{"paper_id":"6405c1b7-c316-4ed4-a270-69e5d3178b15","body_text":"Real-Time Cell Analysis Reveals Distinct Roles of S100A4 in Regulating\nProliferation, Migration, and Invasion of JAR Choriocarcinoma Cells\nBin Yu1,2#, Hai-Gang Ding3#, Feng Zhang1,2, Hong-Mei Lin1,2, Gui-Yu Xia1,2, Ye-Jun\nJiang1,2, Jian Zhao1,2, Guo-Ping Li1,2, Jin-Long Ding1,2, Na Ding1,2, Xin-Yue Zhang1,2,\nHai-Tao Pan1,2, Ping Ying1,2\n ，Yao He1,2\n1Shaoxing Maternity and Child Health Care Hospital, Shaoxing, China.\n2Maternity and Child Health Care Affiliated Hospital, Shaoxing University\n3Shaoxing People's Hospital (Shaoxing Hospital of Zhejiang University), Shaoxing,\nChina.\nCorresponding Author:\nPing Ying\nShaoxing Maternity and Child Health Care Hospital, Shaoxing, China.\nE-mail address: yingping923@163.com\nOr to Yao He\nShaoxing Maternity and Child Health Care Hospital, Shaoxing, China.\nE-mail address: heyao27@126.com\n.CC-BY 4.0 International licenseperpetuity. It is made available under a \npreprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in \nThe copyright holder for thisthis version posted February 28, 2026. ; https://doi.org/10.64898/2026.02.26.708094doi: bioRxiv preprint \n\nFunding sources\nThis research was supported by Zhejiang Provincial Natural Science Foundation of\nChina under Grant No.LTGY23H040005 ， No.LTGY23H040004,\nNo.LTGY24H040005， No.LTGY24H040006, No.LKLY26H040001 and the Health\nCommission of Zhejiang Province, China (WKJ-ZJ-2449, 2023KY368, 2025HY1308,\n2025A14016, 2024A14004 ） ， the Science Technology Department of Shaoxing,\nChina (2022A14006, 2025A14003, 2025A14006),the Health Commission of\nShaoxing, China (2024SKY003, 2022KY036, 2022KY038, 2023SKY047).Key\nLaboratory of Reproductive Health of Shaoxing City, Shaoxing Maternity and Child\nHealth Care Hospital, Shaoxing, Zhejiang 312000, China(2023SSY004,\n2023SSY007)\nAuthors' Contribution\n#Bin Yu and Hai-Gang Ding contributed equally to this work as co-first authors. Bin\nYu, Hai-Gang Ding, Hai-Tao Pan, Yao He, and Ping Ying conceived and designed\nthe study. Bin Yu, Hai-Gang Ding, Feng Zhang, Na Ding, Xin-Yue Zhang, and\nHai-Tao Pan performed the experiments, including cell culture, siRNA transfection,\nReal-Time Cell Analysis (RTCA) for proliferation, migration, and invasion assays,\nWestern blotting, and RT-qPCR analysis. Hong-Mei Lin, Gui-Yu Xia, Ye-Jun Jiang,\nJian Zhao, Guo-Ping Li, Jin-Long Ding analyzed and interpreted the data. Bin Yu,\nHai-Gang Ding, Yao He, and Ping Ying drafted the manuscript. All authors reviewed,\nedited, and approved the final version of the manuscript. Ping Ying and Yao He\nsupervised the entire research project and are the corresponding authors.\n.CC-BY 4.0 International licenseperpetuity. It is made available under a \npreprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in \nThe copyright holder for thisthis version posted February 28, 2026. ; https://doi.org/10.64898/2026.02.26.708094doi: bioRxiv preprint \n\nAbstract\nS100A4, a metastasis promoting calcium binding protein, drives tumor progression\nthrough pleiotropic mechanisms, yet its context dependent functions in gestational\nmalignancies remain elusive. To dynamically decode its role in choriocarcinoma\npathogenesis, we leveraged label free real time cell analysis (RTCA) to profile\nmalignant phenotypes in JAR cells following siRNA mediated S100A4\nsilencing, complemented by apoptosis assessment and targeted signaling profiling.