Liquid biopsy is a promising biomarker for cancer detection and prediction of therapy responses. In this study, we developed new antibody-based sandwich assays to measure three extracellular vesicle (EV) subsets that express cell surface vimentin (CSV) and co-express epithelial cell adhesion molecule (EpCAM) or PD-L1 in plasma. Fifty-seven non-small cell lung cancer (NSCLC) patients who underwent complete resection and 89 stage IV NSCLC patients who received anti-PD-1 antibody were enrolled. Correlations between the plasma EV subsets at baseline and various clinicopathological factors were evaluated. Plasma EpCAM/CSV-EV levels were significantly higher in stage I NSCLC patients compared to healthy donors, suggesting this may be a valuable biomarker for detecting early-stage NSCLC. High plasma CSV/CSV-EV levels at baseline were significantly correlated with a poor prognosis after treatment, both in the surgery cohort and the anti-PD-1 antibody cohort, which suggests that this may be useful for predicting the post-treatment prognosis in NSCLC patients. High plasma CSV/PD-L1-EV levels at baseline were significantly correlated with poor postoperative prognosis. However, high plasma CSV/PD-L1-EV levels at baseline were significantly correlated with a favorable clinical response and prognosis following anti-PD-1 antibody treatment in stage IV NSCLC. Therefore, plasma CSV-related EV subsets at baseline may be attractive biomarkers for early cancer detection (EpCAM/CSV), prediction of postoperative prognosis (CSV/CSV), and prediction of clinical responses and prognosis after anti-PD-1 antibody therapy (CSV/PD-L1). These EV subsets can be easily measured repetitively at appropriate timing. These novel liquid biopsies may be additional and complementary diagnostic biomarkers for NSCLC patients.

Lateral olfactory tract usher substance (LOTUS), also known as cartilage acidic protein-1B (Crtac1B), is a potent endogenous antagonist of Nogo receptor type-1 (NgR1). LOTUS exists in both membrane-bound (m-LOTUS) and soluble (s-LOTUS) forms. m-LOTUS inhibits NgR1 by binding to its C-terminal domain (UA-EC domain), thereby blocking ligand interactions, whereas s-LOTUS disrupts NgR1 signaling by interfering with its interaction with the p75 co-receptor. However, the molecular characteristics of s-LOTUS, including the functional domains required for secretion and NgR1 binding, remain unclear. In this study, we identified the N-terminal 35Met-532Pro region as essential for s-LOTUS secretion. While N-type glycosylation patterns remained unchanged, differences in intracellular localization with the Golgi apparatus were observed between secretory and non-secretory forms. The non-secretory form exhibited higher aggregation and ubiquitination levels. Functionally, the soluble 35Met-532Pro fragment bound to both NgR1 and p75, disrupting the NgR1-p75 complex. In contrast, the UA-EC domain, corresponding to the functional domain of m-LOTUS, bound only to NgR1 and did not interfere with p75 interaction. A growth cone collapse assay using cultured olfactory bulb neurons from lotus-deficient embryonic mice demonstrated that exogenous 35Met-532Pro, but not UA-EC, inhibited Nogo66-induced growth cone collapse. These findings indicate that the N-terminal 35Met-532Pro region of s-LOTUS functions as its active domain, antagonizing NgR1 signaling through dual binding to NgR1 and p75. In contrast, the UA-EC domain lacks this antagonistic property, highlighting the distinct functional domains and mechanisms of action between s-LOTUS and m-LOTUS.

