Single-cell analysis reveals cellular and molecular factors counteracting HPV-positive oropharyngeal cancer immunotherapy outcomes.

BACKGROUND: Oropharyngeal squamous cell carcinoma (OPSCC) induced by human papillomavirus (HPV-positive) is associated with better clinical outcomes than HPV-negative OPSCC. However, the clinical benefits of immunotherapy in patients with HPV-positive OPSCC remain unclear. METHODS: To identify the cellular and molecular factors that limited the benefits associated with HPV in OPSCC immunotherapy, we performed single-cell RNA (n=20) and T-cell receptor sequencing (n=10) analyses of tonsil or base of tongue tumor biopsies prior to immunotherapy. Primary findings from our single-cell analysis were confirmed through immunofluorescence experiments, and secondary validation analysis were performed via publicly available transcriptomics data sets. RESULTS: We found significantly higher transcriptional diversity of malignant cells among non-responders to immunotherapy, regardless of HPV infection status. We also observed a significantly larger proportion of CD4+ follicular helper T cells (Tfh) in HPV-positive tumors, potentially due to enhanced Tfh differentiation. Most importantly, CD8+ resident memory T cells (Trm) with elevated KLRB1 (encoding CD161) expression showed an association with dampened antitumor activity in patients with HPV-positive OPSCC, which may explain their heterogeneous clinical outcomes. Notably, all HPV-positive patients, whose Trm presented elevated KLRB1 levels, showed low expression of CLEC2D (encoding the CD161 ligand) in B cells, which may reduce tertiary lymphoid structure activity. Immunofluorescence of HPV-positive tumors treated with immune checkpoint blockade showed an inverse correlation between the density of CD161+ Trm and changes in tumor size. CONCLUSIONS: We found that CD161+ Trm counteracts clinical benefits associated with HPV in OPSCC immunotherapy. This suggests that targeted inhibition of CD161 in Trm could enhance the efficacy of immunotherapy in HPV-positive oropharyngeal cancers. TRIAL REGISTRATION NUMBER: NCT03737968.

A Phase II Open-Label Randomized Clinical Trial of Preoperative Durvalumab or Durvalumab plus Tremelimumab in Resectable Head and Neck Squamous Cell Carcinoma.

PURPOSE: Clinical implications of neoadjuvant immunotherapy in patients with locally advanced but resectable head and neck squamous cell carcinoma (HNSCC) remain largely unexplored. PATIENTS AND METHODS: Patients with resectable HNSCC were randomized to receive a single dose of preoperative durvalumab (D) with or without tremelimumab (T) before resection, followed by postoperative (chemo)radiotherapy based on multidisciplinary discretion and 1-year D treatment. Artificial intelligence (AI)-powered spatial distribution analysis of tumor-infiltrating lymphocytes and high-dimensional profiling of circulating immune cells tracked dynamic intratumoral and systemic immune responses. RESULTS: Of the 48 patients enrolled (D, 24 patients; D+T, 24 patients), 45 underwent surgical resection per protocol (D, 21 patients; D+T, 24 patients). D±T had a favorable safety profile and did not delay surgery. Distant recurrence-free survival (DRFS) was significantly better in patients treated with D+T than in those treated with D monotherapy. AI-powered whole-slide image analysis demonstrated that D+T significantly reshaped the tumor microenvironment toward immune-inflamed phenotypes, in contrast with the D monotherapy or cytotoxic chemotherapy. High-dimensional profiling of circulating immune cells revealed a significant expansion of T-cell subsets characterized by proliferation and activation in response to D+T therapy, which was rare following D monotherapy. Importantly, expansion of specific clusters in CD8+ T cells and non-regulatory CD4+ T cells with activation and exhaustion programs was associated with prolonged DRFS in patients treated with D+T. CONCLUSIONS: Preoperative D±T is feasible and may benefit patients with resectable HNSCC. Distinct changes in the tumor microenvironment and circulating immune cells were induced by each treatment regimen, warranting further investigation.

KPT6566 induces apoptotic cell death and suppresses the tumorigenicity of testicular germ cell tumors.

