Transforming tumoroids derived from ALK-positive pulmonary adenocarcinoma to squamous cell carcinoma in vivo.

Approximately 3-5% of non-small cell lung cancers (NSCLC) harbor ALK fusion genes and may be responsive to anaplastic lymphoma kinase (ALK) tyrosine kinase inhibitors. There are only a few reports on cell lines with EML4-ALK variant 3 (v3) and tumoroids that can be subject to long-term culture (> 3 months). In this study, we established tumoroids (PDT-LUAD#119) from a patient with lung cancer harboring EML4-ALK that could be cultured for 12 months. Whole-exome sequencing and RNA sequencing analyses revealed TP53 mutations and an EML4-ALK v3 mutation. PDT-LUAD#119 lung tumoroids were sensitive to the ALK tyrosine kinase inhibitors (ALK TKIs) crizotinib, alectinib, entrectinib, and lorlatinib, similar to NCI-H3122 cells harboring EML4-ALK variant 1 (v1). Unexpectedly, clear squamous cell carcinoma and solid adenocarcinoma were observed in xenografts from PDT-LUAD#119 lung tumoroids, indicating adenosquamous carcinoma. Immunostaining revealed that the squamous cell carcinoma was ALK positive, suggesting a squamous transformation of the adenocarcinoma. Besides providing a novel cancer model to support basic research on ALK-positive lung cancer, PDT-LUAD#119 lung tumoroids will help elucidate the pathogenesis of adenosquamous carcinoma.

Establishment and characterization of novel high mucus-producing lung tumoroids derived from a patient with pulmonary solid adenocarcinoma.

Among mucus-producing lung cancers, invasive mucinous adenocarcinoma of the lung is a rare and unique subtype of pulmonary adenocarcinoma. Notably, mucus production may also be observed in the five subtypes of adenocarcinoma grouped under the higher-level diagnosis of Invasive Non-mucinous Adenocarcinomas (NMA). Overlapping pathologic features in mucus-producing tumors can cause diagnostic confusion with significant clinical consequences. In this study, we established lung tumoroids, PDT-LUAD#99, from a patient with NMA and mucus production. The tumoroids were derived from the malignant pleural effusion of a patient with lung cancer and have been successfully developed for long-term culture (> 11 months). Karyotyping by fluorescence in situ hybridization using an alpha-satellite probe showed that tumoroids harbored aneuploid karyotypes. Subcutaneous inoculation of PDT-LUAD#99 lung tumoroids into immunodeficient mice resulted in tumor formation, suggesting that the tumoroids were derived from cancer. Xenografts from PDT-LUAD#99 lung tumoroids reproduced the solid adenocarcinoma with mucin production that was observed in the patient's metastatic lymph nodes. Immunoblot analysis showed MUC5AC secretion into the culture supernatant of PDT-LUAD#99 lung tumoroids, which in contradistinction was barely detected in the culture supernatants of NCI-A549 and NCI-H2122 pulmonary adenocarcinoma cells known for their mucin-producing abilities. Here, we established a novel high-mucus-producing lung tumoroids from a solid adenocarcinoma. This preclinical model may be useful for elucidating the pathogenesis of mucus-producing lung cancer.

Patient-derived tumoroid models of pulmonary large-cell neuroendocrine carcinoma: a promising tool for personalized medicine and developing novel therapeutic strategies.

Pulmonary large-cell neuroendocrine carcinoma (LCNEC), a disease with poor prognosis, is classified as pulmonary high-grade neuroendocrine carcinoma, along with small-cell lung cancer. However, given its infrequent occurrence, only a limited number of preclinical models have been established. Here, we established three LCNEC tumoroids for long-term culture. Whole-exome sequencing revealed that these tumoroids inherited genetic mutations from their parental tumors; two were classified as small-cell carcinoma (S-LCNEC) and one as non-small cell carcinoma (N-LCNEC). Xenografts from these tumoroids in immunodeficient mice mimicked the pathology of the parent LCNEC, and one reproduced the mixed-tissue types of combined LCNEC with a component of adenocarcinoma. Drug sensitivity tests using these LCNEC tumoroids enabled the evaluation of therapeutic agent efficacy. Based on translational research, we found that a CDK4/6 inhibitor might be effective for N-LCNEC and that Aurora A kinase inhibitors might be suitable for S-LCNEC or LCNEC with MYC amplification. These results highlight the value of preclinical tumoroid models in understanding the pathogenesis of rare cancers and developing treatments. LCNEC showed a high success rate in tumoroid establishment, indicating its potential application in personalized medicine.

