CDK4/6 inhibition mitigates stem cell damage in a novel model for taxane‐induced alopecia
2019; Springer Nature; Volume: 11; Issue: 10 Linguagem: Inglês
10.15252/emmm.201911031
ISSN1757-4684
AutoresTalveen S. Purba, Kayumba Ng’andu, Lars Brunken, Eleanor Smart, Ellen Sullivan Mitchell, N. M. K. Nik Hassan, Aaron O’Brien, Charlotte E. L. Mellor, Jennifer Jackson, Asim Shahmalak, Ralf Paus,
Tópico(s)Cancer and Skin Lesions
ResumoArticle12 September 2019Open Access Source DataTransparent process CDK4/6 inhibition mitigates stem cell damage in a novel model for taxane-induced alopecia Talveen S Purba Corresponding Author Talveen S Purba [email protected] orcid.org/0000-0003-3735-7735 Centre for Dermatology Research, School of Biological Sciences, University of Manchester & NIHR Biomedical Research Centre, Manchester, UK Search for more papers by this author Kayumba Ng'andu Kayumba Ng'andu Centre for Dermatology Research, School of Biological Sciences, University of Manchester & NIHR Biomedical Research Centre, Manchester, UK Search for more papers by this author Lars Brunken Lars Brunken Monasterium Laboratory – Skin & Hair Research Solutions GmbH, Münster, Germany Search for more papers by this author Eleanor Smart Eleanor Smart orcid.org/0000-0003-4571-4384 Centre for Dermatology Research, School of Biological Sciences, University of Manchester & NIHR Biomedical Research Centre, Manchester, UK Search for more papers by this author Ellen Mitchell Ellen Mitchell Centre for Dermatology Research, School of Biological Sciences, University of Manchester & NIHR Biomedical Research Centre, Manchester, UK Search for more papers by this author Nashat Hassan Nashat Hassan Centre for Dermatology Research, School of Biological Sciences, University of Manchester & NIHR Biomedical Research Centre, Manchester, UK Search for more papers by this author Aaron O'Brien Aaron O'Brien Centre for Dermatology Research, School of Biological Sciences, University of Manchester & NIHR Biomedical Research Centre, Manchester, UK Search for more papers by this author Charlotte Mellor Charlotte Mellor Centre for Dermatology Research, School of Biological Sciences, University of Manchester & NIHR Biomedical Research Centre, Manchester, UK Search for more papers by this author Jennifer Jackson Jennifer Jackson Centre for Dermatology Research, School of Biological Sciences, University of Manchester & NIHR Biomedical Research Centre, Manchester, UK Search for more papers by this author Asim Shahmalak Asim Shahmalak Crown Clinic, Manchester, UK Search for more papers by this author Ralf Paus Corresponding Author Ralf Paus [email protected] [email protected] orcid.org/0000-0002-3492-9358 Centre for Dermatology Research, School of Biological Sciences, University of Manchester & NIHR Biomedical Research Centre, Manchester, UK Monasterium Laboratory – Skin & Hair Research Solutions GmbH, Münster, Germany Dr. Phillip Frost Department of Dermatology & Cutaneous Surgery, University of Miami Miller School of Medicine, Miami, FL, USA Search for more papers by this author Talveen S Purba Corresponding Author Talveen S Purba [email protected] orcid.org/0000-0003-3735-7735 Centre for Dermatology Research, School of Biological Sciences, University of Manchester & NIHR Biomedical Research Centre, Manchester, UK Search for more papers by this author Kayumba Ng'andu Kayumba Ng'andu Centre for Dermatology Research, School of Biological Sciences, University of Manchester & NIHR Biomedical Research Centre, Manchester, UK Search for more papers by this author Lars Brunken Lars Brunken Monasterium Laboratory – Skin & Hair Research Solutions GmbH, Münster, Germany Search for more papers by this author Eleanor Smart Eleanor Smart orcid.org/0000-0003-4571-4384 Centre for Dermatology Research, School of Biological Sciences, University of Manchester & NIHR Biomedical Research Centre, Manchester, UK Search for more papers by this author Ellen Mitchell Ellen Mitchell Centre for Dermatology Research, School of Biological Sciences, University of Manchester & NIHR Biomedical Research Centre, Manchester, UK Search for more papers by this author Nashat Hassan Nashat Hassan Centre for Dermatology Research, School of Biological Sciences, University of Manchester & NIHR Biomedical