Differential effect of cancer-associated fibroblast-derived extracellular vesicles on cisplatin resistance in oral squamous cell carcinoma via miR-876-3p.

Rationale: Platinum-based chemotherapy is commonly used for treating solid tumors, but drug resistance often limits its effectiveness. Cancer-associated fibroblast (CAF)-derived extracellular vesicle (EV), which carry various miRNAs, have been implicated in chemotherapy resistance. However, the molecular mechanism through which CAFs modulate cisplatin resistance in oral squamous cell carcinoma (OSCC) is not well understood. We employed two distinct primary CAF types with differential impacts on cancer progression: CAF-P, representing a more aggressive cancer-promoting category, and CAF-D, characterized by properties that moderately delay cancer progression. Consequently, we sought to investigate whether the two CAF types differentially affect cisplatin sensitivity and the underlying molecular mechanism. Methods: The secretion profile was examined by utilizing an antibody microarray with conditioned medium obtained from the co-culture of OSCC cells and two types of primary CAFs. The effect of CAF-dependent factors on cisplatin resistance was investigated by utilizing conditioned media (CM) and extracellular vesicle (EVs) derived from CAFs. The impacts of candidate genes were confirmed using gain- and loss-of-function analyses in spheroids and organoids, and a mouse xenograft. Lastly, we compared the expression pattern of the candidate genes in tissues from OSCC patients exhibiting different responses to cisplatin. Results: When OSCC cells were cultured with conditioned media (CM) from the two different CAF groups, cisplatin resistance increased only under CAF-P CM. OSCC cells specifically expressed insulin-like growth factor binding protein 3 (IGFBP3) after co-culture with CAF-D. Meanwhile, IGFBP3-knockdown OSCC cells acquired cisplatin resistance in CAF-D CM. IGFBP3 expression was promoted by GATA-binding protein 1 (GATA1), a transcription factor targeted by miR-876-3p, which was enriched only in CAF-P-derived EV. Treatment with CAF-P EV carrying miR-876-3p antagomir decreased cisplatin resistance compared to control miRNA-carrying CAF-P EV. On comparing the staining intensity between cisplatin-sensitive and -insensitive tissues from OSCC patients, there was a positive correlation between IGFBP3 and GATA1 expression and cisplatin sensitivity in OSCC tissues from patients. Conclusion: These results provide insights for overcoming cisplatin resistance, especially concerning EVs within the tumor microenvironment. Furthermore, it is anticipated that the expression levels of GATA1 and miR-876-3p, along with IGFBP3, could aid in the prediction of cisplatin resistance.

Using Comparative Proteomics to Identify Protein Signatures in Clear Cell Renal Cell Carcinoma.

BACKGROUND/AIM: Renal cell carcinoma (RCC) is one of the most commonly diagnosed cancers in the world. Approximately 25-30% of patients identified with initial kidney cancer will have metastasized tumors, thus 5-year survival rates for these patients are poor. Therefore, biomarker research is required to identify and predict molecular signatures in RCC. MATERIALS AND METHODS: To address this, we used a mass spectrometry (MS)-based proteomics approach to identify proteins related to clear cell RCC (ccRCC) tissues from patients with T1G2, T1G3, T3G2, T3G3, and metastatic RCC (mRCC) stages. RESULTS: We identified and quantified 2,608 and 2,463 proteins, respectively, in ccRCC tissue and identified 1,449 differentially expressed proteins (DEPs). Bioinformatics analysis revealed that serpin family A member 3 (SERPINA3) qualified as biomarker for ccRCC progression. Using indirect enzyme-linked immunosorbent assay (ELISA), immunoblotting, and immunohistochemistry assays it was found that SERPINA3 expression levels in ccRCC tissues were much higher in stages before metastasis. CONCLUSION: Comparative proteomics analysis of ccRCC tissues provided new evidence of SERPINA3 association with ccRCC progression.

3D simulation of interosseous interference in sagittal split ramus osteotomy for mandibular asymmetry.

