**3. GK-1 as an anticancer immunotherapy**

In neoplasms, the host is often immunocompromised due to the presence of immunosuppressive cells and molecules in the TM, to prevent the removal of cancer cells [2]. This highlights the relevance of stimulating the host immune response against cancer antigens by administering immunoadjuvants along with chemotherapy, radiotherapy, or surgery [54]. In this regard, small peptides with a nonspecific immunostimulatory response like GK-1, long known to act as vaccine adjuvants, are potentially useful in cancer therapy. The antitumor effect of GK-1 has been studied in melanoma and breast cancer murine models.

#### **3.1. GK-1 in a mouse melanoma model**

Melanoma is the most malignant form of skin cancer, mainly affecting the Caucasian population [55, 56]. Until recently, systemic therapy for metastatic melanoma had been inefficient, with a 5-year survival rate for patients (<30%) [57, 58]. However, new therapies were recently approved to treat melanoma, such as pegylated-interferon-α2b (IFN-α2b) in the adjuvant setting; ipilimumab, an anti-CTLA4 monoclonal antibody, for metastatic disease; vemurafenib, an oral BRAF inhibitor indicated for patients with metastatic melanoma harboring BRAFV600 mutations, and more recently antibodies against PD-1 like pembrolizumab [59–61] and antibodies blocking PD-L1 pathways, as well as inhibitors of the mitogen-activated protein kinase (MAPK) pathway. Additionally, nonspecific immunomodulation by several cytokines (IL-2, IL-12, TNF-α, and IFN-γ) and TLR ligands [62–64] in addition to adoptive transfer approaches have been widely used [65]. For over a decade, DCs have also been used in immunotherapy against various types of cancer [66–68] as an alternative to chemotherapy, by vaccination with DCs loaded with tumor peptides (i.e., MAGE-AX [69–72] and/or with necrotic or apoptotic tumor cells to induce effector tumor-specific T cells [73, 74]). The efficacy of this immunotherapeutic approach was also evaluated against murine melanoma, using GK-1 as an immunostimulant.

GK-1 has been reported to increase the mean survival and significantly delays tumor growth in a melanoma model with B16-F10 cells, showing more necrotic areas along with the presence of numerous neutrophils (**Figure 1**). Neutrophilia inside pulmonary blood vessels was also observed, without evidence of macroscopic or microscopic metastasis. In a melanoma lung metastatic model, GK-1 decreased lymphocyte count, while increased the number of neutrophils and decreased the serum levels of IFN-γ; on the other hand, an increase in the levels of IFN-γ and IL-12 in the intratumor (lung metastases) environment, along with a decrease in IL-17, IL-4, IL-22, and IL-23 was also observed [75, 76]. The antitumor activities of IL-12 have been established in preclinical studies against various tumor cell lines; the increased concentration of the antitumor cytokine IL-12 found in primary tumors may enhance the damage to tumor cells, limiting the number of cancer cells detaching from the primary tumor [77–79]. Its antitumor activity is also mediated by the induction of IFN-γ [67, 78, 80], which upregulated the expression of MHC-I and -II by B16 cells *in vitro*, favoring a cytolytic response in MHC-I-restricted CTL (**Figure 1**) [81]. There is a consensus that the induction of a Th1 profile or the release of cytokines like IFN-γ and TNF-α by T cells is essential for an effective antitumor immune response in

melanoma [82–84]. In fact, IFN-γ released from CTLs has been considered as a potent mediator of the antitumor response in bulky melanoma tumors [17, 85]. In contrast, IL-17 was proved to directly promote tumor growth and angiogenesis [86–88]. Indeed, it has been shown that IL-17 can promote tumor growth by a direct effect on IL-6 induction, which in turn activates STAT3 in both tumor and nontransformed cells in the TM [89]. Finally, IL-23 is an important molecular driver of Th17 cells in humans; IL-23 is increased in several tumors, and the expression of this cytokine antagonistically regulates local inflammatory responses in the TM, as well as the infiltration of epithelial lymphocytes [80]. Thus, the intratumoral subexpression of IL-17

**Figure 1.** GK-1 in a preclinical mouse melanoma model. In a melanoma murine model with B16-F10 cells, GK-1 led to an increase in neutrophils with the increase of IFN-γ and IL-12 cytokines, along with a decrease in IL-17 and IL-23. On the other hand, the MAGE-AX/GK-1 treatment showed an increase in areas of cell death, characterized by eosinophilic

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regions and production of IFN-γ by CD8+ T-cells.

and IL-23 in GK-1-treated mice could explain the reduced tumor progression (**Figure 1**).

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**3. GK-1 as an anticancer immunotherapy**

68 Cancer Survivorship

**3.1. GK-1 in a mouse melanoma model**

an immunostimulant.

has been studied in melanoma and breast cancer murine models.

