Lipid droplets-related Perilipin-3: potential immune checkpoint and oncogene in oral squamous cell carcinoma.

BACKGROUND: Lipid droplets (LDs) as major lipid storage organelles are recently reported to be innate immune hubs. Perilipin-3 (PLIN3) is indispensable for the formation and accumulation of LDs. Since cancer patients show dysregulated lipid metabolism, we aimed to elaborate the role of LDs-related PLIN3 in oral squamous cell carcinoma (OSCC). METHODS: PLIN3 expression patterns (n = 87), its immune-related landscape (n = 74) and association with B7-H2 (n = 51) were assessed by immunohistochemistry and flow cytometry. Real-time PCR, Western blot, Oil Red O assay, immunofluorescence, migration assay, spheroid-forming assay and flow cytometry were performed for function analysis. RESULTS: Spotted LDs-like PLIN3 staining was dominantly enriched in tumor cells than other cell types. PLIN3high tumor showed high proliferation index with metastasis potential, accompanied with less CD3+CD8+ T cells in peripheral blood and in situ tissue, conferring immunosuppressive microenvironment and shorter postoperative survival. Consistently, PLIN3 knockdown in tumor cells not only reduced LD deposits and tumor migration, but benefited for CD8+ T cells activation in co-culture system with decreased B7-H2. An OSCC subpopulation harbored PLIN3highB7-H2high tumor showed more T cells exhaustion, rendering higher risk of cancer-related death (95% CI 1.285-6.851). CONCLUSIONS: LDs marker PLIN3 may be a novel immunotherapeutic target in OSCC.

Immune checkpoint CD161/LLT1-associated immunological landscape and diagnostic value in oral squamous cell carcinoma.

An active host adaptive response is characterized by the existence of programmed cell death protein 1 (PD-1)+ /IFN-γ+ cytotoxic T cells and IFN-γ-induced PD-L1+ tumor cells (TCs), which predicts high response rate to anti-PD-1/L1 therapy. Recently, CD161 and its ligand LLT1 (CLEC2D) have been identified as an emerging checkpoint for immunotherapy. Clarifying its heterogeneous clinical expression pattern and its immune landscape is a prerequisite for maximizing the response rate of CD161 blockade therapy in a specific population of oral squamous cell carcinoma (OSCC) patients. Here, we investigated the expression pattern of CD161/LLT1 and its association with major immunocytes (T cells, B cells, NK cells, and macrophages) by multiplex immunofluorescence, immunohistochemistry, and flow cytometry in 109 OSCC tissues and 102 peripheral blood samples. TCs showed higher LLT1 levels than tumor infiltrating lymphocytes (TILs), whereas CD161 was highly expressed in CD8+ T cells at the tumor front, which was decreased in paracancerous tissue. High expression of TC-derived LLT1 (LLT1TC ) conferred poor clinical outcomes, whereas higher CD161+ and LLT1+ TILs were associated with better prognosis. Meanwhile, patients with high LLT1TC showed a decreased ratio of CD8+ /Foxp3+ T cells in situ, but CD161+ TILs correlated with more peripheral CD3+ T cells. Interestingly, treatment of OSCC patients with nivolumab (anti-PD-1) could restore tumoral CD161/LLT1 signal. Furthermore, an OSCC subgroup characterized by high LLT1+ TCs and low CD161+ CD8+ T cells showed fewer peripheral T cells and a higher risk of lymph node metastasis, leading to a shorter 5-year survival time (29%). More LLT1TC at the invasive front was another risk characteristic of exhausted T cells. In conclusion, in view of this heterogeneity, the LLT1/CD161 distribution pattern should be determined before CD161-based immunotherapy.

ICOSLG-associated immunological landscape and diagnostic value in oral squamous cell carcinoma: a prospective cohort study.

