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 ).

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 ).

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 ).

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.

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 ).

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 ).

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.

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. 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] 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] 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. + cell population that accumulates in response to paclitaxel treatment (see Fig Representative dual fluorescence stain highlights how EU incorporation in the hair matrix is blocked within the pH3cell 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. 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] 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. + bulge following extended paclitaxel organ culture experiments (see Representative immunofluorescence images of heightened cleaved caspase‐3 immunoreactivity (arrows) within the K15bulge 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. + bulge following extended paclitaxel organ culture experiments (see Representative double immunofluorescence images of elevated γH2A.X immunoreactivity (arrows) within the K15bulge 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. 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] 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 arresting proliferating hair matrix keratinocytes and stem/progenitor cells in the G1 phase of the cell cycle should sharply reduce hair follicle keratinocyte vulnerability to mitosis‐targeting taxane cytotoxicity, we turned to the small‐molecule palbociclib, a highly specific inhibitor of the G1 progression kinases CDK4/6, which results in a reversible cell cycle phase‐specific arrest in G1 (Fry et al, 2004). Treatment of human hair follicles ex vivo with 1 μM palbociclib for 24 h resulted in a blockade of DNA synthesis, as marked by a significant decrease in the number of matrix keratinocytes that had incorporated EdU in situ (Appendix Fig S2A). This showed the successful induction of cell cycle arrest through the stalling of G1 progression. In accordance with the induction of a G1‐specific arrest, the total number of cycling cells (Ki‐67+) was reduced within the hair matrix (Appendix Fig S2B). In addition, the fraction of mitotic cells, as marked by pH3 staining, was significantly reduced by CDK4/6 inhibition (Appendix Fig S2C). Importantly, 24‐h palbociclib treatment alone did not increase the number of cleaved caspase‐3+ cells (Appendix Fig S2D). Together, these data show that short‐term administration of palbociclib is not cytotoxic to human scalp hair follicles and successfully arrests G1/S progression in hair matrix keratinocytes (Appendix Fig S2E).

CDK4/6 inhibition blocks the cytotoxic effects of paclitaxel in the human hair follicle matrix Next, we probed how pharmacologically imposed G1 cell cycle arrest influences human hair follicle responses to paclitaxel treatment. Hair follicles were first pre‐incubated with 1 μM palbociclib for a period of 18 h. This incubation period was chosen to account for the calculated time it would take for post‐G1 human hair matrix keratinocytes to complete a single round of proliferation through S‐G2‐M and exit the cell cycle (or re‐enter G1) (Weinstein & Mooney, 1980). Following this pre‐incubation step, hair follicles were then exposed to both paclitaxel and (reapplied) palbociclib for a further 24 h, after which hair follicles were immediately isolated at the 42 h time point (alongside vehicle; palbociclib‐only; and paclitaxel‐only treatment groups; Fig 5A). Figure 5.Palbociclib blocks paclitaxel‐induced mitotic defects and apoptosis in the human hair follicle matrix A. Schematic of experimental design. Hair follicles (HFs) were pre‐incubated with the CDK4/6 inhibitor palbociclib for 18 h, followed by a further incubation period with and without paclitaxel (or paclitaxel alone) for an additional 24 h.

B. N of 6–9 HFs per condition from three patients. Ordinary one‐way ANOVA with multiple comparisons performed. Adjusted P values = 0.0001[***] and 0.0001 [****], respectively. Palbociclib‐only‐ and palbociclib plus paclitaxel dual‐treated HFs show marked reductions in Ki‐67 expression in the hair matrix beyond that seen with just 24‐h treatment (see Appendix Fig S2B ). Data also confirm that Ki‐67 expression is unaffected by paclitaxel treatment (see also Fig 1 A). Analysis performed usingof 6–9 HFs per condition from three patients. Ordinary one‐way ANOVA with multiple comparisons performed. Adjustedvalues = 0.0001[***] and 0.0001 [****], respectively.

C. P value = 0.0001) the number of pH3 + cells in the hair matrix (see also Fig N of 6–9 HFs per condition from three patients. Ordinary one‐way ANOVA with multiple comparisons performed. Data confirming that paclitaxel treatment significantly increases (adjustedvalue = 0.0001) the number of pH3cells in the hair matrix (see also Fig 1 C). This effect was not observed when paclitaxel‐treated hair follicles were pre‐ and co‐incubated with palbociclib. Analysis performed usingof 6–9 HFs per condition from three patients. Ordinary one‐way ANOVA with multiple comparisons performed.

D. P value = 0.0056). Paclitaxel‐treated hair follicles pre‐ and co‐incubated with palbociclib do not show enhanced cleaved caspase‐3 expression, contrasting with paclitaxel‐only treatment. Ordinary one‐way ANOVA with multiple comparisons performed using N of 4–5 HFs per condition. Cleaved caspase‐3 analysis in hair follicles from a patient donor that showed apoptotic sensitivity to paclitaxel treatment (see Fig 3 D and E and results, main text) (adjustedvalue = 0.0056). Paclitaxel‐treated hair follicles pre‐ and co‐incubated with palbociclib do not show enhanced cleaved caspase‐3 expression, contrasting with paclitaxel‐only treatment. Ordinary one‐way ANOVA with multiple comparisons performed usingof 4–5 HFs per condition.