A subset of meningiomas, including the majority of grade II/III and a proportion of grade I tumors, are refractory to repetitive resection and radiotherapy, and are frequently fatal. Reports of effective medical therapy are extremely rare. Literature suggests that immune checkpoint systems might have a role in aggressive meningiomas. A single-arm, phase 2 study was conducted to evaluate the efficacy and safety of nivolumab for meningiomas refractory to surgery and radiotherapy. Nivolumab (480 mg) was administered intravenously every 4 weeks. The primary endpoint was the objective response rate (ORR) determined by a central independent review committee. To avoid premature discontinuation of potentially effective immunotherapy, response was evaluated based on the iRANO (meningioma) criteria, which is based on the RANO (meningioma) criteria (Neuro Oncol 21:26-36, 2019) with the integration of the immune-related response criteria outlined previously (Lancet Oncol 16:e534-e542, 2015). Archival tumor specimens were obtained from all 29 cases. A total of 29 patients started the study therapy. Response assessment by the central review committee was performed for 28 patients: grade I in 5, grade II in 19, andgrade III in 4 by definition of WHO2016. The best overall response was PR in 1, SD in 13, and uPD/cPD in 14. The ORR was 3.6% and PFS-6 was 23.9%. Nivolumab was well tolerated. There was no case with mutations in the DNA mismatch repair genes. One patient who had multiple grade I meningiomas with biopsy-proven lung metastases showed sustained PR following initial radiological progression. The TMB of the tumor was 8.1/MB. Some specific gene sets were shown to be enriched in the PR case. Although nivolumab monotherapy failed to meet the prespecified primary endpoint, our study demonstrated that a subset of patients could benefit from the therapy and that immune-related response criteria are necessary to evaluate immunotherapy for meningiomas.

2084 Background: The majority of meningiomas, which are the most common central nervous system (CNS) tumors, are benign and often cured by surgical resection alone. However, 20%–30% of meningiomas can be malignant tumors of CNS WHO grade II or III that are refractory to repetitive resection and radiotherapy. Moreover, a proportion of grade I meningiomas is associated with an aggressive clinical course reminiscent of grade II tumors. Reports of effective medical therapy for those tumors are extremely rare. Methods: A single-arm, open-label, phase 2 study was conducted to evaluate the efficacy and safety of nivolumab for meningiomas refractory to surgery and radiotherapy. Nivolumab (480 mg) was administered intravenously every 4 weeks and continued until tumor progression or unacceptable toxicity for up to 365 days. The primary endpoint was the objective response rate (ORR) determined by a central independent review committee. With a one-sided significance level of 5%, a power of 80%, a threshold response rate of 5%, and an expected response rate of 20%, the required sample size was calculated to be 27 patients using the exact binomial test. Considering a 10% attrition rate, the target sample size was set at 29. To avoid premature discontinuation of potentially effective immunotherapy, response was evaluated based on the iRANO (meningioma) criteria, which is based on the RANO (meningioma) criteria (Neuro Oncol 21(1):26-36, 2019) with the integration of the immune-related response criteria outlined previously (Lancet Oncol 16(15):e534-e542, 2015). Archival tumor specimens from all 29 cases were obtained for biomarker analyses. Results: A total of 29 patients started the study therapy. Response assessment by the central review committee was performed for 28 patients: grade I meningioma in 5, grade II in 19, and grade III in 4 by definition of the 2016 WHO criteria. The best overall response was PR in 1, SD in 13, and uPD/cPD in 14. The ORR was 3.6% and progression-free survival at 6 months was 23.9%. Biallelic inactivation of the NF2 gene was detected in 20/27 cases (74%), whereas biallelic inactivation of the CDKN2A gene was identified in 7/27 cases (26%). One patient who had multiple grade I meningiomas with biopsy-proven lung metastases showed near CR following initial radiological progression. The TMB of the tumor was 8.1/MB. Next-generation sequencing found that none of the tumors had mutations of the DNA mismatch repair genes. Nivolumab was well tolerated. Conclusions: Although nivolumab monotherapy failed to meet the prespecified primary endpoint, our study demonstrated that a subset of patients could benefit from the therapy and that immune-related response criteria are necessary to evaluate immunotherapy for meningiomas. Clinical trial information: jRCT2031190074 .

[This corrects the article DOI: 10.1371/journal.pone.0016182.].

