Early Lung Carcinogenesis and Tumor Microenvironment Observed by Single-Cell Transcriptome Analysis

Abstract Background: Ground-glass nodules (GGNs) are radiologically defined pulmonary nodules characterized by preserved bronchial and vascular structures in the lung window on chest computed tomography. Lung adenocarcinoma present in the form of persistent GGN is a good model for studying early lung carcinogenesis. We sought to decipher the transcriptome of early lung cancer and its tumor microenvironment from nonsmokers.Methods: Eleven surgical specimens from 6 patients with persistent pure or part-solid GGNs and no smoking or long-term nonsmoking history were obtained and studied by single-cell RNA sequencing analysis.Results: Early lung cancer cells showed enrichment of genes related to small vesicle processing and surfactant homeostasis compared to normal lung epithelial cells, suggesting that the surfactant-related pathway is strongly involved in early lung carcinogenesis. Even in this early stage of lung carcinogenesis, the tumor immune microenvironment was disrupted, with myeloid-derived suppressor cells showing activation of tumor-promoting cytokine pathways, making the tumor microenvironment more permissive for tumor progression and promoting infiltration of regulatory T cells and depletion of CD8+ cytotoxic T cells (TCs) and γδ TCs. Although mucosa-associated lymphoid tissue (MALT) B cells (BCs) and follicular BCs are present in small proportions, they showed increased infiltration in tumor tissues compared to adjacent normal lung tissues. Overexpression of hypoxia-related genes and active suppression of normal angiogenesis were observed in cancer-associated fibroblasts.Conclusions: Changes in the tumor microenvironment that begin very early in lung cancer create an environment prone to immune evasion, suggesting that regulation of such changes is a strategy for inhibiting cancer growth.

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Phosphorylation of SMAD3 in immune cells predicts survival of patients with early stage non-small cell lung cancer

Journal for ImmunoTherapy of Cancer ◽

10.1136/jitc-2020-001469 ◽

2021 ◽

Vol 9 (2) ◽

pp. e001469

Author(s):

Sebastian Marwitz ◽

Carmen Ballesteros-Merino ◽

Shawn M Jensen ◽

Martin Reck ◽

Christian Kugler ◽

...

Keyword(s):

Lung Cancer ◽

Immune Response ◽

T Cells ◽

Tumor Microenvironment ◽

Immune Cells ◽

Early Stage ◽

Small Cell ◽

Small Cell Lung ◽

Pathway Activation ◽

Smad3 Phosphorylation

BackgroundThe interplay of immune and cancer cells takes place in the tumor microenvironment where multiple signals are exchanged. The transforming growth factor beta (TGFB) pathway is known to be dysregulated in lung cancer and can impede an effective immune response. However, the exact mechanisms are yet to be determined. Especially which cells respond and where does this signaling take place with respect to the local microenvironment.MethodsHuman non-small cell lung cancer samples were retrospectively analyzed by multiplexed immunohistochemistry for SMAD3 phosphorylation and programmed death ligand 1 expression in different immune cells with respect to their localization within the tumor tissue. Spatial relationships were studied to examine possible cell-cell interactions and analyzed in conjunction with clinical data.ResultsTGFB pathway activation in CD3, CD8, Foxp3 and CD68 cells, as indicated by SMAD3 phosphorylation, negatively impacts overall and partially disease-free survival of patients with lung cancerindependent of histological subtype. A high frequency of Foxp3 regulatory T cells positive for SMAD3 phosphorylation in close vicinity of CD8 T cells within the tumor discriminate a rapidly progressing group of patients with lung cancer.ConclusionsTGFB pathway activation of local immune cells within the tumor microenvironment impacts survival of early stage lung cancer. This might benefit patients not eligible for targeted therapies or immune checkpoint therapy as a therapeutic option to re-activate the local immune response.

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M1hot tumor-associated macrophages boost tissue-resident memory T cells infiltration and survival in human lung cancer

Journal for ImmunoTherapy of Cancer ◽

10.1136/jitc-2020-000778 ◽

2020 ◽

Vol 8 (2) ◽

pp. e000778 ◽

Cited By ~ 1

Author(s):

Eva M Garrido-Martin ◽

Toby W P Mellows ◽

James Clarke ◽

Anusha-Preethi Ganesan ◽

Oliver Wood ◽

...

