Aladag, Z. | bioRxiv Science

bioRxiv2024

Development of a Robust Gel-Free 3D Culture System for Generating Spheroids from Axolotl Blastema Cells

Regenerative biology seeks to uncover the principles enabling organisms to restore complex tissues and organs. The axolotl (Ambystoma mexicanum), a salamander with unparalleled limb regenerative capacity, remains a premier model system in this field. However, in vitro studies have been limited by the absence of reliable culture platforms for maintaining blastema cell identity and functionality. Here, we present a robust, gel-free 3D culture protocol that enables the formation of spheroids from axolotl blastema cells under fully defined, serum-free conditions. These spheroids preserve the expression of key regenerative markers such as Prrx1, Msx2, and CTGF for at least 10 days in culture, and exhibit cellular stability supported by antioxidant and amino acid supplementation. Importantly, spheroids cultured for 10 days were capable of initiating extra digit formation when transplanted into limb bud after amputation. With unique advantage of fully defined incubation conditions, this technique adds a powerful in vitro platform to existing experimental models of axolotl limb regeneration.

molecular biology↗

bioRxiv2022

Hexokinase 1b is a novel target for non-small-cell lung cancer

Deregulation of glycolysis is common in non-small cell lung cancer (NSCLC). Hexokinase (HK) enzymes catalyze the phosphoryl-group-transfer in glucose metabolism. There are a very few studies that have begun to reveal the connections between glucose metabolism and splicing programs. Unlike HK2 gene, which is expressed as a single transcript, there are several transcripts of the HK1 gene due to alternative splicing. However, the functional differential roles of HK1 isoforms in glucose metabolism and tumor progression are still elusive. Here, we show that primary NSCLC patient tumor cells metabolically differ from the normal lung epithelium where they display predominant expression of one of the HK1 transcripts, hexokinase1b (HK1b). We utilized CRISPR-Cas9 system to selectively target specific HK1b isoform in NSCLC and show that silencing HK1b in NSCLC cells inhibits tumorigenesis through diminishing glycolysis and proliferation. Our findings constitute the first demonstration of the first biochemical distinction between the HK1 splice variants. Finally, HK1b deletion sensitizes NSCLC cells to standard-of-care, cisplatin treatment, and the combination therapy synergistically increases both apoptotic cell death by cisplatin and autophagic cell death by increased formation of LC3-II associated autophagic vesicles and myelinoid bodies. Notably, loss of HK1b leads to cellular DNA damage, further combination with cisplatin therapy showed significantly increased levels of DNA damage. Importantly, we showed that glycolysis and cisplatin resistance can be restored by adding-back HK1b in HK1b knock-out cells. Our findings reveal that targeting HK1b isoform alone or in combination with cisplatin may represent a novel strategy for NSCLC patients.

cancer biology↗