BACKGROUND: The purpose of this study was to evaluate the pattern of predicted interosseous interference and to determine the influencing factor to volume of bony interference using a computer-assisted simulation system. This retrospective study recruited 116 patients with mandibular prognathism who had undergone sagittal split ramus osteotomy (SSRO) with or without maxillary osteotomy. The patients were divided into 3 groups according to the amount of menton (Me) deviation: less than 2 mm (Group 1), 2-4 mm (Group 2), and more than 4 mm (Group 3). Changes in the distal segments following BSSRO and the volume of the interosseous interference between the proximal and distal segments were simulated after matching preoperative occlusion and postoperative expected occlusion with the cone-beam computed tomography data. Ramal inclinations and other skeletal measurements were analyzed before surgery, immediately after surgery, and at least 6 months after surgery. RESULTS: The anticipated interosseous interference was more frequently noted on the contralateral side of chin deviation (long side) than the deviated site (short side) in Groups 2 and 3. More interference volume was predicted at the long side (186 ± 343.9 mm3) rather than the short side (54.4 ± 124.4 mm3) in Group 3 (p = 0.033). The bilateral difference in the volume of the interosseous interference of the osteotomized mandible was significantly correlated with the Me deviation (r = - 0.257, p = 0.009) and bilateral ramal inclination (r = 0.361, p < 0.001). The predictor variable that affected the volume of the osseous interference at each side was the amount of Me deviation (p = 0.010). CONCLUSION: By using the 3D simulation system, the potential site of bony collision could be visualized and successfully reduced intraoperatively. Since the osseous interference can be existed on any side, unilaterally or bilaterally, 3D surgical simulation is necessary before surgery to predict the osseous interference and improve the ramal inclination.

LDHA Desuccinylase Sirtuin 5 as A Novel Cancer Metastatic Stimulator in Aggressive Prostate Cancer.

Prostate cancer (PCa) is the most commonly diagnosed genital cancer in men worldwide. Around 80% of the patients who developed advanced PCa suffered from bone metastasis, with a sharp drop in the survival rate. Despite great efforts, the detailed mechanisms underlying castration-resistant PCa (CRPC) remain unclear. Sirtuin 5 (SIRT5), an NAD+-dependent desuccinylase, is hypothesized to be a key regulator of various cancers. However, compared to other SIRTs, the role of SIRT5 in cancer has not been extensively studied. Here, we revealed significantly decreased SIRT5 levels in aggressive PCa cells relative to the PCa stages. The correlation between the decrease in the SIRT5 level and the patient's reduced survival rate was also confirmed. Using quantitative global succinylome analysis, we characterized a significant increase in the succinylation at lysine 118 (K118su) of lactate dehydrogenase A (LDHA), which plays a role in increasing LDH activity. As a substrate of SIRT5, LDHA-K118su significantly increased the migration and invasion of PCa cells and LDH activity in PCa patients. This study reveals the reduction of SIRT5 protein expression and LDHA-K118su as a novel mechanism involved in PCa progression, which could serve as a new target to prevent CPRC progression for PCa treatment.

Atypical lipomatous tumour/well-differentiated liposarcoma found in the buccal mucosa: A rare case report.

Oral liposarcomas are uncommon diseases, the most predominant histopathological subtype being atypical lipomatous tumour/well-differentiated liposarcoma. In regard to its clinical aspects in the oral cavity, it is challenging to confirm a diagnosis and develop a treatment plan. In this case report, we present a rare case of atypical lipomatous tumour/well-differentiated liposarcoma in the right cheek of a 77-year-old male patient. Conservative surgery was performed considering the histopathological subtype of the neoplasm. Knowledge of the clinical and histopathological characteristics of this rare disease is essential to maintaining function and aesthetics through conservative treatment in older patients.

Factors influencing the intention to engage in cervical cancer preventive behavior in human papillomavirus-infected women: a cross-sectional survey.

PURPOSE: This study investigated the influence of cervical cancer knowledge, human papillomavirus (HPV) knowledge, self-efficacy, and uncertainty on the intention to engage in cervical cancer preventive behavior in HPV-infected women. METHODS: This descriptive correlational study was conducted among 129 adult women aged 20 to 65 years who received positive HPV results at a general hospital in Changwon, Korea. The dataset was analyzed using descriptive statistics, the independent t-test, analysis of variance, the Pearson correlation coefficient, and multiple regression. RESULTS: The mean score for the intention to engage in cervical cancer preventive behavior was high (4.43±0.65). This intention was significantly different according to age at first sexual intercourse (F=7.38, p=.001), HPV type (F=4.79, p=.010), vaccination (t=3.19, p=.002), and condom use (t=3.03, p=.003). The intention to engage in cervical cancer preventive behavior showed significant, weak-to-moderate positive correlations with HPV knowledge (r=.22, p=.012) and self-efficacy (r=.42, p<.001). Self-efficacy (ß=.46, p<.001), first sexual intercourse at <20 years (ß=.45, p<.001), first sexual intercourse at 20-24 years (ß=.29, p=. 018), HPV high- and low-risk group infection (ß=.26, p=.019), HPV high-risk group infection (ß=.26, p=.026), and vaccination (ß=.21, p=.007) significantly influenced the intention to engage in cervical cancer preventive behavior. These variables explained 34.6% of variance in intention. CONCLUSION: Study findings support the need to develop a program that effectively conveys accurate information about cervical cancer prevention to HPV-infected women and helps them enhance self-efficacy to boost the intention to engage in cervical cancer preventive behavior.