Keyword(s):

Lung Cancer ◽

T Cells ◽

Immune System ◽

Single Cell ◽

Memory T Cells ◽

Adaptive Immune System ◽

Tumor Associated Macrophages ◽

Adaptive Immune ◽

Early Lung Cancer ◽

Resident Memory T Cells

BackgroundThe role of tumor-associated macrophages (TAMs) in determining the outcome between the antitumor effects of the adaptive immune system and the tumor’s anti-immunity stratagems, is controversial. Macrophages modulate their activities and phenotypes by integration of signals in the tumor microenvironment. Depending on how macrophages are activated, they may adopt so-called M1-like, antitumor or M2-like, protumor profiles. In many solid tumors, a dominance of M2-like macrophages is associated with poor outcomes but in some tumor types, strong M1-like profiles are linked to better outcomes. We aimed to investigate the interrelationship of these TAM populations to establish how they modulate the efficacy of the adaptive immune system in early lung cancer.MethodsMacrophages from matched lung (non-tumor-associated macrophages (NTAMs)) and tumor samples (TAMs) from resected lung cancers were assessed by bulk and single-cell transcriptomic analysis. Protein expression of genes characteristic of M1-like (chemokine (C-X-C motif) ligand 9) or M2-like (matrix metallopeptidase 12) functions was confirmed by confocal microscopy. Immunohistochemistry related the distribution of TAM transcriptomic signatures to density of CD8+ tissue-resident memory T cells (TRM) in tumors and survival data from an independent cohort of 393 patients with lung cancer.ResultsTAMs have significantly different transcriptomic profiles from NTAMs with >1000 differentially expressed genes. TAMs displayed a strong M2-like signature with no significant variation between patients. However, single-cell RNA-sequencing supported by immuno-stained cells revealed that additionally, in 25% of patients the M2-like TAMs also co-expressed a strong/hot M1-like signature (M1hot). Importantly, there was a strong association between the density of M1hot TAMs and TRM cells in tumors that was in turn linked to better survival. Our data suggest a mechanism by which M1hot TAMs may recruit TRM cells via CXCL9 expression and sustain them by making available more of the essential fatty acids on which TRM depend.ConclusionsWe showed that in early lung cancer, expression of M1-like and M2-like gene signatures are not mutually exclusive since the same TAMs can simultaneously display both gene-expression profiles. The presence of M1hot TAMs was associated with a strong TRM tumor-infiltrate and better outcomes. Thus, therapeutic approaches to re-program TAMs to an M1hot phenotype are likely to augment the adaptive antitumor responses.

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Single-cell transcriptome atlas of lung adenocarcinoma featured with ground glass nodules

Cell Discovery ◽

10.1038/s41421-020-00200-x ◽

2020 ◽

Vol 6 (1) ◽

Author(s):

Tao Lu ◽

Xiaodong Yang ◽

Yu Shi ◽

Mengnan Zhao ◽

Guoshu Bi ◽

...

Keyword(s):

Lung Cancer ◽

T Cells ◽

Lung Adenocarcinoma ◽

Single Cell ◽

Cancer Cells ◽

Signaling Pathways ◽

Stromal Cells ◽

Cell Types ◽

Ground Glass ◽

Lung Carcinogenesis

Abstract As an early type of lung adenocarcinoma, ground glass nodule (GGN) has been detected increasingly and now accounts for most lung cancer outpatients. GGN has a satisfactory prognosis and its characteristics are quite different from solid adenocarcinoma (SADC). We compared the GGN adenocarcinoma (GGN-ADC) with SADC using the single-cell RNA sequencing (scRNA-seq) to fully understand GGNs. The tumor samples of five patients with lung GGN-ADCs and five with SADCs underwent surgery were digested to a single-cell suspension and analyzed using 10× Genomic scRNA-seq techniques. We obtained 60,459 cells and then classified them as eight cell types, including cancer cells, endothelial cells, fibroblasts, T cells, B cells, Nature killer cells, mast cells, and myeloid cells. We provided a comprehensive description of the cancer cells and stromal cells. We found that the signaling pathways related to cell proliferation were downregulated in GGN-ADC cancer cells, and stromal cells had different effects in GGN-ADC and SADC based on the analyses of scRNA-seq results. In GGN-ADC, the signaling pathways of angiogenesis were downregulated, fibroblasts expressed low levels of some collagens, and immune cells were more activated. Furthermore, we used flow cytometry to isolate the cancer cells and T cells in 12 GGN-ADC samples and in an equal number of SADC samples, including CD4+ T and CD8+ T cells, and validated the expression of key molecules by quantitative real-time polymerase chain reaction analyses. Through comprehensive analyses of cell phenotypes in GGNs, we provide deep insights into lung carcinogenesis that will be beneficial in lung cancer prevention and therapy.

