Next, we carried out a network analysis on these APPs using IPA. Interestingly, interleukin-6 was identified as the key upstream regulator of these proteins (Figure 6A), indicating its significant role in inducing acute phase response. To further confirm this assumption, first, IL-6 levels in serum and within tumor were examined via ELISA and RT-PCR. As a result, IL-6 in circulation maintained at a persistent high level in control mice relative to healthy and even extensively increased on the 11th day (Figure 6B). However, cryo-thermal therapy immediately stimulated a considerable expression of IL-6 in serum and within local tumor on the 1st day, much stronger than mice untreated (Figure 6B, Figure 6C), whereas started to decline on the 2nd day and returned to a similar level as the healthy mice (Figure 6B). These findings are consistent with the APPs expression profiles. Next, prior to its peak expression, we neutralized IL-6 with IL-6 antibody in vivo 3 and 24 h post therapy. Serum was collected 2 days post therapy to examine the expression of APPs. Among APPs, ORM, APCS and HP are well documented in researches [40-42]. In addition, ORM1, ORM2 and ORM3 belong to the same family and ORM1 was identified with the most confident peptides in shotgun and PRM target proteomics compared to ORM2 and ORM3. In this case, ORM1, APCS and HP are selected. Western blot analysis showed that their expressions are significantly down-regulated after IL-6 neutralization (Figure 7). All these results indicated that “acute” activation of interleukin-6 was stimulated by cryo-thermal therapy, inducing the “acute” phase response.

 

 

As described, with the accumulating tumor burden, the expression of APPs steeply raised to the most on the last day (Figure 5), indicating the most severity of chronic inflammation. Chronic inflammation is associated with immunosuppression in many tumors [43], predominately induced by tumor infiltrating Tregs to suppress effector T cells and prevent tumor against immune surveillance [44]. The interaction of ICOS /ICOSL can boost Tregs activation, proliferation, and survival [45-47]. In addition, ICOSL could trigger the expression of immunosuppressive Th2 cytokines, such as IL-4, IL-5 and IL-13[48, 49]. Here, both shotgun proteomics discovery and PRM validation analysis revealed that ICOSL was significantly overexpressed during the late stage of tumor development, whereas it always stayed around the base line under “acute” response (Figure 8). This observation suggested that the Th2 immunosuppression may be attenuated by the cryo-thermal therapy induced “acute” response and thus the activation of anti-tumor immunity was favored.

 

Since “acute” is a remarkable profile of cryo-thermal therapy, which leads to the attenuation of immunosuppression, we then interested in how this “acute” signature could benefit the anti-tumor immunity. We further evaluated proteins significantly expressed in the timeframe of “acute” response (2nd to 5th day post therapy) in shotgun proteomics. These proteins include CPB2, CTSL, C2, C8B, CFP, DPP4, MUP3, GRM7, VNN3 and VASN. Interestingly, among them, C2, C8B, CFP are involved in the activation of complement system; CTSL and DPP4 are responsible for T cell activation. All of them appear to regulate innate and adaptive immunity. Hence, we performed PRM target analysis to further examine these proteins. As a result, the significant expression of C2, C8B and CTSL, DPP4 were well in line with the shotgun proteomics (Table S6).

 

Particularly, C2 is the central regulator of classic and lectin complement pathways, and C8B is the component of terminal membrane attack complex (MAC) for cell lysis. These two proteins were reported to play critical roles in tumor cell clearance through phagocytosis and cell lysis, triggering innate immunity [50, 51]. In consistent, both proteins were shown being significantly up-regulated on the 5th day after treatment and returned to the base line, which is in agreement with the “acute” response profile. On the other hand, under chronic inflammation in untreatedmice, these proteins were shown to stay around the base level (Figure 8). All these observations implied that “acute” phase response could re-activate the impaired complement system under chronic inflammation and benefit anti-tumor innate immunity.

