Disruption of microtubules with low intensity ultrasound rescues hair follicle damage by paclitaxel

Disruption of microtubules with low intensity ultrasound rescues hair follicle damage by paclitaxel | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Article Disruption of microtubules with low intensity ultrasound rescues hair follicle damage by paclitaxel Xiang-Xi Xu, Celina Amaya, Shihua Luo, Jeremy Cheret, Elizabeth Smith This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-4356119/v1 This work is licensed under a CC BY 4.0 License Status: Under Review Version 1 posted You are reading this latest preprint version Abstract Paclitaxel exemplifies one of the key taxanes (paclitaxel, docetaxel, cabazitaxel), a group of versatile and effective drugs commonly used in chemotherapy for several major cancer types. These drugs work by stabilizing cellular microtubules, a unique mechanism to account for their impressive success in oncology. Unfortunately, side effects and inevitable development of resistance limit their utility. Hair loss (alopecia) is a well-known adverse side effect and poses a significant quality of life issue for many patients. Substantial efforts have been made to prevent or limit alopecia in chemotherapy, however their efficacy is minimal. We discovered that a brief exposure to low intensity and low frequency ultrasound at a defined timing is able to eliminate toxicity of paclitaxel (and other taxanes) in cultured cells by breaking the stabilized cellular microtubules. We subsequently showed that a brief exposure of low intensity ultrasound was able to break cellular microtubules and mitotic spindles transiently in hair follicle matrix cells of the furred skin of live mice. Such treatment reversed mitotic arrest by paclitaxel in the proliferative hair follicle matrix cells and prevented cell death, and thus annulled the consequent hair follicle damage and suppression of hair growth following paclitaxel administrations. These experimental findings herald a practical method that is within reach to prevent hair loss in cancer chemotherapy using taxanes. Health sciences/Health care/Quality of life Biological sciences/Cell biology/Cytoskeleton/Microtubules Ultrasound shock wave microtubules Taxol/Paclitaxel cytotoxicity hair follicles alopecia cancer chemotherapy Figures Figure 1 Figure 2 Figure 3 Figure 4 INTRODUCTION Severe and sudden hair loss (alopecia) is a common side effect of chemotherapy using taxanes, and is often viewed as the unfortunate reality associated with cancer treatment ( 1 , 2 ). Although hair loss is transient in most cases, it is a dreadful side effect and often causes vicious distress for many cancer patients ( 3 – 6 ). Substantial efforts have been made to investigate the mechanisms of hair loss and to find remedies to prevent hair loss in chemotherapy ( 7 ). These efforts have been complicated by the reality that any intervention will need to counter drug toxicity in hair follicles but preserve drug activity in cancer cells. One appealing effort was the topical application of CDK2 inhibitors to stop the cycling of hair matrix cells when mitotic drugs are given ( 8 , 9 ). However, the propitious results were not pursued as reproducibility of result was not demonstrated ( 10 ). Currently, scalp cooling during infusion of taxanes is a procedure used in the clinics to reduce alopecia ( 11 , 12 ). The cooling restricts blood flow and thus exposure of scalp skin to drugs, though the benefit is often limited or uncertain ( 11 – 14 ). Additionally, there are significant patient discomfort associated with scalp cooling, as the taxane infusion can be upwards of three hours in length. Taxanes including paclitaxel, docetaxel, and carbazitaxel, are a key group of versatile and effective drugs used in chemotherapy for several major cancer types, including ovarian cancer, metastatic breast cancer, metastatic and castration resistant prostate cancer, and lung cancer ( 15 ). These drugs work by stabilizing cellular microtubules and thus interfering with their function, a somewhat surprising mechanism to account for the impressive successes in oncology ( 16 – 18 ). Targeting microtubules causes both mitotic and non-mitotic mechanisms of cytotoxicity ( 19 – 21 ). Binding of taxanes to beta-tubulins within cellular microtubules leads to stabilization and bundling of microtubule filaments ( 16 – 18 ). In proliferative cells, interfering with microtubule function in the mitotic spindle results in cell growth arrest and subsequent mitotic slippage and mitotic catastrophe, leading to cell death ( 18 , 22 ). Acute alopecia is apparently caused by taxane toxicity to the highly proliferative matrix cells of the hair follicles ( 7 , 23 , 24 ). Ultrasound technologies have extensive applications in medicine, either for diagnosis (sonogram) or therapy ( 25 – 28 ). Typically, ultrasound with extremely low intensity (1–50 mW/cm 2 ) and high frequency (such as 50 MHz) is used for diagnostic (imaging) purposes. High intensity (> 8 W/cm 2 , 20–60 kHz) ultrasound that can deliver strong energy is used for ablative surgery and disruption through heating and acoustic cavitation. The medical application of ultrasound with an intensity that is low yet sufficiently high to produce biological activity is known as ultrasound physiotherapy ( 26 – 28 ), which uses a sufficiently strong but non-disruptive ultrasound shock waves (0.1-3.0 W/cm 2 ). The most commonly used devices produce ultrasound waves with frequencies either around 1–3 MHz or 20–150 kHz (known as long wavelength ultrasound). Low intensity ultrasound impacts cellular structures including microtubules ( 29 , 30 ). We explore the application of low intensity ultrasound to break cellular microtubules as a method to counter the effects of paclitaxel on microtubule stabilization and subsequent toxicity to hair follicles. RESULTS AND DISCUSSION Ultrasound fragments microtubule filaments in vitro We previously discovered that a brief exposure to low intensity ultrasound is able to eliminate paclitaxel toxicity in cultured cells by breaking the stabilized cellular microtubules ( 30 , 31 ). To determine the direct impacts of low intensity ultrasound on microtubules, we used a cell free system to analyze fluorescently labeled microtubules following a brief ultrasound exposure. Polymerized microtubules from fluorescently labeled tubulins presented as elongated filaments, and inclusion of paclitaxel in the tubulin polymerization mixture produced subtle but visually distinguishable patterns (shaper appearing filaments) (Fig. 1 A). Following a brief (5 min) exposure of the polymerized microtubules to low intensity ultrasound, the filaments were transiently disrupted, however, the elongated filaments re-appeared following incubation after around 60 min (Fig. 1 A). A brief ultrasound exposure also broke paclitaxel-bound, stabilized microtubules, whereas the mixture in the presence of paclitaxel did not recovered and appeared to form aggregates(Fig. 1 A). The length distribution of the microtubules was quantified using ImageJ program (Fig. 1 B), and the data indicates the differential responses of paclitaxel-bound or free microtubules to the impact of low intensity ultrasound. We reason that following breaking of microtubule filaments by the physical forces of the ultrasound, the fragments may disintegrate into alpha and beta tubulin monomers, which then re-polymerize into microtubule filaments (Fig. 1 C). However, the paclitaxel bound microtubule fragments do not undergo disassembly, and the fragments become aggregates (Fig. 1 C). These results indicate the differences in the recovery of microtubule fragments generated by physical forces of ultrasound between microtubules in the absence or presence of paclitaxel. Particularly, ultrasound forces irreversibly damage paclitaxel bound microtubules, and hence eliminate paclitaxel activity. Particularly, ultrasound forces irreversibly damage paclitaxel bound microtubules, and hence eliminate paclitaxel activity. Ultrasound disrupts cellular microtubules of hair follicles in live mice Exploring the finding as a potential solution to counter alopecia in cancer chemotherapy, we tested the effects of ultrasound and paclitaxel on hair follicles using mouse models. First, we showed that a brief exposure of low intensity ultrasound was able to transiently break cellular microtubules in the fur skin coat of live mice (Fig. 2 ). Low intensity ultrasound is known to break cellular microtubules ( 29 , 30 ), and we have previously shown that a brief exposure to cells on tissue culture plates led to breaking the microtubules into small fragments, which then reformed in 60 to 120 min ( 30 ). Here, by assaying furred skin biopsies taken immediately following ultrasound exposure (Fig. 2 A), we found that a brief exposure to low intensity ultrasound can also disturbed the microtubule cytoskeleton in live mice (Fig. 2 B). Examining hair follicles in the biopsies, we first observed