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Discriminating mild from critical COVID-19 by innate and adaptive immune single-cell profiling of bronchoalveolar lavages

Cell Research ◽

10.1038/s41422-020-00455-9 ◽

2021 ◽

Author(s):

Els Wauters ◽

◽

Pierre Van Mol ◽

Abhishek Dinkarnath Garg ◽

Sander Jansen ◽

...

Keyword(s):

T Cells ◽

Single Cell ◽

Purinergic Signaling ◽

Significant Proportion ◽

Lung Epithelial Cells ◽

Normal Lung ◽

Host Immune System ◽

T Helper ◽

T Helper 1 ◽

Effector Functions

AbstractHow the innate and adaptive host immune system miscommunicate to worsen COVID-19 immunopathology has not been fully elucidated. Here, we perform single-cell deep-immune profiling of bronchoalveolar lavage (BAL) samples from 5 patients with mild and 26 with critical COVID-19 in comparison to BALs from non-COVID-19 pneumonia and normal lung. We use pseudotime inference to build T-cell and monocyte-to-macrophage trajectories and model gene expression changes along them. In mild COVID-19, CD8+ resident-memory (TRM) and CD4+ T-helper-17 (TH17) cells undergo active (presumably antigen-driven) expansion towards the end of the trajectory, and are characterized by good effector functions, while in critical COVID-19 they remain more naïve. Vice versa, CD4+ T-cells with T-helper-1 characteristics (TH1-like) and CD8+ T-cells expressing exhaustion markers (TEX-like) are enriched halfway their trajectories in mild COVID-19, where they also exhibit good effector functions, while in critical COVID-19 they show evidence of inflammation-associated stress at the end of their trajectories. Monocyte-to-macrophage trajectories show that chronic hyperinflammatory monocytes are enriched in critical COVID-19, while alveolar macrophages, otherwise characterized by anti-inflammatory and antigen-presenting characteristics, are depleted. In critical COVID-19, monocytes contribute to an ATP-purinergic signaling-inflammasome footprint that could enable COVID-19 associated fibrosis and worsen disease-severity. Finally, viral RNA-tracking reveals infected lung epithelial cells, and a significant proportion of neutrophils and macrophages that are involved in viral clearance.

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Green synthesis of nanomaterials for early lung cancer detection

Journal of Cancer Science and Therapeutics ◽

10.36879/jcst.18.000102 ◽

2018 ◽

pp. 1-1

Keyword(s):

Lung Cancer ◽

Green Synthesis ◽

Cancer Detection ◽

World Wide ◽

Early Stage ◽

Age Groups ◽

Early Lung Cancer ◽

Lung Cancer Detection ◽

Synthesis Of Nanomaterials

Lung cancer is the foremost cause of cancer-related deaths world-wide [1]. It affects 100,000 Americans of the smoking population every year of all age groups, particularly those above 50 years of the smoking population [2]. In India, 51,000 lung cancer deaths were reported in 2012, which include 41,000 men and 10,000 women [3]. It is the leading cause of cancer deaths in men; however, in women, it ranked ninth among all cancerous deaths [4]. It is possible to detect the lung cancer at a very early stage, providing a much higher chance of survival for the patients.

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P09.05 Plasma CD27, a surrogate of intratumoral CD27-CD70 interaction, correlates with immunotherapy resistance in renal cancer

Journal for ImmunoTherapy of Cancer ◽

10.1136/jitc-2021-itoc8.55 ◽

2021 ◽

Vol 9 (Suppl 1) ◽

pp. A30.1-A30

Author(s):

N Benhamouda ◽

I Sam ◽

N Epaillard ◽

A Gey ◽

A Saldmann ◽

...

