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A novel peptide probe for identification of PLS3-expressed cancer cells Fanghao Shi, Yan Ma, Yixia Qian, Yuehua Wang, Zihua Wang, Minzhi Zhao, and Zhiyuan Hu Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.9b01061 • Publication Date (Web): 11 Jul 2019 Downloaded from pubs.acs.org on July 19, 2019

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Analytical Chemistry

A novel peptide probe for identification of PLS3-expressed cancer cells Fanghao Shi#a,b,c, Yan Ma#a, Yixia Qian a,b,c, Yuehua Wanga, Zihua Wangd, Minzhi Zhaoa and Zhiyuan Hu*a,b,d a

CAS Key Laboratory of Standardization and Measurement for Nanotechnology, CAS

Key Laboratory for Biomedical Effects of Nanomaterials and Nanosafety, CAS Center for Excellence in Nanoscience, National Center for Nanoscience and Technology of China, Beijing 100190, China b

Sino-Danish College, University of Chinese Academy of Sciences, Beijing 100049,

China c

University of Chinese Academy of Sciences, Beijing 100049, China

d

Center for Neuroscience Research, School of Basic Medical Sciences, Fujian

Medical University, Fuzhou 350108, China # The

first two authors contributed equally to the work.

* Author for correspondence: Zhiyuan Hu ([email protected]; Fax: 86-10-82545643)

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Abstract The T-plastin (PLS3) has a significant implication in epithelial-mesenchymal transition (EMT) and breast cancer prognosis. Using one-bead-one-compound library strategy, a novel peptide TP1 (KVKSDRVC) towards PLS3 was screened and exhibited the specificity for identifying PLS3-expressed cancer cells. Moreover, we found Fluorescein isothiocyanate-labelled TP1 (FITC-TP1) could act as a novel probe for EMT-induced cancer cells, preferentially in the leading edge. It also has satisfactory specificity for PLS3-expressed cancer cells spiked in the blood. FITC-TP1 was expected to become a diagnostic tool to identify PLS3-expressed circulating tumor cells and predict prognosis for patients with breast cancer in the future. Keywords: T-plastin, peptide, epithelial-mesenchymal transition, circulating tumor cells

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Introduction In worldwide, breast cancer occupies the most common cancer and the leading cause of cancer death among females1. Despite the therapeutic development over the decades, breast cancer prognosis depends on the occurrence of metastases that still represents an incurable condition2. Thus, novel prognostic strategies are desperately needed for improving the predictive performance. The clinical utility of circulating tumor cells (CTCs) have shed lights on breast cancer prognosis evaluation. Acting as negative prognostic factors associated with impaired clinical outcome, the enumeration of CTCs predicts adverse progression-free survival (PFS) and overall survival (OS) in patients with breast cancer in the context of chemotherapy treatment3, 4. Cytokeratin and CD45, detected in the common method of CTCs identification, separately indicates tumor cells and white blood cells (WBCs)5. However, cytokeratin may fail to identify the CTCs, since the epithelial-mesenchymal transition (EMT) process renders epithelial cells lose their epithelial markers6. Additionally, using antibody probes give rise to limitations: The cells are usually not available for genomic profiling due to the permeability needed to stain for cytokeratin and DAPI; Long-time incubation for antibodis leads to timeconsumption. Thus, much effort has concentrated on developing predictive markers and effective probes to improve the efficacy of the CTCs technology. T-plastin (PLS3), a kind of actin-crosslinking protein, is a novel indicator for predicting long-term prognosis besides lymph node metastasis and tumour volumes in patients with breast cancer owing to its involvement in the EMT regulation of CTCs7. EMT has been associated with metastatic propensity, since EMT-induced cancer cells gain mesenchymal traits, such as loss of apico-basal polarity, lack of adherent junctions and acquisition of invasive capability, and resistance to apoptosis8, 9. Thus, PLS3 is regarded as a metastasis-specific gene for its role in regulating EMT in CTCs10, 11. Moreover, acting as an integrator of cell motility, PLS3 participates in multiple associated cellular events, such as assembly and maintenance of cytoskeleton, the formation of protrusions and Arp2/3-mediated actin-based movement12-14. The remodeling of actin cytoskeleton provides the major force for driving cells motile. On the basis of actin assembly, protrusions like filopodia and lamellipodia produced in the leading edge are regarded as the prerequisite for cell motility15. Being an actin-bundling protein, PLS3 helps convert branched actin filaments into parallel bundles16, underlying the transition between filopodia and lamellipodia at the leading edge17, 18. PLS3, the 3



