Autopsy analysis reveals increased macrophage infiltration and cell apoptosis in COVID-19 patients with severe pulmonary fibrosis.

Clinical data indicates that SARS-CoV-2 infection-induced respiratory failure is a fatal condition for severe COVID-19 patients. However, the pathological alterations of different types of respiratory failure remained unknown for severe COVID-19 patients. This study aims to evaluate whether there are differences in the performance of various types of respiratory failure in severe COVID-19 patients and investigate the pathological basis for these differences. The lung tissue sections of severe COVID-19 patients were assessed for the degree of injury and immune responses. Transcriptome data were used to analyze the molecular basis in severe COVID-19 patients. Severe COVID-19 patients with combined oxygenation and ventilatory failure presented more severe pulmonary fibrosis, airway obstruction, and prolonged disease course. The number of M2 macrophages increased with the degree of fibrosis in patients, suggesting that it may be closely related to the development of pulmonary fibrosis. The co-existence of pro-inflammatory and anti-inflammatory cytokines in the pulmonary environment could also participate in the progression of pulmonary fibrosis. Furthermore, the increased apoptosis in the lungs of COVID-19 patients with severe pulmonary fibrosis may represent a critical factor linking sustained inflammatory responses to fibrosis. Our findings indicate that during the extended phase of COVID-19, antifibrotic and antiapoptotic treatments should be considered in conjunction with the progression of the disease.

Primary pulmonary hyalinizing clear cell carcinoma with fusions of both EWSR1::CREM and IRF2::NTRK3: report of a case with an aggressive behavior.

Primary pulmonary hyalinizing clear cell carcinoma (HCCC) is a rare salivary gland-type tumor newly recognized in recent years, with approximately 21 cases reported to date in the English literature, which constitutes a challenge in pathology diagnosis, particularly in small biopsy specimens. Here, we present a case of pulmonary HCCC diagnosed by computed tomography-guided percutaneous lung biopsy in a 70-year-old man's right lower lung. Although the morphology and immunophenotype of the tumor suggested the diagnosis of mucoepidermoid carcinoma, fluorescence in situ hybridization failed to reveal the rearrangement of MAML2 gene, which is characteristic of mucoepidermoid carcinoma. Instead, further molecular genetic testing showed that the tumor harbored a rare EWSR1::CREM fusion combined with a previously unreported IRF2::NTRK3 fusion. Pulmonary HCCC is commonly regarded as a low-grade malignant tumor with an indolent course, but this case has a different biological behavior, presenting extensive dissemination and metastases at the time of diagnosis, which expands our understanding of the prognosis of this tumor. The patient has had five cycles of combination chemotherapy and has been alive with the tumor for eight months.

A map of the spatial distribution and tumour-associated macrophage states in glioblastoma and grade 4 IDH-mutant astrocytoma.

Tumour-associated macrophages (TAMs) abundantly infiltrate high-grade gliomas and orchestrate immune response, but their diversity in isocitrate dehydrogenase (IDH)-differential grade 4 gliomas remains largely unknown. This study aimed to dissect the transcriptional states, spatial distribution, and clinicopathological significance of distinct monocyte-derived TAM (Mo-TAM) and microglia-derived TAM (Mg-TAM) clusters across glioblastoma-IDH-wild type and astrocytoma-IDH-mutant-grade 4 (Astro-IDH-mut-G4). Single-cell RNA sequencing was performed on four cases of human glioblastoma and three cases of Astro-IDH-mut-G4. Cell clustering, single-cell regulatory network inference, and gene set enrichment analysis were performed to characterize the functional states of myeloid clusters. The spatial distribution of TAM subsets was determined in human glioma tissues using multiplex immunostaining. The prognostic value of different TAM-cluster specific gene sets was evaluated in the TCGA glioma cohort. Profiling and unbiased clustering of 24,227 myeloid cells from glioblastoma and Astro-IDH-mut-G4 identified nine myeloid cell clusters including monocytes, six Mo/Mg-TAM subsets, dendritic cells, and proliferative myeloid clusters. Different Mo/Mg-TAM clusters manifest functional and transcriptional diversity controlled by specific regulons. Multiplex immunostaining of subset-specific markers identified spatial enrichment of distinct TAM clusters at peri-vascular/necrotic areas in tumour parenchyma or at the tumour-brain interface. Glioblastoma harboured a substantially higher number of monocytes and Mo-TAM-inflammatory clusters, whereas Astro-IDH-mut-G4 had a higher proportion of TAM subsets mediating antigen presentation. Glioblastomas with a higher proportion of monocytes exhibited a mesenchymal signature, increased angiogenesis, and worse patient outcome. Our findings provide insight into myeloid cell diversity and its clinical relevance in IDH-differential grade 4 gliomas, and may serve as a resource for immunotherapy development. © 2022 The Pathological Society of Great Britain and Ireland.

