Antibody‐based cell‐surface proteome profiling of metastatic breast cancer primary explants and cell lines

Donnenberg, Vera S.; Zhang, Jayce Jieming; Moravcikova, Erika; Meyer, E. Michael; Lu, Haihui; Carson, Christian T.; Donnenberg, Albert D.

doi:10.1002/cyto.a.23300

Cited by 14 publications

(16 citation statements)

References 32 publications

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“…In our previously published studies on adherent cells , we noted many strongly positive markers after trypsinization. As a follow‐up, we examined the relationship between number of extracellular trypsin sites and intensity of expression of 10 highly expressed markers using 11 adherent cell lines or primary cultures studied at passage 5.…”

Section: Resultsmentioning

confidence: 53%

“…(A) Fluorescence intensity of 10 markers highly expressed on adherent cells after trypsin treatment. Data on adherent cells (panel A) were previously published . A549 and HMLER‐Twist cells were each assayed in independent triplicates.…”

Section: Resultsmentioning

confidence: 99%

“…When these cases were eliminated, the association disappeared. When we revisited the data that we collected on highly expressed markers (≥95%) in trypsin treated adherent cells (11), there was no correlation between the MFI (after trypsinization) and the number of high probability extracellular trypsin cleavage sites (Fig. 6A).…”

Section: Discussionmentioning

confidence: 99%

“…The treatment protocol was based on the procedure that we have used to detach cultured epithelial cells (0.25% trypsin, 2.21 mM EDTA, 5 min at 37°C). We use an even milder treatment to detach mesenchymal cells (0.02%–0.08% trypsin, 2 mM EDTA ). Corver et al compared the effects of EDTA (5 mM) with or without trypsin (0.25%) on 9 surface markers expressed by ovarian cancer cell lines .…”

Section: Discussionmentioning

confidence: 99%

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Flow cytometric detection of most proteins in the cell surface proteome is unaffected by trypsin treatment

et al. 2018

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Flow cytometry is often performed on adherent cells or solid tissues that have been released from their growth substrate or disaggregated by enzymatic digestion. Although detection of strongly expressed cell surface proteins following such procedures indicates that many survive treatment with proteolytic enzymes, applications such as cell surface proteomics involve assessment of the expression of more than 200 proteins and it is important to know how to interpret negative results. To address this problem, we performed flow cytometry-based cell surface proteomic analysis on two non-adherent cell lines, THP1 and K562, after mock and authentic trypsin treatment, according to a widely used protocol to remove adherent cells (0.25% trypsin, 2.21 mM EDTA, 37°C, 5 min). In a single screening experiment, we examined the effect of treatment on mean fluorescence intensity and on the percent of positive cells and determined the false negative rate. Of 164 determinations that were ≥20% positive after mock treatment, 13 (7.9%) were <20% positive after trypsin treatment. Four proteins were chosen for time-course studies (performed in triplicate), confirming initial sensitivity results but revealing significant variability in the magnitude of the trypsin effect. When trypsin sensitivity of individual proteins was examined as a function of the number of predicted high probability extracellular trypsin cleavage sites, we found that the markers that yielded false negatives all had high numbers of sites (>30), but even so, the majority of proteins with high numbers of trypsin sites could still be detected after mild trypsin treatment. We conclude that the great majority of cell surface proteins can be detected after mild trypsin treatment, but that negative results should not be over-interpreted, due to the possibility of false negatives.

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Section: Resultsmentioning

confidence: 53%

Section: Resultsmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

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Flow cytometric detection of most proteins in the cell surface proteome is unaffected by trypsin treatment

et al. 2018

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“…Importantly, critical evaluation of all aspects of cell treatment is pivotal for single cell proteomics or in future maybe also genomics by flow cytometry. Donnenberg et al (this issue, p. 803) evaluated the impact of trypsin treatment on adherent or tissue cells for flow cytometric surface proteome determination.…”

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confidence: 99%

Shapiro's seventh law

Tárnok

2018

Cytometry Pt A

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Identification of inhibitory immune checkpoints and relevant regulatory pathways in breast cancer stem cells

Shi

Yang

2021

Cancer Medicine

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Immune checkpoint blockade (ICB) has become one of the most promising approaches to activating antitumor immunity. However, only a small subset of patients with breast cancer benefit from ICB treatment. To improve the therapeutic effect in the clinic, precision immunotherapy is proposed to accurately eliminate cancer stem cells that contribute to local recurrence or metastasis, but currently little is known about their immunological properties. In this study, breast cancer–specific datasets in The Cancer Genome Atlas were collected and analyzed by using multiple open‐access web servers. We found that the immunophenotype of breast cancer was characterized by a hypoactive immune microenvironment and a low response to immune checkpoint blockade. The innate immune checkpoint CD200 and the adaptive immune checkpoint CD276, respectively, exhibited a strong correlation with basal/stem gene signature and invasiveness gene signature, both of which represent breast cancer stem cells. Wnt, TGF‐β, and Hedgehog signaling, which are recognized as stemness‐related pathways, showed a significant association with the expression of CD200 and CD276, suggesting cancer stem cell–specific immune checkpoints could be downregulated by inhibiting these pathways. Of note, levels of CD200 and CD276 expression were higher in TGF‐β dominant breast cancer than in other immune types of breast cancer. We also identified gene signatures that represent Wnt, TGF‐β, and Hedgehog signaling‐related CD200 and CD276 expression in breast cancer stem cells. For the luminal A subtype, the patient group with a high level of these gene signatures plus a low infiltration of CD8+ T cells, or dendritic cells, or M1 macrophages had poor overall survival. Our study suggested that CD200 and CD276 are candidate inhibitory immune checkpoints in breast cancer stem cells, which are potentially regulated by Wnt, TGF‐β, and Hedgehog signaling. Synergistic inhibition of these stemness‐related pathways may improve the efficacy of ICB treatment targeting breast cancer stem cells in precision immunotherapy.

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Antibody‐based cell‐surface proteome profiling of metastatic breast cancer primary explants and cell lines

Cited by 14 publications

References 32 publications

Flow cytometric detection of most proteins in the cell surface proteome is unaffected by trypsin treatment

Flow cytometric detection of most proteins in the cell surface proteome is unaffected by trypsin treatment

Shapiro's seventh law

Identification of inhibitory immune checkpoints and relevant regulatory pathways in breast cancer stem cells

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