correlative regulated protein of actin cytoskeleton, could therefore become a metastatic target. Using PLS3 as the target molecule have many other advantages over its metastasisrelated function. For example, PLS3 expression linked with the cellular resistance to chemotherapeutic drugs. PLS3 down-regulation enhanced the sensitivity to cisplatin in bladder cancer cells19. Recently, we found that PLS3 down-regulation augmented the sensitivity to paclitaxol by enhancing apoptosis via p38 MAPK signalling pathway20. Besides, it is available for PLS3 detection at all stages of the EMT, that overcomes the limitations of current epithelial markers, like cytokeratins10. In combination with the absent of PLS3 expression in peripheral blood10, 21, PLS3 is an ideal biomarker for CTCs identification. Herein, we screen the positive peptide TP1 specifically targeting PLS3 to probe the PLS3-expressed tumor cells. Peptide probes have more advantages over antibodies: Peptides are easy to synthesize in largescale22; The metastatic cancer cells can be specifically detected using peptide-based probes during metastatic transformation23; Peptides label the cells alive without prior fixation and permeabilization for they have small size, minimal immunogenicity and rapid binding kinetics24-26. After peptide selection, we aim to identify the probe role of TP1 in breast cancer cells during EMT process. Using spike-in assay, we also verify the specificity of TP1 towards cancer cells spiked in the blood.

Experimental section The materials are listed in Supporting information. One-bead-one-compound peptide library synthesis and Magnetic Beads Assisted Screening towards PLS3 The one-bead-one-compound (OBOC) peptide library was synthesized according to the Fmoc strategy solid phase peptide synthesis (SPPS) strategy as previous reported27. For the screening of PLS3-targeting peptide, the recombinant PLS3 protein (Cloud-Clone Corp., Houston, USA) was biotinylated using a biotin labelling kit according to the manufacturer’s instruction. After the incubation of the biotinylated PLS3 protein with the library for 2 hours at 37 °C, 2 mL of streptavidin coated magnetic beads were added to search for positive peptide beads. Using an integrated microfluidic chip, the positive beads coated by magnetic beads were trapped in the microwells while the negative beads were removed under the magnetic field as previous reported28. In addition to the 4


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beads sorting microchannel, the microfluidic chip consisted of a sequencing microarray. Based on the one-well-one-bead microarray, the in situ matrix assisted laser desorption ionization time-of-flight (MALDI-TOF) sequencing was performed on individual peptide beads. The details are listed in Supporting information. Cell culture and transfection MDA-MB-231 (human breast cancer) and MCF-7 (human breast cancer) cell lines were purchased from the Cell Culture Center of Peking Union Medical College (Beijing, China). Both MDA-MB-231 and MCF-7 cells were cultured in DMEM supplemented with 10% FBS. T-Plastin-specific siRNA (Santa Cruz Biotechnology, CA, USA) was used to knock down the PLS3 mRNA (designated as si-PLS3). The Negative Control siRNA (Ribobio Technology, Guangzhou, China) was applied as a negative control (designated as Ncontrol). Using Lipofectamine 2000 (Invitrogen, Carlsbad, CA, USA), cells at 40–60% confluence in 6-well culture dishes were transfected with the respective siRNAs. TP1 as probes for cancer cells imaging in vitro MCF-7 and MDA-MB-231 cells were separately planted in glass-bottom-dish at a cell density of 105 cells/mL. After incubation at 37 ℃ under 5 % CO2 atmosphere for 24 h, the medium was removed and the nuclei were stained with the Hoechst 33342. The cells were incubated with 1 mg/mL FITC-TP1 for 10 min at 4 °C in the dark. The cells were observed using a confocal laser scanning microscope (ZEISS LSM710) with 63×oil immersion objective. Images were analyzed by using ZEN software. Western blot After transfection or other treatment, the cells were harvested and lysed in RIPA buffer (Cell Signaling Technology, Danvers, MA, USA). Using an Enhanced BCA Protein assay kit (Beyotime, Jiangsu, China), the protein concentration was determined. Then, the whole cell lysates were subjected to SDS-PAGE and transferred to polyvinylidene difluoride membranes (Millipore, Billerica, MA, USA). The membranes were incubated overnight at 4℃ with the indicated antibodies at 1:500 dilution (antibodies from Santa Cruz Biotechnology) or 1:1000 dilution (antibodies from BD Transduction Laboratories, Genetex and Cell Signaling Technology). Then, they were incubated for 1 h at room temperature with the horseradish peroxidise-conjugated secondary antibodies at 1:2000 dilution. The immunoreactive bands were developed by using an ECL detection system (Millipore, Billerica, MA, USA). 5