\nEfficient knockdown (verified by qPCR/Western blotting) significantly attenuated\ncellular proliferation (96 hr cell index slope decreased vs. scramble control; p<0.01)\nand suppressed migration capacity (p<0.01). Critically, S100A4 depletion did not\ninduce apoptosis (flow cytometry and cleaved caspase 3/9 blotting confirmed no\nsignificant change), and invasion through Matrigel coated membranes remained\nstatistically unaltered despite comparable experimental rigor. Mechanistically,\nS100A4 silencing triggered adaptive signaling rewiring: IRS1 and PI3K expression\nwere elevated, Akt1 was suppressed, while MEK1/2 remained unchanged suggesting\ncompensatory pathway activation.\nKeywords：\nS100A4；JAR cells； Cell Proliferation； Cell Migration； Real-Time Cell Analysis\n(RTCA)\n.CC-BY 4.0 International licenseperpetuity. It is made available under a \npreprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in \nThe copyright holder for thisthis version posted February 28, 2026. ; https://doi.org/10.64898/2026.02.26.708094doi: bioRxiv preprint \n\n1. Background\nChoriocarcinoma represents one of the most clinically aggressive gestational\nmalignancies, distinguished by its propensity for early vascular invasion and distant\nmetastasis. Despite advances in chemotherapy, patients with refractory or recurrent\ndisease face mortality rates exceeding 30%1, underscoring an urgent need to dissect\nthe molecular mechanisms governing its disseminative traits. Critical among potential\ndrivers is the metastasis-associated calcium-binding protein S100A42, S100A4 is\nupregulated in various cancers and significantly promotes cancer cell proliferation,\nmigration, invasion, and EMT through the regulation of multiple signaling pathways\nand molecular mechanisms3–5, but remain underexplored in trophoblastic contexts. Its\ndysregulation in placental pathologies6 and association with poor prognosis in\nepithelial tumors7 suggest a plausible, yet unverified, function in choriocarcinoma\nprogression.\nCurrent understanding of S100A4’s oncogenicity, however, relies heavily on endpoint\nassays that capture static snapshots of cell behavior—such as fixed-timepoint\nTranswell invasion or colony formation8—while overlooking dynamic phenotypic\nadaptations. These methods cannot resolve kinetic nuances in cellular responses,\npotentially masking context-dependent functional hierarchies. Moreover, the\npredominance of correlative evidence from tissue studies fails to establish causal\nlinks9 between S100A4 expression and specific malignant behaviors. This knowledge\ngap is particularly consequential for choriocarcinoma, where the unique biology of\n.CC-BY 4.0 International licenseperpetuity. It is made available under a \npreprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in \nThe copyright holder for thisthis version posted February 28, 2026. ; https://doi.org/10.64898/2026.02.26.708094doi: bioRxiv preprint \n\ntrophoblastic cells may engender distinct regulatory dependencies compared to\ncarcinomas of epithelial origin10.\nTo address this limitation, we leveraged Real-Time Cell Analysis (RTCA)11, an\nimpedance-based platform enabling continuous, label-free monitoring of cellular\nfunctions. Unlike conventional approaches, RTCA quantifies phenotypic\ndynamics—from proliferation kinetics to migration velocity and barrier\npenetration—at minute-scale resolution under physiologically relevant conditions12.\nWhile S100A4's role in proliferation and migration is established, its impact on\napoptosis and downstream signaling in trophoblastic malignancies remains ambiguous.\nTo comprehensively dissect S100A4's oncogenic mechanisms, we integrated\napoptosis assessment (via flow cytometry and caspase-3/9 blotting) and targeted\nsignaling profiling (IRS1/PI3K/Akt/MEK axes) alongside RTCA phenotyping. This\nmulti-layered approach addresses whether S100A4 silencing triggers compensatory\npathway activation to sustain invasion despite proliferative/migratory suppression. By\nintegrating siRNA-mediated S100A4 silencing with multi-parametric RTCA profiling\nin human choriocarcinoma JAR cells, this study aims to: (1) delineate the real-time\ncontribution of S100A4 to proliferation, migration, and invasion; (2) test the\nhypothesis that S100A4 exhibits phenotype-selective regulation13 rather than uniform\ncontrol across malignant hallmarks. Our approach not only circumvents the artifacts\nof endpoint fixation but also provides unprecedented temporal resolution for\nfunctional stratification in trophoblastic malignancies.