CONTEXT:Cervical cancer (CC) is one of the leading cancers threatening women's health worldwide. Folate is essential for one-carbon metabolism, influencing DNA synthesis and methylation. However, the role of folate status in cervical carcinogenesis is not fully understood. OBJECTIVE:To explore the association between folate deficiency and CC risk in women, and the association between folate supplementation and the regression of cervical intraepithelial neoplasia (CIN). DATA SOURCES:We conducted a comprehensive literature search in PubMed and Ovid databases with no initial restrictions on publication dates initially conducted on June 1, 2023, and updated on January 30, 2025. Mendelian randomization data were obtained from the IEU Open GWAS Project database (https://gwas.mrcieu.ac.uk/). DATA EXTRACTION:Two of the authors independently extracted data using a standardized method and protocol. DATA ANALYSIS:Twenty-five studies with 14 304 participants were included. The lowest quartile of dietary folate intake (odds ratio [OR]: 1.36; 95% CI: 1.18, 1.58), serum folate (OR: 1.54; 95% CI: 1.18, 2.01), and red blood cell (RBC) folate (OR: 1.43; 95% CI: 1.07, 1.93) were associated with a higher CIN and/or CC risk. Dose-response meta-analysis indicated that an additional 240-280.5 μg/day of folate supplementation was associated with a reduction in CIN and/or CC risk by up to 26%. Each 5-nmol/L increase in serum folate and 50-nmol/L increase in RBC folate were associated with 21% and 5% risk reductions, respectively. Folate supplementation showed a significant association with CIN regression (OR: 1.23; 95% CI: 0.88, 1.72). Mendelian randomization supported a reduced CC risk linked to folate intake (OR: 1.000252; 95% CI: 1.000036, 1.000468). CONCLUSION:Our findings suggest that an additional 240-280.5 μg/day of folate supplementation, along with higher serum and RBC folate concentrations, may be associated with reduced CIN and/or CC risk, highlighting the importance of folate in CC prevention. SYSTEMATIC REVIEW REGISTRATION:PROSPERO registration no.: CRD42019123443.

Global cancer data show a continuous rise in cervical cancer incidence and mortality, burdening public health. To effectively mitigate this challenge, elucidating molecular pathogenesis and novel drug targets are critical for advancing clinical care. In this study, abnormally expressed miRNAs in cervical cancer were identified through TCGA data analysis. Expression levels were quantified using qRT-PCR and Western Blot, at both mRNA and protein levels, respectively. miR-141-5p and ACVRL1’s interaction was validated with dual-luciferase assays. Cell behavior was evaluated using assays including CCK-8 assay. In vivo tumor growth was assessed in nude mice xenograft model. Database screening and subsequent validation using cancer cell lines and clinical samples revealed aberrantly elevated expression of miR-141-5p in cervical cancer. High expression of miR-141-5p in SiHa/HeLa cells significantly promoted cellular proliferation, migration, and invasion while concurrently inhibiting apoptosis, whereas its downregulation elicited opposing effects. ACVRL1 was identified as a direct target of miR-141-5p. Overexpressing miR-141-5p markedly upregulated ACVRL1 mRNA and protein levels, while its knockdown significantly reduced ACVRL1 expression at both mRNA and protein levels. Further functional analysis showed that ACVRL1 knockdown significantly impaired cellular proliferation, migration, and invasion while enhancing apoptosis in cervical cancer cells. Rescue experiments confirmed that miR-141-5p exerted regulatory effects on these cellular behaviors, at least partially through ACVRL1. miR-141-5p downregulation significantly suppressed tumor growth in vivo, as evidenced by reduced tumor volume and weight in nude mice. In brief, this study shows that miR-141-5p upregulation in cervical cancer promotes tumor progression via ACVRL1 regulation, indicating its potential in precision oncology.

EGFR, one of the most successful therapeutic targets, has recently been found to exert a novel function for regulating homologous recombination (HR). Activation of HR is the critical event of treatment failure of PARPi in BRCA1/2 wild-type ovarian cancer (OC). Besides, the antitumor effects of biguanides have also been a focus of attention. Here, we discovered that the new biguanide 4C inhibited HR and sensitized BRCA1/2 wild-type OC cells to PARPi by targeting EGFR. Mechanistically, EGFR promoted nuclear accumulation of both BRCA2 and Rad51, and HR activation by competitively inhibiting the binding of BRCA2 and Rad51 to E3 ubiquitin ligase c-Cbl, thereby reducing cancer cell sensitivity to PARPi following ATM-mediated DNA damage signal transmission from the nucleus to the cytoplasm. Interestingly, EGFR was downregulated by 4C, which in turn enhanced the interaction of BRCA2 and Rad51 with c-Cbl. Consequently, BRCA2 and Rad51 were then ubiquitinated and degraded to inhibit HR and increase the sensitivity of OC to PARPi. Thus, these findings reveal that the combination of 4C with PARPi leading to “synthetic lethality” is an effective strategy for treating BRCA1/2 wild-type OC.

