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Published in final edited form as: J Invest Dermatol. 2021 Mar 26;141(9):2291–2299.e2. doi: 10.1016/j.jid.2021.02.754

Long-Term Effects of Sirolimus on Human Skin TSC2-Null Fibroblast–Like Cells

Xiong Cai 1,2,9, Qingyuan Fan 1,3,9, Gi Soo Kang 1,4, Kelsey Grolig 1, Xiaoyan Shen 1,5,6, Eric M Billings 7, Gustavo Pacheco-Rodriguez 1, Thomas N Darling 8, Joel Moss 1
PMCID: PMC9942268  NIHMSID: NIHMS1866582  PMID: 33773987

Abstract

Tuberous sclerosis complex (TSC) is an autosomal-dominant disorder characterized by hamartomatous tumors of the skin, kidneys, brain, and lungs. TSC is caused by mutations in the TSC1 and TSC2 genes, which result in hyperactivation of the mTOR, leading to dysregulated cell growth and autophagy. Rapamycin (sirolimus) shrinks TSC tumors, but the clinical benefits of sirolimus are not sustained after its withdrawal. In this study, we studied the cellular processes critical for tumor formation and growth, including cell proliferation and cell size. TSC2−/− and TSC2+/− cells were isolated from TSC skin tumors and normal-appearing skin, respectively. Cells were incubated with sirolimus for 72 hours. Withdrawal of sirolimus from TSC2−/− cells resulted in a highly proliferative phenotype and caused cells to enter the S phase of the cell cycle, with persistent phosphorylation of mTOR, p70 S6 kinase, ribosomal protein S6, and 4EB-P1; decreased cyclin D kinase inhibitors; and transient hyperactivation of protein kinase B. Sirolimus modulated the estrogen- and autophagy-dependent volume of TSC2−/− cells. These results suggest that sirolimus may decrease the size of TSC tumors by reducing TSC2−/− cell volume, altering the cell cycle, and reprogramming TSC2-null cells.

INTRODUCTION

Patients with tuberous sclerosis complex (TSC) present with hamartomatous tumors of the skin, brain, heart, lungs, eyes, and kidneys (Henske et al., 2016). Common manifestations of TSC involve the skin (e.g., ungual fibromas, shagreen patch, angiofibromas), nervous system (e.g., cortical dysplasias, subependymal giant cell astrocytoma), kidneys (e.g., angiomyolipoma [AML]), and lungs (e.g., lymphangioleiomyomatosis [LAM]) (Budde and Gaedeke, 2012).

TSC is associated with mutations in either the TSC1 or TSC2 genes (Tyburczy et al., 2015), which encode hamartin and tuberin, respectively. Hamartin and tuberin are part of a heterotrimeric complex with TBC1D7, which is a critical negative regulator of the mTOR (Menon et al., 2014). Tuberin is a GAP for Rheb, which activates mTOR in the guanosine triphosphate–bound state. mTOR is a critical nutritional and cellular energy checkpoint sensor and master regulator of multiple cellular functions, including protein synthesis, ribosome biogenesis, cell cycle, cell proliferation, cell size, and autophagy (Laplante and Sabatini, 2012).

In mammalian cells, two structurally and functionally distinct mTOR-containing complexes have been identified: mTORC1 and mTORC2 (Huang and Manning, 2008). mTORC1 controls cell growth by increasing the phosphorylation of several downstream effectors, including two important translational regulators, ribosomal S6K1 and 4E-BP1 (Brown et al., 1995). Amino acids, GFs, hormones, and mitogens that signal to mTOR control cell growth (Liu and Sabatini, 2020), which as a whole is a regulated process involving cell cycle progression, cell size, cell death, and autophagy (Laplante and Sabatini, 2012).

The dysfunction of hamartin and tuberin results in constitutive activation of mTOR, impacting multiple cellular processes involved in cellular growth and cell proliferation (Goncharova et al., 2002; Schipany et al., 2015). TSC2-null cells show a decrease in autophagy (Parkhitko et al., 2011); inhibition of mTOR by sirolimus increased autophagy and cell survival (Parkhitko et al., 2011). An inhibitor of autophagy, chloroquine, may promote cell death (Parkhitko et al., 2011). Constitutive activation of mTORC1 and its downstream effectors (e.g., p70 S6K, S6 ribosomal protein, 4E-BP1) has been seen in LAM and AML (Li et al., 2008).

Pharmacological inhibitors of mTOR, for example, everolimus and sirolimus, have been used to treat AML (Bissler et al., 2008); subependymal giant cell astrocytoma (Krueger et al., 2010), pulmonary LAM (McCormack et al., 2011), lymphatic abnormalities (Taveira-DaSilva et al., 2011), and TSC-associated skin tumors (Allen et al., 2015; Koenig et al., 2018; Wataya-Kaneda et al., 2017, 2015). It is of interest that the worsening of the skin lesions (i.e., AMLs and pulmonary LAM) appears to be affected by hormonal status and the state of autophagy (Henske et al., 2016; Yu et al., 2011). Angiofibromas increase in size and number during puberty, and pulmonary LAM occurs almost exclusively in women, suggesting that TSC lesions are affected by sex hormones (Saxton and Sabatini, 2017; Taveira-DaSilva and Moss, 2015).

mTOR is a central controller of cellular growth, and thus, its inhibitor, sirolimus, can have pleiotropic effects on cells (Liu and Sabatini, 2020; Lloyd, 2013; Tumaneng et al., 2012). In this study, we studied the growth of human TSC2-null cells after incubation and withdrawal of sirolimus. We evaluated the effects of sirolimus; estrogen; and an inhibitor of autophagy, chloroquine, on TSC2+/− and TSC2−/− fibroblast–like cells from the skin. We showed that TSC2 deficiency increased cell volume, whereas sirolimus reduced the volume of TSC2−/−− fibroblast–like cells. Sirolimus suppressed the proliferation of TSC2+/− and TSC2+/− fibroblasts and selectively stimulated the mTOR-activating protein kinase B (Akt) pathway in TSC2−/− fibroblasts but not in TSC2+/− fibroblasts derived from normal skin. Collectively, cellular effects of sirolimus, chloroquine, and estrogens are likely to have pathological and therapeutic implications.