Research Centre, Manchester, UK Search for more papers by this author Aaron O'Brien Aaron O'Brien Centre for Dermatology Research, School of Biological Sciences, University of Manchester & NIHR Biomedical Research Centre, Manchester, UK Search for more papers by this author Charlotte Mellor Charlotte Mellor Centre for Dermatology Research, School of Biological Sciences, University of Manchester & NIHR Biomedical Research Centre, Manchester, UK Search for more papers by this author Jennifer Jackson Jennifer Jackson Centre for Dermatology Research, School of Biological Sciences, University of Manchester & NIHR Biomedical Research Centre, Manchester, UK Search for more papers by this author Asim Shahmalak Asim Shahmalak Crown Clinic, Manchester, UK Search for more papers by this author Ralf Paus Corresponding Author Ralf Paus [email protected] [email protected] orcid.org/0000-0002-3492-9358 Centre for Dermatology Research, School of Biological Sciences, University of Manchester & NIHR Biomedical Research Centre, Manchester, UK Monasterium Laboratory – Skin & Hair Research Solutions GmbH, Münster, Germany Dr. Phillip Frost Department of Dermatology & Cutaneous Surgery, University of Miami Miller School of Medicine, Miami, FL, USA Search for more papers by this author Author Information Talveen S Purba *,1, Kayumba Ng'andu1, Lars Brunken2, Eleanor Smart1, Ellen Mitchell1, Nashat Hassan1, Aaron O'Brien1, Charlotte Mellor1, Jennifer Jackson1, Asim Shahmalak3 and Ralf Paus *,*,1,2,4 1Centre for Dermatology Research, School of Biological Sciences, University of Manchester & NIHR Biomedical Research Centre, Manchester, UK 2Monasterium Laboratory – Skin & Hair Research Solutions GmbH, Münster, Germany 3Crown Clinic, Manchester, UK 4Dr. Phillip Frost Department of Dermatology & Cutaneous Surgery, University of Miami Miller School of Medicine, Miami, FL, USA *Corresponding author. Tel: +44 161 275 5382; E-mail: [email protected] *Corresponding author. Tel: +1 305 243 7870; E-mails: [email protected]; [email protected] EMBO Mol Med (2019)11:e11031https://doi.org/10.15252/emmm.201911031 PDFDownload PDF of article text and main figures. Peer ReviewDownload a summary of the editorial decision process including editorial decision letters, reviewer comments and author responses to feedback. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Abstract Taxanes are a leading cause of severe and often permanent chemotherapy-induced alopecia. As the underlying pathobiology of taxane chemotherapy-induced alopecia remains poorly understood, we investigated how paclitaxel and docetaxel damage human scalp hair follicles in a clinically relevant ex vivo organ culture model. Paclitaxel and docetaxel induced massive mitotic defects and apoptosis in transit amplifying hair matrix keratinocytes and within epithelial stem/progenitor cell-rich outer root sheath compartments, including within Keratin 15+ cell populations, thus implicating direct damage to stem/progenitor cells as an explanation for the severity and permanence of taxane chemotherapy-induced alopecia. Moreover, by administering the CDK4/6 inhibitor palbociclib, we show that transit amplifying and stem/progenitor cells can be protected from paclitaxel cytotoxicity through G1 arrest, without premature catagen induction and additional hair follicle damage. Thus, the current study elucidates the pathobiology of taxane chemotherapy-induced alopecia, highlights the paramount importance of epithelial stem/progenitor cell-protective therapy in taxane-based oncotherapy, and provides preclinical proof-of-principle in a healthy human (mini-) organ that G1 arrest therapy can limit taxane-induced tissue damage. Synopsis Taxane chemotherapy causes alopecia. This study shows how taxanes are toxic to dividing stem cells and transit amplifying cells in the hair follicle. A CDK4/6 inhibitor, that blocks cell division, can antagonise taxane-induced damage in the hair follicle. Paclitaxel and docetaxel, chemotherapies that cause alopecia, promote mitotic defects and apoptosis in proliferating hair follicle stem/progenitor and transit amplifying cell compartments. Mitotic defects are marked by profound increases in phospho-histone H3 immunoreactivity, micronucleation and transcriptional