Immunofluorescence assay MCF-7 cells was planted in glass-bottom-dish at low or high cell density. For wound healing assay, linear scratches were produced in MCF-7 cells with a pipette tip after incubation at 37 ℃ under 5 % CO2 atmosphere for 24 h. The cells were fixed in 4 % paraformaldehyde for 10 min at room temperature and blocked in 5 % BSA. The cells was first incubated with T-plastin at 1:50 dilution for 2 h followed by Alexa Fluor 647conjugated secondary antibody (dilution, 1:500) for 1 h, and then incubated with Ecadherin (dilution, 1:100) and Vimentin (dilution, 1:100) for 2 h at room temperature followed by FITC-conjugated and PE-conjugated secondary antibodies (dilution, 1:50) at room temperature for 1 h. The nuclei were stained with the Hoechst 33342. The cells were observed using a confocal laser scanning microscope (ZEISS LSM710) with 63×oil immersion objective. Images were analyzed using ZEN software. TP1 as probes for cancer cells spiked in the whole blood cells For the isolation of cancer cells spiked in blood, the samples were prepared by separately spiking MDA-MB-231 cells into 1 mL human whole blood cells from healthy donors. The immunomagnetic microbeads (Ademtech, France) conjugated with an EpCAM antibody (Abcam, Cambridge, MA, USA) were used to recognize EpCAM-positive cells in the spiked samples. After isolated under a magnetic field, the beads captured cells was first blocked in 5 % BSA and incubated with CD45 [MEM-28] (dilution, 1:50) for 1 h at room temperature in the dark. After removing the antibody dilution buffer, the nuclei were stained with the Hoechst 33342. The cells were incubated with 0.5 mg/mL FITC-TP1 for 10 min at 4 °C. The cells were observed using a fluorescence microscope (Olympus IX73) and were analyzed by using ZEN software.

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Results and Discussion Design and Construction of PLS3 Targeting OBOC Peptide Library. We designed the peptide library referenced from the sequences of the residues 504610 in PLS3 protein, which included multiple unique peptide segments for antibody binding (UniProtKB - P13797). Hydrophobic and electrostatic interactions and hydrogen bonding were involved in directing the selection of different amino acids at each position in the peptide library. As shown in Figure S1 & Figure 1a, the onebead-one-compound (OBOC) library for PLS3-targeting peptide screening was established by the “pool and split” method27. With the capacity 64, 000, the peptide library was constructed with the sequence X1 X2 X3 X4 X5 X6 X7 X8 in which X1 represents His, Lys, Asp, Arg or Glu residues, X2 represents Leu, Ser, Val, Ile or Phe residues, X3 represents Lys, Ala, Pro, Glu or Gly residues, X4 represents Asp, Ser, Gln or Asn residues, X5 represents His, Arg, Ser or Asp residues, X6 represents Gly, Asn, Ile or Arg residues, X7 represents Trp, Tyr, Val or Pro residues and X8 represents Cys or Pro residues. The peptide library was then incubated with the biotinylated PLS3 and the streptavidin-coated magnetic beads. With the recognition between peptide beads and PLS3 protein, the positive peptide beads were covered by magnetic beads in an affinity-dependent manner. The peptide beads with high affinity for PLS3 could be isolated and trapped utilizing an integrated lab-on-chip system as previous reported28. After the single bead sorting process, the in situ MALDI-TOF sequencing was performed on the microarray to identify the characters of postivie peptides. Using ClustalX2 multiple alignment29, TP1 (KVKSDRVC) emerged from the random sequences. The Screening system was shown in Figure 1b.

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Figure 1. Schematic diagram. (a) The synthesis of the OBOC library towards PLS3. (b) Schematic model presenting the screening of TP1.