Inhibitory effects of temozolomide on glioma cells is sensitized by RSL3-induced ferroptosis but negatively correlated with expression of ferritin heavy chain 1 and ferritin light chain.

Invasive growth of glioblastoma makes residual tumor unremovable by surgery and leads to disease relapse. Temozolomide is widely used first-line chemotherapy drug to treat glioma patients, but development of temozolomide resistance is almost inevitable. Ferroptosis, an iron-dependent form of non-apoptotic cell death, is found to be related to temozolomide response of gliomas. However, whether inducing ferroptosis could affect invasive growth of glioblastoma cells and which ferroptosis-related regulators were involved in temozolomide resistance are still unclear. In this study, we treated glioblastoma cells with RSL3, a ferroptosis inducer, in vitro (cell lines) and in vivo (subcutaneous and orthotopic animal models). The treated glioblastoma cells with wild-type or mutant IDH1 were subjected to RNA sequencing for transcriptomic profiling. We then analyze data from our RNA sequencing and public TCGA glioma database to identify ferroptosis-related biomarkers for prediction of prognosis and temozolomide resistance in gliomas. Analysis of transcriptome data from RSL3-treated glioblastoma cells suggested that RSL3 could inhibit glioblastoma cell growth and suppress expression of genes involved in cell cycle. RSL3 effectively reduced mobility of glioblastoma cells through downregulation of critical genes involved in epithelial-mesenchymal transition. Moreover, RSL3 in combination with temozolomide showed suppressive efficacy on glioblastoma cell growth, providing a promising therapeutic strategy for glioblastoma treatment. Although temozolomide attenuated invasion of glioblastoma cells with mutant IDH1 more than those with wild-type IDH1, the combination of RSL3 and temozolomide similarly impaired invasive ability of glioblastoma cells in spite of IDH1 status. Finally, we noticed that both ferritin heavy chain 1 and ferritin light chain predicted unfavorable prognosis of glioma patients and were significantly correlated with mRNA levels of methylguanine methyltransferase as well as temozolomide resistance. Altogether, our study provided rationale for combination of RSL3 with temozolomide to suppress glioblastoma cells and revealed ferritin heavy chain 1 and ferritin light chain as biomarkers to predict prognosis and temozolomide resistance of glioma patients.

HOXA5 is amplified in glioblastoma stem cells and promotes tumor progression by transcriptionally activating PTPRZ1.

Although the tumorigenic potential of glioma stem cells (GSCs) is associated with multiple molecular alterations, the gene amplification status of GSCs has not been elucidated. Overexpression of HomeoboxA5 (HOXA5) is associated with increased glioma malignancy. In this study, we identify the gene amplification and protein overexpression of HOXA5 in GSCs and its function in regulating GSC maintenance and the downstream transcriptional effector, to explore the significance of HOXA5 amplification/overexpression for GSC identification and prognostic determination. The HOXA5 gene is significantly amplified in glioblastoma (GBM) and is an independent prognostic factor for predicting worse patient outcomes. Specifically, HOXA5 gene amplification and the resultant protein overexpression are correlated with increased proportions of GSCs and enhanced self-renewal/invasiveness of these cells. Disruption of HOXA5 expression impairs GSC survival and GBM tumor propagation. Mechanistically, HOXA5 directly binds to the promoter region of protein tyrosine phosphatase receptor type Z1 (PTPRZ1), thereby upregulating this gene for GSC maintenance. Suppression of PTPRZ1 largely compromises the pro-tumoral effect of HOXA5 on GSCs. In summary, HOXA5 amplification serves as a genetic biomarker for predicting worse GBM outcome, by enhancing PTPRZ1-mediated GSC survival.