In neoplasms, the host is often immunocompromised due to the presence of immunosuppressive cells and molecules in the TM, to prevent the removal of cancer cells [2]. This highlights the relevance of stimulating the host immune response against cancer antigens by administering immunoadjuvants along with chemotherapy, radiotherapy, or surgery [54]. In this regard, small peptides with a nonspecific immunostimulatory response like GK-1, long known to act as vaccine adjuvants, are potentially useful in cancer therapy. The antitumor effect of GK-1

Melanoma is the most malignant form of skin cancer, mainly affecting the Caucasian population [55, 56]. Until recently, systemic therapy for metastatic melanoma had been inefficient, with a 5-year survival rate for patients (<30%) [57, 58]. However, new therapies were recently approved to treat melanoma, such as pegylated-interferon-α2b (IFN-α2b) in the adjuvant setting; ipilimumab, an anti-CTLA4 monoclonal antibody, for metastatic disease; vemurafenib, an oral BRAF inhibitor indicated for patients with metastatic melanoma harboring BRAFV600 mutations, and more recently antibodies against PD-1 like pembrolizumab [59–61] and antibodies blocking PD-L1 pathways, as well as inhibitors of the mitogen-activated protein kinase (MAPK) pathway. Additionally, nonspecific immunomodulation by several cytokines (IL-2, IL-12, TNF-α, and IFN-γ) and TLR ligands [62–64] in addition to adoptive transfer approaches have been widely used [65]. For over a decade, DCs have also been used in immunotherapy against various types of cancer [66–68] as an alternative to chemotherapy, by vaccination with DCs loaded with tumor peptides (i.e., MAGE-AX [69–72] and/or with necrotic or apoptotic tumor cells to induce effector tumor-specific T cells [73, 74]). The efficacy of this immunotherapeutic approach was also evaluated against murine melanoma, using GK-1 as

GK-1 has been reported to increase the mean survival and significantly delays tumor growth in a melanoma model with B16-F10 cells, showing more necrotic areas along with the presence of numerous neutrophils (**Figure 1**). Neutrophilia inside pulmonary blood vessels was also observed, without evidence of macroscopic or microscopic metastasis. In a melanoma lung metastatic model, GK-1 decreased lymphocyte count, while increased the number of neutrophils and decreased the serum levels of IFN-γ; on the other hand, an increase in the levels of IFN-γ and IL-12 in the intratumor (lung metastases) environment, along with a decrease in IL-17, IL-4, IL-22, and IL-23 was also observed [75, 76]. The antitumor activities of IL-12 have been established in preclinical studies against various tumor cell lines; the increased concentration of the antitumor cytokine IL-12 found in primary tumors may enhance the damage to tumor cells, limiting the number of cancer cells detaching from the primary tumor [77–79]. Its antitumor activity is also mediated by the induction of IFN-γ [67, 78, 80], which upregulated the expression of MHC-I and -II by B16 cells *in vitro*, favoring a cytolytic response in MHC-I-restricted CTL (**Figure 1**) [81]. There is a consensus that the induction of a Th1 profile or the release of cytokines like IFN-γ and TNF-α by T cells is essential for an effective antitumor immune response in

**Figure 1.** GK-1 in a preclinical mouse melanoma model. In a melanoma murine model with B16-F10 cells, GK-1 led to an increase in neutrophils with the increase of IFN-γ and IL-12 cytokines, along with a decrease in IL-17 and IL-23. On the other hand, the MAGE-AX/GK-1 treatment showed an increase in areas of cell death, characterized by eosinophilic regions and production of IFN-γ by CD8+ T-cells.

melanoma [82–84]. In fact, IFN-γ released from CTLs has been considered as a potent mediator of the antitumor response in bulky melanoma tumors [17, 85]. In contrast, IL-17 was proved to directly promote tumor growth and angiogenesis [86–88]. Indeed, it has been shown that IL-17 can promote tumor growth by a direct effect on IL-6 induction, which in turn activates STAT3 in both tumor and nontransformed cells in the TM [89]. Finally, IL-23 is an important molecular driver of Th17 cells in humans; IL-23 is increased in several tumors, and the expression of this cytokine antagonistically regulates local inflammatory responses in the TM, as well as the infiltration of epithelial lymphocytes [80]. Thus, the intratumoral subexpression of IL-17 and IL-23 in GK-1-treated mice could explain the reduced tumor progression (**Figure 1**).