The cancer-immunity cycle has been proposed, and immunotherapeutic approaches, such as vaccines using self-antigens and adjuvant, have been employed for a long time, but their therapeutic effects have been limited. However, recent studies have demonstrated that immune checkpoint inhibitors(ICIs)are able to achieve high therapeutic efficacy, in a variety of cancer types. Today, advances in multi-omics technologies, including single-cell RNA sequencing(scRNA-seq), spatial transcriptomics, and multicolor immunostaining technologies, have made it possible to analyze immune cell dynamics at the single-cell level in a greater detail. While CD8+ T cells play a central role in the antitumor immune response, recent findings have revealed the existence of various subsets within the CD8+ T cell population. During the research on T cell exhaustion, the in vivo dynamics of T progenitor exhausted cells(Tpex cells)/stem cell memory T cells(TSCM)have also been elucidated. Tpex/TSCM cells are present in tumor-draining lymph nodes and within tumors and have reported to be an important target for ICIs. Furthermore, interactions between CD4+ T cells, dendritic cells(DCs), B cells, and CD8+ T cells within the tumor microenvironment are crucial for the induction of cytotoxic CD8+ effector T cells. In human tumor tissues, cancer cells exhibit heterogeneous characteristics and the tumor microenvironment varies depending on cancer type, subtypes, and individual patients. To enhance the anti-tumor effects of CD8+ T cells in immunotherapy, it is essential to achieve a more precise understanding of the in vivo dynamics of CD8+ T cells in each patient and to develop strategies for their effective intervention. This knowledge will then be applied to the development of vaccine therapies, combination immunotherapies, and cellular immunotherapy.

PURPOSE The humanized antivascular endothelial growth factor (VEGF) antibody bevacizumab (Bev) is efficacious for the treatment of NF2-related schwannomatosis (NF2), previously known as neurofibromatosis type 2. This study evaluated the safety and efficacy of a VEGF receptor (VEGFR) vaccine containing VEGFR1 and VEGFR2 peptides in patients with NF2 with progressive schwannomas (jRCTs031180184). MATERIALS AND METHODS VEGFR1 and VEGFR2 peptides were injected subcutaneously into infra-axillary and inguinal regions, once a week for 4 weeks and then once a month for 4 months. The primary end point was safety. Secondary end points included tolerability, hearing response, imaging response, and immunologic response. RESULTS Sixteen patients with NF2 with progressive schwannomas completed treatment and were assessed. No severe vaccine-related adverse events occurred. Among the 13 patients with assessable hearing, word recognition score improved in five patients at 6 months and two at 12 months. Progression of average hearing level of pure tone was 0.168 dB/mo during the year of treatment period, whereas long-term progression was 0.364 dB/mo. Among all 16 patients, a partial response was observed in more than one schwannoma in four (including one in which Bev had not been effective), minor response in 5, and stable disease in 4. Both VEGFR1-specific and VEGFR2-specific cytotoxic T lymphocytes (CTLs) were induced in 11 patients. Two years after vaccination, a radiologic response was achieved in nine of 20 assessable schwannomas. CONCLUSION This study demonstrated the safety and preliminary efficacy of VEGFR peptide vaccination in patients with NF2. Memory-induced CTLs after VEGFR vaccination may persistently suppress tumor progression.