ADP-Ribosylation Factor Like GTPase 4C (ARL4C) augments stem-like traits of glioblastoma cells by upregulating ALDH1A3.

Glioma cells with stem cell-like properties are crucial for tumor initiation, progression and therapeutic resistance. Therefore, identifying specific factors in regulating stem-like traits is critical for the design of novel glioma therapeutics. Herein, we reported that ADP-Ribosylation Factor Like GTPase 4C (ARL4C) was highly expressed in glioma stem-like cells (GSLCs). GSLCs, determined by the efficiency of sphere formation in vitro and tumor growth in vivo, was increased by overexpression of ARL4C. ARL4C induced the tumorigenesis through ALDH1A3. Analyses of 325 patient specimens showed that ARL4C was highly expressed in glioblastoma (GBM) as compared with lower grade gliomas. In addition, higher level ARL4C expression in glioma was correlated with poorer progression-free survival and overall survival of patients. Therefore, ARL4C may act as a novel prognostic marker and a therapeutic target for GBM.

Stromal PD-1+ tumor-associated macrophages predict poor prognosis in lung adenocarcinoma.

Immunotherapies targeting programmed cell death protein 1 (PD-1)/PD-1 ligand (PD-L1) axis have been emerging as a promising therapeutic strategy to treat lung cancer. PD-1 is preferentially expressed by activated T lymphocytes; but whether/how its expression by tumor-associated macrophages (TAMs) in lung adenocarcinoma remains elusive. Herein, we investigate the frequency of PD-1 expression on TAMs in mouse allografts by flow cytometry analysis and evaluate the spatial distribution and clinicopathological significance of PD-1+ TAMs in 213 cases of human lung adenocarcinoma specimens by immunohistochemical staining. We find the expression of PD-1 by both mouse and human TAMs. Mouse PD-1+ TAMs possess unique transcriptional profile as compared to PD-1- TAMs. Furthermore, PD-1 is preferentially expressed by CD163+ TAMs in the tumor stroma than those in the tumor islets of lung adenocarcinoma. Stromal PD-1+ TAM infiltration is an independent predictor of reduced survival as determined by univariate (P < .001) and multivariate (P = .023) analysis. Moreover, patients with high stromal PD-1+ TAMs but low tumor cell PD-L1 expression have the shortest survival (P = .0001). Our study demonstrates that PD-1+ TAMs have unique gene expression characteristics and PD-1+ TAMs in the tumor stroma is a potential prognostic factor in lung adenocarcinoma, suggesting that a better understanding of PD-1+ TAMs will be beneficial for immunotherapy of lung adenocarcinoma patients.

CCL8 secreted by tumor-associated macrophages promotes invasion and stemness of glioblastoma cells via ERK1/2 signaling.

Tumor-associated macrophages (TAMs) constitute a large population of glioblastoma and facilitate tumor growth and invasion of tumor cells, but the underlying mechanism remains undefined. In this study, we demonstrate that chemokine (C-C motif) ligand 8 (CCL8) is highly expressed by TAMs and contributes to pseudopodia formation by GBM cells. The presence of CCL8 in the glioma microenvironment promotes progression of tumor cells. Moreover, CCL8 induces invasion and stem-like traits of GBM cells, and CCR1 and CCR5 are the main receptors that mediate CCL8-induced biological behavior. Finally, CCL8 dramatically activates ERK1/2 phosphorylation in GBM cells, and blocking TAM-secreted CCL8 by neutralized antibody significantly decreases invasion of glioma cells. Taken together, our data reveal that CCL8 is a TAM-associated factor to mediate invasion and stemness of GBM, and targeting CCL8 may provide an insight strategy for GBM treatment.

The landscape of immune microenvironment in lung adenocarcinoma and squamous cell carcinoma based on PD-L1 expression and tumor-infiltrating lymphocytes.