Considering the capacity of GK-1 to enhance DC activation [50], BMDCs matured with TNF-α and stimulated with GK-1 and MAGE-AX were administrated to tumor-bearing mice in the melanoma model with B16-F10 cells; the treatment with MAGE-AX/GK-1 increased survival rates, while mice receiving GK-1 alone had a smaller increase in survival. Moreover, the combination MAGE-AX/GK-1 significantly delayed tumor growth and increased cell death areas, characterized by eosinophilic regions within melanomas. Similarly, both GK-1 alone and MAGE-AX/GK-1 increased the production of IFN-γ-producing CD8 cells, while GK-1 increased the percentage of IL-10 producing CD8+ T-cells [90]. The effect of MAGE-AX/GK-1 could be associated with higher levels of CD8+ lymphocytes in peritumoral lymph nodes, which have been correlated with the survival of patients suffering from melanoma and other cancer types [67, 91, 92].

Those changes suppose a TM that could reduce the tumor growth rate, and the concomitant reduction of cancer cell egress by detachment from the primary tumor, which allows them to invade the stroma and break the basement membrane. These changes could explain the reduction of pulmonary metastasis associated with the GK-1 treatment [98]. Additionally, changes in lung microenvironment associated with the GK-1 treatment have been reported. In this sense, a reduction in the concentration of b-FGF, CCL-3, GM-CSF, CCL-2, TNF-α, and CXCL-9, along with an increased concentration of IL-6 has been found [98] (**Figure 2**). These changes could reduce metastasis development, possibly by inhibiting the proliferation of cells that are essential for the growth of secondary tumors, such as macrophage-associated metastasis (MAM) and MDSC [102, 103]. Considering these results, GK-1 could change the tumor microenvironment, inducing an active antitumoral immune response that could lead to a decrease in cancer burden.

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**Figure 2.** GK-1 in a breast cancer model. GK-1 was associated with an increased IL-12 concentration in the primary tumor, which could involve the infiltration of CD8+ T-cells, NK, and NKT cells. IL-12 is a cytokine produced principally by APC, such as monocytes, macrophages, and dendritic cells. This cytokine can induce specific CD8+ T-cells that are primed against tumor antigens and could serve as a tumor-specific CTL. Additionally, in the lungs, the GK-1-treatment induces a reduction in the concentration of b-FGF, CCL-3, GM-CSF, CCL-2, TNF-α, and CXCL-9, along with an increased

concentration of IL-6, which correlates with a minor lung-metastatic burden.

Vera-Aguilera et al. [79] hypothesized that a combined GK1/anti-PD-L1 therapy could synergize and maximize the individual antitumor effect and extend survival. An increased survival was observed in mice treated with GK1/anti-PD-L1, as well as in mice treated with GK-1 or PD-L1 alone. Animals treated with GK1/anti-PD-L1 had smaller tumor masses. Additionally, GK1/anti-PD-L1 decreased the serum levels of IL-4, IL-5, IL-6, and IL-10. The mechanism by which the combined GK1/anti-PD-L1 treatment improved survival rates remains to be determined; however, the expression of PD-1 on T cells has been proved to be upregulated by IL-6 through the signal transducer and activator of transcription 3 (STAT3) [93], a point of convergence for several oncogenic signaling pathways leading to the expression of immunosuppressing molecules [94]. Similarly, the expression of PD-L1 and PD-L2 is also upregulated by numerous mechanisms, including the production of IL-4 and GM-CSF [93]. All these findings point to a possible synergistic mechanism associated with the reversion from an exhausted phenotype.

#### **3.2. GK-1 in a breast cancer model**

Considering the evidence described above, it is now clear that changes in the microenvironment could induce an antitumor response against the primary tumor and reduce the metastatic disease, which could allow us to control cancer progression. In this regard, immunomodulators like GK-1 can be used as anticancer therapies. In 2017, GK-1 was evaluated in a murine model of invasive breast adenocarcinoma, which spontaneously metastasizes to the lungs, liver, brain, and bone, similarly to breast cancer in humans [95–98]. GK-1 was associated with an increased survival in 4T1 tumor-bearing mice and a reduction in the primary tumor volume rate, which was accompanied by an increase of tumor cell death areas with morphologic features associated with necrosis (pyknosis, karyorrhexis, and karyolysis) and apoptosis (apoptotic bodies) at the primary tumors. These findings, along with an increase in IL-12 concentration in the primary tumor, denote deep changes in the TM induced by GK-1 [98], which could involve the infiltration of TCD8+, NK, and NKT cells in the primary tumor [77, 99, 100] (**Figure 2**). As described in the previous section, IL-12 has been associated with antitumor and antiangiogenic activities [100, 101], due to its capacity of inducing the infiltration of TCD8+ cells within tumor tissues [100]. In fact, it has been reported that a combined treatment with tamoxifen and IL-12 enhanced tumor inhibition due to an increase in apoptosis, and reduced tumor growth in a 4T1 cancer murine model [100].

