Anti-PRL-3 Monoclonal Antibody inhibits the Growth and Metastasis of colorectal adenocarcinoma

Background: Colon cancer is the one of leading causes of cancer-related death. Chemotherapy, radiotherapy and immunotherapy will be the mainstream in inoperable advanced cancer in clinics. Precision treatment is still lack in colon cancer. Materials and Methods: We developed a series of mAbs targeting PRL-3 through different types of immunogens. The binding domains of mAbs were identified through the ELISA and Western blotting experiments. The antitumor activity of mAbs was verified by cell proliferation, migration and invasion experiments. Xenograft subcutaneous and metastatic models and patient derived Xenograft (PDX) model were established. Results: mAb 12G12 targeting 77-120AA exhibited inhibition in migration and invasion experiments. 12G12 inhibited the migration of multiple types of cancer cells, including colon cancer, gastric cancer, esophagus cancer, liver cancer, lung cancer and pancreatic cancer cells. 12G12 decreased the tumor growth and metastasis in Xenograft subcutaneous and metastatic tumor model, respectively. The antitumor activity of mAb 12G12 was also confirmed in PDX model of gastric cancer. PRL-3 interacted with Golgi protein TMED10. Knockdown of TMED10 expression attenuated the cell migration triggered by purified GST-PRL-3 protein. Conclusion: Our results confirmed the antitumor activity of mAb 12G12 in colorectal adenocarcinoma and provided a new potential targeted therapy of colon cancer.


Introduction
The strategies of non-surgical therapy of tumor have been moving from chemotherapy or radiation therapy to individual precision therapy including targeted antitumor drugs, small molecular inhibition [1].Multiple drugs targeting tyrosine kinases have been applied in lung cancer and improved the clinical Ivyspring International Publisher outcomes of a significant portion of NSCLC patients during the last ten years [2].In colon cancer, KRAS, NRAS, BRAF and PIK3CA were the well-known driver mutations.Almost 50% colon cancer patients were reported bearing KRAS mutation.Due to the smooth surface of KRAS protein, it has been considerate as "undruggable".AMG510 targeting KRAS G12C was still under assessment in the solid tumor [3,4].Therefore, high specific antibody-based therapy with minor side effect would be a hopeful direction in the gastrointestinal cancer.
Our group devoted into the exploration of the biological function of PRL-3 in gastrointestinal cancer for decades.PRL-3, one member of the PRLs family, has been identified as an oncogene promoting cell migration and invasion in the many solid tumors [5].Our previous studies show that PRL-3 expression was related with stage, lymph node metastasis and poorer prognosis in colon cancer, breast cancer and ovarian cancer [6][7][8][9].In the transgenic mouse model, PRL-3 also participated in the initial process of inflammation-malignant transitions in colon cancer [10].PRL-3 enhanced the self-renewal of colon cancer cells and inhibition of PRL-3 may reduce the distant metastasis [11].PRL-3 played a critical role in T cell acute lymphoblastic leukemia (T-ALL) initiation and progression through promoting leukemia cell proliferation and migration [12,13].PRL-3 serves as a downstream gene of SMARCA2 in t (4;14) multiple myeloma and activation of PRL-3 maintained MYC gene expression.PFI-3 (a BET inhibitor) could dissociated SMARCA2-NSD2 complex from PRL-3 promoter and induced myeloma cell apoptosis [14].Therefore, small molecular compound targeting PRL-3 would be potential therapy for both solid tumors and hematological malignancies.
Recently, a humanized antibody, PRL-3-zumab, was analyzed in the treatment of gastric cancer and liver cancers [15,16].PRL-3-zumab has been in phase 2 clinical trial in Singapore [17], it gave us more confidence to explore therapeutic potential of novel PRL-3 antibodies.In this study, we generated mAb 12G12 which exhibited powerful antitumor activity in the in vitro and in vivo experiments.mAb 12G12 could be a potential antitumor drug in colon cancer.