 

 

Figure 7. The expression of three acute phase proteins after IL-6 neutralization. Mice were injected intraperitoneally with mouse anti-IL-6 Ab 3 and 24 h post cryo-thermal therapy and sacrificed on the 2nd day. Serum was collected for western blot analysis. Protein amount was evaluated and visualized with Ponceau S. Blots were evaluated with Quantity One 1-D. Results are expressed as relative pixel intensity normalized with control group. Data are shown as mean ± SD. ***p < 0.001 by student t test. n=3 with three technical replicates.

 

 

Figure 8. Time course verification of proteins associated with complement system and adaptive immunity using parallel reaction monitoring. The dash line represents the protein expression level in healthy mice. Data are shown as mean ± SD. **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.

 

 

Figure 9. The maturation of DCs and activation of CD4+CD26+ T cells. (A) The percentage of CD86+MHCII+ DCs, gated on CD11c+. Lymphocytes were isolated from spleens 1 day, 3 dayand 5 day post therapy and stained with the following Abs: anti-mouse-CD11c, anti-mouse- MHCII and anti-mouse-CD86. (B) The percentage of CD26+ T cells gated on CD3+CD4+. Lymphocytes were isolated from spleen on the 2nd day post therapy and stained with the following Abs: anti-mouse-CD3, anti-mouse-CD4 and anti-mouse-CD26. Data are shown as mean ± SD. **p < 0.01, ***p<0.001, by student t test, n=3.

 

In addition, CTSL was also observed up-regulated on the 5th day after therapy (Figure 8). CTSL plays important roles in antigen processing and maturation of antigen presenting cells (APCs) [52]. Such up-regulation suggested a possible maturation of APCs (like dendritic cells). Therefore, we performed flow cytometry to examine the maturation of dendritic cells in spleens of treated mice. As a result, matured DCs (CD11c+CD86+MHCII+) showed a significant increase on the 3rd day after therapy and was even higher on the 5th day, while it remained at a relative low level in the untreated mice (Figure 9A). Concomitant with the “acute” response, DPP4 (known as CD26), with a soluble form in circulating system [53], was also shown up-regulated on the 2^nd day after therapy and returned to the base level afterwards (Figure 8). CD26 is a maker of T cell activation which is highly expressed in the activated and antigen-reactive memory T cells. CD26high T cell could produce T-helper 1 (Th1) cytokines to elicit type-1 anti-tumor response (CD4+CD26+ T cell) or exert cytotoxic T lymphocyte activity (CD8+CD26+ T cell) [54-56]. The up-regulation of CD26 on the 2nd day suggested that more T memory cells were activated and proliferated upon “acute” response. To further confirm this suggestion, we performed flow cytometric analysis upon lymphocytes isolated from splenocytes 2 days post therapy. As a result, we found a strikingly increased percentage of CD4+CD26+ T cell (Figure 9B) as compared to that of untreated mice. Therefore, these results confirmed that more CD4+ memory T cells were activated and proliferated upon “acute” response. By this manner, these cells could potentially serve as Th1 cells to initiate and amplify the adaptive immunity against tumor.

 

Since we observed the maturation of DCs and proliferation of Th1 memory T cells, we next

examined the secretion of Th1 cytokines (IL-12 and IFN-γ) to further investigate whether protective Th1 adaptive immunity was activated under “acute” response. IL-12, naturally produced by dendritic cells, is important in directing the development of Th1 cells [57]. The source of IFN-γ is restricted to CD4+ Th1 cells, once antigen-specific immunity develops [58]. Hence, we examined their splenic expression on the 3rd day post therapy using RT-PCR. As shown in Figure 10, both IL-12 and IFN-γ exhibited the highest expression after the therapy, while in the untreated mice, their expression were inhibited compared to both healthy and treated mice. Therefore, the protective T helper type 1 immune response appeared to be activated after the therapy.

 

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.

 

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.

 

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.