arrest. Pharmacological CDK4/6 inhibition potently induces G1 arrest in the human hair follicle. Pharmacological G1 arrest antagonises the mitosis-targeting cytotoxicity taxanes, thereby protecting stem/progenitor cells and transit amplifying cells from damage. Introduction Chemotherapy-induced alopecia is a highly distressing adverse effect of cancer treatment and can persist long after the completion of chemotherapy treatment regimens (Paus et al, 2013). As many as 8% of patients have been found to be at risk of rejecting chemotherapy due to the psychosocial burden imposed by chemotherapy-induced alopecia, which is detrimental to patient self-esteem, body image and quality of life (McGarvey et al, 2001) especially when the effects of chemotherapy are permanent (Freites-Martinez et al, 2019). The only currently available preventive treatment for chemotherapy-induced alopecia is scalp cooling, whose clinical efficacy is as yet unsatisfactory and difficult to predict, especially with taxane chemotherapy-induced alopecia (Friedrichs & Carstensen, 2014; Cigler et al, 2015; Rugo et al, 2017; Rice et al, 2018). Furthermore, scalp cooling does not extend protection against hair loss to other body sites of cosmetic, cultural, religious and psychosocial relevance, e.g. eyebrow, beard or pubic hair. Therefore, novel and effective chemotherapy-induced alopecia prevention strategies need to be urgently developed and translated into clinical practice. This can only be achieved through the generation of promising preclinical data in appropriate human models that are as close as possible to clinical chemotherapy-induced alopecia (Bodó et al, 2007, 2009; Paus et al, 2013; Böhm et al, 2014; Sharova et al, 2014; Yoon et al, 2016). To date, preclinical chemotherapy-induced alopecia research models have been developed to study how doxorubicin and cyclophosphamide damage the human hair follicle (Bodó et al, 2007, 2009; Paus et al, 2013; Böhm et al, 2014; Sharova et al, 2014; Yoon et al, 2016). However, the field currently lacks a model of how taxanes, major current oncotherapeutics used to treat breast and lung cancer, damage the human hair follicle and cause chemotherapy-induced alopecia. The need for such a model is becoming increasingly important, given the abundance of reports describing permanent taxane chemotherapy-induced alopecia (Prevezas et al, 2009; Tallon et al, 2010; Miteva et al, 2011; Palamaras et al, 2011; Kluger et al, 2012; Tosti et al, 2013; Sibaud et al, 2016; Kang et al, 2018; Martín et al, 2018). This is often reported following treatment with docetaxel, which is the subject matter of on-going lawsuits against Taxotere (docetaxel) manufacturer Sanofi (Raymond, 2019). Taxanes are microtubule-stabilising agents whose principle anti-neoplastic mode of action is through the disruption of mitosis, e.g. by promoting chromosome missegregation/cell division on multipolar spindles (Chen & Horwitz, 2002; Abal et al, 2003; Morse et al, 2005; Weaver, 2014; Zasadil et al, 2014). Taxanes are thus presumed to cause hair loss by damaging rapidly dividing matrix keratinocytes and their counterpart stem/progenitor cells, required for healthy hair growth and hair follicle cycling (Paus & Cotsarelis, 1999; Garza et al, 2011; Paus et al, 2013; Purba et al, 2016, 2017a,b; Gao et al, 2019; Huang et al, 2019). However, the effects of taxane chemotherapy on the human hair follicle remain to be systematically examined. Therefore, in the current study we aimed to develop a clinically relevant ex vivo assay for studying and experimentally manipulating taxane toxicology in healthy human hair follicles to elucidate how taxanes cause chemotherapy-induced alopecia. To do so, we used a well-established ex vivo organ culture model (Langan et al, 2015) to dissect how the taxanes paclitaxel and docetaxel damage full-length human anagen VI scalp hair follicles. Specifically, we focused on how the mitosis-targeting cytotoxicity of taxanes affected highly proliferative hair-forming matrix keratinocytes (Purba et al, 2016, 2017a). Furthermore, we also asked whether taxanes damage (relatively slow-cycling) epithelial stem/progenitor cell niches in the hair follicle outer root sheath (Garza et al, 2011; Purba et al, 2017b), especially as