Programmed death 1 (PD-1)/programmed death-ligand 1 inhibitors are commonly used to treat various cancers, including melanoma. However, their efficacy as monotherapy is limited, and combination immunotherapies are being explored to improve outcomes. In this study, we investigated a combination immunotherapy involving an anti-PD-1 antibody that blocks the major adaptive immune-resistant mechanisms, a BRAF inhibitor that inhibits melanoma cell proliferation, and multiple primary immune-resistant mechanisms, such as cancer cell-derived immunosuppressive cytokines, and a Toll-like receptor 7 agonist that enhances innate immune responses that promote antitumor T-cell induction and functions. Using a xenogeneic nude mouse model implanted with human BRAF-mutated melanoma, a BRAF inhibitor vemurafenib was found to restore T-cell-stimulatory activity in conventional dendritic cells by reducing immunosuppressive cytokines, including interleukin 6, produced by human melanoma. Additionally, intravenous administration of the Toll-like receptor 7 agonist DSR6434 enhanced tumor growth inhibition by vemurafenib through stimulating the plasmacytoid dendritic cells/interferon-α/natural killer cell pathways and augmenting the T-cell-stimulatory activity of conventional dendritic cells. In a syngeneic mouse model implanted with murine BRAF-mutated melanoma, the vemurafenib and DSR6434 combination synergistically augmented the induction of melanoma antigen gp100-specific T cells and inhibited tumor growth. Notably, only triplet therapy with vemurafenib, DSR6434, and the anti-PD-1 antibody resulted in complete regression of SIY antigen-transduced BRAF-mutated melanoma in a CD8 T-cell-dependent manner. These findings indicate that a triple-combination strategy targeting adaptive and primary resistant mechanisms while enhancing innate immune responses that promote tumor-specific T cells may be crucial for effective tumor eradication.

Background: We have developed a glypican-1 (GPC1) targeted CAR-T cell with strong anti-tumor effects against solid tumors in vivo (Kato et al 2020 eLife). In general, an antibody is used for detection of target protein expression in the tumor to identify appropriate targets for the CAR-T cell therapy in solid tumors. However, the local conjunction between CAR-T cells and the target is not exactly the same in the antibody and the target because CAR-T cell recognizes the target by single-chain variable fragment (scFv). In this study, we evaluated the anti-GPC1 scFv-Fc fusion protein as a detection tool of GPC1 expression in human cancer cell lines. Methods: The sequences encoding the anti-GPC1 scFv are based on the sequence of anti-GPC1 mAb (clone: 1-12) that recognizes human and mouse GPC1 (Harada et al., 2017). The sequences of anti GPC-1 scFv were cloned into pCAG-Neo mIgG2a-Fc plasmid for the generation of anti GPC1_scFv-mIgG2a_Fc fusion proteins. The plasmids were transfected into 293 T cells and the fusion proteins were purified from their culture supernatants using Antibody TCS Purification Kit. LK2-GPC1 (a GPC1 overexpressing lung carcinoma cell line), TE8 (an esophageal carcinoma cell line with endogenous GPC1) and LK2-mock (a GPC1-negative lung carcinoma cell line) were used for the evaluation of the anti-GPC1 scFv-Fc fusion protein. Results: In FACS assays, fluorescence microscopy analysis and immunohistochemistry staining, GPC1 expression was successfully detected by both anti-GPC1 scFv of VL-VH and VH-VL in LK2-GPC1 and TE-8, and not detected in LK2-mock. Localization of GPC1 expression on the cell membrane was confirmed by fluorescence microscopy. Perspective: Further comparative evaluation between anti-GPC1 antibody and anti-GPC1 scFv-Fc fusion proteins are needed especially in human tumor tissues. We plan to evaluate a relationship between GPC1 positivity in various human tumor tissues detected by anti-GPC1 scFv-Fc fusion protein and CAR-T cell cytotoxicity against the tissue. Citation Format: Takeshi Sawada, Tomonori Yaguchi, Yuko Sakai, Chinatsu Suzuki, Kenji Morii, Masahiko Aoki, Masaru Fukahori, Satoshi Serada, Tetsuji Naka, Jun K. Yamashita, Yutaka Kawakami, Takako E. Nakajima. Evaluation of the anti-GPC1 scFv-Fc fusion protein as a detection tool of GPC1 expression in human cancers [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 907.