RESULTS

Fibroblast-like skin tumor cells were grown from a periungual fibroma (TSC2 c.4830G>A, p.W1610* plus c.1058_1059delTC, TSC−/− cells); normal-appearing skin (TSC2+/− cells, TSC2 c.4830G>A) was grown from the same patient (Li et al., 2011). These cells (Darling et al., 2010) were used to study the effect of sirolimus, estrogen, and chloroquine on cell proliferation and size.

Sirolimus regulates the proliferation and size of TSC human skin fibroblasts

It has been reported previously that there is an increase in AML tumor size and also in the size of skin lesions after withdrawal of sirolimus (Bissler et al., 2008; Darling, 2018; Malissen et al., 2017).

To obtain further insight into the cellular and molecular aspects of sirolimus treatment and withdrawal, we studied the effects of sirolimus on the proliferation of TSC2−/− skin tumor fibroblasts. For control, we used TSC2+/− fibroblasts obtained from the normal-appearing skin of patients. In addition, the effects of sirolimus were studied using pulmonary artery smooth muscle (PASM) cells. We and others have used PASM cells as a control cell population in studies of LAM, the pulmonary disease in TSC, and these cells are sensitive to sirolimus (Kristof et al., 2005). In this study, cells were incubated with different concentrations of sirolimus (0, 0.2, 2, 20, 200, and 2,000 nM) for up to 3 days (Figure 1a). Suppression of cell proliferation by 0.2 nM sirolimus was noted from the second treatment day for TSC2+/− and TSC2−/−fibroblasts and at 20 nM for PASM. After the third treatment day, 0.2 nM sirolimus suppressed the proliferation of all the three cell lines. Thus, sirolimus blocks the proliferation of human TSC2−/− skin cell fibroblasts in a dose-dependent manner.

Figure 1. Effects sirolimus on proliferation and cell size of TSC2+/− and TSC2−/− fibroblasts and PASM cells.

Figure 1.

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(a) Cells were grown for 1 to 3 days without or with increasing concentrations of sirolimus (0.0, 0.2, 2.0, 20.0, and 200.0 nM). * P ≤ 0.05 (vs. 0 nM). Cells were counted at the indicated times. Results are representative of three independent experiments consisting of duplicate plates. (b) TSC2−/− control cells are larger than TSC2+/− controls. A reduction in TSC2−/− cell volume (**P < 0.01) was observed on incubation with 200 nM sirolimus for 48 hours. (c) TSC2+/− fibroblasts volume distribution (measured as the percentage of the total cell population) in control and sirolimus-treated cells, showing no sensitivity. (d) Sirolimus-sensitive TSC2−/− population shifted toward smaller cell volume, with a more narrow population distribution than that of TSC2−/− controls; cell volume is expressed in pl. PASM, pulmonary artery smooth muscle.

Because TSC2 regulates cell size, we compared the size of TSC2+/− and TSC2−/− fibroblasts. We found that TSC2−/− cells were approximately three times larger than TSC2+/− cells (Figure 1b), consistent with the finding in Drosophila, showing that TSC2 regulates eye cell size (Potter et al., 2001; Rosner et al., 2009). We incubated TSC2−/− and TSC2+/− cells with sirolimus (for 48 hours, 200 nM) and determined the cell volume. Sirolimus had no effect on the volume of TSC2+/− cells, whereas there was a reduction in TSC2−/− cell volume (**P < 0.01) (Figure 1b). After sirolimus treatment, TSC2−/− fibroblasts were still about two times larger than the TSC2+/− controls (Figure 1b).

Effect of sirolimus withdrawal on human TSC2−/− skin fibroblasts and PASM

Because sirolimus is used long-term and is withdrawn in many cases because of side effects and/or unexpected events (Darling, 2018), we decided to study the effect of long-term incubation, followed by withdrawal of sirolimus as described in Supplementary Figure S1. After 3 days of incubation of cells in the presence of sirolimus, the drug was removed. The growth of treated and untreated TSC2+/− fibroblasts was similar (Figure 2a). TSC2−/− cells grow more slowly than TSC2+/− cells. Sirolimus-treated TSC2−/− cells and PASM grew more slowly than the untreated cells. However, after replating, sirolimus-treated TSC2−/− fibroblasts grew at a faster rate than the untreated cells. PASM and TSC2+/− fibroblasts proliferated at the same rate as the untreated cells (Figure 2b). Thus, owing to sirolimus treatment, TSC2−/− cells were reprogramed to grow faster than untreated cells.

Figure 2. Effect of sirolimus withdrawal on cell proliferation.

Figure 2.

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(a) After serum starvation, cells were treated with sirolimus for 3 days and then counted, plated, and incubated for up to 3 days with or without sirolimus as indicated (Supplementary Figure S1). For the (a) first round of sirolimus withdrawal, cells were incubated with 200 nM sirolimus (filled symbols) or without (empty symbols) for 3 days. Cells were harvested; plated on 6-well plates (3 × 104 cells per 2 ml medium per well); grown for 1, 2, or 3 days; and counted (first round of plating). (b) For the second round of sirolimus withdrawal, on day 3, after withdrawal of sirolimus, cells were replated in 6-well plates (3 × 104 cells per 2 ml medium per well); grown for an additional 1, 2, or 3 days; and then counted. *P ≤ 0.05 compared with that of the untreated cells. Results are representative of three independent experiments consisting of duplicate plates. PASM, pulmonary artery smooth muscle.

Hyperactivation of the mTOR pathways after sirolimus withdrawal in TSC2−/−

To obtain insights into the regulation of mTOR after sirolimus withdrawal (Laplante and Sabatini, 2009), we analyzed the total protein and phosphorylated proteins involved in the mTOR pathway (Figure 3). We confirmed that the TSC2−/− fibroblasts do not express tuberin, the gene product of TSC2. The levels of tuberin in TSC2+/− and TSC2−/− fibroblasts did not change after incubation with and then withdrawal of sirolimus (Figure 3). However, mTOR protein decreased immediately after the withdrawal of sirolimus in TSC2−/− fibroblasts but increased above the initial levels after the second round of sirolimus withdrawal. This was not the case for TSC2+/− fibroblasts where mTOR levels remain more stable during the first and second rounds of sirolimus withdrawal. Interestingly, the levels of p70, S6, and 4E-BP1 in TSC2−/− fibroblasts increased during the first round of sirolimus withdrawal and remained elevated during the second round.