irreversible stem/progenitor cell damage may lead to permanent chemotherapy-induced alopecia (Paus et al, 2013). Given that the mode of action of taxanes relies upon direct interference with the cell cycle (i.e. mitosis) to initiate tumour cell death (Chen & Horwitz, 2002; Abal et al, 2003; Morse et al, 2005; Weaver, 2014; Zasadil et al, 2014), we further probed in our newly developed taxane chemotherapy-induced alopecia model the working hypothesis that pharmacologically induced cell cycle arrest protects against taxane-induced human hair follicle damage (Shah & Schwartz, 2001; Blagosklonny, 2011; McClendon et al, 2012; Paus et al, 2013; Beaumont et al, 2016). To achieve this, we used the G1 arresting CDK4/6 inhibitor palbociclib, which is used in the treatment of hormone (oestrogen and/or progesterone) receptor-positive HER2-negative breast cancer (Ro et al, 2015). Palbociclib was employed as previous reports have described that pharmacological CDK4/6 inhibition can protect against chemotherapy-induced acute kidney injury (DiRocco et al, 2014; Pabla et al, 2015) and chemotherapy-induced haematopoietic stem cell exhaustion (He et al, 2017). With this experimental approach, we have probed whether palbociclib is a candidate chemotherapy-induced alopecia-preventive lead agent and sought to obtain a proof-of-principle that pharmacologically induced cell cycle arrest can protect transit amplifying matrix keratinocytes and epithelial stem/progenitor cells within their native tissue habitat from chemotherapy-induced apoptosis, ideally without promoting premature hair follicle regression (catagen) (Paus et al, 2013). Results Taxanes induce the massive accumulation of phospho-histone H3+ cells in the anagen matrix of human scalp hair follicles To determine the effects of taxane chemotherapy on the most rapidly proliferating keratinocytes of human scalp hair follicles, i.e. anagen hair matrix keratinocytes (Purba et al, 2016, 2017a), we first treated microdissected, organ-cultured human hair follicles (Langan et al, 2015) with 100 nM paclitaxel for 24 h, i.e. at a dose that resembles reported plasma concentrations of paclitaxel 20 h post-infusion (Zasadil et al, 2014). In situ cell cycle analyses (Purba et al, 2016) revealed that paclitaxel exerts mitosis-specific effects on proliferating human hair follicle matrix keratinocytes, rather than globally inhibiting proliferation. In fact, as a marker of global cell cycle activity, an analysis of the total number of Ki-67+ cells in the hair matrix showed no statistically significant difference between vehicle- and paclitaxel-treated hair follicles (Fig 1A). Moreover, EdU incorporation within the hair matrix ex vivo revealed no significant effect on the number of cells in S-phase (i.e. undergoing DNA synthesis) following 24-h paclitaxel treatment (Fig 1B). Figure 1. Taxanes increase the number of phospho-histone H3+ cells in the human anagen hair follicle matrix A, B. 100 nM paclitaxel treatment of human hair follicles (HFs) in organ culture for 24 h does not significantly affect the total number of Ki-67+ cells (A) and EdU+ cells (B) (S-phase) in the hair matrix. Unpaired t-test performed using N of 9–12 HFs from three patients. C. 100 nM paclitaxel treatment (24 h) significantly (P ≤ 0.0001) increases the number of mitotic phospho-histone H3 (pH3)+ cells in the hair matrix. Welch's t-test performed using N of nine HFs from three patients. D. 100 nM docetaxel treatment (24 h) significantly (P = 0.0004) increases the number of pH3+ cells in the hair matrix. Unpaired t-test performed using N of 8–9 HFs from three patients. E. Representative immunofluorescence images highlight the effects of 24-h 100 nM taxane treatment on (i) Ki-67 expression [paclitaxel]; (ii) EdU incorporation and pH3 immunoreactivity [paclitaxel]; (iii) pH3 immunoreactivity [docetaxel]. 