<p>Supplementary materials and methods and figures</p>

<p>Supplementary Figures S1-9 and Tables S1-3. Supplementary Figure S1 - Effects of knockdown of NUAK2 on melanoma cells and specificity of the p-AKT antibody. Supplementary Figure S2 - Immunohistochemical analyses of p-Akt and NUAK2 expression in clinical specimens. Supplementary Figure S3 - Kaplan-Meier curves for overall survival of Acral and Non-CSD melanoma patients. Supplementary Figure S4 - Knockdown of NUAK2 induces apoptosis in SM2-1 melanoma cells. Supplementary Figure S5 - Immunohistochemistry showing the expression of p21 following inhibition of the PI3K pathway by LY294002. Supplementary Figure S6 - Schematic diagram of the regulation of the cell cycle machinery by NUAK2 and PI3K pathways. Supplementary Figure S7 - Knockdown of CDK2 by siCDK2 in C32 and mel18 melanoma cells. Supplementary Figure S8 - Immunohistochemical analyses of the expression of CDK2 in clinical specimens. Supplementary Figure S9 - Expression of NUAK2, PTEN, p-Akt, Akt and CDK2, and cell proliferation in various melanoma cell lines treated with Roscovitine. Supplementary Table S1 - Clinical parameters and expressions of CDK2, p-Akt and NUAK2 of 56 acral melanomas and 35 non-CSD melanomas with survival information. Supplementary Table S2 - Genomic status of NUAK2 and PTEN gene in each cell line. Supplementary Table S3 - Expression of p27 and CDK2 in primary melanomas with or without over-expression of NUAK2 and p-Akt.</p>

Myo1b is highly expressed in human HNSCC.

Detection of focal lymph node metastasis by IHC for Turbo-GFP.

Schematic model of MIF function in two different types of glioma cells containing wild-type or mutant P53.

BackgroundRecent advances in immune checkpoint blockade (ICB) have improved patient prognosis in mismatch repair-deficient and microsatellite instability-high colorectal cancer (dMMR/MSI-H CRC); however, PD-1 blockade has faced a challenge in early progressive disease. We aimed to understand the early event in ICB resistance using an in vivo model.MethodsWe subcutaneously transplanted the MC38 colon cancer cells into C57BL/6 mice, intraperitoneally injected anti-PD-1 antibody and then isolated ICB-resistant subclones from the recurrent tumors.ResultsComparative gene expression analysis discovered seven genes significantly downregulated in the ICB-resistant cells. Tumorigenicity assay of the MC38 cells knocked out each of the seven candidate genes into C57BL/6 mice treated with anti-PD-1 antibody and bioinformatics analysis of the relationship between the expression of the seven candidate genes and the outcome of cancer patients receiving immunotherapy identified Rtp4, an interferon-stimulated gene and a chaperon protein of G protein-coupled receptors, as a gene involved in ICB resistance. Immunohistochemical analysis of transplanted tumor tissues demonstrated that anti-PD-1 antibody failed to recruit T lymphocytes in the Rtp4-KO MC38 cells. Mouse and human RTP4 expression could be silenced via histone H3 lysine 9 (H3K9) trimethylation, and public transcriptome data indicated the high expression level of RTP4 in most but not all of dMMR/MSI-H CRC.ConclusionsWe clarified that RTP4 could be silenced by histone H3K9 methylation as the early event of ICB resistance. RTP4 expression could be a promising biomarker for predicting ICB response, and the combination of epigenetic drugs and immune checkpoint inhibitors might exhibit synergistic effects on dMMR/MSI-H CRC.

PDF file, 1193K, Figure S1. Snail+ tumor cells frequently metastasize into bone marrow. Figure S2. Differentiation activity of the specific subpopulations isolated from the Snail+ tumor-stimulated BMCs. Figure S3. The Snail+ tumor-induced MSCs promote tumor metastatic seeding into bone. Figure S4. Increase of fstl1 and its receptor dip2a mRNA expressions in Snail+ tumor cells. Figure S5. FSTL1-knockdown suppresses metastatic seeding of human tumor cells into bone. Figure S6. FSTL1-induced cancer bone metastasis mechanism.