Figure 3. Effect of sirolimus on the mTOR pathway of TSC2−/− fibroblasts.

Figure 3.

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Cells were treated with sirolimus (200 nM) or vehicle (0.07% DMSO) for 72 hours and then plated on 6-cm dishes (1.8 × 105 cells per well) and cultured according to the methods at the different times. After incubation, cells were lysed, and protein samples (15 μg) were separated in NuPAGE gels, and western blotting was run to determine the presence and levels of the indicated proteins. Anti-tuberin antibodies showed that TSC2−/− but not TSC2+/− cells lack this protein. Unphosphorylated and phosphorylated forms of mTOR, p70, S6, and 4E-BP1 were determined. The minus sign (−) indicates the untreated cells; the number 3 indicates the cells incubated with sirolimus for 3 days; first round indicates the cells replated and cultured for 1–3 days after the removal of sirolimus; and second round indicates the cells replated and cultured for 1–3 days after the removal of sirolimus. *P ≤ 0.05 (vs. cells untreated). P-4E BP1, phosphorylated 4E BP1; P-mTOR, phosphorylated mTOR; P-p70, phosphorylated p70; P-S6, phosphorylated S6.

Phosphorylation of mTOR mirrored its expression in both cell lines; phosphorylation was dramatically downregulated for both TSC2+/− and TSC2−/− fibroblasts incubated with sirolimus for 3 days. Phosphorylation of mTOR, p70, S6, and 4E-BP1 was decreased after the withdrawal of sirolimus but remained elevated in TSC2−/− cells during the second round of sirolimus withdrawal (Figure 3). These data suggest that there is a persistent hyperactivation of the mTOR pathway after sirolimus withdrawal.

Regulation of the cell cycle of TSC2−/− cells after sirolimus withdrawal

Because several of the affected genes are involved in the cell cycle, we performed cell cycle analysis on TSC2+/− and TSC2−/− fibroblasts. As expected, the percentage of TSC2−/− cells in the S phase was higher after withdrawal of sirolimus than that of the untreated cells, but there were no cell cycle differences between the treated and the untreated TSC2+/− fibroblasts (Figure 4a). This is consistent with previous studies regarding the role of sirolimus in the control of the S–G1 transition (Laplante and Sabatini, 2012). Our data also showed that both the total Akt protein and phosphorylation levels were upregulated in TSC2+/− and TSC2−/− fibroblasts treated with sirolimus for 3 days (Figure 4c). The total Akt protein and phosphorylation levels were dramatically increased for up to 3 days immediately after the withdrawal of sirolimus for both TSC2+/− and TSC2−/− fibroblasts. It was previously shown that Akt phosphorylation level was increased in some tumor cell lines incubated with sirolimus (Chen et al., 2010; Sarbassov et al., 2006; Sun et al., 2005; Wan et al., 2007). From the fourth day after the withdrawal of sirolimus, total Akt protein levels still maintained high levels for TSC2−/− fibroblasts, but the Akt phosphorylation levels decreased dramatically. For TSC2+/− and TSC2−/− fibroblasts, the Akt phosphorylation levels decreased close to the levels seen in the untreated cells. Akt phosphorylation levels did not remain elevated during the second round of sirolimus withdrawal.

Figure 4. Effect of sirolimus withdrawal on cell cycle of TSC2+/− and TSC2−/−.

Figure 4.

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Cells incubated with 200 nM sirolimus or vehicle for 3 days were washed three times in 10 ml PBS, plated in 6-cm plates (1.8 × 105 cells per 7 ml medium per well), and incubated for 3 days. Cells were then replated on 6-cm plates (1.8 × 105 cells per 7 ml medium per well) and grown for an additional 1, 2, or 3 days to determine the cell cycle stages of cells. (a) Cell cycle analysis of TSC2+/− cells after 3 days of sirolimus treatment. (b) Cell cycle analysis of TSC2−/− cells after 3 days after the withdrawal of sirolimus. *P ≤ 0.05 compared with that of the untreated cells. Results are representative of three independent experiments consisting of duplicate plates. (c) Cells treated with sirolimus (200 nM) or vehicle (0.07% DMSO) for 72 hours were gently trypsinized and plated on 6-cm dishes (1.8 × 105 cells per well) as shown in Figure 1. Cells were lysed, protein lysates (15 mg) were separated on NuPAGE gels, and western blot analysis was performed to monitor the expression and phosphorylation of the indicated proteins. Whole-cell lysates were probed with antibodies against phosphorylated Akt (Ser 473), Akt, p21 Waf1/Cip1, p27 Kip1, p15. (a) TSC2+/− fibroblast. (b) TSC2−/− fibroblast. The minus sign (−) indicates the untreated cells; the number 3 indicates the cells treated with sirolimus for 3 days; first round of withdrawal indicates the cells replated for 1e3 days after the removal of sirolimus; and second round of withdrawal indicates the cells that were cultured for 3 additional days after the removal of sirolimus. *P ≤ 0.05 (vs. cells untreated). The experiments were repeated three times. Akt, protein kinase B; P-Akt, phosphorylated protein kinase B.

Because we observed cell cycle S and G2/M phases, we analyzed the expression of two key regulators of the cell cycle, p21 and p27, which are downstream of Akt and are involved in cell cycle control. After incubation with sirolimus for 3 days, p21 and p27 levels were upregulated in TSC2+/− and TSC2−/− fibroblasts after the first day after sirolimus withdrawal and then decreased on day 2. From the third day after the withdrawal of sirolimus, p21 and p27 began to increase gradually close to the level seen in the untreated cells in the case of TSC2+/− fibroblasts. For TSC2−/− fibroblasts, p21 and p27 levels increased, but the levels were still lower than those seen in the untreated cells. The levels of p21 and p27 remained constant during the second round of sirolimus withdrawal.