\n.CC-BY 4.0 International licenseperpetuity. It is made available under a \npreprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in \nThe copyright holder for thisthis version posted February 28, 2026. ; https://doi.org/10.64898/2026.02.26.708094doi: bioRxiv preprint \n\n2. Materials & Methods\n2.1. Cells and cell culture\nJAR cells were maintained in Roswell Park Memorial Institute (RPMI)-1640 medium\n(Gibco, Cat. no. 21875-042) supplemented with 10% fetal bovine serum (Gibco, Cat.\nno. 10099141), under humidified conditions at 37°C with 5% CO₂. When the cells\nreached 80%–90% confluence, they were digested with 0.25% trypsin-EDTA (Gibco,\nCat. no. 25200056) and passaged at a split ratio of 1:2 to maintain them in the\nlogarithmic growth phase for subsequent experiments.\n2.2. Small interference RNA (siRNA) treatment\nCells were seeded into six-well plates in complete medium for 24 h prior to\ntransfection. Transfection was carried out with Lipofectamine 6000 reagent (Beyotime,\nCat. no. C0526) following the manufacturer’s protocol. Cells were transfected with\n20 μM control siRNA or target siRNA oligos (Ribo, Cat. no. SIGS0007750-1).\nTransfected cells were cultured in complete medium at 37°C for 48 h.\n2.3. Real-time cell analysis (RTCA)\nRTCA is a novel label-free analytical technique capable of non-invasively monitoring\ncellular behaviors, including proliferation, migration, invasion, etc. In this study, we\nused the xCELLigence DP RTCA instrument from Agilent Technologies to perform\nrelevant experiment.\n.CC-BY 4.0 International licenseperpetuity. It is made available under a \npreprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in \nThe copyright holder for thisthis version posted February 28, 2026. ; https://doi.org/10.64898/2026.02.26.708094doi: bioRxiv preprint \n\n2.3.1 Proliferation assay\nCells were digested with trypsin for 2 min after transfection, resuspended in complete\nmedium, counted, and adjusted to a concentration of 6×10⁴ cells/mL. 50 μL medium\nwas added to each well of the E-16 culture plate (Agilent, Cat. no.300601010) to\nacquire background signals. After 30 minutes of incubation in a cell culture incubator,\nbaseline calibration of the plate was performed using the RTCA software.\nSubsequently, 100 μL of the cell suspension was added to each well to seed the cells,\nand cell proliferation was monitored until the experiment concluded.\n2.3.2 Migration assay\nCell migration was evaluated by RTCA method on a CIM plate (Agilent, Cat.\nno.05665817001). Briefly, 165 μL of complete medium was added to the lower\nchamber. After assembling the upper and lower chambers, 30 μL of serum-free\nmedium was added to the upper chamber. The plate was equilibrated at 37°C with 5%\nCO₂ for 1 hour, followed by baseline measurement. Then, 100 μL of serum-free cell\nsuspension containing 40,000 cells was added to the upper chamber per well. After\nallowing the plate to settle at room temperature for 30 minutes, real-time impedance\nmeasurements were performed.