An Fc variant with two mutations confers prolonged serum half-life and enhanced effector functions on IgG antibodies.

The pH-selective interaction between the immunoglobulin G (IgG) fragment crystallizable region (Fc region) and the neonatal Fc receptor (FcRn) is critical for prolonging the circulating half-lives of IgG molecules through intracellular trafficking and recycling. By using directed evolution, we successfully identified Fc mutations that improve the pH-dependent binding of human FcRn and prolong the serum persistence of a model IgG antibody and an Fc-fusion protein. Strikingly, trastuzumab-PFc29 and aflibercept-PFc29, a model therapeutic IgG antibody and an Fc-fusion protein, respectively, when combined with our engineered Fc (Q311R/M428L), both exhibited significantly higher serum half-lives in human FcRn transgenic mice than their counterparts with wild-type Fc. Moreover, in a cynomolgus monkey model, trastuzumab-PFc29 displayed a superior pharmacokinetic profile to that of both trastuzumab-YTE and trastuzumab-LS, which contain the well-validated serum half-life extension Fcs YTE (M252Y/S254T/T256E) and LS (M428L/N434S), respectively. Furthermore, the introduction of two identified mutations of PFc29 (Q311R/M428L) into the model antibodies enhanced both complement-dependent cytotoxicity and antibody-dependent cell-mediated cytotoxicity activity, which are triggered by the association between IgG Fc and Fc binding ligands and are critical for clearing cancer cells. In addition, the effector functions could be turned off by combining the two mutations of PFc29 with effector function-silencing mutations, but the antibodies maintained their excellent pH-dependent human FcRn binding profile. We expect our Fc variants to be an excellent tool for enhancing the pharmacokinetic profiles and potencies of various therapeutic antibodies and Fc-fusion proteins.

Anti-PTK7 Monoclonal Antibodies Exhibit Anti-Tumor Activity at the Cellular Level and in Mouse Xenograft Models of Esophageal Squamous Cell Carcinoma.

PTK7 is a catalytically defective receptor protein tyrosine kinase upregulated in various cancers, including esophageal squamous cell carcinoma (ESCC). In previous studies, we observed a positive correlation between PTK7 expression levels and tumorigenicity in various ESCC cell lines and xenograft mice with ESCC KYSE-30 cells. In this study, we analyzed the effects of anti-PTK7 monoclonal antibodies (mAbs) on the tumorigenic activity in KYSE-30 cells and in mouse xenograft models. PTK7 mAb-32 and mAb-43 bind with a high affinity to the extracellular domain of PTK7. PTK7 mAbs significantly reduced three-dimensional cell proliferation, adhesion, wound healing, and migration. PTK7 mAbs also reduce chemotactic invasiveness by decreasing MMP-9 secretion. PTK7 mAbs decreased actin cytoskeleton levels in the cortical region of KYSE-30 cells. PTK7 mAbs reduced the phosphorylation of ERK, SRC, and FAK. In a mouse xenograft model of ESCC using KYSE-30 cells, PTK7 mAbs reduced tumor growth in terms of volume, weight, and the number of Ki-67-positive cells. These results demonstrated that PTK7 mAbs can inhibit the tumorigenicity of ESCC at the cellular level and in vivo by blocking the function of PTK7. Considering the anticancer activities of PTK7 mAbs, we propose that PTK7 mAbs can be used in an effective treatment strategy for PTK7-positive malignancies, such as ESCC.

Nicotine alleviates alcohol-induced memory and long-term potentiation impairment.