Affinity of TP1 towards Living Cancer Cells. The positive peptide TP1 (KVKSDRVC) was de novo synthesized and identified using MALDI-TOF and high-performance liquid chromatography (HPLC) after purification (Fig. S2a-c). To validate their affinity towards PLS3, dissociation constants (KD) between the peptide and PLS3 protein were determined by surface plasmon resonance imaging (SPRi) assay and calculated from kinetic constants by curve-fitting of the association and dissociation rates to real-time binding and washing data. The KD of TP1 towards PLS3 protein was 2.92 × 10−9 mol L−1, demonstrating a high-affinity binding between TP1 and PLS3 protein (Fig. 2a). The KD of a monoclonal antibody of PLS3 towards PLS3 protein was 1.53 × 10−11 mol L−1 (Fig. S3). Next, the fluorescence staining assay was performed to investigate the specificity of TP1 towards PLS3. MDA-MB-231 and MCF-7 cells were chosen as positive and negative model cells in vitro, since MDA-MB-231 cells expressed high levels of PLS3 expression and MCF-7 cells expressed low levels of PLS3 expression (Fig. 2b). We also validated the expression of PLS3 in MDA-MB-231 and MCF-7 cells using immunofluorescence (IF) assay (Fig. S4). As shown in Figure 2c-j, MDA-MB-231 cells incubated with fluorescein isothiocyanate-labelled TP1 (FITC-TP1) showed strong fluorescence intensity on the cell surface, whereas almost no fluorescence 8


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signal was observed in MCF-7 cells. Thus, the specific binding ability confered TP1 a suitable ligand for specific recognition of PLS3 protein.

Figure 2. The binding affinity of FITC-labelled TP1 towards PLS3 in MCF-7 cells and MDAMB-231 cells. (a) SPRi detection of the specific binding capability of TP1 towards PLS3 protein. (b) The expression of PLS3 was detected in MCF-7 and MDA-MB-231 cells using western blotting. β-actin was used as the loading control. (c-e) Confocal images of FITC-TP1 binding to MCF-7 cells. (g-i) Confocal images of FITC-TP1 binding to MDA-MB-231 cells. Scale bar = 20 μm. (f, j) Analysis of the fluorescent intensity along the red arrows in c & g.

Specificity of the TP1 towards PLS3. To comfirm the specificity of the TP1 towards PLS3, the small interfering RNA (siRNA) targeting PLS3 (si-PLS3) was used to knock down the PLS3 expression. Determined by western blotting, si-PLS3 decreased the protein levels of PLS3 in MDA-MB-231 cells (Fig. 3a). Consistent with the expression levels, weak even no signal was detected in the MDA-MB-231 cells transfected with si-PLS3, whereas TP1 preserved the binding ability towards the MDA-MB-231 cells transfected with Ncontrol si-RNA (Fig. 3b-i). The result further validated that PLS3 protein was the specific target of the peptide ligands TP1, which could in turn indicate the presence of PLS3. Additionally, TP1 degraded in the conditioned medium derived from MDA9



MB-231 cells with an estimated half-life of 60.5 min, demonstrating that the probe was stable enough to allow sufficient time for cancer cell imaging (Fig. S5).

Figure 3. The validation for the specificity of TP1 towards PLS3 using RNA interference assay. (a) Western blot of whole-cell extracts from MDA-MB-231 cells transfected with Ncontrol or si-PLS3 for 48 h. β-actin was used as the loading control. Confocal images of FITC-TP1 binding to MDA-MB-231 cells transfected with Ncontrol (b-d) or si-PLS3 (f-h). Scale bar = 20 μm. (e, i) Analysis of the fluorescent intensity along the red arrows in b & f.

FITC-TP1 as probes for labelling EMT-induced cancer cells Inspite of the involvement of aberrant expression of PLS3 in the EMT program in colorectal cancer (CRC) cells11, the correlation between PLS3 expression and EMT in breast cancer was still unknown. To clarify the relationship between PLS3 expression and EMT, we utilized transforming growth factor β1 (TGF-β1), a canonical EMT inducer, to initiate the EMT 30. Agree with our previous report that TGF-β1 could induce EMT in MCF-7 cells31, it also triggered the EMT, namely that it upregulated the mesenchymal marker N-cadherin and downregulated the epithelial marker Ecadherin in MDA-MB-231 cells (Fig. S6). In both positive and negative model cells, elevated expressions of PLS3 were observed with TGF-β1 treatment, revealing that PLS3 was an EMT marker in breast cancer cells (Fig. 4a). As shown in Figure 4b-h, FITC-TP1 could bind on the surface of the EMT-induced cells as well as control cells in MDA-MB-231 cells, suggesting that FITC-TP1 bound stably to PLS3-expressed cancer cells during EMT process. Compared with the control cells (Fig. 4i-k), TGFβ1-induced MCF-7 cells gained strong fluorescence intensity on the plasma membrane of peripherial cells (Fig. 4l-n), indicating that FITC-TP1 acted as probes for EMT induction in the PLS3-negative cells. Since EMT characterizes the cancer cells with high capacity of metastasis and confers chemoresistance in breast cancer32, 10