\n2.3.3 Invasion assay\nTo ensure uniform coating of Matrigel (Corning, Cat no. 354234) in CIM plates,\ncentrifuge tubes and the upper chamber of the CIM-Plate were pre-cooled at 4 ° C\n.CC-BY 4.0 International licenseperpetuity. It is made available under a \npreprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in \nThe copyright holder for thisthis version posted February 28, 2026. ; https://doi.org/10.64898/2026.02.26.708094doi: bioRxiv preprint \n\novernight. Matrigel was thawed at 4 ° C overnight. Matrigel was diluted with\nserum-free medium at a ratio of 1:60 on ice. To each well of the upper chamber, 50 μL\nof the diluted Matrigel was added, followed by removal of 30 μL. The plate was then\nincubated at 37 ° C for 4 hours until Matrigel solidified. The remaining procedures\nwere identical to those of the cell migration assay.\n2.4. Western blot analysis\nAccording to the kit instructions, total protein was extracted from cells with RIPA\nbuffer (Beyotime, cat no. P0013C), and its concentration was determined by the BCA\nassay (Beyotime, cat no. P0012S). Equal amounts of protein were separated by 10%\nSDS- PAGE (Sagon, cat. no. C651101), transferred onto a PVDF membrane\n(Beyotime, cat. no. FFP24), blocked with TBST containing 5% skim milk at room\ntemperature for 1 hour (Beyotime, cat. no. P0233), and subsequently incubated\novernight at 4°C with primary antibodies against Caspase-3 (1:1000; Beyotime, cat\nno. AF1213), Caspase-9 (1:1000; Beyotime, cat no. AF1264), Tublin (1:1000;\nBeyotime, cat no. A T819),IRS1 (1:500; Beyotime, cat no. AF7299), PI3-K (1:1000;\nBeyotime, cat no. AF1966), Akt1 (1:1000; Beyotime, cat no. AF0045), MEK1/2\n(1:1000; Beyotime, cat no. AF1057), GAPDH (1:1000; Beyotime, cat no. AF5009),\nS100A4(1:1000; Beyotime, cat no. AF5291), and β-Actin (1:1000; Beyotime, cat no.\nAF2811). After three washes with TBST, the membrane was incubated with\nhorseradish peroxidase (HRP)-conjugated secondary antibody at room temperature for\n1 hour: HRP-labeled Goat Anti-Mouse IgG (1:1000; Beyotime, cat no. A0216) and\n.CC-BY 4.0 International licenseperpetuity. It is made available under a \npreprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in \nThe copyright holder for thisthis version posted February 28, 2026. ; https://doi.org/10.64898/2026.02.26.708094doi: bioRxiv preprint \n\nHRP-labeled Goat Anti-Rabbit IgG (1:1000; Beyotime, cat no. A0208). Protein bands\nwere visualized using an ECL reagent (Beyotime, cat. no. P0018AS), and band\nintensities were normalized to an internal reference protein for standardization.\n2.5. RT-qPCR analysis\nTotal RNA was isolated with TRIzol (Tiangen, cat. no. DP419), then\nreverse-transcribed into cDNA employing the PrimeScript RT Kit (Tiangen, cat. no.\nKR116-01) and miRNA-specific stem-loop primers targeting specific miRNAs to\nenhance specificity. RT-qPCR assays were conducted using SYBR Premix Ex Taq\n(Tiangen, cat. no. RK145) and the corresponding primers, with normalization to U6\nsmall nuclear RNA. Relative gene expression levels were determined by the 2−ΔΔ\nCt method.\n2.6. Flow Cytometry Analysis\nJAR cells were measured for apoptosis levels using the Annexin V-FITC/PI Apoptosis\nDetection Kit (Yeasen, cat. no. 40302). Two days after transfection of HTR8 cells, the\ncells were collected by treatment with EDTA-free trypsin(Sagon Biotech, cat. no.\nE607003), washed with Pre-cooled PBS and treated according to the kit's instructions.\nBriefly, 1-5×105 cells were suspended in 100 μL of binding buffer and put in a\ncentrifuge tube, 5 μL Annexin V-FITC and 10 μL PI Staining Solution was added, and\nthen incubated in the dark for 10 minutes. Subsequently, 400 μL of binding buffer was\nadded to the tubes and assayed by flow cytometry. Data were processed using FlowJo\n.CC-BY 4.0 International licenseperpetuity. It is made available under a \npreprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in \nThe copyright holder for thisthis version posted February 28, 2026. ; https://doi.org/10.64898/2026.02.26.708094doi: bioRxiv preprint \n\nv10.6.2 software (Treestar, Ashland, OR, USA).