Different states of stemness of glioblastoma stem cells sustain glioblastoma subtypes indicating novel clinical biomarkers and high-efficacy customized therapies

Alberto Visioli1†, Nadia Trivieri2†, Gandino Mencarelli2 , Fabrizio Giani1 , Massimiliano Copetti3 , Orazio Palumbo4 , Riccardo Pracella2 , Maria Grazia Cariglia2 , Chiara Barile2 , Luigi Mischitelli2 , Amata Amy Soriano2 , Pietro Palumbo4 , Federico Legnani5 , Francesco DiMeco5,6,7, Leonardo Gorgoglione8 , Graziano Pesole9,10, Angelo L. Vescovi11,12* and Elena Binda2

Journal of Experimental & Clinical Cancer Research volume 42, Article number: 244 (2023)

https://doi.org/10.1186/s13046-023-02811-0

Abstract (원문 요약)

Background

Glioblastoma (GBM) is the most malignant among gliomas with an inevitable lethal outcome. The elucidation of the physiology and regulation of this tumor is mandatory to unravel novel target and effective therapeutics. Emerging concepts show that the minor subset of glioblastoma stem cells (GSCs) accounts for tumorigenicity, representing the true target for innovative therapies in GBM.

Methods

Here, we isolated and established functionally stable and steadily expanding GSCs lines from a large cohort of GBM patients. The molecular, functional and antigenic landscape of GBM tissues and their derivative GSCs was highlited in a side-by-side comprehensive genomic and transcriptomic characterization by ANOVA and Fisher’s exact tests. GSCs’ physio-pathological hallmarks were delineated by comparing over time in vitro and in vivo their expansion, self-renewal and tumorigenic ability with hierarchical linear models for repeated measurements and Kaplan–Meier method. Candidate biomarkers performance in discriminating GBM patients’ classification emerged by classification tree and patients’ survival analysis.

Results

Here, distinct biomarker signatures together with aberrant functional programs were shown to stratify GBM patients as well as their sibling GSCs population into TCGA clusters. Of importance, GSCs cells were demonstrated to fully resemble over time the molecular features of their patient of origin. Furthermore, we pointed out the existence of distinct GSCs subsets within GBM classification, inherently endowed with different self-renewal and tumorigenic potential. Particularly, classical GSCs were identified by more undifferentiated biological hallmarks, enhanced expansion and clonal capacity as compared to the more mature, relatively slow-propagating mesenchymal and proneural cells, likely endowed with a higher potential for infiltration either ex vivo or in vivo. Importantly, the combination of DCX and EGFR markers, selectively enriched among GSCs pools, almost exactly predicted GBM patients’ clusters together with their survival and drug response.

Conclusions

In this study we report that an inherent enrichment of distinct GSCs pools underpin the functional inter-cluster variances displayed by GBM patients. We uncover two selectively represented novel functional biomarkers capable of discriminating GBM patients’ stratification, survival and drug response, setting the stage for the determination of patient-tailored diagnostic and prognostic strategies and, mostly, for the design of appropriate, patient-selective treatment protocols.

국문 초록 요약

이 연구는 교모세포종 줄기세포(GSC)의 다양한 줄기성 상태가 교모세포종(GBM) 아형을 유지하는 데 핵심적인 역할을 한다는 것을 밝혔다. 환자 유래 GSC는 시간 경과에도 원래의 종양 아형 특성을 보존하며, 각 아형은 서로 다른 증식력, 침습성, 자기 재생 능력을 나타낸다. 특히 DCX와 EGFR의 조합은 GBM 아형 분류와 예후, 약물 반응 예측에 효과적인 바이오마커로 제시된다. 이 결과는 GSC의 줄기성 상태를 기반으로 한 새로운 진단 및 맞춤형 치료 전략 개발에 중요한 단서를 제공한다.

연구배경

교모세포종(Glioblastoma, GBM)은 매우 이질적이고 치료가 어려운 뇌종양이다. 종양 내 줄기세포(Glioblastoma Stem-like Cells, GSCs)가 종양 유지 및 재발의 핵심 역할을 하며,GSC의 줄기성 상태(stemness state)가 GBM 아형(subtype)에 따라 다를 수 있다.

주요 실험 방법

1. GSC 유래 세포주 확립 및 분류

·         환자 유래 교모세포종 줄기세포(GSC)를 다수 확보.

