Associate Professor Stanford University, California, United States
Introduction: Radiation therapy is a key treatment for lung cancer, but the heterogeneous nature of the tumor microenvironment (TME) can significantly affect therapeutic outcomes. Traditional in vitro models often fail to replicate the complex dynamics of the TME, highlighting the need for advanced systems that better mimic in vivo conditions. Tumor-on-a-Chip (ToC) aims to replicate essential aspects of the TME, providing physiological relevance and reducing dependence on animal models. This study presents a ToC model with a 3D matrix for cancer cell growth, focusing on hypoxic tumor conditions, which are known for their reduced sensitivity to radiation. The study explores the creation of a 3D lung tumor-on-a-chip and features heterogeneous hypoxia induction, radiation therapy, DNA damage assessment, colony formation capability after radiotherapy, and LDH release within the tumor model. It highlights the importance of understanding reduced sensitivity to radiation in these microenvironments. Our study is among the few that explore the role of hypoxia in radiation therapy using organ-on-a-chip platforms and 3D models. Additionally, creating a gradient of hypoxia using microfluidics is another innovative aspect of this research. Moreover, the use of an oxygen scavenger provides an effective method for generating a hypoxia gradient, which is challenging to achieve with traditional hypoxia chambers. The study contributes to improving in vitro models and optimizing radiation treatment strategies. A microfluidic chip was designed and fabricated to create the ToC model, incorporating a 3D lung cancer model within a hydrogel, serving as an extracellular matrix (ECM). Figure 1 provides an overview of this study.
Materials and
Methods: The microfluidic chip was fabricated using soft lithography. A549 lung cancer cells were incorporated into a fibrin hydrogel to create the 3D tumor-on-a-chip model. Hypoxic conditions were achieved by adding an oxygen scavenger, sodium sulfite, to the media. The chip was connected to a peristaltic pump to control fluid flow, allowing dynamic environmental manipulation. A gradient of hypoxia was established within the ToC model to mimic the heterogeneous oxygen distribution found in vivo. A hypoxia gradient is created using two inlets for the chip. One inlet introduces a hypoxic medium prepared by adding 1 % (w/v) sodium sulfite, while the other introduces a normal medium. Both are injected into the chip at a flow rate of 10 µL/min. The laminar flow in the microfluidic channels ensures that these two media flow side-by-side with minimal mixing, creating a gradient of oxygen levels across the chip. This setup allows for a controlled simulation of different oxygen conditions within the tumor environment. Radiation therapy was administered using an X-ray irradiator at a dosage of 10 Gy. LDH release was measured 24 hours post-radiation, while the survival fraction was assessed over two weeks using the clonogenic assay. DNA damage was evaluated through gamma-H2AX immunostaining one hour after exposure to X-rays.
Results, Conclusions, and Discussions: A 3D lung tumor-on-a-chip model was created, as shown in Figure 2A. Hypoxic conditions were induced using an oxygen scavenger (sodium sulfite). After radiation therapy, LDH release from tumor cells in hypoxic conditions was significantly lower than in normoxic conditions (Figure 2B-i). Additionally, a clonogenic assay was performed to compare the colony formation capability of tumor cells under normoxic and hypoxic conditions after radiation therapy (Figure 2B ii-iii), confirming the radioresistance of hypoxic tumor cells. DNA damage was evaluated by staining for gamma-H2AX in the normoxic tumor-on-a-chip model, hypoxic conditions, and under a hypoxia gradient. The results showed that DNA damage in hypoxic tumors was significantly less compared to normoxic conditions, as evidenced by gamma-H2AX immunostaining (Figure 2C i-iii). Furthermore, in the presence of a hypoxia gradient using microfluidics, a corresponding gradient in DNA damage was confirmed (Figure 2C-iv). The hypoxia gradient created within the chip led to hypoxia-dependent DNA damage. The areas with lower oxygen levels demonstrated increased radioresistance, while regions with higher oxygen levels exhibited greater DNA damage.
In conclusion, a hypoxic microenvironment was successfully induced using an oxygen scavenger in a 3D Tumor-on-a-Chip platform. The study confirmed the radioresistance of tumor cells to radiation therapy through various assays, including clonogenic assays, LDH release assays, and DNA damage immunostaining. The 3D tumor model, constructed using a fibrin-based hydrogel within the chip, demonstrated radioresistant behavior under hypoxic conditions after exposure to X-rays. This approach enabled the detailed examination of the Tumor-on-a-Chip model in conditions closely mimicking the in vivo tumor microenvironment, offering valuable insights into the effects of radiation therapy on cancer cells in this advanced in vitro system.