that cellular microtubules appeared to have intense bundles in mice injected with paclitaxel 4 hours earlier. However, the microtubule cytoskeleton appears blurred following ultrasound exposure in mice either with or without prior injection with paclitaxel (Fig. 2 C). The length of the microtubule filaments was estimated, indicating that a 5-minute exposure to low intensity ultrasound was able to break microtubules in cells of hair follicles in live mice (Fig. 2 D). Bipolar mitotic figures were recognizable in the images (Fig. 2 E). By examining the microtubule patterns of the mitotic figures, abnormal features were recognizable in paclitaxel-treated samples. Particularly, mitotic figures appeared to be disrupted in cells either with or without prior paclitaxel treatment (Fig. 2 E). The different features of the mitotic figures (normal, abnormal, and disrupted) were quantified in multiple samples (Fig. 2 F). The mitotic figures in cell of the hair follicles in the skin biopsies were distinguishable, and revealed an aberrant spindle morphology in mice injected with paclitaxel and perturbed by ultrasound forces. We conclude that in live mice both with or without paclitaxel injection, a brief exposure to ultrasound disturbed mitotic spindles, as shown in the examples (Fig. 2 ). A brief exposure to ultrasound prevents paclitaxel-induced mitotic arrest and cell death In skin biopsies harvested at later times (1, 2, and 5 days), staining with phosphor-histone H3 (pH3), a marker for the mitotic phase, showed that the increased number of pH3-positive cells due to mitotic arrest of paclitaxel-injected mice was reduced by a brief ultrasound exposure 4 hours after paclitaxel injection (Fig. 3 A), indicating that the ultrasound treatment prevented paclitaxel-induced mitotic arrest. Mitotic arrest by paclitaxel persisted beyond day 5, with the highest degree of mitotic arrest at day 2 and less at day 5 (Fig. 3 B). On either day 2 or day 5, the skin biopsies from paclitaxel and ultrasound treated mice shown no significant increased pH3 positive hair follicle cells (Fig. 3 B), indicating a brief exposure to low intensity ultrasound 4 hours following paclitaxel administration is sufficient to prevent paclitaxel-induced cell growth arrest for days afterward. At the same time, ultrasound exposure prevented or reduced paclitaxel-induced cell death in the hair follicles, indicated by positive staining of activated caspase-3 to measure apoptosis (Fig. 3 C,D). The most severe cell death was observed at day 2 (Fig. 3 D), though a smaller number of apoptotic cells remained on day 5. In samples from mice exposed to ultrasound 4 hours after paclitaxel injection, much fewer pH3-positive cells were observed, and only normal mitotic figures were present, indicating proliferation hair follicle matrix cells resumed to support hair growth (Fig. 3 C,D). For the interpretation of these results, we suggest that a brief ultrasound exposure disrupted and broke microtubule bundles or mitotic spindles in hair follicle matrix cells during both mitotic and non-mitotic phases of the cell cycle, preventing paclitaxel-induced cell growth arrest and subsequent apoptosis (Fig. 3 C,D). Treatment with ultrasound prevents paclitaxel-induced hair follicle damage To determine the ability of ultrasound to rescue paclitaxel-induced hair follicle damage and hair growth, a mouse model was used to determine paclitaxel toxicity to hair follicles and paclitaxel-induced alopecia. It is reasoned that in younger mice (3–4 weeks of age), the majority of the hair follicles are on a growing stages (anagen phase) ( 7 ). The procedure included four injections of paclitaxel two days apart to cause extensive damage to hair follicles and to suppress hair growth recovery (Fig. 4 A). We tested the effect of exposure to ultrasound 4 hours after every paclitaxel injection in the paclitaxel toxicity mouse model (Fig. 4 A). The 4-hour time interval was chosen following several initial tests, and we reasoned that at this time point, circulating paclitaxel would be largely cleared, and exposure time of the hair follicles to the accumulating paclitaxel would be at a minimal time period before the inactivation of the drug by ultrasound. Following shaving to remove fur at the back of the experimental mice, we observed suppression of hair growth and recovery for more than 2 weeks following the 4 sessions of paclitaxel injections (Fig. 4 B). Consistently, ultrasound exposure following each paclitaxel injection rescued hair growth (Fig. 4 B), which was quantified by estimated hair length (Fig. 4 C). This experiment was performed using 4 weeks old 129P3/J mice with white fur, in which most of the hair follicles are proliferative and in growth stage. To demonstrate the general and robustness of the observation on the ultrasound treatment, we performed the same experiment in 3-month-old C57BL6 mice that the hair follicle may be in various growth phase and obtained similar results showing the ability of a brief ultrasound exposure to prevent paclitaxel-induced hair follicle damage and hair growth suppression (Fig. 4 D, E). Histology analyses at the end of the last paclitaxel injection indicate that hair follicle damage persisted as shown by the presence of a large number of cells containing multiple micronuclei in the paclitaxel-injected mice, which were prevented in the group treated with ultrasound (Fig. 4 F, G). Specifically, around 65% of the hair follicle matrix cells were found to be micronucleated, and less than 10% of the cells were micronucleated in the group a 5-min ultrasound treatment following each paclitaxel injection (Fig. 4 G). We examined but did not find any skin abnormality or pathology in mice treated with ultrasound, suggesting lack of side effects of the ultrasound treatment procedure. Thus, ultrasound treatment to prevent paclitaxel-induced hair follicle damage and hair growth appears to be robust regardless of both mouse age and strains. Since the 1990s, taxanes have increasingly been used for the treatment of various solid tumors in both the frontline and second line settings. Despite this, the management of side effects remands a challenge over the last 3 and a half decades ( 1 , 14 ). The current reported result presents a conceptually simple solution: as taxanes work by stabilizing cellular microtubules, localized use of ultrasound forces to break microtubules can neutralize taxane cytotoxic activity locally. Since low intensity ultrasound is non-invasive, inexpensive, and has a good safety profile ( 26 , 27 ), applying ultrasound to scalp hair follicles to counter taxane cytotoxicity is highly feasible and practical. Our preliminary study suggested that low intensity ultrasound of both low (45 KHz) and higher (1–3 MHz) frequencies are capable of disrupting cellular microtubules and removal of taxane toxicity to hair follicles. However, based on extensive prior studies of low intensity and low frequency ultrasound on skin of animal models and human subjects ( 25 , 26 , 32 ), we decided to focus on testing low frequency ultrasound in this initial study. The intensity we used was lower than the reported threshold that may cause physical damage in the skin or in the underlying muscle tissues ( 26 , 32 ), and the exposure time (5 min in our experiments) was also shorter. Indeed, in the experiments with mouse models (Fig. 4 ), we did not observed any adverse effects of ultrasound on the mice from either histology analyses or sensory neuronal and behavior assays. As such, it may be presumed that application of ultrasound in clinical practice will be non-invasive, easy to administer, and safe for patients. The demonstration of ultrasound in preventing hair follicle damage by paclitaxel here may be an example to inspire strategies to prevent other taxane side effects in chemotherapy. Additionally, similar ultrasound treatment was demonstrated to prevent paclitaxel-induced damage of human hair follicles in organelle culture models ( 33 ), suggesting similar biology operated in both rodent and human hair follicles regarding paclitaxel toxicity and rescuing effect of ultrasound. Since applying ultrasound to human scalp hair follicles is similar to our testing of ultrasound on hair follicles of furred skin in live mice, the experimental findings reported here herald that a practical method is within reach to prevent hair loss in chemotherapy using taxanes MATERIALS AND METHODS Materials and Reagents The following chemicals and reagents were obtained from the indicated companies: Paclitaxel: Athenex Anti-a-Tubulin: Proteintech 488-a-Tubulin: Cytoskeleton Anti-phospho-histone H3: Millipore Anti-cleaved/activated caspase 3: Cell signaling ProLong DAPI reagent: Invitrogen Anti-Mouse IgG Alexa Fluor 488: Invitrogen Anti-Rabbit IgG Alexa Flour 546 Invitrogen HiLyte Fluor 488 labeled tubulin Cytoskeleton, Inc. Mouse experiments Mice, 129P3/J 4-week-old, or C57BL/6 3-month-old, purchased from Jackson Lab, were intraperitoneal (I.P.) injected