We next examined the cell cycle inhibitor factor p15. After incubation with sirolimus for 3 days, p15 levels were upregulated in both TSC2+/− and TSC2+/− fibroblasts. After the withdrawal of sirolimus, p15 levels were dramatically downregulated in both TSC2+/− and TSC2−/−fibroblasts for up to 3 days. On the fourth day after the withdrawal of sirolimus, the levels of p15 in TSC2+/− fibroblasts began to increase gradually to approximate the levels seen in the untreated cells. Levels of p15, even after sirolimus withdrawal, were maintained at the levels seen in the cells incubated with the drug.

Estrogens and chloroquine

Treatments of several patient cohorts with mTOR dysfunction have focused on sirolimus, estrogen, and chloroquine to decrease cell proliferation or growth. We evaluated the effects of these agents (e.g., chloroquine) on the volume of TSC2−/− fibroblast–like cells isolated from TSC skin tumors.

To determine whether estrogen influences cell volume, we used the MCF-7 breast cancer cell line as a control. This cell line is female derived, expresses estrogen and progesterone receptors, and has been used extensively to evaluate the effects of estradiol (Coms‚a et al., 2015; Katzenellenbogen et al., 1987). Our results indicate that estrogen increases MCF-7 cell volume and number in both concentration- and time-dependent manner (Supplementary Figure S2). We next examined the effect of estradiol (10 nM) on the volume of TSC2+/− and TSC2−/− fibroblast–like cells and normal human dermal fibroblasts during a 48 hours incubation. Estrogen increased the volume of both TSC2−/− cells and normal human dermal fibroblasts (Figure 5a and c). Interestingly, the volume of TSC2+/− cells did not change appreciably with sirolimus or estradiol treatment (Figure 5b). As shown earlier, sirolimus decreased TSC2−/− cell volume (Figure 5e) and had no effect on TSC2+/− cells (Figure 5d). In normal human dermal fibroblasts, the cell volume also decreased by sirolimus treatment (Figure 5c).

Figure 5. Effect of sirolimus and CQ on cell volume of TSC2+/− and TSC2+/+ cells.

Figure 5.

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(a) Cell volume measured as the percent volume (pl) and compared with that of the vehicle-treated control. Cell volume results were comparable with those of the NHDF with a reduction in TSC2−/− cell volume when incubated with sirolimus (*P < 0.05); there was an increased cell volume with estradiol treatment (*P < 0.05). (b) No significant effects were observed on cell volume from sirolimus or estradiol treatment in TSC2+/− cells. (c) Sirolimus induced ~10% reduction in the NHDF cell volume (*P < 0.05). Estradiol increased cell volume by ~10% (*P < 0.05). (d) TSC2+/− cell volume (pl) increased in the presence of CQ (**P < 0.01); CQ with sirolimus also increased cell volume (*P < 0.05). (e) Sirolimus treatment decreased TSC2−/− cell volume (**P < 0.01); CQ-treated cells have a comparable volume with that of TSC2−/− control cells, and the combination (R + C) reduced the cell volume (*P < 0.05). (f) Narrow distributions in TSC2+/− control and CQ-treated cells with a shift toward larger cell volume in CQ-treated cell population. (g) TSC2−/− control cells are ~3 times larger than TSC2+/− controls and show wider distributions in both TSC2−/− control and CQ-treated populations with more variability in cell volume within the cell population. No significant cell volume differences were detected between TSC2−/− vehicle-control and CQ-treated cells. CQ, chloroquine; NHDF, normal human dermal fibroblast.

We studied the effect of chloroquine, an autophagy inhibitor, on TSC2−/− cell volume (El-Chemaly et al., 2017; Johnson et al., 2015; Parkhitko et al., 2011). Treatment with chloroquine (10 μg/ml) alone increased the volume of TSC2+/− cells (**P < 0.01) during a 48-hour incubation (Figure 5f) but had no effect on TSC2−/− cell volume (Figure 5g).

Because a combination of sirolimus and chloroquine has been suggested for the treatment of LAM (El-Chemaly et al., 2017) and potentially TSC, we tested the effect of this drug combination on cell volume. Relative to vehicle-control cells, there was a volume increase (*P < 0.05) observed in TSC2+/− cells treated with the drug combination (Figure 5d and e). Chloroquine alone was ineffective in changing TSC2−/− cell volume, although the combination treatment reduced the volume of TSC2−/− cells but not significantly (Figure 5d and e). The histogram reveals a clear shift toward increased cell volume in the chloroquine-treated TSC2+/− cells (Figure 5f). The histogram displays a wider, more variable distribution in the TSC2−/− vehicle-control cell volume than in the TSC2+/− vehicle controls (Figure 5g). These data demonstrate that the TSC2−/− cells were more sensitive to sirolimus than to chloroquine treatment (Figure 5e and g), whereas TSC2+/− cells were more sensitive to chloroquine than to sirolimus treatment (Figure 5d–g). Overall, our findings show an increase in TSC2+/− cell volume after chloroquine treatment with no effect on the volume of TSC2−/− cells. However, TSC2−/− cell volume was reduced by sirolimus treatment and the combination of sirolimus and chloroquine.

DISCUSSION

Our study demonstrates that TSC2−/− skin tumor cells but not TSC2+/− normal skin fibroblasts and TSC2+/+ smooth muscle cells proliferated markedly more rapidly than untreated cells after sirolimus treatment and withdrawal. Consistent with this finding, cell cycle analysis showed a greater percentage of TSC2−/− cells but not of TSC2+/− cells in S and G2/M phases after sirolimus treatment and showed that sirolimus decreased the larger cell size of TSC2−/− cells. Thus, sirolimus controls TSC2−/− cell proliferation, cell size, and cell cycle, and a 6-day exposure to sirolimus followed by drug withdrawal affects proliferation.

Cell growth is critical in neoplastic development, which is controlled at the cellular level by the regulation of multiple processes such as proliferation, cycle, size, and autophagy (Hanahan and Weinberg, 2011). Our data show that only TSC2−/− cells treated with sirolimus proliferated faster than the untreated cells, starting on the third day after the withdrawal of sirolimus. The levels of total and phosphorylated proteins of mTOR and its downstream substrates p70S6, S6, and 4E-BP1 increased only in TSC2−/− cells starting on the third day after the withdrawal of sirolimus (Figure 3). After the withdrawal of sirolimus, phosphorylated Akt levels increased for up to 3 days, with a more pronounced effect seen in TSC2−/− cells than in TSC2+/− cells (Figure 4). Therefore, activated Akt may explain in part the survival of TSC2+/− and TSC2−/− cells incubated with sirolimus (Sun et al., 2005).