A randomized, open-label, phase 2 trial assessed fuzuloparib (a PARP inhibitor) plus apatinib (a vascular endothelial growth factor receptor [VEGFR] inhibitor) versus fuzuloparib alone in patients with recurrent ovarian cancer (ROC). Patients with ROC who had failed ≥ 2 lines of platinum-based chemotherapy were enrolled. Patients with primary platinum resistance were also eligible. Patients were randomized 1:1 to receive fuzuloparib plus apatinib or fuzuloparib. The primary endpoint was objective response rate (ORR). As of Jan 11, 2024, 76 eligible patients were enrolled (39 with fuzuloparib plus apatinib and 37 with fuzuloparib). The ORR was 38.5

Ankyrin repeat domain 49 (ANKRD49) is highly expressed in non-small cell lung cancer (NSCLC) and promotes tumor cell invasion and metastasis by activating the p38/ATF-2/MMP or JNK/ATF-2/c-Jun/MMP pathways. Although epithelial-mesenchymal transition (EMT) is critical for tumor dissemination, the role of ANKRD49 in EMT remains unclear. This study aimed to investigate the effects of ANKRD49 on EMT in NSCLC and to elucidate its underlying molecular mechanisms. The expression levels of ANKRD49 and EMT markers were analyzed in lung adenocarcinoma (LUAD) tissues using tissue microarrays (TMAs) combined with immunohistochemistry (IHC). The role of ANKRD49 in EMT was assessed in vitro and in vivo through real-time qPCR, Western blot, and IHC. Key signaling molecules were quantified by real-time qPCR, Western blot, enzyme-linked immunosorbent assay (ELISA) and IHC. Transcriptional regulation of TGF-β1 by PKNOX1 was verified using luciferase reporter assays and chromatin immunoprecipitation (ChIP). Protein-protein interactions between ANKRD49 and PKNOX1 were examined by co-immunoprecipitation (Co-IP) and immunofluorescence microscopy. TMA analysis revealed a significant negative correlation between ANKRD49 and E-cadherin expression, whereas positive correlations were observed with α-smooth muscle actin (α-SMA) and TGF-β1. ANKRD49 overexpression promoted EMT, whereas its knockdown suppressed EMT in both cellular and animal models. Furthermore, ANKRD49 upregulation increased TGF-β1 levels, which are transcriptionally regulated by PKNOX1, while ANKRD49 silencing had the opposite effect. Mechanistically, ANKRD49 physically interacts with PKNOX1, and PKNOX1 binds to the TGF-β1 promoter, elevating TGF-β1 expression and subsequently activating SMAD signaling. Consistently, ANKRD49 and PKNOX1 levels were positively correlated in LUAD tissues. ANKRD49 facilitates EMT in NSCLC by interacting with PKNOX1 to activate the TGF-β1/SMAD pathway. PKNOX1 mediates this process through transcriptional regulation of TGF-β1.

Cervical cancer (CC) ranks as the fourth most common malignant tumor threatening women's health globally, with the highest mortality rate among female malignant tumors. Owing to insufficient CC screening coverage, along with the complexity and individual variability in carcinogenesis, tumor recurrence, and drug resistance, achieving satisfactory therapeutic outcomes and prognoses for CC patients remains challenging. Consequently, elucidating the mechanisms underlying CC and developing novel diagnostic and therapeutic strategies is of utmost importance for women's health worldwide. Recent studies have shown that circular RNAs (circRNAs) are closely associated with CC progression. As a new class of non-coding RNAs (ncRNAs) characterized by a unique closed-loop structure and high stability, circRNAs can function as tumor suppressors or oncogenic factors through various molecular mechanisms, highlighting their potential as diagnostic and prognostic biomarkers. This review summarizes the biogenesis and functions of circRNAs; examines how dysregulated circRNAs are involved in biological processes such as proliferation, invasion, metastasis, cell death, cellular metabolism, and drug resistance in CC cells; evaluates their potential as diagnostic and prognostic biomarkers for CC; analyzes the challenges of the current research; and explores their clinical therapeutic prospects.