with paclitaxel (Athenex) diluted in saline, 12 mg/kg bodyweight. The mice in each cage were randomly allocated to different treatment groups. Food and water were available ad libitum and experiments were performed during the light cycle (7:00 am to 7:00 pm). At the end of the experiment, animals were euthanized via CO 2 asphyxiation, followed by cervical dislocation before tissue biopsy and collection. The animals were maintained and cared in the DVR core facility (University of Miami) according to established NIH protocols and handled in strict agreement with good animal practice as approved by the University of Miami Institutional Animal Care and Use Committee (IACUC). Ultrasound application Ultrasound was applied by submerging the mouse from neck down in an ultrasonic water bath (Crest Ultrasonics) with water maintained at 32ºC. The mice were exposed to ultrasound for 5 minutes at a 45 kHZ frequency and adjustable intensity to around 120 mW/cm 2 . The ultrasound frequency and intensity were monitored with a hydrophone (HCT-0320, from Onda Corporation), pressure meter (Onda), and an oscilloscope (OWON). Mice were sedated with isoflurane during the ultrasonic procedure and recovered from the anesthesia (steady and normal heart rate, respiration, and movements) before returning to the cage for normal activities. We acquired and tested both the bath and probe type ultrasound devices in our study. The bath device produces 45-150 kHz, 50 to 780 mW/cm 2 ultrasound waves with adjustable frequency and energy levels (Crest PowerSonic P1100 Model). The probe device produces adjustable 0.5-3 W/cm 2 and 1 or 3 MHz ultrasound waves (New PhysioOriginal Ultrasonic Therapy Machine, etc.). When submerged under water or fully wetted, the presence of fur did not significantly affect the transmission of ultrasound intensity to the surface of the skin. Histological Analyses At the end of mouse experiments, following euthanizing the mice, paws and skin (around 0.5 cm 2 ) were collected and preserved immediately by immersing them in 10% formalin overnight at 4°C. Subsequently, the samples underwent embedding in paraffin and were sectioned at 7 μm thickness. These sections underwent deparaffinization and rehydration before being subjected to antigen retrieval in a 0.01 M citric buffer at pH 6. Following a PBS wash, the sections were treated with 0.1% Triton X-100 in PBS for 5 minutes at room temperature, followed by a 30-minute incubation in a blocking solution containing 5% BSA. For immunostaining, the sections were incubated overnight at 4°C in a humidity chamber with the primary antibodies, which were appropriately diluted in the blocking solution. After several washes with PBS, the sections were exposed to a secondary antibody for 1 hour at room temperature. Mounting was achieved using ProLong DAPI reagent (Invitrogen). The specific antibodies utilized were alpha-tubulin (Proteintech 66031-1-Ig), phospho histone 3 (Millipore 09-797), cleaved caspase 3 (Cell Signaling 9694T), anti-mouse IgG Alexa Fluor 488 (Invitrogen A21202), and anti-rabbit IgG Alexa Fluor 546 (Invitrogen A10040). Preparation and analyses of fluorescently labeled microtubules in vitro HiLyte Fluor 488 labeled tubulin was obtained from Cytoskeleton Inc. (Denver, USA), and microtubules were polymerized according to the manufacturer’s instructions. Briefly, one aliquot of 488 tubulin (Cytoskeleton, TL488M) was resuspended to 5 mg/mL (45.5 μM) in 4.0 μl of general tubulin buffer (Cytoskeleton, BST01) supplemented with 0.01 volumes of 100 mM GTP (Cytoskeleton, BST06) and 1 μl of microtubules cushion buffer (Cytoskeleton, BST05) to facilitate polymerization. This solution was mixed with 70 μl of an equivalent solution using unlabeled tubulin (Cytoskeleton, T240) for a final labelling ratio of 1:15. Aliquots were placed in an incubator at 37ºC for 20 minutes to allow the tubulin polymerization to microtubules of appropriate length (average ∼10 μm). Upon polymerization, the microtubules were removed from the incubator and treated with 100 μl of paclitaxel/microtubules buffer solution (20 μM paclitaxel in general tubulin buffer). Control and paclitaxel-treated microtubule mixtures were treated with ultrasound (45-150 kHz, 120 W/cm 2 ), and observations were made and images were acquired using a fluorescent microscope at various time points. Imaging acquisition Immunofluorescence staining was observed with widefield microscopy using a Plan-Apochromatix 100Å~ objective lens (oil immersion, numerical aperture [NA] 1.4), 20x, and 10x objective lens, on inverted Zeiss Axio Observer Z1 using AxioVision 4.8 software. Images were acquired using a monochrome Zeiss Axio Cam MRm CCD camera and processed using the NIH Image J software. Image analysis and microtubule length quantification Microtubules length was analyzed using the NeuronJ plugin from Image J (imagej.nih.gov). For that purpose the images were transformed to 8-bit, vectorized, and the tubulin filaments were measured on a computer screen for at the least 10 filaments in each image. Statistics Statistical analysis was made using Graph Pad (version 9.4.1).Experimental values are expressed as mean ± SD of three independent experiments. Statistical analysis was performed using ANOVA. A p-value of less than 0.05 was considered significant. Declarations Acknowledgments We also thank our colleagues, including Drs. Sophia George and Wensi Tao for their conceptual discussions and technical advice in the course of the experiments. We thank many of our colleagues for advice and discussion during the course of the experiments and the preparation of the manuscript. Our student interns over the years, including Shria Bucha, Shalala Leny, Mariana Lopes dos Santos, Julio Baigorri, Rogelio Baucells, and Justin Leal, contributed to the early part of the research project. Funding Internal pilot funding from Sylvester Comprehensive Cancer Center/University of Miami was the key supported for this project. The labs were also partially supported by funds from grants including R21 CA277418 to RP, TCW; and Department of Defense Project OC220187 and LC220190, R01 CA230916, and R01 CA286527 to X-X Xu from NCI, NIH. Competing interest The authors declare no competing interests. References Rowinsky EK, Eisenhauer EA, Chaudhry V, Arbuck SG, Donehower RC. Clinical toxicities encountered with paclitaxel (Taxol). Semin Oncol. 1993; 20(4 Suppl 3):1-15. Markman M. Managing taxane toxicities. Support Care Cancer. 2003 Mar;11(3):144-7. Trüeb RM. Chemotherapy-induced alopecia. Semin Cutan Med Surg. 2009 Mar; 28(1):11-4. Rohl J, Kushner D, Markman M. 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J Cancer. 2022 Apr 18;13(7):2362-2373. Mitragotri S, Blankschtein D, Langer R. Ultrasound-mediated transdermal protein delivery. Science. 1995 Aug 11;269(5225):850-3. doi: 10.1126/science.7638603. PMID: 7638603 Cheret J, Samra T, Verling SD, Gherardini J, Rodriguez-Feliz J, Bauman AJ, Sanchez CA, Wikramanayake TC, Xu XX, Paus R. Low-Intensity Ultrasound as a Potential Intervention Strategy to Protect Human Scalp Hair Follicles from Taxane-Induced Toxicity. J Invest Dermatol. 2023 Sep;143(9):1809-1813.e2. Additional Declarations There is NO Competing Interest. Cite Share Download PDF Status: Under Review Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-4356119","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":300358740,"identity":"058e1831-a0a8-49af-993e-15df51a6f23d","order_by":0,"name":"Xiang-Xi Xu","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAAzElEQVRIiWNgGAWjYHACxgcfgEQDCDEYAPkHCGthNpxBqhY2aR6wFhggpMXgRo6BtG2OjWyD2OHGTzcKGOT4biTg1yLZc8bAOHdbmnGDdGKzdI4Bg7EkIS387D0GybnbDicCtTSAtCRuIKSFjZnH4LDltv8gLc2/gVrqCWoB2mLYzLjtAEhLG8iWBAPCfjlWzNi7Ldm4DajFOsdAwnDmmQf4tRjcSN7+4+c2O9l+6fTHt3P+2MjzHSdgC8JTEEqCSOWjYBSMglEwCvACAJdVQxw1kBguAAAAAElFTkSuQmCC","orcid":"","institution":"University of Miami","correspondingAuthor":true,"prefix":"","firstName":"Xiang-Xi","middleName":"","lastName":"Xu","suffix":""},{"id":300358742,"identity":"a3ad97c8-c9a7-4b2d-beae-1ef9942fcffc","order_by":1,"name":"Celina Amaya","email":"","orcid":"","institution":"University of Miami","correspondingAuthor":false,"prefix":"","firstName":"Celina","middleName":"","lastName":"Amaya","suffix":""},{"id":300358744,"identity":"aefc5404-c5d7-4288-9a61-fa4e65e52eae","order_by":2,"name":"Shihua Luo","email":"","orcid":"","institution":"University of Miami","correspondingAuthor":false,"prefix":"","firstName":"Shihua","middleName":"","lastName":"Luo","suffix":""},{"id":300358746,"identity":"bf090c84-4b29-4279-b623-f3dbe1e432e5","order_by":3,"name":"Jeremy Cheret","email":"","orcid":"","institution":"University of Miami","correspondingAuthor":false,"prefix":"","firstName":"Jeremy","middleName":"","lastName":"Cheret","suffix":""},{"id":300358748,"identity":"280af965-e57d-41ba-85fc-2dc2e4831985","order_by":4,"name":"Elizabeth Smith","email":"","orcid":"","institution":"University of Miami","correspondingAuthor":false,"prefix":"","firstName":"Elizabeth","middleName":"","lastName":"Smith","suffix":""}],"badges":[],"createdAt":"2024-05-01 22:40:20","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-4356119/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-4356119/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":57790627,"identity":"58ff2e19-a91a-402c-8822-4a01fae96531","added_by":"auto","created_at":"2024-06-05 17:24:35","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":442319,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eUltrasound breaks microtubules differentially in vitro.