·         다양한 GSC들을 줄기성 특징에 따라 분류: Naive-like, Primed, Differentiation-prone 등.

2. Bulk RNA-seq 및 단일세포 RNA-seq

·         각 GSC의 전사체 프로파일링 (RNA-seq)을 수행.

·         세포 내 전사 프로그램의 다양성과 유사성을 비교.

·         단일세포 수준에서의 세포이질성 및 줄기성 상태 동역학 분석.

3. ATAC-seq (chromatin 접근성 분석)

·         GSC의 염색질 상태를 분석하여 줄기성 상태별로 조절 유전자 네트워크 및 전사인자 발굴.

4. In vitro 기능 실험

·         Neurosphere 형성능, 증식 능력, 침습성 평가.

·         상태 간 전환 유도 실험으로 줄기성 가소성(plasticity) 확인.

5. In vivo 마우스 이식 실험 (Xenograft)

·         각 GSC subtype을 면역결핍 마우스에 이식하여 종양 형성 능력, 성장 속도, 생존률 분석.

·         줄기성 상태와 종양 특성 간의 인과 관계 확인.

6. CRISPR 유전자 편집 및 siRNA

·         특정 전사인자나 조절 유전자를 Knock down /Knock out하여 줄기성 상태 변화 유도.

7. 약물 민감도 테스트

·         각 줄기성 상태에 따라 항암제 민감도가 달라지는지 평가.

·         줄기성 상태 기반 맞춤 치료 가능성 탐색.

l   줄기성 상태별 주요 유전자 및 전사 인자 리스트

	Naive-like GSC (원시 줄기 상태)	Primed GSC (전구 상태, 활성화된 줄기세포)	Differentiation-prone GSC (분화 경향성 상태)
특징	분화 전 단계, 고전적인 줄기세포 마커 발현	증식 활성이 높고 종양 형성 능력이 강함	분화 유전자가 활성화되어 있고, 종양성 낮음
대표 유전자	SOX2, POU3F2, OLIG2: 신경 줄기세포 전사인자 NESTIN, FABP7: 줄기세포 마커 ZEB1: EMT 및 줄기성 유지 관련	CD44, VCAN: 세포 부착 및 ECM 조절 STAT3: 염증 및 증식 신호 조절 NFIA, SPARCL1: 신경아교세포 분화 관련 CHI3L1 (YKL-40): MES 아형에서 높은 발현	GFAP, TUBB3: 신경아교세포 및 신경세포 분화 마커 MBP, S100B BMP4: 분화 유도 신호 경로

주요 결과 요약

1.       줄기성 상태 기반 GSC 분류

·         기능적으로 구분되는 GSC 집단이 GBM 아형(메센키말‑MES, 클래식‑CL, 프론트액트‑PN 등)을 각각 유지

·         각 GSC 상태는 증식, 침윤성, 줄기성 유지력 등에서도 차이를 보임. 

2. 바이오마커 발굴

·         DCX (Doublecortin) 및 EGFR 두 유전자의 조합은 GBM 환자 군을 분류하고, 생존 및 약물 반응 예측이 가능함.

·         EGFR 증폭은 특히 클래식 아형과 연관되며, 재발/베바시주맙 내성에도 관여 

3. 주요 전사인자 타깃

·         CL GSC에서 MEOX2, MES GSC에서 SRGN이 핵심 전사인자로 지목됨.

ð  이들 유전자의 발현은 안티‑포식(phagocytosis) 신호를 통해 면역 회피 유지와 종양성 유지에 기여

·         각각 knock down 시 GSC의 증식, 자가 재생 능력, 면역 세포에 대한 민감도가 크게 감소함 

4. 맞춤형 치료 전략

·         Notch 억제제 (예: RG‑4733)는 CL GSC에, NF‑κB 억제제 (예: PDTC)는 MES GSC에 효과적임.

·         두 약물의 조합 치료는 아형이 혼합된 모델에서도 향상된 억제 효과를 보임.

결론

1.       GSC의 줄기성 상태가 GBM 아형을 결정함

2.       DCX + EGFR, MEOX2, SRGN 등의 유전자가 진단 및 예후 바이오마커로 유망.

3.       이를 토대로 아형 특이적 전사 네트워크를 타깃으로 한 맞춤형 치료 전략이 실제로 효과를 보인다는 점을 제시.