This multicenter, randomized, open-label, exploratory Phase 2 trial assessed SHR-2004, an anti-FXI antibody, for venous thromboembolism (VTE) prevention in patients undergoing ovarian cancer surgery-a population at particularly high thrombotic risk conducted at 25 sites in China. Patients with newly diagnosed Stage III-IV or recurrent ovarian cancer scheduled for surgery were randomly assigned (1:1) to receive subcutaneous SHR-2004 (180 mg on Day 1 and 120 mg on Day 15) or enoxaparin-rivaroxaban (subcutaneous enoxaparin 40 mg once daily during hospitalization, followed by oral rivaroxaban 10 mg once daily after discharge until Day 28). The primary efficacy outcome was the composite of VTE events within 28 days. The primary safety outcome was major bleeding (MB) or clinically relevant nonmajor bleeding (CRNMB) from first dose to 28 days. Between February 19, 2024 and September 17, 2024, 198 patients received assigned treatment (90 with SHR-2004, 108 with enoxaparin-rivaroxaban) and were included in the safety analysis. Among 181 patients who completed protocol-mandated ultrasonography, VTE occurred in 11 (12.6%) patients in the SHR-2004 group and 19 (20.2%) in the enoxaparin-rivaroxaban group (difference -8.4%, 95% CI -19.9 to 3.1) by Day 28. MB/CRNMB rates were similar between groups (SHR-2004: 2/90 [2.2%]; enoxaparin-rivaroxaban: 2/108 [1.9%]). In this study, SHR-2004 showed comparable efficacy to enoxaparin-rivaroxaban for preventing postoperative VTE in patients undergoing ovarian cancer surgery, with a similar low risk of MB or CRNMB. The twice-monthly dosing schedule offers a potential advantage over daily anticoagulation, supporting further investigation in adequately powered Phase 3 trials. Trial Registration: ClinicalTrials.gov identifier: NCT06220123.

Small cell neuroendocrine carcinoma of the cervix (SCNECC) is a rare, aggressive cervical cancer with poor prognosis. This study explored molecular drivers of SCNECC progression and identified potential therapeutic targets. Proteomic analysis was conducted to identify differentially expressed proteins (DEPs) in SCNECC. Subsequent GO and KEGG enrichment analyses were performed to delineate key DEPs. Functional assays, including CCK-8, colony formation, cell cycle, and apoptosis assays, were conducted to assess the roles of target genes. Expression levels of target genes were validated using qRT-PCR, western blot, and immunohistochemistry (IHC). Proteomic profiling revealed 2333 DEPs in SCNECC, comprising 2168 upregulated and 165 downregulated proteins. GO and KEGG analyses revealed that the upregulated DEPs were predominantly enriched in processes occurring within the cell nucleus, such as DNA replication, reflecting heightened proliferative activity in SCNECC. IHC confirmed FEN1 overexpression. Functional assays showed that FEN1 knockdown suppressed cell viability, colony formation, and cell cycle, promoted apoptosis, and impeded tumor growth in mice. SC13, a FEN1 inhibitor, also had similar effects. Furthermore, silencing FEN1 markedly reduced PIK3CA, PCNA, and BCL-2 levels, while elevating Caspase-9 expression. CONCLUSION: FEN1 overexpression fuels SCNECC progression via PCNA regulation, positioning FEN1 as a promising SCNECC therapeutic target.