AIMS: The aim of this study was to investigate the tumor microenvironment immune types (TMIT) based on tumor cell programmed cell death ligand 1 (PD-L1) expression and tumor-infiltrating lymphocytes (TILs) distribution and whether distinct TMIT subtypes (TMIT I, PD-L1high /TILhigh ; TMIT II, PD-L1low /TILlow ; TMIT III, PD-L1high /TILlow ; and TMIT IV, PD-L1low /TILhigh ) differentially affect clinical outcomes of patients with lung adenocarcinoma (LAC) and squamous cell carcinoma (SCC). METHODS AND RESULTS: Immunohistochemistry (IHC) was applied to evaluate the expression of PD-L1 and the spatial distribution of programmed cell death 1 (PD-1) and CD8 TILs on the surgically resected specimens from 205 cases of LAC and 149 cases of SCC. PD-1 and CD8 TILs were more frequently distributed in SCC than those in LAC, regardless of their infiltrating in the tumor islets or stroma. The density of TILs was a poor prognostic factor in LAC but a favorable one in SCC. PD-L1 levels and its clinical prognostic significance differed in LAC vs SCC. LAC patients with TMIT III and SCC patients with TMIT I had the longest survival, respectively (P = .0197 and .0049). Moreover, TMIT stratification based on tumor cell PD-L1 expression and stromal CD8+ TILs could be considered as an independent prognostic factor of SCC patients' survival as determined by both univariate and multivariate analysis. CONCLUSION: Our study indicates that different type of TMIT provides its specific microenvironment with diverse impact on survival of LAC and SCC patients and highlights the importance of the integrative assessment of PD-L1 status and TILs' spatial distribution to predict patients' prognosis.

Hybrids by tumor-associated macrophages × glioblastoma cells entail nuclear reprogramming and glioblastoma invasion.

Hybrid formation is a fundamental process in normal development and tissue homeostasis, while the presence and the biological role of hybrids between tumor-associated macrophages (TAMs) and glioblastoma (GBM) cells remain elusive. In this study, we observed that TAM-GBM cell hybrids existed in human GBM specimens as demonstrated by co-expression of glioma biomarkers (GFAP, IDH1R132H and PDGFRA) and macrophage biomarkers (CD68 and CD14). Furthermore, TAM-GBM cell hybrids could also be found in C57BL/6 mice orthotopically inoculated with mouse GBM cells labeled with RFP and after co-culture of bone marrow-derived macrophages from GFP-expressed mice with RFP-labeled GBM cells. The hybrids underwent nuclear reprogramming with unique gene expression profile as compared to parental cells. Moreover, glioma invasion-associated genes were enriched in the hybrids that possessed higher invasiveness, and more hybrids in the invasive margin of GBM were observed as compared to GBM core area. Our data demonstrate the presence of TAM-GBM cell hybrids that enhance GBM invasion. With a better understanding of TAM-GBM cell hybrids, new therapeutic strategies targeting GBM will be developed to treat GBM patients.

Tumour-associated macrophages secrete pleiotrophin to promote PTPRZ1 signalling in glioblastoma stem cells for tumour growth.

Intense infiltration of tumour-associated macrophages (TAMs) facilitates malignant growth of glioblastoma (GBM), but the underlying mechanisms remain undefined. Herein, we report that TAMs secrete abundant pleiotrophin (PTN) to stimulate glioma stem cells (GSCs) through its receptor PTPRZ1 thus promoting GBM malignant growth through PTN-PTPRZ1 paracrine signalling. PTN expression correlates with infiltration of CD11b+/CD163+ TAMs and poor prognosis of GBM patients. Co-implantation of M2-like macrophages (MLCs) promoted GSC-driven tumour growth, but silencing PTN expression in MLCs mitigated their pro-tumorigenic activity. The PTN receptor PTPRZ1 is preferentially expressed in GSCs and also predicts GBM poor prognosis. Disrupting PTPRZ1 abrogated GSC maintenance and tumorigenic potential. Moreover, blocking the PTN-PTPRZ1 signalling by shRNA or anti-PTPRZ1 antibody potently suppressed GBM tumour growth and prolonged animal survival. Our study uncovered a critical molecular crosstalk between TAMs and GSCs through the PTN-PTPRZ1 paracrine signalling to support GBM malignant growth, indicating that targeting this signalling axis may have therapeutic potential.