PRLs mAbs development
We developed PRLs mAbs using three different types of immunogen: purified prokaryotic expression protein, prokaryotic plasmid pVAX1-Igκ-PRL-3(K-P3) and peptide cross-linked with KLH (KLH, Keyhole Limpet Hemocyanin).Generation of prokaryotic proteins and procedures of plasmid immunization were reported previously [18].For the peptide immunization, we choose the non-homogenous sequence on PRL-3 (compare with PRL-1 and PRL-2) for the peptide design.The sequence of Peptide 1: PGKVVEDWLSLVKAKFCEAEFDQVHFQPLPPAVV KLSDALC-KLH (77-95AA).The sequence of Peptide 2: RLRFKDPHTHKTRCCVMEFDQVHFQPLPPAVV KLSDALC-KLH (108-124AA).Mixture of two peptide (50 µg of each) with complete Freund's adjuvant (Sigma, F5881) was subcutaneously injected into Balb/c-nude mice.Two booster immunizations of peptide mixture with incomplete Freund's adjuvant (Sigma, F-5506) were taken on 3 weeks later.Tail blood was obtained for the titer examination.Hybridoma clones was developed by fusion of SP2/0 myeloma cells with Balb/c-nude mouse spleen-derived B cells which had high titer.

Cell proliferation, migration and invasion assays
Cell Counting Kit (CCK)-8 (C0037, Beyotime, Shanghai, China) were used for cell proliferation experiments.3 ×10 4 of indicated colon cancer cells were re-suspended in 100 µl complete medium, and seed in the 96-well plates in triplicate.At 0, 24, 48, 72, and 96 hours, the medium was discarded and 100 µl fresh medium containing 10 µl CCk-8 was added to each well.After 2 hours incubation at 37℃, the spectrophotometer (Thermo Fisher Scientific) was used to measure the absorbance of each well at OD450nm.
For transwell chamber assays, 200 µl of re-suspended cells (2×10 5 /ml for migration and 10×10 5 /ml for invasion) were seeded on the upper chamber of each transwell (Becton Dickinson, San Jose, CA, USA), and 800 µl medium containing 10% FBS was added to the lower chamber.Cells were cultured at 37℃ for 24 hour (for migration) or 48 hours (for invasion).The cells penetrated the upper chamber were fixed in cold methanol, stained in 0.1% crystal violet, and counted in at least 6 randomly selected field under microscope.The 8-week old Balb/c-nude mice were subcutaneously implanted with 2×10 6 LoVo cells in 200 µL of the PBS suspension to establish the xenograft subcutaneous tumor model.mAb 12G12 or IgG (200 µg in 100 µL) were administrated intravenously twice a week for 4 weeks (11).Mice were all sacrificed on day 31.Tumor size was measured using a caliper and tumor volume were calculated using the formula T vol =1/2 × larger diameter × (smaller diameter) 2 .

Xenograft subcutaneous tumor model and xenograft metastatic model
The 8-week-old Balb/c-nude mice were intravenously implanted with 5×10 5 LoVo cells in 200 µL of PBS suspension to establish the lung metastasis model.Following this, 12G12 (400 µg in 200 µL) or PBS (200 µL) were administrated intravenously twice a week for 3 weeks.The weight of mice was monitored every 7 days.All the mice were sacrificed on day 60, and the lung tissues were filled with the Bouin's solution before the detachment [6].

Patients-derived xenograft (PDX) model
Balb/c-nude mice (4-6 weeks old, body weight, 18-20 g) were maintained and treated in accordance with Biomedical Ethical Committee of Peking University Cancer Hospital & Institute.Gastric cancer tissues from three patients were removed under sterile conditions.Tumor tissues were washed three times with a phosphate buffered saline (PBS) solution containing 2% penicillin/streptomycin, and necrotic tissue was removed.For PDX passaging, the tumor tissue was cut into 2 mm × 2 mm × 1 mm pieces and inoculated subcutaneously on the right side of the dorsal front of mice.Sixteen mice were randomly divided into PBS group and 12G12 group.After three day of tumor inoculation, PBS or 12G12 were injected twice a week for 2-3 weeks.Tumors were harvested once they reached the endpoint (> 10 mm in the largest dimension).Tumors were dissected, weighed, and photographed.Tumor volume was calculated using the formula (tumor volume = length × width 2 /2).

