研究背景：

在某些病理条件下，机体可能发生髓外造血现象。然而，对于实体肿瘤来说其内部是否存在髓外造血，目前尚不明确。近期的一些研究揭示了血管周细胞具有祖细胞的特性且能在特定的培养体系或微环境中分化为脂肪细胞、软骨细胞、成骨细胞和骨骼肌细胞等多种细胞。肿瘤微环境（tumor microenvironment，TME）是由多种肿瘤相关细胞、炎症因子和新生血管等组成的复杂微环境，其中，肿瘤的新生血管为肿瘤提供养料和氧气，在肿瘤的生长和转移过程中起着至关重要的作用。肿瘤血管周细胞分布在肿瘤微环境中，可以被血管内皮细胞分泌的细胞因子PDGF-B募集并包覆在血管内皮细胞的外侧，在肿瘤血管的生成和稳定过程中发挥着关键作用。本研究旨在探讨肿瘤微环境中的血管周细胞是否具有髓外造血的潜能，以及在特定炎症因子或细胞因子的作用下，是否能够实现红系样定向造血。此外，还将重点研究血管周细胞在转分化为造血样细胞过程中受到的关键分子调控机制。

研究目的：

本研究旨在探讨病理条件下，尤其是富含PDGF-B的肿瘤微环境中，血管周细胞髓外造血发生及其潜在分子机制。

研究方法：

通过全基因组表达谱分析PDGF-B处理后的小鼠来源的血管周细胞基因变化；采用NG2-CreERT2; R26R-tdTomato小鼠对血管周细胞进行遗传追踪并分析其在体内外的分化潜能；使用流式细胞术（Fluorescence activated cell sorter，FACS）、单细胞测序和集落形成实验对分选的细胞进行鉴定分析；使用吉姆萨及血红蛋白特异性染色分析集落中的细胞类型；使用定量聚合酶链反应（quantitative polymerase chain reaction，qPCR）、酶联免疫吸附测定（enzyme-linked immunosorbent assay，ELISA）、细胞磁珠分选和免疫染色检测红细胞生成素（erythropoietin，EPO）的水平；使用PDGFR阻断剂及EPO阻断剂研究潜在的分子机制；采用骨髓移植方法构建CD45.1（骨髓）/CD45.2（受鼠）嵌合型小鼠模型。

研究结果：

通过PDGF-B处理的血管周细胞全基因组表达谱分析，结果显示：血管周细胞表现出造血干细胞和祖细胞样特征，PDGF-B刺激后的血管周细胞在基因表达水平表现出骨髓红系细胞的特性。

1）采用PDGF-B过表达的路易斯肺癌（Lewis Lung Cancer，LLC）细胞系和小鼠纤维肉瘤（T241）细胞系建立荷瘤小鼠模型，结果显示：PDGF-B可刺激癌症相关成纤维细胞（cancer-associated fibroblasts，CAF）产生高水平的EPO。

2）通过遗传示踪及FACS分析，结果显示：在高表达PDGF-B的肿瘤组织中NG2⁺血管周细胞具备转分化为干/祖细胞样细胞的能力，表现出明显的干性特征，并可以分化为红细胞样细胞。

3）通过单细胞测序和集落形成实验，结果显示：在PDGF-B刺激下，肿瘤组织中的NG2⁺周细胞形成红细胞样集落，并具备红系细胞分化能力，从肿瘤中分离的NG2⁺血管细胞形成了与典型的骨髓造血不同的红细胞祖细胞；使用联苯胺染色及FACS对细胞集落进行检测，结果显示：这些集落呈现出明显的血红蛋白阳性信号或表达红细胞的标志物：Ter119和CD71。

4）采用PDGFR阻断剂及EPO阻断剂处理NG2⁺周细胞，结果表明PDGFRβ信号在转分化过程及EPO在调控红系分化和成熟中的重要作用。

5）通过分析CD45.1/CD45.2嵌合型荷瘤小鼠的表型，结果显示：红系样细胞来源于血管周细胞。

6）采用高表达PDGF-B的人类鳞状细胞癌A431细胞系构建小鼠肿瘤模型；结果显示：PDGF-B可以促进肿瘤微环境中的血管周细胞发生转分化，产生红系样细胞。

研究结论：

本研究提出了肿瘤相关的自发造血（cancer associated spontaneous hematopoiesis, CASH）的新概念，揭示了血管周细胞在肿瘤微环境中转分化为红细胞样细胞的分子机制。该研究为今后肿瘤靶向治疗提供了新策略和潜在的分子靶点。

