Multilabel immunofluorescence and antigen reprobing on formalin-fixed paraffin-embedded sections: novel applications for precision pathology diagnosis

Pan, Jie; Thoeni, Cornelia; Muise, Aleixo M.; Yeger, Herman; Cutz, Ernest

doi:10.1038/modpathol.2016.52

Cited by 17 publications

(15 citation statements)

References 24 publications

(42 reference statements)

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“…Accordingly, re-assessing Cx expression along with a deep characterization of immune cell infiltration in human tumors may, in our opinion, definitively solve the complexity of the mixed findings related to Cxs as a useful diagnostic method (Table 1 ). Precision-medicine diagnostic tools, such as multilabel immunofluorescence on formalin-fixed paraffin-embedded sections ( 195 ) are suitable for evaluation of Cx expression and localization in different cells from the tumor microenvironment. Although much of the data discussed in this review come from in vivo studies, several of the most exciting findings remains to be validated on accurate and specific physiological models.…”

Section: Discussionmentioning

confidence: 99%

Mind the Gaps in Tumor Immunity: Impact of Connexin-Mediated Intercellular Connections

et al. 2017

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Gap junctions (GJs)-mediated intercellular communications (GJICs) are connexin (Cx)-formed plasma membrane channels that allow for the passage of small molecules between adjacent cells, and are involved in several physiopathological processes, including immune responses against cancer. In general, tumor cells are poorly coupled through GJs, mainly due to low Cx expression or reduced channel activity, suggesting that Cxs may have tumor suppressor roles. However, more recent data indicate that Cxs and/or GJICs may also in some cases promote tumor progression. This dual role of Cx channels in tumor outcome may be due, at least partially, to the fact that GJs not only interconnect cells from the same type, such as cancer cells, but also promote the intercellular communication of tumor cells with different types of cells from their microenvironment, and such diverse intercellular interactions have distinctive impact on tumor development. For example, whereas GJ-mediated interactions among tumor cells and microglia have been implicated in promotion of tumor growth, tumor cells delivery to dendritic cells of antigenic peptides through GJs have been associated with enhanced immune-mediated tumor elimination. In this review, we provide an updated overview on the role of GJICs in tumor immunity, focusing on the pro-tumor and antitumor effect of GJs occurring among tumor and immune cells. Accumulated data suggest that GJICs may act as tumor suppressors or enhancers depending on whether tumor cells interact predominantly with antitumor immune cells or with stromal cells. The complex modulation of immune-tumor cell GJICs should be taken into consideration in order to potentiate current cancer immunotherapies.

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Section: Discussionmentioning

confidence: 99%

Mind the Gaps in Tumor Immunity: Impact of Connexin-Mediated Intercellular Connections

et al. 2017

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“…These hyperplastic/hypertrophic effects are reminiscent of the effects of chronic hypoxia on NEBs in vivo and in vitro 9. The use of our novel method of multilabel immunofluorescence on formalin-fixed, paraffin-embedded sections permitted visualization of both cytoplasmic and nuclear epitopes in the same cells and thus was instrumental for the assessment of potential mechanisms of NEB cell hyperplasia 14. Our findings suggest that differentiation from precursor cells is the predominant mechanism for NEB cell hyperplasia as documented by increased expression of Mash1 gene, a critical transcription factor for PNEC/NEB cell differentiation 15.…”

Section: Discussionmentioning

confidence: 99%

“…The stripping of the previous immunolabeled target and re-probing for a new antigen on the same section was performed according to previously reported procedures 14. Briefly, the slides were dipped in 100% xylene for a few seconds to remove the sealant and the coverslip.…”

Section: Methodsmentioning

confidence: 99%

“…To study the potential mechanisms of NEB cell hyperplasia, we used a novel immunohistochemical method applied to formalin-fixed paraffin sections allowing multilabel immunofluorescence with simultaneous detection of cytoplasmic and nuclear epitopes on the same sections 14. Using this method, we examined the expression of an NEB cell cytoplasmic neuroendocrine cell marker, synaptophysin (SYP), together with nuclear immunolocalization of neurogenic genes: mammalian achete–scute homolog (Mash1), prospero homeobox 1 (Prox1), essential for NEB cell differentiation during development and postnatally;15–17 a proliferation marker Ki67; and hypoxia-inducible factor (HIF)-1alpha, the transcriptional mediator of hypoxia-induced expression of numerous genes involved in O 2 homeostasis 18.…”