Alcohol and nicotine are routinely abused together. There is one plausible explanation that comorbidity can alleviate alcohol-induced cognitive impairment. However, the mechanism involved is not known. The aim of this report was to evaluate the interactive effects of alcohol coadministration with nicotine on hippocampal memory and long-term potentiation (LTP). C57BL/6 mice were distributed into 4 treatment groups: control, alcohol, nicotine, and alcohol plus nicotine. All mice received tap water or alcohol solution and saline or nicotine. In water maze test, the alcohol group showed significant decreases in hippocampus function and acquisition training than control, nicotine, and combined treatment groups. The alcohol group also showed a significantly shorter latency in entering the foot-shock compartment than control, nicotine, and combined treatment groups in a passive avoidance test. Theta burst stimulation was adopted to induce concrete LTP in CA1 field recording using hippocampal slice. Alcohol alone administration failed to maintain LTP. Nicotine alone administration did not alter hippocampal LTP. There were no negative effects of alcohol on hippocampal LTP in mice administrated with nicotine. The current study successfully demonstrated beneficial effects of nicotine on alcohol induced memory impairment accompanied by hippocampal LTP impairment after one week of co-administration and one-day withdrawal.

Analysis of the fluid contents of simple bone cyst in the mandible.

Description of simple bone cyst (SBC) content has been controversial. This study aimed to assess and give a clearer picture of the SBC cavity contents. Between 2014 and 2016, 19 patients with SBC verified by histopathological examination were included in this study. SBC cavity content was investigated using clinical, radiographic, surgical, and laboratory findings. The difference in components among cavity fluid, blood, and serum was evaluated using a paired sample t-test for statistical analysis. All 19 SBC cases radiographically and surgically revealed a fluid-filled cavity. The patients' average age was 21.3 ± 13.2 years, with no sex predominance found. SBCs were found mostly in the anterior mandible (n = 12, 63.2%). All lesions were filled with clear straw-colored or blood-colored floods with low concentration. Although the fluid components were similar to those in the blood and serum in the laboratory analysis, the statistical analysis revealed that the fluid components were not significantly different only for eosinophil (p = 0.43) and basophil (p = 0.06) counts as blood components and sodium (p = 0.76), potassium (p = 0.08), and chloride (p = 0.13) concentration as serum components. The results show that SBC is a fluid-filled cavity, with the cavity fluid being more likely similar to serum rather than blood regarding internal components.

Giant lipoma of the tongue: A case report and review of the literature.

This report presents the case of a 49-year-old man who presented with giant masses that had recently grown on the bilateral sides of the tongue. A clinical examination revealed rubbery yellowish lesions protruding from the tongue. A panoramic radiograph showed an enlarged soft tissue shadow of the tongue. Computed tomography showed well-defined circumscribed mass exhibiting a homogeneous low density on the bilateral sides of the tongue. On magnetic resonance images, the masses showed a high signal intensity on T1-weighted images and iso-signal intensity with partially hyperintense margin on fat-suppressed T2-weighted images. Surgical excision was performed, and a histopathologic examination confirmed the diagnosis of lipoma. The patient recovered well with no sign of recurrence. A giant lipoma is defined as a lipoma larger than 5 cm in diameter. A literature review of giant lipomas of the tongue is also presented herein.

Therapeutic effects of TM4SF5-targeting chimeric and humanized monoclonal antibodies in hepatocellular and colon cancer models.

The transmembrane 4 L six family member 5 (TM4SF5) is aberrantly expressed in hepatocellular and colorectal cancers, and has been implicated in tumor progression, suggesting that it could serve as a novel therapeutic target. Previously, we screened a murine antibody phage-display library to generate a novel monoclonal antibody, Ab27, that is specific to the extracellular loop 2 of TM4SF5. In this study, we evaluated the effects of chimeric Ab27 using cancer cells expressing endogenous TM4SF5 or stably overexpressing TM4SF5 in vivo and in vitro. Monotherapy with Ab27 significantly decreased tumor growth in liver and colon cancer xenograft models, including a sorafenib-resistant model, and decreased the phosphorylation of focal adhesion kinase (FAK), p27Kip1, and signal transducer and activator of transcription 3 (STAT3). No general Ab27 toxicity was observed in vivo. Combination treatment with Ab27 and sorafenib or doxorubicin exerted higher antitumor activity than monotherapy. In addition, we humanized the Ab27 sequence by the complementarity-determining region (CDR) grafting method. The humanized antibody Ab27-hz9 had reduced immunogenicity but exhibited target recognition and antitumor activity comparable with those of Ab27. Both Ab27 and Ab27-hz9 efficiently targeted tumor cells expressing TM4SF5 in vivo. These observations strongly support the further development of Ab27-hz9 as a novel therapeutic agent against liver and colorectal cancers.

