(578) Single-cell RNA sequencing and CFTR functional analysis of human eccrine sweat glands establish a viable method for investigating CF genotypic-phenotypic heterogeneity

Background: Chloride concentration in sweat is an essential diagnostic tool for cystic fibrosis and a valuable measure of response to CFTR modulator treatment, generally correlating with key features of CF such as lung disease and pancreatic exocrine severity. However, several exceptions to this relationship have emerged, e.g., F312del and S1455X lead to elevated sweat chloride but near normal lung function when in trans with a CF-causing CFTR variant [1]. To understand the effect of these variants upon CFTR function in eccrine sweat gland (ESG), we have developed a method to characterize ESGs dissected from skin biopsies using single-cell RNA sequencing (scRNA-seq) and short circuit current measurement.

Methods: We received 2 4 mm underarm punch biopsies from a healthy donor (IRB#00119007). From these biopsies, we micro-dissected 7 large ESGs. These glands were dissociated into a single-cell suspension by incubating in collagenase and hyaluronidase overnight followed by treating with dispase, elastase, and trypsin. cDNA library preparation was done with the 10X 3’ v3.1 kit and a NovaSeq 6000 was used to sequence. Seurat was used for analysis. The remaining cells were grown in primary culture by conditional reprogramming using reagent Y and co-culture with irradiated 3T3 cells. CFTR function was measured on passage 2 in Ussing Chambers. Both symmetrical and asymmetrical buffers were tested, cAMP and ATP were used to stimulate chloride channel activity, and Inh-172 was added either on apical or basal side for a quantitative measure of CFTR function.

Results: For scRNA-seq, we captured approximately 3,000 cells and separated them into all expected eccrine sweat gland cells type – clear cells, dark cells, myoepithelial cells, and ductal cells – as well as fibroblasts, keratinocytes, and immune cells. Hypergeometric testing for co-expression with CFTR revealed significant associations with clear cell markers AQP5 (p = 9.84E-12) and CA2 (p = 4.13E-11) as well as ductal cell markers KRT77 (p = 1.88E-3) and S100P (p = 2.01E-4. In primary ESG cultures, amiloride sensitive ENaC activity was higher in symmetric buffer than in asymmetric buffer (7.1±0.1 vs 1.9±0.2 µA/cm²). Interestingly, using asymmetric buffer higher CFTR function was observed when Inh-172 was added to the basal side (11.1±4.0 µA/cm²) compared to the apical side (1.1±4.0 µA/cm²) which recapitulates that CFTR is functional at both apical and basolateral surfaces of the primary ESG cultures, as observed in intact ESGs [2].

Conclusions: We show that CFTR expression occurs in the clear and ductal cells of the ESG and that CFTR functions on both apical and basolateral cell surfaces, consistent with prior findings [2]. Our approach suggests that skin punch biopsies are a minimally invasive technique for studying CFTR expression and function in ESGs of individuals with CF and CF related diseases.

Acknowledgements: Thank you Gloria Seho-Ahiable, Sai Talluru, Meredith Dorminey, Gabrielle Cox, and our anonymous donor for your contributions to this work.

References: [1] Raraigh KS, Paul KC, Goralski JL, Worthington EN, Faino A V, Sciortino S, et al. CFTR bearing variant p.Phe312del exhibits function inconsistent with phenotype and negligible response to ivacaftor. JCI Insight. 2022;7(6).



[2] Wang XE, Reddy MM, Quinton PM. Effects of a new cystic fibrosis transmembrane conductance regulator inhibitor on Cl- conductance in human sweat ducts. Exp Physiol. 2004;89(4):417–25.