Background:Cluster of Differentiation-4(CD4),Cluster of Differentiation-8(CD8), and interleukin-10 (IL-10) have long been considered to be related to cervical cancer, but the exact relationship remains unclear. Few studies investigated the relationship between CD4,CD8,IL-10, and high-risk human papillomavirus (HPV) with risk of cervical intraepithelial neoplasia (CIN). Objective:Our aim is to evaluate the relationship between CD4, CD8, IL-10, and high-risk HPV infection with the risk of CIN, as well as their interactions on CIN. Design:In 2014-2015, a cross-sectional study of screening data was conducted among 2285 women aged 19-65 years who participated in an ongoing community-based cohort of 40,000 women in Shanxi, China. Using categorical and spline analyses to evaluate the relationship between local vaginal fluids of CD4,CD8,CD4/CD8,IL-10, and CIN risk. A total of 1,503 controls were followed up until January 31, 2019. A nested case-control study was used to assess the relationship between vaginal lavage CD4, CD8, CD4/CD8, and IL-10 levels and the risk of CIN progression. Results:After adjusting for possible confounding factors,CD4 and CD8 levels were positively related to CIN risk (the 1st versus 4th quartile CD4,CD8 OR = 0.45[0.34, 0.60] and 0.34[0.26, 0.45] for CIN1, 0.32 [0.21, 0.48] and 0.24 [0.16, 0.38] for CIN2/3). Increased CD4 and CD8 levels were positively related to the occurrence of CIN(P-overall<0.01).CD4/CD8 levels and the risk of CIN1 followed a nonlinear "U-shape" (P-nonlinear <0.01). IL-10 levels and the risk of CIN1 followed a nonlinear "n-shape"(P-nonlinear <0.01).IL-10 levels were inversely related to the occurrence of CIN2/3(OR = 3.87, [2.49, 6.00],P-overall<0.01). The highest risk of CIN was observed in women with high-risk HPV, whose CD4 and CD8 levels were the highest(P-interaction < 0.01).Patients with the lowest IL-10 levels(IL-10 ≤ 53.17pg/ml) who are positive for high-risk HPV infection have the highest risk of CIN2/3(OR = 18.46,[9.33-36.51]). Nested case-control analysis observed a positive relationship between CD4,CD8 levels, and risk of CIN progression (CD4 OR = 0.34,[0.13, 0.94];CD8 OR = 0.27, [0.09, 0.79]),and an opposite relationship between IL-10 levels and risk of CIN progression (OR = 2.92, [1.09, 7.84]). Conclusions:Local vaginal CD4 and CD8 levels were positively correlated with CIN risk, and IL-10 levels were inversely correlated with CIN2/3, whether or not with high-risk HPV infection in Chinese women.

5522 Background: Enlonstobart is a PD-1 inhibitor that has demonstrated durable anti-tumor activity and acceptable safety in patients with PD-L1 positive recurrent/metastatic cervical cancer in a multicenter, single-arm, open-label, phase II study. At primary cutoff date (May 27, 2023), the overall survival (OS) was not reached. Here, we report results from a pre-planned further follow-up (August 20, 2024). Methods: Eligible patients were ≥18 years old with PD-L1-positive (combined positive score ≥ 1) cervical cancer who had progression during or after or intolerance to the first-line platinum-based therapy. A total of 107 patients received enlonstobart 240 mg every two weeks for up to 24 months or until disease progression, intolerable toxicities, or other study discontinuation criteria were met. At the updated cutoff date, analyses of progression-free survival (PFS) and OS in the full analysis set (FAS), which consisted of all patients who had received at least one dose of enlonstobart treatment, and the per protocol set (PPS), which consisted of all patients in the FAS who had at least one available postbaseline tumor assessment were conducted. Results: At the cutoff date of August 20 2024, the median follow-up time was 15.84 months (range 0.4 ~ 35.6 months). In FAS, the median PFS was 3.06 (95 % CI 2.23–6.90) months. The median OS was 19.38 (95 % CI 14.95–25.40) months and the estimated OS rate was 68.14 % (95 %CI 58.20–76.19) at 12 months, 50.78 % (95 % CI 40.46–60.20) at 18 months, and 42.84 % (95 % CI 32.76–52.53) at 24 months. In PPS, the median PFS was 3.81 (95 % CI 2.63–7.49) months. The median OS was 21.26 (95 % CI 15.44–27.66) months and the estimated OS rate was 71.53 % (95 %CI 61.48–79.40) at 12 months, 53.32 % (95 % CI 42.64–62.87) at 18 months, and 44.98 % (95 % CI 34.50–54.89) at 24 months. Conclusions: Enlonstobart monotherapy showed a promising survival in patients with PD-L1 positive recurrent/metastatic cervical cancer, whose disease experienced progression after first-line platinum-based therapy. Clinical trial information: NCT04886700 .

