Single-Cell Study Links Cell Death Patterns to Tumor Diversity in Liver Cancer
Summary
The researchers scrutinized 254 hepatocellular carcinoma (HCC) specimens, employing tissue microarrays and a mathematical model to assess cellular heterogeneity using surrogate markers and apoptotic cell death marker CASP3. Utilizing density contour mapping and spatial proximity analysis, they investigated the influence of apoptotic cell epicenters on tumor diversity and patient prognosis. They discovered regions, termed CASP3+ cell archipelagos, exhibiting greater cellular diversity, impacting survival rates. Additionally, in vitro studies revealed increased cell death and CASP3 expression led to heightened therapy resistance and hypoxic growth. These findings support the hypothesis that escalated apoptotic cell death contributes to augmented tumor heterogeneity and unfavorable patient outcomes.
Research Criteria
This scholarly inquiry seeks to elucidate the associations between apoptotic cell death, spatial organization, and tumor molecular heterogeneity using single-cell in situ hybridization. Examining 254 hepatocellular carcinoma specimens, researchers employed tissue microarrays and a mathematical model to quantify cellular diversity using surrogate markers and CASP3 as an apoptotic indicator. Investigating the effects of apoptotic cell epicenters on tumor diversity and prognosis, they applied density contour mapping and spatial proximity analysis. Additionally, they devised a selectively controlled in vitro cell death model with CRISPR/CRISPR-associated 9 to assess therapeutic response and hypoxic growth.
Fig.1 Experimental design. (Khatib, 2022)
Sample Type
Tumor samples from human HCC cohorts.
Result—Selection of Heterogeneity Markers to Model Tumor Diversity
The chromatic delineation methodology encompasses the employment of Tissue Microarrays (TMAs) comprising juxtaposed neoplastic and non-neoplastic specimens within the exploratory assemblage (LCI), with subsequent RNAScope staining application and confocal microscopy employed to capture visual representations. Quantification was facilitated by Indica Labs' HALO software, and this investigative process was replicated for the confirmatory cohort. Individualized cellular identification of respective biomarkers is signified by discrete color-coded points (CASP3 in verdant, CDKN3 in crimson, and PRC1 in violet), with cellular nucleus recognition achieved via 4',6-diamidino-2-phenylindole (DAPI) staining and concurrent HALO software-rendered estimations pertaining to cytoplasmic periphery. Mean copy number quantification for each molecular indicator-CASP3, CDKN3, and PRC1-executed in both assemblages, revealed considerable disparities in expression within neoplastic samples vis-à-vis non-neoplastic counterparts, suggesting fluctuating extents of apoptotic cellular demise and heterogeneity, thereby substantiating the sufficiency of these molecular markers in modeling the multifariousness of each neoplastic specimen at the unicellular echelon.
Fig.2 Single cell in situ hybridization profiling of HCC tumors from separate patient cohorts. (Khatib, 2022)
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Reference
- Khatib, S. A.; et al. Single-cell biology uncovers apoptotic cell death and its spatial organization as a potential modifier of tumor diversity in HCC. Hepatology. 2022, 76(3): 599-611