A clinical concern of sirolimus therapy is that mTOR inhibition may result in the activation of the phosphatidylinositol 3 kinase–Akt signaling pathway, thus protecting cells from apoptosis (Sun et al., 2005) and the induction of microRNAs that regulate cell proliferation (Trindade et al., 2013). Because our data clearly demonstrated that TSC2−/− cells continued to grow at a low rate even in the presence of sirolimus and proliferate more rapidly after withdrawal of sirolimus, it would be reasonable to consider the combination of sirolimus with other drugs in clinical trials. The combination of sirolimus and the autophagy inhibitor, chloroquine, has been shown to exert synergistic effects on inhibiting the survival of TSC2-null cells and the growth of TSC2-null xenograft tumors (Parkhitko et al., 2011). The combination of the drugs was very well-tolerated by patients and did not show secondary effects.

Our results are consistent with several reports from clinical trials of sirolimus in TSC and LAM, where the affected cells have TSC2 mutations. Sirolimus therapy for patients with TSC and LAM for 12 months resulted in shrinkage of AMLs (Bissler et al., 2008) and marked stabilization of lung function (McCormack et al., 2011), but these clinical benefits in the case of renal AMLs were partially reversed and, in the case of lung function, were not sustained, with equivalent rates of decline observed after the discontinuation of sirolimus (McCormack et al., 2011). Thus, it is possible that patients who are on and off sirolimus may have detrimental effects on lung function.

Cell cycle regulation appears to be cell dependent and is affected by many factors (e.g., GFs), which adds another complexity to understanding cell cycle control in TSC2−/− fibroblast–like cells. We observed differences between TSC2+/− and TSC2−/− cells in the CDKIs p21, p27, and p15 after sirolimus withdrawal (Figure 4). Although the levels of the CDK4 and CDK6 inhibitors p21 and p27 (Sherr and Roberts, 1999) were differentially regulated in TSC2+/− cells from TSC2−/− cells during the withdrawal (second round), the proliferative state of TSC2−/− cells can be explained by the CDKI decrease. CDK4 and CDK6 inhibitors are targets of the Akt pathway (Knudsen and Witkiewicz, 2017) and are key cell cycle regulators (Manning and Cantley, 2007). Because we observed less activation of Akt during the increase of CDKIs, our data suggest that the regulation of p21 and p27 in TSC2−/− skin tumor cells is independent of Akt activation. Expression of p15 in both TSC2+/− and TSC2−/− fibroblasts was initially increased with sirolimus treatment but decreased on sirolimus withdrawal. This regulation prompted us to speculate that CDKIs could be used in conjunction with sirolimus.

Our findings demonstrate that mTOR plays a role in regulating the volume of TSC2−/− fibroblasts, as previously demonstrated in other cell types (Lloyd, 2013; Rosner et al., 2009). Clinical studies showed that sirolimus also decreased the size of TSC tumors (e.g., angiofibromas, shagreen patches) (Budde and Gaedeke, 2012; Nathan et al., 2015). Although we may see a change in size due to decreased individual TSC2−/− cell volume, skin tumor fibroblasts comprise only a small percentage of the total tumor area, with most (e.g., shagreen patches, angiofibromas) being occupied by collagen. Thus, even if the fibroblasts decreased in size by 50%, this would cause only a few percent decrease in total tumor volume. Therefore, there must be remodeling of collagen and decreased angiogenesis to reduce skin tumor size. These findings at the cellular level may explain the use of sirolimus as an effective, cytostatic, and volume-reducing treatment for TSC skin tumors and AML (Bissler et al., 2008; Teng et al., 2014).

Estrogen treatment increased the volume of TSC2−/− skin cells without any effect on TSC2+/− cells. Studies have reported the presence of estrogen receptors (e.g., ER-α, ER-β) in a variety of skin cells (e.g., melanocytes, keratinocytes, fibroblasts), and these receptors have important roles in skin aging, cancer, and pigmentation (Haczynski et al., 2004, 2002). 17b-Estradiol stimulated rapid cytoskeletal rearrangement in human dermal fibroblasts, which corresponded to changes in cell shape and potential change in cell volume (Yu et al., 2009). At the cellular level, estrogens appear to control cell proliferation and mobilized ELT3 cells with TSC2 dysfunction to the lungs in a mouse metastasis model (Yu et al., 2009). Estrogenic effects are mediated through their receptors (ERs) and can potentially increase the volume of smooth muscle cells (El-Hashemite et al., 2005; Finlay et al., 2004) and enhance the proliferation of breast carcinoma cell lines (e.g., MCF-7 cells) (Coms‚a et al., 2015; Katzenellenbogen et al., 1987). Thus, it would appear that skin tumor size could be decreased by antiestrogen therapy.

The problem with sirolimus is that although it typically causes cell shrinkage, it does not show toxicity to TSC2-null cells. It was proposed that autophagy is activated in these cells as a survival mechanism (Barnes et al., 2010). We observed that chloroquine alone had no effects on TSC2−/− fibroblasts but effectively decreased cell volume when combined with sirolimus. Chloroquine, an autophagy inhibitor, has been studied as a potential cancer therapy (Hanahan and Weinberg, 2011; Parkhitko et al., 2011). Chloroquine treatment alone increased the volume of TSC2+/− cells without any effect on TSC2−/− cells, although the combination treatment (chloroquine and sirolimus) decreased TSC2−/− cell volume, and combination therapies are well-tolerated (Govindarajan et al., 2012; Nadiminti and Arbiser, 2005).

Immediate challenges of sirolimus therapy include regrowth of AMLs and a decline of lung function seen after discontinuation of the drug (Bissler et al., 2008; McCormack et al., 2011). More importantly, inhibition of mTORC1 by sirolimus strongly induces autophagy, which may promote the survival of TSC2-null cells (Parkhitko et al., 2011). Thus, a clear understanding of the molecular pathways impacted by sirolimus may help to develop targeted therapies.