20-μm scale. Data information: Error bars are standard error of the mean. Values plotted represent the mean number of positive cells counted per HF analysed. Source data are available online for this figure. Source Data for Figure 1 [emmm201911031-sup-0002-SDataFig1.pdf] Download figure Download PowerPoint However, paclitaxel promoted a large and significant increase in the number of cells labelled positively with the mitosis-specific marker phospho-histone H3 (pH3) (Crosio et al, 2009; Purba et al, 2016; Fig 1C). Treatment with 100 nM docetaxel for 24 h also induced a profound increase in the number of pH3+ cells in the anagen hair matrix, confirming this effect on the human hair follicle as a shared feature of taxanes (Fig 1D). These results reveal that taxanes promote the abnormal accumulation of mitotic (i.e. pH3+) keratinocytes in the hair matrix, signifying mitotic arrest, without affecting G1/S cell cycle progression (Fig 1Ei–iii). Together, these observations are consistent with the recognized mitosis-specific cytotoxicity of taxanes (Jordan et al, 1996; Chen & Horwitz, 2002; Abal et al, 2003; Morse et al, 2005; Weaver, 2014; Zasadil et al, 2014; Mitchison et al, 2017) and validate the usefulness of our ex vivo model for studying taxane toxicity in a rapidly proliferating, healthy human mini-organ. Taxanes promote micronucleation, transcriptional arrest and apoptosis in hair matrix keratinocytes To examine the nuclear morphology of matrix keratinocytes following 24-h paclitaxel and docetaxel treatment, we stained nuclei with Hoechst 33342. Paclitaxel promoted the extensive accumulation of irregular and shrunken nuclei that localised specifically to the most proliferative region of the hair matrix (Fig 2A; i.e. predominantly below the critical line of Auber; Purba et al, 2016, 2017a). Docetaxel treatment (24 h) also promoted the formation of irregular nuclear bodies within the hair matrix, albeit not to the extent seen following paclitaxel treatment (Fig 2B–E). These nuclear abnormalities are likely a consequence of mitosis defects, i.e. chromosome missegregation (Chen & Horwitz, 2002; Abal et al, 2003; Morse et al, 2005; Weaver, 2014; Zasadil et al, 2014), giving rise to micronucleated cells. This well-defined taxane-induced phenomenon (Morse et al, 2005; Mitchison et al, 2017) is a hallmark of "mitotic catastrophe", whereby failed mitosis ultimately leads to cell death or senescence (Vakifahmetoglu et al, 2008; Vitale et al, 2011). Figure 2. Taxanes induce micronucleation in the human anagen hair follicle matrix A, B. The presence of micronucleated cells in the hair matrix in paclitaxel- and docetaxel-treated (100 nM, 24 h) hair follicles (HF) compared to vehicle is significant (P ≤ 0.0001). Mann–Whitney U test performed using N of 12–13 HFs (paclitaxel) and 8 HFs (docetaxel) from three patients. Error bars are standard error of the mean. C. Hoechst 33342 staining of healthy cell nuclei comprising the hair matrix (lined) and dermal papilla in untreated (vehicle) human HFs. 20-μm scale. D. Paclitaxel treatment (100 nM, 24 h) induces the formation of micronucleated bodies, as visualised by Hoechst 33342 staining (arrows), localising to the proliferative region of the hair matrix. i—20-μm scale; ii—10-μm scale. E. 100 nM docetaxel treatment was also seen to promote the formation of micronucleated bodies (arrows). 10-μm scale. Source data are available online for this figure. Source Data for Figure 2 [emmm201911031-sup-0003-SDataFig2.pdf] Download figure Download PowerPoint Next, as a toxicological read-out parameter, we analysed how paclitaxel treatment affects in situ global RNA synthesis in the hair matrix through the detection of ethynyl uridine (EU) incorporated during ex vivo human hair follicle organ culture, using the recently described methodology (Purba et al, 2018). The incorporation of EU was found to be significantly decreased within keratinocytes of the proliferative hair matrix (Fig 3A and B). However, this did not represent a generalised, hair follicle-wide systemic RNA synthesis inhibitory effect (e.g. as seen following broad spectrum CDK inhibition in the human hair follicle; Purba et al, 2018). Instead, the population of cells in the hair matrix that failed to incorporate EU was mainly restricted to cells demarcated by pH3 immunoreactivity (Fig 3C). This demonstrates that RNA transcription is attenuated in abnormally dividing/arrested hair matrix keratinocytes following paclitaxel treatment. This could contribute towards the cytotoxicity of taxanes in the hair follicle, as transcriptional arrest during abnormal mitosis may promote cell death (Blagosklonny, 2007). Figure 3. Paclitaxel blocks nascent transcription and significantly increases cleaved caspase-3 immunoreactivity in hair matrix keratinocytes A. Nascent RNA synthesis, as detected by ethynyl uridine (EU) incorporation, is blocked within clusters of nuclei in the hair matrix (arrows) following paclitaxel treatment (100 nM, 24 h). 