Keyword(s):

T Cells ◽

T Cell ◽

Tumor Microenvironment ◽

Single Cell ◽

Solid Tumors ◽

Tumor Cells ◽

Principal Investigator ◽

Cell Phenotype ◽

Metastatic Rcc ◽

Research Grant

BackgroundCD70, a costimulatory molecule on antigen presenting cells, is known to activate CD27-expressing T cells. CD27-CD70 interaction leads to the release of soluble CD27 (sCD27). However, persistent interaction of CD27 and CD70 such as in chronic infection may exhaust the T cell pool and promote apoptosis. Surprisingly, our analysis based on TCGA database show that clear cell renal cell carcinoma (ccRCC) expresses the highest levels of CD70 among all solid tumors. Despite the important clinical efficacy of immunotherapy by anti-PD-1 in RCC patients, the overall response to anti-PD1 remains modest. The relationship between the CD27-CD70 interaction in the RCC and the response to immunotherapy is still unclear.Materials and MethodsTo study the CD27 and CD70 expression in the tumor microenvironment (TME), FFPE tumor tissues from 25 RCC patients were analysed using multiplex in situ immunofluorescence. 10 fresh RCC tumor samples were collected to analyse the phenotype of CD27+ T cells by flow cytometry and 4 samples were proceeded for single-cell RNA-seq analysis. A cohort of metastatic RCC patients (n = 35) treated by anti-PD-1 were enrolled for the measurement of plasma sCD27 by ELISA and the survival analysis is also realized.ResultsIn the TME, we demonstrated that CD27+ T cells interact with CD70-expressing tumor cells. In fresh tumors from RCC patients, CD27+ T cells express higher levels of cleaved caspase 3 (a classical marker of apoptosis) than CD27- T cells. We confirmed the apoptotic signature (BAX, FASLG, BCL2L11, CYCS, FBXO32, LGALS1, PIK3R1, TERF1, TXNIP, CDKN2A) of CD27+ T cells by single-cell RNAseq analysis. CD27+T cells also had a tissue resident memory T cell phenotype with enriched gene expression of ITGAE, PRDM1, RBPJ and ZNF683. Moreover, CD27+T cells display an exhaustion phenotype with the expression of multiple inhibitory receptors gene signature (PDCD1, CTLA4, HAVCR2, LAG3, etc). Besides, intratumoral CD27-CD70 interaction significantly correlates with plasma sCD27 concentration in RCC (p = 0.0017). In metastatic RCC patients treated with anti-PD-1, higher levels of sCD27 predict poor overall survival (p = 0.037), while it did not correlate with inflammatory markers or clinical prognostic criteria.ConclusionsIn conclusion, we demonstrated that sCD27, a surrogate of T cell dysfunction in tumors likely induced by persistent interactions of CD27+T cells and CD70-expressing tumor cells, is a predictive biomarker of resistance to immunotherapy in mRCC. To our knowledge, this is the first report showing that a peripheral blood biomarker may reflect certain aspects of the tumor-host interaction in the tumor microenvironment. Given the frequent expression of CD70 and CD27 in solid tumors, our findings may be further extended to other types of tumors. CD70-CD27 interaction could thus be considered as a mechanism of tumor escape, but also a novel therapeutic target in cancers.Disclosure InformationN. Benhamouda: None. I. Sam: None. N. Epaillard: None. A. Gey: None. A. Saldmann: None. J. Pineau: None. M. Hasan: None. V. Verkarre: None. V. Libri: None. S. Mella: None. C. Granier: None. C. Broudin: None. P. Ravel: None. B. Jabla: None. N. Chaput: None. L. Albiges: None. Y. Vano: None. O. Adotevi: None. S. Oudard: B. Research Grant (principal investigator, collaborator or consultant and pending grants as well as grants already received); Modest; SIRIC CARPEM, FONCER. E. Tartour: B. Research Grant (principal investigator, collaborator or consultant and pending grants as well as grants already received); Modest; Fondation ARC, INCA PLBio, Labex Immuno-Oncology, SIRIC CARPEM, FONCER, IDEX université de Paris, Inserm Transfert.

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Single-cell RNA Sequencing Reveals Immunosuppressive Myeloid Cell Diversity and Restricted Cytotoxic Effector Cell Trafficking and Activation During Malignant Progression in Glioma

10.1101/2021.09.24.461735 ◽

2021 ◽

Author(s):

Sakthi Rajendran ◽

Clayton Peterson ◽

Alessandro Canella ◽

Yang Hu ◽

Amy Gross ◽

...

