Introduction:: The tumor microenvironment (TME) plays a vital role in cancer progression, necessitating a deeper understanding of its cellular mechanisms for precise treatment. Previous research has emphasized the significance of the interaction between the extracellular matrix (ECM) and tumors in influencing cancer invasion and angiogenesis. Collagen I, a major ECM component, strongly impacts tumor motility and is linked to cancer prognosis. Irregular bundled collagen fibers in patient tissues promote increased invasion length, suggesting a connection between collagen conformation, ECM stiffness, and invasion.

Recently, researchers increasingly used three-dimensional (3-D) cell culture systems to investigate cancer mechanisms, drug resistance, and immune regulation within TME. These models better mimic human tissue structure, particularly when embedded in ECM-mimetic biomaterials. Our study builds on this approach by investigating the correlation between ECM and cancer cell invasion using lung cancer spheroids in a 3-D collagen gel system.

By adjusting collagen gel concentration, we observed changes in spheroid invasion area and morphology based on ECM stiffness. Higher concentration collagen gels resulted in altered invasion patterns compared to lower concentration gels.

Our research aims to delve further into the impact of ECM-cancer cell interactions on angiogenesis. We plan to incorporate vessel endothelial cells to explore how these factors influence angiogenesis within the TME.

This comprehensive approach seeks to gain valuable insights into the complex interplay between ECM, cancer cell invasion, and angiogenesis. Understanding these interactions will contribute to the development of more targeted cancer therapies that consider the dynamic TME and its role in tumor progression.

Materials and Methods:: 1. Spheroid Generation

Two types of non-small lung cancer cell lines, H1975 (CRL-5908, ATCC, US) and CSL1-GFP (kind gift from Hui-Wen Chen lab) were mixed with fibroblast cell line HFL-1(CCL-153, ATCC, US) in 1:1 ratio, forming spheroids with total 10k cell number by non-adherent method. H1975 and HFL-1 were stained using CellTracker Green CMFDA and Red CMTPX (C7025, C34552；Invitrogen, US), and CLS1-GFP possess green fluorescence protein itself.

2. Invasion assay

Collagen I (3 mg/ml, A1048301, Gibco, US) was neutralized by using 0.1M NaOH, 10x PBS (70011044, Gibco, US), dH₂O to obtain a final pH value 7.2-7.4. Spheroids were then mixed with neutralized collagen gel solution and seeded onto 24 well plates, forming domes (Fig. 1) with final collagen gel concentration of 0.5 mg/ml or 1.5 mg/ml. Media were then added to each well and refreshed every 2 days. Images were recorded through fluorescence phase contrast microscope every 24 hrs and traced for 8 days. The invasion area and maximum radius of invasion were then calculated through ImageJ. Invasion rate was calculated by the following formula

Invasion rate = (Day N Invasion area - Day 0 Invasion area)/ Day 0 Invasion area


Results, Conclusions, and Discussions::

Current Results
The results of invasion assay are shown in figure 2 and 3. H1975/HFL-1 spheroids exhibited a lower invasion rate and maximum radius compared to CLS1/HFL-1 spheroids. In addition, both types of spheroids showed a decreased invasion rate when cultured in 1.5 mg/ml collagen gels compared to those in 0.5 mg/ml collagen gels. We found a distinct invasion morphology of the H1975/HFL-1 spheroids that were seeded in 1.5 mg/ml collagen gels, where H1975/HFL-1 spheroids exhibited a more collective invasion pattern. We also found a consistent observation across all spheroid groups: fibroblasts initially migrated and invaded into the surrounding collagen gel during the early invasion process (Fig. 4), suggesting fibroblast’s role in the early stage of invasion.

Conclusions & Discussion
In conclusion, our study demonstrated that the invasion rate and maximum radius of spheroids were decreased when embedded in 1.5 mg/ml collagen gel compared to 0.5 mg/ml collagen gel. Moreover, we found the fibroblasts in spheroids exhibited an early invasion into the ECM proceeding the cancer cells. These results illustrated the interaction between ECM and spheroids, which is affected by surrounding ECM conformation and spheroid composition.

To build upon these findings, our future work aims to investigate the impact of ECM on angiogenesis through tumor cells. We plan to combine spheroids embedded in different concentration collagen gels with vessel endothelial cells to create a model, mimicking the TME. This approach will enable us to observe formation of large, tubule-like structure by endothelial cells in the outer channel, while spheroids will be surrounded by ECM in the inner channel (Fig. 5). Furthermore, we aim to explore effects of cytokines such as TGF-β and lysyl oxidase, on ECM stiffness, invasion, and angiogenesis. Understanding how these factors influence the TME may provide valuable insights into the underlying mechanisms of cancer progression.

Overall, these findings may offer crucial insights into the mechanisms that drive cancer progression, and in turn, provide potential targets for therapeutic interventions in the future. By unraveling the complexities of the tumor microenvironment, we hope to contribute to advancements in cancer research and ultimately improve patient outcomes.


Acknowledgements (Optional): :

References (Optional): :