20-μm scale. B. Quantitative analysis highlights a significant (P ≤ 0.0001) decrease in the number of EU+ nuclei following 24-h paclitaxel treatment. Welch's t-test performed using N of 11–12 hair follicles (HFs) from three patients. C. Representative dual fluorescence stain highlights how EU incorporation in the hair matrix is blocked within the pH3+ cell population that accumulates in response to paclitaxel treatment (see Fig 1). 10-μm scale. D. Cleaved caspase-3 expression in the hair matrix following 24-h paclitaxel treatment. 20-μm scale. E. 100 nM paclitaxel treatment significantly (P = 0.0016) increases the number of cleaved caspase-3+ cells in the hair matrix after 24 h. Mann–Whitney U test performed using N of 16–18 HFs from five patients. Data information: Values plotted represent the mean number of positive cells counted per HF analysed. Error bars are standard error of the mean. Source data are available online for this figure. Source Data for Figure 3 [emmm201911031-sup-0004-SDataFig3.pdf] Download figure Download PowerPoint Paclitaxel treatment significantly increased the number of apoptotic (cleaved caspase-3+) cells within the proliferative hair matrix following 24-h treatment (Fig 3D and E). However, 24-h paclitaxel treatment did not immediately increase cleaved caspase-3 immunoreactivity in hair follicles from all donors, in contrast to the consistent accumulation of pH3+ cells in all hair follicles treated with paclitaxel for 24 h, irrespective of donor (Fig 1C). This suggests substantial interindividual variability in the sensitivity of hair matrix keratinocytes to switch on the apoptotic machinery, e.g. as a reflection of the local balance of Bcl-2 and Fas expression in the hair matrix (Müller-Röver et al, 1999; Sharov et al, 2004; Sharova et al, 2014) following the mitosis-targeting damage inflicted by paclitaxel. This could correspond to the highly variable severity of hair loss seen in the clinic in response to identical chemotherapy regimens (Chung et al, 2013; Paus et al, 2013). To dissect and model the early effects of taxanes on the human hair follicle beyond the initial 24-h treatment period, hair follicles treated with paclitaxel and docetaxel were washed out of drug-containing medium and permitted to continue in organ culture for an additional 24- to 48-h period. Analysis at this time point showed consistent and sustained increases in the number of pH3+ and cleaved caspase-3+ cells in the hair matrix (Appendix Fig S1A–F), indicating lasting hair follicle cytotoxicity imposed by taxanes even after drug washout. Taxanes induce the accumulation of cleaved caspase-3+ and pH3+ cells within the stem/progenitor-rich outer root sheath Hair follicle epithelial stem/progenitor cell damage has never been documented for taxane chemotherapy, yet would plausibly explain the permanency of hair loss in taxane chemotherapy-induced alopecia (Paus et al, 2013; Gao et al, 2019). Therefore, we next investigated the effect of anti-mitotic taxane chemotherapy on the proliferative, yet slower cycling, stem/progenitor cell-containing outer root sheath of human anagen VI scalp hair follicles (Purba et al, 2017b). We found that treatment of hair follicles with paclitaxel or docetaxel significantly increased the number of cleaved caspase-3+ and pH3+ cells in the outer root sheath (Appendix Fig S1G–K). Dual immunofluorescence staining for cleaved caspase-3 or pH3, alongside the hair follicle epithelial stem/progenitor cell marker keratin 15 (K15) (Cotsarelis, 2006; Purba et al, 2014) in paclitaxel-treated hair follicles, showed that accumulating cleaved caspase-3+ and pH3+ cells localise within and immediately adjacent to K15+ expressing cells of the bulge stem cell region and proximal bulb outer root sheath progenitor compartment (Fig 4Ai–ii) (Purba et al, 2014, 2015). Figure 4. Taxanes induce apoptosis and mitotic defects within human hair follicle K15+ epithelial stem/progenitor cell niches A. Paclitaxel treatment (Ai) promotes mitotic arrest (orange arrows/pH3+ cells) and apoptosis (white arrows/caspase-3+ cells), within the K15+ bulge and K15+ proximal bulb outer root sheath (pbORS) stem/progenitor cell compartments of the human hair follicle (HF). 