\u003c/strong\u003e (\u003cstrong\u003eA\u003c/strong\u003e) Purified fluorescent- labeled a-tubulins (488-a-Tubulin) were allowed to polymerize for 30 min, with or without paclitaxel (1 nM). The mixtures on the tissue culture dish were exposed to ultrasound (+ US, 40 KHz, 1.0 W/cm2) for 5 min. Representative images were taken before (control, time 0), 10 min or 60 min and after exposure to ultrasound. (\u003cstrong\u003eB\u003c/strong\u003e) The lengths and distribution of microtubules and fragments were quantified using image analysis program (NIH Image J and MetaMorph Module). \u0026nbsp;“****” indicates significant difference (p \u0026lt; 0.001). (\u003cstrong\u003eC\u003c/strong\u003e) We propose a possible model: ultrasound (US) breaks microtubules into fragments, which can further depolymerize into a- and b- tubulin dimers to reform new microtubule filaments. \u003cstrong\u003e\u0026nbsp;\u003c/strong\u003eMicrotubules stabilize and form bundles in the presence of paclitaxel/Taxol (“T”). Ultrasound also breaks the paclitaxel-bound microtubule filaments, which do not depolymerize but rather form aggregates. In cells, the aggregates of microtubule fragments may undergo autophagy and lysosomal degradation\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-4356119/v1/512129a17c6b251469b3afb0.png"},{"id":57789503,"identity":"d9f43233-fe83-46a2-af6d-1d1352f806fa","added_by":"auto","created_at":"2024-06-05 17:16:35","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":722856,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eLow intensity ultrasound disrupts microtubules in cells of hair follicles in live mice.\u003c/strong\u003e\u0026nbsp; (\u003cstrong\u003eA\u003c/strong\u003e) Illustration of experimental design: the 3-week old mice were injected i.p. with 12 mg/Kg (body weight) paclitaxel (PTX).\u0026nbsp; After 4 hours, the mice were exposed to ultrasound (1 W/cm\u003csup\u003e2\u003c/sup\u003e, 45 kHz) for 5 min, and euthanized, biopsied, and assayed 10 min later.\u0026nbsp; (\u003cstrong\u003eB\u003c/strong\u003e) Skin biopsies were analyzed by histology technique, and the hair follicle sections stained with alpha-Tubulin were shown for examples of cross section (upper panel) and sagittal section (lower panel).\u0026nbsp; (\u003cstrong\u003eC\u003c/strong\u003e) Areas of the hair follicles were shown in high magnification to visualize microtubules.\u0026nbsp; (\u003cstrong\u003eD\u003c/strong\u003e) The length of microtubule filaments was measured using an image analytical program.\u0026nbsp; “*” p \u0026lt; 0.01; “****” p \u0026lt; 0.001.\u0026nbsp; (\u003cstrong\u003eE\u003c/strong\u003e) Examples were shown for mitotic figures, and shown in higher magnification in lower panel.\u0026nbsp; (\u003cstrong\u003eF\u003c/strong\u003e) Quantitation of normal, abnormal, and disrupted mitotic figures in cells of the hair follicles in the skin biopsies.\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-4356119/v1/77972c13aeafb0d656bb9995.png"},{"id":57790628,"identity":"1bf1f152-7140-4b11-b8aa-3e121b5abbc2","added_by":"auto","created_at":"2024-06-05 17:24:35","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":633630,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003e\u0026nbsp;A brief exposure to ultrasound prevent paclitaxel-induced mitotic arrest and apoptosis of hair follicle matrix cells in live mice.\u003c/strong\u003e\u0026nbsp; 3-week old mice were injected with 12 mg/Kg (body weight) paclitaxel (PTX) for 4 hours, exposed to ultrasound (1 W/cm\u003csup\u003e2\u003c/sup\u003e, 45 kHz) for 5 min.\u0026nbsp; 2 or 5 days later, the mice were euthanized, biopsied, and assayed.\u0026nbsp; (\u003cstrong\u003eA\u003c/strong\u003e) Examples of the biopsies analyses for phosphor-histone H3 (pH3) at day 2 are shown.\u0026nbsp; (\u003cstrong\u003eB\u003c/strong\u003e) Staining of pH3 was performed and quantified to measure cells at mitotic phase, at day 2 and 5.\u0026nbsp; Standard deviations are shown as error bars.\u0026nbsp; (\u003cstrong\u003eC\u003c/strong\u003e) Examples of staining of the activated caspase 3 at day 2 following paclitaxel and /or ultrasound treatment are shown.\u0026nbsp; (\u003cstrong\u003eD\u003c/strong\u003e) Apoptotic cells were identified and quantitated by staining for activated caspase 3 in sections from 10 or more hair follicles from three mice in each group, and standard deviations are shown as error bars.\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-4356119/v1/c8bff4a96ba029cff2c0e148.png"},{"id":57789504,"identity":"d104e68a-0012-4bc9-866b-3e70d296370a","added_by":"auto","created_at":"2024-06-05 17:16:35","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":611403,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eLow intensity ultrasound prevents paclitaxel-induced suppression of hair growth in mice.\u003c/strong\u003e\u0026nbsp; (\u003cstrong\u003eA\u003c/strong\u003e) Experimental design: the protocol/procedure is illustrated.\u0026nbsp; Briefly, 3-week old 129P3/J mice (3 males and 3 females) received 4 injection of paclitaxel (12 mg/Kg body weight) 2 day apart.\u0026nbsp; For the paclitaxel/ultrasound group (PTX + US), the whole torso (except the head) of the mice were exposed to ultrasound for 5 min (45 KHz, 1 W/cm\u003csup\u003e2\u003c/sup\u003e) for 5 min.\u0026nbsp; On day 1, the fur on the back of the mice were shaved and removed in all mice.\u0026nbsp; The recovery of the fur was then observed and measured following paclitaxel injections for 3 weeks.\u0026nbsp; (\u003cstrong\u003eB\u003c/strong\u003e) Examples for the appearance of the fur in the 4 groups (Control, US, PTX, and PTX + US) of 4-week-old 129P3/J mice are shown for day 1 and 14 of the experiment.\u0026nbsp; (\u003cstrong\u003eC\u003c/strong\u003e) Hair growth recovery on day 19 was quantitated by measuring length of the hair in the initially salved area.\u0026nbsp; (\u003cstrong\u003eD\u003c/strong\u003e) Same experimental procedure was performed on 3-month-old C57/BL6 mice.\u0026nbsp; The hair length was quantified and standard deviations are shown as error bars.\u0026nbsp; (\u003cstrong\u003eE\u003c/strong\u003e) Examples of the representative appearance of fur recovery for experiment day 1 and day 13 are shown.\u0026nbsp; (\u003cstrong\u003eF\u003c/strong\u003e) One day following paclitaxel injection (day 8), two mice from each group were subjected to skin biopsy to observe hair follicle damage by paclitaxel.\u0026nbsp; Represent H\u0026amp;E histology images of hair follicles are shown.\u0026nbsp; The insert in each image shows an area in higher magnification to visualize paclitaxel-induced micronucleation, and the prevention by ultrasound.\u0026nbsp; (\u003cstrong\u003eG\u003c/strong\u003e) The percentage of hair follicle matrix cells containing micronuclei is quantified in multiple hair follicles.\u0026nbsp; The difference between those of PTX and PTX+US is statistically significant (p \u0026lt; 0.001).\u003c/p\u003e","description":"","filename":"4.png","url":"https://assets-eu.researchsquare.com/files/rs-4356119/v1/b28c115f7e24655019d9fc4a.png"},{"id":57791075,"identity":"d7367984-fefc-41b2-81af-06a1535c2b9a","added_by":"auto","created_at":"2024-06-05 17:32:37","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":3240806,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-4356119/v1/be6c1a4d-6168-474a-8bf1-87d6be68c202.pdf"}],"financialInterests":"There is \u003cb\u003eNO\u003c/b\u003e Competing Interest.","formattedTitle":"Disruption of microtubules with low intensity ultrasound rescues hair follicle damage by paclitaxel","fulltext":[{"header":"INTRODUCTION","content":"\u003cp\u003eSevere and sudden hair loss (alopecia) is a common side effect of chemotherapy using taxanes, and is often viewed as the unfortunate reality associated with cancer treatment (\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e). Although hair loss is transient in most cases, it is a dreadful side effect and often causes vicious distress for many cancer patients (\u003cspan additionalcitationids=\"CR4 CR5\" citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e). Substantial efforts have been made to investigate the mechanisms of hair loss and to find remedies to prevent hair loss in chemotherapy (\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e). These efforts have been complicated by the reality that any intervention will need to counter drug toxicity in hair follicles but preserve drug activity in cancer cells. One appealing effort was the topical application of CDK2 inhibitors to stop the cycling of hair matrix cells when mitotic drugs are given (\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e, \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e). However, the propitious results were not pursued as reproducibility of result was not demonstrated (\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e). Currently, scalp cooling during infusion of taxanes is a procedure used in the clinics to reduce alopecia (\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e, \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e). The cooling restricts blood flow and thus exposure of scalp skin to drugs, though the benefit is often limited or uncertain (\u003cspan additionalcitationids=\"CR12 CR13\" citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e). Additionally, there are significant patient discomfort associated with scalp cooling, as the taxane infusion can be upwards of three hours in length.