Immunohistochemistry
Tissue samples from xenograft tumors (including xenograft subcutaneous tumor model, xenograft metastatic model and PDX model) were fixed in 4% formaldehyde for 24 hours, processed to the paraffin, and sectioned.For immunohistochemistry, 5 µm sections were deparaffinized in xylene and hydrated in alcohol.Endogenous peroxidase activity was then blocked by incubation in 3% hydrogen peroxide-methanol for 10 minutes.For the antigen recovery, the section was heated in a citrate buffer (10 mM Sodium Citrate, pH 6.0) for 15 minutes.After blocking, the primary antibodies against caspase-3(1:1000) (Lot: #9662, Cell Signaling), cleaved caspase-3 (5A1E) (1:400) (Lot: #9664, Cell Signaling) was applied at 4℃ overnight.EnVision+ TM (Dako) was used as the secondary antibody.Antibody binding was visualized by a standard streptavidin immunoperoxidase reaction.These sections were counterstained with hematoxylin.
The score of immunohistochemistry was graded by histochemistry score (H-Score) system.Evaluation was carried out independently by two experienced pathologists.The H-score system was the combination of proportional and intensity of positive tumor cells.H score = ΣPi(i+1).Pi, the ratio of the positive staining cell in all the tumor cells; i, the staining intensity.

Co-immunoprecipitation and GST pull-down
HCT116 cells were transiently transfected with GST-PRL-3, myc-TMED10 and vector for 48 hours.Cells were lysed in buffer (50 mM Tris-HCl, pH 7.5, 150 mM NaCl, 1 mM EDTA and 0.5% NP-40) containing complete protease inhibitors (Roche, USA), and the lysates were used for GST pull-down with Glutathione Sepharose or immunoprecipitation with anti-myc antibody [10].The precipitants were subjected to Western blotting with specific antibodies.Visualization was achieved with chemiluminescence.

Statically analysis
The SPSS (version 26) software package (SPSS Inc, Chicago, IL) was used for statistical analysis.
Qualitative data are expressed as the mean ± standard deviation.Difference between groups were compared by one-way ANOVA or Student's t-test or χ2 test.GraphPad Prism 6 software (GraphPad Software, San Diego, CA) was used for statistical figure or chart display.All experiments were repeated at least three times with consistent results.P-value < 0.05 was considered statistically significant.

mAb 12G12 inhibited colon cancer cell motility in a dose-dependent manner
To further verify the application of mAb 12G12, we utilized PBS (blank control), IgG (negative control) and different dose of 12G12 mAbs (0.5µg/ml, 2.0µg/ml and 10.0µg/ml) in the migration and invasion experiments.mAb 12G12 (2.0 µg/ml) significantly inhibited the migration and invasion of all these 3 colon cancer cells (migration inhibition rate: HCT116, 28.1% ± 2.0%; LoVo, 64.6% ± 7.92%; SW480, 19.5% ± 1.7%; invasion inhibition rate: HCT116, 51.0% ± 1.2%, LoVo, 55.5% ± 6.6%; SW480, 38.3% ± 4.4%) (Figure 3A and 3B).Loaded 12G12 to 10 µg/ml barely further increased the inhibition of cell migration or invasion in all these cell lines.12G12 significantly inhibited proliferation of HCT116 and SW480 cells compared with PBS and IgG (Supplementary Figure 3A).However, we did not observe anti-proliferative ability of 12G12 in LoVo cells in three independent experiments.Our previous studies reported the dual effects of PRL-3 on the cancer cell proliferation, as both overexpression and knockdown of PRL-3 in colon cancer cells could inhibit cell proliferation through distinct mechanisms [10].These in vitro experiments indicated that mAb 12G12 may be utilized as a potential antitumor drug.

mAb 12G12 inhibited colon cancer tumor growth and metastasis to Lung in mice model
To assess the application of mAb 12G12 in vivo, we developed xenograft Subcutaneous tumor model implanted with LoVo cells (Figure 4A).mAb 12G12 dramatically decreased the tumor weight (0.44 ± 0.06 g VS 0.31 ± 0.09 g, p = 0.04) and volume compared to IgG control (Figure 4B and 4C).We didn't observe the loss of body weight in all the groups (p = 0.577, Figure 4D).HE staining confirmed the malignant cell assembles in situ (Figure 4E).Representative images and quantitative score indicated that caspase-3 and cleaved caspase-3 were higher in mAb 12G12 groups than in IgG group (caspase-3: 0.35 ± 0.14 VS 0.025 ± 0.05, p = 0.024; cleaved caspase-3: 0.45 ± 0.087 VS 0.15 ± 0.05, p = 0.032).PRL-3 expression was found in all the groups and there was not significant difference between IgG group and 12G12 group (0.15 ± 0.05 VS 0.35 ± 0.13, p = 0.190) (Figure 4E-4H).
Lung metastasis is frequently observed in colon cancer and associated with poor prognosis.Consistent with the in vitro results, 12G12 reduced the metastatic foci on lung compared to PBS control (4.50 ± 2.88 VS 21.17 ± 15.10, p = 0.024) (Figure 4I and 4J).There was no significant change of body weight (Figure 4K).Metastasis foci in the lung was also confirmed by the HE staining (Figure 4L).

