*2.1. Physical Disruption and Stimulation of Tumor Cells to Obtain Whole Tumor Cells*

TCLs prime antitumor immunity and exhibit immune tolerance against self-antigens. Live tumor cells as a source of antigens could be less immunogenic since these cells contain or secrete factors such as vascular endothelial growth factor, soluble FAS ligand, and MHC class I chain-related proteins A and B, which suppress the function of DCs and T cells [7]. Figure 2 demonstrates the summary of cell lysis by external factors, and their conditions are indicated in Table 1.

Simply, TCLs are generated by repeated freeze-and-thaw cycles (Figure 2A), and protein fragments from the whole tumor cell population are obtained. The development of ice crystals during freezing, and the subsequent concentration upon thawing, results in the physical rupture of cellular bodies [37]. This repeated process facilitates the large-scale release of inflammatory proteins [38]. In general, these protein fragments are DAMPs, including heat shock protein (HSP) and high-mobility group box-1 (HMGB-1), which are classified as class I DAMPs [39]. HSPs and HMGB-1 directly bind and trigger Tolllike receptor (TLR) 2 and TLR4, which are the PRRs located in immune cell membranes. Activated TLR2 and 4 initiate NF-kB and interferon regulatory factors via the myeloid differentiation primary response 88-dependent pathway and toll/interleukin-1 receptor domain-containing adapter-inducing interferon-ß-dependent pathways. Through this, pro-inflammatory cytokines (e.g., IL-1ß, IL-6, and IFN) are released from DCs and stimulate T cell immunity with presented tumor-specific antigens using MHC molecules. [40].

The subsequent physical treatment of tumor cells, such as sonication, is optionally introduced to facilitate the homogeneity of the prepared TCLs. Nano-scale TCLs could be obtained using only sonication (Figure 2B) [29]. Additionally, ultraviolet (UV) irradiation is also commonly used to prepare TCLs by inducing immunogenic cell death (ICD) (Figure 2C) [30]. UV irradiation (1500 µW/cm<sup>2</sup> for 10 min) of TC-1 tumor cells results in both apoptosis and necrosis, and the TCLs from these UV-pulsed DCs exhibit significant surface expression of CD86, CD80, and MHC II molecules. The UV irradiation of tumor

cells also generates effective TCL modulators for inducing an antitumor immune response by further enhancing CD8<sup>+</sup> cell populations. *Pharmaceutics* **2022**, *14*, x FOR PEER REVIEW 4 of 26

**Figure 2.** Schematic illustration of preparation of TCLs via various conditions. (**A**) Repeated freeze– thaw cycle could induce necrosis of tumor cells and release immunogenetic molecules (e.g., DAMPs, TAAs), (**B**) sonication generates nano-sized fragments, average particle size as measured by Nano Sight, (**C**) UV irradiation for facilitated cellular uptake, (**D**) heat shock evokes release of HSP70 in tumor cells, (**E**) oxidation rapidly induces tumor cell death and generates advantageous immunogenic molecules, (**F**) PKHB1 peptide-derived TSP-1 interacts with CD47 and activates the atypical caspase-independent and calcium-dependent signaling in cell death. (**B**) and (**F**) are reproduced with permission from Refs. [29,33,36]. **Figure 2.** Schematic illustration of preparation of TCLs via various conditions. (**A**) Repeated freeze– thaw cycle could induce necrosis of tumor cells and release immunogenetic molecules (e.g., DAMPs, TAAs), (**B**) sonication generates nano-sized fragments, average particle size as measured by Nano Sight, (**C**) UV irradiation for facilitated cellular uptake, (**D**) heat shock evokes release of HSP70 in tumor cells, (**E**) oxidation rapidly induces tumor cell death and generates advantageous immunogenic molecules, (**F**) PKHB1 peptide-derived TSP-1 interacts with CD47 and activates the atypical caspaseindependent and calcium-dependent signaling in cell death. (**B**,**F**) are reproduced with permission from Refs. [29,33,36].

both apoptosis and necrosis, and the TCLs from these UV-pulsed DCs exhibit significant surface expression of CD86, CD80, and MHC II molecules. The UV irradiation of tumor cells also generates effective TCL modulators for inducing an antitumor immune response


Simply, TCLs are generated by repeated freeze-and-thaw cycles (Figure 2A), and **Table 1.** Condition for preparation of TCLs.

by further enhancing CD8+ cell populations.