A surgical case of pulmonary adenocarcinoma in the right upper lobe associated with a systemic artery-to-pulmonary artery fistula.

A 52-year-old female never-smoker with an abnormal shadow in the right lung detected on radiography was referred to our institution. Contrast-enhanced computed tomography revealed an irregular nodule in the upper lobe of the right lung, suggestive of a pulmonary vascular abnormality. Angiography revealed a direct communication between the right internal mammary artery (IMA) and the right upper lobe pulmonary artery branches, with dilated and tortuous vascular proliferation. As multiple branch arteries were seen flowing into the upper lobe from the IMA, transcatheter selective embolization of these vessels and right upper lobectomy by video-assisted thoracoscopic surgery were performed. Contrary to the clinical diagnosis, the pathological finding was a pulmonary adenocarcinoma of the right upper lobe. Additional lymph node dissection was performed later. We report an extremely rare and unprecedented case of pulmonary adenocarcinoma fed by the right IMA, with a literature review.

FOXA2 Cooperates with Mutant KRAS to Drive Invasive Mucinous Adenocarcinoma of the Lung.

The endoderm-lineage transcription factor FOXA2 has been shown to inhibit lung tumorigenesis in in vitro and xenograft studies using lung cancer cell lines. However, FOXA2 expression in primary lung tumors does not correlate with an improved patient survival rate, and the functional role of FOXA2 in primary lung tumors remains elusive. To understand the role of FOXA2 in primary lung tumors in vivo, here, we conditionally induced the expression of FOXA2 along with either of the two major lung cancer oncogenes, EGFRL858R or KRASG12D, in the lung epithelium of transgenic mice. Notably, FOXA2 suppressed autochthonous lung tumor development driven by EGFRL858R, whereas FOXA2 promoted tumor growth driven by KRASG12D. Importantly, FOXA2 expression along with KRASG12D produced invasive mucinous adenocarcinoma (IMA) of the lung, a fatal mucus-producing lung cancer comprising approximately 5% of human lung cancer cases. In the mouse model in vivo and human lung cancer cells in vitro, FOXA2 activated a gene regulatory network involved in the key mucous transcription factor SPDEF and upregulated MUC5AC, whose expression is critical for inducing IMA. Coexpression of FOXA2 with mutant KRAS synergistically induced MUC5AC expression compared with that induced by FOXA2 alone. ChIP-seq combined with CRISPR interference indicated that FOXA2 bound directly to the enhancer region of MUC5AC and induced the H3K27ac enhancer mark. Furthermore, FOXA2 was found to be highly expressed in primary tumors of human IMA. Collectively, this study reveals that FOXA2 is not only a biomarker but also a driver for IMA in the presence of a KRAS mutation. SIGNIFICANCE: FOXA2 expression combined with mutant KRAS drives invasive mucinous adenocarcinoma of the lung by synergistically promoting a mucous transcriptional program, suggesting strategies for targeting this lung cancer type that lacks effective therapies.

The complexity of EGFR exon 19 deletion and L858R mutant cells as assessed by proteomics, transcriptomics, and metabolomics.

Most lung adenocarcinoma-associated EGFR tyrosine kinase mutations are either an exon 19 deletion (19Del) or L858R point mutation in exon 21. Although patients whose tumors contain either of these mutations exhibit increased sensitivity to tyrosine kinase inhibitors, progression-free and overall survival appear to be longer in patients with 19Del than in those with L858R. In mutant-transfected Ba/F3 cells, 19Del and L858R were compared by multi-omics analyses including proteomics, transcriptomics, and metabolomics. Proteome analysis identified increased plastin-2, TKT, PDIA5, and ENO1 expression in L858R cells, and increased EEF1G expression in 19Del cells. RNA sequencing showed significant differences between 19Del and L858R cells in 112 genes. Metabolome analysis showed that amino acids, adenylate, guanylate, NADPH, lactic acid, pyruvic acid glucose 6-phosphate, and ribose 5-phosphate were significantly different between the two mutant cells. Because GSH was increased with L858R, we combined osimertinib with the GSH inhibitor buthionine sulfoximine in L858R cells and observed synergistic effects. The complexity of EGFR 19Del and L858R mutant cells was demonstrated by proteomics, transcriptomics, and metabolomics analyses. Therapeutic strategies for lung cancer with different EGFR mutations could be considered because of their different metabolic phenotypes.