MATERIALS AND METHODS

Cell lines and patient specimens

TSC2+/− and TSC2−/− fibroblasts were grown from skin tumors (i.e., angiofibromas, periungual fibromas) or normal-appearing skin biopsies of three patients diagnosed with TSC (Li et al., 2011, 2005); PASM cells were obtained from Lonza (Walkersville, MD). Patients provided written informed consent and were enrolled in the clinical protocols approved by the National Heart, Lung, and Blood Institute Institutional Review Board (protocols 00-H-0051, 95-H-0186, 96-H-0100).

Cell growth

Smooth muscle cells and TSC2+/− and TSC2−/− fibroblasts were grown in Smooth Muscle Cell Growth Medium −2 and Mesenchymal Stem Cell Growth Medium (Lonza), respectively. TSC2+/− and TSC2−/− fibroblasts (3 × 104 cells/ml) were grown in mesenchymal stem cell growth media.

Long-term effects of sirolimus

To determine the long-term effects of sirolimus and the effects of the drug withdrawal, we followed the scheme shown in Supplementary Figure S1.

Estrogen effect

Cells were plated in phenol red–free DMEM media containing 10% charcoal-treated fetal bovine serum supplemented with penicillin (100 U/ml) and streptomycin (100 mg/ml). Cell cultures were maintained for 48 hours before any treatment or measurement. Estrogen (17β-estradiol) was dissolved in ethanol (Stock solution 1 mM, Sigma-Aldrich, St. Louis, MO), and the cells were incubated at the indicated times and 17β-estradiol concentration. In separate experiments, cells were incubated for 48 hours with sirolimus (200 nM) or chloroquine (10 μg/ml) or both (sirolimus and chloroquine).

Supplementary Material

1

ACKNOWLEDGMENTS

We thank Martha Vaughan (National Heart, Lung, and Blood Institute, National Institutes of Health, Bethesda, MA) who before her passing participated in helpful discussions and critical review of the manuscript. This study was funded in part by the Intramural Research Program of the National Heart, Lung, and Blood Institute of the National Institutes of Health and by the National Institutes of Health grant RO1 CA100907 (TND). We thank the LAM Foundation and the Tuberous Sclerosis Alliance for their assistance in recruiting patients for our studies.

Abbreviations:

Akt

protein kinase B

AML

angiomyolipoma

LAM

lymphangioleiomyomatosis

PASM

pulmonary artery smooth muscle

TSC

tuberous sclerosis complex

Footnotes

CONFLICT OF INTEREST

The authors state no conflict of interest.

Statistical analysis

See the Supplementary Materials and Methods.

SUPPLEMENTARY MATERIAL

Supplementary material is linked to the online version of the paper at www.jidonline.org, and at https://doi.org/10.1016/j.jid.2021.02.754.

Data availability statement

Not datasets are available for this manuscript.