Section: Introductionmentioning

confidence: 99%

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Hyperplasia and hypertrophy of pulmonary neuroepithelial bodies, presumed airway hypoxia sensors, in hypoxia-inducible factor prolyl hydroxylase-deficient mice

Pan

Bishop

Ratcliffe

et al. 2016

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Pulmonary neuroepithelial bodies (NEBs), presumed polymodal airway sensors, consist of innervated clusters of amine (serotonin) and peptide-producing cells. While NEB responses to acute hypoxia are mediated by a membrane-bound O2 sensor complex, responses to sustained and/or chronic hypoxia involve a prolyl hydroxylase (PHD)–hypoxia-inducible factor-dependent mechanism. We have previously reported hyperplasia of NEBs in the lungs of Phd1−/− mice associated with enhanced serotonin secretion. Here we use a novel multilabel immunofluorescence method to assess NEB distribution, frequency, and size, together with the number and size of NEB cell nuclei, and to colocalize multiple cytoplasmic and nuclear epitopes in the lungs of Phd1−/−, Phd2+/−, and Phd3−/− mice and compare them with wild-type controls. To define the mechanisms of NEB cell hyperplasia, we used antibodies against Mash1 and Prox1 (neurogenic genes involved in NEB cell differentiation/maturation), hypoxia-inducible factor-1alpha, and the cell proliferation marker Ki67. Morphometric analysis of (% total lung area) immunostaining for synaptophysin (% synaptophysin), a cytoplasmic marker of NEB cells, was significantly increased in Phd1−/− and Phd3−/− mice compared to wild-type mice. In addition, NEB size and the number and size of NEB nuclei were also significantly increased, indicating that deficiency of Phds is associated with striking hyperplasia and hypertrophy of NEBs. In Phd2+/− mice, while mean % synaptophysin was comparable to wild-type controls, the NEB size was moderately increased, suggesting an effect even in heterozygotes. NEBs in all Phd-deficient mice showed increased expression of Mash1, Prox1, Ki67, and hypoxia-inducible factor-1alpha, in keeping with enhanced differentiation from precursor cells and a minor component of cell proliferation. Since the loss of PHD activity mimics chronic hypoxia, our data provide critical information on the potential role of PHDs in the pathobiology and mechanisms of NEB cell hyperplasia that is relevant to a number of pediatric lung disorders.

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“…These samples include healthy control, IBD control with normal GI histology and without validated variants from infants. Patient’s biopsies from Spain (UBR5 patient) and BC (TTC7A patient 44 ) infants were obtained with Ethics approval and informed consent as previously summarized 58 .…”

Section: Methodsmentioning

confidence: 99%

The E3 ubiquitin ligase UBR5 interacts with TTC7A and may be associated with very early onset inflammatory bowel disease

Dhingani

Guo

Pan

et al. 2020

Sci Rep

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Very early onset inflammatory bowel disease (VEOIBD) denotes children with onset of IBD before six years of age. A number of monogenic disorders are associated with VEOIBD including tetratricopeptide repeat domain 7A (TTC7A) deficiency. TTC7A-deficiency is characterized by apoptotic colitis in milder cases with severe intestinal atresia and immunodeficiency in cases with complete loss of protein. We used whole exome sequencing in a VEOIBD patient presenting with colitis characterized by colonic apoptosis and no identified known VEOIBD variants, to identify compound heterozygous deleterious variants in the Ubiquitin protein ligase E3 component N-recognin 5 (UBR5) gene. Functional studies demonstrated that UBR5 co-immunoprecipitates with the TTC7A and the UBR5 variants had reduced interaction between UBR5 and TTC7A. Together this implicates UBR5 in regulating TTC7A signaling in VEOIBD patients with apoptotic colitis.

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Multilabel immunofluorescence and antigen reprobing on formalin-fixed paraffin-embedded sections: novel applications for precision pathology diagnosis

Cited by 17 publications

References 24 publications

Mind the Gaps in Tumor Immunity: Impact of Connexin-Mediated Intercellular Connections

Mind the Gaps in Tumor Immunity: Impact of Connexin-Mediated Intercellular Connections

Hyperplasia and hypertrophy of pulmonary neuroepithelial bodies, presumed airway hypoxia sensors, in hypoxia-inducible factor prolyl hydroxylase-deficient mice

The E3 ubiquitin ligase UBR5 interacts with TTC7A and may be associated with very early onset inflammatory bowel disease

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