Landscape of surfaceome and endocytome in human glioma is divergent and depends on cellular spatial organization

Governa et al., Proceedings of the National Academy of Sciences, 2022

DOI: 10.1073/pnas.2114456119

Therapeutic strategies directed at the tumor surfaceome (TS),including checkpoint inhibitor blocking antibodies, antibody drug conjugates (ADCs), and chimeric antigen receptor T (CAR-T) cells, provide a new armament to fight cancer. However, a remaining bottleneck is the lack of strategies to comprehensively interrogate patient tumors for potential TS targets. Here, we have developed a platform (tumor surfaceome mapping [TS-MAP]) integrated with a newly curated TS classifier (SURFME) that allows profiling of primary 3D cultures and intact patient glioma tumors with pre-served tissue architecture. Moreover, TS-MAP specifically identi-fies proteins capable of endocytosis as tractable targets for ADC sand other modalities requiring toxic payload internalization. In high-grade gliomas that remain among the most aggressive forms of cancer, we show that cellular spatial organization (2D vs. 3D) fundamentally transforms the surfaceome and endocytome(e.g., integrins, proteoglycans, semaphorins, and cancer stemcell markers) with general implications for target screening approaches, as exemplified by an ADC targeting EGFR. TheTS-MAP platform was further applied to profile the surfaceome and endocytome landscape in a cohort of freshly resected glio-mas. We found a highly diverse TS repertoire between patient tumors, not directly associated with grade and histology, which highlights the need for individualized approaches. Our data pro-vide additional layers of understanding fundamental to the future development of immunotherapy strategies, as well as procedures for proteomics-based target identification and selection. The TS-MAP platform should be widely applicable in efforts aiming ata better understanding of how to harness the TS for personalized immunotherapy

1. 연구 배경

glioblastoma는 치료 저항성이 강하고 재발률이 높은 악성 뇌종양이다. 최근 항체치료, antibody-drug conjugate, CAR-T cell therapy처럼 세포 표면 단백질을 표적으로 하는 치료 전략이 주목받고 있지만, 실제 환자 종양에서 어떤 표면 단백질을 표적으로 삼아야 하는지는 아직 명확하지 않다.

이 논문은 glioma 세포의 surfaceome, 즉 세포 표면에 존재하는 단백질군과, 그중 실제로 세포 안으로 internalization 되는 endocytome을 체계적으로 분석했다. 단순히 표면에 발현되는 단백질을 찾는 것을 넘어, 약물 전달이나 ADC 치료에 중요한 “세포 안으로 들어갈 수 있는 표적”을 함께 찾았다는 점이 이 논문의 주된 연구 내용이라 할 수 있다.

2. 연구 목적

이 연구의 목적은 환자 유래 glioma 조직과 3D glioma culture에서 종양 표면 단백질을 직접 분석할 수 있는 플랫폼을 개발하는 것이다. 저자들은 이를 TS-MAP, tumor surfaceome mapping이라고 하였으며, 이 연구는 glioma 세포가 2D monolayer로 자랄 때와 3D spheroid 형태로 자랄 때 표면 단백질 구성이 달라지는지 확인하였다.  실제 종양은 2D culture보다 3D 조직 구조에 가깝기 때문에, 세포의 공간적 조직화가 drug target discovery에 영향이 다를것이라 유추 하였다.

3. 연구 방법

세포 표면 단백질을 선택적으로 biotinylation한 뒤, 이를 분리하여 LC-MS/MS 기반 proteomics로 분석했다. 이 과정을 통해 표면에 노출된 단백질을 정량적으로 확인할 수 있었다.또한 표면 단백질이 시간이 지나면서 세포 안으로 들어가는지를 보기 위해 endocytosis fraction을 따로 분석했다.  단순한 surfaceome뿐 아니라, 실제 drug payload delivery에 적합한 endocytosis-capable target을 선별할 수 있었다.

분석 대상은 primary human GBM cell culture, 2D culture, 3D spheroid, mouse glioma model, 그리고 freshly resected patient glioma tissue였다. 추가로 SURFME classifier를 만들어, mass spectrometry 결과 중 진짜 세포막/세포표면 단백질에 해당하는 후보들을 걸러냈다.

4. TS-MAP 플랫폼 개발

첫 번째 주요 결과는 TS-MAP이 glioma surfaceome과 endocytome을 안정적으로 분석할 수 있다는 점이다. 해당 논문에서는 세포 표면 biotinylation이 2D culture뿐 아니라 3D spheroid에서도 잘 작동한다는 것을 확인했다.

또한 biotinylated surface proteins가 CD63-positive endolysosomal vesicle로 이동하는 것을 live imaging으로 보여주어, 표면 단백질의 internalization이 실제 endocytosis를 반영한다는 점을 검증했다. 이는 TS-MAP이 단순한 표면 발현 분석을 넘어서, 약물 전달에 중요한 internalization target까지 찾을 수 있는 플랫폼임을 의미한다.

5. 2D와 3D glioma culture의 차이

이 논문의 가장 중요한 메시지는 glioma 세포의 표면 단백질 구성이 2D culture와 3D spheroid에서 크게 다르다는 것이다.

3D spheroid로 자란 glioma 세포에서는 많은 cell-surface proteins의 internalization이 증가했다. 특히 integrins, proteoglycans, semaphorins, cancer stem cell marker와 관련된 단백질들이 3D 환경에서 달라졌다.