REFERENCES

  1. Allen MM, Douds JJ, Liang SX, Desouki MM, Parkash V, Fadare O. An immunohistochemical analysis of stathmin 1 expression in uterine smooth muscle tumors: differential expression in leiomyosarcomas and leiomyomas. Int J Clin Exp Pathol 2015;8:2795–801. [PMC free article] [PubMed] [Google Scholar]
  2. Barnes EA, Kenerson HL, Jiang X, Yeung RS. Tuberin regulates E-cadherin localization: implications in epithelial-mesenchymal transition. Am J Pathol 2010;177:1765–78. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Bissler JJ, McCormack FX, Young LR, Elwing JM, Chuck G, Leonard JM, et al. Sirolimus for angiomyolipoma in tuberous sclerosis complex or lymphangioleiomyomatosis. N Engl J Med 2008;358:140–51. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Brown EJ, Beal PA, Keith CT, Chen J, Shin TB, Schreiber SL. Control of p70 s6 kinase by kinase activity of FRAP in vivo. Nature 1995;377:441–6. [DOI] [PubMed] [Google Scholar]
  5. Budde K, Gaedeke J. Tuberous sclerosis complex-associated angiomyolipomas: focus on mTOR inhibition. Am J Kidney Dis 2012;59:276–83. [DOI] [PubMed] [Google Scholar]
  6. Chen XG, Liu F, Song XF, Wang ZH, Dong ZQ, Hu ZQ, et al. Rapamycin regulates Akt and ERK phosphorylation through mTORC1 and mTORC2 signaling pathways. Mol Carcinog 2010;49:603–10. [DOI] [PubMed] [Google Scholar]
  7. Comșa Ș, Cîmpean AM, Raica M. The story of MCF-7 breast cancer cell line: 40 years of experience in research. Anticancer Res 2015;35:314–54. [PubMed] [Google Scholar]
  8. Darling TN. Topical sirolimus to treat tuberous sclerosis complex (TSC). JAMA Dermatol 2018;154:761–2. [DOI] [PubMed] [Google Scholar]
  9. Darling TN, Pacheco-Rodriguez G, Gorio A, Lesma E, Walker C, Moss J. Lymphangioleiomyomatosis and TSC2−/− cells. Lymphat Res Biol 2010;8:59–69. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. El-Chemaly S, Taveira-Dasilva A, Goldberg HJ, Peters E, Haughey M, Bienfang D, et al. Sirolimus and autophagy inhibition in lymphangioleiomyomatosis: results of a Phase I clinical trial. Chest 2017;151:1302–10. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. El-Hashemite N, Walker V, Kwiatkowski DJ. Estrogen enhances whereas tamoxifen retards development of Tsc mouse liver hemangioma: a tumor related to renal angiomyolipoma and pulmonary lymphangioleiomyomatosis. Cancer Res 2005;65:2474–81. [DOI] [PubMed] [Google Scholar]
  12. Finlay GA, York B, Karas RH, Fanburg BL, Zhang H, Kwiatkowski DJ, et al. Estrogen-induced smooth muscle cell growth is regulated by tuberin and associated with altered activation of platelet-derived growth factor receptor-beta and ERK-1/2. J Biol Chem 2004;279:23114–22. [DOI] [PubMed] [Google Scholar]
  13. Goncharova EA, Goncharov DA, Eszterhas A, Hunter DS, Glassberg MK, Yeung RS, et al. Tuberin regulates p70 S6 kinase activation and ribosomal protein S6 phosphorylation. A role for the TSC2 tumor suppressor gene in pulmonary lymphangioleiomyomatosis (LAM). J Biol Chem 2002;277:30958–67. [DOI] [PubMed] [Google Scholar]
  14. Govindarajan B, Willoughby L, Band H, Curatolo AS, Veledar E, Chen S, et al. Cooperative benefit for the combination of rapamycin and imatinib in tuberous sclerosis complex neoplasia. Vasc Cell 2012;4:11. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Haczynski J, Tarkowski R, Jarzabek K, Slomczynska M, Wolczynski S, Magoffin DA, et al. Human cultured skin fibroblasts express estrogen receptor alpha and beta. Int J Mol Med 2002;10:149–53. [PubMed] [Google Scholar]
  16. Haczynski J, Tarkowski R, Jarzabek K, Wolczynski S, Magoffin DA, Czarnocki KJ, et al. Differential effects of estradiol, raloxifene and tamoxifen on estrogen receptor expression in cultured human skin fibroblasts. Int J Mol Med 2004;13:903–8. [PubMed] [Google Scholar]
  17. Hanahan D, Weinberg RA. Hallmarks of cancer: the next generation. Cell 2011;144:646–74. [DOI] [PubMed] [Google Scholar]
  18. Henske EP, Jóźwiak S, Kingswood JC, Sampson JR, Thiele EA. Tuberous sclerosis complex. Nat Rev Dis Primers 2016;2:16035. [DOI] [PubMed] [Google Scholar]
  19. Huang J, Manning BD. The TSC1-TSC2 complex: a molecular switchboard controlling cell growth. Biochem J 2008;412:179–90. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Johnson CE, Hunt DK, Wiltshire M, Herbert TP, Sampson JR, Errington RJ, et al. Endoplasmic reticulum stress and cell death in mTORC1-overactive cells is induced by nelfinavir and enhanced by chloroquine. Mol Oncol 2015;9:675–88. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Katzenellenbogen BS, Kendra KL, Norman MJ, Berthois Y. Proliferation, hormonal responsiveness, and estrogen receptor content of MCF-7 human breast cancer cells grown in the short-term and long-term absence of estrogens. Cancer Res 1987;47:4355–60. [PubMed] [Google Scholar]
  22. Knudsen ES, Witkiewicz AK. The strange case of CDK4/6 inhibitors: mechanisms, resistance, and combination strategies. Trends Cancer 2017;3:39–55. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Koenig MK, Bell CS, Hebert AA, Roberson J, Samuels JA, Slopis JM, et al. Efficacy and safety of topical rapamycin in patients with facial angiofibromas secondary to tuberous sclerosis complex: the TREATMENT randomized clinical trial. JAMA Dermatol 2018;154:773–80. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Kristof AS, Pacheco-Rodriguez G, Schremmer B, Moss J. LY303511 (2-piperazinyl-8-phenyl-4H-1-benzopyran-4-one) acts via phosphatidylinositol 3-kinase-independent pathways to inhibit cell proliferation via mammalian target of rapamycin (mTOR)- and non-mTOR-dependent mechanisms. J Pharmacol Exp Ther 2005;314:1134–43. [DOI] [PubMed] [Google Scholar]
  25. Krueger DA, Care MM, Holland K, Agricola K, Tudor C, Mangeshkar P, et al. Everolimus for subependymal giant-cell astrocytomas in tuberous sclerosis. N Engl J Med 2010;363:1801–11. [DOI] [PubMed] [Google Scholar]
  26. Laplante M, Sabatini DM. mTOR signaling at a glance. J Cell Sci 2009;122:3589–94. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Laplante M, Sabatini DM. mTOR signaling in growth control and disease. Cell 2012;149:274–93. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Li S, Takeuchi F, Wang JA, Fan Q, Komurasaki T, Billings EM, et al. Mesenchymal-epithelial interactions involving epiregulin in tuberous sclerosis complex hamartomas. Proc Natl Acad Sci USA 2008;105:3539–44. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Li S, Takeuchi F, Wang JA, Fuller C, Pacheco-Rodriguez G, Moss J, et al. MCP-1 overexpressed in tuberous sclerosis lesions acts as a paracrine factor for tumor development. J Exp Med 2005;202:617–24. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Li S, Thangapazham RL, Wang JA, Rajesh S, Kao TC, Sperling L, et al. Human TSC2-null fibroblast-like cells induce hair follicle neogenesis and hamartoma morphogenesis. Nat Commun 2011;2:235. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Liu GY, Sabatini DM. mTOR at the nexus of nutrition, growth, ageing and disease. Nat Rev Mol Cell Biol 2020;21:183–203. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Lloyd AC. The regulation of cell size. Cell 2013;154:1194–205. [DOI] [PubMed] [Google Scholar]