\n.CC-BY 4.0 International licenseperpetuity. It is made available under a \npreprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in \nThe copyright holder for thisthis version posted February 28, 2026. ; https://doi.org/10.64898/2026.02.26.708094doi: bioRxiv preprint \n\n3. Result\nFirst, we aimed to downregulate the mRNA expression of S100A4 in JAR cells using\nsiRNA technology. To this end, JAR cells were transfected with S100A4-specific\nsiRNA sequences and indicated as siS100A4/JAR cells, whereas JAR cells transfected\nwith a scrambled siRNA sequence (serving as a negative control) were appointed as\nCtrl/JAR cells. RT-qPCR analysis confirmed a significant reduction in S100A4\nmRNA expression in the transfected JAR cells. Western blotting was subsequently\nperformed to evaluate the protein expression of S100A4 in these cells (Fig. 1a).\nFollowing siRNA-mediated silencing, the expression of S100A4 protein was\nmarkedly decreased (Fig. 1b).\nFig. 1. S100A4 expression is decreased in siS100A4 -transfected JAR cells. (a) RT-qPCR analysis\nof S100A4 expression in siCtrl and siS100A4/ JAR cells. β-actin was used for normalization (b)\nWestern blot analysis of S100A4 expression in siCtrl and siS100A4/ JAR cells. β-actin is used as a\nloading control.\n.CC-BY 4.0 International licenseperpetuity. It is made available under a \npreprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in \nThe copyright holder for thisthis version posted February 28, 2026. ; https://doi.org/10.64898/2026.02.26.708094doi: bioRxiv preprint \n\nReal-time cell analysis (RTCA) is a novel technique that employs real-time cellular\nmonitoring to detect the proliferation, migration, and invasion of cells during cell\nculture. It enables uninterrupted, label-free, and real-time analysis of cells throughout\nthe experimental process. Currently, RTCA is widely applied by researchers\nworldwide across many diverse research fields. We evaluated the proliferation\ndynamics of S100A4-knockdown JAR cells using xCELLigence RTCA. Cell index\nprofiles showed that S100A4-siRNA-transfected JAR cells exhibited decreased\nproliferation capacities (Fig. 2).\nFig. 2. Knockdown of S100A4 inhibited cell proliferation in JAR cell line. Real time cell analysis\nshowing that knockdown of S100A4 inhibited cell proliferation of JAR cells.\nCell migration, a fundamental property of cells, occurs in numerous physiological and\npathological processes, wherein migrating cells play a critical role in tissue\nmorphogenesis during development, post-developmental tissue repair, and the support\nof tumor invasion and metastasis14. Similarly, to evaluate the effect of S100A4\n.CC-BY 4.0 International licenseperpetuity. It is made available under a \npreprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in \nThe copyright holder for thisthis version posted February 28, 2026. ; https://doi.org/10.64898/2026.02.26.708094doi: bioRxiv preprint \n\nknockdown on the migration and invasion capacities of JAR cells, we transfected JAR\ncells with siRNA-S100A4. Following transfection, both siS100A4-transfected JAR\ncells and control cells (scrambled siRNA-transfected) were seeded into CIM-Plates.\nThe migration and invasion dynamics of these cells were then monitored in real-time\nusing the RTCA system. The results showed that compared with the control group,\nknockdown of S100A4 reduced the migration ability of JAR cells (Fig.3), but did not\nreduce their invasion ability (Experimental results of the liposome reagent control\ngroup and the knockdown of S100A4 experimental group were similar, Fig.4).