels of ISCs remained low and increase with ageing
The binary system escargot-Gal4 [esg-Gal4]-driven GFP was used to mark ISCs and EBs with GFP [34] (Fig. 1A-A'; Fig. 1B). To quantify the ROS levels in ISCs, we measured the intracellular ROS levels in vivo using dihydroethidium (DHE), a redox-sensitive dye that exhibits increased fluorescence intensity when oxidized (Owusu-Ansh et al., 2008). Cells in the ISCs had significantly lower ROS than their neighboring differentiated ECs ( Fig. 1A'', D). Similarly, we found lower superoxide levels in the ISCs using MitoSox red staining ( Fig. 1B'), a unique fluorogenic dye that allows for selective detection of superoxide production in the mitochondria [35]. Nrf2/CncC is a major regulator of the cellular redox state (Hochmuth et al., 2011). To test whether the low levels of ROS in the ISCs is regulated by the Nrf2/CncC, we assessed GFP reporter based on the upstream regulatory sequences of the CncC target gene gstD1 (gstD1::GFP) in the posterior midgut. We observed that gstD1::GFP was . CncC thus appeared to be required in ISCs to maintain low levels of ROS. Previous studies observed age-associated elevated ROS levels and loss of epithelial homeostasis in wild-type flies, where ISCs progressively over-proliferated in aging animals [36]. We examined ISC proliferation rates in the intestinal epithelium by assessing the frequency of phosphorylated histone H3-positive (pH3 + ) cells. Indeed, the ROS levels and proliferation rates of ISCs at Day 21 adult flies were higher than those at Day 7 ( Fig. 1D and E), implicating that the degeneration of the intestinal epithelium was intensified with ageing.

Perturbation of ROS levels in ISCs barely disrupted the intrinsic homeostasis under physiological condition
As a low intracellular concentration of ROS has been proposed critical for stemness and pluripotency of stem cells [1], we tested whether changes of ROS affected the ISCs homeostasis. We utilized the Temporal and Regional Gene Expression Targeting (TARGET) system to spatially and temporally regulate the level of ROS in ISCs (and EBs) [37]. We overexpressed ROS-scavenging enzymes through expression of a UAS-CncC transgene [38], a UAS-Catalase transgene or a UAS-Sod2 transgene [39], specifically in the esg + cells, and found no reduction of ROS levels in esg + cells (Fig. 2A). These data suggested that the ROS levels of ISCs under physiological condition were probably very low that further reduction might be difficult. Meanwhile, ISCs number and overall phenotype resembled wild-types (Fig. 2C). In parallel, when CncC, Catalase and Sod2 were knocked down via a UAS-CncC RNAi transgene [38], a UAS-Catalase RNAi transgene [40], or a UAS-Sod2 RNAi transgene [40], ROS levels in ISCs could be significantly upregulated (Fig. 2B). However, ISCs number and overall phenotype still resembled those of wild-types (Fig. 2C), implicating an intrinsic homeostatic range of ROS in ISCs.

Depletion of notch resulted in ISC-like tumor model and elevated ROS levels
ROS have been recently proposed to be involved in tumor progression. Previous studies revealed that cancer cells often exhibit increased generation of ROS compared with normal cells [11]. To test the role of redox regulation in tumor development, we firstly constructed a tumor-like pathological model caused by the depletion of N. When N-RNAi [41] was induced in ISCs under the control of esg-Gal4, all intestines were able to develop into tumor-like phenotype (Fig. 3A, B-B ′ ). In line with previous reports, the intracellular ROS levels in tumor-like ISCs increased significantly (Fig. 3G), which was accompanied by a gradual increase in proliferation rates (Fig. 3C-F', H), suggesting that increased ROS stress in cancer cells may in turn further contribute to hyperproliferation of ISCs and tumor formation.

