Background:
To advance the implementation of the 3R principles and improve the physiological relevance of human in vitro tumor models, continuous optimization and validation of complex vascularized 3D tumor platforms is required. In this context, the cellular origin and biological suitability of endothelial cells (EC) used for vascularization remain largely underexplored. While human umbilical vein endothelial cells (HUVEC) are widely used due to their availability and robust angiogenic potential, they may not adequately recapitulate the tissue-specific vascular phenotypes observed in human tumors in vivo.

 

Hypothesis/Objective:
Tissue-specific EC populations may represent a promising strategy to improve model physiology, whereas patient-derived and induced pluripotent stem cell (iPSC)-based systems display an important route for the animal-free validation of these model systems.

 

Methods:
Macrovascular human aortic EC (HAoEC), microvascular human dermal EC (HDMVEC), and tumor-relevant human mammary vascular EC (HMVEC) were evaluated as physiologically relevant alternatives and compared to HUVEC. All endothelial populations were stably transduced with fluorescent proteins to enable longitudinal fluorescence imaging and fluorescent-activated cell sorting (FACS).

To investigate 3D vascular network formation under prolonged dynamic culture conditions, individual endothelial cell populations were embedded with fibroblasts in collagen-fibrin hydrogels either with or without HCC1937 breast cancer spheroids in previously established mesoscopic bioreactors compatible with multiple imaging modalities (1,2). Vascular network formation and remodeling were longitudinally monitored by fluorescence microscopy over 15 days. At the endpoint, vessel functionality was assessed by fluorescent microbead perfusion and validated by confocal microscopy, followed by bulk RNA sequencing of endothelial, stromal, and tumor cells.

To establish a patient-specific validation platform, breast cancer organoids were generated from patient biopsies. Peripheral blood mononuclear cells obtained from the same patients were reprogrammed into iPSC and subsequently differentiated into mesenchymal stem cells (RFP iMSC) and EC (GFP iEC). The resulting cell populations were cultured in static 3D hydrogels for up to 23 days as a precursor for future integration into the bioreactor platform.

 

Results:
In tube formation assays, HUVEC displayed the highest angiogenic potential, followed by HMVEC and HDMVEC, whereas HAoEC exhibited the lowest degree of network formation.

Within the dynamic bioreactor platform, functional vascular networks were successfully established using HUVEC, HMVEC, and HAoEC. Longitudinal imaging further demonstrated that the presence of HCC1937 breast cancer spheroids markedly stabilized vascular networks, shown by increased or maintained vascular parameters between days 7 and 15, whereas tumor-free cultures showed progressive network regression after day 7. HDMVEC failed to form networks under dynamic conditions, irrespective of tumor presence. Here, the absence of a functional network led to a decrease in tumor size over time. Among all endothelial populations, HMVEC promoted the strongest tumor growth and exhibited the most pronounced tumor-vascular interactions, suggesting enhanced tissue-specific crosstalk.

Initial transcriptomic analysis of tumor-free HAoEC networks showed a preservation of endothelial identity with enrichment of arterial and angiogenic tip-cell signatures, while accompanying fibroblasts displayed matrix-remodeling and inflammatory phenotypes.

Finally, patient-specific iPSC-derived iEC formed consistently expanding vascular networks during 23 days of static 3D culture. Continuous increases in vascular parameters, close interactions with patient-matched breast cancer organoids, and perivascular localization of iPSC-derived iMSC demonstrated the feasibility of establishing fully patient-specific vascularized tumor models as a precursor for future implementation in the bioreactor platform.

 

Conclusion:
EC origin profoundly influences vascular network formation, stability, and tumor-vascular interactions in advanced breast cancer models. Tissue-specific EC populations represent experimentally feasible alternatives to HUVEC and may improve model physiology. Further imaging studies will be performed to complement and spatially validate bulk RNA sequencing findings, while patient-specific iPSC-derived vascular components provide a foundation for imaging-based investigation of personalized tumor biology and therapy responses.

 

Image/Figure:
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Image/Figure Caption:
Alternative endothelial cell populations show stable network formation, tumor spheroid interaction, and distinct endothelial phenotypes within imaging-compatible mesoscopic bioreactors. (A) Experimental workflow for the generation and longitudinal characterization of vascularized breast cancer bioreactors. Endothelial cells (ECs) were stably transduced with fluorescent proteins prior to dynamic culture. Tumor spheroids consisting of HCC1937 breast cancer cells and normal human dermal fibroblasts (nHDFs) were generated seven days before bioreactor assembly. Vascular network formation was longitudinally monitored during 15 days of dynamic culture followed by functional analysis, histology, and RNA sequencing. (B) Representative tube formation assays demonstrating distinct angiogenic capacities of individual EC populations. Human umbilical vein endothelial cells (HUVEC) displayed the highest angiogenic potential characterized by the largest vessel area, followed by human mammary vascular endothelial cells (HMVEC) and human dermal microvascular endothelial cells (HDMVEC), whereas human aortic endothelial cells (HAoEC) exhibited only limited tube formation with increased numbers of endpoints. Following initial characterization, stably transduced EC populations were incorporated into imaging-compatible mesoscopic bioreactors where EC self-assembled into functional vascular networks. Functionality was validated by perfusion with fluorescent microbeads. (C) Representative fluorescence images of vascular networks generated from individual EC populations cultured in the absence (left) or presence (right) of HCC1937 tumor spheroids at days 7 and 15. HUVEC, HAoEC, and HMVEC formed extensive vascular networks by day 7. In tumor-free cultures, networks progressively regressed during prolonged culture, whereas the presence of tumor spheroids markedly stabilized vascular structures. HMVEC displayed the most pronounced tumor-vascular interactions. HDMVEC failed to establish functional vascular networks irrespective of tumor presence. (D) Quantification of longitudinal changes in vascular parameters between days 7 and 15. Dashed boxplots represent tumor-free networks, whereas solid boxplots indicate tumor-containing networks. (i) Relative changes in vessel area (%) demonstrate pronounced regression of tumor-free networks, while HUVEC and HMVEC networks remained largely stable in the presence of tumor spheroids. HAoEC-derived networks even exhibited an increase in vessel area over time. (ii) Relative changes in branching points/mm² indicating enhanced vascular stability, network expansion, and continued remodeling in tumor-containing cultures. (E) Relative changes in HCC1937 spheroid size between days 7 and 15. Tumor spheroids increased in size in the presence of HUVEC, HAoEC, and HMVEC networks, with HMVEC-containing cultures supporting the strongest tumor growth. In contrast, the inability of HDMVEC to establish vascular networks resulted in reduced tumor growth and spheroid shrinkage. (F) Transcriptomic analysis of isolated and FACS-sorted HAoEC-derived vascular networks cultured in the absence of tumor spheroids. EC subtype enrichment analysis revealed enrichment of arterial, capillary, and particularly angiogenic tip-cell signatures. The heatmap of the 15 most highly expressed genes demonstrated sustained expression of endothelial identity and angiogenesis-associated markers including RGCC, APLN, CXCR4, ESM1, ANGPT2, VWF, SOX17, and KDR, indicating preservation of biologically relevant endothelial phenotypes during prolonged dynamic culture.

 

Author
Alexandra Raab
RWTH Aachen University