 

 

As described above, “acute’ environment is suggested to be the key driver to switch tumor microenvironment from immunosuppressive to immunostimulatory state. In addition, the combination of cryo/hyperthermia showed prolonged survival rate and reduced metastasis compared with cryotherapy or hyperthermia alone. In this case, we are interested in the “acute” profile in different therapeutics and the relationship with anti-tumor immunity. First, we compared the expression of three well documented APPs (ORM1, APCS and HP) [40-42] in serum 2 days post different treatments on tumors, which are 1) cryo-thermal therapy, 2) cryotherapy, 3) hyperthermia and 4) control without treatment. Western blot analysis showed that cryo-thermal therapy induced the strongest acute response as indicated by the highest expression of selected APPs (Figure 12). As described above, “acute” response could lead to DC maturation and Th1 adaptive immune response in early stage. To better compare the immunological effect stimulated by “acute” response, we next tested the adaptive immune response in later stage. As a result, cryo-thermal therapy elicited a significant protective immune response with the most accumulated CD8+ and CD4+ T lymphocytes in spleen 14 days post treatment (Figure 13A, 13B). In addition, the immunosuppressive state was most inhibited indicated by the maximally reduced MDSC (Gr1+CD11b+) (Figure 13C). These findings suggested that as a combination of cryo/hyperthermia, cryo-thermal therapy established the strongest “acute” environment, which could benefit the stimulation of the most pronounced anti-tumor adaptive immune response. Moreover, to delineate the "unique" immunological effect of cryo-thermal therapy on tumor tissues, we further examined the “acute” profile and corresponding immune response in heathy mice under this specific therapy. As shown, cryo-thermal on healthy mice (N+A) induced “acute” response; however, such “acute” pattern was much weaker than that applied on tumors (Figure 12). We assumed that the “danger” signal released from tumors could boost the host “acute” inflammation. In addition, cryo-thermal therapy induced the most pronounced protective T lymphocyte response on tumors, whereas it was not changed much in healthy mice, regardless of treatment or not, indicating that such immune response was not effectively stimulated without the presence of antigen released from tumors (Figure 13A, 13B). As shown, MDSC, with remarkable immunosuppressive abilities in cancer, was largely reduced after cryo-thermal therapy on tumors; however, it was induced under “acute” response in healthy mice, which could quickly differentiate into mature myeloid cells predominantly participating in innate immune response, while have a minimal impact on the adaptive immune response [72] (Figure 13 C).

 

 

Figure 12. The expression of acute phase proteins through different energy-based therapeutics applied on tumors and healthy mice. Tumor bearing mice were treated with 1) cryo-thermal therapy, 2) cryotherapy, 3) hyperthermia and 4) control (no treatment). Healthy mice with and without cryo-thermal were set as another controls. 2 days post therapy, serum was collected for western blot analysis. Protein amount was evaluated and visualized with Ponceau S. Blots were evaluated with Quantity One 1-D. Results are expressed as relative pixel intensity normalized with healthy group. Data are shown as mean ± SD. **p<0.01, ***p < 0.001 by one way ANOVA with Dunnett's test and each group was compared with cryo-thermal therapy. Healthy mice with and without treatment were analyzed by student t test. n=3 with three technical replicates.

 

 

Figure 13. The protective adaptive immune response and immunosuppression arising from different energy-based therapeutics applied on tumors and healthy mice. Tumor bearing mice were treated with 1) cryo-thermal therapy, 2) cryotherapy, 3) hyperthermia and 4) control (no treatment). Healthy mice with and without cryo-thermal were set as another controls. Lymphocytes were isolated from spleens 14 days post therapy. (A) The percentage of CD3+CD4+ T lymphocytes. (B) The percentage of CD3+CD8+ cytotoxic T lymphocytes. (C) The percentage of Gr1+CD11b+ MDSC cells. Data are shown as mean ± SD. *p<0.05, **p<0.01, ***p < 0.001 by one way ANOVA with Dunnett's test and each group was compared with cryo-thermal therapy. Healthy mice with and without treatment were analyzed by student t test. n=3.

 

Taken together, we concluded that cryo-thermal therapy has a better immunological effect over cryo/hyperthermia alone and such effect was unique on tumors.

