TPX2 Derived Vaccine to Inhibit Tumorigenesis and Metastasis in Triple Negative Breast Cancer

Background: triple negative breast cancer (TNBC) lacks of treatment approaches rather than other subtypes of breast cancer. However, it characterizes the highest the level of tumor inltrating lymphocyte (TIL) than other subtypes, indicating the possibility of immunotherapy. Method: female BALB/c background mice are immunized with a TPX2 derived vaccine 4 consecutive times (once a week) from 6 weeks old, and then 4T1 cells are transplanted to the #4 mammary fatpad. Surface tumor volume and No. of lung metastasis were recorded. TIL and splenocyte was collected for T cell subgroup analysis by methods of ow cytometry and IFN-γ ELISPOT. Result: as a result, TPX2 derived vaccine shows moderate effect, shrinking the tumor volume from 804.4 to 504.5 mm 3 , and decreasing the number of lung metastasis from 16.6 to 6.0 in control group compared to vaccine group. CD8+ T cell ratio are obviously increased in TIL between vaccine group and control group (5.87% vs 3.37%, P=0.0012). And vaccine could induce strong immune response both in tumor site (87.6 vs 7.0, p=0.0004) and system (28.2 vs 3.8, p (cid:0) 0.0001) through IFN-γ ELISPOT. Conclusion: our result indicated that TPX2 derived vaccine may be an effective approach to inhibit TNBC tumorigenesis and metastasis.


Introduction
Breast cancer is the leading malignant tumor among women over the world (1). The causes are still unclear, although it is suggested that tumor development is related to obesity, hormone disorder, and genetic inheritance such as BRCA1 mutation. As a subtype, triple negative breast cancer (TNBC) accounts for approximately 15-20% of all breast cancer, which is characterized by rare expression of estrogen receptor (ER), progesterone receptor (PR), and human epidermal growth factor receptor 2 (Her2). Meanwhile, since lacking of targeted therapy(anti-Her2 therapy such as trastuzumab) and anti-endocrine therapy, the approaches of TNBC are limited, contributing to the worst prognosis of TNBC in all breast cancer. However, it has been reported that TNBC owns the highest level of tumor in ltrating lymphocyte (TIL) rather than other kinds of breast cancer, indicating the possible strategy of immunotherapy (2). Fortunately, it has been proven by a trial that immune check point inhibitor (programmed cell death ligand-1, PDL-1) could be bene cial for partial patients (20%-30%) in late stage TNBC in rst line, when combined with chemotherapy(3). Therefore, new approaches, especially new immunotherapy method, are required to improve the clinical outcome of TNBC.
We have previously studied that TPX2 protein is highly expressed in TNBC; what's more, TPX2 level is negatively related to the prognosis of TNBC, while positively related to the lymph node and distant metastasis (4). It is believed that TPX2 over expression could result high chromosome instability (CIN), subsequently causing TNBC tumorigenesis and metastasis (5,6). Meanwhile, TPX2 inducing high CIN could help TNBC escape from interferon (IFN) dependent T cell anti-tumor effect (5). It has also been reported that over expressed TPX2 inducing high CIN could decrease the expression of main histocompatibility complex (MHC) and its relating proteins (such as TAP1, B2M) (7). All these indicate that TPX2 may serve an important role in MHC signaling and anti-tumor immune processing.
Herein, we report a vaccine composed of 4 separate peptide epitopes from TPX2 protein, which shows moderate effect on BALB/c mouse model transplanted with 4T1 cells. Our result suggests that TPX2 derived vaccine is effective in prohibition of tumorigenesis and metastasis, based on inducing CD8 + T cell in ltration to tumor site, and activating strong imune response both in tumor site and system. IFN-γ ELISPOT: TIL was extracted by tumor dissociation kit (Nwbiotec, Beijing), following the company's guide. Spleen was crushed and then ltered through a 70um cell strainer (Absin) and lysed by ACK lysis buffer for eliminating red blood cell, then supernant was collected as splenocyte. 2.0 × 10 5 cells (TIL or splenocyte, seperately) were plated into each well of a 96 well plate coated with anti IFN-γ antibody (MabTech, OH) and vaccine(all 4 peptides) or control. Then after 72 h, plates were washed and a secondary biotinylated anti-mouse antibody (MabTech, OH) was added and incubated over night at 4℃. Then plate was washed by PBS and HRP streptavidin was added to each well and incubated for 1 h.

Material And Method
Then AEC substrate was added, incubated until the color change. Then the plate was washed and dry, and read by automated plate reader system (CTL Technologies, Shaker Heights, OH).
Flow cytometry: single TIL or splenocyte was detected using ow cytometry for expression of surface markers such as CD4, CD8, CD25 (eBioscience), and intracellular marker of FoxP3 by the instrument of company (BD). Data were analyzed using FlowJo software (Tree Star, Ashland, OR).
Statistic: tumor was measured by vernier caliper when nodule palpable and parameter was recorded by maximum diameter (A) and minimum diameter (B) and calculated by the formula volume = 1/2AB 2 (8). Data were analyzed by t-test.