E. Immunofluorescence data representing how palbociclib and/or paclitaxel affect (i) Ki‐67 expression, (ii) pH3 immunoreactivity and (iii) cleaved caspase‐3 expression. 20‐μm scale. Data information: Values plotted represent the mean number of positive cells counted per HF analysed. Error bars are standard error of the mean. 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 5 [emmm201911031-sup-0006-SDataFig5.pdf] Palbociclib‐only treatment for a period nearing 2 days caused a dramatic reduction in Ki‐67 expression within matrix keratinocytes (Fig 5B), well beyond the decreases seen after 24 h of treatment (Appendix Fig S2B). This result is consistent with the report that proteasome‐mediated degradation of Ki‐67 occurs following CDK4/6 inhibition and G1 arrest (Sobecki et al, 2017). The elimination of Ki‐67 expression in matrix keratinocytes by CDK4/6 inhibition was also apparent in the dual palbociclib/paclitaxel treatment group (Fig 5B). Consequently, pharmacological G1 arrest blocked the abnormal accumulation of pH3+ cells in the hair matrix that is otherwise strongly induced in paclitaxel‐only treated hair follicles (Fig 5C). In accordance with this, palbociclib pre‐treatment also prevented an increase in the number of cleaved caspase‐3+ cells within the hair follicles of a donor sensitive to early paclitaxel‐induced apoptosis (Fig 5D). Together, these in situ data, dissected within a complex and highly proliferative human mini‐organ, show that the mitosis‐targeting effects of paclitaxel chemotherapy are rendered ineffective by targeted G1 phase‐specific arrest using the CDK4/6 inhibitor palbociclib (Fig 5Ei–iii). These results therefore provide a proof‐of‐principle that therapeutic cell cycle arrest approaches have the potential to protect hair matrix keratinocytes from taxanes and thus prevent hair follicle damage leading to chemotherapy‐induced alopecia.

Palbociclib‐induced G1 arrest is reversible in the hair matrix We next probed the reversibility of CDK4/6 inhibition. To achieve this, we repeated hair follicle organ cultures as defined above with the addition of a drug washout step at 42 h and a subsequent 24‐ to 48‐h culture period free from palbociclib (Fig 6A). Figure 6.Transient CDK4/6 inhibition emphasises the cell cycle dependency of paclitaxel cytotoxicity in the human hair follicle A. Schematic of experimental design. Hair follicles (HFs) were pre‐treated with palbociclib for 18 h and then further incubated either with or without paclitaxel for a further 24 h. Subsequently, drug‐containing media was washed out and HFs were left in organ culture for an additional 24‐ to 48‐h period.

B. N of 8–13 HFs from three patients. No significant differences in EdU incorporation were found in any experimental conditions compared to vehicle. DNA synthesis in the hair matrix resumed in the palbociclib‐only‐ and palbociclib plus paclitaxel dual‐treated conditions following washout (see also Appendix Fig S3 ). Ordinary one‐way ANOVA with multiple comparisons performed usingof 8–13 HFs from three patients.

C. + cells was seen in the hair matrix of HFs dual treated with paclitaxel and palbociclib following drug washout, but this was not significant, whereas paclitaxel‐only treatment significantly increased the number of cleaved caspase‐3 + cells in the hair matrix (adjusted P value = 0.0003). Palbociclib‐only treatment did not increase the number of cleaved caspase‐3 + cells in the hair matrix following drug washout. Ordinary one‐way ANOVA with multiple comparisons performed using N of 8–12 HFs from three patients. Caspase‐3 vehicle vs. paclitaxel data are also used in Appendix Fig A trending increase in the number of cleaved caspase‐3cells was seen in the hair matrix of HFs dual treated with paclitaxel and palbociclib following drug washout, but this was not significant, whereas paclitaxel‐only treatment significantly increased the number of cleaved caspase‐3cells in the hair matrix (adjustedvalue = 0.0003). Palbociclib‐only treatment did not increase the number of cleaved caspase‐3cells in the hair matrix following drug washout. Ordinary one‐way ANOVA with multiple comparisons performed usingof 8–12 HFs from three patients. Caspase‐3 vehicle vs. paclitaxel data are also used in Appendix Fig S1E

D. P value = 0.0316) increase in the number of pH3 + cells in the matrix after washout. A significant (adjusted P value = 0.0278) increase in the number pH3 + cells was seen in HFs dual treated with both paclitaxel and palbociclib following drug washout. Palbociclib treatment alone showed no significant change in the number of pH3 + hair matrix keratinocytes after drug washout. Ordinary one‐way ANOVA with multiple comparisons performed using N of 8–12 HFs from three patients. pH3 vehicle vs. paclitaxel data are also used in Appendix Fig HFs treated with paclitaxel alone showed a significant (adjustedvalue = 0.0316) increase in the number of pH3cells in the matrix after washout. A significant (adjustedvalue = 0.0278) increase in the number pH3cells was seen in HFs dual treated with both paclitaxel and palbociclib following drug washout. Palbociclib treatment alone showed no significant change in the number of pH3hair matrix keratinocytes after drug washout. Ordinary one‐way ANOVA with multiple comparisons performed usingof 8–12 HFs from three patients. pH3 vehicle vs. paclitaxel data are also used in Appendix Fig S1B

