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Background: Cancer metastasis is the main cause of mortality in cancer patients. However, the drugs targeting metastasis processes are still lacking, which is partially due to the short of effective in vitro model for cell invasion studies. The traditional 2-D culture method cannot reveal the interaction between cells and the surrounding extracellular matrix during invasion process, while the animal models usually are too complex to explain mechanisms in detail. Therefore, a precise and efficient 3-D in vitro model is highly desirable for cell invasion studies and drug screening tests. Methods: Precise micro-fabrication techniques are developed and integrated with soft hydrogels for constructing of 3-D lung-cancer micro-environment, mimicking the pulmonary gland or alveoli as in vivo. Results: A 3-D in vitro model for cancer cell culture and metastasis studies is developed with advanced micro-fabrication technique, combining microfluidic system with soft hydrogel. The constructed microfluidic platform can provide nutrition and bio-chemical factors in a continuous transportation mode and has the potential to form stable chemical gradient for cancer invasion research. Hundreds of micro-chamber arrays are constructed within the collagen gel, ensuring that all surrounding substrates for tumor cells are composed of natural collagen hydrogel, like the in vivo micro-environment. The 3-D in vitro model can also provide a fully transparent platform for the visual observation of the cell morphology, proliferation, invasion, cell-assembly, and even the protein expression by immune-fluorescent tests if needed. The lung-cancer cells A549 and normal lung epithelial cells (HPAEpiCs) have been seeded into the 3-D system. It is found out that cells can normally proliferate in the microwells for a long period. Moreover, although the cancer cells A549 and alveolar epithelial cells HPAEpiCs have the similar morphology on 2-D solid substrate, in the 3-D system the cancer cells A549 distributed sparsely as single round cells on the extracellular matrix (ECM) when they attached to the substrate, while the normal lung epithelial cells can form cell aggregates, like the structure of normal tissue. Importantly, cancer cells cultured in the 3-D in vitro model can exhibit the interaction between cells and extracellular matrix. As shown in the confocal microscope images, the A549 cells present round and isolated morphology without much invasion into ECM, while starting from around Day 5, cells changed their shape to be spindle-like, as in mesenchymal morphology, and then started to destroy the surrounding ECM and invade out of the micro-chambers. © The Author(s) 2022. Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visithttp:// creat iveco mmons. org/ licen ses/ by/4. 0/. The Creative Commons Public Domain Dedication waiver (http:// creat iveco mmons. org/ publi cdoma in/ zero/1. 0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data. Open Access *Correspondence: fanqh@iphy.ac. cn; plwmd1967@126.com 1 Department of Cardiothoracic Surgery, Huashan Hospital, Fudan University, Shanghai, P.R. China 3 Beijing National Laboratory for Condensed Matter Physics and Laboratory of Soft Matter and Biological Physics, Institute of Physics, Chinese Academy of Sciences, Beijing, China Full list of author information is available at the end of the article Page 2 of 9 Jiang et al. BMC Cancer (2022) 22:438 Introduction Cancer metastasis is composed of complex processes, including decreased cell–cell adhesion, increased cell motility, invasion of cancer cells into surrounding tissues, and finally the colonization and formation of secondary tumors [1–3]. The invasion of cancer cells is very hard to be controlled, thus cancer metastasis is the most lethal factor for tumor patients, especially for lung cancer patients [2, 4]. Currently, there is few drug targeting the molecules in metastasis processes, partially due to the lacking of effective in vitro model, which can mimic cancer cells invasion as in vivo [1, 5]. The traditional 2-D culture system cannot reveal the interaction between cells and the surrounding extracellular matrix (ECM) [6–8], while the animal models usually are too complex to explore mechanisms in detail. Therefore, it is important to find an effective in vitro model to study the invasion mechanism and also to quantify the invasion phenomena, which is important for providing a precise evaluation platform for clinical therapy or drug screening [9–12]. There have been some in vitro models for cancer invasion studies. The fundamental method is on 2-D substrate, called wound healing assay or scratch assay. In this method, a wound was made by scratching the cell monolayer in a straight line with a sterile pipette tip. Then cell migration toward the wound center would be monitored, and the cell migration speed is analyzed to indicate the cell-invasion ability. This method is easy for handling and can present cell migration results. However, it can only show the cell migration ability, but not exactly the ability of cell invasion through the surrounding matrix as in vivo [13]. Therefore, it is still a partial way to evaluate cell invasion. Another popular quasi-3D method for invasion studies is the Boyden chamber (Transwell) assay. Cells are separated in two neighboring chambers by a thin layer of porous material. Cells can squeeze through or digest the thin porous layer, and migrate to another chamber, like the cell invasion into a distant region [14]. However, the separating 2-D layer is not like the ECM of the real tissue, thus lacking of the ability for precisely revealing the metastasis process [5, 15]. A lot of microfluidic models have been constructed to study the lung cancer cells, e.g., the designed microfluidic chip with multi-chambers to assist the formation of cancer cell spheroids [16], and the chip to assist the coculture of lung cancer cells and cancer-associated fibroblasts [17]. However, these models only provide 2-D culture substrates for cells, lacking of the ECM, thus they cannot reveal the important interactions between cancer cells and surrounding ECM during invasion. Some 3-D in vitro microfluidic models also emerge in recent years, which contain the hydrogels as ECM for cell culture. A common method is to mix the isolated cancer cells or pre-formed spheroids within the hydrogels [18], which can present the interaction between cells and ECM, but cannot fully recapitulate the tissue structures, such as the gland and alveoli. To mimic the interface within the gland in vivo, a 3-D microfluidic model has been developed in the previous work [9, 19]. Generally, a microfluidic system fabricated with biocompatible polydimethylsiloxane (PDMS) is composed with central hydrogel platform and four micro-channels surrounding the platform [20, 21]. Natural hydrogel type I collagen was filled in the central platform of the 3-D model as ECM for culturing tumor cells, because collagen is the most abundant ECM component in human tissues [22]. Especially, hundreds of micro-chambers were fabricated within the Collagen gel, mimicking the structure of gland as in vivo. The hydrogel platform with micro-chambers is the pivotal component in the 3-D model because it mimics the ECM structures of the tumor microenvironment and provides high-throughput units for tests [6, 23, 24]. However, collagen hydrogel is also very soft and fragile, composed of more than 90% of water [25]. Therefore, it is very hard to construct micro-features on the soft collagen gel. In previous studies, the collagen hydrogels integrated in the microfluidic system usually are with high concentrations of more than 6 mg/ml, to ensure the hydrogel stiffness and strength, avoiding being damaged during the fabrication process. However, ECM in vivo usually has much lower stiffness than that of the 6 mg/ml collagen gel, especially in certain soft tissues, such as the lung gland and alveoli. If the ECM stiffness is too high, it will be impossible for observing the cancer cells invading into the hydrogel and starting the metastasis. It is likely that lung cancer cells would be confined within the microchambers without any invasion, like other normal cells. Or it will take too long time for invasion studies if lung cancer cells are confined in collagen micro-chambers Conclusions: A 3-D in vitro model is constructed for cancer cell invasion studies, combining the microfluidic system and micro-chamber structures within hydrogel. To show the invasion process of lung cancer cells, the cell morphology, proliferation, and invasion process are all analyzed. The results confirmed that the micro-environment in the 3-D model is vital for revealing the lung cancer cell invasion as in vivo.