Introduction

Metabolic reprogramming is a hallmark of cancer, with cells relying on aerobic glycolysis for energy production, even under oxygenated conditions (Warburg effect). Lactate dehydrogenase A (LDHA) converts pyruvate to lactate, supporting cancer cell growth and is linked to poor prognosis and therapy resistance. Meanwhile, lactate dehydrogenase B (LDHB) promotes metastasis by increasing metabolic flexibility. This study aims to establish a head and neck squamous cell carcinoma (HNSCC) cell line with double knockout of LDHA and LDHB to investigate how metabolic impairment affects tumor aggressiveness.

Materials and methods

After selecting top-performing lipid nanoparticles (LNPs), HNSCC cell lines (SAS, UD-SCC 5) were transfected with LNPs containing CRISPR/Cas9 with green fluorescent protein (GFP) and guide RNAs targeting LDHA/B. GFP-positive cells were purified by fluorescence-activated cell sorting and subjected to single-cell cloning to achieve monoclonality. Knockout was confirmed by Western blot and Sanger sequencing, and cell proliferation was monitored for one month post-transduction.

Results

Stable LDHA/B double knockout HNSCC cells were successfully generated, as confirmed by the absence of LDHA and LDHB proteins through Western blotting and the verification of targeted gene disruptions via Sanger sequencing. Additionally, our findings demonstrate that the knockout of LDHA and LDHB impairs tumor cell proliferation in vitro.

Discussion

This study established a stable LDHA/B knockout HNSCC cell line using CRISPR/Cas9-LNPs. This knockout HNSCC model cell line serves as a valuable tool for investigating the effects of metabolic disturbances on tumor aggressiveness and holds potential for improving therapies for aggressive cancers such as HNSCC.

Nein