mAb 12G12 inhibited tumor growth in patient derived xenograft (PDX) model
We further investigated the efficacy of mAb 12G12 in gastric cancer PDX model.The detail clinical pathological information of patients was listed in the Supplementary Table 1.mAb 12G12 dramatically inhibited the tumor growth in all three PDX models (Supplementary Figure 4 A and 4B).PRL-3 level in tissue sections was determined by IHC and the presence of malignant cells in situ was confirmed by HE staining.Levels of caspase-3 and cleaved caspase-3 were significantly increased in mAb 12G12-treated groups (Supplementary Figure 4C).Taken the results of xenograft subcutaneous tumor model and PDX model together, mAb 12G12suppressed tumor growth was associated with the triggering of apoptosis.

mAb 12G12 inhibited cell migration in a TMED10-dependent manner
It has been reported that PRL-3 protein could "inside-out" externalize on the cell surface and exosomal membrane [15], however the mechanism remains elusive.Our previous study reported that PRL-3 localized in cytoplasm and nucleus by interacting with telomere protein RAP1 [10,21].Yeast two-hybrid screening identified a panel of PRL-3 partners [21,22], one of which was identified as the Golgi protein TMED10 (Transmembrane p24 trafficking protein 10 Gene).CoIP and GST pull-down experiments showed that PRL-3 interacted with TMED10 (Figure 5A).Knockdown of TMED10 expression by siRNA was confirmed by Western blotting (Figure 5B).In the subsequent experiments we combined 3 knockdown siRNAs together to maximize the inhibition of TMED10 expression.We found that exogenously purified GST-PRL-3 could promote cell migration and invasion (Figure 5C and  5D).After silencing TMED10 expression by siRNA, GST-PRL-3-promoted migration and invasion were abolished (Figure 5C and 5D).Similarly, mAb 12G12-inhibited cell migration was abrogated (Figure 5E).It indicated that the effects of PRL-3 protein or mAb 12G12 on cell motility could be TMED10-dependent.Next, we detected the levels of PRL-3-related signaling proteins in the cells under treatment with GST-PRL-3 plus mAb 12G12 (Figure 5F).We found that showed that 12G12 was able to inhibit the phosphorylation of p65, AKT and ERK, which was activated by PRL-3 protein (Figure 5F).These results were consistent with the role of PRL-3 in activating diverse signaling pathways [23].mAb 12G12 suppressed tumor in a TMED10 dependent and through NFκB-AKT pathway.

