Hybrid nanocomplexes of rare‑earth nanoparticles and cholesterol inhibit CD44⁺ CSCs in Ehrlich carcinoma, reducing tumor growth 75 % and extending survival

Abstract

Targeting cancer stem cells (CSCs) is central to contemporary oncology. Using the Ehrlich carcinoma (EC) model, we identified CD44⁺/CD24⁻ cells as the dominant CSC subpopulation, exhibiting a ten‑fold higher tumorigenicity than CD44⁻ cells. A hybrid nanocomplex comprising GdYVO₄:Eu³⁺ orthovanadate nanoparticles and cholesterol markedly suppressed tumor growth. In vivo treatment reduced the CD44^high cell fraction by 74.7 % relative to controls, increased CD117⁺ stromal cells, and extended survival by 3.5‑fold.

Background

Malignant growth remains one of the most urgent challenges in medicine. Recent advances hinge on the discovery of CSCs, which resist conventional therapies and drive relapse. CD44, CD24, and CD117 are key phenotypic markers in various cancers, including breast carcinoma (BC) and the transplantable Ehrlich carcinoma (EC). The CD44^high subset, particularly CD44⁺/CD24⁻, has been repeatedly shown to drive tumor initiation and progression. Understanding its biology is essential for developing selective therapeutics.

Methods

Eight‑month‑old female Balb/C mice were used under approved ethical protocols. EC cells were cultured in the peritoneal cavity (PC) to restore native phenotypic traits. Flow cytometry (BD FACS Calibur) quantified CD44, CD24, CD117, and Sca‑1 expression; CD44^high cells were defined by fluorescence >10³ on a logarithmic scale.

Immunomagnetic sorting isolated the CD44⁺ fraction (90 % purity). Tumorigenicity was assessed by intraperitoneal injection of 3 × 10⁶ cells/animal, with tumor growth monitored after 7 days. Dose–response studies employed 3 × 10⁶, 3 × 10⁵, 3 × 10⁴, and 3 × 10³ cells per mouse.

Hybrid nanocomplexes (1.30 g/L GdYVO₄:Eu³⁺ NPs + 0.55 g/L cholesterol) were synthesized via aqueous colloidal methods and characterized by TEM (size <100 nm). EC cells were pre‑treated for 3 h with either NPs alone (option 1) or the full hybrid (option 2) at a ratio of 100 µL NPs per 900 µL cell suspension. Post‑incubation, cells were washed and re‑injected (3 × 10⁶ cells/animal). Tumor burden, CD44^high/CD117⁺ ratio, and survival to day 20 were recorded.

Results

Flow cytometry confirmed a heterogeneous EC population: CD44⁺/CD24⁻ (≈30 %), CD44⁺/CD24⁺, CD44⁻/CD24⁺, and Sca‑1⁺ cells. CD44⁺ cells produced 23‑fold higher ascitic cell counts and 105‑fold higher tumor incidence than CD44⁻ cells at 3 × 10⁶ injections, establishing them as CSCs.

CD117⁺ stromal cells inversely correlated with tumor growth; the CD44^high/CD117⁺ ratio dropped 4‑fold when CD44⁻ cells were used, whereas it remained high (0.02 RU) with CD44⁺ inocula.

Pre‑treatment with NPs alone reduced CD44^high cells by ~50 % and inhibited tumor growth by 59.4 % (P < 0.05). The full hybrid nanocomplex achieved a 74.7 % reduction in tumor growth, a 10‑fold drop in the CD44^high/CD117⁺ ratio, and increased median survival from 12 to 42 days (3.5‑fold).

Discussion

Our findings confirm that CD44^high/CD24⁻ cells constitute the CSC core in EC, driving tumor initiation even at 10⁴ cells/mouse. CD117⁺ cells appear to restrain CSC expansion, as evidenced by the inverse relationship between CD44^high/CD117⁺ ratio and tumor burden. The hybrid nanocomplex exerts dual action: nanoparticles directly impair CSC viability, while cholesterol targets tumor‑cell membranes via SR‑B1 and Cav‑1, enhancing delivery. This synergy explains the superior antitumor efficacy relative to nanoparticles alone.

These results support the CD44^high/CD117⁺ ratio as a prognostic marker and suggest that nanotherapeutics can be tailored to disrupt CSC–microenvironment crosstalk. Future studies should evaluate systemic delivery and potential translation to human solid tumors.

Conclusions

The EC model comprises a heterogeneous mix of tumor and microenvironment cells, with CD44⁺/CD24⁻ cells acting as the primary CSCs.
CD44⁺ cells exhibit a ten‑fold higher tumorigenicity than CD44⁻ cells, driving ascitic tumor growth.
Hybrid nanocomplexes of GdYVO₄:Eu³⁺ NPs and cholesterol reduce tumor growth by 75 % and extend survival 3.5‑fold by selectively targeting CD44⁺ CSCs.
Reduction of the CD44^high/CD117⁺ ratio correlates with therapeutic response, proposing it as a diagnostic and prognostic indicator.