Neuroendocrine tumor secondary to pulmonary hypoplasia: A case report.

Pulmonary hypoplasia is diagnosed during the perinatal period and is a cause of death in newborns. However, these developmental abnormalities are diagnosed in adulthood in some cases. A 70-year-old male smoker was diagnosed with stage IIIA pulmonary adenocarcinoma in the right upper lobe with right middle lobe hypoplasia. He subsequently underwent right upper and middle lobectomy with lymph node dissection by video-assisted thoracoscopic surgery. In addition to an invasive adenocarcinoma in the right upper lobe, pathological examination of the hypoplastic lobe revealed neuroendocrine hyperplasia, as well as tumorlets and a typical carcinoid. Eight cases of pulmonary neuroendocrine tumors that developed from pulmonary hypoplasia have been reported to date. Interestingly, all but one case occurred in the right middle lobe. Neuroendocrine cell hyperplasia has been reported to develop in hypoplastic lungs postnatally; therefore, we speculated that the lesion was the origin of these neuroendocrine tumors. Moreover, the pathological findings suggested that atelectasis was involved in the pathogenesis of this rare condition. In adults, when lobar hypoplasia is diagnosed, neuroendocrine tumors should be anticipated.

Targeting ROR1 in combination with osimertinib in EGFR mutant lung cancer cells.

Lung cancer that exhibits epidermal growth factor receptor (EGFR) gene mutation is sensitive to EGFR-tyrosine kinase inhibitors (TKIs), such as osimertinib. Receptor tyrosine kinase-like orphan receptor 1 (ROR1) may be involved in overcoming EGFR-TKI resistance. Growth inhibition, colony formation, apoptosis, and mRNA/protein levels in four osimertinib-sensitive and resistant cell lines transfected with small interfering RNA (siRNA) targeting ROR1 (siROR1) were evaluated. Cell growth and colony formation were suppressed and apoptosis was increased in all cell lines treated with siROR1. Although EGFR, AKT, and ERK phosphorylation were not suppressed in all cell lines, TGF-ß2, AXL, CDH2, PARP1, PEG10, and TYMS mRNA expression levels were reduced. The combination of osimertinib with siROR1 was effective for the four cell lines, particularly in the two osimertinib-sensitive lines. In conclusion, targeting ROR1 in combination with osimertinib in EGFR mutant lung cancer may be a novel therapeutic option.

Clinical application of a lung cancer organoid (tumoroid) culture system.

Despite high expectations for lung tumoroids, they have not been applied in the clinic due to the difficulty of their long-term culture. Here, however, using AO (airway organoid) media developed by the Clevers laboratory, we succeeded in generating 3 lung tumoroid lines for long-term culture (>13 months) from 41 lung cancer cases (primary or metastatic). Use of nutlin-3a was key to selecting lung tumoroids that harbor mutant p53 in order to eliminate normal lung epithelial organoids. Next-generation sequencing (NGS) analysis indicated that each lung tumoroid carried BRAFG469A, TPM3-ROS1 or EGFRL858R/RB1E737*, respectively. Targeted therapies using small molecule drugs (trametinib/erlotinib for BRAFG469A, crizotinib/entrectinib for TPM3-ROS1 and ABT-263/YM-155 for EGFRL858R/RB1E737*) significantly suppressed the growth of each lung tumoroid line. AO media was superior to 3 different media developed by other laboratories. Our experience indicates that long-term lung tumoroid culture is feasible, allowing us to identify NGS-based therapeutic targets and determine the responsiveness to corresponding small molecule drugs.

Bioelectrical impedance analysis for perioperative water management in adult cardiovascular valve disease surgery.

PURPOSE: Bioelectrical impedance analysis (BIA) has been used recently to measure the body water of patients with acute heart failure. We used BIA in this study to better understand, and possibly identify a predictive marker for, perioperative water behavior in cardiac surgery patients. METHODS: We measured body water and studied its behavior in 44 patients undergoing surgery for cardiac valvular disease at our hospital. Measurements included the levels of extracellular water (ECW), intracellular water (ICW), and total body water, the edema index (EI), and the ratio of ECW to total body water. The first measured EI was defined as the "preoperative EI" and the maximum as the "peak EI". RESULTS: A negative correlation was found between the preoperative EI and the preoperative estimated glomerular filtration rate (eGFR) (R = 0.644, p < 0.001). Positive correlations were found between the peak EI and the ICU stay (R = 0.625, p < 0.001), the peak EI and the ventilation time (R = 0.366, p < 0.01), and the preoperative EI and the ICU stay (R = 0.464, p = 0.026). CONCLUSION: The EI is possibly a predictive marker for perioperative water management in cardiac surgery.