Testicular germ cell tumors (TGCTs) frequently affect adolescent and young adult males. Although TGCT is more responsive to cisplatin-based chemotherapy than other solid tumors, some patients are nonresponders, and following treatment, many patients continue to experience acute and long-term cytotoxic effects from cisplatin-based chemotherapy. Consequently, it is imperative to develop new therapeutic modalities for treatment-resistant TGCTs. Peptidyl-prolyl isomerase (Pin1) regulates the activity and stability of many cancer-associated target proteins. Prior findings suggest that Pin1 contributes to the pathogenesis of multiple human cancers. However, the specific function of Pin1 in TGCTs has not yet been elucidated. TGCT cell proliferation and viability were examined using cell cycle analysis and apoptosis assays following treatment with KPT6566, a potent, selective Pin1 inhibitor that covalently binds to the catalytic domain of Pin1. A xenograft mouse model was used to assess the effect of KPT6566 on tumor growth in vivo. KPT6566 effectively suppressed cell proliferation, colony formation, and ATP production in P19 and NCCIT cells. Further, KPT6566 induced apoptotic cell death by generating cellular reactive oxygen species and downregulating the embryonic transcription factors Oct-4 and Sox2. Finally, KPT6566 treatment significantly reduced tumor volume and mass in P19 cell xenografts. The Pin1 inhibitor KPT6566 has significant antiproliferative and antitumor effects in TGCT cells. These findings suggest that Pin1 inhibitors could be considered as a potential therapeutic approach for TGCTs.

Motion Estimation and Hand Gesture Recognition-Based Human-UAV Interaction Approach in Real Time.

As an alternative to traditional remote controller, research on vision-based hand gesture recognition is being actively conducted in the field of interaction between human and unmanned aerial vehicle (UAV). However, vision-based gesture system has a challenging problem in recognizing the motion of dynamic gesture because it is difficult to estimate the pose of multi-dimensional hand gestures in 2D images. This leads to complex algorithms, including tracking in addition to detection, to recognize dynamic gestures, but they are not suitable for human-UAV interaction (HUI) systems that require safe design with high real-time performance. Therefore, in this paper, we propose a hybrid hand gesture system that combines an inertial measurement unit (IMU)-based motion capture system and a vision-based gesture system to increase real-time performance. First, IMU-based commands and vision-based commands are divided according to whether drone operation commands are continuously input. Second, IMU-based control commands are intuitively mapped to allow the UAV to move in the same direction by utilizing estimated orientation sensed by a thumb-mounted micro-IMU, and vision-based control commands are mapped with hand's appearance through real-time object detection. The proposed system is verified in a simulation environment through efficiency evaluation with dynamic gestures of the existing vision-based system in addition to usability comparison with traditional joystick controller conducted for applicants with no experience in manipulation. As a result, it proves that it is a safer and more intuitive HUI design with a 0.089 ms processing speed and average lap time that takes about 19 s less than the joystick controller. In other words, it shows that it is viable as an alternative to existing HUI.

Distinct exhaustion features of T lymphocytes shape the tumor-immune microenvironment with therapeutic implication in patients with non-small-cell lung cancer.

BACKGROUND: Reinvigoration of T-cell exhaustion with antibodies has shown promising efficacy in patients with non-small-cell lung cancer (NSCLC). However, the characteristics of T-cell exhaustion with regard to tumor-infiltrating lymphocytes (TILs) are poorly elucidated in NSCLC. Here, we investigated the exhaustion status of TILs in NSCLC patients at the intraindividual and interindividual levels. METHODS: We obtained paired peripheral blood, normal adjacent tissues, peritumoral tissues, and tumor tissues from 96 NSCLC patients. Features of T-cell exhaustion were analyzed by flow cytometry. T cells were categorized according to their programmed cell death-1 (PD-1) expression (PD-1high, PD-1int, and PD-1neg cells). Patients were classified based on the presence or absence of discrete PD-1high CD8+ TILs. Production of effector cytokines by CD8+ TILs was measured after T-cell stimulation with or without antibodies against immune checkpoint receptors. RESULTS: Progressive T-cell exhaustion with marked expression of exhaustion-related markers and diminished production of effector cytokines was observed in PD-1high CD8+ TILs compared with PD-1int and PD-1neg CD8+ TILs. Patients with distinct PD-1high CD8+ TILs (PD-1high expressers) exhibited characteristics associated with a favorable anti-PD-1 response compared with those without these lymphocytes (non-PD-1high expressers). Combined inhibition of dual immune checkpoint receptors further restored effector cytokine production by CD8+ TILs following T-cell stimulation. PD-1high CD8+ T lymphocyte populations in the peripheral blood and tumors were significantly correlated. CONCLUSIONS: T-cell exhaustion was differentially regulated among individual patients and was prominent in a subgroup of NSCLC patients who may benefit from PD-1 blockade or combined blockade of other immune checkpoint receptors.