Those changes suppose a TM that could reduce the tumor growth rate, and the concomitant reduction of cancer cell egress by detachment from the primary tumor, which allows them to invade the stroma and break the basement membrane. These changes could explain the reduction of pulmonary metastasis associated with the GK-1 treatment [98]. Additionally, changes in lung microenvironment associated with the GK-1 treatment have been reported. In this sense, a reduction in the concentration of b-FGF, CCL-3, GM-CSF, CCL-2, TNF-α, and CXCL-9, along with an increased concentration of IL-6 has been found [98] (**Figure 2**). These changes could reduce metastasis development, possibly by inhibiting the proliferation of cells that are essential for the growth of secondary tumors, such as macrophage-associated metastasis (MAM) and MDSC [102, 103]. Considering these results, GK-1 could change the tumor microenvironment, inducing an active antitumoral immune response that could lead to a decrease in cancer burden.

Considering the capacity of GK-1 to enhance DC activation [50], BMDCs matured with TNF-α and stimulated with GK-1 and MAGE-AX were administrated to tumor-bearing mice in the melanoma model with B16-F10 cells; the treatment with MAGE-AX/GK-1 increased survival rates, while mice receiving GK-1 alone had a smaller increase in survival. Moreover, the combination MAGE-AX/GK-1 significantly delayed tumor growth and increased cell death areas, characterized by eosinophilic regions within melanomas. Similarly, both GK-1 alone and MAGE-AX/GK-1 increased the production of IFN-γ-producing CD8 cells, while GK-1 increased the percentage of IL-10 producing CD8+ T-cells [90]. The effect of MAGE-AX/GK-1 could be associated with higher levels of CD8+ lymphocytes in peritumoral lymph nodes, which have been correlated with the survival of patients suffering from melanoma and other

Vera-Aguilera et al. [79] hypothesized that a combined GK1/anti-PD-L1 therapy could synergize and maximize the individual antitumor effect and extend survival. An increased survival was observed in mice treated with GK1/anti-PD-L1, as well as in mice treated with GK-1 or PD-L1 alone. Animals treated with GK1/anti-PD-L1 had smaller tumor masses. Additionally, GK1/anti-PD-L1 decreased the serum levels of IL-4, IL-5, IL-6, and IL-10. The mechanism by which the combined GK1/anti-PD-L1 treatment improved survival rates remains to be determined; however, the expression of PD-1 on T cells has been proved to be upregulated by IL-6 through the signal transducer and activator of transcription 3 (STAT3) [93], a point of convergence for several oncogenic signaling pathways leading to the expression of immunosuppressing molecules [94]. Similarly, the expression of PD-L1 and PD-L2 is also upregulated by numerous mechanisms, including the production of IL-4 and GM-CSF [93]. All these findings point to a possible synergistic mechanism associated with the rever-

Considering the evidence described above, it is now clear that changes in the microenvironment could induce an antitumor response against the primary tumor and reduce the metastatic disease, which could allow us to control cancer progression. In this regard, immunomodulators like GK-1 can be used as anticancer therapies. In 2017, GK-1 was evaluated in a murine model of invasive breast adenocarcinoma, which spontaneously metastasizes to the lungs, liver, brain, and bone, similarly to breast cancer in humans [95–98]. GK-1 was associated with an increased survival in 4T1 tumor-bearing mice and a reduction in the primary tumor volume rate, which was accompanied by an increase of tumor cell death areas with morphologic features associated with necrosis (pyknosis, karyorrhexis, and karyolysis) and apoptosis (apoptotic bodies) at the primary tumors. These findings, along with an increase in IL-12 concentration in the primary tumor, denote deep changes in the TM induced by GK-1 [98], which could involve the infiltration of TCD8+, NK, and NKT cells in the primary tumor [77, 99, 100] (**Figure 2**). As described in the previous section, IL-12 has been associated with antitumor and antiangiogenic activities [100, 101], due to its capacity of inducing the infiltration of TCD8+ cells within tumor tissues [100]. In fact, it has been reported that a combined treatment with tamoxifen and IL-12 enhanced tumor inhibition due to an increase in apopto-

sis, and reduced tumor growth in a 4T1 cancer murine model [100].

cancer types [67, 91, 92].

70 Cancer Survivorship

sion from an exhausted phenotype.

**3.2. GK-1 in a breast cancer model**

**Figure 2.** GK-1 in a breast cancer model. GK-1 was associated with an increased IL-12 concentration in the primary tumor, which could involve the infiltration of CD8+ T-cells, NK, and NKT cells. IL-12 is a cytokine produced principally by APC, such as monocytes, macrophages, and dendritic cells. This cytokine can induce specific CD8+ T-cells that are primed against tumor antigens and could serve as a tumor-specific CTL. Additionally, in the lungs, the GK-1-treatment induces a reduction in the concentration of b-FGF, CCL-3, GM-CSF, CCL-2, TNF-α, and CXCL-9, along with an increased concentration of IL-6, which correlates with a minor lung-metastatic burden.