Targeting ROR1 in combination with pemetrexed in malignant mesothelioma cells.

OBJECTIVE: Receptor tyrosine kinase-like orphan receptor 1 (ROR1) is overexpressed in a subset of malignant cells. However, it remains unknown whether ROR1 is targetable in malignant mesothelioma (MM). Therefore, in this study, we investigated the effects of ROR1 inhibition in mesothelioma cells. MATERIALS AND METHODS: Growth inhibition, colony formation, apoptosis, and mRNA/protein levels using siRNA-transfected MM cells were evaluated. Cluster analysis using Gene Expression Omnibus repository of transcriptomic information was also performed. RESULTS: Our results indicated that in three (H2052, H2452, and MESO-1) among four MM cell lines, ROR1 inhibition had anti-proliferative and apoptotic effects and suppressed the activation of AKT and STAT3. Although growth inhibition by siROR1 was minimal in another mesothelioma cell line (H28), colony formation was significantly suppressed. Microarray, quantitative polymerase chain reaction, and Western blot analyses showed that there were differences in the suppression of mRNA and proteins between H2452 and H28 cells transfected with siROR1 compared with those transfected with control siRNA. Cluster analysis further showed that MM tumors had relatively high ROR1 expression, although the cluster in them was different from that in MM cell lines. Thymidylate synthase, a target of pemetrexed, was downregulated in H2452 cells transfected with siROR1. Accordingly, a combination of pemetrexed with siROR1 was found to be effective in the three MM cell lines we studied. CONCLUSION: Our findings may provide novel therapeutic insight into the treatment of advanced MM.

A case report of left lower lobe segmentectomy for pulmonary metastasis from retroperitoneal liposarcoma.

BACKGROUND: The principle treatment for retroperitoneal liposarcoma is surgical resection, however there are many cases of recurrence. In addition to local recurrence, retroperitoneal liposarcoma, particularly dedifferentiated liposarcoma is known to occasionally cause lung metastases. CASE REPORT: A 72-year-old woman with a diagnosis of retroperitoneal liposarcoma and probable right upper lobe early pulmonary adenocarcinoma underwent sequential local tumor resection and right upper lobectomy. Twenty months after liposarcoma resection, a computed tomography (CT) scan of the chest revealed a nodule located in the left lower lobe. A CT-guided biopsy was performed and she was subsequently diagnosed with pulmonary metastasis from retroperitoneal liposarcoma. The nodule enlarged chronologically, however a left lower lobectomy could not be performed because respiratory function after the right upper lobectomy was not sufficient. Therefore, in order to preserve the left superior segment (S6), the basal segments (S8+S9+S10) were resected. Seven months after the surgery, she is living a self-reliant life without recurrence of liposarcoma. CONCLUSION: Here we have reported a case of pulmonary metastasis from retroperitoneal liposarcoma following limited surgery. In cases where respiratory function is limited, lower lobe segmentectomy can be an effective treatment. For the treatment of a single pulmonary metastasis from retroperitoneal liposarcoma, metastasectomy was considered to be effective as long as no local recurrence was seen after initial primary tumor resection.

Development of an integrated CRISPRi targeting ΔNp63 for treatment of squamous cell carcinoma.

TP63 encodes TAp63, which is functionally similar to the tumor suppressor TP53, and ΔNp63, which lacks the transcription-activating domain of TAp63 and appears potently oncogenic in squamous cell carcinomas (SCCs). In this study, we developed an integrated CRISPR interference (CRISPRi) system to selectively suppress ΔNp63 (CRISPRiΔNp63). We engineered this CRISPRi using tandemized guide RNA expression cassettes that targeted the 50 to 100 bp downstream of the transcription start site of ΔNp63 in combination with inactivated Cas9 linked to the transcription repression module Krüppel-associated box repressor domain. The plasmid vector harboring CRISPRiΔNp63 repressed ΔNp63 transcription in lung and esophageal SCC cells. Likewise, Ad-CRISPRiΔNp63, an all-in-one adenoviral vector containing the tandemized gRNAs and dCas9/KRAB expression cassette suppressed ΔNp63 expression in SCC cells. Ad-CRISPRiΔNp63 also effectively decreased cell proliferation and colony formation and induced apoptosis in lung and esophageal SCC cells in vitro and significantly inhibited tumor growth in a mouse lung SCC xenograft model in vivo. These results indicate that ΔNp63 suppression using CRISPRiΔNp63 may be an effective strategy for treating lung and esophageal SCC.

