**4. In vitro studies of cellular autophagy**

### **4.1. 2D and 3D establishment using CRC cell lines**

CRC cell lines such as HT29, SW48, HCT116, and SW480 are adherent cell lines. By default, they will grow in normal tissue-cultured flat-bottom plates or flask in a monolayer, which is a 2D structure. However, monolayer morphology is not the natural appearance of all the established cell lines. Therefore, scientists have developed 3D models to bridge the difference between in vitro and in vivo experiments. For 3D model establishment, the cells were seeded into a round-bottom ultra-low-attachment 96-well plate and allowed to settle down and grow. The cells were observed every day until clear spheroids can be seen. It is important to capture images every day until clear spheroids can be seen. Some cell lines such as SW480 might not form clear rounded spheroids. Once the spheroids are formed, it is important to not disrupt spheroid formation when handling it in situations such as changing media. This is because the cells were seeded in ultra-low-attachment plate, which means they no longer attach to the bottom of the well. Careless handling of the spheroids might cause disruption to it or worse, the loss of spheroids. Although the process of 3D formation is straight forward, not all cell lines are able to form spheroids. Such example is the SW48, which when seeded into the well, remained in multiple clusters instead of forming spheroids as shown in **Figure 3**. Through the formation of 3D models, scientists can better study the gene expressions and cell behaviors [52], as well as carry out drug response experiments on a model that better mimics the natural tumor morphology in the human body [53]. 3D models can also be cocultured with other cells

Cell-Based Assays for Evaluation of Autophagy in Cancers

http://dx.doi.org/10.5772/intechopen.80088

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Starvation is one of the well-known methods that can positively induce autophagy for autophagy-related studies. Common methods of starvation include removal of amino acids as well as serum from the media. The effect of autophagy can be detected as early as 1 hour after removal of the amino acids from the media [54]. This effect can be studied using either

Autophagy-related activities can be inhibited by treating cell lines with inhibitors. In the market, there are multiple types of inhibitors that are manufactured to target specific pathways that are related to autophagy in cells. As shown in **Table 1** below, these inhibitors with known

Cytotoxicity experiments should be carried out by researchers when using such inhibitors as they can reduce the viability of cells tested on. This can be carried out through serial-diluted inhibitors using MTT or any luminescence-based viability assays. The effect of inhibition can then be determined using the safe range of concentration obtained through the cytotoxicity

siRNAs have been developed by companies that specifically targets different genes that produce important proteins that play roles in the activation of autophagy. Some examples of currently available siRNAs are those that target Atg3, Atg5, Atg7, Atg10, Atg12, Atg13, Atg14, and Atg101. When researchers first receive the siRNA, the user should run an optimization experiment to determine a few characteristics of the siRNA. The toxicity of such siRNA should first be tested, and the IC50 should be determined to better understand what concentration range might work the best and have the least impact on cell viability. Cytotoxicity assays such as

**Name Mechanism References** LY294002 PI3K inhibitor [55] 3-methyladenine PI3K inhibitor [56] Wortmannin PI3K inhibitor [57] SBI-0206965 ULK-1 inhibitor [58] Spautin-1 USP10 and USP13 inhibitors [59] SAR405 Vsp18 and Vsp34 inhibitor [60] NSC195058 ATG4 inhibitor [61]

to emulate the tumor microenvironment and investigate cell-cell interactions.

Western blot or immunofluorescence (i.e. p62 accumulation).

assay followed by Western blot or immunofluorescence.

**4.2. Effect of starvation on autophagy**

**4.3. Autophagy inhibitors and treatments**

mechanisms can be used in vitro.

**4.4. siRNA knockdown for autophagy**

**Table 1.** Autophagy inhibitors that can be used in vitro.

**Figure 3.** Spheroid formation of colorectal cancer (CRC) cell lines. 1 × 10<sup>5</sup> cells were seeded into round-bottom ultralow-attachment 96-well plate and incubated at 37°C at 5% CO2 with 95% humidity. The cells were observed every day until spheroids were formed. The images above were taken at 72 hours after seeding. HCT116 and HT29 both formed a round spheroid in the well, while SW480 formed an irregular spheroid. No spheroid was formed 72 hours after seeding.

tumor morphology in the human body [53]. 3D models can also be cocultured with other cells to emulate the tumor microenvironment and investigate cell-cell interactions.

## **4.2. Effect of starvation on autophagy**

Starvation is one of the well-known methods that can positively induce autophagy for autophagy-related studies. Common methods of starvation include removal of amino acids as well as serum from the media. The effect of autophagy can be detected as early as 1 hour after removal of the amino acids from the media [54]. This effect can be studied using either Western blot or immunofluorescence (i.e. p62 accumulation).