Withaferin A mitigates metastatic traits in human oral squamous cell carcinoma caused by aberrant claudin-1 expression.

Abnormal expression of claudin-1 (CLDN1) has important roles in carcinogenesis and metastasis in various cancers. The role of CLDN1 in human oral squamous cell carcinoma (OSCC) remains unknown. Here, we report the functional role of CLDN1 in metastasis of human OSCC, as a potential target regulated by withaferin A. From gene expression profiling with microarray technology, we found that the majority of notable differentially expressed genes were classified into migration/invasion category. Withaferin A impaired the motility of human OSCC cells in vitro and suppressed metastatic nodule formation in an in vivo metastasis model, both associated with reduced CLDN1. CLDN1 overexpression enhanced metastatic nodule formation in vivo, resulting in severe metastatic lesions in lung tissue. Moreover, CLDN1 expression was positively correlated to lymphatic metastasis in OSCC patients. The impaired motility of human OSCC cells upon withaferin A treatment was restored by CLDN1 overexpression. Furthermore, upregulation of let-7a induced by withaferin A was inversely correlated to CLDN1 expression. Overall, these give us an insight into the function of CLDN1 for prognosis and treatment of human OSCC, substantiating further investigation into the use of withaferin A as good anti-metastatic drug candidate.

SIRT5 Directly Inhibits the PI3K/AKT Pathway in Prostate Cancer Cell Lines.

BACKGROUND/AIM: Prostate cancer (PCa) is the most commonly diagnosed genital cancer in men globally. Among patients who develop advanced PCa, 80% are affected by bone metastasis, with a sharp drop in survival rate. Despite efforts, the details of mechanisms of metastasis of PCa remain unclear. SIRT5, an NAD+-dependent deacylase, is hypothesized to be a crucial regulator of various cancers. The role of SIRT5 in cancer has not been extensively studied compared to other SIRTs. In this study, we showed significantly decreased levels of SIRT5 in PC-3M, a highly aggressive PC-3 cell variant. MATERIALS AND METHODS: We characterized the differentially expressed proteins between parental and SIRT5 KO PC-3 cells using quantitative proteomics analysis. RESULTS: A significant increase in expression of interleukin-1ß (IL-1ß) in SIRT5 KO cells was observed, and the PI3K/AKT/NF-ĸB signaling pathway was found significantly elevated in SIRT5 KO cells by the Gene Ontology annotation and KEGG pathway functional enrichment analysis. Moreover, we confirmed that SIRT5 can bind PI3K by immunoprecipitation analysis. CONCLUSION: This study is the first to demonstrate a relationship between SIRT5 and PCa metastasis, suggesting that SIRT5-mediated inhibition of the PI3K/AKT/NK-kB pathway is reduced for secondary metastasis from bone to other tissues.

Facilitating Reparative Dentin Formation Using Apigenin Local Delivery in the Exposed Pulp Cavity.

Apigenin, a natural product belonging to the flavone class, affects various cell physiologies, such as cell signaling, inflammation, proliferation, migration, and protease production. In this study, apigenin was applied to mouse molar pulp after mechanically pulpal exposure to examine the detailed function of apigenin in regulating pulpal inflammation and tertiary dentin formation. In vitro cell cultivation using human dental pulp stem cells (hDPSCs) and in vivo mice model experiments were employed to examine the effect of apigenin in the pulp and dentin regeneration. In vitro cultivation of hDPSCs with apigenin treatment upregulated bone morphogenetic protein (BMP)- and osteogenesis-related signaling molecules such as BMP2, BMP4, BMP7, bone sialoprotein (BSP), runt-related transcription factor 2 (RUNX2), and osteocalcin (OCN) after 14 days. After apigenin local delivery in the mice pulpal cavity, histology and cellular physiology, such as the modulation of inflammation and differentiation, were examined using histology and immunostainings. Apigenin-treated specimens showed period-altered immunolocalization patterns of tumor necrosis factor (TNF)-α, myeloperoxidase (MPO), NESTIN, and transforming growth factor (TGF)-ß1 at 3 and 5 days. Moreover, the apigenin-treated group showed a facilitated dentin-bridge formation with few irregular tubules after 42 days from pulpal cavity preparation. Micro-CT images confirmed obvious dentin-bridge structures in the apigenin-treated specimens compared with the control. Apigenin facilitated the reparative dentin formation through the modulation of inflammation and the activation of signaling regulations. Therefore, apigenin would be a potential therapeutic agent for regenerating dentin in exposed pulp caused by dental caries and traumatic injury.