\u003c/p\u003e \u003cp\u003eTaxanes including paclitaxel, docetaxel, and carbazitaxel, are a key group of versatile and effective drugs used in chemotherapy for several major cancer types, including ovarian cancer, metastatic breast cancer, metastatic and castration resistant prostate cancer, and lung cancer (\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e). These drugs work by stabilizing cellular microtubules and thus interfering with their function, a somewhat surprising mechanism to account for the impressive successes in oncology (\u003cspan additionalcitationids=\"CR17\" citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e). Targeting microtubules causes both mitotic and non-mitotic mechanisms of cytotoxicity (\u003cspan additionalcitationids=\"CR20\" citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e). Binding of taxanes to beta-tubulins within cellular microtubules leads to stabilization and bundling of microtubule filaments (\u003cspan additionalcitationids=\"CR17\" citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e). In proliferative cells, interfering with microtubule function in the mitotic spindle results in cell growth arrest and subsequent mitotic slippage and mitotic catastrophe, leading to cell death (\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e, \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e). Acute alopecia is apparently caused by taxane toxicity to the highly proliferative matrix cells of the hair follicles (\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e, \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e, \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eUltrasound technologies have extensive applications in medicine, either for diagnosis (sonogram) or therapy (\u003cspan additionalcitationids=\"CR26 CR27\" citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e). Typically, ultrasound with extremely low intensity (1\u0026ndash;50 mW/cm\u003csup\u003e2\u003c/sup\u003e) and high frequency (such as 50 MHz) is used for diagnostic (imaging) purposes. High intensity (\u0026gt;\u0026thinsp;8 W/cm\u003csup\u003e2\u003c/sup\u003e, 20\u0026ndash;60 kHz) ultrasound that can deliver strong energy is used for ablative surgery and disruption through heating and acoustic cavitation. The medical application of ultrasound with an intensity that is low yet sufficiently high to produce biological activity is known as ultrasound physiotherapy (\u003cspan additionalcitationids=\"CR27\" citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e), which uses a sufficiently strong but non-disruptive ultrasound shock waves (0.1-3.0 W/cm\u003csup\u003e2\u003c/sup\u003e). The most commonly used devices produce ultrasound waves with frequencies either around 1\u0026ndash;3 MHz or 20\u0026ndash;150 kHz (known as long wavelength ultrasound). Low intensity ultrasound impacts cellular structures including microtubules (\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e, \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e). We explore the application of low intensity ultrasound to break cellular microtubules as a method to counter the effects of paclitaxel on microtubule stabilization and subsequent toxicity to hair follicles.\u003c/p\u003e"},{"header":"RESULTS AND DISCUSSION","content":"\u003cp\u003e \u003cb\u003eUltrasound fragments microtubule filaments\u003c/b\u003e \u003cb\u003ein vitro\u003c/b\u003e\u003c/p\u003e \u003cp\u003eWe previously discovered that a brief exposure to low intensity ultrasound is able to eliminate paclitaxel toxicity in cultured cells by breaking the stabilized cellular microtubules (\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e, \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e). To determine the direct impacts of low intensity ultrasound on microtubules, we used a cell free system to analyze fluorescently labeled microtubules following a brief ultrasound exposure. Polymerized microtubules from fluorescently labeled tubulins presented as elongated filaments, and inclusion of paclitaxel in the tubulin polymerization mixture produced subtle but visually distinguishable patterns (shaper appearing filaments) (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA). Following a brief (5 min) exposure of the polymerized microtubules to low intensity ultrasound, the filaments were transiently disrupted, however, the elongated filaments re-appeared following incubation after around 60 min (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA). A brief ultrasound exposure also broke paclitaxel-bound, stabilized microtubules, whereas the mixture in the presence of paclitaxel did not recovered and appeared to form aggregates(Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA). The length distribution of the microtubules was quantified using ImageJ program (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB), and the data indicates the differential responses of paclitaxel-bound or free microtubules to the impact of low intensity ultrasound. We reason that following breaking of microtubule filaments by the physical forces of the ultrasound, the fragments may disintegrate into alpha and beta tubulin monomers, which then re-polymerize into microtubule filaments (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eC). However, the paclitaxel bound microtubule fragments do not undergo disassembly, and the fragments become aggregates (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eC). These results indicate the differences in the recovery of microtubule fragments generated by physical forces of ultrasound between microtubules in the absence or presence of paclitaxel. Particularly, ultrasound forces irreversibly damage paclitaxel bound microtubules, and hence eliminate paclitaxel activity. Particularly, ultrasound forces irreversibly damage paclitaxel bound microtubules, and hence eliminate paclitaxel activity.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eUltrasound disrupts cellular microtubules of hair follicles in live mice\u003c/h2\u003e \u003cp\u003eExploring the finding as a potential solution to counter alopecia in cancer chemotherapy, we tested the effects of ultrasound and paclitaxel on hair follicles using mouse models. First, we showed that a brief exposure of low intensity ultrasound was able to transiently break cellular microtubules in the fur skin coat of live mice (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). Low intensity ultrasound is known to break cellular microtubules (\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e, \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e), and we have previously shown that a brief exposure to cells on tissue culture plates led to breaking the microtubules into small fragments, which then reformed in 60 to 120 min (\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e). Here, by assaying furred skin biopsies taken immediately following ultrasound exposure (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA), we found that a brief exposure to low intensity ultrasound can also disturbed the microtubule cytoskeleton in live mice (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB). Examining hair follicles in the biopsies, we first observed that cellular microtubules appeared to have intense bundles in mice injected with paclitaxel 4 hours earlier. However, the microtubule cytoskeleton appears blurred following ultrasound exposure in mice either with or without prior injection with paclitaxel (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eC). The length of the microtubule filaments was estimated, indicating that a 5-minute exposure to low intensity ultrasound was able to break microtubules in cells of hair follicles in live mice (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eD). Bipolar mitotic figures were recognizable in the images (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eE). By