 

Metastatic breast cancer remains a big challenge in clinical practice. Based on thermal therapeutic strategy, we previously developed the cryo-thermal therapy as an energy-based cancer intervention. This strategy has shown a good therapeutic efficacy with prolonged lifespan and reduced metastasis (Figure 1). In this study, serum glycoproteome comparison using shotgun proteomics analysis followed by PRM target validation was carried out to dissect the distinct protein profiles between treated and untreated mice on multiple samples (94) and time points (8), in order to better understand the molecular mechanism of the anti-tumor activity on a system-wide view. In general, targeted proteins exhibited consistent significant expression with that in shotgun proteomics, to some extent, the enhanced sensitivity and selectivity of PRM assay provides better quantitative information for some proteins, such as ORM1, ORM3, APCS, ITIH3, PRG4, SELL, MUP3, MGAM and MFAP4. More importantly, such high throughput target proteomics allowed us to validate proteins on a large scale (multiple samples and proteins), which was not suitable using traditional approaches such as western blot and ELISA, due to limitations from time-consuming and antibody dependence.

 

In this study, we found that “acute” response was strongly induced by cryo-thermal therapy with “acute” and high expression of a series of acute phase proteins and consequently reverse the tumor chronic pro-tumorigenic environment. “Acute’ response is generally occurred as an immediate and evident inflammation in response to stress. In tumor progress, chronic environment is often induced with little visible inflammatory reaction, whereas it becomes apparent and severe upon extensive tumor expansion [73]. As the critical upstream regulator of acute response, IL-6 was also identified with an “acute” profile in our study, whose reduced level decreased the magnitude of “acute” response. Previously, “acute” IL-6 induced by adipose-derived stromal/stem cells has been proved to mediate the good therapeutic effects in acute lung injury [74]. In addition, fever-range thermal stress induced “acute” activation of IL-6 has been indicated with anti-tumor activity through boosting T lymphocyte trafficking [75-77] and amplifying the killing cytotoxic effector of CD8+ effector T cells toward tumor cells [78]. However, the sustained elevated IL-6 concentration in circulating has been observed in numerous cancers and is a prognostic indicator of poor outcome [79-81]. Hence we hypothesized that “acute” IL-6 induced “acute” response plays a key role in anti-tumor activity.

 

In this study, we proposed a model that IL-6 induced “acute” response could mediate anti-tumor activity in three aspects, which could interact with each other and result in the good therapeutic efficacy. They are 1) Th2 immunosuppressive response breaking, shifting pro-tumorigenic phenotype toward 2) tumoricidal innate and Th1 adaptive immunity activation and 3) up-regulation of tumor progression and metastatic inhibitors. The details were described below (Figure 14).

 

