β1 integrin regulation

Integrins are heterodimeric adhesion receptors mediating adhesion to extracellular matrix proteins and to other cells. Integrins are important in embryonic development, structural integrity of connective tissue, blood thrombus formation, and immune defense system. Integrins are transmembrane proteins whose ligand binding capacity (activity) is regulated by large conformational changes. Extracellular ligand binding or intracellular effector binding to integrin cytoplasmic face regulate integrin activity. Integrins are thus able to mediate bi-directional signaling. Integrin function is also regulated by intracellular location. Integrins are constantly recycled from endocytic vesicles to plasma membrane, and this has been shown to be important for cell migration and invasion as well. Deregulation of integrin functionality can lead to deleterious illnesses, such as bleeding or inflammatory disorders. It is also evident that integrin deregulation is associated with cancer progression. In this study, a novel Beta1 integrin associating protein, Rab21, was characterized. Rab21 binding to integrin cytoplasmic tail was shown to be important for Beta1 integrin endo- and exocytosis – intracellular trafficking. It was furher shown that this interaction has an important role in cell adhesion, migration, as well as in the final step of cell division, cytokinesis. This work showed that abrogation of Rab21 function or β1 integrin endocytic traffic, can lead to defects in cell division and results in formation of multinucleated cells. Multinucleation and especially tetraploidy can be a transient pathway to aneuploidy and tumorigenesis. This work characterized chromosomal deletions in rab21 locus in ovarian and prostate cancer samples and showed that a cell line with rab21 deletion also had impairment in cell division, which could be rescued by Rab21 re-expression. The work demonstrates an important role for Rab21 and Beta1 integrin traffic regulation in cell adhesion and division, and suggests a probable associaton with tumorigenesis. In this study, Beta1 integrin activity regulation was also addressed. A novel cell array platform for genome-scale RNAi screenings was characterized here. More than 4500 genes were knocked-down in prostate cancer cells using siRNA-mediated silencing. The effects on Beta1 integrin activity were analyzed upon knock-downs. The screen identified more that 400 putative regulators of Beta1 integrin activity in prostate cancer. In conclusion, this work will help us to understand complex regulatory pathways involved in cancer cell adhesion and migration.Siirretty Doriast

Clinical Impact of Tumor-Associated Macrophage and T-Cell Contents in Diffuse Large B-Cell Lymphoma

Peer reviewe

Small GTPase Rab21 regulates cell adhesion and controls endosomal traffic of β1-integrins

Dynamic turnover of integrin cell adhesion molecules to and from the cell surface is central to cell migration. We report for the first time an association between integrins and Rab proteins, which are small GTPases involved in the traffic of endocytotic vesicles. Rab21 (and Rab5) associate with the cytoplasmic domains of α-integrin chains, and their expression influences the endo/exocytic traffic of integrins. This function of Rab21 is dependent on its GTP/GDP cycle and proper membrane targeting. Knock down of Rab21 impairs integrin-mediated cell adhesion and motility, whereas its overexpression stimulates cell migration and cancer cell adhesion to collagen and human bone. Finally, overexpression of Rab21 fails to induce cell adhesion via an integrin point mutant deficient in Rab21 association. These data provide mechanistic insight into how integrins are targeted to intracellular compartments and how their traffic regulates cell adhesion

Clinical Impact of Immune Cells and Their Spatial Interactions in Diffuse Large B-Cell Lymphoma Microenvironment

Purpose: Tumor-infiltrating immune cells have prognostic sig-nificance and are attractive therapeutic targets. Yet, the clinical significance of their spatial organization and phenotype in diffuse large B-cell lymphoma (DLBCL) is unclear. Experimental Design: We characterized T cells, macrophages, and their spatial interactions by multiplex IHC (mIHC) in 178 patients with DLBCL and correlated the data with patient demo-graphics and survival. We validated the findings on gene expression data from two external DLBCL cohorts comprising 633 patients. Results: Macrophage and T-cell contents divided the samples into T cell-inflamed (60%) and noninflamed (40%) subgroups. The T cell-inflamed lymphoma microenvironment (LME) was also rich in other immune cells, defining immune hot phenotype, which did not as such correlate with outcome. However, when we divided the patients according to T-cell and macrophage contents, LME char-acterized by high T-cell/low macrophage content or a correspond-ing gene signature was associated with superior survival [5-year overall survival (OS): 92.3% vs. 74.4%, P = 0.036; 5-year progres-sion-free survival (PFS): 92.6% vs. 69.8%, P = 0.012]. High pro-portion of PD -L1-and TIM3-expressing CD163- macrophages in the T cell-inflamed LME defined a group of patients with poor outcome [OS: HR = 3.22, 95% confidence interval (CI), 1.63-6.37, P-adj = 0.011; PFS: HR = 2.76, 95% CI, 1.44-5.28, P-adj = 0.016]. Furthermore, PD-L1 and PD-1 were enriched on macrophages interacting with T cells. Conclusions: Our data demonstrate that the interplay between macrophages and T cells in the DLBCL LME is immune checkpoint dependent and clinically meaningful.Peer reviewe