Result
Anti-TPX2 vaccine inhibited tumorigenesis and lung metastasis in TNBC Surface tumor at mice fat pad was recorded when nodule palpable. After 8 days of 4T1 tumor cell injection, most of the tumor nodules could be touched in fat pad of control group (adjuvant group). After 4 weeks, ucler occurs at the surface of some tumor nodules, then mice were sacri ced. The mean tumor volume was 504.5 mm 3 in vaccine group (n = 5) compared to 804.4 mm 3 in adjuvant group (n = 5) with a p-value of less than 0.0001 on day 29 after 4T1 tumor cell injection (Fig. 1A). It has been demostrated that 4T1 cell transplanted to BALB/c female mice could mimic the metastasis behaivor of human TNBC, such as lung metastasis (9). So that the number of tumor nodule metastasized to lung surface was also calculated in our study. the number of lung metastasis in control group was 16.6, compared to 6.0 in vaccine group with a p value of 0.0003 (Fig. 1B). The representitive pathology image of primary tumor site and lung metastasis were shown in Fig. 1C and D. As a conculsion, our result indicated that TPX2 derived vaccine could inhibit TNBC tumorigenesis and lung metastasis in 4T1 cell transplanted to BALB/c mouse model.

Anti-TPX2 vaccine increased CD8 + T cell in ltrating in tumor site
We then examined T cell subgroup and ratio in TIL by using tumor dissociation kit. As expected, CD8 + T cell ratio in all CD3 + T cell was obviously increased in vaccine group (5.87%) rather than control group (3.38%) with a p value of 0.0012 ( Fig. 2A). Interestingly, CD4 + T cell ratio in all CD3 + T cell was also increased in vaccine group (24.55%) than in control group (18.44%) with a p value of 0.0003 (Fig. 2B).
Anti-TPX2 vaccine inducing strong immune response both in tumor site and system IFN-γ ELISPOT was performed to detect the immune responese of vaccine. As expected on TIL, it revealed that TPX2 derived vaccine could induce obvious immune response in vaccine group (87.6 spot/well) compared to control group (7.0 spot/well, p = 0.0004) (Fig. 3A). Meanwhile, TPX2 derived vaccine could also induce high immune response in system; IFN-γ ELISPOT of splenocyte indicated 28.2 spot/well in vaccine group compared to 3.8 spot/well in control group(p<0.0001) (Fig. 3B).

Discussion
In our manuscrpit, we have demostrated that TPX2 derived vaccine is effective in BALB/c background mice transplanted with 4T1 cell line, through leading CD8 + T cell in ltration to tumor and inducing strong immune response in tumor site and system.
Our experiment indicates that TPX2 derived vaccine could induce CD8 + T cell in ltration in tumor site. It has been demostrated that the prognosis of TNBC is positvely associated with TIL and its subpopulation CD8 + T cell; on the contrary, the poliferation of TNBC is related to decreasing of CD8 + T cell in TIL (10)(11)(12). So it is reasonable to suggest that elevating CD8 + T cell in ltration in tumor site could bene t the prognosis of TNBC. In our study, TPX2 derived vaccine successfully induced CD8 + T cell in ltrating in tumor site and stimulated strong immune response and thus showed moderate effect in prohibition of tumorigenesis. However, it has also been suggested that CD8 + T cell with functional T cell receptor (TCR) only take few parts in all CD8 + T cells of TIL, even though in some "hot carcinoma"(13). That means using vaccine could stimulate the formation of antigen speci c TCR, subsequently increase TCR speci c CD8 + T cells. Our data also suggest that TPX2 derived vaccine could induce strong immune response in system, and then decrease lung metastasis. It is easy to understand that vaccine works systematically, and to some extent modi ed the tumor evironment in system and then inhibit the metastasis of TNBC.
There is no obvious side effect observed in our study. The body weight of mice in vaccine group showed no decreasing after vaccine injection (data not shown). Meanwhile, no autoimmue response occurred in our vaccine group. However, there are also some limitations in our study. Firstly, our study and TPX2 derived vaccine was only suitable for BALB/c background mice; since human has totally different MHC background compared to mice, it is easy to understand that our vaccine is only t for mice, rather than for human kind. Secondly, in our study, it demostrated that Treg in TIL seems to be unexpectedly evoked, indicating that our vaccine works in an immune-supressed microenviroment, which could inhibit the effect of vaccine.

Conclusion
In conclusion, our work has supplied a TPX2 derived vaccine which showed moderate effect in a TNBC mouse model. Our vaccine could induce high immune response in tumor site and system, and increase the in ltration of CD8 + in tumor site. Further work should be done to overcome the unexpected evocation of Treg, and deliever it to human kind.