이 결과는 target screening을 2D culture만으로 수행하면 실제 종양에서 중요한 표적을 놓칠 수 있다는 것을 보여준다. 즉, glioma drug delivery target을 찾을 때는 세포가 실제 종양처럼 조직화된 3D 환경에서 분석하는 것이 중요하다.

6. Endocytome의 의미

Endocytome 분석은 이 논문에서 특히 중요한 부분이다. 항체치료나 CAR-T처럼 표면에 붙는 것만으로 작동하는 치료도 있지만, ADC나 nanoparticle drug delivery처럼 약물이 세포 안으로 들어가야 하는 전략에서는 표적 단백질의 internalization 능력이 매우 중요하다. TS-MAP을 통해 표면에 존재하는 단백질과 실제로 세포 안으로 들어가는 단백질을 구분했다. 따라서 이 플랫폼은 단순한 biomarker discovery가 아니라, drug delivery target discovery에 직접적으로 연결될 수 있다.

7. Patient Glioma 분석

저자들은 TS-MAP을 실제 freshly resected patient glioma tissue에 적용했다. 총 10명의 glioma 환자 조직을 분석했으며, low-grade oligodendroglioma, anaplastic astrocytoma, primary GBM, recurrent GBM, gliosarcoma 등 다양한 병리학적 유형이 포함되었다.

분석 결과, 환자마다 surfaceome profile이 매우 달랐다. 흥미롭게도 이 차이는 단순히 WHO grade나 histology만으로 설명되지 않았다. 같은 high-grade glioma라도 표면 단백질 구성이 다를 수 있다는 뜻이다.

이는 glioma 치료에서 universal target 하나를 찾는 접근보다, 환자별 surfaceome/endocytome을 기반으로 한 personalized targeting 전략이 필요하다는 점을 보여준다.

8. Tumor Microenvironment까지 포함한 분석

이 연구는 종양세포만 보는 데서 멈추지 않고, glioma tissue 안에 존재하는 immune cells, tumor-associated macrophages, endothelial cells 같은 tumor microenvironment 구성요소도 함께 고려했다.

저자들은 tissue section과 single-cell suspension에서 CD45-positive immune cells, CD68-positive macrophages, CD31-positive endothelial cells, EGFR-positive tumor cells 등이 biotinylation 되는 것을 확인했다. 이는 TS-MAP이 종양세포뿐 아니라 종양 미세환경 내 다양한 세포군의 표면 단백질 분석에도 활용될 수 있음을 의미한다.

9. 주요 결론

이 논문은 glioma의 surfaceome과 endocytome이 세포의 공간적 조직화에 따라 크게 달라진다는 것을 보여준다. 2D culture에서 얻은 표면 단백질 정보는 실제 3D tumor architecture를 충분히 반영하지 못할 수 있다.

또한 환자 glioma 조직 간 surfaceome heterogeneity가 매우 크기 때문에, glioma 표적치료와 drug delivery 전략은 환자별로 달라져야 할 가능성이 높다. TS-MAP은 이러한 personalized target discovery를 가능하게 하는 플랫폼으로 제시된다.

Tumor surfaceome mapping (TS-MAP) of glioma tumors. (A) Schematic outline of procedures for uncovering the surfaceome and endocytome in intact patient, mouse tumors, primary GBM 2D and 3D-spheroid cultures (A1). (A2) A workflow was established for reversible biotinylation, TS enrichment by high-affinity HPLC, and LC-MS/MS, integrated with high-resolution imaging using a GaAsP array-confocal detector (Airyscan) and FACS. (A3) An in-house TS classifier (SURFME) was curated for filtering and categorization of bona fide membrane proteins, identifying potential target candidates for further validation by immunofluorescence analyses of matched tumor sections, and pilot ADC treatment experiments in vitro. (B) Quantification of surfaceome and endocytome in primary GBM cell cultures grown in 2D and 3D. Representative histograms from FACS analysis of nonbiotinylated (control), total surface biotinylation (surface), residual cell-surface signal following MesNa treatment (surface + MesNa), and endocytosed surface proteins (internalized) in U3065 (Upper) and U3082 (Lower) GBM cells, grown in 2D or 3D, as indicated. (C) Quantitative analysis of the experiment presented in B with endocytosed proteins expressed as a fraction of the total surfaceome protein abundance. Data are presented as mean ± SD from three independent experiments each performed in triplicates. MFI, median fluorescence intensity. (D) High-resolution Airyscan imaging of surface and endocytosed biotinylated proteins (green) in GBM cells grown in 2D or 3D (shown in representative brightfield images), as indicated. (Scale bars, 5 μm [U3065 2D], 2 μm [U3065 2D Inset], 50 μm [U3065 and U3082 3D, Left], and 20 μm [U3065 and U3082 3D, Left].) (E) Airyscan imaging of endocytosed biotinylated proteins (green) and the early endosome marker EEA1 (magenta) in GBM cells grown in 2D or 3D, as indicated, after 1.5 h of endocytosis. (Scale bars, 5 μm, and 2 μm for Insets.) (F) Airyscan imaging for visualization of endocytosed surface proteins in the membrane of endolysosomal vesicles, as indicated by CD63-mCherry (magenta). Images were captured 45 min after initiation of endocytosis (see also Movies S1 and S2). (Scale bars, 2 μm.) (D–F) Shown are representative images from at least three independent experiments. White squares indicate zoomed areas shown in Insets. White arrows indicate colocalization.