Cryo-thermal therapy, by applying through rapid freezing followed by heating, would result in severe thermal and mechanical stresses to local tumor. The severe stress could thus stimulate a rapid and vast generation of IL-6 through macrophages influx. Such “acute” activation of IL-6 rapidly induces the “acute” phase response, generating an “acute” microenvironment, and disrupting the chronic inflammation in tumor site. Such “acute” microenvironment restricts the expression of ICOSL around the baseline, well controlling the Th2 immunosuppressive response (like Tregs, IL-4, IL-5, and IL-13) induced by chronic environment and favoring a protective immunity hub. Under this “acute” environment, acute phase proteins (APPs) are up-regulated, facilitating the phagocytosis of tumor cells and clearance of injurious agents through opsonisation or secreted pathogen recognition receptors [36, 82]. In addition, complement system is also activated, further improving the clearance of damaged tumor cells through phagocytosis, and facilitating the lysis of invading tumor cells via the formation of the terminal membrane attack complex (MAC) [83] to release the metastatic risk. By such manner, anti-tumor innate immunity is initiated. Subsequently, complement system could drive the innate toward adaptive immunity to promote the anti-tumor immune response. It has been shown that its products, such as anaphylatoxins (C3a, C5a) [84, 85], C1q and C3 fragment [86-88] can directly induce dendritic cells (DCs) maturation and promote the development of effector T cells. This is in line with our observation that CTSL was up-regulated and more DCs were maturated on the similar day to complement system activation (3rd and 5th day). In addition, it has been reported that “acute” IL-6 could limit the activity of virus-specific Tregs, thereby facilitating the activity of virus-specific memory CD4+ T cells[89]. Consistently, in our study, an increased number of CD26+CD4+ memory T lymphocytes were activated and proliferated accompanied by the“acute” response, favoring the Th1 anti-tumor adaptive immunity. To be noted, in our study, activated memory T cells appeared a bit earlier than the matured DCs augmentation (2nd day vs 3rd day). In fact, memory T cells have lower need of MHC-peptide for activation than naïve T cells and response more fast and robust [90]. Such rapid response allows memory T to attack the tumor cells as early as possible. Furthermore, DC producing IL-12 contributes to the development of Th1 that generates high amount of IFN-γ to further promote Th1 polarization, while suppressing Th2 cell differentiation, favoring the protective Th1 adaptive immune response. Notably, “acute” response started to resolve with reduced production of IL-6, declined expression of APPs on 2nd day and less leukocyte trafficking. Such resolution is critical, not only to maintain the homeostasis of hostile environment, but also bridge the gap between innate and adaptive immunity for the optimal development of adaptive immunity [91].

 

What’s more, “acute” micro-environment activated the secretion of tumor progression and metastatic inhibitory proteins by host cells to reduce metastasis. Finally, with the struggle of above three aspects, “acute” response induced by cryo-thermal therapy switches the Th2 immunosuppressive, pro-tumorigenic environment to a Th1 immunostimulatory, tumoricidal phenotype, protecting the host from accumulated tumor burden, reducing metastasis and recovering the host metabolism in the end.

 

To be noted, APPs studied in our work are relative high abundant proteins, which benefit their identification and quantification in cryo-thermal therapy and other biological events, like in cryotherapy or hyperthermia treatment of cancer. However, their “acute” profiles are markedly different. Cryo-thermal therapy induced the strongest “acute” response and elicited the most pronounced immunological effect compared to cryotherapy and hyperthermia alone. Moreover, cryo-thermal therapy on healthy tissue induced weaker “acute” response and failed to stimulate effective immunological effect compared with tumors. In this case, we suggested that the magnitude of “acute” and the release of “danger” signals play a key role in determining the efficacy of anti-tumor immunity.

 

 

Figure 14. The schematic graph of “acute” response mediated anti-tumor activity induced by cryo-thermal therapy. The cryo-thermal therapy performed by rapid switch between freezing and heating causes a severe thermal and mechanical stresses to local tumor, leading a rapid and vast generation of IL-6 by macrophage influx. The “acute” IL-6 induced “acute” response could mediate anti-tumor activity in three aspects, which could interact with each other and enhance the anti-tumor effect. They are 1) Th2 immunosuppression breaking, 2) driving anti-tumor innate and Th1 adaptive immunity activation, characterized by complement system activation, more matured DCs and CD4+ memory T cell accumulation, as well as the secretion of Th1 cytokines to promote the differentiation of naïve T cells to Th1 cells, and 3) up-regulation of tumor progression and metastatic inhibitory proteins. In addition, “acute” resolution is critical, not only to maintain the homeostasis of hostile environment, but also bridge the gap between innate and adaptive immunity for the optimal development of adaptive immunity. Finally, “acute” response mediated anti-tumor activity leads to the recovered liver metabolism and normal physiological function. In addition, tumor recurrence is reduced and metastasis is inhibited. Proteins in red are up-regulated, green are down-regulated.

 

In summary, using comprehensive discovery proteomics analysis followed by target proteomics validation at a large scale sample size, we performed a system-based high throughput analysis to better understand the biological response induced by cryo-thermal therapy and reveal the stimulation of tumor-specific immunity at the molecular level. The cryo-thermal therapy induced strongest “acute” response helped boost systematic anti-tumor immune response in the host for a stronger immunogenicity environment, which enabled more effective cure of metastatic tumor.