Background

Under certain pathological conditions, extramedullary hematopoiesis (EMH) can occur in the body. However, whether EMH exists in solid tumors with high oxygen demands remains unclear. Recent studies have revealed that pericytes possess progenitor cell characteristics and can differentiate into various cell types, including adipocytes, chondrocytes, osteoblasts, and skeletal muscle cells under specific culture systems or microenvironments. The tumor microenvironment (TME) is a complex milieu composed of various tumor-associated cells and inflammatory factors. Among them, pericytes and the cytokine platelet-derived growth factor-B (PDGF-B), secreted predominantly by vascular endothelial cells, play critical roles in the recruitment of pericytes and their coverage of microvessels.

This study aims to investigate whether pericytes in the TME possess the potential for extramedullary hematopoiesis and whether they can undergo erythroid-oriented hematopoiesis under the influence of specific inflammatory factors or cytokines. Furthermore, the study will focus on elucidating the key molecular regulatory mechanisms underlying the transdifferentiation of pericytes into hematopoietic-like cells.

Purpose

The aim of this study was to investigate whether pericytes have the ability to regulate hematopoiesis and its potential mechanisms under pathological conditions, especially in the PDGF-B-rich tumor microenvironment (TME).

Methods

Whole-genome expression profiling was performed on pericytes derived from mice following PDGF-B treatment; NG2-CreERT2; R26R-tdTomato transgenic mice were used for genetic lineage tracing to analyze the in vivo and in vitro differentiation potential of pericytes; Flow cytometry (FACS), single-cell RNA sequencing, and colony-forming unit (CFU) assays were employed to characterize and analyze sorted cells; Giemsa staining and hemoglobin-specific staining were used to identify cell types within the colonies; Quantitative polymerase chain reaction (qPCR), enzyme-linked immunosorbent assay (ELISA), magnetic bead cell sorting, and immunostaining were utilized to measure erythropoietin (EPO) levels; PDGFR inhibitors and EPO inhibitors were applied to investigate the potential molecular mechanisms; A bone marrow transplantation model was established using CD45.1 (bone marrow donor)/CD45.2 (recipient) chimeric mice.

Results

Whole-genome expression profiling of NG2⁺ pericytes treated with PDGF-B revealed that these cells exhibited characteristics similar to hematopoietic stem and progenitor cells. NG2⁺ pericytes stimulated by PDGF-B displayed gene expression signatures typical of bone marrow-derived erythroid cells.

1) Tumor-bearing mouse models were established using PDGF-B-overexpressing Lewis lung carcinoma (LLC) cells and T241 mouse fibrosarcoma cells. The results demonstrated that PDGF-B could stimulate cancer-associated fibroblasts (CAFs) to produce high levels of EPO.

2) Genetic lineage tracing and FACS analysis showed that NG2⁺ pericytes in PDGF-B-enriched tumor tissues acquired stem/progenitor-like properties and exhibited clear stemness characteristics. These cells were capable of differentiating into erythroid-like cells.

3) Single-cell RNA sequencing and colony-forming assays indicated that NG2⁺ pericytes in tumor tissues formed erythroid-like colonies under PDGF-B stimulation and demonstrated erythroid differentiation potential. The NG2⁺ cells isolated from tumors gave rise to erythroid progenitors distinct from those in bone marrow hematopoiesis. Benzidine staining and FACS analysis of the colonies showed a strong hemoglobin-positive signal and expression of erythroid markers.

4) Treatment of NG2⁺ pericytes with PDGFR inhibitors and EPO inhibitors confirmed the essential role of PDGFRβ signaling in the transdifferentiation process and the critical function of EPO in regulating erythroid differentiation and maturation.

5) Analysis of the CD45.1/CD45.2 chimeric tumor-bearing mice demonstrated that these erythroid-like cells originated from pericytes rather than from bone marrow hematopoietic cells.

6) Tumor models were established using A431 human squamous cell carcinoma cells with high PDGF-B expression. The results showed that PDGF-B promoted the transdifferentiation of pericytes in the tumor microenvironment into erythroid-like cells.

Conclusions

This study proposes a novel concept of Cancer-Associated Spontaneous Hematopoiesis (CASH) and elucidates the molecular mechanism by which pericytes within the tumor microenvironment transdifferentiate into erythroid-like cells. These findings provide new insights into tumor biology and suggest potential molecular targets for future tumor therapies.