Keyword(s):

T Cells ◽

Tumor Microenvironment ◽

Single Cell ◽

Immune Cell ◽

Myeloid Cell ◽

Malignant Progression ◽

Low Grade ◽

Cell Trafficking ◽

High Grade ◽

Single Cell Rna Sequencing

Low grade gliomas (LGG) account for about two-thirds of all glioma diagnoses in adolescents and young adults (AYA) and malignant progression of these patients leads to dismal outcomes. Recent studies have shown the importance of the dynamic tumor microenvironment in high-grade gliomas (HGG), yet its role is still poorly understood in low-grade glioma malignant progression. Here, we investigated the heterogeneity of the immune microenvironment using a platelet-derived growth factor (PDGF)-driven RCAS (replication-competent ASLV long terminal repeat with a splice acceptor) glioma model that recapitulates the malignant progression of low to high-grade glioma in humans and also provides a model system to characterize immune cell trafficking and evolution. To illuminate changes in the immune cell landscape during tumor progression, we performed single-cell RNA sequencing on immune cells isolated from animals bearing no tumor (NT), LGG and HGG, with a particular focus on the myeloid cell compartment, which is known to mediate glioma immunosuppression. LGGs demonstrated significantly increased infiltrating T cells, CD4 T cells, CD8 T cells, B cells, and natural killer cells in the tumor microenvironment, whereas HGGs significantly abrogated this infiltration. Our study identified two distinct macrophage clusters in the tumor microenvironment; one cluster appeared to be bone marrow-derived while another was defined by overexpression of Trem2, a marker of tumor associated macrophages. Our data demonstrates that these two distinct macrophage clusters show an immune-activated phenotype (Stat1, Tnf, Cxcl9 and Cxcl10) in LGG which evolves to an immunosuppressive state (Lgals3, Apoc1 and Id2) in HGG that restricts T cell recruitment and activation. We identified CD74 and macrophage migration inhibition factor (MIF) as potential targets for these distinct macrophage populations. Interestingly, these results were mirrored by our analysis of the TCGA dataset, which demonstrated a statistically significant association between CD74 overexpression and decreased overall survival in AYA patients with grade II gliomas. Targeting immunosuppressive myeloid cells and intra-tumoral macrophages within this therapeutic window may ameliorate mechanisms associated with immunosuppression before and during malignant progression.

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COL1A1 is a prognostic biomarker and correlated with immune infiltrates in lung cancer

PeerJ ◽

10.7717/peerj.11145 ◽

2021 ◽

Vol 9 ◽

pp. e11145

Author(s):

Qishun Geng ◽

Zhibo Shen ◽

Lifeng Li ◽

Jie Zhao

Keyword(s):

Lung Cancer ◽

T Cells ◽

Dendritic Cell ◽

Cd4 T Cells ◽

Malignant Tumors ◽

Functional Enrichment ◽

Expression Level ◽

Normal Lung ◽

Hub Genes ◽

Clinical Prognosis

Objective Lung cancer (LC) is one of the top ten malignant tumors and the first leading cause of cancer-related death among both men and women worldwide. It is imperative to identify immune-related biomarkers for early LC diagnosis and treatment. Methods Three Gene Expression Omnibus (GEO) datasets were selected to acquire the differentially expressed genes(DEGs) between LC and normal lung samples through GEO2R tools of NCBI. To identify hub genes, the DEGs were performed functional enrichment analysis, the protein–protein interaction (PPI) network construction, and Lasso regression. Then, a nomogram was constructed to predict the prognosis of patients with carcinoma based on hub genes. We further evaluated the influence of COL1A1 on clinical prognosis using GSE3141, GSE31210, and TCGA database. Also, the correlations between COL1A1 and cancer immune infiltrates and the B7-CD28 family was investigated via TIMER and GEPIA. Further analysis of immunohistochemistry shown that the COL1A1 expression level is positively correlated with CD276 expression level. Results By difference analysis, there were 340 DEGs between LC and normal lung samples. Then, we picked out seven hub genes, which were identified as components of the risk signature to divide LC into low and high-risk groups. Among them, the expression of COL1A1 is highly correlated with overall survival(OS) and progression-free survival (PFS) (p < 0.05). Importantly, there is a moderate to strong positive relationships between COL1A1 expression level and infiltration level of CD4+ T cells, Macrophage, Neutrophil, and Dendritic cell, as well as CD276 expression level. Conclusion These findings suggest that COL1A1 is correlated with prognosis and immune infiltrating levels, including CD4+ T cells, Macrophage, Neutrophil, and Dendritic cell, as well as CD276 expression level, indicating COL1A1 can be a potential immunity-related biomarker and therapeutic target in LC.