 

Additional File 1:

Supplementary Table 1.

http://www.thno.org/v06p0773s1.xlsx

Additional File 2:

Supplementary Table 2.

http://www.thno.org/v06p0773s2.xlsx

Additional File 3:

Supplementary Table 3.

http://www.thno.org/v06p0773s3.xlsx

Additional File 4:

Supplementary Table 4.

http://www.thno.org/v06p0773s4.xlsx

Additional File 5:

Supplementary Table 5.

http://www.thno.org/v06p0773s5.xlsx

Additional File 6:

Supplementary Table 6.

http://www.thno.org/v06p0773s6.xlsx

Additional File 7:

Supplementary Figures.

http://www.thno.org/v06p0773s7.pdf

 

 

A1BG: Alpha-1-B glycoprotein; APCs: Antigen presenting cells; APCS: Serum amyloid P-component; APPs: Acute phase proteins; C2: Complement component 2; C8B: Complement component 8, beta polypeptide; CFP: Complement factor properdin; CST6: Cystatin E/M; CTSL: Cathepsin L1; DDA: Data-dependent acquisition; DC: Dendritic cell; DKK3: Dickkopf-related protein 3; DMEM: Dulbecco’s Modified Eagle’s Medium; DPP4: Dipeptidyl-peptidase 4; FBS: Fetal bovine serum; FDR: False discovery rate; HCD: Higher-energy collisional dissociation; HP: Haptoglobin; ICOSL: ICOS ligand; IL-4: Interleukin 4; IL-5: Interleukin 5; IL-6: Interleukin 6; IL-12: Interleukin 12; IL-13: Interleukin 13; IFN-γ: Interferon gamma; IPA: Ingenuity Pathway Analysis; iTRAQ: Isobaric tags for relative and absolute quantitation; MDSC: Myeloid-derived suppressor cells; MFAP4: Microfibrillar-associated protein 4; MGAM: Maltase-glucoamylase; MUP3: Major urinary protein 3; NAPSA: Napsin-A; ORM1: Alpha-1-acid glycoprotein 1; ORM2: Alpha-1-acid glycoprotein 2; ORM3: Alpha-1-acid glycoprotein 3; PON1: Serum paraoxonase/arylesterase 1; PRG4: Proteoglycan 4; PRM: Parallel reaction monitoring; SELL: L-selectin; sLIFR: Soluble leukemia inhibitory factor receptor; SRM: Selected reaction monitoring; TAMs: Tumor associated macrophages; TFE: Trifluoroethanol; Th1: T-helper 1; TPP: Trans-Proteomic Pipeline; Tregs: T regulatory cells

 

The authors wish to thank Wei Zhang (Thermo Fisher Scientific), ChengPin Shen (Shanghai

CloudScientific Technology Co., Ltd), Samuel Bader, Luis Mendoza, Kristian Swearingen and Patrick Flores (Institute for System Biology) for their support of mass spectrometer’s operation and data analysis. We also would like to acknowledge Dr. Jie Hao (Shanghai Center for Systems Biomedicine) for help with statistical and bioinformatics data analysis and Dr. Xin Ku for critical comments during manuscript preparation. This work was funded in part by the American Recovery and Reinvestment Act (ARRA) funds through National Institutes of Health from the NHGRI grant RC2HG005805, the NIGMS grants R01GM087221, S10RR027584 and 2P50GM076547 to the Center for Systems Biology, the National Science Foundation MRI grant 0923536. This work was also supported by the National Natural Science Foundation of China (11275126, U1532116) and China Scholarship Council.

 

TX, PL, WY, and RLM conceived and designed the research. LXX was the project leader. TX conducted the experiments and data analysis. TX and WY wrote the manuscript. YZ and LY helped design and modify the glycocapture and iTRAQ experiments. LY helped analysis the MS data. KL performed the RT-PCR experiments.

 

The authors have declared that no competing interest exists.

 

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