Dysregulation of TFH-B-TRM lymphocyte cooperation is associated with unfavorable anti-PD-1 responses in EGFR-mutant lung cancer.

Patients with non-small cell lung cancer (NSCLC) with epidermal growth factor receptor (EGFR) mutations exhibit an unfavorable response to PD-1 inhibitor through unclear mechanisms. Hypothesizing that EGFR mutations alter tumor-immune interactions, we compare tumor-infiltrating lymphocytes between EGFR mutant (EGFR-MT) and wild type (EGFR-WT) tumors through single-cell transcriptomic analysis. We find that B cells, CXCL13-producing follicular helper CD4+ T (TFH)-like cells, and tissue-resident memory CD8+ T (TRM)-like cells decreased in EGFR-MT tumors. The NOTCH-RBPJ regulatory network, which is vital for persistence of TRM state, is perturbed, and the interactions between TFH and B cells through the CXCL13-CXCR5 axis disappear in EGFR-MT tumors. Notably, the proportion of TRM-like cells is predictive for anti-PD-1 response in NSCLC. Our findings suggest that the impairment of TFH-B-TRM cooperation in tertiary lymphoid structure formation, accompanied by the dysregulation of TRM homeostasis and the loss of TFH-B crosstalk, underlies unfavorable anti-PD-1 response in EGFR-MT lung tumors.

Performance Index for Extrinsic Calibration of LiDAR and Motion Sensor for Mapping and Localization.

Light Detection and Ranging (LiDAR) is a sensor that uses a laser to represent the surrounding environment in three-dimensional information. Thanks to the development of LiDAR, LiDAR-based applications are being actively used in autonomous vehicles. In order to effectively use the information coming from LiDAR, extrinsic calibration which finds the translation and the rotation relationship between LiDAR coordinate and vehicle coordinate is essential. Therefore, many studies on LiDAR extrinsic calibration are steadily in progress. The performance index (PI) of the calibration parameter is a value that quantitatively indicates whether the obtained calibration parameter is similar to the true value or not. In order to effectively use the obtained calibration parameter, it is important to validate the parameter through PI. Therefore, in this paper, we propose an algorithm to obtain the performance index for the calibration parameter between LiDAR and the motion sensor. This performance index is experimentally verified in various environments by Monte Carlo simulation and validated using CarMaker simulation data and real data. As a result of verification, the PI of the calibration parameter obtained through the proposed algorithm has the smallest value when the calibration parameter has a true value, and increases as an error is added to the true value. In other words, it has been proven that PI is convex to the calibration parameter. In addition, it is able to confirm that the PI obtained using the proposed algorithm provides information on the effect of the calibration parameters on mapping and localization.

Tumor-Infiltrating Regulatory T-cell Accumulation in the Tumor Microenvironment Is Mediated by IL33/ST2 Signaling.