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Unbiased Selection of Peptide–Peptoid Hybrids Specific for Lung Cancer Compared to Normal Lung Epithelial Cells

ACS Chemical Biology ◽

10.1021/acschembio.5b00592 ◽

2015 ◽

Vol 10 (12) ◽

pp. 2891-2899 ◽

Cited By ~ 15

Author(s):

Jaya M. Matharage ◽

John D. Minna ◽

Rolf A. Brekken ◽

D. Gomika Udugamasooriya

Keyword(s):

Lung Cancer ◽

Epithelial Cells ◽

Lung Epithelial Cells ◽

Normal Lung ◽

Lung Epithelial ◽

Selection Of

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The Nutritional Cytokine Leptin Promotes NSCLC by Activating the PI3K/AKT and MAPK/ERK Pathways in NSCLC Cells in a Paracrine Manner

BioMed Research International ◽

10.1155/2019/2585743 ◽

2019 ◽

Vol 2019 ◽

pp. 1-8 ◽

Cited By ~ 1

Author(s):

Fengzhou Li ◽

Shilei Zhao ◽

Tao Guo ◽

Jinxiu Li ◽

Chundong Gu

Keyword(s):

Lung Cancer ◽

Tumor Microenvironment ◽

Western Blot ◽

Signaling Pathways ◽

Cancer Progression ◽

Molecular Mechanisms ◽

Lung Fibroblasts ◽

Normal Lung ◽

Serum Leptin ◽

Coculture System

Purpose.Leptin is a nutritional cytokine encoded by the obesity gene whose concentration in the tumor microenvironment is closely related to the occurrence and progression of cancer. However, previous evidence has suggested that there is no clear relationship between serum leptin concentrations and lung cancer progression. Cancer-associated fibroblasts (CAFs), the most abundant component of the tumor microenvironment in a variety of solid tumors, were recently reported to produce leptin. Therefore, it was inferred that leptin is most likely to affect non-small-cell lung cancer (NSCLC) through an autocrine and paracrine mechanism. In the current study, we investigated the paracrine effect and mechanism of leptin produced by CAFs on NSCLC by establishing a novel in vitro cell coculture system.Methods.A noncontact coculture device was designed and made by 3D printing. CAFs and paired normal lung fibroblasts (NLFs) from 5 patients were successfully isolated and cocultured with two NSCLC cell lines in a coculture system. The background expression of leptin was detected by western blot. The in situ expression of leptin and its receptor (Ob-R) in NSCLC tissues and paired normal lung tissues was analyzed by immunohistochemistry. Furthermore, we downregulated the expression of leptin in CAFs and assessed changes in its promotion on NSCLC cells in the coculture system. Finally, changes in the phosphorylation of ERK1/2 and AKT were examined to investigate the molecular mechanisms responsible for the paracrine promotion of NSCLC cells by leptin.Results.Leptin was overexpressed in nearly all five primary CAF lines compared with its expression in paired NLFs. IHC staining showed that the expression of leptin was high in NSCLC cells, slightly lower in CAF, and negative in normal lung tissue. Ob-R was strongly expressed in NSCLC cells. The ability of A549 and H1299 cells to proliferate and migrate was enhanced by high leptin levels in both the cocultured fibroblasts and the culture medium. Furthermore, western blot assays suggested that the MAPK/ERK1/2 and PI3K/AKT signaling pathways were activated by leptin produced by CAFs, which demonstrated that the functions of paracrine leptin in NSCLC are as those of the serum leptin to other cancers.Conclusion.Leptin produced by CAF promotes proliferation and migration of NSCLC cells probably via PI3K/AKT and MAPK/ERK1/2 signaling pathways in a paracrine manner.

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