Unique gene patterns lead to distinct functional phenotypes and chemosensitivity profiles among subclones obtained from a single glioblastoma cell line

Abstract

One of the characteristics of malignant tumors is heterogeneity, which refers to the molecular or genetic differences among progeny cells during tumor growth. This heterogeneity contributes to variations in the tumor growth rate, invasive ability, drug sensitivity, and prognosis. To gain a deeper understanding of the molecular background underlying tumor heterogeneity, we construct monoclonal cell lines derived from the glioblastoma (GBM) cell line U87-MG by limiting dilution assays. The selected CF5 and G11 subclones exhibit completely different cell morphologies and, more importantly, distinct functional phenotypes. CF5 exhibits stronger proliferative properties and chemoresistance, whereas G11 shows greater motility and invasion. Transcriptomic sequencing reveals great differences in gene expression among the CF5, G11, and U87 cell lines, and downregulated genes in individual clones are significantly enriched in gene sets related to extracellular matrix function. ITGA11 and ITGA6, as research subjects, are demonstrated to exclusively regulate functional phenotypes and chemotherapy sensitivity in CF5 or G11 cells. In U87 cells, combined knockdown of these two genes significantly inhibits tumor growth and increases chemotherapy sensitivity, but knockdown of either gene alone does not. In summary, these data reveal that even under uniform growth conditions, the heterogeneity of tumor cells and their diverse genetic backgrounds remain significant and persistent. This finding is crucial for accurately identifying tumor-related genes and their functional phenotypes, and a thorough understanding of the genetic and molecular background underlying tumor heterogeneity is essential for comprehensive cancer treatment.

1. Introduction

Glioblastoma multiforme (GBM) is one of the most prevalent malignant brain tumors known for its profound tumoral heterogeneity and dismal therapeutic outlook [1,2]. Current treatment protocols involving surgical resection, radiotherapy, and pharmacological intervention with agents such as temozolomide (TMZ) have improved short-term survival rates in patients [3,4]. However, heterogeneity among GBM cells within a given tumor increases the risk of tumor recurrence, which ultimately leads to treatment failure [5–8]. Thus, a comprehensive understanding of GBM tumor heterogeneity is imperative for formulating efficient novel treatment modalities aimed at suppressing tumor progression and improving patient survival rates.

Tumor heterogeneity, which encompasses genetic, epigenetic, phenotypic, and functional variability, has emerged as a primary determinant of unfavorable prognoses and outcomes [9,10]. Numerous studies on GBM have revealed clear genetic diversity among single GBM cells, with individual tumors harboring distinct subgroups of corresponding cells [11,12]. These single-cell-derived clones from glioma cells exhibit unique morphological, genetic, and differentiation features. While numerous innovative techniques have been devised to assess single-cell heterogeneity at the gene and protein levels, most methodologies are predominantly geared toward genomics without effective integration of genomics and phenotypic studies.

The differences in integrin expression among different cell subtypes are closely linked to their genetic and functional heterogeneity, which influences crucial tumor processes such as adhesion, invasion, and vascular growth [13–15]. Integrins, notably integrin α6, are potential biomarkers for GBM and are closely related to tumor heterogeneity [16–18]. Although targeting these specifically expressed integrins has shown promise in improving GBM treatment outcomes, the intricate interplay between integrins and tumor cells poses challenges and limits their study [19,20]. Therefore, understanding integrin expression in diverse clones is imperative for patient stratification and the optimization of novel therapies.