Regulatory T cells (Treg) are enriched in the tumor microenvironment (TME) and suppress antitumor immunity; however, the molecular mechanism underlying the accumulation of Tregs in the TME is poorly understood. In various tumor models, tumor-infiltrating Tregs were highly enriched in the TME and had significantly higher expression of immune checkpoint molecules. To characterize tumor-infiltrating Tregs, we performed bulk RNA sequencing (RNA-seq) and found that proliferation-related genes, immune suppression-related genes, and cytokine/chemokine receptor genes were upregulated in tumor-infiltrating Tregs compared with tumor-infiltrating CD4+Foxp3- conventional T cells or splenic Tregs from the same tumor-bearing mice. Single-cell RNA-seq and T-cell receptor sequencing also revealed active proliferation of tumor infiltrating Tregs by clonal expansion. One of these genes, ST2, an IL33 receptor, was identified as a potential factor driving Treg accumulation in the TME. Indeed, IL33-directed ST2 signaling induced the preferential proliferation of tumor-infiltrating Tregs and enhanced tumor progression, whereas genetic deletion of ST2 in Tregs limited their TME accumulation and delayed tumor growth. These data demonstrated the IL33/ST2 axis in Tregs as one of the critical pathways for the preferential accumulation of Tregs in the TME and suggests that the IL33/ST2 axis may be a potential therapeutic target for cancer immunotherapy.

Peripheral natural killer cells and myeloid-derived suppressor cells correlate with anti-PD-1 responses in non-small cell lung cancer.

Inhibition of immune checkpoint proteins like programmed death 1 (PD-1) is a promising therapeutic approach for several cancers, including non-small cell lung cancer (NSCLC). Although PD-1 ligand (PD-L1) expression is used to predict anti-PD-1 therapy responses in NSCLC, its accuracy is relatively less. Therefore, we sought to identify a more accurate predictive blood biomarker for evaluating anti-PD-1 response. We evaluated the frequencies of T cells, B cells, natural killer (NK) cells, polymorphonuclear myeloid-derived suppressor cells (PMN-MDSCs), mononuclear myeloid-derived suppressor cells (M-MDSCs), and Lox-1+ PMN-MDSCs in peripheral blood samples of 62 NSCLC patients before and after nivolumab treatment. Correlation of immune-cell population frequencies with treatment response, progression-free survival, and overall survival was also determined. After the first treatment, the median NK cell percentage was significantly higher in responders than in non-responders, while the median Lox-1+ PMN-MDSC percentage showed the opposite trend. NK cell frequencies significantly increased in responders but not in non-responders. NK cell frequency inversely correlated with that of Lox-1+ PMN-MDSCs after the first treatment cycle. The NK cell-to-Lox-1+ PMN-MDSC ratio (NMR) was significantly higher in responders than in non-responders. Patients with NMRs ≥ 5.75 after the first cycle had significantly higher objective response rates and longer progression-free and overall survival than those with NMRs <5.75. NMR shows promise as an early predictor of response to further anti-PD-1 therapy.

Single-cell transcriptome analysis reveals TOX as a promoting factor for T cell exhaustion and a predictor for anti-PD-1 responses in human cancer.

BACKGROUND: T cells exhibit heterogeneous functional states in the tumor microenvironment. Immune checkpoint inhibitors (ICIs) can reinvigorate only the stem cell-like progenitor exhausted T cells, which suggests that inhibiting the exhaustion progress will improve the efficacy of immunotherapy. Thus, regulatory factors promoting T cell exhaustion could serve as potential targets for delaying the process and improving ICI efficacy. METHODS: We analyzed the single-cell transcriptome data derived from human melanoma and non-small cell lung cancer (NSCLC) samples and classified the tumor-infiltrating (TI) CD8+ T cell population based on PDCD1 (PD-1) levels, i.e., PDCD1-high and PDCD1-low cells. Additionally, we identified differentially expressed genes as candidate factors regulating intra-tumoral T cell exhaustion. The co-expression of candidate genes with immune checkpoint (IC) molecules in the TI CD8+ T cells was confirmed by single-cell trajectory and flow cytometry analyses. The loss-of-function effect of the candidate regulator was examined by a cell-based knockdown assay. The clinical effect of the candidate regulator was evaluated based on the overall survival and anti-PD-1 responses. RESULTS: We retrieved many known factors for regulating T cell exhaustion among the differentially expressed genes between PDCD1-high and PDCD1-low subsets of the TI CD8+ T cells in human melanoma and NSCLC. TOX was the only transcription factor (TF) predicted in both tumor types. TOX levels tend to increase as CD8+ T cells become more exhausted. Flow cytometry analysis revealed a correlation between TOX expression and severity of intra-tumoral T cell exhaustion. TOX knockdown in the human TI CD8+ T cells resulted in downregulation of PD-1, TIM-3, TIGIT, and CTLA-4, which suggests that TOX promotes intra-tumoral T cell exhaustion by upregulating IC proteins in cancer. Finally, the TOX level in the TI T cells was found to be highly predictive of overall survival and anti-PD-1 efficacy in melanoma and NSCLC. CONCLUSIONS: We predicted the regulatory factors involved in T cell exhaustion using single-cell transcriptome profiles of human TI lymphocytes. TOX promoted intra-tumoral CD8+ T cell exhaustion via upregulation of IC molecules. This suggested that TOX inhibition can potentially impede T cell exhaustion and improve ICI efficacy. Additionally, TOX expression in the TI T cells can be used for patient stratification during anti-tumor treatments, including anti-PD-1 immunotherapy.