Discussion
The role of PRL-3 has been concerned for almost 20 years and its role in tumor initiation and metastasis has been demonstrated in both our previous study and by other groups [5,10,23].PRL-3 promoted tumor progression through various pathways, such as PI3K-AKT-ERK, NFκB and Jak2-STATs pathways [21,[24][25][26].Recent researches showed that PRL-3 also participated in the regulation of tumor microenvironment via recruiting B cell, NK cells and macrophages [16].Our previous study confirmed that DNA vaccine with plasmid encoding PRL-3 triggered CTL and T helper type 1 immune response [18].These studies revealed that targeting PRL-3 is not only hopeful in monoclonal antibody therapy but also in adjuvant therapy with immune inhibitions.Using different immunogens, including prokaryotic protein, prokaryotic plasmids and peptides, we generated 13 different mAbs targeting PRL proteins.Through antibody specificity identification, we got 3 antibodies which specifically recognized PRL-3.mAb 12G12 is the "orphan" antibody probably targets the phosphorylation domain of PRL-3, which was different from binding domain of commercial clone 318 (Figure 1D).We tried to connect the anti-metastasis (Supplementary Figure 2) and anti-proliferation efficiency (Supplementary Figure 3) of mAb 12G12 to the endogenous PRL-3 status in tumor cells.Unfortunately, mAb 12G12 consistently inhibited the cell migration and invasion of colon cancer, gastric cancer and esophagus cancer independent of PRL-3 expression.For example, compared to HCT116, LoVo cells have lower PRL-3 expression, but mAb 12G12 has higher suppression of cell migration.Furthermore, the inhibitory efficiency of mAb 12G12 was negatively correlated with PRL-3 expression in lung cancer (lower expression of PRL-3 in PG, higher suppression of cell migration) and pancreatic cancer (higher expression of PRL-3 in ASPC-1, lower suppression of cell migration) (Supplementary Figure 2B-2G).Our previous study and other group's reports indicated that the prospect of PRL-3 research may hide in the tumor microenvironment, including secreting factors, exosomes, and immune cells [15,25,27].Therefore, we believe that mAb 12G12 may be applied in the treatment of solid tumors through different mechanisms.Besides 12G12, mAbs 8B7 and D3 also suppressed the cell migration and/or invasion in colon cancer cell lines (Figure 2).As we all know, preclinic antitumor drug development is full of uncertain factors.Our study potentially increased the success rate of developing antitumor mAbs to PRL-3.
Other group reported that antibody targeting PRL-3 eliminated PRL-3+ liver tumors and gastric tumors in vivo [15,16].In our study, mAb 12G12 suppressed the tumor growth of colon cancer cell-derived and gastric cancer tissue-derived mice model.12G12 also inhibited lung metastasis of colon cancer cells (Figure 4 and Supplementary Figure 4).Levels of caspase-3 and cleaved caspase-3 were elevated upon mAb 12G12 treatment (Figure 4E-4G and Supplementary Figure 4C), indicating that mAb 12G12 suppressed the tumor growth by triggering apoptosis pathways.Notably, 12G12 had negligible effect on body weight of mice in the in vivo experiments, suggesting the safety of using this mAb.
PRL-3 zumab, a hopeful antitumor drug, could be targeted to exosomal membrane and trigger the ADCC/ADCP cascade.Our previous study showed that PRL-3 protein translocates from cytoplasm to nuclei, even telomere end by forming a PRL-3-RAP1-TRF2 complex [10].We found that TMED10, a trafficking protein localized on the cell membrane, could directly interact with PRL-3 (Figure 5A).Knockdown of TMED10 not only counteracted PRL-3-promoted migration and invasion, but also blocked the 12G12's inhibitory effect on cell migration (Figure 5C-5E).It was revealed that TMED10 functions as a protein channel for vesicle entry and secretion.TMED10 also regulated the cytokine IL1β secretion [28,29].There is also a cross-talk between cytokines and PRL-3 expression on tumors.Overexpression of PRL-3 induced the secretion of IL1α in a NF-κB-JAK-STAT3 dependent manner.Inhibition of IL1α level suppressed the PRL-3-promoted cell migration [23].Therefore, we supposed that TMED10 may mediate translocation of PRL-3 to plasma membrane, thereby facilitating recognition by mAb 12G12.
Collectively, our results confirmed the antitumor activity of mAb 12G12 in colorectal adenocarcinoma.Considering the role of PRL-3 in tumor immune microenvironment and the success of immunotherapy in clinics, mAb 12G12 combine with immune inhibitor, chemotherapy or radiotherapy should also be evaluated in the future.
Animal study was approved by the Biomedical Ethical Committee of Peking University Cancer Hospital & Institute (License Number: EAEC 2019-05) and performed along established institutional animal welfare guidelines concordant with the US guidelines (NIH Publication #85-23, revised in 1985).

Figure 3 .
Figure 3. mAb 12G12 inhibited colon cancer cell motility in a dose-dependent manner.Transwell chamber migration assays (A) and Matrigel invasion assays (B) of HCT116, LoVo and SW480 cells with different 12G12 dosages (2.0 µg/ml, 5.0µg/ml,10.0µg/ml).PBS and IgG (10.0µg/ml) treatment were set as blank and negative control.The experiments were repeated three times.The values are the mean and standard deviation of normalize the number of each group with PBS.*, p <0.05; **, p <0.01; ***, p <0.001.

Figure 4 .
Figure 4. mAb 12G12 inhibited colon cancer tumor growth and metastasis to lung in mouse models.A-G Effects of mAb 12G12 on the tumor growth in Balb/c-nude mice.The mice were intravenously injected with antibody (10 mg/kg) or 40 mg/kg control IgG twice a week for 4 weeks at 5 day after intravenous injection of 5×10 6 LoVo cells.All mice were euthanized and harvested.A. Representative photographs of tumors.B and C. Average tumor weights (B) and volume (C) monitored every 4 days