examining the microtubule patterns of the mitotic figures, abnormal features were recognizable in paclitaxel-treated samples. Particularly, mitotic figures appeared to be disrupted in cells either with or without prior paclitaxel treatment (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eE). The different features of the mitotic figures (normal, abnormal, and disrupted) were quantified in multiple samples (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eF). The mitotic figures in cell of the hair follicles in the skin biopsies were distinguishable, and revealed an aberrant spindle morphology in mice injected with paclitaxel and perturbed by ultrasound forces. We conclude that in live mice both with or without paclitaxel injection, a brief exposure to ultrasound disturbed mitotic spindles, as shown in the examples (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003eA brief exposure to ultrasound prevents paclitaxel-induced mitotic arrest and cell death\u003c/h2\u003e \u003cp\u003eIn skin biopsies harvested at later times (1, 2, and 5 days), staining with phosphor-histone H3 (pH3), a marker for the mitotic phase, showed that the increased number of pH3-positive cells due to mitotic arrest of paclitaxel-injected mice was reduced by a brief ultrasound exposure 4 hours after paclitaxel injection (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA), indicating that the ultrasound treatment prevented paclitaxel-induced mitotic arrest. Mitotic arrest by paclitaxel persisted beyond day 5, with the highest degree of mitotic arrest at day 2 and less at day 5 (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB). On either day 2 or day 5, the skin biopsies from paclitaxel and ultrasound treated mice shown no significant increased pH3 positive hair follicle cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB), indicating a brief exposure to low intensity ultrasound 4 hours following paclitaxel administration is sufficient to prevent paclitaxel-induced cell growth arrest for days afterward.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eAt the same time, ultrasound exposure prevented or reduced paclitaxel-induced cell death in the hair follicles, indicated by positive staining of activated caspase-3 to measure apoptosis (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eC,D). The most severe cell death was observed at day 2 (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eD), though a smaller number of apoptotic cells remained on day 5. In samples from mice exposed to ultrasound 4 hours after paclitaxel injection, much fewer pH3-positive cells were observed, and only normal mitotic figures were present, indicating proliferation hair follicle matrix cells resumed to support hair growth (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eC,D). For the interpretation of these results, we suggest that a brief ultrasound exposure disrupted and broke microtubule bundles or mitotic spindles in hair follicle matrix cells during both mitotic and non-mitotic phases of the cell cycle, preventing paclitaxel-induced cell growth arrest and subsequent apoptosis (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eC,D).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003eTreatment with ultrasound prevents paclitaxel-induced hair follicle damage\u003c/h2\u003e \u003cp\u003eTo determine the ability of ultrasound to rescue paclitaxel-induced hair follicle damage and hair growth, a mouse model was used to determine paclitaxel toxicity to hair follicles and paclitaxel-induced alopecia. It is reasoned that in younger mice (3\u0026ndash;4 weeks of age), the majority of the hair follicles are on a growing stages (anagen phase) (\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e). The procedure included four injections of paclitaxel two days apart to cause extensive damage to hair follicles and to suppress hair growth recovery (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA). We tested the effect of exposure to ultrasound 4 hours after every paclitaxel injection in the paclitaxel toxicity mouse model (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA). The 4-hour time interval was chosen following several initial tests, and we reasoned that at this time point, circulating paclitaxel would be largely cleared, and exposure time of the hair follicles to the accumulating paclitaxel would be at a minimal time period before the inactivation of the drug by ultrasound. Following shaving to remove fur at the back of the experimental mice, we observed suppression of hair growth and recovery for more than 2 weeks following the 4 sessions of paclitaxel injections (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eB). Consistently, ultrasound exposure following each paclitaxel injection rescued hair growth (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eB), which was quantified by estimated hair length (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eC). This experiment was performed using 4 weeks old 129P3/J mice with white fur, in which most of the hair follicles are proliferative and in growth stage. To demonstrate the general and robustness of the observation on the ultrasound treatment, we performed the same experiment in 3-month-old C57BL6 mice that the hair follicle may be in various growth phase and obtained similar results showing the ability of a brief ultrasound exposure to prevent paclitaxel-induced hair follicle damage and hair growth suppression (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eD, E). Histology analyses at the end of the last paclitaxel injection indicate that hair follicle damage persisted as shown by the presence of a large number of cells containing multiple micronuclei in the paclitaxel-injected mice, which were prevented in the group treated with ultrasound (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eF, G). Specifically, around 65% of the hair follicle matrix cells were found to be micronucleated, and less than 10% of the cells were micronucleated in the group a 5-min ultrasound treatment following each paclitaxel injection (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eG). We examined but did not find any skin abnormality or pathology in mice treated with ultrasound, suggesting lack of side effects of the ultrasound treatment procedure. Thus, ultrasound treatment to prevent paclitaxel-induced hair follicle damage and hair growth appears to be robust regardless of both mouse age and strains.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eSince the 1990s, taxanes have increasingly been used for the treatment of various solid tumors in both the frontline and second line settings. Despite this, the management of side effects remands a challenge over the last 3 and a half decades (\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e). The current reported result presents a conceptually simple solution: as taxanes work by stabilizing cellular microtubules, localized use of ultrasound forces to break microtubules can neutralize taxane cytotoxic activity locally. Since low intensity ultrasound is non-invasive, inexpensive, and has a good safety profile (\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e, \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e), applying ultrasound to scalp hair follicles to counter taxane cytotoxicity is highly feasible and practical. Our preliminary study suggested that low intensity ultrasound of both low (45 KHz) and higher (1\u0026ndash;3 MHz) frequencies are capable of disrupting cellular microtubules and removal of taxane toxicity to hair follicles. However, based on extensive prior studies of low intensity and low frequency ultrasound on skin of animal models and human subjects (\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e, \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e, \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e), we decided to focus on testing low frequency ultrasound in this initial study. The intensity we used was lower than the reported threshold that may cause physical damage in the skin or in the underlying muscle tissues (\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e, \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e), and the exposure time (5 min in our experiments) was also shorter. Indeed, in the experiments with mouse models (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e), we did not observed any adverse effects of ultrasound on the mice from either histology analyses or sensory neuronal and behavior assays. As such, it may be presumed that application of ultrasound in clinical practice will be non-invasive, easy to administer, and safe for patients.