An EGFR ligand promotes EGFR-mutant but not KRAS-mutant lung cancer in vivo.

EGFR ligands (e.g., EGF and TGFA) have been shown to be clinically associated with poor survival in lung cancer. Since TGFA itself initiates autochthonous tumors in liver, breast, and pancreas but not in the lung in transgenic mice in vivo, it would appear that an EGFR ligand may not initiate but rather promote lung cancer. However, it has not been proven in vivo whether lung cancer is promoted by an EGFR ligand. Using transgenic mouse models conditionally expressing EGFRL858R or KrasG12D with TGFA (an EGFR ligand) in lung epithelium, we determined that TGFA promoted the growth of EGFRL858R-lung tumors in airway regions but not that of KrasG12D-lung tumors. Analysis of TCGA datasets identified ΔNp63 and AGR2 as potential key tumor-promoting regulators, which were highly induced in the TGFA-induced EGFRL858R-lung tumors. The expression of AGR2 was positively correlated with the expression of TGFA in human EGFR-mutant lung adenocarcinomas. The expression of TGFA in human EGFR-mutant lung adenocarcinomas but not in the EGFR wild-type lung adenocarcinoma was associated with poor survival. These results suggest that targeting EGFR ligands may benefit patients who carry EGFR-mutant lung tumors but will not benefit patients with KRAS-mutant lung tumors.

Three-Component Repurposed Technology for Enhanced Expression: Highly Accumulable Transcriptional Activators via Branched Tag Arrays.

In the past few years, several types of artificial transcriptional activator, based on CRISPR-Cas9, have been developed and refined. Of these, in synergistic activation mediator and SunTag systems, the effector proteins, expressed in trans, can be recruited to the target sites via the MS2 RNA-binding system and GCN4-scFv antibody system, respectively. Here, we report a strong transcriptional activation system achieved by fusing GCN4 repeat to MS2 coat protein to accumulate numbers of activators, fused to scFv antibodies. By targeting the CDH1 gene, we show that our novel system, named "TREE," results in a greater effect of activating exogenous reporter and endogenous gene. Moreover, by targeting another gene, RANKL, we consistently show the superiority of the TREE system with fewer single-guide RNAs compared to conventional systems. Our TREE system is a promising tool for transcriptional activation and can potentially contribute to other dCas9-mediated technologies such as epigenome editing and chromosome visualization.

Targeted silencing of SOX2 by an artificial transcription factor showed antitumor effect in lung and esophageal squamous cell carcinoma.

SOX2 is a transcription factor essential for early mammalian development and for the maintenance of stem cells. Recently, SOX2 was identified as a lineage specific oncogene, recurrently amplified and activated in lung and esophageal squamous cell carcinoma (SCC). In this study, we have developed a zinc finger-based artificial transcription factor (ATF) to selectively suppress SOX2 expression in cancer cells and termed the system ATF/SOX2. We engineered the ATF using six zinc finger arrays designed to target a 19 bp site in the SOX2 distal promoter and a KOX transcriptional repressor domain. A recombinant adenoviral vector Ad-ATF/SOX2 that expresses ATF/SOX2 suppressed SOX2 at the mRNA and protein levels in lung and esophageal SCC cells expressing SOX2. In these kinds of cells, Ad-ATF/SOX2 decreased cell proliferation and colony formation more effectively than the recombinant adenoviral vector Ad-shSOX2, which expresses SOX2 short hairpin RNA (shSOX2). Ad-ATF/SOX2 induced the cell cycle inhibitor CDKN1A more strongly than Ad-shSOX2. Importantly, the ATF did not suppress the cell viability of normal human cells. Moreover, Ad-ATF/SOX2 effectively inhibited tumor growth in a lung SCC xenograft mouse model. These results indicate that ATF/SOX2 would lead to the development of an effective molecular-targeted therapy for lung and esophageal SCC.

Gene signature driving invasive mucinous adenocarcinoma of the lung.