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33,

the prognostic role of CTCs-derived PLS3 may correlate with its expression levels

varying with EMT spectrum.

Figure 4. FITC-TP1 as probes for cancer cells imaging during EMT. (a) Western blot of whole-cell extracts for PLS3 in MDA-MB-231 cells and MCF-7 cells stimulated with TGF-β1 (5 ng/mL) for 24 h. β-actin was used as the loading control. Confocal images of FITC-TP1 binding to MDA-MB-231 cells untreated (b-d) or treated (f-h) with TGF-β1 or binding to MCF-7 cells untreated (i-k) or treated with TGF-β1 (l-n). Scale bar = 20 μm.

FITC-TP1 as probes for labelling EMT-induced cells in the leading edge Inspite of the identification of the EMT-induced cells at the periphery shown in Figure 4l-n, FITC-TP1 failed to label the membrane of the central MCF-7 cells with TGF-β1 treatment. Combined with the specificity of TP1 towards PLS3, this portion of cells were regarded as PLS3 absent. Thus we supposed that TGF-β1 failed to increase PLS3 expression in the cells that going through incomplete EMT34. To further validate the expression of PLS3 and the existence of incomplete transition, the cells were triple-stained with antibodies of PLS3, E-cadherin and Vimentin (a marker of mesenchyal phenotype). Compared with the control cells, EMT was triggered accompanied by the increased Vimentin expression and decreased E-cadherin expression (Fig. 5a, b). Additionally, the expression of PLS3 was up-regulated. The result implied that PLS3 absence or an incomplete transition wasn’t the reason why FITC-TP1 failed to label the central MCF-7 cells. Interestingly, we found that PLS3 as well as Vimentin strikingly assembled in the edge of the TGF-β1-induced MCF-7 11



cells (Fig. 5b). This staining pattern was similar to that observed in the cells at high density. PLS3 and vimentin were strongly concentrated in the border of the cells at high density (Fig. S7). According to the result shown in Figure 2 & Figure 3, FITCTP1 exhibited a good specificty towards PLS3 protein that FITC-TP1 could label the cells with high expression of PLS3, but fail to label the cells with PLS3 absence. Herein, the strong staining in the border of the EMT-induced cells with antibody suggested a large amount of PLS3 proteins assembling in the edge, which may explained why FITC-TP1 only stained the margin of peripherial cells in Figure 4l. On two-dimensional culture substrates, filopodia and lamellipodia were utilized to drive the prostrution of the leading edge of the cells moving into the surroundings35. As is reported, fimbrin and Vimentin colocalized in the filopodia and podosomes in macrophage cells13. We assumed that FITC-TP1 identified only the leading edge where the protrusion formed in the EMT-induced MCF-7 cells. To prove the assumption, the scratch wound healing assay was conducted to determine the distribution of Vimentin and PLS3 in the leading edge. During the progress of wound healing, the cells bordering the wound form dynamic actin protrusions and become motile36. MCF-7 cells produced only very slight PLS3 accumulation along the wound edge, while TGF-β1 induced strong stainings of PLS3 and Vimentin in the leading edge (Fig. 5c, d). The enlarged images of single cells in the leading edge in Figure 5d were furthur obtained using high-resolution microscopy (Fig. 5e). As TGF-β1 contributed to enhanced motility and wound healing 37, the result suggested that PLS3 assembled in the leading edge of motile cells. In addition, FITC-TP1 could also bind in the leading edge of the TGF-β1-induced motile cells bordering the scratch (Fig. 5f, g). Moreover, the enlarged images shown in Figure 5h exhibited the ability of peptide probe in detecting the single cells of the leading edge. Taken together, PLS3 has a role in showing the leading edge of motile cells during wound healing and FITC-TP1 migh be a useful tool for identifying living motile cells with EMT-induction.