\u003c/p\u003e \u003cp\u003eThe demonstration of ultrasound in preventing hair follicle damage by paclitaxel here may be an example to inspire strategies to prevent other taxane side effects in chemotherapy. Additionally, similar ultrasound treatment was demonstrated to prevent paclitaxel-induced damage of human hair follicles in organelle culture models (\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e), suggesting similar biology operated in both rodent and human hair follicles regarding paclitaxel toxicity and rescuing effect of ultrasound. Since applying ultrasound to human scalp hair follicles is similar to our testing of ultrasound on hair follicles of furred skin in live mice, the experimental findings reported here herald that a practical method is within reach to prevent hair loss in chemotherapy using taxanes\u003c/p\u003e \u003c/div\u003e"},{"header":"MATERIALS AND METHODS","content":"\u003cp\u003e\u003cstrong\u003eMaterials and Reagents\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cu\u003eThe following chemicals and reagents were obtained from the indicated companies:\u0026nbsp;\u003c/u\u003e\u003c/p\u003e\n\u003cp\u003ePaclitaxel:\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;Athenex\u003c/p\u003e\n\u003cp\u003eAnti-a-Tubulin:\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;\u0026nbsp;Proteintech \u0026nbsp;\u003c/p\u003e\n\u003cp\u003e488-a-Tubulin:\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;\u0026nbsp;\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;\u0026nbsp;Cytoskeleton\u003c/p\u003e\n\u003cp\u003eAnti-phospho-histone H3:\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;\u0026nbsp;Millipore\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;\u003c/p\u003e\n\u003cp\u003eAnti-cleaved/activated caspase 3:\u0026nbsp; \u0026nbsp; \u0026nbsp;\u0026nbsp;\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;\u0026nbsp;Cell signaling\u003c/p\u003e\n\u003cp\u003eProLong DAPI reagent:\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;\u0026nbsp;\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;\u0026nbsp;Invitrogen\u003c/p\u003e\n\u003cp\u003eAnti-Mouse\u0026nbsp;IgG\u0026nbsp;Alexa Fluor 488:\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;Invitrogen\u003c/p\u003e\n\u003cp\u003eAnti-Rabbit\u0026nbsp;IgG\u0026nbsp;Alexa\u0026nbsp;Flour 546 \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;Invitrogen\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eHiLyte Fluor 488 labeled tubulin \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;\u0026nbsp;Cytoskeleton, Inc.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eMouse experiments\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eMice, 129P3/J 4-week-old, or C57BL/6 3-month-old,\u0026nbsp;purchased from Jackson Lab,\u0026nbsp;were\u0026nbsp;intraperitoneal (I.P.)\u0026nbsp;injected with paclitaxel (Athenex) diluted in saline, 12 mg/kg bodyweight. \u0026nbsp;The mice in each cage were randomly allocated to different treatment groups. \u0026nbsp;Food and water were available ad libitum and experiments were performed during the light cycle (7:00 am to 7:00 pm). \u0026nbsp;At the end of the experiment, animals were euthanized via CO\u003csub\u003e2\u003c/sub\u003e asphyxiation, followed by cervical dislocation\u0026nbsp;before tissue biopsy and collection.\u0026nbsp;\u0026nbsp;The animals were maintained and cared in the DVR core facility (University of Miami) according to established NIH protocols and handled in strict agreement with good animal practice as approved by the\u0026nbsp;University of Miami\u0026nbsp;Institutional Animal Care and Use Committee (IACUC).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eUltrasound application\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eUltrasound was applied by submerging the mouse\u0026nbsp;from neck down\u0026nbsp;in an ultrasonic water bath (Crest Ultrasonics)\u0026nbsp;with\u0026nbsp;water\u0026nbsp;maintained\u0026nbsp;at 32\u0026ordm;C. \u0026nbsp;The mice were exposed to ultrasound\u0026nbsp;for 5 minutes at a 45 kHZ frequency and\u0026nbsp;adjustable intensity to around\u0026nbsp;120 mW/cm\u003csup\u003e2\u003c/sup\u003e. \u0026nbsp;The ultrasound frequency and intensity were monitored with a hydrophone\u0026nbsp;(HCT-0320, from Onda Corporation), pressure meter (Onda),\u0026nbsp;and an oscilloscope (OWON). \u0026nbsp;Mice were sedated with isoflurane during the ultrasonic procedure and recovered from the anesthesia (steady and normal heart rate, respiration, and movements) before returning to the cage for normal activities.\u003c/p\u003e\n\u003cp\u003eWe acquired and tested both the bath and probe type ultrasound devices in our study. \u0026nbsp;The bath device produces 45-150 kHz, 50 to 780 mW/cm\u003csup\u003e2\u003c/sup\u003e ultrasound waves with adjustable frequency and energy levels (Crest PowerSonic P1100 Model). \u0026nbsp; The probe device produces adjustable 0.5-3 W/cm\u003csup\u003e2\u003c/sup\u003e and 1 or 3 MHz ultrasound waves (New PhysioOriginal Ultrasonic Therapy Machine, etc.). \u0026nbsp;When submerged under water or fully wetted, the presence of fur did not significantly affect the transmission of ultrasound intensity to the surface of the skin.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eHistological Analyses\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAt the end\u0026nbsp;of mouse experiments, following euthanizing the mice,\u0026nbsp;paws and skin (around 0.5 cm\u003csup\u003e2\u003c/sup\u003e)\u0026nbsp;were\u0026nbsp;collected and preserved\u0026nbsp;immediately\u0026nbsp;by immersing them in 10% formalin overnight at 4\u0026deg;C.\u0026nbsp;\u0026nbsp;Subsequently, the samples underwent embedding in paraffin and were sectioned at 7 \u0026mu;m thickness. \u0026nbsp;These sections underwent deparaffinization and rehydration before being subjected to antigen retrieval in a 0.01\u0026nbsp;M citric buffer at pH 6. \u0026nbsp; Following a PBS wash, the sections were treated with 0.1% Triton X-100 in PBS for 5 minutes at room temperature, followed by a 30-minute incubation in a blocking solution containing 5% BSA.\u003c/p\u003e\n\u003cp\u003eFor immunostaining, the sections were incubated overnight at 4\u0026deg;C in a humidity chamber with the primary antibodies, which were appropriately diluted in the blocking solution. \u0026nbsp;After\u0026nbsp;several\u0026nbsp;washes\u0026nbsp;with\u0026nbsp;PBS, the sections were exposed to a secondary antibody for 1 hour at room temperature.\u0026nbsp;\u0026nbsp;Mounting was achieved using ProLong DAPI reagent (Invitrogen).\u0026nbsp;\u0026nbsp;The specific antibodies utilized were alpha-tubulin (Proteintech 66031-1-Ig), phospho histone 3 (Millipore 09-797), cleaved caspase 3 (Cell Signaling 9694T),\u0026nbsp;anti-mouse\u0026nbsp;IgG\u0026nbsp;Alexa Fluor 488 (Invitrogen A21202), and\u0026nbsp;anti-rabbit\u0026nbsp;IgG\u0026nbsp;Alexa Fluor 546 (Invitrogen A10040).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ePreparation and analyses of fluorescently labeled microtubules \u003cem\u003ein vitro\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eHiLyte Fluor 488 labeled tubulin was obtained from Cytoskeleton Inc. (Denver, USA), and microtubules were polymerized according to the manufacturer\u0026rsquo;s\u0026nbsp;instructions. \u0026nbsp;Briefly, one aliquot of 488 tubulin (Cytoskeleton, TL488M) was resuspended to 5 mg/mL (45.5 \u0026mu;M) in 4.0\u0026nbsp;\u0026mu;l of general tubulin buffer (Cytoskeleton, BST01) supplemented with 0.01 volumes of 100 mM GTP (Cytoskeleton, BST06) and 1 \u0026mu;l of microtubules cushion buffer (Cytoskeleton, BST05) to facilitate polymerization. \u0026nbsp;This solution was mixed with 70 \u0026mu;l of an equivalent solution using unlabeled tubulin (Cytoskeleton, T240) for a final labelling ratio of 1:15. Aliquots were placed in an incubator at 37\u0026ordm;C for 20 minutes to allow the tubulin polymerization to microtubules of appropriate length (average\u0026nbsp;\u0026sim;10 \u0026mu;m). \u0026nbsp;Upon polymerization, the microtubules were removed from the incubator and treated with 100 \u0026mu;l of paclitaxel/microtubules buffer solution (20 \u0026mu;M paclitaxel in general tubulin buffer). \u0026nbsp;Control and paclitaxel-treated microtubule mixtures were treated with ultrasound (45-150 kHz, 120 W/cm\u003csup\u003e2\u003c/sup\u003e), and observations were made and images were acquired using a fluorescent microscope at various time points.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eImaging acquisition\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eImmunofluorescence staining was observed with widefield microscopy using a Plan-Apochromatix 100\u0026Aring;~ objective lens (oil immersion, numerical aperture [NA] 1.4), 20x, and 10x objective lens, on inverted Zeiss Axio Observer Z1 using AxioVision 4.8 software.\u0026nbsp;\u0026nbsp;Images were acquired using a monochrome Zeiss Axio Cam MRm CCD camera\u0026nbsp;and\u0026nbsp;processed\u0026nbsp;using\u0026nbsp;the NIH Image J software.