Tumor microenvironment dictates regulatory T cell phenotype: Upregulated immune checkpoints reinforce suppressive function.

BACKGROUND: Regulatory T (Treg) cells have an immunosuppressive function in cancer, but the underlying mechanism of immunosuppression in the tumor microenvironment (TME) is unclear. METHODS: We compared the phenotypes of T cell subsets, including Treg cells, obtained from peripheral blood, malignant effusion, and tumors of 103 cancer patients. Our primary focus was on the expression of immune checkpoint (IC)-molecules, such as programmed death (PD)-1, T-cell immunoglobulin and mucin-domain containing (TIM)-3, T cell Ig and ITIM domain (TIGIT), and cytotoxic T lymphocyte antigen (CTLA)-4, on Treg cells in paired lymphocytes from blood, peritumoral tissue, and tumors of 12 patients with lung cancer. To identify the immunosuppressive mechanisms acting on tumor-infiltrating Treg cells, we conducted immunosuppressive functional assays in a mouse model. RESULTS: CD8+, CD4+ T cells, and Treg cells exhibited a gradual upregulation of IC-molecules the closer they were to the tumor. Interestingly, PD-1 expression was more prominent in Treg cells than in conventional T (Tconv) cells. In lung cancer patients, higher levels of IC-molecules were expressed on Treg cells than on Tconv cells, and Treg cells were also more enriched in the tumor than in the peri-tumor and blood. In a mouse lung cancer model, IC-molecules were also preferentially upregulated on Treg cells, compared to Tconv cells. PD-1 showed the greatest increase on most cell types, especially Treg cells, and this increase occurred gradually over time after the cells entered the TME. PD-1 high-expressing tumor-infiltrating Treg cells displayed potent suppressive activity, which could be partially inhibited with a blocking anti-PD-1 antibody. CONCLUSIONS: We demonstrate that the TME confers a suppressive function on Treg cells by upregulating IC-molecule expression. Targeting IC-molecules, including PD-1, on Treg cells may be effective for cancer treatment.

Identification of ganglioside GM2 activator playing a role in cancer cell migration through proteomic analysis of breast cancer secretomes.

Cancer cell secretomes are considered a potential source for the discovery of cancer markers. In this study, the secretomes of four breast cancer (BC) cell lines (Hs578T, MCF-7, MDA-MB-231, and SK-BR-3) were profiled with liquid chromatography-tandem mass spectrometry analysis. A total of 1410 proteins were identified with less than 1% false discovery rate, of which approximately 55% (796 proteins) were predicted to be secreted from cells. To find BC-specific proteins among the secreted proteins, data of immunohistochemical staining compiled in the Human Protein Atlas were investigated by comparing the data of BC tissues with those of normal tissues. By applying various criteria, including higher expression level in BC tissues, higher predicted potential of secretion, and sufficient number of tandem mass spectra, 12 biomarker candidate proteins including ganglioside GM2 activator (GM2A) were selected for confirmation. Western blot analysis and ELISA for plasma samples of healthy controls and BC patients revealed elevation of GM2A in BC patients, especially those who were estrogen receptor-negative. Additionally, siRNA-mediated knockdown of GM2A in BC cells decreased migration in vitro, whereas the overexpression of GM2A led to an increase in cell migration. Although GM2A as a diagnostic and prognostic marker in BC should be carefully verified further, this study has established the potential role of GM2A in BC progression.