Though invasive mucinous adenocarcinoma of the lung (IMA) is pathologically distinctive, the molecular mechanism driving IMA is not well understood, which hampers efforts to identify therapeutic targets. Here, by analyzing gene expression profiles of human and mouse IMA, we identified a Mucinous Lung Tumor Signature of 143 genes, which was unexpectedly enriched in mucin-producing gastrointestinal, pancreatic, and breast cancers. The signature genes included transcription factors FOXA3, SPDEF, HNF4A, mucins MUC5AC, MUC5B, MUC3, and an inhibitory immune checkpoint VTCN1/B7-H4 (but not PD-L1/B7-H1). Importantly, induction of FOXA3 or SPDEF along with mutant KRAS in lung epithelium was sufficient to develop benign or malignant mucinous lung tumors, respectively, in transgenic mice. FOXA3 and SPDEF induced MUC5AC and MUC5B, while HNF4A induced MUC3 in human mucinous lung cancer cells harboring a KRAS mutation. ChIP-seq combined with CRISPR/Cas9 determined that upstream enhancer regions of the mucin genes MUC5AC and MUC5B, which were bound by SPDEF, were required for the expression of the mucin genes. Here, we report the molecular signature and gene regulatory network driving mucinous lung tumors.

A Case of Birt-Hogg-Dubé (BHD) Syndrome Harboring a Novel Folliculin (FLCN) Gene Mutation.

BACKGROUND Birt-Hogg-Dubé (BHD) syndrome is an autosomal dominant disorder clinically characterized by pulmonary cysts, spontaneous pneumothorax, renal cell cancer, and skin fibrofolliculomas. The disorder is caused by germline mutations in the FLCN gene. CASE REPORT A 56-year-old female was admitted to our hospital with a diagnosis of bilateral spontaneous pneumothorax. A computed tomography (CT) scan of the chest revealed bilateral multiple bullae predominantly located in the subpleural and mediastinal areas in the bilateral upper and lower lobes. Although she was cured by thoracic cavity drainage, she underwent resection of bilateral lung bullae because she had a prior history of right pneumothorax at 37- and 45-years of age. She had no signs of renal tumor but had fibrofolliculoma in her face and a family history of pneumothorax, we therefore suspected BHD syndrome. DNA sequence analyses determined that there was a two base pair deletion in exon 4 of the FLCN gene, confirming the diagnosis of BHD syndrome. CONCLUSIONS Here we report a case of BHD syndrome with a previously unreported FLCN mutation.

Aqueous-Medium Carbon-Carbon Bond-Forming Radical Reactions Catalyzed by Excited Rhodamine B as a Metal-Free Organic Dye under Visible Light Irradiation.

The utility of rhodamine B as a water-soluble organic photocatalyst was studied in the cascade radical addition-cyclization-trapping reactions under visible light irradiation. In the presence of (i-Pr)2NEt, the electron transfer from the excited rhodamine B to perfluoroalkyl iodides proceeded smoothly to promote the carbon-carbon bond-forming radical reactions in aqueous media. When i-C3F7I was employed as a radical precursor, the aqueous-medium radical reactions proceeded even in the absence of (i-Pr)2NEt. In these reactions, the direct electron transfer from the excited singlet state of rhodamine B would take place. Furthermore, the cleavage of the C-I bond in less reactive i-PrI could be achieved by the reductive electron transfer from the excited rhodamine B, which was confirmed by the fluorescence quenching of rhodamine B with the addition of i-PrI.

Novel Midkine Inhibitor iMDK Inhibits Tumor Growth and Angiogenesis in Oral Squamous Cell Carcinoma.

Midkine is a heparin-binding growth factor highly expressed in various human malignant tumors. However, its role in the growth of oral squamous cell carcinoma is not well understood. In this study, we analyzed the antitumor effect of a novel midkine inhibitor (iMDK) against oral squamous cell carcinoma. Administration of iMDK induced a robust antitumor response and suppressed cluster of differentiation 31 (CD31) expression in oral squamous cell carcinoma HSC-2 cells and SAS cells xenograft models. iMDK inhibited the proliferation of these cells dose-dependently, as well as the expression of midkine and phospho-extracellular signal-regulated kinase in HSC-2 and SAS cells. Moreover, iMDK significantly inhibited vascular endothelial growth factor and induced tube growth of human umbilical vein endothelial cells in a dose-dependent fashion. These findings suggest that midkine is critically involved in oral squamous cell carcinoma and iMDK can be effectively used for the treatment of oral squamous cell carcinoma.