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Figure 5. FITC-TP1 as probes for labelling EMT-induced cells in the leading edge. Triplestainings with antibodies of PLS3, Vimentin and E-cadherin in MCF-7 cells at low density untreated (a) or treated (b) with TGF-β1 (5 ng/mL) for 24 h . Distributions of PLS3, Vimentin and E-cadherin in MCF-7 cells bordering the wound edge untreated (c) or treated (d) with TGF-β1. 5 × enlarged views of the boxed area from (d) are shown in (e). Blue: nuclei; green: PLS3; orange: Vimentin; red: E-cadherin. Confocal images of FITC-TP1 binding to MCF-7 cells bordering the 13



wound edge untreated (f) or treated (g) with TGF-β1. 5 × enlarged views of the boxed area from (g) are shown in (h). Blue: nuclei; green: FITC-TP1. Scale bar = 20 μm.

FITC-TP1 as probes for cancer cells imaging in spike-in assay After revealing the probe role of FITC-TP1 during EMT, we tested whether FITCTP1 was appropriate for labelling CTCs. To evaluate the specificity of TP1 towards PLS3 in the blood samples, the MDA-MB-231 cells were spiked in the human whole blood cells to mimic CTCs samples. Using conventional magnetic enrichment method, EpCAM-positive MDA-MB-231 cells labelled with anti-EpCAM beads were isolated from the spiked samples under a magnetic field38, 39. Identify with the previous report, MDA-MB-231 cells represented CD45-negative and CD45-positive WBCs represented PLS3-negative using IF assay (Fig. S8). As shown in Figure 6a-h, FITC-TP1 bound on the surface of MDA-MB-231 cells but failed to label WBCs. The schematic diagram illustrating the probe function of TP1 for cancer cells imaging was shown in Figure 6i. The results indicated that FITC-TP1 could preferentially probe PLS3-expressed cancer cells in the blood samples and FITC-TP1 might act as a novel probe to label CTCs.

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Figure 6. PLS3 detection in MDA-MB-231 cells spiked in the whole blood cells by FITC-TP1 peptide. (a-d) White blood cells (WBCs) was identified by the CD45(PE-DyLight™ 594) antibody. Nuclei were stained with Hoechst 33342, and the merged image is an overlay of the Hoechst 33342(blue), CD45 (red), and PLS3 (green) images. Scale bar=100 µm. (e-h) Enlarged views of stained cells are shown in MDA-MB-231 cells. (i) Schematic illustration of FITC-TP1 as a probe for labelling PLS3-expressed cancer cells during EMT progress or in spiked blood samples.

Conclusions In summary, a novel peptide TP1 with high affinity towards PLS3 protein was screened out using OBOC strategy based on a lab-on-chip system. The FITC-TP1 bound stably to PLS3-expressed cells during EMT process. In PLS3-negative cells, FITC-TP1 could probe the EMT-induced cells at the periphery. Using wound healing assay, we found PLS3 assembled in the leading edge of motile cells with EMT induction, and that FITC-TP1 identified the leading edge of motile cells with EMTinduction as a probe. FITC-TP1 could also recognize PLS3-expressed cancer cells in the peripheral blood, it has potential to become a diagnostic tool for PLS3-expressed 15



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CTCs. We expected that the FITC-TP1 could be used in clinical for identifying the PLS3-epressed CTCs associated with the prognosis for patients with breast cancer. Acknowledgements This work was supported by the National Natural Science Foundation of China under Grant numbers 21775031 and 31870992; Beijing Municipal Natural Science Foundation under Grant numbers L172035 and 2172056; Key Research Program of the Chinese Academy of Sciences under Grant number KFZD-SW-210.

Conflicts of interest There are no conflicts to declare.

Supporting Information. The Supporting Information is available free of charge on the ACS Publications website. Extended materials and methods, synthesis process of the OBOC peptide library towards PLS3, the structural formula and identification of TP1, SPRi detection of the binding capability of anti-PLS3 towards PLS3 protein, confocal images of PLS3 in MDA-MB-231 and MCF-7 cells, distributions of peptide concentrations in the fresh or conditioned medium at different time, TGF-β1 induced EMT in MDA-MB-231 cells, TP1 as probes for identifying the edge of cells at high density and double immunostaining of PLS3 and CD45 in WBCs and MDA-MB-231 cells supplied as Supporting Information.

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