Background: We previously reported that stroma cells regulate constitutive and inductive PD-L1 (B7-H1) expression and immune escape of oral squamous cell carcinoma. ICOSLG (B7-H2), belongs to the B7 protein family, also participates in regulating T cells activation for tissue homeostasis via binding to ICOS and inducing ICOS+ T cell differentiation as well as stimulate B-cell activation, while it appears to be abnormally expressed during carcinogenesis. Clarifying its heterogeneous clinical expression pattern and its immune landscape is a prerequisite for the maximum response rate of ICOSLG-based immunotherapy in a specific population. Methods: This retrospective study included OSCC tissue samples (n = 105) to analyze the spatial distribution of ICOSLG. Preoperative peripheral blood samples (n = 104) and independent tissue samples (n = 10) of OSCC were collected to analyze the changes of immunocytes (T cells, B cells, NK cells and macrophages) according to ICOSLG level in different cellular contents. Results: ICOSLG is ubiquitous in tumor cells (TCs), cancer-associated fibroblasts (CAFs) and tumor infiltrating lymphocytes (TILs). Patients with high ICOSLGTCs or TILs showed high TNM stage and lymph node metastasis, which predicted a decreased overall or metastasis-free survival. This sub-cohort was featured with diminished CD4+ T cells and increased Foxp3+ cells in invasive Frontier in situ, and increased absolute numbers of CD3+CD4+ and CD8+ T cells in peripheral blood. ICOSLG also positively correlated with other immune checkpoint molecules (PD-L1, CSF1R, CTLA4, IDO1, IL10, PD1). Conclusion: Tumor cell-derived ICOSLG could be an efficient marker of OSCC patient stratification for precision immunotherapy.

Lipid Droplet-Related PLIN2 in CD68+ Tumor-Associated Macrophage of Oral Squamous Cell Carcinoma: Implications for Cancer Prognosis and Immunotherapy.

Background: PLIN2 (adipose differentiation-related protein) belongs to the perilipin family and is a marker of lipid droplets (LDs). Numerous types of tumor exhibit a high PLIN2 level, but its tumorigenic or tumor-suppressive role has been in debate. Recently, LDs serve as innate immune hubs and show antimicrobial capacity. We here aimed to investigate the heterogeneous functions of PLIN2 in the tumor microenvironment and immune regulation. Methods: This retrospective study included 96 oral squamous cell carcinoma (OSCC) samples and analyzed the spatial distribution of PLIN2 by immunohistochemistry (IHC) and LD level by oil red O staining. A total of 21 serial sections were obtained to analyze the relationship between PLIN2 and immune cells by IHC and immunofluorescence (IF). Single-cell sequencing was used to analyze the cell locations of PLIN2. The values of diagnosis and prognosis of PLIN2 were also evaluated. Tumor Immune Estimation Resource (TIMER), cBioPortal databases, and IHC analysis were used to investigate the relationship between PLIN2 and OSCC immune microenvironment. Results: PLIN2 was mainly expressed in tumor-infiltrating immunocytes (TIIs) of OSCC. Patients with high PLIN2 harbored more cytoplastic LDs. CD68+ tumor-associated macrophages (TAMs), instead of T cells and B cells, were found to be the main resource of PLIN2 in OSCC stroma and lung, pancreas, prostate, and testis. However, CD56+ NK cells also showed less extent of PLIN2 staining in OSCC. Moreover, patients with a high PLIN2 level in immune cells had a higher TNM stage and were susceptible to postoperative metastasis, but the escalated PLIN2 level in invasive tumor front independently predicted shorter metastasis-free survival. Furthermore, a high PLIN2 presentation in the microenvironment induced immune suppression which was featured as less infiltration of CD8+ T cells and more CD68+ TAMs and Foxp3+ Tregs, accompanied by more immune checkpoint molecules such as CSF1R, LGALS9, IL-10, CTLA-4, and TIGIT. Conclusion: CD68+ TAM-derived PLIN2 might participate in regulating immune balance of OSCC patients, which provides new insight into immune checkpoint therapy.

Therapeutic Erythrocytapheresis Is Effective in Treating High Altitude Polycythemia on the Qinghai-Tibet Plateau.