Use of composite protein database including search result sequences for mass spectrometric analysis of cell secretome.

Mass spectrometric (MS) data of human cell secretomes are usually run through the conventional human database for identification. However, the search may result in false identifications due to contamination of the secretome with fetal bovine serum (FBS) proteins. To overcome this challenge, here we provide a composite protein database including human as well as 199 FBS protein sequences for MS data search of human cell secretomes. Searching against the human-FBS database returned more reliable results with fewer false-positive and false-negative identifications compared to using either a human only database or a human-bovine database. Furthermore, the improved results validated our strategy without complex experiments like SILAC. We expect our strategy to improve the accuracy of human secreted protein identification and to also add value for general use.

Discovery of melanotransferrin as a serological marker of colorectal cancer by secretome analysis and quantitative proteomics.

To discover serological colorectal cancer (CRC) markers, we analyzed cell line secretome to gather proteins of higher potential to be secreted from tissues into circulation. A total of 898 human proteins were identified, of which 62.2% were predicted to be released or shed from cells. The identified proteins were compared with tissue proteomes to find candidate proteins whose expressions were elevated in tumor tissues compared with normal tissues as revealed by (i) quantitative proteomic analysis based on cICAT and mTRAQ or (ii) data mining of immunohistochemical images piled in Human Protein Atlas database. By applying various stringent criteria, 11 candidate proteins were selected. Among these, we validated an significant increase (p = 0.0018) of melanotransferrin (TRFM) at the plasma level of CRC patients through Western blotting, using 130 plasma samples containing 30 healthy controls, 80 CRC patients, and 20 patients of other diseases. Finally, we measured the expression level of TRFM in 325 plasma samples containing 77 healthy controls and 228 CRC patients (34.6 ± 4.2 ng/mL and 67.0 ± 6.4 ng/mL, p < 0.0001) through ELISA and demonstrated the area under the receiver operating characteristic curve of 0.723 (p < 0.0001) with a 92.5% specificity, 48.2% sensitivity, and 95.7% positive predictive value. Furthermore, unlike CEA and PAI-1, up-regulation of TRFM in pathological stages I & II groups compared with stages III & IV groups lead us to expect the use TRFM for early-stage diagnosis of CRC. In this study, we suggest TRFM as a potential serological marker for CRC and expect our discovery strategy to help identify highly cancer-specific and body-fluid-accessible biomarkers.

High mobility group box-1 is phosphorylated by protein kinase C zeta and secreted in colon cancer cells.

High mobility group box-1 (HMGB1), a nuclear protein, is overexpressed and secreted in cancer cells. Phosphorylation on two different nuclear localization signal regions are known to be important for the nuclear-to-cytoplasmic transport and secretion of HMGB1. However, little is known about the biochemical mechanism of HMGB1 modifications and its subsequent secretion from cancer cells. To identify the specific enzyme and important sites for HMGB1 phosphorylation, we screened the protein kinase C (PKC) family in a colon cancer cell line (HCT116) for HMGB1 binding by pull-down experiments using a 3XFLAG-HMGB1 construct. Strong interactions between atypical PKCs (PKC-ζ, λ, and ι) and cytoplasmic HMGB1 were observed in HCT116 cells. We further identified the most critical PKC isotype that regulates HMGB1 secretion is PKC-ζ by using PKC inhibitors and siRNA experiments. The serine residues at S39, S53 and S181 of HMGB1 were related to enhancing HMGB1 secretion. We also demonstrated overexpression and activation of PKC-ζ in colon cancer tissues. Our findings suggest that PKC-ζ is involved in the phosphorylation of HMGB1, and the phosphorylation of specific serine residues in the nuclear localization signal regions is related to enhanced HMGB1 secretion in colon cancer cells.