Adverse prognostic impact of regulatory T-cells in testicular diffuse large B-cell lymphoma

Objectives Testicular diffuse large B-cell lymphoma (T-DLBCL) is a rare and aggressive extranodal lymphoma. We have previously shown that high content of tumor-infiltrating lymphocytes (TILs) and PD-1 expressing TILs associate with better survival in T-DLBCL. In this study, we have further characterized distinct TIL subtypes and their proportions in association with patient demographics and survival. Methods We used multiplex immunohistochemistry to characterize TIL phenotypes, including cytotoxic T-cells (CTLs; CD8(+), OX40(+), Granzyme B+, Ki-67(+), LAG-3(+), TIM-3(+), PD-1(+)), CD4(+)T-cells (CD3(+), CD4(+), TIM-3(+), LAG-3(+)), regulatory T-cells (Tregs; CD3(+), CD4(+), FoxP3(+)), and T helper 1 cells (Th1; CD3(+), CD4(+), T-bet(+)) in 79 T-DLBCLs, and correlated the findings with patient demographics and outcome. Results We observed a substantial variation in TIL phenotypes between the patients. The most prominent CD8(+)TILs were Ki-67(+)and TIM-3(+)CTLs, whereas the most prominent CD4(+)TILs were FoxP3(+)Tregs. Despite the overall favorable prognostic impact of high TIL content, we found a subpopulation of T-bet(+)FoxP3(+)Tregs that had a significant adverse impact on survival. Lower content of CTLs with activated or exhausted phenotypes correlated with aggressive clinical features. Conclusions Our results demonstrate significant variation in TIL phenotypes and emphasize the adverse prognostic impact of Tregs in T-DLBCL.Peer reviewe

Prognostic Impact of Tumor-Associated Macrophages on Survival Is Checkpoint Dependent in Classical Hodgkin Lymphoma

Tumor microenvironment and immune escape affect pathogenesis and survival in classical Hodgkin lymphoma (cHL). While tumor-associated macrophage (TAM) content has been associated with poor outcomes, macrophage-derived determinants with clinical impact have remained undefined. Here, we have used multiplex immunohistochemistry and digital image analysis to characterize TAM immunophenotypes with regard to expression of checkpoint molecules programmed cell death ligand 1 (PD-L1) and indoleamine 2,3-dioxygenase 1 (IDO-1) from the diagnostic tumor tissue samples of 130 cHL patients, and correlated the findings with clinical characteristics and survival. We show that a large proportion of TAMs express PD-L1 (CD68+, median 32%; M2 type CD163+, median 22%), whereas the proportion of TAMs expressing IDO-1 is lower (CD68+, median 5.5%; CD163+, median 1.4%). A high proportion of PD-L1 and IDO-1 expressing TAMs from all TAMs (CD68+), or from CD163+ TAMs, is associated with inferior outcome. In multivariate analysis with age and stage, high proportions of PD-L1+ and IDO-1+ TAMs remain independent prognostic factors for freedom from treatment failure (PD-L1+CD68+/CD68+, HR = 2.63, 95% CI 1.17–5.88, p = 0.019; IDO-1+CD68+/CD68+, HR = 2.48, 95% CI 1.03–5.95, p = 0.042). In contrast, proportions of PD-L1+ tumor cells, all TAMs or PD-L1− and IDO-1− TAMs are not associated with outcome. The findings implicate that adverse prognostic impact of TAMs is checkpoint-dependent in cHL

Checkpoint protein expression in the tumor microenvironment defines the outcome of classical Hodgkin lymphoma patients

Emerging evidence indicates a major impact for the tumor microenvironment (TME) and immune escape in the pathogenesis and clinical course of classical Hodgkin lymphoma (cHL). We used gene expression profiling (n = 88), CIBERSORT, and multiplex immunohistochemistry (n = 131) to characterize the immunoprofile of cHL TME and correlated the findings with survival. Gene expression analysis divided tumors into subgroups with T cell-inflamed and -noninflamed TME. Several macrophage-related genes were upregulated in samples with the non-T cell-inflamed TME, and based on the immune cell proportions, the samples clustered according to the content of T cells and macrophages. A cluster with high proportions of checkpoint protein (programmed cell death protein 1, PD-1 ligands, indoleamine 2,3 dioxygenase 1, lymphocyte-activation gene 3, and T-cell immunoglobulin and mucin domain containing protein 3) positive immune cells translated to unfavorable overall survival (OS) (5-year OS 76% vs 96%; P = .010) and remained an independent prognostic factor for OS in multivariable analysis (HR, 4.34; 95% CI, 1.05-17.91; P = .043). cHL samples with high proportions of checkpoint proteins overexpressed genes coding for cytolytic factors, proposing paradoxically that they were immunologically active. This checkpoint molecule gene signature translated to inferior survival in a validation cohort of 290 diagnostic cHL samples (P < .001) and in an expan-sion cohort of 84 cHL relapse samples (P = .048). Our findings demonstrate the impact of T cell-and macrophage-mediated checkpoint system on the survival of patients with cHL.Peer reviewe