OBJECTIVE:Investigate the efficacy/safety of ociperlimab (anti-TIGIT monoclonal antibody [mAb]) + tislelizumab (anti-PD-1 mAb) in recurrent/metastatic (R/M) cervical cancer (CC). METHODS:Patients had R/M CC, received ≥1 prior chemotherapy, and were not amenable to curative treatment. In stage 1, 80 patients were randomized 1:1 to ociperlimab 900 mg + tislelizumab 200 mg every 3 weeks (cohort 1) or tislelizumab monotherapy (cohort 2). In stage 2, 98 additional patients were enrolled in cohort 1. Primary endpoint was blinded independent review committee-assessed objective response rate (ORR) by RECIST v1.1 for PD-L1+ subgroup and all-comers in cohort 1. RESULTS:Between March 2 and December 15, 2021, 178 patients were enrolled, and all were treated (cohort 1: 138; cohort 2: 40). ORR of cohort 1 PD-L1+ subgroup and all-comers were 27.4% (95% CI 18.2%-38.2%) and 23.2% (16.4%-31.1%), respectively. In cohort 1, median progression-free survival (PFS) was 3.0 months (95% CI 2.6-4.9) (all-comers) and 4.1 months (95% CI 2.7-6.9) (PD-L1+); median overall survival was 12.2 months (95% CI 9.9-16.6) (all-comers) and 16.4 months (95% CI, 10.4 months-not estimable) (PD-L1+). 70.3% of cohort 1 had ≥1 treatment-related adverse event (TRAE); 18.1% experienced ≥1 grade ≥3 TRAE. Immune-mediated AEs occurred in 35.5% of cohort 1. CONCLUSIONS:In patients with R/M CC who had received prior chemotherapy, ociperlimab + tislelizumab has promising antitumor activity in both all-comers and PD-L1+ subgroup, supporting further investigation of immune-modulating agent combinations for R/M CC. TRIAL REGISTRATION:ClinicalTrials.gov Identifier: NCT04693234; https://clinicaltrials.gov/study/NCT04693234?term=NCT04693234&rank=1; EudraCT: https://eudract.ema.europa.eu/2020-004657-77.

Although minor cytological abnormalities predict a low risk of high-grade lesions, their high reporting rates lead to a considerable number of high-grade lesion cases. We carried out this study to analyze the immediate risk and 5-year cumulative risk of high-grade cervical lesions in high-risk human papillomavirus (Hr-HPV)-positive patients with minor cytological abnormalities and to investigate the clinical significance of minor cytological abnormalities during follow-up in our single-center. A total of 1892 patients with positive Hr-HPV, cytology result of atypical squamous cells of undetermined significance (ASC-US) or low-grade squamous intraepithelial lesion (LSIL) and also underwent colposcopy and biopsy were selected to analyze the immediate risk of high-grade cervical lesions. Besides, a total of 832 patients with baseline histological results of CIN1 or below and 5-year follow-up data available were further used to analyze the 5-year cumulative risk of high-grade cervical lesions. The immediate incidence rates of CIN3 + in the ASC-US and LSIL groups were 6.27

Hepatocellular carcinoma (HCC) is the predominant form of liver cancer and ranks as the third leading cause of cancer-related mortality globally. The liver performs a wide range of tasks and is the primary organ responsible for metabolizing harmful substances and foreign compounds. Oxidative stress has a crucial role in growth and improvement of hepatocellular carcinoma (HCC). Nuclear factor erythroid 2 (1)-related factor 2 (Nrf2) is an element that regulates transcription located in the cytoplasm. It controls the balance of redox reactions by stimulating the expression of many genes that depend on antioxidant response elements. Nrf2 has contrasting functions in the normal, healthy liver and HCC. In the normal liver, Nrf2 provides advantageous benefits, while in HCC it promotes harmful effects that support the growth and survival of HCC. Continuous activation of Nrf2 has been detected in HCC and promotes its advancement and aggressiveness. In addition, Activation of Nrf2 may lead to immune evasion, weakening the immune cells’ ability to attack tumors and thereby promoting tumor development. Furthermore, chemoresistance in HCC, which is considered a form of stress response to chemotherapy medications, significantly impedes the effectiveness of HCC treatment. Stress management is typically accomplished by activating specific signal pathways and chemical variables. One important element in the creation of chemoresistance in HCC is nuclear factor-E2-related factor 2 (Nrf2). Nrf2 is a transcription factor that regulates the activation and production of a group of genes that encode proteins responsible for protecting cells from damage. This occurs through the Nrf2/ARE pathway, which is a crucial mechanism for combating oxidative stress within cells.

