In summary, we suggested that “acute” response could elicit innate and Th1 adaptive immunity, reversing the tumor-mediated immunosuppressive environment toward an anti-tumor hub.
Figure 10. The secretion of Th1 cytokines on the 3rd post therapy. Spleens were extracted on the 3rd post therapy and the expression of IL-12 and IFN-γ were examined by RT-PCR. (A) The expression of IL-12. (B) The expression of IFN-γ. Data are shown as mean ± SD. *p < 0.05, ***p < 0.001 by student t test. Triple replicates assay.
“Acute” environment could benefit the upregulation of tumor metastatic inhibitory proteins
In addition to the strongly stimulated “acute” response by cryo-thermal therapy, tumor metastasis inhibition was also observed (Figure 1D). We then ask how such “acute” environment affects tumor metastasis. Here, tumor associated proteins significantly changed in shotgun proteomics (CST6, DKK3, NAPSA, A1BG) were targeted using PRM. Another protein sLIFR, exhibiting a non-significant but high level after therapy in shotgun analysis, was also included for PRM analysis due to its role in tumor progression (Additional File 7: Figure S7). By PRM, all proteins were shown up-regulated during the late stage post therapy (Figure 11A, Additional File 7: Figure S6B). Notably, CST6 was reported as the inhibitor of CTSL [59], which is a lysosome protease and could be secreted to extracellular space to enhance tumor invasion and metastasis [60]. Hence, we also targeted CTSL in PRM and found its down-regulation post therapy, which is opposite to the pattern of CST6 (Figure 11A).
Among them, CST6 and DKK3 are well accepted tumor suppressor proteins [61-63]; soluble LIFR and NAPSA were reported with capability to arrest tumor growth and metastasis [64]; although A1BG shows controversial profiles in different cancers [65-68], it does associate with tumor progression. Interestingly, all of these up-regulated proteins localized in the extracellular space. Taken together, we proposed that the cryo-thermal therapy induced “acute” environment stimulates the expression of tumor progression and metastatic inhibitory proteins and further augment their secretion from host cells to circulation, which counteract the negative impact of chronic inflammation on cancer malignance and prevent tumor metastasis.
“Acute” response induced anti-tumor activity ended up with the recovery of host physiology
It is well accepted that excessive and uncontrolled inflammatory mediators would cause organ failure. In untreated mice, lung swelling was accompanied by the increasing tumor metastasis, indicating its disordered function. However, the lung size was similar to normal in post therapy mice (Figure 1D). In our shotgun proteomics analysis, we found a significant up-regulation of MFAP4 protein on the last day after therapy. MFAP4 was related to elastogenesis in lung. Deficiency of MFAP4 in mice was shown to develop a spontaneous loss of lung function [69, 70]. Therefore, we carried out PRM analysis on MFAP4 for further evaluation. As a result, MFAP4 was consistently expressed at a low level and decreased over time in response to tumor burden, while up-regulated and returned to the base line (Figure 11B) post therapy, which is generally consistent with shotgun proteomics results (Table S6). Therefore, the malfunction of lung upon tumor progression could be reversed by the cryo-thermal therapy under a mechanism of the anti-tumor activity mediated by the “acute” response.
In addition, chronic inflammation could result in changes of lipid metabolism and decreased gluconeogenesis [71]. Hence, significant proteins associated with glucose and lipid metabolism were examined using PRM. These proteins are MUPs, MGAM and PON1 (Figure 11B, Additional File 7: Figure S6C). Similar to that in the shotgun proteomics analysis (Additional File 6: Table S6), these proteins were shown to remain at low levels and decreased over time under chronic environment. However, they were increased to the normal level after cryo-thermal therapy. Overall, it is suggested that the metabolism was distorted during tumor progression, while it could successfully be restored under “acute”environment.
Figure 11. Time course verification of proteins associated with tumor progression and metabolism using parallel reaction monitoring. The dash line represents the protein expression level in healthy mice. Data are shown as mean ± SD. *p < 0.05, **p < 0.01, ***p < 0.001 by two-way ANOVA with the Bonferroni correction. n=5~6 for each condition, except that on the 28th day, only 3 mice left in the control group.