\nFig. 3 Real time cell analysis showing that knockdown of S100A4 inhibited cell migration of JAR\ncells\n.CC-BY 4.0 International licenseperpetuity. It is made available under a \npreprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in \nThe copyright holder for thisthis version posted February 28, 2026. ; https://doi.org/10.64898/2026.02.26.708094doi: bioRxiv preprint \n\nFig. 4 Real time cell analysis showing that knockdown of S100A4 did not reduce their invasion\nability in JAR cells.\nAnd then ， the effect of S100A4 knockdown on apoptosis in JAR cells was\ninvestigated by flow cytometry and western blot, as shown in Figure 5. Both western\nblot (Fig. 5a) and flow cytometry(Fig. 5b) results demonstrated that S100A4\nknockdown had no significant effect on apoptosis in JAR cells.\nFig. 5 The apoptosis status of JAR cells following S100A4 knockdown. (a) Western blot analysis\nof apoptosis markers (caspase-3, caspase-9); (b) Flow cytometry analysis of Annexin V/PI\nstaining. Both assays showing no significant effect of S100A4 knockdown on apoptosis.\n.CC-BY 4.0 International licenseperpetuity. It is made available under a \npreprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in \nThe copyright holder for thisthis version posted February 28, 2026. ; https://doi.org/10.64898/2026.02.26.708094doi: bioRxiv preprint \n\nStudies have demonstrated that S100A4 silencing promotes corneal burn wound\nhealing through inhibition of the PI3K/Akt/mTOR pathway15. Additionally, S100A4\nhas been shown to be regulated by the PI3K/Akt signaling pathway in cancer16. The\npotential involvement of S100A4 in the altered biological behaviors of JAR cells\nmight be associated with the PI3K/AKT signaling pathway. Therefore, we performed\nWestern blot analysis to assess the expression status of key proteins in this pathway.\nResults showed that after knocking down the expression of S100A4 in JAR cells,\nIRS1 and PI3K protein expression was upregulated, whereas Akt1 expression was\ndownregulated (Fig 6).\nFig. 6 The expression of key proteins in the PI3K/AKT signaling pathway following S100A4\nknockdown in JAR cells.\n.CC-BY 4.0 International licenseperpetuity. It is made available under a \npreprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in \nThe copyright holder for thisthis version posted February 28, 2026. ; https://doi.org/10.64898/2026.02.26.708094doi: bioRxiv preprint \n\n4. Discussion\nThis study suggests that S100A4 may exert phenotype-selective regulatory effects in\nchoriocarcinoma. siRNA-mediated S100A4 knockdown significantly suppressed\ncellular proliferation and migration , but exhibited minimal impact on Matrigel\ninvasion. Notably, both the lipofectamine control group and S100A4-knockdown\ngroup showed comparable invasion capacity, yet both were lower than the untreated\ncell control—indicating potential non-specific suppression from transfection reagents.\nThis relative stability of the invasive phenotype contrasts with the canonical paradigm\nof S100A4 as a universal metastasis coordinator in epithelial cancers17. Notably,\nsiRNA-mediated S100A4 knockdown did not induce significant apoptosis in JAR\ncells, as evidenced by concordant flow cytometry (Annexin V/PI staining) and\nWestern blot data (unchanged cleaved caspase-3/9 levels).\nThe invasion resilience may relate to trophoblast-specific developmental programs.