Countering microtubule stabilization within hair follicles in ovarian cancer chemotherapy

Specific Aims (Revised 1-29-2024) For treating ovarian cancer, paclitaxel is the front-line drug used in combination with cisplatin/carboplatin, and is also used alone in a dose dense schedule for recurrent cancer. A highly effective anti-microtubule agent, paclitaxel also causes dose-limiting adverse effects. In particular, as many as 80% of patients receiving paclitaxel develop alopecia, which severely impacts patients’ quality of life. We have discovered that a brief exposure to low-intensity ultrasound can protect various types of proliferating cells from the cytotoxic activity of paclitaxel and cell death in vitro by transiently disrupting paclitaxel-induced formation of rigid and stabilized microtubule bundles. We hypothesize that, through this mechanism, low-intensity ultrasound may prevent alopecia (locally) without compromising the efficacy of systematic cancer therapy. The objectives of the proposal are to test the hypothesis, using human scalp hair follicle organ cultures and suitable mouse models. The initial basic experiments are supported by an DOD pilot funding, and the R01 support will further extend the goals and work, and solidify the results and conclusions, which will guide optimal design of a protocol for clinical trial in the near future. Specific Aim 1. Investigate mechanisms: breaking of the drug-stabilized microtubules and cellular mechanisms in countering paclitaxel activity. Sub-Aim 1-1: In cell-free system, we will investigate the fragmentation of polymerized microtubules by low intensity ultrasound, with and without the presence of paclitaxel. Sub-Aim 1-2: In human scalp hair follicle organ culture, we will study ultrasound impacts on microtubules: their breaking, aggregation of fragments, and degradation by autophagy and in lysosomes. Sub-Aim 1-3: In human scalp hair follicle organ culture, we will study the ultrasound rescuing of cells death by paclitaxel + carboplatin combination, which is commonly used in the treatment of ovarian cancer. Sub-Aim 1-4: We will also verify the ability of ultrasound to counter additional taxanes, including docetaxel and cabazitaxel, which are used in treatment of metastatic breast and prostate cancer, respectively. Sub-Aim 1-5: We will study a serious of time courses, from immediate (5 min), to long term (7 days) of the effects of paclitaxel and/or ultrasound in the human scalp hair follicle organ cultures. The hair follicle organ culture system will enable our intricate analyses using microscopy and cell biology approaches to interrogate both temporary and permanent paclitaxel-induced damage, and this aim will identify underlying protective mechanisms of ultrasound therapy. Specific Aim 2. Verify the prevention of hair follicle damage and study the mechanism of ultrasound in countering paclitaxel cytotoxicity in live mice. Sub-Aim 2-1: We will carry out extensive analyses to verify the biology and mechanisms in live mice. Sub-Aim 2-2: In human scalp hair follicle organ culture, we will study the ultrasound rescuing of cells death by paclitaxel + carboplatin combination, which is commonly used in the treatment of ovarian cancer. Sub-Aim 2-3: We will also verify the ability of ultrasound to counter additional taxanes, including docetaxel and cabazitaxel, which are used in treatment of metastatic breast and prostate cancer, respectively. Sub-Aim 2-4: Single cell RNA-seq technology will be used to analyze hair follicles to determine the overall expression profiles of various cell types impacted by paclitaxel and/or ultrasound. Sub-Aim 2-5: The study in mouse models also will determine in long term (3 to 6 months) impacts of drugs (carboplatin and paclitaxel) and ultrasound on hair follicles and skin in mice, to determine any potential side effects of ultrasound treatment. Specific Aim 3. Determine the ability of ultrasound to prevent paclitaxel-caused hair follicle damage and loss in a mouse model grafted with human scalp hair, and the long-term consequences. Sub-Aim 3-1: We have established a humanized alopecia mouse model by transplanting human scalp haired skin onto immune compromised mice. We will test if ultrasound exposure can prevent hair follicle damage and hair loss caused by paclitaxel treatment in the model. Sub-Aim 3-2: In addition to analyze the acute (1 to 7 days following drug and ultrasound treatment) effects, the experiments will also include long term (3 to 6 months after procedure) effects to verify the mechanisms uncovered in mouse models, and to determine if any potential side effects and safety of ultrasound treatment on human hair follicles. Sub-Aim 3-3: With additional recourse, we will be able to study an increase number of mice implanted with human hair follicles to obtain strong statistical results. This will also allow us to compare and determine the potential differential responses of the hair follicles to paclitaxel and/or ultrasound in correlation with genders, age, and racial ancestral, to determine if biological differences exist. The aims and experiments in the proposed project will help to translate and develop a basic science discovery into clinical practice, to establish a procedure to prevent hair loss in cancer treatment. If successful, the project will have major impact to the quality of life issue for cancer patients.