Abstract Objectives This study aims to develop an innovative, deep model for thymoma risk stratification using preoperative CT images. Current algorithms predominantly focus on radiomic features or 2D deep features and require manual tumor segmentation by radiologists, limiting their practical applicability. Methods The deep model was trained and tested on a dataset comprising CT images from 147 patients (82 female; mean age, 54 years ± 10) who underwent surgical resection and received subsequent pathological confirmation. The eligible participants were divided into a training cohort (117 patients) and a testing cohort (30 patients) based on the CT scan time. The model consists of two stages: 3D tumor segmentation and risk stratification. The radiomic model and deep model (2D) were constructed for comparative analysis. Model performance was evaluated through dice coefficient, area under the curve (AUC), and accuracy. Results In both the training and testing cohorts, the deep model demonstrated better performance in differentiating thymoma risk, boasting AUCs of 0.998 and 0.893 respectively. This was compared to the radiomic model (AUCs of 0.773 and 0.769) and deep model (2D) (AUCs of 0.981 and 0.760). Notably, the deep model was capable of simultaneously identifying lesions, segmenting the region of interest (ROI), and differentiating the risk of thymoma on arterial phase CT images. Its diagnostic prowess outperformed that of the baseline model. Conclusions The deep model has the potential to serve as an innovative decision-making tool, assisting on clinical prognosis evaluation and the discernment of suitable treatments for different thymoma pathological subtypes. Key Points • This study incorporated both tumor segmentation and risk stratification. • The deep model, using clinical and 3D deep features, effectively predicted thymoma risk. • The deep model improved AUCs by 16.1pt and 17.5pt compared to radiomic model and deep model (2D) respectively.

Cervical cancer(CCa)is a substantial global health concern,and its lymph node metastasis(LNM)significantly di-minishes patients'survival rates.1 Hence,a thorough investigation into the molecular mechanisms driving this progression is vital.Integrin alpha 2(ITGA2)plays critical roles in various tumorigenic processes via cancer-related signaling pathways.2 In this study,we advanced the un-derstanding of ITGA2's influence on CCa by revealing its role in promoting CCa LNM.Mechanistically,ITGA2 up-reg-ulates SNAIL,instigating epithelial-mesenchymal transi-tion(EMT)and activating the protein kinase B(AKT)/mammalian target of rapamycin(mTOR)pathway,thereby augmenting CCa LNM.Remarkably,the inhibitor E7820,by down-regulating ITGA2 expression,shows promise in attenuating EMT and LNM of CCa.Therefore,E7820 could be developed as a potential therapeutic agent for the treatment of CCa LNM.

Hexavalent chromium [Cr(VI)], one common environmental contaminant, has long been recognized as a carcinogen associated with lung cancer, but roles and mechanisms of Cr(VI)-induced epigenetic dysregulations in carcinogenesis remain to be investigated. In this study, we identified that RNA m5C methyltransferase NSUN2 was significantly upregulated in Cr(VI)-transformed cells and lung tissues of Cr(VI)-exposed mice. Inhibition of NSUN2 reduced cell proliferation, migration, colony formation and tube formation abilities. We found NSUN2-mediated m5C modification induced metabolic reprogramming and cell cycle by promoting the mRNA stabilities of ME1, GLUT3 and CDK2. In addition, knockdown of NSUN2 attenuated tumorigenesis and angiogenesis in vivo. RNA m5C reader ALYREF was identified to be involved in NSUN2-mediated m5C modification in Cr (VI)-induced carcinogenesis. Further study showed that EP300 induced NSUN2 upregulation through transcriptional activation by inducing histone modification at H3K27ac site for regulating Cr(VI) carcinogenesis. Our findings demonstrated novel role and mechanism of NSUN2 and epigenetic changes by increasing the RNA m5C modification that are important for Cr (VI)-induced carcinogenesis through NSUN2/ALYREF pathway. NSUN2, ALYREF, ME1, GLUT3 or/and CDK2 may be used as potential new biomarkers or/and therapeutic target(s) in the future.