Experience of patients diagnosed as asymptomatic COVID-19 after dental treatment.

BACKGROUND: The potential risk of coronavirus disease 2019 (COVID-19) transmission from asymptomatic COVID-19 patients is a concern in dental practice. However, the impact of this risk is not well documented to date. This report describes our dental clinical experience with patients who did not exhibit symptoms of COVID-19 but were later confirmed as positive for COVID-19. CASE PRESENTATION: Of the 149,149 patients who visited the outpatient clinic of KNUDH and the 3291 patients who visited the Oral and Maxillofacial Surgery Clinic of KNUH, 3 were later confirmed as having COVID-1 between 1 February 2020 and 28 February 2021. Owing to close contact with these patients during their treatments, 46 dental and medical staff had to undergo quarantine from the date of the patients' confirmation of COVID-19 infection. CONCLUSION: The presented cases showed the potential existence of asymptomatic COVID-19 patients after dental treatment with aerosol-generating procedures. Clinicians should be aware of the infection prevention measures and try to protect healthcare personnel from secondary infection of COVID-19 during dental treatments.

The impact of COVID-19 on the injury pattern for maxillofacial fracture in Daegu city, South Korea.

BACKGROUND: This study aimed to analyze the impact of COVID-19 on oral and maxillofacial fracture in Daegu by comparing the demographic data in 2019 and 2020, retrospectively. We collected data from all patients having trauma who visited the emergency room for oral and maxillofacial fractures. METHODS: This retrospective study was based on chart review of patients who visited the emergency department of Kyungpook National University Hospital in Daegu, South Korea from January 1, 2019, to December 31, 2020. We conducted a comparative study for patients who presented with maxillofacial fractures with occlusal instability during pre-COVID-19 era (2019) and COVID-19 era (2000) with demographics and pattern of injuries. RESULTS: After the outbreak of COVID-19, the number of monthly oral and maxillofacial fractures, especially sports-related oral and maxillofacial fractures, decreased significantly. Also, the number of alcohol-related fractures increased significantly. In addition, as the number of monthly confirmed cases of COVID-19 increases, the incidence of fracture among these cases tends to decrease. CONCLUSIONS: The COVID-19 pandemic has changed the daily life in Korea. Identifying the characteristics of patients having trauma can provide a good lead to understand this long-lasting infectious disease and prepare for future outbreaks.

Differential Angiogenic Potential of 3-Dimension Spheroid of HNSCC Cells in Mouse Xenograft.

The experimental animal model is still essential in the development of new anticancer drugs. We characterized mouse tumors derived from two-dimensional (2D) monolayer cells or three-dimensional (3D) spheroids to establish an in vivo model with highly standardized conditions. Primary cancer-associated fibroblasts (CAFs) were cultured from head and neck squamous cell carcinoma (HNSCC) tumor tissues and co-injected with monolayer cancer cells or spheroids into the oral mucosa of mice. Mice tumor blood vessels were stained, followed by tissue clearing and 3D Lightsheet fluorescent imaging. We compared the effect of exosomes secreted from 2D or 3D culture conditions on the angiogenesis-related genes in HNSCC cells. Our results showed that both the cells and spheroids co-injected with primary CAFs formed tumors. Interestingly, vasculature was abundantly distributed inside the spheroid-derived but not the monolayer-derived mice tumors. In addition, cisplatin injection more significantly decreased spheroid-derived but not monolayer-derived tumor size in mice. Additionally, exosomes isolated from co-culture media of FaDu spheroid and CAF upregulated angiogenesis-related genes in HNSCC cells as compared to exosomes from FaDu cell and CAF co-culture media under in vitro conditions. The mouse tumor xenograft model derived from 3D spheroids of HNSCC cells with primary CAFs is expected to produce reliable chemotherapy drug screening results given the robust angiogenesis and lack of necrosis inside tumor tissues.