  33. Malissen N, Vergely L, Simon M, Roubertie A, Malinge MC, Bessis D. Long-term treatment of cutaneous manifestations of tuberous sclerosis complex with topical 1% sirolimus cream: a prospective study of 25 patients. J Am Acad Dermatol 2017;77:464–72.e3. [DOI] [PubMed] [Google Scholar]
  34. Manning BD, Cantley LC. AKT/PKB signaling: navigating downstream. Cell 2007;129:1261–74. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. McCormack FX, Inoue Y, Moss J, Singer LG, Strange C, Nakata K, et al. Efficacy and safety of sirolimus in lymphangioleiomyomatosis. N Engl J Med 2011;364:1595–606. [DOI] [PMC free article] [PubMed] [Google Scholar]
  36. Menon S, Dibble CC, Talbott G, Hoxhaj G, Valvezan AJ, Takahashi H, et al. Spatial control of the TSC complex integrates insulin and nutrient regulation of mTORC1 at the lysosome. Cell 2014;156:771–85. [DOI] [PMC free article] [PubMed] [Google Scholar]
  37. Nadiminti U, Arbiser JL. Rapamycin (sirolimus) as a steroid-sparing agent in dermatomyositis. J Am Acad Dermatol 2005;52(Suppl. 1):17–9. [DOI] [PubMed] [Google Scholar]
  38. Nathan N, Wang JA, Li S, Cowen EW, Haughey M, Moss J, et al. Improvement of tuberous sclerosis complex (TSC) skin tumors during long-term treatment with oral sirolimus. J Am Acad Dermatol 2015;73:802–8. [DOI] [PMC free article] [PubMed] [Google Scholar]
  39. Parkhitko A, Myachina F, Morrison TA, Hindi KM, Auricchio N, Karbowniczek M, et al. Tumorigenesis in tuberous sclerosis complex is autophagy and p62/sequestosome 1 (SQSTM1)-dependent. Proc Natl Acad Sci USA 2011;108:12455–60. [DOI] [PMC free article] [PubMed] [Google Scholar]
  40. Potter CJ, Huang H, Xu T. Drosophila Tsc1 functions with Tsc2 to antagonize insulin signaling in regulating cell growth, cell proliferation, and organ size. Cell 2001;105:357–68. [DOI] [PubMed] [Google Scholar]
  41. Rosner M, Fuchs C, Siegel N, Valli A, Hengstschläger M. Functional interaction of mammalian target of rapamycin complexes in regulating mammalian cell size and cell cycle. Hum Mol Genet 2009;18:3298–310. [DOI] [PMC free article] [PubMed] [Google Scholar]
  42. Sarbassov DD, Ali SM, Sengupta S, Sheen JH, Hsu PP, Bagley AF, et al. Prolonged rapamycin treatment inhibits mTORC2 assembly and Akt/PKB. Mol Cell 2006;22:159–68. [DOI] [PubMed] [Google Scholar]
  43. Saxton RA, Sabatini DM. mTOR signaling in growth, metabolism, and disease. Cell 2017;168:960–76. [DOI] [PMC free article] [PubMed] [Google Scholar]
  44. Schipany K, Rosner M, Ionce L, Hengstschläger M, Kovacic B. eIF3 controls cell size independently of S6K1-activity. Oncotarget 2015;6:24361–75. [DOI] [PMC free article] [PubMed] [Google Scholar]
  45. Sherr CJ, Roberts JM. CDK inhibitors: positive and negative regulators of G1-phase progression. Genes Dev 1999;13:1501–12. [DOI] [PubMed] [Google Scholar]
  46. Sun SY, Rosenberg LM, Wang X, Zhou Z, Yue P, Fu H, et al. Activation of Akt and eIF4E survival pathways by rapamycin-mediated mammalian target of rapamycin inhibition. Cancer Res 2005;65:7052–8. [DOI] [PubMed] [Google Scholar]
  47. Taveira-DaSilva AM, Hathaway O, Stylianou M, Moss J. Changes in lung function and chylous effusions in patients with lymphangioleiomyomatosis treated with sirolimus. Ann Intern Med 2011;154. 797–293. [DOI] [PMC free article] [PubMed] [Google Scholar]
  48. Taveira-DaSilva AM, Moss J. Clinical features, epidemiology, and therapy of lymphangioleiomyomatosis. Clin Epidemiol 2015;7:249–57. [DOI] [PMC free article] [PubMed] [Google Scholar]
  49. Teng JM, Cowen EW, Wataya-Kaneda M, Gosnell ES, Witman PM, Hebert AA, et al. Dermatologic and dental aspects of the 2012 International Tuberous Sclerosis Complex Consensus Statements. JAMA Dermatol 2014;150:1095–101. [DOI] [PMC free article] [PubMed] [Google Scholar]
  50. Trindade AJ, Medvetz DA, Neuman NA, Myachina F, Yu J, Priolo C, et al. MicroRNA-21 is induced by rapamycin in a model of tuberous sclerosis (TSC) and lymphangioleiomyomatosis (LAM). PLoS One 2013;8:e60014. [DOI] [PMC free article] [PubMed] [Google Scholar]
  51. Tumaneng K, Russell RC, Guan KL. Organ size control by Hippo and TOR pathways. Curr Biol 2012;22:R368–79. [DOI] [PMC free article] [PubMed] [Google Scholar]
  52. Tyburczy ME, Dies KA, Glass J, Camposano S, Chekaluk Y, Thorner AR, et al. Mosaic and intronic mutations in TSC1/TSC2 explain the majority of TSC patients with no mutation identified by conventional testing. PLoS Genet 2015;11:e1005637. [DOI] [PMC free article] [PubMed] [Google Scholar]
  53. Wan X, Harkavy B, Shen N, Grohar P, Helman LJ. Rapamycin induces feedback activation of Akt signaling through an IGF-1R-dependent mechanism. Oncogene 2007;26:1932–40. [DOI] [PubMed] [Google Scholar]
  54. Wataya-Kaneda M, Nakamura A, Tanaka M, Hayashi M, Matsumoto S, Yamamoto K, et al. Efficacy and safety of topical sirolimus therapy for facial angiofibromas in the tuberous sclerosis complex : a randomized clinical trial. JAMA Dermatol 2017;153:39–48. [DOI] [PubMed] [Google Scholar]
  55. Wataya-Kaneda M, Tanaka M, Yang L, Yang F, Tsuruta D, Nakamura A, et al. Clinical and histologic analysis of the efficacy of topical rapamycin therapy against hypomelanotic macules in tuberous sclerosis complex. JAMA Dermatol 2015;151:722–30. [DOI] [PubMed] [Google Scholar]
  56. Yu J, Parkhitko A, Henske EP. Autophagy: an ‘Achilles’ heel of tumorigenesis in TSC and LAM. Autophagy 2011;7:1400–1. [DOI] [PMC free article] [PubMed] [Google Scholar]
  57. Yu JJ, Robb VA, Morrison TA, Ariazi EA, Karbowniczek M, Astrinidis A, et al. Estrogen promotes the survival and pulmonary metastasis of tuberin-null cells. Proc Natl Acad Sci USA 2009;106:2635–40. [DOI] [PMC free article] [PubMed] [Google Scholar]

Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

1
NIHMS1866582-supplement-1.pdf (225.9KB, pdf)

Data Availability Statement

Not datasets are available for this manuscript.

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