High endothelial venules (HEV) are specialized blood vessels that mediate lymphocyte trafficking to lymph nodes. Tertiary lymphoid structures (TLS) are ectopic lymphoid formations that develop in inflamed, infected or tumoral tissues. TLS contain HEV and B cell follicles surrounded by a T-cell zone and are characterized by abundant chemokine expression. The presence of TLS and HEV in solid tumors is positively correlated with patient survival in many cancer types, and may even be predictive of better response to immune-checkpoint blockade. However, the molecular mechanisms underlying the intratumoral HEV and TLS formation remain unclear. Using murine syngeneic tumor models, we found that systemic activation of lymphotoxin beta receptor (LTBR) with an agonistic antibody induced tumor-specific HEV formation, increased dendritic cell (DC) and T cell infiltration, and enhanced T cell activation in the tumor. In vitro assays revealed that LTBR agonism directly upregulated activation and maturation markers in bone marrow-derived DCs and promoted DC-mediated CD4 and CD8 T cell activation. Single agent LTBR agonist treatment attenuated Colon26 tumor growth in a CD8 T cell-dependent manner. In combination treatment studies, LTBR agonism further augmented anti-tumor efficacy of anti-PD1 and of CAR-T therapy. Notably, LTBR agonist mAb treatment upregulated the expression of TLS-related chemokines and induced TLS-like structures in approximately 20% of treated tumors. To enhance TLS induction, we generated syngeneic tumor models engineered to express several cytokines/chemokines and performed an in vivo screen for tumor-associated TLS formation. We identified some factors that in combination with LTBR agonism induced B cell enrichment and immature TLS formation, while others promoted robust TLS induction and anti-tumor effect. TLS formation induced by combined LTBR agonism and cytokine expression is associated with augmented anti-tumor responses to anti-CTLA-4 treatment. By studying anti-tumor mechanisms of LTBR agonism-mediated HEV and TLS formation, this work informs the future therapeutic strategies to boost T cell infiltration and activation in solid tumors. Citation Format: Disi An, Guoying Chen, Wei Wang, Katja Mohrs, David DiLillo, Christopher Daly, Gavin Thurston, John Lin, Namita Gupta, Mickey Atwal, Frank Kuhnert. Boosting anti-tumor immunity by promoting high endothelial venule and tertiary lymphoid structure formation in solid tumors via LTBR agonism. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 4134.

Supplementary Methods and Supplementary Figure 1-20: Supplementary Figure 1 shows the growth of the tumors after orthotopic transplantation. Supplementary Figure 2 shows that over-expression of SIX1 promotes lymphangiogenesis and lymph-node metastasis of C33a cells in vivo. Supplementary Figure 3 shows the assay of cytokine and growth factor with antibody-based array. Supplementary Figure 4 shows the expression of the genes coding for the lymphangiogenic factors. Supplementary Figure 5 shows that SIX1 promotes VEGF-C expression in C33a cells. Supplementary Figure 6 shows that SIX1 promotes migration and tube-formation of HLECs. Supplementary Figure 7 shows the knock-down of VEGF-C in cervical cancer cells. Supplementary Figure 8 shows that the promotional effect of SIX1-expressing tumor cells on the migration and tube-formation of HLECs in vitro is mediated by VEGF-C. Supplementary Figure 9 shows that the promotional effect of SIX1-expressing tumor cells on lymphangiogenesis in vivo is mediated by VEGF-C. Supplementary Figure 10 shows that the promotional effect of SIX1-expressing C33a cells on lymphatic-vessel is mediated by VEGF-C. Supplementary Figure 11 shows that the promotional effect of SIX1 on lymph-node metastasis in vivo is mediated by VEGF-C. Supplementary Figure 12 shows the differences in gene expression levels between tumors in vivo and tumor cells cultured in vitro. Supplementary Figure 13 shows the immunohistochemical analysis of TGF-βs in tissue microarray of human CSCC specimens. Supplementary Figure 14 shows that SIX1 enhances the effect of TGF-β1 on VEGF-C production by tumor cells and tube-formation of HLECs in vitro. Supplementary Figure 15 shows that TGF-β contributes to the differences in VEGF-C expression between tumors in vivo and tumor cells cultured in vitro. Supplementary Figure 16 shows that TGF-β signaling is required for SIX1 to promote lymphangiogenesis and lymph-node metastasis in vivo. Supplementary Figure 17 shows that knocking down the expression of SMAD2 or SMAD3 or TβR1 suppresses TGF-β1 induced VEGF-C expression. Supplementary Figure 18 shows that higher expression of SIX1 correlates with higher expression of TβR1 in human CSCC specimens. Supplementary Figure 19 shows the binding of SIX1 to the VEGF-C promoter. Supplementary Figure 20 shows that knocking down the expression of SMAD2 and SMAD3 does not affect the binding of SIX1 to the VEGF-C promoter.