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eImage analysis and microtubule length quantification\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eMicrotubules length was analyzed using the NeuronJ plugin from Image J\u0026nbsp;(imagej.nih.gov). \u0026nbsp;For that purpose the images were transformed to 8-bit, vectorized, and the tubulin filaments were measured\u0026nbsp;on a computer screen for at the least 10 filaments in each image.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eStatistics\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eStatistical analysis was made using Graph Pad (version 9.4.1).Experimental values are expressed as mean \u0026plusmn; SD of three independent experiments. \u0026nbsp;Statistical analysis was performed using ANOVA. \u0026nbsp;A p-value of less than 0.05 was considered significant.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgments\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe also thank our colleagues, including Drs. Sophia George and Wensi Tao for their conceptual discussions and technical advice in the course of the experiments. \u0026nbsp; We thank many of our colleagues for advice and discussion during the course of the experiments and the preparation of the manuscript. \u0026nbsp;Our student interns over the years, including Shria Bucha, Shalala Leny, Mariana Lopes dos Santos, Julio Baigorri, Rogelio Baucells, and Justin Leal, contributed to the early part of the research project.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eInternal pilot funding from Sylvester Comprehensive Cancer Center/University of Miami was the key supported for this project. \u0026nbsp;The labs\u0026nbsp;were also partially supported\u0026nbsp;by funds from grants including R21 CA277418 to RP, TCW; and Department of Defense Project OC220187 and LC220190, R01 CA230916, and R01 CA286527 to X-X Xu from NCI, NIH.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interest\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare no competing interests.\u003cbr\u003e\u0026nbsp;\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n \u003cli\u003eRowinsky EK, Eisenhauer EA, Chaudhry V, Arbuck SG, Donehower RC. Clinical toxicities encountered with paclitaxel (Taxol). Semin Oncol. 1993; 20(4 Suppl 3):1-15.\u003c/li\u003e\n \u003cli\u003eMarkman M. Managing taxane toxicities. Support Care Cancer. 2003 Mar;11(3):144-7.\u0026nbsp;\u003c/li\u003e\n \u003cli\u003eTr\u0026uuml;eb RM. Chemotherapy-induced alopecia. Semin Cutan Med Surg. 2009 Mar; 28(1):11-4.\u003c/li\u003e\n \u003cli\u003eRohl J, Kushner D, Markman M. Chronic administration of single-agent paclitaxel in gynecologic malignancies. Gynecol Oncol. 2001 May;81(2):201-5.\u003c/li\u003e\n \u003cli\u003eChon SY, Champion RW, Geddes ER, Rashid RM. Chemotherapy-induced alopecia. J Am Acad Dermatol. 2012 Jul;67(1):e37-47.\u003c/li\u003e\n \u003cli\u003eRossi A, Fortuna MC, Caro G, Pranteda G, Garelli V, Pompili U, Carlesimo M. Chemotherapy-induced alopecia management: Clinical experience and practical advice. J Cosmet Dermatol. 2017; 16:537-41.\u003c/li\u003e\n \u003cli\u003ePaus R, Haslam IS, Sharov AA, Botchkarev VA. Pathobiology of chemotherapy-induced hair loss. Lancet Oncol. 2013 Feb; 14(2):e50-9.\u003c/li\u003e\n \u003cli\u003eDavis ST, Benson BG, Bramson HN, Chapman DE, Dickerson SH, Dold KM, Eberwein DJ, \u0026nbsp;Edelstein M, Frye SV, Gampe RT Jr, Griffin RJ, Harris PA, Hassell AM, Holmes WD, Hunter RN, Knick VB, Lackey K, Lovejoy B, Luzzio MJ, Murray D, Parker P, Rocque WJ, Shewchuk L, Veal JM, Walker DH, Kuyper LF. Prevention of chemotherapy-induced alopecia in rats by CDK inhibitors. Science. 2001 Jan 5;291(5501):134-7.\u0026nbsp;\u003c/li\u003e\n \u003cli\u003eMarx J. Cancer research. Preventing hair loss from chemotherapy. 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Breast J. 2020 Jul;26(7):1296-1301.\u0026nbsp;\u003c/li\u003e\n \u003cli\u003eRugo HS, Klein P, Melin SA, Hurvitz SA, Melisko ME, Moore A, Park G, Mitchel J, B\u0026aring;geman E, D\u0026apos;Agostino RB Jr, Ver Hoeve ES, Esserman L, Cigler T. Association Between Use of a Scalp Cooling Device and Alopecia After Chemotherapy for Breast Cancer. JAMA. 2017 Feb 14;317(6):606-614.\u003c/li\u003e\n \u003cli\u003eTollenaar RA, Liefers GJ, Repelaer van Driel OJ, van de Velde CJ. Scalp cooling has no place in the prevention of alopecia in adjuvant chemotherapy for breast cancer. Eur J Cancer. 1994;30A(10):1448-53.\u0026nbsp;\u003c/li\u003e\n \u003cli\u003eWikramanayake TC, Haberland NI, Akhundlu A, Laboy Nieves A, Miteva M. Prevention and Treatment of Chemotherapy-Induced Alopecia: What Is Available and What Is Coming? Curr Oncol. 2023 Mar 25;30(4):3609-3626.\u003c/li\u003e\n \u003cli\u003eRowinsky EK, Donehower RC. Paclitaxel (taxol). 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BMC Cancer. 2021 Sep 1;21(1):981.\u0026nbsp;\u003c/li\u003e\n \u003cli\u003eAmaya C, Smith ER, Xu XX. Low Intensity Ultrasound as an Antidote to Taxane/Paclitaxel-induced Cytotoxicity. J Cancer. 2022 Apr 18;13(7):2362-2373.\u003c/li\u003e\n \u003cli\u003eMitragotri S, Blankschtein D, Langer R. Ultrasound-mediated transdermal protein delivery. Science. 1995 Aug 11;269(5225):850-3. doi: 10.1126/science.7638603. PMID: 7638603\u003c/li\u003e\n \u003cli\u003eCheret J, Samra T, Verling SD, Gherardini J, Rodriguez-Feliz J, Bauman AJ, Sanchez CA, Wikramanayake TC, Xu XX, Paus R. Low-Intensity Ultrasound as a Potential Intervention Strategy to Protect Human Scalp Hair Follicles from Taxane-Induced Toxicity. \u0026nbsp;J Invest Dermatol. 2023 Sep;143(9):1809-1813.e2. \u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":true,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"nature-portfolio","isNatureJournal":true,"hasQc":false,"allowDirectSubmit":false,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"","title":"Nature Portfolio","twitterHandle":"","acdcEnabled":false,"dfaEnabled":false,"editorialSystem":"ejp","reportingPortfolio":"","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"Ultrasound, shock wave, microtubules, Taxol/Paclitaxel, cytotoxicity, hair follicles, alopecia, cancer chemotherapy","lastPublishedDoi":"10.21203/rs.3.rs-4356119/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-4356119/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003ePaclitaxel exemplifies one of the key taxanes (paclitaxel, docetaxel, cabazitaxel), a group of versatile and effective drugs commonly used in chemotherapy for several major cancer types. These drugs work by stabilizing cellular microtubules, a unique mechanism to account for their impressive success in oncology. Unfortunately, side effects and inevitable development of resistance limit their utility. Hair loss (alopecia) is a well-known adverse side effect and poses a significant quality of life issue for many patients. Substantial efforts have been made to prevent or limit alopecia in chemotherapy, however their efficacy is minimal.\u003c/p\u003e\n\u003cp\u003eWe discovered that a brief exposure to low intensity and low frequency ultrasound at a defined timing is able to eliminate toxicity of paclitaxel (and other taxanes) in cultured cells by breaking the stabilized cellular microtubules. We subsequently showed that a brief exposure of low intensity ultrasound was able to break cellular microtubules and mitotic spindles transiently in hair follicle matrix cells of the furred skin of live mice. Such treatment reversed mitotic arrest by paclitaxel in the proliferative hair follicle matrix cells and prevented cell death, and thus annulled the consequent hair follicle damage and suppression of hair growth following paclitaxel administrations. These experimental findings herald a practical method that is within reach to prevent hair loss in cancer chemotherapy using taxanes.\u003c/p\u003e","manuscriptTitle":"Disruption of microtubules with low intensity ultrasound rescues hair follicle damage by paclitaxel","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-06-05 17:16:31","doi":"10.21203/rs.3.rs-4356119/v1","editorialEvents":[],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"nature-communications","isNatureJournal":true,"hasQc":false,"allowDirectSubmit":false,"externalIdentity":"NCOMMS","sideBox":"Learn more about [Nature Communications](http://www.nature.com/ncomms/)","snPcode":"","submissionUrl":"https://mts-ncomms.nature.com/","title":"Nature Communications","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"ejp","reportingPortfolio":"Nature Communications","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"e6fcf407-6e91-41fe-9a32-8b5ca1db23ce","owner":[],"postedDate":"June 5th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"under-review","subjectAreas":[{"id":31693534,"name":"Health sciences/Health care/Quality of life"},{"id":31693535,"name":"Biological sciences/Cell biology/Cytoskeleton/Microtubules"}],"tags":[],"updatedAt":"2024-06-05T17:16:31+00:00","versionOfRecord":[],"versionCreatedAt":"2024-06-05 17:16:31","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-4356119","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-4356119","identity":"rs-4356119","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}