Systems pathology by multiplexed immunohistochemistry and whole-slide digital image analysis

The paradigm of molecular histopathology is shifting from a single-marker immunohistochemistry towards multiplexed detection of markers to better understand the complex pathological processes. However, there are no systems allowing multiplexed IHC (mIHC) with high-resolution whole-slide tissue imaging and analysis, yet providing feasible throughput for routine use. We present an mIHC platform combining fluorescent and chromogenic staining with automated whole-slide imaging and integrated whole-slide image analysis, enabling simultaneous detection of six protein markers and nuclei, and automatic quantification and classification of hundreds of thousands of cells in situ in formalin-fixed paraffin-embedded tissues. In the first proof-of-concept, we detected immune cells at cell-level resolution (n = 128,894 cells) in human prostate cancer, and analysed T cell subpopulations in different tumour compartments (epithelium vs. stroma). In the second proof-of-concept, we demonstrated an automatic classification of epithelial cell populations (n = 83,558) and glands (benign vs. cancer) in prostate cancer with simultaneous analysis of androgen receptor (AR) and alpha-methylacyl-CoA (AMACR) expression at cell-level resolution. We conclude that the open-source combination of 8-plex mIHC detection, whole-slide image acquisition and analysis provides a robust tool allowing quantitative, spatially resolved whole-slide tissue cytometry directly in formalin-fixed human tumour tissues for improved characterization of histology and the tumour microenvironment.Peer reviewe

CDX2 Loss With Microsatellite Stable Phenotype Predicts Poor Clinical Outcome in Stage II Colorectal Carcinoma

Current risk factors in stage II colorectal carcinoma are insufficient to guide treatment decisions. Loss of CDX2 has been shown to associate with poor clinical outcome and predict benefit for adjuvant chemotherapy in stage II and III colorectal carcinoma. The prognostic relevance of CDX2 in stage II disease has not been sufficiently validated, especially in relation to clinical risk factors, such as microsatellite instability (MSI) status, BRAF mutation status, and tumor budding. In this study, we evaluated the protein expression of CDX2 in tumor center and front areas in a tissue microarrays material of stage II colorectal carcinoma patients (n=232). CDX2 expression showed a partial or total loss in respective areas in 8.6% and 10.9% of patient cases. Patients with loss of CDX2 had shorter disease-specific survival when scored independently either in tumor center or tumor front areas (log rank P=0.012; P=0.012). Loss of CDX2 predicted survival independently of other stage II risk factors, such as MSI status and BRAF mutation status, pT class, and tumor budding (hazard ratio=5.96, 95% confidence interval=1.55-22.95; hazard ratio=3.70, 95% confidence interval=1.30-10.56). Importantly, CDX2 loss predicted inferior survival only in patients with microsatellite stable, but not with MSI-high phenotype. Interestingly, CDX2 loss associated with low E-cadherin expression, tight junction disruption, and high expression of ezrin protein. The work demonstrates that loss of CDX2 is an independent risk factor of poor disease-specific survival in stage II colorectal carcinoma. Furthermore, the study suggests that CDX2 loss is linked with epithelial-to-mesenchymal transition independently of tumor budding.Peer reviewe

Digital image analysis of multiplex fluorescence IHC in colorectal cancer recognizes the prognostic value of CDX2 and its negative correlation with SOX2

Flourescence-based multiplex immunohistochemistry (mIHC) combined with multispectral imaging and digital image analysis (DIA) is a quantitative high-resolution method for determination of protein expression in tissue. We applied this method for five biomarkers (CDX2, SOX2, SOX9, E-cadherin, and beta-catenin) using tissue microarrays of a Norwegian unselected series of primary colorectal cancer. The data were compared with previously obtained chromogenic IHC data of the same tissue cores, visually assessed by the Allred method. We found comparable results between the methods, although confirmed that DIA offered improved resolution to differentiate cases with high and low protein expression. However, we experienced inherent challenges with digital image analysis of membrane staining, which was better assessed visually. DIA and mIHC enabled quantitative analysis of biomarker coexpression on the same tissue section at the single-cell level, revealing a strong negative correlation between the differentiation markers CDX2 and SOX2. Both methods confirmed known prognostic associations for CDX2, but DIA improved data visualization and detection of clinicopathological and biological associations. In summary, mIHC combined with DIA is an efficient and reliable method to evaluate protein expression in tissue, here shown to recapitulate and improve detection of known clinicopathological and survival associations for the emerging biomarker CDX2, and is therefore a candidate approach to standardize CDX2 detection in pathology laboratories.Peer reviewe