INTRODUCTION: High altitude polycythemia (HAPC) is a common chronic disease at high altitudes. It is characterized by excessive erythrocytosis (≥190 g·L-1 in females or ≥210 g·L-1 in males). HAPC severely jeopardizes the health status of plateau dwellers. The Qinghai-Tibet plateau, with an elevation above 4000 m, is the highest plateau in the world. Both Han and Tibetan populations residing there face the threat of HAPC. Therapeutic erythrocytapheresis (TE) was introduced to Tibet as an alternative to phlebotomy in 2015. METHODS: In this study, we retrospectively analyzed 155 patients with HAPC treated with TE in Tibet. Routine blood testing values before and after TE were compared to evaluate treatment efficacy. The efficiency rate, defined as the rate of increase in red blood cell depletion attained by TE compared with 450 mL whole blood phlebotomy, was calculated using whole blood volume and hematocrit before and after treatment and used to identify patients who maintained a normal hemoglobin level in the year after the TE procedure. RESULTS: On average, TE reduced red blood cell levels by 1.5×1012·L-1, hemoglobin concentration by 52 g·L-1, and hematocrit by 14% (P<0.001 for each). Patients who underwent TE with an efficiency rate ≥1.9 were more likely to maintain a normal hemoglobin level in the following year than those who underwent TE with an efficiency rate <1.9 (90 vs 28%, P<0.01). CONCLUSIONS: TE is a feasible therapeutic method to treat HAPC on the Qinghai-Tibet plateau. The efficiency rate is a useful tool to predict the expected interval between TE procedures.

Low-dose decitabine priming with intermediate-dose cytarabine followed by umbilical cord blood infusion as consolidation therapy for elderly patients with acute myeloid leukemia: a phase II single-arm study.

BACKGROUND: Treatment of acute myeloid leukemia (AML) in elderly patients remains a great challenge. In this prospective single arm study (ChiCTR-OPC-15006492), we evaluated the efficacy and safety of a novel consolidation therapy with low-dose decitabine (LD-DAC) priming with intermediate-dose cytarabine (ID-Ara-C) followed by umbilical cord blood (UCB) infusion in elderly patients with AML. METHODS: A total of 25 patients with a median age of 64-years-old (60-74-years-old) who achieved complete remission (CR) after induction chemotherapy were enrolled in the study. RESULTS: The 2-year actual overall survival (OS) rate and leukemia-free survival (LFS) was 68.0 and 60.0%, respectively. The hematological and non-hematological toxicity were mild to moderate, and only one patient died in remission due to infection with possible acute graft versus host disease (aGVHD). Compared to a concurrent cohort of patients receiving conventional consolidation therapy, the study group tended to have an improved OS and LFS (p = 0.046 and 0.057, respectively), while the toxicity was comparable between the two groups. CONCLUSIONS: This study suggested the novel combination of LD-DAC, ID-Ara-C, and UCB infusion might be an optimal consolidation therapy for elderly patients with AML, and a prospective phase III randomized study is warranted to confirm this observation. TRIAL REGISTRATION: This single-arm phase II clinical trial in elderly AML patients was registered prospectively at www.chictr.org.cn (identifier: ChiCTR-OPC-15006492 ) on June 2, 2015.

The nature of mutations induced by replication­transcription collisions.

The DNA replication and transcription machineries share a common DNA template and thus can collide with each other co-directionally or head-on. Replication­transcription collisions can cause replication fork arrest, premature transcription termination, DNA breaks, and recombination intermediates threatening genome integrity. Collisions may also trigger mutations, which are major contributors to genetic disease and evolution. However, the nature and mechanisms of collision-induced mutagenesis remain poorly understood. Here we reveal the genetic consequences of replication­transcription collisions in actively dividing bacteria to be two classes of mutations: duplications/deletions and base substitutions in promoters. Both signatures are highly deleterious but are distinct from the previously well-characterized base substitutions in the coding sequence. Duplications/deletions are probably caused by replication stalling events that are triggered by collisions; their distribution patterns are consistent with where the fork first encounters a transcription complex upon entering a transcription unit. Promoter substitutions result mostly from head-on collisions and frequently occur at a nucleotide that is conserved in promoters recognized by the major σ factor in bacteria. This substitution is generated via adenine deamination on the template strand in the promoter open complex, as a consequence of head-on replication perturbing transcription initiation. We conclude that replication­transcription collisions induce distinct mutation signatures by antagonizing replication and transcription, not only in coding sequences but also in gene regulatory elements.