20-μm scale. (Aii) High magnification montage demonstrating mitotic arrest (orange arrow) and apoptosis/caspase-3 positivity (white arrow) within the K15+ bulge. 10-μm scale. B. Representative immunofluorescence images of heightened cleaved caspase-3 immunoreactivity (arrows) within the K15+ bulge following extended paclitaxel organ culture experiments (see Materials and Methods). 50-μm scale. C. Graph showing significantly (P = 0.029) increased K15/caspase-3 double-positive cells within the bulge following extended paclitaxel HF organ cultures. Welch's t-test performed using N of 8–9 HFs from three patients. D. K15+ cells of the human HF bulge express Ki-67 during extended organ culture experiments. Paclitaxel treatment did not significantly affect the number of bulge K15/Ki-67 double-positive cells. Unpaired t-test performed using N of 8–9 HFs from three patients. E. Representative double immunofluorescence images of elevated γH2A.X immunoreactivity (arrows) within the K15+ bulge following extended paclitaxel organ culture experiments (see Materials and Methods). 50-μm scale. F. γH2A.X analysis showing a significant (P = 0.0065) increase in the number of cells with DNA double-strand breaks in the K15+ bulge following extended organ culture experiments. Mann–Whitney U test performed using N of 5–6 HFs from two patients. Data information: Values plotted represent the mean number of positive cells counted per HF analysed. Error bars are standard error of the mean. pbORS, proximal bulb outer root sheath. EC, extended cultures. Source data are available online for this figure. Source Data for Figure 4 [emmm201911031-sup-0005-SDataFig4.zip] Download figure Download PowerPoint An analysis of the number of cleaved caspase-3+ cells following extended human hair follicle organ cultures (see Materials and Methods) revealed that paclitaxel significantly increases apoptosis in the K15+ bulge (Fig 4B and C). Consistent with the effects observed in the hair matrix, paclitaxel did not significantly affect the number of Ki-67+ cells in the K15+ bulge (Fig 4D) (proliferation in the bulge is enhanced during ex vivo organ culture; Purba et al, 2017b). In addition, γH2A.X analysis (Mah et al, 2010) also showed that paclitaxel treatment significantly increases DNA damage in the K15+ bulge (Fig 4E and F), possibly because of prolonged mitotic arrest (Ganem & Pellman, 2012). Together, these data provide the first evidence that proliferating stem/progenitor cell populations of human anagen VI hair follicles located in distinct compartments of the outer root sheath (Purba et al, 2017b) are indeed damaged by taxane chemotherapy, at least under ex vivo conditions. This damage could play a pivotal role in the pathobiology of permanent taxane-induced alopecia and calls for the rapid development of hair follicle stem cell-protective strategies in the management of this form of chemotherapy-induced alopecia (Paus et al, 2013) to curb the alarming rise in reported cases (Prevezas et al, 2009; Tallon et al, 2010; Miteva et al, 2011; Palamaras et al, 2011; Kluger et al, 2012; Tosti et al, 2013; Kang et al, 2018; Martín et al, 2018). Targeted pharmacological inhibition of CDK4/6 induces G1 arrest in proliferating human hair matrix keratinocytes ex vivo We next aimed to identify a suitable small molecule capable of potently and specifically inducing cell cycle arrest in matrix keratinocytes during hair follicle organ culture that could be employed to counteract the mitosis-targeting cytotoxicity of taxanes documented above. In this context, cell cycle arrest therapy has previously been advocated, but a prominent paper proposing this strategy in a rodent model of chemotherapy-induced alopecia (Davis et al, 2001) was later withdrawn (Davis et al, 2002). Therefore, proof-of-principle for this potential chemotherapy-induced alopecia management strategy remains to be demonstrated, namely in human scalp hair follicles. Arguing that arres
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