*2.3. Preparation of Tumor Cell Membranes*

As a source of TCLs, several studies primarily focused on the production of tumor cell membrane components. Since cell membrane contents participate in protein–protein interactions in immune system processes, inflammatory responses, and chemokine signaling pathways, tumor cell membrane proteins (e.g., CD44, MUC, CD98, and integrin) could be used as tumor-specific antigens and receptors to effectively trigger immune responses in cancer therapy [50]. Centrifugation has been used to isolate cell membrane proteins from tumor cells. For purification, (1) the physical disruption of collected cells by homogenization or freeze–thaw cycles with lysis buffer, (2) centrifugation at low speed (1000–2000 RCF) to separate cellular debris and nuclei contained in the pellet, and (3) ultracentrifugation at high speed (100,000–200,000 RCF) to separate all membrane fractions from soluble proteins in the supernatant, have generally been performed (Figure 3A). Additional sucrose treatment provides a density gradient for obtaining membrane fractions (Figure 3B). The resuspension of membrane fractions within sucrose results in further separation of cell surface membranes, mitochondrial membranes, and other types of membrane components [51]. Isolated cell membrane components obtained through this centrifugation process could be further incorporated into various template biomaterials. For instance, a biomimetic antitumor nanovaccine was fabricated via the coating of membrane components onto calcium pyrophosphate inorganic NP templates (Figure 3C) [10]. This inorganic carrier platform to deliver TCL membrane-derived antigens consisted of (1) cell membrane fragments, isolated from sucrose-dependent separation, that promoted specific immune reactions as antigens, and (2) biocompatible calcium phosphate templates as immune adjuvants that stimulated innate immunity by activating the NLRP-3 inflammasome and the production of cytokines (e.g., IL-1ß) [52] for T cell-based responses. Therefore, the dual functionality of calcium pyrophosphate nanoparticles coated with antigen-rich TCL membranes could improve the antigen presentation of DCs, as well as provide adjuvant effects, dramatically increasing the expression of DC surface markers and the subsequent proliferation of CD8<sup>+</sup> T cells (Figure 3D).

**Figure 3.** Schematic illustration of tumor cell membrane isolation and application in anticancer immunotherapy. (**A**) Cell membrane isolation using ultracentrifugation to obtain the total cell membrane component, (**B**) sucrose-dependent isolation for separating the cell surface membrane components, (**C**) design of tumor cell membrane-coated CaPyro nanoparticle, and (**D**) anticancer immunity mechanism using these NPs. All subfigures were reproduced with permission from Refs. [10,51]. **Figure 3.** Schematic illustration of tumor cell membrane isolation and application in anticancer immunotherapy. (**A**) Cell membrane isolation using ultracentrifugation to obtain the total cell membrane component, (**B**) sucrose-dependent isolation for separating the cell surface membrane components, (**C**) design of tumor cell membrane-coated CaPyro nanoparticle, and (**D**) anticancer immunity mechanism using these NPs. All subfigures were reproduced with permission from Refs. [10,51].

### **3. Role of DCs in Cancer Immunotherapy. 3. Role of DCs in Cancer Immunotherapy**

### *3.1. Phenotype of Dendritic Cells 3.1. Phenotype of Dendritic Cells*

[58].

DCs located in the spleen and various lymphoid tissues generally exhibit unique immune functions in activating T cells through antigen presentation [53,54]. However, the interaction between DCs and T cells occurs only in mature stages of DCs, which depends upon successful antigen uptake. DCs mostly exist in an immature state, but sufficient antigen uptake initiates a change to the mature state. During the functional maturation process, changes in the morphological and phenotypic characteristics of DCs influence immune system activity [55]. Mature DCs (mDCs) with a rough surface and multiple pseudopodia, and immature DCs (iDCs) with a spherical and smooth structure, exhibit different phagocytotic and migration abilities [56]. Therefore, when phagocytosis and endocytosis preferentially occur in the immature state, the morphological conversion (i.e., more dendritic structure) and optimization of antigen presentation by DCs occur sequentially. Then, these mDCs with a higher level of MHC molecules quickly migrate to the lymph nodes for 2–3 days, while maintaining their presentation ability, and are ready to stimulate other immune cells [57]. Consequently, mDCs can initiate and maintain adaptive immunity (including antigen specificity, humoral immunity mediated by antibodies, antigen-specific cellular immunity and memory) through a pathophysiological network with other immune cells, such as T cells, B cells, and NK cells DCs located in the spleen and various lymphoid tissues generally exhibit unique immune functions in activating T cells through antigen presentation [53,54]. However, the interaction between DCs and T cells occurs only in mature stages of DCs, which depends upon successful antigen uptake. DCs mostly exist in an immature state, but sufficient antigen uptake initiates a change to the mature state. During the functional maturation process, changes in the morphological and phenotypic characteristics of DCs influence immune system activity [55]. Mature DCs (mDCs) with a rough surface and multiple pseudopodia, and immature DCs (iDCs) with a spherical and smooth structure, exhibit different phagocytotic and migration abilities [56]. Therefore, when phagocytosis and endocytosis preferentially occur in the immature state, the morphological conversion (i.e., more dendritic structure) and optimization of antigen presentation by DCs occur sequentially. Then, these mDCs with a higher level of MHC molecules quickly migrate to the lymph nodes for 2–3 days, while maintaining their presentation ability, and are ready to stimulate other immune cells [57]. Consequently, mDCs can initiate and maintain adaptive immunity (including antigen specificity, humoral immunity mediated by antibodies, antigen-specific cellular immunity and memory) through a pathophysiological network with other immune cells, such as T cells, B cells, and NK cells [58].
