Cancer-associated fibroblasts (CAFs) are abundant components of the tumor microenvironment in the skin of early-stage mycosis fungoides (MF) patients. Evidence suggests CAFs promote the progression of early-stage MF to late-stage MF by suppressing the tumor surveillance function of the immune system. Currently, little is known about the CAFs responsible for immune evasion and disease progression in MF or how this differs by patient demographics or stage of disease. As CAFs are heterogeneous cells, investigating CAFs requires novel molecular techniques that account for the diversity of cell populations and can determine the location of critical subtypes. The Research Training Plan will use the novel molecular techniques of single-cell RNA sequencing (AIM 1) and spatial RNA transcriptomics (AIM 2) to uncover the subtypes and location of CAFs responsible for immune evasion and MF progression from a diverse patient population. In Aim 1, Dr. Johnson will work with mentor Dr. Winston Timp and advisor Dr. Ron Sweren to obtain patient samples, dissociate whole skin tissue, and perform single-cell RNA sequencing on patients from diverse stages and demographics in the K99 phase. Dr. Johnson will train with advisors, Drs. Elana Fertig and Stephanie Hicks to develop computational analysis and apply statistical models to identify subtypes of CAFs based on gene expression. Advisor Dr. Jaroslaw Jedrych will provide guidance with immunohistochemistry verification of gene biomarkers. The R00 phase will include the analysis to identify biomarkers relevant to MF disease diagnosis and prognosis in this diverse patient population. In Aim 2, Dr. Johnson will work with mentor Dr. Winston Timp and collaborator Dr. Elana Fertig to execute spatial RNA sequencing techniques and acquire computational analysis to integrate single-cell RNA sequencing data with spatial RNA sequencing data to locate the subtypes of CAFs identified in AIM 1 in the K99 period. The R00 period will further explore all cells within the tumor microenvironment to uncover candidate cytokines and receptors responsible for crosstalk pathways between the MF tumor cells and the CAF subtypes of interest. Dr. Johnson has had some prior training in single-cell RNA sequencing and analysis and expertise in skin biology. The Career Development Plan is tailored to enable Dr. Johnson to develop computational analytical skills and refine her statistical analysis of single-cell and spatial data while gaining valuable background skills through coursework. Mentor Dr. Winston Timp is a leading expert in sequencing and genomics. Advisors Dr. Stephanie Hicks, an expert in single-cell statistical analysis, and Dr. Elana Fertig, an innovator in computational analysis of integrative transcriptomics, offer complementary expertise. The JHU Department of Dermatology and Biomedical Engineering environment is an outstanding collaborative setting with excellent core facilities and biorepositories infrastructure. In summary, the s...