Background Stress keratin 17 (K17) is an intermediate filament keratin induced during would healing, inflammatory diseases, papillomavirus infection as well as in cancers. Our prior work described an immune-regulatory role of K17 in three distinct mouse cancer models.1–3 We showed K17 expression decreased CXCL9 expression in the tumor microenvironment (TME), reduced T cell infiltration, and contributed to disease progression.1–3 Additionally, K17 conferred resistance to immune checkpoint blockade (ICB) treatment in a HNC mouse model and in patients.2 In current study, we identify tumor-intrinsic factors regulated by K17 that affects immune response in the TME to uncover novel mechanisms underlying K17-mediated immune regulation. Methods We used MOC2 cells as a syngeneic mouse HNC model. K17 was knocked out from parental MOC2 cells by Crisper/Cas9 and a single cell clone of K17KO cells was generated. Bulk RNA sequencing of WT cells and K17KO cells were performed in triplicate. This K17KO clone and parental MOC2 cells were injected subcutaneously into C57BL/6 and NSG mice to confirm the rejection of K17KO cells was only observed in immunocompetent mice. MOC2 tumors and K17KO tumors growing in C57BL/6 mice were collected at day 7 post implantation, at the peak of K17KO tumor growth for single cell RNA seq analyses. Results Our prior scRNA-seq analysis of sorted CD45+ cells showed that myeloid cells are the major producers of CXCL9 among immune cells in MOC2 tumors. And macrophages from WT tumors expressed less Nos2 (M1 marker) and more Arg1 (M2 marker) compared to those from K17KO tumors. Our new scRNA-seq data encompassing all cells in TME confirmed that CXCL9 expression is mainly detected in the myeloid compartment, and is low in fibroblasts and tumor cells. Additionally, single cell analysis of epithelial cells from tumors and bulk RNA seq of tumor cells in tissue culture identified 148 differentially expressed genes in common that were upregulated in WT tumor cells both in vitro and in vivo. Among these genes were CCL2 and galectin-3, known to contribute to M2-macrophage polarization.4 5 We therefore hypothesize that K17 expression in tumor cells upregulates CCL2 and galectin-3 expression, which in turn directs M2 polarization of macrophages to induce immune evasion in head and neck cancers. Conclusions We identified CCL2 and galectin-3 as tumor factors regulated by K17 expression. Validation studies are needed to confirm their protein expressions and their direct role in tumor-macrophage communications. References Wang W, Uberoi A, Spurgeon ME, Gronski E, Majerciak V, Lobanov A, Hayes M, Loke A, Zheng ZM, Lambert PF. Stress keratin 17 enhances papillomavirus infection-induced disease by downregulating T cell recruitment. PLoS Pathog, 2020 Jan 22;16(1):e1008206. Wang W, Lozar T, Golfinos AE, Lee D, Gronski E, Shaw EW, Hayes M, Bruce JY, Kimple RJ, Hu R, Harari PM, Xu J, Keske A, Sondel PM, Fitzpatrick MB, Dinh HQ, Lambert PF. Stress Keratin 17 Expression in Head and Neck Cancer Contributes to Immune Evasion and Resistance to Immune-Checkpoint Blockade. Clinical Cancer Research, 2022 May 27;OF1-OF16. doi: 10.1158/1078-0432. PMID: 35621713. Wang W*, Spurgeon ME*, Pope A, McGregor S, Shaw EW, Gronski E, Lambert PF. Stress keratin 17 and estrogen support viral persistence and modulate the immune environment during cervicovaginal murine papillomavirus infection. Proceedings of the National Academy of Sciences. 2023 March 14; https://doi.org/10.1073/pnas.2214225120 (*Co-First Authors) Alison C MacKinnon, Sarah L Farnworth, Philip S Hodkinson, Neil C Henderson, Kirsten M Atkinson, Hakon Leffler, Ulf J Nilsson, Christopher Haslett, Stuart J Forbes, Tariq Sethi. Regulation of Alternative Macrophage Activation by Galectin-31. J Immunol 15 February 2008;180(4):2650–2658. https://doi.org/10.4049/jimmunol.180.4.2650 Elena Sierra-Filardi, Concha Nieto, Ángeles Domínguez-Soto, Rubén Barroso, Paloma Sánchez-Mateos, Amaya Puig-Kroger, María López-Bravo, Jorge Joven, Carlos Ardavín, José L Rodríguez-Fernández, Carmen Sánchez-Torres, Mario Mellado, Ángel L Corbí. CCL2 Shapes Macrophage Polarization by GM-CSF and M-CSF: Identification of CCL2/CCR2-Dependent Gene Expression Profile. J Immunol 15 April 2014;192(8):3858–3867. Ethics Approval Experiments were approved and performed in accordance with guidelines approved by the Association for Assessment of Laboratory Animal Care, at the University of Wisconsin Medical School. This study was approved by the University of Wisconsin School of Medicine and Public Health Institutional Animal Care and Use Committee under protocol number M005871.