**2. Cancer metastasis**

At present, metastasis is known as the result of a complex multistep cell-biological process collectively known as the invasion-metastasis cascade. It involves the changes of cancer cells in the physical position from the primary tumor to distant or adjacent sites of dissemination, and the colonization of the "seed cells" by adapting to the alien tissue microenvironments. More specifically, during metastatic progression, the first change is the cellular adhesion and morphology of cancer cells are reduced by EMT. Then, cancer cells improved the capability of invading the normal tissue surrounding (local invasion). Next, cancer cells make a way into (intravasation) and out of (extravasation) systemic circulation such as the lymphatic or circulatory system to land at distant sites. In this step, surviving cancer cells are termed circulating tumor cells. Lastly, the cancer cells proliferate and colonize in an unknown tissue microenvironment of different distant organs (**Figure 1**).

#### **2.1 The invasion-metastasis cascade**

#### *2.1.1 Local invasion*

The local invasion of cancer cells is the foundation for metastatic cancer process. Local invasion refers to the entry of cancer cells into the surrounding tumorassociated stroma, subsequently entering the adjacent normal parenchymal tissue.

#### **Figure 1.**

To invade the stroma, cancer cells must first break the basement membrane (BM) located at the interface of the epithelial tissue and connective tissue. The specialized extracellular matrix (ECM) can exchange material and regulate tissue growth, differentiation, and regeneration [16]. When these cancer cells invade the stroma, they have to be confronted with diverse cancer-associated stromal cells, including myofibroblasts, fibroblasts, adipocytes, endothelial cells, and plenty of bone marrow-derived cells (BMDCs) (i.e., macrophages, mesenchymal stem cells) [17]. Then, these stromal cells are able to enhance the aggressiveness of cancer cells through various cytokines. Secretion of interleukin-6 (IL-6) by cancer-associated fibroblasts (CAFs), stimulates the migration and invasiveness of colorectal cancer cells by the STAT3-LRG1 axis [18]. Increased IL-4 in endothelial cells can lead to enhanced invasiveness of liver cancer cells via the ERK-AKT signaling axis [19]. Besides, colorectal cancer cells secret IL-4 to promote M2-like tumor-associated macrophage (TAM) polarization [20]. These findings suggest there exists a positive-feedback loop in the tumor microenvironment, which is that cancer cells maintain high inflamed surroundings, and these stromal cells further enhance the malignant characteristics of cancer cells.

Researchers have observed various patterns of invasion when cancer cells infiltrate the substrates of adjacent tissues. Due to the dissemination of cancer cells, as individuals and collectives, these researchers divide migrations into individual cell migration and collective cell migration. Both types of migration are simultaneously present in many cancers [21]. In cancer progression, the plastic changes of numerous cancer cells are shown by morphological and phenotypical conversions, such as EMT and its reverse process the mesenchymal-epithelial transition (MET) [22], the collective-amoeboid transition (CAT) [23], the mesenchymal-amoeboid transition (MAT) [24]. Among these conversions, EMT has been increasingly

*Overview of metastatic Cascade. During metastatic progression, the first change is that the cellular adhesion and morphology of cancer cells are reduced by epithelial-mesenchymal transition (EMT). Then, the ability of cancer cells to invade the surrounding normal tissue (local invasion) is increased. Next, cancer cells make a way into (intravasation) and out of (extravasation) systemic circulation such as the lymphatic or circulatory system to land at a distant site. In this step, surviving cancer cells are termed circulating tumor cells. Lastly, the cancer cells proliferate and colonize an unknown tissue microenvironment of different distant organs. This figure was created with BioRender.com.*

considered a crucial and indispensable stage in the cancer metastatic process over the last decade [22], despite the studies of Seyfried et al. in the VM mouse model of systemic metastasis suggesting that EMT is unnecessary for the initial cancer metastasis [25, 26].

EMT is a cellular process activated by master transcription regulators, including-ZEB1, ZEB2, Twist, Slug, and Snail, which enhance cell motility and migration ability to invade stroma. Besides, transforming growth factor (TGF)-β has proved to be a strong inducer of EMT by collaborating with other signaling pathways, especially the RAS-MAPK cascade [27]. Moreover, increasing emerging evidence shows the potent roles in invasion and EMT of many long noncoding RNAs (lncRNAs), such as lncRNA MEG3, lncRNA PNUTS, and lncRNA MIR100HG [28–30]. During this process, cells lost epithelial characteristics and markers like E-cadherin and cytokeratin, instead of gaining mesenchymal characteristics and markers like vimentin, N-cadherin, and fibronectin [22]. In addition to cancer metastasis, EMT has been involved in different cancer stages, including cancer initiation, malignant progression, cancer stemness, and drug resistance [31].