E. Representative fluorescence images of EdU and pH3/cleaved caspase‐3 staining in paclitaxel‐only‐ and palbociclib plus paclitaxel dual‐treated HFs following drug washout. Where DNA synthesis has resumed following reversal of the G1 arrest, paclitaxel cytotoxicity is emergent. 20‐μm scale. Data information: Values plotted represent the mean total number of cells counted per HF analysed. Error bars are standard error of the mean. Please also see supporting data in Appendix Fig Data information: Values plotted represent the mean total number of cells counted per HF analysed. Error bars are standard error of the mean. Please also see supporting data in Appendix Fig S3 and S4 . HS, hair shaft; wo, washout. Source data are available online for this figure. Source Data for Figure 6 [emmm201911031-sup-0007-SDataFig6.pdf] In the hair matrix of palbociclib‐only treated hair follicles, EdU incorporation resumed in the majority (66%) of cases following drug washout (Fig 6B; Appendix Fig S3A). This demonstrates that CDK4/6 inhibition is reversible (Fry et al, 2004) in human hair follicle matrix keratinocytes. Furthermore, palbociclib‐only treated hair follicles did not show increased cleaved caspase‐3 immunoreactivity (Fig 6C; Appendix Fig S3B), indicating that transient G1 arrest is not cytotoxic to hair matrix keratinocytes.

Paclitaxel requires active proliferation to exert hair matrix cytotoxicity and may be retained in the hair follicle We then analysed hair follicles treated with both palbociclib and paclitaxel, after washout and culture in drug‐free medium (Fig 6A), and found that DNA synthesis also resumed within the hair matrix in this treatment group (Fig 6B). This resumption of cell cycle progression was met with a paclitaxel‐induced accumulation of pH3+ cells within the hair matrix (Fig 6D). This shows that paclitaxel can exert an anti‐mitotic effect as soon as the cell cycle resumes and progression to mitosis is permitted (Fig 6E). This effect occurred despite the removal of paclitaxel from the culture medium, which suggests drug retention of paclitaxel in hair follicle keratinocytes, or otherwise could be indicative of existing damage that only becomes apparent following resumed cell proliferation. Notably, paclitaxel has been described to be retained in cancer cells for 1 week in vitro and in vivo (Mori et al, 2006). Resumed cell proliferation in the combined palbociclib and paclitaxel treatment group, post‐drug washout, resulted in a trending (yet not significant) increase in the number of cleaved caspase‐3+ cells in the hair matrix (Fig 6C). The lack of statistical significance in this treatment group post‐washout, in contrast to paclitaxel‐only treated hair follicles, can be partly attributed to the lack of a G1 arrest reversal in the hair matrix of a small number of hair follicles. Indeed, in the few hair follicles treated with palbociclib and paclitaxel that showed little to no EdU incorporation following drug washout, no corresponding accumulation of cleaved caspase‐3+ or pH3+ cells was observed (Appendix Fig S4). Together, these results show how cell proliferation is necessary for paclitaxel to exert any anti‐mitotic and pro‐apoptotic effects within the hair matrix and that paclitaxel may be retained in hair follicle keratinocytes.

Paclitaxel and palbociclib do not promote catagen in human hair follicles during ex vivo organ culture Hair follicles respond to chemotherapy with distinct changes in hair follicle cycling, i.e. either by prolonged maintenance in anagen (“dystrophic anagen” pathway) or by rapid, premature induction of apoptosis‐driven hair follicle regression (“dystrophic catagen” pathway; Paus et al, 1994, 2013; Hendrix et al, 2005). To determine therefore how palbociclib and paclitaxel treatments influence the hair cycle during ex vivo culture, we staged treated hair follicles at the indicated 42 h and 66–90 h time points (Appendix Fig S5). Morphological analysis showed that paclitaxel treatment did not promote hair follicles to enter catagen (Appendix Fig S5). This indicates that, despite massive paclitaxel‐induced mitotic defects and apoptosis, taxane chemotherapy initially promotes the “dystrophic anagen” pathway (Paus et al, 2013). Palbociclib‐only treatment also did not result in catagen induction ex vivo, perfectly in line with the observation that palbociclib does not induce apoptosis in the hair matrix (Fig 6C; Appendix Fig S2D). This demonstrates that palbociclib can be effectively used to induce a reversible arrest that is non‐toxic and does not promote catagen, and thus permits growth to resume in anagen upon discontinuation of drug application (as suggested by resumed EdU incorporation in the hair matrix after palbociclib removal; Fig 6B). Therefore, temporary G1 arrest therapy by palbociclib is unlikely to promote (clinically undesired) catagen development and the associated subsequent inevitable hair loss (telogen effluvium).