The antitumor effects of Z-100 have been reported in several papers [
7‐
10]. In this study, the antitumor effect of Z-100 was dose-dependent and, although 1 mg/kg was considered the optimal dose, Z-100 significantly inhibited tumor growth even at 0.01 mg/kg. With the exception of one non-clinical study, there have been no previous reports on the antitumor effects of Z-100 at doses as low as 0.01 mg/kg. The lone study that used such low doses of Z-100 examined its antitumor effects on an inguinal lymph node metastasis model [
11]. This prior study and the present study are similar in that both used “pre-administration” of Z-100 at low doses. In the study on inguinal lymph node metastasis, Z-100 administration was started at the same time as tumor injection into the plantar region; however, because the tumor did not metastasize into inguinal lymph nodes until 2–3 weeks later, the strategy can be considered “pre-administration” of Z-100 for treatment of cancer metastasis in the inguinal lymph nodes. Therefore, administering Z-100 prior to cancer exacerbation may be key to maximizing its effects. While it is not possible to prophylactically administer medicines against cancer development in clinical practice, it is possible to “pre-administer” them to prevent metastasis, recurrence or relapse. Thus, Z-100 may be clinically effective for inhibiting malignant transformation of cancer.
Our in vivo experimental system, in which Z-100 showed high efficacy, made it possible to investigate tumor infiltrating cells, leading to the identification of infiltrating CD8
+ T cells. Further analysis showed that these infiltrating CD8
+ T cells were effector memory cells, and that an anti-CD8 antibody abolished the antitumor effects of Z-100. Thus, there is no doubt that the antitumor effects of Z-100 are mediated by CD8
+ T cell infiltration. That administration of Z-100 increased the proportion of infiltrating CD8
+ T cells suggests that the tumors had been converted to hot tumors, and that Z-100 could be a hot tumor inducer. Immunotherapies that include an anti-PD-1 antibody have been reported to be highly effective against hot tumors [
1,
4,
21], which may explain why the combination of Z-100 and anti-PD-1 antibody was effective in the report by Kobayashi et al. [
14]. Further, we previously reported that Z-100 increases IL-12p40 production [
13]. IL-12p40 is known bind to IL-12p35 to form the heterodimer IL-12, which is involved in antitumor immunity [
22]. Some studies have suggested that inducing IL-12 leads to the formation of hot tumors [
23,
24], while another report indicated that suppressing IL-12p40 leads to cold tumors [
25]. Therefore, it is possible that the inability of Z-100 to increase IL-12p40 production in IL-12p40 KO mice may have prevented tumors from becoming hot tumors. However, it is questionable whether administration of IL-12 would have the same effect as administration of Z-100. Although administration of IL-12 has been reported to have anti-tumor effects, toxicity has also been observed [
26‐
30]. Given that Z-100 has very mild toxicity in clinical practice [
17], its effects may differ from those of IL-12 administration. Z-100 stimulation has been reported to increase TNF-α, IL-1β, IFN-γ, and IL-2 from immune cells [
9,
10,
13,
31], the production of which may underlie the different activities of Z-100 and IL-12. We also previously reported that Z-100 increases IL-12p40 production from M1-like macrophages differentiated from mouse bone marrow cells and human CD14
+ cells in the presence of GM-CSF [
13]. In that report, peptidoglycans were present in the Z-100, and commercially available peptidoglycans have been shown to increase IL-12p40 production from these cells. Further, knockdown of nucleotide-binding oligomerization domain 2 (NOD2) suppressed this Z-100-mediated increase in IL-12p40 production. Therefore, we concluded that the peptidoglycans in Z-100 led to production of IL-12p40 via NOD2. Thus, peptidoglycans from
Mycobacterium tuberculosis and NOD2 agonists could also be hot tumor inducers. Further studies are needed to confirm this theory.
In this study, we used the oral squamous cell carcinoma cell line Sq-1979. In general, squamous cell carcinoma is known as a hot tumor-like cancer, while melanoma is known as a cold tumor-like cancer [
32]. In a previous study, Z-100 suppressed tumor growth and increased survival in a subcutaneous injection model of B16 melanoma [
10]. Z-100 has also shown an inhibitory effect on lung metastasis models of B16 melanoma [
9]. In addition, Z-100 suppressed metastasis in a lymphatic metastatic model of B16 melanoma and increased the number of immune cells in lymph nodes [
11]. These reports suggest that Z-100 is also effective against cold tumors.
In summary, this study found that "pre-administration" of Z-100 induced potent antitumor effects, and that Z-100 acts by inducing the infiltration of CD8+ T cells into tumors. This suggests that Z-100 may convert tumors into hot tumors for more favorable response to immunotherapy. Thus, Z-100 is expected to have antitumor effects in clinical practice, including in combination with various immunotherapies.