Depletion of β-integrin resulted in ISC loss phenotype and elevated ROS levels
Tumor cells are usually displaced from their matrix niches in the early stages of metastasis. The data described above suggested that high level of ROS was essential in ISCs over-proliferation in the N RNAi ISC  tumors. We then examined whether it was similar in metastasis stages. Attachment to extracellular matrix (ECM) is required for the survival and proliferation of normal epithelial cells. Epithelial tumor cells, however, often acquire "anchorage independence", a property that may contribute to their ability to invade and grow in foreign environments. We first examined the role of matrix attachment of ISCs. Previous studies have shown integrin-mediated cell adhesion is required for ISC maintenance [42]. We utilized the TARGET system to deplete mys in ISCs to form matrix-deprived model. Having confirmed the functionality of the RNAi lines by the characteristic wing blistering phenotype observed upon mys depletion (Brower and Jaffe, 1989) ( Fig. 4A and B), we found that depletion of mys subunit resulted in loss of ISCs in normal fly from Day7 after shift to the restrictive temperature (Fig. 4C-E). To assay the redox state in ISCs that are still alive, we monitored endogenous ROS levels in vivo by using DHE. Interestingly, ROS levels increased moderately in ISCs, similar to that in N RNAi ISC tumors (Fig. 5F). Next, we utilized the TARGET system to deplete both N and mys in ISCs to establish the matrix-deprived tumor model, wherein metastatic tumor initiating cells were mimicked. A severe ISC loss phenotype was observed (Fig. 4G) and the ROS levels of the surviving N-and mys-depleted ISCs increased remarkably (Fig. 4H).

ROS manipulations modified N RNAi ISC-like tumors
As we observed similar moderately elevated ROS levels in both tumor-like ISCs (N RNAi ) and extracellular matrix (ECM)-deprived ISCs (mys RNAi ), we wondered whether there was difference underneath the similarity. When CncC, Catalase or SOD2 was overexpressed in the N RNAi context, tumor formation was significantly prevented (Fig. 5A-E) albeit the ROS levels were not decreased at the late stage of tumor formation (Fig. 5F). On the other hand, when ROS levels were elevated ( Figure 5L) upon silencing of CncC, Catalase or SOD2 in the N RNAi context, we observed an increase of doublets of ISCs in the case of CncC RNAi or SOD2 RNAi (Fig. 5 G-H ′ , J, K) and even small tumor-like clusters in the case of Catalase RNAi in the early tumor induction stage (Fig. 5I-I'). Taken together, these results suggested that the increased ROS aggravated hyperproliferation of ISCs and tumor formation in N RNAi tumors, implicating a tumor-promoting role of ROS in such context.

Differential ISC redox levels modified Mys RNAi -mediated ISC loss phenotype
To explore the possibility that moderately high level of ROS caused by mys RNAi was the cause of ISCs loss, we reduced ROS levels upon expressing of CncC and Catalase in the mys RNAi fly. Surprisingly, we found that downregulation of ROS levels aggravated the loss phenotype of ISCs (Fig. 6A-E). In contrast, upregulation of ROS levels (Fig. 6J) partially suppressed the ISCs loss ( Fig. 6F-I, K). Collectively, these results suggested that ROS has a pro-surviving capacity in the ECMdeprived ISCs.
However, reducing ROS ( Figure 6M) in the ECM-deprived N RNAi ISCs prevented ISCs loss ( Figure 6L, N), which was opposite to those simple ECM-deprived ISCs, suggesting that excessive amounts of ROS could be toxic to the cells and antioxidants might promote the survival of tumorinitiating cells that lack extracellular attachment. These results indicated an unanticipated mechanism for tumor cell survival in altered matrix environments through antioxidants.