\nPhysiological extravillous trophoblast invasion relies on mechanical deformation\nthrough vascular endothelia rather than protease-dependent matrix degradation18. The\npreserved transmigration capacity of knockdown cells through 8- μ m pores\n(simulating endothelial fenestrations) supports the existence of S100A4-bypass\nmechanisms in choriocarcinoma.Notably, S100A4, as a calcium-binding protein,\ndirectly interacts with non-myosin IIA (NMIIA) to regulate cellular motility and\ninvasiveness19. However, the loss of S100A4 may be compensated through other\nmechanisms, such as maintaining cytoskeletal remodeling and cellular migratory\n.CC-BY 4.0 International licenseperpetuity. It is made available under a \npreprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in \nThe copyright holder for thisthis version posted February 28, 2026. ; https://doi.org/10.64898/2026.02.26.708094doi: bioRxiv preprint \n\ncapacity via the high expression of MYH9 and ITGB1. This compensatory\nmechanism may involve cytoskeletal reorganization and activation of signaling\npathways, thereby sustaining normal cellular functions and adaptive responses.\nTherapeutically, while targeting S100A4 effectively controls proliferation and\nmigration phenotypes, its limited efficacy against invasion necessitates combinatorial\napproaches. In the context of choriocarcinoma, the inhibition of mechanotransduction\npathways has shown promise in overcoming drug resistance and enhancing\ntherapeutic efficacy. For example, the inhibition of brain-derived neurotrophic\nfactor/tyrosine kinase B signaling suppresses choriocarcinoma cell growth, indicating\nthe potential of targeting signaling pathways in this cancer type20. Additionally,\nmetformin has been shown to regulate autophagy via LGMN to inhibit\nchoriocarcinoma, further supporting the role of metabolic and mechanotransduction\npathways in cancer therapy21. Mechanistically, S100A4 silencing triggered\nparadoxical signaling adaptations: IRS1 and PI3K expression increased, while Akt1\ndecreased and MEK1/2 remained unchanged (Fig. 6).\nLimitations include: (1) Molecular evidence for compensatory pathways needs in vivo\nvalidation; (2) Clinical correlation between S100A4 expression and invasive\nphenotypes remains unestablished. Collectively, our data reveal a phenotype-signaling\nhierarchy: S100A4 silencing primarily disrupts proliferative/migratory programs via\nAkt1 suppression, while compensatory IRS1/PI3K elevation and MEK pathway\n.CC-BY 4.0 International licenseperpetuity. It is made available under a \npreprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in \nThe copyright holder for thisthis version posted February 28, 2026. ; https://doi.org/10.64898/2026.02.26.708094doi: bioRxiv preprint \n\nstability preserve invasion. This functional decoupling underscores the need for\ncombinatorial targeting (e.g., S100A4 inhibition + PI3K/mTOR blockade) to fully\nsuppress choriocarcinoma dissemination.\n.CC-BY 4.0 International licenseperpetuity. It is made available under a \npreprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in \nThe copyright holder for thisthis version posted February 28, 2026. ; https://doi.org/10.64898/2026.02.26.708094doi: bioRxiv preprint \n\nReferences\n1. Barber, E. L., Schink, J. C. & Lurain, J. R. Hepatic metastasis in gestational\ntrophoblastic neoplasia: patient characteristics, prognostic factors, and outcomes.\nJ Reprod Med 59, 199–203 (2014).\n2. Boye, K. & Maelandsmo, G. M. S100A4 and metastasis: a small actor playing\nmany roles. Am J Pathol 176, 528–535 (2010).\n3. Zuo, Z. et al. Interplay between Trx-1 and S100P promotes colorectal cancer cell\nepithelial-mesenchymal transition by up-regulating S100A4 through AKT\nactivation. J Cell Mol Med 22, 2430–2441 (2018).\n4. Li, F. et al. S100A4-MYH9 Axis Promote Migration and Invasion of Gastric\nCancer Cells by Inducing TGF-β-Mediated Epithelial-Mesenchymal Transition. 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Gene 853, 147090 (2023).\n.CC-BY 4.0 International licenseperpetuity. It is made available under a \npreprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in \nThe copyright holder for thisthis version posted February 28, 2026. ; https://doi.org/10.64898/2026.02.26.708094doi: bioRxiv preprint","source_license":"CC-BY-4.0","license_restricted":false}