*4.4. Natural Components*

*4.3. 3D Polymeric Gel* 

utilized as a cancer vaccine platform.

Some natural compounds possess sufficient adjuvant efficacy to trigger DC activation. Previous studies have used LPS, a membrane component of Gram-negative bacterial cell walls, because of its adjuvant effect on the activation of TLR4 signaling pathways and the CD4<sup>+</sup> T cell response [136]. Hence, a series of studies have investigated exogenous signaling via TLR4 on immune cells, and have tried to design TLR4 agonists as vaccine adjuvants [137–139]. LPS was reported to interact with TLR-4 in DCs, inducing multiple intracellular signaling cascades to express extracellular signal-regulated kinase, c-Jun N-terminal kinase, p38 mitogen-activated protein kinases, and NF-κB, and affected the production of IL-12 [140]. However, the single-use of LPS for immune activation might evoke vaccine reactogenicity, and induce improper signaling direction for DC activation and further vaccination [141,142].

Despite LPS-mediated immune activation, a high level of immunosuppressive cytokine secretion (such as IL-10) is usually observed. Therefore, other cellular components in bacterial cells could be used for the upregulated expression of immunoactivators, with

reductions in immunosuppressive cytokines to deliver the TCLs [27]. For example, the empty envelope of Gram-negative bacteria (i.e., bacterial ghosts (BGs)) with intact cell surface structures exhibited strong adjuvant properties for the induction of DC maturation, and carried TCLs as immune adjuvants in the empty inner core. Facilitated by co-administration with IFN-γ, these TCL-loaded BGs showed superior DC maturation (i.e., upregulated expression of DC maturation markers, including CD86, CD80, and MHC II) compared to treatment with LPS. The secretion of Th1-polarizing cytokine IL-12p70 in DCs was also increased by TCL-loaded BGs with IFN-γ, whereas the level of pro-tolerogenic cytokine IL-10 was decreased. Moreover, the expression of immunoglobulin-like transcript 3, an inhibitory receptor used to establish suppressor T cells by inducing tolerance [143], was also decreased in DCs treated with TCL-loaded BGs. These results demonstrate that the TCL-loaded BGs could potentially overcome immunosuppressive and pro-tolerogenic effects on various cancer types as an effective inducer of Th1-polarized CD4<sup>+</sup> and associated CD8<sup>+</sup> T cell-mediated antitumor immunity.

The β-glucan particles (GPs) derived from yeast (e.g., *Saccharomyces cerevisiae*) are another example of natural compound-based fabrication of a TCL carrier (Figure 5B) [144]. Since the 1,3-β-glucan outer shell can provide receptor-mediated phagocytic uptake by cells expressing β-glucan receptors, GPs can be used for the APC-targeted delivery of soluble payloads [145]. Various potential functions of GPs, such as the stimulation of pathogens invading the body, sustained antigen release, facile internalization into APCs, and PAMPlike signaling, could induce robust immune activation. Through a similar encapsulation of antigens into the inner hollow cavity of GPs, the induction of safe immunogenicity by an engineered pathogen-mimicking system, and long-term interaction via the sustained release of cargo antigens, could be achieved. Therefore, Hou et al. [28], developed GPs encapsulating murine colon adenocarcinoma cell (MC38) lysates with additional stimulation provided by a CpG TLR9 agonist. In addition, the co-incorporation of poly-L-arginine improved the protection against challenge from live tumor cells in animal models when co-injected with tumor antigens, and also promoted the in vivo charging of MHC II<sup>+</sup> APCs [146,147]. This GP platform was internalized in up to 70% of the DCs by energy-dependent and dectin-1 receptor-mediated endocytosis, and the sustained release of the cargos resulted in the significantly higher expression of CD86 than that of the LPS controls. Moreover, NLR pyrin domain-containing protein 3 inflammasome-mediated DC activation was also confirmed by increased cleaved caspase-1 p10 (10 kDa) levels in GP-treated BMDCs, and the correlated IL-1ß secretion [148]. A summary of whole-TCL delivery platforms using liposomes, 3D polymeric gel, and natural components are indicated in Table 3.



3D, three dimensional; CTL, cytotoxic T lymphocytes; DOX, doxorubicin; TCL, tumor cell lysate; NIR, nearinfrared radiation; TLR, Toll-like receptor; PEV, poly(L-valine); PEA, poly(L-alanine); LN, lymph node; GM-CSF, granulocyte-macrophage colony-stimulating factor; Ab, antibody; DCs, dendritic cells; RGD, Arg-Gly-Asp; IFN, interferon; ILT3, immunoglobulin-like transcript 3; NLRP3, nucleotide-binding oligomerization domain 3.