Discussion
Tissue homeostasis is regulated by tightly controlled division and differentiation of multipotent somatic stem cells that give rise to different cell types of a given tissue. These processes need a precisely regulated ROS levels. In our work, we found that ISCs maintain low intracellular ROS levels and prevent excessive proliferation in normal intestine. Neither upregulation nor downregulation of ROS levels significantly affected ISCs, implicating that the self-renewal of ISCs in young flies could maintain in a homeostatic state. However, old intestinal epithelia exhibit significantly elevated ROS levels in ISCs, which is related to age-related increase in ISC proliferation rates and disruption of epithelial homeostasis.
Mounting evidence suggests that many types of cancer cell have increased levels of ROS [43]. Tumor suppressor and oncogenic pathways frequently mutated in cancer commonly result in increased accumulation of ROS [44][45][46][47]. An increase in ROS is associated with abnormal cancer cell growth and reflects a disruption of redox homeostasis. The Notch signaling pathway regulates cell fate determination during development and alterations of the Notch signaling can lead to a variety of disorders including human malignancies. In this work, depletion of N could induce an ISCs-like tumor phenotype, which facilitated us to study the role of ROS in tumor. We observed that increased oxidation of ISCs in N-RNAi conditions was accompanied by elevated proliferation rates, and we further found that tumor formation could be obviously prevented when we reduce ROS levels in ISCs-like tumor cells through overexpression of oxidant scavenger proteins. Our work suggested that increased ROS stress in cancer cells may contribute to hyperproliferation of tumor initiating cells and tumor formation, which raises the possibility that regulation of ROS could be a potential therapy for Notch-related cancer [48]. Furthermore, we observed that there was no tumor formation throughout our experiment when we downregulated the fatty acid oxidation (FAO) pathway in N-RNAi conditions (data not shown), which was in line with a previous report that cancer cells deprived of glucose maintain ATP production through FAO [49].
Normal epithelial cells require matrix attachment for survival and the ability of tumor cells to survive outside their natural extracellular matrix (ECM) niches during metastasis is dependent on acquisition of anchorage independence [50]. What roles ROS plays in this progress remains elusive. To address this question, we genetically knockdown mys in ISCs to mimic a ECM-deprived model, and we found ISCs number declined dramatically. This is consistent with previous work [51]. Unexpectedly, downregulation of ROS levels aggravated ISCs loss phenotype, and the survived mys RNAi ISCs all had elevated levels of ROS. Previous studies revealed that detachment of epithelial cells from ECM causes an ATP deficiency due to loss of glucose transport [51]. Indeed, we also found that ISCs loss phenotype could be rescued after feeding the mys-RNAi flies high-glucose food (data not shown). More evidence is needed to confirmed whether the loss of ISC was caused by ATP deficiency. Overall, our results suggest that moderate elevation of ROS in ECM-deprivation model is not the main cause of ISCs loss but a stress response during detachment from ECM. Previous studies also revealed that increase ROS would be correlated with anoikis protection of endothelial cells during cell detachment from ECM [52,53]. Taken together, we speculate that ROS is a key signaling molecules eliciting pro-survival signals protection from ISCs anoikis in the absence of ECM contact.
Increased ROS levels are considered to be closely linked to the accelerated formation of metastasis, and the removal of ROS is a rational strategy to inhibit metastasis. However, there are also controversial results reported thus far, and ROS should have diverse effects on tumor metastasis [21]. Through depletion of N and mys in ISCs, which resembled metastatic tumor cells, we observed severe ISCs loss symptoms. A previous study in the fly midgut also found that reducing integrins in Nor Apctumor cells inhibited their growth, and demonstrated that tumor cells competed with ECs for attachment to the basement membrane, via integrine-mediated adhesion [25]. It is noteworthy that high levels of ROS in Nmystumor cells (higher than N RNAi or mys RNAi alone) were observed in our research. Consistently, matrix detachment associated with tumorigenesis lead to excess production of ROS [54][55][56][57][58], which can damage cellular components and compromise cell viability [59]. Collectively, we hypothesize that excessive accumulation of ROS, beyond the cellular tolerant threshold, is the main cause of ISCs loss phenotype in ECM-deprived tumor model. This interpretation is supported by the fact that antioxidants can rescue ISCs loss phenotype in ECM-deprived tumor model. Collectively, these results suggested that antioxidant scavenges excessive ROS and may promote the survival of tumor cells in pathological matrix environments and thus enhance malignancy.
Taken together, our results revealed the intracellular redox level is a critical cell fate determinant of stem cells and tumor cells under various conditions and suggested that the antioxidant-based intervention of stem cells and tumors should be formulated with caution according to the specific situations (Fig. 7). Fig. 7. Summary of the levels and regulations of ROS in ISCs under different conditions N depletion induced a moderate increase of intrinsic ROS generation (red arrow), leading to a hyperproliferation of ISCs and tumor formation. Downregulation of ROS in tumor cells could suppressed the hyperproliferation phenotype. ECM-deprivation caused by mys depletion could lead to ISCs loss phenotype, also induced a moderate intrinsic increase of ROS generation (red arrow). Further increased ROS in turn facilitated the survival of ECM-deprived ISCs while reduced ROS exacerbated the loss of ECM-deprived ISCs. N-and mys-depleted ISCs, which resembled metastatic tumor cells, harbored even higher ROS levels that were toxic (two red arrows) and were subjected to more severe oxidative stress, which could be partially prevented by ectopic supply of antioxidant enzymes. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)